Horticultural Reviews (Volume 35)

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HORTICULTURAL REVIEWS Volume 35

edited by

Jules Janick Purdue University

HORTICULTURAL REVIEWS Volume 35

Horticultural Reviews is sponsored by: American Society of Horticultural Science International Society for Horticultural Science

Editorial Board, Volume 35 Marie-Christine Daunay Ian Merwin Ed Stover

HORTICULTURAL REVIEWS Volume 35

edited by

Jules Janick Purdue University

Copyright # 2009 by All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientiﬁc, Technical, and Medical business with Blackwell Publishing. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and speciﬁcally disclaim any implied warranties of merchantability or ﬁtness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of proﬁt or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publication Data: ISBN 978-0470-38642-2 ISSN 0163-7851 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

Contents

Contributors Dedication 1. Genetic Resources and Domestication of Macadamia

ix xiii 1

Craig M. Hardner, Cameron Peace, Andrew J. Lowe, Jodi Neal, Phillip Pisanu, Michael Powell, Adele Schmidt, Chris Spain, and Kristen Williams I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Wild Genetic Resources Germplasm Domestication Genetics of Key Scion Selection Traits Propagation and Rootstock Traits Cultivar Utilization Summary Acknowledgments Literature Cited

2. Pomegranate: Botany, Horticulture, Breeding

4 8 32 56 92 95 105 108 108

127

D. Holland, K. Hatib, and I. Bar-Ya’akov I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Taxonomy and Morphology Origin and Genetic Resources Horticulture Breeding Health Beneﬁts Concluding Remarks Acknowledgments Literature Cited

128 129 134 141 172 175 177 178 178

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CONTENTS

3. Daylily: Botany, Propagation, Breeding

193

Surinder K. Gulia, Bharat P. Singh, Johnny Carter, and Robert J. Griesbach I. II. III. IV. V. VI. VII.

Introduction Botany Anatomy and Physiology Horticulture Genetics Conclusion Literature Cited

4. Horseradish: Botany, Horticulture, Breeding

193 194 196 203 207 214 214

221

Ashraf Shehata, Richard M.S. Mulwa, Mohammad Babadoost, Mark Uchanski, Margaret A. Norton, Robert Skirvin, and S. Alan Walters I. II. III. IV. V. VI.

Introduction History Botany Horticulture Breeding Literature Cited

5. 1-Methylcyclopropene: Mode of Action and Relevance in Postharvest Horticulture Research

222 223 227 234 247 255

263

Wendy C. Schotsmans, Robert K. Prange, and Brad M. Binder I. II. III. IV. V. VI. VII.

Introduction Ethylene Response Pathway Physiological Processes Affected Side Effects Summary and Future Research Needs Acknowledgments Literature Cited

266 268 277 295 299 299 300

6. Postharvest Biology and Technology of Cucurbits

315

Steven A. Sargent and Donald N. Maynard I. II. III. IV.

Introduction Crops Conclusions Literature Cited

316 319 345 346

CONTENTS

vii

7. Physiological Disorders of Grape: Bunch Stem Necrosis and Early Bunch Stem Necrosis

355

Andrea H. Pickering, Ian J. Warrington, and David J. Woolley I. II. III. IV. V. VI.

Introduction Physiology of Berry Growth and Development Bunch Stem Necrosis Early Bunch Stem Necrosis Summary and Conclusions Literature Cited

8. Plug Transplant Technology

356 358 365 382 385 389

397

Daniel J. Cantliffe I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Importance of the Plug Industry Plug Production Technology Organic Plug Production Postharvest Handling of Plugs Associated Production Techniques Mechanization Conclusions and Prospects Literature Cited

9. A History of Grafting

397 400 401 418 420 422 425 427 428

437

Ken Mudge, Jules Janick, Steven Scoﬁeld, and Eliezer E. Goldschmidt I. II. III. IV. V. VI. VII.

Introduction Natural Grafting Historical Evidence History of Clonal Rootstocks Graft Hybrids Conclusion Literature Cited

438 445 449 475 478 485 487

Subject Index

495

Cumulative Subject Index

497

Contributor Index

523

Contributors

Mohammad Babadoost Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA I. Bar-Ya’akov Section of Deciduous Fruit Trees Sciences, Newe Ya’ar Research Center, Agricultural Research Organization, PO Box 1021, Ramat Yishay, 30095, Israel Brad M. Binder Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706, USA Daniel J. Cantliffe University of Florida/IFAS, Horticultural Sciences Department P O Box 110690, Gainesville, FL 32611, USA Johnny Carter Agriculture Research Station, Fort Valley State University, Fort Valley, Georgia 31030, USA Eliezer E. Goldschmidt R. H. Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem, PO Box 12 Rehovot 76100, Israel Robert J. Griesbach United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA Surinder K. Gulia Agriculture Research Station, Fort Valley State University, Fort Valley, GA 31030, USA Craig M. Hardner School of Land, Crop and Food Science, University of Queensland, St Lucia, 4068, Australia K. Hatib Section of Deciduous Fruit Trees Sciences, Newe Ya’ar Research Center, Agricultural Research Organization, PO Box 1021, Ramat Yishay, 30095, Israel Doron Holland Section of Deciduous Fruit Trees Sciences, Newe Ya’ar Research Center, Agricultural Research Organization, PO Box 1021, Ramat Yishay, 30095, Israel Jules Janick Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA Andrew J. Lowe Department of Ecology and Evolution, School of Earth and Environmental Science, North Terrace, University of Adelaide, Adelaide, 5005 Australia Donald N. Maynard Gulf Coast Research & Education Center, University of Florida/IFAS, Wimauma, FL 33598, USA

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Ken Mudge USA

CONTRIBUTORS

Department of Horticulture, Cornell University, Ithaca, NY 14853,

Richard M.S. Mulwa Department of Crops, Horticulture and Soils, Egerton University, PO Box 536, Egerton, Kenya. Jodi Neal Department of Ecosystem Management, University of New England, Madwick Drive, Armidale, 2351, Australia Margaret A. Norton Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Cameron Peace Department of Horticulture and Landscape Architecture, Washington State University, 39 Johnson Hall, Pullman, WA 99164, USA Andrea H. Pickering The Horticulture and Food Research Institute of New Zealand Limited, Tennent Drive, Palmerston North, 4442, New Zealand Phillip Pisanu Department for Environment and Heritage, PO Box 39, Kingscote, 5223, Australia Michael Powell Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, 4558, Australia Robert K. Prange Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia, B4N 1J5 Canada Steven A. Sargent Horticultural Sciences Department, University of Florida/ IFAS, Gainesville, FL 32611, USA Adele Schmidt Current address: Department of Zoology, University of Melbourne, Parkville, 3010, Australia Wendy C. Schotsmans Agriculture and Agri-Food Canada, Atlantic Food and Horticulture Research Centre, 32 Main Street, Kentville, Nova Scotia, B4N 1J5 Canada Steven Scoﬁeld U.S Department of Agriculture, Agricultural Research Service Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA Ashraf Shehata Floriculture and Garden Design Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt Bharat P. Singh Agriculture Research Station, Fort Valley State University, Fort Valley, GA 31030, USA Robert Skirvin Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Chris Spain School of Integrative Biology, Faculty of Biological and Chemical Sciences, University of Queensland, St Lucia, 4067, Australia Mark Uchanski Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA S. Alan Walters Department of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USA.

CONTRIBUTORS

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Ian J. Warrington Institute of Natural Resources, Massey University, PO Box 11 222, Palmerston North, 4442 New Zealand Kristen Williams CSIRO Sustainable Ecosystems, Tropical Forest Research Centre, PO Box 780, Atherton, 4883, Australia David J. Woolley Institute of Natural Resources, Massey University, PO Box 11 222, Palmerston North, 4442 New Zealand

Allan Ross Ferguson

Dedication: Allan Ross Ferguson

This volume is dedicated to Allan Ross Ferguson in recognition of his outstanding contributions to kiwifruit research, both by his own work and through synthesis of knowledge from the studies of others. Ross was born in 1943 in Morrinsville, a small New Zealand country town. He attended Gisborne Boys’ High School and ﬁnished as Dux in 1961. He graduated BSc (ﬁrst honors) in biochemistry and botany in 1965 at Victoria University, Wellington, with a prestigious university senior scholarship. While still an undergraduate, he spent a summer vacation on a Fruitgrowers’ Federation Studentship in the Fruit Research Division, DSIR, at Mount Albert Research Centre (MARC) in Auckland. It was one of those small events that change a life. He enjoyed the culture of the laboratory; it in turn valued his obvious intellect and character enough to head-hunt him. In January 1965 he joined Fruit Research Division. Although the organizational name has since changed four times, the location (MARC) and the course of Ross’s work did not, and his high productivity was not interrupted. Ross spent that ﬁrst summer studentship with me, and from that time we were closely associated in work and friendship for more than 30 years. During his ﬁrst three years at Fruit Research Division, Ross carried out Ph.D. studies on the nitrogen metabolism of Spirodela oligorrhiza, enrolled in the Cell Biology Department of Auckland University. During this early part of his career, Ross was a biochemist of great promise. Within his ﬁrst seven years he had made three major contributions toward understanding nitrogen nutrition in plants. He was among the very ﬁrst to clearly demonstrate enzyme induction in plants, to demonstrate permease induction in plants, and to start outlining the control mechanisms operating in the various nitrogen assimilation pathways. All three areas subsequently became major research ﬁelds, and Ross’s early work can now be seen as truly pioneering. Had he continued, he would surely have become a major international ﬁgure in biochemistry. But that was to change. In about 1973, E.G. Bollard left research to become division director. He had already started a small fertilizer trial on the new and small kiwifruit plantings in the Bay of Plenty, and he asked Ross to take it over. Over xiii

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the next seven years, there was an explosive increase in the size of the industry, and DSIR devoted increasing resources to studying kiwifruit biology. Ross progressively shifted from nitrogen biochemistry to the slow process of collecting knowledge and understanding about the biology of kiwifruit. His earliest kiwifruit work established its mineral nutrient requirements. He developed nutrient budgets for kiwifruit, identifying the risk of potassium deﬁciency in cropping orchards. He also used the kiwifruit vine to study the phenomenon of xylem sap bleeding; his three papers on the subject greatly improved our understanding of mineral nutrient transport in deciduous plants in general. But that was just a beginning. Among Ross’s characteristics has been a marked thoroughness in what he does. Once his kiwifruit research was under way, he began a major thrust to acquire all existing information in any language on kiwifruit. Where the material was in other than English, he arranged for translations to be made. Thus for the ﬁrst time ever, much of the material written on kiwifruit was gathered in one place and could be put into perspective. Ross prepared a comprehensive series of reviews, which underpinned his subsequent work and that of colleagues around the world. The ﬁrst appeared as ﬁve chapters in the seminal work, Kiwifruit: Science and Management. Others were published in subsequent years, including two comprehensive reviews of kiwifruit botany in Horticulture Reviews. At a more general level, he was able to unravel the fascinating story of the domestication of the kiwifruit, one of the few crops to have its history recorded in such detail. There was a second area where he led. In about 1978, China started to lift the bamboo curtain: One of the ﬁrst science delegations allowed out visited Australia and New Zealand, including MARC. There were reciprocal invitations from the Chinese: E.G. Bollard represented the Royal Society of New Zealand on the ﬁrst and Ross Ferguson was on the second in 1981. He met up with senior Chinese biologists, particularly Liang Chou-Fen (Guilin), who at the time was the leading Actinidia taxonomist in China, the home of the Actinidiaceae and the source of the original kiwifruit. They hit it off. Liang Chou-Fen visited MARC in 1983, and he and Ross wrote a taxonomic revision of our kiwifruit in 1984. The Chinese greatly value long-standing friendships, and Ross has retained his status there throughout the intervening 25 years. He has an unrivaled access from the western world into Chinese work on Actinidia and an unparalled knowledge of it. Key institutions have been the Guangxi Institute of Botany (the late Liang Chou-Fen) and the Wuhan Institute of Botany (Huang Hongwen). His laboratory has

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hosted a number of Chinese colleagues and he has coauthored several papers with them, giving them an entre´e to the English-language literature. Perhaps not at quite the same exclusive level, he has had a long-standing and rewarding collaboration with the Italian kiwifruit scientists (particularly through the universities of Bologna and Udine), more signiﬁcant these days now that Italy produces more kiwifruit than New Zealand. His early efforts to establish links with China and to resolve much confusion about Actinidia taxonomy were vital to the future of kiwifruit breeding. His research group now holds the most comprehensive individual collection of Actinidia species outside China, providing the raw material for HortResearch’s breeding program, culminating in the recent commercialization of Actinidia chinensis ‘Hort16A’ (marketed as ZESPRITM GOLD Kiwifruit), which is becoming as important to the New Zealand kiwifruit industry as the original ‘Hayward’, Actinidia deliciosa. Ross has recently used ﬂow cytometry to estimate chromosome numbers and establish ploidy, partly to unravel the paths of evolution in the Actinidia genus and partly to suggest proﬁtable lines for future interspeciﬁc hybridization and breeding of new fruit types. These studies of ploidy have helped to identify, and sometimes resolve, major problems in the interspeciﬁc breeding program. Ross’s encyclopedic knowledge made him the obvious choice as the International Registrar, Kiwifruit Cultivars. Various honors have come his way. He has often been an invited speaker at international meetings. In 1990, he was elected a fellow of the Royal New Zealand Institute of Horticulture (elevated to Associate of Honour in 1998), a fellow of the New Zealand Society for Horticultural Science in 1992, awarded a New Zealand Science and Technology Medal in 1995, awarded a Achievement Award, HortResearch 2000, elected a fellow of the Royal Society of New Zealand in 2000 and made an ofﬁcer, New Zealand Order of Merit in the Queen’s Birthday Honours in 2007. But two contributions that deserve recognition fall under the radar. Over his career he has been one of the hardest-working and most effective internal referees of his colleagues’ work at MARC and of papers submitted to international journals. And he has found the time to supply growers and the general public with many informative and digestible popular articles. In another facet, he has contributed considerable time and skill to New Zealand societies that interface between science and the public. He fully deserves all those honors, just as he deserves this present one. Outside the laboratory, his life has been characterized by a very wide range of interests, rather in the nature of a renaissance man. Ross has a

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deep interest in the history of science, particularly botanical exploration, and an abiding interest in the arts. He is a regular patron of the various music and art events in Auckland and has built up a signiﬁcant collection of New Zealand art. He has been particularly kind and helpful to the many people who have come to visit him from overseas, and frequently has offered them accommodation in his own home, sometimes for very long spells. He has a huge interest in the world and the people around him, so much so that his friends and colleagues have a stock phrase they often use: ‘‘Ask Ross, he will know.’’ There’s a lot more to his world than kiwifruit! Roderick L. Bieleski Fellow, HortResearch, New Zealand

1 Genetic Resources and Domestication of Macadamia Craig M. Hardner School of Land, Crop and Food Science University of Queensland St. Lucia, 4068, Australia Cameron Peace Department of Horticulture and Landscape Architecture Washington State University 39 Johnson Hall Pullman, WA 99164 USA Andrew J. Lowe Department of Ecology and Evolution School of Earth and Environmental Science North Terrace University of Adelaide Adelaide, 5005, Australia Jodi Neal Department of Ecosystem Management University of New England Madwick Drive Armidale, 2351, Australia Phillip Pisanu Department for Environment and Heritage PO Box 39 Kingscote, 5223, Australia

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 1

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Michael Powell Faculty of Science, Health and Education University of the Sunshine Coast Maroochydore DC, 4558, Australia Adele Schmidt Department of Zoology University of Melbourne Parkville, 3010, Australia Chris Spain School of Integrative Biology Faculty of Biological and Chemical Sciences University of Queensland St. Lucia, 4067, Australia Kristen Williams CSIRO Sustainable Ecosystems Tropical Forest Research Centre PO Box 780 Atherton, 4883, Australia I. INTRODUCTION A. Botany B. Horticulture II. WILD GENETIC RESOURCES A. Taxonomy 1. Families, Tribes, and Gondwanan Origin 2. Morphology and Phylogenetics B. Cytogenetics C. Species Distributions and Hybrid Zones 1. Macadamia integrifolia 2. Macadamia jansenii 3. Macadamia ternifolia 4. Macadamia tetraphylla 5. Interspecific Hybridization D. Ecology 1. Habitat and Structural and Floristic Characteristics 2. Rainfall, Climate, and Soils 3. Abundance and Population Dynamics E. Genetic Structure and Dynamics of Native Populations 1. Genetic Structure of Natural Populations 2. Mechanisms of Gene Flow F. Conservation Status of Wild Populations 1. In Situ Conservation 2. Ex Situ Conservation

1. GENETIC RESOURCES AND DOMESTICATION OF MACADAMIA III. GERMPLASM DOMESTICATION A. Hawaii 1. Initial Introductions 2. First Orchards 3. Scion Selection Program 4. Further Introduction of Australian Germplasm 5. Summary of Pedigree Relationships B. Australia 1. Early Seedling Orchards 2. 1950 Seedling Surveys 3. Norm Greber Selections 4. Miscellaneous Australian Selections 5. Hidden Valley Plantations Program 6. Australian Macadamia Breeding Program C. Other Programs 1. California 2. South Africa 3. Kenya 4. Others D. Genetic Structure of Domesticated Germplasm 1. Use of Molecular Markers 2. Influences on Genetic Structure 3. Wild Genetic Diversity Represented in Cultivation IV. GENETICS OF KEY SCION SELECTION TRAITS A. Tree Structure B. Flowering Phenology C. Fruit Set and Arrangement D. Yield 1. Age of First Crop 2. NIS Yield per Tree E. Nutrition Utilization F. Abnormal Vertical Growth G. Phenology of Fruit Drop H. Pest and Disease Resistance I. Stick-tights J. Nut Characteristics 1. Nut Size 2. Nut Shape 3. Nut Defects 4. Kernel Recovery K. Attributes of Kernel Quality 1. Raw Kernel Visual Appearance 2. Oil Content and Percentage First-Grade Kernel 3. Kernel Size 4. Percentage of Whole Kernels 5. Bitter Kernels 6. Quality Attributes of Roasted Kernel 7. Shelf Life L. Performance in Extreme Environments

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V. PROPAGATION AND ROOTSTOCK TRAITS A. Germination and Seedling Growth B. Rooting and Growth of Cuttings C. Graft Compatibility D. Rootstock Effects on Scion Performance VI. CULTIVAR UTILIZATION A. Scion Cultivars 1. Hawaii 2. Australia 3. South Africa 4. China 5. Other Countries B. Rootstocks VII. SUMMARY VIII. ACKNOWLEDGMENTS IX. LITERATURE CITED

I. INTRODUCTION Macadamia F. Muell is a long-lived evergreen tree of subtropical and tropical origin (Maiden 1888; Cheel and Morrison 1935; Stephenson 1990a; Nagao and Hirae 1992). The embryo of the mature fruit produced by two of the Australian species (M. integrifolia Maiden & Betche and M. tetraphylla L.A.S. Johnson) is a high-valued edible kernel that is the basis of an expanding world industry. Macadamia kernels are consumed as roasted snack food, chocolate-coated confectionary, in bakery products and ice cream, and as oil (Cavaletto 1981; Stephenson 1990b; Stephenson 2005) and ﬁt the characteristics of a luxury good, where demand is elastic with income (Osman 1982; Surono 1987). Macadamia is the only member of the Australian ﬂora to have been domesticated as an internationally commercial food crop. Knowledge of genetic resources has important consequences for management, crop development, and breeding. However, this knowledge is not well documented for macadamia. In this review, we collect and evaluate literature from disparate sources to describe the distribution, structure, and status of the wild germplasm and the origin, important selection criteria, and utilization of the domesticated resource. A. Botany The fruit of the macadamia is described as a follicle (Francis 1928; Hartung and Storey 1939); being a ‘‘dry dehiscent fruit formed from one carpel and having a longitudinal line of dehiscence’’ (Strohschen 1986).

1. GENETIC RESOURCES AND DOMESTICATION OF MACADAMIA

5

It is composed of an inner kernel, comprising a small subglobose embryo and two large semiglobose cotyledons, encased by a thick and woody outer testa (shell) and a ﬁbrous outer pericarp (husk) (Strohschen 1986). The shell is usually extremely hard (Jennings and Macmillian 1986; Naimi-Jamal and Kaupp 2007). Parenchyma cells in the mature embryo contain abundant oil bodies (Walton and Wallace 2005), and the oil content of fresh mature kernels is around 76% (Saleeb et al. 1973; USDA 2006), making macadamia the highest oil-yielding commercial nut (Strohschen 1986). Most of the oil is composed of monounsaturated fats (78% of total lipids), primarily oleic (18:1, 58% of total lipids) and palmitoleic (16:1, 17% of total lipids) acids (Cavaletto et al. 1966; Saleeb et al. 1973; USDA 2006). This is the highest concentration of palmitoleic acid in any natural food (Bridge and Hilditch 1950; Cavaletto 1980; Colquhoun et al. 1996). Saturated fats comprise 16% of the total lipid component. Sugar content at maturity is around 5%, with most (97 to 99% of total sugars) being nonreducing sugars (Cavaletto et al. 1966; USDA 2006; McConchie et al. 2007b; McConchie et al. 2007a; Wall and Gentry 2007). Mature embryos of M. ternifolia F. Muell. contain high levels of cyanogenic glycosides at maturity (Dahler et al. 1995). In contrast, the levels of cyanogenic glycosides are high in the developing embryos of M. integrifolia prior to shell hardening, but decline by fruit maturity. Cotyledons of germinating seeds and the tissues of young seedlings of M. integrifolia, M. ternifolia, and M. tetraphylla also contain very high levels of cyanogenic glycosides (Dahler et al. 1995) and may be adaptations to reduce herbivory (Dahler et al. 1995; O’Neill 1997). Macadamia ﬂowers are borne on a rachis that may contain 100 to 300 ﬂowers, each approximately 10 mm in length (Urata 1954; Ito 1980). Mature trees ( > 15 years of age) may produce approximately 10,000 racemes. Anthesis in the main production areas of Australia occurs over a period of approximately 5weeks (depending on cultivar) from early September to early October (Moncur et al. 1985; Boyton and Hardner 2002).In contrast, the period of ﬂowering in Hawaii extends over a protracted period of up to 30 weeks between November and May with three distinct peaks that are distinguishable between late January and early April (Nagao and Sakai 1988; Nagao and Sakai 1990; Nagao et al. 1992; Nagao et al. 1994). Flowers are pollinated by insects—in Australia, primarily European honeybees (Apis mellifera) and native bees (Trigona spp.) (Heard 1994; Wallace et al. 1996). About 10% of the ﬂowers set fruit (Sakai and Nagao 1985), and crosspollination increases initial set (Sedgley et al. 1990) and generally

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ﬁnal yield (Ito and Hamilton 1980; Trueman and Turnbull 1994a; Wallace et al. 1996; McConchie et al. 1996). During a short period 2 to 3 weeks after anthesis, developing fruit abscise at a high rate, coincident with a high rate of growth in the size of remaining fruit (Sakai and Nagao 1985; Trueman and Turnbull 1994b). High rates of abscission are also observed 5 to 7 weeks and again around 10 weeks after anthesis (Sakai and Nagao 1985; Trueman and Turnbull 1994b). Generally, only low rates of abscission occur after this period and appear to be a consequence of pest or disease attack (Sakai and Nagao 1985; Trueman and Turnbull 1994b). Growth in fruit size continues to approximately 12 to 15 weeks after anthesis (Sakai and Nagao 1985; Nagao and Hirae 1992; Trueman and Turnbull 1994b) with shell hardening also complete by this time (Jones 1937, 1939, 1994b; Trueman and Turnbull 1994b). Fruit mass continues to increase to approximately 23 weeks after anthesis (Trueman and Turnbull 1994a; McConchie et al. 1996). Oil content of the developing embryo is initially low until 12 to 15 weeks, after which the rate of oil accumulation increases rapidly, reaching a plateau at approximately 23 to 25 weeks after anthesis (Jones 1937, 1939; Baigent 1983; McConchie et al. 1996; Trueman et al. 2000). Initial studies appear to have assumed that splitting of the husk indicated fruit maturity (Cheel and Morrison 1935; Wills 1939; Leverington 1958); however, more recent studies indicate that the husk dehiscence occurs well after maximum oil content of the kernel has been reached and generally after the fruit have abscised from the tree (Trueman et al. 2000). The period of mature fruit drop in Australia is between March and July (approximately 24 to 46 weeks after anthesis), although this may extend to overlap with ﬂowering in September (Nagao and Hirae 1992; Boyton et al. 2002; Hardner 2005). Fruit abscission in Hawaii occurs between August and April (Ito 1984; Nagao and Hirae 1992). This, combined with the extended ﬂowering period, leads to the presence of fruit at different development stages on the tree at the same time in this environment. B. Horticulture Macadamias were recognized by aboriginal culture prior to colonization. The name goojabarigh (Bailey 1901) is the local aboriginal name of the species indigenous to northern Queensland. Farther south, the local aboriginal name for the macadamia species that occurs in the Mount Bauple area is jindilli (Gross 1995); kindal kindal is the term used by the Aborigines for the macadamia that grows in the northeast of New South

1. GENETIC RESOURCES AND DOMESTICATION OF MACADAMIA

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Wales (Maiden 1888; Cheel and Morrison 1935). In the Pine Rivers, north of Brisbane where two species co-occur, burrwang is reportedly the local indigenous people’s name for macadamia (Wagner-Wright 1995). Common European names for macadamia include smooth shell macadamia, rough shell macadamia, Queensland nut, Bopple nut, bauple nut, popple nut, Australian nut, bush nut, and gympie nut (Francis 1928; Cheel and Morrison 1935; Wills 1939; Hamilton and Storey 1956; Leverington 1958, 1971). Although macadamias used in commercial plantations are derived from species indigenous to Australia (Gross 1995), the crop was initially commercialized in Hawaii (Wagner-Wright 1995). Currently, macadamia is produced in several tropical and subtropical regions, primarily Australia, Hawaii, southern and central Africa (South Africa, Kenya, and Malawi), and Central and South America (Guatemala and Brazil) (Piza et al. 2006), with some development in southeast Asia (Thailand and China) (Supamatee et al. 1992; Xiao et al. 2002b; Venkatachalam and Sathe 2006). World production of macadamia kernels in 2005 was estimated at 28,000 tonnes(t) (Piza et al. 2006), up 115% from 13,000 t in 1995 (USITC 1998). Currently, macadamia represents 1.3% of the world nut meat market (INC 2006). Macadamia trees are commonly propagated by grafting selected scions onto seedling rootstocks (Stephenson 1990a; Nagao and Hirae 1992), although cuttings and clonal rootstocks have also been used (Stephenson 1990a; Trochoulias 1992; Wiid and Hobson 1996; Bell 1996). However macadamia propagated as cuttings are less stable in the ﬁeld than plants on seedling rootstocks (Hamilton and Fukunaga 1959; Hobson 1971; Phiri 1985; Nagao and Hirae 1992; Trochoulias 1992). Similar observations have been reported for tissue-cultured plants (Xiao et al. 2002a). Orchards are generally established with selected cultivars grafted onto seedling rootstocks at densities between 100 to 350 trees/ha (Stephenson 1990a; Nagao and Hirae 1992), although higher densities of 667 trees/ha have been used (Trochoulias and Burnside 1987). In contrast, the Kenyan macadamia production system is characterized by many small land owners each growing only a few trees (Gathungu and Likimani 1975; Onsongo 2006). Grafted trees usually begin bearing between 3 and 6 years of age (Stephenson 1990a; Oosthuizen 1992; Nagao and Hirae 1992) and may be commercially productive for at least 40 to 60 years (Hamilton and Fukunaga 1959). Common horticulture practices of fertilization, weed, pest and disease control, and canopy management are implemented (Stephenson 1990a; Nagao and Hirae 1992; Stephenson and Trochoulias 1994; Hardner et al. 2006). In some areas irrigation is applied, but

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this is not a universal practice (Trochoulias and Johns 1992; Stephenson and Trochoulias 1994). In commercial orchards, mature fruit are generally allowed to abscise and fall from the tree for harvesting. Fruits are either mechanically or hand-harvested from the ground at regular intervals to reduce the incidence of kernel deterioration (Leverington 1962a; Mason 1983; Mason and Wells 1984; Liang et al. 1996). After harvesting, the husk is mechanically removed, and, prior to processing, nuts are dried to around 1.5% kernel moisture content using a regime of initially low temperature ( < 40 C) to reduce kernel browning (Prichavudhi and Yamamoto 1965; Mason and McConachie 1994; Mason 2000). Dried nuts are mechanically cracked to extract the kernel (mature embryo). The term kernel recovery refers to the mass of kernel extracted per mass of nut in shell (NIS). Sorting pre- and postcracking is used to remove unacceptable product and to grade kernels into product styles (Mason and McConachie 1994; Mason 2000). Raw kernel may be further processed using oil or air-dry roasting (Moltzau and Ripperton 1939; Leverington 1962a; Winterton 1966; Mason 1987; Mason and McConachie 1994). Consumer surveys have indicated a strong preference for roasted kernel as snack food compared to raw kernel (O’Riordan et al. 2005). Macadamia production requires a large initial investment in terms of land, purchase of grafted trees, farm machinery, infrastructure, and in some areas, irrigation (Hardner et al. 2006). The major costs of production are land rental (38%), general ﬁxed costs (20%), and orchard establishment (9%) (Hardner et al. 2006). The major costs of processing nuts to raw kernel are cracking (30%), sorting (20%), and packing (22%). II. WILD GENETIC RESOURCES A. Taxonomy 1. Families, Tribes, and Gondwanan Origin. The ancestors of Macadamia can be traced to a group of primitive rain forest plants ancestral to the modern Proteaceae Juss. family. These ﬁrst appear in the palynological record during the late Cretaceous, around 100 million years ago, when Australia was still part of the great southern landmass, Gondwanaland (Ramsay 1963; Johnson and Briggs 1963; Johnson and Briggs 1975; Boland 1984). Despite having their evolutionary roots in the rain forest, the Proteaceae is now not well represented in these ecosystems, with most species being adapted to dryer, ﬁreprone habitats (Johnson and Briggs 1963).

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9

The Proteaceae is part of an ancient group of angiosperms (ﬂowering plants) in subclass Magnoliidae (dicotyledons) that comprises approximately 1,500 species in 80 genera, of which 900 species in 50 genera are found in Australia (Harden 1990). The center of diversity of the family appears to be have been in the part of Gondwanaland that is now Australia (Johnson and Briggs 1975). Other well-known Proteaceae genera include Grevillea R.Br. ex Knight, Banksia L.f., Hakea Schrad. & J.C. Wendl., Protea L., and Leucadendron R.Br., all of which are cultivated for their inﬂorescences (Harden 1990; Criley 1998; Sedgley 1998; Coetzee and Littlejohn 2001; Ben-Jaacov and Silber 2006). In a recent classiﬁcation of Proteaceae (Weston and Barker 2006), the genus Macadamia is located in the tribe Macadamieae, together with several other Australian genera, including Athertonia L.A.S. Johnson & B.G. Briggs, Catalepidia P.H. Weston, Gevuina Molina and Hicksbeachia F. Muell., many of which also produce sizable nuts (Stace et al. 1998). Although Floydia L.A.S. Johnson & B.G. Briggs had been included in Macadamieae (Johnson and Briggs 1975), it has been moved to the tribe Roupaleae Meisn. (Weston and Barker 2006), which is in the same subfamily of Proteaceae. The center of origin of this tribe is probably eastern Australia and neighboring landmasses that once formed part of eastern Gondwanaland (Johnson and Briggs 1975). The present-day distribution of the tribe includes Australia, some Paciﬁc islands, South America, and South Africa (Venkata Rao 1970; Johnson and Briggs 1975). Macadamia is closely aligned with two other genera, Brabejum L. (1 species endemic in southern Africa) and Panopsis Salisb. (11 species endemic to tropical and subtropical America) in the subtribe Macadamiinae (Johnson and Briggs 1963, 1975; Venkata Rao 1970; Gross 1995; Weston and Barker 2006). 2. Morphology and Phylogenetics. Species of Macadamia have been informally grouped into two clades based on morphological and geographic afﬁnities (Johnson and Briggs 1975). A recent classiﬁcation of Proteaceae that also incorporates molecular data (Weston and Barker 2006) suggests the genera is paraphyletic and includes the other genera from the subtribe Panopsis and Brabejum as subclades. Six species from New Caledonia described as Macadamia (Virot 1968; Hamilton 1970) have since been placed in Virotia L.A.S. Johnson & Briggs, within the subtribe Virotiinae of Macadamieae (Weston and Barker 2006). The ‘‘southern clade’’ (coastal central and southern Queensland and northern New South Wales) comprises four species, and the ‘‘northern clade’’ has ﬁve species, distributed in northern Australia and Sulawesi, Indonesia. All Macadamia species generally have the form of a small to

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medium-size tree, up to 40 m (Gross 1995). The leaves are simple and sclerophyllous with or without spinose margins. Flowers are formed as a conﬂorescense and the fruit is a globular follicle. The Southern Clade. The four species of the ‘‘southern clade,’’ M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii C.L. Gross & P.H. Weston, are naturally found in a narrow region along the eastern coast of Australia, between 152 and 154 E longitude and 25 and 29 S latitude. The ﬁrst botanical specimens were of M. ternifolia (Fig. 1.1A) collected by the Australian explorer Ludwig Leichhardt in 1843 (Smith 1956) from the Conondale Range region. Despite earlier awareness, Macadamia was not formally described until 1857 by Ferdinand von Mueller, based on material collected with Walter Hill from the Pine River valley north of Brisbane (Fig. 1.1B). The genus was dedicated to John Macadam, the honorary secretary (and later president) of the Philosophical Institute of Victoria (von Mueller 1857). John Macadam is also famous for his role as one of the umpires in the ﬁrst recorded game of Australian Rules Football (Blainey 1990). Mueller called the taxon ‘‘Macadamia ternifolia’’; however, his type specimen included material of both M. ternifolia and what was subsequently classiﬁed as M. integrifolia (Fig. 1.1B), which lead to much subsequent confusion (Smith 1956). This herbarium sheet does not include any fruit, and although a drawing of the fruit is presented in the formal description of the species (von Mueller 1857), it appears more like the fruit of a Grevillea species than that of any macadamia. Several taxonomic treatments of the group followed (Storey 1959). Maiden and Betche classiﬁed the smooth-leafed variant as M. integrifolia in 1897 (Maiden and Betche 1897) (Fig. 1.1C). This holotype was described as being collected from Camden on the central coast of New South Wales (NSW), although this is well outside the natural range of the species and may have come from a cultivated individual (Johnson 1954). Two years later they revised it

3

Fig. 1.1. Herbarium sheets of Macadamia species. (A) Herbarium specimen of the thenunnamed M. ternifolia collected by Ludwig Leichhardt in 1843. Reproduced with permission from the archives of the Royal Botanic Gardens, Melbourne. (B) Herbarium specimen used to describe Macadamia ternifolia, including the Holotype (upper right) and a second specimen (lower left) later identiﬁed as M. integrifolia. Reproduced with permission from the archives of the Royal Botanic Gardens, Melbourne. (C) Macadamia integrifolia holotype. Reproduced with permission from the archives of the Royal Botanic Gardens, Sydney. (D) Macadamia tetraphylla holotype. Reproduced with the permission of the Royal Botanic Gardens, Sydney.

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to M. ternifolia var. integrifolia, having observed ‘‘all degrees of transition between the two leaf forms’’ (Maiden and Betche 1899). Up until 1954, specimens of M. tetraphylla were considered as either M. integrifolia or M. ternifolia (Smith 1956) due to their spinose leaf margins, which was the major character for species resolution at the time. Macadamia tetraphylla was eventually classiﬁed as a separate species in 1954 (Johnson 1954) (Fig. 1.1D). Finally in 1956, the species was resolved into the three taxa recognized today, M. integrifolia, M. tetraphylla, and M. ternifolia (Smith 1956). Two previously described species, M. lowii F.M. Bailey and M. minor F.M. Bailey, are recognized as synonyms of M. ternifolia. The fourth species of the southern clade, M. jansenii, was discovered more recently by Ray Jansen (Gross and Weston 1992) and therefore was not involved in the earlier taxonomic confusion of the genus. In broad terms, the cultivated species M. integrifolia and M. tetraphylla are medium-size trees (attaining heights of 6 to 18 m and 3 to 18 m respectively) (Gross 1995), and bear large edible nuts. In contrast, the wild relatives M. ternifolia and M. jansenii are smaller trees (up to 8 m and 6 to 9 m, respectively) (Gross 1995), with small, bitter, inedible nuts, attributable to the presence of cyanogenic glycosides (Dahler et al. 1995). Several morphological characters can be used to distinguish between the four southern clade species (Table 1.1), although the description of a shell thickness of up to 1 cm for M. integrifolia seems a little excessive compared to Leverington (1962a), who reports a maximum shell thickness of 0.7 cm across 94 genotypes. The major advantage of morphological descriptors lies in their ease of detection. However, some of these traits are visible only at certain times of the year (e.g., leaf ﬂush color, ﬂoral and fruit descriptors) or at reproductive maturity (ﬂoral and fruit descriptors). Considerable morphological variation within each species, and overlap between them, can make visual classiﬁcation difﬁcult (Johnson 1954). Most of the leaf descriptors are not valid for identifying young seedlings, as juvenile states for most characters are similar across the species. In the adult form also, leaves of M. integrifolia may have spiny margins and leaf dimensions resembling those of M. ternifolia or hybrids of M. integrifolia and M. tetraphylla. Environmental effects can cause large variation in some characters, such as leaf and nut size. Specimens of M. tetraphylla with white ﬂowers instead of the characteristic pink-red hue are occasionally observed (McConachie 1980; Gross 1995). Overlap in other characters is also observed, including leaves per whorl, leaf dimensions, nut size, and shell thickness (Johnson 1954).

13

Creamy white

2.5–3.1 2.4–3.0 Smooth 6–10

Floral Flower color

Fruit Nut dimensions (cm) Shell texture Shell thickness (mm) 2.6–3 1.6–2.4 Pebbled/rough 2–6

Pink

Usually 4 7–30 1.4–6 High Oblong to oblanceolate Always many Acute or subacute Truncate, attenuate 0–4 Pink to red

M. tetraphylla

1.6 1.2 Smooth 1

Pink

Usually 3 9–12.5 2–3.5 Medium high Narrowly ovate Few Acute, mucronate Attenuate 4–10 Pink to red

M. ternifolia

1.4–1.8 1.1–1.6 Smooth 0.8–1.5

Creamy brown

3 10–17.5 2.5–5 Medium Oblanceolate None Acute to attenuate Attenuate or cuneate 2–14 Green

M. jansenii

a Etymology: Macadamia: after John Macadam (1827–1865), secretary of the Philosophical Institute of Victoria; integrifolia: ‘‘entire leaves’’— leaf margins not (as) spinose as in M. tetraphylla; tetraphylla: ‘‘four leaves’’— leaves in whorls of four; ternifolia: ‘‘three leaves’’— leaves in whorls of three; jansenii: after R.C. Jansen (1941–1997), a naturalist who ﬁrst collected the species. Source: Descriptions from Gross (1995), Stanley and Ross (1986), and Storey (1959).

Usually 3 6.5–14 2–6.5 Low Ovate to obovate Few or none Acute to obtuse Very shortly attenuate 6–18 Green

M. integrifolia

Leaf No. per whorl Dimensions (cm) Ratio length: width Shape Margin spines Apex Base Petiole length (mm) Flush color

Character

Table 1.1. Morphological differences between the four Macadamia species of the Australian southern clade. These characters are for mature specimens.a

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A potential cause of the considerable morphological variation observed is interspeciﬁc hybridization (Johnson 1954; Smith 1956). The morphological state of F1 Macadamia hybrids is typically intermediate for most characters (Storey and Saleeb 1970). Some individuals may be latergeneration hybrids or backcrosses, from natural hybrid zones or from sites of cultivation. Extensive interspeciﬁc hybridization renders the species status of many individuals highly ambiguous when assessed solely by morphology. The gradation of leaf forms observed by Maiden and Betche (1897) in M. integrifolia to M. ternifolia may have been due to the presence of natural hybrids of the two species (Storey 1965b). No clear relationships between the four southern clade species are obvious from morphological comparisons (see Table 1.1). While M. ternifolia and M. jansenii are often considered similar because their small bitter nuts are not suitable for cultivation and because of their relatively smaller height, other features group the species in different ways. M. ternifolia and M. tetraphylla share a common feature in their pink-red leaf ﬂushes and ﬂowers. All but M. tetraphylla have whorls usually of three leaves. All but M. jansenii and some specimens of M. integrifolia have some degree of spines on their leaf margins. Molecular marker studies have shed further light on genetic afﬁnities and phylogenetic relationships between southern clade species. In an isozyme study, Sharp and Playford (1997) found M. ternifolia and M. jansenii to be relatively closely related, as were M. integrifolia and M. tetraphylla. However, the inclusion of interspeciﬁc hybrids between the latter pair of species probably confounded the relationships among the species. Another isozyme study by Aradhya et al. (1998) concluded that M. ternifolia is either a conspeciﬁc variant or a close relative of M. integrifolia but that M. tetraphylla was more closely related to M. integrifolia than to M. ternifolia. Unfortunately, this analysis probably was compromised by the inclusion of hybrids and the omission of M. jansenii. At the very least, the results clearly demonstrated that the three species form a species complex. Results from a combined randomly ampliﬁed DNA ﬁngerprinting (RAF; Waldron et al. 2002) and sequence tagged microsatellite site (STMS) marker study (Peace et al. 2002) suggested that M. integrifolia and M. tetraphylla were sister species, and there was greater afﬁnity between M. ternifolia and M. jansenii than with the other two species. This work was extended to include a wider range of germplasm for the four main species of the southern clade and was careful to exclude hybrids from the analysis (Peace 2005). The most closely related species pair in this analysis was M. integrifolia and M. tetraphylla, with M. ternifolia being

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15

more closely related to this species pair than to M. jansenii, which was the genetic outlier of the four species. However, this study included M. ternifolia accessions from only half the known natural range of the species and only two specimens of M. jansenii. A more comprehensive survey of germplasm of these two species is required to deﬁnitely resolve relationships between the four species of the southern clade. The Northern Clade. The ‘‘northern clade’’ of Macadamia includes ﬁve species. Three species are native to far north Queensland: M. whelanii F.M. Bailey, M. claudiensis C.L. Gross & B. Hyland and M. grandis C.L. Gross & B. Hyland (Gross 1995). Two other species have been reported from the tropical island province of Sulawesi, Indonesia, where M. hildebrandii Steenis has a wide distribution and M. erecta has been recorded at high altitude (Sleumer 1955; McDonald and Ismail 1995). A major distinction between the southern and northern clade macadamias is the branched conﬂorescence of the latter (Gross 1995; McDonald and Ismail 1995). In addition, adult leaves of the far north Queensland and Sulawesi species occur in whorls of four or more, and leaf margins are always spineless (Gross 1995; McDonald and Ismail 1995). Nuts of these ﬁve species tend to be larger than those of the southern clade species (Gross 1995; McDonald and Ismail 1995). Kernels of M. whelanii are known to contain cyanogenic glycosides (Gross 1995), similar to M. ternifolia and M. jansenii. However, this characteristic is not shared with M. claudiensis, M. hildebrandii, and M. erecta (McDonald and Ismail 1995). For example, M. hildebrandii reportedly produces fruit with edible kernels that have good eating qualities (Sleumer 1955). Such information is not available for M. grandis (Gross 1995). The size of M. grandis trees in the wild are similar, or larger, than for M. integrifolia and M. tetraphylla (Gross 1995; McDonald and Ismail 1995). Johnson and Briggs (1975) suggested that the Sulawesi species evolved from an Australian progenitor around 15 million years ago when the two landmasses were still connected. Such a progenitor is likely closely allied to the northern clade of Australian macadamias, given the close morphological similarities between members of this group compared to the southern clade taxa. Limited isozyme evidence suggests that Hicksbeachia pinnatifolia (which belongs to a different subtribe) is more closely related to the southern clade Macadamia than is M. hildebrandii (Aradhya et al. 1998). If so, it brings into question afﬁnities between Macadamia and genera such as Panopsis and Brabeium that are within the same subtribe as Macadamia.

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A molecular marker analysis (RAF) of several Proteaceae species (Peace 2005) conﬁrmed that the northern clade species, M. whelanii and M. claudiensis, are not closely related to the southern clade Macadamias or to each other. In accordance with current taxonomy, but in contrast to the above-mentioned isozyme results, the RAF study indicated that other species from the Macadamieae tribe, including Hicksbeachia pinnatifolia, which is native to southeast Queensland and northern NSW, are no more genetically similar to the southern clade Macadamias than are M. whelanii and M. claudiensis (Peace 2005). A further more detailed phylogenetic investigation of the tribe is warranted. The remainder of this review focuses predominantly on the commercially developed Macadamia species and their close relatives in the southern clade. B. Cytogenetics All Macadamia species surveyed to date are reported to be diploid with a haploid chromosome number of 14: M. integrifolia (Ramsay 1963; Storey and Saleeb 1970); M. integrifolia, M. tetraphylla, and M. ternifolia (Storey 1965b); and M. integrifolia, M. tetraphylla (Storey and Saleeb 1970). A single report of polyploidy has been made (IPBGR 1986), but no details were provided to enable veriﬁcation. Hybridization between M. tetraphylla and M. integrifolia does not appear to disrupt normal chromosome pairing or disjunction, and the chromosome number of F1 progeny remains n ¼ 14 (Storey and Saleeb 1970). In the most recent review of the cytological data for 188 species in 65 genera of Proteaceae, Stace et al. (1998) suggests that the genera of subfamily Grevilleoideae are almost entirely diploid, with chromosome base numbers of n ¼ x ¼ 10, 11, 12, 13, and 14 and with two observed instances of triploidy. They argued against an earlier hypothesis that this chromosome series represented ‘‘paleo-polyploidy’’ from an ancestral genome of x ¼ 5 or 7 (e.g., Venkata Rao 1970; Johnson and Briggs 1975) and instead suggested that members of the Proteaceae are derived from an ancestral genome of x ¼ 12 or 21, with 24 chromosome arms (fundamental number [FN] ¼ 24). The ancestral grevilleoid genome of x ¼ 14 is probably of Gondwanan origin, and consists of 10 metacentric and 4 short telocentric chromosomes (Stace et al. 1998). Many members of the tribe Macadamieae appear to have retained this original genome: n ¼ x ¼ 14 for Macadamia, Brabeium, and Floydia, with n ¼ x ¼ 13 (fusion of a telocentric and a metacentric chromosome) for Hicksbeachia and Gevuina (Stace et al. 1998). However, they also suggest that

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additional chromosomal evolution subsequent to this origin is possible. Chromosome numbers for Athertonia, Panopsis, and several other members of tribe Macadamieae have not yet been reported. While information on chromosome size in Macadamia is not available, ﬁve other genera surveyed from subfamily Grevilleoideae have relatively small chromosomes (Stace et al. 1998). In particular, the genome size of Brabeium stellatifolium, in the same subtribe as Macadamia, was the smallest reported (mean chromosome length of 1.0 mm; (Stace et al. 1998). Having such small grevilleoid chromosomes is evidence against a paleo-polyploid origin, as is the lack of additional isozyme loci in Australian genera of Proteaceae (Stace et al. 1998). The number of isozyme loci in Macadamia reported by Vithanage and Winks (1992) and Aradhya et al. (1998) also appears consistent with a diploid rather than ancient tetraploid origin. Stace et al. (1998) suggest that if paleo-polyploidy has occurred in ancestral Proteaceae, molecular genetic investigation in genera such as Macadamia may reveal (extensive) gene silencing, which would have occurred through the process of ‘‘diploidization.’’ C. Species Distributions and Hybrid Zones The most up-to-date information on the distribution of southern clade Macadamia species is provided by a recent ﬁeld collection for ex situ conservation and assessment that was based on initial surveys of Queensland’s Environmental Protection Agency databases and herbarium records (Hardner et al. 2004). With the exception of M. jansenii, which occurs at a single site in the Bulburin State Forest near Miriam Vale, central east Queensland (Gross and Weston 1992), the southern clade Macadamia are distributed in a narrow band parallel to the coast between Lismore in northern NSW and Mount Bauple in southeast Queensland (Hardner et al. 2004; Peace 2005) (Fig. 1.2). Within this natural range, the clade occurs in lowland subtropical rain forest among coastal valleys and foothills. The three southern species occupy separate though overlapping parts of this geographic range. The natural distribution of M. ternifolia and M. integrifolia is conﬁned to southeast Queensland, and M. tetraphylla mainly occurs in northern NSW, extending into the coastal valleys of the Gold Coast hinterland in southern Queensland (Barry and Thomas 1994; Hardner et al. 2004; Peace 2005). 1. Macadamia integrifolia. This is the most widely distributed of the southern Macadamia species (Barry and Thomas 1994) (Fig. 1.2).

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Fig. 1.2. Natural distribution of southern species of Macadamia and natural hybrids. Dotted lines indicate the extent of hybrid zones.

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It occurs parallel to the coast extending from Mount Bauple near Maryborough in the north to the NSW–Queensland border in the south (Hardner et al. 2004; Peace 2005), a linear distance of approximately 275 km. Frequently sympatric with M. ternifolia and occasionally M. tetraphylla, M. integrifolia occurs in subtropical lowland rainforest communities (Harden 1990), ranging from complex to simple notophyll vine forest and microphyll-notophyll vine forest (Barry and Thomas 1994, Table 2). 2. Macadamia jansenii. This species is known from a single site in the Pine Creek catchment of Bulburin State Forest, in central eastern Queensland (Gross and Weston 1992), and is over 150 km north from the nearest population of any other southern clade species of Macadamia. The population comprises approximately 30 individuals, located on the moderately graded lower slopes of a narrow gully containing an intermittently running tributary (Barry and Thomas 1994). 3. Macadamia ternifolia. This species extends from Goomboorian approximately 18 km northeast of Gympie, to Mount Nebo approximately 10 km west-southwest of Samford in southern Queensland (Hardner et al. 2004; Peace 2005). Macadamia ternifolia occurs across approximately 150 km from north to south and, in its southern range, overlaps with the northern range of M. integrifolia (Fig. 1.2). 4. Macadamia tetraphylla. M. tetraphylla occurs in the coastal rain forests of the Richmond and Tweed River catchments in northeast NSW, and extends north to Mount Wongawallan in southeast Queensland (Barry and Thomas 1994). In its northern range its distribution overlaps M. integrifolia, where hybrids are frequently found at sympatric sites (Peace 2003) (Fig. 1.2). This species is often found in small remnants of the former Big Scrub (Holmes 1987; Lott and Duggin 1993), albeit as small populations, and has also been recorded in riparian rain forest in this region (Pisanu 2001). Northern outlier records of M. tetraphylla on the Sunshine Coast may be historical plantings (Hardner et al. 2004; Peace 2005). It may be that before widescale clearance of the Big Scrub, M. tetraphylla had a more continuous and higher-density distribution than today. Certainly the species is frequently found in a myriad of small remnant patches that now comprise less than 1% of the landscape (Pisanu 2001). 5. Interspeciﬁc Hybridization. Hybridization between M. integrifolia and M. tetraphylla readily occurs both in cultivation, and naturally

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where the species co-occur (Storey 1959; Barry and Thomas 1994; Hardner et al. 2000; Peace 2005). Natural hybrids between M. integrifolia and M. ternifolia have also been widely found in areas of sympatry (Peace 2005). Originally evidence for hybridization was based on observations of trees with intermediate morphology (Smith 1956; Storey 1959). More recently, however, DNA marker analysis has conﬁrmed that natural hybrids do occur (Peace 2005). Due to allopatry, hybrids between other species pairs in the southern clade have not been recorded in the wild, although hybrids between all pairs of species have been synthesized artiﬁcially (Hardner et al. 2000). Natural populations of the two cultivated species, M. integrifolia and M. tetraphylla, are sympatric south of Brisbane in southeast Queensland, and it is common to ﬁnd trees displaying intermediate morphologies. However, in no cases have specimens of both pure species been found in the same population, and in the middle of the natural hybrid zone is a small region where only hybrids occur (Peace 2005). This natural hybrid zone was believed to be restricted in area of only a few square kilometers (Storey and Saleeb 1970; McConachie 1980; Hardner et al. 2000), but a more recent survey has determined that the zone extends over at least 20 km, and perhaps much farther (Peace 2005). It is possible that the zone has been extended by human disturbance, such as the removal of a 10-km-wide eucalypt belt in the Gold Coast hinterland in the early 1900s that acted to promote increased pollen ﬂow between populations and species (Wills 1961). DNA analysis (RAF) of individuals in this hybrid zone has identiﬁed a full range of potential genotypic combinations between the two species, with a clear gradation from pure M. integrifolia to pure M. tetraphylla types from north to south (Peace 2005). Such a pattern of variation indicates that interspeciﬁc hybrids are fertile and have been segregating as later-generation hybrids and/or backcrossing to pure species types for many generations. The genetic distinctness of M. integrifolia and M. tetraphylla means that they should continue to be recognized as sound species, and F1 (and later-generation hybrids) between the two can be identiﬁed easily using multilocus DNA analysis (Peace 2005). The hybrid zone between M. integrifolia and M. ternifolia is similar, although hybridization between these species may not be so extensive and was identiﬁed only recently. Even though the two species were known to coexist over a greater geographic range than M. integrifolia and M. tetraphylla, trees with intermediate morphology had not been reported (Storey 1965b; McConachie 1980). However, a recent survey located several macadamia populations that included both M. ternifolia

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21

and M. integrifolia trees and at least one possible hybrid, all in the Pine River/Samford Valley area (Hardner et al. 2004; Peace 2003). DNA analysis of these trees with species-speciﬁc markers (Peace 2005) conﬁrmed the existence of several interspeciﬁc hybrids, and the cooccurrence of specimens of the pure species, in contrast to that found for the M. integrifolia/M. tetraphylla hybrid zone. Although fewer individuals were analyzed than from the M. integrifolia/M. tetraphylla hybrid zone, a range of intermediate genotypes was identiﬁed including F1, later-generation segregants, and/or backcrosses (Peace 2005). This further indicates that F1 hybrids are fertile and that hybridization between the two species has occurred over many generations in this zone. Controlled crosses between M. ternifolia and M. jansenii, and M. ternifolia and M. integrifolia have produced viable progeny (Hardner et al. 2000). No attempts were made to cross M. tetraphylla with either M. ternifolia or M. jansenii, as ﬂowering times did not overlap. However, given the cross-compatibility among the other pairings of species in the southern clade, it is likely that M. tetraphylla completes the group of fully cross-compatible species. Attempts to hybridize M. integrifolia with the northern clade species M. whelanii or M. claudiensis were unsuccessful (Hardner et al. 2000). Pollen from the northern clade species appeared to germinate on M. integrifolia styles, but pollen tube growth was arrested before reaching the ovule. Graft compatibility within, but incompatibility between, has been demonstrated for the southern and northern clade species (Storey and Frolich 1964), further evidence that the northern clade macadamia species are closely allied with each other but not with the southern clade species. The high degree of reproductive compatibility among the southern species suggests that hybridization may pose a threat to the integrity of the pure species in the wild, particularly considering that the cultivated germplasm in Australia largely coincides with the native distribution of three main species of the southern clade. D. Ecology 1. Habitat and Structural and Floristic Characteristics. The southern clade Macadamia species are native to the subtropical lowland rain forest of northern NSW and southeast Queensland. In Queensland, Webb’s (1968) structural-physiognomic classiﬁcation (e.g., Sattler and Williams 1999) is mainly used to describe plant communities; Floyd’s (1990) structural-physiognomic-ﬂoristic classiﬁcation is often used in NSW. Some reports of Macadamia habitat refer to both systems (e.g., Barry and Thomas 1994).

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Table 1.2. Common rain forest types associated with three common southern clade Macadamia species. Macadamia spp. M. integrifolia

Structural classiﬁcation Complex Notophyll Vine Forest Notophyll Vine Forest Araurcarian Notophyll Vine Forest

M. tetraphylla

Notophyll Vine Forest Mixed Notophyll Vine Forest

M. ternifolia

Complex Notophyll Vine Forest

Araurcarian Notophyll Vine Forest

Subtropical rainforest ﬂoristic alliance Argyrodendron trifoliolatum dominant Argyrodendron actinophyllum dominant A. actinophyllum and Araucaria cunninghamii A. trifoliolatum dominant Cupaniopsis anacardioide Acmena spp. A. trifoliolatum dominant or Argyrodendron trifoliatu and Dissilaria baloghioides alliance A. actinophyllum and Araucaria cunninghamii

More rarely in notophyll gallery rain forest or complex notophyll riparian vine forest Source: Structural classiﬁcations after Webb (1968) and subtropical rain forest subformation ﬂoristic alliances after Floyd (1990).

Tropical and subtropical rain forest of the lowlands typically have three or more tree layers, with or without emergents, whereas at higher altitudes and latitudes, one or two distinct vegetation layers are more common (Webb and Tracey 1994). Under the classiﬁcation of Floyd (1990), rain forests where Macadamia is most common are subtropical types, usually dominated by Argyrondendron species (Booyongs), coastal rain forest on basalt dominated by Cupaniopsis anacardioides (Tuckeroo), and rain forest with Araucaria (Hoop Pine) as an emergent tree (Table 1.2). Floyd’s (1990) ﬂoristic alliances are equivalent to a number of types deﬁned on the basis of structure (various forms of notophyll vine forest) (Table 1.2). Notophyll rain forests contain species where the majority of leaves are approximately 6 to 8 cm long. Tropical rain forest tends to be comprised of species with mesophyll leaves (12.7 cm long or larger) compared to subtropical forms, and temperate rainforests typically have smaller leaves (on the order of 2.5 cm long, after Webb 1968). A variety of plant lifeforms and features are characteristic of subtropical rain forest. These include a multilayered billowing

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canopy, stranglers, palms, plant buttressing, epiphytes, woody vines, large-leaved herbs, and ground vines (Floyd 1990; Hunter 1991). Dryer subtropical rain forests generally have two tree strata, an upper layer with scattered emergents such as hoop pine and a lower continuous stratum. Leaves are commonly compound, thick and hard, and usually less than 7.5 cm long (microphyll, after Webb 1968). Stranglers and woody vines are common, but plant buttressing and large epiphytes are rare. The shrub layer is well developed and prickly, and the herb layer is sparse (Floyd 1990; Hunter 1991). 2. Rainfall, Climate, and Soils. The subtropical rain forest in which the southern clade Macadamia typically are found occurs in warm, humid locations, where annual rainfall is high (> 1300 mm), reliable and uniformly distributed or with summer maxima. The wettest months in northern NSW and southeast Queensland are January, February, or March. The driest months are August or September. Average annual rainfall is between 1120 and 2351 mm. Temperatures in the region tend to be moderate, with average minima between 13 and 16.4 C and maxima between 22.4 and 27.1 C. January is generally the hottest month and July the coolest (Commonwealth Bureau of Meteorology 2003). Subtropical rain forests are found below 600 m elevation on Cainozoic igneous rocks, especially on basalt and rhyolite in the McPherson and Main ranges of southern Queensland (Sattler and Williams 1999) and on the volcanic geology of the Tweed shield (RACAC 1996). Rain forest is mostly found at low altitude and is replaced with warm-temperate rain forest with increasing altitude or latitude (Floyd 1990). Rain forest is common on high-fertility soils such as red krasnozems and brown prairie soils that are rich in nutrients like phosphorous and calcium, essential for rain forest growth (Floyd 1990). Occurring over a range of substrates and topographic positions where there is high soil nutrient status and good drainage (Barry and Thomas 1994), M. integrifolia has the largest geographic range and may also have the greatest environmental amplitude of the southern Macadamia species. M. integrifolia is most commonly found on high-nutrient volcanic (basalt and diorite) and alluvial soils that are slightly acid (pH 5.5 to 6.5) and has been recorded at altitudes between 5 and 340 m, on slopes ranging from steep to level (Barry and Thomas 1994), and on north, southeasterly, and west aspects. Found on high-fertility volcanic soils, M. tetraphylla also occurs to a lesser extent on alluvial deposits (e.g., upper Mullumbimby Creek) or weathered volcano-lithic rocks in the Burringbar Range (Pisanu 2001). Within the Tweed and Richmond River catchments, M. tetraphylla

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occurs at the base of the rhyolite cliffs of the border ranges, on the lower south- and east-facing slopes of Mount Warning (Pisanu 2001). Soils are well drained (Barry and Thomas 1994), with textures from clayey sand to loams or silty clay, and soil pH between 4.98 and 5.87 (Pisanu 2001). The species is found at altitudes between 10 and 460 m (Barry and Thomas 1994) but mostly around 150 m (Gross 1995), and on moderate to steep slopes on north, south, east, and west aspects (Pisanu 2001). Generally found on soils derived from volcanic parent material, mostly basalt but also trachyte, andesite, tuff, and rhyolite, M. ternifolia is also known to occur at the interface between sandstone and basalt (Barry and Thomas 1994). Soils tend to be well-drained sandy loams to light clays, and pH ranges between 5.5 and 7.0 (Barry and Thomas 1994). This species is found at altitudes between 100 and 320 m but mostly below 200 m, usually on moderate to steep hill slopes and foot slopes (Barry and Thomas 1994). It may have a more restricted habitat preference than the other two species as it has mostly been recorded in south-facing gullies. Macadamia jansenii is found in a single moderately steep gully at 350 m elevation with east-southeast aspect. The geology is Muncon volcanics, a mixed intermediate and basic lava volcanic/sedimentary complex. Soils are dark brown sandy clay loams with good drainage, about 40% rock fragments on the surface, and pH 7.0 (Barry and Thomas 1994). 3. Abundance and Population Dynamics. Patterns of abundance within populations of Macadamia species appear to vary. Macadamia integrifolia and M. ternifolia occur sparsely in their natural habitat, with individual plants widely separated (Barry and Thomas 1994; Neal 2007). In contrast, M. tetraphylla distribution is described as clumped, with very few individuals dispersed between clumps (Pisanu 2001). It is not clear, however, whether these patterns are natural phenomena or an artifact of clearing and habitat fragmentation. Several studies on the population dynamics of wild Macadamia suggest that mature populations are demographically stable, having low rates of mortality and recruitment, with recruitment increasing in response to disturbance. Pisanu (2001) found that survivorship within M. tetraphylla populations was high at all growth stages with low mortality at mature stages, suggesting that populations have some level of resilience to periodic disturbance. Populations in small fragments were found to be increasing at slow rates, whereas populations within contiguous forest did not change over a three-year period (Pisanu 2001). Similarly, an investigation into the population dynamics and

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demography of M. integrifolia (Neal 2007) observed increased site-level fecundity and recruitment in small and medium-size habitat fragments compared to larger remnants. No evidence of differences in mortality levels between fragment sizes was found, suggesting stronger population growth in the smaller fragments compared to the more intact sites. E. Genetic Structure and Dynamics of Native Populations 1. Genetic Structure of Natural Populations. Molecular marker studies have provided insights into the patterns of genetic diversity in remnant wild populations of Macadamia. The ﬁrst survey of genetic diversity used isozymes (Sharp and Playford 1997), and clustered southern M. integrifolia and northern M. tetraphylla populations together and separately from northern M. integrifolia and southern M. tetraphylla populations. The authors concluded that this pattern was caused by the inclusion of hybrid populations in the southern M. integrifolia region, which confounded the true relationship between the species regions. As part of a more recent molecular marker study, 165 genomic loci (RAF) were screened (Peace 2005) for 274 accessions of the National Macadamia Germplasm Collection, comprising most of the geographic range of the four southern clade species (Hardner et al. 2004). The four species could be clearly distinguished using this marker set, and hybrids were removed from the analysis of the pure species. M. integrifolia populations exhibited a signiﬁcant isolation-bydistance effect over the range of the species. (Proximate populations were more genetically similar than more distant populations.) Populations from a northern M. integrifolia group around the Mary River valley (including the Mount Bauple and Amamoor regions) were partially differentiated from a southern group of populations from the Pine Rivers district south to the Gold Coast hinterland. However, the overall measure of genetic differentiation between populations was moderate (Gst ¼ 0:233) and indicates historical gene exchange between proximate populations (Peace 2005). M. tetraphylla populations overall exhibited lower regional differentiation (Gst ¼ 0:143), indicating higher levels of historical gene ﬂow between populations (Peace 2003). No signiﬁcant isolation-by-distance effect was observed. In the regions sampled for M. ternifolia, this species exhibited greater regional and population differentiation, but less diversity within populations, than M. integrifolia sampled in the same region (Peace 2005). However, approximately two-thirds of the natural distribution of M. ternifolia (mostly the northern range) were not surveyed and so remain uncharacterized. Little is known of genetic character of the only

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known population of M. jansenii. However, the two accessions of this species that were assessed in the RAF marker analysis were as genetically distant as any two M. ternifolia accessions, and not particularly closer than any two accessions within the other two species, suggesting that the single known population of M. jansenii contains appreciable genetic diversity (Peace 2005). Two microsatellite marker studies have recently been completed independently for M. tetraphylla and M. integrifolia. A study of M. tetraphylla (Spain 2006) screened six populations from the Mount Warning caldera to the Lennox Head area of NSW for four microsatellite loci (Schmidt et al. 2006). Moderate to high levels of genetic diversity (He ¼ 0:422) and low adult population differentiation (y ¼ 0.016) were found, indicating high historic gene ﬂow between populations. Four microsatellite loci (Waldron et al. 2002; Peace et al. 2003; Schmidt et al. 2006) were used to screen 10 populations of M. integrifolia in the Amamoor and Samford regions of Queensland (Neal 2007). High levels of adult tree diversity (He ¼ 0:77) and low genetic differentiation between populations at Amamoor (Fst ¼ 0:069) and Samford (Fst ¼ 0:047) were also evident in this species. 2. Mechanisms of Gene Flow. The low population differentiation observed in the above molecular marker studies of M. integrifolia and M. tetraphylla suggests that gene-ﬂow mechanisms are sufﬁcient to have maintained a network of interbreeding populations over a large area of suitable habitat (Spain 2006; Neal 2007). Considering the difference in scale of sampling between the microsatellite studies and the RAF marker studies, the levels of gene ﬂow are comparable, and indicate high historical gene ﬂow for both species between proximate populations (5 to 50 km) but more restricted gene ﬂow among more dis- tant populations (> 50 km). In general, mid- to understory shrubs and trees are expected to have effective gene-ﬂow mechanisms to maintain genetic contact between disparate individuals (Ward et al. 2005). This hypothesis could be explored further in macadamia by using biparental markers to assess the relative contribution of pollen and seed dispersal. Certainly, Macadamia species appear adapted to low-density living. Although most current knowledge of the reproductive biology of Macadamia is based on cultivated trees growing within the natural range, it probably closely approximates that occurring in the wild. However, it is likely that individuals within natural populations are inﬂuenced by a wider range of complex ecological factors (Neal 2007), and thus the actual reproductive behavior of these plants may be quite different from that of the relatively precocious individuals growing in

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commercial plantations that have been developed through selection and horticulture inputs. In orchard studies, M. integrifolia has been observed to possess a partial gametophytic self-incompatibility mating system (Sedgley et al. 1990). This should effectively promote outcrossing and limit selffertilization in natural populations. As predicted, a survey of openpollinated progeny arrays from two remnant M. integrifolia stands (Neal 2007) conﬁrmed that progeny are almost solely the result of outcrossing, with no selﬁng or biparental inbreeding observed. Outcrossing experiments in orchards observed higher rates of fertilization and nut set when cultivars were outcrossed, though some viable seed is still produced after selﬁng (Sedgley et al. 1990; Meyers 1997; Vithanage et al. 2003). However, in natural populations of M. integrifolia, no optimal crossing distance or effect of genetic relationship (including selﬁng) on fruit set was observed, suggesting that fruit set in wild M. integrifolia populations is likely to be resource limited rather than pollen limited (Neal 2007). Similar to that in cultivated orchards (Heard and Exley 1994; Wallace et al. 1996), both introduced honey bees and native stingless bees have been observed visiting M. integrifolia ﬂowers in natural populations (Neal 2007). However, in M. tetraphylla, only honey bees were observed foraging ﬂowers at nine sites over a three-year period (Pisanu 2001). Studies of pollen ﬂow in orchards suggest that cross-pollination can occur over hundreds of meters across rows (Vithanage et al. 2003). In natural populations, pollination distances over several kilometers have been observed using paternity analysis (Neal 2007). Despite concerns that the introduced honeybee A. mellifera may pose a threat to many native plant species by altering pollination and increasing inbreeding (Gross 2001), this hypothesis is not supported in M. integrifolia given that almost complete outcrossing was observed in the wild (Neal 2007). It may be that the stronger ﬂight and increased potential for pollen carryover of A. mellifera (Ghazoul et al. 1998) can even lead to increased pollination distances compared to native pollinators. Water, gravity, and animals have also been proposed as potential dispersal vectors of Macadamia fruit (Pisanu 2001; Peace 2005). Pisanu (2001) found that dispersal of M. tetraphylla seeds was linked to slope angle with distinctive seedling shadows down-slope of large trees and with a mean distance of seedlings to adults of 3.81 m. All seed germination occurred in close proximity to adult trees, with 77% of seeds germinating within two meters of an adult during the threeyear period of study (Pisanu 2001). The presence of small M. tetraphylla populations and individual plants along creek beds downstream of large populations of these species in the upper catchments suggests that

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limited dispersal by ﬂood events may occur (McConachie 1980; Pisanu 2001; Peace 2005). Gallery notophyll vine forest has been identiﬁed as habitat for M. integrifolia and M. ternifolia (Environmental Protection Agency 2005b), and a signiﬁcant number of Macadamia population records are located adjacent to watercourses, providing support for this hypothesis (Pisanu 2001). However, the commercial practice of using water to separate mature nuts (sinkers) from low-quality nuts (ﬂoaters) does not suggest that mature viable fruit ﬂoat in water. It may be that swift ﬂoodwaters are the only effective mechanism by which macadamia seeds are carried downstream. Rodents (Rattus rattus, Uromys caudimaculatu) are predators of Macadamia seeds in Australian orchards (Horskins and Wilson 1999), with signiﬁcant levels of nuts removed to adjacent habitats (Elmouttie and Wilson 2005), suggesting that rats may have a role in the dispersal of nuts in the wild. Pisanu (2001) found evidence of rodent seed predation at all M. tetraphylla study sites with 25 to 100% of seeds taken in a ﬁeld seed removal trial. However, there is little evidence of hording of nuts by rats, as almost all nuts found in rat burrows in native habitat adjacent to commercial orchards were damaged (Elmouttie and Wilson 2005). There is limited information of the role of birds and other mammals in the dispersal of macadamia fruit in natural populations (McConachie 1980; Peace 2005). The role of humans in the precolonization dispersal of Macadamia is unknown. Macadamia nuts were a food source for indigenous peoples both in Indonesia and Australia (Gross 1995; Hill and Baird 2003). M. hildebrandii, M. integrifolia, M. tetraphylla, and M. whelanii are all recorded as being eaten or used for their oil (Gross 1995), and it has been suggested that Aborigines may have transported Macadamia nuts over long distances (McConachie 1980).

F. Conservation Status of Wild Populations 1. In Situ Conservation Status of Macadamia Habitat. Protection of habitat is critical to Macadamia conservation. The region that contains the southern Macadamia clade is currently experiencing sustained growth in both the agriculture and urban-industrial sectors, with increasing pressure on remaining areas of native vegetation and wildlife populations (Hardner et al. 2004). A high proportion of the former extent of lowland subtropical rain forest throughout the range of southern Macadamia has been cleared. For example, in the combined Tweed, Byron Coast,

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Richmond River administration areas, less than 30% of the original land cover comprises undisturbed native vegetation (NSW Parks and Wildlife Service 1993). Much of the central and eastern part of this region was formerly covered by the ‘‘Big Scrub,’’ an area of continuous subtropical lowland rain forest extending from the townships of Bryon Bay to Lismore (Holmes 1987). Currently less than 1% of the Big Scrub remains, comprising a disjunct network of small remnants totaling 550 ha across the Lismore Plateau (Lott and Duggin 1993). Similarly, in southeast Queensland, approximately 40% of the original extent of subtropical rain forest remains (WWF Australia 2004; Accad et al. 2006). In southeast Queensland, one of the four regional ecosystem types described as habitat for southern Macadamia (12.3.1) (Environmental Protection Agency 2005b) is represented by less than 10% of its original extent and is classiﬁed as Endangered (Accad et al. 2006). Loss of Macadamia-suitable habitat has been greatest on private property, where less than 24% of the original extent remains (Accad et al. 2006). As of 2003, 37% of remnant Macadamia habitat was located on private property, with a further 41% located within relatively unprotected state forests (subject to timber exploitation). Currently, 21% of remnant Macadamia habitat is located within conservation areas (Accad et al. 2006). Impact of Habitat Fragmentation. Much of the remnant wild Macadamia occurs as small ( < 50 individuals) fragmented populations surrounded by cleared habitat (Hardner et al. 2004; Neal 2007). The impact of habitat fragmentation on the population demography, associated community diversity, and genetic diversity of Macadamia has been examined recently. Spain (2006) surveyed six fragmented populations of M. tetraphylla of varying size and disturbance. The genetic diversity of seedling cohorts within stands was positively correlated with population size. However, while there was no correlation with population inbreeding and population size, level of inbreeding was signiﬁcantly correlated with density of adult trees. This may indicate a potential biparental inbreeding effect, as spatial genetic structure (where proximate individuals are more genetically similar than those farther away) was evident at all sites. Compared to mature trees, genetic differentiation in the seedling cohort increased (from 0.016, p ¼ 0:23, to 0.061, p < 0:0001, respectively), indicating increased genetic drift due to a reduction in gene ﬂow of the seedling cohort compared to mature trees, a probable consequence of the fragmentation process. Diversity of the ﬂoristic community associated with M. tetraphylla was not signiﬁcantly related to fragment size, but a

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signiﬁcant correlation between disturbance level and species paucity and the incidence of invasive species was evident. No correlation was found between the community diversity of species and the genetic diversity of M. tetraphylla. A second study (Neal 2007) surveyed 10 plots of M. integrifolia within rain forest patches of varying size and isolation. Rather unintuitively, stronger population growth rates were evident in small fragments compared to plots located in medium- and large-habitat blocks, as a result of increased site-level fecundity and recruitment, but with no apparent change in short-term mortality. Resource availability, particularly increased lights levels in small fragments, is the most likely cause of this observed effect. Population viability may also beneﬁt from the long life span of the species and observed resilience of adult plants to disturbance, potentially buffering populations against stochastic events (Neal 2007). In contrast to the study of M. tetraphylla (Spain 2006), Neal (2007) found that heterozygosity estimates in M. integrifolia were comparable across sites and cohorts, independent of fragmentation status. However, allelic diversity was correlated with fragment size. In addition, small M. integrifolia sites displayed increased differentiation, decreased interpopulation gene ﬂow, and higher genetic similarity between individuals compared to plots in medium and large fragments. At two study sites where open-pollinated progeny arrays were surveyed, there was little evidence of inbreeding, and paternity analysis of open pollinated progeny arrays demonstrated long-distance gene ﬂow between sites that were separated by 2.8 km, suggesting that despite fragmentation, M. integrifolia can maintain genetic connectivity over a wide geographic area (Neal 2007). Pollination by introduced honeybees in small fragments may actually facilitate gene ﬂow across the landscape due to increased foraging distances and greater capacity for pollen carryover compared to native pollinators, an effect observed in other species (Dick 2001). In summary, both studies identiﬁed detrimental genetic effects of fragmentation for two Macadamia species that are likely to be shared by all species demonstrating similar life history characteristics. These impacts include decreased genetic diversity within fragments and decreased gene ﬂow between fragments. In both studies, a potential effect of intrapopulation genetic structure was identiﬁed as a potential inbreeding threat to small populations. However, despite these ﬁndings, species of Macadamia appeared to maintain high levels of genetic diversity even within small fragments, indicating that small fragments retain conservation value. In addition, smaller fragments exhibited increased demographic growth and potential for long-distance gene ﬂow.

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Supplementary planting in small fragments, or increasing connectivity between remnants through habitat restoration, is expected to reverse some of the observed fragmentation impacts and would be among the best strategies to preserve the species in situ, particularly in highly fragmented landscapes (Neal 2007). Legislative Protection. At the national level, Australian legislation and policies deﬁne measures for the conservation of species and communities under the Environment Protection and Biodiversity Conservation Act 1999 (Department of Environment and Heritage 2006). The country is further obligated as signatory to treaties under the Convention on Biological Diversity 1992, which requires consideration of a global strategy for plant conservation, protection of ecosystems, natural habitats, and the maintenance of viable populations of species in natural surroundings (Secretariat of the Convention on Biological Diversity 2005). At the global level, the conservation status of species is assessed and deﬁned under the International Union for the Conservation of Nature (IUCN) Red List of Threatened Species (Standards and Petitions Working Group 2006). The southern Macadamia species are not currently red-listed because most plant taxa listed in the 1997 IUCN Red List of Threatened Plants (Walter and Gillett 1998) have not yet been evaluated against the revised Red List Criteria (IUCN 2006). At the regional level, southern Macadamia species are listed under the Queensland Nature Conservation Act 1992 (Environmental Protection Agency 2005a) and the NSW Threatened Species Conservation Act 1995 (Department of Environment and Conservation 2006). Under relevant Australian jurisdictions and the 1997 IUCN Red List, M. jansenii is classed as Endangered, because it is known only from a very small population with very restricted distribution. The remaining three species are classed as Vulnerable because of population declines attributed to clearing and fragmentation of lowland subtropical rain forest throughout their geographic range. 2. Ex Situ Conservation. A collection of cuttings from over 370 trees across more than 70 sites (including native populations, old planted populations, and stands of unknown origin) has been used to establish the National Macadamia Germplasm Collection as an extensive core collection of the major species of the southern Macadamia clade (Hardner et al. 2004; Peace 2005). This collection has been planted in orchard trials to conserve a large sample of the genetic variation and evaluate the material for introduction into future breeding programs. An obvious exclusion is M. jansenii, although the small size of the only

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known population of this species may limit collection through conventional methods.

III. GERMPLASM DOMESTICATION Genetic improvement in macadamia has delivered long-term commercial gains to macadamia production (Hamilton and Ito 1984; Stephenson and Gallagher 2000) and has underpinned the success and expansion of the industry throughout the world (Hamilton and Ito 1984; Stephenson 1990a; Nagao and Hirae 1992; Allan 1993). However, as the crop has only recently been domesticated, with cultivars only a few generations from the wild, macadamia germplasm is relatively underdeveloped, and much potential for genetic improvement appears to exist. Detail of the origin and pedigree of domesticated germplasm is scant with most only available in industry publications. Recent development of DNA marker methodology (Aradhya et al. 1998; Steiger 2003; Steiger et al. 2003; Peace 2005; Peace et al. 2005; Schmidt et al. 2006) has assisted with the elucidation of genetic relationships in the domesticated germplasm. A review of published and unpublished literature identiﬁed over 900 cultivar names representing 500 apparently distinct genetic entities (Hardner and McConchie 1999). However, only selections that have had some commercial or historical signiﬁcance are considered here. A. Hawaii Despite the Australian origins of the plant, macadamia was initially commercialized in Hawaii, and germplasm improvement is considered as having played a major role in this development (Hamilton and Storey 1956; Shigeura and Ooka 1984; Hamilton and Ito 1986; Nagao and Hirae 1992; Wagner-Wright 1995). In addition, Hawaiian cultivars are responsible for much of the current world production (Hamilton and Fukunaga 1970; Allan 1989; Ito and Hamilton 1989; Stephenson 1990a; Peace et al. 2005; Tay 2006). 1. Initial Introductions. The ﬁrst introduction of macadamia to Hawaii was on the island of Hawaii by William Herbert Purvis sometime between 1881 and 1885 (Hamilton and Storey 1956; Hamilton and Fukunaga 1959; Shigeura and Ooka 1984; Wagner-Wright 1995) (Fig. 1.3). The origin of these seeds is uncertain, although DNA proﬁling suggests that the germplasm was sourced from the Mount Bauple region (Peace 2005).

33

Fig. 1.3. Recorded origins of domesticated macadamia germplasm. Dotted lines represent uncertainty of lineage, and OP signiﬁes open pollination. Horizontal relationships are not necessarily reﬂective of chronological order of selection.

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It is also reported that this sample contained germplasm producing small bitter nuts (Shigeura and Ooka 1984), although it is unknown if these represented what is known today as M. ternifolia. A second independent introduction of M. integrifolia was made in 1892 to the island of Oahu by Captain Robert Alfred Jordan, who reportedly was given some locally collected seeds during a visit to a friend in Pimpama, south of Brisbane (Shigeura and Ooka 1984; Wagner-Wright 1995). However, recent DNA proﬁling suggests that wild origin of this germplasm is from populations around the Amamoor region (Peace 2005), approximately 300 km north of Pimpama (Fig. 1.3) (see later discussion). This introduction reportedly produced six trees (Wagner-Wright 1995) and was considered the principal source of the ﬁrst of the Hawaiian commercial cultivars (Storey 1965b), although both the Purvis and Jordan germplasm sources appear to have given rise to important cultivars (Peace 2005). M. tetraphylla was used in reforestation plantations on the island of Hawaii in 1892 to 1894 by the Territorial Board of Agriculture and Forestry and thus represents a third early introduction of macadamia into Hawaii (Shigeura and Ooka 1984; Wagner-Wright 1995). No details of the source can be found, but it is has been suggested they came from the Murwillumbah area in northeast NSW (McConachie 1980; Wagner-Wright 1995). There are suggestions of other introductions of macadamia in the early 20th century (Wagner-Wright 1995), but no other information conﬁrming this could be found. 2. First Orchards. From 1910, the potential of macadamia as a crop was considered in Hawaii, and by 1912, the Hawaii Agricultural Experiment Station (HAES) had begun distributing seedlings for commercial plantings (Wagner-Wright 1995). The ﬁrst commercial orchards were established in the 1920s by the Honokaa Sugar Company at Mauka Kea on the island of Hawaii and by the Hawaiian Macadamia Nut Company (HMNC) in 1925 at Nutridge on the island of Oahu and Keauhou on the island of Hawaii (Ito 1983; Shigeura and Ooka 1984; Wagner-Wright 1995). The orchard at Mauka Kea was reportedly established with seed collected from the Purvis introduction of M. integrifolia (Shigeura and Ooka 1984) (Fig. 1.3). Some authors suggest the Nutridge orchard was planted with both the Jordan and Purvis seedlings (Shigeura and Ooka 1984; Wagner-Wright 1995), although others (Urata 1954) record that only the Purvis germplasm was used. The Keauhou orchard was planted with over 7,000 seedlings of both M. tetraphylla and M. integrifolia, although the M. tetraphylla trees were later removed (Shigeura and Ooka 1984;

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Wagner-Wright 1995). Numerous other small orchards were also established (Wagner-Wright 1995). From the early development of the industry in Hawaii, M. integrifolia was the preferred species (Ripperton et al. 1938; Cavaletto 1983; Shigeura and Ooka 1984). M. tetraphylla trees were considered to bear spasmodically and be more susceptible to insect attack. The shape of nuts was considered to be unsuitable (oblong) and shells harder and denser than M. integrifolia. The quality of kernels was reportedly more variable after oil roasting, although the taste was considered sweeter and more pronounced (Ripperton et al. 1938; Cavaletto 1983; Shigeura and Ooka 1984; Wagner-Wright 1995). In contrast, M. integrifolia nuts were considered to have thinner shells and more consistent response under oil roasting (Wagner-Wright 1995). No M. tetraphylla trees have been planted in Hawaii since about 1939 (Hamilton and Storey 1956), and existing M. tetraphylla trees were either eliminated or top-worked (Wagner-Wright 1995). 3. Scion Selection Program. The development of reliable grafting technology (Jones and Beaumont 1937; Shigeura and Ooka 1984) created the possibility of reducing the variability of seedling material and exploiting the full genetic variation available. This is considered to be one of the turning points in the history of the crop (Hamilton and Fukunaga 1973; McConachie 1980; Shigeura and Ooka 1984; WagnerWright 1995). It has been suggested that clonal orchards produce three to ﬁve times that of seedling orchards (Hamilton and Fukunaga 1959). A scion selection program was initiated by the Hawaiian Agricultural Experiment Station (HAES) between 1934 and 1936 by surveying existing commercial seedling orchards to identify elite trees for further testing (Hamilton and Storey 1956; Hamilton and Ito 1984; Shigeura and Ooka 1984; Wagner-Wright 1995). Initial selection of promising orchard seedlings was based on observations of tree structure and vigor, production, apparent pest and disease resistance, nut characteristics, kernel recovery, and kernel characteristics (Beaumont 1937; Hamilton and Fukunaga 1973; Shigeura and Ooka 1984), although there is little detail on how these were assessed and integrated to compare candidates. In 1935 and 1936, the HAES made the ﬁrst selections from seedling orchards, all M. integrifolia by morphology (Hamilton and Storey 1956; Wagner-Wright 1995). By 1938, nuts from 19,000 trees had been evaluated to select 41 promising cultivars (Wagner-Wright 1995). This was reduced to ﬁve selections for establishment of clonal orchards over six sites for evaluation of productivity (Hamilton and Fukunaga 1959,

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1973; Shigeura and Ooka 1984; Wagner-Wright 1995). Approximately 8,000 seeds were also collected from these selections for establishment of progeny trials (Wagner-Wright 1995). In 1948, ﬁve cultivars were released and given Hawaiian names to associate them with their origin (Hamilton and Ito 1984; Shigeura and Ooka 1984; Wagner-Wright 1995) (Fig. 1.3). ‘Keauhou’ (HAES 246) is the oldest Hawaiian cultivar, ﬁrst selected in 1935. The others were ‘Pahau’ (HAES 425), ‘Nuuanu’ (HAES 336), ‘Kohala’ (HAES 386), and ‘Kakea’ (HAES 508). Following the release of the ﬁrst set of cultivars, the selection program was continued and expanded to the screening of seedlings in orchards or progeny plantings (Hamilton and Ito 1976). Between 1934 and 1984, an estimated 120,000 orchard seedlings and progeny plantings had been surveyed to give over 900 selections (Hamilton and Ito 1976, 1984). Most of these were discarded after what is described as preliminary screening and evaluation procedures (Hamilton and Fukunaga 1973; Hamilton and Ito 1976). While some selections have proved commercially valuable, the shortcomings of this process have been described (Hamilton and Fukunaga 1973); standards used for macadamia selection were later considered variable and arbitrary, were based mainly on superﬁcial observation of the original seedling trees and nuts produced, and testing for yield, quality and suitability was often incomplete. The most promising selections were grafted and established in trial plantings to objectively evaluate their productivity under orchard conditions (Hamilton and Fukunaga 1973; Hamilton and Ito 1976). Released cultivars were also planted as checks. Selections were assessed for vigor, tree habit, presence of stick-tights, number of fruit per raceme, productivity on favorable and unfavorable sites, nut size, kernel recovery, percentage of ﬁrst-grade kernel, and raw kernel appearance (Hamilton and Ito 1976). Details of these selection criteria are discussed later. Techniques for controlled crossing to produce full-sib families were developed in Hawaii (Urata 1954); however, it has been reported that while 300 crosses were made, they failed to produce progeny with desirable characteristics (Stephenson 1990a). By 1990, a total of 14 cultivars had been named and released by HAES (Nagao and Hirae 1992). In addition to the initial ﬁve cultivars, ‘Ikaika’ (HAES 333) and ‘Wailua’ (HAES 475) were released in 1952, ‘Keaau’ (HAES 660) in 1966, ‘Kau’ (HAES 344) in 1971, ‘Mauka’ (HAES 741) and ‘Makai’ (HAES 800) in 1977, and ‘Purvis’ (HAES 294) and ‘Pahala’ (HAES 788) in 1981 (Hamilton et al. 1981; Hamilton and Ito 1984; Shigeura and Ooka 1984). The ﬁnal named cultivar from the program ‘Dennison’ (HAES 790) was released in 1990 (Hamilton and Ito 1990).

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Two HAES selections have been named by others. ‘HAES 791’, which was rejected in Hawaii because of poor structure and prolonged ﬂowering under tropical conditions, was found to be suitable to South African conditions (Blight 1989). It was originally given the name ‘Richard’ in South Africa (Blight 1989) but later named ‘Fuji’ in Hawaii (Peace et al. 2005). ‘Jordan’ (HAES 426) was named as an ornamental cultivar in California (Brooks and Olmo 1978). Other selections made in Hawaii outside the HAES evaluations are also recorded (Urata 1954; Hamilton and Ito 1976; Shigeura and Ooka 1984; Wagner-Wright 1995), in particular ‘Honokaa Special’, ‘Chong 3’, and ‘Bond 23’. There has been no release of cultivars since 1990, although promising later selections have been made available for commercial utilization without ofﬁcial release and are commonly known by their HAES selection number (Ito and Hamilton 1989; Stephenson et al. 1995; Nagao et al. 2003). Few new seedlings have been planted out for evaluation (Mehlenbacher 2003). 4. Further Introduction of Australian Germplasm. A second wave of introductions was made from the 1940s through the 1950s (Hamilton and Fukunaga 1962). These authors record that the ﬁrst successful importation of scion wood was made in 1949 by the University of Hawaii of a reputedly highly productive clone from a grower in southern Queensland. This, however, was of M. tetraphylla type and not productive in Hawaii. A further six clones were imported from Australia in 1950 and 1951, and one M. tetraphylla and two M. integrifolia types were imported from Queensland in 1952. In 1954, Dr. Beaumont of the HAES visited Australia and collected 34 scions of which 24 were successfully propagated in Hawaii. A further 24 scions were imported after 1955 (Hamilton and Fukunaga 1962). By 1962, 30 of the introductions had fruited, with 21 being of M. tetraphylla or hybrid type. Of those fruiting, ‘HAES 685’ (‘B21’ and ‘Teddington’ in Leverington 1962a) was the only introduction listed as a promising variety, and ‘HAES 666’ (‘B5’ or ‘Rickard’ in Leverington 1962a) was kept for further observation. This group also included ‘HAES 695’ (or ‘NSW-44’), which although discarded in Hawaii (Hamilton and Fukunaga 1962), would later be taken to California and named as ‘Beaumont’. It has been suggested that the Australian M. integrifolia selections introduced into Hawaii had a tendency to bear earlier than the introduced M. tetraphylla selections (Hamilton and Fukunaga 1962). However, there has been little impact of these introductions on the Hawaiian breeding program (Hamilton and Fukunaga 1973), apart as parents for several newer selections (see below).

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Table 1.3. Summary of HEAS cultivars. Name

HAES no.

Keauhou Pahau Nuuanu Kohala Kakea Ikaika Wailua Keaau Kau Mauka Makai Purvis Pahala

246 425 226 386 508 333 475 660 344 741 800 294 788

Dennison

790

Referencea

Source

Release year

Keauhou orchard Keauhou orchard Keauhou orchard Keauhou orchard Nutridge orchard Nutridge orchard

1948 1948 1948 1948 1948 1952 1952 1966 1971 1977 1977 1981 1981

1,2 1,2 1,2 1,2 1,2 3,4 5 4, 6 3, 4, 7 4, 5 4, 8 7, 9, 10 9, 10, 11

1990

9

Glaisyer Orchard, Kauai Nutridge orchard Glaisyer orchard, Kauai OP progeny of ‘Keauhou’ Nutridge orchard Originally reported as OP progeny of ‘Jordan’ OP progeny of ‘Keauhou’

1 ¼ Storey 1963; 2 ¼ Shigeura and Ooka 1984; 3 ¼ Hamilton and Ito 1984; 4 ¼ Aradhya et al. 1998; 5 ¼ Hamilton and Storey 1956; 6 ¼ Steiger et al. 2003; 7 ¼ Wagner-Wright 1995, 8 ¼ Ito and Hamilton 1989; 9 ¼ Ito and Hamilton 1990; 10 ¼ Brooks and Olmo 1983; 11 ¼ Hamilton et al. 1981.

a

5. Summary of Pedigree Relationships. The historical records and known pedigrees of the cultivars suggest they may be between two and four generations from the wild (Table 1.3, Fig. 1.3). ‘Kakea’, ‘Ikaika’, ‘Kau’, and ‘Purvis’ were selected from among seedling planted in the Nutridge orchard (Shigeura and Ooka 1984; Wagner-Wright 1995). Recent DNA marker studies indicates that ‘Kakea’, ‘Ikaika’, and ‘Purvis’ share greater afﬁnity among themselves compared to ‘Kau’ (Peace 2005). ‘Keauhou’ was selected from the Keauhou orchard (Shigeura and Ooka 1984; Wagner-Wright 1995). ‘Keaau’ and ‘Mauka’ were selected from the Glaisyer orchard in Lawai valley on the island of Kauai (Hamilton and Ito 1977a; Vithanage and Winks 1992; Wagner-Wright 1995; Aradhya et al. 1998) (Table 1.3) and appear closely related from molecular marker analysis (Peace 2005). No record of the germplasm used to establish these orchards is available. Several of the Hawaiian cultivars are advanced generation selections from open-pollinated progeny of early cultivars. The cultivar ‘Keauhou’ is reported as the seed parent ‘Makai’, which was selected from openpollinated progeny planted at the Waiakea Experimental Farm (Hamilton and Ito 1977a; Ito and Hamilton 1989; Aradhya et al. 1998), and ‘Dennison’, which was selected from similar progeny planted at the University of Hawaii Waimanalo Research Station (Hamilton and Ito

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39

1990). ‘HAES 804’ and ‘HAES 835’ are also reported as open-pollinated progeny of ‘Keauhou’ (Ito and Hamilton 1989). DNA marker analysis (Peace 2005) has conﬁrmed all of these cases of ‘Keauhou’ parentage and has identiﬁed other undescribed progeny from this parent (‘HAES 783’ and ‘HAES 828’). ‘Pahala’ was originally reported as an open-pollinated progeny of ‘Jordan’ (Hamilton et al. 1981; Brooks and Olmo 1983). The parentage of ‘Jordan’ is not recorded; however, it appears to have been originally selected from the Keauhou orchard (Brooks and Olmo 1978). Others (Aradhya et al. 1998; Steiger et al. 2003) may have misunderstood this description, as they have attributed the parentage of ‘Pahala’ to the cultivar ‘Keauhou’. ‘Honokaa Special’ is reported as the seed parent of ‘HAES 814’ (Vithanage and Winks 1992). There is little detail on the orchard origin of the maternal parent, although it was most likely selected from the Mauka Kea planting of the Honokaa Sugar Company (Wagner-Wright 1995), which was established with predominately Purvis germplasm (Urata 1954; Shigeura and Ooka 1984). ‘HAES 816’ is an openpollinated progeny of ‘HAES 666’ (Ito and Hamilton 1989), which is identiﬁed by others (Hamilton and Fukunaga 1962; Leverington 1962a) as the Australian selection ‘Rickard’. and ‘Teddington’ (‘B21’ or ‘HAES 685’) is reportedly the mother of the open-pollinated selection ‘HAES 856’ (Aradhya et al. 1998). ‘Keaau’ is reportedly the maternal parent of the open-pollinated ‘HAES 915’ (Ito and Hamilton 1989). B. Australia 1. Early Seedling Orchards. Until the mid-1960s, orchards in Australia were established with seedling material (Wills 1961; Leverington 1962a, 1971). Planting of macadamia in Australia reportedly began around the 1860s in areas coincident with the natural distribution of the species, with seed most likely sampled from the surrounding natural populations (McConachie 1980) (Fig. 1.3). The world’s ﬁrst commercial macadamia orchard was of M. tetraphylla and was planted sometime between 1878 and 1888, at Rous Mill, near Lismore, NSW (McConachie 1980). By 1900, there were ﬁve M. tetraphylla orchards in NSW but no recorded orchards in Queensland, but many specimen trees in parks and gardens (McConachie 1980). The ﬁrst orchard in Queensland (of 30 M. tetraphylla trees) was planted in about 1910, while the ﬁrst large commercial orchard in Queensland was planted in 1917 (McConachie 1980). Orchard plantings increased in both NSW and Queensland through the 1910s to 1930s (Wills 1939; Willis 1961; McConachie

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1980). NSW orchards were entirely of the local M. tetraphylla species until about 1931, whereas both species were planted in Queensland before this time (McConachie 1980). A high proportion of selections from a survey of Australian orchards in the early 1950s were M. tetraphylla types (Leverington 1962a), consistent with a bias toward this species in these early plantings. It has been suggested that the ﬁrst Australian orchard at Rous Mill was the source of much of the seed for these early orchards (McConachie 1980). The presence of old M. tetraphylla seedling plantings north of Brisbane (Hardner et al. 2004; Peace 2005), distant from wild populations of this species but within the natural distribution of natural M. integrifolia populations, illustrates the wide distribution of the germplasm. Nurseries in the early 1900s may have played a major role in the distribution of genetic material, although few records are available that trace the origin of germplasm. By the mid-1930s, a nurseryman in Brisbane, Walter Petrie, had selected and named some of his parent trees (including ‘Smooth Queen’, ‘Eggshell’, ‘Pearl’, ‘Comet’, ‘Rough King’, ‘Planet’, ‘Large Everbearer’, and ‘Large Queen’) (Petrie 1935; Trochoulias et al. 1989) and seedling trees were sold under the name of their seed parent (Ian McConachie pers. comm.). The cultivar ‘Don’ may be a synonym of Petrie’s ‘Large Queen’ (Trochoulias et al. 1989), although it could also be a seedling selection from this parent. The origin of this early nursery material is unclear, but it probably encompassed both species and may be hybrids (Trochoulias et al. 1989). It is unlikely these original parental selections have survived (Storey 1963), although there are later reports in the literature of ‘Eggshell’ (Trochoulias et al. 1989) and an accession ‘Smooth Queen’ is reported in the USDA Germplasm Repository in Hawaii (Aradhya et al. 1998). 2. 1950 Seedling Surveys. Australian attempts at clonal grafting were not succ-essful until the mid-1950s (Leigh 1968; Leverington 1971; McConachie 1980). The poor uniformity in Australian seedling orchards and the success of the Hawaiian industry stimulated an interest in discovering elite genetic material in Australian orchards (Leverington 1962a). Evaluation of seedling trees in Australia reportedly began in 1948 (Storey 1963), with Leverington (1962a, 1971) reporting a survey of Queensland and NSW orchards being undertaken in 1952 by state Agriculture departments to identify elite individuals based on observed tree vigor, growth characteristics, cropping habit, potential yield, and nut quality/shape, although no detail of how these criteria were assessed or integrated is given. It is also reported (Hamilton and Fukunaga 1962)

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that Dr. J.H. Beaumount from HAES further encouraged these surveys during a visit to Australia in 1954. Ninety-four individuals were selected for further evaluation based on nut characteristics including: (1) shape and size, (2) thickness of shell wall, (3) kernel diameter, (4) kernel color, (5) quality of kernel after the removal of mould and insect damage, and (6) palatability after oil roasting (Leverington 1962a). Forty Queensland selections were given a preﬁx to identify the origin of the material, which ranged from Maryborough to the Gold Coast Hinterland (I and T—Currumbin; X—Victoria Point; S—Manly; H and P—Gilston, J—Flaxton, G—Eight Mile Plains, N—Tamborine; B—Maryborough; M—Maleny; D—Greber at Amamoor). Most of the selections were identiﬁed as M. tetraphylla (only ‘H2’, ‘B5’, ‘B6’, ‘B10’, and ‘B22’ were identiﬁed as M. integrifolia). Fifty-four NSW selections were identiﬁed only by a number, including ‘NSW-44’, which was a hybrid selection from a property at Highﬁelds (Vithanage and Winks 1992), west of Casino, outside the natural distribution of the species. This selection would later be named in California as ‘Beaumont’ (Storey 1965a). Other NSW selections were from Carool and Stokers Siding (Leverington 1962a). The number of selections was reduced by rejecting candidates with small kernel diameter, then using kernel recovery and percentage ﬁrstgrade kernel (Leverington 1962a). The processing properties of the roasted and salted kernels were also evaluated (Leverington 1971). Names were given to several of the initial selections (Anon 1961): ‘Ardrey’ (‘J4’), ‘Amamoor’ (‘D8’), ‘Collard’ (‘L4’), ‘Colliston’ (‘H1’), ‘Elimbah’ (‘F1’), ‘Flaxton’ (‘J3’), ‘Greber’ (‘D1’) (different from the Malawi selection, ‘D1’, as assessed by Peace 2005), ‘Hinde’ (‘H2’), ‘Howard’ (‘L1’), ‘Maroochy’ (‘J6’), ‘Oakhurst’ (‘B20’, identiﬁed as M. integrifolia type in Anon. 1961 but as M. tetraphylla in Leverington 1962), ‘Rickard’ (‘B5’), ‘Stephenson’ (‘H3’), ‘Sewell’ (‘N3’), ‘Teddington’ (‘B21’ in Leverington 1962a or ‘HAES 685’ in Hamilton and Fukunaga 1962, described as M. integrifolia M. tetraphylla by Storey (1963) and M. tetraphylla by Leverington 1962a and Hamilton and Fukunaga 1962), and ‘Tinana’ (‘B6’). ‘Renown’ (‘D4’ in Leverington 1962a but linked to this name by Storey and Hopﬁnger 1974) was also included in this list. The cultivar ‘Powell’ or ‘Powell’s Pride’ (‘P1’ in Leverington 1962a) is recorded as an Australian selection by Dr. J.H. Beaumont when he visited Australia in 1954 (Storey 1963). These selections are probably only one to three generations from the wild, depending on the sources of the seeds for the establishment of the original orchards (Fig. 1.3). ‘Hinde’ is the only cultivar of this program currently in commercial use; it is the preferred rootstock in the Australian

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industry (Stephenson 1990a; Trochoulias 1992b). Details are not available on the source of the germplasm used to establish the Gilston orchard where this cultivar was selected, but DNA marker analysis identiﬁed this cultivar as pure M. integrifolia, apparently originating from the southern part of the range of this species (Peace 2005). 3. Norm Greber Selections. In addition to the selections made by the Queensland Department of Agriculture and Stock in the mid-1950s from his property, selections undertaken by Norm Greber form another major group of Australian germplasm (Fig. 1.3). These were derived from open-pollinated seed collected from wild populations and from his, and others’, seedling orchards and backyard trees (Trochoulias et al. 1989; Vithanage and Winks 1992). Historical records suggest that selections ‘Own Choice’, ‘NRG’, and ‘Greber’ originate from the same seed lot collected from nearby wild M. integrifolia populations in the Amamoor Creek valley near Gympie (Trochoulias et al. 1989; Vithanage and Winks 1992). It is unknown how many maternal trees comprised this wild seed lot. ‘Renown’ and ‘Nutty Glen’ are recorded as being selections from Norm Greber’s farm in Amamoor; however, as these are hybrid types, the seed for these selections could not have come entirely, if at all, from local populations. It has been suggested that seedlings for the Amamoor orchard were supplied by Walter Petrie (Peace 2003). Other cultivars of Norm Greber, ‘Greber Hybrid’ and ‘Own Venture’, are recorded as full sibs, with ‘Own Choice’ and ‘Renown’ as parents (Trochoulias et al. 1989), although it is unknown if and how hybridization was controlled. A series of selections assigned ‘X’ also originated from progeny plots planted in Norm Greber’s backyard at Beerwah, although the source of seed is unclear (Trochoulias et al. 1989). This is not to be confused with the ‘X’ preﬁx used for earlier selections from Victoria Point (Leverington 1962a). The preﬁx ‘NG’ has also been used to identify these selections (e.g., Stephenson et al. 1995). Storey (1965b) also recorded ‘Eggshell’ (‘D3’), indicating this was a selection from the Greber property; however, there are no records of how this relates to the nursery seed parent tree of the same name listed by Petrie (1935). Parentage analysis using DNA markers (Peace 2005) has been used to disentangle some of the relationships within the Greber germplasm. An identical DNA proﬁle across a large number (over 100) of dominant and codominant markers and multiple genotypes suggests that the germplasm tested as ‘Own Choice’ may be the same as that of ‘HAES 772’. This is supported by reports that these two cultivars have similar morphology (Vithanage and Winks 1992). While ‘Own Choice’ is of

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43

M. integrifolia type, ‘NRG’ has been classiﬁed as of hybrid type according to morphology and DNA marker assessment of species composition (Peace 2005). This fact suggests that, if this cultivar had indeed been sampled from nearby wild populations, the seed tree was pollinated by foreign germplasm, possibly from the nearby orchard of the breeder Norm Greber, most likely ‘Renown’ (Peace 2005). Percentage analysis also conﬁrmed that ‘Own Venture’ is probably a seedling of ‘Own Choice’ but not ‘Renown’, and ‘Greber Hybrid’ is probably not a direct seedling of either (Peace 2005). From a dendrogram based on DNA markers, ‘X3’, ‘X4’, and ‘X8’ clustered in a hybrid group that included ‘Beaumont’ but separate to another hybrid group containing ‘Renown’, ‘NRG’, ‘Greber Hybrid’, and known progeny of Renown (Peace 2005), indicating a different genetic background. Historical records suggest that ‘X4’ is a hybrid selection from Walter Petrie (Trochoulias et al. 1989). 4. Miscellaneous Australian Selections. There are records of several miscellaneous cultivars in Australia from the 1940s to the 1990s (Trochoulias et al. 1989; Vithanage and Winks 1992). ‘Kopp’, ‘Heilscher’, and ‘Daddow’ are recorded as selections from backyard or farm plantings around the Maryborough region propagated from M. integrifolia seed collected from wild populations around Mount Bauple or the headwaters of Tinana Creek (Fig. 1.3), although there are some reservations that M. integrifolia occurs naturally in this area (Ian McConachie pers. comm.). According to DNA marker analysis, ‘Heilscher’ most likely did originate from natural populations of this region. However, ‘Kopp’ appeared to have a mixed heritage, and ‘Daddow’ was determined to be derived from more southerly native M. integrifolia populations, one of the few cultivars identiﬁed as such, and more related to ‘Hinde’ than any other cultivar (Peace 2005). ‘Release’ and ‘Mason 97’ were from separate properties near Gympie, ‘Armanasco’ is recorded as from a property to the south of Brisbane, and ‘Probert’ is from near Mapleton (Vithanage and Winks 1992). ‘McGregor’ is an obscure Australian selection (Storey 1963) with little information of its origin. Numerous other Australia selections have been described. ‘HAES 680’ was selected by Dr. J.H. Beaumont on a trip to Australia in 1954 (Hamilton and Fukunaga 1962; Wagner-Wright 1995). ‘Rickard selection’ (‘HAES 687’) was also collected on this trip, presumably from Rickard’s property in Maryborough identiﬁed in the 1950 Australian survey (Leverington 1962a). No details are available on selections identiﬁed as ‘Imbil’ and ‘Jackman’ (Hamilton and Fukunaga 1962).

44

C. M. HARDNER ET AL.

Several cultivars were recorded as selections by Dr. W.B. Storey from the University of California when he visited Australia in 1960. ‘Currumbin’ (Z1), ‘Tomewin’ (Z2), and ‘Taylor’s Triumph’ (Z3) were selected from a property in the Currumbin valley (Storey 1963, 1965b; Storey and Hopﬁnger 1974). Other Australian selections include ‘Collins’ from Redland Bay, ‘Duranbah 95’ from northern NSW, ‘Mammoth’ (I1 in Leverington 1962), ‘Nelson’ (also ARN) from Stokers Siding (reported in Leverington 1962 but not given a designation), ‘Tallebudgera’ (T) from along Tullebudgera creek, and ‘Wilson-10’ from Mount Tamborine (Storey and Hopﬁnger 1974). ‘Rankine’ (also known as ‘HY’) is considered a hybrid type, and ‘The Pocket’ is M. integrifolia (Storey 1965b). 5. Hidden Valley Plantations Program. A breeding program was initiated at Hidden Valley Plantations in 1972 (Bell and Bell 1987) (Fig. 1.3). Early in the program, seedlings produced from open-pollinated seeds from high-yielding seedling parent trees were evaluated, but the program has progressed to evaluation of open-pollinated progenies from named cultivars and preliminary selections, through to progenies from semicontrolled crosses of certain cultivars. More than 25 characteristics are included in a weighted selection scheme. These include resistance to husk spot, kernel mass, tree structure, cropping, shape of kernel, color of kernel, and kernel sticking (assumed adherence to shell). The performances of standard cultivars are used as checks for evaluation (e.g., ‘Keauhou’ for ﬁeld and ‘Makai’ for kernel characteristics, Bell and Bell 1987). Much of the assessment of these characters relies on visual assessment by trained operators; some characters are not well deﬁned, some confound measurement and importance, and in some cases the relationship between the character and importance is not linear. Several cultivars from this program have been released. ‘A4’ and ‘A16’ were the ﬁrst plants to achieve Plant Breeders Rights (PBR) in Australia (Bell et al. 1988). These are both open-pollinated progeny from ‘Renown’, and recent DNA marker analysis has indicated that they are full sibs, with ‘Own Choice’ as the pollen parent (Peace 2005) (Fig. 1.3). A third cultivar, ‘A38’, has also been given PBR status and is the openpollinated progeny of ‘Own Choice’ (Hidden Valley Plantations 1994). Other cultivars have been released and used in commercial orchards without PBR. ‘A29’ (Bell and Bell 1987), ‘A104’, and ‘A199’ are openpollinated progeny of ‘Renown’ (Vithanage and Winks 1992), not ‘Own Choice’ as reported by Aradhya et al. (1998) (H. Bell pers. comm.), although A199 appears to be a seedling of both ‘Renown’ and ‘Own Choice’ from DNA marker analysis (Peace 2005). ‘Own Choice’ is the

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mother of open-pollinated progeny ‘A90’ (Vithanage and Winks 1992) and ‘A203’ (Aradhya et al. 1998), and ‘A268’ is an open-pollinated seedling of ‘Kau’ (H. Bell pers. comm.), the latter conﬁrmed by DNA marker analysis (Peace 2005). 6. Australian Macadamia Breeding Program. A major breeding program was initiated in 1996 to produce cultivars suited to Australian conditions (Hardner and McConchie 1999; Mehlenbacher 2003; Hardner et al. 2005). This program is based on a quantitative genetic approach, where pedigree relationships and experimental design are used to increase accuracy of predicted genetic values and a formal selection index is used to trade off differences in multiple traits across multiple candidates (Hardner and McConchie 1999). Candidate cultivars identiﬁed in the program are vegetatively propagated for further testing in regional cultivar trials (Hardner and McConchie 2003; Hardner et al. 2005). The major selection objectives of this program are tree size, precocity, average rate of yield increase, proportion of reject NIS, total kernel recovery, proportion of reject kernel, proportion of marketable whole kernel, and marketable kernel size (Hardner and McConchie 1999, 2003; Hardner et al. 2005, 2006). Elite selections are identiﬁed using a selection index, with an index value calculated for each candidate as a linear combination of the genetic value of the individual for each selection objective, weighted by the importance of the trait (Hardner et al. 2006). Trait weights are derived as the change in a proﬁtability index (i.e., proﬁt/costs) for an economic model due to a unit change in the level of the trait. This model includes the costs of production of a 100-ha orchard over a 20-year planning horizon from orchard establishment, the cost of processing the nuts, and the price of a range of raw kernel styles sold by the factory (Coverdale et al. 1999; Hardner et al. 2006). Net present value is used to account for the timing of costs and income over a long planning horizon. These economic weights are based on current production systems that may not be applicable in 10 to 20 years when trees come into commercial production. However, the future can be uncertain, and models of current production systems provide a useful structure for examination of future scenarios. A linked pedigree also enables the breeding program to respond quickly to changes in the relative importance of selection objectives, as elite genotypes can be quickly identiﬁed under alternative scenarios (Hardner and McConchie 1999). Genetic values of the breeding objective traits are predicted using genetic correlations with the traits that have been directly assessed on

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the progeny (Hardner and McConchie 1999). Juvenile-mature correlations for different objectives indicate that selection can be made for kernel recovery, percentage whole kernels, and kernel size within two years of the ﬁrst crop, but several years of yield are required to select for yield (Hardner et al. 2001, 2002). These results support selection for long-term yield by eight years after planting. Two cycles of crossing have been undertaken (1993–1994 and 1997– 1999) among 40 Hawaiian and Australian cultivars, and about 5000 seedlings have been planted across 14 sites in three of the major growing areas in Australia (Hardner and McConchie 2003; Mehlenbacher 2003). Mixed-model statistical methods (Henderson 1984) will be employed to combine data across sites and years and predict the genetic values of individuals across or within growing regions (Hardner and McConchie 1999). Release of cultivars from the ﬁrst breeding cycle is predicted for 2012 (Mehlenbacher 2003), 15 years after the planting of the ﬁrst progeny trials. In comparison, the initial cultivar release in Hawaii (in 1948) was 23 years after the initial seedlings had been planted in the production orchards (1925, Shigeura and Ooka 1984). The quantitative genetic approach, where gain is achieved by increasing accuracy of selection, also contrasts with previous strategies adopted in macadamia, where gain was achieved through large population size and high selection intensity but low accuracy. This quantitative approach is not well suited to traits that are vaguely deﬁned or rely on personal judgments. Nevertheless, a formal quantitative approach enables all available information to be combined objectively for prediction of genetic value and identiﬁes gaps in knowledge and assumptions that are made to ﬁll these gaps. This approach also provides a comprehensive structure for review and modiﬁcation, which is important for institutional breeding programs. C. Other Programs 1. California. The origins of Californian cultivars are relatively obscure. M. integrifolia was reportedly introduced into California in about 1879, with other introductions in following years and the introduction of M. tetraphylla in the early 1890s or 1900s (Storey 1957, 1965b; Ferguson and Arpaia 1990) (Fig. 1.3). Both species were used in ad hoc plantings from San Francisco to the Mexican border prior to 1946. Early Australian selections (described in Leverington 1962a) were introduced in the early 1960s (Storey 1964). There has been no comprehensive program to develop new cultivars for

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California (Ferguson and Arpaia 1990), although by the 1950s several local cultivars of M. integrifolia (‘Arcia’, ‘Faulkner’, ‘Parkey’) and M. tetraphylla (‘Burdick’, ‘Hall’, ‘Santa Ana’) types had been identiﬁed (Storey 1963; Steiger et al. 2003). Other local Californian cultivars include ‘Pierce’, ‘Kirsch’, ‘Tanner’, ‘Bays’, and ‘Limonera’ (Schroeder 1994). The cultivar ‘Cate’ is a M. tetraphylla–type selection that was propagated from a seedling growing in Malibu in 1958 and was planted in California during the 1970s (James 1978). ‘Beaumont’ and ‘Jordan’, originally selected in Australia and Hawaii respectively, were named in California as cultivars for ornamental use (Storey 1965a; Brooks and Olmo 1983). 2. South Africa. South African macadamia germplasm can be traced back to cultivated germplasm from Australia, Hawaii, and California (Fig. 1.3). The seed used to establish the ﬁrst orchards in 1930s were reportedly imported from Hawaii (Peace et al. 2005). This seed gave rise to an indigenous selection, ‘Nelmak 1’, which is of hybrid type, and it is believed that other South African cultivars, ‘Nelmak 2’ and ‘Nelmak 26’, are progeny of ‘Nelmak 1’ (Peace et al. 2005). M. integrifolia and M. tetraphylla seeds were also imported from Australian nurseries (Petrie and others) in 1935 and used to produce seedlings for orchard establishment. The South African selections ‘R14’, ‘W148’, and ‘W266’ are reportedly derived from these introductions (Peace et al. 2005). Seeds of the Californian M. integrifolia selection ‘Faulkner’ were reportedly introduced from Hawaii in the 1970s and were the source of a series of ‘F’ selections (Peace et al. 2005). 3. Kenya. Macadamias were introduced into Kenya in 1946, and plantations in Kenya prior to 1973 were established with seedling material (Gathungu and Likimani 1975). A selection program was initiated in 1971 to identify superior trees for grafting. Criteria for selection were: (1) vigorous growth, (2) spreading structure with wide crotch angles, (3) consistent yields, (4) short ripening period, (5) resistance to pest and diseases, (6) nut size, (7) kernel shape, (8) kernel recovery, (9) oil content, and (10) health implications (Gathungu and Likimani 1975). The preference for a spreading habit is in contrast to the Hawaiian preference for an upright form. The authors suggested that hybrids could also be used to reduce oil content but increase sweetness. Seven Kenyan selections have been published: ‘Kiambaa T22’, ‘Chania H28’ and ‘H29’, ‘BHL 1’, ‘BHL 2’, ‘BHL 3’, and ‘BHL 6’ (Gathungu and Likimani 1975). The extent of adoption of this material is unclear. These selections have an average oil content between 76.5% and 82%,

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between 80% and 100% ﬁrst-grade kernel, average kernel recovery between 33% and 36%, average nut diameter between 23 and 27 mm, average kernel diameter between 17 and 21 mm, and sucrose content between 1.68% and 2.64% (Gathungu and Likimani 1975). 4. Others. A number of cultivars have been developed in several other countries. The cultivar ‘Yonik’ (13/3) was selected in Israel from progeny raised from seeds described as ‘Kona-778’ type that were introduced from Hawaii in 1966 (Kadman and Slor 1982). ‘HAES 778’ is the M. integrifolia selection ‘Faulkner’ from California (Aradhya et al. 1998), but further research is required to conﬁrm the relationship between these two cultivars. A series of Brazilian selections has been recorded in the literature. The selections ‘Keaudo’ (IAC 2-23), ‘Keaufa’ (IAC 4-21), ‘Keaumi’ (IAC 4-20), and ‘Keaure’ (IAC 4-18) are reported as open-pollinated sibs from ‘Keauhou’ (Ojima et al. 1976; Barbosa et al. 1991). ‘Kakea’ is reportedly the seed parent of ‘Kakedo’ (IAC 4-10) and ‘Kakere’ (IAC 5-10) (Ojima et al. 1976). No details are reported for the parentage of the Brazilian selections ‘Aloha’, ‘Campinas A’, ‘Campinas B’, ‘Campinas F’, ‘Campinas H’ and ‘Waiado’ (Barbosa et al. 1991; de Sa 1991; Aradhya et al. 1998; Sacramento et al. 1999). Seeds were introduced into Thailand in 1953 (Supamatee et al. 1992) from unknown sources. Several selections from Thailand (‘Kau Kor #1’ and ‘Kau Kor #2’) have been recorded (Steiger et al. 2003). Two indigenously developed cultivars have been reported from Mexico (Quintas 2006) without further detail of parental origin. Macadamia seed was introduced into New Zealand in the 1890s, and a range of cultivars was introduced in the 1970s (Richardson and Dawson 1991). A private company has made a number of local selections, which are given preﬁxes ‘PA’ and ‘PB’. A breeding project is reportedly under way in the Panxi region of China, with particular emphasis on M. tetraphylla germplasm for cold resistance (Xiao et al. 2002b).

D. Genetic Structure of Domesticated Germplasm 1. Use of Molecular Markers. Molecular marker technology is a power- ful tool for analyzing genetic relationships among cultivars. Several markers systems have been used to quantify genetic diversity within sets of macadamia cultivars, enabling comparisons of relatedness between various domesticated origins and determination of the likely causes of cultivars gene-pool differentiation.

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Isozyme Marker Studies. The ﬁrst molecular study in macadamias employed nine isozyme loci to survey 74 cultivars that were placed into 10 groups based on the combinations of four alleles detected at the phosphoglucoismerase (pgi) locus (Vithanage and Winks 1992). However, the distribution of cultivars from different selection origins and species type among these groups was not consistent. Germplasm analysis was extended using 16 isozyme loci to group 40 cultivars into seven apparent groups (Aradhya et al. 1998). At the highest level of organization, the cultivars grouped clearly as either M. integrifolia or M. tetraphylla, but several known hybrid-type cultivars (‘Renown’, ‘Beaumont’, and ‘A16’) complicate the further detail of the organization. Five groups (1a to 1e) were relatively closely associated and together were classiﬁed as M. integrifolia, although one of these groups (1c) contained mostly hybrids. The two M. integrifolia groups with the largest number of individuals, and mostly of Hawaiian selection origin (1a and 1b), were very closely related compared to the other groups. The authors used this apparent separate grouping as evidence that the two early introductions of M. integrifolia to Hawaii (by Purvis and Jordan) were from genetically distinct ancestral populations. However, this is not convincing, given the little diversity and subjective demarcation between the two groups. Alternative interpretations are that the close afﬁnity between the two groups suggests that cultivars were derived from only one of the germplasm sources (‘Purvis’ or ‘Jordan’), or that the two sources were not from distinct natural origins. The authors also asserted that Australian selections probably represent a different genetic background (natural origin) and selection history to Hawaiian selections. However, several Australian cultivars clustered within groups 1a and 1b, while groups 1c, 1d, and 1e included many hybrids and other cultivars with ambiguous species status, indicating that at least some of the differences between groups were due to the inclusion of M. tetraphylla germplasm ancestry in cultivars. DNA Marker Studies. In a third marker study (Vithanage et al. 1998), 76 mostly M. integrifolia genotypes were screened with random ampliﬁed polymorphic DNA (RAPD) and codominant STMS markers, although no clear arrangement was observed other than single accessions of M. tetraphylla, M. ternifolia, and M. jansenii each appearing very distinct from the M. integrifolia and hybrid cultivars, similar to observations in the isozyme studies. Cultivars domesticated within Hawaii and Australia were distributed throughout the dendrogram. A survey with 105 ampliﬁed fragment length polymorphism (AFLP) markers of 24 accessions of the cultivated species (and three

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accessions of related species) identiﬁed two distinct groups of Hawaiian cultivars within M. integrifolia (Steiger et al. 2003). However, the speciﬁc cultivar composition of these groups was different to the isozyme study of Aradhya et al. (1998). The authors compared the overall diversity in the macadamia germplasm with coffee and papaya, ﬁnding it higher in macadamia (Steiger et al. 2003), although it is likely the diversity among the macadamia cultivars was inﬂated by the inclusion of multiple species. Thirty cultivars were separated into four groups using a principal component analysis of the allelic variation of 175 RAF (randomly ampliﬁed DNA ﬁngerprinting) and STMS codominant marker alleles and 230 dominant RAF markers (Peace et al. 2002). Species composition weighted % of species speciﬁc markers was calculated using 134 alleles speciﬁc to M. integrifolia and 34 speciﬁc to M. tetraphylla (Peace et al. 2004). Species speciﬁcity of a marker was determined by surveying the frequency of the markers in groups of cultivars that had been characterized as pure species from morphology (Peace 2005). Using this methodology, the four groups of cultivars corresponded to M. integrifolia—Hawaiian selection origin; M. integrifolia—Australian selection origin; hybrids—Australian selection origin; and M. tetraphylla—Australia selection origin. Species status (M. integrifolia, hybrid, or M. tetraphylla) was clearly the major determinant of genetic differences (Peace et al. 2002). This methodology was improved (Peace 2005) by determining species speciﬁcity of markers from the National Macadamia Germplasm Program collection (Hardner et al. 2004) of 274 accessions from 58 wild populations. Individual genotypes were initially assigned to one of six species types based on morphology (M. integrifolia, M. tetraphylla, M. jansenii, M. ternifolia, M. integrifolia M. tetra-phylla hybrid, M. integrifolia M. ternifolia hybrid), and species-speciﬁc markers were then identiﬁed as those that were only represented in wild populations of one of the species but not in those of other species or in hybrid populations. This separates classiﬁcation of marker species speciﬁcity from their implementation in the study of species relationships in the domestication germplasm. The species-speciﬁc markers were then used to survey the genetic diversity of 83 cultivars and selections (Peace 2005). Cultivars were distributed in eight distinct clusters—four of M. integrifolia, three clusters of hybrids, and one M. tetraphylla, with six to 26 individuals per group. Principal component analysis with separation of cultivars in two dimensions was also used to display this grouping arrangement (Peace 2005). The species speciﬁcity of the markers allowed the species composition of the genotypes to be quantiﬁed

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(Peace et al. 2001, 2005; Peace 2005). A study of South African cultivars (Peace et al. 2005) was a subset of this larger study. The two RAF primers used for the cultivar and wild germplasm survey (Peace 2005) were chosen for their ability to amplify at least one microsatellite marker, and thus the information generated by these primers were RAMiFi markers (randomly ampliﬁed microsatellite ﬁngerprinting, Peace et al. 2004). In total, 165 dominant markers and nine codominant microsatellite markers were used (Peace 2005). These markers were also employed to verify/deduce the parentage or identity of certain cultivars within the 85-cultivar set (Peace et al. 2002; Peace 2005; Peace et al. 2005), the results of which are described within the domestication history sections. For this purpose, the microsatellite markers were the most useful, while the accompanying dominant markers provided abundant accessory information (Peace 2005). The Hawaiian M. integrifolia cultivars formed two distinct clusters. Cluster 1 contained ‘Keauhou’ and its known offspring, ‘Ikaika’ and ‘Kakea’, and ‘HAES 816’, a Hawaiian selection from open-pollinated progeny of an Australian selection introduced into Hawaii. Cluster 2 contained ‘Kau’, ‘Keaau’, ‘Mauka’, ‘HAES 814’, and an old Australian selection, ‘Own Choice’. Aradhya et al. (1998) also found ‘Kau’, ‘Keaau’, and ‘Mauka’ to have very similar genetic proﬁles. Clusters 3 and 4 contained a range of Australia M. integrifolia selections. Clusters 5, 6, and 7 contained cultivars that are mixtures of the two species, including the Australian cultivars ‘A4’ and ‘A16’ with their maternal parent ‘Renown’. Cluster 7 appeared to share greater afﬁnity with M. tetraphylla. The last cluster contained cultivars of pure M. tetraphylla origin. Comparison of Marker Results. Clustering of macadamia species and cultivars from the various marker systems studies was compared by Peace et al. (2004). Six sets of marker data (two isozyme, two of STMS, and one each of RAPD and RAMiFi) were obtained for a common set of 14 macadamia individuals. These individuals consisted of nine cultivars typically regarded as M. integrifolia (the Hawaiian-bred ‘Keauhou’, ‘Ikaika’, ‘Kau’, ‘Kakea’, ‘Keaau’, ‘Mauka’, ‘Makai’, and ‘HAES 816’ and the Californian-bred ‘Faulkner’), three regarded as M. integrifolia M. tetraphylla hybrids (‘A4’, ‘A16’, and ‘Beaumont’), and one accession each of M. tetraphylla and M. ternifolia. The exact accessions used for the latter two species varied for some of the marker studies. Matrices of pairwise genetic distance were calculated, dendrograms were produced, and the matrices were compared using Mantel matrix correlation (Peace et al. 2004).

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The six dendrograms revealed some overall trends. Most marker systems identiﬁed the M. tetraphylla and M. ternifolia accessions as the most distantly related individuals. The three hybrid cultivars tended to cluster together and separately to the M. integrifolia cultivars. The M. integrifolia ‘Faulkner’ appeared distinct from the other cultivars of that species. The eight M. integrifolia cultivars of Hawaiian selection origin tended to form a separate cluster. The correlation among the relationships of cultivars for three of the marker sets (isozyme from Aradhya et al. 1998; RAPD from Vithanage et al. 1998; RAMiFi from Peace et al. 2002) was signiﬁcant and greater than 0.6 (Peace et al. 2004). These three sets of marker systems produced similar clustering arrangements of cultivars that were also consistent with expectations, and Peace et al. (2004) concluded that these markers were more robust. There was little correlation of the genetic relationships from each of the three other marker studies with any other study. For two of the other marker systems (isozyme from Vithanage and Winks 1992; STMS from Vithanage et al. 1998), the differentiation of individuals was more divergent, including intermixing in the dendrogram of individuals from different species. The STMS and RAMiFI marker data sets of Peace et al. (2002) produced the largest genetic distances and thus appeared to better be able to distinguish among genotypes (Peace et al. 2004). 2. Inﬂuences on Genetic Structure. The combined results of genetic marker studies, particularly where species status of cultivars was considered, suggest that germplasm organization of cultivated macadamia is determined primarily by species status (i.e., whether an individual is one of the pure species or a hybrid) and species composition (the proportion of each constituent species within a hybrid) (Peace 2005). Natural origin is considered the second most important factor followed by breeding/selection origin. Species and Hybrids in Cultivation. In all marker studies to date, pure M. integrifolia and pure M. tetraphylla cultivars were observed to be the most genetically separated individuals, with hybrids (where identiﬁable as such) intermediate. Species composition calculations indicated that increasing amounts of one species over the other primarily determined the overall placement of hybrid cultivars (Peace 2005). Cultivars all along the scale from pure M. integrifolia to pure M. tetraphylla have been identiﬁed by genetic marker analysis (Peace et al. 2002, 2005; Peace 2005). This continuity in species composition of the domesticated germplasm suggests that hybrids are fully fertile in

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cultivation. According to these analyses, and consistent with assessment by morphology, hybrid cultivars are particularly common in the Australian and South African macadamia industries (Peace et al. 2005; Peace 2005). However genetic marker analyses also suggest that the species type of many macadamia cultivars is misclassiﬁed by morphology, particularly those with small M. integrifolia or M. tetraphylla compositions, including several common cultivars of Australian selection origin (Peace 2005). Further research is required to determine the effects the various proportions of each species detected within cultivars might have on their performance. Particular mention is made of the cultivar ‘Fuji’, which is the only macadamia genotype, cultivated or otherwise, identiﬁed as a trispecies hybrid (Peace 2005; Peace et al. 2005). Species composition calculations suggest that the M. ternifolia composition of ‘Fuji’ is approximately onequarter, at least two generations removed from its M. ternifolia ancestor (Peace 2005). The M. ternifolia possessed by ‘Fuji’ is an unusual phenomenon for macadamia, and demonstrates the opportunities that may exist from greater exploration of the wild genetic resources of the genus. Several of the characteristics of this cultivar may have been derived from its M. ternifolia heritage (Peace 2005; Peace et al. 2005). In Hawaii and South Africa, ‘Fuji’ trees are reportedly small, spindly trees (Blight 1989), similar to the stature of wild M. ternifolia (Gross 1995). This supports the view that this cultivar is susceptible to wind damage in exposed areas but could be ideal for high-density planting (Blight 1989). The absence of bitter kernels (presence of cyanogenic compounds), which is normally found in wild M. ternifolia, is likely to be due to the recessive gene action of this trait (Hardner et al. 2000). Native Origin of Cultivars. Peace (2005) deduced natural origins of cultivars by linking the presence of particular markers of apparent restricted geographic origin in wild populations with their presence in cultivars and identifying the most closely related wild populations and speciﬁc wild accessions for each cultivar from both cluster analysis and raw genetic distance values. Although there are limitations to this method, given the small population sizes sampled for the wild accessions included in the analysis and the difﬁculty, particularly for cluster analysis, in determining origins for cultivars derived from mixing between natural gene pools, the outcomes were clear for certain cultivar groups. The northernmost regions of the native range of M. integrifolia were implicated as contributing the most to the genetic background of cultivars of the world’s macadamia industry, including the Hawaiian germplasm groups, many Australian cultivars, and cultivars from several

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other countries (Peace 2005). In most cases, these assignments corresponded to the historical records of the source of these cultivars. The evidence from the molecular studies in macadamia is in conﬂict with the generally accepted view that the Jordan germplasm is derived from the southern range of M. integrifolia. In the ﬁrst instance, Hawaiian cultivars tend to cluster together in these marker studies, suggesting that only one or two germ pools were sampled in the initial introduction. Second, the results in Peace (2005) indicate that the two clusters containing the named Hawaiian selections from the original 1920s orchards are both associated with the northern range (Mount Bauple/Amamoor) of M. integrifolia, with cluster 1 sharing greater afﬁnity with the Amamoor region and cluster 2 appearing to be more closely aligned with the Mount Bauple region. This is supported by the inclusion in cluster 2 of ‘Heilscher’, an Australian selection that reportedly came from the Mount Bauple area. Third, Pimpama, the reported origin of the Jordan collection, lies at the opposite end of the species range some 300 km to the south within the hybrid zone of M. integrifolia and M. tetraphylla (Fig. 1.3). Further work is required to reconcile this conﬂict. It may be that the seeds for the Pimpama trees were sourced from the northern distribution. Alternatively, none of the Jordan germplasm may be represented in the domesticated Hawaiian germplasm. It has been reported that the Purvis germplasm was collected from the Mount Bauple area (McConachie 1980). If this is correct, this germplasm may be represented by cluster 2 in Peace (2005). The Australian cultivars ‘Daddow’ and ‘Hinde’ were the only pure M. integrifolia cultivars identiﬁed with natural origins entirely or predominantly in the southern M. integrifolia regions (Peace 2005). The Tweed River valley in the central part of the native range of M. tetraphylla was most implicated by RAMiFi markers as the source of cultivars of that species (Peace 2005). Hybrid cultivars from the three major cultivated hybrid germplasm did not appear to have arisen from any one region, suggesting that hybrids in cultivation are artiﬁcial species combinations and not directly sampled from the natural hybrid zone (Peace 2005). Selection and Breeding. Breeding/selection origin clearly does not adequately describe the organization of genetic diversity in macadamia (Peace 2005). Cultivars of Hawaiian and South African origin were spread among four different clusters; three cultivars from Malawi were not particularly related and were each located in different clusters; Australian cultivars were in every major cluster; and even selections from the same Australian program of Norm Greber were in ﬁve different

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clusters. Germplasm exchange in recent generations appears to account for the lack of geographical continuity in the clustering of macadamia cultivars. Breeding programs, such as those in Hawaii and Greber’s, have included germplasm from several sources; this has resulted in some individuals having little genetic afﬁnity despite undergoing a common selection regime. Intermixing of germplasm through domestication appears to have given rise to some groups of germplasm where cultivars within the groups are more closely related to each other than to any wild accession surveyed (Peace 2005). These represent novel germplasm that apparently does not occur in the wild. It is suggested that most of the widely planted cultivars in Australia, Hawaii, or South Africa are of this germplasm type (Peace 2005). 3. Wild Genetic Diversity Represented in Cultivation. Domestication appears to have captured a large proportion of the neutral genetic diversity present in recent collections from the remnant wild populations. Peace (2005) calculated that the 83-cultivar set surveyed contained half the genetic diversity (measured as the number of observed polymorphic dominant markers and codominant marker alleles) of the wild accessions of the National Macadamia Germplasm Collection from the three main species of the southern clade of Macadamia. Alternatively, the proportion is approximately two-thirds when measured as average heterozygosity of dominant markers, presumably higher due to the inclusion of many hybrid cultivars that arose in cultivation rather than being directly sampled from wild gene pools. More M. integrifolia diversity is represented in cultivation than for other Macadamia species (Table 1.4).

Table 1.4. Proportion of the genetic diversity contained with the National Macadamia Germplasm Collection that is represented within the 10 most-plant cultivars in the three largest macadamia-producing regions in the world. Proportion of wild germplasm diversity (%) Average heterozygosity of dominant markers Cultivar group Pure M. integrifolia Pure M. tetraphylla Both cultivated species All species

Hawaii Australia 15 0 25 22

Source: Adapted from Peace 2005.

79 18 47 43

Polymorphic markers and alleles

S. Africa

Hawaii

89 30 57 55

28 0 15 11

Australia S. Africa 50 12 31 23

46 19 34 27

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Low genetic diversity (average heterozygosity; number of polymorphic markers) was detected in the two clusters dominated by Hawaiianderived cultivars (0.013 and 0.026; 18 and 23), compared to other clusters (0.050 to 0.083; 29 to 46). This supports the hypothesis of low genetic diversity within Hawaiian germplasm (Peace 2005), which is in contrast to the discussion in Aradaya et al. (1998). Interestingly, the introduction of new germplasm into the Hawaiian breeding program in the 1950s does not appear to have increased genetic diversity appreciably. For example, selections ‘HAES 814’, ‘HAES 816’, and ‘HAES 856’ were seedlings of trees other than the ﬁrst-generation Hawaiian cultivars but were still very closely related to other Hawaiian cultivars, apparently due to a similar native region of origin (Peace 2005). The diversity of germplasm utilized in commerical production is expected to be much less than that described by Peace (2005) for the large 83-cultivar set, as many of these are rare with very limited planting. Genetic diversity within only the most widely planted cultivars of the three largest-growing regions was calculated separately and found to be about between 40% to 80% less than that in the larger cultivar set (Peace 2005; Table 1.4). The most widely planted Hawaiian cultivars contain a low proportion of total available diversity, even for M. integrifolia. Although Hawaiian M. integrifolia cultivars form the bulk of the orchard trees in Australia and South Africa, genetic diversity in cultivation is considerably higher in these countries due to the popularity of cultivars from other sources. South Africa has the most diversity, as it incorporates the most M. tetraphylla germplasm and a minor amount from M. ternifolia (from the cultivar ‘Fuji’). Given the short history of domestication in macadamia, it is unlikely that the domesticated germplasm represents the only source of elite genetic material in macadamia. There appears considerable opportunity to capture large gains through exploring the wider diversity available in the genus.

IV. GENETICS OF KEY SCION SELECTION TRAITS There are many biological traits of interest for genetic improvement in macadamia (Bell 1983; Hardner and McConchie 1999, 2003; Stephenson and Gallagher 2000). However, often these traits are poorly deﬁned, and there is limited information on their inheritance (Hardner and McConchie 1999; Hardner et al. 2001). Response to selection is determined by the extent of genetic variation in a trait and the intensity of selection (Falconer 1989). There are a few reports of genetic parameters estimated from cultivar trials (Hardner

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et al. 2001, 2002). However, because these cultivars have been selected, the magnitude of genetic variation may be underestimated compared to a progeny population (Falconer 1989). Studies reporting signiﬁcant differences between cultivars provide other sources of evidence for the existence of genetic variation. However, it is difﬁcult to assess the importance of cultivar differences that are reported without signiﬁcance testing. In addition, some studies report means for unbalanced designs that are biased due to the absence of information from particular sites. Reported observations of cultivar performance gained from familiarity with the crop are similarly difﬁcult to evaluate but can provide additional information on possible extent of genetic variation. In this review we examine a wide range of published data on cultivar performance. To assist with the summary and comparison of these results, data were accumulated across studies and analyzed using a mixed-model Restricted Maximum Likelihood (REML) approach to account for unbalance in the data (Patterson and Thompson 1971). Several publications have presented masses of data. These are obviously valuable for understanding the genetic data of macadamia; however, the analysis and summary of this information is beyond the scope of this publication. A. Tree Structure Tree structure has been deﬁned in terms of vigor, habit, tree size, and canopy density, although methods for quantifying most of these characteristics have not been developed, and assessment relies on trained assessors (Stephenson et al. 1995). A set of standard descriptors for tree structure have been developed (e.g., Domingo et al. 2004), which could be used to assist consistency among studies. Early selections in Hawaii favored vigorous trees with round or coneshaped habit (Hamilton and Ito 1977a), and spreading habit was favored in a Kenya selection program (Gathungu and Likimani 1975). In contrast, an upright habit was favored in the later Hawaiian selections, as this was considered more suitable for higher planting densities and increased early returns (Hamilton and Fukunaga 1973; Hamilton and Ito 1977a; Hamilton et al. 1981). The preference for upright trees is also followed in Australia (Stephenson and Gallagher 2000). The economic effect of tree size has been modeled by linking planting density to canopy width at age 10 (Hardner et al. 2006). Cultivars with larger canopy width are planted at wider densities so that the age at which canopies touch is maintained at 10 years. This has a large negative

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impact on proﬁtability, given all other traits are unchanged. An alternative approach could be to assume a common planting density and let tree size determine timing of orchard operations, particularly canopy management. There is also an interest in ultra-high-density plantings (e.g., 5 3 m, Trochoulias and Burnside 1987; Stephenson 1990a); however, the relationship of tree size with proﬁtability may not be the same as described above for orchards at coventional densities, due to differences in the production system and cost structures. Individual broad sense heritability of canopy width is moderate (H ¼ 0:3), and the correlation of cultivars across environments was high (Hardner et al. 2002). Canopy width was also genetically correlated with stem girth (rg ¼ 0:6). In Australia, ‘A4’ had the smallest predicted mean (4.2 m) for 40 cultivars over two sites compared to ‘Keauhou’ and ‘Makai’ (5.7m) (Hardner et al. 2002). ‘A16’, ‘HAES 814’, ‘Daddow’, ‘NG18’, ‘Own Venture’, ‘HAES 849’, ‘Keaau’, ‘Mauka’, and ‘Kau’ were intermediate. Cultivar means for canopy width at 10 years (Hardner et al. 2006) was correlated with width at 14 years averaged across four sites (Stephenson and Gallagher 2000) (rcv ¼ 0:6). Reports of tree structure from ﬁeld observations indicate that ‘Keauhou’ and ‘Kakea’ are spreading trees with round canopies and ‘Kau’, ‘Keaau’, ‘Mauka’, ‘Pahala’, and ‘Makai’ are upright (O’Mara 1977; Hamilton et al. 1981; Hamilton and Ito 1984). It has also been suggested that ‘Makai’ requires a higher intensity of tree training at a young age to develop a good structured tree (Ito and Hamilton 1989). Stem girth has been examined by some authors (Allan 1989; Supamatee et al. 1992; Stephenson et al. 1995). In Australia, heritability of stem girth calculated from a trial of 40 cultivars across four sites was low to moderate (H ¼ 0:2), but cultivar performance was highly correlated across sites (Hardner et al. 2001). To account for unbalance, a-REML analysis was undertaken on data presented for stem girth of 10 cultivars across seven locations in Thailand with four replications at each location (Supamatee et al. 1992). This analysis indicated that ‘Keaau’ was signiﬁcantly more vigorous than ‘Own Choice’ and an indigenous hybrid cultivar, but there was no signiﬁcant difference among the other cultivars. The analysis could not test for genotype-by-environment interaction (G E) as no within-site error was reported. In Hawaii, ‘Ikaika’ and ‘Kakea’ are considered more vigorous than ‘Kau’ and ‘Keauhou’ based on general ﬁeld observations (Hamilton and Fukunaga 1959; Hamilton and Ito 1984). An open canopy density may be important for penetration of light (Huett 2004) and spray application; however, the density of canopies is difﬁcult to quantify, and there is a lack of studies demonstrating a

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measurable impact. Reports based on ﬁeld observations classify the canopy of ‘Kau’ as dense and state that the canopy of ‘Daddow’ becomes ‘denser with age’. ‘Keaau’, Mauka’, ‘HAES 816’, and ‘A16’ are reported as having moderate-dense canopies, and ‘Keauhou’ as only having a moderate canopy density. Others (O’Mara 1977) describe the canopy of ‘Hinde’ as open. Resistance to wind damage is also an important consideration in Australia (Stephenson and Gallagher 2000), China (Lu et al. 1998b), and Hawaii (Shigeura and Ooka 1984), where tropical cyclones and typhoons can cause severe damage. Spreading trees with wide crotch angles are considered more susceptible to wind damage (Hamilton and Ito 1984) although this has not been quantitatively demonstrated. Direct assessment of resistance of macadamia to wind damage was undertaken in southern China (Lu et al. 1998a, 1998b, 2004). For a wind strength between 7 and 9 on the Beaufort scale, there were signiﬁcant differences in damage among cultivars with ‘Own Choice’ the most resistant, followed by ‘Kau’, ‘Ikaika’, ‘Keauhou’, and ‘Makai’. Differences disappeared at wind strengths above 11. Strong wind can cause immediate loss of yield and long-term damage to the tree (Lu et al. 1998b). Yield was reduced by 60% to 70% in the years following wind damage, and yield recovered in only 50% of trees in the second year after damage. These quantitative differences among cultivars are supported by observations in Hawaii (Hamilton and Ito 1984; Ito and Hamilton 1989). ‘Hinde’ is also considered susceptible to wind damage (O’Mara 1977), and ‘Fuji’ is considered susceptible in exposed areas in Hawaii (Blight 1989). B. Flowering Phenology Genetic variation in the length of ﬂowering period may have consequences for the opportunity for cross-pollination, and the extent to which indiscriminate environment events that are adverse for pollination (e.g., rain) may impact on the reproductive capacity of a tree. In Hawaii, an association between length of ﬂowering period and length of harvest period has been suggested (Nagao and Hirae 1992). Individual broad sense heritability of individual trees calculated from a study of 20 cultivars at a single site over a single season in Australia indicated that there are strong differences among cultivars for the date of the commencement of ﬂowering of individual racemes (H ¼ 0:87) with lower genetic variation for duration of ﬂowering of an individual raceme (H ¼ 0:53) (Boyton and Hardner 2002). Early-ﬂowering cultivars include ‘HAES 842’, ‘HAES 814’, ‘Kau’, and ‘Keauhou’, contrasted

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with later ﬂowering cultivars ‘A4’, A16, and ‘NG8’. However, variability in timing of the commencement of ﬂowering among individual racemes and the duration of ﬂowering within a cultivar (within and between trees) is larger than the variability among cultivars, suggesting opportunity for overlap of ﬂowering between cultivars. In Hawaii, ‘Keaau’ reportedly ﬂowers over a tightly deﬁned period compared to ‘Kakea’, which tends to have a diffuse pattern (Nagao and Hirae 1992). Further knowledge on the impact of pollination availability on productivity is required to investigate the implications of differences in ﬂowering phenology among cultivars. C. Fruit Set and Arrangement The large number of ﬂowers in a raceme provides the opportunity for multiple fruit per raceme. In Hawaii, there is a preference for fruits in clusters of 10 to 20 nuts (Hamilton and Ito 1977b), although the rationale for this is not apparent. Alternatively, a large number of fruit per raceme may reduce the effectiveness of spraying for pest and disease control. Differences among cultivars in the number of mature fruit set per raceme tagged at ﬂowering have been reported under Australian conditions (McConchie et al. 1997; Boyton et al. 2002). Fruit set per raceme from controlled pollination was highest for ‘HAES 849’ (8.6) and ‘Mauka’ (8.2); intermediate for ‘Kau’ (5.8); lower for ‘Keaau’ (5.0), ‘A16’ (4.9), ‘HAES 816’ (4.6), ‘HAES 814’ (4.6); and lowest for ‘Own Venture’ (3.5), Daddow (3.3), ‘Keauhou’ (2.8), ‘HAES 781’ (2.8), ‘HEAS 842’ (1.7), and ‘A4’ (1.6) (McConchie et al. 1997; Meyers 1997). Differences among these groups were signiﬁcant. In an alternative study under natural pollination (Boyton et al. 2002), fruit set ranged from 2.2 per raceme for ‘Kau’ to 0.3 for ‘HAES 816’, although this calculation also included racemes that failed to produce fruits (47% of racemes tagged at anthesis). However, the ranking of cultivars in these studies may be different from that for number of fruits per raceme at maturity where no account is made of the number of failed racemes. In Australia, ‘A38’ reportedly sets up to 30 mature fruit per raceme (Hidden Valley Plantations 1994). D. Yield Yield is one of the fundamental traits for selection in macadamia (Cull 1978; Winks 1983; Hardner et al. 2006). The general pattern of yield in macadamia is commencement of production between age three and six years, followed by a general increase with age, leveling to a plateau

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at later ages (Nagao and Hirae 1992; Mayer et al. 2006). Results from several cultivar trials suggest that, in general, yield reaches a plateau around 12 years of age at densities of 8 9 m (Ito et al. 1983), 5 10 m (Ito et al. 1998), and 5 9 m (Nagao et al. 2003). Planting density may affect yield (Oosthuizen 1992; Mayer et al. 2006), although there is little evidence to support a later age decline in yield in crowded orchards (Hardner et al. 2000; McFadyen et al. 2004). In addition, yields may vary by 60% due to seasonal inﬂuences (Hardner et al. 2000; Mayer et al. 2006). This complexity makes a quantitative deﬁnition of yield difﬁcult (Winks et al. 1986). Parameters that have been used to describe yield are: age of ﬁrst crop (Hardner et al. 2006), NIS per tree at a certain age (Stephenson and Gallagher 2000; Hardner et al. 2006), and total or average yield over a particular period (Stephenson et al. 1999; Hardner et al. 2006). The linear rate of the increase in yield has been used to describe yield during the accumulation phase of production (Stephenson and Gallagher 2000; Hardner et al. 2006). The complexity of describing yield has led some (Stephenson 2001) to suggest that physiological studies may provide a better platform for understanding of the trait, thereby enabling in greater selection response. There has been an interest in developing a productivity index that relates yield to tree size to enable the comparison of yield across different ages and management scenarios (Winks 1986) and identify trees with higher yield per hectare (Hardner et al. 2002). The best regression between tree size and yield was achieved by describing tree size as the vertically projected area of the canopy (Chapman et al. 1986; Winks et al. 1986). Hardner et al. (2002) reported a productivity index calculated as cumulative yield divided by the horizontal projection of canopy area at age 10. Use of a productivity index to select trees for yield per hectare or compare trees at different ages requires the assumption that the ratio between yield and tree size is constant across ages or sites, but this has not been veriﬁed. For comparison across studies, care is also needed to understand exactly what is being reported for yield. Yield may be reported as wet nut in husk (e.g., Nagao et al. 2003) or wet nut in shell (e.g., Ito and Hamilton 1987) with moisture content of over 20% (Stephenson 1990a; Wall and Gentry 2007). Alternatively, yield may be expressed as nut in shell (NIS) at a constant moisture content of 10% or 1.5% kernel moisture content, as this is the level nuts are generally dried to for cracking and processing (Stephenson 1990a; Mason and McConachie 1994). In some studies, yield is assessed after nuts that have fallen prior to the completion of oil accumulation have been removed from the site (Hardner et al. 2002).

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Alternatively, this material may be included and may lead to an overestimation of commercial yield (e.g., Piza et al. 2006). In other cases, the inclusion or exclusion of such material is not reported. Some studies report yield estimates from single-tree plot designs (e.g., Stephenson et al. 1996). The competitive environment of these trials is likely to be highly variable compared to production orchards, which generally have a single cultivar planted along each planting row. Studies are required to verify the accuracy of these designs. 1. Age of First Crop. An earlier age of bearing is generally considered a desirable characteristic in a cultivar as it can increase early orchard returns (Hardner and McConchie 1999). Commencement of production at three to four years after planting is considered desirable in Australia (Stephenson and Gallagher 2000). However, there is little quantitative information on the genetic architecture of this trait in macadamia. There was little range in the predicted effects for age of ﬁrst crop for 20 cultivars over two sites (0.2 to 0.1) (Hardner et al. 2006). The authors note that there was a large interaction between cultivar and site for this trait although further detail was not presented. Field observations suggest that ‘Ikaika’ (Hamilton and Ito 1984) and ‘Kakea’ (Hamilton and Fukunaga 1959) are particularly precocious compared to other Hawaiian cultivars. This, however, was not observed for ‘Ikaika’ when trialed in the Panxi region of China (Xiao et al. 2002a). ‘Beaumont’, ‘HAES 814’, and particularly ‘Fuji’ are considered precocious cultivars in South Africa (Blight 1989; Allan et al. 1999). 2. NIS Yield Per Tree. The selection criteria employed in the early Hawaiian program prior to the mid-1980s for yield was a minimum annual production of 45 kg per tree at age 8 in favorable sites and 35 kg per tree in less favorable (e.g., high temperature or wind, soil and drainage problems) sites (Hamilton and Ito 1976). This threshold was later increased to 68 kg at year 10 in favorable sites (Hamilton and Ito 1986). In contrast, a consistent yield of two tonnes per ha at 10 years (16 kg per tree at 10 8 m spacing) has been recommended as the benchmark for cultivars in Australia (Stephenson and Gallagher 2000). Broad sense individual heritability (H) for annual yield to 10 years of nut in shell at 10% moisture content ranged between 0.06 and 0.18 for a trial of 40 cultivars across four sites in Australia (Hardner et al. 2002), considerably lower than canopy width and nut and kernel characteristics (Hardner et al. 2001), which were also examined. This suggests that assessment of yield on trees outside controlled trials or using a low number of replicates may not accurately estimate genetic potential.

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Genetic correlations were high (0.7 and 1.0) among yield in successive years (Hardner et al. 2001). Yield at age 4 was not highly correlated with yield from 6 to 10 years of age (rcv ¼ 0:49 to 0.11), although genetic correlations between years were greater in later years. Correlations were higher when yield was expressed as cumulative yield, due to an averaging over annual variation. A high correlation of cumulative yield to age 7 with cumulative yield to year 10 (rcv ¼ 0:9) has been used to develop strategies for early selection in macadamia (Hardner et al. 2001). This study also indicated that there was no genetic correlation between yield and tree size (rcv ¼ 0:1) although there was a high within-tree correlation. This result has also been observed in other studies (Chapman et al. 1986). Several reports have demonstrated difﬁculty in ﬁnding signiﬁcant differences in yield among cultivars (Ito et al. 1983; Winks et al. 1987; Stephenson et al. 1995; Nagao et al. 2003), supporting the observation of low heritability for this trait. There was no signiﬁcant difference between the top 21 cultivars for cumulative yield from 4 to 8 years of age and no signiﬁcant difference between the top 16 cultivars for average yield over the same period (Stephenson et al. 1995) in the same trial used by Hardner et al. (2002) to estimate genetic parameters for yield. Higher heritability (H ¼ 0:5) has been reported for a productivity index of cumulative yield to 10 years per square meter of projected canopy area (Hardner et al. 2002). In addition, signiﬁcant differences were found between nine cultivars for the regression of vertically projected canopy area and yield (Winks et al. 1986). Cultivar means for the productivity index were highly correlated with estimates of intercept of the regression of yield on tree size (rcv ¼ 0:99) compared to those for slope (rcv ¼ 0:33). There is little quantitative data on the presence of genotype-byenvironment interactions for yield in macadamia. Genetic correlations of 40 cultivars over four sites were variable between years but were higher and more consistent for cumulative yield, except for correlations with one particular site (Hardner et al. 2002). In contrast, there was much lower G E for the productivity index of yield per square meter of projected canopy area, suggesting this parameter may be more efﬁcient for selection than yield per se. These data were also used to examine the stability of cultivar means across sites and years (Stephenson et al. 1995), where a general trend was reported for higher-yielding cultivars to be more variable across sites and years. However, this analysis does not account for the low accuracy of the predicted means, which are based on a maximum of four replications.

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Differential response of cultivars to altitude has been reported for Hawaii (Hamilton and Ito 1984; Ito and Hamilton 1989; Nagao and Hirae 1992) and Kenya (Gathungu and Likimani 1975), although no data were presented. Yield data for several cultivars at different altitudes has been presented (Ito and Hamilton 1987); however, this was based on only one to three replications of each cultivar at each site and is likely to be highly inaccurate. The physiological basis of apparent differences in productivity with altitude is poorly understood. A number of other studies have reported yield at younger ages during the accumulation phase of production (Winks 1983; Ito et al. 1991; Supamatee et al. 1992; McCubbin and Lee 1996; Swanepoel and Hobson 1999; Lu et al. 2004). However, the variability in the quality of the data makes it difﬁcult to integrate the results for comparison. In particular, estimates of production based on results from a single year or from very early in the bearing life of a tree may not be indicative of tree performance at later ages. Yield for trees that are reaching the mature phase of production have been reported for a number of cultivars across a number of studies (Ito et al. 1983; Winks et al. 1987; Phiri 1985; Stephenson et al. 1995, 1999; Nagao et al. 2003), although some results have been not been included here (e.g., O’Mara 1977; Ito and Hamilton 1987) as they were based on only two to three replications of each individual. The rank of cultivars tends to be similar across studies. In Hawaii, the average annual yield between 10 and 16 years was signiﬁcant higher for ‘Kau’ (47 kg WNIS per year) and ‘Keauhou’ (46 kg) compared to ‘Keaau’ (38 kg) with ‘Kakea’ (44 kg) and ‘Ikaika’ (42 kg) intermediate (Ito et al. 1983). Similarly, in an Australian study of 40 cultivars (Stephenson et al. 1999), the cultivars with the highest average annual yield of NIS between 12 and 14 years were ‘Kau’ (21 kg NIS per year) and ‘Keauhou’ (20 kg NIS per year); ‘Keaau’ was one of the lower-yielding cultivars (16 kg) in Australia. Other low-yielding cultivars in the Australian study included: ‘A4’ (12 kg), ‘A16’ (13 kg), ‘HAES 816’ (14 kg), ‘HAES 814’ (15 kg), ‘HAES 849’ (15 kg), and ‘HAES 835’ (16 kg). ‘Mauka’ (19 kg), ‘Daddow’ (18 kg), ‘Own Venture’ (18 kg), and ‘NG18’ (17 kg) were intermediate. In a later Hawaiian study (Nagao et al. 2003), ‘Kau’ produced the highest average annual yield between 10 and 13 years (45 kg WNIS per year). In agreement with the Australian results, the lowest yields were for ‘Mauka’ (29 kg), ‘HEAS 849’ (27 kg), and ‘Pahala’ (25 kg). Other smaller studies in Malawi (Phiri 1985) and Australia (Winks et al. 1987) have also demonstrated the superior yield of ‘Keauhou’ and the relatively low yield of ‘Keaau’. The ranking of cultivars for mature yield in Stephenson et al. (1999) is in general

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agreement with average total yield of NIS to 10 years (Stephenson et al. 1995), although at earlier ages the yield of ‘Daddow’, ‘NG18’, and ‘Own Venture’ were equal to that of ‘Kau’ and ‘Keauhou’ and the yields of ‘HAES 814’ and ‘HAES 849’ were intermediate. However, this study also reported that there was no signiﬁcant difference among the top 15 cultivars, suggesting more replication is required to accurately identify the relative yield of cultivars. Differences in absolute yield between Hawaii and Australia may in part be due to the difference in the production system between the two countries, in particular the extended ﬂowering and harvest seasons in Hawaii, where nuts are present on the tree all year round (Nagao and Hirae 1992), or different methods of assessment. There seems little support for differential performance of cultivars in Hawaii and Australia as suggested by some authors (Cull 1978; Stephenson et al. 1995). E. Nutrition Utilization Field observations of variability in symptoms of nutrient deﬁciencies among cultivars have been used to suggest a genetic basis to the efﬁciency of nutrient utilization (Hamilton and Fukunaga 1959). However, no experimental data has been provided to offer strong support for this hypothesis. In a limited on-farm trial, no signiﬁcant differences were found in leaf nutrient content of 12-year-old bearing trees of ‘Keauhou’, ‘Ikaika’, ‘Kakea’, and ‘Keaau’ (Pire et al. 2002). Yamaguchi (2003) suggest that ‘Purvis’ has a higher demand for nitrogen compared to ‘Keauhou’, ‘Kakea’, and ‘Keaau’, with ‘Ikaika’ and ‘Kau’ requiring less. A similar pattern is reported for phosphorus, although it is considered ‘Kakea’ and ‘Keaau’ have higher demands for this nutrient than ‘Keauhou’. No genetic variation for potassium demand was suggested. In contrast, others (Stephenson and Cull 1986; Robinson et al. 1997; Huett and Vimpany 2007) suggest ‘Kau’ requires more nitrogen than ‘A4’, ‘Kakea’ ‘Mauka’, ‘Keauhou’, ‘Hinde’, and ‘Keaau’. It has also been suggested that ‘Own Choice’ may be particularly susceptible to copper deﬁciency (O’Mara 1977). Further quantitative information on the interaction between nutrition and the physiological processes of the tree is required to build these results into a selection program. F. Abnormal Vertical Growth ‘‘Abnormal vertical growth’’ is a term used to describe a disorder of excessive vertical growth and reduction or absence of ﬂowering that has been reported in Australia, South Africa, and Costa Rica (O’Farrell and

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Searle 2003). There is reportedly a higher frequency of the disorder in ‘Kau’ and ‘Mauka’, and symptoms have been observed in ‘Keauhou’, ‘Kakea’, ‘Keaau’, and ‘Makai’, although not in ‘A4’ or ‘A16’ (O’Farrell and Searle 2003). Further work is required to quantitatively describe the disorder, and assess its genetic basis and interactions with environmental factors, before genetic improvement can be attempted. G. Phenology of Fruit Drop Variability in the length of fruit drop may impact proﬁtability of macadamia production where fruit are harvested from the ground following natural abscission. Harvesting is a major cost of production (Nagao and Hirae 1992; Hardner et al. 2006). As fruit are harvested at 2 to 6 weekly intervals to maintain quality (Leverington 1962a; Mason 1983; Mason and Wells 1984; Liang et al. 1996), increased length of the period over which fruit drop will increase these costs. It has also been suggested that lateness of fruit drop may affect ability to control pest and diseases (Stephenson and Gallagher 2000). M. tetraphylla selections reportedly have a much shorter fruit drop season than M. integrifolia selections (Leverington 1958, 1962a). Fruit drop patterns have been quantiﬁed using a generalized logistic function (Hardner et al. 2005a), although the authors report convergence problems for a number of samples, suggesting that an alternative model should be explored. Stephenson et al. (1995) deﬁned harvest period for selection as early (> 90% of the crop dropped over ﬁrst four months of mature nut drop), mid (> 90% of nut dropped over the ﬁrst six months), and late (>10 % of crop remaining in tree after six months). Based on ﬁeld experience, ‘Keaau’ is described as having a short harvest period and ‘Kakea’ is considered to have a long drop period (Hamilton and Ito 1984; Ito and Hamilton 1989; Stephenson and Gallagher 2000). ‘Own Choice’ is reported to be a late-dropping cultivar (O’Mara 1977). The phenology of fruit drop in macadamia can be manipulated through the use of ethephon (Jones et al. 1996; Trueman et al. 2002). Differential response among ﬁve cultivars to application of ethephon approximately nine months after anthesis has been reported, with greater fruit drop in cultivars that had commenced natural abscission by the time of application (‘HAES 814’, ‘HAES 842’, and ‘HAES 849’) compared to ‘A16’ and ‘Own Venture’ (Salter et al. 2003). The authors suggest that the impact of ethephon is related to the phenological stage of the cultivar, although ethephon was applied only at a single date in this study. This study also reports a signiﬁcant effect of cultivar on leaf

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loss after ethephon application, but this was unrelated to the effect on fruit abscission. H. Pest and Disease Resistance Numerous pests and diseases appear to have coevolved with Macadamia in its natural habitat. However only a small number affect cultivation in Australia. For example, of the 150 or more insect species that are hosted by Macadamia (Gallagher et al. 2003), fewer than 10 are regarded as orchard pests of economic importance (Huwer and Maddox 2003). Pests and diseases may account for substantial crop losses through fruit abscission prior to the completion of kernel development or direct damage to the kernel (Leverington 1958; Waite et al. 1999; Jones 2002), and there is an interest in developing resistance in cultivars as part of an integrated pest management strategy (e.g., Jones and Caprio 1992). In contrast, some studies imply pest damage is unrelated to genotype and is mainly a consequence of variable management (Leverington 1962a; Stephenson 2001). A full understanding of the pest and disease cycles is required to develop resistant cultivars. In Australia, nut-borer (Cryptophlebia ombrodelta, also called litchi fruit moth, Jones 1994a) is a major pest and can cause premature fruit drop prior to completion of shell hardening and oil ﬁlling (Ironside 1982; Waite et al. 1999). Tropical nut borer (Hypothenemus obscurus) causes kernel damage by attacking abscised fruit on the ground (Jones and Caprio 1992; Jones et al. 1996). Attack to developing fruit by fruit spotting bug (Amblypelta nitida) can lead to abortion of the immature fruit or kernel damage if it occurs later in the season (Waite et al. 1999; Gallagher et al. 2003). In Hawaii, the koa seedworm (Cryptophlebia illepida) can cause fruit drop prior to completion of oil accumulation (premature fruit drop) if attacked prior to shell hardening, although little actual kernel damage has been reported (Jones and Caprio 1992; Nagao et al. 2003). Southern green stink bug (Nezara viridula) is capable of piecing the shell during any stage in fruit development and after abscission, resulting in damage to the kernel (Nagao and Hirae 1992; Jones and Caprio 1994; Shearer and Jones 1996; Wright et al. 2003; Golden et al. 2006). Experimental evidence suggests that fruits that are mature or near mature do not necessarily abscise following feeding by N. viridula (Jones and Caprio 1994). The actual extent of crop loss due to pest and disease attack may depend on timing of attack in the crop cycle (Waite et al. 1999). In a study in Hawaiian orchards, macadamias appeared able to compensate for the removal of up at least 30% of fruit prior to 150 days postanthesis

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(Tobin et al. 1997). Crop loss due to insect attack, however, may be underestimated if based on the proportional mass of kernels that are damaged; this ignores the amount of kernel that otherwise would be produced if the kernel had developed normally (Jones and Caprio 1992). Measurement of damage levels as the proportion of nuts with damaged kernel may present a more realistic measure of the impact of pest damage (Jones and Caprio 1992; Golden et al. 2006). Genetic variation in the extent of nut borer damage has been reported (Villiers 1977) (Table 1.5). Timing and degree of husk and shell hardness is thought to be related to the penetration ability of the larvae (Jones et al. 1992; Campbell et al. 2005). Genetic variation for husk hardness has been demonstrated, with hardness being highest for ‘Ikaika’, ‘HAES 816’, and ‘Fernleigh Special’ (Campbell et al. 2005). Differential rates of shell hardening among cultivars have also been observed (Jones 1994b), suggesting possibilities for manipulation through selection. However, alternative methods (e.g., parasitoids, Waite et al. 1999) may be more efﬁcient than genetic improvement for managing the impact of this pest. Cultivar differences in kernel damage from tropical nut borer have been reported (Jones and Caprio 1992; Jones et al. 1996) (Table 1.5). Kernel damage was signiﬁcantly higher for ‘Keaau’ (26% after four Table 1.5. Susceptibility of macadamia cultivars and selections to insect pests and tendency for stick-tights in Hawaii. Cultivar

Tropical nut borer

Southern stinkbug

Keauhou Purvis Ikaika Kau Kakea Keaau Mauka Pahala Makai 816 835 849 856 A4 A16 A38

Medium High Low Medium Medium High High High Low High Medium

Medium Low Low Medium Medium High Medium Medium Low High Medium

Low High Medium

High High Medium

Koa seedworm Medium-high High

Medium-high Low Medium-high Low

Medium-high

Stick-tights Low High-very high Low-high Low-medium High Low-medium Low-high Low-very high Low High Low-medium Low-high Low-medium Very high High High

Source: Hamilton and Fukunaga 1973; Hamilton and Ito 1984; Ito and Hamilton 1989; Jones et al. 1996; Jones 2002; Nagao et al. 2003.

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weeks on the ground) and ‘Kau’ (18%) than ‘Ikaika’ (9%) and ‘Mauka’ (5%). The percentage damage of kernel from ‘Keauhou’ (12%) was not signiﬁcantly different from that for the other cultivars. However, at high pest pressures, all cultivars experienced similar high levels of kernel damage (Jones et al. 1996). There is good evidence that shell thickness is the causal mechanism of resistance as there was a good ﬁt of a power relationship between these two variables (R2 ¼ 0:84) (Jones et al. 1996). In contrast, the Pearson correlation coefﬁcient between values of insect (and mold) damage and shell thickness reported for 94 Australian selections (Leverington 1962a) was not signiﬁcant (r ¼ 0:28), although this analysis confounds genetic and nongenetic effects. No experimental evidence has been published to verify a differential response of cultivars to fruit spotting bug in macadamia. Waite et al. (1999) observed no difference in response of ‘Kau’ and ‘Makai’ to fruit spotting bug exposure. However, variation in damage for the related Amblypelta l. lutescens among cashew cultivars (Peng et al. 2005) suggests that further investigations could uncover genetic variation for this trait in macadamia. In Hawaii, variation among cultivars has been observed for the percentage of kernels with damage from southern green stink bug (Jones and Caprio 1992) (Table 1.5). The cultivars ‘Purvis’ and ‘Makai’ had signiﬁcantly less damage than the average; ‘Kau’, ‘Mauka’, and ‘Pahala’ did not differ signiﬁcantly from the average level of damage; and ‘HAES 816’ and ‘HAES 856’ exhibited signiﬁcantly more damage. It has been suggested that shell thickness and the rate of shell hardening may contribute to resistance (Jones and Caprio 1992; Nagao et al. 2003). Based on ﬁeld observations and experience, the susceptibility of 12 common cultivars to the three major insects in Hawaii has been reported (Table 1.5); however, these observations suggest that susceptibility for the different pests is not genetically correlated. However, the use of general terms and descriptive language makes further analysis difﬁcult. Observations of high incidence of kernel damage in ‘HAES 816’ appear to conﬁrm its susceptibility (Nagao et al. 2003). Anthracnose is a disease of the husk and leaves (Hamilton and Storey 1956) and can be a particular problem in humid areas and when annual rainfall is greater than 1800 mm (Hamilton and Fukunaga 1959). A suggestion has been made that cultivars that are resistant to anthracnose have low stick-tights (Hamilton and Fukunaga 1970; Hamilton et al. 1981; Ito and Hamilton 1989). The cultivars ‘Keauhou’, ‘Kakea’, ‘Ikaika’, and ‘Wailua’ are considered to have good to excellent resistance to anthracnose (Hamilton and Storey 1956). ‘Pahala’ is considered to have moderate resistance (Hamilton et al. 1981).

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Husk spot is considered a signiﬁcant disease of macadamia in Australia, causing premature nut drop prior to completion of oil ﬁlling (Stephenson 1990a; Mayers 1991). There are no reliable ﬁgures for the economic impact of the disease, although some estimates range from 30% to 40% of crop loss. It is difﬁcult to evaluate the accuracy of reported cultivar differences in disease susceptibility/tolerance without a clearer understanding of the relationship between disease severity and economic impact (Drenth 2004). I. Stick-tights Stick-tight nuts is a condition where the connective tissue between the stem and the fruit dies and nuts remain on the tree after the end of the harvest season until the husk rots and the old nuts fall (Nagao and Hirae 1992; Jones 2002). A link between stick-tights and high levels of pest and disease loads has been suggested (Jones and Caprio 1992; Jones et al. 1992, 1996), apart from the direct impact of crop loss. Absence or a very low occurrence of these nuts is preferred in Hawaiian (Hamilton and Ito 1984; Nagao and Hirae 1992; Jones 2002) and Australian (Stephenson and Gallagher 2000) selections. There is little information on the biology of this condition, and no studies have assessed the extent of stick-tights. A role for anthracnose has been suggested, but data are not available to establish this link. Reports of differences among cultivars suggest a genetic basis for this trait (Table 1.5), although the apparent large variability within a cultivar also suggests an environmental component to variation. Cultivars that have been described as producing stick-tights are ‘Pahala’, ‘Kakea’, ‘Ikaika’ (Hamilton et al. 1981) ‘Own Choice’, and, to some extent ‘Hinde’ (Stephenson 1990a), although stick-tights have not been observed for ‘Own Choice’ in trials in China (Lu et al. 2004). A clearer understanding of the condition, how it can be objectively measured, and the impact on the production system are required to enable inclusion in selection decisions. J. Nut Characteristics 1. Nut Size. Several nut characteristics have been considered as selection criteria in the development of macadamia germplasm. The Hawaiian program preferred cultivars that produced uniform mediumsize nuts with 140 to 150 nuts per kg (6.5–7.0 g per nut) (Hamilton and Ito 1976, 1977b), although a wider range of nut size (130–190 nuts per kg) was considered acceptable in later selections (Hamilton and Ito

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1984, 1986). The importance of nut size may be related to cracking efﬁciency, although this could be because crackers are designed for a particular size range, so that a higher frequency of damage may occur in consignments where the range of nut size is large (Liang 1980). Sorting prior to cracking, or an improvement in cracker technology, may reduce the importance of this trait. Based on commercial experience, it is also suggested that nuts smaller than 19 mm are difﬁcult to handle, resulting in higher labor costs (Leverington 1958, 1962a, 1971; Gathungu and Likimani 1975). Nut size has been found to be a highly heritable trait (individual broad sense heritability, H ¼ 0:63), with little G E across locations or ages (Hardner et al. 2001). A signiﬁcant genetic correlation between nut and kernel mass is also reported. Others (Beaumont 1937) report a signiﬁcant phenotypic correlation between nut size and kernel mass. High heritability of nut size is supported by ﬁeld observations that seedlings germinated from seed selected for their small size produce a high proportion of small fruits (Gathungu and Likimani 1975). Cultivars with small nut sizes are ‘HAES 814’ (5.0 g), ‘Keaau’ (5.5– 5.7 g), and ‘NG18’ (5.8 g); cultivars with large nuts include ‘Own Venture’ (8.1 g), ‘A4’ (7.1 g), ‘Makai’ (7.1 g), and ‘Kau’ (7.0–7.6 g) (Hamilton and Ito 1984; Stephenson et al. 1995). ‘Purvis’ is reported as having a average size nut (6.5 g) in Hawaii (Hamilton and Ito 1984), although in Australia this cultivar produces large nuts (7.2 g), similar in size to ‘Makai’ (Stephenson et al. 1995). 2. Nut Shape. Round nuts are considered easier to crack and grade than ovoid nuts (Leverington 1962a, 1971; Winterton 1968). Twin nuts, where two hemispherical nuts are formed, are considered rejects as they do not crack well (Cavaletto 1981). However, no studies quantify these characteristics or the impact of variation on costs of production. M. tetraphylla reportedly produces a higher frequency of ovoid nuts (Leverington 1958), but little is known of the extent of genetic variation within the species. 3. Nut Defects. Nuts that exhibit signs that the process of germination (i.e., opening of the suture in the shell) has commenced represent a source of crop loss, as the appearance and taste of germinating kernel is considered unacceptable, and the opening in the shell may permit entrance of disease organisms. Fruit may germinate on the tree or after the fruit has dropped to the ground, prior to harvesting. Germination in susceptible cultivars has been linked to the occurrence of wet weather; however, it has also been suggested that increased harvesting

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frequency may minimize the occurrence of this defect (Hamilton and Ito 1984). A variability in nursery germination percent may be correlated with susceptibility to germination prior to harvesting (Hardner and McConchie 2006). Field experience suggests that ‘Keaau’, ‘Pahala’, and ‘Beaumont’ are prone to germination, particularly in wet weather (Hamilton and Ito 1984; Allan 1989; Ito and Hamilton 1989; Hardner and McConchie 2006). Open microplyes are also considered unacceptable. At anthesis in macadamia, the nucellus is incompletely surrounded by the outer integuments, and a micropyle is formed 10 to 11 weeks later (Strohschen 1986). Usually a white enamel micopylar plug forms as the shell hardens (Francis 1928; Strohschen 1986); however, in some genotypes, the micropyle can be open at maturity (Stephenson 1990a; Nagao and Hirae 1992). This opening can allow entry of insects, molds, and moisture, thereby making the kernel unacceptable. It has been reported that 10% of nuts from ‘Keauhou’ may be affected by this defect (Stephenson 1990a). 4. Kernel Recovery. Kernel recovery, or kernel percent, is one of the easiest traits to assess and one of the most commonly reported. It can be deﬁned simply as the percentage mass of nut that is the kernel (i.e., the embryo) and is used to calculate the expected mass of kernel from a given mass of nuts. Kernel recovery has a direct impact on the production system as ﬁxed costs of production and processing per unit weight of kernel are lower with higher kernel recovery (Hardner et al. 2006). However, it has been suggested that cultivars with high kernel recovery have thinner shells, and, as discussed, thin-shelled cultivars are more susceptible to insect damage, preharvest germination, insect and rat damage, and kernel damage during cracking (Leverington 1958, 1962a, 1971; Gathungu and Likimani 1975). The actual detail of how the trait is assessed, and therefore its meaning, may vary among studies. The mass of nuts may be wet NIS at ﬁeld moisture (e.g., Ito et al. 1983; Nagao et al. 2003), 10% moisture content (e.g., Hardner et al. 2002), or NIS dried to 1.5% kernel moisture content (e.g., McCubbin and Lee 1996; Swanepoel and Hobson 1999). It has been reported (Leverington 1962a) that kernel recovery assessed from wet nuts may be higher than kernel recovery assessed after dehydration. Although the variability in assessment methods differences may affect absolute values, a study with 14 commercial cultivars indicated that the genetic correlation among kernel recovery calculated from wet NIS and NIS at 1.5% kernel moisture content is high (0.95) (Hardner et al. 2005b). In other studies, reject (e.g., mold and insect) nuts may be removed prior to assessment of nut mass (Leverington 1962a;

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Stephenson 2000). Kernel recovery will tend to be higher in this case if reject nuts have a lower kernel recovery than the sample average. The status of kernels included in numerator of the kernel recovery equation may also differ among studies. Some studies use the total mass of kernel (Stephenson et al. 1995; Hardner et al. 2001), while others (e.g., Leverington 1962a) remove unsound kernel, including those affected by insects, mold, or germination, prior to measurement of kernel mass, as these defects are not considered to be under genetic control. However, sound kernel recovery may not accurately represent kernel recovery in the absence of these defects, particularly if the levels of unsound kernel are high, as nuts containing unsound kernel are included in the denominator. Adherence of pieces of kernel to the inside of the shell after cracking may occur (Leverington 1958), and whether or not this is included in the assessment of kernel mass may affect how kernel recovery is calculated. Kernel recovery of 36% was recommended as the minimum for selecting cultivars in Australia (Stephenson and Gallagher 2000), while in Hawaii the selection threshold ranged between 34% (Cavaletto 1983), and 37% or 38% (Hamilton and Ito 1977b). Several studies report higher kernel recovery of nuts collected from M. tetraphylla compared to M. integrifolia. In a sample of 94 selections from Australian orchards, the average kernel recovery for M. tetraphylla selections was 37% compared to 30% for M. integrifolia selections (Leverington 1962a, 1971). Saleeb et al. (1973) reported kernel recovery of 45% for M. tetraphylla and 39% for M. integrifolia selections and cultivars, some of which were a subset of the previous study. These authors also reported the shell of the nuts was signiﬁcantly thinner in the middle and top for M. tetraphylla cultivars. Whether these results are affected by selection is difﬁcult to determine. Total kernel recovery was found to be highly heritable in a trial of 40 cultivars assessed over four sites in Australia analyzed using a mixed model approach (H ¼ 0:6, Hardner et al. 2001). This study also observed no detectable G E with site or age for kernel recovery. In contrast, a stability analysis (sensu Pritts and Luby 1990) with an extended data set (two additional sites) suggested the kernel recovery of some cultivars was unstable across sites and ages (Stephenson et al. 1999). The difference between these studies may be that the regression approach used in the stability analysis did not take into account the error of prediction of the cultivar mean. To summarize and compare published kernel recovery for cultivars across a range of studies from Hawaii (Ito and Hamilton 1983; Ito et al. 1983; Ito and Hamilton 1989; Ito and Iyo 1992; Ito et al. 1998; Nagao

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et al. 2003), Australia (Winks et al. 1987; Stephenson et al. 1999; Stephenson 2001), South Africa (Allan 1989; Oosthuizen et al. 1989; McCubbin and Lee 1996; Swanepoel and Hobson 1999), Brazil (Barbosa et al. 1991; Sacramento et al. 1995; Piza et al. 2006), Malawi (Phiri 1985), and China ((Xiao et al. 2002b), a REML analysis (as described earlier in this section on selection criteria) was undertaken. Sites across the different studies were grouped into locations for the analysis. Grouping of sites in Hawaiian studies was based on altitude, while the grouping of sites in other areas used geographical proximity. Countries were treated as ﬁxed, and cultivar and location within country were treated as random. Data from multiple locations across multiple years enabled the construction of an error term to test the signiﬁcance of location within country. The overall mean of kernel recovery across the different studies was 35%. Kernel recovery differed signiﬁcantly among country and studies but was highly heritable (H ¼ 0:6), identical to that found in the previous Australian study. The interaction between cultivar and country or location within country was not signiﬁcant, again conﬁrming the results from the Australia study that the relative performance of cultivars across environments for kernel recovery is highly stable. This agrees with general observations that the characteristics of Australian selections introduced into Hawaii in the 1950s were similar in both countries (Hamilton and Fukunaga 1962). The signiﬁcant effect of location within country indicates that cultivar means may be biased if all cultivars are not represented at each location and the analysis does not account for this imbalance. There is reasonable separation of the predicted cultivar means from this analysis (Table 1.6); however, the precision of the test between the cultivars could be improved by increasing the representation of cultivars across countries. Kernel recoveries for ‘A4’, ‘HAES 849’, ‘HAES 816’, and ‘A16’ are signiﬁcantly higher than most of the named Hawaiian cultivars (‘Purvis’, ‘Makai’, ‘Dennison’, ‘Kau’, ‘Keauhou’, and ‘Ikaika’). There are no signiﬁcant differences among the named Hawaiian cultivars, except that the kernel recovery of ‘Pahala’ and ‘Keaau’ are signiﬁcantly greater than that for ‘Kau’, ‘Keauhou’, and ‘Ikaika’. These results are in general agreement with published standards for these cultivars (Hamilton and Ito 1984), allowing for the difﬁculty in detecting signiﬁcant differences in this analysis. Interestingly, ‘A4’, ‘A16’, and ‘Beaumont’, which have relatively high kernel recoveries, grouped in the hybrid clusters in the analysis of genetic diversity (Peace 2005), consistent with the expectation of high kernel recovery for M. tetraphylla.

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Table 1.6. Predicted cultivar means across six countries for kernel recovery, percentage ﬁrst grade kernel, kernel mass, and percentage whole kernels. Cultivar A4 849 816 A16 814 Keaau Pahala Beaumont NG18 Own Venture Mauka Kakea Daddow Purvis Makai 835 Dennison 856 Kau Keauhou Ikaika lsd (0.95)

Kernel recovery (%)

1st grade (%)

41.9 39.1 38.9 38.1 37.9 36.0 36.0 35.9 35.8 34.9 34.5 34.2 33.9 33.7 33.4 32.1 31.8 31.5 31.5 31.2 30.5 4.0

97 91 90 94 92 92 93 95 92 92 91 91 92 93 95 94 93 93 92 86 92 7

Kernel mass (g) 3.2 2.8 2.9 2.9 1.9 2.1 2.3 2.3 2.4 2.9 2.3 2.2 2.4 2.6 2.5 2.2 2.2 2.5 2.3 2.4 2.2 0.4

Wholes (%) 50 62 62 60 46 48 51 44 63 58 47 – 48 60 57 68 – 45 55 46 – 10

Kernel recovery may also be correlated with other important selection traits. A signiﬁcant correlation was found among phenotypic values reported for kernel recovery and shell thickness (r ¼ 0:70) across 93 (mostly M. tetraphylla) preliminary selections from seedling orchards in Australia (Leverington 1962a). However, no correlation was found between kernel recovery and percentage insect (and mold) kernel damage (r ¼ 0:05), although other studies have demonstrated a strong relationship between shell thickness and damage from Hypothenemus obscurus (see earlier discussion). There is also a moderate genetic correlation between kernel recovery and kernel mass. K. Attributes of Kernel Quality Kernel quality is considered an important selection objective in macadamia improvement (Cavaletto 1977, 1981; Hamilton and Ito 1984; Nagao and Hirae 1992; Gallagher et al. 1998; Hardner et al. 2006); however, its meaning can be vague and inconsistent. Quality can be

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conceptually deﬁned as the value judgment made by the consumer about a product based on available cues within the personal and situational context (Steenkamp 1990). The perception of quality by the consumer is a particularly important factor inﬂuencing food choice of luxury goods (Tsai 2005), such as macadamia. This perception may inﬂuence immediate purchase decisions, and reinforce product perceptions to support future purchase (Steenkamp 1990; Grunert 2002). In this review, kernel quality is taken to mean the combination of kernel attributes that inﬂuence consumer food choice and not a speciﬁc kernel attribute, as sometimes used in the literature (e.g., percentage ﬁrst-grade kernel). Different sectors of the macadamia supply chain impose quality standards on the product, although, generally, macadamia quality standards are determined by the perceived cues of consumer preference for the roasted snack food product (Cavaletto 1981). A plump, light golden whole kernel, with crisp texture and delicate fresh ﬂavor, and free of visual imperfection, is considered to represent the highest quality of roasted snack product (Cavaletto 1981). However, the importance of different kernel quality attributes may vary with product, market, and consumer. Sensory attributes of odor, appearance, ﬂavor, and texture play important roles in developing and reinforcing quality concepts for the consumer (Moskowitz 1995), although other attributes such as price and health beneﬁts may also be important (Jaeger 2006). 1. Raw Kernel Visual Appearance. The visual appearance of raw kernel has been used as a major criterion of kernel quality in macadamia (Leverington 1962a; Cavaletto 1977; Shimabukuro 1984), presumably based on experience and perceptions that these correlate with the kernel quality of the ﬁnal product, although there has been little explicit testing of this association. Attributes that give the kernel an appearance inconsistent with the assumed ideal kernel appearance may be regarded as imperfections, and hence of lower quality (Tsai 2005). In addition, the visual appearance of raw kernels may be used as a cue for other undesirable sensory experiences. In this context, a raw kernel that is plump, white to cream colored, and without visual defect is considered to produce roasted kernels with the highest quality (Winterton 1968; Leverington 1971; Hamilton and Ito 1977b; Trochoulias 1995). A range of visual attributes are considered to impact on kernel quality. The presence of mold or insect-damaged kernel is obviously unacceptable from a food safety perspective (Leverington 1958, 1971). General discoloration of the kernel has been associated with deterioration on the orchard ﬂoor due to delayed harvesting (Liang et al. 1996). Other forms

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of kernel discoloration, such as dark rings (also called onion rings, Swanepoel and Hobson 1999) and off-color (darkened) tops or bases, are also considered to be unacceptable (Cavaletto 1977, 1981; Hamilton and Ito 1977b; 1977, Simabukuro 1984). Alternatively, others (Leverington 1971) suggested that a light gray base in raw kernel may not be objectionable, if it was subsequently masked by the roasting treatment. However, it is unclear if basal discoloration is deﬁned by the absolute color of the base or relative to the overall color of the kernel, which may also be variable. It has been suggested that the basal discoloration may be the result of absorption of tannins from the shell (Leverington 1971). In some seasons, ‘Mauka’ may produce some level of discolored kernel (Stephenson 1990a). The occurrence of overall gray discoloration of kernel has been reported and linked with the infection by the bacteria Enterobacter cloacae in kernels at ﬁeld moisture content and the production of off ﬂavor and odors that can spoil entire batches (Nishijima et al. 2007). The appearance of a yellow, brown, orange, or green strip on the kernel apex following drying is described and attributed to germination (Leverington 1962a, 1971; Guthrie et al. 2004). Nuts exhibiting open cracks in the shell typical of germination generally are removed prior to cracking. It is unknown if, at what level, and when undesirable textures and tastes develop throughout the progress of germination, although cyanogenic glucosides, which impart a bitter taste, are elevated in M. integrifolia and M. tetraphylla kernels that have commenced germination (Dahler et al. 1995). Small kernels with a shriveled and deformed appearance have been reported, and this is considered to be due to low oil content of the kernel (immaturity) (Ripperton et al. 1938; Leverington 1962a; Cavaletto 1977; Himsteadt 2002; Guthrie et al. 2004). Shriveled kernel can be a visual cue that the kernel may be susceptible to overroasting and have an objectionably hard texture, conditions commonly associated with lowoil-content kernels. The oil content, mass, and size of kernel classiﬁed as shriveled was signiﬁcantly lower (46%, 1.3 g, 11 mm) compared to kernel classiﬁed as sound (76%, 2.4 g, 14 mm), although the oil content of some kernel classiﬁed as shriveled was near what would be expected to produce acceptable roasted product (70%) (Ripperton et al. 1938; Mason and Wells 1984). The adherence of the dark lining of the inner shell to the kernel, or of kernel to the inside of the shell, is also considered unacceptable (Leverington 1958, 1962a). Physical damage to raw kernels can be considered to detract from the quality of the product and has been reported to lead to undesirable localized browning of the kernel (Wallace et al. 2001).

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Increases in the levels of raw kernel with visual imperfections result in increased sorting costs and increased ﬁxed costs of production per unit mass of acceptable kernel (Hardner et al. 2006). This assumes that all kernels with visual imperfections are rejected; however, kernels with minor degrees of visual imperfections may only be downgraded for use in products that do not demand high visual quality. There is little information on the genetic architecture of visual kernel disorders. This may be due in part to the problems of applying repeatable and objective assessment methods. Most disorders require visual assessment. Human sensory assessments are prone to bias and can be variable if these are not conducted with a controlled and structured approach (Sidel and Stone 1991; Meilgaard et al. 1999). Often thresholds are used to deﬁne reject, unsound, commercial, or sound kernel (e.g. Cavaletto 1981; Liang et al. 1996; Swanepoel and Hobson 1999; Stephenson 2000), but description of these thresholds is generally not given or is simply referenced as ‘‘standard commercial practice’’ (e.g., Liang et al. 1996; Swanepoel and Hobson 1999), making comparison among studies difﬁcult. While grades are useful to facilitate the ﬂow of information among different sectors, any classiﬁcation scheme is dependent on the ability to measure the attribute. Greater accuracy and hence ability to manage is achieved by replacing subjective assessment methods with those that are based on objective measures (Erickson 1994). NIR (near-infrared) technology has successfully been applied to the discrimination of nonreject kernels from kernels that were classiﬁed as immature, discolored, insect damaged, and moldy, but was not able to differentiate among other disorder classes (Guthrie et al. 2004) and was not tested against kernels with less severe forms of these disorders. Reﬁnements of instrumental methods eventually may provide an objective means for assessment of kernel visual imperfections. The lack of repeatable assessment methods means that only general observations of differences among cultivars developed by familiarity with the product have been reported. Kernels produced by M. tetraphylla genotypes reportedly tend to be darker with a grayish base compared with M. integrifolia, which tends to be white (Leverington 1958). Cultivars that have been noted as having discolored base include ‘Ikaika’ (Winks 1983) and ‘HAES 849’ (Stephenson and Gallagher 2000). As discussed, genetic variation in germinability under nursery conditions may indicate a genetic basis to the occurrence of visual germination disorders of the kernel (Hardner and McConchie 2006). Differences in the proportion of kernel that were shriveled among samples taken from seedling selections have been attributed to genetic variation (Leverington

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1962a); however, the timing of collection of these samples is unknown, as is the relative size of nongenetic effects for these attributes. Signiﬁcant differences among cultivars for gray kernel discoloration (linked to Enterobacter cloacae infection) have been reported (Nishijima et al. 2007) with ‘Keauhou’ having the lowest incidence of gray kernel discoloration compared to ‘Kau’ and ‘Kakea’ in nuts sampled from two Hawaiian orchards and inoculated in the laboratory. This, however, is in contrast to ﬁeld observations that gray discoloration occurs at a higher frequency in commercial kernels from ‘Keauhou’ (Nishijima et al. 2007). Further research is require to develop an understanding of the relationship between variability in biological characteristics that may affect susceptibility to infection, such as physical nut structure and phenology, and the inheritance of these characteristics. General terms have been used to describe raw kernel quality of individual cultivars. Stephenson and Gallagher (2000) describe ‘A4’ as attractive; ‘Daddow’, ‘A16’, and ‘HAES 814’ as good color; ‘Keaau’, ‘Mauka’, and ‘HAES 781’ as cream to beige in color; ‘Keauhou’, ‘HAES 842’, and ‘HAES 816’ as variable; ‘HAES 849’ as beige to light brown; and ‘Kau’ as darker than the other kernels. This is similar to descriptions by Winks (1983) for ‘Keaau’ (excellent) and ‘Daddow’ (excellent) and ‘Keauhou’ (good). Bell and Bell (1987) also describe the appearance of ‘A16’ and ‘A4’ as good along with that of ‘A268’, and they considered ‘A199’ excellent. However, it is difﬁcult to use these observations for selection, as they depend on the preferences of the observers, which may not be consistent across studies. Some studies report measures of percentage unsound kernel (e.g., Swanepoel and Hobson 1999; Stephenson 2000; Stephenson and Gallagher 2000). While this is an attempt to quantify the extent of kernel quality, it includes all forms of quality disorders. However, the heritability of an aggregated trait will be low, unless all traits are highly correlated genetically. A low heritability means apparent differences between candidates are due to nongenetic variation that genetic selection cannot exploit. Clearly more work is required to develop objective and repeatable methods to assess of attributes of the visual appearance of raw kernel and determine their genetic basis. 2. Oil Content and Percentage First-Grade Kernel. A relationship between the oil content (as assessed by speciﬁc gravity) of raw M. integrifolia kernels and the acceptability of oil-roasted kernels has been established (Ripperton et al. 1938; Mason and Wills 1983) and applied as a selection criterion in macadamia improvement (Hamilton and Ito 1984; Stephenson et al. 1999). Initial studies by Ripperton et al.

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(1938) demonstrated that raw M. integrifolia kernels less than 1.000 g/g speciﬁc gravity (SG) (estimated oil content of 72%) were light golden in color, with a mild nutty ﬂavor and crisp texture, and considered the most acceptable when (presumably oil) roasted. These were classiﬁed as ﬁrst-grade kernels. Kernels between 1.000 and 1.025 g/g SG (estimated oil content of 72%–68% oil) were described as having tendency to be somewhat dark in color, with off ﬂavors and a spongy texture, and were considered suitable only for confectionary or bakery products (secondgrade kernel). Raw kernels higher than 1.025 g/g SG were small in size, with a shriveled base and hard texture, and on roasting became very dark with an unpleasant burned ﬂavor. These were considered acceptable only for oil products (third grade). First-grade kernel is also referred to as No. 1 kernel (Ito and Hamilton 1980; Allan et al. 1999) or ﬂoaters (Ito et al. 1998). These relationships were conﬁrmed by a later study using a hedonic sensory panel with kernels taken from two ground harvests of ‘Keauhou’ throughout the Australian season (Mason and Wills 1983). However, a reanalysis of the data for kernel oil content by speciﬁc gravity presented in two studies indicates that the relationship between oil content and speciﬁc gravity is not consistent across the four different sets of kernels (M. integrifolia —Ripperton et al. 1938; M. tetraphylla—Ripperton et al. 1938; ‘Keauhou’ harvest 1— Mason and Wells 1983; ‘Keauhou’ harvest 2—Mason and Wells 1983). The intercept of the linear regression is signiﬁcant lower for the ‘Keauhou’—harvest 2 (256.8) compared to the M. tetraphylla data (304.1), and the ‘Keauhou’—harvest 1 (285.7) and the M. integrifolia data (285.6) are intermediate and not signiﬁcantly different from the other intercepts. The slope of the ‘Keauhou’—harvest 2 regression (182.3) is signiﬁcantly less negative than the other regressions (‘Keauhou’ harvest 1 ¼ 209:7; M. integrifolia ¼ 213.5; M. tetraphylla 231.3). The consequence of these results is that predicted oil content of kernels at SG¼1.000 differs signiﬁcantly among the different sets of kernel being 76.0% for the ‘Keauhou’—harvest 1 regression, 74.5% for the ‘Keauhou’—harvest 2 regression, 72.8% for the M. tetraphylla regression, and 72.1% for the M. integrifolia regression. This means, for example, that if the M. integrifolia regression is applied to the kernels from the ﬁrst harvest of ‘Keauhou’ in Australia, kernels with an actual oil content between 72 and 76% would be predicted to have an oil content below 72%. The most common method used to describe the level of ﬁrst-grade kernel for selection is the percentage of kernel that are above SG ¼ 1.000 (percentage of ﬁrst-grade kernel). It is usually determined as the percentage mass of kernels that ﬂoat in water (Ripperton et al. 1938;

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Cavaletto 1981; Mason and Wills 1983). In some studies (e.g., Swanepoel and Hobson 1999), all kernel is included in the sample for assessment of percentage ﬁrst-grade kernel, while in other studies (e.g., Leverington 1962a), spoiled kernel, such as insect damaged or moldy kernel, are removed prior to evaluation. Timing of sampling may also have important implications for estimation of percentage ﬁrst-grade kernel. For example, percentage of ﬁrst-grade kernel has been assessed in some cases using samples taken at peak fruit drop (e.g., Leverington 1962a; Stephenson et al. 1995). However, this may overestimate the percentage of ﬁrst-grade kernel, if the crop is collected over the entire fruit drop season and includes kernel near the start of the season, where oil content may be more variable (Ito and Hamilton 1983; Ironside 1987). A threshold of 95% percentage of ﬁrst-grade kernel is used as a standard for cultivar recommendation (Hamilton and Ito 1986; Ito 1995; Stephenson and Gallagher 2000). In addition, cultivars that have a stable production of ﬁrst-grade kernel across different environments are considered particularly valuable under the highly variable Australian growing conditions (Stephenson et al. 1995). This review highlights the uncertainties of using percentage of ﬁrstgrade kernel as a selection criterion. First, the relationships between kernel quality and oil content were established using oil roasting; however, the response of kernels under oil roasting may not be the same as under air roasting. Lighter air roasting can be used to manage some roasting disorders (Cull 1978; Mason 1987), particularly if the target consumers do not have a strong preference for darker-roasted kernels (e.g., O’Riordan et al. 2005). Second, the signiﬁcant variability in the relationship between speciﬁc gravity and oil content among kernel samples discussed suggests the percentage of ﬁrst-grade kernel may not be accurate at differentiating between the potential of genotypes to produce high-quality roasted product. Finally, a relationship between roasting response and oil content does not conﬁrm variability in oil content as the causal factor, as it may be a surrogate for another correlated compound that is directly involved in the roasting reactions. There is conﬂicting evidence for a difference in oil content between M. tetraphylla and M. integrifolia. No signiﬁcant difference in percentage of oil content of a range of kernels sampled from openpollinated progeny of the two species was found when determined directly through extraction (Saleeb et al. 1973), although the SG of the M. tetraphylla was lower, in agreement with the regression analysis presented earlier. In contrast, a lower oil content for M. tetraphylla is reported by Winterton (1968), although it is unknown if this was

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determined by applying the M. integrifolia regression to M. tetraphylla, which, as demonstrated, would predict a lower percentage of ﬁrstgrade kernel for M. tetraphylla. In addition, Leverington (1971) reports a larger variability in percentage of ﬁrst-grade kernel among M. tetraphylla genotypes, suggesting that the nuts had been collected prior to the completion of oil accumulation (Cameron McConchie pers. comm.). Studies of oil accumulation in macadamia (Jones 1937, 1939; Baigent 1983; McConchie et al. 1996; Trueman et al. 2000) have been undertaken using seed collected from M. integrifolia seedlings and cultivars, but little is known of the oil accumulation pattern in M. tetraphylla. Differences in oil proﬁle between the two species have also been reported with percentage of the unsaturated oleic (18:1) and eicosenoic (20:1) fatty acids signiﬁcantly higher in kernels from M. integrifolia, while levels of stearic (18:0) and arachidic (20:4) fatty acids were lower (Saleeb et al. 1973). Percentage of ﬁrst-grade kernel is under weak genetic control (H ¼ 0:2) compared to other nut and kernel traits in a study of 40 cultivars planted at four locations in Australia (Hardner et al. 2001). This is in agreement with the results from a REML analysis undertaken across a range of published cultivar values: Australia (Winks et al. 1987; Stephenson et al. 1999; Stephenson 2001), Hawaii (Ito and Hamilton 1983, 1989; Ito et al. 1983, 1998; Nagao et al. 2003), South Africa (Allan 1989; Swanepoel and Hobson 1999), Brazil (Sacramento et al. 1995) (H ¼ 0:16). The average ﬁrst-grade kernel across all studies was 92%. First-grade kernel differed signiﬁcantly among countries, studies, and locations. In contrast to the smaller Australian study, the REML analysis indicates the ranking of cultivars for ﬁrst-grade kernel is sensitive to environmental variation. A stability analysis with an extended data set of the Australian study (two additional sites, Stephenson et al. 1995) also suggested cultivars with low overall percentage of ﬁrst-grade kernel were more sensitive to environmental variation. Sensitivity of ﬁrst-grade kernel to environmental variation, particularly to temperature, water deﬁcit, and management practices, has been suggested in other studies (Radspinner 1970;. Stephenson and Gallagher 1986; Allan 1989; Supamatee et al. 1992; Stephenson and Trochoulias 1994; Stephenson et al. 2000; Stephenson 2003). The limited separation of cultivar means from the REML analysis (Table 1.6) is a consequence of the low heritability of the trait and the presence of a sizable G E component of variation. ‘A4’ is the highest ranked cultivar for percentage ﬁrst-grade kernel and is signiﬁcantly different from all named Hawaiian cultivars except ‘Makai’, which has been described as producing high-quality kernel (Hamilton and Ito 1984).

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‘Keauhou’ is the lowest-ranked cultivar for ﬁrst-grade kernel, in agreement with previous observations (Hamilton and Ito 1984), although it is only signiﬁcantly different from ‘Makai’ and ‘A4’. ‘‘Cultivar’’ means for percentage of ﬁrst-grade kernel were only weakly correlated with those for average nut mass (rg ¼ 0:40, P > 0.01), but the correlation with kernel recovery was not signiﬁcant (Hardner et al. 2001). These results are conﬁrmed by the REML analysis undertaken here (rcv ¼ 0:17). Recovery of ﬁrst-grade kernel, which is the ratio of ﬁrst-grade kernel mass to nut mass, is reported by some studies instead of percentage of ﬁrst-grade kernel (Supamatee et al. 1992; Ito and Iyo 1992; Ito 1995). A cross-study REML analysis undertaken of these values, and values calculated from the previous studies that report both kernel recovery and percentage of ﬁrst-grade kernel, indicates that the genetic control of this trait was intermediate to these two traits (H ¼ 0:36). The presence of interactions of cultivar with country and cultivar with location in this analysis agrees with previous reports of G E for this trait (Ito 1995). It appears that variation in kernel recovery is the main driver of differences in ﬁrst-grade kernel recovery among cultivars as the correlation between these two traits is close to unity (rcv ¼ 0:98); the correlation with percentage of ﬁrst-grade kernel and ﬁrst-grade kernel recovery is lower (rcv ¼ 0:48). 3. Kernel Size. Kernel size is a commonly reported character of cultivars, but the importance of its role in selection is unclear. Sorting costs may be greater with smaller kernel (Leverington 1962a; Winterton 1968; Hardner et al. 2006), and small kernels may be more susceptible to cracker damage (Leverington 1962a, 1971) and overroasting (Storey and Kemper 1960). Kernels less than 1.5 g are considered too small for processing (Supamatee et al. 1992). It has been suggested, however, that large kernels may be prone to underroasting due to incomplete heat penetration to the center (Leverington 1962a; Winterton 1968). Data are not provided to support this hypothesis, and it may be possible to avoid underroasting through modiﬁcation of the roasting process. It has also been suggested kernels greater than 3.5 g are too large for packaging in cans and bottles (Supamatee et al. 1992). Kernel size in part deﬁnes different raw kernel styles, which differ in value (Hardner et al. 2006) and may be important for marketing; the suggestion is that a few large kernels in a packet are less attractive than a large number of smaller kernels (Leverington 1962a, 1971). Large kernels were favored when consumers were surveyed for their preferences for individual kernels (O’Riordan et al. 2005), but this may not be the same as size preferences when a given mass of kernel is examined (Cameron

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McConchie pers. comm.). The ideal size of kernels for commercial use is reportedly 18 to 22 mm in diameter and 2 to 3 g in mass (Leverington 1962a, 1971; Supamatee et al. 1992; Ito 1995). Saleeb et al. (1973) found no signiﬁcance difference in size of kernels collected from M. tetraphylla and M. integrifolia cultivars. However, this result may not represent the natural variability between the two species, as kernel size is highly heritable and was probably included in the selection history for these cultivars. Average kernel mass is commonly used to describe kernel size. Mass is easier to measure than kernel size, and there is a strong phenotypic correlation between these two traits (Beaumont 1937). Average kernel mass usually is measured by weighing a sample of kernel and dividing this mass by the number of kernels present in the sample. However, as a signiﬁcant relationship between kernel size and oil content has been established (Mason and Wills 1983), average kernel mass may be biased downward if immature kernels are present in the sample. Average kernel size has been reported in all the studies listed earlier for kernel and ﬁrst-grade kernel recovery (except Ito et al. 1983). Again, the results of a REML analysis of the data in these studies are consistent with other studies that report high heritability (H ¼ 0:6), limited G E (Hardner et al. 2001), and moderate correlation with kernel recovery (rcv ¼ 0:48 in Hardner et al. 2001; rcv ¼ 0:67 across the 17 studies included in the REML analysis) (Table 1.6). The low G E found in these analyses contradicts suggestions by others (Ito 1995) that cultivars should be selected for speciﬁc sites with respect to kernel size. The named Hawaiian cultivars tend to have smaller kernels. There is no signiﬁcant difference among these cultivars except that ‘Keaau’ kernels are on average smaller than ‘Purvis’ (Table 1.6). The cultivars ‘A4’, ‘HAES 816’, Own Venture’, and ‘A16’ have signiﬁcantly larger kernels on average than all the named Hawaiian cultivars except ‘Purvis’ and ‘Makai’. 4. Percentage of Whole Kernels. At cracking, and possibly after, some macadamia kernels split along the line that separates the two cotyledons, producing half kernels. The percentage of whole kernels can inﬂuence kernel value as this trait partially deﬁnes product styles that vary in price (Wallace et al. 2001; Walton and Wallace 2005; Hardner et al. 2006). In addition, particular market segments may prefer whole kernels (Hardner et al. 2001). In contrast, some earlier authors did not consider the production of halves to be a disadvantage (Leverington 1971).

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Percentage of whole kernels is generally assessed by measuring the mass of kernels in a sample that are whole after cracking (Stephenson 2000). A moderate heritability for this trait has been reported (H ¼ 0:3, Hardner et al. 2001), while a reduced REML analysis of the limited number of studies that report percentage whole kernel (Barbosa et al. 1991; Swanepoel and Hobson 1999; Stephenson et al. 1999; Stephenson 2001; Nagao et al. 2003; Walton and Wallace 2005) indicates a strong genetic control for this trait (H ¼ 0:8). Differences in cuticular structure at the break zone between the two cotyledons were observed between ‘HAES 835’ and ‘Mauka’ and related to differences in percentage of whole kernel (Walton and Wallace 2005). Narrow cuticles, denser and more numerous electron-dense objects (possible storage protein bodies), and less cuticle convolutions were associated with a higher percentage of whole kernels. Further work is required to conﬁrm this association over more genotypes. The percentage of whole kernel may be affected by the use of different crackers (Rodrigues et al. 1998; and to some extent Wallace et al. 2001); however, little is known about the interaction between cultivar and cracker. Small differences in percentage of whole kernels among crackers were reported for a sample of ‘A38’ nuts, but there were no differences in a sample of ‘Keauhou’, and differences between cultivars was much larger than differences between crackers (Wallace et al. 2001). While it is suggested that genetic variation for nut size may result in genetic variation for percentage of whole kernels, as differences in nut size may affect cracker efﬁciency (Liang 1980; Tang et al. 1982), there is no genetic correlation between these two traits (Hardner et al. 2001). Cultivar means for percentage of whole kernels are not correlated with kernel recovery (rcv ¼ 0:1), percentage of ﬁrst-grade kernel (rcv ¼ 0:1), or average kernel mass (rcv ¼ 0:3) (Table 1.6), consistent with previous studies (Hardner et al. 2002). Across a range of studies, ‘HAES 835’, ‘NG18’, ‘HAES 816’, ‘HAES 849’, ‘Purvis’, ‘A16’, Own Venture’, and ‘Makai’ produced signiﬁcantly more wholes than ‘Keauhou’, ‘HAES 814’, ‘HAES 856’, and ‘Beaumont’ (Table 1.6). 5. Bitter Kernels. Rarely, seedlings arise that produce bitter kernels due to elevated levels of cyanogenic glucosides (Dedolph and Hamilton 1959; Young and Hamilton 1966), which also occur in M. ternifolia (Dahler et al. 1995). Production of bitter kernels in grafted scions taken from seedlings known to produce bitter kernels conﬁrms the genetic control of this attribute (Young and Hamilton 1966). Interspeciﬁc controlled crossings have indicated that the gene action is recessive (Hardner et al. 2000).

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6. Quality Attributes of Roasted Kernel. Roasting improves the odor, appearance, texture, and ﬂavor of macadamias (O’Riordan et al. 2005). Roasting can be undertaken either by immersion in oil (Moltzau and Ripperton 1939; Mason et al. 1995) or using dry air (Winterton 1962; Wesley et al. 2007), although there is little information relating the effect of different treatments under these two methods. It has been suggested that optimum kernel quality is achieved by roasting in oil at 127 C for 15 minutes; lighter roasting produces less desirable kernel (Dela Cruz et al. 1966). Direct testing of Australian consumers found no signiﬁcant difference in overall liking among four air-roasting treatments (135 C, 12 min; 135 C, 18 min; 155 C, 5 min; 155 C, 8 min). However, consumers preferred the appearance of lighter-roasted kernels over that for medium-roasted kernels, but the odor and ﬂavour of mediumroasted kernels (O’riordan et al. 2005). Overall kernel color is the most common attribute used to describe the quality of roasted macadamia kernel. In addition, defects such as localized or an extreme darkening of the kernel may become apparent after roasting, commonly referred to as after-roasting darkening (ARD) (Cavaletto 1980; Albertson et al. 2006). Internal browning of kernels also can occur following roasting, if high initial temperatures are used to dry nuts that have a high moisture content, although this defect also may occur simply following particular unfavorable drying conditions (Prichavudhi and Yamamoto 1965). Kernel color usually is assessed as time to reach a desired level of color as judged by an operator (Isaacs et al. 1998) or the color after a deﬁned roasting treatment assessed using color cards, ﬂatbed scanners, or a Minolta color meter (Lemmer et al. 1998; Albertson et al. 2005; McConchie et al. 2007a; Wall and Gentry 2007). Roasted kernels may be allocated to different products, with light-colored kernels used for snack food, darker kernels tending to be used in confectionary and chopped nut products, and very dark kernels rejected (Leverington 1971), although this may depend on market preferences. A number of authors report observations of a difference in quality of roasted kernel between the two species (Moltzau and Ripperton 1939; Leverington 1958, 1962a, 1971; Cavaletto 1980, 1983). Raw kernels of M. integrifolia are described as being light in color that changes to golden brown on roasting, while the roasted color of M. tetraphylla is considered more variable with kernels browning faster on roasting. Roasted M. tetraphylla kernels are also reportedly ﬁrmer and harder in texture, with a sweeter but variable ﬂavor, in contrast to the crisp and delicate texture and mild and uniform ﬂavor reported for roasted M. integrifolia kernels

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(Leverington 1971). These observations have been used to recommend that the two species should be separated for processing (Winterton 1968; Leverington 1971; Cavaletto 1983) and that M. tetraphylla kernels should be oil-roasted at a lower temperature to avoid charring (Moltzau and Ripperton 1939; Leverington 1971). The development of the industry has concentrated on M. integrifolia germplasm, partly on the basis of these results, although a preference for M. tetraphylla kernels has been reported (Ripperton et al. 1938; Leverington 1963; Gathungu and Likimani 1975). The difference in roasting performance between the two species has been attributed to a higher sugar content of M. tetraphylla (6–8%) compared to M. integrifolia (4%) (Winterton 1968; Cavaletto 1980, 1983) Amino acids have been implicated in the roasting process in macadamia (Albertson et al. 2006), and a signiﬁcantly higher absolute content for M. integrifolia has been reported, although there is virtually no difference in amino acid proﬁle (Saleeb et al. 1973). A recent study, however, has suggested that more detailed reconsideration of the M. tetraphylla germplasm is warranted (McConchie et al. 2007c). Although these authors found signiﬁcant differences for change in color with roasting among three M. integrifolia cultivars, a hybrid cultivar, and ﬁve accessions M. tetraphylla from the wild, differences could not be grouped on the basis of species status, except at extreme roast conditions that would not be commercially acceptable. In addition, the authors report no signiﬁcant difference in sucrose content between the different germplasm types and very low overall levels of reducing sugars. A more thorough analysis using germplasm sampled from the wild is warranted to fully characterize species differences. Several other studies report differences among cultivars in the appearance of roasted kernels (Isaacs et al. 1998; Lemmer et al. 1998; McDonagh 2003; McConchie et al. 2007d; Wall and Gentry 2007) and further demonstrate the difﬁculty of determining roasting quality from species status. A generalized linear model analysis was undertaken of the means presented in Isaacs et al. (1998) for percentage of roasting rejects and roasting time for eight cultivars (‘A16’, ‘A4’, ‘Mauka’, ‘Makai’, ‘Hinde’, ‘Heilscher’, ‘Keauhou’, and ‘Kau’) stored under ﬁve conditions and oil roasted to a standard color. Percentage roasting rejects were signiﬁcantly greater for ‘A16’ (7.3%—hybrid) and ‘Keauhou’ (5.9%—M. integrifolia) compared to ‘A4’ (3.5%—hybrid) and ‘Heilscher’ (0.6%—M. integrifolia), with ‘Mauka’ (5.6%— M. integrifolia), ‘Makai’ (4.7%—M. integrifolia), ‘Hinde’ (4.5%— M. integrifolia), and ‘Kau’ (4.1%—M. integrifolia) intermediate. However, results for roasting time from this study appear to be biased

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as there is a signiﬁcant correlation with appearance (as judged by a hedonic sensory panel) and roasting, which would be not expected if the treatment of roasting to a standard color was applied without bias. The correlation among cultivar means for roasting rejects and appearance preference was not signiﬁcant. The sensitivity of ‘Keauhou’ to roasting has been noted in other studies. Using a color chart to assess differences in color, the cultivars ‘Keauhou’, ‘Pahala’, and ‘Kakea’ were reported as being more variable in color and darker than ‘Mauka’ and ‘Fuji’ when oil or dry roasted to a common time (Lemmer et al. 1998). The visual response of cultivars considered hybrids (‘Nelmak 1’, ‘Nelmak 2’, ‘Nelmak 26’, and ‘Beaumont’) was similar to that of ‘Keauhou’, and ‘Kakea’. In a separate study (McDonagh 2003), ‘Keauhou’ was consistently darker (as assessed using color density calculated from a scanned image) than ‘A38’ and ‘A16’ when roasted under a range of times and temperatures. This is supported in a more recent and comprehensive study (McConchie 2006b) that found roasted ‘Kau’ and ‘Keauhou’ kernels were signiﬁcantly darker (as assessed using color meter) than kernels from ‘HEAS 849’ and ‘A16’. This study also found signiﬁcant differences among cultivars for preroast (raw) color and the change in color with roasting, although it is difﬁcult to determine if this is correlated with preroast color. Further work is required to determine if post-roast color can be managed through sorting based on raw kernel color. Signiﬁcant cultivar differences in darkening after extreme roasting conditions have been reported (Albertson et al. 2005), with ‘Own Venture’ exhibiting less extreme reaction than the other cultivars examined (‘A16’ and ‘HAES 814’). No signiﬁcant differences in reducing sugar content and internal color of kernels after were found among ‘Kakea’, ‘Keauhou’, ‘Kau’, and ‘Keaau’ (Wall and Gentry 2007). There is conﬂicting evidence on the signiﬁcance of genetic variation for other sensory attributes in macadamia. Although a thorough descriptive sensory analysis of macadamia using a trained sensory panel found signiﬁcant differences in odor, ﬂavor, aftertaste, and texture of roasted kernels between air roasting and aging treatments, no signiﬁcant effect of source, which encompassed a range of cultivar, geographic, and management variability in Australia, was found (O’Riordan et al. 2005). This supports suggestions that only minor ﬂavor differences among cultivars exist (Cavaletto 1983). Signiﬁcant differences in texture preference were also not found among eight cultivars assessed by a hedonic sensory panel (Isaacs et al. 1998), following an analysis of the published means. In contrast, a REML analysis of means for texture

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preference assessed by a hedonic panel presented in Gallagher et al. (1998) for a larger study of kernels from 18 cultivars stored at different conditions both prior to and post roasting (assumed to oil roasting to speciﬁc color), indicated signiﬁcant differences among cultivars. Signiﬁcant differences among cultivars for ﬂavor preference were also apparent following a REML analysis of means presented in both studies (Gallagher et al. 1998; Isaacs et al. 1998). The analysis of the larger study data also indicated that raw cultivar means for texture and ﬂavor preferences were signiﬁcantly correlated with preferences for roasted kernels (texture: rcv ¼ 0:85; ﬂavor: rcv ¼ 0:87). The texture of ‘HAES 816’, ‘HAES 849’, and ‘Own Venture’ were signiﬁcantly preferred in the larger study over kernels from ‘Keauhou’, ‘A4’, ‘HAES 842’, ‘Daddow’, ‘HEAS 814’, ‘A16’, ‘Kau’, ‘Mauka’, and ‘Keaau’, which produced the least preferred kernel, although there were no signiﬁcant differences in texture among eight cultivars (which included ‘A4’, ‘A16’, ‘Kau’ ‘Keauhou’, and Mauka’) in the smaller roasting study (Isaacs et al. 1998). Flavor of roasted kernels from ‘Mauka’, ‘Keaau’, ‘HAES 849’, ‘HAES 816’, ‘HAES 781’, ‘Keauhou’, and ‘NG13’ were signiﬁcantly preferred over the ﬂavor of ‘NG18’, ‘HAES 842’, ‘Daddow’, ‘A16’, and ‘A4’ in Gallagher et al. (1998). The ranking of ‘Keauhou’, ‘Kau’, ‘A16’, and ‘A4’ was similar in the smaller study (Isaacs et al. 1998), although the preference for ‘Mauka’ was inconsistent, as it was the least favored in the smaller study. While not tested in the larger study, kernel from ‘Makai’ was the most preferred for ﬂavor in Isaacs et al. (1998). There was no correlation among cultivar means for texture and ﬂavor preferences in Gallagher et al. (1998) (rcv ¼ 0:2). However, it is difﬁcult to compare results from across hedonic studies and relate these to consumer preferences (Mialon and Murray 2001). Attempts have been made to use a single measure to describe differences in kernel quality among cultivars (Hamilton and Ito 1984; Gallagher et al. 1998; Nagao et al. 2003). There were signiﬁcant differences among cultivars in overall quality preference from a hedonic sensory assessment when cultivar means presented in Gallagher et al. (1998) were analyzed using the REML approach. Cultivar means were not correlated with preferences for ﬂavor preferences (rcv ¼ 0:34), and the correlation with texture preferences was only slightly signiﬁcant (rcv ¼ 0:50). Preference for the quality of ‘HAES 849’ and ‘HAES 816’ kernels was signiﬁcantly higher than for ‘Kau’, ‘A16’, ‘Keaau’, and ‘A4’. However, these measures suffer from the deﬁciencies of the hedonic sensory approach discussed earlier in that they may not predict well the preferences of target markets or consumers in general.

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The quality of roasted kernel have been described using general terms from ‘‘fair’’ to ‘‘excellent’’ (Hamilton and Fukunaga 1970, 1973; Hamilton and Ito 1976, 1986; Hamilton 1984) or using kernel rating system (1 ¼ fair to 4 ¼ excellent) (Hamilton and et al. 1981; Hamilton and Ito 1986; Nagao et al. 2003). The quality of ‘Pahala’ is described as 3.6 or ‘‘excellent’’ in comparison with ‘Purvis’ (3.4) and ‘Keauhou’ (2.9) (Hamilton et al. 1981). In a separate study (Nagao et al. 2003), the kernel quality rating of ‘Makai’ was also high (3.5 compared to 3.3 for ‘Pahala’). ‘HAES 849’ had the lowest quality of cooked kernels (3.1), but this does include ‘Keauhou’. The cooked quality of ‘Makai’ has also been described as ‘‘excellent’’ in other studies (Hamilton and Fukunaga 1973; Hamilton and Ito 1984, 1986). Other cultivars considered to produce kernels with ‘‘excellent’’ cooked quality include ‘Keaau’, ‘Mauka’, ‘Dennison’ (Hamilton and Fukunaga 1970, 1973; Hamilton and Ito 1976, 1984, 1986). The kernel quality of ‘Kau’ was considered ‘‘excellent’’ by some (Hamilton and Fukunaga 1973; Hamilton and Ito 1976) but dropped to ‘‘very good’’ in Hamilton and Ito (1984 and 1986). ‘Purvis’ was also considered ‘‘very good’’ by Hamilton (1984) but ‘‘excellent’’ two years later by Hamilton and Ito (1986). The reported quality of ‘Kakea’ was variable from ‘‘fair’’ in Hamilton and Fukunaga (1970), to ‘‘good’’ (Hamilton and Fukunaga 1973), ‘‘very good’’ (Hamilton and Ito 1976), and ‘‘excellent’’ (Hamilton and Ito 1984, 1986). Cooked kernel from ‘Ikaika’ was consistently considered ‘‘fair.’’ Again, these terms are subjective and difﬁcult to compare across studies and use for selection, particularly if trade-offs are required among several traits. 7. Shelf Life. The quality of roasted kernels may be compromised by the development of unpleasant ﬂavors due to changes in chemical composition of the kernel with age (rancidity) (Himsteadt 2002; O’Riordan et al. 2005), despite the fact that the extracted oil of macadamia being highly resistant to rancidity (Saleeb et al. 1973). The storage life of roasted M. integrifolia kernels is considered to be longer than for M. tetraphylla (Leverington 1958, 1962a). It is suggested that the poorer shelf life of M. tetraphylla kernels is a consequence of undercooking, as kernels from this species may be roasted using lighter conditions in an attempt to manage their perceived sensitivity to roasting. However, this hypothesis has not been conﬁrmed in later trials (Mason et al. 1995). Free-fatty acids and peroxide values have been used as measures of the level of rancidity in macadamia kernels, although these measures may not correlate well with sensory perceptions of rancidity or

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staleness (Frankel 1998; Himsteadt 2002; Mason et al. 2004). Maximum values in industry guidelines are 0.5% and 3 to 5 meq/kg respectively (Mason et al. 2004). Season, kernel size, and processing method were found to have greater inﬂuences on peroxide values compared to the species type of a cultivar (pure M. integrifolia versus hybrid) (Luttig and Kruger 1999). In contrast, the effect of cultivar was highly signiﬁcant effect (free-fatty acid PrðFÞ < 0:001; peroxide values PrðFÞ ¼ 0:009) when the means of the storage-roasting trial of eight cultivars (Isaacs et al. 1998) were reanalyzed. However, the lack of signiﬁcant interaction with cultivar and storage treatment suggests that differences among the cultivar samples prior to storage were maintained throughout the trial. Although scant, published evidence does not support a genetic basis in macadamia of susceptibility to rancidity. No signiﬁcant effect of source (representing different cultivars from different farms), or a interaction between source and aging treatment, was detected on sensory perception of rancidity (O’Riordan et al. 2005). In addition, no signiﬁcant difference was detected among the ﬂavor preference of three cultivars (‘Ikaika’, ‘Keauhou’, and ‘Kakea’) following storage (Dela Cruz et al. 1966). Further, the REML analysis of the hedonic studies already outlined (Gallagher et al. 1998; Isaacs et al. 1998) found no signiﬁcant interaction between cultivar and storage treatment for hedonic preference, indicating that all cultivars respond the same way to aging. It has been suggested that genetic variability in the proﬁle of antioxidants could be used to select for cultivars less susceptible to ﬂavor deterioration with aging (Mason 2000). However, levels of antioxidants are low in macadamia and probably not effective for the stability of kernel ﬂavor (Cavaletto 1980; Rosenthal et al. 1984; Kaijser et al. 2000; Himsteadt 2002; Wu et al. 2004). L. Performance in Extreme Environments There has been interest in development of cultivars that perform well in cold environments (Xiao et al. 2002b). Many consider M. tetraphylla germplasm better suited to cooler environments (Cavaletto 1983; McCubbin and Lee 1996; Wiid and Hobson 1996; Allan et al. 1999; Xiao et al. 2002a). ‘Beaumont’, ‘Own Choice’, and ‘Hinde’ reportedly perform well in cooler environments of inland China (Xiao et al. 2002a, 2002b; Zheng and Zhang 2002), New Zealand (Gordon 1987; Richardson and Dawson 1991; Warren 2003), and South Africa (Allan 1993). In a trial of 10 cultivars across a range of environments in Thailand from latitudes 7.5 to 19.8 N, altitude 100 to 1300 m, average annual rainfall from 1,050

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to 3,200 mm, and maximum temperature from 23 to 33 C, ‘Kakea’ was identiﬁed as the most susceptible cultivar to high temperatures (Supamatee et al. 1992). There are a range of physiological processes affected by high and low temperatures and interactions with other environmental variables (Stephenson and Trochoulias 1994; Huett 2003). Further work is required to develop a quantitative understanding of how these processes impact productivity and nut and kernel characteristics, so that they can be manipulated through breeding and selection.

V. PROPAGATION AND ROOTSTOCK TRAITS Elite cultivars of macadamia are commonly propagated by grafting onto seedling rootstocks, and less commonly using clonal rootstocks or own rooted cuttings (Stephenson 1990a; Nagao and Hirae 1992; Trochoulias 1992; Bell 1996; Hardner and McConchie 2006). Clonal propagation of rootstock provides greater control of genetic variation and can lead to more uniform orchards (Howard 1987). A. Germination and Seedling Growth Horticultural experience with macadamia is that germination of nuts is usually spread over several months with germination occurring from four weeks (Storey and Kemper 1960) to ﬁve (Wills 1939; Hamilton 1957) or eight months (Ojima et al. 1976) after sowing. Genetic variation in germinability (percentage germination) and rate of germination has been reported (Hamilton 1957; Ojima et al. 1976; Kadman and Joffe 1981; Hardner 2004; Hardner and McConchie 2006). In a nursery study on rootstock propagation of 15 genotypes (Hardner 2004; Hardner and McConchie 2006), germinability after six months was highest for ‘HAES 849’, ‘D4’ (also known as ‘Renown’), ‘Mauka’, and ‘Beaumont’ and lowest for ‘A268’, ‘A38’, and ‘Keauhou’. High germinability of ‘Beaumont’ nuts has also been reported by others (Allan 1989). Germinability for ‘Hinde’ (currently the favored seedling rootstock in Australia), ‘Kau’, ‘HAES 781’, ‘HAES 814’, ‘HAES 816’, ‘HAES 842’, ‘A16’, and ‘NG8’ was intermediate (Hardner 2004; Hardner and McConchie 2006). No general difference between M. integrifolia and hybrid cultivars was observed. It has been suggested that nuts from M. tetraphylla germinate faster than those from M. integrifolia (Phiri 1985; Nagao and Hirae 1992) or that thin-shelled nuts germinate faster (Wills 1939; Leverington 1962a; Nagao et al. 2003). It is possible that alternative nursery condition may produce different results.

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M. tetraphylla seedlings reportedly grow faster and are more uniform (Phiri 1985; Hamilton 1988; Nagao and Hirae 1992; Trochoulias 1992), enabling grafting to occur six months earlier than expected with M. integrifolia rootstocks (Hamilton 1988). Signiﬁcant genetic variation in nursery growth rate among seedlings from 15 cultivars has been reported (Hardner 2004; Hardner and McConchie 2006). One year after potting up, seedlings from ‘Beaumont’ were the most vigorous. The least-vigorous seedlings were progeny from ‘HAES 849’, ‘Keauhou’, ‘HAES 842’, ‘HAES 781’, and ‘Kau’, with the height of ‘HAES 814’, ‘NG8’, ‘HAES 816’, Mauka’, ‘A38’, ‘A16’, ‘Hinde’, ‘A268’, and ‘Renown’ intermediate. Growth of progeny was not correlated with the germinability of the nuts of the cultivar. B. Rooting and Growth of Cuttings Several studies report signiﬁcant differences in rooting success among cuttings collected from different cultivars (Cormack and Bate 1977b; Hardner 2004; Hardner and McConchie 2006). Cuttings taken from ‘Beaumont’ consistently demonstrate high rooting success (Cormack and Bate 1977b; Cruz-Castillo et al. 2000; Hardner 2004; Hardner and McConchie 2006). In a survey of 12 cultivars propagated as cuttings (Hardner 2004; Hardner and McConchie 2006), strike was superior for ‘Beaumont’ (80%), ‘A268’ (76%) and ‘NG8’ (70%), and ‘HAES 814’ (68%). Rooting success of cuttings from ‘Ikaika’ was also comparable to ‘Beaumont’ (Cormack and Bate 1977b). These authors also considered ‘Keauhou’ and ‘Elimbah’ moderately easy to root (Cormack and Bate 1977b). This is in agreement in part with Hardner and McConchie (2006), who report rooting success for ‘Keauhou’ (59%) to be similar to ‘Mauka’ (61%), ‘A16’ (61%), ‘Kau’ (54%), and ‘HAES 781’ (55%). These studies also identiﬁed ‘Kakea’ and ‘Keaau’ (Cormack and Bate 1977b) and ‘HAES 842’ (40%), ‘HAES 816’ (34%), and ‘HAES 849’ (23%) (Hardner and McConchie 2006) as recalcitrant germplasm. The relationship between rooting response of cultivars and stem carbohydrate levels of the mother plant is variable (Cormack and Bate 1977b), and no correlation has been demonstrated between the average strike success of cuttings from a cultivar and the germinability of seeds (Hardner and McConchie 2006). In general, it is difﬁcult to ﬁnd support in these results for the hypothesis that Hawaiian-derived cultivars are more difﬁcult to root than Australian selections, as suggested by others (Bell 1996). Although some work has been undertaken to develop tissue culture methods for clonal propagation macadamia (Mulwa and Bhalla 2000,

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2007), there is no information on genetic differences in tissue propagation success. These authors report identical correspondence between the marker proﬁle of stock plants and tissue-cultured plantlets, and compare this with RAPD polymorphisms detected in similar studies in other species, to suggest that clonal identify is maintained with propagation using auxiliary bud proliferation from single nodes. However, while promising, these results do not suggest that other nonsampled loci are unaffected. Variation in nursery growth due to genetic differences has been reported in several studies (Cormack and Bate 1977a; Hardner 2004; Hardner and McConchie 2006). These studies indicate that cuttings from cultivars that have a high strike success also tend be vigorous in the nursery (rcv ¼ 0:6, Hardner and McConchie 2006), and less vigorous cuttings tend to be have a lower root mass and are more variable in vigor (Cormack and Bate 1977a). C. Graft Compatibility Grafting of rootstock and scions of M. tetraphylla, M. ternifolia and M. integrifolia has been reported to be successful in any combination (Storey and Frolich 1964). Signiﬁcant genetic variation for budding success of scion and rootstock has been reported across a range of genotypes (Hardner 2004; Hardner and McConchie 2006). The effect of scion genotype was larger than the effect of rootstock genotype; however, this is likely to be confounded with nongenetic effects as generally all scions from the same cultivar were budded on the same day in this study. Budding success was superior for ‘A268’ (51%) ‘NG8’ (29%), and ‘HAES 814’ (23%) compared to ‘Kau’, ‘Mauka’, ‘HAES 816’ (all 6%), and ‘HAES 842’ (2%). No effect of rootstock type (clonal or seedling) on scion budding success was found. Rootstocks with low (< 10%) average take across several scions were ‘Mauka’, ‘HAES 842’, and ‘A16’, compared to ‘Beaumont’ (34%), which was the superior rootstock for budding success. Although this study also reports no effect of rootstock vigor on budding success, some selection for this trait was undertaken prior to propagation. D. Rootstock Effects on Scion Performance Despite the impact of rootstocks in other crops, particularly in apple (Rom and Carlson 1987), there is little quantitative evidence of strong rootstock effects in macadamia. Reviews of industry publications (Phiri 1985; Nagao and Hirae 1992) suggest M. tetraphylla rootstocks are

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less susceptible to disease and have a better root system compared to M. integrifolia rootstocks; however, there are little data to support these hypotheses, and certainly rigorous ﬁeld experiments with genetic material representative of the two species are lacking. Scions on M. tetraphylla stocks reportedly produce higher yields (Hamilton 1988; Nagao and Hirae 1992), although no signiﬁcant difference in yield was observed in a ﬁeld trial of ﬁve Hawaiian M. integrifolia cultivars propagated as cuttings (own-roots) or on M. tetraphylla seedling rootstocks (Phiri 1985). Overgrowth of M. integrifolia scions on M. tetraphylla rootstocks, or ‘‘later-age incompatibility,’’ has been observed (Hamilton 1988). Cracks in the trunk at the graft union may also be present and provide an entry point for disease (Hamilton 1988). However, there are no data available on the extent of this syndrome or the effect on production or other traits (Hamilton 1988). It has been suggested that rootstock genotype may affect nutrient accumulation, and variability in macadamia orchards has been attributed to genetic variation among seedling rootstocks (Nagao and Hirae 1992). Again, there are little data available to enable these hypotheses to be examined. In a limited ﬁeld trial with two macadamia cultivars (Trochoulias 1992a), differences in yield between rootstock genotypes propagated as seedlings or cuttings were not consistent across years, and no differences in kernel traits were observed. No signiﬁcant effect of rootstock on early ﬁeld growth height (at two years after planting) was found in a trial of 12 cultivars propagated as own-rooted cuttings or grafted onto clonal and seedling rootstocks of the same 12 cultivars (plus three additional seedling rootstock cultivars), although signiﬁcant scion effects were detected (Hardner and McConchie 2006). Further quantitative information is required on the effects of rootstock on production, nut characteristics, and kernel quality attributes (Hamilton 1988; Hardner and McConchie 2006).

VI. CULTIVAR UTILIZATION Cultivar utilization must consider a range of important criteria (Hamilton and Fukunaga 1959; Hardner and McConchie 1999; Stephenson and Gallagher 2000). However, as discussed, much of this information for the various selection criteria is descriptive, making comparison among cultivars, and hence accurate selection, difﬁcult. This uncertainty in the performance of cultivars is likely to be a major issue limiting the potential of macadamia production.

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A. Scion Cultivars 1. Hawaii. Cultivar recommendations in Hawaii were developed using a culling approach to selection and the standard described above for yield, tree structure, number of nuts per cluster, nut size, kernel recovery, percentage of ﬁrst-grade kernel, and kernel size (Hamilton and Fukunaga 1973; Hamilton and Ito 1976, 1984, 1986). However, there is little detail on rationale for the recommendation of speciﬁc cultivars. Following the release of the ﬁrst ﬁve cultivars from the Hawaiian selection program in 1948, three of the ﬁve recommended cultivars in 1948 (‘Pahau’, ‘Nuuanu’, and ‘Kohala’) were no longer on the recommended list by 1953 (Wagner-Wright 1995). There is no record of the reasons for the rejection of these cultivars. ‘Kakea’ is considered to be reasonably hardy and consistent with upright and rounded (but not spreading) canopy, producing exceptional yields and kernels of high quality, but can produce stick-tights and has a long harvest period (Hamilton and Fukunaga 1970; Hamilton and Ito 1976, 1984). Recommended cultivars in 1956 were ‘Keauhou’, ‘Wailua’, ‘Kakea’, and ‘Ikaika’ (Hamilton and Storey 1956), although ‘Wailua’ (released in 1952) was dropped by 1959 (Hamilton and Fukunaga 1959), again for unknown reasons. ‘Ikaika’ is described as hardy and precocious, but later age yields tend not to be as great as other cultivars (Hamilton and Ito 1984). By 1970, ‘Keaau’ had been added to the list of standard cultivars for Hawaii, which also included ‘Keauhou’, ‘Kakea’, and ‘Ikaika’ (Hamilton and Fukunaga 1970). ‘Keaau’ is described as being favored for an upright growth habit, outstanding nut and kernel characteristics, and short harvest period, but has a problem with germination of nuts in wet conditions (Hamilton and Ito 1984). After the release of new cultivars in the 1970s, ‘Keauhou’ was dropped from recommended cultivars in Hawaii because of variable kernel quality (Hamilton and Ito 1984; Nagao and Hirae 1992), presumably percentage of ﬁrst-grade kernel. Certainly, as discussed, ‘Keauhou’ has a lower percentage of ﬁrst-grade kernels and may produce a high frequency of roast rejects under certain roast conditions, but the ﬂavor and texture of the kernel is similar to that of other Hawaiian cultivars. ‘Keauhou’ may require different processing conditions from some other common cultivars, and this may be unsuitable for commercial operations. The cultivar is considered to produce good yields but has a broadly spreading tree structure and is susceptible to wind damage (Hamilton and Ito 1984).

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By 1984, ‘Ikaika’ had also been dropped from the list of recommended cultivars, which at this stage included ‘Purvis’, ‘Kau’, ‘Kakea’, ‘Keaau’, ‘Mauka’, ‘Pahala’, and ‘Makai’ (Hamilton and Ito 1984). ‘Purvis’ is described as good cropping, with high percentage of ﬁrstgrade kernel and kernels of exceptionally good quality and ﬂavor (Hamilton and Ito 1984). ‘Kau’ is considered more upright, hardier, and more wind resistant than ‘Keauhou’, but with better kernel quality (Hamilton and Ito 1984; Stephenson 1990a). ‘Mauka’ is regarded as hardy, with upright growth and higher kernel recovery and percentage of ﬁrst-grade kernel compared to ‘Kau’ (Hamilton and Ito 1984). ‘Pahala’ is also considered to be narrow and upright, with high kernel recovery and good kernel quality (Hamilton and Ito 1984). ‘Makai’ reportedly resembles ‘Keauhou’ in tree form, yield, and nut characteristics but is considered to produce kernels of outstanding quality. Of the newer selections, ‘HAES 816’ was rejected in Hawaii due to high incidence of stick-tights and ‘HAES 849’ due to thinner shells and low yields (Nagao et al. 2003). Cultivar recommendation in Hawaii also considered site suitability (Nagao and Hirae 1992). ‘Ikaika’ was particularly favored for poorerquality sites, where soil fertility was low or suffered exposure to the wind (Hamilton and Fukunaga 1959; Hamilton and Ito 1984). The altitude range present in the Hawaiian islands stimulated an interest in the suitability of cultivars to 600 m elevation and above (Ito et al. 1990; Nagao and Hirae 1992). In Hawaii, cultivars reported as having a wide range of suitability to elevations up to 610 m include ‘Kau’, ‘Keaau’ ‘Pahala’, ‘Makai’, and ‘HAES 816’. ‘Dennison’ is considered better than other cultivars below 150m, and ‘Purvis’ and ‘HAES 835’ are less suitable at elevations above 450 m. ‘Mauka’ is reportedly more suited to elevations above 200 m and ‘856’ to high elevations up to 670 m. There is however, no information on what data were used in for these recommendations. It has been suggested that the main drivers of grower adoption of recommended cultivars in Hawaii were suitability to location, grower preference (Hamilton and Fukunaga 1973), and availability of budwood (Hamilton and Ito 1984). It has also been suggested that the popularity of ‘Kau’ may be due in part to the attractive and distinctive tree form of this cultivar (Ito and Hamilton 1989). Similar to other horticultural crops, it was reported that exaggerated and misleading claims were commonly encountered (Hamilton and Fukunaga 1973). The impact of insect damage on crop loss, and the apparent presence of genetic variability for susceptibility, has led some to strongly suggest that resistance to insect damage should be included in selection

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decisions (Jones 2002). However, an increase pest resistance needs to be balanced against variability in other key selection traits. The majority of orchards in Hawaii are planted with HAES-released cultivars, with limited areas planted with cultivars selected outside this group (e.g., ‘Chong 6’ and ‘Honokaa Special’) (Hamilton and Fukunaga 1970; Hamilton and Ito 1977b; Yamaguchi 2006). ‘Keauhou’, ‘Ikaika’, and ‘Kakea’ were reportedly the major cultivars planted in older orchards in 1989 (Ito and Hamilton 1989), no doubt due to their popularity during the expansion phase of the industry prior to 1980 (Yamaguchi 2006). However, ‘Kau’, ‘Keaau’, and ‘Mauka’ were preferred for establishment of new orchards after 1980 (Ito and Hamilton 1989; Yamaguchi 2006). By 2003, ‘Pahala’, ‘Makai’, and ‘Purvis’ were the most common cultivars in the younger orchards (Nagao et al. 2003), although the majority of the orchard estate remained planted with ‘Keauhou’, ‘Ikaika’, ‘Kau’, ‘Kakea’, and ‘Keaau’ (Yamaguchi 2006). 2. Australia. Cultivar utilization in Australia prior to the 1980s was hampered by lack of reliable data, particular for Australian conditions (Winks 1983; Stephenson 1990a). The development of the Australian industry has largely been based on Hawaiian cultivars, mainly because information on their performance, albeit in Hawaii, was available (Winks 1983; Stephenson 1990a). The early Hawaiian cultivars ‘Keauhou’ and ‘Kakea’ were available in Australia by the early 1960s (McConachie 1980). By the early 1980s, the cultivars ‘Keaau’, ‘Kau’, ‘Mauka’, ‘Makai’, ‘Purvis’, and ‘Pahala’, and three other HAES selections (‘HAES 781’, ‘HAES 794’, and ‘Dennison’) had been introduced (Winks 1983). Other HAES selections (705, 762, 772, 783, 789, ‘Fuji’, 795, 804, 807, 814, 815, 816, 828, 835, 836, 837, 842, and 849) became available in Australia in the late 1980s (Winks et al. 1987). Several authors suggest the performance of Hawaiian cultivars in Australia is poorer than their performance in Hawaii, particularly for yield and kernel quality (Cull 1978; Winks 1983; Hamilton and Ito 1986; Trochoulias and Burnside 1987; Stephenson 1990a), implying these cultivars are less suited to Australian growing conditions. For example, ‘Kakea’ is considered intolerant of the hot and dry conditions in Australia, although this cultivar is considered hardy in Hawaii (see earlier discussion, Stephenson 1990a). While ‘Kau’ was highly regarded in Hawaii, it reportedly has not performed as well in Australia (Stephenson 1990a; Gallagher et al. 1998), particularly due to low kernel recovery, erratic yields in some environments (Stephenson and Gallagher 2000), and susceptibility to ‘‘abnormal vertical growth’’ (O’Farrell and Searle 2003, see earlier discussion).

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Australian experience appears to conﬁrm the Hawaiian experience with ‘Ikaika’, of poor later-age productivity (Stephenson 1990a). ‘Makai’ produces high-quality kernel under Australian conditions similar to its performance in Hawaii (Stephenson 1990a). ‘Keauhou’ is reported as having similar variable kernel quality to that found in Hawaii, and produces nuts with high incidence of open micropyles under some Australian conditions (Stephenson 1990a). However, it is one of the most widely planted cultivars in Australia and is considered an industry standard, in contrast to its status in Hawaii (Stephenson et al. 1997; Stephenson 1990a). The proposition that relative performance of cultivars is different between Hawaii and Australia is challenged by the similarity in relative ranking for yield of the limited number of cultivars planted in both locations. Whether the difference in performance between Australia and Hawaii demonstrates the potential of selection for local suitability, or simply reveals the limits of the Australian environment, requires further investigation. There is little information on the utilization of Australian selections prior to 1990 (Winks 1983; Winks et al. 1987; Stephenson et al. 1995), with only ‘Own Choice’ and ‘Hinde’ having been recorded as being of commercial signiﬁcance (Stephenson 1990a). ‘Own Choice’ is described as an upright tree, although slightly spreading, that crops heavily and produces high-quality kernel but can suffer a high incidence of sticktights (Stephenson 1990a). ‘Hinde’ was considered more suitable to cooler environments and was popular prior to 1990 (Stephenson 1990a; Hardner et al. 2006). A series of cultivars trials established over six sites in 1984–1985 greatly expanded the knowledge of cultivar performance in Australia (Winks et al. 1987; Stephenson et al. 1995, 1999; Stephenson and Gallagher 2000; Hardner et al. 2001, 2002; Mayer et al. 2006). Further cultivar trials were established in 1992, 1995, and 1996 (Stephenson 2001). By 2000, the most widely planted cultivars in Australia were reportedly ‘Keauhou’, ‘Kakea’, ‘Ikaika’, ‘Makai’, ‘Keaau’, ‘HAES 849’, ‘Hinde’, A4’, ‘A16’, and ‘A38’ (Peace et al. 2000). All cultivars that were utilized in the 1980s in the Australian industry were considered to have at least one major defect including (in order of importance): yield, quality, poor tree habit, stick-tights, excessive length of fruit drop period, low yield at a young age, susceptibility to insect and disease, susceptibility to early germination, susceptibility to heat stress, excessive premature nut drop 5 to 8 weeks after anthesis, and incidence of open micropyles (Stephenson 1990a). More recently, other selection criteria were identiﬁed, including attributes affecting kernel quality such as ﬂavor, texture appearance, shelf life, percentage of whole kernels, and kernel size (Hardner and McConchie 1999), although the extent of

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genetic control, and thus the potential for changing these through genetic selection, is unknown. Recommendations for cultivars in Australia have been made by initially rejecting cultivars with serious defects and then considering yield per tree of ﬁrst-grade kernel (Stephenson and Gallagher 2000). Thresholds for 22 desirable characteristics have been described: robust, compact and open habit; resistant to wind damage; tolerance of suboptimal conditions but responsive to good management; absence of stick-tight nuts; absence of pregermination in nuts or kernel; tolerance of major pest or diseases; short-harvest season, with 80% to 90% of the harvest completed within six months of mature nut fall; precocious, with bearing by three or four years after planting; at least 1 kg/year increase in NIS yield per tree from age of ﬁrst crop to reach at least 6.5 kg per tree by 10 years; NIS remains in husk after it falls from the tree; easy separation of nuts from husk and no husk adhering to nut after dehusking; nuts regular and round; no nuts smaller than 18 mm in diameter; sound kernel recovery greater than 36%; high and stable ﬁrstgrade kernel (over 95%); high percentage of whole kernels; regular round kernels; kernel color uniform and free from discoloration; even color after roasting; and acceptable sensory quality to processors, marketers, and consumers. However, as some of these selection criteria are not well deﬁned or quantiﬁed, accuracy of predicting cultivar performance is likely to be low. In addition, application of thresholds over such a large number of selection criteria is likely to lead to reduce gain compared to a selection index (Cotterill and Dean 1990). Based on evaluation of cultivars across six sites, combined with expert knowledge from growers, ‘Mauka’, ‘HAES 783’, ‘ HAES 814’, ‘HAES 842’, ‘HAES 849’, ‘Daddow’, and ‘A16’ were recommended as acceptable across the Australian industry in 2000 (Stephenson 2000). Recommendations of speciﬁc cultivars for particular regions in Australia were also made based on trial results (Stephenson et al. 1995; Stephenson and Gallagher 2000), although the data for these recommendations are limited as each region was represented only by a single site, and there was only a maximum of four replications for each cultivar at each site. The selection index developed to identify elite selections in the Australian Macadamia Breeding Program (see discussed earlier) has been applied to the evaluation of 20 cultivars over two of the trial sites in Stephenson et al. (1995) (Hardner et al. 2006). Economic weights for eight traits (canopy width at 10 years—m; age of ﬁrst crop—year; average rate of yield increase during the accumulation phase of production—kg/year; percentage of reject NIS—kg NIS/100 kg NIS; total kernel recovery—kg kernels/100 kg NIS; percentage reject

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kernel—kg kernels/100 kg kernels; percentage if marketable kernels— kg kernels/100 kg kernels; average grade of marketable whole kernel— mm) were calculated as the change in relative proﬁtability (proﬁt/total costs) of an economic model of production and processing costs, and the value of raw kernel. The model indicates that to offset the reduction in value of a 1% point lower total kernal recovery, a cultivar would require a canopy width of 0.1 m less, a rate of yield increase more than 0.1 kg/year higher, 1.3% less reject NIS, 2.1% less reject kernel, 10% more wholes or an average kernel size of 10 mm smaller. Applying the economic weights to 20 cultivars tested over two sites in subtropical Australia (southeast Queensland and northern NSWs) suggested that the top ﬁve cultivars for this region based on these traits are ‘HAES 849’, ‘Own Venture’, ‘HAES 814’, ‘A4’, and ‘HAES 804’ (Hardner et al. 2006) This study illustrates the importance of the selection based on overall performance. ‘HAES 849’ was ranked only tenth for average rate of yield increase and tree size but had the third lowest age to ﬁrst yield, the second highest kernel recovery, and the third highest percentage of whole kernels. The cultivar with the highest yield was ‘HAES 344’, but this cultivar was the sixth largest cultivar and had poor kernel recovery and percentage of whole kernels. Cultivar rankings were reportedly robust to a 20% change in land costs, other production costs, processing costs, and kernel prices (Hardner et al. 2006). The importance of different criteria for selection is determined not only by the value of the economic weight but also on the ability to change the trait through selection (i.e., heritability). Average rate of yield increase, canopy width, and total kernel recovery were the largest contributors to the variation in the index value. In contrast, the index was only marginally affected by differences in proportion of whole kernel, kernel size, and age to ﬁrst yield. These results could be used to prioritize the assessment of traits for selection. Other selection criteria, which may or may not be important, were not included in this analysis (e.g., tree structure, nut size and shape, pest and disease resistance, ﬂower and nut drop phenology, visual appearance of raw kernel, quality of roasted kernels, or shelf life). However, cultivars that produce high-quality nuts and kernel may not be suitable if production characteristics are unfavorable (Cull 1978). 3. South Africa. Graft-wood of the older Hawaiian cultivars became available in South Africa by 1969 following earlier introductions of these cultivars (Allan 1995). More recent cultivars and selections were introduced in the 1970s. The cultivar ‘Beaumont’ was introduced into South Africa from California in 1968 (Wiid and Hobson 1996).

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Similar to experiences in Australia, early Hawaiian cultivars (‘Keauhou’, Kakea’, and Ikaika’) reportedly did not perform well in South Africa, although ‘Kau’ and ‘Keaau’ are considered better (Allan 1993). It is suggested that M. integrifolia cultivar types are less productive under cooler subtropical conditions of South Africa than cultivars of hybrid origin (Wiid and Hobson 1996; McCubbin and Lee 1996; Allan et al. 1999). The high quality of raw kernel from some M. tetraphylla and hybrid selections has also been used to suggest that these may be more suited to cooler areas, although further testing of roasted kernel product is required (Allan 1993). Cultivar recommendations in South Africa are based on Hawaiian kernel quality standards (i.e., average kernel mass between 2–3 grams, greater than 34% kernel recovery, and 95% ﬁrst-grade kernel), resistance to anthracnose, fairly uniform shell thickness with no open micropyle, even round shape of the nut, limited variation in nut size, round kernel, absence of basal discoloration or discolored rings, roasting ability, shelf life, yield per tree of greater than 45 kg NIS at 10 years of age, resistance to stink bug, lack of soft kernel, time of ﬂowering, harvest season, and tree shape and branching habit (Allan 1989; Oosthuizen et al. 1989). However, it is not clear how some of these standards are deﬁned, assessed, and prioritized. In 1989, ‘Keaau’, ‘Kau’, ‘Kakea’, ‘Keauhou’, and ‘Ikaika’ were recommended for both the southern Lowveld and Soutpansberg growing areas (Oosthuizen et al. 1989) based on standards of nut size, kernel recovery greater than 33%, kernel mass between 2 and 3 grams, greater than 75% oil content of kernels, and productivity determined from four trees of each cultivar at two locations. ‘Nelmak 2’ was also only recommended for the southern Lowveld and ‘Selection 26’ only for Soutpansberg. By the 1990s, ‘Mauka’, ‘Pahala’, and ‘Makai’ were considered to be superior in South Africa to ‘Keauhou’, Ikaika’, ’Kakea’, ‘Purvis’, and ‘Cate’, based on superior kernel quality and reasonable yield (Allan et al. 1999). Others cultivars considered superior were ‘Keaau’ and ‘Beaumont’ in particular, and ‘Kau’, ‘HAES 781’, ‘HAES 814’, ‘HAES 816’, and ‘Nelmak 2’ (Allan et al. 1999). In contrast, others (McCubbin and Lee 1996) consider ‘A4’, ‘A16’, and ‘Beaumont’ superior to ‘Kau’, ‘Mauka’, ‘HAES 816’, and ‘Makai’, primarily because of precocity. Some concerns have been expressed about a high proportion of sticktights, germination, and the vigorous growth of ‘Beaumont’ (Allan 1989; McCubbin and Lee 1996). It was suggested that this cultivar may be more suitable to particular production systems of hand harvesting or as a temporary tree in high-density plantings (McCubbin and Lee

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1996), although other work has suggested this cultivar is productive at later ages in high-density plantings (Wiid and Hobson 1996). The limited availability of reliable yield data for South Africa makes recommendations difﬁcult to evaluate. A proﬁtability index was calculated to assess cultivars based on average NIS price, yield at six or eight years after planting, sound kernel recovery, percentage of ﬁrst-grade kernel, and tree spacing (Swanepoel and Hobson 1999). This is the value of NIS production per hectare with the assumption that all unsound kernels are rejected, all kernels greater than 1.000 speciﬁc gravity will produce kernel with no value, and there are no differences in costs associated with the variability in these traits. A REML analysis (to account for unbalance of cultivars across sites) of the proﬁtability values published in this study indicates that the value of production from ‘HAES 814’, ‘Nelmak 2’, ‘A4’, and ‘Beaumont’ was signiﬁcantly superior to the other cultivars examined (‘Fuji’, ‘A16’, ‘ Kau’, ‘Pahala’, Mauka’, Keaau’, ‘HAES 816’, ‘Purvis’, ‘HAES 789’, ‘HAES 862’, and ‘Makai’). However, these recommendations are made with limited data and may not accurately reﬂect cultivar performance. The main cultivars in commercial orchards in South Africa by 1999 were ‘Keauhou’, ‘Fuji’, Nelmak 2’, ‘Keaau’, and ‘Kau’ (Swanepoel and Hobson 1999). The cultivar ‘Beaumont’ has also been planted widely throughout the country and is also popular as clonal rootstock (Bell 1996; Wiid and Hobson 1996; Hardner and McConchie 2006). 4. China. A range of Hawaiian (all major releases) and Australian (‘Hinde’, ‘Own Choice’, ‘A4’, and ‘A16’) cultivars were introduced into China in the 1970s (Xiao et al. 2002b). During the 1980s, these cultivars were used to establish orchards in coastal areas (Guanxi, Ueng Nang, Shichuan, Hainan, and Fujien provinces); however, these orchards suffered extensive cyclone damage (Lu et al. 1998b; Xiao et al. 2002a, 2002b). Since 1997, new plantings have been undertaken in the inland areas (Uengnang and Shichuan provinces), although the cooler temperatures and high rainfall in these areas may limit macadamia productivity (Xiao et al. 2002a). ‘Hinde’, ‘Own Choice’, and ‘Beaumont’ were observed to be tolerant of cold and wind and to produce good yields in the Panxi region of the Shichuan province (Zheng and Zhang 2002; Xiao et al. 2002a, 2002b). ‘Hinde’ is reportedly vigorous with yields of 8 to 10 kg per tree at nine years in experimental trials, and is considered very hardy to cold wind and drought but susceptible to poor soil. ‘Beaumont’ is considered precocious and suitable for inland and mountainous areas in China. ‘Own Choice’ is favored as it is more

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resistant to wind (Lu et al. 1998b, 2004) and is reported to be resistant to drought (Xiao et al. 2002a). Most of the main Hawaiian cultivars except ‘Pahala’ were not favored due to poor ﬂowering at age four (Xiao et al. 2002a). ‘Makai’ exhibits poor growth in China. Similar to conclusions developed in South Africa, experience in China suggests that M. tetraphylla genotypes are more suitable for these cooler environments and M. integrifolia cultivars should be ignored (Xiao et al. 2002b). 5. Other Countries. There is limited information about the utilization of genetic material in other macadamia-producing countries. Hawaiian cultivars reportedly dominated the orchard estate in Brazil in the 1990s, with the ﬁve most common cultivars being ‘Kau’, ‘Kakea’, ‘Keaau’, ‘Mauka’, and ‘Makai’ (Sacramento et al. 1995). M. tetraphylla cultivars (‘Elimbah’ and ‘Cate’) were preferred in California because the species is considered more suitable to the cooler climate (Cavaletto 1983). Several elite M. tetraphylla and hybrid selections have been identiﬁed for Kenya (Gathungu and Likimani 1975). Hybrid cultivars also appear popular in New Zealand, with 75% planted to ‘Beaumont’ and smaller plantings of ‘Renown’ in 1991, although orchards with ‘Own Choice’ and some Hawaiian cultivars have also been established (Gordon 1987; Richardson and Dawson 1991; Warren 2003). Cultivar utilization appears to be hampered by limited evaluation trials. In addition, kernels produced by M. tetraphylla selections may not be as commercially acceptable as kernel from the M. integrifolia cultivars that dominate the market (Hamilton 1988). B. Rootstocks M. tetraphylla was favored for seedling rootstocks in Hawaii from the 1960s (Storey 1976; Hamilton 1988; Nagao and Hirae 1992; Trochoulias 1992), probably due to perceived superior nursery performance (Hamilton 1988; Stephenson 1990a; Trochoulias 1992) and stronger root system (Wagner-Wright 1995). However, observations of ‘‘later age incompatibility symptoms’’ prompted a conversion to M. integrifolia (Hamilton 1988; Nagao and Hirae 1992). M. tetraphylla seedling rootstocks were also used in Australian in the 1970s, apparently because of faster and more even germination and growth (Stephenson 1990a). ‘Eggshell’ was reportedly used as a source of seedling rootstocks for the expansion of the Australian industry by the CSR company in the mid-1960s (Trochoulias et al. 1989), although the relationship with the early Australian seed parent of the same name (Petrie 1935) is not known. Seedling progeny from

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‘Renown’, a hybrid cultivar, became popular in the 1980s (Trochoulias 1992), but since the early 1990s, the majority of Australian orchards have been established with seedling rootstocks from ‘Hinde’ (Stephenson 1990a; Trochoulias 1992; Hardner and McConchie 2006). This cultivar is reportedly favored because it has a broad stem that is considered advantageous for grafting at an early age (Stephenson 1990a). A hybrid cultivar ‘Beaumont’ has been used as a clonal rootstock in South Africa due to its high strike success and vigorous nursery growth (Wiid and Hobson 1996 and earlier discussion). Orchards in California have been reportedly established with M. tetraphylla rootstocks, primarily because this species is considered more suitable to cooler environments (Hamilton 1988). There may also be the potential to use M. ternifolia, which is generally smaller than M. tetraphylla and M. integrifolia, as a dwarﬁng rootstock (Hardner et al. 2000; Peace 2005), although work is required to test for the transmission of cyanogenic properties from the rootstock to the scion, which has been reported for other seedling material (Hamilton and Young 1966). Currently there is insufﬁcient information to support selection for rootstocks based on effects on scion performance (Storey 1957; Hobson 1971; Hamilton 1988; cf. Huett 2003; Hardner and McConchie 2006). As such, performance of rootstocks in the nursery will continue to be the dominant rationale for choice among rootstock genotypes.

VII. SUMMARY Macadamia is an iconic Australian plant. Most species are endemic, the genus is one of the few current rain forest representatives of the ancient Gondwanan family Proteaceae, the plant has important cultural meanings for the indigenous peoples of Australia, it is the only member of the Australian ﬂora that has become an international commercial food crop, and Australia is the world leader in the production of this highly valued nut. Genetic improvement has supported the development of the industry in Hawaii and its expansion worldwide, and has delivered substantial gains across a range of traits. This is particularly true for traits that are highly heritable and easy to measure, such as nut size, kernel recovery, and kernel size. In some cases, although no quantitative method has been used to measure traits, high heritability has enabled identiﬁcation of cultivars that are easy to propagate, have an upright structure, and are free of bitter kernels.

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Breeding generally has been undertaken following the conventional method of intense selection among seedling progeny followed by clonal replication of a reduced number of candidates. However, selection among the seedlings has commonly used phenotypic performance without controlling environmental variation; hence selection accuracy for genetic effects is likely to be low, particularly for traits with low heritability. While selection accuracy is expected to be high in the clonal trials, selection intensity is generally low, as generally only a few candidates are evaluated. It may be possible that greater gains could be achieved with a different balance between selection intensity and accuracy in the different testing phases. Although there is a general understanding in macadamia of selection traits and their interaction with the production system, much of this information is imprecise and based on anecdotal knowledge or limited data. This lack of detailed understanding is likely to have restricted the opportunities of improving key selection criteria. For example, this review has highlighted uncertainties associated with traits such as sticktights and some attributes of kernel quality. Methods of assessment of traits may also vary among studies, making results difﬁcult to compare for selection decisions. Selection response may also be compromised by the limited information on genetic architecture of many traits, particularly when mass selection strategies are implemented for traits with low heritability. These traits are improved more efﬁciently through quantitative approaches. Further, it is often difﬁcult to deduce the relative importance of different traits in selection programs. This can lead to a waste of selection pressure on traits with little importance, compromising gain in traits that actually can impact on the production system, and has the potential to introduce personal biases in selection decisions that are difﬁcult to evaluate. This is particularly so for kernel quality attributes, where there are very little data on appearance attributes that can be used for selection. This chapter has underlined the limited genetic diversity of the Hawaiian germplasm that is the basis of much of the world industry. Given the short selection history of this germplasm and the relatively weak selection pressure (due to low accuracy), it is highly unlikely this material represents the only source of elite germplasm available in Macadamia. Opportunity to make signiﬁcant advances may exist by increasing the genetic base of breeding programs by introduction of novel germplasm unrepresented in the Hawaiian gene pool. Gene pool diversity and avoidance of inbreeding could be managed using information from the neutral genetic marker studies on the genetic structure of the domesticated and wild germplasm. This study

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could be extended to examine if there is an association between variation in selection traits and marker variation, particularly species composition. There is a need to better understand the performance of many selection criteria across a wider germplasm pool for management of current genetic resources and future genetic improvement. Selection origin does not offer an adequate description of the relatedness of germplasm or genetic performance. In addition, species status is not a consistent indicator of genotype performance. In particular, major gaps in the knowledge of the behavior of commercially important traits in the M. tetraphylla germplasm is likely to hamper the utilization of this resource in current improvement programs. Similarly, systematic evaluation is required to determine whether there are useful traits in other two species of the macadamia southern clade, M. ternifolia and M. jansenii, that can be introduced through hybridization with the cultivated species. A better understanding of the relative performance of germplasm in diverse environments may provide opportunities for more efﬁcient utilization of the genetic resources of macadamia. Experience suggests that M. tetraphylla germplasm is suited to cooler climates. However, work is required to conﬁrm that this is general and that the use of this germplasm does not compromise the beneﬁts that could be gained from the use of alternative genetic material. In addition, data on the performance of common cultivars across different environments are needed to evaluate the hypothesis that Hawaiian cultivars are less suited to environments foreign to their selection origin. In this review, data from several studies were integrated to demonstrate that the relative performance of cultivars across countries was very stable for kernel recovery, but cultivar ranking for ﬁrst-grade kernel was inconsistent across these environments. This approach could be extended to other key selection traits, particularly yield. The limited development of the genetic resources of macadamia means the existing wild populations of the species are an extremely valuable resource for future genetic improvement. However, these populations currently are highly fragmented. An ex situ collection of samples from many of the known populations has already been established. The most up-to-date knowledge of the distribution of the three main species of the southern clade has been published in this review. The next important step in conservation of the wild populations is to develop more comprehensive knowledge of this distribution. Detailed knowledge of the genetic structure and dynamics of these populations, combined with ecological and demographic studies, is

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required to underpin the management of these populations for national and international beneﬁt.

VIII. ACKNOWLEDGMENTS This review is dedicated to the memory of Henry Bell (1927–2008), a pioneer of the modern Australian macadamia industry. Henry was passionate about improving macadamia horticulture and made major contributions to conservation of wild germplasm, propagation technology, selection methods, high-density planting, and genetic improvement through the development of a private breeding program that produced the A varieties that are have now been widely adopted thoughout Austrlia and overseas. Henry was always happy to share his time and wealth of knowledge with any similar passionate soul, and the success of macadamia is in no small part a consequence of his enthusiasm and foresight for the crop. Many people have contributed to the production of this review. Foremost we wish to thank Kaye Guidetti of CSIRO Information Technology Services who, along Patrick Ledwith and Robyn Mills (also CSIRO ITS), provided enormous assistance in gathering the obscure and difﬁcult-to-access publications cited in this review. Carl Davies of CSIRO Plant Industry prepared Fig. 1.3 and assisted with the ﬁnal version of Fig. 1.1a and 1.1b. Anfernee Tseng and Sharon de Wit assisted with translation of several non-English articles. We also wish to thank Russ Stephenson, David Mayer, Andre Drenth, Andrew Miles, Olufemi Akinsanmi, Ruth Huwer, Craig Maddox, and Lisa McFadyen, who made contributions to an initially planned larger review of macadamia natural history, utilization, and horticulture. We hope this second phase of the review of macadamia will be published in the near future. Last, we wish to thank our families for their continued support throughout the long days of thought and synthesis.

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Stephenson, R.A., and B.W. Cull. 1986. Standard leaf nutrient levels for bearing macadamia trees in south-east Queensland. Scientia Hort. 30:73–82. Stephenson, R.A., and E.C. Gallagher. 1986. Effects of temperature during latter stages of nut development on growth and quality of macadamia nuts. Scientia Hort. 30:219–225. Stephenson, R.A., and E.C. Gallagher, 2000. Selecting better macadamia varieties. Queensland Department of Primary Industries, Brisbane. Stephenson, R.A., E.C. Gallagher, and V.J. Doogan. 1997. Leaf nitrogen as a guide for fertilising macadamia. Austral. J. Expt. Agr. 37:599–604. Stephenson, R.A., E.C. Gallagher, V.J. Doogan, and D.G. Mayer. 2000. Nitrogen and environmental factors inﬂuencing macadamia quality. Australian J. Expt. Agr. 40:1145–1150. Stephenson, R.A., E.C. Gallagher, P.J. OHare, and D. Firth. 1999. New macadamia cultivars to 2010 and beyond. Hort. Res. Develop. Corp., Sydney. Stephenson, R.A., E.C. Gallagher, and C.W. Winks. 1995. The breeding, selection and development of new macadamia cultivars for the Australian industry. Hort. Australia Ltd., Sydney. Stephenson, R.A., and T. Trochoulias. 1994. Macadamia. pp. 147–163. In: B. Schaffer and P.C. Andersen (eds.), Handbook of environmental physiology of fruit crops, -Vol. 2: Subtropical and tropical crops. CRC Press, Boca Raton, FL. Storey, W.B. 1957. The macadamia in California. J. Florida State Hort. Soc. 70:333–338. Storey, W.B. 1959. History of the systematic botany of the Australian species of macadamia. California Macadamia Soc. Yearb. 5:68–78. Storey, W.B. 1963. The named varieties of Macadamia. California Macadamia Soc. Yearb. 9:67–74. Storey, W.B. 1965a. Beaumont: A new dual purpose macadamia variety. California Macadamia Soc. Yearb. 11:19–25. Storey, W.B. 1965b. The ternifolia group of macadamia species. Pac. Sci. 19:507–514. Storey, W.B. 1976. Subtropical and tropical fruit and nut crops in California, USA. Acta Hort. 57:53–62. Storey, W.B., and E.F. Frolich. 1964. Report on the graft compatibility of macadamia. California Macadamia Soc. Yearb. 10:54–58. Storey, W.B., and J.A. Hopﬁnger. 1974. Report on the tetraphylla introductions from Australia. California Macadamia Soc. Yearb. 20:47–55. Storey, W. B., and W.C. Kemper. 1960. A study of macadamia seed germination. California Macadamia Soc. Yearb. 6:1–5. Storey, W.B., and W.F. Saleeb. 1970. Interspeciﬁc hybridization in macadamia. California Macadamia Soc. Yearb. 16:75–85. Strohschen, B. 1986. Contributions to the biology of useful plants. 4. Anatomical studies of fruit development and fruit classiﬁcation of the macadamia nut (Macadamia integrifolia Maiden and Betche). J. Appl. Bot. Food Qual. 60:239–247. Supamatee, S., P.J. Ito, and D. Jalichan. 1992. Early performance of Australian and Hawaiian macadamia cultivars in Thailand. pp. 107–111. In: Proc. 1st Intl. Macadamia Res. Conf., 28–30 July 1992. Kailua, HA. Surono, S.S. 1987. A comparative analysis of consumer demand for macadamia nuts in Honolulu and Los Angeles. Ph.D. diss., Univ. Hawaii. Swanepoel, J.F., and M.E. Hobson. 1999. Preliminary yield potential assessment and nut quality of macadamia cultivars. pp. 145–154. In: Proc. 1st Intl. Acadamia Symp. in Africa, Mpumulanga Parks Board, 27–30 Sept. 1999, Nelsprint, South Africa. Tang, G.P., T. Liang, and F. Munchmeyer. 1982. A variable deformation macadamia nut cracker. Trans. ASAE 26:1506–1511.

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Tay, K. 2006. Guatemala tree nuts macadamia nuts 2006: gain report. www.fas.usda.gov/ gainﬁles/200603/146187037.pdf. 2006. Tobin, M.E., A.E. Koehler, and R.T. Sugihara. 1997. Effects of simulated rat damage on yields of macadamia trees. Crop Prot. 16:203–208. Trochoulias, T. 1992. Rootstock type affects macadamia performance. Acta Hort. 296:147– 152. Trochoulias, T. 1995. Effect of cultural practices on macadamia nut quality. Acta Hort. 370:93–98. Trochoulias, T., and A. Burnside. 1987. Close spaced macadamia. p. 221. In: Proc. 2nd Australian Macadamia Res. Workshop, 15–19 Sept. 1987, Bangalow, Australia. Trochoulias, T., and G.G. Johns. 1992. Poor response of macadamia (Macadamia integrifolia Maiden and Betche) to irrigation in a high rainfall area of subtropical Australia. Australian J. Expt. Agr. 32:507–512. Trochoulias, T., C.W. Winks, and C. Heselwood. 1989. An historical perspective on variety evaluation in Australia from interviews with Norm Greber, patron of the Society. Australian Macadamia Soc. News Bul. 16:17–19. Trout, S.A., and R.E. Leverington. 1962. The processing of macadamia nuts in Australia. pp. 801–809. In: Proc. 1st Intl. Congr. Food Sci. Technol., 18–21 Sept. 1962, London. Trueman, S. J., C.A. McConchie, and C.G.N. Turnbull. 2002. Ethephon promotion of crop abscission for unshaken and mechanically shaken macadamia. Australian J. Expt. Agr. 42:1001–1008. Trueman, S.J., S. Richards, C.A. McConchie, and C.G.N. Turnbull. 2000. Relationships between kernel oil content, fruit removal force and abscission in macadamia. Australian J. Expt. Agr. 40:859–866. Trueman, S.J., and C.G.N. Turnbull. 1994a. Effects of cross-pollination and ﬂower removal on fruit set in macadamia. Ann. Bot. 73:23–32. Trueman, S.J., and C.G.N. Turnbull. 1994b. Fruit set, abscission and dry matter accumulation on girdled branches of macadamia. Ann. Bot. 74:667–674. Tsai, S.P. 2005. Impact of personal orientation on luxury-brand purchase value—an international investigation. Intl. J. Market Res. 47:429–454. Urata, U. 1954. Pollination requirements of macadamia. Tech. Bul. Hawaii Agr. Expt. Sta. 22:1–40. U.S. Department of Agriculture. 2006. USDA national nutrient database for standard reference, release 19. www.nal.usda.gov/fnic/foodcomp/cgi-bin/list_nut_edit.pl. U.S. International Trade Commission 1998. Macadamia nuts: economic and competitive conditions affecting the U.S. industry. USITC, Washington. Venkatachalam, M., and S.K. Sathe. 2006. Chemical composition of selected edible nut seeds. J. Agr. Food Chem. 54:4705–4714. Venkata Rao, C. 1970. Studies in the Proteaceae. Proc. Indian Natl. Sci. Acad. 36:345–363. Villiers, E.D. 1977. Macadamia pests. Information Bul.—Citrus Subtrop. Fruit Res. Inst. 60:10–11. Virot, R. 1968. Prote´ace´es. pp. 5–254. In: A. Aubreville (ed.), Flore de la Nouvelle Cale´donie et De´pendances, Vol. 2. Muse´um National d’Histoire Naturelle, Paris. Vithanage, V., C.[M.] Hardner, K.L. Anderson, N.[M.] Meyers, C.[A.] McConchie, and C. [P.] Peace. 1998. Progress made with molecular markers for genetic improvement of macadamia. Acta Hort. 461:199–208. Vithanage, V., K. O’Connor, C.[P.] Peace, N.[M.] Meyers, R.L. Forrester, and C.A. McConchie. 2003. Use of molecular markers in macadamia: managing pollination in orchards. pp. 73–76. In: Proc. 2nd Intl. Macadamia Symp., 29 Sept.–4 Oct. 2003, Tweed Heads, Australia.

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2 Pomegranate: Botany, Horticulture, Breeding D. Holland, K. Hatib, and I. Bar-Ya’akov Section of Deciduous Fruit Trees Sciences Newe Ya’ar Research Center Agricultural Research Organization PO Box 1021 Ramat Yishay, 30095, Israel I. INTRODUCTION II. TAXONOMY AND MORPHOLOGY A. Botanical Classification B. Vegetative Growth C. The Flower D. The Fruit E. Juvenility and Age of Fruiting III. ORIGIN AND GENETIC RESOURCES A. Origin and Cultivating Regions B. Collections and Germplasm IV. HORTICULTURE A. Cultivars 1. India 2. Iran 3. China 4. Turkmenistan and Tajikistan 5. Turkey 6. Israel 7. Spain 8. United States 9. Georgia 10. Tunisia 11. Egypt 12. Saudi Arabia and Iraq 13. Vietnam 14. Morocco 15. Sicily, Italy Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 127

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D. HOLLAND, K. HATIB, AND I. BAR-YA’AKOV B. Irrigation C. Fertilization D. Tree and Orchard Design E. Plant Protection F. Weed Control G. Fruit Physiological Disorders H. Postharvest BREEDING HEALTH BENEFITS CONCLUDING REMARKS ACKNOWLEDGMENTS LITERATURE CITED

I. INTRODUCTION Pomegranate (Punica granatum L., Punicaceae) is an ancient, beloved plant and fruit. The name ‘‘pomegranate’’ follows the Latin name of the fruit Malum granatum, which means ‘‘grainy apple.’’ The generic name Punica refers to Pheonicia (Carthage) as a result of mistaken assumption regarding its origin. The pomegranate and its usage are deeply embedded in human history, and utilization is found in many ancient human cultures as food and as a medical remedy. Despite this fact, pomegranate culture has always been restricted and generally considered as a minor crop. The pomegranate tree requires a long, hot and dry season in order to produce good yield of high-quality fruit. Pomegranates are native to central Asia, but since the pomegranate tree is highly adaptive to a wide range of climates and soil conditions, it is grown in many different geographical regions including the Mediterranean basin, Asia, and California. Recent scientiﬁc ﬁndings corroborate traditional usage of the pomegranate as a medical remedy and indicate that pomegranate tissues of the fruit, ﬂowers, bark, and leaves contain bioactive phytochemicals that are antimicrobial, reduce blood pressure, and act against serious diseases such as diabetes and cancer. These ﬁndings have led to a higher awareness of the public to the beneﬁts of the pomegranate fruit, particularly in the western world, and consequently to a prominent increase in the consumption of its fruit and juice. The development of industrial methods to separate the arils from the fruit and improvement of growing techniques resulted in an impressive enlargement of the extent of pomegranate orchards. New orchards are now planted in the traditional growing regions as well as in the southern hemisphere in South America, South Africa, and Australia.

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II. TAXONOMY AND MORPHOLOGY A. Botanical Classiﬁcation Punicaceae contains only two species, Punica granatum L. and P. protopunica Balf. f. 1882. Punica protopunica is endemic to the Socotra Island (Yemen) and is the only congeneric relative of P. granatum species currently is cultivated (Zukovski 1950; Levin and Sokolova 1979; Guarino et al. 1990; Mars 2000; Levin 2006). Based on xylem anatomy, P. protopunica has been suggested as the ancestral of the genus (Shilkina 1973). The n ¼ x chromosome number is 8 (Yasui 1936; Darlington and Janaki Ammal 1945; Raman et al. 1971; Sheidai and Noormohammadi 2005) or 9 (Darlington and Janaki Ammal 1945). B. Vegetative Growth Pomegranate is a shrub that naturally tends to develop multiple trunks and has a bushy appearance. When domesticated, it is grown as a small tree that grows up to 5 m. Under natural conditions, it can sometimes grow up to more than 7 m; at the other extreme, in severe natural environment, one can ﬁnd creeping bush varieties (Levin 2006). In addition, there are dwarf cultivars that do not exceed 1.5 m (Levin 1985, 2006; Liu 2003). Most of the pomegranate varieties are deciduous trees. However, there are several evergreen pomegranates in India. Singh et al. (2006) reported deciduous Indian varieties and identiﬁed 16 genotypes that behaved as evergreen in Rajasthan India. Sharma and Dhilom (2002) evaluated 30 evergreen cultivars in Punjab India. There are clearly prominent differences among pomegranate varieties with respect to leaf shed. Some evergreen cultivars shed their leaves in higher elevations and colder climates (Nalawadi et al. 1973) and should be regarded as conditionally deciduous. The young branches from the vegetative growth of the recent year are numerous and thin. The color of the bark of young branches depends on the variety. In some, bark color varies from pink to purple, while in others it is light green with pink-purple spots or stripes. Upon maturation, the pink color of the branch starts to disappear, and in the second year, the bark will become light gray that darkens as the tree matures (Goor and Liberman 1956). The bark of the old tree tends to split, and in certain cases it is detached from the trunk. The wood color is light yellow. Young branches sometimes have thorns at their tips that are visible already in the axils in the young bloom. The young

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branches are polygonal (quadrangular). As the branches mature, they become round. Young leaves tend to have a reddish color that turns green when the leaf matures. In varieties with young pink-purple bark, this color appears also on the sheath and the petiole, on the lower part of the central vein, and in the leaf margins. Leaves have an oblanceolate shape with an obtuse apex and an acuminate base. Mature leaves are green, entire, smooth, and hairless with short petioles. They usually have a special glossy appearance (particularly at the upper part of the leaf) and contain idioblasts with secretory substances that have not yet been identiﬁed (Fahan 1976). The leaves are exstipulate, opposed and pairs alternately crossing at right angles. Some varieties have 3 leaves per node arranged at 120 degrees and even 4 leaves per node on the same tree (2 opposed leaves per node) (Moreno 2005). C. The Flower Flowering occurs about 1 month after bud break on newly developed branches of the same year, mostly on spurs or short branches. Flowers can appear solitary, pairs, or clusters. In most cases, the solitary ﬂowers will appear on spurs along the branches while the clusters are terminal. In the northern hemisphere, ﬂowering occurs in April-May. However, ﬂowering may continue until end of summer, particularly in young trees. Such ﬂowers are fertile, but the fruit will not properly mature because the trees enter the cooler season and the dormancy period in Mediterranean climatic conditions. Flowering and the consequent fruit set last about 1 month. During this period, there are three waves of ﬂowering (Ben-Arie et al. 1984; Shulman et al. 1984; El Sese 1988; Assaf et al. 1991b; Hussein et al. 1994; Mars 2000). In evergreen cultivars in southern India, ﬂowering season was observed in three periods: June, October, and March (Nalwadi et al. 1973) or throughout the year (Hayes 1957). In the early balloon stage, the ﬂower resembles a small pear with a greenish color on its basal part and reddish color on its apex or entirely dark red. As the ﬂower matures, it develops an orange-red to deep red sepal color, which varies among different varieties. The petals are orange-red or pink and rarely white (Feng et al. 1998; Wang 2003; Levin 2006; Beam Home 2007). Several pomegranate cultivars from India, Russia, China, and Turkmenistan were reported as ornamental pomegranates that are ‘‘double ﬂowered’’ (Iskenderova 1980, 1988; Feng et al. 1998; Wang 2003; Levin 2006). These cultivars have an unusually high petal number and petal color. Some of these cultivars

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are fertile and produce edible fruit while others are infertile. Nalawadi et al. (1973) deﬁned 10 stages for ﬂower development. According to these authors, the time required for completion of ﬂower bud development in Indian cultivars is between 20 and 27 days (Nalawadi 1973; Josan 1979a). We found a good correlation between the color of the sepals and the ﬁnal color of the fruit skin. Usually cultivars with deep-red fruit skin will have a darker-red ﬂower. Pomegranate ﬂowers develop into one of two types of ﬂowers normally produced by pomegranates: hermaphrodite ﬂowers (‘‘vase shape’’) (Plate 2.1A) and male ﬂowers (‘‘bell shape’’) (Plate 2.1B). Both types have several hundred stamens. The bell-shape ﬂower has a poorly developed or no pistil and atrophied ovaries containing few ovules and is infertile. Therefore, the bell shape ﬂower is referred as a male ﬂower and will drop without fruit set. The vase-shape ﬂower is fertile with a normal ovary capable of developing fruit. The stigma of the hermaphrodite is at the anthers height or emerging above them. This position allows for self-pollination as well as pollination by insects. The factor that determines the fruit set capacity is the number of vase-shape ﬂowers. Therefore, cultivars with higher vase-shape to bell-shape ratio will have a higher fruit yield potential. The percentage of the vase-shape ﬂowers among the Israeli cultivars is 43% to 66% (Assaf et al. 1991b). Other studies in India indicate 53% to 80% ratios for Indian local cultivars (Nalawadi et al. 1973). An intermediate third type of a ﬂower has been described that has short style and a developed ovary which is sometimes fertile (Goor and Liberman 1956; Nalawadi et al. 1973; Assaf et al. 1991b). The sepals, 5 to 8 fused in their base, form a red ﬂeshy vase shape. The sepals will not drop with fruit set but will stay as an integral part of the fruit as it matures, generating a fruit crowned with a prominent calyx. The ﬂower has 5 to 8 petals. Their number usually equals the number of sepals. The petals, which alternate with the sepals, are separated and have a pink-orange to orange-red color depending on the variety. The petals are obovate, very delicate, and slightly wrinkled. The multiple long stamens are inserted into the calyx walls in a circle and frequently number more than 300 per ﬂower. They have an orangered ﬁlament and yellow bilocular anthers that remain attached to the prominent calyx. Nectaries are located between the stamens and the ovary base (Fahan 1976). The carpels vary in number but are usually eight superimposed in two whorls. They form a syncarpic ovary and are arranged in two layers. Josan et al. (1979b) studied anthesis and receptivity of stigma. These authors report that the time taken by the ﬂowers to complete anthesis was 3 to 5 hours. The stigma attained

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Plate 2.1. Pomegranate cultivars diversity and fruit development. A. Vase-shape ﬂower; B. Bell-shape ﬂower; C–E. Different stages of fruit color development in three cultivars: C. ‘C13’, D. ‘P.G.116-17’, E. ‘P.G.127-28’, 1. May, 2. June, 3. August, 4. October (NadlerHassar et al. unpublished); F. Fruit diversity of pomegranate cultivars grown in Israel. (See insert for color representation of this plate).

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receptivity one day before anthesis and remained in receptive condition up to the second day after anthesis. The pomegranate is both self-pollinated and cross-pollinated by insects, mainly bees. Wind pollination is reported to occur but infrequently (Morton 1987). Emasculation and bagging studies on Indian, Turkmen, Israeli, and Tunisian pomegranate cultivars indicate that pomegranate ﬂowers can self-pollinate and produce normal fruit (Nalawadi et al. 1973; Karale et al. 1993; Mars 2000; Levin 2006; Holland et al. unpubl.). It was noted, however, that the degree of fruit set by self-pollination varies among different pomegranate cultivars (Levin 1978; Kumar et al. 2004). In hermaphrodite ﬂowers, 6% to 20% of pollen may be infertile; in male ﬂowers, 14% to 28% are infertile. The size and fertility of the pollen vary with the cultivar and season (Morton 1987). D. The Fruit The fruit develops from the ovary and is a ﬂeshy berry. The nearly round fruit is crowned by the prominent calyx. The apex of this crown is almost closed to widely opened, depending on the variety and on the stage of ripening. The fruit is connected to the tree with a short stalk. Following fruit set, the color of the sepals’ skin in the developing fruit changes continuously from the prominent orange-red to green. In later stages of fruit maturation, the color will change again until it reaches its ﬁnal characteristic color as the fruit ripens. The external color ranges from yellow, green, or pink overlain with pink to deep red or indigo to fully red, pink or deep purple cover, depending on the variety and stage of ripening (Plate 2.1 C1–C4, D1–D4, E1–E4). There are some exceptional cultivars, such as the black pomegranate which acquires its black skin very early and remains black until ripening time (Plate 2.1 E1–E4). The skin (leathery exocarp) thickness varies among pomegranate cultivars. The multi-ovule chambers (locules) are separated by membranous walls (septum) and ﬂeshy mesocarp. The chambers are organized in a nonsymmetrical way. Usually the lower part of the fruit contains 2 to 3 chambers while its upper part has 6 to 9 chambers. The chambers are ﬁlled with many seeds (arils). The arils contain a juicy edible layer that develops entirely from outer epidermal cells of the seed, which elongate to a very large extent in a radial direction (Fahan 1976). The sap of these cells develops a turgor pressure that preserves the characteristic external shape of these cells. The color of the edible juicy layer can vary from white to deep red, depending on the variety. Levin (2006) reports that occasionally metaxenia is observed such that there are several seeds of different color within an individual pomegranate. The arils vary in size

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and the seeds vary in hardness among different varieties. Varieties known as seedless actually contain seeds that are soft. There is no correlation between the outer skin color of the rind and the color of the arils. These colors could be very different or similar, depending on the variety. The external outer skin color does not indicate the extent of ripening degree of the fruit or its readiness for consumption because it can attain its ﬁnal color long before the arils are fully ripened. The fruit ripens 5 to 8 months after fruit set, depending on the variety. The most pronounced difference in ripening time among cultivars is not derived from the differences in ﬂowering dates but rather from the time required to ripening from anthesis. E. Juvenility and Age of Fruiting The pomegranate has a relatively short juvenile period compared to other fruit trees, such as citrus, members of the Rosaceae, and nuts. When grown from seeds, a small proportion of pomegranate seedlings will develop ﬂowers in their ﬁrst year of growth (Terakami et al. 2007; Holland et al. unpubl.). In their second year, these plants will bear fruits (Fig. 2.1a,b). Most seedlings will ﬂower and bear fruit in their second or third year. The fruit color characteristics of juvenile plants will stay similar to those of mature pomegranate trees, although the ﬁrst-year fruits are usually smaller. The ability to ﬂower and to bear fruit in very young seedlings was also noted in ‘Nana’, a dwarf type (Terakami et al. 2007). It is noteworthy, however, that there is a physiological difference between young plants established from seeds (juvenile) and young plants established from cuttings of mature plants. Among perennial plants, the time required for seedlings to ﬂower is not necessarily identical to the time required for young plants established from cuttings of mature plants. In pomegranates, these two physiologically different periods last for a similar duration, while in other species, the time length required for ﬂowering could vary dramatically. In citrus, for example, the juvenile period for seedlings may extend up to 5 to 7 years while trees prepared by grafting of mature cuttings will set fruit in about 3 years.

III. ORIGIN AND GENETIC RESOURCES A. Origin and Cultivating Regions Wild pomegranates are growing today in central Asia from Iran and Turkmenistan to northern India. Pomegranate is considered as native

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Fig. 2.1. (a) Six-month-old seedling of ‘Nana’ var. bearing fruits; (b) Fruit-bearing F1 population in their second year.

to these regions. N.I. Vavilov stated that the pomegranate origin is in the Near East. A.P. de Candolle determined Iran and its surrounding as its origin (Goor and Liberman 1956). Goor and Liberman (1956) deﬁned southwest Asia as the pomegranate origin. Culture of pomegranate began in prehistoric times. It is estimated that pomegranate domestication began somewhere in the Neolithic era (Levin 2006; Still 2006). Pomegranates are thought to have been domesticated initially in the Transcaucasian-Caspian region and northern Turkey (Zohary and Spiegel-Roy 1975; Harlan 1992). Evidence for using pomegranates in the Middle East is dated at over 5,000 years ago. Pomegranate artifacts and relics dating to 3000 BCE and on were found in Egypt, Israel Armenia, and Mesopotamia (Goor and Liberman 1956; Still 2006; Stepanyan 2007). Carbonized fragments of pomegranate rinds dating from early Bronze Age were found in Jericho and Arad, Israel (Still 2006), in Nimrod, Lebanon (Still 2006), in Egypt (Still 2006), and in

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Armenia (Stepanyan 2007). Pomegranates were introduced throughout the Mediterranean region to the rest of Asia to North Africa and to Europe. They traveled to the Indian peninsula from Iran about the ﬁrst century CE and were reported growing in Indonesia in 1416. The Greeks and the successor empires distributed the pomegranate all over Europe. Spanish sailors brought pomegranates to the New World, and Spanish Jesuit missionaries introduced pomegranates into Mexico and California in the 1700s (Goor and Liberman 1956; Morton 1987). The ability of pomegranate trees to adjust to variable climatic conditions is reﬂected in the wide distribution of the wild form throughout Eurasia to the Himalayas (Levin 2006). The optimal climatically growth conditions for pomegranate exist in Mediterranean-like climates. These include high exposure to sunlight; mild winters with minimal temperatures not lower than 12 C; and dry hot summers without rain during the last stages of the fruit development (Levin 2006). Under such conditions, the fruit will develop to its best size and optimal color and sugar accumulation without the danger of splitting. Pomegranate is cultivated today throughout the world in subtropical and tropical areas in many different microclimatic zones. Commercial orchards of pomegranate trees are now grown in the Mediterranean basin (North Africa, Egypt, Israel, Syria, Lebanon, Turkey, Greece, Cyprus, Italy, France, Spain, Portugal) and in Asia (Iran, Iraq, India, China, Afghanistan, Bangladesh, Myanmar, Vietnam, Thailand; and in the former Soviet republics: Kazakhstan, Turkmenistan, Tajikistan, Kirgizstan, Armenia, and Georgia). In the New World, pomegranates are grown in the United States and Chile. New orchards are now established in South Africa, Australia, Argentine, and Brazil. B. Collections and Germplasm Pomegranate collections of wild and domesticated accessions were reported to be in Asia, Europe, North Africa, and North America. Still (2006) based on data of Frison and Servinsky (1995) lists the sites and numbers of pomegranate accessions in Albania, Cyprus, Italy, Spain, France, Germany, Hungary, Israel, Portugal, Russia, Tunisia, Turkey, Turkmenistan, Ukraine, the United States and Uzbekistan. Mars (2000) adds India, Morocco, Greece, Egypt, and Tajikistan. The larger collections are in Garrygala, Turkmenistan, and St. Petersburg, Russia (Still 2006). Four important centers not mentioned in these lists include the Iranian collection in Tehran, Saveh, Yazd, and Markazi (Fadavi et al. 2006; Zamani et al. 2007) and China’s collection. The reported pomegranate germplasm collections are listed in Table 2.1. The accessions

Table 2.1. Pomegranates germplasm collections in the world.

Country Azerbaijan China China India India

Iran

Iran Israel

Russia

Tajikistan Thailand Turkmenistan

Tunisia Turkey Turkey

Turkey

Ukraine Ukraine U.S.A.

Uzbekistan Uzbekistan

Location

No. accessions

Reference

Unknown Different provinces Yunnan 3 collections (unknown locations) National Bureau of Plant Genetic Resources Regional Station, Phagli, Shimla Agricultural research stations of Saveh (Markazi province) and Yazd (Yazd province) Yazd Newe Ya’ar Research Center, Agricultural Research Organization, Yizre’el Valley N.I. Vavilov Research Institute of Plant Industry, St. Petersburg Unknown 5 locations in Chiang Mai, 1 in Bangkok Turkmenian Experimental Station of Plant Genetic Resources, Garrygala 2 collections, 1 in Gabes, South Tunisia Alata Horticultural Research Institute, Erdemli Plant Genetic Resources Department, Agean Agricultural Research Institute, Izmir Cukurova Universiy, Adana

200–300 238 At least 25 At least 30 each 90

Levin 1995 Feng et al. 2006 Yang et al. 2007 Mars 2000

More than 100

Fadavi et al. 2006

About 760 67

Zamani et al. 2007 Bar-Ya’akov et al. 2003, 2007

800

Frison and Serwinski 1995

200–300 29

Levin 1995 Thongtham 1986

1,117

Levin 2006

63

Mars and Marrakchi 1999 Onur 1983; Onur and Kaska 1985 Frison and Serwinski 1995

Unknown Nikita Botanical Gardens, Yalta, Crimea U.S. National Clonal Germplasm Repository, Davis, CA Unknown Schroeder Uzbek Research Institute of Fruit Growing, Viticulture, and Wine Production, Tashkent, Glavpochta

200–300 370

More than 70 158

33

Rana et al. 2007

Ozguven et al. 1997; Ozguven and Yilmaz 2000 Levin 1995 Yezhov et al. 2005

Almost 200

Stover 2007; USDA 2007

200–300 Unknown

Levin 1995 Zaurov et al. 2004

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include pomegranate trees that are classiﬁed wild, semiwild, or cultivated. In this review, cultivars are deﬁned as cultivated pomegranates that have been selected and have a name. Varieties are all other pomegranates types for which there is no information concerning their cultivation. The origin of pomegranate is considered by some authors to be in Central Asia, parts of which are in Iranian territory. The Iranian collection is of special interest since it is expected to contain some of the more diverse pomegranate varieties. Talebi Baddaf et al. (2003) reported low variability among Iranian genotypes using random ampliﬁcation of polymorphic DNA (RAPD). However, RAPD analysis of 24 Iranian cultivars reported by Zamani et al. (2007) indicated a high level of polymorphism among genotypes. The Indian collection is also interesting since wild pomegranates were reported to be grown on the slopes of the Himalayas and because most of the evergreen pomegranate cultivars reported today originate from India (Singh et al. 2006). Thus, it is expected that pomegranate germplasm from India might include highly genetically diverse pomegranate varieties. Previous reports indicate at least three Indian collections of pomegranate germplasm (Gulick and Van Sloten 1984; Mars 2000). A survey of wild pomegranates from western Himalaya was recently reported by Rana et al. (2007). They report on a highly diverse collection of 90 accessions that is being conserved and characterized in the National Bureau of Plant Genetic Resources Regional Station ﬁeld gene bank. In China, there are several reports of diverse germplasm resources (Feng et al. 2006; Yang et al. 2007). Of 238 cultivars grown in different provinces, 50 are recently bred (Feng et al. 2006). One of these collections is in Yunnan (Yang et al. 2007). RAPD analysis of 25 pomegranate accessions showed that their genetic background is complex and difﬁcult to classify. The RAPD data disagreed with the traditional taxonomy based on ﬂavor, petal color, and skin and aril color. The collection from Garrygala, Turkmenistan, is of special interest since it contains specimens collected from a geographical region that is a part of the central Asian region considered as the origin of pomegranate. This collection contains wild material (Levin 1981) as well as pomegranate cultivars collected from cultivated regions. The high morphological diversity among its specimens includes dwarf and decorative specimens and accessions varieties that differ in shape, color, resistance to splitting, date of ripening, taste, juice content, and seed size (Levin 1994). The collection in Garrygala contains also

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specimens from Transcaucasia and foreign countries such as Spain, the United States, Iran, Tajikistan, and India. Levin (1995) reports the establishments of 200 to 300 accessions for collection in each country in Azerbaijan, Ukraine, Tajikistan, and Uzbekistan. Mirzaev et al. (2004) report on the Uzbek pomegranate collection located in the Schroeder Uzbek Research Institute of Fruit Growing, Viticulture, and Wine Production in Tashkent. Yezhov et al. (2005) report on a collection of 370 accessions in the Nikita Botanical Gardens in the Ukraine established by Nina K. Arendt. This collection includes accessions from central Asia, Transcaucasia, Iran, Afghanistan, Spain, Italy, and the United States. Despite the high diversity expected from central Asian pomegranate varieties, ampliﬁed fragment length polymorphism (AFLP) analysis of 65 pomegranate accessions representing the central and west Asia regions as well as Russia and the United States suggested a narrow variation and lack of signiﬁcant genetic divergence (Aradhya et al. 2006). Consequently Yilmaz et al. (2006) have also found narrow genetic base in pomegranate genotypes selected from different regions of Turkey using RAPD analysis. In Turkey, a collection of more than 180 accessions was established in Alata Horticultural Research Institute. This collection contains genotypes from the Mediterranean, Aegean, South Eastern, and Bitlis Turkish regions. Onur (1983) and Onur and Kaska (1985) report on 72 cultivars. Ozguven et al. (1977 Ozguven and Yilmaz 2000) report on 33 cultivars in Cukurova University Adana, and Frison and Serwinski (1995) report on 158 pomegranate accessions in Izmir. Wild pomegranate survey and morphological analysis on populations from the southeast regions of Armenia was reported by Stepanyan (2007). However, it is unclear whether a live collection was established. In Tunisia, Mars and Marrakchi (1999) reported the establishment of two collections containing 63 accessions that represent 20 local landraces collected from different growing regions in Tunisia. The Tunisian collection was reported as highly divergent (Mars and Marrakchi 1999; Mars 2000). Genetic analyses of Tunisian cultivars report high polymorphism using RAPD and AFLP analyses (Hasnaoui et al. 2006; Jbir et al. 2006). A collection of 29 cultivars was established in Thailand (Thongtham 1986). Only ﬁve specimens of this collection are from Thailand; the rest are from India, the United States, Israel, Russia, Iran, Spain, and Italy. The Israeli collection of pomegranate in Newe Ya’ar Research Center contains 67 accessions. Most of the specimens in the collection are from Israel, including semiwild accessions, and the rest are introduced

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from the United States, Spain, China, India, and Turkey. All of these accessions are grown today in a single plot in Newe Ya’ar Research Center to validate their morphological characteristics excluding the effects of microclimatic conditions. All of the accessions in the Israeli collection were analyzed for their phenological and morphological characteristics (Assaf et al. 1991a; Bar-Ya’akov et al. 2003; Bar-Ya’akov et al. 2006). Some of the data are documented in the Israel Gene Bank for Agricultural Crops in the Agricultural Research Organization in Bet Dagan (http://igb.agri.gov.il/). The Israeli germplasm collection was analyzed for its content of antioxidant constituents. A comparative study among different specimens of the collection was reported on the levels of ellagic acid, punicalagin, punicalin, and galagic acid, the major contributors to antioxidant activity (Tzulker et al. 2007). The chemical analysis was followed by a genetic analysis based on inter-small sequence repeats (ISSR) technique in order to characterize the 36 genotypes of the collection. Although it was possible to demonstrate a substantial amount of polymorphism among the Israeli genotypes, additional techniques such as SSR and AFLP should be developed for pomegranates to signiﬁcantly advance the study of pomegranate divergence and evolutionary relationships. As pointed out by Still (2006), much more sophisticated and elaborate research in pomegranate genomics will be required to reliably assess evolutionary relationships among different pomegranate accessions and relate genetic markers to morphological characteristics. It is highly probable that there is considerable redundancy among accessions. The U.S. National Clonal Germplasm Repository in Davis, California, has almost 200 pomegranate accessions. The collection includes accessions from all over the world including Turkmenistan, Russia, Iran, and Japan (Stover 2007; USDA 2007) A signiﬁcant proportion of the accessions is comprised of pomegranate cultivars introduced from foreign countries. It is expected that in many cases, the true origin of the cultivars is obscured. Clearly the Davis, California, collection, the Garrygala, Turkmenistan, collection, the Newe Ya’ar, Israel, collection, and most probably other collections contain specimens that originated from other countries. Levin indicated that selected accessions from his collection were distributed to the United States and Israel (Mars 2000; Levin 2006; Stover 2007). The Thai collection and the Indian collection also were reported to contain accessions from other countries. In the local collection in Israel, for example, one can ﬁnd accessions that have the same name but are actually different varieties (homonyms), and likely there are instances where identical cultivars are called by different names

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(synonyms). Efforts toward eliminating redundancy within the collections of each country and between countries will help to assess the divergence among pomegranate accessions. In this respect, the evolvement of landraces of introduced foreign accessions in the new hosting country should be considered.

IV. HORTICULTURE A. Cultivars Interesting pomegranate cultivars were reported from several locations all over the world, including Europe (Spain, France, Italy, Greece, and Cyprus), Asia (Turkey, Turkmenistan, Kirgizstan, Azerbaijan, Iran, India, China, Russia, Israel), and North Africa (Morocco, Tunisia, Egypt). The botanical differences between wild pomegranates and cultivated pomegranates are not obvious except for P. protopunica. Pomegranate cultivars were spread throughout different regions and continents, and it is probable that some of the pomegranate cultivars acquired different names in different countries and are in fact the same basic genotypes. On several occasions, a clue to the origin of the cultivar is embedded within its name. For example, the cultivars ‘Kaboul’ or ‘Kandahary’ in the Indian collections hint to their possible origin from from the Afghan cities of Kabul and Qandahar. Similarly in the collection from Turkmenistan, one can ﬁnd names such as ‘Afghansky’, ‘Washingtonsky’, ‘Iran 29-3’, and ‘Kalifornijsky’ (Levin 1996). The name of pomegranate in Chinese is ‘‘An Shi Liu’’, which means ‘‘the fruit of Kabul’’, reﬂecting its origin in Afghanistan (Fazzioli and Fazzioli 1990). Despite the difﬁculty in assessing the authenticity of cultivars and their distinguishing characteristics, some cultivars are clearly distinguished. Different cultures favor different fruit characteristics, and cultivar selection reﬂects these differences. For example, in India, most people dislike acidic fruit, and nonacidic cultivars have been selected. In Israel, most people originating from western European countries prefer sweet-sour cultivars, such as ‘Wonderful’. Israelis originating from Middle East countries usually prefer nonacidic cultivars with very soft seeds, such as ‘Malisi’. Thus, part of the variability in pomegranate cultivars in the world is a reﬂection of the different tastes and priorities in each country. Most of the cultivars known today are selections from an unknown origin, mostly chance seedlings or mutations collected from places where there are no records documenting their origin. However, some

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cultivars are the result of deliberate crosses. Such cultivars were reported particularly from India (Keskar et al. 1993; Samadia and Pareek 2006), China (Zhao et al. 2006; Yang et al. 2007), and to a smaller extent Israel (Holland et al. 2006; Bar-Ya’akov et al. 2007). In these countries, active breeding programs were reported, and are described in Section V. 1. India. Some of the better-known Indian cultivars (‘Ganesh’, ‘Mridula’, ‘Bhagwa’) share common characteristics. These include sweet ﬂavor low-acid, small to medium fruit size, and thin rind. Unlike pomegranate cultivars from other countries, quite a signiﬁcant proportion of Indian cultivars originate from active breeding programs. ‘Ganesh’ is perhaps the best-known Indian cultivar (Purohit 1986; Sonawane and Desai 1989; Aulakh 2004; Singh 2004). This evergreen cultivar has very soft seeds. The arils are red and the taste is low-acid and sweet. The highest yield of marketable fruits is in January, but cropping could be achieved in October, March-April, May-June, June-August, or July-September (Sonawane and Desai 1989). The skin color of ‘Ganesh’ is green to orange-yellow, depending on the season, and the fruit size is small. However, fruit thinning can enhance the fruit size signiﬁcantly and fruit above 350 g is achievable. The yield and juice content of ‘Ganesh’ is good (Aulakh 2004; Singh 2004), but depend on whether the trees are grown under intensive agricultural conditions or under inadequate agricultural treatment. The yellow-green color of ‘Ganesh’, its tendency to split, and its relatively lower quality are reasons why it is not used a great deal as an export cultivar. ‘Ganesh’ was extensively used in India for breeding and crosses with other cultivars, such as ‘Kabul’ (large fruit, yellow red skin, sweet, hard seeds); ‘Jyoti’ and ‘Bedana’ (medium-size fruit, brownish skin, sweet, soft seeds) (Nageswari et al. 1999); ‘Nana’ and ‘Kabul Yellow’ (Jalikop 2003; Jalikop et al. 2005). Two Indian cultivars, ‘Mridula’ (‘Arkta’) and ‘Bhagwa’ (‘Kesar’) (Vasantha Kumar 2006), are most extensively used for export, particularly to Europe. These cultivars have an appealing red skin and aril color and are soft seeded. Their taste is low-acid, sweet with a relatively small size (200 to 300 g). The rind is relatively thin, which is a weakness, because they are amenable to physical damage. ‘Bhagwa’ is more prone to physical damage. ‘Mridula’, ‘Bhagwa’ and ‘Ganesh’ are evergreen cultivars. They are exported to Europe usually in January–February.

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The enormous area of the Indian peninsula and its very divergent climatic zones clearly require cultivars that are adapted to each of the regions. Additional Indian cultivars include ‘Alandi’, ‘Muskat’ (Wavhal and Choudari 1985; Purohit 1986), ‘Jalore’, ‘Jodhpur Red’, ‘Dholka’ (large fruit, yellow red skin, sweet, hard seeds, evergreen), ‘Bassein’, ‘Malta’, ‘Kandhari’ (large fruit, deep red skin, pink-blood red arils, subacid, hard seeds) (Singh 2004), ‘Guleshah’, ‘Molus’, ‘Sharin’, ‘Anar’ (Aulakh 2004), ‘Jylothi’, ‘Bedana’, ‘Bosco’ (Nageswari et al. 1999), ‘Srinagar Special’ (Misra et al. 1983), ‘Chawla’, ‘Nabha’, and ‘Achikdana’ (Kumar and Khosla 2006). 2. Iran. About 760 genotypes, specimens, and cultivars were reported in the Yazd pomegranate collection (Behzadi Shahrbabaki 1997; Zamani et al. 2007). Since these specimens were brought from many provinces, synonyms or obvious similarities in appearance were observed among these specimens (Zamani et al. 2007). Zamani et al. (2007) list 24 Iranian genotypes. Varasteh et al. (2006) listed ﬁve commercially important Iranian cultivars and discussed their fruit characteristics and their potential for further breeding. ‘Malas-e-Saveh’, ‘Rabab-e-Neyriz’, ‘Malas-e-Yazdi’, ‘Sishe Kape-Ferdos’, and ‘Naderi-eBudrood’ were considered valuable (Varasteh et al. 2006). These cultivars are late ripening, medium to large size with thick red rind and red arils. In addition, several other late Iranian cultivars were noted: ‘Ardesstani Mahvalat’, ‘Bajestani Gonabad’, ‘Ghojagh Ghoni’, ‘Khazr Bardaskn’, ‘Malas Yazd’, ‘Galou Barik’, ‘Bajestan’, ‘Zagh’, ‘Shavar Daneh Ghermez’, ‘Seﬁd’, ‘Togh Gardan’, and ‘Esfahani Daneh Ghermez’ (Varasteh et al. 2006; Iran Agro Food 2007). ‘Alack’ is an early Iranian cultivar that ripens in late August to early September and is used for export. The season for this cultivar lasts until 15 September (Van der Wiel 2007a). Zamani et al. (2007) report on ‘Alak Shirin’ (sweet) and ‘Alak Torsh’ (sour); both are red, small sized, with hard seeds. ‘Maykhosh’ is a late export cultivar that can be picked until the end of December (Van der Wiel 2007a). 3. China. Chinese cultivars are characterized by a very large variability and sometimes unusual features, such as spur-type growth habit, double ﬂowers, and white ﬂowers. Chinese cultivars vary from small to very large. Taste could be sour or sweet. Some of the Chinese cultivars are very early, beginning in early August, and some are late, ending the season in November. Evergreen cultivars are also known (Dong 1997). Most Chinese cultivars are either selections from unknown origin or seedlings of known cultivars.

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‘87-Qing 7’ is an early-bearing, productive spur-type cultivar that is one of the very few published reports of a mutation in pomegranate (Liu et al. 1997). Aside from its commercial importance, ‘87-Qing 7’ and its parent ‘Qingpitian’ might be of special help for gene mapping and functional gene analysis. ‘Duanzhihong’, selected from a local orchard, is another spur-type cultivar from Xingcheng (Liu 2003). This cultivar has a compact bush; it ripens in the end of August: Fruit size is above 340 g on average with bright red skin color; arils are pinkish red. Feng et al. (1998) evaluated 30 Chinese cultivars and identiﬁed four superior cultivars: ‘Dabaitian’ (sweet, white skin, white ﬂowers), ‘Heyinruanzi’ (sweet-sour, green skin, red ﬂowers), ‘Tongpi’ (sweetsour, green skin, red ﬂowers), and ‘Bopi’ (sweet-sour, red skin, red ﬂowers). The ‘Teipitian’ cultivar was reported to be popular with a very large fruit that can reach more than 1.5 kg; skin is yellow-green and the arils are bright red (Dong and Yang 1994). Two cultivars (‘Linxuan 8’ and ‘Lintong 14’) are from the Lintong area in China: ‘Linxuan 8’ ripens in September and has soft seeds; ‘Lintong 14’ matures toward October (Sun et al. 2004). ‘Taishan Dahong’, an early cultivar, is a seedling of an unknown origin from the southern foothills of Tai Mountain, Shaanxi (Sun et al. 2004). One of the common pomegranate cultivars from Sichuan province is ‘Qingpiruanzi’ (Diao 2004), which matures in mid- to late August and has large fruit, soft seeds, and pink arils. Other cultivars with commercial importance were reported from Longyang district in Baoshan municipality (Zhou 2005). Three promising cultivars (‘Baishuijing’, ‘Chuanshiilu’, and ‘Hongshuijing’) were selected. Other cultivars from Hui Li County in the Sichuan province are ‘Ping Di’ and ‘Jian Di’; both have green skin and soft seeds (Sichuan Hui Li Pomegranates Association 2007). ‘Yushiliu 1’, ‘Yushiliu 2’, and ‘Yushiliu 3’ are red-skinned and highly adapted to sandy alkaline soils (Feng et al. 2000). From these samples, it is apparent that in China, cultivars were mostly chosen based on size, juice content, seed softness, and time of ripening. Pink color of skin and arils was not a reason for disqualiﬁcation. Chinese cultivars with unusual number of petals (‘‘double ﬂower’’) and petal color were considered suitable cultivars for ornamental purposes. The color of these cultivars varies from red-pink to pink-white, and some are fertile and produce edible fruit. Examples of double-ﬂowered selections with good eating quality include ‘Honghuachongbai’ and ‘Baihuachongbai’ (Feng et al. 1998). The double-ﬂowered ‘Mudanhua’ has peonylike ﬂowers and was noted for its long ﬂowering season, from early May to late October (Wang 2003).

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4. Turkmenistan and Tajikistan. The Turkmen collection in Garrygala is of special interest due to its size, variability, and geographical location. Levin (1996) provided a detailed report on the various qualities and characteristics of the Turkmen pomegranate fruit. The Turkmen varieties were categorized by size, taste, skin color, aril color, seed softness, productivity, tendency to split and to diseases, storage capabilities, sugar content, juice content, and time of ripening. Cultivars combining early ripening, good ﬂavor, and chemical com-position were noted (Levin and Levina 1986). Kzyl Anar’ and ‘Achik-Dona’ from the Garrygala collection were also tested in Tajikistan together with ‘Bashkalinski’, ‘Desertnyi’, ‘Shainakskii’, and ‘Podarok’ (Ivanova 1982). ‘Kzyl Anar’ has relatively small fruits (200 to 250 g), hard seeds, and red or dark-cherry arils. ‘Acik-Dona’ has a medium-size (Ivanova 1982) to large-size (Levin 1996) fruits with hard seeds and pale crimson to red arils, along with high yield. Published records of commercial Turkmen cultivars in Turkmenistan or in any other countries appear to be lacking. Some of the Turkmen cultivars were sent to Israel and to the United States (Mars 2000; Levin 2006; Stover 2007). 5. Turkey. Few reports on Turkish cultivars were published. Among the known Turkish cultivars are ‘Cekirdksiz’, ‘Ernar’, ‘Fellahyemez’, ‘Hatay’, ‘Hicaznar’, ‘Izmir 1’, ‘Izmir 1264’, ‘Izmir 1265’, ‘Janarnar’, ‘Katrbas’, ‘Lefan’, ‘Mayhos II’, ‘Mayhos IV’, ‘Silifke Asisi’, and ‘Yufka Kabuk’ (Ozguven and Yilmaz 2000; Ozguven et al. 2006). There are several selections of ‘Hicaznar’, ‘Izmir’, and ‘Silifke’ differentiated by numbers. ‘Hicaznar’ is a red cultivar that is considered a high producer. The fruit has a sweet-sour taste and hard seeds and is somewhat similar to ‘Wonderful’. ‘Lefan’ is a selection from Hatay with yellow skin, large arils, a sweet-sour ﬂavor, and very hard seeds. ‘Janarnar’ has red skin, red arils, sweet-sour ﬂavor, and hard seeds. ‘Izmir 26’ has a sweet ﬂavor. It appears that most of the Turkish cultivars are sweet-sour, and the preferred color is red. 6. Israel. Israeli pomegranate varieties are unexpectedly diverse compared to the small size of the country (Plate 2.1F). More than 50 pomegranate accessions were found in Israel. These accesssions are very divergent in their fruit external and internal appearance, growth habit, ripening time, taste, and seed softness. The outside skin color of the Israeli cultivars varies from deep purple to yellow-pink, or green. and eight are grown commercially. All the Israeli accessions were given identity numbers in recognition that synonyms and homonyms were

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likely among the names when collected. Based on comparisons in a single orchard in Newe Ya’ar certain pomegranate cultivars are very different from one another but have the same name (e.g., ‘Hershkovich’) while others that are identical got different names (e.g., ‘Wonderful’). These cultivars were identiﬁed as valuable for commercial growth: ‘P.G.116-17’, ‘Wonderful’ (‘P.G.100-1’ and ‘P.G.101-2’), ‘P.G.128-29’ (‘Akko’), ‘Shani-Yonay’, ‘Rosh Hapered’, ‘P.G.127-28’ (‘Black’), ‘P.G.118-19’ (‘Hershkovich’), and ‘Malisi’ (‘P.G.106-7’) (Plate 2.2 A–G). Traditionally, three types of pomegranate cultivars were grown in Israel, ‘Rosh Hapered’ ‘Malisi’, and ‘Red Lufani’ (‘Shara’bi’). The ﬁrst two cultivars have pink arils and their taste is sweet without any sourness. ‘Rosh Hapered’ has a large fruit, large arils, hard seeds, and pink skin. This cultivar is traditionally used in the Jewish holidays since it ripens at the end of August and in the past was considered one of the main early cultivars for export. ‘Malisi’ has soft seeds, light pink arils, and yellow-pink to green skin. ‘Malisi’ is grown only in small plots and is not used for export. ‘Red Lufani’ is a synonym of ‘Wonderful’. The main cultivar that is grown today is ‘P.G.101-2’ (‘Wonderful’). This cultivar was reported to be imported from the United States about 100 years ago (Goor and Liberman 1956). This large-size pomegranate ripens in the beginning of October. It is a sweet-sour pomegranate with red arils and skin when fully ripened. There are many ‘Wonderful’ selections in Israel. Seven were characterized in the Newe Ya’ar collection. The landraces vary in their time of ripening, their external color, the timing of skin color appearance during fruit development, and the degree of seed hardness. Among the ‘Wonderful’ landraces, ‘Kamel’ is the most colorful cultivar (Plate 2.2G). This cultivar has a full red skin color that develops much earlier than regular ‘Wonderful’. It is very productive cultivar and produces high-quality fruits. As the export market increased and demand for early red cultivar strengthened, two additional early red cultivars were introduced to commercial cultivation: ‘Akko’ and ‘Shani-Yonay’ (Holland et al. 2007). Both are soft-seeded cultivars with sweet/sourless taste and red skin color. Their appealing look and good taste makes them the leading early Israeli export cultivars. ‘Akko’ differs from ‘Shani-Yonay’ in growth habit and shape of fruit and tree. Both cultivars produce small-medium fruit of average size of 300 to 400 g. Two cultivars ‘P.G.116-17’ and ‘P.G.118-19’ (‘Hershkovich’) ripen between the late ‘Wonderful’ and the early cultivars. ‘P.G.116-17’ is today the best export cultivar. It has a large fruit, large red arils, and an appealing red skin. An additional Israeli cultivar just recently introduced to commercial growth is the black ‘P.G.127-28’. This cultivar has a deep purple-black skin with red

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Plate 2.2. Pomegranate cultivars. A. ‘Rosh Hapered’; B. ‘P.G.127-28’; C. ‘P.G.116-17’; D. ‘P.G.118-19’ (‘Hershkovich’); E. ‘Wonderful’; F. ‘Shani-Yonay’; G. ‘Kamel’; H. ‘P.G.128-29’ (‘Akko’); I. ‘Emek’. (See insert for color representation of this plate.)

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soft-seeded arils. It produces a small fruit that matures in November in Newe Ya’ar conditions and is the latest ripening cultivar tested. The unusual skin color, the very late ripening time, and the soft red arils make ‘P.G.127-28’ an appealing late cultivar. 7. Spain. At least 40 Spanish cultivars were reported in the literature. Melgarejo divides these cultivars into three groups: sweet, sweet-sour, and sour (Melgarejo et al. 2000). Some of the common commercial cultivars include ‘Mollar de Elche’ and its selections ‘ME1’ (‘Mollar de Elche No. 1’), ‘ME5’, ‘ME6’, ‘ME14’, ‘ME15’, ‘ME16’, and ‘ME17’, ‘Agria de albatera’, ‘Agria de Blanca’, ‘Agridulce de Ojos’ (‘ADO’), ‘Albar de Bianca’ (‘BA’), ‘Borde de Albatera’ (‘BA’) and its selection ‘BA1’, ‘Borde de Blanka’ (‘BB’), ‘Casta del Reino de Ojos’ (‘CRO’) and its selection ‘CRO1’, ‘Mollar de Albatera’ (‘MA’) and its selection ‘MA4’, ‘Mollar de Orihuela’ (‘MO’) and its selection ‘MO6’, ‘Pinon Duro de Ojos’ (‘PDO’), ‘Pinon Tierno Agridulce de Ojos’ and its selections ‘PTO1’ (‘Pinon Tierno de Ojos No. 1’), ‘PTO2’ and ‘PTO7’, ‘San Felipe de Bianca’ (‘SFB’), and ‘Valencian No. 1’ (‘VA1’) (Melgarejo et al. 1995, 2000 Hernandez et al. 2000; Legua et al. 2000a, 2000b; Martinez et al. 2006). Some Spanish cultivars like ‘Mollar de Elche’, ‘Borde de Albatera’, ‘Pinon Tierno de Ojos’, ‘Casta del Reino de Ojos’, and others have several selections or clones, which are actually landraces. A different number associated with the cultivar name indicates the different landraces within each cultivar (Melgarejo et al. 1995, 2000; Hernandez 2000; Legua et al. 2000a, 2000b). The best-known Spanish cultivar is the landrace ‘Mollar de Elche’, which produces sweet fruit with soft seeds. The outside color is pink-red and the arils are red. ‘Mollar de Elche’ ripens in October-November. Martinez et al. (2000) indicate that ‘ME14’ and ‘ME15’ have the highest yield. Another sweet cultivar is ‘Valencian’. ‘Agridulce de Ojos’ and ‘Pinon Tierno de Ojos’ are sweet-sour (Melgarejo et al. 2000), and ‘Agria de Albatera’, ‘Agria de Blanca’ (Melgarejo et al. 1995), ‘Borde de Albatera’, and ‘Borde de Blanca’ are sour (Melgarejo et al. 2000). ‘PTO2’ and ‘CRO1’ have shown a high juice content (Martinez et al. 2006). Many of the Spanish cultivars have been subjected to detailed studies of their sugar and organic acid content (Legua et al. 2000; Melgarejo et al. 2000), oil content of their seeds, and fatty acid composition of the oil seed (Melgarejo et al. 1995; Hernandez et al. 2000). It was found that citric acid and malic acid were the predominant organic acids (Melgarejo et al. 2000). The predominant fatty acid in the oil seed was polyunsaturated (n-3) linolenic acid followed by oleic acid (Melgarejo et al. 1995).

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Under the conditions of Newe Ya’ar, Israel, the Spanish cultivars ‘ME17’, ME20’, ‘VA1’, and ‘PTO1’ were found to have a pink-yellow skin color and light-pink aril color. Overall, the colors of the Spanish cultivars in Israel were poor and unattractive compared to their colors in Spain. ‘ME’ selections ripen at the beginning of October in Israel. It appears that the examined Spanish cultivars are sensitive to their environmental conditions with respect to color intensity. 8. United States. Pomegranates in the New World probably were imported by travelers from Europe. There is a relatively limited number of pomegranate cultivars in the United States ‘Wonderful’, the most important cultivar, originated in Florida and was discovered in Porterville, California, about 1896 (LaRue 1980). This cultivar is the most widely planted commercial pomegranate cultivar in California. The fruit is a large with red arils, sweet-sour taste, and semihard seeds, and it ships well. The external appearance of the fruit is very appealing with red glossy color. The several Israeli landraces of ‘Wonderful’ are either ‘Wonderful’ seedlings (most likely) or sports. It is unclear whether the American ‘Wonderful’ is genetically distinguishable from any of the Israeli ‘Wonderful’ landraces. The American ‘Wonderful’ fruit is much harder and less prone to mechanical aril extraction than the Israeli landraces, but these differences could reﬂect variations in growth condi-tions. ‘Wonderful’ is also grown in western Europe and Chile (Stover and Mercure 2007). ‘Early Wonderful’ is a sport of ‘Wonderful’ found in California (Stover and Mercure 2007). It ripens about two weeks before ‘Wonderful’ and acquires a red skin color much earlier than the original one (California Rare Fruit Growers Inc. 1997). The quality of the juice is inferior to the original ‘Wonderful’. Another commercial cultivar is ‘Early Foothill’, an early cultivar with red skin and aril color. It is much smaller than ‘Wonderful’ and its fruit quality is not as good. A cultivar grown in the United States to a much lesser extent is ‘Granada’, a ‘Wonderful’ sport (Stover and Mercure 2007). This early-maturing cultivar ripens in mid-August. The quality of the fruit and juice is not considered as good and its commercial value is limited. ‘Ruby Red’ is a cultivar with similar size and ripening time as ‘Wonderful’ but stores less well than ‘Wonderful’. Other cultivars grown to a limited extent are ‘Balegal’, ‘Cloud’, ‘Fleshman’, ‘Crab’, ‘Francis’, ‘Green Globe’, ‘Home’, ‘King’, ‘Phoenicia’, ‘Sweet’, and ‘Utah Sweet’ (California Rare Fruit Growers 1997). Several ornamental pomegranate cultivars, such as ‘California Sunset’, are being sold in the United States. At least two originated in Japan. These include the

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‘‘double-ﬂower’’ cultivars: ‘Nochi Shibori’ and ‘Toyosho’, according to the Davis depository list (USDA 2007). 9. Georgia. Several cultivars were reported in Georgia, including ‘Pirosmani’, ‘Gruzinskii No. 1’, ‘Gruzinskii No. 2’, ‘Vedzisur’i, ‘Lyaliya’, ‘Tengo’, ‘Imeretis Sauketeso’, ‘Bukistsikhe’, ‘Khorsha’, ‘Zugdidi’, ‘Erketuli’, ‘Forma No. 1’, ‘Forma No. 15’, ‘Forma No. 70’, ‘Shirvani’, ‘Apsheronskii Krasnyi’, ‘Burachnyi’, ‘Rubin’, ‘Frantsis’ ‘Sulunar’, ‘Kyrmyz Kabukh’, ‘Shiranar’, Shakhanar’, and ‘Gyuleisha Krasnaya’ (Trapaidze and Abuladze 1989; Alkhazov and Chakvetadze 1991; Vesadze and Trapaidze 2005). These cultivars were noted for their resistance to splitting: ‘Apsheronskii Krasnyi’, ‘Burachnyi’, ‘Frantsis’, ‘Kyrmyz Kabukh’, ‘Lyaliya’, ‘Pirosmani’, ‘Rubin’, ‘Shirvani’, and ‘Verdzsuri’ (Trapaidze and Abuladze 1989; Vesadze and Trapaidze 2005). The highest juice content was found in ‘Sulunar’, ‘Pirosmani’, ‘Vedzisuri’, and ‘Imeretis Sauketeso’ (Vesadze and Trapaidze 2005). 10. Tunisia. Almost all the pomegranate fruits produced in Tunisia are consumed locally, and the cultivars grown in traditional orchards are not of the best quality. Just a few local cultivars are planted in new orchards (Mars and Marrakchi 1999). Among the Tunisian cultivars one can ﬁnd ‘Gabsi’ (the main cultivar, sweet); ‘Tounsi’ (sweet, late ripening); ‘Zehri’ (sweet, ripens end of August or beginning of September); ‘Cheﬂi’ (sweet, poor skin color, big nice arils); ‘Mezzi’, ‘Jebali’, ‘Garoussi’ (sweet-sour, green skin); ‘Garoussi’; ‘Kalaii’ (sweet, poor skin color, big nice arils); ‘Zaghouani’; ‘Andalousi’ (sweet); and ‘Bellahi’ (Mars and Marrakchi 1999; F. Abed Elhadi pers. commun.) 11. Egypt. Four Egyptian cultivars were documented in the literature: ‘Arabi’, ‘Manfaloty’, ‘Nab ElGamal’, and ‘Wardy’ (Abu-Taleb et al. 1998; Saeed 2005). ‘Manfaloty’ was more sensitive to salt stress while ‘Nab ElGamal’ was the best with respect to loss of chlorophyll in response to elevated salt concentration in the irrigation water (Saeed 2005). ‘Manfaloty’ (or ‘Manfaloot’) has large, juicy dark-red arils and ripens from the end of August or the beginning of September (Van der Wiel 2000b). Apart from these cultivars, ‘Granada’ is used in Egypt as an early cultivar. It is unclear whether the Egyptian ‘Granada’ is identical to the American ‘Granada’, although its early ripening season suggests that they are similar (Van der Wiel 2000b). 12. Saudi Arabia and Iraq. Very little information is available on pomegranates from Saudi Arabia and Iraq. ‘Ahmar’ (red), ‘Aswad’ (black),

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and ‘Halwa’ (sweet) were reported as important in Iraq and ‘Mangulati’ in Saudi Arabia (Morton 1987). 13. Vietnam. ‘Vietnamese’ is an evergreen cultivar from Vietnam. It has orange ﬂowers, bright red skin color, and small and juicy arils (Jene’s Tropical Fruit 2006). 14. Morocco. About 17 pomegranates clones and cultivars were reported by Oukabli et al. (2004) from Meknes, including ‘Gjeigi’, ‘Dwarf ever Green’, ‘Grenade Jaune’, ‘Gordo de Javita’, ‘Djeibali’, and ‘Onuk Hmam’. 15. Sicily, Italy. Six Sicilian pomegranate selections were reported by Barone et al. (2001): ‘Dente di Cavallo’, ‘Neirana’, ‘Profeta’, ‘Racalmuto’, ‘Ragana’, and ‘Selinunte’. The local accessions were considered less attractive than the Spanish cultivars. B. Irrigation Although pomegranates enjoy heat and thrive in arid and semiarid areas, they need regular irrigation throughout the dry season to reach optimal yield and fruit quality (Sulochanamma et al. 2005; Levin 2006; Holland et al. unpubl.). The pomegranate fruit requires heat for its development. Sulochanamma et al. (2005) found that drip irrigation had positive effects on pomegranate growth parameters such as tree height, stem diameter, and plant spread. Positive effect was also noted on fruit yield and fruit weight (Prasad et al. 2003; Shailendra and Narendra 2005; Sulochanamma et al. 2005). In most growing areas where commercial growth of pomegranates is practiced, some sort of irrigation is required. In Israel, irrigation usually starts in late April and lasts throughout the summer, producting yields of 25 to 45 t/ha. Similar data are reported from California. Drip irrigation is used most commonly in these orchards, although some growers prefer sprinklers (which cause difﬁculties in weed control). Most of the large commercial orchards in Israel, India, and the United States utilize drip irrigation methods. In experiments done in India and Iran, drip irrigation saved up to 66% of water compared to surface irrigation (Behnia 1999; Chopade et al. 2001). The total amount of water for pomegranate irrigation in Israel for the entire season is 5,000 to 6,000 m3/ha, depending on the type of soil and the weather conditions. Daily irrigation is practiced during the irrigation season. The amount of daily irrigation is calculated as

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percentage of daily water loss measured by evaporation from Class A evaporation pan. The percentage of water compensation varies according to ﬁeld conditions. Computerized irrigation yields better results and allows for better control of water quantities and time intervals between successive water applications. Computerized irrigation is of special importance when fertilization and other treatments are applied through the water. There are few reports on the effect of irrigation levels and the time and interval of water application on yield and quality of pomegranate fruit. Control of irrigation timing and seasonal application are important not only for better growth and yield of the pomegranate trees but also are used to control time of ripening. For example, in India, timing of irrigation is used to control and optimize the yielding season of evergreen pomegranates (Sunawane and Desai 1989). By applying different irrigation regimes, it was possible to direct the desirable time of fruit yield in Indian pomegranates. In view of the global warming phenomenon and the increasing water shortage experienced in many arid and semiarid regions that are the most suitable regions for pomegranate growth, water availability and irrigation are of major considerations in pomegranate culture. Therefore, many more efforts will be required to develop optimal and effective irrigation methods that are suitable for pomegranate growth. One direction toward this goal is the development of a computer program for calculating pomegranate drip irrigation. This program calculates the irrigation and fertilizer requirements (Gimenez et al. 2000). One of the most important issues concerning pomegranate irrigation is the ability to use alternative water sources, particularly recycled water and saline water. Usage of recycled water is strongly connected to salinity since quite often salinity increases in recycled water (Raviv et al. 1998). Pomegranates are amenable to irrigation with saline water. In Israel, several desert orchards in the Negev Highlands and in the southern Arava are irrigated with saline water. The level of salinity in the water of these orchards ranges between 2.5 to 4.0 dS/m. Under these conditions in Israel, Israeli and Turkmen cultivars grew to produce normal yield and fruit qualities without apparent damages on the trees. Production using saline waters requires constant irrigation to leach the salt and prevent the detrimental effects of increased salinity. One of the side effects of such practice is higher vegetative growth, which should be controlled in trees that grow too fast. Pomegranate trees were irrigated with 4,000 and 6,000 parts per million (ppm) saline water. Under these conditions, the saline water negatively

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affected various vegetative growth factors. Differences among different cultivars were observed, and application of Paclobutrazol was reported to reduce salinity damage (Saeed 2005). Salinity tolerance among 10 commercial Iranian cultivars in pots was reported by Tabatabaei and Sarkhosh (2006). In this experiment, the authors indicate pronounced differences to irrigation with saline water among pomegranate cultivars. The mechanisms responsible for pomegranate tolerance to saline water are not yet fully understood. However, it is well documented that pomegranate tissues accumulated sodium, chlorine, and potassium in response to irrigation with saline water and that the concentration of these ions was increased with increased concentrations of salt in the irrigation water (Doring and Ludders 1987; Naeini et al. 2004, 2006). These authors indicated tolerance to saline water up to concentration levels of 40 mM NaCl in the water. Above this concentration, growth parameters such as the length of the main stem, length and number of internodes, and the area of leaf surface were severely affected (Naeini et al. 2006). The data just mentioned suggests that the pomegranate tolerance to salinity is due to resistance of its tissues to higher levels of salt rather than ability to prevent penetration of ions into its tissues. Recycled water is now relatively abundant in several regions in Israel. Some pomegranate orchards are irrigated with recycled water after secondary or tertiary treatment. It appears that pomegranate trees respond well to irrigation with recycled water. Positive response to irrigation with recycled water was also reported by Levin (2006) in pomegranate orchards in Turkmenistan. As high-quality water becomes less available and more expensive, it is expected that recycled water will become a common irrigating practice in arid areas. C. Fertilization The available data on pomegranate fertilization is very limited. Most of the reported fertilization experiments were conducted in India and few in Turkey and Iran. A common practice in Israel is to supply fertilizers via the irrigation system (Blumenfeld et al. 2000). When surface irrigation combined with fertilizers was compared to drip irrigation with solid soluble fertilizers, the drip irrigation proved to be the better treatment (Firake and Kumbhar 2002). In Israel, the recommended quantity for nitrogen is 200 kg/ha and for potassium (potassium oxide) is 300 kg/ha (Kosto et al. 2007). About 60 kg/ha of phosphorus (phosphorus pentoxide) is recommended.

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Nitrogen is applied with the beginning of growth through the entire irrigation period up until two weeks before harvest. However, additional nitrogen fertilization is supplied after harvest in early-ripening cultivars. Excessive or late applications of nitrogen may delay fruit maturity and color development (LaRue 1980). Potassium is applied throughout the irrigation season. Phosphorus is applied as phosphoric acid or in complete fertilizer mixtures. When phosphoric acid is used, applications are in two phases, the ﬁrst at the beginning of the season and the second at the end. The last application is also used to clean the irrigation system. When a complete fertilizer mixture is used, it is applied throughout the nitrogen fertilization season (Kosto et al. 2007). Foliar application of potassium chloride and potassium sulfate for maintaining optimal levels of potassium were reported by Muthumanickam and Balakrishnamoorthy (1999). Microelements, such as zinc, iron, and manganese, applied on leaves have resulted in increased yield and juice content (Balakrishnan et al. 1996). A crucial step toward more educated application of fertilizers is to determine the standard levels of macro- and microelements in pomegranate leaves of important commercial cultivars. In Israel, such data are not yet available; however, a survey of four different orchards resulted in these values: 1.99% N, 0.22% P, 1.07% K, 2.97% Ca, 0.25% Mg, 0.02% Na, 0.76% Cl, 23 ppm B, 75 ppm Fe, 33% Mn, 11 ppm Cu (F. Abed Elhadi, pers. commun.). Prasad and Mali (2003) have shown that the ratio of aril weight to total fruit weight is linearly correlated with the rates of supplied nitrogen while total soluble solids (TSS) were not affected. The dependence of yield, fruit weight, aril number, aril volume, pH, acidity, and TSS on nitrogen, manganese, and potassium were studied by Panahi and Amiri (2006) in Iran. It was shown that potassium application increased fruit weight, signiﬁcantly increasing the yield. D. Tree and Orchard Design Unlike most other fruit trees, use of rootstocks with pomegranates is not a common practice. Pomegranates are very easy to root from cuttings, and this is the major method for pomegranate propagation. Orchard establishment can be done by directly planting the cuttings in the soil or by planting potted nursery trees. The latter method sometimes is preferred because it assures a better uniformity and establishing success of the trees. Pomegranates are bushy plants that tend to produce multiple suckers which sprout from the stem either underground or aboveground. The

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traditional way of growing pomegranates is the multiple trunk method. In this practice, the tree is allowed to develop 3 to 5 main trunks that sprout from the ground level. The branches are trained to grow as an open vase (Blumenfeld et al. 2000). The height of the tree is typically maintained below 4 m. This method helps maintain productive branches through many years and helps to cope with pathogens inﬂicted by stem borers, such as Euzophera sp. When a branch is lost, a shoot is trained as a replacement branch. The disadvantage of multiple trunks is that it complicates many cultivation practices such as pruning, spraying, removal of unwanted growth, and fruit harvesting. The plants tend to produce many new branches in their interior, and the bushy new growth is amenable to aphid attacks. With the development of effective chemicals against stem borers, today it is possible to rely on the singlestem method, which has several variations. The most common practice today in modern orchards in Israel is to train a single trunk up to about 30 cm. The trunk is than split to 3 or 4 main branches, and the tree is trained as a vase shape to a height of 3.5 to 4 m. Properly irrigated and fertigated orchards trained in this way often produce more than 30 tonnes per hectare on the average. One of the main problems in pomegranate production is the tendancy for young branches to bend from fruit weight in the ﬁrst years of production, distrupting tree structure and causing ground-contact of fruit. For this reason, it is a common practice to tie up branches or to shorten the main branches by pruning. Frequently it is necessary to support the branches, particularly when there is a heavy load of fruit on the young branches. Light is a very important factor in pomegranate bearing and fruit quality. Therefore, summer pruning is required to remove suckers and new branches that appear continuously on the exposed trunks. Winter pruning is used mostly when there is a need to induce new growth, eliminate broken or intrusive branches, and/or control the tree height. In California, pomegranate trees are frequently pruned in the ﬁrst years of growth to strengthen the main trunk and leading branches. The branches growing from the main trunk are maintained relatively short, and the tree is topped by machine to keep control of its height. This method requires intensive labor, and sometimes the trees are overexposed to sunlight, which may cause fruit sunburn. To provide optimal light for fruit development, pomegranate trees are planted with wide spacing (typically 6 4 m) despite relatively small tree size. Some growers plant denser orchards (6 2 m) to obtain higher yields in early years with removal of alternate trees in later years.

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E. Plant Protection Pomegranates are prone to various pests and plant diseases that include insects, fungi, and bacteria. Primary pests and diseases vary between the different geographical regions. While some pests are a big problem in one place, they are unharmful or absent from other regions. However, some pests and diseases are common to most of the pomegranategrowing regions. The list of the main pomegranate pests is provided in Table 2.2. Among the main insects that attack the pomegranate stems and trunk are the bark beetle Island pinhole borer or shothole borer Xyleborus perforans Wollaston 1857 (Coleoptera: Scolytidae), the stem borer moth Euzophera sp. (Lepidoptera: Pyralidae) (Blumenfeld et al. 2000; Jagginavar and Nalik 2005), and the tea shothole borer Euwallacea (Xyleborus) fornicatus Eichhoff 1868 (Coleoptera: Scolytidae) (Mote and Tambe 1990). Effective insecticides for the management of these insects were reported (Mote and Tambe 1990; Blumenfeld et al. 2000; Jagginavar and Nalik 2005). The bark-eating caterpillar Indarbela quadrinotata Walker 1856 (Lepidoptera: Cossidae) is known as pomegranate pest in India (Shevale 1991). Leopard moth Zeuzera pyrina Linnaeus 1761 (Lepidoptera: Cossidae) was reported as pome-granate trunk borer in Spain (Juan et al. 2000), Turkey (Ozturk et al. 2005), and Israel (Goor and Liberman 1956). In China, Zeuzera coffeae Nietner 1861 (Lepidoptera: Cossidae) was reported as an important pomegranate pest (Ma and Bai 2004). Aphids are serious and widespread pests in pomegranate orchards. Young pomegranate leaves are highly susceptible to aphid attacks. Aphids Aphis punicae Passerini 1863 (Aphididae: Homoptera) (Blumenfeld et al. 2000) and the cotton aphid Aphis gossypii Glover 1877 (Hemiptera: Aphididea) (Juan et al. 2000; Carroll et al. 2006) tend to attack the leaves in early spring and secrete honeydew, which attracts ants and sooty mold (a charcoal-black fungus of several genera) that appears as a black cover on the surface of the infected leaves, branches, and fruits. The fungus is not pathogenic but can cause major damage, particularly to young trees. The ash whiteﬂy Siphoninus phillyreae Haliday 1835 (Hemiptera: Aleyrodidae) was reported to be a pest on pomegranate leaves in several countries, including Turkey (Ozturk et al. 2005), Egypt (Mesbah 2003), Venezuela (Arnal and Ramos 2000), India (Shevale and Kaulgud 1998), and California (Carroll et al. 2006). Some scales were reported as pomegranate pests. Among them are Japanese wax scale Ceroplastes japonicus Green (Hemiptera: Coccidae)

157

Branches

Black scale

Hemiptera: Coccidae

Saissetia oleae Olivier 1791

Japanese wax scale

None

Nigra scale

Lepidoptera: Cossidae Hemiptera: Coccidae

Leopard moth

Lepidoptera: Cossidae

Parasaissetia Hemiptera: Coccidae nigra Nietner 1861

Zeuzera coffeae Nietner 1861 Ceroplastes japonicus Green

Bark-eating caterpillar

Bark beetle, island pinhole borer, or shothole borer Pomegranate stem borer moth Tea shothole borer

Common name

Lepidoptera: Cossidae

Lepidoptera: Pyralidae Coleoptera: Scolytidae

Euzophera sp.

Euwallacea (Xyleborus) fornicatus Eichhoff 1868 Indarbela quadrinotata Walker 1856 Zeuzera pyrina Linnaeus 1761

Coleoptera: Scolytidae

Xyleborus perforans Wollaston 1857

Branches and trunk

Order: family

Pest scientiﬁc name

Pest target

Worldwide

Southern Europe, Mediterranean, Southeast Asia, Central Asia, USA Southeast Asia, Australia Southern Europe, central Asia, China Worldwide

From Japan south to New Guinea and west to India, U.S.A. India

Israel

India, Portugal, USA

Known distribution

Table 2.2. Pest list of pomegranate and their distribution in pomegranate-growing regions.

(Continued)

Jadhav and Ajri 1992; Shevale and Kaulgud 1998 Goor and Liberman 1956; Juan et al. 2000; Carroll et al. 2006

Ma and Bai 2003

Goor and Liberman 1956; Juan et al. 2000; Ozturk et al. 2005 Ma and Bai 2004

Shevale 1991

Blumenfeld et al. 2000 Mote and Tambe 1990

Jagginavar and Nalik 2005

Reference for damage

158

Leaves

Leaves and branches

Coccus pseudomagnoliarum Kuwana 1914 Ceroplastes sinensis Del Guercio 1900

Branches

Aphididae: Homoptera

Hemiptera: Aphididae

Aphis gossypii Glover 1877

Homoptera: Pseudococcidae

Cotton aphid

None

Citrus mealybug

Crapemyrtle scale

Chinese wax scale

Hemiptera: Coccidae

Hemiptera: Coccidae

Citricola scale

Common name

Hemiptera: Coccidae

Order: family

Aphis punicae Passerini 1863

Eriococcus lagerstroemiae Kuwana 1907 Planococcus citri Risso

Pest scientiﬁc name

Pest target

Table 2.2. (Continued):

Reference for damage

Blumenfeld et al. 2000; Juan et al. 2000; Mani and Krishnamoorthy 2000; Ozturk et al. 2005 Blumenfeld et al. 2000 Southern Europe, Mediterranean, Near East, Central Asia, Southeast Asia Southern Europe, Juan et al. 2000; Carroll East Mediterranean, et al. 2006 Southeast Asia, Australia, South America, USA, South Africa

Worldwide

Southern Europe, Carroll et al. 2006 Near East, Caucasus, Australia, USA Southern Europe, Juan et al. 2000 Mediterranean, Near East, Australia, South America, USA, South Africa India, China Zhao et al. 1998

Known distribution

159

Fruit

Common Guava Blue

Cotton Bollworm moth Honeydew moth

Lepidoptera: Lycaenidae

Lepidoptera: Noctuidae Lepidoptera: Phycitidae

Apomyelois Pyralidae: ceratoniae Zeller Phycitinae 1839 (or Actomeylois ceratoniae)

Cornelians

Lepidoptera: Lycaenidae

Deudorix epijarbas Moore 1858 Virachola isocrates Fabricius 1793 or Deudorix isocrates Fabricius 1793 Helicoverpa armigera Hu¨bner 1805 Cryptoblabes gnidiella Millie´re

Carob moth (or the date moth)

None

Moth

Lepidoptera: Arctiidae Lepidoptera: Lycaenidae

Creatonotos gangis Linnaeus 1763 Virachola livia Klug 1834

Ash whiteﬂy

Hemiptera: Aleyrodidae

Siphoninus phillyreae Haliday 1835

South Europe, North Africa, Southwest Asia, Central and South America, USA Europe, Near East, USA

Worldwide

India

India, Sri Lanka, Burma, Australia

Southeast Asia, Australia North Africa, East Mediterranean

Worldwide

(Continued)

Moawad 1979; Mirkarimi 2000; Ozturk et al. 2005; Carroll et al. 2006

Blumenfeld et al. 2000; Juan et al. 2000

Teggelli et al. 2002

Shevale and Kaulgud 1998; Arnal and Ramos 2000; Mesbah 2003; Ozturk et al. 2005; Carroll et al. 2006 Raghunath and Butani 1977 Goor and Liberman 1956; Awadallah et al. 1971; Wisam and Mazen 2000 Zaka-ur-Rab 1980; Divender and Dubey 2005 Morton 1987; Shevale and Kaulgud 1998; Karuppuchamy et al. 2001

160

Platynota stultana Walsingham 1884 Lobesia botrana Denis & Schiffermu¨ller Ceratitis capitata Wiedemann 1824 Pseudococcus maritimus Erhorn

Fruit

Pseudococcus comstocki Kuwana Drosicha quadricaudata Green 1922 Scirtothrips dorsalis Hood 1919 Brevipalpus lewisi McGregor 1949

Pest scientiﬁc name

Pest target

Table 2.2. (Continued):

Comstock mealybug

Mealy bug

Chilli thrips

Citrus ﬂat mite

Homoptera: Margarodidae Thysanoptera: Thripidae Prostigmata: Tenuipalpidae

Mediterranean fruit ﬂy Grape mealybug

Tephritidae: Dacinae Homoptera: Pseudococcidae

Homoptera: Pseudococcidae

Omnivorous leafroller moth Grapevine moth

Common name

Lepidoptera: Tortricidae Lepidoptera: Tortricidae

Order: family

South Europe, Near East, North Africa, Southeast Asia, Australia, USA

Southeast Asia, Australia, USA

India, Sri Lanka

Mediterranean basin, Central Asia, Southeast Asia, South America, USA, South Africa Asia, Australia, USA

South Europe, former USSR, Near East, North Africa. Worldwide

USA

Known distribution

Carroll et al. 2006

Bagle 1993; Shevale and Kaulgud 1998

Rawat et al. 1989

Carroll et al. 2006

Juan et al. 2000; Ozturk et al. 2005 Carroll et al. 2006

LaRue 1980; Carroll et al. 2006 Vasil’eva and Sekerskaya 1986

Reference for damage

161

Roots

Leaves

Tenuipalpus Prostigmata: punicae Tenuipalpidae Pritchard & Baker 1958 Tenuipalpus Prostigmata: granati Sayed Tenuipalpidae 1946 Tenuipalpus Prostigmata: (Brevipalpus) Tenuipalpidae youseﬁ Nassar & Ghai 1982 Eutetranychus Acarina: orientalis Klein Tetranychidae 1936 Meloidogyne Tylenchida: incognita Kofoid Heteroderidae White 1919 Chitwood 1949 and Meloidogyne javanica Treub 1885 Chitwood 1949 Most of the world

India

South Europe, Asia, Africa, Australia, USA Worldwide

Mite

Red mite

Oriental red mite

Root knot nematode

Near East, South Europe, Southeast Asia

Mite

LaRue 1980; Verme 1985; Siddiqui and Khan 1986; Juan et al. 2000; Shelke and Darekar 2001

Kumawat and Singh 2002

Ram and Singal 1990

Blumenfeld et al. 2000

Khosroshahi 1984; Juan et al. 2000

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in Sichuan, China (Ma and Bai 2003); nigra scale Parasaissetia nigra Nietner 1861 (Hemiptera: Coccidae) in Maharashtra, India (Jadhav and Ajri 1992; Shevale and Kaulgud 1998); black scale Saissetia oleae Olivier 1791 (Hemiptera: Coccidae) in Spain, California, and Israel (Goor and Liberman 1956; Juan et al. 2000; Carroll et al. 2006); citricola scale Coccus pseudomagnoliarum Kuwana 1914 (Hemiptera: Coccidae) in California (Carroll et al. 2006); and Chinese wax scale Ceroplastes sinensis Del Guercio 1900 (Hemiptera: Coccidae) (Juan et al. 2000) and crapemyrtle scale Eriococcus lagerstroemiae Kuwana 1907 (Hemiptera: Coccidae) in Shandong, China (Zhao et al. 1998). The larvae of the moth Creatonotos gangis Linnaeus 1763 (Lepidoptera: Arctiidae), which is found in southeast Asia and Australia, was found feeding on pomegranate leaves. This pest caused extensive defoliation of the trees (Raghunath and Butani 1977). Insect pests of the fruit can cause major problems in those regions where the insects exist. One of these pests is the pomegranate butterﬂy Virachola livia Klug 1834 (Lepidoptera: Lycaenidae), which penetrates the fruit in early stages of fruit development and causes arils rot (Goor and Liberman 1956; Awadallah et al. 1971; Wisam and Mazen 2000). The pomegranate fruit borers Cornelians Deudorix epijarbas Moore 1858 (Lepidoptera: Lycaenidae) (Zaka-ur-Rab 1980; Divender and Dubey 2005) and the common guava blue Virachola isocrates Fabricius 1793 (or Deudorix isocrates Fabricius 1793) (Lepidoptera: Lycaenidae) (Morton 1987; Shevale and Kaulgud 1998; Karuppuchamy et al. 2001) are important pomegranate pests in east Asia, especially in the Indian peninsula. The butterﬂy lays eggs on ﬂower buds and the calyx of developing fruits, and in a few days the caterpillars enter the fruit by way of the calyx. These fruit borers may cause loss of an entire crop unless the ﬂowers are sprayed (Morton 1987). The cotton bollworm moth Helicoverpa armigera Hu¨bner 1805 (Lepidoptera: Noctuidae) was also reported as fruit borer in India (Teggelli et al. 2002). The honeydew moth Cryptoblabes gnidiella Millie´re (Lepidoptera: Phycitidae) causes crown rot toward ripening and storage (Blumenfeld et al. 2000; Juan et al. 2000). The carob moth Apomyelois ceratoniae Zeller 1839 (Pyralidae: Phycitinae) (or the date moth Actomeylois ceratoniae) is known to damage pomegranate fruits in many countries: California (Carroll et al. 2006), Saudi Arabia (Moawad 1979), Iran (Mirkarimi 2000), and Turkey (Ozturk et al. 2005). The omnivorous leafroller moth Platynota stultana Walsingham 1884 (Lepidoptera: Tortricidae) causes fruit damage in California where two fruits touch or a leaf touches the fruit, but the effect is usually minor (LaRue 1980; Carroll et al. 2006). The grapevine moth Lobesia botrana Denis & Schiffermu¨ller

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(Lepidoptera: Tortricidae) attaks pomegranate trees in Crimea, Ukraine (Vasil’eva and Sekerskaya 1986). Recently increased damages in Israeli pomegranate orchards were reported to be inﬂicted by the Mediterranean fruit ﬂy Ceratitis capitata Wiedemann 1824 (Tephritidae: Dacinae) (I. Kosto, pers. commun.). It is thought that the recent damages were caused by a secondary attack of the ﬂy after the initial fruit penetration by V. livia. C. capitata was reported as pomegranate pest in Spain and in Turkey (Juan et al. 2000; Ozturk et al. 2005). Citrus mealybug Planococcus citri Risso (Homoptera: Pseudococcidae) (Blumenfeld et al. 2000; Juan et al. 2000; Mani and Krishnamoorthy 2000; Ozturk et al. 2005), grape mealybug Pseudococcus maritimus Erhorn (Homoptera: Pseudococcidae), and Comstock mealybug Pseudococcus comstocki Kuwana (Homoptera: Pseudococcidae) (Carroll et al. 2006) might cause damage by settling between two fruits or inside the crown. Rot can occur where the mealybugs secrete honeydew. The mealy bug Drosicha quadricaudata Green 1922 (Homoptera: Margarodidae) was recorded on wild pomegranate in Himachal Pradesh, India (Rawat et al. 1989). Thrips can cause damage to fruit and tree. Reports from India indicate such damages by the thrips Scirtothrips dorsalis Hood 1919 (Thysanoptera: Thripidae) (Bagle 1993; Shevale and Kaulgud 1998). Proper pest management can easily solve thrips damages. Mites can attack pomegranate leaves, especially the Tenuipalpus punicae Pritchard & Baker 1958 (Prostigmata: Tenuipalpidae) and Tenuipalpus granati Sayed 1946 (Prostigmata: Tenuipalpidae). Severe damage might result in defoliation (Khosroshahi 1984; Blumenfeld et al. 2000; Juan et al. 2000). The citrus ﬂat mite Brevipalpus lewisi McGregor 1949 (Prostigmata: Tenuipalpidae) causes ‘‘alligator skin’’ damage to pomegranate rind in California, which makes the fruit nonmarketable (Carroll et al. 2006). The red mite Tenuipalpus (Brevipalpus) youseﬁ Nassar & Ghai 1982 (Prostigmata: Tenuipalpidae) was recorded in India (Ram and Singal 1990). The oriental red mite Eutetranychus orientalis Klein 1936 (Acarina: Tetranychidae) heavily infests pomegranate in Rajasthan, India (Kumawat and Singh 2002). Reports from Spain, California, India, and Libya mentioned nematodes as a problem, especially in sandy soils. The main pests are the root knot nematode Meloidogyne incognita Kofoid and White 1919 Chitwood 1949 and Meloidogyne javanica Treub 1885 Chitwood 1949 (LaRue 1980; Verme 1985; Siddiqui and Khan 1986; Juan et al. 2000; Shelke and Darekar 2001). Fungi and bacteria are responsible for several serious pomegranate diseases. The list of pomegranate diseases is given in Table 2.3.

164 Leaf black spot

Fungus Bacteria

Ceuthospora phyllosticta C. Massalongo Xanthomonas axonopodis pv. punicae

Pseudocercosporella granati Rawla Deighton 1976 Discosia punicae Shreem. & M. Reddy Cercospora sp.

Twig dieback

Fungus

Pleuroplaconema sp.

Leaf spot Leaf spot Fruit spot

Fungus Fungus Fungus

Twig dieback

White root rot

Fungus

Dematophora nectarix Hartig

Dry rot

Dry root rot

Fungus Fungus

Dry rot

Fungus

Coniella granati Hebert & Clayton 1963 Sutton 1969

Canker

Fungus

Zythia versoniana Sacc. Sacc. 1884 Phomopsis sp. Fusarium solani

Wilt

Damage

Fungus

Fungus or bacterium

Ceratocystis ﬁmbriata Ellis and Halsted

Scientiﬁc Name

India

India

India

India

Not speciﬁed

Not speciﬁed

Israel

India, Greece

India

Not speciﬁed

China

India, China

Regions reported

Table 2.3. Diseases list of pomegranate and their distribution in pomegranate-growing regions.

Upasana and Verma 2002; Vijai and Indu 2005 Mahla and Ashok 1989 Shreemali and Reddy 1971 Morton 1987; Reddy et al. 2005

Morton 1987

Sharma 1998, Tziros and TzavellaKlonari 2007 Sztejnberg and Madar 1979 Morton 1987

Kore and Mitkar 1993

Somasekhara and Wali 2000; Huang et al. 2003 Morton 1987; Tang et al. 1998 Morton 1987

Reference for damage

165

Leaf spot

Fungus

Fruit rot

Fungus

Fungus

Glomerella cingulata Fruit rot

Fruit rot

Fruit rot

Fungus

Fungus

Leaf spot

Fungus

Coniella granati Sacc. Petr. & Syd. Phytophthora sp.

Setosphaeria rostrata K.J. Leonard 1976 Alternaria alternate Fr. Keissl. 1912

Leaf spot

Fungus

Alternaria alternate Fr. Keissl. 1912 Aspergillus niger Tiegh. 1867

Scab

Fungus

Sphaceloma punicae

Fruit spot

Fungus

Colletotrichum gloeosporioides

India

India, Spain

Turkey

Greece, Spain, USA

India

India

India

China

India

More et al. 1989; Juan et al. 2000; Sushma and Sharma 2006 Singh and Chohan 1972

LaRue 1980; Juan et al. 2000; Tziros et al. 2007 Yildiz and Karaca 1973

Raghuwashi et al. 2005

Raghuwashi et al. 2005

Jamadar et al. 2000; Reddy et al. 2005 Morton 1987; Zheng et al. 2004 Raghuwashi et al. 2005

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Relatively high incidence of pomegranate wilt caused by the fungus Ceratocystis ﬁmbriata Ellis and Halsted was reported in Karnataka and Maharashtra provinces in India (Somasekhara and Wali 2000) and in Yunnan, China (Huang et al. 2003). The initial disease symptoms were yellowing and wilting of the leaves on a single branch. Severely affected plants show brown discoloration of roots and stems (Somasekhara and Wali 2000). Pomegranate canker caused by Zythia versoniana Sacc. Sacc. 1884, which attacks the fruit, branches, and trunk, was reported in China (Tang et al. 1998). Morton reports this fungus as causing pomegranate disease as well (Morton 1987). Antifungal treatment with carbendazin was reported to be effective against the disease. Dry rot may be caused by Phomopsis sp. (Morton 1987), Fusarium solani (dry root rot) (Kore and Mitkar 1993), or Coniella granati Hebert & Clayton 1963 Sutton 1969 (Sharma 1998, Tziros and Tzavella-Klonari 2007). Dematophora nectarix Hartig is the causal agent of white root rot in pomegranate in Israel (Sztejnberg and Madar 1979). Twig dieback may be caused by either Pleuroplaconema sp. or Ceuthospora phyllosticta C. Massalongo (Morton 1987). Leaf spot diseases are caused by the infection of fungi or bacteria and, if not treated, they can cause leaf blight and defoliation. Bacterial black spot disease caused by Xanthomonas axonopodis pv. punicae (Upasana and Verma 2002; Vijai and Indu 2005). Pseudocercosporella granati Rawla Deighton 1976 (Mahla and Ashok 1989) and Discosia punicae Shreem. & M. Reddy (Shreemali and Reddy 1971) cause fungus leaf spot. Fruit spot disease is caused by Cercospora sp. (Morton 1987; Reddy et al. 2005) or Colletotrichum gloeosporioides (Jamadar et al. 2000; Reddy et al. 2005). Sphaceloma punicae scab was identiﬁed in China (Zheng et al. 2004) and also mentioned by Morton (1987). Some other patho-gens were mentioned in India as fruit and leaf spot associated diseases: Alternaria alternate Fr. Keissl. 1912, Aspergillus niger Tiegh. 1867andSetosphaeriarostrataK.J.Leonard1976(Raghuwashietal.2005). Fruit rot diseases are the result of several fungi infections: among them Alternaria alternata Fr. Keissl. 1912 (LaRue 1980; Juan et al. 2000; Tziros et al. 2007), Coniella granati Sacc. Petr. & Syd. (Yildiz and Karaca 1973), Phytophthora sp. (More et al. 1989; Juan et al. 2000; Sushma and Sharma 2006) and Glomerella cingulata (Singh and Chohan 1972). F. Weed Control Pomegranates are grown primarily in arid regions and require regular irrigation. Irrigation also encourages weed growth that competes with

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trees for water and soil nutrients. In addition, weeds can host a wide array of damaging pomegranate pests and might disturb efﬁcient pest control. Weed control in pomegranates is particularly important in younger trees. Later on, as the trees mature, the shade will inhibit the weeds, and the intensity of weed management procedures is reduced. Weed control should be modiﬁed according to weed composition in the speciﬁc orchard. Several herbicides are used to control weeds in pomegranate orchards. Goal1 (Oxyﬂuorfen) and simazine (triazine) are used as preemergence weed killers. Both are used to control a wide spectrum of annual broadleaf weeds and grasses. Postemergance, Roundup (Glyphosate) is used as a nonselective herbicide to control grasses and weeds while 2,4-dichlorophenoxyacetic acid (2,4-D) derivatives (phenoxy herbicides) are used to control broad leaf weeds. The combination of both postemergent materials is used when needed (Blumenfeld et al. 2000). Extensive use of these herbicides may result in tree damage; therefore, care should be taken when using them. Bucsbaum et al. (1982) reported phytotoxicity when herbicides were applied to pomegranate grown in pots but not when applied to ﬁve-year-old pomegranates grown in the orchard. These authors found that the best weed control was obtained with simazine, oryzalin (sulfonamide herbicide, a selective preemergence herbicide used for control of annual grasses and broadleaf weeds) and terbutryne (s-triazine herbicide, a selective herbicide used for control of annual grasses and broadleaf weeds) (Bucsbaum et al. 1982). Usage of glyphosate to control Cuscuta monogyna L., which heavily infested pomegranate trees in Iran, was reported by Saied et al. (2003). Lack of experience growing pomegranate in many areas and small area in cultivation limit both registration and recommendations for herbicide applications to pomegranate orchards. The availability of efﬁcient herbicides is currently relatively limited, and work should be done to broaden the spectrum of certiﬁed chemicals for use in pomegranate orchards. A common practice today in modern pomegranate orchards in Israel is to use polythene mulches. Such mulches conserve soil moisture, reducing water consumption by 20% to 25% (Aulakh and Sur 1999; Ravid et al. 2004), and signiﬁcantly reduce weed population by 20% to 26% compared to controls (Aulakh and Sur 1999; Ravid et al. 2004). G. Fruit Physiological Disorders Fruit splitting and sunburn may affect pomegranate fruits and sometimes cause signiﬁcant commercial damage. Fruit splitting actually

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may be regarded as the last stage of normal pomegranate fruit developmental process where the fruit is spreading its seeds. Most known pomegranate cultivars will eventually split when they overripen. Some cultivars, such as the Israeli cultivars ‘P.G.131-32’, and ‘P.G.118-19’ and some cultivars from the pomegranate collection in Saveh (Tabatabaei and Sarkhosh 2006), tend to split in much earlier stages of fruit development or in higher frequencies than others. ‘Shirvan’, ‘Burachni’, and ‘Asperonskii Krasnyi’ were found to be resistant to splitting (Trapaidze and Abuladze 1989), suggesting that at least some aspects of fruit splitting in pomegranates are genetically controlled independently from environmental conditions. This assumption is also corroborated by Hepaksoy et al. (2000). The extent of fruit splitting is signiﬁcantly reduced by regular irrigation, particularly by drip irrigation (Prasad et al. 2003). It is known that rainfall on mature pomegranate fruits following the end of the dry season can induce rapid fruit splitting. Therefore, splitting is a problem in regions where the fruit maturation overlaps a rainy season. There are indications from Israel and from Turkey that shading may induce fruit splitting, most probably by changing the water balance due to lower radiation (Yazici and Kaynak 2006; Y. Shahak pers. commun.). A few reports indicate that spraying with gibberelic acid (GA3) at 150 ppm or with benzyl adenine (BA) at 40 ppm could signiﬁcantly reduce fruit splitting (Sepahi 1986; Mohamed 2004; Yilmaz and Ozguven 2006). Other studies indicate that application of boron may reduce fruit split (Singh et al. 2003; Shiekh and Rao 2006). The cause for sunburn is the combined action of high solar radiation, low humidity, and high temperatures. Yazici and Kaynak (2006) indicated that solar radiation between 220 J/cm2 to 324 J/cm2 as highly correlated with fruit surface temperatures and that fruit surface temperatures that cause sunburn vary between 41 C and 47.5 C. In Israel, late cultivars such as ‘Wonderful’ that ripen in autumn and are exposed throughout the summer to strong solar radiation and hot temperatures are much more susceptible to sunburn. Early cultivars such as ‘Akko’ and ‘ShaniYonay’ are less susceptible. It is not yet known whether there are differences among cultivars with respect to sunburn sensitivity and whether skin color is a factor in this respect. Studies conducted by Yazici et al. indicated that 35% shading and application of Kaoline are effective in reducing sunburn on pomegranate fruit (Melgarejo et al. 2004; Yazici and Kaynak 2006). For the ‘Hicaznar’ cultivar, Kaolin treatment proved to be the best method as it also increased color of the fruits (Yazici and Kaynak 2006).

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H. Postharvest A review on postharvest biology and technology of pomegranate was recently published by Kader (2006). The review summarizes the current knowledge on the pomegranate morphological characteristics, composition and compositional changes during maturation and ripening, quality indices, and postharvest biology. This section will mention some of the major points concerning postharvest biology of the pomegranate fruit in addition to the new postharvest technologies developed and practiced today in Israel. As the pomegranate fruit matures on the tree, a reduction in the titratable acidity and parallel increase in TSS, pH, and color intensity is observed (Kader 2006). Once the fruit is harvested, it keeps respiring at a relatively low rate. This rate is decreased with time after the harvest. Storage at low temperature can keep respiration rate under 8 ml CO2 per kg min1. Due to relatively low respiration rates and low amount of ethylene evolved (0.2 ml per kg min1), the pomegranate fruit is classiﬁed as nonclimacteric. The pomegranate fruit increases its respiration rate and ethylene production immediately after exposure to ethylene. However, the effect of ethylene treatment on respiration rapidly declines. Ethylene treatments did not affect signiﬁcantly fruit parameters of harvested fruit such as color, TSS, pH or titratable acidity. These data indicate that the pomegranate fruit will not mature postharvest and should be harvested only when fully mature. The major problems in pomegranate storage are loss of fruit weight, fruit size reduction, skin damages such as husk scald (browning of the skin surface) (Or-Mizrahi and Ben-Arie 1984; Ben-Arie and Or 1986; Deﬁlippi et al. 2006), and development of crown and fruit rot (Adaskaveg and Forster 2003; Tedford et al. 2005). Gray mold caused by Botrytis cinerea Whetzel and rot caused by Penicillium implicatum Biourge 1923, Rhizopus arrhizus Fischer 1892, and Alternaria solani Sorauer 1896 are storage diseases of pomegranate fruit (Kanwar and Thakur 1973; Vyas and Panwar 1976; Morton 1987; Labuda et al. 2004; Palou et al. 2007). In California, Botrytis cinerea, which causes postharvest decay, is the primary limiting factor for long-term storage (Adaskaveg and Forster 2003; Tedford et al. 2005). Fenhexamid and ﬂuidioxonil treatments were shown to be very effective in reducing natural incidence of gray mold caused by B. cinerea. To prevent development of fungicide resistance in these pests, a combination of sanitation treatments with chlorine

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and fungicides dip was recommended before cold storage (Adaskaveg and Forster 2003). Palou et al. (2007) indicated synergistic effects between antifungal treatments and controlled atmosphere (CA) of 5 kPa O2 þ 15 kPa CO2 in ‘Wonderful’ pomegranates artiﬁcially inoculated with B. cinerea. A combination of waxing with antifungal treatments was suggested by Sarkale et al. (2003) and by Ghatge et al. (2005) to extend the shelf life and the quality of pomegranate in cold storage and ambient conditions. Pretreatment of pomegranates with hot water at 45 C was shown to reduce chilling injury and electrolyte and K leakage (Artes et al. 2000; Mirdehghan and Rahemi 2005). Heat treatment was also shown to be effective in maintaining the nutritive and functional properties of pomegranate fruit after a long period of storage (Mirdegahan et al. 2006) and in reducing pomegranate moth damage (Moghadam and Nikkhah 2005). Pomegranate fruits can be kept well for long time after harvesting. Experimental data showed that fruits could be stored between 0 C and 4.5 C at 85% relative humidity for several months (Mukerjee 1958; Kader et al. 1984; Or-Mizrahi and Ben-Arie 1984). Saxena et al. (1987) found that time of harvest, temperature, and oxygen level signiﬁcantly affected husk scald. These authors found that delaying harvest reduced the percentage of fruit developing husk scald. Low oxygen and low temperatures inhibited husk scald, probably by inhibiting enzyme-dependent oxidation processes (Or-Mizrahi and Ben-Arie 1984). A combination of low oxygen levels (2%–3%), low temperature (2 –6 C), and late harvest were found optimal to reduce chilling injuries while preserving taste qualities. Low oxygen levels may cause anaerobic respiration, which in turn causes the accumulation of fermentive volatiles (Kader et al. 1984; Or-Mizrahi and Ben-Arie 1984). Therefore, 5% oxygen was suggested as a compromise oxygen level where skin damage is inhibited while fermentive volatiles are not produced (Kader 2006). The effectiveness of CO2 in inhibiting scald development in addition to its fungicidic effects led Hess-Pierce and Kader (2003) to recommend 5% oxygen þ 15% CO2 as the optimal CA for pomegranate storage at 7 C and 90% to 95% relative humidity (Kader 2006). Ranjbar et al. (2006) demonstrated that polyethylene bag wraps signiﬁcantly reduced weight loss and improved appearance of the fruit following storage. In Israel, several long storage experiments were conducted by Porat et al. (2005, 2006, 2007). These authors have developed new storage technology (modiﬁed atmosphere packaging) that involves the usage of special bags (Xtend1) which have small pores (microperfoation) (Porat et al. 2006; Sachs et al. 2006). These

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bags result in the development of 5% CO2 and 12% to 14% O2 within the bag surrounding the fruit. The Xtend packaging reduces weight loss from 7% to 3.5%, reduces scald from 38% to between 2% and 11%, and reduces crown decay when pomegranate fruit were stored at 6 C for 16 weeks. Using either the Xtend packaging technique just described or CA conditions of 2% O2 þ 3% CO2 at 6 C permitted storage of pomegranate fruit for to four to ﬁve months with acceptable commercial quality. Antifungal pretreatment of the pomegranate fruit was recommended before storage began. Data on storage experiments that was reported by Hess-priece and Kader (2003) and Porat et al. (2005, 2006) were based on the cultivar ‘Wonderful’. As mentioned in the cultivar section, the fruit qualities of the Israeli ‘Wonderful’ and the American ‘Wonderful’ are different. It was also demonstrated that different pomegranate cultivars contain different levels of secondary metabolites that have antioxidant activities (Tzulker et al. 2007). These in turn could potentially change the sensitivity of pomegranate fruit to skin damage and pathogen attack. Therefore, care should be taken with respect to storage conditions in each geographical region and for each cultivar. Currently many new cultivars are being introduced to commercial growth in addition to ‘Wonderful’. Therefore, special postharvest experiments should be carried separately for each cultivar. Apart from consumption of pomegranates as fresh fruit, they are also used for other purposes, such as isolated arils, juice, wine, and healthpromoting agents. One of the newest developments in pomegranate culture is an efﬁcient commercial method to extract intact arils (Rodov et al. 2005; Shmilovich et al. 2006). Several machines were developed, but the most efﬁcient one is able to produce more than 1 ton of arils a day (Shmilovich et al. 2006). This development requires some new studies in order to prolong the shelf life of the arils and to preserve them either as fresh or frozen product. Such studies are only just beginning. Juice is produced industrially from either crushing whole pomegranate fruit or isolated arils. Some manufacturers preferred the isolated arils because the juice is less bitter and tastes better to many people. The byproduct of the aril and juice industry are the remnants of the fruit skin, membranes, and seeds. The fruit skins and membranes are rich in elagitannins, which have a wide array of health-promoting bioactivities (Seeram et al. 2006a), and their extracts have a commercial value for humans and for animal feed. The seeds are a source of oil that contains a rare combination of unsaturated fatty acids (Seeram et al. 2006b) and sterols. Seeds powder is a common component of some Indian food recipes.

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V. BREEDING Most pomegranate cultivars grown today are the result of human selection from naturally occurring varieties. Until recent years, pomegranates were selected according to the demands of local consumers and not for export. Therefore, the main cultivars found today reﬂect the local priorities of each country or region. Examples are the traditional Indian and Spanish cultivars that are characterized by their soft seeds and low-acid taste. Increased world demand and economic importance of pomegranate exports should signiﬁcantly inﬂuence pomegranate selection criteria, which will have an increasing role in pomegranate breeding. Traditionally pomegranates were selected on the basis of their juice content, fruit size, colors, yield, and taste. The preferences of skin or aril color and taste were not identical in all countries. Export considerations raise the importance of ripening time, skin and aril color, taste, and health beneﬁts as prioritized by consumers in the end markets. The most intensive breeding projects based on crosses between cultivars and aimed toward pomegranate improvement were reported from India. Breeding of pomegranates in India was done for disease resistance (Jalikop et al. 2005; Jalikop et al. 2006), low acidity and high fruit quality under hot arid environment (Samadia and Pareek 2006); fruit yield (Manivannan and Rengasamy 1999); juice production (Jalikop and Kumar 1998); aril color (Wavhel and Choudhari 1985); fruit weight, ﬂesh color, seed size, and juice content (Karale et al. 1979); TSS (Choudhari and Shirsath 1976); and seed softness (Jalikop and Kumar 1998). Two of the main commercial export cultivars from India, ‘Mridula’ and ‘Bahgwa’, are the result of a selection from progenies of a cross between ‘Ganesh’ and the red ‘Gul Shah Red’ pomegranate cultivar from Russia (Mahatma Phule Agricultural University 2007). ‘Ganesh’ itself is an evergreen selection from ‘Alandi’ (Jalikop 2003) that produces a soft-seeded fruit with poor fruit quality. ‘Mridula’ and ‘Bahgwa’ combine the red skin color, seed softness, and evergreen habit of growth from their parents. Since very little is known on the heritability of desirable traits in pomegranates, few experiments were conducted to study the inheritance of some important features, such as acidity, seed hardness, and aril color. From crosses between ‘Daru’ and ‘Ganesh’ or ‘Daru’ and ‘Ganesh’ progenies, it was found that high acidity was always dominant to low acidity, pink aril color was dominant to white color, and hard-seeded nature was dominant to soft (Jalikop et al. 2005). Recessive markers for yellow foliage color and rosette-forming habit of growth, originated from a mutant of ‘Kabul Yellow’, were used for breeding and for studying the mode of

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pollination of pomegranate (Jalikop 2003). ‘Daru’ was used as a parent in breeding pomegranate cultivars tolerant to bacterial nodal blight (Xanthomonas campestris pv. parthenii). These authors indicated that the resistance to bacterial nodal blight is controlled by recessive genes (Jalikop et al. 2005). For many fruit attributes, soft and semisoft seeded pomegranate cultivars share similarities, whereas hard-seeded pomegranates are distinctively different (Jalikop and Kumar 1998). For example, the hard-seeded cultivars as a group had signiﬁcantly higher fruit weight and volume than semisoft and soft-seeded pomegranates. Soft-seeded pomegranates were recommended by Jalikop and Kumar (1998) as parents for developing high-juice cultivars due to their signiﬁcantly higher content of juice. Karale and Desai (2000) measured heterosis for fruit characters manifested by the individual hybrids over midparental value. They found that heterosis values were maximal for juice weight and aril weight percentage. Inheritance of fruit characteristics such as skin and aril color, taste and seed softness was studied using several combinations of crosses between the cultivars ‘Fellahyemez’, ‘Ernar’, and ‘Hicaznar’. When the sweet-sour ‘Hicaznar’ was crossed with the sweet cultivar ‘Ernar’, about 40% of the progenies were sour; when both parents were sweet, about 90% of the progenies were sweet (Ataseven Isik 2006). Breeding for frost resistance was reported from Turkmenistan by Levin (1979). Hybrid seedlings with good frost resistance were achieved following successive crosses (Levin 2006). In China, several pomegranate cultivars have been obtained by breeding. These include the early-ripening ‘Yushiliu 4’ (Zhao et al. 2006), the soft-seeded ‘Hongmanaozi’ (Zhao et al. 2007), good-quality fruit ‘Zaoxuan 018’ and ‘Zaoxuan 027’ (Wang et al. 2006) and ‘Duo Hong 1’, ‘Duo Qing 11’, and ‘Duo Bai 2’ (Liang and Cheng 1991). A breeding project in Israel was initiated in 2002. Breeding objectives are dictated predominantly by the demands of the European markets and exploit the principal advantages of the Israeli cultivars: early ripening, good color, and soft seeds. The project is aimed toward extending the pomegranate season, particularly by producing very early and very late ripening cultivars. In addition, appealing skin and aril color (particularly bright red color) are desirable features. Breeding was initiated by selecting seedlings from open pollination of known cultivars. So far, the cultivar ‘Emek’ was released from screening these populations (Plate 2.2I). ‘Emek’ is a very early cultivar that ripens in mid-August. It has a pink-red skin and bright red arils. The seeds are soft and the taste is sweet and low acid. ‘Emek’ ripens earlier than ‘Shani-Yonay’ (Holland et al. 2007), and its average weight is higher

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than ‘Shani-Yonay’. Another new Israeli cultivar is ‘Kamel’, which was selected in Newe Ya’ar. ‘Kamel’ is essentially a very similar cultivar to ‘Wonderful’ with respect to fruit quality and tree growth habit. Its most distinguishing trait is the dark-red skin color, which appears about a month earlier than in ‘Wonderful’. ‘Emek’, ‘ShaniYonay’, and ‘Kamel’ were submitted for registration in Israel and abroad. Recently new breeding projects based on deliberate crosses were initiated in Israel. These crosses aim at obtaining very early ripening cultivars that are tolerant to the negative effect of heat on pomegranate anthocyanin content in the skin and the aril. Two populations of crosses between ‘Wonderful’ and evergreen cultivars from an Indian origin were established. About 400 seedlings for each population were planted (Fig. 2.1b). These populations and their F2 selfed progenies will also serve to study the inheritance of important traits such as anthocyanin content in the skin and arils of the pomegranate fruit and the inheritance of the evergreen phenotype. In addition to traditional crosses, other methods used in pomegranate breeding include chemical mutagenesis (Shao et al. 2003; Matuskovic and Micudova 2006), gamma irradiation (Kerkadze 1987), and genetic transformation (Terakami et al. 2007). Pomegranate tetraploids have been produced through colchicine treatment of shoots (Shao et al. 2003). The tetraploid plants that were generated had shorter roots, wider and shorter leaves, and ﬂowers with enlarged diameter as compared to diploid pomegranates. Kerkadze (1987) reported on the generation of cultivar ‘Karabakh’ by using gamma irradiation. The tetraploid pomegranates produced viable pollen. Agrobacterium mediated genetic transformation of pomegranate was recently reported by Terkami et al. (2007). Pomegranate cultivars developed through genetic engineering are not expected in the near future due to severe restrictions on commercial usage of genetically modiﬁed plants and because transformation systems have not been developed for commercially important cultivars. However, the development of transformation systems in ‘Nana’ (Terkami et al. 2007) is expected to be useful as a model system to study genetic manipulation of pomegranate, in identifying important pomegranate genes for future exploitation, and for deciphering the function of genes in pomegranates. There are very few reports on molecular genetic work done with pomegranate. Only a handful of genes were isolated from P. granatum and deposited in Genebank (http://www.ncbi.nlm.nih.gov/Genbank/). Most of the genes deposited are those involved in production of unsaturated fatty acids, genes that encode for parts of ribosomal RNA,

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the mitochondrial matR gene, and the rbcL genes encoding for the large subunit of ribulose-1-5-bisphosphate carboxylas. About 10 genes involved in anthocyanin biosynthesis pathways from several cultivars of pomegranates have been isolated (D. Holland et al. unpubl.). A comparative work is now being conducted to study the level of their expression and the structural differences of these genes among pomegranate cultivars that display prominent differences in skin and aril colors. Molecular markers, such as AFLP, RAPD, and ISSR, were reported by several groups. Although Jbir and Zamani concluded that pomegranates are highly polymorphic (Jbir et al. 2006; Zamani et al. 2007), others concluded that the degree of polymorphism in pomegranates was surprisingly low (Talebi Baddaf et al. 2003; Aradhya et al. 2006; Yilmaz et al. 2006). In some of these studies, the apparent phenotypical differences observed among pomegranate cultivars were not reﬂected in the polymorphism of the molecular markers. Obviously many more markers should be isolated from pomegranates to make them useful for breeding and for evolutionary studies. Work in this direction involves the construction of pomegranate genomic libraries potentially containing microsatellites. Up until now about 26 SSR primer pairs were used for screening pomegranate genotypes (Hasnaoui et al. 2006; Mehranna et al. 2006).

VI. HEALTH BENEFITS Ancient cultures understood the health-promoting effects of the pomegranate tree. Products from all parts of the pomegranate tree, including the fruit, bark, ﬂowers, roots, and leaves, were used for medical treatments of a wide list of diseases and ailments of humans. A detailed review of modern studies on pomegranate and human health was published in recent book (Seeram et al. 2006a). Modern chemical analysis of bioactive phytochemicals produced by the pomegranate tree is just beginning. Potentially active phytochemicals found in pomegranates include sterol and terpenoids in the seeds, bark, and leaves; alkaloids in the bark and leaves; fatty acids and triglycerides in seed oil; simple gallyol derivatives in the leaves; organic acids in the juice; ﬂavonols in the rind, fruit, bark, and leaves; anthocyanins and anthcyanidins in the juice and rind; and catechin and procyanidins in rind and juice (Seeram et al. 2006b). The level of these compounds in the pomegranate tree may change during the development of the tree, during fruit maturation, under different environmental and cultivation conditions, and between pomegranate cultivars. Tzulker et al. (2007)

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showed a large variation among pomegranate cultivars with respect to the level of polyphenols, antioxadative activity, and the corresponding content of phytochemicals, such as elagic acid, galagic acid, punicalin and punicalagin. Disease targets of pomegranates include coronary heart diseases, cancer (skin, breast, prostate, and colon), inﬂammation, hyperlipidemia, diabetes, cardiac disorders, hypoxia, ischemia, aging, brain disorders, and AIDS (Shishodia et al. 2006). Modern medical research assessed the bioactivity of pomegranate juice and various pomegranate extracts against the diseases just described and helped to identify some of the molecular mammalian components that are targets for pomegranate phytochemicals. Some of these components include metalloproteinases, vascular endothelial growth factor, lipoygenase, mitogen-activated protein kinase, migration inhibitory factor, c-Jun-N-terminal kinase (JNK), and extracellular signal regulated kinase (ERK1/2) (Shishodia et al. 2006). In fact, most of the pomegranate literature published today focuses on the health beneﬁts of the pomegranate tree. However, little information about the phytochemical constituents responsible for the observed activities or about the bioavailability of the suspected active compounds is provided. Bioavailability is an important issue since some of the main active constituents in pomegranates are rapidly degraded in the body and their physiological levels become negligible. In vivo experiments with atherosclerotic mice indicated that pomegranate juice consumption has antiatherogenic properties with respect to all three related components of atherosclerosis: plasma lipoproteins, arterial macrophages, and blood platelets (Aviram et al. 2000). Experiments with human patients showed that consumption of pomegranate juice for two weeks decreased angiotensin-convertin enzyme (ACE) activity by 36% and small but signiﬁcant reduction was found in systolic blood pressure (Aviram and Dornfeld 2001). The importance of pomegranate juice is reﬂected not only in its high level of antioxidants as compared to other plant sources but also on its wide range of human and animal target components. Antiatherogenic activity was correlated with antioxidant activity and polyphenol content, but very little evidence is available on direct detection of the chemical nature of this activity. A correlation was found between the ability to prevent low-density lipoprotein (LDL) oxidation and the level of antioxidant activity. Antioxidant activity in turn was found to be highly correlated with hydrolysable tannins, particularly punicalagin (Kulkarni et al. 2007; Tzulker et al. 2007). Punicalagin, which is produced in pomegranates, was shown to possess pharmacological attributes including anti-inﬂammatory, antiproliferative, apoptotic,

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and antigenotoxic properties (Lin et al. 1999; Chen et al. 2000; Seeram et al. 2005; Adams et al. 2006). Certain ﬁelds of cancer research related to pomegranate products attained phase II clinical trials. In men with rising prostate-speciﬁc antigen (PSA) following surgery or radiation for prostate cancer, consumption of pomegranate juice signifantly prolonged PSA doubling time (Pantuck et al. 2006). These results were consistant with corresponding laboratory effects on prostate cancer in vitro as measured by cell proliferation and apoptosis as well as oxidative stress. The authors conclude that the results warrant further studies with placebocontrolled treatments. In this respect, there is evidence that ellagic acid, caffeic acid, luteolin, and punicic acid synergistically inhibit the proliferation and invasion of PC-3 prostate cancer cells across MatrigelTM artiﬁcial membranes (Lansky et al. 2005). Among the oldest known pomegranate health beneﬁts are its activities against infectious diseases (Jayaprakasha et al. 2006). Antibacterial activity of pomegranate extracts was demonstrated against a wide array of bacteria, fungi, and viruses. The active phytochemicals in pomegranates are found to be tannins and alkaloids (Jayaprakasha et al. 2006). Punicalagin was found to have an antimicrobial activity in addition to the other bioactivities already described (Burapadaja and Bunchoo 1995; Machado et al. 2002; Jayaprakasha et al. 2006). Antimicrobial activity was recently attributed to compounds extracted from pomegranate juice that include the anthocyanins pelargonidin-3galactose, cyanidin-3-glucose, the ﬂavonoids quercetin and myricetin, and gallic acid (Naz et al. 2007).

VII. CONCLUDING REMARKS Impressive advances in scientiﬁc and agricultural work on pomegranates have been achieved in recent years. Research in biology and medicine have discovered some of the molecular sites in mammalian systems at which pomegranate phytochemicals are acting and corroborated some traditional knowledge on pomegranate as an important medical plant. Most of the current published work on pomegranates is on their medical aspects and only a small fraction focused on the physiology and biology of the pomegranate tree. Most of the available clinical data today on pomegranates was obtained by usage of crude extracts, partially puriﬁed fractions of tissue extracts, or pomegranate juice. The consumption of fresh fruit and its therapeutic effects require additional studies. Much more analytical chemical

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work will be required to identify the chemical nature of the active compounds and their mode of action. Relative to other crop plants, the knowledge of the genetics, physiology, and biology of pomegranate trees is poor. Almost no work has been published on pomegranate fruit development, ﬂower development, induction of ﬂowering, root physiology, and stress responses. All of these ﬁelds are important for improving pomegranate crops and producing higher yields and healthier fruit. More research effort and more advances have been realized in commercial production methods, postharvest technology, and fruit processing of pomegranate. There is little published molecular work on pomegranates. No databases of expressed sequence tags (ESTs), sequenced genes, or genetic maps were reported. Breeding of pomegranates is done today in few centers, mainly in Iran, India, Turkey, Israel, and China. Most of the breeding projects are based on traditional crosses; few reports exist on the use of more advanced technologies in breeding. A step in this direction is the development of a genetic transformation system in pomegranates, which is essential for gene functional analysis and for exploring the biology of the pomegranate tree. The pomegranate fruit is now sold and recognized almost everywhere in the world, and increased demand requires a concomitant improvement in production and quality. New modern orchards have been planted in the southern hemisphere as well as in the traditional growing areas. The renewed interest in the tree and its products followed by the increase in its commercial importance are expected to increase the amount of research of this interesting and ancient culture.

VIII. ACKNOWLEDGMENTS We thank the Israel Gene Bank for Agricultural Crops of the Agricultural Research Organization, Bet Dagan, and the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development for ﬁnancial support. We also thank the National Council for Research and Development of the Israel Ministry of Science and Development for ﬁnancial support in the establishment of the genetic collection.

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3 Daylily: Botany, Propagation, Breeding Surinder K. Gulia, Bharat P. Singh, and Johnny Carter Agriculture Research Station Fort Valley State University Fort Valley, GA 31030 USA Robert J. Griesbach United States Department of Agriculture Agricultural Research Service Beltsville, MD 20705 USA

I. INTRODUCTION II. BOTANY A. History B. Systematics III. ANATOMY AND PHYSIOLOGY A. Roots, Stems, and Leaves B. Inflorescences and Flowers IV. HORTICULTURE A. Propagation B. Pests and Diseases C. Cultivar Registration and Awards V. GENETICS A. Genome and Ploidy Level B. Flower Color Inheritance C. Biotechnology VI. CONCLUSION VII. LITERATURE CITED

I. INTRODUCTION Daylilies (Hemerocallis spp., Hemerocallidaceae) are herbaceous perennials grown extensively as ornamental plants in home gardens Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 193

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and commercial land inﬂorescences worldwide. Their botanical name, Hemerocallis, is derived from the Greek words hemero (‘‘a day’’) and callis (‘‘beauty’’) referring to the fact that each ﬂower lasts only one day (Panavas et al. 1999). However, multiple buds on the inﬂorescences provide bloom over a number of weeks. Ancient Chinese and Japanese used daylilies for their roots, leaves, and ﬂowers as both food and medicine. Daylily buds contain more protein and vitamin C than green beans and asparagus and vitamin A equivalent to asparagus (Erhardt 1992). All parts of the plant are edible and consumed either dried or fresh (e.g., young shoots are cooked as vegetables in China while ﬂowers and bud are delicacies in the cuisines of several southeast Asian countries). Daylily can be consumed in various preparations, such as chicken with daylily, daylily soup, daylily casserole, deep-fried daylilies, and steamed daylily. Recent sensory evaluation and consumer preference studies (Knight et al. 2004; Pollard et al. 2004) also support the potential food value of daylilies. In addition, daylily roots and crowns are used as a pain reliever, a diuretic, an antidote to arsenic poisoning, and an anticancer agent (American Hemerocallis Society 2007). Daylily ﬂowers are known to possess antioxidant properties (Mao et al. 2005) and cyclooxygenase inhibitory activities (Cichewicz and Nair 2002). Daylilies are easy to grow, have attractive ﬂower colors and shapes, and the plant has an attractive growth habit. They are tolerant to drought, ﬂooding, and heat stress and grow well in most soil types under full sun or light shade. Besides of their esthetic value, they are used to help control soil erosion along highways and water channels (Munson 1989; Garber 2004). Daylilies thrive well over a large climatic range in North America, from southern Florida to northern Canada (Peat and Petit 2004). Daylilies, however, are unsuitable as cut ﬂower or potted plant due to short ﬂower life. Market value of daylilies in United States together with other perennials was estimated at $571 million in 2002 (U.S. Dept. of Agriculture [USDA] 2003).

II. BOTANY A. History According to Chinese oral traditions, reference to daylily (known as Hsuan Ts’ao) dates back to 2697 BCE (Kitchingman 1985), but the ﬁrst written record appears in the canonical writings of Confucius dating

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back to about 551–479 BCE (Barnes 2004). Hu (1968a) found the ﬁrst reference of H. fulva in the writing of the Chou dynasty dating back to 112–255 BCE, where it was grown for food and medicine (Kitchingman 1983). About 300 BCE, daylily was brought from the Far East to Europe by the silk and spice traders. By 25 BCE, H. ﬂava was known to the Greeks, Romans, Egyptians, and Africans (Baker 1937). In 1597, John Gerard was the ﬁrst English herbalist who used the name daylily to designate the Chinese Hsuan Ts’ao (Hu 1968a). By 1620, H. ﬂava and H. fulva daylilies were cultivated in England (Stout 1934), they found their way to the United States in the 18th and 19th centuries (Garber 2004). The pre-Linnnaean name of daylily was Ephemeron from the Greek epi (‘‘upon’’) and hemera (‘‘day’’). In 1753, Linnaeus retained hemera and added calos (‘‘beauty’’) and created a new generic name Hemerocallis (Eddison 1987). Many books describe the early history of daylily including information on species classiﬁcation, propagation, and cultural practices (Stout 1986; Munson 1989; Erhardt 1992). Schabell (1990) has reviewed the history of daylily cultivation in detail. B. Systematics Hemerocallis is native to Asia throughout China, northern India, Japan, and Korea. The ﬁrst extensive taxonomical study of Hemerocallis was carried out by Stout (1941); however, he died before his complete monograph could be published. Using Stout’s draft manuscript, ShiuYing Hu (1968b) published the ﬁrst monograph with a key to 23 species separated into three groups. In 1969, Hu recognized two additional species: H. tazaifu (Hu 1969a) and H. darrowiana (Hu 1969b). Erhardt (1992) developed a more elaborate classiﬁcation of daylily species separating them into ﬁve groups: fulva, citrina, middendorﬁi, nana, and multiﬂora (Table 3.1). Erhardt recognized only 20 species (Table 3.2). He did not recognize H. graminea, H. luteola, or H. littorea. Since 1992, two additional species have been recognized: H. hongdoensis (Chung and Kang 1994) and H. taeanensis (Kang and Chung 1997). In 1985, Dahlgren et al. separated Hemerocallis from the Liliaceae and placed them within their own family, the Hemerocallidaceae. Hemerocallidaceae differ from Liliaceae in the shape of their seeds, placement of their nectarines, and type of their roots. Hemerocallidaceae seeds are black and round shaped while Liliaceae seeds are brown and ﬂat. Nectaries are located in the walls of the ovary in Hemerocallidaceae but

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Table 3.1. Classiﬁcation of Hemerocallis species and common characteristics of each group. Species group Citrina

Fulva

Species H. H. H. H. H.

Middendorfﬁi H. H. H. Nana H.

Multiﬂora

H. H.

Common characteristics

altissima, H. citrina, Inﬂorescences are multiple branched, coreana, H. lilioasphodelus, ﬂowers mostly yellow, nocturnal minor, H. pedicellata, habit, fragrant with long perianth thunbergii, H. yezoensis tubes. aurantiaca, H. fulva Inﬂorescences are branched, ﬂowers brownish-red (fulvous dye) color, diurnal habit, roots have spindleshaped swellings. dumortieri, H. esculenta, Inﬂorescences are nonbranched, exaltata, H. hakuunensis, ﬂowers orange color, diurnal habit, middendorfﬁi bracts short, broad and overlapping. forrestii, H. nana Inﬂorescences are nonbranched, inﬂorescences max. 50 cm long, ﬂowers reddish-orange color, diurnal habit, perianth tube shorter than 1 cm; not winter-hardy. micrantha, H. multiﬂora, Inﬂorescences have many branches, plicata orange to orange-yellow color, diurnal habit, ﬂowers on short stalks smaller than 7 cm, tubes less than 2 cm long.

Source: Erhardt 1992.

at the base of the perigonial leaves in Liliaceae. Unlike Liliaceae, Hemerocallidaceae do not grow from true bulbs. Molecular approaches are helping to more accurately deﬁne Hemerocallis taxa (Noguchi and De-Yuan 2004; Noguchi et al. 2004). For example, it appears that H. citrina var. vespertina originated from at least three different lineages that invaded the Japanese archipelagoes separately through different routes (Noguchi and De-yuan 2004). Juerg Plodeck and Jianping Zhuang Plodeck (2003) have a Web site that describes Hemerocallis systematics in detail (www.hemerocallis-species.com/).

III. ANATOMY AND PHYSIOLOGY The anatomy and physiology of daylily plants was reviewed in detail by Voth et al. (1968). Daylily plants are composed of rammets, commonly called fans, that consist of an underground thickened stem, roots, rhizomes, leaves, and ﬂowering inﬂorescences. The underground stem, commonly called a crown, contains the apical meristem. The crown is

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Table 3.2. List of Hemerocallis species with an overview of their trait’s description. Name of species H. altissima

H. aurantiaca

H. aurantiaca ‘Major’

H. citrina

H. citrina var. vespertina H. coreana H. darrowiana H. dumortieri

H. esculenta H. exaltata

H. forrestii

H. fulva

H. graminea

H. hakuunensis H. hongdoensis

Description of traits M–La; dor.; noc., fr.; ﬂowers on inﬂorescence 120–200 cm tall; pale yellow ﬂower; > 30 ﬂowers per inﬂorescence; ﬂower diam. 7.5 cm; tall; fading in hot sun EM; ev.; diu.; ﬂowers on inﬂorescence 60–90 cm tall; orange with red tinge ﬂower; 6–8 ﬂowers per inﬂorescence; ﬂower diameter > 12 cm EM; ev.; diu.; sl. fr.; ext.; Re.; ﬂowers on inﬂorescence 60–90 cm tall; yellow-orange; 5–10 ﬂowers per inﬂorescence; ﬂower diameter > 12cm M–MLa; dor.; noc.; ﬂowers on inﬂorescence 100–115 cm tall; pale yellow ﬂower; 30–70 ﬂowers per inﬂorescence; ﬂower diameter > 12cm M–MLa; dor.; noc., sl.fr.; ﬂowers on inﬂorescence 180 cm tall; light yellow ﬂower; 30–70 ﬂowers per inﬂorescence; fading in full sun EM–M; dor.; diu.; fr.; ﬂowers on inﬂorescence 50–80 cm tall; yellow ﬂower; 50–80 ﬂowers per inﬂorescence M–MLa; dor.; diu.; yellow ﬂower; 2 ﬂowers per inﬂorescence EE ; dor.; diu.; ﬂowers on inﬂorescence 15–60 cm tall; orange ﬂower; sepal outside brownish red; 2–4 ﬂowers per inﬂorescence EM; dor.; diu.; ﬂowers on inﬂorescence 60–90 cm tall; orange ﬂower; 5–6 ﬂowers per inﬂorescence EM–M; dor.; diu.; ﬂowers on inﬂorescence 120–150 cm tall; orange ﬂower; branching only at its apex; thick inﬂorescences, recurving sepals, and broad petals EM; dor.; diu.; ﬂowers on inﬂorescence 30–40 cm tall; orange-red or orange (two forms); pedicel 2–3 cm long, not everywhere winter-hardy; low growing; pedicels EM; dor.; diu.; ﬂowers on inﬂorescence 60–90 cm tall; orange with red tinge; H. fulva ‘Kwanso’ and H. fulva ‘Flore Pleno’ have double ﬂowers; H. fulva var. rosea has rose-red ﬂowers; H. fulva var. littorea shows semi-evergreen to evergreen behavior; recurving tepals; eye, bitone, wavy margins; median stripe; nerves; fulvous-red and rose; medium to long tube; a few H. fulva varieties can show up to 100 ﬂowers per inﬂorescence EM; dor.; diu., ext.; ﬂowers on inﬂorescence up to 75 cm tall; strong orange ﬂower; 2–3 ﬂowers per inﬂorescence; ﬂower diameter > 10 cm; grasslike leaves M; dor.; diu.; ﬂowers on inﬂorescence 85–100 cm tall; orange, ﬂowers 6-11 ﬂowers per inﬂorescence M; dor.; diu.; ﬂowers on inﬂorescence 60–90 cm tall; orange-yellow ﬂower; 1-cm-long pedicel; 5–17(–23) ﬂowers per inﬂorescence (continued)

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Table 3.2. (Continued) Name of species H. lilioasphodelus H. micrantha H. middendorfﬁi

H. minor

H. multiﬂora

H. nana

H. pedicellata H. plicata

H. taeanensis H. thunbergii

H. yezoensis

Description of traits E-EM; dor.; noc.–diu., fr., ext.; ﬂowers on inﬂorescence 76 cm tall; light yellow ﬂower; makes runners, trumpet form –; dor.; diu.; – cm; orange ﬂower; 4 ﬂowers per inﬂorescence; very small tepals EE; dor.; diu.; Re.; ﬂowers on inﬂorescence 60–90 cm tall; orange ﬂower; the 2 bracts are broad oval and overlapping at base; dwarf plant; up to 10 ﬂowers per inﬂorescence E–EE; dor.; diu.; fr.; ext.; ﬂowers on inﬂorescence 45–60 cm tall; yellow ﬂower; pedicels several cm long; dwarf habit; grasslike leaves; 2–5 ﬂowers per inﬂorescence M–MLa; dor.; diu.; ﬂowers on inﬂorescence 60–120 cm tall; orange ﬂower; 75–100 ﬂowers per inﬂorescence; repeatedly branched; 0.8–1.2-cm-long pedicels; broad petals; trumpet shape E; dor.; diu.; ﬂowers on inﬂorescence 15–30 cm tall; reddishorange ﬂower; only 1 ﬂower per inﬂorescence; very small tube; not everywhere winter-hardy; dwarf – ; dor.; diu.; ﬂowers on inﬂorescence 55–65 cm tall; red-orange ﬂower; pedicel length 2–4.5 cm E -; dor.; diu.; ﬂowers on inﬂorescence 25–55 cm tall; orange-yellow ﬂower; 0.5–2 cm long pedicels; 5-11 ﬂowers per inﬂorescence EM; dor.; diu.; ﬂowers on inﬂorescence 30-70 cm tall; orange-yellow ﬂower; 0.2–3-cm-long pedicels M–MLa; dor.; noc.; fr.; ext.; ﬂowers on inﬂorescence 100–115 cm tall; lemon-yellow ﬂower; green throat; ﬂower diameter up to 10 cm; 1–2-cm-long pedicels; 4-20 ﬂowers per inﬂorescence; rufﬂed tepals and broad petals – ; dor.; diu., sl.fr.; ﬂowers on inﬂorescence 40–85 cm tall; lemon-yellow ﬂower; ﬂower diameter up to 10 cm; up to 3-cm-long pedicels; 4–12 ﬂowers per inﬂorescence

Flower time: EE ¼ extra early; E ¼ early; EM ¼ early midseason; M ¼ midseason; MLa ¼ late midseason; La ¼ late; VLa ¼ very late; Winter behavior: dor. ¼ dormant, deciduous; sev. ¼ semi-evergreen; ev. ¼ evergreen; Flower characteristics: noc. ¼ nocturnal; diu. ¼ diurnal; sl. fr. ¼ slightly fragrant; fr. ¼ fragrant; ext. ¼ extended; dbl. ¼ double; Re. ¼ rebloom. – ¼ Information unavailable. Source: Erhardt 1992; Plodeck 2002.

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Fig. 3.1. Daylily plant showing various parts with emphasis on ﬂoral biology. (Source: Iowa State University, 2006. University Extension publication available at www. extension.iastate.edu/Publications/RG303.pdf#search¼22RG303.)

top-shaped and develops a shallow depression on its upper surface (Voth et al. 1968). The leaves and roots arise from this depression. Contractile roots keep the crown underground (Putz 1998). A typical daylily plant is illustrated in Fig. 3.1. A. Roots, Stems, and Leaves Daylily roots can be ﬁbrous or rhizomatous (Voth et al. 1968). The difference in the root system is one criterion for separating daylilies into different species. The roots may form ﬂeshy tuberlike structures, as in case of H. citrine. In H. minor and H. nana, the roots thicken only near their ends, indicating that these species are related to each other.

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Roots of H. dumortieri are cylindrical; those of H. fulva are spindle shaped. Daylilies are drought-resistant because of two root characteristics: The rhizomatous roots can store large amount of water and ﬁbrous roots can exploit soil water fully. Putz (1998) describes a contractile behavior of H. fulva roots pulling the cryptocorm downward, thus not allowing the roots to get too close to the soil surface and be exposed to harsh weather conditions while dormant. Daylily leaves cannot be divided into distinct blade, petiole, and base regions (Voth et al. 1968). In general, they are grasslike in appearance and are arranged ‘‘fanlike’’ in two compact ranks (distichous). The leaves may stand erect, arch outward, or bend over near their tips or tend to fold along midrib. Daylilies exhibit three types of growth habit commonly referred to as dormant, semievergreen, and evergreen. Dormant types resume growth in spring when day temperature becomes warm enough to support growth and stop growing in autumn when days shorten and temperature cools. Before leaves die down, a compact resting bud is formed in the crowns of the plant. Within the compact bud, the leaves are protected from freezing and dehydration during winter. Dormant behavior in daylilies can be induced by short day-length or low temperature. In the fall, one or more axillary vegetative buds are formed, which remain dormant until spring. Evergreen daylilies grow throughout the year and do not form compact buds under either short day-length or low temperature. When exposed to freezing temperature, the leaves die, as does the entire plant in many instances. Semi-evergreen cultivars do not fully ﬁt under either dormant or evergreen category, and growth behavior is commonly dictated by the prevailing environmental conditions. Many modern hybrids are in this class, resulting from the cross between evergreen and dormant types. B. Inﬂorescences and Flowers Daylily stems gives rise to 1 to 3 ﬂower inﬂorescences per year. The inﬂorescences may have either an apical or axillary origin. The time of ﬂoral initiation varies greatly in daylilies. Depending on the cultivar, inﬂorescences can be initiated from July through December (Arisumi and Frazier 1968). One initiated, the inﬂorescence development is arrested until the next growing season in both evergreen and dormant types. Certain cultivars, commonly referred to as reblooming, can initiate and elongate more than one inﬂorescence in succession during a single growing season.

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The daylily inﬂorescence is a bostryx or modiﬁed cyme so that the right- or left-hand branch is the more vigorous than the apex (Voth et al. 1968). The inﬂorescences vary in length from 4 cm (H. darrowiana) to 200 cm (H. altissima); and can be erect, arched, or bowed down toward the ground under weight of their bloom, as is in case of H. multiﬂora. Not all species have branched inﬂorescences. In H. nana, the unbranched inﬂorescences carry a solitary bloom. In addition, the shape and arrangement of bracts on the inﬂorescence can be useful in identifying species. For example, the bracts in H. middendorfﬁi are broad, oval shaped, and overlap each other (Hu 1968b). As each ﬂower lasts only for a day, ﬂower bud count is very important in daylily for prolonged bloom. Most daylily cultivars bloom for two to four weeks. Some cultivars, commonly known as bud builders, have an indeterminant inﬂorescence that continues to produce ﬂower buds throughout the season. Daylilies are monoclinous plants (i.e., both pistils and stamens occur within the same ﬂower). Flower parts consist of six segments: the inner three segments are petals while the outer three segments are sepals. The throat where the ﬂower meets the stem is often a different color from the rest of the bloom. Protruding from the throat are six stamens terminating in anthers. Daylily pollen is normally brown in color but may also be reddish or yellow. In the center of stamens, the pistil is noticeably longer than the ﬁlaments and consists of a stigma that is connected through the style to the ovary within the perianth tube. The perianth is tubular from the ovary to the point where stamens are attached. The stigma itself consists of three small bulbous thickenings that exude a sticky substance during the optimum period of pollination. After pollination, the pollen tubes grow rapidly down the style, reaching the micropyle in 5 to 8 hr (Stout and Chandler 1933; Arisumi 1962). Pollen tube growth is then arrested for about 8 hr after which growth continues. Fertilization occurs 36 to 48 hr after pollination. After fertilization, seed capsule pods form that are either round or elongated elliptical and contain six ribs that open in pairs. Capsules contain three layers of black seeds that are either round or elliptical with a small raised point. Capsules typical ripen in about 50 days after pollination. The ﬂowers of daylily cultivars ﬂowers come in several colors, color patterns, and forms. The colors range in shades and color patterns differ in intensity or type within or between sepal and petal. There are also wide variations in ﬂower size, and forms may vary from informal to circular or triangular. The ﬂower and other characteristics are covered in more detail in Table 3.3.

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Table 3.3. List of major traits and their types taken into consideration for daylily hybridization. Major traits Flower color variations

Flower dusting and dotting Flower color distribution Flower patterning Flower throat color Flower throat size Flower shape

Flower Flower Flower Flower

shape of segment edges texture size

Flowering habit Flowering season Growth habit Leaf forms Ploidy level Disease

Types Near-white, light yellow, strong yellow, orange, copper, peach or melon, ﬂesh tones, brown, rose-pink, rose-red, pale red, deep red, dark mahogany red (almost black), purple and absolute black. Plain color, dotting, diamond dusting and gold dusting Self-colored, blended, polychrome, bitone, reverse bitone, bicolor and reverse bicolor Simple, eyed, banded, halo, watermark, edged tipped and picotee Yellow, orange, and green Small, large and dilated Side view: ﬂat, trumpet, ﬂaring, recurved and double. Front View: circular, triangular, star-shaped, spider, orchid-shaped and informal Rounded, pointed, pinched and twisted Tailored ribbed and rufﬂed Rippled or ribbed, smooth or waxy Miniature ( < 7.5 cm), small 97.5–11.5), large (11.5–17.5 cm) and giant ( > 17.5) Diurnal (day blooming), extended blooming and nocturnal (night blooming) Extra early, early, early–midseason, midseason, late midseason, late, very late Branching habit: dwarf ( < 30 cm), small (30–50 cm), medium (51–80 cm) and tall ( > 80 cm) Overwintering: deciduous, evergreen and semi-evergreen Diploid, triploid and tetraploid Resistance or susceptible to rust

Source: Erhardt 1992.

Daylilies complete their reproductive cycle in a single day. Each ﬂower on a daylily opens in the morning and by late afternoon undergoes rapid senescence. Unlike many ﬂowers, ethylene is not involved in the senescence process in daylily (Lukaszewski and Reid 1989; Lay-Yee et al. 1992). Daylily petals undergo a series of chemical changes before and after the opening of bloom (Guerrero et al. 1998; Panavas et al. 1998; Stephenson and Rubinstein 1998). Upon opening,

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fructan hydrolysis results in an increase in the osmolatity (Bieleski 1993). This increase results in petal and sepal expansion. Speciﬁc activities of cellulase and pectin methylesterase are highest before ﬂower opening, and speciﬁc activities of polygalacturonase and betagalactosidase increase after ﬂower opening. In addition, the activities of proteinases (Stephenson and Rubinstein 1998), RNAses (Panavas et al. 1998), and DNAses (Panavas et al. 1999, 2000) increase almost simultaneously just prior to, or along with, ﬂower opening. Senescence in daylily is characterized by changes in lipid metabolism (Bieleski and Reid 1992) and phloem export of carbohydrates (Bieleski 1995). In daylily, a cDNA for a putative cysteine proteinase has been cloned (Valpuesta et al. 1995; Guerrero et al. 1998), and Rubinstein’s (Panavas et al. 1998, 1999, 2000) group has cloned six additional cDNAs designated as Dsa (daylily senescence associated). One gene (Dsa6), a putative S1-type nuclease, is expressed only in petals and the level of its message increases as the ﬂower opens (Panavas et al. 1999).

IV. HORTICULTURE A. Propagation Daylily propagation and culture practices have been extensively reviewed (Benzinger 1968; Dunwell 1996, 1998, 2000; Black 2003; Garber 2004; Latimer 2004). Daylilies are commercially propagated asexually by dividing the crown. Seed propagation is used for breeding (Munson 1989; Dunwell 1998). Seeds of deciduous daylilies require cold stratiﬁed at 0 to 7 C for 6 to 8 weeks to germinate; the seeds of evergreen daylilies do not require cold treatment (Griesbach and Voth 1957). It usually requires two years to ﬂower from seed. After several years of growth, daylily plants are composed of several crowns that are interconnected. These large plants can be propagated asexually by separating the crowns into individual plants. The annual rate of new crown development is genotype dependant and varies from 1:3 to 1:25, averaging 8:1 (Apps 1995; Dunwell et al. 1995). Thus, it often can take 10 years or more to have adequate number of plants to meet market demand of a newly released cultivar (Dunwell 1998). There are other methods for asexual propagation. For example, a single crown can be cut into several pieces, which usually then develop into new growing points (Erhardt 1992). In addition, small

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shoots may develop from buds on the inﬂorescence. These shoots can be easily removed and rooted into the soil to form new plants. Fully developed proliferations usually take 10 to 30 days to root well, and these plants usually ﬂower within 12 to 15 months (Dunwell et al. 1995; Dunwell 1998). Dunwell (1998) obtained 14 proliferations from a single ‘Lisa My Joy’ plant that had four inﬂorescences. The application of growth regulators such as benzyladenine (BA), benylamino purine (BAP), indoleacetic acid (IAA), and cycocel have been used both to increase the number of shoots that developed from a single bud on the inﬂorescence and to induce dormant buds to develop into shoots (Pickles 1997; Zurles 2002; Leclere et al. 2006). In vitro micropropagation can accelerate vegetative propagation. Daylilies can be micropropagated from young inﬂorescences (Meyer 1976; Pounders and Garton 1996), ﬂower petals (Heuser and Apps 1976), ovaries (Krikorian and Kann 1980, 2002, 2003), suspension culture cells (Krikorian et al. 1981b; Smith and Krikorian 1991), isolated protoplasts (Fitter and Krikorian 1981; Ling and Sauve 1995; Aziz et al. 2003), anther ﬁlaments and immature seed embryos (Gulia and Carter 2007). Recently a liquid bioreactor system was developed for very large-scale propagation (Adelberg et al. 2007). Like most plants, daylily ability to regenerate from tissue culture is dependent on the genotype and source of the explant (Cheng et al. 2004). However, it is not dependent, as some believe, on the ploidy level of the plant (Adelberg et al. 2007). There are reports of successful regeneration of aseptically cultivated plantlets of Hemerocallis ‘Autumn Blaze’ in space (Levine and Krikorian 1992) aboard the shuttle Discovery during a 5-day mission within NASA’s Plant Growth Unit (PGU) apparatus. But in another space experiment (a 132-day experiment on the space station Mir), daylily plants from embryogenic cell cultures produced poor growth and nuclear abnormalities (Krikorian 1999). Pioneering work of Krikorian and Kann (1979) with daylily cells demonstrated totipotency (i.e., the potential to grow new plants from a variety of aseptically cultured tissues and cells). Further work by Ling and Sauve (1995) resulted in regenerated daylily plants from protoplast-derived calli. The protoplasts underwent sustained division to produce multicellular colonies on MS medium supplemented with 0.5 mg/L naphthalene acetic acid (NAA) and 0.5 mg/L BA. Efﬁcient regeneration from adventitious shoots can occur on clusters after subculture (Chen et al. 2005). Paclobutrazol and sucrose levels in the media signiﬁcantly affected starch accumulation, growth value, and dry weight percentage of liquid-cultured meristematic clusters. The

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use of liquid shake cultures for mass proliferation of meristematic clusters followed by regeneration of adventitious shoots on semisolid agar culture could be an efﬁcient system for large-scale micropropagation of daylily (Chen et al. 2005). B. Pests and Diseases Diseases and insects are generally not a serious problem on daylily. Before onset of rust problem, daylilies were considered almost disease and pest free (William-Woodward and Buck 2002). Daylily rust, caused by Puccinia hemerocallidis, was ﬁrst reported in the United States during 2000 concurrently in Georgia and in Tennessee (WilliamWoodward et al. 2001; Windham et al. 2004). By late 2001, daylily rust was identiﬁed in 30 states, and has become a problem throughout United States (Hernandez et al. 2002; Sakhanokho et al. 2004b). Mueller et al. (2003a, b) and Li et al. (2005) studied rust resistance and classiﬁed daylily cultivars into resistant and susceptible groups. Daylily rust has separate asexual and sexual life cycles. In the asexual life cycle, urediospores land on living plant tissue, germinate, and form mycelium within the leaf. Eventually the mycelium forms a mass of urediospores that infect new tissue and result in a buildup of the disease during the growing season. Conditions conducive to spore germination and growth are long periods of leaf wetness; temperatures between 15 and 30 C; and a high relative humidity between of 75% and 80% (Mueller and Buck 2003). At the end of the growing season, the mycelium forms teliospores instead of urediospores. The teliospores lie dormant on dead leaves during the winter. In the spring, the teliospores germinate to produce basidiospores, which do not infect daylily but infect the alternate host Patrinia. In Patrinia, the basidiospores form pycnia in which the sexual stage of the life cycle occurs. The sexual stage of the life cycle results in aeciospores, which infect daylily and start the asexual urediospore stage of the life cycle over again (Hernandez et al. 2002). Even though two spore types, urediospores and teliospores, are detected on daylilies within the United States, no one has yet discovered infected Patrinia plants (Hernandez et al. 2002). In addition, basidiospores have not been detected. Since it appears that daylily rust can not utilize the U.S. native species of Patrinia, it is hoped that the disease will not be a signiﬁcant problem in colder areas of the United States. However, daylily rust has successfully overwintered in the warmer areas of the United States (USDA Zone 7). In warm climates, teliospores are not required for winter survival since

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the asexual urediospore stage can continue through the winter. Some people are also concerned that daylily rust could persist in areas where there is a protective covering of deep and continuous snow throughout the winter or perhaps under a heavy winter mulch. Research is continuing into the biology and winter survival of this disease. Various studies also have been conducted to study effects of application of various fungicides (Buck and Williams-Woodwards 2003; Mueller et al. 2005) and environmental factors such as light, temperature, and leaf wetness (Mueller and Buck 2003) on rust development. An interesting study by Reilly et al. (2005) has shown a correlation between rust susceptibility and nickel. Not only did nickel spray treatment (200 ppm) prevent the infection of clean plants, it also prevented the infection of new leaves formed on diseased plants. Along with rust, daylily plant can also be attacked by diseases such as leaf streak (Aureobasidium microstictum), root-knot nematode (Meliodgyne incognita), soft rot (Erwinia carotovora), and insect pests such as ﬂower thrips (Frankliniella tritici), two spotted spider mites (Tetranychus spp.), aphids and bugs (Lopidea conﬂuenta), slugs and snails (Spencer 1972, 1973). Recently Armillaria root rot in daylily was reported (Schnabel et al. 2005) in South Carolina. Armillaria root rot causes stunting and necrosis of leaves at the tip, thereby lowering the esthetic value of the plant. C. Cultivar Registration and Awards The American Hemerocallis Society (AHS) is the ofﬁcial Hemerocallis registrar (Gretchen Baxter, Registrar, American Hemerocallis Society, PO Box 9887, Greensboro, NC 27429) and has a searchable electronic version their cultivar registration database call Day ‘Dream’ (Daylily Registry Electronic Access Modules) using FilemakerProTM software (www.daylilies.org). As of 2007, over 58,400 cultivars have been registered. Gosukonda et al. (2005) developed artiﬁcial neural networks (ANNs) to predict daylily hybrid patterns from known characteristics of parents used in hybridization. The traits included were height, diameter, foliage, blooming habit, ploidy, and blooming sequence in a cultivar analysis based on 230 genotypes. The results from prediction plots indicated a better accuracy from regression model than ANN models suggesting the need for more data sets within the domain of training of ANN for it to predict hybrid patterns. Thus, there is clear need to develop relational database with data-mining and

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data-visualization capability to provide a uniﬁed information resource to breeders. The American Hemerocallis Society also has a judging system that grants three different cultivar awards (Award of Merit, Honorable Mention and Junior Citations) based on a deﬁned set of standards (Table 3.3). As of 2007, 4,091 cultivars have been awarded (www.daylilies.org/AllAHSAwards06.pdf). In 1985, the All-American Daylily Selection Council (AADSC) was organized to administer a network of test sites throughout North America to evaluate commercially available cultivars for garden performance (www.allamericandaylilies.com). To date, nearly 6,000 cultivars have been evaluated. Daylilies earning the AADSC’s ‘‘AllAmerican’’ designation show superior performance across at least ﬁve USDA hardiness zones.

V. GENETICS A. Genome and Ploidy Level Most Hemerocallis species are diploid with 11 pairs of chromosomes (Takenaka 1929; Stout 1932; Brennan 1992). Tomkins (2003, 2004) estimated the genome size at 4408 Mb (megabase pairs), which is comparatively smaller than barley, oats, wheat, and onion but larger than rice, sorghum, maize, and sugarcane (Fig. 3.2). Several taxa with the H. fulva species complex (H. fulva ‘Europa’, H. fulva ‘Kwanso’, H. fulva var. pauciﬂora Hotta & Matsuoka, and H. fulva var. maculate Baroni) are not diploid but naturally occurring triploids ð2n ¼ 3x ¼ 33Þ (Stout 1932; Chandler 1940). Since there are no known tetraploid forms of any of the species, these triploid species did not arise from tetraploid/diploid crosses. Mostly likely they originated from an unreduced egg cell. Arisumi (1970) roughly estimated that the frequency of unreduced egg cells was 1 in 15,000. He did not ﬁnd a single unreduced pollen grain. In some species, triploids can be created easily through tetraploid/ diploid crosses. However, in daylily, Arisumi (1973) obtained only 29 triploid seedlings in 1,607 diploid-tetraploid pollinations. The frequency of success was doubled when the tetraploid parent was female. There have been a number of reports that amateur breeders have obtained ‘‘triploid’’ plants from tetraploid/diploid crosses; however, most of these ‘‘triploids’’ turn out to be diploid. The tetraploid parent used in these crosses usually was derived from a colchicine-induced tetraploid chimera (R.A. Griesbach pers. commun.).

S. K. GULIA, B. P. SINGH, J. CARTER, AND R. J. GRIESBACH

Onion

Wheat

Oats

Barley

Daylily

Sugarcane

Maize

Tulip poplar

Petunia

Tomato

Sorghum

Grape

Poplar

Rice

18,000 17,000 16,000 15,000 14,000 13,000 12,000 11,000 10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 Arabidopsis

Genome Size (Mbp)

208

Plant Species Fig. 3.2. Histogram showing the genome size of various plant species in comparison to daylily genome. (Source: Database of genome sizes (DOGS) online available at www.cbs. dtu.dk/databases/DOGS/index.html.)

Triploid daylilies are not very fertile due to the formation of multivalent chromosome pairing during meiosis. In H. fulva var. Kwanso, the 11 trivalents formed during meiotic prophase I resulted in 33 univalents during metaphase I and irregular tetrads containing various numbers of nonviable pollen grains (Dark 1932). Even though daylily triploids are not very fertile, they can be used in breeding. Stout (1926) obtained 1% viable seed using H. fulva ‘Europa’ as a female parent and 15% viable seed when using ‘Europa’ as the male parent. The ability of colchicine to induce polyploidy was discovered in 1937 (Blakeslee and Avery 1937) and used in daylily during the 1940s to create tetraploids. The difference between tetraploid and diploid forms of the same daylily is striking. In most instances, the tetraploid ﬂower is larger, heavier in substance, and more richly colored (Stamile 1990; Kehr 1996; Petit and Callaway 2000; Sakhanokho et al. 2003). In 1947, the ﬁrst colchicine-induced tetraploid daylily (‘Brilliant Glow’) was produced by Robert Schreiner, a student at the University of Minnesota, by treating the diploid ‘Cessida’. In 1948, Quinn Buck at

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the University of California reported ﬂowering the tetraploid forms of ‘Soudan’ and ‘Kanapaha’. A year later, Hamilton Traub of the U.S. Department of Agriculture in Maryland ﬂowered the tetraploid form of ‘Mayor Starsynski’ named ‘Tetra Starzynski’ (Traub 1959 and 1960). Not all colchicine-induced tetraploid daylilies produce tetraploid offspring. The daylily meristem has three different apical cell layers (L-I, L-II, L-III). The L-I layer is responsible for forming the epidermis of all organs and forms the mesophyll tissue along the leaf margin. The pollen and seeds originate from the L-II, as do, the entire mesophyll tissue of the petals and sepals and the mesophyll tissue in the outer region of the leaf next to that formed from the L-I. The L-III forms the mesophyll tissue in central region of leaf and does not contribute to any ﬂoral tissue. Colchicine treatment of daylily plants nearly always results in chimeras rather than in pure polyploids (Arisumi 1964). There are three types of chimeras: mericlinal, periclinal, and sectorial. In mericlinal chimeras, the tetraploid tissue occurs in cells along the side of the meristem and results in tetraploid tissue only on one side of the plant. Mericlinal chimeras are unstable and usually revert back to pure diploidy. In periclinal chimeras, the tetraploid tissue occurs in one (or more) of the meristem layers (L-I, L-II, or L-II), in a hand-inglove conﬁguration. Periclinal chimeras are relatively stable. In sectorial chimeras, the tetraploid tissue occurs as a solid section through all apical layers on only one side of the meristem. Thus, cell division products of the tetraploid cells give rise to a section of tetraploid tissue. Sectorial chimeras are relatively stable. When using colchicine-treated plants in breeding, it very important to identify what type of chimera they are and the ploidy of their L-II layer. Besides chromosome counts, stomata size, pollen size (Arisumi 1965), and ﬂow cytometry (Saito et al. 2003) have been used for determining ploidy. The drawback in using stomata size is that it measures the ploidy level of L-I tissue and not L-II tissue. The ﬁrst tetraploids were not as fertile as diploids (Peck and Peck 1969), due the production of multivalents during meiosis. Artiﬁcially chromosome-doubled plants are autotetraploid and form quadravalents during meiosis that can lead to abnormal meiosis (Fig. 3.3). For example in ‘Crestwood Ann’, numerous multivalents during meiosis resulted in lagggers and bridges. Over time, more fertile tetraplopids were selected in breeding. These fertile plants had a normal meiosis. This is similar to what was found over time in maize (Gilles and Randolph 1951). Speciﬁc genes have been described in several species that prevent multivalent formation (Jackson and Casey 1982). Similar

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Fig. 3.3. Meiosis in daylily: (A) ‘Crestwood Ann’ showing metaphase 1; (B) ‘Crestwood Ann’ showing anaphase 1 with abnormal cell-plate formation; (C) ‘Crestwood Ann’ showing anaphase 1 with laggers forming micopollen grain; and (D) ‘Crestwood Ann’ showing anaphase 1 with unequal multivalent separation forming bridge.

genes most likely played a role in developing more fertile tetraploid daylilies. Most modern tetraploid cultivars are now as fertile as their diploids. The ﬁrst major tetraploid breeding program was started in 1955 by Robert A. Griesbach at DePaul University in Chicago, Illinois, and Orville Fay, a retired chemist. They developed a new method of colchicine treatment utilizing germinating seedlings. The seed treatment was easier and more efﬁcient than the whole-plant treatments used by Schreiner, Buck, and Traub (Traub 1959, 1960). Using the seed treatment, one could treat a larger number of plants in the same amount of time. In addition, the seed treatment resulted in a higher frequency of tetraploidy (Griesbach et al. 1963). Besides publishing the treatment method, Griesbach held numerous training sessions to teach the procedure to both commercial and amateur breeders.

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Table 3.4. Point scale standards for judging daylilies. Characteristic Complete plant Garden value (10 pt) Vigor (10 pt) Performance (10 pt) Foliage Inﬂorescence Height (10 pt) Branching/bud count (10 pt) Flower Durability/fragrance (10 pt) Color (10 pt) Form (10 pt) Distinction TOTAL

Points 30

10 20

30

10 100

Source: American Hemerocallis Society, 2008.

In 1961, Fay and Griesbach released four tetraploids: ‘Crestwood Ann’, ‘Crestwood Bicolor’, ‘Crestwod Evening’, and ‘Crestwood Lucy’. Unlike the previously released tetraploid cultivars, the Crestwood cultivars were reasonably priced and widely distributed. The wide distribution of these cultivars, coupled with Griesbach’s training sessions, resulted in a new wave of daylily breeding (Traub 1973). Through the years, reﬁnements in the colchicine treatment procedure have occurred (Arisumi 1964, 1972; Buck 1969; Warner 1969; Chen and Goeden-Kallemeyn 1979; Barr 1990; Brennan and King 2003; Sakhanokho et al. 2004a). One of the reﬁnements by Toru Arimsu of the U.S. Department of Agriculture in Maryland involved treating the exposed vegetative meristem (Arisumi 1964). This very successful method for converting diploid cultivars into tetraploid clones was used by many amateur breeders during the 1960s and 1970s to expand the tetraploid gene pool (Traub 1973). A number of studies have been carried out to determine genetic diversity among daylily populations using isozymes and allozymes (Kang et al. 1998; Kang and Chung 2000), AFLP (Tomkins et al. 2001), and chloroplast DNA (Noguchi et al. 2004) markers. Isozymes studies by Kang and Chung (2000) revealed high level of allozyme variation within 30 populations of ﬁve Hemerocallis species from Korea and low level of allozyme divergence within species. Tomkins et al. (2001) conducted diversity analysis of 19 primary genotypes and 100 cultivars of daylily from different time periods

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using AFLP markers. They observed that there was a slight decrease over time in the genetic diversity of diploid cultivars that were released between 1940 and 1964. From 1965 to 1980, the genetic diversity remained unchanged. Interestingly, the genetic diversity of 40 tetraploid cultivars released from 1980 to 1998 was slightly lower (Nei’s similarity coefﬁcient of 0.850) than that of 21 diploid cultivars released during the same period (0.814). The genetic diversity of the diploid cultivars released during this time period was signiﬁcant lower than that found in the wild species (0.762). These data suggest that there is need to further increase both the tetraploid and diploid gene pools. B. Flower Color Inheritance Flower color in daylily is the result of three pigments: chlorophylls, carotenoids, and ﬂavonoids. Flavonoids can be further divided into copigments (colorless) and anthocyanins (colored). Chlorophylls are located within chloroplasts in the cell cytoplasm. Carotenoids are contained within chromoplasts, whereas ﬂavonoid and betalains are located within the cell vacuoles. The yellow through orange colors of ﬂowers typically are due to the carotenoid pigments, whereas blue to red colors typically are attributed to anthocyanins. Chlorophylls are responsible for green color. Each type of pigment is the result of a different sequence of biochemical reactions. The production of each pigment is independent of the other pigments. In most cases, a defect in the ﬂavonoid pathway has no effect on the carotenoid and chlorophyll pathways and vice versa (Griesbach 1984, 2005). Most daylily ﬂowers derive their color from more than one pigment source (Griesbach and Batdorf 1995). It is very striking to compare the ﬂower color of modern cultivars with that of the species. Modern cultivars come in a wide range of colors, from purple through red and yellow through orange; the ﬂower color of nearly all of the species, however, is limited to yellow through orange. Two taxa have unique colors: Hemerocallis fulva fm. rosea has rosecolored ﬂowers and H. fulva fm. disticha has mahogany-colored ﬂowers. The orange ﬂower color of the wild type H. fulva fm. fulva (Munsell 7.5R 7/14) is the result of a single anthocyanin (cyanidin-3-rutinoside) and two carotenoids (zeaxanthin and lutein) (Asen and Arisumi 1968; Griesbach and Batdorf 1995). The novel mahogany ﬂower color of H. fulva fm. disticha (RHS 171B) is the result of a mutation in the ﬂavonoid pathway leading to delphinidin-3-rutinoside instead of

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cyanidin-3-rutinoside. Similar to H. fulva fm. fulva, H. fulva fm. disticha contains the two carotenoids zeaxanthin and lutein. The rose ﬂower color of H. fulva fm. rosea (Munsell 2.5YR 5/10) is the result of mutation that reduces the concentration of the caroteinoid pigments while maintaining the concentration of anthocyanin cyanidin-3-rutinoside. During the 1960s, a group of breeders (Fay, Griesbach, and Peck) determined the inheritance of a mutation that reduced the concentration of anthocyanin pigments while maintaining the concentration of caroteinoid pigments. They gave this recessive mutation the name melon. In 1934, the New York Botanical Garden released three daylily cultivars with new red ﬂower colors that seen in the wild species (Stout 1942). The cultivar ‘Theron’ with dark red ﬂowers was derived from H. x aurantiaca, H. fulva, and H. ﬂava. The cultivar ‘Red Bird’ with scarlet-red ﬂowers was derived from intrabreeding several unique forms of H. fulva from Chengtu and Kuling, China, with orange-scarlet ﬂowers instead of the typical orange color. The cultivar ‘Rosalind’ with rose-red ﬂowers was derived from intrabreeding three different H. fulva fm. rosea clones. ‘Theron’, ‘Red Bird’, and ‘Rosalind’ led to the development of modern true red, pink, and purple ﬂowered cultivars. C. Biotechnology The tissue culture techniques previously described can be used to produce transgenic daylily plants. Mutants can arise through the tissue culture process. These mutations are called somaclonal (Larkin and Scowcroft 1981). Somaclonal mutations can arise from mutagenic chemicals used in the tissue culture process. In addition, the tissue culture process allows naturally occurring mutant cells to develop into whole plants. For example, a leaf cell containing a mutation in a ﬂower color gene would express that mutation only if it were regenerated into a whole plant. Hemerocallis ‘Yellow Tinkerbelle’ is a somaclonal mutation of H. ‘Eenie Weenie’ that has a more dwarf growth habit (Griesbach 1989). The frequency of somaclonal mutations is reported to be very low in daylily (Krikorian et al. 1981a; Griesbach 1989). Griesbach (1990) has suggested delaying the shoot formation, use of undifferentiated tumorlike callus cells or older callus, and use of auxins as ways of increasing the frequency of somaclonal variants. Regeneration protocols and genetic transformation of daylily (Hemer ocallis spp. ‘Stella de Oro’) by particle bombardment have been achieved (Aziz et al. 2003). Callus cultures initiated from ovules were bombarded with gold particles coated with plasmid-harboring Basta1

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resistance gene. Resulting putative transgenic calli were selected after 2 weeks, and surviving calli regenerated shoots after 2 months. Polymerase chain reaction and Southern blotting were used to conﬁrm independent transformation events. Genetic engineering could prove useful to introduce new traits that do not exits in the gene pool.

VI. CONCLUSION Daylily is an important ornamental crop that also has culinary and medicinal uses. There is scope for further expansion of its use in landscaping because the plant is drought resistant and requires low maintenance. However, due to asexual mode of propagation in daylily through slow-dividing crowns, it has been difﬁcult to meet the market demand for choice new cultivars. The use of tissue culture on commercial scale to accelerate propagation rate may hold the key to solving this problem. It should be kept in mind that separate tissue culture protocols may be needed for individual cultivars. Until now, daylily breeding has been carried out mostly by amateur breeders. Objectives of these breeding efforts were to produce cultivars differing in ﬂower characteristics: notably color, shape, and form of ﬂowers. A large number of diploid and tetraploid daylily cultivars were produced for these purposes. Considering the market potential for daylily, now there is need for genetic studies and widening of gene pool to incorporate value-added traits in new daylily cultivars. Ornamental use of daylily as cut ﬂowers has potential, provided ﬂowers longevity is extended to multiple days. The genes for this purpose are available in other species, but their introgression into daylily is awaited. The lucrative markets of daylily for food and medicine are essentially untapped. In summary, daylily is an important land inﬂorescence plant that holds promise of considerable expanded use for this and other purposes provided its propagation problems are solved and certain needed traits are incorporated into the crop.

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Saito, H., K. Mizunashi, S. Tanaka, Y. Adachi, and M. Nakano. 2003. Ploidy estimation in Hemerocallis species and cultivars by ﬂow cytometry. Scientia Hort. 97:185–192. Sakhanokho, H.F., C. Cheatham, and C. Pounders Jr. 2003. Chromosome doubling via injection of colchicine in Ba-pretreated daylily (Hemerocallis spp). 31st Annual Horticulture Field Day, 2 Oct. 2003, Mississippi State Univ., Poplarville, MS. Sakhanokho, H. F., C. Cheatham, and C. Pounders Jr. 2004a. Evaluation of techniques to induce polyploidy daylilies. Southern Nursery Assoc. Proc. 592–594. Sakhanokho, H.F., W. Copes, L. Nyochembeng, and R. Kelley. 2004b. Molecular approaches to control Puccinia hemerocallidis on daylily. p. 18. In: Meeting abstract. Biotechnol. Symp. 4 June 2004. Alabama A&M Univ., Normal, AL. Schabell, J. 1990. The daylily: 5000 years of glory. Daylily J. 45(4):348–353. Schnabel, G., K.E. Bussey, and K. Bryson. 2005. First report of gallica causing armyllaria root rot in daylily in South Carolina. Plant Dis. 89(6):683. Smith, D.L., and A.D. Krikorian. 1991. Growth and maintenance of an embryogenic cell culture of daylily (Hemerocallis) on hormone-free medium. Ann. Bot. 67:443–449. Spencer, J.A. 1972. Conditions favoring development of bacterial soft rot. Hemerocallis J. 27(3):24. Spencer, J.A. 1973. Colecephalus Hemerocallis [sic], the cause of daylily leaf-streak: morphology, taxonomy, and cultural characteristics. Hemerocallis J. 26(3):12–16. Stamile, P. 1990. From diploid to tetraploid. Daylily J. 45(3):242–249. Stephenson, P., and B. Rubinstein. 1998. Characterization of proteolytic activity during senescence in daylilies. Physiologia Plant, 104:463–473. Stout, A.B. 1926. The capsules, seeds, and seedlings of the orange daylily. J. Heredity 12:243–249. Stout, A.B. 1932. Chromosome numbers in Hemerocallis, with reference to triploidy and secodary polyploidy. Cytologia 3:250–259. Stout, A.B. 1941. Memorandum on a mongraph of the genus Hemerocallis. Herbertia 8:67– 71. Stout, A.B. 1942. Origin and genetics of some classes of red-ﬂowered daylilies. Herbertia 8:161–174. Stout, A.B. 1986. Daylilies. Sagapress, Inc. Millwood, NY. Stout, A.B. and C. Chandler. 1933. Pollen tube behavior in Hemerocallis with special reference to incompatibilities. Bul. Torry Bot. Soc. 60:397–417. Takenaka, Y. 1929. Karyological studies in Hemerocallis. Cytologia 1:76–84. Tomkins, J.P. 2001a. DNA ﬁngerprinting in daylilies: genetic variation among modern cultivars - Part I. Daylily J. 56(2):195–200. Tomkins, J.P. 2001b. DNA ﬁngerprinting in daylilies: genetic variation relationships among species—Part II. Daylily J. 56(3):343–347. Tomkins, J.P. 2003. How much DNA is in daylily? Estimating genome size using ﬂow cytometry. Daylily J. 58(2):205–209. Tomkins, J.P. 2004. Cloning the daylily genome. Continued study at Clemson University in genome analysis of Hemerocallis. Daylily J. 59(3):245–252. Tomkins, J.P., T.C. Wood, L.S. Barnes, A. Westman, and R.A. Wing. 2001. Evaluation of genetic variation in the daylily (Hemerocallis spp.) using AFLP markers. Theor. Appl. Genet. 102:489–496. Traub, H.P. 1959. First decade of Hemerocallis washingtonia. Part 1. Plant Life 15:69–79. Traub, H.P. 1960. First decade of Hemerocallis washingtonia. Part 2. Plant Life 16:111– 120. Traub, H.P., W.Q. Buck, and H.C. Lloyd. 1973. Second decade of Hemerocallis washingtonia, Jan.1, 1959–Dec. 31, 1968. Plant Life 29:125–140.

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U.S. Department of Agriculture National Agriculture Research Service. 2003. 2002 summary of ﬂoricultural crops, US Department of Agriculture, Bellsville, MD. Valpuesta, V., N.E. Lange, C. Cuerrero, and M.S. Reid. 1995. Upregulation of a cysteine protease accompanies the ethylene-insensitive senescence of daylily (Hemerocallis) ﬂowers. Plant Molec. Biol. 28:575–582. Voth, P.D., R.A. Griebach, and J.R. Yeager. 2002. Developmental anatomy and physiology of daylily. pp. 128–129. In: The new daylily handbook for 2002. AHS, Alexandria, VA. Warner, J.E. 1969. Colchicine treatment of adult clones. The Hemerocallis J. 23(2):39–41. William-Woodward, J.L., and J.W. Buck. 2002. Disease and pest of daylily. pp. 222–239. In: F. Gatlin and J. Brennan (eds.). New daylily handbook. American Hemerocallis Society, Kansas City, MO. William-Woodward, J.L., J.F. Hennen, K.W. Parda, and J.M. Fowler. 2001. First report on daylily rust in the United States. Plant Dis. 85:1121. Windham, A.S., M.T. Windham, T.C. Stebbins, W.E. Copes, and L.H. Self. 2004. A ﬁrst report of daylily rust in Tennessee. Southern Nursery Assoc. Proc. 49:231–232.

4 Horseradish: Botany, Horticulture, Breeding Ashraf Shehata, Richard M.S. Mulwa, Mohammad Babadoost, Mark Uchanski, Margaret A. Norton, and Robert Skirvin University of Illinois at Urbana-Champaign Urbana, IL 61801 USA S. Alan Walters Department of Plant, Soil, and Agricultural Systems Southern Illinois University Carbondale, IL 62901 USA

I. INTRODUCTION II. HISTORY III. BOTANY A. Taxonomy and Nomenclature B. Morphology C. Reproductive Biology D. Genetic Structure E. Biochemistry IV. HORTICULTURE A. Production 1. Propagation 2. Field Establishment 3. Field Cultivation 4. Fertilization 5. Diseases, Pests, and Weeds 6. Harvesting 7. Postharvest Technology B. Utilization 1. Therapeutics 2. Culinary Uses 3. Industrial Uses

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 221

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V. BREEDING A. Crop Improvement in the United States B. Germplasm Collection C. Breeding Methods 1. Breeding Cycle 2. Hybridization 3. Increasing Fertility 4. Seedling Evaluation 5. New Cultivar Releases 6. The Future VI. LITERATURE CITED

I. INTRODUCTION Horseradish (Armoracia rusticana P. Gaertner, B. Meyer & Scherbius, Brassicaceae), native to southeastern Europe and western Asia, is a large-leaved, hardy perennial herb that forms a rosette of large, entire leaves, long ﬂowering stalks with small white ﬂowers in a terminal panicle, and thick pungent roots (Bailey and Bailey 1976). Horseradish was known in antiquity as both a medicinal herb and a condiment, and the latter use is the primary one today (Rosengarten 1969). The bitter ﬂavors and pungent aroma of horseradish are a result of sulfurcontaining glucosinolates in the tissues breaking down into isothiocyanates (Li and Kushad 2004). After harvest, the main root can be ground into a processed product, while the side roots are retained for planting the following season. The principal commercial horseradish production areas are located in the United States and Hungary and to a lesser extent in other parts of Europe (Table 4.1), but accurate worldwide production and consumption data are difﬁcult to obtain. Approximately 1,600 ha of horseradish are grown in the United States each year (Table 4.1) with Illinois producing approximately 40% of the total, making it the leading producer (Gerber et al. 1983; Eastburn and Chang 1994; Babadoost et al. 2001). An estimated 10.8 billion kg horseradish is processed each year in the United States (Kumar 2003; Horseradish Information Council 2008). The other major horseradish production areas in the United States are located in Eau Claire, Wisconsin, and Tulelake, California. Smallerscale production is found in other states, including Michigan, Ohio, Pennsylvania, New Jersey, Connecticut, Massachusetts, Oregon and Washington among others (Weber 1949; Bratsch 2006). Many of these states are historic producers of horseradish due to site conditions (soils and climate) and European immigrant settlement patterns. Horseradish

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Table 4.1. Major horseradish-growing countries. Country Austria Canada

Region

Crop area (ha)

Graz, 300 Feldbach Ontario 400

Planting season

Harvesting season

Main cultivars

Yields (kg/ha)

April–May

Sept–Mar

Steirischer

> 8000

April–May

Oct–May

Big Top Western Several Bagamer, Danish Big Top Western Several Big Top Western

6000–18000

Germany Nurnberg Hungary Debrecen, Ujleta USA Californiaa

200 1,200

April–May April–May

Sept–Mar Sept–Nov

400

Year round

Sept–Nov

Illinois Wisconsin

800 400

April–May April–May

Sept–Mar Sept–Mar

> 10,000 7500 4000–8000 9000 9000

a Primarily grown as perennial crop. Source: M. Babadoost and A. Walters, pers. commun.

is also produced internationally, especially in European countries including Germany, Hungary, and Poland. Recently China has also begun commercial production of horseradish, although much of the crop is produced in small farms. This review examines origins, history, botany, horticulture, and genetic improvement of horseradish.

II. HISTORY Early records indicate that horseradish is a native of the temperate regions of eastern Europe and western Asia, where wild types are found growing from Finland and Poland to the regions around the Caspian Sea and the deserts of Cumania (now Romania) and Turkey (De Candolle 1890; Hedrick 1919). From here, horseradish spread to western Europe and across the Atlantic to the New World. Today horseradish has become naturalized in many parts of the world and can be found both cultivated and growing wild. Jews since medieval times have used horseradish as one of the bitter herbs in ceremonies concerned with the feast of Passover celebrating the exodus from Egypt, but horseradish is not a biblical herb nor is there evidence that the plant was used in Egypt (Schaffer 1981). Horseradish has been reported to have grown in Greece in antiquity (Rizza and Harrison 2002) and appears to be referred to by Theophrastus (371–287 BCE) in Enquiry into Plants (Hort 1926) who

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refers to various kinds of radish: ‘‘the Corinthian, that of Cleonae, the Leiothasian, amorea, the Boeotian, The Leiothasian, is called by some the Thracian radish, and it stands the winter best.’’ Pedanius Dioscorides (20–70 CE), a Roman army physician from Anazarbus, Cilicia (now Turkey), in his famous work Latinized as De Material Media written about 65 CE, refers to a wild raphanos (radish) that appears to be horseradish: The wild radish, which the Romans call armoracium, has leaves resembling those of the cultivated, tending rather towards the leaves of charlock [Raphanus raphanistrum] but its root is thin, long, and somewhat sharp. Both the root and the leaves are boiled to eat as potherbs. It warms, it is diuretic, and it is very hot. II.112. (Beck 2005)

The Latin amoracium or amoracia (plural) is subsequently referred to by Columella in De Re Rustica (6.17.8, 12.9.3) published between 61 and 64 CE and Pliny in Historia Naturalis (19.82 20.22) published in 77. Pliny notes that armoracia was called armon in Pontus (Dalby 2003), and armoracia has been retained as the generic name of horseradish. Amoracia is referred to by Palladius in the fourth century. Albertus Magnus in the 13th century refers to wild raphanus. Leonhart Fuchs (1542) referred to horseradish as Raphanus sylvestris (Meyer et al. 1999) (Fig. 4.1). De Candolle (1890) suggested that the word chren was the earliest name used for horseradish in the Slavic languages of eastern Europe, where the crop was endemic. Later, chren was introduced into German and French dialects in the forms of kren, kreen, cran, and cranson. In Germany it is called meerrettich, or ‘‘sea radish,’’ because it grew in coastal areas by the sea. Other similar words for horseradish in western Europe were meerretig, mee-radys, and meridi, all of which mean ‘‘sea radish.’’ The name meer seems to have been misunderstood by the English as ma¨hre (‘‘old horse’’), perhaps reﬂecting the rankness and toughness of the roots (Tucker and DeBaggio 2000). This name may have been corrupted to ‘‘mare-radish’’ and from there transformed to ‘‘horse-radish’’ (Courter and Rhodes 1969). Various names of horseradish from around the world are presented in Table 4.2. The ﬁrst use of the term ‘‘horse radish’’ was made by John Gerard in his famous English herbal (1597) that contains a lengthy entry with a woodcut (Fig. 4.2) and a clear description of the plant. Some believe the English called the plant horseradish in reference to its propensity to rapidly spread in a ‘‘galloping’’ behavior (i.e., it is difﬁcult to control once it has been introduced in an area). Another possible origin for this

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Fig. 4.1. Raphanus sylvestris (Horseradish) plate from the great herbal of Leonhart Fuchs (1542). (Source: Meyer et al. 1999). Table 4.2. International names for horseradish (Armoracia rusticana). Language Arabic Chinese Dutch French German Italian Japanese Portuguese Russian Spanish Swedish

Name Fujl har La gen Mierikswortel Raifort, cran Meerrettich, Kren Barbaforte, ramolaccio Seiyo wasabi Ra´bano picantes Khren Cochlearia, ra´banto picante, ra´bano rusticano Sko¨rbjuggso¨rt

Source: Wiersema and Leon 1999; Tucker and Debaggio 2000.

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Fig. 4.2. Horseradish woodcut from Gerard’s Herball (1597).

herb’s name is that it was ﬁrst called harsh radish because it is so bitter on the tongue (McCann 2004). The horse in horseradish may connote the plant’s size and coarseness, in comparison to the cultivated radish (McCann 2004). Grieve (1931) makes the point that the preﬁx horse is often used in this way, as in horse-mint and horse chestnut. Horseradish was introduced into the United States from Europe by early settlers and became popular in gardens around the New England states in the early 1800s. In 1806, the plant was common in the northeastern United States and listed in a catalog of garden vegetables (Hedrick 1919). By 1840, it was growing wild near Boston (Courter and Rhodes 1969; Horseradish Information Council 2007). Horseradish is now naturalized throughout many areas of North America (Lust 1974).

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Commercial cultivation of horseradish in America began in the 1850s. It was brought to Chicago in 1856 by the Sell family, immigrants from Germany. They gave plants to the Sass family, who initiated the development of the horseradish industry in Chicago (Courter and Rhodes 1969). Immigrants continued spreading horseradish production in the Midwest. By the late 1890s, a thriving horseradish industry had developed in an area of the fertile soils of the Mississippi river valley in Illinois near St. Louis called the river bottoms (Horseradish Information Council 2007). About the same time, a small center of horseradish production was also established in Eau Claire, Wisconsin. According to Weber (1949), homesteaders in the Tulelake region of northern California began cultivating horseradish after World War II, and other areas followed suit with numerous plantings being made in Michigan, Ohio, Pennsylvania, northern New Jersey, Connecticut, Massachusetts, and Washington. In 1909, Illinois had only about 50 ha planted to horseradish. Current estimates put production at 650 to 800 ha. Today the crop areas east of St. Louis, Missouri, are considered the most concentrated horseradish production regions in America. Collinsville, Illinois, advertises itself as the ‘‘Horseradish Capital of the World.’’

III. BOTANY A. Taxonomy and Nomenclature The taxonomy of horseradish has changed over time. Dioscorides calls the plant wild raphnos (from radix, meaning ‘‘root’’); the similar name Raphanus was maintained by the Renaissance herbalist Mattioli and Gerard while the ancient Latin writers Columella and Pliny use armoracia. The word ‘‘armoracia’’ derives from the Celtic ar (‘‘near’’), mor (‘‘the sea’’), rich (‘‘against’’), that is, a plant growing near the sea (Courter and Rhodes 1969). Linnaeus (1753) gave it the botanical name Cochlearia armoracia, based on its long leaves that are supposed to resemble an oldfashioned spoon, or cochleare. The plant was included in the Materia Medica of the London Pharmacopoeias of the 18th century under the name Raphanous rustican, the name given by Gerard (Grieve 1931), Modern taxonomists have placed it in the genus Armoracia (Courter and Rhodes 1969). Manton (1932) observed that the somatic chromosomes of horseradish were similar to those of plants in the genus Rorippa and

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suggested that the genus Rorippa must have given rise to both Armoracia and Nasturtium. In the 1941 edition of Hortus Second, Bailey named it Armoracia rusticana. Later, in the 1949 second edition of his Manual of Cultivated Plants, he called it Armoracia lapathifolia. The name Cochlearia armoracia given by Linnaeus in Species Plantarum (1753) was later changed to Cochlearia rusticana by Lamarck in Flore Francaise (1788). However, this name was later dismissed as an illegitimate and superﬂuous by the committee of the International Code of Botanical Nomenclature (ICBN, Montreal Code) because Cochlearia armoracia L. had been cited as a synonym. Horseradish was also named Armoracia lapathifolia by Gilbert in Flora Lithuanica Inchoata (1782), which was also an invalid name according to the ICBN because the genus was not described and a binomial system was not consistently used in this ﬂora. According to Fosberg (1966), Armoracia was separated from the genus Cochlearia and considered a separate genus based on stigma and fruit pod differences. Cochlearia is characterized by a pronounced capitate stigma and a capsule with a strong midrib plus a lateral network of veins. Armoracia is distinguished by a small capitate stigma and a fruit with a weak or inconspicuous midrib and an indistinct network of veins. The ﬁrst valid published scientiﬁc name of horseradish resulting from the generic name Armoracia was A. rusticana in 1800 by Gaertner, Meyer, and Scherbius (Fosberg 1966; Courter and Rhodes 1969). All modern workers use this binomial. B. Morphology Various types of horseradish have been recognized based on their leaf forms. Type I includes the plants with smooth leaves (‘Bohemian’ types) with tapered leaf bases. Type III plants (‘Maliner Kren’ or ‘Common’ types) typically have crinkled leaves with a heart-shaped leaf base. Type II plants are intermediate between the other two types (Rhodes and Courter 1965a; Courter and Rhodes 1969; Tucker and DeBaggio 2000). Horseradish is a large-leaved, hardy, and glabrous perennial herb that grows to a height of up to 120 cm. The leaves are long-petioled, oblong-ovate, cordate at the base, unevenly crenate, and grow to a length of 30 to 100 cm. The lower cauline leaves have shorter petioles and may be lobed or comb-shaped-pinnate; they have linear-oblong, entire-margined, or serrate sections. The upper cauline leaves have

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Fig. 4.3. Horseradish in ﬂower (left) and at pod set (right).

narrow bases and are mostly sessile, oblong or lanceolate, unevenly crenate to serrate, and obtuse at the apex. The uppermost leaves of the plant are linear or almost entire-margined. Some purple-leafed and variegated ornamental cultivars are also available, though rare (Pleasant 2003). Horseradish plants produce numerous fragrant ﬂowers with 5- to 7-cm-long upright pedicels borne on racemes that have four sepals, four petals, and six tetradynamous stamens (Fig. 4.3). Their sepals are 2.5 to 3 mm long, broadly ovate and with a membranous white margin. Petals are white, 5 to 7 mm long, and broadly obovate. The inner stamens are 2.5 mm long; the outer ones are 1.5 mm long. The stigma is broad, round, and gently two-lobed. Horseradish bears 4- to 6-mmlong, globose to obovate siliques with persistent styles on 20-mm long, upright-spreading stems. The seeds are smooth and brown when mature (Ozgur et al. 2004). The root system of horseradish consists of a long, white, cylindrical or tapering main root that can grow to about 60 cm in loose soils. Several thin lateral roots also form around the main root and near the collar of the crown. Undisturbed, the root system can reach a depth of 3 to 4 m with a lateral spread of about 1 m (Weaver and Bruner,1927) (Fig. 4.4).

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Fig. 4.4. Mature root system of a 10-year-old plant of horseradish (squares are 30 cm2). (Source: Weaver and Bruener 1927).

C. Reproductive Biology The horseradish inﬂorescence is a paniculate raceme of shortpediceled, small white ﬂowers (Rhodes 1977). ‘Big Top Western’ and ‘Bohemian’ types have uniformly elongated racemes that form somewhat rounded panicles, while the lower racemes of ‘Common’ types tend to elongate more than the terminal racemes, giving the

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inﬂorescence the appearance of a corymb (Rhodes et al. 1965a). Compared to ‘Big Top Western’ and ‘Bohemian’ types, the ﬂowers of the ‘Common’ type often fail to develop and abort before opening. Generally, the ﬂowers produced by ‘Common’ are smaller in size than either ‘Big Top Western’ or ‘Bohemian’ types. The second year after signiﬁcant crown development has occurred (i.e., from set root to large primary root), the horseradish plant will naturally ﬂower during April and May in Illinois. Mature roots can be forced under greenhouse conditions, if previously provided two months of cold treatment at 0 to 5 C (Rhodes et al. 1965b). The horseradish fruit is a silique (referred to as a pod) composed of two carpels that are separated by a dividing wall. The carpels are somewhat ﬂattened and dehisce along the two sides at maturity (Stokes 1955). Fruits mature in three to four weeks after successful pollination and fertilization (Weber 1949). Once the pod dehisces, the exposed seeds will either remain attached to the placental wall or detach and drop from the pod. Although mature pods will turn brown in color, dehiscence usually occurs while fruits are still green (Weber 1949). The seeds in the pods are arranged in four rows, with two rows on each side of the division (Stokes 1955). The ovary usually contains 16 to 20 ovules (Weber 1949) with seed numbers ranging from 1 to 6 per fruit (Rhodes 1977; Stokes 1955). Among the horseradish clones used for breeding in Illinois, 0 to 6 seeds per pod are generally observed (Rhodes et al. 1965b). D. Genetic Structure The genus Armoracia includes three species: A. macrocarpa (Waldst. & Kit.) ex Baumg., A. rusticana, and A. sisymbroides (DC.) Cajander (A. Miller pers. commun.). Cultural horseradish (A. rusticana) is suspected of being an interspeciﬁc hybrid, but the species involved are unknown (Brezezinski 1909; Tucker and DeBaggio 2000). Horseradish is a tetraploid (2n ¼ 4x ¼ 32) (Manton 1932; Skalinska et al., 1976; Uhrikova and Majovsky 1980; Mesicek and JavurkovaJarolimova 1992) with very low fertility (Smith 1976; Simpson and Conner-Ogorzaly 1986). Although plants ﬂower copiously, seed production is very low. The plants are believed to be highly self-incompatible. Horseradish often has been described as male sterile (Weber 1949). The problem is difﬁcult to explain. The anatomical structure of the horseradish anther and its walls appears normal and is typical of the Brassicaceae with all layers properly developed and fully functional.

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At maturity, there are no obvious impediments to pollen release (Winiarczyk et al. 2007). Many pollen grains are shrunken; Stokes (1955) reports that only about 60% of horseradish pollen is functional. Based on its sterility, Brezezinski (1909) suggested that horseradish is an interspeciﬁc hybrid between unknown parents. Cytological evidence supports this possibility. For instance, Weber (1949) found partial pairing of chromosomes and other irregularities during microsporogenesis and megasporogenesis. Easterly (1963) reported aneuploidy in meiotic stages of pollen mother cells as well as differing chromosome counts of n ¼ 14 and n ¼ 16 in different plant specimens. Stokes (1955) stated that failure to set seeds following fertilization was due primarily to endosperm-maternal tissue incompatibility and embryo abortion. In spite of low fertility, viable seed can be obtained in horseradish (Brezezinski 1909; Weber 1949; Moravec 1963; Rhodes et al. 1965 a,b). A. rusticana is probably an allotetraploid based on the irregularities commonly observed in chromosome pairing, although some homology will occur between the two genomes allowing the production of viable seed (Horwitz 1983). E. Biochemistry Horseradish roots, when unbroken, are inodorous. The intense pungency and aroma of horseradish results from crushing. grinding, or chewing the cells (Courter and Rhodes 1969). This response is thought to be related to an antiherbivory defense system. Normally, the components that cause the intense pungency are physically separated from one another. Glucosinolates are found primarily in the vacuole (Grob and Matile 1979); the enzyme that causes the reaction, myrosinase, is stored within myrosin grains in myrosin cells. Crushing mixes the sinigrin and 2-phenylethylglucosinolate glucosinolates with myrosinase (Simon et al. 1984), and pungent volatile allyl oils (isothiocyanates) are produced (Acquaah 2002). The enzyme myrosinase has an optimum pH of 7 and shows maximum activity at 37 C over a 20-minute time period. The resulting isothiocyanates are more stable under acidic conditions than neutral (Depree et al. 1999). Thus, to maximize the volatile oils and maximize the ‘‘bite’’ of freshly ground horseradish, it should be ground at approximately 37 C, allowed to stand for 20 minutes, then stabilized by the addition of a mild acid (typically vinegar) (Fig. 4.5). The ground product should be consumed quickly or refrigerated to minimize the loss of the volatile ﬂavor compounds. However, ground horseradish

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Fig. 4.5. Fresh-ground horseradish root produces its characteristic sinus-clearing ‘‘bite’’ through the breakdown of a family of sulfur-containing compounds called glucosinolates. Upon crushing, these glucosinolates are mixed with the enzyme myrosinase to form primarily isothiocyanates (under neutral conditions). These compounds rapidly volatilize. For full ﬂavor, the product must be consumed within a short period of time.

slowly loss its pungency, becomes darkened, and develops off ﬂavors even under refrigeration; this quality loss can be slowed by adding a fat or oil, such as cream (Courter and Rhodes 1969). Li and Kushad (2004) evaluated 27 horseradish root accessions and 9 leaf accessions for glucosinolate content and myrosinase enzyme activity. Eight different glucosinolates were detected in both root and leaf tissues, but four were found in quantity: sinigrin, glucobrassicin, neoglucobrassicin, and gluconasturtin. These authors found that glucosinolate content and myrosinase activity varied widely among accessions. Horseradish roots are also rich in an enzyme called horseradish peroxidase. The enzyme is used in various applications, including analysis of cholesterol and glucose levels in blood, immunoassays, and DNA probes. Horseradish peroxidase levels among horseradish varieties cultivars can be highly variable (Kushad et al. 1999). The horseradish root also contains coumarins (e.g., aesculetin and scopoletin), phenolic acids that are derivatives of caffeic acid and hydroxycinnamic acid (Stoehr and Herrman 1975; Newall et al. 1996). Also present in quantity is ascorbic acid, asparagin, and resin (Karnick 1994; Budavari 1996; Newall et al. 1996).

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IV. HORTICULTURE A. Production 1. Propagation. For commercial root production, horseradish is always vegetatively propagated. Seed propagation is utilized in breeding efforts. Traditional. Horseradish is commercially propagated from root cuttings, called sets. Sets are saved from the fall harvest and stored until the following spring. Sets are cut from primary roots and used for propagation if they show no obvious symptoms of disease. Sets are usually 1 to 2 cm in diameter and 30 to 38 cm long, and are prepared with a straight cut on the proximal end and a slant cut on the distal end to help planters observe polarity in upright planting (USDA 1968). Polarity can be ignored by laying the sets horizontally during planting, but this can delay plant emergence. Micropropagation. Horseradish plants may be produced in tissue culture. This process is especially useful when large numbers of pathogen-free plants are desired or when yield declines due to high disease load in planting stock are experienced. Production of pathogenfree planting stocks was initiated at the University of Illinois for the growers in the Collinsville area of the United States (Uchanski et al. 2004). In vitro cultures were established from small leaf explants (Norton et al. 2001). Apical meristems excised from in vitro plantlets were tested for the presence of Turnip mosaic virus (TuMV) using reverse-transcriptase polymerase chain reaction (RT-PCR) and tested for Verticillium dahliae using PCR (Uchanski 2007). Some pathogenfree plants of each cultivar were placed in cold storage (4 C) to serve as nuclear propagation stock, and the remainder were multiplied for distribution to growers. Tissue-cultured plants multiply readily on Murashige and Skoog (1962) medium supplemented with 2 micromoles per liter of 6-benzylaminopurine (BA). In one experiment, 15 pathogen-free plants of two different cultivars, ‘Illinois Horseradish (ILHR) 15K’ and ‘ILHR 1722’, produced 1,479 and 1,285 plantlets, respectively, in 20 days (Uchanski 2007). To further streamline in vitro propagation of horseradish, studies have been conducted to produce somatic embryos of horseradish in liquid medium (Shigeta and Sato 1994; Nakashimada et al. 1995). Somaclonal variation can present a challenge for crops that are massproduced in plant tissue culture (Skirvin et al. 1994). Shehata (2004)

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conducted ﬁeld tests to detect the level of somaclonal variation among horseradish clones derived from various tissues in vitro. Variation was found to be very low (0.5%) and within acceptable limits. Shehata also reported optimal horseradish shoot proliferation on MS medium supplemented with BA (2 mmol/L). These shoots could be acclimatized within three weeks under mist in a greenhouse with 100% survival. Within six weeks, most plants were ready for ﬁeld planting. Seed Propagation. Seed propagation of horseradish is possible but not commercially viable. Horseradish is highly heterozygous and will not reproduce true to type from seed. In addition, as discussed, horseradish sets seed poorly. Seed propagation is used only in breeding programs (Walters 2007; Doll 2006) (see Section V). 2. Field Establishment. The horseradish plant thrives in deep loam or sandy soil types with good drainage. For the best response to fertilizers, a pH range of 6 to 7.5 is ideal. Organic matter is often added to maintain a good soil structure. Shallow soils or those with hard pans are not suitable as they compromise strong root development and thus may curtail yields. Although perennial by nature, horseradish is generally cultivated commercially as an annual crop. Planting of sets in the eastern United States starts in April and may be carried out up to the end of May, depending on the region. Areas with warmer springs usually adopt earlier planting dates. Under certain circumstances, fall planting may be feasible, as long as it is done early enough (usually late August or September) to allow the crop to establish before the advent of freezing weather. However, this is not yet a common practice among horseradish growers. Fields are prepared for planting with ridgers to create raised beds that will be left open for ease of planting. When planting tissue-cultured plants, the ridges are covered with black polyethylene mulch because of the advantage of weed control and moisture preservation. Ridging increases yield of high-quality roots by ensuring that the soil is loose so that large roots can develop. Ridging also aids root removal at harvest. Planting is done either by hand or using modiﬁed transplanters that place sets in the beds at a 45-degree angle. For hand planting, sets are placed in furrows about 10 cm deep. The spacing adopted is 40 to 60 cm between plants and 75 to 90 cm between rows, giving a plant population of 20,000 to 25,000 plants/ha. When planting sets, irrigation early in the growing season is not mandatory as long as there is adequate moisture in the soil. For tissue-culture plantlets,

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irrigation is essential and should be applied immediately after planting, as these come with a few leaves and may need the initial watering to set them in the soil properly. To ensure greater yields, supplemental irrigation may be applied during the dry months of August through September. 3. Field Cultivation. Two main categories of horseradish roots are grown in Illinois. ‘‘Wild’’ roots are used for sauce production; higherquality, ‘‘lifted’’ roots are sold fresh and can bring considerably higher returns per hectare. Both ‘‘wild’’ and ‘‘lifted’’ roots result from sets that are planted in the spring and managed only for weeds, nutrition, and pests. ‘‘Wild’’ roots are harvested in the fall. These plants tend to have many lateral roots in addition to the main taproot. Additionally, two to three times per growing season, the soil is dug from around the ‘‘lifted’’ roots without disturbing the distal end of the root system, and the crown portion of the plant is physically lifted by hand with a curved metal hook. The crowns are thinned to remove side shoots, any side roots are rubbed off, and the plants are replaced in the ground. This effort results in a very large, unbranched ‘‘number 1’’ root that is harvested in the fall and can bring twice the return per hectare of ‘‘wild’’ roots. 4. Fertilization. Many studies have been conducted to determine the optimal feeding nutrition schedule for horseradish (Poniedzialek 1977; Poniedzialek et al. 1987; Bratsch 2000; Hopen 2001). Horseradish is a high-yielding crop (4.5–9.0 kg/ha.) and a heavy feeder. To maintain proper nutrition, fertilizer is applied based on soil test results, soil type, cultivar, and cropping history. One-half to two-thirds of the annual nitrogen needs for the crop are applied at planting. The balance is surface broadcast as liquid or granules one to two months after planting (Bratsch 2000). For optimal horseradish growth, soil testing should be conducted to ensure the pH is within the range 6 to 6.5. Liming may be necessary to bring soil pH to this range. Manure application is also recommended at 27 to 45 t/ha in the fall. Depending on the soil type, fertilizer applications should supply 100 to 200 kg/ha nitrogen (N), 100 to 150 kg/ha phosphorus (P2O5), 100 to 150 kg/ha potassium (K2O), 1 to 3 kg/ha boron (B), and 30 to 50 kg/ha sulfur (S), respectively. Swiader et al. (1992) recommends nitrogen rates ranging from 110 to 170 kg/ha, with P and K applied according to soil test results. Excessive nitrogen rates should be avoided, as this can result in excessive foliar growth and highly branched, irregular root formation. Horseradish also has

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a high requirement for boron, similar to other members of the Brassicaceae. 5. Diseases, Pests, and Weeds Fungal Diseases. A few pests and diseases are of economic importance in horseradish. However, in recent years, Illinois production has declined as a result of increased disease pressure and its associated decrease in yield (C-FAR 2000). The problem is believed to be related to an internal discoloration of the root tissues, which is caused by Verticillium and Fusarium species (Eastburn and Chang 1994; Babadoost et al. 2004; Babadoost 2006). Other diseases of horseradish include white rust (Albugo candida), leaf spot (Alternaria brassicae, Cercospora armoraciae), bacterial leaf spot (Xanthomonas campestris var. armoraciae), bacterial gall (Agrobacterium tumefaciens), brittle root (Spiroplasma citri), and Turnip mosaic (Turnip mosaic virus) (Hickman and Varma 1968; Eastburn and Weinzierl 1995; Babadoost 2006). Internal root discoloration (IRD) is a disease complex that is described as the most serious challenge to the horseradish industry in the United States and worldwide (Babadoost et al. 2004). The disease makes roots unmarketable and can be a major cause of yield losses when production is carried out on infected ﬁelds. At least three fungal pathogens, Verticillium dahliae, V. longisporum, and Fusarium solani, have been identiﬁed to be the causal agents of IRD (Eastburn and Chang 1994; Babadoost et al. 2004). Symptoms include streaking, rotting, or darkening of the internal root tissues (Fig. 4.6). Root tissues should be naturally pure white; processors have very low thresholds for these symptoms and will reject whole shipments of discolored

Fig. 4.6. Cross section of a healthy horseradish root (left) and a root showing symptoms of internal root discoloration (right).

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roots. At present, no effective control methods are available, and intense research into its control is ongoing (Babadoost and Islam 2006; Babadoost et al. 2007; Uchanski 2007). Control typically relies on avoidance and the use of certiﬁed pathogen-free planting stocks. Virus Diseases. Horseradish is vegetatively propagated by root cuttings and often suffers clonal degeneration, losing vigor and productivity over time. Virus buildup and concomitant yield reductions over time (‘‘running out’’) have been well documented in crops other than horseradish, including strawberry (Stadelbacher 1980), potato (Morel and Muller 1964), and dahlia (Morel and Martin 1952). Virus elimination programs to make certiﬁed plants have restored yields of these crops (Stadelbacher 1980). In horseradish, ‘‘running out’’ of clones may be attributed at least partially to Turnip mosaic virus (TuMV), since the viral load (titer) can increase in each succeeding generation to further weaken the clone (Uchanski et al. 2004). TuMV is the most common virus reported for horseradish (Dana and McWhorter 1932; Herold 1957; Yoshii et al. 1963; Li and Cheo 1964). TuMV and other viruses may play a secondary role in the horseradish disease complex by weakening plants and making them more susceptible to other stresses, such as drought, insects, and nematodes (Horwitz et al. 1985). Hickman and Varma (1968) conducted a virus infection status survey of 47 horseradish clones. These clones originated from several different countries, including Czechoslovakia, Denmark, France, Germany, Israel, Japan, Poland, Sweden, the United Kingdom, and the United States. They identiﬁed unknown viruses using symptomology, electron microscopy, and serology. They detected and identiﬁed sap-transmissible viruses in 30 of the horseradish samples. These included Arabis mosaic virus (53% of the samples), Cabbage black ringspot virus (¼TuMV, 36%), and Cauliﬂower mosaic virus (13%). Hickman and Varma (1968) were able to free the clones of Cabbage black ringspot virus and Cauliﬂower mosaic virus by apical meristem culture on Morel and Muller’s medium (1964). Later, horseradish was freed of TuMV using in vitro meristemming techniques. These plants were ﬁeld tested and yields increased signiﬁcantly (Uchanski et al. 2006). Other Diseases. White rust, caused by the oomycete Albugo spp., is one of the most important foliar diseases of horseradish. This disease occurs in Europe every season. In Illinois, outbreaks of white rust have been reported during the spring and fall months following prolonged periods

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of cool, dewy nights and slightly warmer days (University of IllinoisRPD report 1990; Takashi et al. 2002). The disease progresses rapidly on leaves, causing retardation of root development and undesirable woodiness of the primary root. White rust is controlled by fungicide applications starting during shoot emergence and continuing at 7 to 14-day intervals as long as the weather is cool and moist. In the past 10 years, however, white rust has not been a problem in horseradish growing areas in the United States. Root gall, caused by Agrobacterium tumefaciens, is considered a serious problem in Europe, but its occurrence is not common in North America. Alternaria leaf spot, caused by the fungus Alternaria brassicae, Cercospora leaf spot, caused by the fungus Cercospora armoraciae, and bacterial leaf spot, caused by the bacterium Xanthomonas campestris var. armoraciae, have been reported to occur on horseradish, but their impact on horseradish yield and quality have not been studied in detail. Also, some bacterial species of Pseudomonas and Xanthomonas are usually associated with internally discolored horseradish roots, but they do not appear to be the primary causal agent in of the root discoloration. Brittle root is considered to be one of the most serious diseases that affects horseradish but can be managed by thorough scouting and vector control (Rimmer et al. 2007). The disease is caused by a specialized group of bacteria called the spiroplasmas (Spiroplasma citri) and is restricted to the phloem tissues (Eastburn and Weinzerl 1995). Symptoms of the disease include discoloration (yellowish tan) of the phloem tissues in the roots, yellowing of the leaves, curling of leaf margins, and reduction in root yield and quality. Symptoms usually occur about 40 days after infection. Infected plants are stunted, roots have a brittle texture, and the plant may die before the end of the growing season. Babadoost et al. (2001) conducted a detailed ﬁeld survey of the incidence of horseradish diseases in Illinois. The authors made several important observations from their studies. Most horseradish roots that showed internal discoloration symptoms were infected with one or more of the organisms involved in the disease complex. They also noted that up to 67% of asymptomatic roots harbored pathogenic and nonpathogenic organisms, such as Verticillium spp., Fusarium spp., nonsporulating fungi, and bacteria. Insect Pests. Few insect pests of economic importance have been identiﬁed for horseradish. During the growing season, ﬁelds are scouted weekly for the beet leaf hopper (Circulifer tenellus) because it is the primary vector for brittle root disease (causal agent: Spiroplasma citri). Insecticide applications are advised to control the vector if sweep

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net samples reveal the presence of beet leafhoppers before late July or August (Eastburn and Weinzierl 1995). Other insect pests included in a scouting program are the imported crucifer weevil (Baris lepidii), ﬂea beetles (Epitrix spp.), and diamondback moth larvae (Plutella xylostella). Insects not usually included in a scouting program are false chinch bug (Nysius raphanus), onion thrips (Thrips tabaci), green peach aphid (Myzus persicae), mealy plum aphid (Hyalopterus pruni), cabbage looper (Trichoplusia ni), imported cabbage worm (Pieris rapae), and grasshoppers (many genera). Nematodes. Babadoost et al. (2000) conducted a ﬁeld survey in horseradish ﬁelds in Illinois in 1999 and found no serious nematode infections. In 2002, however, Babadoost et al. (2003) reported a severe outbreak of root knot caused by Meloidogyne incognita in a commercial ﬁeld in Collinsville, Illinois. Furthermore, Walters et al. (2004) conducted a ﬁeld survey for four years in Illinois horseradish ﬁelds and found nematodes in the genera Helicotylenchus, Hoplolaimus, Meloidogyne, Paratylenchus, Pratylenchus, Tylenchorhynchus, and Xiphinema to be prevalent throughout the production region. Generally low soil populations of these nematode species were found, but the potential to cause yield loss via interaction with other pathogens may be of concern. The interaction between plant parasitic nematodes and the horseradish disease complex needs to be addressed in more detail. Weeds. Weeds in horseradish ﬁelds are typically controlled with preemergent herbicides followed by hand-removal, mechanical cultivation, and/or postemergent herbicides. Several herbicides and herbicide combinations have been approved for horseradish (Wahle 2005). If these herbicides were not available, the cost of horseradish production would increase dramatically. Species of concern include pigweeds (Amaranthus spp.), lambsquarters (Chenopodium album), ragweeds (Ambrosia spp.), foxtails (Alopecurus spp.), black nightshade (Solanum ptycanthum), yellow nutsedge (Cyperus esculentus), annual morninglory (Ipomoea spp.), Johnsongrass (Sorghum halepense), and ﬁeld bindweed (Convolvulus arvensis), among others. 6. Harvesting. The greatest increase in horseradish root mass in the United States occurs in late summer and early fall. Therefore, for the highest yields, harvesting usually is carried out in the months of November and December. However, if fall harvesting is not possible, spring harvesting can be done. In most commercial operations,

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Fig. 4.7. One-row modiﬁed potato digger often used for digging horseradish.

harvesting is done after freezing temperatures have killed the foliage. Freezing also physiologically conditions the roots to store properly. When harvesting is done before a killing frost, a rotary cutter usually is used to mow the leaves as close to the soil as possible. Several days are allowed between leaf removal and actual lifting of roots. Harvesting is done using a modiﬁed potato digger set at a depth that allows maximum root recovery (usually about 50 cm) (Fig. 4.7). The numbers of volunteer plants that grow the following cropping season is reduced when more roots are retrieved from the soil. The occurrence of numerous volunteer horseradish plants in ﬁelds reduces the beneﬁt of crop rotation and may maintain or increase levels of pathogens for subsequent horseradish production (Rundle et al. 2007). Harvested roots are transported to packing sheds where they are evaluated visually for internal discoloration and rotting. Unacceptable roots are discarded. Lateral roots are separated from the healthy taproots for next year’s planting stock. Lateral roots are trimmed and packed into plastic-lined pallets for winter storage (1 –2 C) until planted as sets the next spring (Fig. 4.8). Taproots are graded, packed into pallets, and either stored at 1 to 2 C or immediately shipped to processors. 7. Postharvest Technology. To avoid injuries that tend to impair their storage potential, horseradish roots should be handled with care. Roots

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Fig. 4.8. Horseradish production cycle (top); horseradish root set, plant, commercial root (bottom).

can store satisfactorily for 10 to 12 months at 0 C with a relative humidity of 90% to 95%. To ensure maintenance of moisture, the roots can be stored in perforated plastic bags or plastic-lined bins. Roots preferably should be stored in darkness to avoid greening under light exposure. Frequent inspections in storage are essential to arrest any developing decay damage. In previous years, some commercial farms

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stored horseradish over winter in outdoor pits and trenches. Whether roots should be washed and dried before storage is debatable and differs among growers. Our experience indicates that roots stored with their soil on keep better than those that are washed after harvesting. Most growers choose not to wash their roots for cost reasons. B. Utilization 1. Therapeutics. Horseradish has a long history of utilization for medicinal purposes in traditional medicine. Ancient Greeks used horseradish as a rub for low back pain and believed it was an aphrodisiac (Walton 1958). Gerard (1597) claimed that horseradish reduced pain from sciatica, expelled afterbirth, relieved colic, increased urination, and killed worms in children. It was claimed to be an expectorant, soothing for respiratory problems, and may help relieve rheumatism by stimulating blood ﬂow to inﬂamed joints. Bentley and Trimen (1880) stated that grated horseradish root was mixed with honey and warm water for inﬂuenza, and it could be used as poultice by adding cornstarch to fresh horseradish and applying it to the affected areas in a gauze bandage. In 1880, Bentley and Trimen reported, ‘‘It has the same properties as mustard; being a stimulant, a diuretic, and a diaphoretic, when given internally, and rubefacient or even vesicant, when externally applied.’’ According to Grieve (1931), horseradish syrup is effective for hoarseness, provides relief for whooping cough, and when applied externally helps to remove freckles. It has been used as an expectorant cough medicine and was even believed to be a potent cure for everything from rheumatism to dropsy and scurvy. Native Americans used horseradish obtained from settlers to treat toothaches (McCann 2004) while the Cherokee use it as a urinary aid for gravel (kidney stones), as a diuretic, as a gastrointestinal aid to improve digestion, and as a respiratory aid to treat asthma, coughs, and bronchitis (Moerman 1998). The approved modern therapeutic applications for horseradish are based on its long history of use in well-established systems of traditional medicine, pharmacological studies in animals, and its well-documented chemical composition (Sjaastad et al. 1984; Natella 1998). Some German studies have investigated the effects of horseradish on nonspeciﬁc urinary tract infections (Schindler et al. 1960) and the antibacterial action of its essential oils (Kienholz and Kemkes 1960). Horseradish has been approved in Germany for the treatment of infections of the respiratory tract and as supportive treatment in urinary tract infections. In the United States, the root is the active

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ingredient of Rasapen1, a urinary antiseptic drug (Budavari 1996). Horseradish is listed as Generally Recognized as Safe (GRAS) in the FDA Code of Federal Regulations. 2. Culinary Uses. During the Renaissance, horseradish consumption spread from central Europe northward to Scandinavia and westward to England. However, the British did not eat horseradish until 1640. By the late 1600s, horseradish was the standard accompaniment for beef and oysters among most all English people. Both the leaves and roots of horseradish were utilized in dishes. In the spring, the tender leaves frequently were used for greens and were good for that purpose especially when mixed with dock or other wild plants (Medsger 1939). Horseradish is often consumed boiled as a pot herb; it is boiled, the water is drained, and it is boiled a second time to eliminate bitter or harmful substances before consumption. It is believed that horseradish became popular as a condiment in old Europe because there was no refrigeration and its sharp spiciness covered the taste of tainted meats (Vandaveer 2002). Gerard (1597) believed horseradish ‘‘causeth better digestion than mustard.’’ Hill (1952) also reported that it aided digestion and prevented scurvy. English innkeepers are reported to have grown the pungent root to make cordials for reviving exhausted travelers at inns and coach stations (Horseradish Information Council 2007). In modern times, the white pungent roots of horseradish are grated and used to make a condiment (horseradish sauce) with a very strong mustardlike ﬂavor. The white, pungent roots of horseradish are grated and mixed with vinegar to form a condiment often used with boiled meats or ﬁsh and as a ﬂavoring in other recipes (Doll et al. 1999). It can be creamed and is the main ingredient of ‘‘horsey sauce’’ and is often colored red with beets. Once grated, the mashed root develops the pungent ﬂavor from the myrosinase and glucosinolate reaction in water (discussed earlier). This reaction is stopped and stabilized by the addition of vinegar to control the intensity of ﬂavor. In addition to adding ﬂavor to foods, horseradish also has been shown to inhibit spoilage (Ward et al. 1998). Horseradish oil has been shown to be effective in naturally preserving roast beef sample ﬂavor and color (Delaquis et al. 1999). The same glucosinolates and their breakdown products that give horseradish its ﬂavor may also help it to serve as a functional food with approximately 10 times the carcinogen neutralization capabilities as compared to broccoli (Li and Kushad 2004). Additionally, it is a popular and ﬂavorful food additive with no fat and very few calories (Table 4.3).

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Table 4.3. Nutritional content of the horseradish root (serving size ¼ 5 g). Component Energy (kcal) Water (%) Dietary ﬁber (g) Fat (g) Carbohydrate (g) Protein (g) Minerals (mg) Calcium Iron Zinc Manganese Potassium Magnesium Phosphorus Vitamins (mg) Vitamin A Vitamin C Thiamin Riboﬂavin Niacin Vitamin B6 Vitamin E Folate

Amount 2 85 0 0 1 0 3 0 0 0 12 1 2 0 1 0 0 0 0 0 3mg

Source: Rizza and Harrison 2002,

A number of similar condiments are associated with horseradish but are different species. Wasabi (Wasabi japonica Matsum, Brassicaceae) is a plant similar to horseradish and is used to produce a product called Japanese horseradish or wasabi. The ground product is green in color and is served as a condiment or sauce (Fig. 4.9). The stem sometimes is described as a rhizome and is prepared for use by removing all leaves, petioles, offshoots, and roots (Chadwick et al. 1993). Although the ground rhizomes provide a taste similar to horseradish, wasabi is an aquatic plant. This plant typically is found growing along stream beds in mountain river valleys in Japan, but production can also be found in New Zealand, Taiwan, and in the United States (Oregon). Although the fresh rhizome is the most valuable part, both the rhizomes and leaves can be ground as a condiment usually served as a spice in Japanese cuisine. The ground paste often is paired with traditional raw ﬁsh and noodle dishes. True wasabi is very expensive, and most condiments sold as wasabi are

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Fig. 4.9. Fresh-ground wasabi (Wasabia japonica) paste (left) and leaﬂess stems referred to as rhizomes (right). Wasabi has similar ﬂavor components to horseradish but is much more difﬁcult to produce and has a light green coloration. (Source: Chadwick et al. 1993).

actually powdered horseradish root that has been dyed green. The main ﬂavor component wasabi is 2-propenyl isothiocyanate (allyl isothiocyanate). Three compounds are particularly important in the unique ﬂavor of wasabi: 6-methylthioheptyl isothiocyanate, 7-methylthioheptyl isothiocyanate, and 8-methylthioheptyl isothiocyanate (Depree et al. 1999). The methylsulphinylalkyl compounds are found in both wasabi and horseradish, but the methylthioalkyl compounds are found only in wasabi (Depree et al. 1999). The horseradish tree or drumstick tree (Moringa oleifera Lamarck, Moringaceae) is a plant that grows in small areas of the southern foothills of the Himalayas. The roots taste similar to horseradish and can be used as a substitute (University of Leicester 2004). 3. Industrial Uses. In addition to the culinary value of horseradish, recent research has been aimed at identifying other uses for the crop, including peroxidase production (Kushad et al. 1999), an antimicrobial preservative in food (Delaquis et al. 1999), and deodorization of swine manure from feed lot operations (Govere et al. 2005). The long-established industrial use for horseradish has been in the extraction of horseradish peroxidase, an enzyme widely used in clinical diagnostics and immunoassays. However, more industrial applications for the plant are being developed. Minced horseradish roots have proven to be more effective in the decontamination of

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phenol-polluted industrial waste waters than the pure peroxidase enzyme extracted from the roots; up scaling of this application is currently under way (Dec and Bollag 1994). More recently, minced horseradish roots have been utilized in the deodorization of animal farm wastes, particularly the deodorization of swine manure in combination with hydrogen peroxide (Govere et al. 2005). This work is also on pilot trials in farmers’ ﬁelds in Pennsylvania (Dec 2006). These novel uses for horseradish may open new markets and have the potential to beneﬁt growers worldwide. Another suggested use for horseradish includes weed suppression via the allelopathic response of glucosinolates in soil (Li and Kushad 2004). Glucosinolates and their byproducts can act as biofumigants when used as green manure, thus showing potential for weed control in organic production.

V. BREEDING Although horseradish has been in cultivation for more than two millennia in southeastern Europe (De Candolle 1890) and has been commercially produced in the United States since the 19th century (Rhodes 1977), the continuous development of new, improved horseradish cultivars has been limited. The most probable reason for the low amount of breeding work with horseradish is that although horseradish plants ﬂower profusely, fertility is low. The lack of adequate natural seed production through traditional breeding methods has been an impediment to the development of new cultivars. A. Crop Improvement in the United States Prior to the 20th century, the only way to improve horseradish was to select and plant root cuttings from the most desirable plants (Rhodes et al. 1965b). However, some breeding efforts have been made in the United States, primarily in Wisconsin and Illinois (Weber 1949; Rhodes et al. 1965a, Chris Doll pers. commun.). For many years, horseradish was believed to be sterile (Courter and Rhodes 1969) and therefore impossible to improve by traditional sexual crosses. Luther Burbank (1914) wrote: The horseradish does, indeed, bloom with the greatest profusion. But the blossoms prove sterile. The plant has entirely and probably forever lost the power of producing seed. I have elsewhere [made] a joking offer of one thousand dollars an ounce for horseradish seed. Of course I knew that no

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horseradish seeds were to be had, yet I would gladly have given then, and I would glad pay, at the rate of $1000 an ounce for horseradish seed. But there is not the remotest probability that any one will ever legitimately claim the prize..

Although viable seeds have been reported, there are many factors related to failure to set viable seeds (Weber 1949; Stokes 1955). To overcome spontaneous embryo abortion, Ozgur et al. (2004) reported a simple method to perform horseradish embryo rescue using plant tissue culture. The authors used MS medium with no plant growth regulators to rescue embryos derived from horseradish crosses and selfpollinations. They found that rescued immature embryos germinated readily in vitro; control seeds (conventional germination in soil) germinated either poorly or not at all. This simple method allows horseradish breeding efforts to progress where they otherwise would have been restricted by embryo abortion. Wisconsin. Crossing of horseradish clones (primarily ‘Bohemian’ and ‘Common’ types) was ﬁrst initiated in the United States by Weber in 1947 from germplasm collected at commercial farms and non-cultivated areas in Wisconsin (Weber 1949). Seedlings selected from these ﬁrst crosses were developed for use by Wisconsin growers as well as for making additional crosses. However, this horseradish breeding initiative lasted only a few years. Illinois. Some of the Wisconsin clones were obtained by Dr. M.B. Linn of the University of Illinois in the early 1950s to study disease resistance in horseradish. In the mid-1950s, Dr. N.F. Oebker added to this collection with plants from Ohio and three clones from Russia. When Dr. Oebker left the University of Illinois, Dr. A.M. Rhodes assumed responsibility for the horseradish collection, expanded accession efforts, and began making crosses. The program was initiated by using the previously collected materials as well as collecting horseradish germplasm from various parts of Europe, seedlings originally bred at the University of Wisconsin, and other sources from the United States By 1969, germplasm had been added to the collection from Austria, Bulgaria, Canada, England, Hungary, Denmark, Sweden, Czechoslovakia, Iowa, Vermont, and Illinois. From 1959 through 1982, seedlings of crosses were grown on the University of Illinois South Farm for initial evaluation, and promising selections were then moved to southwestern Illinois for ﬁeld testing and evaluation by commercial growers. Breeding ceased with

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Dr. Rhodes’s retirement in 1982, but ﬁeld evaluation of seedlings continued until breeding was resumed in 1993 by C. Doll. Dr. J. Juvik was the administrator of the University of Illinois horseradish germplasm collection during this interim period but did not make any new crosses. However, the Illinois program resumed in 1993 with crosses made by C. Doll, and he continued as program leader until 1997. At this time, the horseradish breeding program was turned over to Dr. A. Hamblin (University of Illinois) and R. Gerstenecker (horseradish grower) until 2003. During this time, random ampliﬁed polymorphic DNA markers (RAPDs) were used to explore the diversity of the Illinois horseradish collection (Hamblin et al. 2002). From 2003 to 2005, the program was again administered by C. Doll. Dr. S.A. Walters (Southern Illinois University) took over the horseradish breeding program in 2005 and is currently the program leader. Until the middle to late 1960s, horseradish production in the United States was conﬁned mainly to three cultivars, ‘Common’, ‘Swiss’, and ‘Big Top Western’ (Rhodes 1977), although ‘Sass’ was also grown in limited amounts in Illinois due to its high-yield ability (Courter and Rhodes 1969). Due to this narrow genetic base, a horseradish breeding program was initiated in Illinois to develop additional cultivars to prevent widespread losses to diseases, insects, or other possible causes (Rhodes 1965a, Rhodes 1965b). Although ‘Big Top Western’ is still produced in California and Wisconsin (Table 4.1), the horseradish breeding program in Illinois has provided new cultivars to the horseradish industry every few years since the 1960s (Chris Doll pers. commun.). These cultivars constitute as much as 99% of those that are grown in Illinois today. Currently there are many cultivars that growers use for planting each year, with new material released each year from the Horseradish Growers of Illinois (HGI) breeding program. Certain cultivars are preferred by speciﬁc growers, although most growers typically grow four to ﬁve or more. Most growers try to limit the number of cultivars grown due to problems such as maintaining genetic purity of a particular clone that often arises from maintaining set stock of several different clones. Currently, the most widely grown cultivars in Illinois include: ‘15K’, ‘22C’, ‘1038’, ‘1573’, ‘1590’, ‘1722’, ‘7586’, ‘D25-E2’, and ‘D18-E1’ (Dorris et al. 2007). These are unavailable except to the Horseradish Growers of Illinois, because all grower members contributed funds to their development. The current overall goal of the Illinois program is to develop commercially acceptable horseradish cultivars with increased IRD complex resistance along with high-quality and high-yielding roots through traditional breeding.

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B. Germplasm Collection The horseradish germplasm collection was established in the mid1950s by the University of Illinois (Atibalentja and Eastburn 1998). Today it is still maintained at the same location but is a joint repository between the Horseradish Growers of Illinois and the University of Illinois. The horseradish germplasm collection contains about 140 clones either imported from various places around the world (primarily Europe) or developed in the United States (M. Kushad pers. commun.). This assortment of horseradish germplasm is the only such collection in the world for this crop. Furthermore, germplasm materials are added to the collection continually, with most being highly advanced clones from the breeding program and to a lesser extent, new clones collected from various parts of Europe and the United States. This collection contains about 45 clones from various European countries, 15 clones from North America, and 90 clones developed through the Illinois horseradish breeding program. Although the germplasm has been evaluated for resistance to various diseases, results have indicated that 0% and 5% of the materials in the collection are resistant to TuMV (Horwitz et al. 1985) and V. dahliae (Atibalentja and Eastburn 1998), respectively. In Illinois, the problem of nonviable seed was originally overcome by following the suggestions of Weber (1949) by using ‘Common’ as the female parent and ‘Bohemian’ or ‘Big Top Western’ as the male parent (Rhodes et al. 1965b). The original crosses at the University of Illinois were made in an effort to combine characters such as improved disease resistance, better root quality, and increased yields (Rhodes et al. 1965b). Although the ‘Common’ type of horseradish only produces sparse ﬂowers and no functional pollen, they are partially female fertile and produce a small quantity of seed when a crossed with a proper male parent (Stokes 1955). Due to an active breeding program in Illinois, funded primarily through the Horseradish Growers of Illinois, new cultivars have regularly been released since the 1960s. This has provided growers multiple cultivars from which to choose, although certain cultivars are preferred over others due to some internal root discoloration resistance, yields, or exceptional quality characteristics. However, after a period of about 10 years, most horseradish cultivars normally run their course, as quality, vigor, and yielding ability becomes less over time compared to previous years’ plantings. Since both ‘Common’ and ‘Big Top Western’ types have been widely utilized for new cultivar development in Illinois, the speciﬁc leaf

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shapes that are characteristic for each type are now difﬁcult to distinguish due to crossbreeding between the two types (Bratsch 2006). Although some self-pollination of the breeding materials used today probably occurs at a low rate, cross-pollination between breeding materials is still important to obtain optimal seed set.

C. Breeding Methods 1. Breeding Cycle. Because horseradish cultivars are highly heterozygous clones, the only way to maintain a particular selection is through asexual propagation; and this is accomplished by using root cuttings. The breeding methods used for horseradish are somewhat similar to other asexually propagated crops. New genetic combinations are made by cross-pollinating with other cultivars or germplasm materials and seedlings are evaluated after vegetative propagation. The typical cycle for breeding, evaluation, and introduction is about eight years. The procedure is: (1) seed development and collection from a particular cross or outcrossed clone; (2) seedling evaluation under ﬁeld conditions; (3) seedlings that are chosen are further evaluated and selected for three additional years in the ﬁeld; (4) ﬁeld increase of those clones making it through the ﬁeld evaluation and selection process, with growers making the ﬁnal determination to cultivar status; (5) tissue culture and ﬁeld increase of new cultivars (done the same year); and (6) new cultivars become available to commercial growers for planting. Generally, about 10% to 15% of materials are selected during each cycle and passed on to the next stage. The ﬁeld selections are primarily based on IRD resistance, root quality (smooth roots), set production, direction of root development in the soil, and yield potential. A clone showing any symptoms of IRD during the selection process is immediately discarded from the program, even if it shows great potential in other important characters. Once a new cultivar has been identiﬁed, it must be multiplied rapidly to achieve high plant numbers in a short period of time; this is accomplished through in vitro propagation methods (Norton et al. 2001) followed by subsequent rooting under mist and then ﬁeld increase. In the Illinois breeding program, the development, selection, and introduction of new cultivars is a joint effort among the Horseradish Growers of Illinois, the University of Illinois, and Southern Illinois University. The growers provide the breeding program with their time, labor, land, and monies. However, the operation of this program differs somewhat

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from most crop-breeding programs in that no formal release is made (Rhodes 1977). 2. Hybridization. Hybridization of horseradish has been achieved primarily by the polycross (natural intercrossing) and proven cross methods in Illinois. However, the polycross method is used most often since it is the simplest and easiest way to produce new genetic combinations. Intercross blocks for the polycross method are developed depending on the ﬁnal objectives for cultivar development. In the current breeding program, two intercross blocks are developed each year. One block is used primarily to intercross commercial cultivars and advanced clones, while the other has commercial cultivars, advanced clones, and germplasm materials with resistance to Verticillium dahliae. The IRD complex is the most important problem facing eastern U.S. horseradish growers. To help combat this problem, germplasm currently is being introgressed from resistant sources in the development of commercial-type cultivars. Last, a limited number of crosses are made each year between two superior cultivars and/or advanced clones under greenhouse conditions to obtain seedlings that have a good chance at becoming advanced selections or cultivars. For the targeted pollinations between two clones, no emasculations are done prior to hand-pollinations since plants are reputedly self-sterile. Pollen is transferred by shaking and rubbing open ﬂowers of the male parent onto the stigmatic surfaces of the female parent over a period of 2 to 3 days. Both breeding systems can be repeated further by intermating the superior clones that were recently produced. Regardless of breeding method, 2-year-old roots (or crowns) from selected plant materials are used since these will produce ﬂowers. Crowns usually are planted in late March to early April, with ﬂowers appearing in about 5 to 6 weeks (depending on spring temperatures). Seeds are ready to harvest by early to mid-June. Seed pods are collected, the seeds are cleaned and packeted, and seed packets are placed into plastic containers and stored at 2 to 5 C until planting. Also ongoing each year is crown development from planted set roots of the clones that will be used the following year to develop seed from speciﬁc crosses in the greenhouse or will be placed into the polycross block. 3. Increasing Fertility. Although the production and use of seed is important only for the breeder to develop new genetic combinations, the lack of adequate seed production has hindered horseradish breeding efforts in many parts of the world. Winiarczyk et al. (2007)

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reported from Poland that slightly greater numbers of horseradish seed can be obtained through cross-breeding plants from geographically remote areas, although the use of cross-breeding still results in the production only of few seed. In Wisconsin, Stokes (1955) indicated that cross-pollination is needed to achieve the highest seed set in horseradish, as self-incompatibility prevents signiﬁcant seed development. Weber (1949) also indicated that the production of fertile lines of horseradish through cross-pollination and selection would greatly stimulate improvement research with this plant. Furthermore, research aimed at obtaining horseradish plants capable of sexual reproduction could result in higher genetic diversity, better adaptation to various environmental conditions, and pest resistance (Winiarczyk et al. 2007). Weber (1949) found that seed development will occur on the ‘Common’ type if functional pollen from the ‘Bohemian’ type is placed on receptive ‘Common’ stigmas, and the cross between these two types of horseradish is the most effective way of collecting viable seed. The ‘Common’ type is completely pollen-sterile, although about 5% of the ovules contain normal functional gametophytes (Weber 1949). The ‘Bohemian’ type often has functional pollen (Weber 1949; Stokes 1955). Although horseradish seed can be produced today easily from the cultivars, breeding lines, or other germplasm materials used in making crosses, the number and viability of seed obtained differs among crosses that are made. 4. Seedling Evaluation. Depending on seed numbers obtained the previous year, about 15 to 20 F1 families directly relating to 3,500 to 7,000 seedlings are evaluated each year in the Illinois program. The seed for these families result from polycross nurseries or speciﬁc crosses made in the greenhouse. In previous years, fewer seedlings were evaluated, starting with about 150 seedlings per year during the ﬁrst few years of the breeding program and about 1,200 seedlings per year once large viable seed numbers were obtained (Rhodes 1977). During early March, seeds are germinated, and about 3 weeks later, seedlings are transplanted into plastic seedling trays. These plants are grown under greenhouse conditions until the 3- to 6-leaf stage, with plants then hardened off in an outdoor cold frame or high tunnel for about one week prior to ﬁeld planting. Seedlings are transplanted into raised beds in early May, allowed to grow for about six months, and dug with a one-row modiﬁed potato digger in late October to mid-November. If a signiﬁcant freeze event has not occurred by harvest, the foliage of seedlings and other breeding

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materials are mowed prior to digging. Seedlings are placed into large plastic-lined bins according to the particular cross or outcrossed parent. Bins are placed into cold storage (2 –5 C), with evaluations and selections made within 2 to 3 weeks of harvest. The ﬁeld evaluation and selection process is similar for ﬁrst-, second-, and third-generation clonal selections, although these clones are replicated and the selections are made directly in the ﬁeld. All selections are wrapped in plastic bags and placed into cold storage (2 –5 C) for about 6 months until spring planting. 5. New Cultivar Releases. Clones that are selected during the ﬁnal year of the selection process are increased in the ﬁeld the following year. This increase results in large root numbers, which allow growers to evaluate the overall IRD resistance, quality, variability, and yield for each clone. The growers judge the possible new cultivar materials provided to them based on multiple quality characters as well as yield potential. Once growers make a decision on those that will be elevated to cultivar status, the clone is placed into tissue culture to ensure that it is pathogen free prior to ﬁeld increase and grower distribution. The tissue culturing and subsequent ﬁeld increase usually is done in the same year, with grower distribution occurring the following year. Once superior genotypes are obtained, these are preserved and perpetuated by growers through vegetative propagation of root cuttings. 6. The Future. There is still signiﬁcant variability in Illinois germplasm for developing new and improved cultivars. However, although the current primary aim of the horseradish breeding program is to develop cultivars with high levels of IRD complex resistance along with other quality and yield characters using traditional breeding techniques, molecular methods to assist in the breeding process should be incorporated to support new cultivar development in the future. Protoplast fusion techniques or in vitro embryo rescue (Izu 1989; Ozgur et al. 2004) might facilitate the development of hybrids between horseradish clones that were previously incompatible. Greater heterosis should result from crosses involving parents that are the most distantly related. The production of horseradish somatic embryos in liquid medium (Shigeta and Sato 1994; Nakashimada et al. 1995) may further streamline the in vitro propagation of horseradish. Once somatic embryos are produced, they can be encapsulated in alginate calcium gel to produce ‘‘artiﬁcial seeds’’ that could be mass-produced without the need for costly hand-transfers (Skirvin et al. 2007). Then the ‘‘seeds’’ could be

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moved directly to a greenhouse without a rooting step. Using this system, new cultivar releases could be produced en masse for ﬁeld plantings and commercial-scale cultivation of certiﬁed pathogen-free plantlets. Atibalentja and Eastburn (1998) indicated that there is potential for breeding improved horseradish cultivars that combine resistance to Verticillium dahliae (found in speciﬁc germplasm materials) and the high-yield and quality characters of commercial clones. Currently, introgressing resistance from these speciﬁc lines into commercially acceptable horseradish cultivars is a major focus of the program, as Verticillium dahliae is considered one of the primary pathogens of the IRD complex. There is a lack of basic biological and genetic information about horseradish. To achieve the greatest amount of seed development, the amount of self-pollination (or level of self-incompatibility due to male sterility) and outcrossing that occurs in current cultivars and various breeding materials needs to be determined. Additionally, genetic relationships among breeding lines, germplasm materials, and cultivars need to be determined, so that the greatest possible gains can be made in speciﬁc crosses. Molecular techniques such as ampliﬁed fragment length polymorphisms are currently being used to determine genetic relationships between horseradish clones, although traditional techniques also can be reexamined with horseradish (Rhodes et al. 1969) A determination of the genetic relationships among clones will prevent inadvertent crossing between two clones with similar genetic backgrounds, thus avoiding severe loss in plant vigor due to inbreeding. Although horseradish has been successfully transformed (Mano and Matsuhashi 1995), no speciﬁc cultivars have been developed using this process.

VI. LITERATURE CITED Acquaah, G. 2002. Horticulture: Principles and practices. 2nd ed. Prentice-Hall, Englewood Cliffs, NJ. Atibalentja, N., and D.M. Eastburn. 1998. Verticillium dahliae resistance in horseradish germplasm from the University of Illinois collection. Plant Dis. 82:176–180. Babadoost, M. 2006. Development of internal discoloration of horseradish root in commercial ﬁelds. Horseradish Research Review and Proceedings from the Horseradish Growers School, Jan. 26, 2006. Univ. Illinois Ext., pp. 5–6. Babadoost, M., W. Chen, A.D. Bratsch, and C.E. Eastman. 2004. Verticillium longisporum and Fusarium solani: two new species in the complex of internal discoloration of horseradish roots. Plant Pathol. 53:669–676.

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Simpson, B.B., and M. Conner-Ogorzaly. 1986. Economic botany: plants in our world. McGraw-Hill, New York. Sjaastad, O.V., A.K. Blom, and R. Haye. 1984. Hypotensive effects in cats caused by horseradish peroxidase mediated by metabolites of arachidonic acid. J. Histochem, Cytochem. 32:1328–1330. Skalinska, M., J. Jankum, and H. Wcislo. 1976. Further studies in chromosome numbers of Polish angiosperms, 11th contribution. Acta Biologica Cracoviensia, Series Botanica 19: 107–148. Skirvin, R.M., K.D. McPheeters, and M. Norton. 1994. Sources and frequency of somaclonal variation. HortScience 29:1232–1237. Skirvin, R.M., M.A. Norton, R. Mulwa, A. Shehata, M. Uchanski, and W. Wannarat. 2007. Somatic embryogenesis of horseradish (Armoracia rusticana) plants. Horseradish Research Review and Proceedings from the Horseradish Growers School, Jan. Univ. Illinois Ext., pp. 23–24. Smith, P.M. 1976. Horseradish. pp. 305–306. In: N.W. Simmonds (ed.), Evolution of crop plants. Longman, New York. Stadelbacher, G.J. 1980. Strawberry nursery production and plant certiﬁcation. pp. 223–227. In: N.F. Childers (ed.), The strawberry: varieties, culture, pests and control, storage, marketing, Proc. Nat. Strawberry Conf., St. Louis, MO. Stoehr, H., and K. Herrman. 1975. Phenolic acids of vegetables III. Hydroxycinnamic acids and hydroxybenzoic acids of root vegetables. Z. Lebensmittel-Untersuchung Forschung 159:219–224. Stokes, G.W. 1955. Seed development and failure in horseradish. J. Hered. 46:15–21. Swiader, J.M., G.W. Ware, and J.P. McCollum. 1992. Producing vegetable crops. Interstate Publishers, Danville, IL. Takashi, H., I. Seigo, and N. Eiji. 2002. White rust of horseradish caused by Albugo sp. Bul. Hokkaido Prefectural Agr. Expt. Sta.. 82:83–88. Tucker, A.O., and T. DeBaggio. 2000. The big book of herbs: A comprehensive illustrated reference to herbs of ﬂavor and fragrance. Interweave Press, Loveland, CO. Uchanski, M.E. 2007. Yield and quality of pathogen-free horseradish (Armoracia rusticana) planting stock. Ph.D. diss. Univ. Illinois, Urbana-Champaign. Uchanski, M.E., M.A. Norton, and R.M. Skirvin. 2006. In vitro disease elimination: ﬁndings and future research. Horseradish Research Review and Proc., Horseradish Growers School, Jan. Univ. Illinois Ext., pp. 45–48. Uchanski, M., R.M. Skirvin, and M.A. Norton. 2004. The use of in vitro thermotherapy to obtain turnip mosaic virus-free horseradish plants. Acta Hort. 631:175–179. Uhrikova A., and J. Majovsky. 1980. In: A. Love (ed.), IOPB Chromosome Number Reports LXIX. Taxon 29:703–730. University of Illinois-RPD report. 1990. White rusts of vegetables. Dept. Crop Sci., Univ. Illinois. RPD 960. University of Leicester. 2004. Moringa oleifera Lam. www.mobot.org/gradstudents/olson/ oleifera.html. 29 June. U.S. Department of Agriculture 1968. Commercial growing of horseradish. Crops Research Division, ARS. Leaﬂet no. 547. U.S. Department of Agriculture 2000. Tony Bratsch, materials coordinator. Crop proﬁle for horseradish in Illinois. http://pestdata.ncsu.edu/cropproﬁles/docs/ILhorseradish.html 20 March. Vandaveer, C. 2002. How could horseradish help the environment? www.killerplants. com/plants-that-changed-history/20020820.asp. 9 November.

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Wahle, E. 2005. 2004 evaluation of herbicides for use in horseradish production. Horseradish Research Review and Proc. Horseradish Growers School, Jan. Univ. Illinois Ext., pp. 36–40. Walters, S.A. 2007. Horseradish breeding at Southern Illinois University for 2007. Horseradish Research Review and Proceedings from the Horseradish Growers School, Jan. 25, 2007. Univ. Illinois Ext., pp. 5–8. Walters, S.A., J.P. Bond, M. Babadoost, D.I. Edwards, and Z.A. Handoo. 2004. Plant-parasitic nematodes associated with horseradish in Illinois. Nematropica 34:191–197. Walton, A.H. 1958. Aphrodisiacs from legend to prescription. Paperback Library, New York. Ward, S.M., P.J. Delaquis, R.A. Holley, and G. Mazza. 1998. Inhibition of spoilage and pathogenic bacteria on agar and pre-cooked roast beef by volatile horseradish distillates. Food Res. Intl. 31(1):19–26. Weaver, J.E., and W. Bruner. 1927. Horse-radish. Root development of vegetable crops. McGraw-Hill, New York, pp. 150–154. Weber, W.W. 1949. Seed production in horseradish. J. Hered. 40:223–227. Wiersema, J.H., and B. Leon. 1999. World economic plant, a standard reference. CRC Press, Boca Raton, FL. Winiarczyk, K., D. Tcho´rzewska, and J. Bednara. 2007. Development of the male gametophyte of an infertile plant, Armoracia rusticana Gaertn. Plant Breed. 126:433–439. Yoshii, H., M. Sugiura, and T. Iwata. 1963. Studies on the daikon mosaic virus (DMV), the Japanese strain of turnip mosaic virus. Proc. Assoc. Plant Protection Kyushu 1:26.

5 1-Methylcyclopropene: Mode of Action and Relevance in Postharvest Horticulture Research Wendy C. Schotsmans and Robert K. Prange Agriculture and Agri-Food Canada Atlantic Food and Horticulture Research Centre 32 Main Street Kentville, Nova Scotia B4N 1J5 Canada Brad M. Binder Department of Horticulture University of Wisconsin 1575 Linden Drive Madison, WI 53706 USA

ABBREVIATIONS AND ACRONYMS PLANT SPECIES BINOMIALS I. INTRODUCTION A. Scope of Review B. History II. ETHYLENE RESPONSE PATHWAY A. Overview of the Signal Transduction Pathway B. Receptor Structure and Function C. Control of Receptor Levels D. Recovery of Ethylene Sensitivity after 1-MCP Treatment E. Number of Receptors that Need to Be Blocked III. PHYSIOLOGICAL PROCESSES AFFECTED A. Ethylene Biosynthesis

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 263

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IV.

V. VI. VII.

W. C. SCHOTSMANS, R. K. PRANGE, AND B. M. BINDER B. Respiration Rate C. Pigment Metabolism 1. Chlorophylls 2. Carotenoids 3. Flavonoids 4. Enzymatic Browning 5. Nonenzymatic Browning D. Cell Wall Metabolism E. Aroma Metabolism F. Antioxidants SIDE EFFECTS A. Physiological Disorders 1. Superficial Scald 2. Internal Breakdown Disorders B. Stress Responses 1. Chilling Injury 2. Pathogen Attack 3. Wounding SUMMARY AND FUTURE RESEARCH NEEDS ACKNOWLEDGMENTS LITERATURE CITED

ABBREVIATIONS AND ACRONYMS 1-MCP ACC ACO ACS ADH AFS CA CAT Cel CTR EGase EIL EIN EREBP ERF ERS ETR Exp FOL GAF

1-methylcyclopropene 1-aminocyclopropane-1-carboxylic acid ACC oxidase ACC synthase alcohol dehydrogenase a-farnesene synthase controlled atmosphere catalase cellulase constitutive triple response endo-b-1,4-glucanase EIN3-like ethylene insensitive ethylene response element binding protein ethylene response factor ethylene response sensor ethylene response expansin Fusarium oxysporum f.sp. lycopersici cyclic guanosine monophosphate, adenylyl cyclase, formate hydrogen lyase transcription activator

5. 1-METHYLCYCLOPROPENE

GST MAPK NR PAL PE PERE PG PK PL PME POX PPO rin SAM SCF E3 SIMK SIMKK SOD TF a-ara a-gal a-gluc a-man b-gal b-gluc b-xyl

glutathione S-transferase mitogen-activated protein kinase/extracellular signal-regulated kinase never ripe (also referred to as LeETR3) phenylalanine ammonia lyase pectin esterase primary ethylene response element polygalacturonase protein kinase pectate lyase pectin methyl esterase peroxidase polyphenoloxidase ripening inhibitor tomato mutant S-adenosyl-methionine Scp1, Cullin1, Ringbox1, F-Box containing ubiquitin ligating enzyme salt stress inducible MAPK SIMK kinase superoxide dismutase jasmonate regulated transcription factor a-L-arabinofuranosidase a-D-galactosidase a-D-glucosidase a-D-mannosidase b-D-galactosidase b-D-glucosidase b-D-xylosidase

PLANT SPECIES BINOMIALS Dk Fa Le Ma Md Pa Pc Pp Vv

Diospyros kaki (persimmon) Fragaria ananassa (strawberry) Lycopersicon esculentum esculentum or Solanum lycopersicon (tomato) Musa acuminata (banana) Malus domestica or Malus sylvestris subsp. mitis (apple) Persea americana (avocado) Pyrus communis (pear) Prunus persica (peach or nectarine) Vitis vinifera (grape)

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I. INTRODUCTION A. Scope of Review Postharvest physiologists are regularly asked if 1-methylcyclopropene (1-MCP) treatment will ‘‘work’’ for a certain commodity on which it has not been tested. Although the initial response might be that its effect is unknown, this response reﬂects disinterest. It is preferable to formulate an answer better reﬂecting why a straight answer to this question is impossible. The answer is both simple and complex. Simple in that if the commodity’s quality and development is affected by ethylene in any way, 1-MCP will have an effect. This effect will be determined by what process is ethylene-driven and regulated to the extent of the sensitivity of that process to ethylene and its importance in the physiology of the product. And the answer is complex in that the type and size of the effect depends on numerous factors, such as the type of commodity (fruit, vegetable, or ﬂower); the plant species and cultivar; the way in which ethylene promotes, inhibits, and regulates the process you are trying to control; and the speciﬁcity of the process you want to affect. The positive results obtained in several crops have resulted in investigations into the usefulness of 1-MCP that have generated an enormous amount of information. A database search in April 2007 resulted in 480 articles about 1-MCP published since the year 2000. This research has shown that 1-MCP can reduce ethylene production, respiration, softening, color change, aroma production, and the occurrence of physiological disorders. Treatment with 1-MCP can thus increase storage life of numerous fruits with treatment efﬁcacy depending on such factors as the concentration of 1-MCP used, the species and cultivar, storage condition and duration, and maturity of the fruit before application. Most horticultural commodities that were tested do respond to 1-MCP with the biggest effects found in climacteric fruit and those with very speciﬁc responses to exogenous ethylene (Huber et al. 2003). There are also processes and fruits and vegetables in which there is no 1-MCP effect. Watkins (2006b) divided the research using 1-MCP into two blocks: research where 1-MCP is used as a tool to further investigate the role of ethylene in plant physiology, and research where 1-MCP is used as a commercial technology to improve the maintenance of product quality. A synthesis of this information up to 2005 according to horticultural product and type of effect can be found on a Web site (www.hort.cornell.

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edu/mcp/) from Cornell University dedicated to 1-MCP (Watkins and Miller 2005). More information on practical concentrations, treatment conditions, and responses can be found in previous reviews for certain ﬂoricultural (Serek et al. 2006) and edible horticultural products (Blankenship and Dole 2003; Li et al. 2003; Sisler and Serek 2003; Serek et al. 2004, 2006; Zhai et al. 2005; Watkins 2006a,b). The majority of the publications discussing 1-MCP fall under the second kind of research identiﬁed by Watkins (2006b). These publications report the changes in selected quality attributes of speciﬁc fruit using certain 1-MCP concentrations and treatment conditions. Although useful, this does not increase our knowledge of the working principles of 1-MCP or the physiology of the different products. In this review, we have chosen to focus our attention on the ﬁrst kind of research identiﬁed by Watkins (2006b), that is, on articles that elucidate the physiological processes affected by 1-MCP and thus are under direct or indirect ethylene regulation. While reading through this review, it will become clear that the focus is on fruits and vegetables, not ﬂoriculture. For a review covering the control of ethylene responses in ﬂowers, please refer to Serek et al. (2006). In this review, according to nomenclature convention, species binomials were added for genes, transcripts, or proteins unless they are from Arabidopsis thaliana.

B. History The importance of ethylene in plant physiology was ﬁrst noted at the beginning of the 20th century (Neljubow 1901), and it has intrigued scientists ever since. In the 1960s, the molecular requirements for ethylene action were investigated, and Burg and Burg (1967) proposed that a metal is involved in ethylene action and that ethylene analogs are active in the same order as they bind to silver. To enable identiﬁcation and closer study of the ethylene receptor, the search was on for ethylene analogs that would bind more strongly to the receptor (Blankenship 2003). These studies led to the discovery of cyclic oleﬁns such as 2,5-norbornadiene as possibilities (Sisler and Pian 1973). From this point to the actual discovery of 1-MCP, many problems arose, as described in previous reviews (Sisler and Serek 1997; Blankenship 2003; Sisler 2006). Cyclopropenes were found to bind to the ethylene receptor and counteract and delay ethylene responses. The collaborative efforts between the laboratories

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of Edward Sisler (Department of Biochemistry) and Sylvia Blankenship (Department of Horticultural Science) at North Carolina State University into ethylene physiology resulted in the discovery of 1-MCP. The ﬁrst mention of this compound dates back to two 1996 publications by Sisler et al. (1996a, b) and a patent (Sisler and Blankenship 1996) describing the use of cyclopropenes including 1-MCP as inhibitors of ethylene action. All cyclopropenes seem to be active, but some are required in higher concentrations than others to have the same effect. 1-MCP was chosen for further commercialization since it was more active than 3-methylcyclopropene and 3,3dimethyl-cyclopropene and more stable than cyclopropene (Sisler and Serek 1997; Sisler et al. 2001). 1-MCP is commercialized as a stable powder in which it is in a complex with g-cyclodextrin (Watkins 2006b). Once dissolved in water, 1-MCP is easily released as a gas. For use on ornamentals, 1-MCP is marketed as EthylBloc1 by Floralife, Inc. (Walterboro, South Carolina). For use on all edible horticultural products, global use rights were obtained by AgroFresh, Inc., a subsidiary of Rohm and Haas (Springhouse, Pennsylvania), which markets 1-MCP under the trade name SmartFreshTM (Sisler and Serek 2003). Food use registration for 1-MCP has been obtained in most countries with considerable horticultural industry. Although Sisler et al. (1996a, b) chose 1-MCP out of the cyclopropenes as the best candidate for further study and commercialization for use on horticultural produce, other cyclopropenes are being developed (Sisler et al. 2003; Sisler and Serek 2003). Among these are 3-methylcyclopropene (Sisler et al. 1999); 1-hexylcyclopropene (Kebenei et al. 2003); 1-octylcyclopropene (Kebenei et al. 2003; Buanong et al. 2005); 1-ethylcyclopropene (Feng et al. 2004); 1-propyl-cyclopropene (Feng et al. 2004); 1-cyclopropenylmethyl butyl ether (Saleh-Lakha et al. 2004); and 1-decylcyclopropene (Buanong et al. 2005). In this review, however, we focus only on 1-MCP.

II. ETHYLENE RESPONSE PATHWAY Since 1-MCP inhibits ethylene perception by binding to the ethylene receptors to block the effects of endogenous and exogenous ethylene (Sisler et al. 1996a), we provide an overview on what is known about ethylene signal transduction with a focus on the receptors. For longer reviews on the signal transduction pathway and the ethylene receptors, see Li and Guo (2007) and Hall et al. (2007).

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A. Overview of the Signal Transduction Pathway The currently accepted theory on ethylene action is that of a negative regulator model of ethylene receptor function (Hall et al. 1999; Bleecker and Kende 2000; Binder and Bleecker 2003) and a largely linear ethylene response pathway leading from hormone perception by the receptor to transcriptional regulation in the nucleus (Guo and Ecker 2004). According to current models, responses to ethylene are mediated by a family of receptors that have homology to bacterial two-component receptors (Chang et al. 1993) and affect a signal transduction chain responsible for activation of transcription factors and genes responsible for the vast range of ethylene responses. In these models, the ethylene receptors form a physical complex with a kinase, CTR1 (constitutive triple response), that negatively regulates the response pathway in the absence of ethylene (Kieber et al. 1993; Clark et al. 1998; Cancel and Larsen 2002; Gao et al. 2003; Huang et al. 2003). There is genetic evidence that ethylene responses require the presence of the EIN2 (ethylene-insensitive) membrane protein (Alonso et al. 1999). Two transcription factors, EIN3 and the EIN3-like protein EIL1, appear to act downstream of EIN2 and are required for most, if not all, long-term ethylene responses (Chao et al. 1997; Alonso et al. 2003; Binder et al. 2004). It is now clear that the protein levels of EIN3 and EIL1 are controlled in an ethylene-dependent manner via an SCF E3 (Skp1, Cullin1, Ringbox1, F box ubiquitin-protein ligase) complex containing the EBF1 and EBF2 (EIN3-binding F box) proteins for selective ubiquitination (Guo and Ecker 2003; Potuschak et al. 2003; Yanagisawa et al. 2003; Gagne et al. 2004; Binder et al. 2007). A number of possible feedback mechanisms also have been identiﬁed, but less detail is known about their function and regulation and will not be discussed here. Current models (Fig. 5.1) for ethylene signaling posit that the receptor/CTR1 complex are signaling in air and inhibiting downstream components (Binder and Bleecker 2003). Ethylene binding to the receptors inhibits the receptor/CTR1 complex, and downstream components are released from inhibition. This alters the activity of the EIN2 protein via an unknown mechanism. The altered activity of EIN2 either directly or indirectly causes a reduction in the ubiquitination of EIN3/EIL1, resulting in increased levels of these transcription factors and activation of the transcriptional cascade. This primary ethylene signal transduction chain seems to be common to all ethylene responses across plant species.

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Fig. 5.1. Inverse agonist and cooperative receptor model of ethylene action. (a) At low ethylene concentrations, unoccupied receptors activate CTR1, which suppresses ethylene responses. Primary signaling is through ETR1 and ERS1. ETR2, EIN4, and ERS2 signal less effectively to CTR1 but stimulate ETR1 and ERS1. (b) When the concentration of the hormone increases, ethylene binds to the receptors, which leads to deactivation of the receptors and the release of inhibition by CTR1. The dominant-insensitive alleles etr1–1 (c) and ein4–1 (d) are locked in the active state because they cannot bind ethylene. The etr1–1 receptor continues to activate CTR1 and inhibit the ethylene response pathway even when the remaining receptors are inhibited by ethylene. The ein4–1 receptor continues to activate CTR1, perhaps through interaction with ETR1 and ERS1, even in the presence of ethylene. (e) In triple-receptor null plants containing only ETR1 and ERS1, the reduced receptor number results in insufﬁcient activation of ETR1 and ERS1. This, in turn, leads to a reduced activation of CTR1 and constitutive ethylene responses. (f) In the doublereceptor null plants that lack ETR1 and ERS1, the remaining receptors are incapable of maintaining sufﬁcient activation of CTR1 because the primary signaling receptors are missing. (Adapted from Binder and Bleecker 2003.)

The transcription factors from the primary ethylene signal transduction chain (EIN3/EIL1) in turn activate other transcription factors (Fig. 5.2) such as ERF1 (ethylene response factor) from the plantspeciﬁc EREBP (ethylene response element binding protein) family of transcription factors that interact with ethylene-responsive genes (Solano et al. 1998) which encode effector proteins necessary in a

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wide variety of ethylene responses, from disease resistance to differential cell growth (Guo and Ecker 2004). This suggests that at least two rounds of transcriptional activation are required for responses to ethylene. Many genes are up- and down-regulated when plants are treated with ethylene (Schenk et al. 2000; Van Zhong and Burns 2003), including a subset of the receptors (Hua et al. 1998). ERF1 also regulates the jasmonate-mediated defense response. From this point on, other transcription factors formed through developmental and/or other hormone-signaling pathways integrate into the ethylene-signaling pathway (Lorenzo et al. 2003). B. Receptor Structure and Function Ethylene receptors are membrane-bound, disulﬁde-linked proteins that use copper as a cofactor for ethylene binding (Schaller et al. 1995; Rodrı´guez et al. 1999). It is thought that when ethylene binds to the copper cofactor, it changes the coordination state of the copper resulting in conformational changes in the receptor. The number of receptor isoforms varies from species to species (Mita et al. 1998; Sato-Nara et al. 1999; Yamasaki et al. 2000; Terajima et al. 2001; Klee 2002; Rasori et al. 2002; Shibuya et al. 2002; El-Sharkawy et al. 2003; Ma and Wang 2003; Gallie and Young 2004; Yau et al. 2004; Arora et al. 2006; Tanase and Ichimura 2006; Bustamante-Porras et al. 2007; Fernandez-Otero et al. 2007; Wang and Kumar 2007). In Arabidopsis, where most research on the receptors has been conducted, there are ﬁve receptor isoforms: ETR1 (ethylene response), ETR2, EIN4, ERS1 (ethylene response sensor), and ERS2. We know they are the receptors because all ﬁve isoforms can bind ethylene with high afﬁnity (Schaller and Bleecker 1995; Hall et al. 2000; O’Malley et al. 2005), and speciﬁc mutations in any of these isoforms confer dominant ethylene insensitivity in the plant (Bleecker et al. 1988; Chang et al. 1993; Hua and Meyerowitz 1998; Hua et al. 1998; Sakai et al. 1998; Hall et al. 1999). Five of the tomato (Le, Lycopersicon esculentum esculentum) receptor isoforms (O’Malley et al. 2005) and the product of Synechocystis slr1212 (Rodrı´guez et al. 1999) also have high afﬁnity for ethylene binding. Based on bioinformatics analysis, all ethylene receptors are predicted to contain three N-terminal transmembrane a-helices, a GAF (cyclic guanosine monophosphate, adenylyl cyclase, formate hydrogen lyase transcription activator) domain, and a kinase domain. Some isoforms also contain a receiver domain (Wang et al. 2006b). The ethylenebinding domain is found in the three transmembrane helices (Schaller and Bleecker 1995; Rodrı´guez et al. 1999; O’Malley et al. 2005). While

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Salt stress Salicylic acid Elicitors Viral infection

CTR1 SIMKK

SIMK EIN2 Jasmonate

EIN5 EIN6

EIN3

?

Ub/26S

EIL1

TF?

ERF1 Ethylenespecific EREBPs

PERE

? Nucleus

ERF1

Effectors GCC-box

PDF1.2

Defense responses

HLS1

?

Differential cell growth Current Opinion in Plant Biology

Fig. 5.2. A model for the ethylene response pathway in the regulation of gene expression. Ethylene gas is perceived by a family of endoplasmatic reticulum–associated ethylene receptors. CTR1 is proposed to be activated by the unoccupied receptors via physical interaction with them, and ETR and CTR1 function is inhibited upon binding of ethylene to the receptors. A MAPK module, consisting of salt stress–inducible MAPK (SIMK) and SIMK kinase (SIMKK), is proposed to act downstream of CTR1. Downstream components in the ethylene pathway include several positive regulators (EIN2, EIN5, EIN6 and the transcription factors EIN3 and EIL1). The level of EIN3 is controlled by ethylene, possibly

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Fig. 5.3. A current model is that 1-MCP suppresses the ethylene response pathway by permanently activating ethylene receptors. (a) When 1-MCP binds to a highly expressed receptor like ETR1, it keeps ETR1 in the active conformation. This leads to continuing activation of CTR1, even when the remaining receptors are inhibited by ethylene. (b) When 1-MCP binds to EIN4, EIN4 is locked in the active state. This keeps ETR1 and ERS1 activated in the presence of ethylene. (Adapted from Binder and Bleecker 2003.)

kinase activity has been observed in receptors from Arabidopsis and tobacco (Gamble et al. 1998, 2002; Xie et al. 2003; Moussatche and Klee 2004; Zhang et al. 2004), where examined, this activity appears unnecessary for ethylene signaling (Gamble et al. 2002; Wang et al. 2003; Xie et al. 2006). 1-MCP is a competitive inhibitor for ethylene binding (Hall et al. 2000), suggesting that it acts at the site for ethylene binding (Fig. 5.3). Analyses of mutations in the transmembrane domain of ETR1 have revealed that certain residues are crucial for ethylene binding (Schaller and Bleecker 1995; Hall et al. 1999; Rodrı´guez et al. 1999). Of particular interest is the fact that Cys65 in ETR1 is crucial for coordinating Cu(I); mutating this residue results in a protein incapable of binding copper or

3

via the proteasome (Ub/26S). The primary ethylene signaling pathway components (from ETR to EIL1) are required for all known ethylene responses and seem to respond only to ethylene. Several EREBP transcription factors are known to be immediate targets of EIN3/ EIL1, which can bind to a primary ethylene response element (PERE) in the promoters of EREBP genes. One EREBP, called ERF1, is also involved in jasmonate-mediated gene regulation. An unidentiﬁed jasmonate-regulated transcription factor (TF) may also bind to the promoter of ERF1 to activate its expression. The promoter of ERF1 would then integrate signals from both the ethylene-and jasmonate-signaling pathways. Many EREBP proteins are known to regulate gene expression through interaction with a cis-element called the GCC-box, which is found in several ethylene-responsive genes, including PDF1.2 and HOOKLESS1 (HLS1). These genes encode effector proteins that are needed to execute a wide variety of ethylene responses, from disease resistance to differential cell growth. ‘‘?’’ represents an unknown factor or element. Arrows and t-bars represent positive and negative effects, respectively. Solid lines indicate effects that occur through direct interaction. Dotted lines indicate effects that have not yet been shown to occur through direct interaction. (From Guo and Ecker 2004. Copyright Elsevier 2004.)

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ethylene (Rodrı´guez et al. 1999). These earlier observations have been extended by a recent study on the ethylene binding domain of ETR1 (Wang et al. 2006b). Using mutational analysis, the authors found that speciﬁc amino acids (including Cys65) in the middle of helices I and II are required for ethylene binding. Other residues located in helices I and III result in a receptor that does not turn off even when ethylene binds the mutated receptor. It is unclear whether the residues important for ethylene binding are also required for binding of 1-MCP. All the receptors contribute to signaling (Bleecker et al. 1988; Chang et al. 1993; Hua et al. 1995, 1998; Sakai et al. 1998; Hall et al. 1999), and there is functional overlap between the different isoforms (Hua and Meyerowitz 1998). However, they are not entirely redundant in function. Based on sequence comparisons of the ethylene binding domains from plants and cyanobacteria, the ethylene receptors in plants can be divided into two subfamilies (Guo and Ecker 2004; Wang et al. 2006b). At least in Arabidopsis, subfamily I (ETR1 and ERS1) receptors contribute more to signaling than subfamily II (ETR2, EIN4, and ERS2) receptors (Zhao et al. 2002; Hall and Bleecker 2003; Wang et al. 2003; Guo and Ecker 2004; Xie et al. 2006). This has led to the suggestion that subfamily II receptors mainly function via subfamily I receptors (Binder and Bleecker 2003). There is also evidence that speciﬁc receptor isoforms have unique roles, particularly in physiological response (Seifert et al. 2004; Binder et al. 2006; Kevany et al. 2007). The importance of subfamily I over subfamily II receptors is not true for all species (Kevany et al. 2007), which highlights the importance of studying a variety of species. C. Control of Receptor Levels The negative agonist model for ethylene function predicts that ethylene response and sensitivity increases when the number of receptors decreases. Thus, the control of ethylene receptor levels, synthesis, and tissue location could play an important role in speciﬁc ethylene responses and help us better understand how to use 1-MCP. Studies on tomato indicate that ethylene receptor levels likely control the ethylene response (Klee 2002; Kevany et al. 2007) where the degradation of speciﬁc receptor isoforms controls fruit ripening (Kevany et al. 2007). Receptor transcript levels have been studied in a variety of fruits during development and ripening as well as after treatment with either ethylene or 1-MCP. The pattern of these changes varies from species to species and even cultivar to cultivar. In ripening apple (Md, Malus domestica) and peach (Pp, Prunus persica) fruit, an increase in

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receptor transcripts (MdETR1, MdERS1, PpETR1, PpERS1) occurs in association with increments in ethylene biosynthesis (Dal Cin et al. 2006); the main difference between the two fruits is situated mainly in the higher level of constitutive MdETR1 and MdERS1 gene expression in apple compared with PpETR1 and PpERS1 in peach. Treatment with 1-MCP reduces MdERS1 and MdETR1 expression but has no signiﬁcant effect on PpERS1 and PpETR1 expression (Dal Cin et al. 2005, 2006). However, other researchers found MdERS1 and MdERS2 expression to be reduced whereas MdETR1 was little affected by 1-MCP treatment (Tatsuki and Endo 2006). The reasons for this discrepancy are not clear but might be found in the use of a different cultivar; ‘Orin’ and ‘Fuji’ were used by Tatsuki and Endo (2006) whereas Dal Cin et al. (2005, 2006) used ‘Golden Delicious’. Treatment of developing peach with 1-MCP, however, does not affect PpETR1 transcription, while it down-regulates PpERS1, indicating that developmental signals also play a role in the ethylene signaling pathway (Rasori et al. 2002). In avocado (Pa, Persea americana), the transcription of PaERS1 is suppressed to trace levels (Owino et al. 2002), and in grapes (Vv, Vitis vinifera), VvETR1 transcript levels are reduced by 1-MCP (Chervin et al. 2005). In tomato, changes in the transcription of receptor genes LeETR1 and LeETR2 during ripening are minor but do follow the ethylene production pattern. NR (or LeETR3) mRNA accumulation does not follow this pattern, and the transcription of these genes is not inﬂuenced by 1-MCP treatment. Accumulation of LeETR4/5/6 mRNA increases rapidly in control fruit whereas it is reduced to the basal level in 1-MCP-treated fruit (Tassoni et al. 2006) for 8 days, after which transcription increases. A more recent publication by Kevany et al. (2007) compared changes in receptor transcript levels with changes in receptor proteins levels and shows a more complex picture. They conﬁrm that LeETR1 and LeETR2 do not change much during ripening and are not affected by 1-MCP, but at the onset of ripening, NR, LeETR4, and LeETR6 transcription increases whereas LeETR5 transcript level does not change much. Even more interesting and opposite to the changes in receptor transcript levels, NR, LeETR4, and LeETR6 protein levels are highest during fruit development and decline at the onset of ripening. This decline is prevented by 1-MCP and stimulated by exogenous ethylene, indicating an ethylene-induced degradation of receptor proteins during ripening (Kevany et al. 2007). However, this increased receptor degradation might occur only at high levels of ethylene (Kevany et al. 2007), since at low levels of ethylene, there is a correlation between ETR2 transcript levels and ETR2 protein levels

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(Chen et al. 2007), and in air, there is a correlation between total receptor transcript levels and ethylene binding levels in Arabidopsis (O’Malley et al. 2005). D. Recovery of Ethylene Sensitivity after 1-MCP Treatment Another prediction of the negative agonist model for ethylene signaling is that recovery after the removal of ethylene occurs either by dissociation of ethylene from the receptors or synthesis of new receptors or a combination of both. 1-MCP is believed to bind irreversibly to the ethylene receptor complex, but as Blankenship and Dole (2003) pointed out, there is a lack of supporting data for the assumption that the return of ethylene sensitivity therefore must be due to the appearance of new binding sites. The existence of a recovery of ethylene sensitivity mechanism is supported by the effectiveness of renewed exposure to 1-MCP in tomato (Hoeberichts et al. 2002), where the effect of a single 1-MCP treatment on ripening lasts 5 to 7 days but is prolonged to 12 to 16 days by renewed exposure. In peach, a single dose of 1-MCP does not alter ethylene biosynthesis (Mathooko et al. 2001), but pulse treatment delays the induction of ethylene biosynthesis. Immature ‘Bartlett’ (‘Williams’) pears (Pc, Pyrus communis) also beneﬁt from retreatment with 1-MCP after a period of storage as long as ripening has not set in (Ekman et al. 2004). The return of ethylene sensitivity has been well documented in several fruits (Blankenship and Dole 2003; Watkins 2006a,b), but evidence for the increase of receptor sites or an explanation as to what happens exactly was not provided until research by Tassoni et al. (2006) with 1-MCP treated tomato, where ripening was delayed for 8 days at which point ethylene sensitivity returned. During those 8 days, transcription of LeETR4/5/6 reduced to a basal level followed by an increase to a similar level as in control fruit (Tassoni et al. 2006). This conﬁrms the hypothesis that the site regeneration system exists, but it does not yet provide insight into the triggers for this regeneration. E. Number of Receptors that Need to Be Blocked A further question scientists have is how many receptors there are and how many need to be ﬁlled with 1-MCP or other ethylene analogs to have a speciﬁc effect. This seems to be highly fruit speciﬁc, as illustrated by the enormous number of publications concerning the optimal concentration of 1-MCP and treatment duration (Blankenship and Dole 2003; Li et al. 2003; Sisler and Serek 2003; Serek et al. 2004, 2006; Zhai et al. 2005; Watkins 2006a,b). In banana (Ma, Musa acuminata), a minimal

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concentration (300 nL L-1) is required to extend shelf life, and high concentrations (30 mL L-1) can impair sufﬁcient ripening (Klieber et al. 2003). In parsley (Petroselinum crispum), too-low concentrations (0.01 mlL-1) result in an acceleration of senescence whereas adequate concentrations (10 ml L-1) efﬁciently retard leaf senescence (Ella et al. 2003b). A possible explanation for this accelerated senescence in the presence of low concentrations of 1-MCP was found in relief from ethylene autoinhibition by a small amount of receptors being bound by 1-MCP and locked in the activated state. However, there was not enough 1-MCP to occupy all the ethylene receptors. The small amount of produced ethylene therefore could inactivate receptors lacking 1-MCP and initiate the signal transduction chain leading to enhanced ethylene production (Ella et al. 2003b). This overview should make it clear that ethylene signal transduction is complex. There are many questions to be answered, and 1-MCP can be a signiﬁcant help in elucidating these questions. Given the fact that there are varying numbers of receptor isoforms in different species with distinct organ distribution and nonredundant function, it is very difﬁcult to make concrete predictions regarding physiological outcomes when using 1-MCP or other ethylene response inhibitors.

III. PHYSIOLOGICAL PROCESSES AFFECTED A. Ethylene Biosynthesis One of the most important consequences of blocking the ethylene signal transduction pathway by 1-MCP is the inhibition of ethylene production. The inhibition of ethylene production is most likely to occur through two key enzymes in the ethylene production pathway (Fig. 5.4). ACC synthase (ACS) is involved in the conversion of S-adenosyl-methionine (SAM) to ACC, which is then converted to ethylene by ACC oxidase (ACO) (Wang et al. 2002). In general, a reduction in ethylene biosynthesis by 1-MCP treatment is accompanied by down-regulation of ACO expression and/or activity (Mathooko et al. 2001, 2004; Hoeberichts et al. 2002; Owino et al. 2002; Deﬁlippi et al. 2005b; Dal Cin et al. 2006; Zhang et al. 2006) plus downregulation of ACS expression and activity in apple (Deﬁlippi et al. 2005b; Dal Cin et al. 2006), avocado (Owino et al. 2002), banana (Zhang et al. 2006), but not in peach (Mathooko et al. 2001, 2004). This indicates that the differential effect of 1-MCP treatment on ethylene biosynthesis is likely through ACS. Additionally, recovery of ethylene

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Protein Synthesis

Methionine

HCOOH ATP

SAM synthetase

Adenine

PPi + Pi

Methylated Acceptors

NH3 H3C

COO–

S

5”-methyladenosine

CH2 O Ade HO OH

Spermidine/Spermine biosynthesis pathway

ACS

SAM

Pi

Inhibitor ?

(high activity or stable protein) malonyl-ACC +

–OOC

Ptase

C H2C

Inhibitor ACS ?

CH2

ACC O2

H

H C

H

Calcium?

(low activity or unstable protein?)

Reactive oxygen species?

ACO

CO2 + HCN

Kinase

NH3

C H

Transcription regulation

Harmones?

ethylene Stresses: Pathogen infections, Wounding, Ozone, UV-B, etc. Fig. 5.4. Biosynthetic pathway and regulation of ethylene: The ﬁrst step is the formation of SAM by SAM synthetase from methionine. SAM is converted to ACC by ACS under most conditions, which is the rate-limiting step in ethylene biosynthesis. ACC is the immediate precursor of ethylene. ACO catalyzes the ﬁnal step of ethylene synthesis using ACC as substrate. Transcriptional regulation of both ACS and ACO is indicated by dashed arrows. Reversible phosphorylation of ACS is hypothesized and may be induced by unknown phosphatases (Ptase) and kinases, the latter presumably activated by stresses. Both native and phosphorylated forms (ACS-Pi) of ACS are functional, although the native ACS may be less stable or active in vivo. A hypothetical inhibitor is associated with ACS at the carboxyl end and may be dissociated from the enzyme if it is modiﬁed by phosphorylation. (Adapted from Wang et al. 2002. Copyright American Society of Plant Biologists 2002.)

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sensitivity in mountain papaya (Vasconcellea cundinamarcenensis) is followed by an increase in ACO activity, but ethylene concentration does not increase to the same level compared with control fruit due to the limited availability of ACC (Moya-Leo´n et al. 2004), indicating that ACS expression does not recover as fast as ACO expression. Both these enzymes (ACS and ACO) are encoded by a multigene family of up to eight ACS genes and four ACO genes in tomato (Nakatsuka et al. 1998), some of which are regulated by ethylene. Inhibition of ethylene production after treatment with 1-MCP is therefore either through reduced expression of one or more of these ethylene-regulated genes or through reduced activities of their respective enzymes. The fact that not all of the genes are ethylene-regulated means that 1-MCP will be effective only in those cases (or developmental stages) where these speciﬁc genes and their product are important. Ethylene biosynthesis can be divided into two distinct phases/ systems (Fig. 5.5). System 1 is functional during vegetative growth, under negative feedback regulation by ethylene and responsible for producing basal ethylene levels that are detected in all tissues, including those of nonclimacteric fruits. System 2 operates during the ripening of climacteric fruits and senescence of some petals and is under positive feedback regulation (Alexander and Grierson 2002). For tomato, there is ample evidence that some of the genes and products responsible for

Fig. 5.5. Model proposing the differential regulation of LeACS gene expression during the transition from system 1 to system 2 ethylene synthesis in tomato. Never ripe (Nr) mutant cannot perceive ethylene due to a mutation in the ethylene-binding domain of the NR ethylene receptor. The symbols ve (negative) and þve (positive) refer to the action of ethylene on signaling pathways resulting in repression (ve) or stimulation (þve) of LeACS gene expression. [Source: Adapted from Barry et al. 2000. (Copyright American Society of Plant Biologists 2000.) Alexander and Grierson 2002.]

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W. C. SCHOTSMANS, R. K. PRANGE, AND B. M. BINDER

ethylene biosynthesis are more abundant in the preclimacteric stage (LeACS1, LeACS3, LeACS6, LeACO1, and LeACO4) when system 1 of ethylene regulation is operational. Transition to system 2 at the climacteric stage is accompanied with increased expression of LeACS2, LeACS4, LeACO1, and LeACO4 (Nakatsuka et al. 1998; Alexander and Grierson 2002). As system 1 is under negative feedback regulation by ethylene, an increase in ethylene production (e.g., during ripening) will inhibit system 1, decreasing the basal level of ethylene production; system 2 is under positive feedback regulation and will be enhanced by the presence of ethylene. We can assume that treatment with 1-MCP shuts down system 2 but also removes the negative feedback regulation of system 1 up to the climacteric stage, when system 1 is no longer operational. This might explain why ethylene production is not always completely stopped and why ripening can still continue after 1-MCP treatment. If the developmental trigger for certain processes (color change, cell wall degradation, and aroma development) arises and the base level of ethylene is available, certain ACS genes still can be expressed and ACC production resumed. Banana has a very speciﬁc biphasic climacteric pattern, different from other climacteric fruits. The ethylene and respiratory peak seen in other climacteric fruits is followed a few days later by a second smaller peak. It is no surprise then that in banana, the two distinct and simultaneous systems of ethylene production coexist. 1-MCP treatment blocks the autocatalytic pathway (system 2) as observed through decreased transcription of MaACS and accumulation of ACC as well as a reduction in the respiratory and ethylene peak; 1-MCP treatment does not affect the basal level (system 1) of MaACO transcription and activity and ethylene production (Pathak et al. 2003). The different regulation of the various genes in the gene families means that it is becoming more critical for the general understanding of 1-MCP and ethylene action to specify which gene speciﬁcally is downregulated after 1-MCP treatment. It is also important to control other factors (temperature, damage) during experiments or commercial treatment due to the general physiology of the speciﬁc fruit. For instance, 1-MCP treatment on nectarines (Pp, Prunus persica) is effective at higher temperatures (25 C) with a strong decrease in ethylene production and lower PpACS, PpACO1, and PpACO2 transcription relative to the control; 1-MCP is completely ineffective at lower temperature (4 C) with an increase in ethylene production and higher PpACS, PpACO1 and PpACO2 transcription relative to the control (Bregoli et al. 2005). The hypothesis behind this effect was that at low temperatures, there is either an inadequate binding to ethylene

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receptors or slower regeneration of receptors or a cold stress–induced ethylene production. Since the receptor proteins predicted from DNA sequence are similar in different fruits (Wang et al. 2006b), it seems unlikely that 1-MCP binds well at low temperatures in some fruits with the expected reduction of ethylene biosynthesis (de Wild et al. 2003) and not in others. Indirect conﬁrmation of this point is found in the observation that exogenously expressed receptors from both Arabidopsis and tomato have similar binding afﬁnities for ethylene (O’Malley et al. 2005), suggesting that receptors from a variety of fruits are likely to have similar afﬁnities for 1-MCP. Cold stress–induced ethylene production is a much more likely explanation through the ‘‘wounding’’ induced by the cold, which, in peach, directly activates transcription of PpACS1 and PpACO1 (Mathooko et al. 2001). Wounding tomato fruit results in increased expression of LeACS2, LeACS6, and LeACO1, even if fruit are treated with 1-MCP prior to wounding (Yokotani et al. 2004), indicating that the stimulation of these genes through wounding is independent of ethylene. Note that the genes activated are LeACS2, linked with system 2 and up-regulated by ethylene, and LeACS6, linked with system 1 and down-regulated by ethylene (Nakatsuka et al. 1998). This would ensure continued production with (through LeACS2) or without (through LeACS6) the presence of ethylene and thus also the presence of 1-MCP. A similar pattern was seen in peach (i.e., wounding peach activates transcription of PpACS1 and PpACO1 independent of de novo protein synthesis). This fact means that these genes are primary response genes and that the wounding signal is transmitted to the nucleus via preexisting components in the cell and not mediated by ethylene (Mathooko et al. 2001). Additionally, during peach ripening, a single dose of 1-MCP does not alter PpACS1 and PpACO1 transcription (Mathooko et al. 2001), but pulse treatment with 1-MCP does delay PpACS1 transcription and inhibits PpACO1 transcription. This fact implies that PpACS1 is not under positive feedback regulation by ethylene, showing a similarity with tomato, where LeACS1 is linked with system 1 (Nakatsuka et al. 1998), which is under negative feedback regulation by ethylene. There is conﬂicting evidence in avocado (Owino et al. 2002), where positive feedback regulation of the PaACS1 gene and negative feedback regulation of the PaACS2 gene by ethylene occurs, while PaACO exhibits positive feedback regulation by ethylene and is also induced by wounding. Additionally, although 1-MCP application inhibits ACS and ACO activity and PaACS1 transcription, and suppresses PaACO transcription, discontinuation of the treatment leads to superinduction of PaACS1 and PaACO transcripts (Owino et al. 2002). It is not clear whether the difference (PaACS1 is under positive

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W. C. SCHOTSMANS, R. K. PRANGE, AND B. M. BINDER

feedback regulation and PpACS1 is not) is due to a species-speciﬁc mechanism of ethylene biosynthesis regulation or if there is some other reason for this difference. Ethylene is not necessarily the initiator for the onset of ripening. For example, fruit of persimmon (Dk, Diospyros kaki) held in ambient lowhumidity conditions (40%–60% relative humidity) has a biphasic ethylene production pattern, with an initial increase during the ﬁrst two days and a second increase on the sixth and eighth day after harvest (Nakano et al. 2002). During the initial increase in ethylene, the calyx produces more ethylene accompanied by increased expression of DkACS2 compared with the pulp, where only a small amount of ethylene is detected. The second increase in ethylene production is limited to the pulp and accompanied by increased ACC content and expression of DkACS1, DkACS2, and DkACO1 genes. The initial ethylene production in the calyx is likely a response to water stress and not to the presence of ethylene. This was later conﬁrmed since 1-MCP suppresses only the second increase in ethylene production (Nakano et al. 2003). The hypothesis is that ethylene initially produced in the calyx diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis (Nakano et al. 2003). Suppression of ethylene production by 1-MCP is accompanied by decreased activities of ACS and ACO with greater inhibition of ACS (Ortiz et al. 2006). The differential effect of 1-MCP treatment on ethylene biosynthesis depending on fruit, maturity, and ripening stage does not exclude an effect of 1-MCP on other quality aspects. In postclimacteric tomatoes, there is no reduction of ethylene production after 1-MCP treatment, but color development, softening, and increases in the ratio of soluble solids content/acidity are inhibited by 1-MCP treatment (Guillen et al. 2005). Earlier, we mentioned that persimmon has a biphasic ethylene production pattern; the initial increase in ethylene production is not ethylene-driven and not affected by 1-MCP treatment, but the second increase in ethylene production is ethylene-driven and suppressed by 1-MCP treatment (Nakano et al. 2003). This could explain why Ortiz et al. (2005) did not ﬁnd a suppression in ethylene evolution after treatment of persimmon with 1-MCP. However, 1-MCP does delay persimmon softening, loss of soluble solids content, weight loss, and color development (Nakano et al. 2003; Ortiz et al. 2005). B. Respiration Rate Respiration rate is one of the ﬁrst metabolic reactions discussed when evaluating ripening. The respiration rate of most treated products either

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decreases with 1-MCP treatment or ripening-related increases in respiration rate are delayed by 1-MCP (Watkins 2006b), especially in climacteric fruits where ethylene production and respiration accompany each other. However, there are exceptions; 1-MCP has no signiﬁcant effect on respiration rate of the nonclimacteric sweet cherry (Prunus avium), whereas respiration is stimulated by exogenous ethylene (Gong et al. 2002), indicating that some receptors remain unaffected by 1-MCP or that there has been no induction of receptors that 1-MCP can bind to. Fresh-cut products present a different challenge. 1-MCP treatment of intact ‘Gala’ apples delays but does not reduce ethylene production and respiration of the apple slices (Bai et al. 2004) after 14 days of storage, which may indicate that wound and induced respiration are not ethylene-driven. C. Pigment Metabolism Color of fruits and vegetables is the ﬁrst criterion used by consumers to assess quality of fruit and vegetables. These colors are a function of the presence of four primary pigment classes in the fruit: chlorophylls, carotenoids, ﬂavonoids, and betalains. Additionally, pigments are formed during discoloration reactions in which phenols are oxidized forming brown pigments (Kays 1999). Besides the minimum color requirements at harvest, color changes are used as a measure of ripening evolution. Although most publications regarding 1-MCP mention its effects on color change, less information has been published regarding 1-MCP effects on pigment metabolism. 1. Chlorophylls. Loss of greenness, or increased yellowing, in most products is inhibited or delayed by 1-MCP (Watkins 2006b) through decreased or delayed chlorophyll degradation (Ella et al. 2003a; Golding et al. 2003; Hershkovitz et al. 2005; Moretti et al. 2005; Opiyo and Ying 2005; Wang et al. 2006a), decreased gene expression of chlorophyllase (Ella et al. 2003a), reduced activity of chlorophyllase (Ella et al. 2003a; Forney et al. 2003; Gong and Mattheis 2003), and reduced activity of peroxidase (POX) (Forney et al. 2003; Gong and Mattheis 2003; Hershkovitz et al. 2005). Complete inhibition of chlorophyll degradation could be positive in leafy vegetables and some other green products, where loss of greenness is the main issue. However, in most products, unmasking of other colored pigments that are concealed due to the presence of chlorophyll is an important aspect in the ripening process and is needed to ensure consumer appreciation,

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making it essential that 1-MCP delays chlorophyll degradation but does not completely inhibit it. Degreening is not always under strict ethylene control. In green oroblanco (Citrus grandis C. paradisi) peel tissue, 1-MCP and thus ethylene have only a small effect on the induction of chlorophyllase enzyme activity (Porat et al. 2001). In cucumbers (Cucumis sativus), 1-MCP treatment prior to exposure to ethylene does not prevent degreening, indicating that breakdown of chlorophyll in the cucumber skin may be triggered by developmental factors and not by endogenous ethylene (Nilsson 2005). Ethylene independent degreening (degreening after 1-MCP treatment) in apple has been achieved through treatment with methyl jasmonate (Fan and Mattheis 1999), a senescence-promoting substance (Ueda and Kato 1980), possibly through the interaction of jasmonate-regulated transcription factors (Fig. 5.2) with the ethylene response pathway. 2. Carotenoids. Carotenoid biosynthesis is delayed by 1-MCP treatment during peach (Cecchi et al. 2005) and tomato ripening (Moretti et al. 2001, 2005). In tomato, control fruit stored for 17 days have 190% more total carotenoids than fruit treated with 1-MCP (Moretti et al. 2001). Gene expression and activity of the enzymes (Fig. 5.6) phytoene synthase (involved in the ﬁrst step of carotenoid biosynthesis, where phytoene is produced), and phytoene desaturase (converts phytoene into the colorless phytoﬂuene and the yellow -carotene) is induced by ethylene and thus delayed by 1-MCP in apricot (Prunus armeniaca) (Marty et al. 2005). In contrast, 1-MCP does not affect the apricot’s gene expression and activity of the enzymes -carotene desaturase (catalyzes the conversion of -carotene into orange neurosporene and red lycopene) and b-lycopene cyclase (responsible for the formation of b-carotene and its derivative xanthophylls from lycopene) (Marty et al. 2005). In many cases, it is not desirable to block pigment production completely since the color development is an intrinsic part of the ripening process and is the ﬁrst aspect consumers use to decide whether to purchase a speciﬁc fruit. This is where the applied concentration of 1-MCP becomes critical. Use of the appropriate concentration delays accumulation of lycopene and carotenoids in cherry tomato; excessive concentrations inhibit the accumulation of lycopene and carotene to the point where the fruit never reaches the color of control fruit (Opiyo and Ying 2005). 3. Flavonoids. Anthocyanin accumulation is transiently inhibited by 1-MCP treatment in grape skins (Chervin et al. 2005) and strawberry

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Fig. 5.6. Carotenoid pathway and b-carotene metabolism. (Marty et al. 2005.)

(Fa, Fragaria ananassa) (Jiang et al. 2001) but not in immature ‘Fuji’ apple (Mattheis et al. 2004), pear (MacLean et al. 2007), and sweet cherries (Mozetic et al. 2006). Unlike carotenoids, the location of 1-MCP control on ﬂavonoid biosynthesis has not been extensively studied. Even though ﬂavonoid concentration is not affected by 1-MCP treatment in pear, the transcription of two key ﬂavonoid biosynthetic enzymes (phenylalanine ammonia-lyase and chalcone synthase) is inhibited, indicating that pear fruit does not biosynthesize ﬂavonoids postharvest but carbon from the ﬂavonoid biosynthesis is diverted into the production of hydroxycinnamic acids, such as chlorogenic acid. The accumulation of the latter is temporarily decreased by ethylene treatment (MacLean et al. 2007). However, there are indications that for grape, the effect of 1-MCP on anthocyanin accumulation might be through decreased sucrose accumulation (Chervin et al. 2006), since 1-MCP treatments reduce the

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transcription of a sucrose transporter. In Arabidopsis, sucrose acts as a signal molecule up-regulating anthocyanin biosynthesis (Solfanelli et al. 2006). 4. Enzymatic Browning. A ﬁrst type of browning is enzymatic browning, which occurs when phenolic compounds are oxidized by polyphenoloxidase (PPO) to o-quinones, which form brown-colored polymers (Mathew and Parpia 1971; Mayer 1987). Ascorbic acid, a natural antioxidant, can prevent this by converting o-quinones back to diphenols (Franck et al. 2007). The ultimate cause of this reaction needs to be looked for elsewhere, as the phenolic compounds and the enzymes that oxidize them are normally separated by membranes (Larrigaudie`re et al. 1998); therefore, the causes of browning must be sought in processes affecting membrane integrity (Franck et al. 2007). Enzymatic surface browning and loss of acceptable visual quality of pineapple (Ananas comosus) slices is reduced by treatment with 1-MCP due to a reduction of the hydrolysis of endogenous ascorbic acid (Budu and Joyce 2003). In loquat (Eriobotrya japonica) fruit, reduction of enzymatic ﬂesh browning by 1-MCP is related to a decrease in PPO activity, a delay in the decline of total phenolic content, and a delay in the increase of relative electrical conductivity and loss of compartmentalization in fruit tissue (Cai et al. 2006a). 5. Nonenzymatic Browning. Another type of discoloration is nonenzymatic browning due to a Maillard reaction between reducing sugars and the a-amino groups of nitrogenous compounds at high processing temperatures (> 150 C), forming melanoidins, for instance, during potato (Solanum tuberosum) processing (Schallenberger et al. 1959). When potato tubers are continuously treated with ethylene during storage, tuber sprouting is prevented through inhibition of visible sprout cell differentiation and elongation (Prange et al. 1998). This also causes ethylene-induced fry color darkening, induced by an increased conversion of starch to sugars after exposure to ethylene (Daniels-Lake et al. 2005). Treatment with 1-MCP before the ethylene is applied prevents this fry color darkening without blocking ethylene control of tuber sprouting (Prange et al. 2005). The explanation for this result can be found in the location and differentiation of the tissue involved. Sprouts grow when metabolically active meristematic cells in the tuber eyes multiply through mitosis and new ethylene binding sites are produced, which is why continuous ethylene treatment is necessary. Fry tissue is cut from the pith and cortex of the tuber, where cells are less metabolically active compared with the tuber eyes and where ethylene

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binding sites are less likely to be produced (Prange et al. 2005). Ethylene provided after 1-MCP treatment would therefore have an effect on tuber sprouting but not on fry color, since in the latter all receptors are likely to be occupied by 1-MCP.

D. Cell Wall Metabolism Softening in fruit during ripening is a complex process consisting of simultaneous biosynthesis and breakdown of the various components of the cell wall, including loosening of the network by expansion (Exp), breakdown of the matrix pectins, and degradation of the hemicellulose and cellulose frames as well as ligniﬁcation. Most of the fruits that have been researched so far retain ﬁrmness in response to treatment with 1-MCP; the magnitude and duration of this response varies with fruit species, cultivar, and maturity stage (Huber et al. 2003). Although the main structure of cell walls is similar for all fruits and some of the ripening-related cell wall modiﬁcations are observed in most fruits, texture differs in fruit species due to differences in the extent and the timing of the modiﬁcations of the cell wall architecture. Speciﬁc cell wall modiﬁcations occur in certain fruits, giving each fruit its speciﬁc ripe texture; for instance, apple versus apricot versus avocado. The cell wall is not the only component involved in ﬁrmness and softening; the water status of the fruit or the turgor pressure in the cells plays an important role. Although the potential role of 1-MCP on turgor maintenance has not been studied speciﬁcally, there are indications that it does have an inﬂuence as water loss during storage of apple and pear is signiﬁcantly reduced after 1-MCP treatment (Baritelle et al. 2001). Wall-localized enzymes and structural proteins—that is, Exp, endo-b-1, 4-glucanase (EGase), endo-b-1,4-mannase, pectate lyase (PL), pectin methyl esterase (PME), polygalacturonase (PG), and b-galactosidase (b-gal)—are the prime candidates to control softening of ﬂeshy fruits (Huber et al. 2003). Recent work on the effect of 1-MCP treatment on these enzymes is summarized in Table 5.1. In general, 1-MCP treatment delays or decreases transcription, translation, or activity of the enzymes and structural proteins that were investigated. However, as shown in the table, every author measures different enzymes and structural proteins on different fruits and uses different methodology to quantify the effect of 1-MCP. Although all these results have merit, a concerted effort to elucidate more details of the mechanisms is desirable. Some comparisons can be made from the information available.

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Table 5.1. Regulation of cell wall modifying enzymes and structural proteins by 1-MCP. Fruit Avocado

Banana

Source Feng et al. 2000; Jeong et al. 2002; Jeong and Huber 2004 Lohani et al. 2004

Trivedi and Nath 2004 Kiwifruit

Boquete et al. 2004

Mango Nectarine

Sane et al. 2005 Dong et al. 2001; Lurie et al. 2002a

Pear

Hiwasa et al. 2003a Hiwasa et al. 2003b Trinchero et al. 2004

Mwaniki et al. 2005 Persimmon Kubo et al. 2003

Tomato

Itai et al. 2003

Enzyme/ Protein

Transcription

Translation

Activity

EGase PG

# #

Cel1 PG PL PME Exp1

# # # #

a-ara b-gal b-xyl ExpA1 EGase PE PG EGase PG Exp2, 3, 5, 6 a-ara a-gal b-gal b-gluc b-xyl b-gal Cel3 Exp2 PG1 a-ara b-xyl

#

" – # – # #

# # # " –

#

# # #

# # # # # #

A downward arrow (#) indicates a decrease or delay, an upward arrow (") indicates an increase, a dash (–) indicates no effect, and no signal means it was not measured.

Treatment with 1-MCP delays or decreases transcript and protein accumulation of MiExpA1 in mango (Mangifera indica) (Sane et al. 2005) as well as transcript accumulation of DkExp2 (Kubo et al. 2003), MaExp1 (Trivedi and Nath 2004), and PcExp2, 3, 5, 6 (Hiwasa et al. 2003b). Expansin weakens the links between xyloglucan and the cellulose microﬁbrils; suppressing this action would impair the accessibility of the cell wall for the different wall hydrolases (Brummell 2006), making expansin action a key part in cell wall metabolism. PG transcription and enzyme activity also decrease after 1-MCP treatment in a range of fruits (Table 5.1). Nevertheless, even though

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PaPG activity is completely suppressed in avocado for up to 24 days, ﬁrmness still declines nearly 80% (Jeong and Huber 2004). 1-MCP treatment prevents the positive feedback or ethylene-induced increase in enzyme activity, but it also removes negative feedback (i.e., in nectarine the activity of PpPG and PpPE [pectin esterase] is positively regulated, whereas PpEGase is negatively regulated by ethylene) (Lurie et al. 2002a). This can lead to problematic ripening after 1-MCP treatment through impaired control of the normal sequence of cell wall hydrolysis. In nectarines, expression of PpPG and PpPE is inhibited by 1-MCP during storage and expression of PpPG is inhibited during subsequent ripening (Table 5.1). Conversely, transcription of PpEGase is enhanced by 1-MCP whereas in control fruit and fruit treated with ethylene, PpEGase transcription is low throughout the storage and poststorage ripening period (Dong et al. 2001). The imbalance in expression of PpPG and PpPE will result in an imbalance in the softening process. In addition, 1-MCP removes the negative control that ethylene exerts on the expression of PpEGase, subsequently leading to abnormal softening and the occurrence of severe disorders. A possible explanation would be that normal softening involves mainly solubilization of pectins while the cellulose-hemicellulose network stays intact. An increase in PpEGase transcription will result in breakdown of that network. A similar negative feedback mechanism is present in tomato, the expression of Lea-ara (a-L-arabinofuranosidase) in ripe fruit increases after 1-MCP treatment (Itai et al. 2003) whereas that of Leb-xyl (b-D-xylosidase) is not affected (Table 5.1). This negative ethylene feedback may not be present in pears (Hiwasa et al. 2003a), where 1-MCP treatment does not affect the expression pattern of PcEGase genes but softening is delayed concurrent with lower expression of PcPG genes (Table 5.1). Cultivar dependency of the effects of 1-MCP on cell wall–degrading enzymes was illustrated in apricots (Botondi et al. 2003), with a more signiﬁcant softening reduction in ‘Ceccona’ apricots by 1-MCP treatment compared with ‘San Castrese’ apricots through differential effects on the activity of several of the important enzymes (Table 5.2). In ‘Ceccona’ apricots, 1-MCP treatment lowers the activity of a-gal (a-D-galactosidase), b-gal, b-xyl, a-man (a-D-mannosidase), and b-gluc (b-D-glucosidase) and increases the activity of a-gluc (a-D-glucosidase); in ‘San Castrese’ apricots, howevers 1-MCP treatment only has a suppressing effect on PME and a-man, thus making the overall reduction of softening smaller compared with ‘Ceccona’ apricots. These last two examples again stress that although ethylene might be a strong controlling factor in certain processes, it is not necessarily the

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Table 5.2. Enzyme activity of cell wall modifying enzymes after 1-MCP treatment in ‘Ceccona’ and ‘San Castrese’ apricots. Enzyme a-gal a-gluc a-man b-gal b-gluc b-xyl PME

Apricot cultivar —————————————————– Ceccona San Castrese # " # # # # –

– – # – – – #

A downward arrow (#) indicates a decrease or delay, an upward arrow (") indicates an increase, and a dash (–) indicates no effect. Source: Botondi et al. 2003.

only factor, a point also illustrated by research combining 1-MCP treatment and air or controlled atmosphere (CA) storage of apple. A combination of 1-MCP treatment and CA storage is more effective in delaying apple softening compared with 1-MCP treatment and air storage (Watkins et al. 2000; DeLong et al. 2004; Bai et al. 2005), indicating that part of the softening process in apples is not purely ethylene-driven but there are other limiting factors. Research into the control of softening by ethylene has been difﬁcult since the changes that happen during ripening include increases in ethylene; thus, it is difﬁcult to attribute these changes to the general ripening process (developmental) or the mere presence of ethylene. In other words, it is difﬁcult to determine if ethylene is a cause or not. Additionally, the expression of some genes is stimulated while others are suppressed on onset of fruit ripening when ethylene production is induced. 1-MCP has been used extensively to differentiate between nonethylene developmental control and ethylene control. In strawberry, expression of Fab-gal3 is normally high during fruit growth (green fruit), where it allows turgor-driven cell growth and expansion through reversible cell wall loosening; expression of Fab-gal3 is minimal during the later stage of maturation and ripening (Balogh et al. 2005). Thus, logically, 1-MCP treatment only has an effect on Fab-gal3 expression in the green stage (Table 5.3). 1-MCP treatment signiﬁcantly up-regulates Fab-gal3 expression, indicating that it is under negative feedback regulation by ethylene. The opposite is true for FaPL-B, highly expressed during ripening when it cuts b-1,4 linked galacturonosyl residues of pectins from the middle lamella and

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Table 5.3. Effect of 1-MCP treatment on transcript accumulation of cell wall modifying enzymes in strawberry at different maturity stages from immature (green) to ripe (red).

Enzyme ADH b-gal GST PK PL

Maturity stage —————————————————————————————– Green White Pink Red – " – – –

" – – – –

# – # # –

# – # # #

A downward arrow (#) indicates a decrease or delay, an upward arrow (") indicates an increase, and a dash (–) indicates no effect. Source: Balogh et al. 2005.

primary cell wall resulting in the maceration of plant tissue. FaPL-B is ripening-speciﬁc and down-regulated by 1-MCP (Table 5.3) and thus under positive ethylene control (Balogh et al. 2005). Furthermore, FaADH (alcohol dehydrogenase), FaGST (glutathione S-transferase), and FaPK (protein kinase) transcription are ripening-induced and down-regulated by 1-MCP (Table 5.3), indicating ethylene-dependent transcription regulation (Balogh et al. 2005). Different members of the b-gal gene family in ‘La France’ pear fruit are abundant at different stages during fruit development and ripening (Table 5.4), indicating they have different developmental and hormonal regulation characteristics (Mwaniki et al. 2005). PcGAL1 and PcGAL4 are found only in mature fruit and upon onset of fruit ripening initiated by increasing ethylene production; PcGAL6 and PcGAL7 are abundant during fruit growth while transcription decreases during fruit ripening when ethylene concentrations are high. The study of the effect of 1-MCP treatment on the gene expression of these different b-Gal isozymes makes it possible to distinguish them as partly upregulated (PcGAL1 and PcGAL4), partly down-regulated (PcGAL6 and PcGAL7), or not affected (PcGAL2, PcGAL3 and PcGAL5) by 1-MCP and thus ethylene. Additionally, PcGAL3, PcGAL5, PcGAL6, and PcGAL7 are more involved in rapid cell division and/or expansion; PcGAL1 and PcGAL4 are involved in ripening-associated cell wall disassembly; and PcGAL2 is involved in both (Mwaniki et al. 2005). These results again stress that ethylene is not the only factor responsible for cell wall modiﬁcation control; there are other triggers, such as developmental initiation, that control the ethylene dependency (Mwaniki et al. 2005). Similar differential regulation of b-Gal isozymes

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Table 5.4. Effect of 1-MCP treatment on expression of different members of the b-gal gene family (PpGal1–7). b-gal gene number Attribute

1

2

3

4

5

6

7

1-MCP effect Expressed during Fruit expansion Storage Ripening Involved in Rapid cell division or expansion Ripening associated cell wall disassembly

#

–

–

#

–

"

"

– – þ

þ – –

þ – –

– þ þ

þ – –

þ – –

þ – –

– þ

þ þ

þ –

– þ

þ –

þ –

þ –

A downward arrow (#) indicates a decrease or delay, an upward arrow (") indicates an increase, a dash (–) indicates no effect or no involvement, and a plus sign (þ) indicates involvement. Source: Mwaniki et al. 2005.

by ethylene and 1-MCP has been found in avocado (Tateishi et al. 2007) and ‘Charentais’ cantaloupe melon (Cucumis melo var. cantalupensis) (Nishiyama et al. 2007). Although normally ripening is associated with softening and loss of ﬁrmness, this is not always the case. Toughening of harvested green asparagus (Asparagus ofﬁcinalis) due to lignin deposition is reduced (Liu and Jiang 2006) by 1-MCP treatment. In loquat fruit, ﬁrmness tends to increase in storage due to ligniﬁcation. Although this was believed to be a chilling injury symptom in previous studies, Cai et al. (2006b) showed that the increase in ﬁrmness in loquat fruit during ripening is associated with an increase in the lignin content due to increases in activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase, and POX as well as a decrease in cellulose content due to an increase in cellulase (Cel) activity and that the activity of all these enzymes is ethylene-regulated. Treatment with 1-MCP does not completely block the activity of the various enzymes, but 1-MCP considerably slows the increase in activity during normal ripening. The effect of 1-MCP on PAL and POX activity in peach is quite different; PAL activity increases and the normal increase in POX activity is delayed (Liu et al. 2005). Cell wall metabolism not only inﬂuences expansion and softening of fruit, it also plays a role in the abscission of fruits, ﬂowers, and leaves. It can be delayed using 1-MCP treatments, as shown in cherry tomato (Beno et al. 2004), where 1-MCP delays fruit abscission through reduced expression of different EGases (e.g., LeEGase1, LeEGase2, LeEGase3, LeEGase5, LeEGase7, and LeEGase8). In citrus fruit, the

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same effect is not seen, low concentrations of 1-MCP have no effect, and a high concentration results in a signiﬁcant increase in calyx abscission (Cronje et al. 2005). Leaf abscission of unrooted poinsettia cuttings during transport at higher temperatures ( > 18 C) can be reduced by 1-MCP treatment, but this coincides with higher ethylene production, which may cause a problem during longer transport times if the efﬁcacy of 1-MCP decreases over that time (Faust and Lewis 2005). E. Aroma Metabolism Ethylene induces biochemical, physical, and chemical changes resulting in increased protein synthesis and changes in enzyme activity and is considered to be a major trigger in the aroma production process in climacteric fruits (Deﬁlippi et al. 2004, 2005a, b). Ripening-related aroma production starts after the climacteric rise of ethylene production, and typical ‘‘ripe’’ ﬂavor compounds are produced only after ripening has been initiated by ethylene (Tressl et al. 1975). Therefore, an effect of 1-MCP on aroma development is very likely. Use of 1-MCP (Lurie et al. 2002b; Deﬁlippi et al. 2004, 2005b) has helped greatly to identify the physiological mechanisms behind the ethylene regulation of aroma production. During normal ripening of ‘Anna’ apples (Lurie et al. 2002b), the acetate and butyrate esters increase greatly and alcohols and aldehydes decrease. 1-MCP-treated apples retain more alcohols, aldehydes, and b-damascenone volatiles than untreated apples do. This likely explains why volatile production by 1-MCPtreated apples is lower compared with untreated controls (Fan and Mattheis 2001; Kondo et al. 2005) and suggests that ethylene action is needed for adequate volatile production. Results from 1-MCP treatment of fruit indicate ethylene regulation of ester production through ethylene-dependent gene expression and activity of alcohol acyl-coenzyme A transferase in apple (Deﬁlippi et al. 2005b; Li et al. 2006a, b) and ‘Charentais’ melon (Flores et al. 2002; El-Sharkawy et al. 2005). Further research on ‘Charentais’ using 1-MCP treatment to determine ethylene dependence more speciﬁcally indicates that this last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably through differentially regulated alcohol acetyltransferases (Flores et al. 2002). At the same time, no effect is observed on expression levels and activity of ADH (Deﬁlippi et al. 2005b) or lipoxygenase (Deﬁlippi et al. 2004), the latter resulting in only slight differences in the levels of alcohol and aldehyde volatiles. In ‘Ceccona’ apricot, 1-MCP treatment has a slightly different

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W. C. SCHOTSMANS, R. K. PRANGE, AND B. M. BINDER

effect on the volatile proﬁle, reducing the synthesis of lactones and promoting the rise of terpenols (Botondi et al. 2003). The type of esters produced also is affected by 1-MCP treatment, with straight-chain ester production decreasing more than that of branched-chain esters (Mattheis et al. 2005), which is similar to what happens in controlled atmosphere storage (Lopez et al. 2000; Saevels et al. 2004). This indicates a similar control factor for at least part of the biochemical processes, where ethylene and volatile production, enzyme activity levels, and precursor availability (Deﬁlippi et al. 2005a) are differentially affected by ethylene regulation. The repression of ester production by 1-MCP continues for 8 days of poststorage ripening after 14 weeks in cold storage, and the apple aroma does not reach the same level as in control fruit (Li et al. 2006a). This concurs with previous research; 1-MCP treatment of apples almost completely prevents volatile production during shelf life following an 8-week cold-storage period (Xuan and Streif 2005); although aroma production increases after 18 weeks of storage, it does not reach the levels of the controls in air storage. In some cases, 1-MCP treatment can prevent unpleasant ﬂavors and odors that would develop in controlled-atmosphere storage, such as the unpleasant spicy ﬂavor developing in ﬁgs (Ficus carica) after 7 storage days (D’Aquino et al. 2003) or dimethyl trisulﬁde production, resulting in an off odor in broccoli (Brassica oleracea) ﬂorets (Forney et al. 2003) F. Antioxidants As mentioned in previous reviews, research on the effect of 1-MCP treatment on antioxidants is scarce and research into the mechanisms behind these effects is even more so. 1-MCP treatment reduces loss of ascorbic acid in guava (Psidium guajava) fruit (Hassan 2005), mango (Cocozza et al. 2004), minimally processed pineapple (Budu and Joyce 2003) and preserves total water-soluble antioxidant capacity of apple (MacLean et al. 2003). Ascorbic acid and H2O2 levels are not signiﬁcantly different in 1-MCP-treated apples (Larrigaudie`re et al. 2005; Vilaplana et al. 2006) and pears (Larrigaudie`re et al. 2004b, 2005) compared with controls. In pears, the enzymatic antioxidant potential or the activity of the enzymes involved in neutralizing active oxygen species by transforming radicals into H2O2 (by superoxide dismutase [SOD]) and subsequently into water (by catalase [CAT], POX, and ascorbate-POX) is enhanced by 1-MCP treatment (Larrigaudie`re et al. 2004b; Fu et al. 2007), indicating greater resistance to oxidative stress and a direct or indirect relation with

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ethylene. In apple, the activity of SOD is similar for 1-MCP-treated and control fruit; CAT activity of 1-MCP-treated apples increases sharply initially but then slowly decreases to the same level as the control fruit; whereas POX activity increases following 1-MCP treatment and remains higher compared with the control fruit (Larrigaudie`re et al. 2005; Vilaplana et al. 2006). IV. SIDE EFFECTS In this section, we discuss some consequences of the physiological changes brought on by 1-MCP treatment. These include discoloration reactions, physiological disorders, and stress responses that are not directly affected by 1-MCP treatment; however, the processes leading up to development of the disorder are, and the end result is that there is a change in the plants response to certain conditions after 1-MCP treatment.

A. Physiological Disorders 1. Superﬁcial Scald. Superﬁcial scald is characterized by uneven browning of the skin and is caused by the oxidation products of a-farnesene, probably the conjugated trienes or trienols (such as 6-methyl-5-hepten-2-one) that disrupt cell walls and cause browning of the skin (Anet 1972). The rate-limiting enzyme during synthesis of a-farnesene via the mevalonic acid pathway is a-farnesene synthase (Rupasinghe et al. 2000b). The incidence of superﬁcial scald in apples and pears can be reduced or eliminated using 1-MCP through the reduction of accumulation of 6-methyl-5-hepten-2-one (Fan et al. 1999; Rupasinghe et al. 2000a; Watkins et al. 2000) as well as a-farnesene (Fan et al. 1999; Watkins et al. 2000; Argenta et al. 2003; Apollo Arquiza et al. 2005; Haines et al. 2005; Lurie et al. 2005; Isidoro and Almeida 2006) linked to an inhibition of up-regulation of the MdAFS1 (a -farnesene synthase) (Lurie et al. 2005; Pechous et al. 2005) or PcAFS1 gene (Gapper et al. 2006). In other cases, development of superﬁcial scald is not prevented by 1-MCP treatments, but the severity of the symptoms is reduced (i.e., in ‘Conference’ pears), due to lower production of a-farnesene (Rizzolo et al. 2005). 2. Internal Breakdown Disorders. The effect of 1-MCP on the occurrence of physiological disorders varies; there are as many positive as

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negative reports, as summarized in Watkins and Miller (2005). As physiological disorders often are due to multiple factors, the action of 1-MCP on this kind of alterations is complex. As a general feature, when the disorder is related only to senescence, 1-MCP treatment through its action on ethylene action would likely be beneﬁcial. This is the case, for example, for internal breakdown and senescence in pear (Calvo 2003), core browning and senescent breakdown in ‘Redcort Cortland’, ‘Redmax’, and ‘Summerland McIntosh’ apples (DeLong et al. 2004); and other types of internal breakdown related to senescence (Larrigaudie`re et al. 2004a). However, most reports do not offer explanations or identify speciﬁc processes or enzymes affected by 1-MCP. Some do; in ‘Yali’ pears, the inhibition of core browning after 1-MCP treatment is linked to higher activity of antioxidant enzymes (CAT, SOD, and POX) (Fu et al. 2007). Disorders apparently caused by 1-MCP treatment also have been described. One disorder is a brown, necrotic depression of the skin of ‘Granny Smith’ apple (Zanella 2003), likely related to calcium deﬁciency. Another one in ‘Golden Delicious’ apple is described as diffuse skin browning and is related to higher POX activity, is clonedependent, and is related to the sensitivity of the clone to russeting (Larrigaudie`re et al. 2007). B. Stress Responses 1. Chilling Injury. Chilling injury symptoms can be enhanced in banana (Jiang et al. 2004) and reduced in avocado fruit (Hershkovitz et al. 2005), persimmon (Salvador et al. 2006), pineapple (Selvarajah et al. 2001), tangerine (Citrus reticulata) and grapefruit (Citrus paradisi) (Dou et al. 2005) by 1-MCP treatment. In oranges (Citrus sinensis), 1-MCP does not affect chilling injury (Dou et al. 2005). The reduction of chilling injury symptoms by 1-MCP treatment is linked with a reduction in mesocarp discoloration, PPO and POX activities in avocado fruit (Hershkovitz et al. 2005), and with delayed ascorbic acid and soluble solids decline in pineapple (Selvarajah et al. 2001). In avocado fruit, 1-MCP does not reduce external chilling injury (skin blackening) whereas it does inhibit internal chilling injury symptoms (diffuse ﬂesh discoloration), suggesting that ethylene plays a role in the internal manifestation of chilling injury but not in the external symptom development (Woolf et al. 2005). 2. Pathogen Attack. Ethylene signaling is involved in several plant defense-related processes, such as the production of phytoalexins,

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pathogenesis-related proteins, the induction of the phenylpropanoid pathway, and cell wall alterations (Diaz et al. 2002). Ethylene also stimulates the development of necrosis and in many cases the hypersensitive response in tomato (Ciardi et al. 2001; Diaz et al. 2002). The latter is a defense mechanism whereby a rapid necrosis of cells at the site of pathogen infection due to H2O2 accumulation is followed by a local and systemic activation of defense-related genes (Diaz et al. 2002) in order to limit pathogen growth through the plant (Ciardi et al. 2001). It is clear that this rapid cell death is detrimental to the further invasion of biotrophs (parasitic pathogens) but beneﬁcial for the invasion of necrotrophs (pathogens that require dead cells). Thus, depending on the type of pathogen, removing this ethylene-induced defense response by 1-MCP treatment would have different results. In tomato, the key ethylene receptor in this process seems to be LeETR4 (Ciardi et al. 2001). Ethylene also might be involved in the plant’s response to pathogens through the activation of pathogenesis-related proteins. These pathogenesis-related proteins do not defend the plant against infection but will affect pathogen spread and can have very different functions. In pepper (Capsicum annuum) plants, the resistance induced by Fusarium oxysporum f.sp. lycopersici (FOL) has been linked to an increase in chitinase activity as well as an increase in cell wall–bound phenolics and is ethylene-driven. Thus, pepper plants previously infected with FOL and treated with 1-MCP can become infected with Phytophthora capsici, Verticillium dahliae, or Botrytis cinerea whereas FOL-induced resistance normally prevents this (Diaz et al. 2005). Again, this ethylene control is not necessarily through positive feedback; in tomato, the proteinase inhibitor I gene expression is induced by 1-MCP pretreatment, which results in an increased susceptibility to infection by Botrytis cinerea (Diaz et al. 2002). One must be careful when attributing preliminary rotting after 1-MCP treatment to the inhibiting effect of 1-MCP on speciﬁc processes, such as the production of natural antifungal compounds in ripening fruit, without giving actual evidence that these processes are in fact inhibited. For instance, in avocado (Adkins et al. 2005) and banana (Bagnato et al. 2003), 1-MCP treatment doubles the time between harvest and eating ripeness, but also results in a higher percentage of body rots and stem-end rots in avocado (Adkins et al. 2005) and crown rot in banana (Bagnato et al. 2003) before they are ripe. This could be due to differences in antifungal compounds but also to the longer time the rots had to develop since no control fruit were held for the same length of time.

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In strawberries, increased production of CO2 by 1-MCP-treated fruit is associated with the earlier onset of rots (Bower et al. 2003) and comparatively low levels of phenolics are associated with the decreased disease resistance of the fruit (Jiang et al. 2001). In other cases, the 1-MCP effect is obvious, but the mechanism behind it is not identiﬁed. High concentrations of 1-MCP enhance the development of decay in ‘Fallglo’ tangerines, ‘Hamlin’ and ‘Valencia’ oranges, and white ‘Marsh’ grapefruit (Dou et al. 2005) whereas lower concentrations do not. 1-MCP treatment is associated with slightly higher severity of external blemishes in papaya (Carica papaya) and custard apple (Annona squamosa A. cherimola); slightly higher rot severity in avocado, custard apple, and papaya; and at least double the severity of stem rots in mango, relative to fruit not treated with 1-MCP (Hofman et al. 2001). There are also numerous cases where 1-MCP treatment is beneﬁcial and prevents decay. 1-MCP prevents early senescence and decay in pear (Calvo 2003) and reduces decay in tomato (Sun et al. 2003; Guillen et al. 2005), guava (Hassan 2005), and peach (Liu et al. 2005). For the latter, this coincides with enhanced activity of PPO, PAL, and POX in fruit inoculated with Penicillium expansum. PPO is involved in the phenolics pathway and PAL and POX in the lignin pathway; there are only few microorganisms that can break down lignin, making ligniﬁcation a very effective defense strategy. And in some cases, 1-MCP does not affect decay at all; for example, blue and gray mold decay of pears is not altered by 1-MCP treatment (Lafer 2005). From the previous paragraphs, it is clear that the results vary even within the same fruit type, depending on the 1-MCP concentrations used and differences in experimental design. The same variability can be seen for fresh-cut fruit. Microbial counts are generally unaffected by 1-MCP in stored papaya slices or slices prepared at each sampling (Ergun et al. 2006) and in ‘Crispin’ apple slices (Rupasinghe et al. 2005) whereas microbial counts are suppressed in ‘Empire’ apple slices (Rupasinghe et al. 2005). In sliced ‘Gala’ apple, decay development on the cut surface is promoted after 1-MCP treatment (Bai et al. 2004). 3. Wounding. As with other physiological processes, ethylene is not necessarily the only signal to induce stress-related responses. Exposure to 1-MCP decreases the accumulation of phenolic compounds and subsequent tissue discoloration of whole heads or leaves of iceberg lettuce (Saltveit 2004) but does not interfere with the wound-induced increase in phenolic content of the tissue. This fact indicates that wounding and ethylene act independently in the induction of

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phenylpropanoid metabolism and the accumulation of phenolic compounds. V. SUMMARY AND FUTURE RESEARCH NEEDS Ethylene is a naturally occurring growth regulator in plants that plays an important and necessary role in normal plant growth and development. Human intervention in the role of ethylene by blocking its actions through 1-MCP can produce certain desirable horticultural responses, but blocking ethylene action should not be assumed to always be beneﬁcial to plant growth and development. Therefore, it is not surprising in some cases that blocking ethylene action with 1-MCP has a wide array of effects on plant tissue, not all of them beneﬁcial. In many cases, varying observations for different fruits and vegetables or cultivars found in the literature are the result of both the researcher’s approach, leading to differences in the measurement of various compounds, and also of presumed ethylene dependence of certain processes. The vast amount of differing results does not indicate a failure of 1-MCP to work in some cases but reﬂects how little we know regarding the physiology of the materials we work with. With 1-MCP, we have an extremely powerful tool to investigate this physiology and more speciﬁcally to investigate which plant processes are ethylene-mediated and where in the genetic and biochemical pathways this ethylene mediation comes into play. The goal of the research program that resulted in 1-MCP was to ﬁnd a research tool that would help elucidate the role of ethylene in plants. 1-MCP has deservedly been successful in this regard. It will continue to provide the best means, in combination with creative research minds, to illuminate the interplay between ethylene and plant growth and development. We believe that a concerted effort by a number of the laboratories currently performing experiments on 1-MCP would result in a more efﬁcient use of time and other resources. Consequently, our understanding of the working principles of 1-MCP as well as ethylene will improve since every discovery concerning 1-MCP increases our knowledge regarding the physiology of fruit and vegetables.

VI. ACKNOWLEDGMENTS Agriculture and Agri-Food Canada contribution number: 2340. *Wendy Schotsmans was a Canadian Government Laboratories Visiting Fellow

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Trinchero, G.D., G.O. Sozzi, F. Covatta, and A.A. Fraschina. 2004. Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of ‘Bartlett’ pears. Postharvest Biol. Technol. 32:193–204. Trivedi, P.K., and P. Nath. 2004. MaExp1, an ethylene-induced expansin from ripening banana fruit. Plant Sci. 167:1351–1358. Ueda, J., and J. Kato. 1980. Isolation and Identiﬁcation of a senescence-promoting substance from wormwood (Artemisia absinthium L.). Plant Physiol. 66:246–249. Van Zhong, G., and J.K. Burns. 2003. Proﬁling ethylene-regulated gene expression in Arabidopsis thaliana by microarray analysis. Plant Mol. Biol. 53:117–131. Vilaplana, R., M.C. Valentines, P. Toivonen, and C. Larrigaudiere. 2006. Antioxidant potential and peroxidative state of ‘Golden Smoothee’ apples treated with 1-methylcyclopropene. J. Am. Soc. Hort. Sci. 131:104–109. Wang, B.G., W.B. Jiang, H.X. Liu, L. Lin, and J.H. Wang. 2006a. Enhancing the post-harvest qualities of mango fruit by vacuum inﬁltration treatment with 1-methylcyclopropene. J. Hort. Sci. Biotechnol. 81:163–167. Wang, K.L.-C., H. Li, and J.R. Ecker. 2002. Ethylene biosynthesis and signaling networks. Plant Cell 14:131–151. Wang, W., A.E. Hall, R. O’Malley, and A.B. Bleecker. 2003. Canonical histidine kinase activity of the transmitter domain of the ETR1 ethylene receptor from Arabidopsis is not required for signal transmission. PNAS 100:352–357. Wang, W.Y., J.J. Esch, S.H. Shiu, H. Agula, B.M. Binder, C. Chang, S.E. Patterson, and A.B. Bleecker. 2006b. Identiﬁcation of important regions for ethylene binding and signaling in the transmembrane domain of the ETR1 ethylene receptor of Arabidopsis. Plant Cell 18:3429–3442. Wang, Y., and P.P. Kumar. 2007. Characterization of two ethylene receptors PhERS1 and PhETR2 from petunia: PhETR2 regulates timing of anther dehiscence. J. Expt. Bot. 58:533–544. Watkins, C.B. 2006a. 1-methylcyclopropene (1-MCP) based technologies for storage and shelf life extension. Int. J. Postharvest Technol. Innov. 1:62–68. Watkins, C.B., 2006b. The use of 1-methylcyclopropene (1-MCP) on fruits and vegetables. Biotechnol. Adv. 24:389–409. Watkins, C.B., J.F. Nock, and B.D. Whitaker. 2000. Responses of early, mid and late season apple cultivars to postharvest application of 1-methylcyclopropene (1-MCP) under air and controlled atmosphere storage conditions. Postharvest Biol. Technol. 19:17–32. Watkins, C.B., and W.B. Miller. 2005. A summary of physiological processes or disorders in fruits, vegetables and ornamental products that are delayed or decreased, increased, or unaffectedbyapplicationof1-methylcyclopropene(1-MCP).www.hort.cornell.edu/mcp/. Woolf, A.B., C. Requejo Tapia, K.A. Cox, R.C. Jackman, A. Gunson, M.L. Arpaia, and A. White. 2005. 1-MCP reduces physiological storage disorders of ‘Hass’ avocados. Postharvest Biol. Technol. 35:43–60. Xie, C., J.S. Zhang, H.L. Zhou, J. Li, Z.G. Zhang, D.W. Wang, and S.Y. Chen. 2003. Serine/ threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. Plant J. 33:385–393. Xie, F., Q. Liu, and C.K. Wen. 2006. Receptor signal output mediated by the ETR1 N terminus is primarily subfamily I receptor dependent. Plant Physiol. 142:492–508. Xuan, H., and J. Streif. 2005. Effect of 1-MCP on the respiration and ethylene production as well as on the formation of aroma volatiles in ‘Jonagold’ apple during storage. Acta Hort. 682:1203–1210.

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6 Postharvest Biology and Technology of Cucurbits Steven A. Sargent Horticultural Sciences Department University of Florida/IFAS Gainesville, FL 32611 USA Donald N. Maynard Gulf Coast Research & Education Center University of Florida/IFAS Wimauma, FL 33598 USA

I. INTRODUCTION A. Economic Value B. Harvest and Postharvest Technology II. CROPS A. Watermelon (Citrullus lanatus) 1. Nutritional Value 2. Quality Indices 3. Harvest Maturity Indices 4. Handling, Packing, and Storage 5. Postharvest Diseases/Disorders 6. Fresh-Cut and Food Safety B. Cucumber (Cucumis sativus) 1. Nutritional Value 2. Quality Indices 3. Harvest Maturity Indices 4. Handling, Packing, and Storage 5. Postharvest Diseases/Disorders 6. Fresh-Cut and Food Safety C. Melon (Cucumis melo) 1. Nutritional Value 2. Quality Indices 3. Harvest Maturity Indices 4. Handling, Packing, and Storage Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 315

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5. Postharvest Diseases/Disorders 6. Fresh-cut and Food Safety D. Squash and Pumpkin (Cucurbita maxima, C. moschata, and C. pepo) 1. Nutritional Value 2. Quality Indices 3. Harvest Maturity Indices 4. Handling, Packing, and Storage 5. Postharvest Diseases/Disorders 6. Fresh-Cut and Food Safety E. Chayote (Sechium edule) F. Wax Gourd (Benincasa hispida) G. Bitter Melon (Momordica charantia) H. Smooth Luffa (Luffa aegyptiaca), Angled Luffa (Luffa acutangula) III. CONCLUSIONS IV. LITERATURE CITED

I. INTRODUCTION The Cucurbitaceae, or gourd, family includes several crops of signiﬁcant economic and nutritional importance. Cultivated species originated from the subtropical and tropical Americas, Africa, and Asia. Cucurbits are mainly herbaceous and frost-sensitive annuals, having a vining growth habit with tendrils. The usually yellow or white ﬂowers are monoecious, and the pistillate ﬂowers have an inferior ovary containing three carpels that form a single to multiseeded, ﬂeshy berry called a pepo (Anon. 1976). Water is the principal component of fresh cucurbit fruit, and the moisture content of mesocarp tissue varies from 86% to 96%. This review focuses on the postharvest biology and technology of edible cucurbits. A. Economic Value Cucurbits account for signiﬁcant production worldwide. China is the principal producing country, followed by Turkey, India, and Iran. The Ukraine, United States, Spain, and Egypt are other major cucurbit producers (Table 6.1). Watermelon is the most economically important cucurbit crop grown in the United States, with a total production value of $433.4 million grown on 57,911 ha in 2006 (Table 6.2). Watermelon production rose by 14.1% from 2004 to 2006, whereas muskmelon, cucumber, and honeydew production remained relatively stable (U.S. Dept. of Agriculture [USDA] 2007c). Watermelon also had the highest per-capita consumption (6.4 kg), followed by muskmelon (4.6 kg) (Table 6.2). Cucumber had the highest value of

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Table 6.1. Principal cucurbit producing (Mt) countries, 2005. Rank Crop

First

Second

Third

Fourth

Fifth

Cucumberz Melony Pumpkinx Watermelon

China China China China

Turkey Turkey India Turkey

Iran Iran Ukraine Iran

Russia Spain USA USA

USA USA Egypt Egypt

z

Includes gherkins. Includes muskmelon. x Includes squash. Source: FAOSTAT 2007. y

these crops, averaging $0.11/kg, 38% higher that either muskmelon or honeydew and 120% higher than watermelon. In 2004, the United States imported over $280 million in melon crops, and exports totaled just under $100 million (Boriss et al. 2006). Mexico furnishes most watermelon and other melons, while Guatemala and Costa Rica supply most imported muskmelon. Canada imports almost all melons exported from the United States. Imports of cucumbers for pickles has increased from 1% of total consumption in 1990 to 11% in 2006 with India accounting for about half of the imported product (Lucier and Jerardo 2007). Several cucurbits contribute signiﬁcantly to human health, including watermelon, muskmelon, and orange-ﬂeshed squash and pumpkins. Thorough compilations of the nutritional composition of selected cucurbits are available (FAO 2007; USDA 2007b). Since the late 1980s, increasing consumer demand for convenient, nutritious, and safe foods has driven a rapid rise in the amount of fresh produce that is Table 6.2. U.S. production, value and per capita consumption of selected cucurbit crops 2006.

Crop Watermelon Muskmelon Cucumber Honeydew Squash

Harvested area (ha)

Total production (t)

57,911 36,300 22,237 9,227

1,908,399 897,024 449,873 228,520 54,295z

Total production value (millions $)

Average price ($/kg)

Per capita consumption (kg)

433.4 340.1 249.9 90.7

0.05 0.08 0.11 0.08

6.4 4.6 2.9 0.9

z Squash calculated from total reported domestic shipments; other data unavailable. Source: Adapted from USDA 2007c.

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processed into fresh-cut products in ready-to-eat portions. In the United States, more than 10% of all fresh produce currently is processed into some form of fresh-cut product, amounting to sales of over $12 billion (USDA 2007). Results of a consumer survey reported that 75% of respondents considered themselves regular purchasers of fresh-cut produce, buying an item at least once per month. Fresh-cut cucurbits in order of usage are watermelon, melon, squash, and cucumber (The Packer 2002). B. Harvest and Postharvest Technology Most cucurbits are hand-harvested because fruit are produced over time on trailing vines that require sequential harvests for maximum yields. Cucumbers for pickling are the exception; modern hybrids have been developed to produce high yields for once–over mechanical harvest. Still, many pickling cucumbers are hand-harvested or employ harvesting aids of various farm-built design. Harvesting aids usually consisting of moving belts on either side of a central self-propelled vehicle with facilities for grading and packing are commonly used for many cucurbits that require multiple harvests. Most harvesting aids are farm or locally produced to the speciﬁcations of individual growers. Cucurbits other than winter squash, pumpkin, and watermelons are quite perishable and require cooling shortly after harvest to maintain quality (Kader 2002). Recommended cooling methods for some cucurbits are shown in Table 6.3. Storage life for the most perishable cucurbits is as short as a week to several months for pumpkins and

Table 6.3. Cooling methods for cucurbits. Cucurbit Cucumber Melon Casaba, crenshaw, honeydew Muskmelon Squash Summer Winter Watermelon

Cooling methodz R, FA, FA-EC FA, R HC, FA, PI R, FA, FA-EC R R, FA, HC

z FA ¼ Forced air cooling, FA-EC ¼ Forced air evaporative cooling, HC ¼ Hydrocooling, R ¼ Room cooling, PI ¼ Package icing Source: Kader 2002.

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Table 6.4. Recommended storage conditions and storage life of cucurbits. Storage conditions

Cucurbit Bitter melon Chayote Cucumber, slicing Cucumber, pickling Luffa Melon (see Table 6.11) Pumpkin Squash, summer Squash, winter Tropical pumpkin Watermelon

Temperature ( C)

Relative humidity (%)

Approximate storage life

10–12 7 10–12 4 10–12

85–90 85–90 85–90 95–100 90–95

2–3 weeks 4–6 weeks 10–14 days 7 days 1–2 weeks

12–15 7–10 12–15 10–13 10–15

50–70 95 50–70 50–70 90

2–3 months 1–2 weeks 2–3 months 2–3 months 2–3 weeks

Source: Kader 2002.

winter squash with proper temperature and humidity management (Table 6.4). To retard ripening, the ethylene action inhibitor 1-methylcyclopropene (1-MCP) shows promise to signiﬁcantly reduce postharvest losses for many horticultural crops, including most cucurbits (Blankenship and Dole 2003; Watkins 2006; Huber 2008). Gaseous or liquid forms of 1-MCP applied pre- or postharvest extend quality by retarding ripening in climacteric fruits and by protecting nonclimacteric fruits from ethylene-induced senescence. However, several factors impact the efﬁcacy and practicality of commercial use of 1-MCP, including concentration and time/temperature of exposure, stage of maturation, and cultivar. 1-MCP will continue to be applied to more crops and value-added products as protocols are developed for these factors in conjunction with other postharvest technologies.

II. CROPS This section reviews the nutritional, quality, and handling aspects of the principal cucurbit crops grown worldwide. A. Watermelon (Citrullus lanatus) Watermelon is native to southern Africa, with its center of origin in present-day Botswana. Wild types, often with bitter ﬂesh, still can be

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found in that area, and the fruit frequently serves as a ‘‘living canteen’’ in times of prolonged drought. Watermelon plants are monoecious annuals with long trailing thin and angular vines that bear branched tendrils and lobed leaves. Watermelon ﬂowers, which are smaller and less showy than those of many other cucurbits, are borne solitary in leaf axils and remain open for only one day. Staminate ﬂowers appear ﬁrst and outnumber pistillate ﬂowers by about 7:1. Pollination is effected mostly by honeybees (Wehner 1996). Fruit vary in weight from 1 kg to over 100 kg, but market types are usually between 2 and 14 kg. Fruit shape is round to elongated, and rind color is light to dark green, often with a typical striping pattern that identiﬁes the cultivar or type. The majority of watermelons sold have dark pink to deep-red ﬂesh, but there is a niche market for yellow and orange-ﬂeshed watermelons (Maynard 2001). China is the leading watermelon-producing country with harvested area of more than 2 million hectares (Table 6.5). In comparison, Turkey, which is the second most important watermelon-producing country, had only 137,000 ha in 2005. Other important producers of watermelons are the Russian Federation, Iran, Brazil, Egypt, and the United States (FAOSTAT 2007). In recent years, the availability of seedless (triploid) watermelon cultivars has changed consumer preferences in the United States so that now over 90% of watermelons sold are seedless and command a higher price than seeded watermelons (Maynard et al. 2007; USDA 2007a). Seedless watermelons are available in all types, and smallersize fruit with thin rinds are increasing in popularity. The decreased demand for seeded watermelons has led to the release of special pollenizer watermelon cultivars to increase triploid fruit set. Although

Table 6.5. Principal watermelon producing countries, 2005. Country China Turkey Russian Federation Iran Brazil Egypt United States NA: Data unavailable. Source: FAOSTAT 2007.

Harvested area (1,000 ha) 2,014.5 137.0 112.0 100.0 75.0 62.0 55.2

Production (million t) 69.3 3.8 NA 2.2 NA 1.5 1.7

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more expensive than standard hybrids, the pollenizer cultivar is planted in-row and eliminates the need for a second diploid cultivar, thus increasing the population of triploid plants by up to one-third (Freeman and Olson 2007). 1. Nutritional Value. Watermelon supplies signiﬁcant nutrition, being an excellent source of vitamins A and C. Watermelon is a rich natural source of the nonnutritive phytochemical citrulline. Citrulline, a nonessential amino acid, is found in many of the cucurbits but is highest in watermelon, where it was ﬁrst discovered. Citrulline is present in all parts of the watermelon and is highest in the ﬂesh on a fresh weight basis (0.5 to 3.7 mg/g) (Rimando and Perkins-Veazie 2005). Citrulline is a component of the nitric oxide system in humans and acts as a vasodilator, improving the oxygen-holding capacity of blood. Citrulline may prove to be useful in cardiovascular health and control of obesity (Wu et al. 2007; Curis et al. 2005). Until recently, lycopene, a red pigment, was thought only to be an important contributor to visual quality. However, it is now known to have the highest singlet oxygen-quenching capacity in vitro among the common dietary carotenoids. Inverse relationships have been reported between lycopene intake and incidences of cancers of the prostate, pancreas, and to a certain extent stomach. In some studies, lycopene was the only carotenoid associated with risk reduction. Lycopene also may help prevent cardiovascular disease, through anti-inﬂammatory activity and prevention of foam cell formation (Collins and PerkinsVeazie, 2006; Raﬁ et al. 2007). Although watermelon contains approximately 30% more lycopene than tomato, the latter plays a larger role in cancer risk reduction due to higher per-capita consumption, 74.8 kg, compared to 6.4 kg for watermelons (Clinton and Giovannucci 1998; Maynard 2001). Red-ﬂeshed watermelon fruit contained 4,000 to 12; 000 mg lycopene / 100 g fresh weight, whereas orange-ﬂeshed and yellow-ﬂeshed fruit contained 370 to 420 mg/100 g and 10 to 80 mg/100 g fresh weight, respectively (Perkins-Veazie et al. 2002b; Perkins-Veazie et al., 2006a; USDA 2007b). Within the red-ﬂeshed types, fruit of diploid hybrid (seeded) cultivars generally had higher lycopene concentrations than fruit of diploid open-pollinated (seeded) cultivars. Triploid (seedless) cultivar fruit had lycopene concentrations equal to or higher than those in fruit of diploid hybrid (seeded) cultivars. Seeded and seedless miniwatermelons (2.6–3.4 kg) were higher in lycopene than large-type watermelons (Perkins-Veazie et al. 2006a). In a larger test, 18 miniwatermelon cultivars were grown in North Carolina, South Carolina,

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north and south Florida production areas in the eastern United States. Lycopene contents were consistently 20% to 60% higher for watermelons grown in Florida, possibly due to lower number of fruit/plant or to higher nitrogen application rates that favored vine growth (PerkinsVeazie et al. 2006b). Fruit maturity also affects lycopene content. At peak ripeness, fruit had higher lycopene concentrations than unripe (7 days preripe) or overripe (7 days postripe) fruit (Perkins-Veazie et al. 2002a, 2006c). Minimally processed watermelon fruit lost about 10% of its lycopene after 7 or 10 days storage at 2 C (Perkins-Veazie et al. 2002a). 2. Quality Indices. U.S. Fancy grade watermelon must have ‘‘very good internal quality,’’ deﬁned as greater than 10% soluble solids content, be properly shaped and mature according to type, and be free from damage caused by sunburn, whiteheart, hollowheart, decay, or bruises. Seedless types may not have more than 10 mature seeds exposed on eight cut surfaces after a lengthwise and a crosswise cut (USDA 2006). 3. Harvest Maturity Indices. Watermelon reaches harvest maturity from 75 to 95 days after planting, depending on the cultivar and local growing conditions (Maynard and Hochmuth 2007). Since there is no abscission zone at the stem end, the harvest crew uses an external indicator of minimal ripeness, namely when the epidermal groundspot becomes light yellow (Pratt 1971). Soluble solids content, analyzed destructively from ﬂesh samples, varies from 8.1% for unripe miniwatermelon, increasing to 11.1% when ripe (PerkinsVeazie et al. 2006b). Other indicators of ripeness have been compiled (Table 6.6). 4. Handling, Packing, and Storage. Watermelons often are harvested onto trailers or gondolas for transport to a central location, where they are graded and manually transferred into plastic or corrugated pallet bins for direct shipment to the buyer. Often these same pallet bins are displayed on the supermarket ﬂoor. They also may be packed in the ﬁeld and shipped in bulk directly to buyers. Watermelons from the Caribbean area are packed in corrugated cartons that are palletized for shipment. Watermelon is classiﬁed as a nonclimacteric fruit; at 20 C, the respiration rate is low (6–9 mg/kg-hr) and ethylene production is extremely low (<1:0 mL/kg-hr) (Elkashif et al. 1989). Therefore, unless pulp temperatures are extremely elevated at harvest, the fruit is roomcooled to 10 to 15 C, with an anticipated storage life of 14 to 21 days.

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Table 6.6. Subjective and objective indicators of watermelon maturity. Index Fruit maturity Tendril appearance Abscission zone Epidermis Mesocarp indicators Color

External indicators 30–35 days from pollination Tendril closest to fruit is dried None According to type; yellow groundspot

Undermature is pink in the locule ﬁrst, then pink all over; overmature has orange cast; color expands into the rind (rind looks thinner). Lycopene Undermature has 20–50% less, depending on cultivar and days from full maturity; overmature will have slightly more, the same, or 10% less. Soluble solids content Difﬁcult to separate mature from overmature; sometimes higher SSC in locule compared to heart in slightly undermature; locations are equal in mature; heart slightly higher in overmature. 9.8 Brix (‘Millionaire’; oval seedless) 10.8 Brix (‘Xite’; mini, seedless) pH Increases slightly from undermature to overmature. Texture (ﬁnger/mouth feel) Mealy in overmature; slimy in very overmature. Ripe fruit may be crunchy or crisp, depending on cultivar. Flavor (taste) Undermature has a ‘‘green’’ or cucumber-like ﬂavor; overmature has pumpkin ﬂavor.

Source: Adapted from Pratt 1971; Rubatsky and Yamaguchi 1997; Perkins-Veazie et al. 2006a; Maynard 2001.

Sugar concentration in placental tissue decreased during storage of ‘Charleston Gray’ watermelon as a function of increased storage temperature (Chisholm and Picha 1986). Exposure to ethylene gas during storage or transport accelerates senescent processes in nonclimacteric cucurbits. Placental tissue of ripe watermelon had severe breakdown (watersoaking) when exposed to extremely low concentrations of ethylene (1 mL/L) (Risse and Hatton 1982). Elkashif and Huber (1988) reported two- to threefold increases in watermelon electrolyte leakage rates and greater breakdown in cell wall ultrastructure after 6 days exposure to 50 mL/L ethylene at 18 C. They later noted that this same ethylene exposure accelerated placental senescence in both immature and mature watermelons (Elkashif et al. 1989). However, a prestorage treatment of ripe watermelon to the ethylene-action inhibitor 1-methylcyclopropene (1-MCP: 18 hr; 5 mL/L) completely arrested the development of watersoaking during 3-week subsequent exposure to 50 mL/L ethylene at 13 C (Mao et al. 2006).

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Chilling injury symptoms, notably pitting on the epidermis, are induced by storage below 10 to 15 C. When small, round watermelons were subjected to a 3-day/26 C preconditioning treatment prior to storage at 1 C, fewer chilling injury symptoms developed than those not receiving the pretreatment (Risse et al. 1990). Intact watermelons held at 5 C showed a slight loss in lycopene, indicating a possible chilling injury symptom. In contrast, watermelon held at room temperature (20 –21 C) gained in lycopene and b-carotene (PerkinsVeazie and Collins 2006). 5. Postharvest Diseases/Disorders. Watermelon is susceptible to a number of plant pathogens that cause postharvest decays during storage and shipping (Table 6.7). Important disorders induced during production are blossom-end rot caused by calcium deﬁciency and misshaped fruit due to poor pollination. These and other watermelon fruit defects have been reviewed by Maynard and Hopkins (1999). 6. Fresh-Cut and Food Safety. Fresh-cut watermelon has a porous texture, making it difﬁcult to rinse after processing, and shelf life is typically 3 to 4 days under commercial conditions. Therefore, wholefruit sanitation and cooling to 5 C prior to and during processing were found to be critical to signiﬁcantly reduce microbial growth during subsequent storage at 3 C (Durigan et al. 1996). Sliced pieces stored Table 6.7. Common postharvest decays and causal organisms of cucurbits. Common name Alternaria rot Anthracrose Bacterial soft rot Bacterial fruit rot Belly rot Black rot Blue mold Choanephora rot Cottony leak Fusarium rot Phytophthora rot Rhizopus rot Scab Sclerotinia rot Sour rot (waxy rot) Southern blight

Organism Alternaria alternata Colletorichum orbiculare Erwinia caratovora Acidovorax avenae subsp. citrulli Rhizoctonia solani Didymella bryoniae Penicillium spp. Choanephora cucurbitarum Pythium spp. Fusarium spp. Phytophthora capsici Rhizopus stolonifer Cladosporim cucumerinum Sclerotinia sclerotiorum Geotrichum candidum Sclerotinia rolfsii

Commonly affected crops Cucurbits Cucurbits Cucurbits Watermelon, melon Cucumber Winter squash Cucumber, melon Summer squash Cucurbits Cucurbits Cucurbits Cucurbits Cucurbits Cucurbits Cucurbits Cucurbits

Source: Adapted from Snowdon (1992); Zitter, et al. (1996); Hopkins et al. (2000).

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at 3 C had less juice leakage than those stored at 1 C or 5 C, and utilization of these procedures extended postharvest life to 8 days. Cultivars may vary in the proportion of usable product for fresh cut. Cartaxo (1998) determined that storage at 3 C and a controlled atmosphere of 5% O2 and 10% CO2 extended postharvest life to 15 days by controlling microbial growth and retaining selected quality parameters (overall appearance, ﬂavor, and mouth feel). Fonseca et al. (2004) reported that the respiration rate of fresh-cut watermelon increased during storage at <14% O2 concentration, indicating the tissue was stressed. In contrast to the beneﬁcial effect of 1-MCP on maintaining cellular integrity and ﬁrmness of whole fruit cited earlier, 1-MCP (10 mL/L) applied prior to processing of fresh-cut watermelon did not maintain ﬁrmness during subsequent storage (Mao et al. 2006). A rigorous plan incorporating Good Agricultural Practices and Best Management Practices is essential in minimizing the risk of product contamination by human pathogens or noxious materials during harvest and handling operations (USDA). Cross-contamination during handling or process operations must be avoided since pathogens survive well in moist tissues. It was reported that Campylobacter jejuni survived at least 6 hr on the surfaces of cut watermelon held at room temperature, conditions similar to those found on salad bars (Castillo and Escartin 1994). B. Cucumber (Cucumis sativus) Cucumber originated in the Indian subcontinent, with secondary centers of diversity in China and the Near East, where wild types still can be found. It was probably domesticated in Asia and is cultivated in many diverse forms. The ﬁrst horticultural types were selected in the 1700s following introduction into Europe. They were brought to the Americas by Christopher Columbus, and Native Americans were growing cucumbers from Florida to Canada by the early 16th century. (Whitaker and Davis 1962; Robinson and Decker-Walters 1997; Maynard and Maynard 2000). There are currently ﬁve horticultural types of cucumbers grown in the ﬁeld or under protected culture. Two primary ﬁeld types are slicing and pickling cucumbers, although production under protected culture is on the rise in Mexico. European (Dutch or English) cucumber is the traditional greenhouse-grown type, while in recent years production of minicucumber and Beit Alpha types has increased, mainly under protected culture. Minicucumber cultivars originate from Dutch or other seed sources, while Beit Alpha cultivars originate

326

S. A. SARGENT AND D. N. MAYNARD Table 6.8. Principal cucumber/gherkin producing countries, 2005. Country China Turkey Cameroon Russian Federation Iran United States

Harvested area (1,000 ha)

Production (million t)

1,553.1 NA 100.0 87.0 80.0 69.2

26.6 1.7 NA 1.3 1.4 1.0

NA: Data unavailable. Source: FAOSTAT 2007.

from Israeli seed sources (Shaw et al. 2007). Fruit morphology serves to distinguish among these types. Slicing cucumber fruit are seeded, white-spined, smooth, cylindrical with tapered ends, and have a length:diameter ratio of about 4.0. Pickling cucumbers are also seeded, may have black or white spines, have a somewhat warty appearance, and have a length:diameter ratio of 3.0. European, minicucumber, and Beit Alpha types are parthenocarpic, gynoecious, and seedless. China is the most important cucumber- and gherkin- (pickling cucumber) producing country with more than 1.5 million ha in cultivation (Table 6.8). Other important cucumber/gherkin-producing countries in 2005 were Turkey, Cameroon, Russian Federation, Iran, and the United States (FAOSTAT 2007). 1. Nutritional Value. Cucumber is 96% water and contains relatively low amounts of vitamin A and C (FAO 2007; USDA 2007b). Accordingly, fresh and pickled cucumbers are prized for their crisp texture and cooling effect rather than for their nutritional attributes. 2. Quality Indices. Cucumber fresh quality standards are deﬁned for both packing and receiving points, detailing grade requirements based on freedom from defects, shape, length, and diameter (USDA 1958). At least one major cucurbit breeding program has incorporated postharvest quality characteristics such as weight loss, ﬁrmness, appearance, ﬂavor, and carpel separation into evaluation of cultigens (Wehner et al. 2000). Standards for pickling cucumbers (USDA 1936) specify dimensions and freedom from damage, decay, and disease. 3. Harvest Maturity Indices. Cucumber is prized for crisp texture and juicy mesocarp and is therefore harvested immature, being selected by

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size and shape. Following fruit set, pickling cucumbers are ready for harvest after about 5 days (Peirce 1987), slicing cucumbers after 15 to 18 days (Maynard and Hochmuth 2007), European after 10 to 12 days (Hochmuth 2001), and minicucumber/Beit Alpha after 4 to 6 days (Shaw et al. 2007). Days from pollination to fruit harvest are temperature dependent. In the United States, both ﬁeld-grown and greenhouse-grown (European) cucumbers are harvested according to similar grade standards that specify acceptable shape, color, ﬁrmness, and freedom from several defects. Field-grown slicing cucumber must have a minimum length of 15.2 cm and must not exceed a maximum diameter of 6.0 cm (USDA 1958); the diameter of pickling cucumber ranges from 2.7 to 5.1 cm depending on use (USDA 1936; Peirce 1987). European greenhouse cucumber has a minimal length requirement of 28 cm and has a pack standard that requires individual fruit to be enclosed in shrink-wrap ﬁlm (USDA 1985b). Although no ofﬁcial U.S. grade standards exist for minicucumber and Beit Alpha cucumber, expectations of buyers and consumers relate to typical features of these types: dark green color, ﬁrm, and narrow diameter which is generally 2.0 to 4.0 cm. Both types are clipped at harvest leaving a very short peduncle. Twelve minicucumber and Beit Alpha cultivars were grown under protected culture in the mild/humid winter climate of north Florida; those with the highest yield and best postharvest storage quality were resistant to powdery mildew: ‘Manar’, ‘Alamir’, ‘LD CB845’, and ‘General’ (Hochmuth et al. 2004). Grown under similar conditions, those with the best production characteristics were ‘Manar’, ‘Sarawat’, and ‘Tornac’ (Shaw et al. 2007). 4. Handling, Packing, and Storage. Following harvest into ﬁeld bins, slicing and pickling cucumber types for fresh market are washed, graded, mechanically sized, and waxed on packing lines. Greenhousegrown types typically are packed unwashed. European cucumber is graded and shrink-wrapped prior to packing, while minicucumber and Beit Alpha types are graded and packed directly into shipping containers. Cucumber stores well up to 14 days at 10 to 12 C and 85% to 90% relative humidity. It is classiﬁed as a nonclimacteric fruit according to respiratory activity. Respiration rates for slicing cucumber and Beit Alpha cucumber have been reported at 23 to 29 mg/kg-hr and 4 to 9 mg/kg-hr at 10 C, respectively (Saltveit 2004: Villalta and Sargent 2004). Moisture loss is of concern during handling since immature cucumber fruit have underdeveloped cuticles. In the United States,

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slicing cucumbers typically are coated with edible wax and hydrocooled prior to packing, while European greenhouse cucumbers are covered with shrink-wrap plastic ﬁlm to control moisture loss and stem-end shriveling. In contrast, minicucumber and Beit Alpha cucumber are less susceptible to weight loss than the European cucumbers and are not shrink-wrapped or waxed (Lamb et al. 2001). Stapleton et al. (2002a) reported ‘Alexander’ Beit Alpha cucumber had good quality following 14 days storage at 10 , 12.5 , or 15 C without waxing or plastic overwrap, although fruit treated by either method had less weight loss. After 21 days storage, unwaxed fruit from all temperatures were reported to have ‘‘grassy’’ off ﬂavor, and waxed fruit stored at 15 C developed bitterness in the locular tissue. Medium-size ‘Sarig’ and ‘Alexander’ Beit Alpha cucumber (2.9–3.5 cm) stored for 14 days at 10 C were ﬁrmer and had less weight loss than large-size, 3.5to 4.1-cm fruit (Stapleton et al. 2002b). Slicing and pickling types beneﬁt from storage at low oxygen concentrations ð <4%Þ, but no beneﬁt has been reported for the other types (Saltveit 2004). Several postharvest physical stresses shorten the postharvest life of cucumber. Minicucumber stored at either 5 or 7.5 C developed visible chilling injury symptoms after 7 and 14 days, respectively, while those stored at 12.5 C progressively developed epidermal protuberances between 14 and 21 days (Villalta et al. 2003). Tolerance to chilling injury was reported to be maternally inherited and most likely associated with the chloroplast genome (Chung et al. 2003). Kang et al. (2002) reported that cucumber grown in a greenhouse with an average daytime temperature of 32 C and subsequently stored at 10 C was more resistant to chilling injury and remained ﬁrmer than when grown at 27 C. Tissues stressed by physical impacts during harvest and handling rapidly develop watersoaking. Transmission electron microscopy revealed cellular disorganization in bruised cucumber tissue after 48 hr of storage at 23 C, as starch granules disappeared and dense inclusions in chloroplasts appeared (Abbott, et al. 1991). A positive correlation between bruise severity and peroxidase activity was reported for pickling cucumbers (Miller and Kelly 1989). Stress from storage in elevated CO2 (60% at 25 C for 24 hr) stimulated spikes in respiration and ethylene production of slicing cucumber that was attributed to increased peroxidase activity (Mathooko et al. 1995). Cucumbers produce very minute amounts of ethylene (< 1.9 mL/kg-hr at 20 C; Saltveit 2004; Villalta 2005) and are very sensitive to ethylene. A 12-hr exposure of minicucumber (‘Manar’) to 25 mL/L ethylene at 12 C induced necrotic lesions and decay within 72 hr (Sargent et al. 2002). In

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a later study, exposure to 10, 5, or 1 mL/L ethylene at 10 C caused premature softening after 6, 9, and 12 days, respectively (Villalta and Sargent 2004). However, pretreatment of slicing and/or Beit Alpha-type cucumber with gaseous (Lima et al. 2005) or liquid (Alleoni et al. 2006) forms of 1-MCP (100 ZL/L) maintained tissue integrity during constant exposure to 100 mL/L ethylene up to 8 days. 5. Postharvest Diseases/Disorders. Cucumber is susceptible to several postharvest diseases that are summarized in Table 6.7. Disease resistance was reported to be imparted by the addition of silicon to the nutrient solution in greenhouse-grown cucumbers in British Columbia (Samuels et al. 1993). However, excess silica that accumulated in trichomes and epicuticular wax resulted in a noticeably rougher epidermis and duller appearance. Despite these slightly negative effects, this practice has been widely adopted in that industry. Fruit quality is affected by several nutritional disorders during growth and development. Under severe conditions, calcium deﬁciency appears as water-soaked lesions in the blossom end of the fruit that later are expressed as blossom-end rot (Ho and Adams 1994). Carpel separation from mesocarp tissue forms air cavities at the fruit stem end (Frost and Kretchman 1989). Water stress conditions in the ﬁeld cause fresh pickling cucumbers to develop pillowy fruit disorder, characterized by white, porous areas in mesocarp tissue that appear as water-soaked regions following fresh-pack pickling process (Thomas and Staub 1992). Boron deﬁciency symptoms in minicucumber cause small fruits to abort; symptoms on older fruits include stunted and curved growth and ‘‘mottled-yellow longitudinal streaks which develop into corky markings (scurﬁng) along the skin’’; these symptoms are most severe at the blossom end (Cresswell and James 1998). Phosphorus deﬁciency in European seedless cucumbers caused cell membranes to become more permeable, increasing electrolyte leakage and inducing a stress-related burst in respiration, reducing postharvest life (Knowles et al. 2001). Light intensity and spectral quality are correlated to fruit quality of European seedless cucumbers grown in greenhouses, particularly as encountered in northern climates during winter months. Poor light quality limited chlorophyll synthesis, which hastened the onset of yellowing during postharvest handling (Lin and Jolliffe 1996). 6. Fresh-Cut and Food Safety. Pathogenic Listeria and Salmonella species have been isolated from samples of raw cucumber (Beuchat 1995). Therefore, avoidance of contamination of fresh cucumbers

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must be a primary concern during production, harvest, handling, and processing operations. C. Melon (Cucumis melo) There are three principal groups of commercial melons: Cantalupensis, Reticulatus, and Inodorus (Anon. 1976; Robinson and Decker-Walters 1997; Rubatzky and Yamaguchi 1997; Goldman 2002). Each group has many types and cultivars; heirloom cultivars offer an array of unique types within each group (Goldman 2002). Melons of the Cantalupensis group are popular in France but are seldom found commercially in the United States. Fruit are generally round with prominent ribs and sutures; netting if any is scarce. Most are orange-ﬂeshed and aromatic and do not abscise from the vine when ripe. ‘Charentais’ is a popular smooth type, but there are warty types, such as ‘Prescott Fond Blanc’. The Reticulatus type is the commonly cultivated melon found in the United States. Fruit are round or oval, tan or straw-colored, and usually weigh 1 to 3 kg. They are typically well netted with a musky odor, and are orange-ﬂeshed. The types grown in the western United States are round and usually unribbed, and those grown in the East may have pronounced ribbing. Fruits abscise from the vines when mature and are properly called muskmelon and Persian melons. The term ‘‘cantaloupe’’ often is used commercially in the United States but is horticulturally incorrect. Various hybrid melons, such as the green-ﬂeshed ‘Galia’, are often considered in this group. The Inodorus group are referred to as winter melons because the hard rinds helps preserve them until winter arrives. They include Honeydew, Crenshaw, Casaba, and Juan Canary types. As their name indicates, they lack fragrance but are very sweet. Melons in the Inodorus group also are andromonoecious, and fruit are round to oval, white to yellow at maturity, smooth or wrinkled and not netted. Fruit usually weigh 2 to 4 kg, and most do not slip at maturity (Robinson and Decker-Walters 1997; Maynard and Maynard 2000), although many of the newer hybrids do slip at harvest maturity. Melon is thought to have originated in southern Africa where landraces abound. However, diverse local populations are also found in India and the Middle East, suggesting that these areas may be centers of origin as well. Sweet melons were ﬁrst noted in Europe in the 15th century following widespread dispersal throughout Asia, Africa, and the Middle East, after which Christopher Columbus brought melon to the Americas.

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Table 6.9. Principal muskmelon/other melon producing countries, 2005. Country China Turkey Iran United States Spain Romania

Harvested area (1,000 ha)

Production (million t)

578.5 103.0 80.0 44.7 NA 36.0

15.1 1.7 1.2 1.2 1.2 NA

NA: Data unavailable. Source: FAOSTAT 2007.

China is now the leading melon-producing country with over 15 million tonnes from 578,000 ha (Table 6.9). Other important melonproducing countries in 2005 were Turkey, Iran, the United States, Spain, and Romania (FAOSTAT 2007). 1. Nutritional Value. Melons provide pharmacological levels of vitamin C and b carotene as well as folic acid and potassium (Lester and Crosby 2002; USDA 2007b). Differences in b carotene and phenolic concentrations were reported between a netted muskmelon cultivar (‘Cruiser’) and a nonnetted orange-ﬂeshed muskmelon cultivar (‘Orange Dew’), with the nonnetted cultivar having higher concentrations and, therefore, more antioxidant value (Hodges and Lester 2006). 2. Quality Indices. Soluble solids concentration varies in melons: muskmelon: 9% to 12% soluble solids (Bower et al. 2002); honeydew: 9% to 17% soluble solids (Lester and Shellie 1992), ‘Galia’: 11% to 12% soluble solids (Artes et al. 1993). ‘Galia’ melons are known to taste sweeter than other melons, possibly due to more juice or to a lower acidity or astringency rather than a soluble solids concentration higher than other specialty melons. Artes et al. (1993) reported ‘Galia’ to have a soluble solids concentration of 10.8%, similar to that for three other melon types (‘Piel de Sapo’, ‘Amarillo’, and ‘Tendral’), but total titratable acidity for ‘Galia’ was 50% to 75% lower (0.054% citric acid) than the other cultivars. Nondestructive epidermal spectral data of immature- and mature-harvested ‘Juan Canary’ melons was compared to destructive quality parameters (chlorophyll content, yellow pigments, soluble solids content, and ﬁrmness), showing promise to nondestructively predict soluble solids concentration (Forbus et al. 1992). Sucrose and total sugars increased as much as

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50% in ‘Earl’s Knight’ muskmelon when plants were heated to 25 – 30 C from 3 to 17 days after anthesis (Kano, 2006). Plant nutrition also affects fruit quality. Pacheco (1996) reported higher soluble solids content and better ‘Galia’ fruit appearance from those fruit harvested from plants treated with increased preplant application of potassium (140 kg/ha was determined as the optimal rate for marketable yield and fruit size). Potassium plays a critical role in the transport of sucrose into muskmelon fruit. Regular foliar applications of potassium prior to harvest increased fruit soluble solids concentration, ﬁrmness, ascorbic acid, and b-carotene content (Lester et al., 2005). Low potassium concentrations in cucurbits are a serious limiting factor to postharvest quality. Potassium plays a key role in maintaining plasma membrane integrity via the cytoplasm, thus slowing disruption of cellular functions (Lester et al. 1998). Preharvest calcium application increased mesocarp calcium concentration and quality in honeydew but not in muskmelon fruit (Lester and Grusak 2004), and postharvest dipping of honeydew melon in calcium solutions was effective in delaying senescence (Lester and Grusak 2001). In California, shifting from water-intensive furrow irrigation to drip irrigation had no negative effects on muskmelon yield or quality (Hartz 1997). Use of saline irrigation water had no signiﬁcant effects on ‘Galia’ melon fruit quality or yield (Mendlinger and Pasternak 1992). Preharvest spray application of AVG (aminoethoxyvinylglycine) on muskmelon slightly delayed initial development of the abscission zone and caused leaf chlorosis but did not affect fruit quality at harvest; although AVG-treated fruits had up to 30% less ethylene production than untreated fruit, there was no effect on quality during storage (Shellie 1999). Application of AVG as a soil injection increased ﬁrmness of muskmelon but did not affect soluble solids content (Leskovar et al., 2006). Honeydew melons exposed to direct sunlight had subepidermal pulp temperatures up to 48 C in tests in California, leading to solar yellowing (‘‘sunburn’’) (Lipton et al. 1987). Synthesis of yellow pigment was reported for honeydew melons exposed to high levels of solar radiation, which can be partially offset with protective coatings of clay-based materials (Forney 1990). Greenhouse-grown ‘Haon’ and ‘Polidor’ plants with greater leaf area and restricted growth rate due to cooler night temperatures produced melons with higher soluble solids concentrations (Welles and Buitelaar 1988). 3. Harvest Maturity Indices. Primarily classiﬁed as climacteric fruits according to respiratory rate and ethylene production, melons must be

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Table 6.10. Appearance criteria for optimal harvest maturity of selected melon types. Melon type

Stem abscission zone

Muskmelon Honeydew Galia Charantais

Fully formed Cultivar dependent Fully formed None

Epidermal color Green, netted White to cream Light yellow, some green, netted Smooth sutures

Sources: Artes et al. 1993; Bower et al. 2002; Evensen 1983; Fallik et al. 2001; Lester and Shellie 1992.

harvested after the completion of physiological maturity to ensure proper postharvest ripening. Several methods are used to determine harvest maturity. For example, days after anthesis to harvest for muskmelon are 42 to 46 (Maynard and Hochmuth 2007). Visual indicators are also used, including development of a stem abscission zone, epidermal color, and development of netted surface (Table 6.10). However, Kendall and Ng (1988) reported that some nonnetted genotypes did not produce ethylene until 20 days postharvest, suggesting that these types were not clearly climacteric. Evensen (1983) found that muskmelon had best postharvest quality and storage life when harvested with green epidermis and at full slip (complete formation of the stem scar); those harvested at half-slip stage had poorer ﬂavor following storage and ripening. For honeydew melon, the best postharvest quality was obtained using these parameters at harvest: white epidermal color, soluble solids above minimally required value, and typical fruit shape, as determined by length-to-diameter ratio (Lester and Shellie 1992). Aroma volatile compounds also signiﬁcantly affect melon ﬂavor with more than 250 volatiles reported in the literature. An extensive study of muskmelon harvest maturity revealed that virtually no aroma volatiles associated with melon ﬂavor (esters, acetates, and alcohols) were produced until quarter-slip stage of harvest maturity, and these increased thereafter through full-slip stage (Beaulieu 2006). In this same study, aldehydes, associated with ‘‘green’’ or ‘‘grassy’’ ﬂavor, decreased from 6% of total volatiles at quarter-slip stage to about 1% at full-ripe stage. Epidermal (ground) color is also an important indicator of harvest maturity. In ‘Galia’ melons, highest aroma volatile concentrations and higher soluble solids contents were found in fruit harvested with yellow ground color and slight green areas, a ripeness stage that permits transport to distant markets while maintaining high quality (Fallik et al. 2001). Charantais melons are considered mature when the suture becomes smooth and the fruit cheeks are ﬁlled (Bower et al.

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2002). These authers also reported that melons ripened on the vine had lower respiration and ethylene production rates than those harvested preclimacteric. Harvest maturity affects resistance to physical impacts and compression in muskmelon. Fruit harvested at full slip withstood up to 120-cm drop onto a conveyor belt, while half-slip-harvested fruit were ﬁrmer but were less resilient and bruised or cracked when dropped 90 cm; (Foster et al. 1979). 4. Handling, Packing, and Storage. In California and Arizona, melons are directly packed into shipping containers. In more humid areas, ﬁeld-grown melons typically are loaded into ﬁeld bins, open trailers, or gondolas and transported to a central facility, where the containers are tipped, allowing the melons to roll into a water bath or onto conveyors for washing, grading, and packing. Greenhouse-grown melons are harvested into lugs and packed directly into shipping cartons. Recommended storage temperatures vary by melon type (Table 6.11). Respiratory and ethylene production maxima for melon at 5 C range from 8 to 10 mg/kg-hr and 10 to 100 mL kg-hr, respectively. Mature orange- and green-ﬂeshed muskmelon can be treated with exogenous ethylene (50 to 100 mL/L) 24 to 72 hr after harvest to stimulate uniform initiation of ripening (Reid 2002), as is also commercially done for banana, mango, and tomato. Muskmelon at three-quarter-slip or less are precooled using forced-air cooling or hydrocooling; those at full slip can also tolerate cooling with package icing. Casaba, Crenshaw, Honeydew, and Persian melons generally are room-cooled. The ethylene-action inhibitor 1-MCP retards ripening and softening of ‘Galia’ melons for up to 8 days when harvested preclimacteric and for 2 days when harvested fully ripe (Ergun et al. 2005). Epidermal color of Table 6.11. Recommended storage conditions for selected melon types.

Melon type

Storage temperature ( C)

Relative humidity (%)

Expected Controlled atmosphere (%) storage life (days) Oxygen Carbon dioxide

Muskmelon Whole: 2–7 Galia and charantais Fresh cut: 1 Honeydew Whole: 10

95 > 95% 95

10–14 3–5 21

Fresh-cut: 5 Whole: 10

90–95

6–10 21

Casaba, crenshaw, and juan canary

3–6 6–15 10–20 NA Not Not recommended recommended 5% 5% NA NA

NA: Data unavailable. Sources: Artes et al. 1993; Fallik et al. 2001; Shellie and Lester 2004.

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fruit treated with 1-MCP at preclimacteric stage never developed past light yellow, although mesocarp tissue quality was normal. Unwrapped muskmelon softened by about 40% with 6% fresh weight loss, while shrink-wrapped fruit lost only 0.3% fresh weight and were considered ﬁrm (11 Newtons) (Lester and Bruton 1986). In another study, Collins et al. (1990) noted excellent appearance of shrink-wrapped muskmelon following 21 days storage at 4 C; however, trained taste panelists rated ﬂavor and aroma inferior to that of unwrapped fruit. Modiﬁed atmosphere packaging is effective in delaying senescence for whole and fresh-cut muskmelon and honeydew (Table 6.11). Condensation within sealed packaging can promote decay on whole fruit during shipping. However, the condensation issue has been solved by sealing melons chilled to 4 C in atmospheric gas evacuated plastic box liners. This is the commercial practice currently used in Central American melon-exporting countries. Storage below minimum safe temperature results in development of chilling injury symptoms. External symptoms include sunken areas that become infected with saprophytic microorganisms. Internal tissues are affected by chilling injury, causing discoloration of mesocarp tissue in muskmelon. Abnormal ripening due to chilling injury results in off ﬂavors and aromas. However, honeydew melon exposed to high solar radiation accumulated more unsaturated lipid fatty acids in epidermal tissue (Forney 1990) and lower amounts of 1-aminocyclopropane-1-carboxylic acid (ACC) (Lipton et al. 1987), possible reasons for induced tolerance during storage to what would have otherwise been chilling temperatures. Ethylene was implicated as contributing to chilling injury in transgenic ‘Charentais’ fruit that were developed to produce almost no ethylene (Ben-Amore et al. 1999). These melons did not develop chilling injury despite 21 days storage at 2 C, whereas fruit from wild-type plants developed chilling injury. When treated with ethylene at ambient temperatures, the transgenic fruit developed chilling injury symptoms during subsequent storage. Antisense technology was used to transform parental lines of ‘Galia’ melon to reduce ethylene production during ripening (Nun˜ez-Palenius et al. 2006). Fruit from the transformed male parental line was harvested at full-slip stage and was ﬁrmer and had lower ethylene production than fruit from the wild-type plants at the same harvest maturity. 5. Postharvest Diseases/Disorders. Melons are susceptible to several postharvest diseases so sanitation and temperature management are

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critical in suppressing them as long as possible during storage and handling (Table 6.7). Postharvest heat treatment shows promise for decay control in muskmelon. Full-slip fruits heat-treated for 3 minutes at 57 C by immersion and shrink-wrapped maintained good quality and low surface decay rates up to 20 days at 4 C (Lester 1989). However, irradiation treatment (2 kiloGrays) was ineffective in controlling decay and accelerated fruit softening, weight loss, and electrolyte leakage. In a later study, Mayberry and Hartz (1992) applied heat treatment to muskmelons for 3 minutes with 60 C water prior to storage at 3 C. Treated fruit had excellent appearance and ﬁrmness and showed no stem-end decay up to 28 days of storage while untreated control fruits were unmarketable. Following immersion in 52 C water for 2 minutes, ‘Galia’ melons were free from decay up to 8 days at 20 C; however, off ﬂavors were noted (Teitel et al. 1989). Dry heating the surface of the melons to 52 C did not reduce decay. Teitel et al. (1991) found that ‘Galia’ melons could be dipped for 1 to 2 minutes in 55 C water and immediately wrapped with PVC plastic ﬁlm; acceptable quality was maintained for 9 days at 18 C. Heat injury symptoms were characterized as ranging from small pits to general browning up to 50% of the fruit surface. 6. Fresh-Cut and Food Safety. Melons along with watermelon comprise the majority of fresh-cut fruits. As with other products, efﬁciency and proﬁts are dependent on the proportion of usable pulp that can be sold as fresh-cut, and the edible portion varies with melon type. Artes et al. (1993) reported edible portions as 62% of whole fruit for ‘Piel de Sapo’, 63% for ‘Amarillo’, 58% for ‘Galia’, and 53% for ‘Tendral’. Storage at 4 C maintained acceptable quality in fresh-cut muskmelon for 4 days and honeydew melon for 14 days, based on ratings of trained sensory panelists and microbial counts (O’Connor-Shaw et al. 1994). Successful efforts to extend postharvest life of fresh-cut melon involve combinations of treatments, including more effective sanitation, use of calcium solutions, and modiﬁed-atmosphere packaging. Sapers et al. (2001) found that washing whole muskmelon with 5% hydrogen peroxide was more effective in sanitizing the rind than 1000 mL/L chlorine or other commonly used sanitizing treatments. Luna-Guzman et al. (1999) dipped muskmelon cylinders for 1 minute in 2.5% calcium chloride at 60 C, which signiﬁcantly retarded mesocarp softening during storage at 5 C and extended postharvest life to 15 days. Calcium propionate and calcium amino acid chelate are superior to calcium chloride in maintenance of fresh-cut melon ﬂavor quality and shelf stability (Saftner et al. 2003, 2006).

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Muskmelon cubes stored in a modiﬁed-atmosphere package (5% O2 10% CO2) suppressed microbial growth more than those held in air at 5 C (Bai et al. 2001). Packages ﬂushed with the gas mixture had higher visual quality than those in which the product gas exchange modiﬁed the internal atmosphere. Whole ‘Galia’ melons were treated with 1 mLL1 gaseous 1-MCP at 20 C for 24 hr then cut into cubes and stored at 5 C (Ergun et al. 2007). Cutting treated and untreated melon induced a similar wound response of ethylene production after 1 day, whereas intact fruit had continuous basal ethylene production throughout storage. Melon pretreatment with 1-MCP retarded mesocarp softening and development of water-soaking in fresh-cut melon up to 10 days. Avoidance of contamination in conjunction with proper sanitation and temperature control is also critical in reducing the potential for contaminating fresh-cut melons with human pathogens. Escherichia coli O157:H7 multiplied on muskmelon rinds and cut surfaces for up to 3 weeks at 25 C (Del Rosario and Beuchat 1995). Following inoculation with a cocktail of Salmonella spp. and storage for 5 hr at room temperature, fresh-cut muskmelon had higher microbial populations than either fresh-cut watermelon or honeydew melon (Ukudu and Sapers 2007). Once inoculated, whole melon surfaces are very difﬁcult to sanitize, particularly the netted types. Whole muskmelon inoculated with E. coli spp. had lower populations after immersion in 1000 mL/L chlorine solution than in hydrogen peroxide, although efﬁcacy decreased with increased delay following inoculation (Ukuku et al. 2001). And neither of these treatments completely sanitized contaminated surfaces. Muskmelon and honeydew cubes inoculated with C. botulinum were stored for 9 days; at 15 C toxin was detected, whereas inoculated cubes stored at 7 C had no toxin present (Larson and Johnson 1999). The toxin was also associated with severe fruit decay, although the presence of background ﬂora appeared to limit growth of C. botulinum. D. Squash and Pumpkin (Cucurbita maxima, C. moschata, and C. pepo) The economically important species of Cucurbita (C. maxima, C. moschata, and C. pepo) are native to the subtropical and tropical Americas. The American species quickly became established throughout the world and largely replaced the Old World gourds such as bottlegourd (Legenaria siceraria) and the snake gourds (Trichosanthes spp.) as human food sources. There is considerable overlap in the common names of the three principal Cucurbita species. Crops commonly called squash or

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pumpkin are included in Cucurbita. The decorative gourds (C. pepo) are the source of least confusion since they are easily identiﬁed by appearance. Squash and pumpkins often are used interchangeably depending on local custom. As a general rule, pumpkins are round or nearly so whereas squash are irregularly shaped. The decorative or Halloween pumpkin (C. pepo) is always referred to by that name. Within squash, there are summer and winter types. The summer types (cocozelle, crookneck, straightneck, scallop, vegetable marrow, and zucchini) are fast growing, have soft rinds, are consumed when the fruit is immature, and are quite perishable (Paris 2001). Very immature fruits (e.g., baby squash types) and the edible blossoms comprise a very small portion of commercially handled squash but are in high demand by upscale restaurants and command high prices. Winter squash are harvested when the fruits and seeds are fully mature. The durable rinds provide several months of storage life in contrast to summer squash, which must be consumed within two weeks or less. Summer squash are mostly determinate or have a bush growth habit; winter squash are mostly indeterminate or vining in growth habit. The principal Cucurbita spp. may be further grouped according to horticultural traits. Fruit shape and color and rind durability are the main discriminating characteristics. Some of the types are arbitrary and of historical interest only. For example, cushaw squash, winter crookneck squash, and marrow squash are not commonly grown, but they may be regionally important. There is a lively interest in maintaining stocks of heirloom squash that offer unique qualities (Goldman 2006). The gourds and pumpkins of C. pepo are grown mostly for ornamental rather than culinary purposes and are increasing in economic importance in the United States. Show pumpkins (C. maxima) are grown exclusively for competition in the heaviest-fruit contests held in various parts of the United States. In 2007, the winning pumpkin grown by Joe Jutras of North Scituate, Rhode Island, USA, weighed an astounding 766 kg (Raver 2007). China is the leading producer of pumpkin/squash, with India a close second (Table 6.12). China produced 28% of the annual world production between 2003 and 2005 (Lucier and Dettmann 2007). Other important countries in pumpkin/squash production in 2005 were Cameroon, Cuba, Ukraine, the United States, and Egypt (FAOSTAT 2007). 1. Nutritional Value. Orange-ﬂeshed winter squash provide an excellent source of vitamin A. Two compact tropical pumpkin

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Table 6.12. Principal pumpkin/squash producing countries, 2005. Country India China Cameroon Cuba Ukraine United States Egypt

Harvested area (1,000 ha) 360.0 308.2 115.0 95.0 55.0 40.2 NA

Production (million t) 3.5 5.8 NA NA 1.1 0.9 0.7

NA: Data unavailable. Source: FAOSTAT 2007.

hybrids (Cucurbita moschata) were recently released and reported to contain 49 and 57 mg/kg fresh weight total carotenoids in the pulp, higher than that reported for butternut squash (45 mg/kg fresh weight) (Maynard et al. 2002). Most winter squash provide useful sources of vitamin C (FAO 2007; USDA 2007). 2. Quality Indices. Quality characteristics vary by squash type, including characteristic epidermal and stem color, shape, and dimensions according to applicable grade standards (USDA 1983, 1985a). Summer squash are harvested immature, when the fruit are small and the rind has a distinctive sheen. Winter squash are harvested on reaching physiological maturity or during ripening, when the fruit has reached a minimal level of acceptable quality. Silverleaf whiteﬂy (Bemisia argentifolii) must be controlled during production of all squash types since feeding on leaves induces leaf silvering and fruit color blanching, signiﬁcantly reducing marketability (Maynard and Cantliffe 1989). Instead of typical medium to dark-green coloration, zucchini fruit appear light green, acorn squash are mottled green and/or yellow, and golden acorn squash are white. 3. Harvest Maturity Indices. In warm weather (>25 C mean temperature), summer squash fruits grow rapidly, requiring harvest daily or every other day for tenderness and highest quality. Fruit should be harvested 3 to 4 days after pollination (DAP) for zucchini and 5 to 7 DAP for summer squash (Maynard and Hochmuth 2007). When harvested after this time, the rinds become dull in overmaturity with a concomitant loss of quality. Hydroponically grown baby squash were harvested about 2 DAP when fruits reached < 10 cm in length for

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zucchini and yellow squash and < 4 cm in diameter for scallop types; all types weighed from 20 to 30 g/fruit (Shaw and Cantliffe 2004). Yellow squash blossoms (‘Dixie’) had best postharvest life when harvested with petals fully extended but closed; male ﬂowers were clipped with 25 mm peduncle, and female ﬂowers were detached from the immature fruit with a gentle twist (Villalta et al. 2004). Winter or hard-shelled squash are so named because they are grown to maturity, requiring 60 to 110 days from planting to produce a marketable product. Harvest maturity for pumpkin is very cultivardependent and is from 80 to 110 days from planting, while buttercup and butternut require 60 to 70 days from planting (Maynard and Hochmuth 2007). Harvest maturity also affects postharvest quality. Although somewhat cultivar-dependent, harvest maturity of acorn squash was critical to yield and eating quality; dry matter continued to accumulate until 30 DAP, and soluble solids content increased as much as 50% from 25 DAP until 55 DAP at seed maturity (Loy 2006). In another study, immature-harvested spaghetti squash ( <42 DAP) were reported to have poor edible quality following cooking (Edelstein et al. 1989). 4. Handling, Packing, and Storage. Delicate summer squash are harvested into shallow ﬁeld bins that must be free of sand and- or other foreign material to prevent abrasions or are ﬁeld-packed. The bins are transported to a packing facility, where they are either tilted to pour the squash into water or immersed so that the fruit ﬂoat out to minimize transfer impacts and abrasions. Squash harvested onto mobile units (‘‘mule trains’’) either are packed directly into shipping containers or are run over a short packing line for washing and sizing prior to packing. Winter squash and pumpkins also may be harvested into bins, trailers, or gondolas for transport to a central packing facility, to a receiver, or into a storage facility. Recommended storage temperatures for summer squash are 5 C and 10 C for zucchini and yellow straightneck or crookneck, respectively (McCollum 2002). Due to perishability, summer squash are rapidly cooled using hydrocooling or forced-air cooling. Baby yellow ‘Sunray’ summer squash stored in vented polystyrene ‘‘clamshell’’ containers maintained acceptable quality up to 14 days at 10 C (Brew et al. 2006). Closed blossoms of yellow ‘Dixie’ summer squash stored well at 2.5 C for 7 days in clamshell containers (Villalta et al. 2004). Summer squash are susceptible to chilling injury. ‘Dixie’ yellow crookneck squash developed surface pitting and had increased water loss during storage at 2 C. After 3 days storage, this stress stimulated

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ethylene production (from undetectable to 1.5 ml/kg-h), and after 6 days, respiration began to rise (McCollum 1989). Chilling injury causes development of necrotic tissue below the epidermis of yellow summer squash. Storage in low oxygen (4%) minimized development of chilling injury in zucchini squash at 2.5 C; however, after transfer to air at 10 C for 2 days, chilling injury symptoms became apparent. Severe off ﬂavors developed after storage at 2.5 C or 10 C; some were even more apparent after cooking (Mencarelli et al. 1983). Yellow summer squash quality became unacceptable from 6 to 20 days at 5 C and 85% relative humidity (RH) with those having the Gene B (yellow epidermal RH pigmentation) being most susceptible to chilling injury and shrivel hereafter (Sherman et al. 1987). Winter squash, unlike summer squash, have a long postharvest life of 1 to 3 months or more, with ideal temperatures reported for pumpkin and butternut squash from 10 to 13 C and relative humidity of 50% to 70% (Whitaker and Davis 1962; Wien 1997; Decker-Walters and Walters 2000). Room cooling is generally adequate for winter squash. Respiration and ethylene production are low for all of these nonclimacteric squash. Butternut squash is the rare winter squash seriously affected by moisture loss during storage, leading to hollowneck disorder. Hollowneck is characterized by the gradual development of internal open areas in the neck of the fruit. It does not become visible externally until weight loss exceeds 10%, at which point internal symptoms are severe (Francis and Thomson 1964). Curing (drying) winter squash at ambient or elevated temperatures prior to storage did not extend postharvest life or reduce decay but rather promoted weight loss (Schales and Isenberg 1963). Controlled-atmosphere storage (1% O2, 7% CO2) maintained green color and suppressed decay of buttercup (Brecht 2002) but is not recommended for other whole squash/pumpkin types. High humidity in controlled-atmosphere storage may adversely affect squash quality. 5. Postharvest Diseases/Disorders. A number of diseases affect squash and pumpkins (Table 6.7). Hot-water treatments did not reduce decay or extend postharvest life of winter squash. ‘Delica’ was immersed in 50 C water for up to 12 minutes; after 12 weeks of storage, there was no difference from untreated fruit (Arvayo-Ortiz et al. 1994). 6. Fresh-Cut and Food Safety. Storage of fresh-cut summer squash is recommended at 5 C; reducing oxygen to 0.25 to 1.0% was also reported to be beneﬁcial (Saltveit 2001). Fresh-cut tropical pumpkin (Cucurbita moschata) can be stored at 0 C.

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E. Chayote (Sechium edule) Chayote, also known as mirliton or vegetable pear, originates in Guatemala and Mexico, and today large-scale production occurs in Costa Rica, Guatemala, and Brazil as well as in other tropical areas. Because the plant is short-day with respect to ﬂowering, production is restricted to subtropical and tropical areas of the world; in frost-free areas, plants are perennial. The fruit bears a single seed that may sprout within the fruit (vivipary), and either the entire fruit or the excised seed is used for propagation. Commercial fruit are generally pear-shaped, from light to dark green, and about 10 cm long. The fruit is consumed as a vegetable and is similar to scallop summer squash in texture and ﬂavor. Young shoots and leaves as well as portions of the tubers also are consumed. The fruit is also used in the food industry as an ingredient in sauces and ﬁllings (Robinson and Decker-Walters 1997). The vining growth habit requires that the plant be trained on an overhead trellis so workers can walk beneath the crop and easily identify and harvest fruit ready for market. Chayote should be harvested immature ﬁrm, with tender, smooth skin (absence of spines) and natural gloss (Aung et al. 1996), about 35 days from fruit set (Stephens 2003). Chayote is a minor contributor nutritionally and contains 24 kcal per 100 g cooked pulp (USDA 2007b). Common postharvest chayote diseases are found in Table 6.7. Following harvest, fruit are transported, sometimes via a miniature train system, to a packinghouse for sorting and placement in shipping containers. They are often wrapped individually in tissue paper or polyethylene bags to prevent abrasion of the tender skin. Storage is recommended at from 5 to 10 C and 85% to 95% RH. The respiration rate is low (4 mg/kg/hr), and room cooling is normally adequate. Chayote can be stored from 4 to 6 weeks (Stephens 2003). When stored at 25 , 15 , or 10 C, chayote lost 1.3%, 0.5% and 0.02% of the fresh weight per day respectively; use of a polyvinyl chloride (PVC) ﬁlm overwrap retarded weight loss by 80% to 90% at these same temperatures (Aung et al. 1996). F. Wax Gourd (Benincasa hispida) The wax gourd is named for the white waxy bloom that covers the fruit surface. It is also known as Chinese winter melon, ash gourd, and Chinese preserving melon, among others. Although India and Indochina have the greatest diversity, the centers of origin are believed to be Japan and Indonesia. Wax gourds thrive in areas with a long, warm growing

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season and will withstand rain during production. China and India are the greatest producers by far, and wax gourds can be found throughout Asia and wherever people of Asian descent have immigrated. Wax gourd is the only cultivated cucurbit within the genus Benincasa and is found together with watermelon, bottle gourd, luffa, and lesser cucurbits in the tribe Benincasea. Wax gourd shares many characteristics with Cucurbita, the squash, but differs in certain critical ﬂower characteristics, centers of origin, and chromosome number. Wax gourd provides minor nutritional value (Wills et al. 1984; USDA 2007b). Because of its mild ﬂavor, wax gourd is complimented by many foods. The mature fruit ﬂesh can be eaten as a vegetable or its seed cavity can be hollowed out and used as a container for soup made with other vegetables, ﬁsh, or meat. During growth, the young shoots and leaves may be used as greens. The wax gourd often serves as the centerpiece for festive occasions (Nayer and More 1998). Wax gourd has a heavy waxy epicarp, and the mesocarp is white, crisp, juicy, and mild in ﬂavor. There are two types, round and oblong, and some types grow as large as 2 m long and 1 m wide, weighing as much as 35 kg (National Acad. Sci. 1975). The fruit are produced at intervals along the trailing vines. The fruit may be harvested at any stage of maturity. When harvested immature, it is prepared similar to summer squash. At full maturity, the waxy bloom is developed and the fruit is harvested on reaching the desired weight, or it may be left in the ﬁeld until needed or until market conditions favor harvest. Wax gourd stores well for several months. Soga et al. (2004) reported that wax gourd should be stored above 12 C; those stored for 5 months below that temperature developed chilling injury, characterized by surface pitting and decreased ascorbic acid content. Common postharvest diseases are listed in Table 6.7. G. Bitter Melon (Momordica charantia) Bitter melon is known by several names, including bitter gourd, bitter cucumber and balsam pear. It is native to the tropics of southeast Asia and India and is very popular in many Asian countries (Robinson and Decker-Walters 1997). Most likely it was introduced to the New World from Africa in the 17th or 18th century. Bitter melon plants are perennials that usually are cultured as annuals. The fruit has a typical warty appearance, and the shiny epidermis color can vary in shades of green, but should be uniform and free from splitting, which is a sign of overmaturity (Zong et al. 1995). There are three

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Table 6.13. Bitter melon types and quality parameters. Type

Fruit length (cm)

Small Long

10 to 20 30 to 60

Triangular, cone-shaped

9 to 12

External appearance Dark green Light green; medium protuberances Light to dark green; prominent tubercles

Flavor Very bitter Slightly bitter Medium to strongly bitter

Source: Cantwell et al. 1996.

horticultural types of bitter melon, classiﬁed according to the parameters in Table 6.13. Momordicine is the alkaloid responsible for the bitter ﬂavor, and immature-harvested fruit generally is less bitter than matureharvested fruit. As with wax gourd, the tender shoots and leaves of bitter melons can be prepared as cooked greens. The sliced pulp and seeds are soaked in water to remove bitterness prior to preparation as boiled, curried, fried, or pickled Asian dishes. Bitter melon is an excellent source of ascorbic acid, providing 84.0 mg/100 g F.W. (USDA 2007b). Medicinal attributes of bitter melon have been extensively explored in cancer cell cultures and animal studies. The leaves and fruit appear to prevent overexpression of a P-glycoprotein, which reduces drug accumulation in cancer cells and decreases lipids, leading to reduced adiposity in animals (Chan et al. 2005). Bitter melon may be useful in prevention or alleviation of diabetes, but therapeutical uses still need to be closely studied (Basch et al. 2003). The very long vines are trained on an inverted U-shaped trellis to facilitate harvest. The fruit is harvested from immature to mature, but prior to yellowing of the fruit and seed maturation, which is about 14 days after fruit set (Cantwell et al. 1996). Respiration and ethylene production rates are low at 15 mL/g-hr and 0.1 to 0.3 ZL/g-hr, respectively (Zong et al. 1995). Fruit stored at 10 C and 85% to 90% RH remains in good condition for 2 to 3 weeks. Forced-air cooling or hydrocooling are appropriate cooling methods. Common postharvest diseases are listed in Table 6.7. Bitter melon is prepared like immature squash. Sliced fruit exhibit wound-induced increases in respiration and ethylene production for 2 days following slicing, and a maximum of 4 days postharvest life during storage at 2 C (Wang et al. 2007). H. Smooth Luffa (Luffa aegyptiaca), Angled Luffa (Luffa acutangula) Smooth luffa is also known as loofah, sponge gourd, vegetable sponge, dishcloth gourd, and rag gourd, while angled luffa is referred to as

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ridged gourd, angled gourd, ridged luffa, and Chinese okra. Both species probably originated in India and are widely grown throughout tropical Asia and South America, particularly Guatemala, Colombia, and the Caribbean, where the smooth type is the most common (Robinson and Decker-Walters 1997; Nayer and More 1998). The immature fruit is the main food use and is prepared like summer squash, although young shoots and leaves may be used as greens. If grown to full maturity, fruit of smooth luffa produce phytosponges. Following harvest, the outer fruit wall and inner pulp are removed and the remaining ﬁber is dried to produce the sponge. The edible portion of angled luffa is reported to be quite low at 54% compared to other horticultural crops; it is also low in nutrients (Wills et al. 1984). Luffa vines are very large, lending themselves to training on a stout vertical trellis that encourages the development of straight fruit. Immature fruit are harvested when about 15 cm long to avoid accumulation of bitter compounds. It should have a fresh appearance with no physical injury. Optimal storage of luffa is from 10 to 12 C and 90% to 95% RH. Respiration and ethylene production are low, 19.0 mL/L and <0:1ZL/ghr, respectively, at 10 C; at this temperature, 14 days pos-tharvest life can be anticipated (Zong et al. 1995). Common postharvest diseases are listed in Table 6.7.

III. CONCLUSIONS Crops from the cucurbit family are of worldwide importance, contributing to the economic and nutritional well-being of people from virtually every nation. While signiﬁcant amounts of cucurbits are grown by large-scale operations destined for international markets, these crops also play a vital role for small farmers in local economies. Future postharvest research must continue to address relevant issues at both of these levels. By necessity, this requires collaborative, multidisciplinary approaches ranging from basic to applied sciences. These priority areas of postharvest biology and technology should be considered for cucurbit crops: Development of more in-depth understanding of basic postharvest physiology and genomics related to nutrition, medicine, ripening, and senescence Evaluation of new and underexploited cucurbit crops with economic potential

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Collaboration with breeding programs to incorporate new germplasm with better postharvest quality and nutrition Collaboration with engineers to apply new technologies in automation, packaging, and product quality tracking/traceback from ﬁeld through consumer levels Collaboration with microbiologists and plant pathologists to ensure safe and secure crops

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7 Physiological Disorders of Grape: Bunch Stem Necrosis and Early Bunch Stem Necrosis Andrea H. Pickering The Horticulture and Food Research Institute of New Zealand Limited Tennent Drive Palmerston North 4442, New Zealand Ian J. Warrington and David J. Woolley Institute of Natural Resources Massey University, PO Box 11 222 Palmerston North 4442, New Zealand

I. INTRODUCTION II. PHYSIOLOGY OF BERRY GROWTH AND DEVELOPMENT A. Inflorescence B. Flower C. Pollination D. Berry Growth and Veraison E. Ripening 1. Cation Accumulation 2. Plant Growth Substances III. BUNCH STEM NECROSIS A. Symptoms B. Possible Causes 1. Environmental 2. Mineral Nutrition in Vine and Berry 3. Plant Growth Regulators 4. Canopy Development 5. Xylem Development C. Application of Various Minerals and Plant Growth Regulators 1. Potassium 2. Calcium

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3. Magnesium 4. Nitrogen 5. Plant Growth Regulators IV. EARLY BUNCH STEM NECROSIS A. Symptoms B. Possible Causes C. Application of Various Minerals and Plant Growth Regulators 1. Nutrients 2. Plant Growth Regulators V. SUMMARY AND CONCLUSIONS VI. LITERATURE CITED

I. INTRODUCTION There are many physiological disorders in grapes (Vitis spp., Vitiaceae), and some may affect only certain cultivars of seeded as compared to seedless grapes. As these disorders are not the result of a pest, bacterial, or viral infection, and are therefore the result of the vine reacting to environmental conditions and/or management practices, the causes, and therefore control, of some of these disorders often are not fully understood. Many are thought to be nutrient related. Symptoms of magnesium, calcium, and boron deﬁciency often can be recognized and corrected if displayed in the vine. Other problems, such as sunburn and hail or frost damage, are also easily recognizable. There may be management procedures that can reduce the effect of these adverse factors on the crop. However, in some disorders, the cause is not so easily determined. In some such cases, it is suggested that a nutrient imbalance or certain environmental condition is the cause of the disorder, but proving this can be complicated, due to the varying environmental conditions from season to season and the considerable variability observed between vines. The initial symptoms of some physiological disorders affect the vine itself; in others, the symptoms directly affect only the bunch. Such disorders include berry shrivel, shatter, bunch stem necrosis (BSN), and early bunch stem necrosis (EBSN). This review looks speciﬁcally at BSN and EBSN. BSN and EBSN are two physiological disorders that occur in grapes around the world. They can be detrimental to both the wine and table grape industries, causing large losses in production. BSN can affect grape and, therefore, wine quality and yield (Ureta et al. 1981). EBSN can dramatically reduce berry set and cause a large reduction in yield. In some cases, crop losses of more than 60% have been reported for BSN

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(Rumbos 1989). EBSN has been reported to have caused as much as 90% crop loss in some Muscat varieties (Jackson 1988). Symptoms of BSN can occur any time after the beginning of the lag stage of fruit growth when the concentrations of most plant growth regulators are low and sugar accumulation begins to increase. This is also the time when the osmotic value of the berries is higher than that of the rachis (StellwaagKittler 1975, 1983). Symptoms occur on the peduncle, rachis, and berries and continue to develop progressively from veraison until harvest (Theiler 1975b; Stellwaag-Kittler 1983). Due to its international occurrence, BSN is known by many names. These include Stiella¨hme in Germany (‘‘stalk necrosis’’, also used), le desse`chement de la raﬂe in France (‘‘drying (or withering) of the peduncle’’), and palo negro in Chile. Other English names include waterberry used in the United States and shanking used in New Zealand (Jackson and Coombe 1995). However, some references state that waterberry and palo negro are similar to but not the same disorders as BSN (Pearson and Goheen 1988). Throughout this review, the term ‘‘BSN’’ will be used to describe the disorder being discussed. The ﬁrst known reports of a disorder matching the symptoms of BSN in Europe was in 1937 (Osterwalder 1937) and in Australia in 1953 (Coombe and Allan 1953). The ﬁrst record of BSN in Californian vineyards was by Bioletti in 1923 (cited in Weaver 1976; Morrison and Iodi 1990). Interestingly, Weaver (1976) states that there are in fact two conditions that the name waterberry covers. The ﬁrst, which Bioletti (cited in Weaver 1976) described, was stated to be caused by overcropping and therefore undernourishment of the berries and occurred only at the tips of the clusters. The symptoms included berries with lack of sugar, color, ﬂavor, and shipping quality, but Weaver (1976) did not mention any necrosis. The second condition had the same symptoms as the ﬁrst but did include necrosis of the pedicel, could occur in all parts of the bunch, and was found not to be caused by overcropping (Kasimatis 1957). This second disorder was also found to be directly associated with the blockage of xylem vessels in the pedicel by tyloses (gummy or resinous secretions [Soule 1985]). The extent of this blockage determined the degree of deterioration in berry development, and it was not until the blockage was almost complete that necrosis of the pedicel occurred (Kasimatis 1957). It is therefore difﬁcult to determine if either or both of these conditions are in fact the same condition as that termed BSN, but it is very likely that the second condition is the same as BSN. Early bunch stem necrosis, also known as inﬂorescence necrosis (IN), is a different disorder from BSN. It appears that the ﬁrst report of

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this disorder was as late as 1988 when Jackson and Coombe (1988) described it (Jackson 1994). They called the disorder EBSN due to its similarity to BSN. Therefore, this is the term which will be used to describe this particular disorder in this review. EBSN affects the pedicels, rachis, and peduncles any time from pedicel elongation up to and including ﬂowering, and not after veraison, as in the case of BSN (Jackson 1988; Jackson and Coombe 1988). Some researchers have observed that it is more common a week or two before ﬂowering (Jackson and Coombe 1988). BSN has been extensively studied in many countries, particularly France and Germany, and consequently much of the information on this disorder is not published in English. This appears to have resulted in duplication of research into this disorder. Early in the research into BSN, the small amount of information available was published as a part of local bulletins on growing grapes for the wine industries in France and Germany. From there the literature was generally reviewed and published only as the introduction to a scientiﬁc paper. However, Capps (1999) produced an extensive review of much of the material for a master’s thesis. This review has attempted to gather as much of the literature on BSN into one place, no matter what language it was published in, and therefore includes the information in Capps’s (1999) master’s review. Some articles are not cited in this review due to the extensive repetition of some information. EBSN has been less extensively researched, possibly due to the recent observation of this disorder. Therefore, there is less literature available and generally most of what is available has been published in English. Jackson and Coombe (1988) produced an excellent overview of this disorder. This review has used and expanded on their overview to include more recent work into this disorder. II. PHYSIOLOGY OF BERRY GROWTH AND DEVELOPMENT The reproductive and vegetative anatomy of grape vines is well documented (Weaver 1976; Kennedy 2002; Dry and Coombe 2004; Mullins et al. 2004) and is reported here only brieﬂy in order to give a general overview of normal development and to deﬁne terms. A. Inﬂorescence Inﬂorescence primordial initiation occurs in the season prior to the season in which the ﬂowers and berries are formed (Winkler 1965; Pratt 1971; Weaver 1976; Jackson and Schuster 2001). It begins around the

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time of ﬂowering and continues throughout that season until veraison (Dry and Coombe 2004). Therefore, environmental conditions around that time can affect the next season’s ﬂowering and crop load. Warm sunny weather during the time of inﬂorescence initiation is required for a large crop in the following season (Jackson and Schuster 2001). Soon after bud break, once the current season’s canes have elongated to the node number at which the inﬂorescence is situated, the form of the inﬂorescence can be seen with the naked eye (Winkler 1965). From bud swell through bud break until ﬂowering, inﬂorescence primordia growth resumes from the previous season. The peduncle and rachis of the inﬂorescence develop, lengthening and expanding in width, and ﬂower formation occurs (Dry and Coombe 2004). Although Jackson and Coombe (1995) state that the term ‘‘peduncle’’ includes ‘‘the central axis (rachis), the stems of the laterals, and the pedicels’’ of the inﬂorescence and later, the bunch, Soule (1985) deﬁnes the peduncle as ‘‘a stalk of a ﬂower cluster (inﬂorescence)’’, the rachis as ‘‘the extension of the peduncle, to which the pedicels are attached if present’’; and the pedicel as a ‘‘stalk supporting a single ﬂower.’’ Winkler (1965) also states very clearly that ‘‘the main axis of the cluster is called the rachis. Branches arise from the rachis at irregular intervals and divide to form the pedicels which bear the individual ﬂowers. The region of the rachis extending from the shoot to its ﬁrst branch is called the peduncle, or ‘stem.’’’ Throughout this review, the terms used to describe the different parts of the inﬂorescence and bunch will be the three separate terms as described by Winkler (1965) (Fig. 7.1). Some studies have used the term ‘‘bunch stem.’’ It is unclear, whether their authors mean the peduncle, rachis, or both. Consequently when discussing particular literature, the term used by the original authors is employed. Peduncle Pedicel

Rachis

Inflorescence/bunch

Individual flower or berry Fig. 7.1. Stylized drawing of a grape inﬂorescence/bunch showing the various parts: peduncle, rachis, and pedicel.

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B. Flower There has been some discrepancy in whether ﬂower initiation can occur prior to dormancy or if it occurs at bud burst in the following spring. A review by Pratt (1971) on the reproductive anatomy of the grape vine has reported that ﬂower initiation on the inﬂorescence can occur at any time from the summer before to the spring of ﬂowering, depending on environmental factors. Gerrath (1992) published a table summarizing results of studies into the time of initiation of the ﬂoral primordia. This table shows that some studies have found ﬂoral primordia prior to dormancy. However, most studies have found that ﬂoral primordium formation occurs around the time of bud burst in the following spring (Scholeﬁeld and Ward 1975; Srinivasan and Mullins 1976; Swanepoel and Archer 1988; Gerrath 1992; Mullins et al. 2004). The contradictions among these various studies may be due to the different techniques used, different cultivars, the difference between some male and female ﬂowers, or varying environmental factors. The petals of a grape ﬂower are green and fused at the top to form a cap called the corolla or calyptra (Weaver 1976; Jackson and Schuster 2001; Dry and Coombe 2004). At ﬂowering, the calyptra separates from the rest of the ﬂower at the base near the pedicel and falls off (Weaver 1976). This is called cap fall and is considered to be the time of ﬂowering (anthesis) (Jackson and Schuster 2001; Dry and Coombe 2004). C. Pollination Most V. vinifera cultivars have perfect (hermaphroditic) ﬂowers (Pratt 1971). In the wild, many are still dioecious, with separate male and female plants, but when domesticated, perfect ﬂowers appear to have been selected to avoid having unproductive males in a vineyard (Dry and Coombe 2004), although in some commercial vineyards, female varieties are still pollinated by hermaphrodites. The ﬂowers are pollinated by the wind and can also self-pollinate (Jackson and Schuster 2001). If weather conditions around ﬂowering are wet, caps may not fall totally from the ﬂower. Although pollination can still occur, fruit set may be adversely affected (Weaver 1976). D. Berry Growth and Veraison Many authors have used different methods to describe grape berry growth. These include classiﬁcation due to color and chemical change, cell metabolism, and diameter, length, volume or weight measurements

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(Pratt 1971). Eichhorn and Lorenz developed a comprehensive scheme that included all growth stages of the vine (referenced in Dry and Coombe 2004). It is known as the E-L system with 47 numbers detailing the growth stages from winter bud to leaf fall. The E-L number for the following stages are: ﬂowering—23, berry set—27, veraison—35, harvest—38 (Dry and Coombe 2004). ‘‘Veraison’’ in French means the commencement of berry color change and has become a term used to describe the events at the end of the lag stage in berry growth when, among other things, berries soften, sugars increase, malic acid decreases, color changes, and berry growth accelerates again (Coombe and Hale 1973; Dry and Coombe 2004). By using berry growth measurements, it can be shown that berry growth follows a double sigmoid curve from anthesis to maturity with (1) a period of rapid growth, (2) a period of slow growth (termed the lag stage), and (3) a ﬁnal period of rapid growth (Harris et al. 1968; Pratt 1971; Weaver 1976; Jackson and Schuster 2001). However, other authors have either condensed this into two phases of growth or expanded it into four stages. This depends on whether they separate out the lag stage or not in their deﬁnition, or add an extra stage at the beginning when there is little berry growth, and berry drop and nucellus growth occur (E-L 23–27) (Pratt 1971). For the sake of simplicity, here three stages are considered making up the two phases of berry growth. Many authors continue to include the lag stage in their discussions even when considering there to be two phases of growth. Phase One is therefore from anthesis to veraison (E-L 23–33), when cell division and cell expansion both account for the growth in the berry (Harris et al. 1968; Weaver 1976). Cell division has generally stopped by approximately 40 days after bloom, with it ﬁrst ceasing in the placenta and inner pericarp 7 to 11 days after full bloom (FB), in the outer pericarp 19 to 20 days after FB, and ﬁnally in the hypodermis and epidermis 32 to 38 days after FB (Pratt 1971). This timing is different for different cultivars and different environments. These ﬁgures obtained from Pratt’s review (1971) were sourced from work carried out on ‘Delaware’, ‘Campbell Early’, ‘Muscat Bailey A’, and ‘Koshu’. This growth phase is called berry formation (Dry and Coombe 2004) and includes Stages I and II. The seeds attain their full size in this phase, but the endosperm and embryo are incompletely developed (Pratt 1971; Dry and Coombe 2004). The end of this phase is characterized by Stage II, the lag stage, in which it is thought that parts of the embryo are differentiated (Pratt 1971) and little extra growth in the berry occurs. The lag stage sometimes can be brief or prolonged, depending on environmental conditions and whether it is a seeded or

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nonseeded cultivar (Weaver 1976). It has been suggested that this lag stage is due to a reduction in the sink strength of berry ﬂesh for assimilates (Zhang et al. 2003). This may be due to the reduction in gibberellins in the berry (Bhullar and Dhillon 1974). At this stage, the seeds become much stronger sinks than the ﬂesh of the berry (Zhang et al. 2003). Phase Two begins at veraison and continues through to maturity (E-L 34–38). Berry growth is due to cell expansion with the importation of sugars through the phloem. This phase is called berry ripening (Dry and Coombe 2004). E. Ripening From veraison onward, the berry content changes as part of the normal ripening process. The concentration of soluble solids stays relatively constant prior to veraison and then increases sharply (Hrazdina et al. 1984; Morrison and Iodi 1990; Dry and Coombe 2004) as both the glucose and fructose concentrations increase and tartaric and malic acid concentrations decline rapidly until harvest (Downton and Loveys 1978; Cawthon and Morris 1982; Crippen and Morrison 1986). The reduction in acids results in the titratable acidity also declining after veraison (Morrison and Iodi 1990). Tartaric acid has a high initial concentration after anthesis and steadily declines throughout the season, whereas malic acid has a low initial concentration after anthesis, increases sharply until veraison, and then declines rapidly (Hrazdina et al. 1984; Gutierrez-Granda and Morrison 1992; Dry and Coombe 2004). The pH, which is generally constant prior to veraison, increases sharply from postveraison until harvest (Morrison and Iodi 1990; Gutierrez-Granda and Morrison 1992; Dry and Coombe 2004). Xylem connections between the berry and the vine become discontinuous near veraison (Fig. 7.2) but are not thought to disappear completely (Creasy and Lombard 1993). Studies have shown that although initially water ﬂows predominantly in the peripheral xylem system, once the discontinuity occurs, water ﬂow in the xylem occurs in the axial system, which is less conductive (During et al. 1987). However, this is thought not to occur in the cultivar ‘Shiraz’ (Rodgiers et al. 2000). This changes the berry water relations, with berries pre-veraison being more susceptible to water stress than post-veraison berries (Creasy and Lombard 1993). The change in water ﬂow in the xylem is thought to be due to the appearance of breaks in the xylem tracheid wall membranes in the peripheral bundles (During et al. 1987;

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Phloem

Relative rates of water flow into the berry via xylem or phloem vascular elements

Xylem Berry formation

Berry size

Berry ripening

Engustment Veraison Lag phase Periods when compounds accumulate Malate

Tartrate

Sugar

Anthocyanin

Aroma compouds

80

100

Pericarp cell division Setting

0

20

40

60

120

Days after flowering Flowering

ºBrix 4

7

10

14

18

36

37

22

26

E-L 23

27

29

31

32

33

34 35

38

39

Seed size (mm)

5 Testa Nucellus

3

Endosperm Embryo 1 4 days

14 days

28 days

42 days

98 days

Fig. 7.2. Appearance of berries at 10-day intervals revealing the two successive sigmoidal growth curves of a grape berry, designate ‘‘berry formation’’ and ‘‘berry ripening’’. Three generalized x-axes are shown: days after ﬂowering, approximate juice Brix values during ripening, and developmental growth stages using modiﬁed E-L system. The key growth stages and the approximate timing of the accumulation of major solutes are shown. At bottom: Scale drawings of anatomical features in the longitudinal sections of developing grape seeds at days 4, 14, 28, 42 and 98 days after ﬂowering. (Source: Dry and Coombe 2004.)

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Findlay et al. 1987). It has been hypothesized that this rupture is due to the rapid expansion of the berry in the ﬁnal growth stage (During et al. 1987). However, Creasy et al. (1993) found that dye uptake was affected around the time of softening, which occurred before the rapid increase in growth. The hypothesis of xylem rupture has also been questioned lately with a new hypothesis of a loss of hydrostatic gradient in the berry apoplast causing the decrease in xylem ﬂow (Bondada et al. 2005). A rise in sugar accumulation has also been found to coincide with the decline in dye uptake, which is the suggested timing of xylem dysfunction (Creasy et al. 1993). The reduction in xylem ﬂow due to the dysfunction is thought to cause the resulting increase in phloem translocation in normal ripening fruit (During et al. 1987). This increase in phloem translocation may therefore result in the increase in sugar accumulation and also the change in cation accumulation in the berry (During et al. 1987). 1. Cation Accumulation. Potassium (K) concentration is initially high in the berry after anthesis but declines throughout Stage I, remains constant during the lag stage, and increases during Stage III after veraison (Hrazdina et al. 1984; Crippen and Morrison 1986; Morrison and Iodi 1990; Gutierrez-Granda and Morrison 1992). The potassium content per berry continues to increase through the ﬁnal stage of ripening, indicating that potassium movement into the berry is still possible (Esteban et al. 1999). Calcium (Ca) concentration increases up to veraison but steadily declines throughout Stage III, with the decline leveling off toward harvest (Hrazdina et al. 1984; Morrison and Iodi 1990; Creasy et al. 1993; Esteban et al. 1999). This decline in calcium concentration is due mainly to the increase in berry size and the cessation of calcium being imported into the berry, as calcium content per berry increases up until veraison, then remains largely constant throughout the ripening period (Hrazdina et al. 1984; Esteban et al. 1999). Magnesium concentration declines until veraison and then remains relatively constant throughout Stage III (Hrazdina et al. 1984), with berry magnesium content continuing to increase throughout this stage (Esteban et al. 1999). The decrease in xylem ﬂow, through either rupture of xylem vessels in the peripheral vascular system of the grape or a loss of hydrostatic gradient in the berry apoplast, is thought to be the reason that calcium inﬂux is reduced during ripening. The increase in potassium concentration after veraison is associated with the increased phloem translocation as, although potassium moves in the xylem and phloem,

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it is present in very high concentrations in the phloem sap (Mengel and Kirkby 1987). The potassium and calcium concentrations in the peduncle follow a similar pattern to that of concentrations in the berry, although potassium concentration decreases again close to maturity and calcium increases slightly. Magnesium concentration increases up until veraison, decreases rapidly, and then increases again toward maturity (Stellwaag-Kittler 1975). Cocucci et al. (1988) found that reduced berry number had no affect on potassium concentration in the rachis of the bunch, but calcium concentration does decrease with a reduction in berry number. 2. Plant Growth Substances. Auxin-like substances have been found at FB in the berry. Studies have found that they increase slowly until a maximum is reached at the end of Stage II and then decrease sharply (Alleweldt and Hifny 1972; Bhullar and Dhillon 1974; Zhang et al. 2003). Application of exogenous auxin substances before the onset of the second rapid growth stage (Stage III), such as benzothiazole-2oxyacetic acid (BTOA), prolongs the lag stage and therefore delays the onset of ripening (Hale 1968). Gibberellic acid (GA3) has not been found in the berry at FB but has been found to increase after FB to reach a peak before Stage II, then decrease until there was no gibberellic activity after Stage II (Bhullar and Dhillon 1974). Abscisic acid (ABA) has been found to be present before ﬂowering but then to decline progressively (Lilov and Angelova 1977) and then to be present in low concentrations until gibberellin and auxin concentrations begin to drop. After this, ABA has been found to increase slowly until Stage II (Bhullar and Dhillon 1974; Zhang et al. 2003). During Stage II, ABA concentration has been found to increase sharply to a peak after veraison, then decline again throughout Stage III until harvest (Coombe and Hale 1973; Bhullar and Dhillon 1974; Downton and Loveys 1978).

III. BUNCH STEM NECROSIS Bunch stem necrosis (BSN) incidence is dependent on grape cultivar, rootstock (Stellwaag-Kittler 1975; Theiler 1976; Scienza 1982; Boselli et al. 1983) and vineyard (Boselli et al. 1986), with some cultivars and vineyards demonstrating consistently high incidences of the disorder (Stellwaag-Kittler 1975; Jahnl 1983). Susceptible cultivars include ‘Cabernet Sauvignon’, ‘Riesling’, ‘Gewu¨rztraminer’, ‘Chasselas’, ‘Carignan’, ‘Grenache’, and the table cultivars ‘Muscat de Hambourg’ and

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‘Cardinal’. Less susceptible cultivars include ‘Merlot’, ‘Pinot’ cultivars, and ‘Sylvaner’ (Clement 1977; Clement 1978a). High-vigor rootstocks or those with a tendency toward magnesium deﬁciency, such as SO4, 161-49C, and 45-53M tend to show a higher BSN incidence (Delas et al. 1976; Clement 1978a; Lupton 1985). This correlation is consistent with the studies into magnesium and the incidence of BSN, which is discussed later. The incidence of BSN can also be higher in some seasons compared to others (Holzapfel and Coombe 1995). A. Symptoms Initial symptoms of BSN include the appearance of dark-brown sunken necrotic spots on the pedicel, rachis, or peduncle (Delas et al. 1976; Theiler 1976; Theiler 1986a). They can be of varying shape and size, and if they remain as small spots, are harmless (Stellwaag-Kittler 1983). These initial symptoms are called primary symptoms (Theiler 1976). However, if the necrosis spreads to girdle the entire stem, then secondary symptoms occur (Delas et al. 1976; Theiler 1976; Haub 1986). The ﬁrst cells to become necrotic are the stomata, epidermis, and hypodermis of the grape peduncle, pedicel, or rachis (Fig. 7.3) (Delas et al. 1976; Theiler 1976; Brendel et al. 1983). At the cellular level, it appears that the middle lamella is initially affected (Jahnl 1975). The necrosis then spreads to the cortical tissue and ﬁnally to the phloem cells in more severe cases (Theiler 1976; Brendel et al. 1983). Berries adjacent to the affected rachis show visual symptoms and are typically dull and opaque in appearance and soft in texture; juice

Fig. 7.3. Left: healthy stomata opening in the epidermis. Right: necrotized (dead) stomata opening of a rachis, where the primary symptoms of BSN can develop (Theiler 1975b).

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analyses shows that they lack sugar and ﬂavor due to disruption to the phloem cells (Delas et al. 1976; Theiler 1976; Theiler 1986a). If the necrosis spreads to girdle the entire part of the affected area, the rachis then may desiccate and may either abscise or remain on the bunch in a shriveled or completely dry condition (Delas et al. 1976; Jackson and Schuster 2001) (Figs. 7.4, 7.5, and 7.6). The tips of the rachis and wings (shoulders) are areas where this condition most frequently develops (Stellwaag-Kittler 1983). The pattern of necrosis development corresponds with the pattern of polyphenol distribution in the cortical parenchyma (Stellwaag-Kittler 1975), which may be the effect of the necrosis. Botrytis cinerea infection symptoms can also be confused with BSN, but there are many differences between the disorders (Delas et al. 1976). As Botrytis is caused by a fungus, often the conidia are visible (Delas et al. 1976; Pearson and Goheen 1988). Furthermore, the margins of the necrotic tissue caused by Botrytis are not as well deﬁned

Fig. 7.4. A healthy bunch (left) and BSN affected bunch (right). Necrosis of rachis and shriveling of berries can be seen in the lower three-quarters of the affected bunch.

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Fig. 7.5. BSN-affected bunch. The distal end is exhibiting BSN symptoms with rachis necrosis and shriveled berries. The demarcation between healthy and necrotized tissue is evident.

as in BSN and can also occur at any stage in the development of the berry (Delas et al. 1976; Pearson and Goheen 1988). However, often the presence of BSN then predisposes bunches to Botrytis infection and often both are present (Bolay et al. 1966; Delas et al. 1976). Before symptoms develop, BSN-affected berries cannot be distinguished from healthy berries by either their size or chemical composition. BSN-affected berries, however, expand at a much slower rate after veraison (Morrison and Iodi 1990). The rapid increase in the concentration of soluble solids in the ﬁrst two weeks after veraison in healthy berries is not seen in BSN-affected berries, but there is a slow increase later in development (Morrison and Iodi 1990). However, BSN-affected berries never reach the Brix concentrations of healthy berries (Osterwalder 1943; Ureta et al. 1981; Morrison and Iodi 1990). This trend is also seen in berry pH (Morrison and Iodi 1990) and in the phenolic compounds proanthocyanidins and anthocyanins

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Fig. 7.6. Necrosis and shriveled berries on a rachis branch in the middle of a bunch. As the necrosis has not girdled the primary axis of the rachis, berries distal to the branch are not exhibiting visual symptoms of BSN.

(Ureta et al. 1981). The decrease in titratable acidity is also delayed in BSN-affected berries, and concentrations never drop to that of healthy berries (Morrison and Iodi 1990). Tartaric acid concentrations do not drop in BSN-affected berries as in healthy berries (Ureta et al. 1981) due to the reduced expansion of the berry and therefore a reduced dilution effect (Morrison and Iodi 1990). However, there is a further accumulation of tartaric acid in BSN-affected berries not seen in healthy berries (Morrison and Iodi 1990). The concentration of potassium in the juice of BSN-affected berries does not increase as it would in normal berries (Morrison and Iodi 1990). Calcium concentration also increases greatly in BSN-affected berries (Morrison and Iodi 1990). Generally potassium and calcium concentrations in rachis samples increase throughout the season. Some studies have found little difference between susceptible and nonsusceptible cultivars (Cocucci et al. 1988)

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while others have found a resistant cultivar to have a higher potassium concentration in the rachis (Scienza 1982). However, the continued accumulation of calcium and reduced accumulation of potassium in BSN-affected berries is considered to be due to the normal loss of xylem ﬂow not occurring in BSN-affected bunches (Osterwalder 1943; Morrison and Iodi 1990). The slower, postveraison growth rate may be related to the lack of dysfunction. It has been hypothesized that there must be some physiological change in BSN-affected berries before softening, in order for the xylem to remain continuous (Creasy et al. 1993). Concentrations of certain plant growth regulators have also been studied to determine their concentrations in healthy and affected bunches. Ruiz and Moyano (1994) found that, in the peduncle and rachis of BSN-affected bunches, putrescine concentration was higher than in healthy bunches. In that study, putrescine concentration had an increasing gradient from relatively healthy tissue toward the necrotized area of the bunch. Although putrescine synthesis can occur from the decarboxylation of arginine, high concentrations of nitrogen are not considered to be the sole reason behind the high concentrations of putrescine (Ruiz and Moyano 1994), as potassium deﬁciency has been demonstrated to increase putrescine concentrations in plants (Davies 1995). The plant growth regulator ABA has also been implicated in BSN incidence. It is often higher in the peduncle of BSN-affected berries, although there appears to be no correlation between the incidence of BSN and ABA concentration in the peduncle (Holzapfel and Coombe 1997). ABA concentrations have also been found to be higher in the seeds of affected berries; ABA concentrations decreased in value from affected berries, to unaffected berries on affected bunches, to unaffected bunches on affected vines, with healthy bunches on healthy vines having the lowest ABA value (Broquedis and Bouard 1981). However, studies have shown that removing the berries after fruit set resulted in there being no symptoms of BSN (Scienza and Fregoni 1978; Theiler and Coombe 1985), and yet ABA concentrations in the rachis of bunches without berries reached higher values before veraison than bunches with berries (Scienza and Fregoni 1978). In the preceding studies, the concentrations of cations and plant growth regulators were assessed after the visual symptoms of BSN had begun to appear. Thus, it is likely that these changes are a consequence of the physiological disruption caused by BSN development rather than being the cause of the disorder.

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B. Possible Causes 1. Environmental. The severity of BSN varies dramatically not only among vineyards but also between seasons (Holzapfel and Coombe 1995). BSN is not caused by any pathogen (Stellwaag-Kittler 1975; Haub 1986) and, due to the apparent inﬂuence the environment has on the severity of BSN (Stellwaag-Kittler 1975), it is now generally accepted that BSN is a result of the grapevine reacting to its surrounding environmental conditions. These conditions include climate, site, soil management including fertilization, and pruning as well as other factors, such as cultivar and rootstock (Theiler 1975a). However, exactly what combination of these factors consistently leads to BSN is still not proven. Rain events at certain times have been associated with high incidences of BSN (Boselli et al. 1983). In France and Austria, a high frequency of rain events around veraison was found to be highly correlated with the incidence of BSN (Brechbuhler 1987; Redl 1987; Baldacchino-Reynaud 2000). However, in Australia, Germany, and Italy, this was not the case (Hartmair 1975; Boselli et al. 1986, 1987; Holzapfel and Coombe 1995). Theiler and Muller (1986) found that a high amount of rain over the ﬂowering period increased the incidence of BSN. In Greece, high incidences of BSN were associated only with high rainfall if it followed a long period of dry weather (Rumbos 1989). During rain events, temperatures usually drop and light levels are lower, both of which have been implicated in the incidence of BSN (Koblet et al. 1997). Studies in Germany and France have found a negative correlation between average midday temperature at ﬂowering (Theiler 1983, 1986b; Theiler and Muller 1986), or at the ﬁrst-growth phase of the berries, and BSN incidence (Boselli et al. 1987; Baldacchino-Reynaud 2000), but this was not found to be true in Australia (Holzapfel and Coombe 1995). In Australia, temperature in the 20 days before ﬂowering and the week over which veraison occurred had the highest inverse relationship with BSN incidence (Holzapfel and Coombe 1995). However, in Austria, where no correlation of average midday temperature around ﬂowering with BSN incidence was found, there was a relationship between average daily temperature and maximum daily temperature from ﬂowering to a berry diameter of 2 to 3 mm and BSN incidence. In contrast to other studies, this relationship was positive (Redl 1987). Perez and Gaete (1986) found that vines grown under shade conditions had signiﬁcantly more BSN incidence than those grown

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in full sunlight. Theiler and Muller (1986) also found a signiﬁcant inverse relationship between mean duration of sunshine during ﬂowering and BSN incidence. Pickering (2006) determined that it was during the three-week period immediately following ﬂowering that shade increased the incidence of BSN, both when the shade occurred in the current and in the previous growing season. This postﬂowering period was considerably more responsive to shade than shading during periods either prior to ﬂowering or closer to veraison. This susceptibility of the post-ﬂowering period was conﬁrmed in both ﬁeld and controlled-environment studies. Rain events also result in an increase in air humidity. Nicolli et al. (1977) concluded that high relative humidity affected BSN incidence more than either maximum or minimum temperature. Jordan (1985) found a twofold increase in the incidence of BSN associated with a twofold increase in relative humidity for vines grown in controlledenvironment rooms. However, when Pickering (2006) carried out an experiment similar to Jordan (1985), no correlation between relative humidity and BSN could be found. Leaf damage due to hailstorms has also been found to increase BSN incidence (Koblet et al. 1997). These various studies, although often contradictory, do indicate that environmental conditions around certain physiological events in berry development, particularly around ﬂowering, can inﬂuence the incidence of this disorder. 2. Mineral Nutrition in Vine and Berry. It has been hypothesized that BSN is the consequence of a deﬁciency in calcium and/or magnesium (Hartmair and Grill 1965; Haub 1986; Baldacchino-Reynaud 2000), and BSN has, therefore, been considered to be a disorder similar to other calcium-related disorders, such as bitter pit and watercore in apples and blossom-end rot in tomatoes and peppers (Boselli and Fregoni 1986; Haub 1986). Because high concentrations of potassium tend to compete negatively against the uptake of calcium and magnesium (Mengel and Kirkby 1987), it was hypothesised that high values of potassium would cause calcium and magnesium deﬁciencies. An imbalance between potassium, magnesium, and/or calcium in the rachis and leaf tissue often has been reported as the cause of BSN (Brechbuhler 1975; Donna 1985; Cocucci et al. 1988). Nahdi et al. (1993) found that BSN was related to an imbalance in the potassium/ magnesium (K/Mg) ratio and that an optimum ratio in the petiole at veraison was 4. Other studies have found no correlation between leaf nutrition and BSN incidence (Boselli et al. 1986; Pickering 2006), or

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only that it was able to be used for a prognosis of BSN at certain locations (Boselli et al. 1987). Hartmair (1975) found a positive correlation between the ratio of K/(CaþMg) in the peduncle and BSN, and stated that an increase in this ratio, due to a decrease in calcium or magnesium or an increase in potassium, resulted in an increase in BSN incidence. Other studies also have found that tissue from susceptible cultivars showed a higher K/Ca ratio than that in tissue from less susceptible cultivars (Cocucci et al. 1988). Also, healthy-appearing rachis tissue from diseased bunches showed a higher K/Ca ratio than healthy tissue from unaffected bunches of the same cultivar (Feucht et al. 1975). Lauber and Koblet (1967) found that at the ﬁrst signs of BSN, the bunch stems had higher K/Mg, K/Ca, and K/(CaþMg) ratios than healthy stems; in healthy tissue that had been treated with foliage sprays and had no signs of BSN symptoms, the ratios were much smaller. Feucht et al. (1975) also found the ratio of K/Ca to be high in tissue taken from rachis that were determined by an electron microscope to be developing BSN, even though visual symptoms had not appeared. The ratio was highest in the parenchyma cells of the cortex, but not as high in the xylem and on the surface of the rachis. The ratio became higher as the disorder progressed. However, Christensen and Boggero (1985) found that due to a reduction in potassium and consistent concentrations of calcium and magnesium, that the ratio of K/(CaþMg) was lower in the rachis of BSN affected bunches. Fregoni and Scienza (1975) found that a resistant cultivar, ‘Barbera’, consistently had a higher K/(CaþMg) ratio in the bunch stem than the susceptible cultivar, ‘Bonarda’. Shin et al. (1984) found that in the two cultivars they were investigating, all three ratios of K/Ca, K/Mg and K/(CaþMg) were higher in healthy vines compared to vines with BSN. Yet other studies have found no correlation between nutrient content of the peduncle and rachis and the occurrence of BSN (Claus 1965; Redl 1983; Pickering 2006). Potassium. Jordan (1985) reported that vines with BSN demonstrated a higher potassium concentration in the leaf tissue than vines without BSN. However, it was the rate at which potassium accumulated that was better correlated with BSN incidence: a high rate resulted in high BSN incidence (Jordan 1984). However, Spring et al. (1999) investigated potassium concentration in the leaf tissue of two cultivars of varying BSN incidence; the cultivar with higher BSN incidence did not consistently demonstrate higher leaf potassium concentration across all rootstocks studied compared to the other cultivar.

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Christensen and Boggero (1985) did not ﬁnd a correlation with the concentration of potassium in the petiole and BSN incidence. Shin et al. (1984) found that in one cultivar, the potassium concentration in the petiole was higher in vines with BSN, and yet in another cultivar, the potassium concentration was lower in vines with BSN. However, when looking at the potassium concentration in the rachis, Shin et al. (1984) found that in both cultivars, the potassium concentration was lower in BSN-affected bunches. Other studies (Scienza 1982; Christensen and Boggero 1985; Ruiz and Moyano 1994) have also found that potassium concentrations were lower in the rachis of BSN-affected bunches, whereas Redl (1983) found the opposite with potassium concentration being higher. Scienza and Fregoni (1978) also found that in the rachis of bunches without berries and that did not demonstrate BSN, potassium concentration was lower; therefore, potassium concentration was higher in the BSN-affected bunches. Some research has found that potassium concentration is lower in BSN-affected berries (Osterwalder 1943; Morrison and Iodi 1990), but Scienza (1982) found that a cultivar resistant to BSN had lower potassium concentration in the berry juice compared to a susceptible cultivar. The results from these various studies demonstrate that there is no consistent relationship between potassium concentration in the leaves, petioles, rachis, or berries and the incidence of BSN across all cultivars and rootstocks. Conclusions have ranged from stating that there is a positive relationship to stating a negative relationship between potassium concentration and the incidence of BSN. Calcium. Early in the study of BSN, it was thought that disease incidence was due to a deﬁciency of calcium in cells (Hartmair and Grill 1965; Alleweldt and Hifny 1972), as BSN symptoms are similar to those of other calcium deﬁciency symptoms (Schaller 1983). High calcium concentrations have also been found in leaves from cultivars that are less susceptible to BSN compared to more susceptible cultivars (Spring et al. 1999). Wood from affected vines also had lower calcium concentrations than that from healthy vines (Hartmair and Grill 1965). However, Christensen and Boggero (1985) did not ﬁnd a correlation between petiole calcium concentration and BSN incidence. At veraison, unaffected berries and resistant cultivars have slightly higher concentrations of calcium than BSN-affected berries and susceptible cultivars (Scienza 1982; Morrison and Iodi 1990). Berry respiration rate of less susceptible cultivars can also be higher, which is hypothesized as the reason the calcium concentration was higher (Boselli and Volpe 1990). Foliar fertilizers that contain magnesium and

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reduce BSN incidence have also been found to increase the calcium content of the rachis (Redl and Weindlmayr 1985). On bunches where the berries had been removed and BSN symptoms did not occur, the calcium concentration in the peduncle was also higher than on bunches with berries and BSN symptoms (Scienza and Fregoni 1978). Calcium concentration was also found to be higher in berries containing more seeds (Boselli et al. 1995). Haub (1986) stated that ‘‘diseased stalks’’ have 20% less calcium on average than healthy ones. However, in an extensive survey involving many cultivars, Redl (1983) did not ﬁnd a correlation between rachis calcium concentration and BSN incidence, but did ﬁnd higher calcium concentrations in the peduncles of affected bunches. Christensen and Boggero (1985) also found no relationship between rachis calcium concentration and BSN incidence. Boselli et al. (1995) found that calcium concentration was higher in a cultivar that was more susceptible to BSN incidence (‘Croatina’) when compared to a cultivar that was less susceptible (‘Barbera’), yet Scienza (1982) found the exact opposite. Therefore, like the results for potassium, those for calcium concentration are inconsistent, and no conclusive relationship between calcium concentration and BSN incidence has been found. Magnesium. Some studies have shown that BSN-affected bunches tend to have lower concentrations of magnesium in the rachis and the vines with affected bunches also exhibiting lower magnesium values in the leaf blade (Redl 1983; Spring et al. 1999). Spring et al. (1999) showed that a cultivar that consistently demonstrated high BSN incidence (‘Chasselas’) had lower leaf magnesium values than the cultivar ‘Gamay a` Pully’, which consistently had low BSN incidence. However, Brechbuhler and Meyer (1988) found that although applications of nitrogen to the soil also increased magnesium concentrations in the leaves, the incidence of BSN also increased. Other studies have found no correlation between magnesium concentration in the leaves and leaf petioles and BSN incidence (Jordan 1984; Christensen and Boggero 1985). Haub (1986) reported 40% lower magnesium concentration in BSNaffected bunch stems compared to healthy ones. Scienza (1982) found that a susceptible cultivar also had lower magnesium concentrations in the rachis compared to a resistant cultivar. The uptake of magnesium from a solution also appears to be cultivar dependent, with the more susceptible cultivars having a lower capacity to take up magnesium (Schimansky 1983). Applications of foliar fertilizers that decrease BSN have also been found to increase magnesium content in the rachis. On

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bunches where berries had been removed and no BSN symptoms occurred, magnesium concentrations in the peduncle were higher than in peduncles with berries and exhibiting BSN symptoms (Scienza and Fregoni 1978). Christensen and Boggero (1985) found no correlation between magnesium concentration in the rachis and BSN incidence. Therefore, the majority of studies do demonstrate a relationship where low magnesium concentrations in the rachis and peduncle are associated with a higher incidence of BSN. However, it appears likely that leaf and petiole magnesium concentrations are not related to BSN incidence and therefore are not good indicators for determining the cause of this disorder. Nitrogen. When comparing two cultivars with differing BSN incidence, Spring et al. (1999) found that the cultivar with the lower incidence (‘Gamay a` Pully’) also demonstrated higher nitrogen concentrations in the leaves than the cultivar with the higher incidence (‘Chasselas’). However, Jordan (1985) reported that vines with a high incidence of BSN had higher nitrate values in leaf tissue than vines without BSN; yet in the season before, Jordan (1984) found no such evidence. Brechbuhler and Meyer (1988) also found that an increase in leaf nitrogen concentrations due to nitrogen application to the soil increased BSN incidence. Christensen and Boggero (1985) found highincidence areas to have higher petiole nitrogen than low-incidence areas. Ammonium (NH4) concentrations in the rachis have been shown to be higher in BSN-affected bunches (Redl 1983; Christensen and Boggero 1985; Ruiz and Moyano 1994). Foliar-applied substances that decrease the incidence of BSN have also been found to decrease the berry nitrogen content. However, high nitrogen concentrations in the rachis and berry are not always found to be associated with BSN (Ruiz and Moyano 1993; Capps and Wolf 2000). Holzapfel and Coombe (1997) found a negative correlation between released-NH4 and BSN but no relationship between free-NH4 and BSN. Christensen and Boggero (1985) did not ﬁnd a correlation between nitrate and BSN incidence. Due to the conﬂicting results among studies, it was suggested that high ammonium values in BSN-affected rachis may in fact be the result of some dysfunction of nitrogen metabolism associated with BSN (Ruiz and Moyano 1993) or a secondary effect related to the senescence of the peduncle tissue (Keller and Koblet 1995). Therefore, as with potassium and calcium concentrations, there appears to be no clear, consistent relationship between nitrogen concentrations in the leaves, rachis, and berries and BSN incidence.

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Increasing the organic matter in the soil has also been found to reduce the incidence of BSN (Frisullo and Faretra 2003), as has growing a ground cover between rows rather than leaving the soil bare (Koblet and Lauber 1968; Perret and Koblet 1973; Hinkel 1992). 3. Plant Growth Regulators. The plant growth regulator ABA has been shown to be an important hormone in the process of senescence in plants. This and the fact that high ABA concentrations have been found in the peduncle and berries of BSN affected bunches have led to the hypothesis that BSN may be caused by an imbalance in hormone metabolism during the ripening of the berries (Baldacchino et al. 1987a). 4. Canopy Development. Vine canopy development and the training systems used to grow vines have been shown to inﬂuence the incidence of BSN. Early in the investigations into BSN incidence, it was reported that BSN was more prevalent in vigorous vines that had been over pruned, overwatered, overfertilized with nitrogen or potassium, or had heavy crop loads (Bolay et al. 1965; Delas et al. 1976; Cline 1987). Later, Moreno and Pavez (2000) found a positive correlation between high leaf layer number (LLN), low canopy gaps, and excessive growth during the veraison to harvest period and the incidence of BSN. However, other studies (Pickering et al. 2004; Pickering 2006) found that it was the vine growth in the three weeks immediately after ﬂowering that affected the incidence of BSN, and possibly the measures of vigor later in bunch development were an indication of what had occurred earlier. Theiler (1975b) also found that vigorous vine growth increased BSN incidence while weak growth reduced it. Training systems that prune to a high trunk height have resulted in high BSN incidence (Hifny 1971; Redl 1984), as have those training systems that encourage the vigor of individual canes (Hifny 1971; Redl 1984). Brendel and Hofmann (1983) found that by decreasing dormant bud number on canes, BSN incidence increased in cultivars ‘Riesling’, ‘Thurling’, and ‘Ehrenfelser’. However, they also found that increasing dormant bud number on canes increased BSN incidence for the cultivars ‘Kanzler’, ‘Optima’, and ‘Scho¨nburger’. Reports on BSN published in the 1970s suggested the use of training systems to reduce vine vigor and therefore BSN incidence (Clement 1978b). Holzapfel and Coombe (1995) found that vines that were spurpruned developed considerably more BSN than vines that were minimally pruned. Redl (1988) found that a training system traditionally used in Austria that resulted in an improved leaf exposure decreased the incidence of BSN on ‘Green Veltliner’. This training

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system also resulted in larger leaves, increased leaf area, and longer shoots, although it may not necessarily have resulted in a more vigorous canopy. Severely root pruning vines, which resulted in a signiﬁcant decrease in vine vigor, also resulted in a signiﬁcant decrease in BSN incidence (Pickering et al. 2004). Pickering (2006) found that this reduction in vine vigor and BSN incidence persisted for the three years of that particular study. Some studies have found BSN to be more prevalent on vines with less vigorous shoots (Scienza and Fregoni 1978; Song et al. 2003). Shin et al. (1984) also found similar results for the two cultivars ‘Golden Queen’ and ‘Himrod Seedless’. Becker (1990) found the vigorous cultivar ‘FR946-60’, which is a cross between European and American vines, to be resistant to BSN. The number of bunches per vine has been found to be inversely correlated with BSN incidence (Nicolli et al. 1977), especially when training systems were compared (Holzapfel and Coombe 1995). However, this correlation was not robust when the year susceptibility was compared with the crop loads in those years (Holzapfel and Coombe 1995). Some studies have also found that defoliation around the bunches, a common practice in New Zealand around veraison time to control Botrytis, actually increases the incidence of BSN (Koblet et al. 1969). Therefore, although high vigor has been positively correlated with a high incidence of BSN, the results are not always consistent. However, the description of ‘‘vigor’’ is not always precise, and the time that the vigor of the vines is measured is not always stated or consistent across all studies. 5. Xylem Development. During and Lang (1993) found that in BSNsusceptible cultivars, the xylem development was suppressed just distal to each node of the peduncle, creating a restriction in the xylem system. The cross-sectional area of the xylem was reduced, and only small primary vessels were present in this area. Cultivars that were less susceptible did not demonstrate such a limitation. The restriction demonstrated a high hydraulic resistance that in turn would affect the transport of nutrients through the xylem. From this work, they developed a screening procedure for new cultivars to determine if they may be susceptible to BSN or not. During and Lang (1993) also found that the extent of xylem development close to the node was generally less in the distal and lateral branches, which are areas more prone to BSN (compared to the proximal branches of a bunch).

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In trials investigating how ﬂowers and berries inﬂuence peduncle health, it was found that removal of ﬂowers resulted in the death of the peduncle, but removal of set berries did not have this effect. Peduncles with berries removed stayed green and turgid until the end of the growing season, and symptoms of BSN did not develop (Theiler and Coombe 1985). The ﬁnal cross-section of deberried peduncles was less than that of attached berry peduncles. The peduncle obtains 75% of its ﬁnal cross-sectional area by the middle of ﬂowering (Theiler and Coombe 1985), and therefore it appears that the presence of the berry inﬂuences xylem development. Theiler and Coombe (1985) found that by applying GA3 to the peduncle during fruit set, not only did the peduncle remain healthy with no symptoms of BSN, but the area of the metaxylem was also signiﬁcantly increased. Another plant growth regulator, a-naphthalene acetic acid (NAA), when applied to deﬂowered bunches had the same effect as GA3 in that peduncles remained healthy; when applied to intact bunches, however, it did not decrease the incidence of BSN as GA3 did. It also did not increase the area of the metaxylem (Theiler and Coombe 1985). Therefore, it may be a plant growth regulator, or a combination of plant growth regulators, produced in the berry that inﬂuences xylem development and also the incidence of BSN. C. Application of Various Minerals and Plant Growth Regulators 1. Potassium. Sandy soils fertilized with potassium demonstrated higher values of BSN in France (Baldacchino-Reynaud 2000), yet Cooper et al. (1987) found no effect on BSN incidence by applying potassium to soils in Chile. Jordan (1985) also found no effect of high potassium in a nutrient solution on BSN incidence. In Italy, the practice of plowing in organic matter increased the exchangeable potassium in the soil but reduced the incidence of BSN (Frisullo and Faretra 2003). Applying potassium directly to the bunch by spraying potassium chloride (KCl) does signiﬁcantly increase berry potassium concentration, although there is no effect when potassium nitrate (KNO3) is used instead (Failla et al. 1996). Application of substances containing potassium sulphate (K2SO3) have been found to increase the incidence of BSN (Rumbos 1989). As studies into potassium concentration and BSN incidence do not demonstrate a consistent relationship, it is not surprising that studies into the application of potassium and this disorder are also not consistent.

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2. Calcium. Some studies have shown that application of calcium, either as a soil fertilizer or as a foliar spray, can reduce the incidence of BSN in some cultivars (Stellwaag-Kittler and Haub 1964; Cline 1987). Hartmair and Grill found that the application of lime reduced the incidence of BSN (Hartmair and Grill 1965). Applying calcium chloride (CaCl2) either by itself or in conjunction with magnesium chloride (MgCl2) can also reduce BSN incidence (Lauber and Koblet 1967). Application of calcium to the bunch by spraying CaCl2 signiﬁcantly increases berry calcium concentration (Failla et al. 1996). However, the effectiveness varies from year to year, and in some cases and some cultivars, has no control over the disorder (Jordan 1985; Cline 1987; Capps and Wolf 2000). Christensen and Boggero (1985) also referenced work that found that calcium nitrate (Ca(NO3)2) sprays increased the incidence of BSN. They also found that dipping bunches in various solutions containing calcium did not affect the incidence of BSN. As with potassium, the inconsistencies within these studies are similar to the inconsistencies within studies of tissue calcium. 3. Magnesium. Many studies have found that foliar application of substances containing magnesium can reduce the incidence of BSN (e.g., Lauber and Koblet 1967; Koblet et al. 1969; Brechbuhler 1975, 1991; Schaller 1977; Haub 1983; Cline 1987). Application of magnesium can occur through many different products, but magnesium sulfate (MgSO4) and magnesium nitrate (Mg(NO3)2) have been found to be very effective (Beetz and Bauer 1983; Fabre et al. 1983; Rumbos 1989). Jordan (1985) reported that spraying bunches with magnesium ﬁve times from berry set to veraison achieved good control of BSN. Good control was also achieved with just two applications near veraison (Theiler 1979; Beetz and Bauer 1983; Haub 1986), although Theiler (1979) recommended an additional spray after bloom in the case of persistent severe occurrences. Better control of BSN was obtained when magnesium sprays were directed at the bunches rather than the entire vine (Theiler 1980; Haub 1986), as foliar sprays have been found to be ineffective. However, Christensen and Boggero (1985) referenced work that found Mg(NO3)2 sprays increased the incidence of BSN. They also found that dipping bunches in various solutions containing magnesium had no effect on the incidence of BSN. The presence of calcium or potassium in some solutions has been found to impede the uptake of magnesium, but this was counteracted by increasing the amount of magnesium in the solution or by spraying the bunches more often (Schimansky 1983).

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Overall however, these studies are in agreement with those carried out on tissue magnesium and the incidence of BSN, where low rachis and berry magnesium concentrations were correlated with a high incidence of the disorder. 4. Nitrogen. Results concerning the application of nitrogen on the incidence of BSN are as conﬂicting as the results obtained from applications of other mineral nutrients. Studies have shown that the application of nitrogen or high nitrogen in the soil solution around bloom time increases BSN incidence (Gysi 1983; Christensen and Boggero 1985; Brechbuhler and Meyer 1988; Perret et al. 1994; Keller et al. 2001). The application of di-ammonium phosphate ((NH4)2HPO4) directly to the bunches has also been found to increase BSN incidence (Christensen and Boggero 1985). However, Capps and Wolf (2000) found that the application of nitrogen fertilizer, including around bloom time, increased veraison rachis nitrogen concentration and decreased BSN incidence. They also found that in years with elevated tissue nitrogen, BSN incidence was low. 5. Plant Growth Regulators. The application of gibberellins (GAs) around ﬂowering to bunches or directly to the rachis has been shown to reduce BSN incidence (Alleweldt and Hifny 1972; Beetz and Bauer 1983; Haub 1983; Theiler and Coombe 1985; Pickering 2006). Application before and during ﬂowering resulted in small or ‘‘shot’’ berries, but application during fruit set affected fruit development only slightly (Theiler and Coombe 1985). Pickering (2006) found that application of GA3 immediately after fruit set reduced the incidence of BSN and increased berry size. Hifny (1971) also found that GAs applied 43 days after ﬂowering reduced BSN incidence. Therefore, timing of gibberellin application for BSN control is important in order to reduce the possible negative side effects. Some studies have shown that the auxins NAA, b-indole acetic acid (IAA), and 3-indolebutyric acid (IBA) have no affect on BSN incidence (Theiler and Coombe 1985; Pickering 2006). However, other studies found that IAA reduced BSN, but not to such an extent as GAs (Hifny 1971; Alleweldt and Hifny 1972). NAA reduced the amount of secondary symptoms but not primary. Cooper et al. (1987) also found a reduction in BSN incidence due to NAA, although this was not statistically signiﬁcant. Pickering (2006) found that the application of naphthylphthalamic acid (NPA), an auxin transport inhibitor, did not affect the incidence of BSN any differently from the application of IAA. In an experiment where ﬂowers were removed and various plant

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growth regulators were applied to determine the effect on peduncle death, Theiler and Coombe (1985) found that the application of NAA and GA3 prevented peduncle death after deﬂowering, and GA3 increased peduncle thickening by increasing the cross-sectional area of metaxylem. This thickening occurred no matter when the GA3 was applied. Alleweldt and Hifny (1972) found that the application of GA3 caused the development of numerous, nonligniﬁed xylem cells. They also found that GA3 in combination with IAA gave rise to robustly walled parenchyma cells, sclerenchyma cells, and ligniﬁed metaxylem. Holzapfel and Coombe (1998) found that certain substances can cause the appearance of necrotic tissue similar to that found in BSN. Agmatine had the highest potency for inducing BSN. Ammonium sulfate ((NH4)2SO4), BTOA, ABA-M (a 50/50 mixture of ABA and t-ABA), and ABA all induced BSN symptoms to the same degree, though less than agmatine. All except (NH4)2SO4 caused large increases in the concentrations of ABA extracted from bunch stems, and it was therefore thought that ABA may play an important role in inducing BSN. However, the correlation between extracted ABA and BSN incidence was not strong. Baldacchino et al. (1987c) also found that application of ABA around veraison at concentrations of 25 mM and above induced symptoms similar to that of BSN. The concentration of ABA required to induce BSN in a susceptible cultivar such as ‘Cabernet Sauvignon’ was lower than that required in a more tolerant cultivar, such as ‘Merlot’ (Baldacchino et al. 1987b). A strong positive correlation between BSN incidence and putrescine in bunch stems has been found by Ruiz and Moyano (1994). Ruiz et al. (2004) also found similar results in table grapes demonstrating the ‘‘soft berry’’ problem. Holzapfel and Coombe (1998) hypothesized that the effect of agmatine in inducing BSN was not due to an increase in free ammonium ion concentrations but rather the catabolism of ammonia to putrescine.

IV. EARLY BUNCH STEM NECROSIS As with BSN, EBSN is not caused by a fungal infection and is considered to be a physiological disorder (Jackson and Coombe 1988). Also, the severity of EBSN appears to be cultivar and site dependent as in BSN (Jackson and Coombe 1988). These cultivars have been shown to be susceptible to EBSN (in decreasing order of susceptibility): ‘Alicante’, ‘Brown Muscat’, ‘Muscat Hamburg’, ‘Muscat Ottonel’,

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‘Queen of the Vineyard’, ‘Black Hamburg’, and ‘Italia’. However, ‘Malbec’, ‘Pinot Noir’, ‘Merlot’, and ‘Meunier’ have also demonstrated a susceptibility to EBSN. Cultivars that are reported as having low incidence include ‘Schuyler’, ‘Cabernet Sauvignon’, ‘Pearl of Csaba’, ‘Fiesta’, ‘Chasselas’, ‘Flora’, ‘Cardinal’, ‘Riesling’, ‘Chardonnay’, and ‘Pinotage’ (Jackson and Coombe 1988). In addition, studies into EBSN have used ‘Riesling’, ‘Cabernet Sauvignon’, and ‘Gewu¨rztraminer’, all of which demonstrated EBSN symptoms in those trials (Jackson 1991). A. Symptoms Initial symptoms of EBSN begin as the pedicel elongates and include individual ﬂower bud drop with the pedicels still attached (Jackson and Coombe 1988). Brown necrotic areas can be seen at the base of the pedicels on the ﬂowers that have dropped off (Jackson 1988), and this is what appears to be the cause of the ﬂower bud drop, not the formation of an abscission zone (Jackson and Coombe 1988). Later entire sections of the inﬂorescence may shrivel and die and may or may not drop off (Jackson 1988; Jackson 1994). B. Possible Causes Much less wosrk has been carried out to investigate the possible causes of EBSN. However, some studies have shown that stress, whether water or nutrient stress, prior to ﬂowering has increased the incidence of EBSN (Jackson 1988, 1991). Low nitrogen availability during bloom has been reported to increase EBSN incidence in ‘Cabernet Sauvignon’ (Keller and Hrazdina 1997) but not in ‘Mu¨ller-Thurgau’ grapevines (Keller and Koblet 1994, 1996). Jordan (1989) found a positive relationship of ammonium ion content and the severity of EBSN. However, Keller and Koblet (1994) did not ﬁnd a relationship between nitrogen supply and EBSN and therefore concluded that the substantial amounts of ammonia could not have been derived from excessive nitrogen uptake and reduction. Instead, they hypothesized that the excessive amounts of ammonia may have been from increased glutamate dehydrogenase activity due to the remobilization of carbohydrates during periods of reduced carbohydrate availability. Light intensity appears to be implicated in the development of EBSN, as it has been shown to increase as light intensity decreases (Jackson 1988, 1991; Keller and Koblet 1994). Trials have found that 50% shading doubles the incidence of EBSN compared to unshaded vines, and at 90% shading, all bunches showed EBSN symptoms

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(Jackson and Coombe 1988). Low light conditions of 30 mmol m2 s1 have been reported to induce EBSN in ‘Mu¨ller-Thurgau’ grapevines, although moderate light conditions of 140 mmol m2 s1 did not (Keller and Koblet 1996). EBSN also appears to be more prevalent in the central part of the vine above the trunk, especially when this area is heavily shaded (Jackson and Coombe 1988). As with BSN, high-vigor vines also tend to increase the incidence of EBSN; this is thought to be due to an increase in shading around the inﬂorescence (Jackson and Coombe 1988). However, severely decreasing the number of leaves also increases the incidence of EBSN (Jackson 1991). It has also been observed that more EBSN occurs on cooler sites at high altitudes compared to warmer sites (Jackson and Coombe 1988). Other factors thought to predispose vines to EBSN are root restriction and precipitation (Jackson 1994). C. Application of Various Minerals and Plant Growth Regulators 1. Nutrients. Unlike BSN, where the application of calcium chloride to bunches occasionally has been found to decrease the symptoms of the disorder, some trials have found that when applied before ﬂowering, calcium chloride actually can induce symptoms indistinguishable from EBSN (Jackson and Coombe 1988). Diammonium phosphate has also been found to produce not only BSN-like symptoms, as already discussed, but also EBSN-like symptoms if applied before ﬂowering (Jackson and Coombe 1988). With both calcium chloride and diammonium phosphate, the cations calcium and ammonium respectively were found to be the causal agents in developing the EBSN symptoms, not the anion components (Jackson and Coombe 1988). In further studies where ammonium nitrate (NH4NO3), ammonium sulphate ((NH4)SO4), potassium nitrate (KNO3), and potassium sulphate (K2SO4) were applied to try to induce symptoms of EBSN, it was found that the NO3, SO42, or Kþ ions were not associated with the development of EBSN symptoms and that therefore it was the NH4þ ion that is involved in the development of EBSN (Gu et al. 1994). It was suggested that the cations were entering the tissue and causing physiological effects the same as or similar to EBSN, as a burning effect of the cations on the young tissue was discounted after further study (Jackson and Coombe 1988). 2. Plant Growth Regulators. Ethephon is a chemical that releases ethylene and is used to stimulate ripening in fruit. In some studies, it has been found to enhance berry retention if applied to the top 10 to

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20 cm of vine shoots, but if used incorrectly, it can increase the incidence of EBSN (Jackson 1994). Jackson (1991) found that EBSN incidence increased after applying Ethephon to vines prior to and during 20% to 30% ﬂowering. There was some evidence that applying Ethephon later, and only to the leaves, may not increase the incidence to such a degree as application during the early development of the inﬂorescence (Jackson 1991). The application of IAA, NAA, benzyladenine, and GA3 has been reported to not affect the incidence of EBSN (Jackson 1991). Although much of the literature indicates similarities between BSN and EBSN, it appears that impacts of various substances and the timing of their effectiveness actually may differ between the two disorders. However, work in either disorder may give further insight into the other.

V. SUMMARY AND CONCLUSIONS BSN and EBSN are both physiological disorders. BSN is detrimental to grape quality as berry ripening is retarded and berry quality compromised, while EBSN reduces berry set and therefore crop load. Symptoms of BSN include dark necrotic spots on the peduncle, rachis, or pedicel of the bunch post-veraison, and berries become typically dull and opaque in appearance and soft in texture. Lesions may grow to girdle the entire affected area of a pedicel or rachis; if this occurs, desiccation of berries distal to the necrotic tissue may follow with shriveled berries either abscising or remaining on the bunch. Sugars and pH in berry juice remain low and acid concentrations remain high, resulting in berries not attaining commercial maturity. There is further accumulation of calcium, but the accumulation of potassium that usually accompanies berry ripening does not occur. It is thought that this is due to xylem dysfunction not occurring as is surmised to occur in healthy bunches, and therefore xylem and phloem ﬂows remain at concentrations found pre-veraison. Putrescine and ABA are also higher in BSN-affected berries compared to healthy berries. Symptoms of EBSN include brown, necrotic areas that can be seen at the base of the pedicels and begin to appear as the pedicel elongates and up until berry set. Entire sections of the inﬂorescence may shrivel and die and may or may not drop off. Susceptibility to both BSN and EBSN depends on cultivar, rootstock, vineyard, and season. Low temperature, rain, and shade at varying times during bunch and inﬂorescence development, respectively, have

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all been associated with the disorders, although different publications often conﬂict. Nonetheless, Pickering (2006) clearly demonstrated that the period immediately following ﬂowering is the most critical development time in the predisposition to BSN. Shade imposed in the ﬁeld and placement of vines in controlled-environment conditions during the early stages of the ﬁrst phase of berry development signiﬁcantly increased the incidence of BSN, while application of GA3 to the bunch and a reduction in vine growth during this time could reduce the incidence. During this early phase in berry development, the number, size, shape of cells, and cell wall properties in the ﬂesh and skin are determined; development of the phloem, parenchyma, and collenchyma in the peduncle and rachis continues; and most of the seed development occurs. Therefore, it is clear that factors adversely affecting cell division and expansion in the peduncle, rachis, berry, and/or seed during this early stage may have detrimental effects on ﬁnal berry quality and the incidence of BSN. Pickering (2006) hypothesized that it was competition between vegetative and reproductive growth during the early stage of berry development that inﬂuenced the incidence of BSN and factors that favored vegetative growth immediately after ﬂowering would predispose bunches to BSN. Whether the competition between the two competing sinks was for nutrients or carbohydrates was not determined. However, evidence suggests that EBSN is a result of a depleted carbohydrate supply (Keller and Koblet 1994, 1995). Inconsistencies among BSN studies may be the result of varying vine responses to a combination of environmental factors that result in competition between reproductive and vegetative sinks. For example, in studies where rain pre-ﬂowering was found to increase BSN incidence, it may be the increase in vegetative growth post-ﬂowering, and therefore the increase in competition between vegetative and reproductive sinks, that increased the incidence of BSN. It has been hypothesized that BSN is a calcium and/or magnesium deﬁciency, and high ratios of K/Ca, K/Mg or K/(CaþMg) have been implicated in BSN development. Nitrogen concentration is also thought to lead to BSN and EBSN; however, again, the literature on these relationships with both BSN and EBSN is conﬂicting. Applications of magnesium and/or calcium have been found to decrease BSN, although the success with soil and foliage applications often has been limited. Sprays directed at the bunch alone have proven to be most effective, although, again, the literature is conﬂicting with varying results in different vineyards, in different seasons, and on different cultivars. However, where sprays target the bunch area

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directly, nutrients may be delivered to the areas where they are required sooner than when applied to the soil or foliage and therefore be more likely to counteract any subsequent deﬁciency impacts. Conversely, application of calcium chloride has been found to increase the incidence of EBSN. Application of nitrogen has in some cases increased BSN, while in others it has decreased BSN incidence. Studies have hypothesized that, in the case of EBSN, it is the NH4þ ion that causes EBSN development, probably as a result of an imbalance between nitrogen and carbohydrate concentrations rather than due to excessive amounts of nitrogen (Keller and Koblet 1994, 1995). Depleted or reduced carbohydrate concentrations rather than just competition for carbohydrates between vegetative and reproductive sinks may also contribute to predisposing bunches to BSN in some cases, which may explain the conﬂicting results between nitrogen and BSN incidence among studies. Conﬂicting nutrient results may also be due to the timing of sampling, which in most studies on BSN tend to be around veraison. By this time, although not physically obvious, BSN may already have begun to affect nutrient concentrations in the bunch; therefore, any correlations found are most likely to be a consequence and not a cause of BSN. If BSN is the result of a nutrient disorder, then measurement of nutrient concentrations and ratios around the critical time identiﬁed by Pickering (2006) is likely to provide a better understanding of what may be occurring. Analyzing the nutrient concentrations in speciﬁc zones of tissues within the bunch may also provide a clearer indication as to whether BSN is the result of a nutrient disorder because bulk sampling may mask any possible deﬁciencies. The application of ABA directly to bunches has caused BSN-like symptoms while some auxin-like substances can decrease BSN incidence. The application of gibberellins have also reduced BSN incidence, although at certain times in berry development, this application may be detrimental to both fruit set and berry development. It is thought that the decrease in BSN incidence due to gibberellin application is through increased xylem differentiation (Theiler and Coombe 1985). More susceptible cultivars tend to have reduced xylem formation and therefore function, creating a restriction in the xylem for substances being transported into the developing bunch. It is reported that seeded berries do not respond to applications of gibberellins; it is thought that seeded cultivars have sufﬁcient endogenous gibberellin for full development (Coombe 1973). However, seeded cultivars have responded to the application of gibberellin (Beetz and Bauer 1983; Theiler and Coombe 1985;

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Pickering 2006), which possibly suggests that these cultivars may have suboptimal concentrations of gibberellins after anthesis and hence the reduction of BSN when exogenous gibberellin is applied. As gibberellins increase cell division and expansion, a lack of endogenous gibberellin may lead to a reduction in cell division in the young fruit, resulting in low potential sink activity and the inability to compete effectively against vegetative sinks. Pickering (2006) hypothesized that not only did certain events have to occur immediately after ﬂowering to predispose bunches to BSN, but certain conditions around veraison also had to occur in order for the disorder to manifest. Gibberellin concentration has been found to decrease during anthesis and then increase again until the beginning of the lag stage (Zhang et al. 2003). Therefore, if susceptible cultivars generally have a low concentration of gibberellins, once the bunches are predisposed to BSN, the continued suboptimal concentrations of gibberellin may result in a higher manifestation of BSN. Thus, application of exogenous gibberellin before veraison would reduce BSN incidence, which has been found (Hifny 1971; Alleweldt and Hifny 1972). The lack of effect of IAA, NAA, and GA3 on the incidence of EBSN is in contrast to some studies on BSN. However, this may be a result of how these hormones act within the developing inﬂorescence. Ethephon, which releases ethylene, has, however, been shown to increase EBSN if applied prior to or early on during ﬂowering, and application to the entire vine rather than just the leaves appears to increase the incidence of EBSN (Jackson 1991). If reduced carbohydrate supply is a causal factor for EBSN, Ethephon may be reducing the carbohydrate supply to the developing inﬂorescence by either reducing vegetative growth too much before ﬂowering and therefore reducing the overall supply of carbohydrates, or reducing the sink strength of the inﬂorescence for carbohydrates when the chemical is applied to the entire vine including the developing inﬂorescence. Although less work has been carried out on EBSN, it appears that identifying the underlying mechanism behind the development of this disorder may be closer at hand. Due to the similarity of the two disorders, this fact may indicate that work into BSN may have been investigating the wrong areas or that these two disorders are less alike than was originally assumed. However, the manifestation of EBSN symptoms occurs much earlier in the development of the bunch. Therefore, there is a shorter time frame in which factors can inﬂuence the incidence of EBSN and thus a shorter time frame in which research into this disorder needs to concentrate. Narrowing down the time

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frame for the predisposition of BSN to the postﬂowering period is clearly a distinct advance in the understanding of this disorder and the further direction of new research into its control.

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Scholeﬁeld, P.B., and R.C. Ward. 1975. Scanning electron microscopy of the developmental stages of the Sultana inﬂorescence. Vitis 14:14–19. Scienza, A. 1982. Recent information on the causes of stalk withering. Vignevini 9:15–30. Scienza, A., and M. Fregoni. 1978. The relationship between the number of berries in the cluster and grapevine rachis desiccation. Vignevini 5:31–37. Shin, K.C., J.Y. Moon, J.S. Choi, and S.B. Kim. 1984. Studies on the causes of grape stalk necrosis. Research Reports, Ofﬁce of Rural Development, S. Korea. Horticulture 26: 10–14. Song, G., M. Ryou, and S. Seok. 2003. Causes and control methods for waterberry in ‘Kyoho’ grapevines. J. Korean Soc. Hortic. Sci. 44:688–691. Soule, J. 1985. Glossary for horticultural crops. John Wiley & Sons, New York. Spring, J.L., J. Aerny, and J.P. Ryser. 1999. Inﬂuence of the rootstock on mineral nutrition of the scion. First results of an experiment with Chasselas and Gamay in the Leman Basin. Revue Suisse de Viticulture, d’Arboriculture et d’Horticulture 31:321–327. Srinivasan, C., and M.G. Mullins. 1976. Reproductive anatomy of the grapevine (Vitis vinifera L.): origin and development of the Anlage and its derivates. Ann. Bot. (Lond.) 38:1079–1084. Stellwaag-Kittler, F. 1975. Studies to elucidate the causal mechanism of stalk withering in grapes. Mitt. Klosterneuburg 25:3–18. Stellwaag-Kittler, F. 1983. External symptoms of stalk necrosis on grapes. Mitt. Klosterneuburg 33:94–99. Stellwaag-Kittler, F., and G. Haub. 1964. Die Stiellahme der Trauben. Der Deutsche Weinbau 20:844–846. Swanepoel, J.J., and E. Archer. 1988. The ontogeny and development of Vitis vinifera L. cv. Chenin blanc inﬂorescence in relation to phenological stages. Vitis 27:133–141. Theiler, R. 1975a. Factors affecting stalk atrophy—their importance for the occurrence of the symptoms. Mitt. Klosterneuburg 25:37–44. Theiler, R. 1975b. Grape stalk atrophy—studies from 1970 to 1973. Schweiz. Zeit. Obst Weinbau 111:18–28. Theiler, R. 1976. Stalk necrosis of grapes. Schweiz. Zeit. Obst Weinbau 112:130–139. Theiler, R. 1979. Prophylactic cultural and control measures to reduce stalk necrosis. Schweiz. Zeit. Obst Weinbau 115:118–120. Theiler, R. 1980. Stem atrophy—prognosis and control methods for 1980. Schweiz. Zeit. Obst Weinbau 116:499–508. Theiler, R. 1983. Forecast of the incidence of stem atrophy on grapevine (results from 1978 to 1982 and forecast for 1983). Schweiz. Zeit. Obst Weinbau 119:522–532. Theiler, R. 1986a. Stalk necrosis of grapes. Obstbau Weinbau 23:59–62. Theiler, R. 1986b. Stem atrophy—forecast of incidence and control in 1986. Schweiz. Zeit. Obst Weinbau 122:536–537. Theiler, R., and B.G. Coombe. 1985. Inﬂuence of berry growth and growth regulators on the development of grape peduncles in Vitis vinifera. Vitis 24:1–11. Theiler, R., and H. Muller. 1986. Relationships between climatic factors and the incidence of stalk necrosis in Riesling Sylvaner. Vitis 25:8–20. Ureta, F., J.N. Boidron, and J. Bouard. 1981. Inﬂuence of dessechement de la raﬂe on grape quality. Am. J. Enol. Vitic. 32:90–92. Weaver, R.J. 1976. Grape growing. John Wiley & Sons, New York. Winkler, A.J. 1965. General viticulture. Univ. California, Berkeley. Zhang, X., G. Luo, R. Wang, J. Wang, and D.G. Himelrick. 2003. Growth and development responses of seeded and seedless grape berries to shoot girdling. J. Am. Soc. Hort. Sci. 128:316–323.

8 Plug Transplant Technology Daniel J. Cantliffe University of Florida/IFAS Horticultural Sciences Department PO Box 110690 Gainesville, FL 32611-0690 USA

I. INTRODUCTION II. IMPORTANCE OF THE PLUG INDUSTRY III. PLUG PRODUCTION TECHNOLOGY A. Cell Size B. Transplant Age C. Media D. Irrigation and Nutrition E. Light and Temperature F. Conditioning IV. ORGANIC PLUG PRODUCTION V. POSTHARVEST HANDLING OF PLUGS VI. ASSOCIATED PRODUCTION TECHNIQUES A. Seed Germination B. Seed Pelleting, Priming, and Grafting C. Potential Plug Plant Biological Enhancement Treatments VII. MECHANIZATION VIII. CONCLUSIONS AND PROSPECTS IX. LITERATURE CITED

I. INTRODUCTION In 1925, Memoir 87, W.E. Loomis of Cornell University stated: ‘‘The acreage of vegetables produced from transplanted plants, the value of such crops, and the very considerable cost of growing the plants, give the practice of transplanting a high place in the economics of vegetable production.’’ This publication, Loomis’s doctoral dissertation, under Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 397

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the direction of H.C. Thompson, was a milestone in better understanding various factors that affected transplant root and shoot growth responses, hardening, and speciﬁc tests for various crops transplanted to the ﬁeld. In all his experiments, Loomis relied on the stage of maturity rather than transplant age. He overseeded and thinned in greenhouse beds, pots, or ﬂats. His tomato transplant spacing experiments utilized pots of singulated plants conﬁgured in a wooden ﬂat. Although the pot size would be large for today’s standards, the results from these experiments provided the foundation to present-day plug technology. Plugs deﬁned as containerized transplants (Styer and Koranski 1997), have been used by ornamental and vegetable transplant growers since the 1960s. Their greatest advantages are that they can be grown individually in a cell and can be mechanized. The production of plugs is best suited for protected culture where water and fertilizer can be strictly controlled. The plug itself can be ‘‘sized’’ according to the species and/or the size of the plant desired. By doing so, the plug can be grown based on the customer’s needs as well as the equipment needed for transplanting. The great advantage of the system is that the root system remains undisturbed from growing the plug to transplanting in the ﬁeld. Furthermore, diseases to roots and foliage can be minimized when plants are grown under protected culture. Other advantages for plugs are less time and labor to transplant, more uniform plant growth, ability to singulate seed at planting, and reduced disease spread; disadvantages include greater cost and the need for more highly trained people and facilities to grow the plugs. The use of media in large-size singulated cells increases the weight and root volume of each seedling. This, in turn, increases costs of both production and transportation from the growing facility to the customer. The plug industry in the United States and globally grew rapidly due to the invention of the ‘‘speedy seedling’’ that inventor George Todd Sr. termed Speedling1. Todd founded Speedling, Inc., in Sun City, Florida, in the early 1970s. He was a vegetable grower who grew summer cauliﬂower in New York and winter production in Florida. The cauliﬂower crop, normally grown from bare-rooted transplants in the open ﬁeld, was generally nonuniform, grew slowly after transplanting and was often infected with diseases and root rots. To achieve transplants that were uniform and that would start growing as soon as they were planted, Todd invented a reusable Styrofoam ﬂat that had cells which were an inverted pyramid with a hole at the bottom so that water and excess fertilizer could drain and which would help ‘‘air-prune’’ roots as

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they reached the opening. The Speedling1 ﬂat was designed to be a onesize rectangular tray that could be sown by vacuum seeders then moved to a greenhouse for growing on waist-high aluminum rails. The plants could be irrigated and fertilized mechanically by overhead boom spray misters. The tray was patented in the United States in 1970 and since then in numerous other countries. The concept and ability to mechanize this system was an immediate great success. Speedling not only grew plants for Leisey and Todd Farms (Bud Leisey was George Todd’s partner) but also started to sell to neighbors. The business grew almost overnight, and Speedling transplants were soon sold up and down the east coast of the United States. In 1973, the Speedling, Inc. production model was franchised through license agreements. The system consisted of a lightweight, multicelled growing container, soilless mix, an open-sided greenhouse system with 80% space utilization, and a mobile overhead watering system, sold to other growers in Florida, such as, Zellwin Farms in Zellwood, and to other transplant producers in the United States, Mexico, Israel, and Australia. In total, Todd started transplant nurseries in 20 other countries. The plug concept continued to grow rapidly in this fashion on a global basis. In mid-1984, Todd opened Speedling II in Bushnell, Florida, with a new concept of using ﬂotation of the Styrofoam Speedling ﬂats in an ebb-and-ﬂow system of irrigation and nutrient management. By 1989, Speedling had 500,000 square feet in production in Bushnell, then added in 1991 another 500,000 square feet in Nipomo, California. The new system allowed almost complete use of the greenhouse ﬂoor but was expensive to construct, as all ﬂoors and large shallow pools were made with poured concrete. The production houses were constructed to allow easy movement of ﬂats. Water was recycled, and root systems were kept in water for extended periods, causing roots to protrude from the bottom of the cell. After some initial work by Leskovar et al. (1994), a system was developed to air-prune roots to mimic the initial Speedling I setup in Sun City, Florida. One of the unique features of Speedling II was that it was constructed over 80 km from the major tomato and pepper production regions in Florida. Further, water contacted foliage only when pesticides or supplemental fertilizer were applied. This helped eliminate many of the foliar diseases often encountered in the Speedling I overhead irrigation system. Speedling I was located immediately adjacent to tomato and pepper ﬁelds, which invited diseases and insects to the transplant plug houses; Speedling II largely avoided this problem. Speedling later expanded to several

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locations in Texas and California to be closer to major vegetable production; in the 1990s, it expanded to China.

II. IMPORTANCE OF THE PLUG INDUSTRY Plugs have been used extensively for bedding plant production as well as vegetables. Japan, the United States, and the Netherlands produce $3.5 billion worth of plugs, with $2.5 billion in the United States alone (Hamrick 2005). Pansy and impatiens are the two most important bedding plants grown in the United States, while petunias, geraniums, marigolds, begonias, salvia, dianthus, and catharanthus are important contributors to the economic importance of plug crops. All these crops are produced via seeds, and the use of plugs greatly expanded their sales. Bedding plants, similar to the vegetable transplant industry, were mostly grown in the ﬁeld at high plant densities, dug from the soil, and then packed in boxes and sold as bare-root transplants. The inexpensive seeds were from open-pollinated cultivars and broadcast sown. All major seed companies shifted to F1 hybrids during this period wherein seed costs skyrocketed (1 kg hybrid tomato might cost $350,000 today versus $44 for 1 kg of open-pollinated tomato seeds in 1985). The high costs for seeds require that transplant growers essentially get 100% emergence and a completely uniform plant stand. Many others developed plug systems, some bringing new concepts to the industry as well as some ‘‘reinventing’’ the Speedling system. Tray design has been a key to the long-term success of the plug industry. Trays must accommodate as many cells as possible without wasting space; they must be inexpensive, durable, and reusable. Styrofoam or plastic have been the key components of plug trays. Plug production of vegetables, bedding plants, cut ﬂowers (started from seed), tobacco, and trees are worldwide industries, and their importance is increasing each year. In 1965, no plants were produced from plugs; by 1997, 25 billion plants per year were produced worldwide from plugs; in 2001, over 40 billion plants from plugs; and Carlson (2001) estimated that over 1 trillion plugs would be produced by 2010. Producers of plugs are generally specialists in what they do; thus, most growers purchase their plugs from large nursery operations. Today’s plug producer must be knowledgeable in seed testing, seed treatments and germination, greenhouse mechanization, and integrated pest management (IPM) as well as all other components of production (Gordon 2006). This chapter explores the technology

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required to produce plug transplants. Much of the information is an update or expansion of the information contained in Styer and Koranski’s 1997 book, Plug and Transplant Production.

III. PLUG PRODUCTION TECHNOLOGY The switch from ﬁeld to greenhouse production and open-pollinated to hybrid cultivars has created a need and constant thrust for new innovative technology to economically sustain plug producers. The high labor costs for plug producers in the United States, Europe, and Japan has given rise to automation of planting, growing, and harvesting plugs. Growing plugs in warm climates has the advantage of reduced heating and lighting costs; however, transport of the product to distant markets becomes a major cost. Vegetable plug producers, with lowervalue crops, keep production simple, while ornamental plug producers typically are heavy on facilities and equipment. The range of sophistication runs from wood- or metal-framed plastic greenhouses with boom mist systems for fertigation, gravel ﬂoors, and no heating capability, to computer-controlled glass greenhouses with overhead curtains, under-bench heating, full concrete ﬂoors, and high-intensity discharge lamps. Prices for construction may range in excess of $250 per square meter or as low as $10, depending on the level of technology. In a sense, a plug production facility is similar to ﬂoor space in a shopping mall. To maximize proﬁts, the space must be ﬁlled 100% of the time, and rapid turnover of the product is an absolute necessity. Therefore, utilization of over 90% of the ﬂoor space with ﬂats that are 100% ﬁlled with crops that can be grown in fewer than 4 to 6 weeks can lead to greater proﬁts for the plug producer. Keeping the houses ﬁlled 12 months of the year and automation for labor reduction adds to the proﬁts of successful plug producers. A. Cell Size Choice of tray cell size (number and/or volume) to grow plugs depends on several factors, including economics, plant growth rates, and customer demands. Cell size varies from 72 to 800 cells per standard tray (53.7 27.5 cm) (Hamrick 2005). Cell sizes can vary by both the diameter (round cells) or size-length (square cells) and depth of the cell. Size then relates to cell volume, which controls the amount of media used as well as water holding capacity. It is obviously less expensive on per-plant basis to grow and ship a small-volume cell than a larger one.

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The smaller the cell size dimensions in the tray, the more units a grower can produce per unit area since plugs will ﬁnish faster (ready for sale) and more plugs can grow per m2 in the greenhouse space. However, getting an ornamental crop ready for sale from plugs from small cells will take longer—generally a week longer for each decrease in cell size from a standard 288 (cell) tray—since the plug will have to be planted into larger pots (Hamrick 2005). Because of small seed size and slower growth rates, begonia and celery are started in very small cells then transferred to larger cells as the plant and root systems develop. Generally container type—expandable polystyrene tray, injectionmolded tray, or vacuum-formed tray—has no inﬂuence on transplant growth parameters (Marsh and Paul 1988). In some species such as Pinus palustris, older seedlings might become too large for some container types (South et al. 2005). Likewise, container shape— inverted pyramid, cylinder, or cylinder with bottom lip—had no effect on the performance of marigold (Latimer 1991). However, it has been well documented that plugs grown in large cells are taller and have greater dry weights than those grown in small cells (Cantliffe 1993; NeSmith and Duval 1998). Capsicum pepper, tomato, eggplant, watermelon, broccoli, cauliﬂower (Cantliffe 1993), cabbage (Marsh and Paul 1988), onion (Leskovar and Vavrina 1999), Douglas-ﬁr (Haase et al. 2006), marigold (Latimer 1991), coleus, celosia, and pansy (Nam et al. 2003), and muskmelon (Walters et al. 2005) were reported to have earlier yields and more rapid early transplant growth when grown in larger cells. Total yields are not always affected by cell size, especially in crops with multiple harvests (Vavrina 2001; Walters et al. 2005). When ‘South Bay’ lettuce transplants were produced in Speedling cells from 2 to 40 cm3, harvest was delayed approximately 2 weeks for plants grown in the 2-cm3 cell size (Nicola and Cantliffe 1996). Yield in the ﬁeld from the 2-cm3-cell plants was reduced compared to the other larger transplant cell sizes. Plugs are a viable alternative to bare root- and cold-stored frigo strawberry transplants (Bish et al. 1997b; Durner et al. 2002). Hochmuth et al. (2006a) reported that strawberry plug plants could be established with 2711 L ha1 water while it took 9.4 million L ha1 water if the common practice of sprinkler irrigation was used on barerooted transplants. Large (10-mm-diameter crowns) plug plants consistently led to greater early yields than plants from medium (8-mm-diameter crowns) or small (6-mm-diameter crowns) plugs and bare-rooted transplants (Hochmuth et al. 2006b). Bish et al. (1997a and 2003) also found that larger (300 cm3) cell volumes led to greater plug size of strawberries than those grown from smaller plugs (75 and

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150 cm3), but the larger container sizes did not result in consistently higher yields. As container size increases, root and shoot biomass, including leaf area, increase (Cantliffe 1993). If root volumes are reduced, water and nutrient uptake might likewise be reduced, thus leading to decreased shoot size. Watermelon plugs grown in an 18-cm3-volume cell were small at planting and yielded less than plants grown from plugs produced in 80-cm3 cells (Lin and Latimer 1995). This same trend was reported by Graham et al. (2000), wherein plants from watermelon plugs in 31-cm3 cells had lower yields than those produced in 350-cm3 cells. Weston and Zandstra (1986) have shown that plugs with larger root systems can better survive transplant shock. However, it should again be emphasized that plugs grown in large cell volumes will require a longer time to mature in the greenhouse for direct ﬁeld transplanting. Thus, transplant age becomes a factor for consideration. B. Transplant Age A major interactive factor related to studies on cell size is the effect that transplant age has on the ﬁnal product. Getting a positive response from using a large cell size may require that the plant be older before pulling (Leskovar and Vavrina 1999). Liptay (1987) grew processing tomato ‘H-2653’ in cell sizes of 3, 5.5, 10 and 35 cm3 for 4, 5, 6, and 7 weeks. Regardless of plant age, 35-cm3 plugs led to the earliest yields. When smaller cell sizes were used, the older 6- and 7-week-old transplants led to earlier yields. Transplant age did not affect total yield. In similar studies by Weston and Zandstra (1986), ‘Pik-Red’ tomatoes were sown on the same day into Speedling cells of six different volumes from 4.4 to 39.5 cm3 and pulled for planting 28 days later. Again, 28-day-old transplants from larger cells were earlier but total yields were unaffected. Thus, at least with tomato, if earliness is not a factor, smaller cell volumes would provide better returns to cost. In a subsequent study using only an 18.8-cm3 Speedling cell size, Weston and Zandstra (1989) grew ‘Pik-Red’ tomato plugs for 3, 4, 5, and 6 weeks. The 4- and 5-week-old plugs were earlier than the 3- and 6-week-old plugs. Total yields were the same for 3-, 4-, and 5-week-old-plugs. Leskovar et al. (1991) produced tomato plugs that were 2, 3, 4, 5, and 6 weeks old, then grew them on growers’ ﬁelds using two types of irrigation. The plugs were commercially produced by Speedling II, Inc. Typically, tomato producers preferred 6-week-old plugs. Their results concluded that there was no improvement in yields by using the traditional older

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transplants. They recommended that younger transplants might be used to achieve rapid seedling establishment with minimal transplant production costs. In a review on tomato transplant age, Vavrina and Orzolek (1993) concluded that ‘‘after more than 60 years of transplant age research, it appears that transplants of 2 to 13 weeks can produce comparable yields, depending on the many factors involved in commercial production.’’ Similarly, Techniculture type (plugs designed for rapid mechanical transplanting) lettuce plugs had less head weight variability among 13- and 19-day-old transplants than 25-day-old transplants (Wurr and Fellows 1986). Also, 3- to 6-week-old Chinese cabbage plugs had similar yields (Kratley et al. 1982). Plug age of 30, 40, or 50 days had no effect on total yields of pepper (Weston 1988). Plug age at transplanting also had little effect on yields of cabbage, cauliﬂower, and broccoli (Wurr et al. 1986; Jones et al. 1991). Walters et al. (2005) held muskmelon transplants for 1, 2, or 4 weeks after emergence and found that yields from plants pulled 2 weeks from emergence were greater than those from plants pulled 1 or 4 weeks after emergence. However, it is important to consider that pulling young transplants with small root systems may be impractical in some species, as plugs may lose media from the cell, resulting in problems during planting as well as reducing transplant shock tolerance. NeSmith (1994) grew plug muskmelon transplants for 2, 4, 6, and 8 weeks and found no differences among yields. Results for squash by the same author (NeSmith 1993) were similar to muskmelon. Plugs grown for 10, 14, or 21 days grew more rapidly after transplanting than those grown for 28 or 35 days; however, transplant age had little inﬂuence on subsequent yields. Older plugs were harder to handle at transplanting; thus 21-day old plants were recommended. Leskovar and Vavrina (1999) recommended use of 8 to 10-week-old onion plug transplants compared to 5-, 6-, 11-, or 12-week-old plugs. The younger transplants led to larger bulb yield when grown in a 20-cm3 cell compared to a 6.5-cm3 cell; however, they had reduced survival rates in the ﬁeld. Sweet potatoes, like strawberry runners, are commonly produced from cuttings. Plug transplants that were 11 or 15 days old resulted in greater yields of storage roots than those from vine cuttings (Islam et al. 2006), but transplant age had no inﬂuence on the results. The authors felt that the use of sweet potato plug transplants would be economical due to greater marketable yields of storage root with lower production costs.

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Transplant age has little inﬂuence on total yields of a diverse number of vegetable crops. The situation for optimizing crop time (age) for ornamental plugs is largely dependent on container size. Obviously the greenhouse will be more efﬁcient and proﬁtable with more crop turns (Styer and Koranski 1997). Crop time is dependent on temperature, light, water, nutrients, use of chemical growth regulators, and cell volume. In seasons or areas when light and temperatures are low, plug crops take longer. If water or nutrients are rationed, the plug crop will take longer, as it will when growth regulators are used to control plant height or root development. Regardless of all these factors, crop time in the plug greenhouse is controlled by cell volume. As the cell volume increases, crop time increases. Thus, a grower needs to know how long it takes to produce and ﬁnish a certain size of plug in the container. The larger the plug sizes, the shorter the ﬁnishing time for a potted crop coming from the greenhouse. Based on these principles, ornamental and bedding plant plugs may be produced in a southern location with good light quality and temperature and then transported to a northern market to be ﬁnished closer to the point of sale. If time to ﬁnish the crop is limited, generally a larger plug is used. Costs for the plug may increase but costs to ﬁnish the crop decreases. When plugs are ﬁnished at the same location where they are produced, crop cycles can be conﬁgured to best use the greenhouse facility on a 10-month basis. This is done by altering plug sizes to obtain a ﬁnished product, many times in a standard-volume container. To illustrate this pattern of production, tomatoes were grown in plug trays containing 200, 406, or 648 cells for 1 to 6 weeks, then moved to a 48-cell ﬂat (Marr and Jirak 1990). Seedlings grown in 648-plug trays were smaller at weeks 5–6, while seedlings from the 200-cell trays were larger throughout the 6-week period than the other two tray sizes. Yield from tomato plant plugs decreased when the plugs were held 6 or 7 weeks in the 406-plug tray compared to moving them to the 48-cell market pack after weeks 1 to 5. C. Media Considerations on how to choose media for plug production is thoroughly reviewed in Styer and Koranski’s (1997) book. Physical characteristics of the media are important for water and nutrient holding capacity as well as proper aeration for optimum root growth. Consistency and availability of the media components must also be considered. Thus, both physical and chemical components of media

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must be understood, and both play a key role in optimizing shoot and root growth. Choosing a media for plug production also must be based on economic considerations. Costs for ingredients may optimize plant growth, but costs for the product(s) may not be economically viable. Cell volume then becomes a key economic factor with regard to the quantity of media required to grow the plugs. Additionally, small cell volumes can create problems with moisture retention, aeration, and soluble salt buildup. The type of irrigation method (overhead or ebb and ﬂow) will also dictate media decisions. Choosing the proper media for plug production may be the key factor for success or failure of plant growing. Media controls aeration, moisture-holding capacity, pH, nutrient retention and uptake, and soluble salts buildup. Media relates directly to considerations for plug cell size, irrigation and fertilization, plug conditioning, and transplant age properties. Media inﬂuences vegetative and root growth rates, and quantitatively it is a major cost factor for any plug production system (Argo 1998). A growing media must be sterile and may or may not be inert, but it should have cation exchange capacity (CEC). The CEC is associated with pH and nutrient buffering capacity (Argo 1997a). For this reason, many plug production operations use peat as a major part of media makeup. On a weight basis, the CEC of sphagnum peat is about nine times that of a loamy mineral soil. However, due to the low-bulk density of the peat, the effective CEC is lower than that of mineral soil. Potentially inexpensive mineral soils cannot be used for media mixes because they are inconsistent, not sterile, extremely heavy, and have low porosity. Thus, all media are artiﬁcial mixes with additives such as perlite, polystyrene, rockwool, bark, coconut coir, or vermiculite. These additives increase aeration or water-holding capacity (Argo and Biernbaum 1994). Normally, some type of liming material is added to adjust pH to 6.2 to 6.8 and to supply calcium and/or magnesium (Styer and Koranski 1997). When growing pine and birch seedlings in plugs, Heiskanen and Rickala (1998, 2000) observed no beneﬁt for root growth or subsequent establishment by adding perlite or sand to a pure sphagnum peat media. Cedar plugs grew better in media that contained a higher peat content than one that contained 75% bark (Derby and Hinesley 2005). This fact was directly related to the greater water-holding capacity of the peat mix. Various alternates to using peat as the major constituent of plug media have been investigated. Peat is not a renewable resource and

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has become quite expensive compared to other substrates. Abad et al. (2005) compared physical properties of 13 coconut coir dusts collected from Asia, the Americas, and Africa to sphagnum peat by mixing various portions of different-size fractions and varying the degree of husk grinding, screen size at grading, or aging the coir. They found the coir and peat varied considerably in particle size and suggested that irrigation schedules would have to be altered on a cropby-crop basis. Arenas et al. (2002) produced tomato plugs on 16 media combinations with peat, coir, perlite, or vermiculite. Plugs grown in greater than 50% coir had reduced growth compared to those grown in peat. They associated high nitrogen (N) immobilization by microorganisms and a high carbon:nitrogen ratio with the reduced performance of the high-coir media. Gruda and Schnitzler (2004a,b) used wood ﬁber substrates as a substitute for peat in a plug mix. The wood ﬁber as a substrate had similar volume weight and total pore space as peat but lower water retention. The wood ﬁber media drained quickly; however, it had to be compressed to minimize substrate loss. Tomato transplants grew as well in either wood ﬁber or peat substrate (Gruda and Schnitzler 2004b). Increasing volume weight (by compression) led to a reduction in root growth. The authors concluded that wood ﬁbers were a good alternative to peat in the media and that moderate compression was needed to reduce the rate of drainage. Vermicompost has been used successfully to produce vegetable plugs (Paul and Metzger 2005). The process uses certain earthworm species (Eismia fetida) to stabilize organic wastes, giving rise to an odorless, peatlike material with excellent moisture-holding capacity, structure, and available nutrients. Growth of tomato, eggplant, and pepper plugs grown in a commercial peat-based mix (Metro mix 360; Scotts, Inc., Maysville, Ohio) was improved by adding 10% or 20% of Vermicompost (made from worm-worked cattle manure). Vavrina (1999) used spun cellulose acetate as a replacement for peat: vermiculite in the plug mix. Tomato plugs had reduced fresh weight, dry weight, and height, while muskmelon plants had reduced dry weights in the ﬁeld after 20 and 45 days posttransplanting when grown in the cellulose acetate media. Total yield of both crops were unaffected; however, tomato harvesting was delayed from plugs grown on the cellulose acetate media. Based on these results, the author felt that the cellulose acetate could be used as an alternative media since it is relatively inexpensive. Charred rice hulls have also been used as a supplement to commercial media containing peat (Braz et al. 2003).

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Growth of lettuce plugs on the amended media was the same as that from commercial media. Composted poultry litter (pine sawdust þ poultry litter; a mixture of chicken manure and bedding material) was added (50:50 v/v) to a commercial peat:vermiculite mix. Growth of collard, broccoli, cabbage, and tomato transplants was similar in either media (Gruertal et al. 1997). Uniformly composted waste materials are potentially, suitable additives to the media. Sewage sludge compost has also been used as additives to or replacement for peat. Sludge from a high-metal source led to poor tomato and cabbage plant growth (Sterrett et al. 1982). The use of lowmetal sludge (residential limed raw sewage sludge) compost as 50% of the media was an effective organic component of the growing media. Marketable yields and metal content of muskmelon, cabbage, and tomato were the same as a 100% peat/vermiculite mix (Sterrett 1983). More recently, similar results were reported in Greece with tomato and eggplant transplants grown in as high as 75% sludge and 25% peat (Bletsos and Gantidis 2004). At a 50:50 sludge-peat ratio, plant growth exceeded that of plants grown in 100% peat. None of these studies examined pore space, water-holding capacity, or aeration of the media. The ratio of water to air held in the media is controlled by particle size and pore space distribution (Argo 1997b). Capillary pores ( < 0.3 mm) retain water after irrigation, while noncapillary pores (> 0.3 mm) provide root aeration (Argo 1998). Thus, it is important to provide an adequate mix of both capillary and noncapillary pores in the media. Bish et al. (1997a) studied the effects of particle size and media aeration on root and crown growth of strawberry plugs fertigated with a capillary mat system. They found that optimal transplant growth and quality was dependent on media aeration and that media particle size could be altered, based on container volume, to achieve optimal media aeration. When lettuce plugs were grown in a peat/vermiculite media that was compressed (1.5 times normal weight) or not compressed, the compression of the media led to increased shoot growth when the plugs were grown in 19.3- or 39.7-cm3 cells (Nicola and Cantliffe 1996). The authors concluded, however, that compression of the media in the cells was not justiﬁed because it was more costly and did not beneﬁt yield in the ﬁeld. The composition of the media directly affects water-holding capacity and plant nutrition. Therefore, irrigation type, timing, and quantity must be based on the media used. Nutritional practices are then implemented once irrigation and media are determined.

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D. Irrigation and Nutrition Plugs can be irrigated above the plants (overhead) or subirrigation (ﬂoatation) via ebb and ﬂow. The costs vary considerably. Overhead irrigation usually requires a boom with nozzles, while the more expensive ebb-and-ﬂow system requires concrete ﬂoors and a recycling system for the water. The advantage of the latter system is that no water is applied to the plug foliage; therefore, there is reduced potential for development and spread of diseases. Also, water and nutrients are recycled, reducing groundwater pollution, thus saving water and fertilizer. Management of the irrigation timing and volume directly relates to managing the nutrient supply. The most important part of plug growing is adequate root development. Irrigation and nutrient management controls root growth as well as shoot growth. Overhead irrigation moves water and nutrients from the top of the plug cell to the drain hole, while ﬂoatation irrigation moves water from the bottom of the cell by capillary until the cells are saturated. Although the type of irrigation system may alter root morphology in the plug, total yields of many crops are not affected by the type of irrigation (Leskovar and Cantliffe 1993; Leskovar et al. 1994; Franco and Leskovar 2002). Flotation systems improved uniformity of bell and jalapeno peppers and tomato (Leskovar and Boales 1995; Leskovar 1998). Root systems need to be pruned at the drain opening at the bottom of the cell. This is commonly achieved by air pruning, either on the T-rail system for overhead irrigated plugs or via extending the ‘‘dry’’ period for subirrigated plugs. Tomato transplants grown with ﬂotation had excessive root growth protruding from the bottom of the container (Leskovar et al. 1994). Roots had to be mechanically pruned, a practice that reduced productivity in the ﬁeld. Similar results were reported for sweet corn produced in a ﬂotation system (Frantz and Welbaum 1995). Controlling root growth is a major step to ﬁnishing a plug that will ‘‘pull’’ easily and survive in the ﬁeld. Lettuce plugs grown with overhead irrigation had decreased basal root number, leaf, and root dry mass compared to plugs grown with subirrigation (Nicola et al. 2004). Plugs grown with subirrigation were ready for the ﬁeld earlier than overhead irrigated plugs. Basal root length was greater for overhead than subirrigated jalapeno peppers, but lateral root length was greater for subirrigated plugs (Leskovar and Heineman 1994). Total root elongation was similar for both irrigation treatments. Subirrigation reduced shoot-to-root ratio and promoted plant hardiness.

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The ultimate goal for a plug is to have superior seedling establishment. For dicots, this means adequate development of a taproot, associated with lateral and basal roots, while for monocots a taproot is associated with adventitious and lateral roots (Leskovar and Stoffella 1995). Seasonal conditions of the greenhouse, media, cell size, irrigation, and fertilization patterns all help regulate root growth and development of a plug. Stress stimuli can help regulate root growth. However, in practice, probably the single most signiﬁcant factor regulating root growth in a cell is N nutrition. The interaction of N fertilization with irrigation frequency and type dictates partitioning of carbon between roots and shoots. Too much N can greatly increase plug shoot-to-root ratios. High-irrigation regimes can further promote large ratios. Dufault (1998), reviewing vegetable nutrition reported that 38 papers published since 1940 (58-year period), discussed this topic for 11 vegetable species. Approximately 33% of all the work was devoted to tomato, while the major nutrient researched was N. About 40% of the research recommended N rates in the range of 300 to 400 mg L1 N. Few papers (10%) recommended N fertility rates lower than 100 mg L1. Karchi et al. (1992) reported that the greatest dry root mass and highest root:leaf ratios of lettuce plugs, a leafy crop, resulted with high phosphorus (P) and low N fertilizer ratios. Fertilizer combinations with high N and low P resulted in the greatest leaf weights and lowest root mass. Cantliffe and Soundy (2000) summarized vegetable transplant nutrient and water management and concluded that many of the fertilizer applications have been split: preplant and applied during production. Recent practices for plug production favor increasing the frequency of fertilization to coincide with each irrigation, minimizing potassium applications when vermiculite is used in the media, reducing P applications when peat is used and when P is added in a soluble form, and adding N at levels that control both shoot and root growth. ‘South Bay’ iceburg lettuce plugs were fertigated continuously in a ﬂoatation (subirrigation) system containing N at 0 to 60 mg L1 (Soundy and Cantliffe 2001). Both shoot and root mass were improved by increasing N, but the increase in shoot mass was much greater than for root mass in response to N, resulting in higher shoot-to-root ratios. The effects of N on plug shoot and root growth were consistent regardless of season. The plugs were grown in a peat:vermiculite media and were bigger at transplanting when grown with 60 mg L1 compared to 15 mg L1 N. This led to larger head size at harvest in the

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ﬁeld from plants grown with 60 mg L1 N. Soundy et al. (2005) fertigated ‘South Bay’ lettuce plugs every 1, 2, 3, or 4 days with 0, 30, 60, 90, or 120 mg L1 N in a ﬂoatation irrigation system. As N concentration increased, shoot mass, shoot:root ratios, and leaf N content all increased. Based on subsequent ﬁeld grow-out trials, the authors suggested that fertigation with N at 60 or 90 mg L1, regardless of irrigation frequency up to 4 days apart, led to optimizing root systems in the plug tray and superior yields and head quality of lettuce. Growth of tomato transplants decreased as N concentration in the nutritional system increased from 4 to 60 mmol L1 regardless of light conditions (Basoccu and Nicola 1995). Dry matter of the root, the entire plant, and root:shoot ratios were the greatest at the lowest two N concentrations of 4 and 8 mmol L1. Yields from a crop produced in a greenhouse were generally unaffected by preplant fertilizer concentrations. When celery plugs were grown in a vermiculite:perlite:peat media and slow-release N from 1.25 to 10 g kg1 of medium was added, plant growth and shoot and root dry mass increased; however, it was recommended that an N rate of 1.25 g kg1 was adequate to produce quality transplants (Dufault 1987). Tremblay et al. (1987) produced celery transplants at concentrations of 200, 400, or 600 mg L1 N in a peat-based media and found that the greatest leaf area and dry weight of the plugs came from the 400 mg L1 concentration treatment. No differences were observed for root dry weight or matter while root:shoot ratio decreased as N increased. In subsequent work, the same authors fertilized celery plugs with nutrient solutions containing 150, 250, or 350 mg L1 N in various NO3:NH4 ratios and reported similar results (Tremblay and Gosselin 1989a). Potentially, all N concentrations used were in excess of what was needed to produce quality celery transplants. In a third paper, Tremblay and Gosselin (1989b) used lower N concentrations in fertilizer mix and compared 150 and 350 mg L1 N. Root:shoot dry weight ratios were greater at the lower N rate; there was no difference in root dry weights. Similar to what has been reported for lettuce and celery, tomato plugs improved stem, leaf, leaf area, shoot, and root growth in both fall and spring seasons when N application was increased from 0 to 75 mg L1 over a range of 0, 15, 30, 45, 60, and 75 mg L1 N (Vavrina et al. 1998). Although plugs grown at the 75 mg L1 N concentration were larger than those grown at lower concentrations, yields of extra-large fruits were greater at the 45 mgL1 N and higher rate. Total fruit harvest weights from a fall crop in Florida were greater from the 15–45 mg L1 N

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rates; those from the spring crop were greater from the 45–75 mgL1 N rates. Thus, the authors felt that season may play a role when applying plug N fertilization for optimization of fruit yields in the ﬁeld. Regardless, N levels below 100 mg L1 N produced quality transplants. In earlier work, Weston and Zandstra (1989) fertilized tomato transplants with 100–400 mg L1 N and reported greater early yields in the ﬁeld in Michigan from 200 and 400 mg L1 N; however, there were no differences in total yield. Both P and K have been researched as to their potential effects on plant growth and development of plugs. Generally, when adequate rates of P are supplied to the growing media, little enhancement of transplant growth and performance in the ﬁeld is observed when P levels are increased (Dufault 1987; Tremblay et al. 1987; Weston and Zandstra 1989; Melton and Dufault 1991; Karchi et al. 1992; Soundy et al. 2001a). Soundy et al. (2001a) produced lettuce plugs with ﬂoatation irrigation at 0, 15, 30, 45, or 60 mg L1 P in a peat media, in different seasons of the year, then transferred them to the ﬁeld. Only 30% of the plugs produced at 0 P could be pulled from the transplant ﬂats while over 90% could be pulled when P was added. They reported that pretransplant P hastened maturity and increased lettuce head weight but that the 15 mg L1 P level was adequate to achieve this response. When vermiculite is used in a peat mix media, there is generally little response to K (Melton and Dufault 1991). Lettuce plugs produced in a peat:vermiculite mix with no added K had at least 24 mg L1 water-extractable K and produced yields equivalent to plugs supplied with 15–60 mg L1 K in a ﬂotation irrigation system (Soundy et al. 2001b). E. Light and Temperature Light (quality, intensity, and duration) and temperature are important environmental factors that control plug plant growth. Light may also be used to condition plugs before they are transplanted to more hostile environments. In the past, plug growers have been limited in their efforts to alter these two environmental factors. Using lights to extend or intensify the light period is very expensive and is generally not an option, with the exception of certain high-priced ornamental plant plugs. New photo-selective greenhouse ﬁlms now allow growers to alter light quality inexpensively; the negative effects of light on growth can be used to slow growth down in an effort to control plant size. Improving light quality can improve plug growth. Under a ﬁxed photoperiod, lettuce plug growth and quality was improved by

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increasing the photosynthetic photon ﬂux (PPF) (Kitaya et al. 1998). Fixing the PPF and increasing the photoperiod also improved lettuce plug growth and quality. In order to produce a compact plug, Kitaya et al. (1998) suggested that 200 mmol m2 s1 is needed. These data were obtained under closed chamber conditions (Kozai et al. 2004) wherein light quality, quantity, and duration could be easily altered. Tomato transplants were produced in a closed system and grown in a 24-hour light period at PPF of 200 mmol m2 s1 and compared to those grown under a 16-hour photoperiod at 300 mmol m2 s1 (Ohyama et al. 2005). Both light treatments produced the same daily integrated PPF of 17.3 mol m3. Plugs grown under 24-hour photoperiod had increased fresh and dry weights and leaf area. Electric energy efﬁciency of the closed system was greater under the 24-hour photoperiod due to the reduction in lamps for lighting. In greenhouse experiments, tomato transplants were grown under natural or supplementary lighting of 100 mmol m2 s1. Supplementary lighting occurred from 0500 to 2200 hours and was interrupted during sunny periods in Quebec, Canada (Boivin et al. 1987). Plugs from the supplemental light treatments at transplanting had greater dry weights than those grown under natural light, leading to earlier yields and greater total yields. End-of-day (EOD) treatments of red light given to tomato transplants produced plants that were shorter and had less total leaf length than those not given an EOD (Decoteau and Fiend 1991). Although ﬂower numbers were increased by this treatment, fruit production in the greenhouse was not. Thus, the treatment was effective for controlling plug growth. Takaichi et al. (2000) altered covering ﬁlms on greenhouses in Japan to increase the red/far-red photon ﬂux ration (R/FR) of solar radiation. The high R/FR treatment reduced stem and petiole elongation of muskmelon, tomato, and cucumber. Cucumber, tomato, and bell pepper transplants were grown under clear ﬁlm and two FR-light-intercepting ﬁlms (Cenny et al. 2004; Rajapakse and Li 2004). PPF was adjusted for all ﬁlm treatments. The FR-light-absorbing ﬁlms were effective in reducing stem elongation and producing compact cucumber, bell pepper, and tomato seedlings. Height control was via internode length reduction, not internode number. Signiﬁcant stem elongation could also be achieved by EOD FR exclusion, but continuous FR radiation reduced growth more. In other work, tomato transplants grown in the greenhouse under light-ﬁltering FR plastic ﬁlm were more compact than those grown under clear plastic or no plastic (Evans and McMahon 2004). Total yields and fruit quality were not altered by the FR ﬁlm treatment.

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Greenhouse-grown tomato plug transplants in Italy were given supplemental UV radiation and, depending on dose, signiﬁcantly lowered plant height, leaf area, and shoot dry weight (Del Corso and Lercari 1997). Fruit production in the greenhouse was unaffected by UV treatment. The use of ultraviolet-B (UVB) irradiation was determined to be an effective method to control transplant growth of hot peppers, cucumber, and tomato transplants in Korea (Kwon et al. 2003a,b). Timing of the UVB ﬁrst treatment, duration, intensity, and number of cycles were all important in controlling plant growth without adverse effects. This was found to be a 2 to 4 KJ m2d1 for cucumber and tomato, respectively, and 4 KJ m2d1 at two-day intervals for hot pepper. Farnesi et al. (2004) reported that when UVB lamps in a greenhouse were placed above the seedlings for 0.5 to 2 hours/day, plant height, leaf area, stem, and leaf dry weights were reduced by this treatment in muskmelon, cucumber, squash, and watermelon. Li et al. (2000) looked extensively at responses of bell pepper transplants to photoselective plastic ﬁlms that intercepted FR wavelengths at 760 nm, leading to R:FR ratios from 1.1 to 3.7. The light treatments led to a reduction in plant height and internode length by 10 to 35% as well as a reduction in leaf area and shoot dry weights. Photoselective ﬁlms with an R-FR ratio of 2.2 led to a 30% reduction in plant heights after four weeks of treatment. Controlling plant growth led to more easily transplantable seedlings. Temperature also greatly affects plug growth; however, most of the time, it is the most difﬁcult environmental factor to control due to costs. Regulation of low temperatures is generally easily solved by addition of heat. However, costs for fossil fuels have increased dramatically over the past 10 years, to the point where the cost of using heaters for plug production is essentially prohibitive. For this reason, most of the large commercial plug producers of both ornamentals and vegetable plugs in the United States have moved to warmer climates in southern Florida, Texas, and California. In many locations, especially in the southern United States, high temperatures become more of a problem than low temperatures. Altering high temperatures is costly and many times impossible, generally as the plug houses are passively ventilated. Where wet mats and fans are used, construction, maintenance, and operational costs become a major concern. Further, in areas of high relative humidity, wet mats are less effective and can increase risks from foliar diseases. In an attempt to control plug plant growth of Brussels sprouts, cabbage, and swede, Bakken and Flones (1995) altered the difference

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between day and night temperature (DIF) during production. By creating a negative DIF (lower day temperature than night temperature), transplant hypocotyl and petioles were shorter compared to positive or zero DIF. The Brussels sprouts and cabbage from negative DIF produced more yield in the ﬁeld than those from positive DIF. Maintenance of positive or negative DIF is potentially more economically accomplished when outside temperatures are mild or cool. The house can be opened to reduce temperature or closed to increase temperature. Again, in warm climates, using DIF to control plant growth becomes more problematic. F. Conditioning In the past, transplants and plugs have been ‘‘conditioned’’ before transplanting to harsher conditions of the ﬁeld or production greenhouse. Typically, reducing water and fertilizer (especially N) were used to reduce growth and condition, or ‘‘harden off,’’ transplants. As a result of this practice, newly transplanted plugs generally grow extremely slow, which can result in delayed harvest. Thus, other methods to control plug growth have been investigated. In summary, light and temperature can be altered to reduce plant growth, but this practice can be expensive and may not necessarily lead to hardening of the plant. Dufault (1994) developed pretransplant nutrient conditioning (PNC) with muskmelons and tomatoes wherein the plugs receive high levels of fertilizer during greenhouse production so that they later can tolerate transplant stresses better, recover from transplant shock, and potentially enhance early yields. During PNC, N levels can be as high as 400 mg L1 and then reduced to harden the plants just before the plugs are ﬁeld planted. In other work, PNC also led to an increase in early yield and number of ‘Jubilee’ watermelons harvested but did not affect total yields or fruit size (Graham et al. 2000). These researchers felt that PNC had no advantage for the grower, but increasing cell size for watermelon plugs was conducive to improving early and total yields. Hardening or conditioning of the transplants before ﬁeld planting was of no apparent advantage. In contrasting work, Garton and Widders (1990) reported that growing tomato plugs under a low-fertilizer regimen of 5.4 mM N, 1.0 mM P, and 1.6 mM K led to greater fruit yields than those obtained from plugs grown under a higher-K regimen. Withholding all fertilizer temporarily before transplanting resulted in a major depletion of tissue N and P concentrations, which led to slow posttransplant shoot growth and lower yields.

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For many species, it appears that growing the plug under optimum nutrient conditions is best for posttransplant adaptation and growth. Reducing but not eliminating N 5 to 10 days prior to ﬁeld setting, in conjunction with reduced available water, best hardens plugs. Another method to improve stem strength and plant hardening is to apply mechanical stress to the seedling (Latimer et al. 1988). In comparing shear force as a mechanical stress to drought, broccoli plugs were brushed with a piece of cardboard or subjected to wind twice daily (Latimer 1990). Another group of plants were allowed to visibly wilt for 2 hours per day. All three conditioning treatments reduced leaf dry weight and area, stem dry weight and length, and total shoot and root dry weights. The brushing treatment improved ﬁeld establishment. Latimer et al. (1986, 1991b), Baden and Latimer (1992), and Johjima et al. (1992) used similar brushing techniques on tomato, cucumber, squash, cabbage and eggplant plugs with similar success. In some cases, results from brushing varied; for example, short internode cucumbers were less affected by the brushing than long internode cucumbers (Latimer et al. 1991). Similar to those experimenting with PNC, the author felt that brushing might be an effective alternative to using drought stress or plant growth retardants to condition transplants prior to ﬁeld planting. Similarly, there was a cultivar interaction for broccoli and cabbage when brushed with a wooden closet rod, wherein brushing had no effect on height control of certain cultivars but signiﬁcantly reduced stem length of others (Baden and Latimer 1992). Brushing was also effective in reducing petiole elongation and shoot dry weights of pansy by 25% to 30% without causing plant damage or affecting subsequent ﬂowering (Garner and Langton 1997). Brushing also reduced thrip populations in tomato, eggplant, and watermelon (Latimer and Oetting 1994). Aphid populations were likewise reduced on tomato transplants. Both brushing and drought conditioning reduced plant height, but drought conditioning had no effects on thrip or aphid populations. Bedding plant plug height can be regulated commercially with plant growth retardants, such as Alar or B-Nine (daminozide) (Kuehny et al. 2001). Plant structure is compact, sturdy, and generally darker green. Unfortunately, the label for Alar was removed for use on all food crops in 1989, so the chemical could no longer be used for vegetable plug conditioning. Reduction/removal of water and nutrients as a conditioning practice can lead to a reduction in subsequent early transplant growth; thus, alternative treatments such as brushing and other natural hormones have been investigated in recent years.

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Leskovar and Cantliffe (1992) compared drought stress and abscisic acid (ABA) as conditioning agents to control growth morphology and dry weights of pepper plugs. ABA (104M) applied to leaves 28, 32, or 37 days after seeding reduced leaf growth but did not inﬂuence root growth or total fruit yields. Drought stress conditioning reduced root dry weight, basal root count, and root diameters. The authors recommended ABA as an alternative for drought stress to control plug growth in the nursery. Similar results were obtained by using ABA analogs with tomato, snapdragon, nasturtium, and marigold transplants (Sharma et al. 2005a,b, 2006). Root dips were more effective than foliar applications. Improved resistance to transplant shock was achieved by maintaining higher leaf water content, thus slowing wilting. Seedling treatments with an ABA analog prior to transplanting helped prevent transplant shock without negative longterm growth and yield potential. Goreta et al. (2007) evaluated growth and physiological responses of pepper plugs to foliar applications of ABA, aminoethoxyvinyl glycine (AVG), an ethylene biosynthesis inhibitor, and physical ﬁlm-forming barriers AntiStress, Transﬁlm, and Vapor Gard. Water-deﬁcit tolerance of the pepper plugs occurred only when the plants were treated with ABA. The effectiveness of ABA was associated with improved plant water relations, reduced leaf abscission, and reduced growth rates. Other chemical compounds, such as paclobutrazol (PCB), a gibberellin biosynthesis inhibitor, have been used effectively to condition transplants (Dikshit et al. 2004). Internode length of tomato plugs was reduced 30% when applied to 4-week-old seedlings and 18% when applied to 6-week old seedlings. The percentage of ﬁrstcluster fruits was enhanced by 40% through the PCB treatment. Farnesi et al. (2004) compared PCB treatment to a UV treatment on plug muskmelon, cucumber, squash, and watermelon plug transplants. The UV treatments led to reduced plant height, leaf area, and stem and leaf dry weights. Bish et al. (1996) applied PCB to the shoots of 6-week-old ‘Sweet Charlie’ strawberry transplants at concentrations of 0, 50, 100, 200, and 400 mg L1. Increasing PCB concentrations decreased petiole length and reduced runner growth, both desirable traits for strawberry plug production. Early fruit yields, and thus gross income, were signiﬁcantly improved by PCB treatments. When considering the best and most cost-efﬁcient methods for plug conditioning, many factors come into play, including location time of year (temperature, light, humidity), species, time from plug harvest to planting, and ﬁeld conditions at planting. Withholding water and

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fertilizer is the least expensive conditioning method but may adversely retard early transplant growth in the ﬁeld. Brushing requires management, and can be difﬁcult to maintain on a large scale. Also, plant damage from brushing might lead to disease infection. Chemicals such as PCB and other nonnatural compounds have to be labeled, and the labels must be retained over time for usage. Therefore, ABA, as a natural plant hormone, might offer a bright future for plug producers for conditioning most species under a variety of conditions. The economics of using ABA, when to apply, and the optimum concentrations to apply still need to be worked out for many plug crops. Strawberry plugs are a viable substitute for the traditional bare root transplants commonly used by growers (Bish et al. 2000, 2001; D’Anna et al. 2003; Hochmuth et al. 2006a,b). In order to obtain early production from these plugs, the plants have to be subjected to cooler temperatures and, for winter production, shortened photoperiods (Kirschbaum et al. 1998). This process is also termed ‘‘conditioning.’’ Durney (1998) conditioned three-week old ‘Sweet Charlie’ strawberry plugs in seven 9-hour short days with chilling at night (21 /12 C, day/ night). For early production in January and February, yields were increased, but yields were similar during March and April. During winter production, yields from December to February bring the greatest economic returns to the grower. Bish et al. (2002) grew ‘Sweet Charlie’ strawberry plugs in greenhouses at 35 /25 C (day/night) or 25 /15 C for two weeks prior to ﬁeld planting. Plugs conditioned at 25 /15 C had greater root dry weights at planting and greater yields than those from 35 /25 C. Thus, strawberry plugs seem to do well after transplanting without additional conditioning treatments. Since early ﬂowering and fruiting are essential for winter production systems, the plants need to experience lower temperatures at night (preferably 15 to 20 C) for about 2 weeks prior to transplanting. It is essential that the plugs be actively growing during the conditioning process.

IV. ORGANIC PLUG PRODUCTION Organic producers must use both seeds and transplants that are produced organically, when available. If organic seeds are available, growers must then either obtain or grow plugs organically (Greer 2005). Producing ornamental and vegetable plugs organically is essentially the same as producing them under conventional systems, except that no nonapproved chemicals may be used, meaning that fertilizers must be from approved organic sources, media must be approved, and pest

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management becomes somewhat more difﬁcult. Thus, all the signiﬁcant factors required in order to produce quality plugs are similar for both systems. Approval of media for organic production generally centers on replacements for peat, as this substrate may be contaminated with chemicals (herbicides) and is considered nonsustainable. Compost has been found to be a viable alternate to the use of peat in media used to grow lettuce and cabbage (Burger et al. 1997; Ozores-Hampton and Vavrina 1997; Raviv et al. 1998; Sterrett 2001). Composting food residuals led to plugs of equal quality (size) as a commercial peatbased media (Clark and Cavigelli 2005). When developing and using a composted material as a major component of the media for plug production, all previously discussed characteristics for a quality media apply, such as particle size, water-holding capacity, pH, salinity, and stability (resistance to decomposition). Obtaining suitable fertilizers is a problem for organic plug producers because fertilizers are not always consistently formulated, are not completely water soluble, and may contain higher levels of salts. Larger plug producers supplement preplant fertilizers, through the irrigation system, either overhead or ebb and ﬂow. For either system, it is essential that the fertilizer is completely soluble and will not clog the irrigation system. If this is not the case, then all fertilizer must be added before planting, which prevents strict control of the nutritional system, especially N. Hardening, by reducing N levels, is impossible, and adding nutrients becomes more difﬁcult. Also, many organic materials may burn foliage if applied directly to leaves. Various plantand animal-based fertilizers have been used successfully to produce plugs (Koller et al. 2004; Russo 2005). Although several reports expound on the virtues of composted sewage sludge as an amendment to the media or for nutrient value, it is not permitted to be used under the National Organic Program (NOP) in the United States or in the European Union (EU). Other products, such as Sea Tea (compost tea), which contains Escherichia coli or enterococci bacteria, can be used so long as it has been pretested and meets Environmental Protection Agency recommended guidelines (Russo 2005). Numerous organic fertilizers have been tested recently for use with organic production of plugs, including Sea Tea, Rocket Fuel (100% organic source) (Russo 2005), horn powder (horn and claws of mammals), feather powder (chicken feathers), castor oil cake, malt sprouts, potato protein, maize protein, vinasse (residue of sugar beet processing), lupin, ﬁeld bean seeds, oil pumpkin seed cake, sunﬂower seed cake, rapeseed cake (Koller et al. 2004), Fertrell Super-N, and

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Bioﬂora Fish-O-Mega (Paranjpe et al. 2004). Some, such as horn powder, as well as materials from dried blood, have been restricted because of potential problems with using materials from animals. The levels of success in growing a plug crop with any of these materials is generally related to additional research on formulations, timing of application, concentrations, and species response. Most of these materials will cause leaf burn if applied directly to the foliage or used in too high of a concentration. In the United States, any fertilizer approved by the NOP can be used for organic plug production. Regardless, growing plugs organically is a major challenge due to the inconsistencies of the available organic fertilizer sources, especially with regard to available N and excessive salts.

V. POSTHARVEST HANDLING OF PLUGS Once plugs are grown, they normally have to be shipped; sometimes rather long distances (200–2,000 km). Handling plugs varies, generally according to the distance shipped. For instance, for local shipments (250 km or less), producers generally leave the plug in the growing tray, stacking them on shelves in a semi-open sided trailer and receiving back the empty trays once planted. Plugs shipped long distances are pulled, then packed in cartons or crates. The performance of the plug once transplanted is heavily related to its vigor when it arrives. Many factors can affect plug vigor, including how the plant was conditioned (hardened), size of the plug, species, mechanical injuries, container cell size, age of the plug, length and conditions of transport, and planting conditions (Cantliffe 1993, 1995). Comprehensive studies and reviews on pre- and postharvest practices affecting vegetable transplant quality have been published by Cantliffe (1993) and Leskovar and Cantliffe (1990, 1991) and will not be repeated here. Essentially, some species ship better than others, smaller younger plugs and small container sizes ship better than larger plugs; and short shipping times with refrigeration in aerated containers are best, as is planting under ideal ﬁeld conditions. Kubota and Kozai (1995) developed a model system for plug storing by experimenting with broccoli plant aseptically germinated and cultured in vitro for three weeks. Dry weights of the plantlets during storage were best maintained by keeping the carbon dioxide (CO2) exchange rates close to zero. Transplant quality was best maintained at 2 mmolm2 s1 photosynthetic photon ﬂux density (PPFD) light compensation point at 5 to 10 C without sugar or at 5 C with sugar in the medium. The authors

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felt that such a system incorporating PPFD, CO2 exchange, and temperature would help extend storage of plugs. In follow-up work with eggplant, Kubota et al. (2002) stored 3-week-old plugs for 4 weeks under different combinations of PPF and photoperiod. Continuous light at a PPF close to the light compensation point of 5 mmol m2 s1 gave rise to plugs that had no dry weight or quality losses. When plugs were stored at the same temperatures in the dark, leaf yellowing and signiﬁcant dry weight losses occurred. The authors also examined other light intensities (PPF) and photoperiods, which provided the equal daily integrated PPF of 430 mmol m2 which was achieved at the 5 mmol m2 s1 PPF in a 24-hour photoperiod. The combinations of PPF and photoperiod giving rise to this PPF level produced results similar to maintaining dry weight and quality of the plugs. In more recent work, Kubota et al. (2004) studied transplant quality of 6- to 8-week-old grafted tomato transplants during 2 days of transportation in temperature-controlled trailers. Reducing air temperature inside the trailer from the conventional 18 to 12 C signiﬁcantly improved ﬂowering and fruit of the ﬁrst truss. Leskovar and Cantliffe (1991) had similar ﬁndings for fresh-market tomatoes; transplant quality was improved by storing the plants at 5 C instead of 15 C. Kubota and Kroggel (2006) further explored storing tomato transplants in chambers with visible ﬂower trusses for 4 days at temperatures of 6 , 13 , or 19 C in darkness or with illumination at 12 mmol m2 s1. The lower temperatures with illumination maintained the highest-quality seedlings. The lower temperatures also maintained the highest photosynthetic activity, which was validated by the transportation trail between British Columbia and Arizona mentioned above. When transplants from the 19 C storage treatment were planted in the greenhouse, there was signiﬁcant ﬂower abortion or a delay in fruit development. Sato et al. (2004) improved transplant quality to cabbage seedlings through water stress by withholding water for 1 day before storage, then storing the plants for 6 days at 10 C in the dark. Leaf water potential of the stress plants was reduced, leading to less starch depletion in the stressed plants than the control plants. Most plugs have transportation times from 2 to 4 days. Under poor conditions of high temperature and darkness, the plugs will elongate and lose starch reserves. Ideal transportation/storage conditions include light; however, from a practical standpoint, this is generally impractical due to costs, equipment, and the fact that plugs may be in closed boxes. Thus, reduction of water before shipping in low temperatures may help best to maintain optimum plug quality.

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VI. ASSOCIATED PRODUCTION TECHNIQUES A. Seed Germination Plug growers must be aware of conditions that optimize seed germination and stand uniformity. This is especially important with the high cost seeds of many of today’s F1 hybrids that are grown as plugs. Several books related to plug production cover the subjects of environment and facilities to optimize germination (Styer and Koranski 1997; VanderVelde 2000). Germination in plug trays is dependent on species, moisture, aeration, temperature, and sometimes light. In order to optimize emergence rate and uniformity under conditions of varying external environmental conditions, it is advised that plug trays be placed in germination chambers or rooms that control temperature and humidity and provide light when needed (Styer and Koranski 1997). Aeration is dependent on the media used, depth of planting, cover materials, moisture levels in the media, stacking of the ﬂats, and air exchanges in the germination facility. For these reasons, most seeds are surface planted on top of the media then covered with coarse vermiculture, watered to drainage, stacked with aeration between the ﬂats, and placed in a room at near optimum germination temperature for the species (Cantliffe 1998, 2000). B. Seed Pelleting, Priming, and Grafting Many seeds that are sown for plugs; are small and/or irregularly shaped. Most commercial plug producers use drum vacuum seeders thus, these types of seeds should be pelleted or coated. Pelleting builds up seed size and uniformity by layering the seed with layers of a claytype material and binder. Seeds normally pelleted include lettuce, tomato, pepper, eggplant, onion, celery, begonia, petunia, and lisianthus. Seed coating is accomplished by applying wet solutions containing a dye and many times fungicides or other chemical treatments. The coating does not build up seed size, nor does it change the shape of the seed. The coating makes the seeds more ﬂowable in the seeder. Species that are commonly coated include cucumber, watermelon, squash, melon, marigold, zinnia, dahlia, impatiens, rannunculus, and anemone. Using dyes in the coating or pelleting process allows the seeds to be more visible at sowing. Both processes generally reduce the storability of the seed. Also, pelleted seeds cost more to ship because of the extra weight.

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Pelleting may slow down germination rates but not uniformity. In order to improve both germination rate and uniformity, many seeds are primed, regardless of whether they are pelleted or not. Seed priming process requires that seeds be hydrated and that germination is initiated usually under ideal conditions at reduced temperature (Parera and Cantliffe 1994). After a period of time, germination is stopped, and the seeds are redried to their initial water content. Because of this process, primed seeds should not be stored more than 6 to 9 months. Also, it is generally better for seed quality and enhancement performance if the same company ﬁrst primes the seed, then adds the pellet or coat. If seeds are primed, then redried, coating or pelleting may cause germination to start again as seeds are usually wetted during these processes. Redrying a second time may cause irreversible damage to the primed/pelleted seeds. If radicle growth is initiated during the second wetting process, it will be killed during the dryback process. Parera and Cantliffe (1994) have published a comprehensive review of seed priming. Most, if not all, seeds used for plug production can be primed by commercial ﬁrms specializing in priming and pelleting or by seed companies. Several species that are commonly transplanted from plugs, including tomato, pepper, watermelon, cucumber, melon, and eggplant, are now produced and sold as grafted plants (Lee and Oda 2003). These crops are high value, produced in greenhouses, and highly susceptible to root diseases. Vegetable grafting has been practiced for many years in various Asian countries, such as China, Korea, and Japan. Also, all grafted crops must be sold as plug plants. Compatible rootstocks have been developed for all of the above-mentioned species. Yields have been improved in crops both produced in the ﬁeld and greenhouse, many times in the absence of root disease pressure. A signiﬁcant increase in greenhouse production of vegetables in the Mediterranean area (Spain, Italy, Israel, and Turkey) has led to a signiﬁcant increase in the numbers of grafted plants used in these areas. Recently tomato greenhouse growers in Canada, the United States, and Mexico have adopted the use of grafted plants even though they grow in soilless hydroponic systems and the transplant costs more than standard plug plants. Returns to yield and fruit quality overcome the increased costs for the plug. Production, care, and shipping of the grafted plugs are generally no different from those processes required to produce and ship standard plugs. The importance of grafted plugs, especially for crops grown in the ﬁeld, may continue to increase due to the loss of methyl bromide. The use of robots to handle plug replants and transplanting could reduce costs and may be efﬁcient for large plug producers.

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C. Potential Plug Plant Biological Enhancement Treatments Various plant growth–promoting biological materials have been tested with plug-growing systems in attempts to improve root growth, plant growth, reduce soilborne pathogens, and improve yields. Many trials have used the compounds as seed treatments, while more recent studies have mixed the biologicals directly with the plug medium. There appears to be a potential role for incorporating plant growth– promoting biological materials in plug production systems, especially in early seedling growth; however, extensive research is needed. Populations of plant growth–promoting rhizobacteria (PGPR) were greater on lower and lateral tomato roots following application through soilless medium than through seed treatment (Zhinong et al. 2003). Also, the level of growth promotion of tomato transplants was greater from the medium application than from the seed application. It appeared that the medium application method led to good colonization of the bacteria in the rhizosphere. Adequate colonization has long been a major problem with bacterization and biological plant growth promoters and may now be better applied via the plug transplant. Zhinong et al. (2003) stated that applying PGPR’s for growth promotion as well as biological control of soilborne diseases seems best suited for the more uniform conditions of transplant systems. Vavrina et al. (2004) applied 11 commercially available systemic acquired resistance (SAR) materials with biological control properties, defense proteins, plant activators, organic compounds, modiﬁed fertilizers, or inert compounds to tomato transplants. They found few signiﬁcant SAR growth-enhancing effects. A growth-promoting rhizobacteria, Bacillus subtilis (a PGPR), improved growth of tomato transplants. The SARs did not lead to reduced symptoms from bacterial spot, but four of them did improve root growth when the plants were infested with root gall nematodes after transplanting. Kloepper et al. (2004) tested combinations of B. subtilis and B. amyloligue faciens with chitosan (an organic amendment) and reported signiﬁcant growth enhancement of tomato, pepper, cucumber, and tobacco potentially due to induced resistance of the plants to root-knot nematodes, bacterial spot, and tomato crown and root rots. The PGPR combinations with chitosan that the authors used were commercialized and sold as ‘BioYield’ (Gustafson LLC). The material is incorporated with the mix used to grow the plugs. The growth-promoting bacterium Burkholderia sp. applied to seeds and/or seedlings of tomato, pepper, and cucumber gave varying results according to species and application method (Nowak et al. 2004).

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Bacterization on the seedling was most effective on tomato and pepper, while bacterization of both seeds and seedling worked best on cucumber. Seed bacterization was ineffective on pepper and sometimes effective with tomato and cucumber. The response varied among cultivars regardless of species. Yields of cucumbers as number of fruits per plant and weight per plant were increased by using the bacterium. When the peat-based potting media was treated with liquid seaweed extracts (LSE), fewer cabbage seedlings were infected with Phythium ultimum (damping-off disease) (Dixon and Walsh 2004). The LSE effects were thought to be due to a stimulation of microbe growth that was antagonistic to P. ultimum. Thus, the LSE was considered to be a biostimulant. Growth of 18-day-old tomato seedlings was improved by the addition of a multistrain Trichoderma harzianum to the growing medium (Ozbay et al. 2004). However, plants were not evaluated in yield trials.

VII. MECHANIZATION Development of modern plug transplant production systems over the past 25 years has led to mechanization of much of the system from seedling to plug transplanting. Unfortunately, due to the biological nature of the system, growing plug plants still requires a signiﬁcant amount of labor. The transplant tray is the ﬁrst area that facilitates mechanization, both in seeding and potentially automatic ﬁeld transplanting (Shaw 1993). Until recent times, much of the vegetable plug industry was based on a rectangular reusable Styrofoam ﬂat. The tray ﬁts on aluminum stands or trays can be ﬂoated; it is lightweight, inexpensive, and durable. The tray can be used for automated seeding, and ﬁnished plugs can be either pulled and packed or shipped directly in the tray. Unfortunately, the Styrofoam planter ﬂat cannot be used for automatic ﬁeld transplanting, as it is not strong enough to withstand the rigors of mechanized transplanting. More recently, the industry has been making a shift to stronger, more durable (and still ﬂoatable) plastic trays. Some specialized transplant operations have been custom-planting vegetable plug seedlings using injection-molded plastic trays (Shaw 1993, 1999). In this system, seedlings are pushed downward, or vertically, through the cell bottoms by plungers. These systems allow

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planting up to eight rows at a time with 2 to 3 people instead of 10 to 12 people. Essentially all plug transplant operations have mechanized the seeding process (Styer 2000). Trays are loaded in bulk onto a line where they are ﬁlled with media, dibbled, sown via a vacuum drum seeder, topped with vermiculite (or some such porous coating), watered, and then stacked by hand. Operators have to inspect the line, stack trays to ﬁll, and stack the ﬁlled ﬂats as they come off the line. Generally, at least one person loads seed for the drum vacuum seeder. This process is enhanced if the seeds are pelleted, round, and of reasonable size (not too large or too small). Up to 1,200 trays per hour can be sown so long as the seeds are not oddly shaped, such as zinnia or marigold; needle seeders are generally preferred for these types of seeds. The number of lines and seeders can be increased to increase the volume of trays sown. More sophisticated plug operations use robots to load and unload ﬂats onto benches. Trays can be bar coded to allow for rapid computer identiﬁcation and tracking in the greenhouse. This same system can be used to insure that the correct numbers of ﬂats are sown for each species and cultivar. Once sown, the trays can be stacked on pallets and brought to germination rooms via a forklift. Robotic systems can be used for replugging in order to provide 100% usable plugs (Styer 2000). A computer vision system scans each cell and determines empty cells and unusable seedlings (Beytes 2000). Air ejection removes these cells, and usable seedlings are replugged. Automated robotic repluggers can cut labor costs by 75% for this operation. Once the seeds are germinated but not emerged from the media surface, trays are transported to the production house. Automation in the greenhouse includes irrigation, fertilization, and pest management. This is accomplished by boom devices that fertigate, irrigate, and/or apply chemicals for pest control for both bench-grown or ebb-and-ﬂow growing styles. This level of mechanization allows one grower to care for several transplant houses. Robots have been tested to pull ﬂats from the greenhouse and to pull and pack transplants for shipping. Numerous trials have examined the use of robots in all phases of the plant production system (Simonton 1992; Ting and Giacomelli 1992; Ting et al. 1992; Gao and Cui 1994; Huang et al. 1994; Sakane 1996; Kondo and Ting 1998). Ultra-precise global positioning systems (GPS) have been used to guide tractors to transplant at high speeds (9.8 kmph) (Abidine et al. 2004). The keen interest in mechanization and robots is dampened by only one negative factor: cost. However, due to the high costs for labor, the

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inability to obtain technically oriented labor for these jobs, and the high degree of precision required for the plug transplant system to work, continued improvements in the use of robotics and GPS technology should lead to greater mechanization of this industry over the next 10 years.

VIII. CONCLUSIONS AND PROSPECTS Transplanting vegetables and ornamentals has been done for hundreds of years. However, in the last three decades, the use of plugs has completely transformed the transplant industry and has greatly expanded the amount of plugs being used on a worldwide basis for both ornamental and vegetable species. This dynamic change can be attributed to three main factors: (1) the switch from open-pollinated to hybrid cultivars in the mid- to late 1970s; (2) the increase in popularity of expensive ornamental and bedding plants; and (3) the invention of the Speedling1 plug transplant system, which could be heavily automated. Plug transplant production has become a highly commercialized business, wherein most farmers buy their plugs from professional growers. It has been estimated that by 2010, over 1 trillion plugs will be produced and sold worldwide. Many factors help to produce quality plug transplants. These include the use of high-quality seeds, growing the plugs in a sterile media with good drainage, water-holding capacity, and providing optimal rates of fertility. Further, plugs are germinated under more or less optimal conditions to obtain uniform stands and are grown in protected culture under greenhouse conditions. Rate of plant development, root structure, plant height, and vegetative matter can be tightly controlled under these conditions. By producing plugs away from normal commercial production areas, producers can largely offset disease and insect spread from coming into their greenhouses. Container cell sizes can be adjusted to help produce plant sizes that conform to strict customer demands. Postharvest technology of plugs has come a long way to help producers to be sure only the highest-quality plants reach their customers. Automation of the transplant system likely has been the most neglected area of this exciting new transplant technology. For many commercial farmers, the transplant process still takes quite a bit of labor. Current research has gone into determining methodologies to streamline further both plug production and plug planting. The use of robots for tray ﬁlling, in the production greenhouse, and the

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mechanization of both the planting and growing process, as well as fertilization of the tray and harvest process can further reduce labor requirements of the plug production system. Potentially in the future, expansion of transplant systems that can mechanically place plugs in cells during transplanting will become the general method instead of its present limited highly specialized and highly technical equipment required for today’s multirow planters. The advent of plug transplants have allowed growers of many specialty crops the ability to greatly reduce seed costs, increase stand uniformity, and in many cases increase yields and quality of the products produced. In the future, many more crops may be grown as plug transplants, especially those of high economic value and potentially high seed cost.

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Kozai, T., C. Chun, and K. Ohyama. 2004. Closed systems with lamps for commercial production of transplants using minimal resources. Acta Hort. 630:239–252. Kratky, B.A., J.K. Wang, and K. Kubojiri. 1982. Effects of container size, transplant age, and plant spacing on Chinese cabbage. J. Am. Soc. Hort. Sci. 107:345–347. Kubota, C., and T. Kozai. 1995. Low-temperature storage of transplants at the light compensation point: air temperature and light intensity for growth suppression and quality preservation. Scientia Hort. 61:193–204. Kubota, C., and M. Kroggel. 2006. Air temperature and illumination during transportation affect quality of mature tomato seedlings. HortScience 41:1640–1644. Kubota, C., M. Kroggel, D. Solomon, and L. Benne. 2004. Analyses and optimization of long-distance transportation conditions for high-quality tomato seedlings. Acta Hort. 659:227–231. Kubota, C., S. Seiyama, and T. Kozai. 2002. Manipulation of photoperiod and light intensity in low-temperature storage of eggplant plug seedlings. Scientia Hort. 94:13–20. Kuehny, J.S., A. Painter, and P.C. Branch. 2001. Plug source and growth retardants affect ﬁnish size of bedding plants. HortScience 36:321–323. Kwon, J.K., J.C. Park, J.H. Lee, D.K. Park, and Y.H. Choi. 2003. Effect of UV-B irradiation on overgrowth retardation of plug-grown fruit vegetable transplants. J. Kor. Soc. Hort. Sci. 44:458–463. Kwon, J.K., J.C. Park, J.H. Lee, D.K. Park, Y.H. Choi, and M.A. Cho. 2003. Physiological changes and antioxidant anzyme activities of fruit vegetable plug transplants irradiated with different UV-B intensities. J. Kor. Soc. Hort. Sci. 44:464–469. Latimer, J.G. 1990. Drought or mechanical stress affects broccoli transplant growth and establishment but not yield. HortScience 25:1233–1235. Latimer, J.G. 1991a. Container size and shape inﬂuence growth and landscape performance of Marigold seedlings. HortScience 26:124–126. Latimer, J.G. 1991b. Mechanical conditioning for control of growth and quality of vegetable transplants. HortScience 26:1456–1461. Latimer, J.G., and R.B. Beverly. 1993. Mechanical conditioning of greenhouse-grown transplants. HortTechnology 3:412–414. Latimer, J.G., R.B. Beverly, and B. Blum. 1988. Analysis of shear force data in broccoli transplant studies. HortScience 23:627. Latimer, J.G., T. Johjima, and K. Harada. 1991. The effect of mechanical stress on transplant growth and subsequent yield of four cultivars of cucumber. Scientia Hort. 47:221–230. Latimer, J.G., and R.D. Oetting. 1994. Brushing reduces thrips and aphid populations on some greenhouse-grown vegetable transplants. HortScience 29:1279–1281. Latimer, J.G., T. Pappas, and C.A. Mitchell. 1986. Growth responses of eggplant and soybean seedlings to mechanical stress in greenhouse and outdoor environments. J. Am. Soc. Hort. Sci. 111:694–698. Lee, J.M., and O. Masayuki. 2003. Grafting of herbaceous vegetable and ornamental crops. Horticultural Reviews 28:61–124. Leskovar, D.I. 1998. Root and shoot modiﬁcation by irrigation. HortTechnology 8:510– 514. Leskovar, D.I., and A.K. Boales. 1995. Plant establishment systems affect yield of jalapen˜o peppers. 1st Intl. Symp. Solanaceae Fresh Market. Malaga, Spain. Acta Hort. 412:275– 280. Leskovar, D.I., and D.J. Cantliffe. 1990. Does the initial condition of the transplants affect tomato growth and development? Proc. Fla. State Hort. Soc. 103:148–153.

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Leskovar, D.I., and D.J. Cantliffe. 1991. Tomato transplant morphology affected by handling and storage. HortScience 16:1377–1379. Leskovar, D. I., and D.J. Cantliffe. 1992. Pepper seedling growth response to drought stress and exogenous abscisic acid. J. Am. Soc. Hort. Sci. 117:389–393. Leskovar, D.I., and D.J. Cantliffe. 1993. Comparison of plant establishment method, transplant, or direct-seeding on growth and yield of bell pepper. J. Am. Soc. Hort. Sci. 118:17–22. Leskovar, D.I., D.J. Cantliffe, and P.J. Stoffella. 1991. Growth and yield of tomato plants in response to age of transplants. J. Am. Soc. Hort. Sci. 116:416–420. Leskovar, D.I., D.J. Cantliffe, and P.J. Stoffella. 1994. Transplant production systems inﬂuence growth and yield of fresh-market tomatoes. J. Am. Soc. Hort. Sci. 119:662–668. Leskovar, D.I., and R.R. Heineman. 1994. Growth of ‘TAM-Mild Jalapen˜o-1’ pepper seedlings as affected by greenhouse irrigation systems. HortScience 29:1470–1474. Leskovar, D.I., and P.J. Stoffella. 1995. Vegetable seedling root system: Morphology, development, and importance. HortScience 30:1153–1159. Leskovar, D.I., and C.S. Vavrina. 1999. Onion growth and yield are inﬂuenced by transplant tray cell size and age. Scientia Hort. 80:133–143. Li, S., N.C. Rajapakse, R.E. Young, and R. Oi. 2000. Growth responses of chrysanthemum and bell pepper transplants to photoselective plastic ﬁlms. Scientia Hort. 84:215–225. Liu, A., and J.G. Latimer. Root cell volume in the planter ﬂat affects watermelon seedling development and fruit yield. HortScience 30:242–246. Loomis, W.E. 1925. Studies in the transplanting of vegetable plants. Cornell Univ. Agr. Expt. Sta. Memoir 87. Marr, C.W., and M. Jirak. 1990. Holding tomato transplants in plug trays. HortScience 25:173–176. Marsh, D.B., and K.B. Paul. 1988. Inﬂuence of container type and cell size on cabbage transplant development and ﬁeld performance. HortScience 23:310–311. Melton, R.R., and R.J. Dufault. 1991. Nitrogen, phosphorus, and potassium fertility regimes affect tomato transplant growth. HortScience 26:141–142. Miller, M.N., and C.N. Smith. 1980. The containerized vegetable transplant industry. Institute Food and Ag. Sciences, Univ Fa, Gainesville. Economic Impact Rep. 129. Nam, H.H., J.H. Woo, and K.B. Choi. 2003. Growth response of seedlings of annual bedding plants inﬂuenced by raising period and cell size of plug tray. J. Korean Soc. Hort. Sci. 44:223–227. Naoshi, K., and K.C. Ting. 1998. Robotics for plant production. Artiﬁcial Intelligence Rev. 12:227–243. NeSmith, D.S. 1993. Transplant age inﬂuences summer squash growth and yield. HortScience 28:618–620. NeSmith, D.S. 1994. Transplant age has little inﬂuence on yield of muskmelon (Cucumis melo L.). HortScience 29:916. NeSmith, D.S., and J.R. Duval. 1998. The effect of container size. HortTechnology 8:495– 498. Nicola, S., and D.J. Cantliffe. 1996. Increasing cell size and reducing medium compression enhance lettuce transplant quality and ﬁeld production. HortScience 31:184–189. Nicola, S., J. Hoeberechts, and E. Fontana. 2004. Studies on irrigation systems to grow lettuce (Lactuca sativa L.) tranplants. Acta Hort. 631:141–148. Paranjpe, A.V., D.J. Cantliffe, and R.L. Koenig. 2004. Developing a system to produce organic plug transplants for organic strawberry production. Proc. Fla. State Hort. Soc. 117:276–282. Parera, C.A., and D.J. Cantliffe. 1994. Presowing seed priming. Hort. Rev. 16:109–141.

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9 A History of Grafting Ken Mudge Department of Horticulture Cornell University Ithaca, NY 14853 USA Jules Janick Department of Horticulture and Landscape Architecture Purdue University West Lafayette, IN 47907 USA Steven Scofield U.S. Department of Agriculture, Agricultural Research Service Department of Agronomy Purdue University West Lafayette, IN 47907 USA Eliezer E. Goldschmidt R.H. Smith Institute of Plant Sciences and Genetics in Agriculture Hebrew University of Jerusalem PO Box 12 Rehovot 76100, Israel

I. INTRODUCTION A. Definitions B. Uses of Grafting 1. Vegetative Propagation 2. Avoidance of Juvenility 3. Cultivar Change 4. Creation of Unusual Growth Forms 5. Repair 6. Size Control 7. Biotic and Abiotic Stress Resistance

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II. III.

IV. V.

VI. VII.

K. MUDGE, J. JANICK, S. SCOFIELD, AND E. E. GOLDSCHMIDT 8. Transfer of Infectious Agents 9. Physiological Studies NATURAL GRAFTING HISTORICAL REVIEW A. Mesopotamia B. Biblical and Talmudic Sources C. Ancient Greece and Persia 1. Greece 2. Persia D. Rome E. China F. Medieval and Islamic Period 1. Medieval Europe 2. Islamic Period G. Renaissance Europe H. Early Modern Europe I. Nineteenth Century 1. French Wine Industry 2. Citrus Problems J. Twentieth Century 1. In Vitro Grafting 2. Herbaceous Vegetable and Ornamental Grafting. 3. Flower Bud Grafting 4. Mukibat Grafting of Cassava HISTORY OF CLONAL ROOTSTOCKS GRAFT HYBRIDS A. Graft Chimeras B. Graft Transformation CONCLUSION LITERATURE CITED

I. INTRODUCTION Since the origins of agriculture, the progressive domestication of food crops has been intimately related to a series of innovations in plant propagation. In the Fertile Crescent, 10,000 to 12,000 years ago, nomadic peoples subsisted in part by collecting the seeds of autogamous (self-pollinating) wild grasses (emmer and einkorn wheat, barley) and pulses (lentils, chickpeas, peas). Unintentional selection for traits useful to humans, such as nonshattering, uniform ripening, and other characteristics known as the domestication syndrome, progressed relatively rapidly. This resulted in some measure of food security, which contributed to social stability and the decline of nomadism. Although fruits, nuts, and other tree-related foods and ﬁbers were an important part of the diet, it was only several thousands

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of years later that domestication of woody plants began (Childe 1958; Zohary and Spiegal Roy 1975; Janick 2005). Unlike the grains and pulses domesticated much earlier, most useful woody species are highly heterozygous and do not come true to type from seed, which precluded rapid genetic improvement by seedling selection. What made tree domestication possible was the mastery of cloning via asexual propagation. Initially this involved cloning of genetically superior individuals of species that rooted easily from cuttings or layering or propagated by offshoots. The modiﬁcation and adoption by early agriculturists of these techniques allowed for the domestication of ﬁg, grape, pomegranate, and olive in the third or fourth millennium (Zohary and Spiegel-Roy 1975); all of which root easily from cuttings, and date palm, which was propagated by division of offshoots. The domestication of woody species that do not root easily from cuttings, such as apples, pears and plums, did not come until the discovery of grafting, at least several thousand years later, about the beginning of the ﬁrst millennium BCE. Thus, grafting is a pivotal technology in the history of temperate fruits and probably inﬂuenced their movement from Central Asia to Europe (Juniper and Maberly 2006). However, when and where detached scion grafting, which made possible the domestication of a new range of fruit trees, was invented is not clear. A. Deﬁnitions Grafting can be deﬁned as the natural or deliberate fusion of plant parts so that vascular continuity is established between them (Pina and Errea 2005) and the resulting genetically composite organism functions as a single plant. Two adjacent intact plants or different branches of the same plant can become naturally or intentionally grafted together; deliberate grafting involves inserting a previously cut shoot into an opening in another plant growing on its own root system (detached scion grafting). The term scion (cyon) refers to the shoot piece or bud cut from a donor plant that will grow into the upper portion of the grafted plant, The terms stock, under stock, or rootstock all refer to the plant that receives and fuses with the scion and functions as the root system of the grafted plant. Stock is synonymous with either rootstock or under stock, but the latter two differ with respect to their conﬁguration within the grafted plant. Rootstock implies that only the root system of the composite plant is derived from the original stock, whereas the term under stock is used when the lower portion of the grafted plant includes not only the root system but also some portion of the shoot system on which the scion is grafted. In the simplest case, a grafted plant usually consists of a single

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Fig. 9.1. Single-worked and double-worked grafts in fruit trees. (Source: Janick 1983).

graft union between a stock and a single scion. However, in a process called double working, a three-part grafted plant is constructed consisting of three genetically distinct parts, (rootstock, interstock, and scion) separated in linear sequence by two graft unions (Fig. 9.1). From a genetic perspective, grafting involves the creation of a compound genetic system by uniting two (or more) distinct genotypes, each of which maintains its own genetic identity throughout the life of the grafted plant. For example, a scion of a red-ﬂowering rose grafted on a white rose stock will continue to produce red roses rather than pink (hybrid) roses. However, controversial claims of graft ‘‘hybridization’’ have persisted, and new information on gene silencing cased by the transmission of RNA across the graft union suggests that grafting could have genetic consequences (see Section V). Another important genetic consideration related to grafting concerns the limits of compatibility. That taxomomic afﬁnity is the arbiter of which species can be grafted successfully onto any other has often been misunderstood through the history of grafting (Pease 1933). Broadly speaking, interclonal/intraspeciﬁc grafts are nearly always compatible, interspeciﬁc/intrageneric grafts are usually compatible, intrageneric/intrafamilial grafts are rarely compatible, and interfamilial grafts are essentially always incompatible. These generalizations are complicated by the observation that the degree of taxonomic afﬁnity necessary for compatibility varies widely across different taxa.

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For example, sugar maple (Acer sacharrum) cultivars can be grafted on any sugar maple seedling (interclonal/intraspeciﬁc), but in the case of red maple (Acer rubrum), grafting between different cultivars or seedlings is often incompatible. The interspeciﬁc/intrageneric combination of almond (Prunus amygdalus)/peach (P. persica) is compatible, but almond/apricot (P. armeniaca) is not. A comprehensive review by Nelson (1968) assessing the compatibility/incompatibility of many different stock/scion combinations across a wide range of species, genera, and families is useful not only horticulturally but also to our understanding of the physiological nature of compatibility. Andrews and Marquez (1993) recognize four potential factors that may contribute to incompatibility: (1) cellular recognition, (2) wounding response, (3) growth regulators, and (4) incompatibility toxins. In addition to graft incompatability, graft failure also can be caused by anatomical mismatching, poor craftsmanship, environmental conditions, and disease (Hartmann et al. 2002). B. Uses of Grafting Grafting is now a well-developed practice that has many horticultural and biological uses. One of the most ancient of horticultural craft secrets, grafting has had a wide impact on many practices and continues to be a force with implications for current technology. The many uses of grafting are listed next. 1. Vegetative Propagation. From the uncertain origins of grafting to the present day, the principal use of grafting has been for vegetative propagation of species that are otherwise difﬁcult to propagate asexually. Through this process of cloning, it is assured that the ramets (vegetative offspring) are genetically identical to the scion donor tree. In the case of scions grafted onto most seedling under stocks (apomictic seedlings are the exception), only the scion-derived top of the grafted plant is clonal. 2. Avoidance of Juvenility. Seedlings begin as juvenile plants, which are, by deﬁnition, incapable of ﬂowering. In woody plants, this period of juvenility typically lasts several years in fruit trees to several decades in forest species, before the plant undergoes a transition to the mature phase, when it becomes capable of ﬂowering. The next generation of seedlings is juvenile. The long delay in ﬂowering of these juvenile seedlings is economically undesirable. Fruit producers can overcome this delay in ﬂowering by grafting a scion from a mature tree onto any

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rootstock (seedling or mature), because a mature scion maintains its ﬂowering state in a graft to a juvenile seedling. 3. Cultivar Change. Cultivars of various fruit trees go in and out of style, especially as breeders produce improved ones. Cultivar change can be speeded up by taking advantage of the mature root system. Where the old tree is healthy, new scions can be grafted on scaffold branches of an established tree, a process known as topworking. A related practice is to use grafting to ‘‘build’’ a tree with multiple cultivars. When selfincompatibility is a problem, as in cherry and apple, a pollinizer can be grafted to achieve cross pollination within a single tree. 4. Creation of Unusual Growth Forms. In the ornamental nursery trade, it is a common practice to graft a scion from dwarf or weeping cultivar onto a tall straight stem of a compatible understock to mimic an arborescent growth habit. Tree roses can be formed by double working using a shrubby garden rose scion, a Multuﬂora de la Grifferaie interstock, to form a straight trunk, and ‘Dr. Huey’ rootstock. Grafting to create unusual growth forms in a practice called arborsculpture involves intertwining and grafting together the stems of two or more plants in order to create domes, chairs, ladders, and other fanciful sculptures (Fig. 9.2). 5. Repair. In cases where a root pathogen or bark damage (girdling) adversely affects an established tree, inarching seedlings around the base of the injured tree can effectively save the tree. Bridge grafting also can be used to repair a girdled stem. Brace grafting can be used to strengthen trees by internal grafts between branches. 6. Size Control. Certain rootstocks will result in dwarﬁng or invigoration of the scion cultivar. In apple, a single scion cultivar grafted onto the full range of size-controlling rootstocks can result in trees ranging from 2 to 10 m in height. In other species, certain interspeciﬁc scion/stock combinations will result in dwarﬁng, such as pear on quince and orange (Citrus sinensis) on trifoliate orange (Poncirus trifoliata). 7. Biotic and Abiotic Stress Resistance. Just as rootstocks have been selected for controlling the size of the scion, rootstocks also have been selected for resistance to various diseases, pests, and abiotic stresses. Rootstocks have been identiﬁed or selected for resistances to viral diseases (tristeza in citrus), bacterial diseases (ﬁre blight on apple),

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Fig. 9.2. Arborsculpture of Alex Erlandson’s Tree Circus made in the 1940s. (Photos top and bottom left and bottom right courtesy of R. Reames. Photo top right courtesy of Jon Covello.)

fungal diseases (collar rot caused by Phytophthora on apple), nematodes (Meloidogyne on peach and walnut), and insect pests (Phylloxera on grape). In the last case, grafting Vinifera grapes onto native American rootstocks species and hybrids saved the European wine industry in the 19th century (see Section III.I.1). Deliberate selection of rootstock resistance to Eriosoma lanigerum, the wooly

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apple aphid, allowed apple production in Australia and New Zealand where this pest was abundant (see Section IV). Recent studies also indicate that so-called orchard replant disease syndromes, in which fruit trees fail to establish successfully when planted into old orchard sites, can be mitigated by selection of resistant or tolerant rootstocks (Leinfelder and Merwin 2006). Unlike size control, rootstock resistances to these diseases and pests are not transmitted to the scion cultivar, but instead improve the health and survival of the rootsystem before and/or after grafting. There are also rootstocks selected for tolerance to excessively wet or droughty soils, alkaline or acidic soils, and other potentially stressful abiotic stresses. 8. Transfer of Infectious Agents. Virus indexing is the converse of employing virus-resistant rootstocks to prevent viral diseases. Many horticulturally useful plants may harbor devitalizing but otherwise asymptomatic viruses, while other related species readily exhibit symptoms to the same virus. Virus indexing to identify asymtomatic viruses involves grafting a piece of the suspect (asymptomatic) plant onto the susceptible (symptomatic) stock. Since all viruses are graft transmissible, the stock will exhibit symptoms if the virus is present in the scion. Although virus indexing is a reliable diagnostic tool, it is used less frequently since the advent of the more sensitive and more speciﬁc enzyme-linked immunosorbant assay (ELISA) or an assay based on the polymerase chain reaction (PCR). Grafting is used to transfer a phytoplasma (cell wall-less bacterium) to modify the growth habit of poinsettia, which induces a desirable branching (compact) growth habit. In all modern poinsettia cultivars, branching is induced by grafting an ‘‘infecting’’ scion onto a new cultivar. Similarly, the presence of latent viruses in certain apple rootstocks may actually improve the performance of scions grafted onto those rootstocks, compared to virus-free clones. For example, apple trees on virus-free EMLA.9 rootstocks are usually more vigorous and less precocious and productive than the same scions grafted onto other M.9 clones, such as M9.337, in which latent viruses have not been eliminated (Autio et al. 2001). 9. Physiological Studies. Grafting has been widely used in physiological and genetic studies to determine the transfer of mobile elements in plants. This includes translocation of alkaloids and secondary metabolites (Wilson 1952); transfer of the ﬂowering stimulus (ﬂorigen) (Zeevaart 2006); transfer of a potato tuberization stimulus (tuberin) (Ewing and Struik 1992); transfer of growth substances such

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as cytokinin from roots to shoots (Foo et al. 2007); and the transfer of RNAs (Tournier et al. 2006) (see Section V). Despite this extensive list of useful functions, grafting has not been without its critics. L. H. Bailey, the esteemed proponent of scientiﬁc horticulture and advocate for grafting, wrote in The Nursery Book (1914): ‘‘The opinion is commonly expressed by horticultural writers that graftage is somehow vitally pernicious and that its effect on the plant must be injurious . . . akin to magic and entirely opposed to the laws of nature.’’ Jonathan Chapman (1779–1845), the legendary Johnny Appleseed who roamed the frontiers of the upper midwestern United States planting apple nurseries by seed, is reported to have felt that cutting into a tree was cruel, despite the fact that grafting of apples was widely practiced at that time (Tully 2007). More recently, some proponents of organic agriculture proposing a new ethic for plant culture have questioned both tissue culture and grafting (Lammerts Van Bueren. 2007). To this day, despite considerable advances in the science and technology of plant propagation, grafting still plays an important role in the production of horticultural crops, including fruits, ornamentals, and vegetables (Lee and Oda 2003). Even though the origins of grafting are obscure, the signiﬁcance of its discovery to crop domestication and agriculture throughout recorded history is quite clear. The ancient and modern history of grafting and its impact on horticulture then, now, and in the future are the focus of this review. The carpentry, anatomy, and art of grafting (see Garner 1988) are discussed only peripherally. II. NATURAL GRAFTING Although grafting usually is considered a horticultural practice that involves the deliberate manipulation of stock and scion to form a graft union, both shoot and root grafting occur naturally (Fig. 9.3). It is possible that deliberate approach grafting by early agriculturists came about from an attempt to mimic the natural fusion of two plants or different parts of the same plant that occurs in natural grafting. Juniper and Maberly (2006) have speculated that grafting may have come about by observing natural grafts that occurred in shelters made from arching live poles arranged in a circle and tied together at the top. When poles of an easy-to-root species (e.g. Salix spp., Ficus spp.) are driven into moist ground, they will root, and if they are tied tightly together, they may also ‘‘naturally’’ (nondeliberately) graft to each other. The same essential requirements that exist for successful detached scion grafting—compatibility, cambial alignment/contact, pressure,

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Fig. 9.3. Natural grafting. (A) Root grafting of yellow birch. (Photo by K. Mudge) (B) Root grafting of apple. (Photo by Janick 2005) (C) Natural root autografting of strangler ﬁg on sable palm. (Photo by K. Mudge) (D) Shoot grafting of oak. (Photo by R. Uva).

and avoidance of desiccation—apply to natural grafting. The concept of discrete stock and scion is ambiguous in natural grafting when both sides remain attached to a common root system, in the case of self- or auto grafting, or when each remains attached to its own independent root system, in the case of natural grafting between two plants. Natural stem grafting is far less likely to occur than natural root grafting, even though the former would be more obvious and easily detected. Roots cross or otherwise come into contact with each other as they elongate. Embedded within a soil matrix, they are held ﬁrmly together as they increase in diameter, resulting in increased pressure between the two. Only when a branch of one tree becomes

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wedged in the fork of another is the prolonged contact under pressure likely to persist long enough for graft union formation to occur. Exceptions to the generalization that aboveground natural grafting is infrequent are vines such as Hedera helix (English ivy) and the aerial roots of strangler ﬁgs, which fuse readily in part because they become tightly appressed against the substrate (tree trunk or wall) on which they are growing (Fig. 9.3D). Natural shoot grafting has been reported in Alnus oregano (Rigg and Harrar 1931; cited in La Rue 1934), Hedera helix (Millner 1932, cited in La Rue 1934), red pine, the East African shrub Maeura triphylla, black cherry, star magnolia, live oak, and white oak, sugar maple, and sycamore (K. Mudge unpubl.). Natural root grafting has been widely reported and extensively studied because it is common and affects forest ecology. Graham and Bornman (1966) cited 150 species forming natural root grafts, although Larue (1934) indicated that natural root grafting was scarce in Larix laricina, Populus tremuloides, Fraxinus nigra, and Prunus serotina. Natural root grafting has also been observed in strangler ﬁg, white pine, Populus spp., sugar maple, beech, and yellow birch (K. Mudge, unpubl.). The vast majority of reports of natural root grafting involve two individuals of the same species (intraspeciﬁc), but several reports of natural grafting between different species (interspeciﬁc) include several species of oak, maple, and pine (Graham and Bornman 1966). Interfamilial combinations have been reported between Betula alleghaniensis and Ulmus Americana (Larue 1934); and B. alleghaniensis and Acer saccharinum (Graham and Bornman 1966). However, reports of interfamilial grafts must be regarded with skepticism, since there are no veriﬁable reports of compatible interfamilial grafts. Based on dissection of apparent graft unions between cedar and hemlock, Eis (1972) found that invariably both were separated by layers of bark and hence were not true graft unions. Natural root grafting can inﬂuence forest ecology in a number of ways: (1) effects on tree distribution and dominance within a stand; (2) effects on a surviving tree after an intergrafted tree adjacent to it is cut down or otherwise dies; (3) mechanical stabilization against wind throw; and (4) disease transmission across graft unions. It can be observed in a natural or managed forest stand that trees of a given species tend to be clumped rather than evenly distributed (Graham and Bornman 1966), which suggests that tree-to-tree interactions are to some extent complementary rather than strictly competitive. Eis (1972) concluded that natural root grafting is a means by which cooperativity occurs, to the extent that a dominant tree could provide resources

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(carbohydrate, water, minerals) to its subordinate neighbors, allowing them to persist under low light or drought stress. He speculated that the root systems and lower boles of suppressed trees within a clump may obtain most of their carbohydrate from the dominant tree to which they are root grafted. The common occurrence of living stumps also supports the cooperative model. Unlike most angiosperm tree species, stumps of most conifers, including white pine, do not regenerate from epicormic or adventitious shoots (P. Smallidge pers. commun.), and hence their persistence cannot be attributed to photosynthates from newly regenerated shoots. Bornman (1966) observed that stumps sometimes persist and continue to grow, for years or even as long as decades, when they are root grafted to adjacent trees but not when they occur singly, suggesting that carbohydrates translocated via root grafts from the standing tree to the stump are responsible for the persistence of the latter. In this sense, living stumps can be considered hemiparasites on the trees to which they are grafted (Bornman 1966). It should be noted that mycorrhizal interconnections may account to some extent for persistence of living stumps, but these are unlikely to play more than a minor role. It has been suggested that another beneﬁcial effect of intergrafting of the root systems of a pair or a cluster of trees is the resulting stabilization of individual trees against wind throw (Loehle and Jones 1990). Basnet et al. (1993) found that intergrafted trees of tabonuco (Dacryodes excelsa) underwent signiﬁcantly less hurricane damage than isolated trees. Loehle and Jones (1990) considered the adaptive signiﬁcance of root grafting in trees from an evolutionary perspective. They concluded that the assimilates gained by a suppressed tree through grafting to a dominant tree could keep the former alive longer so that it could reproduce or until it was released by a change in the surrounding canopy. Clumping of trees within a forest stand and the persistence of living stumps are positive ecological consequences of natural root grafting. Grafting between adjacent trees can have substantial negative consequences, however, related primarily to the spread of diseases. Two of the best-known and probably most consequential examples are transmission of dutch elm disease (DED) and of oak wilt disease. The causal organism for DED is the fungus Ophiostoma ulmi (Brasier 1991). Above-ground transmission is via the elm bark beetle, Hylurgopinus ruﬁpes, but root grafts are another major means of transmission (Epstein 1978). Control of root graft transmission requires trenching on a regular basis between trees, with or without fumigation in the trenches. Epstein (1978) considered failure to deal with root graft transmission to be the major reason that many DED control

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attempts in the midwestern United States have failed. Oak wilt, caused by Ceratocystis fagacearum, is another serious fungal disease transmitted by root grafts. Epstein (1978) reviewed a number of other root graft–transmitted fungal pathogens (Trametes pini, Armillaria mellea, Polyporus schweinitzii, Endothia gyrosa, Fomes annosus, and Phytophthora lateralis). A bacterial disease called citrus variegated chlorosis is transmitted between citrus rootstocks via natural root grafts (He et al. 2000). The viroid ASVBd, causal agent of avocado sunblotch disease (Dodds et al. 2001), and xyloporosis, a viral disease of citrus (Epstein 1978), are transmitted via natural root grafts. This discussion of natural grafting would be incomplete without consideration of the symbiotic association between mistletoes (parasitic plants in the order Santalales) and a wide range of woody plant species. Parasitic mistletoes are not usually included in the literature on natural grafting, but the intimate associations between stock and scion and between mistletoe and host suggests that they share some common features anatomically and physiologically. In fact, in the mistletoe literature, the union between the parasitic plant and its host tree is sometimes referred to as a ‘‘graft’’ union (Thoday 1956). This is particularly interesting considering that the taxonomic range of compatibility between mistletoe and host is much greater than the taxonomic limits for plant to plant grafting. In mistletoes, interfamilial associations with host trees are common, whereas interfamilial graft combinations do not occur in woody plants (Mendel 1953). It would appear that much could be learned about the nature of graft compatibility by studying mistletoe/host plant associations. Nonetheless, there is at least one critical difference between the mistletoe ‘‘grafts’’ and nonparasitic plantto-plant grafts. In both there is apoplastic continuity across the union (Cotzee and Fineran 1989), but nonparasitic plant-to-plant grafts are also interconnected symplastically via plasmadesmata (Tidemann 1989). Plasmadesmata have not been convincingly demonstrated at the interface between mistletoe and its plant host (Cotzee and Fineran 1989). Other systems that may shed some light on the nature of graft compatibility include pollen/stigma interaction and human tissue compatibility.

III. HISTORICAL EVIDENCE A. Mesopotamia The evidence that grafting was used in ancient Mesopotamia is murky at best. Harlan (1995) speculates that grafting developed in Eurasia

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before classical times but offers no speciﬁc evidence. Circumstantial evidence for grafting is suggested by Harris et al. (2002) and Juniper and Maberly (2006) based on the text of a Sumerian cuneiform fragment dated approximately 1800 BCE. The text, translated and interpreted by Lion (1992), refers to the transport of grapevine shoots to be replanted at Mari (present-day Iraq). However, Lion’s commentary does not mention grafting per se, and there is no reason to surmise that the vine shoots were intended as scions, because grape shoots root readily from hardwood cuttings, and cutting propagation was the standard method of grape propagation throughout the Middle East. The suggestion of Juniper and Maberly that gradual salinization of agricultural soils may have motivated farmers to graft edible but salt-intolerant grapes onto salt-tolerant wild grape rootstocks is a fascinating conjecture and if true would represent the ﬁrst known use of grafting to take advantage of speciﬁc rootstock effects. B. Biblical and Talmudic Sources The Hebrew Bible, one of the most important of ancient documents, is composed of numerous books written over 1,000 year period (ca. 1400– 400 BCE), including law, prophecy, poetry and parables, many of which relate to plants and agriculture. Grafting is not speciﬁcally mentioned, but many references appear to suggest that it was practiced. The several biblical texts that allude to grafting concern the grapevine as a parable and refer to reversion from cultivated to wild types: ‘‘And I planted you ‘Sorek’, all true seed, and how did you revert into a wild, alien vine’’ (Jeremiah 2:21). This could be construed as an undesired mutation that appeared in the grapevine; more likely it can be understood as an outgrowth of the stock on which the grape was grafted. A similar text is from Isaiah 5: 1–2: ‘‘My beloved had a vineyard in a very fruitful hill. He digget it and cleared it of stones, and planted it with choice vines; he built a watchtowere in the midst of it, and hewed out a wine vat in it; and he looked for it to yield grapes, but it yielded wild grapes.’’ The book of Leviticus 19:19, dated to about 1400 BCE, states: ‘‘Thou shalt not sow thy ﬁeld with two kinds of seed.’’ This is the basic admonition against mixing seeds of different kinds or sowing them in close proximity, which was discussed in detail in the Talmudic tractate Kilayim of the Mishna (commentaries on biblical law composed in the third century CE.) Although the Mishna explicitly states (Kilayim 1:7) ‘‘It is unlawful to graft tree on tree, vegetable on vegetable, tree on vegetable or vegetable on tree’’ [if they belong to a different species], it is questionable whether grafting was originally included in the prohibition

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of mixing mentioned in Leviticus. The art of grafting may have not yet been practiced in ancient biblical times. Greek and Roman sources (see Section II.C,D) indicate that grafting was well known and widely practiced the Mediterranean region by the ﬁfth century BCE and during the Talmudic–Hellenistic times, when the Mishna was composed. In the Mishna, grafting and layering of grapevines appear as a common practice (Tractate Orla’ 1:5) and planting, layering, and grafting are often described together as regular methods of fruit tree propagation (Tractate Sheviith’ 2:6; Sota 8:2). One paragraph of the Mishna (Kilayim 1:4) lists several fruit tree stock/scion combinations, mostly from the Rosaceae: ‘‘And in trees: grafting pear with krustomal [a kind of pear], or quince with Crataegus is permissible. However, grafting of apple with wild pear, peach with almond or Ziziphus vulgaris with Ziziphus spina-Cristi, in spite of their similarity, is forbidden’’ [translated from Feliks (1967)]. In several Talmudic parables, marriage is compared to grafting; thus we ﬁnd that marriage of a scholar into a noble family is to be praised, comparable to a graft between high-quality grape cultivars; whereas marriage of a scholar into a family of illiterates is as unacceptable as a graft between quality grapes and wild grapes (Talmud Bavli Pesachim 49a). However, according to Talmudic sources (Yerushalmi Kilayim 1:7), the verse ‘‘Your sons like olive seedlings surrounding your table’’ (Psalms 128:3) is interpreted ‘‘Just like olive trees, which are never grafted, so your offspring will be ﬂawless.’’ This may indicate that even where permissible, grafting was understood as some kind of breeding. This idea is actually stated clearly by the Talmudic scholar Shmuel (third century CE) who ﬁnds a hint to the prohibition of interspeciﬁc grafting in Leviticus 19:19: ‘‘Thou shalt not sow thy ﬁeld with two kinds of seed, nor breed two kinds of animals.’’ Thus, just as it was forbidden to breed two kinds of animals, it was forbidden to graft two plant species (Talmud Bavli Kidushin 39a). A kinship between grafting and sexual intercourse is borne out also by the reasoning of the prohibition of mixing provided by Maimonides (see Section III.F). The notion that breeding can lead to the appearance of a new species is also mentioned in Talmudic sources (Yerushalmi Kilayim 1:4), just as in the contemporary Roman sources, as is discussed in detail by Feliks (1967). The grafting of etrog (citron, Citrus medica) onto lemon (C. limon) or other citrus stock is still a matter of contention among orthodox Jews. Citron is an important component in the feast of Tabernacles (Succoth) and is still highly prized as a gift for orthodox Jews during this joyous holiday (from which the feast of Thanksgiving derives). From its

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introduction to Palestine by the second century BCE, citron was the only citrus fruit in that region until lemon arrived in the seventh century CE. At some point after its introduction, lemon was being used as a rootstock for citron. Based on Jewish law, interspeciﬁc grafting (or ‘‘hybridization’’) was not permitted. In the 16th century, that law was speciﬁcally applied to citron grafted onto lemon, resulting in attempts to locate ‘‘pure’’ citron trees that have never been grafted or sexually hybridized. In a recent study involving citron populations from Yemen, Morocco, Israel, and Italy, using randomly ampliﬁed polymorphic DNA (RAPD) and sequence characterized ampliﬁed regions (SCAR) molecular analyses conclude that there has been no genetic introgression by lemon or other citrus species (Nicolosi et al. 2005). Grafting is speciﬁcally mentioned in the New Testament (Christian Bible). In Romans 11:24, it is used as an analogy for how Gentiles can become one with Israel: ‘‘For if you were cut off from what is by nature a wild olive tree, and grafted, contrary to nature, into a cultivated olive tree, how much more will these natural branches be grafted back into their own olive tree?’’ A similar and clearly derivative passage on grafting is found in the Book of Mormon (Jacob 5:17–18), which implies a belief that the fruit of the scion could be drastically improved by the root stock: And it came to pass that the Lord of the vineyard looked and beheld the (‘‘tame,’’ not wild) tree in which the wild olive branches had been grafted; and it had sprung forth and begun to bear fruit. And he beheld that it was good; and the fruit thereof was like unto the natural (‘‘tame’’) fruit. And he said unto the servant: Behold, the branches of the wild tree have taken hold of the moisture of the root thereof, that the root thereof hath brought forth much strength; and because of the much strength of the root thereof the wild branches have brought forth tame fruit.

C. Ancient Greece and Persia 1. Greece. The earliest veriﬁable written account of grafting is from the Hippocratic treatise, On the Nature of the Child, thought to have been written in about 424 BCE by one or more of the followers of Hippocrates, hence referred to ‘‘Pseudo Hippocrates’’ (Meyer 1854; Vochting 1892; Lonie 1981; Mendel 1953): Some trees however, grow from grafts implanted into other trees: they live independently on these, and the fruit which they bear is different from that of the tree on which they are grafted. This is how: ﬁrst of all the graft

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produces buds, for initially it still contains nutriment from its parent tree, and only subsequently from the tree in which it was engrafted. Then, when it buds, it puts forth slender roots in the tree, and feeds initially on the moisture actually in the tree on which it is engrafted. Then in course of time it extends its roots directly into the earth, thorough the tree on which it was engrafted: thereafter it uses the moisture which it draws up from the ground. (Lonie 1981)

The passage suggests that grafting was a common technique at that time and thus must be centuries older than the fourth century BCE. In this treatise, the embryonic and subsequent development of a child is related to that of a seed. Unlike many later references to grafting over the next two millennia, the author recognizes that a grafted plant produces fruit true to the scion cultivar rather than the stock. At the time, this was considered a paradox, consistent with the belief that each species nourished itself by drawing from the earth a species-speciﬁc ﬂuid through its roots. This is the theory of ‘‘speciﬁc ﬂuid’’ as described by Lonie (1981). The paradox is as follows: ‘‘The rootstock of a grafted plant obtains its nourishment (speciﬁc ﬂuid) directly from the soil. The scion would not have direct access to its own speciﬁc ﬂuid, but only that of the rootstock, and hence it would be transformed by this rootstock ﬂuid into the rootstock variety.’’ To explain this paradox, the author proposed that rather than the scion being merely joined to the stock, it was rooted directly into the stock. The roots of this scion ‘‘cutting’’ grew down through the stem of the stock directly into the soil. There it was able to take up and transport its species-speciﬁc ﬂuid and hence maintain its species (genetic) identity, giving rise to fruit of the scion cultivar rather than that of the stock (Fig. 9.4). This idea of speciﬁc ﬂuids is one that persisted, at least in some circles, until the seventeenth century in England. The same paradox was confronted by Robert Sharrock’s (1672) in his book The History of the Propagation & Improvement of Vegetables by the Concurrence of Art and Nature, although he suggested another ‘‘metaphysical’’ solution to the problem, which will be discussed in Section III.H. Theophrastus (371–287 BCE), a student of Aristotle and now considered the father of botany, reiterated the view of Pseudo Hippocrates that grafting is ‘‘propagation in another tree’’ (the stock as substrate for rooting of the scion) rather than mere juxtaposition. He discussed grafting techniques, such as the importance of avoiding desiccation by cutting the stock and scion precisely so that they ﬁt together tightly and the core is not exposed to drying. This was the reason for wrapping the stock/scion junction with layers of lime bark,

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Fig. 9.4. The theory of the preservation of graft identity from Pseudo-Hippocrates: scion rooting through the graft union permits the extraction of its speciﬁc ‘‘ﬂuid’’ from the soil.

plastered mud, and hair. In extreme cases, ‘‘set a pot of water over it and let the water drip.’’ No mention is made of what we would call genetic/taxonomic aspect of compatibility but rather the importance of stock and scion having ‘‘like bark’’ and time of bud break. Theophrastus comments on dwarf fruit trees, which were likely to have been introduced to Greece by Alexander the Great during his conquest of Asia Minor. Among these was a small, low-growing type of apple. This may have been predecessor to the current dwarﬁng apple rootstock ‘Malling 8’ (Tukey 1964). 2. Persia. The origins of Persian gardens may date to the fourth millenium BCE based on evidence of pottery. Remnants of the gardens

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of Cyrus the Great date to 500 BCE. Persia was at the crossroads of importations of fruits from the East. Juniper and Mabberly (2006) suggest that grafting likely originated in Persia, but they present no direct evidence. D. Rome During the Roman era, a number of authors wrote about grafting as a common agricultural practice. Marcus Porcius Cato (234–148 BCE), the earliest of the Latin writers, is his famous work De agri cultura, described various methods of grafting that are still used with different fruit crops, including cleft and approach grafting as well as budding. He described another curious technique that combines grafting and layering: With an awl bore a hole through the vine which you are grafting, and ﬁt tightly to the pith two vine shoots of whatever variety you wish, cut obliquely. Join pith to pith, and ﬁt them into the perforation, on each side. Have these shoots each two feet long; drop them to the ground and bend them back toward the vine stock, fastening the middle of the vine to the ground with forked sticks and covering with dirt. Smear all these with the kneaded mixture, tie them up and protect them in the way I have described for olives. (Hooper 1935)

Marcus Terrentius Varro (167–27 BCE) addressed the issue of the limits of stock/scion compatibility directly: ‘‘you cannot, for instance, graft a pear on an oak, even though you can on an apple.’’ He goes on to describe the curious view of soothsayers that multiple cultivars grafted onto a single understock ‘‘attracts the lightning and turns into as many bolts as there are varieties.’’ He indicates that a scion of a tree of better type should be grafted onto a lesser one. It seems likely that approach grafting was perhaps the earliest method of deliberate grafting, and preceded detached scion grafting. Varro describes approach grafting as a method that had recently been developed, in cases ‘‘where the trees stand close to each other.’’ A particularly interesting commentary on the genetic limits of compatibility (or lack thereof) was put forth by Publius Verglius Maro (known as Virgil, 70–19 BCE). Virgil, best known for his epic poem the Aeneid, later spent seven years writing a didactic collection of fourth ‘‘books’’ on farming called the Georgics (a Greek word meaning ‘‘tillage, agriculture, and rural affairs’’), which was akin to an almanac for a gentleman farmers. A passage in the Georgics states: ‘‘But the rough arbutus with walnut-fruit is grafted; so have barren planes ere now stout apples borne, with chestnut-ﬂower the beech, the mountain-ash

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with pear-bloom whitened o’er, and swine crunched acorns ‘neath the boughs of elms.’’ This passage makes clear that at least some of Virgil’s knowledge of grafting was not ﬁrsthand because his understanding of the limits of compatibility was decidedly incorrect. A compellingly poetic verse describing cleft grafting is presented next in the famous translation by John Dryden (1631–1700) (Virgil 1953). But various are the ways to change the state Of plants, to bud, to graff, to inoculate For, where the tender rinds of trees disclose Their shooting germs, a swelling knot there grows: Just in that space a narrow slit we make, Then other buds from bearing tress we take; Inserted thus, the wounded rind we close, In whose moist womb the admitted infant grows. But when the smoother bole from knots is free; We make a deep incision in the tree. And in the solid wood the slip inclose; The battening bastard shoots again and grows; And in short space the laden boughs arise; With happy fruit advancing to the skies. The mother plant admires the leaves unknown Of alien trees and apples not her own.

Several decades later, Caus Plinius Secondus, known as Pliny the Elder (23–79 CE), wrote about graft compatibility, demonstrating a lack of understanding similar to that of Virgil, when he described ‘‘inoculation’’ (budding) of ﬁg and apple, but goes on to remark that: ‘‘Thus far has Nature been our instructor in these matters.’’ He gave precise instructions for grafting grape vines, and advised: ‘‘Engraft moist places from a white grape, dry places from a black’’ (Book XVII, Bostock and Riley 1855). However, Pliny gives a logical explanation of wide grafts in a discussion of the ﬁrst discovery of grafting: Nature has also taught us the art of grafting by means of seed. We see a seed swallowed whole by a famished bird; when softened by the natural heat of the crop, it is voided, with the fecundating juices of the dung, upon some soft couch formed by a tree; or else, as is often the case, is carried by the winds to some cleft in the bark of a tree. Hence it is that we see the cherry growing upon the willow, the plant upon the laurel, the laurel upon the cherry, and fruits of various tints and hues all springing from the same tree at once. It is said too, that the jack-daw, from its concealment of the seeds of plants in holes which serve at its store-house, give rise to a similar result. (Book XVII, Bostock and Riley 1855).

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Fig. 9.5. Detached scion grafting from a third-century mosaic from St. Roman-en-gal, Vienne, France. (Source: Janick 2005).

A Roman mosaic found in St. Roman-en-gal, Vienne, France, from the third century CE displaying a series of agricultural events during the calendar depicts detached scion grafting (Fig. 9.5). This image is the oldest deﬁnitive image of grafting. Rutilius Tauros Aemilianus Palladius (fourth century CE) authored a 14-book work Opus agriculturae of which the last book, in elegiac verses, is on grafting (Owen 1897). Crops include grape, olive, pear, pomegranate, apple, peach, medlar, citron, plum, carob, ﬁg, mulberry, service tree (Sorbus), cherry, almond, pistacio, chestnut, and walnut. Compatibility is reported for olive with wild olive; pear with quince, medlar, wild ash (fallacious), chestnut (fallacious); apple with medlar, service tree (Sorbus), plum (fallacious), willow (fallacious), plane-tree (fallacious), peach (fallacious), plum (fallacious), poplar (fallacious), chestnut (fallacious); plum with chestnut (fallacious); carob with other fruits (?); mulberry with ﬁg (fallacious), ash (fallacious), beech (fallacious), chestnut (fallacious), terebinth (wild pistachio) (fallacious); service tree (Sorbus) and quince (fallacious); cherry with plum, plane tree (fallacious), poplar (fallacious); almond with plum, chestnut (fallacious); pistachio with almond (fallacious); chestnut with willow

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(fallacious); and walnut with arbutus (fallacious). The high number of fallacious (incompatible) combinations indicates that Palladius was not writing from personal experience. E. China Reports of Chinese agriculture prior to the 14th century BCE are largely mythological because historical records from China begin very sparsely in about 1200 or 1250 BCE and do not get rich until about 400 BCE (Chang 1965; Arbuckle 1994; R. MacNeal pers. commun.). Pollan (2001) simply asserts that grafting originated in China sometime during the second millennium BCE. One dubious claim for the early origins of grafting is based on the mention of citrus fruits in the Chinese literature during the reign of Ta Yu (ca. 2205–2197 BCE). Cooper and Chapot (1977) mention that chu (a generic term for kumquats and small-fruited mandarins) are indigenous to the ancient Yanchow region and probably were included in tributes. Accounts of chu in early Chinese herbals indicate that they were a wild-growing loose-skinned fruit. One early (but unfortunately undated) Chinese tradition has chu grafted onto chih (Poncris trifoliata). Hartmann et al. (1997) put the date at 1560 BCE, but no evidence is presented. Juniper and Maberly (2006) speculate that grafting of mulberry may have originated in association with the development of a Chinese silk industry about 300 BCE, although it is not clear what would have motivated the development of this relatively elaborate means of cloning because the mulberry (Morus alba) is easily rooted from cuttings (Hartmann et al. 2002) and by layering (Anon. 2007a). The earliest (indirect) credible evidence that grafting was being practiced in China may have been written about in the ﬁrst century BCE in The Book of Fan Sheng-Chih Shu. According to Shih Sheng-Han (1959), the original book was lost long ago and only excerpts have been preserved in later agricultural treatises, especially in the all-comprehensive Ch’I Min Yao Shu (Essential Techniques for the Farming Populace) by Jia Sixie in the 6th century CE, and translated into English by Shih Sheng-Han (1962). According to Jia Sixie, the earlier Book of Fan Sheng describes grafting of bottle gourd (Lagenaria siceraria). The practice involved planting 10 seeds in one hole, tying the emerging stems together so that they fuse (approach graft), and then pruning to a single shoot, which would remain attached to 10 root systems. The purpose may have been to increase fruit size or lengthen the production season by delaying root system decline due to fungal diseases (10 root systems hold out longer than 1), rather than

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invigoration of the scion since bottle gourd is extremely vigorous to begin with (H.C. Wien pers. commun.). Most of the recommendations regarding the techniques and timing of grafting by Jia Sixie are reasonable and sound, but his advice regarding the limits of graft compatibility is not always accurate. The recommendation to graft pear onto ‘‘crab apple’’ (Pyrus phaeocarpa) or P. betulifolia is reasonable because they are different species within the same genus. However, he describes the combination of Pyrus sp (scion) and either jujube (Zizyphus sp.) or pomegranite (Punica granatum L.) rootstock. These species are not in the same family and hence are certain to be incompatible since there are no veriﬁable reports of successful interfamilial grafts in woody plants. Advice is given in Ch’I Min Yao Shu pertaining to the use of grafting in the open orchard compared to the courtyard of the home: ‘‘when grafting pear trees in an orchard, scions should be inserted sidewise; in a courtyard, insert upwards—sidewise scions facilitate fruit-picking while upright growth makes no trouble with the buildings.’’ The author goes on to say that scions taken near the stump give betterlooking trees but fruit later. This sensitivity to horticultural esthetics is in contrast to his disdain for beauty for its own sake: ‘‘Flowers may certainly be pleasant for the eye, but (empty) blooms in spring without substantial autumn fruits are vain and fraudulent things. So there is no need to record them.’’ F. Medieval and Islamic Period 1. Medieval Europe. From the surviving records of agriculture from ancient Greece and Rome, it is clear that grafting was well known and widely practiced in the Mediterranean region by at least the ﬁfth century BCE (see Section III.C). It is likely that the art of grafting survived in Christian monasteries after the fall of the Roman Empire. Grafting is mentioned in King Alfred’s English translation of Pope Gregory’s Regula Pastoralis around 897 CE (Juniper 2006). In the thirteenth century, Albert of Bollstadt (Albertus Magnus) wrote in De Vegetabilibus that grafting was one of the means by which to bring about cultivar improvement from wild species (Biewer 1992). He proposed that plant life was mutable and that new species could be produced by grafting. 2. Islamic Period. During the decline of the Roman Empire after the ﬁfth century, a period often referred to as the Dark Ages, the Arabs were the major successors of the Greeks and Romans in all areas of science and technology, including horticulture. The Arabs took special

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interest in gardens and gardening, probably because their ancestors originated in desert countries. This love of plants is clearly shown in a special genre of poetry, the rawdiya or (garden poems), which became the most common poetic themes. Islamic gardening was inspired and inﬂuenced by the traditional Persian fruit tree and ornamental gardening. Islamic rulers competed among themselves in the magniﬁcence of their gardens. Basra, Cairo, Damascus, and later on Seville, Cordova, and Valencia were renowned for the size and beauty of their gardens, which preceded those of Christian Europe by several centuries; the earliest records of Italian gardens are from the fourteenth century. Numerous new crop plants were introduced during the Islamic period (Watson 1983); many were initially cultivated as ornamentals in the royal gardens of the Islamic rulers. Grafting undoubtedly was used extensively in the gardens of the Islamic era; Mazaheri (1951) reports that the garden of Il-Khans was directed by a Persian botanist who wrote a book on the grafting of fruit trees. Little is known, however, about developments in the understanding of grafting effects, and superstitions concerning grafting were common during this period. Thus, in his Guide for the Perplexed (Part III; Chapter 37), Maimonides (1137–1205) wrote: When a tree is grafted into another in the time of a certain conjunction of sun and moon, and incense is burned whilst a formula is uttered, that tree will allegedly produce something that will be exceedingly useful. The most remarkable witchcraft, as described in The Nabatean Book of Agriculture [Ibn-Whashiya’s tenth century treatise] was the grafting of an olive branch on a citron tree. . . .They also said that when one species is grafted upon another, the branch which is to be grafted must be in the hand of a beautiful damsel, whilst a male person has disgraceful sexual intercourse with her: during that intercourse the woman grafts the branch into the tree. (Friedlander 1904, slightly edited)

Although considered by Maimonides to be trustworthy, the reliability of Ibn-Wahshiya, who allegedly describes the ‘‘ancient’’ agriculture, has been seriously questioned by modern investigators of Islamic literature. The Nabatean Book of Agriculture has been called a forgery, although the superstition may have long existed (Frazer 1935). G. Renaissance Europe About 1440, Jon Gardener wrote The Feate of Gardening, which included a section on grafting, but the work was never published and only two copies are known today (Harvey 1985; Juniper, 2006). An

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important grafting-related milestone was recorded by Champier in 1472. He mentioned the dwarﬁng Paradise apple for the ﬁrst time in the horticultural literature (Tukey 1964), which implies a fairly sophisticated understanding of stock/scion interactions. Paradise, then as today, was presumably used primarily as a rootstock, not a scion cultivar since it has soft insipid fruit, in order to take advantage of its dwarﬁng effect on the scion and because it roots easily from cuttings. Vegetative propagation maintained its dwarﬁng character because apples do not come true to type from seed. Paradise may be the earliest examples of the use of clonal apple rootstocks in Europe. This is particularly signiﬁcant since Paradise was the progenitor of the modern dwarﬁng rootstocks M.8 and M.9 (originally known as ‘Jaune de Metz’) and other dwarﬁng rootstocks (Webster 2003). By the 16th century, the increased literacy after the widespread adoption of the printing press (invented by Gutenburg in 1440) led to increased demand for and availability of horticultural works that included descriptions of grafting. Some of these included the Boke of Husbandry in England by John Fitzherbert (1531), Ein Neues Pﬂantzbu¨chlin by the Bavarian Johann Domitzer (1531), and De Natura stirpium libri tres authored by the Parisian physician and botanist Jean Ruel (1536). In 1558, the Italian Gambattista della Porta published a four-volume work in Latin entitled called Natural Magick (Porta 1558), which in 1584 was expanded into 21 volumes and translated into English. The subtitle, Wherein Are Set Forth All the Riches and Delights of the Natural Sciences, suggests the tone of the work. His subjects ranged from the fantastic (Of changing metals, Of the generation of animals), to the mundane (Of cookery). Scholars have concluded that he had a propensity for exaggeration and embellishment, ‘‘to bring out the full wonder and marvel of the world’’ (Price 1957). In the third volume, which was titled Of the Production of New Plants, he states in the subsection ‘‘How to make new fruits compounded of many’’: the Fig tree may be incorporated into the Plane tree, and the Mulberry tree,. . . likewise the Mulberry tree into the Chestnut tree, the Turpentine tree, and the White Poplar, whereby you may procure White Mulberries, and likewise the Chestnut tree into a Hazel, and an Oak, and likewise the Pomegranate tree into all trees, for that it is like to a common whore, ready and willing for all comers.’’ Throughout the work Porta acknowledges using information on grafting from ancient scholars, including Virgil and Columella. Porta not only wildly overstated the limits of compatibility between stock and scion, as did Virgil 15 centuries earlier, but he went further with his claims that the stock

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could dramatically alter the nature of the scion. For example, he asserted that mulberry scion on white poplar stock would generate white mulberry fruit. Although Porta’s fantastic claims could easily be dismissed as the work of an uninformed, inexperienced pedant with a penchant for exaggeration, Natural Magick was widely read throughout Europe for over a century (Porta 1659). For example, Nicholas Bonnefons (1658) repeated the claim that grafting mulberry onto popular generated white mulberries. A work by Leonard Mascall in 1572, A Booke of The Arte and Manner How to Plant and Graffe All Sorts of Trees . . . by one of the Abbey of S. Vincent in Fraunc (Mascall 1589), was largely a translation from French to English based on a French manuscript by David Brossar (L’art et Maniere de Semer Pepins et de Faire Pepiniere) (Anon. 2007b). The introduction by Mascall explains: ‘‘there is none that more doth refresh the vital spirits of men, nor more engender admiration in the effects of nature, or that is cause of greater recreation to the weary and traveyled spirit of man, or more proﬁtable to mans life, than is the skill of planting and grafﬁng.’’ In his introduction, Mascall attempts to motivate a broad range of prospective grafters: The poore man may with pleasure ﬁnde Some thing to help his neede So may the rich man reape some fruit Where earth he had but weed The noble man that needeth naught May thereby have at will Such pleasant fruit to serve his life And give each man his ﬁll

The book, although translated from the French, goes on to claim that earlier works on grafting from Greece, Barbarie, Italy, and France ‘‘doeth very small proﬁt for this our Realme of England.’’ Mascall goes on to chide his country men for their lack of diligence: ‘‘which I can blame nothing more then the negligence of our nation, which hath had small care heretofore in planting an graffyng . . . but if we would endeavour our selves thereunto as other countries doe wee might ﬂorish and have many a strange kinde of fruit which now we have oftentimes the want therof.’’ Mascall expressed disdain for those who promoted the idea that the scion could take on the ‘‘nature’’ of the stock. He wrote: ‘‘many which have written that if ye graft the medlar upon the quince tree, they shall

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be without stones, which is abusive and mockery. For I have (saith he) proved the contrary myself.’’ Mascall’s book and many that followed in the late sixteenth and seventeenth centuries, is a practical, hands-on guide for the fruit tree gardener. It makes fewer fantastic claims than Porta about the limits of compatibility. The book recommends grafting cherry to cherry, and apple to apple or to pear, although Mascall suggests some incompatible interfamilial combinations (cherry on crabapple, ﬁg on peach, and apricot on ﬁg). An illustration on the title page of Mascall’s work shows a top graft greatly exaggerated in size, illustrated in Juniper and Maberly (2006, p. 96). Exaggerations of size were not uncommon in illustrations of grafting of the sixteenth and seventeenth centuries (Anon. 1654; Mascall 1589; Sharrock 1672; Markham 1635; Meager 1688; Langford, 1699). H. Early Modern Europe In 1618, William Lawson published a book on orchard gardening that was speciﬁc to the North of England: A New Orchard and Garden: Or, the Best Way for Planting, Grafﬁng, and to Make Any Ground Good for a Rich Orchard, Particularly in the North. Most, if not all, of the seventeenth century books that covered propagation-related topics described various grafting and budding (inoculation) techniques in sufﬁcient detail that, after allowing for the challenge of ‘‘translating’’ early modern English into contemporary English, could be faithfully executed today with reasonable expectation of success. However, as in earlier times, misinformation abounded during this period on the issue of the genetic limits of compatibility. A related consideration was extraordinary claims about the inﬂuence of the stock on the scion (mingling of traits). The principal fruit tree species of consequence to seventeenth century gardeners were apples, pears, medlars, quince, peach, cherry, plum, and apricot, but mulberries and ﬁgs were also mentioned. Most authors got the basics right: apple on apple or crabapple stock, pear on pear or quince stock, and even pear on apple stock, which was acknowledged, as is the case, to be a short lived combination. Thomas Barker (1651), in his book The Country-mans Recreation, or the Art of Planting and Grafﬁng, copied Mascall’s entire section (several pages) on compatibility verbatim without attribution. More advice regarding red apples and other minor (secret) miracles was published in 1654 in The expert gardener; or a treatise containing certaine necessary, secret, and ordinary knowledge by an anonymous

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author (who gave credit to ‘‘sundry Dutch, and French Authors’’; Anon. 1654). In order to make red apple, the reader is advised to graft an apple scion upon an alder or cherry stock. Alternatively, soak the scion in pike’s, blood. The book conveyed many other ‘‘secrets,’’ such as a method for producing cherries without stones that involved using an iron to draw the ‘‘heart and marrow from both sides’’ of the stock cherry tree, followed by anointing with ox dung and then grafting another cherry scion to it. According to this author, there were essentially no limits to compatibility, and apples could be grafted to apple, pear, cherry, willow, ﬁg, and chestnut. Nearly all of the books published during this time were practical agricultural treatises written for the educated practitioner/landowner. A work by Robert Sharrock (1630–1684), The History of the Propagation and Improvement of Vegetables. . ., ﬁrst published in 1659 and reprinted in 1672, was somewhat different. It attempted to be both practical as well to convey the natural history, botany, and natural philosophy related to the propagation of plants. Sharrock drew from his own ‘‘observations made from experience and practice,’’ as did most garden writers of his time, but also incorporated his critical analysis of other published works and his speculations on natural philosophy and religion. ‘‘I gave myself the trouble to run over with my eye all books I could procure of the subjects, not intending to trust any.’’ There he discovered ‘‘a multitude of monstrous untruths . . . in both Latin and English old and new writers.’’ Sharrock was a remarkably ecumenical scholar. In addition to work just mentioned, he wrote at least 12 others books on diverse topics, including theology, natural philosophy, physics, history, and even sexual deviancy. Sharrock was the archdeacon of Winchester Cathedral and fellow of New College, Oxford, and his theological orientation permeates his writing, which was not at all uncommon for scholars of the scientiﬁc revolution. The latter was no doubt reinforced by his friendship with Sir Robert Boyle (Arber 1960), one of the most inﬂuential contributors to the scientiﬁc revolution, best known for Boyle’s gas law ðpV ¼ kÞ. According to the book’s Dedicatory Epistle, Boyle requested Sharrock to write this scientiﬁc treatise on plant propagation. Sharrock embraced a practical as well as a scholarly/experimental approach to the subject, and he speciﬁcally eschewed ‘‘natural Magick and romantic stories.’’ He may have been referring to Porta’s Natural Magick (1584) or more generally to any ‘‘magical’’ explanations of natural phenomenon, but he does mention Gambista della Porta in other sections of the book, unﬂatteringly. The title and subtitle of the book, The History of the Propagation and

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Improvement of Vegetables by the Concurrence of Art and Nature, is elaborated on in the section ‘‘Of Natural Propagation and Seminal Principles Latent from the Creation.’’ He writes: ‘‘Industry and art may bring materials and place them ﬁtly for it, but nature works them. And therefore, as one sayeth, it is the great art of man to ﬁnd out the arts of nature.’’ Sharrock’s ﬁfth chapter is titled Insitions, which refers to grafting and budding. It begins with a core principle necessary for success in any and all types of grafting and budding: ‘‘Grafting is an art of so placing the cyon upon a stock that the sap may pass the stock to the cyon without impediment. . . .The space that is between the bark and the stock is the great channel for conveyance and keeping of sap so that every one that grafts well so orders the manner that these spaces be so laid, that the passage may be easy.’’ Concerning the recurrent issue of the limits of graft compatibility, Sharrock’s views were consistent with some of the more enlightened writers of the time, stressing that: ‘‘The cyon or thing implanted be of like nature to the stock.’’ Rather than simply giving several examples of compatible (e.g., apple/apple) and less compatible (e.g., apple/ pear) combinations, as most earlier authors had done, Sharrock added the caveat: ‘‘But to tell what nearness in every kind is enough, is a matter of greatest Art.’’ In the same chapter, he undertakes a critical analysis of some previous claims regarding compatibility. This section is titled ‘‘Kirchers Experiments Concerning Insitions Examined.’’ Sharrock writes: ‘‘I have tried mulberries on Beech, Quinces, Apples, Pears, Elms, Poplars and by grafting they would not take, and yet he [Kircher] afﬁrms they take easily.’’ In addition to questioning Kircher’s claims of excessively broad limits of compatibility, Sharrock addresses the assertions of several other authors back to and including Porta (1584), that the traits of stock and scion mingle within the new fruit on the scion. Sharrock writes (referring to Kircher): ‘‘mulberries are by conjunction with white Poplars made to be of a white kind and bear white mulberries [a claim made by Porta in 1584]. . . .Pear being grafted on a mulberry bring a red colored pear . . .a white rose grafted upon a red, will bring the Rosa Mundi or a ﬂower both red and white. This I have often prov’d false by mine own trial.’’ Thus he exhibits not only healthy skepticism but introduces ‘‘experimentation.’’ It is tempting to perceive Sharrock as the voice of reason emerging from the intellectual chaos of the Middle Ages, but then he reverts so stunningly in his judgment about compatibility that it is difﬁcult to imagine that this text was written by the same author:

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There are almost inﬁnite stories of strange conjunctions which urge earnestly for credit, some of insitions made upon animal bodies: The Lord of Pieresh had a present made him of a plum-tree branch which bore blossoms and leaves, which sprang from a thorn that grew in the breast of a shepheard, this shepheard having got this thorn by falling upon a plumtree. . . .The bodies of divers beasts be excoriated and planted anew with silk, ﬁne wool, cotton, or the linen. When these new plantations shall have succeeded to any considerable advantage of the planters then we also will leave our vegetabnle and apply our selves by these rarer ways of insition to the improvement of animal bodies.

It is just possible that these lines betray his sense of humor rather than his gullibility because this is the only instance of extreme exaggeration in the book. In many instances, Sharrock goes beyond his own observations and practices or those of his contemporaries and, in the spirit of the scientiﬁc revolution of which he was a part, he speculates on more mechanistic questions that border on natural philosophy. One of the issues he spends a signiﬁcant amount of time on is the paradox originally addressed by Pseudo Hippocrates, about 2,000 years earlier, regarding the essence of plant growth. If a plant of a given species grows by extracting from the soil a ﬂuid speciﬁc to its particular kind (and no other), how then does this ﬂuid taken up by stock serve to provide for the growth of the scion of a different species? Or, as Sharrock put it: ‘‘But how the sap of the stock (suppose a white thorn) can serve to make the wood, bark, leaves and fruit of its cyon, suppose a pear, is a difﬁcult question. For grant there be an elective attraction of sap from the earth, yet how shall a white thorn choose that which is ﬁt for a pear?’’ The Greeks (see Pseudo Hypocrites translation, Section III.C) dealt with this paradox by asserting that the scion rooted itself into the stock and these scion roots grew down, all the way through the stock, and emerged into the soil, where they took up the growth ﬂuid appropriate to the scion species. It is not clear if Sharrock knew of this ‘‘Theory of Speciﬁc Fluids’’ as described by Lonie (1981), but he chose to resolve the paradox in another, more metaphysical way. Sharrock writes, ‘‘when the sap gathered in its roots comes to the place of conjuncture, it is there forced to undergo a total corruption and lapse into the bed of its ﬁrst matter, from whence by a new generation, there arises a new sap, begot in the tree by speciﬁck faculty, which in a pear graff may be called a pear-sap-making power.’’ He goes on to generalize to a broad range of natural phenomena: ‘‘it is equally applicable to all things in the world, each thing being made by some such thing-making power. Diva Colchodea, the grand-general form-making-intelligence.’’

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Fig. 9.6. Different types of grafts (Source: Sharrock 1672).

One particularly useful and practical contribution was the single illustration in Sharrock’s (1672) book (Fig. 9.6) of a stylized tree showing nine different grafting and budding techniques as well as ‘‘circumposition’’ (air layering), all applied to a single tree trunk. This, along with the detailed ﬁgure caption, goes far beyond other books of the 17th century and earlier in providing visual representation of grafting techniques described in the text. Seventeen years later, Langford’s book (1696) included a very similar illustration, obviously derived from Sharrock’s work. At about the same time as Sharrock, the Frenchman Nicholas Bonnefons (1658), author of The French Gardiner (1658), wrote accurately about what could be grafted to what (regarding pears and apples) based on his own experience. When he speculated based on the words of others he, like Sharrock, succumbed to exaggeration: ‘‘There are some curious persons who graffe Queen apple up the white mulberry, and hold that the fruit does surpasse in redness all others.’’

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In other respects, Bonnefons had a very sophisticated understanding of asexual propagation in the service of clonal rootstock production. He writes of the Paradise rootstock in particular: ‘‘on Layers of the tree (called by the French Pommier de Parradis) and in particular the Queen apple does wonderfully prosper upon it, and more red within then those which are graffed upon the free (wild?) stock.’’ He describes rootstock propagation by mound layering, and trench layering methods that are still used today. The use of clonal rootstocks, including the size-controlling Paradise, was more prevalent in France than in England in the late seventeenth century. Neither the comprehensive work of the Englishmen Sharrock (1672) or that of Langford (1696) mentions clonal rootstock production, and Langford in particular recommend using crabapple seedlings as the preferred stock for apple. The English book The Compleat Planter & Cyderist by an anonymous ‘‘Lover of Planting’’ in 1685 (republished by Juniper and Juniper 2001) speciﬁcally mentions the Paradise rootstock. In the eighteenth century, practical advice on grafting was a common theme in gardening books. The sixteenth edition of the The Gardeners Kalendar by Philip Miller (1775) has 14 references to grafting tasks throughout the year. In 1795, Thomas Andrew Knight would present a paper (I. Observations on the grafting of tree) to the Royal Society (Anon. 1844). In North America, the prevailing view was that grafting of fruit trees was time consuming, difﬁcult, and unnecessary given that the primary goal was to produce fruit for hog feed or cider. The third President of the United States, Thomas Jefferson (1743–1826), was an avid fruit tree gardener (170 cultivars of fruit trees). At his home at Monticello, he preferred to grow most fruit trees from seed because he believed they were healthier than grafted trees, but he did have his gardener graft named cultivars of apples or bud cherries in order to preserve their dessert qualities (Hatch 1949). I. Ninteenth Century 1. French Wine Industry. Grapes and olives are the most economically important temperate-zone fruit crops in the world. Because of the importance of wine, grapes have had an (arguably) greater impact on human history and culture than any other fruit crop. Nowhere is the latter more true than in France. Imagine the panic in the French wine industry in the latter half of the ninteenth century when an insect plague nearly wiped out grape production. The insect phylloxera (Dactylosphaera vitifolii) often kills the highly susceptible

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vinifera grape (Vitis vinifera), from which most wines were and still are made. To this day the pest has never been eradicated or otherwise defeated and remains a problem in susceptible wine-grape-producing regions throughout the world. From the French point of view, the phylloxera panic began in 1864 in the southeastern Midi region of France (Campbell 2005). Grape vines were observed to rapidly decline, as leaves turned from yellow to red and rapidly dried up and defoliated, while the root systems collapsed and rotted, resulting in death of the entire vine. It took an additional three years, while the malady continued to spread, before an aphidlike insect infesting the roots was observed to be the culprit. Thanks to detective work of an American entomologist, C. V. Riley, and several French scientists including a botanist, J. E. Planchon, entomologists V. Signoret, J. E. Westwood, and J. Lichtenstein, the pest was determined to have been introduced from eastern North America, where it occurs at low frequency on leaves and roots of American grape species (V. labrusca and other species) without killing the vines. It was not lost upon the scientiﬁc community of the time that phylloxera was an object lesson in the evolutionary theory of natural selection that Charles Darwin had published a few years earlier in On the Origin of Species. Coevolution of the pest and its host in North America has resulted in American grape species with resistance to phylloxera, allowing the two to coexist. Vinifera grapes, however, having never been exposed to the pest, had no natural (genetic) resistance, and quickly succumbed to the onslaught. The phylloxera ‘‘plague’’ eventually destroyed a quarter to a third of all grape vineyards planted in France. In the ﬁrst few years after phylloxera’s appearance on the French grape scene, the response of grape growers was to rip up the affected vines, roots and all, and burn them. Needless to say, this approach was completely unsuccessful, and infestation progressed from the Midi to other grape-producing regions. As it became widely acknowledged that phylloxera originated in North America and that American vines were phylloxera resistant, two opposing socioscientiﬁc movements emerged. The Americainistes, including Planchon and a rich wine connoisseur, Leo Laliman, believed that that importation of American stock should be encouraged either for direct production of wines from American grapes or to serve as rootstocks on which to graft French vines. The opposing point of view of the Sulfuristes, who advocated expensive soil injection with the chemical carbon bisulﬁde, was that American stock should be banned from France entirely lest the pest be

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introduced to regions not yet infested. Furthermore, there was a great deal of prejudice against wines made from American grapes as they were (and by some still are) widely considered unpalatable. It was even feared that the American rootstock would impart an undesirable ‘‘foxy’’ character to wines from Vinifera cultivars grafted onto them. In his engaging narrative The Botanist and the Vintner—How Wine Was Saved for the World, Christopher Campbell (2005) put it this way: ‘‘grafting proud French vines onto alien roots seemed the counsel of miscegenating madmen.’’ Later attempts to cross French and American species in order to obtain phylloxera-resistant hybrids was never more than marginally successful as far as the French were concerned. Eventually the practice became strictly regulated in France and remains so. However, such French-American interspeciﬁc hybrids, although they are of intermediate hardiness, are important to wine production in some parts of the world, including eastern North America. By 1890, the Sulfuristes had largely capitulated to the Americainistes, and grafting French cultivars onto American stocks became widespread. Laliman proclaimed far and wide that he had been the ﬁrst to recommend the grafting solution in 1869, although it has been claimed that the American C. V. Riley was the ﬁrst to suggest it in 1870. During the ensuing years when ‘‘reconstruction’’ (the practice of replanting vineyards with grafted grapes) was in full swing, grafting became all the rage, with people enrolling in special classes and proudly displaying their certiﬁcates of completion. Books and articles on grafting were abundant, and new grafting methods were invented, including machine grafting for mass production of grafted plants. Reconstruction was not without its problems, however. The two American grape species used as rootstocks, V. riparia (‘Riparia Gloire’) and V. rupestris (‘Rupestris St. George’), were not well adapted to the chalky (high pH) soils of the better wine-producing regions of France, resulting in leaf chlorosis due to iron deﬁciency and less-than-optimal performance. In another transatlantic collaboration, a French plant pathologist, Pierre Viala and an American grape enthusiast and plant explorer, T. V. Munson, introduced an American species, V. berlandieri, which was phylloxera resistant as well as tolerant of high soil pH. This might have solved the problem, but V. berlandieri turned out to be unusually difﬁcult to root from dormant cuttings, the standard means of propagation of grape rootstocks. The ultimate solution, which prevails today, was to hybridize V. berlandieri with the easier-to-propagate V. riparia and V. rupestris (Rieger 2006).

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2. Citrus Problems. Evidence of citrus grafting goes back to the Roman era. Gallesio (1811) cites Palladius (ﬁfth century CE) for grafting citron. Grafting of citrus was practiced during the Middle Ages and the Renaissance, while many types of citrus were considered exotic ornamentals and grown in orangeries throughout western Europe (Fig. 9.7). Gallesio (1811) was aware of the ideas that grafting can give rise to the appearance of new species but noted that his observations

Fig. 9.7. Grafting of citrus grown in containers. (From Volkamer 1708).

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did not support such notions. However, the introduction of grafting as a standard procedure in commercial citrus groves took place only during the middle and late 19th century. According to Chapot (1975), the advent of foot-rot, or gummosis, caused by various species of Phytophthora ﬁrst in the Azores Islands in 1842 and later in almost all other citrus-growing countries, obliged the use of scions grafted on rootstocks tolerant of the disease. The sour orange (C. aurantium) was the universally favored citrus rootstock until the breakout of the Tristeza virus epidemic in South America during the 1930s and its subsequent spread to North America and the Mediterranean. Sweet orange grafted on the sour orange rootstock proved to be most susceptible to the Tristeza viral disease, which necessitated the replacement of sour orange by other rootstocks. The susceptibility to Tristeza and other virus and viruslike diseases became a major consideration in modern citriculture. The trifoliate orange (Poncirus trifoliata), which is a deciduous, cold-resistant citrus relative, and resistant to tristeza, is used as a rootstock in cool citrus-growing regions such as Japan. K. Twentieth Century A number of innovations have occurred in grafting technology in the 20th century. These include in vitro grafting, herbaceous vegetable and ornamental grafting, ﬂower bud grafting, and Mukabit grafting of cassava. 1. In Vitro Grafting. One of the most ingenious grafting innovations of the 20th century has been in vitro grafting, which involves grafting a shoot tip about 1.5 mm high from a mature plant onto a seedling rootstock. This technique was developed by Toshio Murashige et al. (1972) with citrus and is now used in many species. The technique is used in citrus to rid plants of viruses and other systemic pathogens. The technique exploits two concepts: (1) meristems are relatively virus and pathogen free; and (2) meristems from mature plants retain the mature phase. Thus, the use of in vitro shoot tip grafting produces plants that are virus free and reproductively mature. This technique overcomes problems of producing virus-free plants from nucellar seedlings or by thermotherapy. Although nucellar seedlings of citrus are both clonal and virus free, the seedlings are juvenile and take many years to ﬂower. In the case of thermotherapy, many viruses, such as exocortis viroid and stubborn virus, are difﬁcult to clean up with this process (Roistacher 2004). In vitro grafting was used in Spain to produce

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virus-free plants of all cultivars and is considered a major factor in improving the Spanish citrus industry (Navarro et al. 1975). A successful technique in citrus involves growing 2-week-old, darkgrown seedlings (often ‘Troyer’ citrange) as rootstocks and 0.14- to 0.17-mm-long shoot tips of any cultivar as scions (Navarro et al. 1975). A scion that includes three-leaf primordia is cut with a razor blade attached to a handle as a scalpel. Shoot tips are inserted in an inverted T made at the top of the decapitated rootstock epicotyl. Highest success is obtained with the shoot tip set on the vascular ring tissue on top of the decapitated epicotyl. Nutrient solution was a modiﬁed Murashige and Skoog media with high sucrose (7.5%). Light is maintained during growth of grafts. 2. Herbaceous Vegetable and Ornamental Grafting. Although the use of grafting of vegetables is an ancient practice (see Section II.E), grafting was not been a common practice in herbaceous vegetables and ornamentals until the 20th century (Lee 2003; Lee and Oda 2003). Information on grafting scions of watermelon (Citrullus lanatus) on bottle gourd (Lagenarias scieraria) rootstock to overcome yield decline was described in the 1920s by Japanese workers in Korea. Since that time there has been an explosion of the use of this technique in watermelon, cucumber, melons, tomato, eggplant, pepper, and ornamental cactus, especially in Korea and Japan. The advantages of vegetable grafting are attributed principally to resistance of the understock to soilborne diseases, such as verticillium wilt, fusarium wilt, bacterial wilt, and nematodes, but also to increase of vigor and stress tolerance. The problems associated with banning methylbromide for soil fumigation have increased vegetable grafting in Europe and the United States, and this technique is now considered to be an environmentally sustainable practice. Grafting of ornamental cactus has created a large industry in Korea. Ten million grafted cacti are exported from that country each year. Because of the increased expenses associated with grafting, grafted seedlings are mostly used in greenhouses and tunnels but also are used in ﬁeld production for watermelon, cucumbers, and tomato. In Korea and Japan, practically all greenhouse production of watermelon, cucumbers, and melons are from grafted plants. The increased use of grafting has resulted in breeding programs to produce speciﬁc diseaseresistant rootstocks, many involving various formerly noncultivated species. Most grafting is done by hand using various modiﬁcations of the detached scion and approach grafting techniques and insertion

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techniques (whip and tongue, cleft, hole insertion, splice), often held together by various clips and pins. Experienced grafters can perform an average of 1,000 grafts per day. Special techniques used to heal the graft union involve environmental control (temperature, light, and humidity). In order to reduce the cost of grafting, various machines and robots have been developed to perform the grafting operation. A simple grafting machine can produce 600 grafts per hour per twoperson team; more costly grafting robots can perform 600 to 1,200 grafts per hour, but highly uniform seedlings are required in order to increase grafting efﬁciency, and robots are costly. The creation of the plug seedling industry produced by specialized nurseries has increased the potential for machine grafting, and the vegetable grafting technique is increasing rapidly throughout the world (Cantliffe 2009). 3. Flower Bud Grafting. A unique use of grafting has been developed in the service of the Taiwan pear industry (Kuniyama 1996; Gemma 2002). The Japanese pear, or nashi (Pyrus pyrifolia), is a favorite in Taiwan, but some cultivars cannot be grown in the subtropical areas there because of insufﬁcient chilling. This is overcome by ﬂower bud grafting. After trees in Japan have received chilling nearly sufﬁcient to break dormancy, budwood is harvested and shipped to Taiwan, where it is held at 2 to 4 C to complete the chilling requirement. The prechilled scions are grafted into ‘Heng Shan’ pear trees and produce two to four marketable fruits per cluster. Grafting must be renewed each year. The technique beneﬁts Japanese growers, who obtain an additional source of revenue from exporting buds, and Taiwanese growers, who proﬁt by early crops of marketable fruit that receive very high prices. 4. Mukibat Grafting of Cassava. Not all modern innovations necessarily involve cutting-edge technology. Mukibat grafting is an example of horticultural innovation by a peasant subsistence farmer. In 1958, a farmer named Mukibat, living on the island of Java, Indonesia, took an extension grafting course and shortly thereafter began to experiment with grafting of cassava, which is an important food crop in Indonesia and many other tropical countries. He grafted an inedible relative of cassava, the Ceara rubber tree (Manihot glaziovii), onto a cassava (Manihot esculenta) stock (De Foresta et al. 1994). The resulting yield of cassava tubers was dramatically increased, on the order of 30% to 100% or more, compared to ungrafted cassava (De Bruijn and Dharmaputra 1974). The potential agronomic signiﬁcance of this enhanced yield for food sufﬁciency is obvious. Unlike apples in

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which selected rootstocks are used to dwarf or otherwise control the size of the scion cultivar (see Section IV), Mukibat grafting uses a selected scion to invigorate the rootstock. Effects of the scion on rootstock vigor have not been generally reported.

IV. HISTORY OF CLONAL ROOTSTOCKS For more than 2,000 years, the most important reason for grafting has been to asexually propagate scion cultivars. This could be accomplished by grafting a scion cultivar onto any compatible seedling rootstock. Across many centuries, however, there was a belief that certain rootstock genotypes affected the scion cultivar differently from others. In some cases, this was based on accurate empirical observation. For example, in China in the sixth century, Jia Sixie wrote in Qi Min You Shu (Sheng-Han 1962) that pear grafted onto ‘‘tang’’ (Pyrus phaeocarpa or P. betulifolia) would produce large pears with ﬁne ﬂesh, as compared to pear grafted onto ‘‘du’’ ( P. calleryana or P. ussuriensis), which were not as good. As early as the 17th century, it was known that quince (Cydonia oblonga) had a dwarﬁng effect on pear, and was the recommended rootstock for espalier pears (Tukey 1964). In other cases, the alleged rootstock effect was based on wishful thinking. For example, Gambista della Porta (1584) wrote that mulberry grafted onto white poplar would produce white mulberry fruit, and in 1563 the anonymous author of The Crafte of Graffynge & Plantynge of Trees (republished by Juniper and Juniper 2001) wrote that an apple grafted onto elm would produce red apples. Whether accurate or otherwise, these alleged rootstock effects involved interspeciﬁc (Pyrus phaeocarpa/P. calleryana), intergeneric (Pyrus/Cydonia), or even interfamilial (Malus/Ulmus) scion/rootstock combinations. Exploitation of the observation that selected scion/ rootstock combinations could have profound effects on scion cultivar development is one of the most signiﬁcant advances in pomology over the last 1,000 years. In modern terminology, such compound genetic systems involve the independent optimization (selection and breeding) and asexual propagation of both the scion genotype and the rootstock genotype. The oldest and even today the most apparent criterion for clonal rootstock optimization has been rootstock effects on scion vigor (size control). For particular horticultural applications, dwarf fruit trees have been an important goal, apparently since ancient Greece. In about the fourth century BCE, Alexander the Great is said to

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have sent low growing dwarf apple trees, ‘‘Spring Apples,’’ to the Lyceum (gymnasium with a wooded area), where Aristotle and later Theophrastus was the director. Given that grafting was known by Theophrastus (Janick 1989) and that this Spring apple was easy to root, it may be that it was used as a dwarﬁng rootstock for other apples. The same low-growing (dwarf) apple was later used by Roman agriculturists, and it is likely that they used it as a dwarﬁng rootstock. Bunyard (1920) felt that the Spring apple was the progenitor of the Paradise apple. Juniper and Mabberly (2006) suggest that apple genotypes that were eventually selected as size-controlling rootstocks migrated from wild populations in the Tian Shan Mountains of Kazakhstan, westward through Persia and Armenia into Europe. They point out that the low-growing, easily rooted ‘French Paradise’ is very similar to the Armenian rootstock ‘Marga Khndzor’. The gene(s) for the horticulturally important dwarﬁng phenotype is (are) being identiﬁed and cloned in New Zealand by S. Gardiner (pers. commun). ‘Paradise’ apple, however it arrived in Europe, was ﬁrst mentioned in print by the Frenchman Champier in 1472 and later by Dalechamps in 1507 (Tukey 1964). ‘French Paradise’ was ﬁrst introduced into England in 1696 and into America during the early 19th Century. Tukey argues that its ease of rooting was initially more important than its dwarﬁng effect to European growers, but why bother to clone a rootstock at all if it was not dwarﬁng? Paradise as described above refers to a dwarﬁng rootstock that is easily propagated asexually, but eventually a distinction arose between ‘French Paradise’ and ‘English Paradise’. ‘French Paradise’ referred to the centuries-old (if not millennia-old) dwarﬁng, easily cloned genotype, whereas ‘English Paradise’ or ‘Doucin’, ﬁrst described in 1519, referred to a somewhat more vigorous (semi-dwarﬁng) rootstock that was more difﬁcult to clone. These genotypes represented some of the genetic diversity that would become the basis for later rootstock selection and breeding during the early 20th century. In the meantime, the issue of genotype nomenclature was becoming increasingly confused. In part this was due to deliberate selection of new genotypes (e.g., ‘Jaune de Metz’, a chance seedling selected in Metz, France, in 1879). More prominently, though, the confusion was due to frequent, location-dependent renaming of established clones (e.g., ‘Red Paradise’, ‘River’s Paradise’, ‘Holstein Doucin’) or due to mislabeling in the nursery. In 1870, Thomas Rivers noted 14 different kinds of ‘Paradise.’ By the early 20th century, this situation had become untenable, and efforts to bring order to chaos were undertaken independently in Germany, the Netherlands, France, and England.

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The rootstock standardization and selection program that got under way at the East Malling Research Station at Kent, England, proved to have a profound effect on apple production that persists today (Tukey 1964). Initially (1912) station director R. Wellington and subsequently (1914) R. G. Hatton assembled a collection of ‘Paradise’ apple rootstocks, including 71 collections from 35 sources including Britain (29), France (3), Germany (1), and the Netherlands (1). Each of these was evaluated with respect to size control, propagation, and productivity. Nine of them were renamed to eliminate confusion and released in 1917 (‘M I’, ‘M II’, ‘M III’, etc.). ‘Doucin’ (‘English Paradise’) was renamed ‘M II’, ‘French Paradise’ as ‘M VIII’, and ‘Jaune de Metz’ as ‘M IX’. Eventually the number of East Malling releases was increased to 27. These were categorized into size classes from Very Dwarf, Semi Dwarf, Vigorous, Very Vigorous. Currently the number after the preﬁx M is presented as an Arabic numeral (Fig. 9.8). Although rootstock disease and pest resistance were not key selection criteria for these initial efforts, they were the focal points of subsequent collaborative efforts. In 1917 (Ferree and Carlson, 1987), the East Malling Research Station and the John Innes Horticultural Institute at Merton, England, participated in collaborative effort to solve a critical problem faced by apple growers in Australia and New Zealand where wooly apple aphid (Eriosoma lanigerium) was a limiting factor in apple production. Capitalizing on progress that had

Fig. 9.8. Size control in apple with clonal rootstocks. (Courtesy Vanessa Gray).

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been made with the EM selections, they crossed these (and a few others) with the ‘Northern Spy’ apple, which was known to be resistant to the wooly apple aphid. By screening 3,758 seedling for wooly aphid resistance and characterizing the survivors for size control, propagation, compatibility, precocity, and ﬁeld performance on two different soils, a total of 15 selections were made, numbered Malling-Merton 101 through 115. Several of these are still in use in various parts of the world. Another rootstock improvement program involving the East Malling Research Station in collaboration with the Long Ashton (LA) Research Station had to do with elimination of latent viruses that had accumulated in the original East Malling (EM) rootstocks over time, resulting in declining vigor and graft incompatibility. By the process of micropropagation combined with heat treatment of the original M series and MM series, the EMLA series were created. As would be expected, virus elimination resulted in increased vigor. Perhaps unexpectedly, the elimination of latent viruses did not always improve horticultural attributes such as scion yield efﬁciency for these rootstocks.

V. GRAFT HYBRIDS The effect of grafting on the genetic integrity of the scion has been an issue in biology and horticulture since antiquity. In the 16th century, Jewish law prohibited the use of citron fruit for the feast of Tabernacles if the citron tree had been grafted onto lemon root stock (Nicolosi et al. 2005). Some rabbis even went so far as to express the view that citron fruit from an ungrafted tree originally propagated from cutting taken from a tree that had been grafted was not permissible. This view was derived in part from a general belief that the rootstock inﬂuences the nature (genotype) of the scion so that a fruit from a grafted tree or from a tree propagated from a grafted tree was in some sense a hybrid between the stock and scion and hence religiously forbidden. William Shakespeare alludes to the inﬂuence of stock on scion in The Winter’s Tale: You see, sweet maid, we marry A gentler scion to the wildest stock And make conceive a bark of baser kind By bud of nobler race. This is an art Which does mend Nature—change it rather; But the art itself is Nature.

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The fact that scions of high-quality fruits maintained their ‘‘noble’’ nature even when grafted on wild (‘‘degenerate’’) plants is a constant biblical theme, and provided anecdotal evidence that rootstock did not profoundly affect fruit morphology. Although it has been widely accepted as a horticultural truism that grafted plants retain their own genetic identity, the Bible is rife with mention of spontaneous development of ‘‘degenerate plants,’’ implying that there could be a genetic effect through the graft union (see Section II.B). Such references mistakenly imply a change in the nature (genotype) of the scion due to the inﬂuence of the stock, when in reality such ‘‘degenerations’’ are more likely due to suckering of the rootstock. Nevertheless, the observed rootstock effects on performance of the scion (e.g., dwarﬁng) were difﬁcult to explain fully and suggested to some a genetic change of the scion associated with grafting. In the 13th century, Albert of Bollstadt (Albertus Magnus) wrote in De Vegetabilibus that grafting was one of the means by which to bring about cultivar improvement from wild species, intuitively believing that the close physical contact between stock and scion ought to have some ‘‘genetic’’ effect, but clear experiments were never performed (Biewer 1992). Furthermore, it was long observed that grafted scions from trees on dwarﬁng rootstocks revert to normal size when regrafted onto nondwarﬁng rootstocks. Thomas Langford (1696) was equivocal about grafting not changing the integrity of the scion: ‘‘if you graff a scion on a stock differing from it in kind, whether the fruit of this new tree will be anything better than the fruit of the tree from whence the scion was taken . . .may be held in the negative. . . because the stock only conveys food and nourishment to the scion and then when the scion hath received it, it converts it perfectly into its own nature.’’ However, he goes on to make a case for the opposing point of view: ‘‘graff in natural bodies is hardly conceivable without some commixture of their natures and there as some reasons from experience that make this probable. 1) seeds take after the stock, and if the seeds are inﬂuenced by the stock, the fruit must also be, and 2) in the same orchard all planted to the same scion variety some are better than others, and this can be attributed to differences in the seedling rootstocks.’’ A. Graft Chimeras The occurrence of plants containing a mixture of phenotype involving sectors of tissue known as graft chimeras (named after a mythological beast, part lion, goat, and dragon) suggested a novel genetic effect of

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grafting. Confusingly, graft chimeras have been referred to as graft hybrids, but we will use the latter term only for alleged graft-induced direct genetic transformation as discussed later. It was long noticed that in rare cases grafting produced new types of plants (horticultural curiosities) composed of mixtures of stock and scion characteristics. In the 16th century, the Italian Giambattista della Porta (1584) declared that the scion of an orange grafted on a lemon stock could produce lemons ‘‘half sweet, half sour.’’ Gervase Markham (1635) stated: ‘‘if you take an apple graft and a pear graft of like bigness and having cloven them join them as one body in grafting, the fruits they bring forth will be half apple and half pear.’’ In 1672, Robert Sharrock described with skepticism an experiment of Kircher, who found that a white rose grafted upon red roses will bring a ‘‘ﬂower both red and white.’’ In 1674, Pietro Nati (1625–1685), director of the Botanical Garden of the University of Pisa, published De malo limonia citrata-aurantia vulgo la bizzarria (On the citron-orange lemon, called the bizzarria), in which he described a lemon-orange ‘‘graft chimera,’’ a tree that bears oranges, lemons, and citrons and combinations thereof. Its leaves and ﬂowers are also a mixture of different citrus. One of the best-known chimeral hybrids arose in 1825, when a Parisian nurseryman grafted Chamaecytisus purpureus onto Laburnum anagyroides. From the junction between stock and scion, an adventitious bud developed, and the shoot that developed from this had leaf and ﬂoral characteristics that were intermediate between the stock and scion phenotype. This oddity, named Laburnocytisus Adamii, was widely regarded a true hybrid of the two ‘‘parental’’ species. Propagated by grafting, the clone is still in existence (Cowles and Chamberlin 1911). These mixtures of two genetic tissues or genotypes have been explained as a special type of genetic mosaic (Marcotrigiano and Gradziel 1997) where the lineage of genetically dissimilar apical cells continues into developing plant organs. Chimeras based on the structure of the meristem may be classiﬁed as sectoral (a wedge of tissue), mericlinal (a wedge of tissue in one or more layers of the meristem), and periclinal, (hand-in-glove type) conﬁned to one or more layers of the apical meristem. The periclinal chimeras are stable. The Bizzaria orange ﬁrst described in 1674 was proved to be a periclinal chimera composed of sour orange and citron (Cowles and Chamberlin 1911). They also summarized evidence that disputed the contention that Laburnocytisis and others were true hybrids. For example, seeds from Laburnocytisis always gave rise to Laburnum seedlings rather that the segregation of phenotypes that would

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expected from a true hybrid. Earlier research by E. Baur (1910, cited in Cowles and Chamberlin 1911) established that Laburnocytisis and other alleged graft hybrids were periclinal hybrids with the outer tissues layers of the stem and other organs consising of cells that were of the Chamaecytisus genotype, whereas cells of the inner core were Laburnum. Since ﬂoral organs, including gametes, arise in the inner core tissue, which was genetically Laburnum, only Laburnum seedlings grew from seed obtained from the graft chimera. Thus the explanation of graft chimeras as mixtures of tissues with genetically preserved identities refuted the contention that grafting induced genetic change at the cellular level. The concept of chimeral engineering involving graft insertion of epidermal cells of an insectresistant genotype has been suggested in Solancaceae (tomato and nightshade) and Brassicaceae (Goffreda et al. 1990; Lindsay et al. 1995). B. Graft Transformation In the early 20th century, genetic dogma proclaimed that although the stock may inﬂuence the phenotype of the scion (e.g., size control), the scion and stock maintain their genetic identity (Bailey 1928). This paradigm, which might be called conservation of genetic identity, was then challenged by reports of graft transformation. The theory of graft transformation has an infamous history in the former Soviet Union. The Russian pomologist and fruit breeder Ivan Vladimirovich Michurin (1855–1935), with a popular reputation similar to Luther Burbank in the United States, made the neo-Lamarkian assertion that genetic effects, could be induced by the environment, including grafting, although this assertion was not based on speciﬁc experiments. The issue became profoundly political when Troﬁm Lysenko, a Soviet crop physiologist best known for his explanation of the vernalization of winter wheat (cold induction of wheat ﬂowering through seed treatment), developed a theory of environmentally induced genetic effects. He held the position that formal genetics based on the gene, which he termed Mendelian-Morganism, was reactionary and bourgeois and contrasted it with Darwinism-Michurinism, the reconciliation of inheritance of acquired characters with Marxist dialectical materialism. Supported by Stalin, the Lysenko faction succeeded in making genetics a political issue (Glass 1948). In 1940, N. I. Vavilov, the plant breeder and botanist best known for his work on the domestication and centers of origin of crop plants, was relieved of his position as head of the Institute of Genetics and imprisoned in 1943. There he later

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died of starvation, to become a martyr for science. In 1964, the physicist Andrei Sakharov spoke out against Lysenko in the General Assembly of the Academy of Sciences and charged him with being ‘‘responsible for the shameful backwardness of Soviet biology and of genetics in particular’’ (Gorelik 2005). Vavilov’s reputation has been restored in Russia, and he is now considered one of the most revered personalities in science. The claims of graft transformation were generally unreproducible by appropriate methods of experimentation (Stubbe 1954; Bohme, 1957; Topoleski and Janick 1963). However, over the past several decades, many papers have been published in peer reviewed journals, such as Science, Proceedings of the National Academy of Science (USA), Genetics, Journal of Heredity, Euphytica, and Theoretical and Applied Genetics, reporting observations that appear to support the concept of graft hybridization, particularly a series of papers published by Japanese researchers using a range of species including red pepper, eggplant, tomato, tobacco, and soybean. The most intensive analysis focused on changes observed in grafted Capiscum annum cultivars with varying fruit morphology (Hirata et. al. 1986; Yagishta 1961; Kashara et al. 1971). This work employed ‘‘mentor grafting’’ in which a very young seedling that is continually defoliated is grafted to a mature rootstock, so that the scion is a sink for stock-derived nutrients. A range of stocklike phenotypic characteristics were observed in the scion fruit, and in some case, these characteristics were transmitted to seedling progeny (Taller 1998). Another Japanese scientist repeated this work (Ohta 1991) with largely similar results. Among the stocklike phenotypes reported to be inherited in the scion progeny were alterations of fruiting direction, fruiting habit, and pericarp color. Ohta states that genetic analysis indicates that these three traits are due to independently inherited Mendelian genes that are highly stable in the cultivars used in the grafting studies. The frequency of transmission of stocklike traits in the progeny of the scion was reported to be highly variable across different experiments, but with an average rate of 0.84%. A second line of experimentation with graft-induced variation involves the generation of cytoplasmic male sterility through grafting of petunia (Frankel 1956; Edwardson and Corbett 1961), sugarbeet (Curtis 1967), and alfalfa (Thompson and Axtell 1978). In these studies, cytoplasmic male sterile plants were used as rootstock and lines expressing nuclear maintainer/restorer genes were the scions. The scions were observed to ﬂower normally; however, a signiﬁcant percentage of progeny plants were scored as male sterile.

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All of these studies run against the mainstream understanding of genetics and are viewed with considerable skepticism. None of the authors cited has proffered an explanation for these observations that can be supported by modern molecular biology. Ohta (1991) speciﬁed that the mechanism responsible for graft-induced variation was ‘‘graft transformation,’’ in which chromatin was translocated from the stock to the scion. A variant of this model was offered by Liu (2006) in a review in which he states: ‘‘I propose that the stock mRNA molecules are being transferred to the scion—then reverse transcribed into cDNA that can be integrated into the genome of the scions germ cells, embryonic cells, as well as the somatic cells of juvenile plants—may be the main mechanism of graft hybridization.’’ The evidence provided for the transfer of chromatin or DNA sequences is highly questionable. Ohta (1991) built his case on a microhistological analysis that he interpreted as showing chromatin masses moving through the cell walls of dying cells. Taller et al. (1998) presented random ampliﬁed polymorphic DNA (RAPD) analyses indicating that a stock-speciﬁc DNA marker could be detected in graft-induced variants; however, it must be noted that standard controls for RAPD experiments were not included in the data presented. It is difﬁcult to assess the signiﬁcance of observations associated with graft transformation given the lack of rigorous experimental characterization and, with the exception of Ohta, the absence of independent experimental replication. If these observations are in fact artifactual, the most likely explanation may be pollen contamination, although efforts to avoid this were noted in some of the papers. Modern plant molecular biology has certainly not provided support for the models involving translocation of DNA from one cell to another. However, before leaving this topic, it is worth considering how the recent insights into RNA-mediated gene silencing might apply to grafting and could provide a plausible mechanism for some of the phenomena associated with graft-induced variation. It is now known that the accumulation of double-stranded RNA (dsRNA) activates a homology-dependent mechanism that cleaves the dsRNA into 21 to 25 base-pair fragments, known as small interfering RNAs (siRNAs). These are utilized to direct the sequence-speciﬁc degradation of mRNA or to suppress transcription via DNA methylation (Baulcombe 2005). A particularly fascinating aspect of this process is that the silencing signal can be propagated through the phloem so that gene silencing occurs elsewhere in the plant (Tournier et al. 2006). It should be noted that such movement would be stimulated in mentor grafting. This was

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demonstrated by experiments in which transgenic tobacco plants expressing green ﬂuorescent protein (GFP) were grafted onto rootstock containing another GFP construct that was silenced. In this case, the rootstock GFP transgene was silenced because it produces dsRNA homologous to the GFP coding sequence. In these grafted plants, the silencing signal is propagated into the scion where GFP expression is consequently abolished via degradation of the mRNA. The demonstration of the transmission of silencing across the graft junction raises the question whether it could be involved in graftinduced variation. Several reports describing graft hybrids claim that graft-induced variation can be inherited. Heritable genetic changes can also result from the RNA-mediated gene silencing mechanism. One of the actions known to result from production of siRNAs is sequencespeciﬁc methylation of DNA. Heritable transcriptional silencing of GFP genes in transgenic tobacco has been observed when siRNAs target methylation to the promoter (Jones et. al 2001). Approximately 30% of seedlings that display GFP silencing will maintain this state throughout their life cycle; plants that remain fully silenced give rise to silenced progeny, the majority of which will eventually revert to being nonsilenced. Although all of this experimentation involves the silencing of transgenes rather than endogenous genes, most aspects of the silencing mechanism apply to naturally occurring genes and transgenes alike. Based on what is now known, RNA-mediated silencing could provide an explanation for variation reported in graft hybrids, provided the phenotypic changes result from gene inactivation. If this mechanism does underlie such variation, several conditions would be predicted to exist in the rootstock and scion. It would be predicted that the gene in the rootstock responsible for triggering the graft-induced variation would be silenced and have a conﬁguration such that it produces dsRNA. If graft-induced variation is heritable, it would also be predicted that the homologous gene, which becomes silenced in the scion, would not be transcribed in the progeny and would display DNA methylation. The point we want to make is that although the topic of graftinduced variation has been surrounded by much controversy and generated much skepticism, aspects of the phenomena associated with it have interesting parallels with RNA-mediated gene silencing. We certainly are not prepared to state that any of the graft-induced changes reported in these papers result from RNA-mediated gene silencing. However, if similar observations could be produced in a model plant like Arabidopsis, the resources would be available to rapidly

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determine which genes were being affected. Given the wealth of tools available for studying RNA-mediated gene silencing, this system could be employed to carry out a rigorous reexamination of graft-induced variation (Brosnan et al. 2007).

VI. CONCLUSION Grafting is an ancient horticultural technology that is essential to modern horticulture. The practice of joining together two living organisms that then function as one has long been considered a mysterious process and craft secret. This review sheds light on the origins of this process and considers how its history relates to current and future advances in our understanding and application of modern grafting. Given the abundance of natural grafting in the wild, it is reasonable to assume that deliberate grafting arose by discovery rather than invention. The earliest deﬁnite written evidence of grafting dates to Pseudo Hippocrates discussion of the graft union, which was written about 412 BCE, indicating that the introduction of grafting predated Alexander’s excursions to the East in the fourth century BCE. This detailed discussion of the graft union suggests that grafting was an established technology at the time. Since grafting is not directly discussed in the Hebrew Bible, it was probably not a common practice in Mesopotamia but derived instead from the East, perhaps in Central Asia. A Chinese report from the sixth century referring back to grafting in China in the ﬁrst century suggests that grafting may have been a common practice there even before this time. Whether the Chinese and Greek references to grafting represents separate independent ‘‘inventions’’ or more likely an Asian discovery that migrated, East and West, along with other components of agricultural technology (Carter 1977) still cannot be resolved. Regardless of modern advances in propagation technology, such as the use of rooting hormones, misting of cuttings, and micropropagation, grafting still remains a common and essential method of plant cloning for a wide range of purposes and a wide range of species.. Not only is grafting still used for traditional tree fruit crops and woody ornamentals, but it is increasingly used for herbaceous vegetables. The goal of much of the modern breeding of disease-resistant rootstocks is to reduce the use of chemical pesticides for disease management, thus contributing to the goal of sustainable agricultural production. During the twentieth century, the independent breeding of clonal fruit tree (especially apple) rootstocks and scion genotypes has

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allowed for the construction, by grafting, of compound genetic systems that allow an unprecedented level of size control and other desirable rootstock and scion characteristics. Despite the fact that size control (dwarﬁng) is one of the most important selection criteria in rootstock breeding, the mechanism by which this occurs is just beginning to be unraveled at the molecular level. Resolving this question will allow even greater control over compound genetic systems. Stock/scion compatibility has been one of the most persistently misunderstood aspects of grafting from its earliest record until at least the 18th century. In about 50 BCE, Virgil wrote ‘‘swine crunched acorns ‘neath the boughs of elms,’’ suggesting an impossible interfamilial (Ulmaceae/Fagaceae) graft combination. Similar fantastic claims were made by the Chinese writer Jia Sixie in the sixth century, the Italian Gambista Dela Porta in the 16th century, and the Englishman Leonard Mascall in 1572, to name a just a few. All these fundamental misunderstandings of the need for taxonomic afﬁnity between stock and scion suggest that those who wrote about grafting historically were not always the ones actually practicing the method, which is a reminder of the gulf between the educated class and their gardeners. There does not appear to be one overall explanation for graft incompatibility among different taxa. We suggest that the anatomical, physiological, and genetic bases for compatibility need to be examined in a wider biological context. The ability of some parasitic higher plants including mistletoes to form an efﬁcient graft union (as some choose to interpret it) is an example of not only interfamilial but interorder ‘‘graft’’ compatibility (e.g., Santalales/Fagales in the case of a mistletoe/oak association). A better understanding of this association could shed some light on the basis of compatibility among nonparasitic plants. Over 4,000 species of angiosperms are able to directly invade and parasitize other plants (Nickrent et al. 1998; Joel et al. 2007). Not only were the limits of graft compatibility consistently misunderstood over millennia; during the same period there were frequent fantastic claims of commingling stock and scion traits, particularly the scion taking on characteristics of the stock. One notable exception appears in the earliest known writings on grafting by Pseudo Hippocrates in 430 BCE, which acknowledged that the stock and scion remained unique. It is now widely accepted that stock and scion each retain their own identity. The introduction of the concept of genetics in the 20th century has made it clear that there is a difference between environmental, external effects and inherent, hereditary cues.

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Thus, while the physiological inﬂuences of grafting are well recognized and indisputable, it is still debatable whether grafting induces genetic changes across the graft union. In the ﬁrst half of the 20th century, persistent claims of heritable graft ‘‘transformation’’ were reported by Michurin, Lysenko, and others, but such claims were treated skeptically by most scientists. Yet claims of graft transformation continue to be reported and published, in reputable journals, and the evidence of genetic effects of grafting have not been unequivocally discounted. The recent recognition that gene silencing signals, perhaps small RNAs, can pass across the graft union has reopened up the issues concerning the genetic effects of grafting. We suggest that the book on this interesting paradox has many chapters that remain unwritten. VII. LITERATURE CITED Andrews, P.K., and C.S. Marquez. 1993. Graft incompatibility. Hort. Rev. 15:183–231. Anon, 1654. The expert gardener; or a treatise containing certaine necessary, secret, and ordinary knowledge in grafting and gardening. Early English Books Online (cited 10 January 2008). Available at http://gateway.proquest.com/openurl?ctx_ver¼Z39.882003&res_id¼xri:eebo&rft_id¼xri:eebo:citation:18775177. Anon. 1844. A selection from the physiological and horticultural papers by the late Thomas Andrew Knight. Longman, Orme, Brown, Green, and Longman, London. (Facsimile edition, ASHS Press, 2001). Anon. 2007a. Morus alba. Agroforestry Tree Database, World Agroforestry Center, Nairobi, Kenya. (cited 10 Janurary 2008). Available at www.worldagroforestry.org/Sites/ TreeDBS/aft/speciesPrinterFriendly.asp?Id¼1170. Anon. 2007b. Herbals and early gardening books. Patten Collection, Arizona State Univ. (cited 10 January 2008). Available at www.asu.edu/lib/speccoll/patten/html/authors. html. Arber, A. 1960. Robert Sharrock (1630–1684): A precursor of Nehemiah Grew (1641–1712) and an exponent of ‘‘Natural Law’’ in the plant world. Isis 51:3–8. Arbuckle, G. 1994. Literacy and orality in early China. Text of lecture at Calgary Institute for the Humanities, Feb. 15, 1994, David C. Lam Institute for East-West Studies (cited 10 January 2008). Available at www.cic.sfu.ca/nacc/articles/litoral/loral.html. Autio, W.R., J.L. Anderson, J.A. Barden, G.R. Brown, R.M. Crassweller, P.A. Domoto, A. Erb, D.C. Ferree, A. Gaus, P.M. Hirst, C.A. Mullins, and J.R. Schupp. 2001. Location affects performance of Golden Delicious, Jonagold, Empire, and Rome Beauty apple trees on ﬁve rootstocks over ten years in the 1990 NC-140 cultivar-rootstock trial. J. Amer. Pom. Soc. 55:138–145. Bailey, L.H. 1914. The nursery-book, a complete guide to the multiplication of plants. Macmillan, New York. Bailey, L.H. 1928. The standard cyclopedia of horticulture. Macmillan, New York. Barker, T. 1651. The country-mans recreation, or the art of planting, grafﬁng, Early English Books Online (cited 10 January 2008). Available at http://gateway.proquest.com/openurl?ctx_ver¼Z39.88-2003&res_id¼xri:eebo&rft_id¼xri:eebo:citation:99866534.

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Subject Index Anatomy & morphology, daylily, 196–203 Daylily, 193–220 Dedication, Allan Ross Ferguson xiii–xvi Disorder, grape physiological, 355–395 Early bunch stem necrosis of grape, 355–395 Ethylene, 1-methylcyclopropene, 263–313 Floricultural crops, daylily, 193–220 Flower and ﬂowering, daylily, 193–220 Fruit: grape, 355–395 pomegranate, 127–191 Fruit crops: grape physiological disorder, 355–395 pomegranate, 127–191 Genetics & breeding: daylily, 207–214 horseradish, 247–255 macadamia, 1–125 pomegranate, 172–175 Germplasm: macadamia, 1–125 pomegranate, 1134–141 Graft and grafting, history, 437–493 Grape physiological disorder, 355–395 Growth substances, 1-methylcyclopropene, 263–313 Health phytochemicals: horseradish, 243–244 pomegranate, 175–177

History, grafting 437–493 Horseradish, 221–265 Macadamia, genetic resources & domestication, 1–125 1-Methylcyclopropene, 263–313 Nut crops, macadamia genetic resources & domestication, 1–125 Physiology, 1-methylcyclopropene, 253–313 Plug transplant technology, 397–436 Pomegranate, 127–191 Postharvest physiology & technology: bitter melon, 343–344 cucumber, 325–330 cucurbits, 315–354 luffa, 344–345 melon, 330–337 1-methylcyclopropene, 263–313 pumpkin, 337–341 squash, 337–341 watermelon, 319–325 wax gourd. 342 Propagation, macadamia, 92–95 Root & tuber crops, horseradish, 35:221–265 Rootstocks: clonal history, 475–478 macadamia, 92–95 Vegetable crops: cucurbit postharvest, 315–354 horseradish, 221–265 plug industry & technology, 387–436

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 495

Cumulative Subject Index (Volumes 1–35) A Abscisic acid: chilling injury, 15:78–79 cold hardiness, 11:65 dormancy, 7:275–277 genetic regulation, 16:9–14; 20–21 lychee, 28:437–443 mango fruit drop, 31:124–125 mechanical stress, 17:20 rose senescence, 9:66 stress, 4:249–250 Abscission: anatomy & histochemistry, 1:172–203 citrus, 15:145–182, 163–166 ﬂower & petals, 3:104–107 mango fruit drop, 31:113–155 regulation, 7:415–416 rose, 9:63–64 Acclimatization: foliage plants, 6:119–154 herbaceous plants, 6:379–395 micropropagation, 9:278–281, 316–317 Actinidia, see Kiwifruit Adzuki bean, genetics, 2:373 Agapanthus, 25:56–57 Agaricus, 6:85–118 Agrobacterium tumefaciens, 3:34 Air pollution, 8:1–42 Alkaloids, steroidal, 25:171–196 Allium: development, 32:329–378 phytonutrients, 28:156–159 Alkekenge, history & iconography, 34:36–40 Almond: bloom delay, 15:100–101 breeding, 34:197–238

in vitro culture, 9:313 postharvest technology & utilization, 20:267–311 wild of Kazakhstan, 29:262–265 Alocasia, 8:46, 57, see also Aroids Alternate bearing: chemical thinning, 1:285–289 fruit crops, 4:128–173 pistachio, 3:387–388 Aluminum: deﬁciency & toxicity symptoms in fruits & nuts, 2:154 Ericaceae, 10:195–196 Amarcrinum, 25: 57 Amaryllidaceae, growth, development, ﬂowering, 25:1–70 Amaryllis, 25:4–15 Amorphophallus, 8:46, 57, see also Aroids Anatomy & morphology: Allium development, 32:329–378 apple ﬂower & fruit, 10:273–308 apple tree, 12:265–305 asparagus, 12:71 cassava, 13:106–112 citrus, abscission, 15:147–156 daylily, 35:196–203 embryogenesis, 1:4–21, 35–40 ﬁg, 12:420–424; 34:127–137 fruit abscission, 1:172–203 fruit storage, 1:314 ginseng, 9:198–201 grape ﬂower, 13:315–337 grape seedlessness, 11:160–164 heliconia, 14:5–13 kiwifruit, 6:13–50

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 497

498 Anatomy & morphology (Continued ) magnetic resonance imaging, 20:78–86, 225–266 orchid, 5:281–283 navel orange, 8:132–133 pecan ﬂower, 8:217–255 plant architecture, 32:1–61 pollution injury, 8:15 red bayberry, 20:92–96 waxes, 23:1–68 Androgenesis, woody species, 10:171–173 Angiosperms, embryogenesis, 1:1–78 Anthurium, see also Aroids, ornamental fertilization, 5:334–335 Antitranspirants, 7:334 cold hardiness, 11:65 Apical meristem, cryopreservation, 6:357– 372 Apple: alternate bearing, 4:136–137 anatomy & morphology of ﬂower & fruit, 10:273–309 bioregulation, 10:309–401 bitter pit, 11:289–355 bloom delay, 15:102–104 CA storage, 1:303–306 chemical thinning, 1:270–300 cider, 34:365–415 crop load, 31:233–292 fertilization, 1:105 ﬁre blight control, 1:423–474 ﬂavor, 16:197–234 ﬂower induction, 4:174–203 fruit cracking & splitting, 19:217–262 fruiting, 11:229–287 functional phytonutrients, 27:304 germplasm acquisition & resources, 29:1–61 in vitro, 5:241–243; 9:319–321 light, 2:240–248 maturity indices, 13:407–432 mealiness, 20:200 nitrogen metabolism, 4:204–246 pollination, 34:267–268 replant disease, 2:3 root distribution, 2:453–456 scald, 27:227–267 stock-scion relationships, 3:315–375 summer pruning, 9:351–375 tree morphology & anatomy, 12:265–305

CUMULATIVE SUBJECT INDEX vegetative growth, 11:229–287 watercore, 6:189–251 weight loss, 25:197–234 wild of Kazakhstan, 29:63–303, 305–315 yield, 1:397–424 Apricot: bloom delay, 15:101–102 CA storage, 1:309 origin & dissemination, 22:225–266 wild of Kazakhstan, 29:325–326 Arabidopsis: molecular biology of ﬂowering, 27:1–39, 41–77 Architecture, plant, 32:1–61 Aroids: edible, 8:43–99; 12:166–170 ornamental, 10:1–33 Arsenic, deﬁciency & toxicity symptoms in fruits & nuts, 2:154 Artemisia, 19:319–371 Artemisinin, 19:346–359 Artichoke, CA storage, 1:349–350 Asexual embryogenesis, 1:1–78; 2: 268–310; 3:214–314; 7:163–168, 171–173, 176–177, 184, 185–187, 187–188, 189; 10:153–181; 14: 258–259, 337–339; 24:6–7; 26:105–110 Asparagus: CA storage, 1:350–351 ﬂuid drilling of seed, 3:21 postharvest biology, 12:69–155 Aubergine, see eggplant Auxin: abscission, citrus, 15:161, 168–176 bloom delay, 15:114–115 citrus abscission, 15:161, 168–176 dormancy, 7:273–274 ﬂowering, 15:290–291, 315 genetic regulation, 16:5–6, 14, 21–22 geotropism, 15:246–267 mango fruit drop, 31:118–120 mechanical stress, 17:18–19 petal senescence, 11:31 Avocado: CA & MA, 22:135–141 ﬂowering, 8:257–289 fruit development, 10:230–238 fruit ripening, 10:238–259 rootstocks, 17:381–429 Azalea, fertilization, 5:335–337

CUMULATIVE SUBJECT INDEX B Babaco, in vitro culture, 7:178 Bacteria: diseases of ﬁg, 12:447–451 ice nucleating, 7:210–212; 11:69–71 pathogens of bean, 3:28–58 tree short life, 2:46–47 wilt of bean, 3:46–47 Bacteriocides, ﬁre blight, 1:450–459 Bacteriophage, ﬁre blight control, 1:449–450 Banana: CA & MA, 22:141–146 CA storage, 1:311–312 fertilization, 1:105 in vitro culture, 7:178–180 Banksia, 22:1–25 Barberry, wild of Kazakhstan, 29:332–336 Bean: CA storage, 1:352–353 ﬂuid drilling of seed, 3:21 resistance to bacterial pathogens, 3:28–58 Bedding plants, fertilization, 1:99–100; 5:337–341 Beet: CA storage, 1:353 ﬂuid drilling of seed, 3:18–19 Begonia (Rieger), fertilization, 1:104 Belladonna, history & iconography, 34:14–19. Biennial bearing. See Alternate bearing Bilberry, wild of Kazakhstan, 29:347–348 Biochemistry, petal senescence, 11:15–43 Bioreactor technology, 24:1–30 Bioregulation, see also Growth substances apple & pear, 10:309–401 Bird damage, 6:277–278 Bitter pit in apple, 11:289–355 Black currant, bloom delay, 15:104 Black pepper, 33:173–266 Blackberry: harvesting, 16:282–298 wild of Kazakhstan, 29:345 Bloom delay, deciduous fruits, 15:97 Blueberry: developmental physiology, 13:339–405 harvesting, 16:257–282 nutrition, 10:183–227

499 Boron: deﬁciency & toxicity symptoms in fruits & nuts, 2:151–152 foliar application, 6:328 nutrition, 5:327–328 pine bark media, 9:119–122 Botanic gardens, 15:1–62 Bramble, harvesting, 16:282–298 Branching, lateral: apple, 10:328–330 pear, 10:328–330 Brassica classiﬁcation, 28:27–28 Brassicaceae, in vitro, 5:232–235 Breeding, see Genetics & breeding Broccoli, CA storage, 1:354–355 Brussels sprouts, CA storage, 1:355 Bulb crops, see also Tulip development, 25:1–70 ﬂowering, 25:1–70 genetics & breeding, 18:119–123 growth, 25: 1–70 in vitro, 18:87–169; 34:427–445 micropropagation, 18:89–113 root physiology, 14:57–88 virus elimination, 18:113–123 Bunch stem necrosis of grape, 35:355–395 C CA storage. see Controlled-atmosphere storage Cabbage: CA storage, 1:355–359 fertilization, 1:117–118 Cactus: crops, 18:291–320 grafting, 28:106–109 reproductive biology, 18:321–346 Caladium, see Aroids, ornamental Calcifuge, nutrition, 10:183–227 Calciole, nutrition, 10:183–227 Calcium: bitter pit, 11:289–355 cell wall, 5:203–205 container growing, 9:84–85 deﬁciency & toxicity symptoms in fruits & nuts, 2:148–149 Ericaceae nutrition, 10:196–197 foliar application, 6:328–329 fruit softening, 10:107–152 nutrition, 5:322–323

500 Calcium (Continued ) pine bark media, 9:116–117 tipburn, disorder, 4:50–57 Calmodulin, 10:132–134, 137–138 Caparis, see Caper bush Caper bush, 27:125–188 Capsicum pepper, see pepper history & iconography, 34:62–74 Carbohydrate: ﬁg, 12:436–437 kiwifruit partitioning, 12:318–324 metabolism, 7:69–108 partitioning, 7:69–108 petal senescence, 11:19–20 reserves in deciduous fruit trees, 10:403–430 Carbon dioxide, enrichment, 7:345–398, 544–545 Carnation, fertilization, 1:100; 5:341–345 Carrot: CA storage, 1:362–366 ﬂuid drilling of seed, 3:13–14 postharvest physiology, 30:284–288 Caryophyllaceae, in vitro, 5:237–239 Cassava: crop physiology, 13:105–129 molecular biology, 26:85–159 multiple cropping, 30:355–500 postharvest physiology, 30:288–295 root crop, 12:158–166 Cauliﬂower, CA storage, 1:359–362 Celeriac, CA storage, 1:366–367 Celery: CA storage, 1:366–367 ﬂuid drilling of seed, 3:14 Cell culture, 3:214–314 woody legumes, 14:265–332 Cell membrane: calcium, 10:126–140 petal senescence, 11:20–26 Cell wall: calcium, 10:109–122 hydrolases, 5:169–219 ice spread, 13:245–246 tomato, 13:70–71 Cellular mechanisms, salt tolerance,16:33–69 Chelates, 9:169–171 Cherimoya: CA & MA, 22:146–147 pollination, 34:266–267

CUMULATIVE SUBJECT INDEX Cherry: bloom delay, 15:105 CA storage, 1:308 origin, 19:263–317 wild of Kazakhstan, 29:326–330 Chestnut: blight, 8:281–336 botany & horticulture, 31:293–349 in vitro culture, 9:311–312 Chicory, CA storage, 1:379 Chilling: injury, 4:260–261; 15:63–95 injury, chlorophyll ﬂuorescence, 23:79–84 pistachio, 3:388–389 China, protected cultivation, 30:37–82 Chlorine: deﬁciency & toxicity symptoms in fruits & nuts, 2:153 nutrition, 5:239 Chlorophyll ﬂuorescence, 23:69–107 Chlorosis, iron deﬁciency induced, 9:133–186 Chrysanthemum fertilization, 1:100–101; 5:345–352 Cider, 34:365–415 Citrus: abscission, 15:145–182 alternate bearing, 4:141–144 asexual embryogenesis, 7:163–168 CA storage, 1:312–313 chlorosis, 9:166–168 cold hardiness, 7:201–238 fertilization, 1:105 ﬂowering, 12:349–408 functional phytochemicals, fruit, 27:269–315 honey bee pollination, 9:247–248 in vitro culture, 7:161–170 irrigation, 30:37–82 juice loss, 20:200–201 navel orange, 8:129–179 nitrogen metabolism, 8:181 practices for young trees, 24:319–372 rootstock, 1:237–269 viroid dwarﬁng, 24:277–317 Classiﬁcation: Brassica, 28:27–28 lettuce, 28:25–27

CUMULATIVE SUBJECT INDEX potato, 28:23–26 tomato, 28:21–23 Clivia, 25:57 Cloche (tunnel), 7:356–357 Coconut palm: asexual embryogenesis, 7:184 in vitro culture, 7:183–185 Cold hardiness, 2:33–34 apple & pear bioregulation, 10:374–375 citrus, 7:201–238 factors affecting, 11:55–56 herbaceous plants, 6:373–417 injury, 2:26–27 nutrition, 3:144–171 pruning, 8:356–357 Colocasia, 8:45, 55–56, see also Aroids Common blight of bean, 3:45–46 Compositae, in vitro, 5:235–237 Container production, nursery crops, 9:75–101 Controlled-atmosphere (CA) storage: asparagus, 12:76–77, 127–130 chilling injury, 15:74–77 ﬂowers, 3:98; 10:52–55 fruit quality, 8:101–127 fruits, 1:301–336; 4:259–260 pathogens, 3:412–461 seeds, 2:134–135 tropical fruit, 22:123–183 tulip, 5:105 vegetable quality, 8:101–127 vegetables, 1:337–394; 4:259–260 Controlled environment agriculture, 7:534–545, see also Greenhouse greenhouse crops; hydroponic culture; protected culture Copper: deﬁciency & toxicity symptoms in fruits & nuts, 2:153 foliar application, 6:329–330 nutrition, 5:326–327 pine bark media, 9:122–123 Corynebacterium ﬂaccumfaciens, 3:33, 46 Cotoneaster, wild of Kazakhstan, 29: 316–317 Cowpea: genetics, 2:317–348 U.S. production, 12:197–222 Cranberry: botany & horticulture, 21:215–249 fertilization, 1:106

501 harvesting, 16:298–311 wild of Kazakhstan, 29:349 Crinum, 25:58 Crucifers phytochemicals, 28:150–156 Cryopreservation: apical meristems, 6:357–372 cold hardiness, 11:65–66 Cryphonectria parasitica. See Endothia parasitica Crytosperma, 8:47, 58, see also Aroids Cucumber: CA storage, 1:367–368 grafting, 28:91–96 Cucurbita pepo, cultivar groups history, 25:71–170 Currant: harvesting, 16:311–327 wild of Kazakhstan, 29:341 Custard apple, CA & MA, 22:164 Cyrtanthus, 25:15–19 Cytokinin: cold hardiness, 11:65 dormancy, 7:272–273 ﬂoral promoter, 4:112–113 ﬂowering, 15:294–295, 318 genetic regulation, 16:4–5, 14, 22–23 grape root, 5:150, 153–156 lettuce tipburn, 4:57–58 mango fruit drop, 31:118–120 petal senescence, 11:30–31 rose senescence, 9:66 D Date palm: asexual embryogenesis, 7:185–187 in vitro culture, 7:185–187 Datura, history & iconography, 34:44–51 Daylength, see Photoperiod Daylily, 35:193–220 Dedication: Bailey, L.H., 1:v–viii Beach, S.A., 1:v–viii Bukovac, M.J., 6:x–xii Campbell, C.W., 19:xiii–xiv Cantliffe, D.J., 33:xi–xiii Cummins, J.N., 15:xii–xv De Hertogh, A.A., 26:xi–xii Dennis, F.G., 22:xi–xii Faust, Miklos, 5:vi–x

502 Dedication (Continued ) Ferguson, A.R., 35: xiiiHackett, W.P., 12:x–xiii Halevy, A.H., 8:x–xii Hess, C.E., 13:x–xii Kader, A.A., 16:xii–xv Kamemoto, H., 24:x–xiii Kester, D.E., 30:xiii–xvii Looney, N.E., 18:xii–xv Magness, J.R., 2:vi–viii Mizrahi, Y. 34:xi–xv Moore, J.N., 14:xii–xv Possingham, J.V., 27:xi–xiii Pratt, C., 20:ix–xi Proebsting, Jr., E.L., 9:x–xiv Rick, Jr., C.M., 4:vi–ix Ryugo, K., 25:x–xii Sansavini, S., 17:xii–xiv Sedgely, M., 32:x–xii Sherman, W.B., 21:xi–xiii Smock, R.M., 7:x–xiii Sperling, C.E., 29:ix–x Stevens, M.A., 28:xi–xiii Warrington, L.J. 31:xi–xii Weiser, C.J., 11:x–xiii Whitaker, T.W., 3:vi–x Wittwer, S.H., 10:x–xiii Yang, S.F., 23:xi Deﬁciency symptoms, in fruit & nut crops, 2:145–154 Deﬁcit irrigation, 21:105–131; 32:111–165 Defoliation, apple & pear bioregulation, 10:326–328 ‘Delicious’ apple, 1:397–424 Desiccation tolerance, 18:171–213 Dieffenbachia, see Aroids, ornamental Dioscorea., see Yam Disease: air pollution, 8:25 aroids, 8:67–69; 10:18; 12:168–169 bacterial, of bean, 3:28–58 cassava, 12:163–164 control by virus, 3:399–403 controlled-atmosphere storage, 3: 412–461 cowpea, 12:210–213 ﬁg, 12:447–479 ﬂooding, 13:288–299 hydroponic crops, 7:530–534 lettuce, 2:187–197

CUMULATIVE SUBJECT INDEX mycorrhizal fungi, 3:182–185 ornamental aroids, 10:18 resistance, acquired, 18:247–289 root, 5:29–31 stress, 4:261–262 sweet potato, 12:173–175 tulip, 5:63, 92 turnip moasic virus, 14:199–238 waxes, 23:1–68 yam (Dioscorea), 12:181–183 Disorder, see also Postharvest physiology: bitterpit, 11:289–355 ﬁg, 12:477–479 grape physiological, 35:355–395 pear fruit, 11:357–411 watercore, 6:189–251; 11:385–387 Dormancy, 2:27–30 blueberry, 13:362–370 fruit trees, 7:239–300 tulip, 5:93 Drip irrigation, 4:1–48 Drought resistance, 4:250–251 cassava, 13:114–115 Durian, CA & MA, 22:147–148 Dwarﬁng: apple, 3:315–375 apple mutants, 12:297–298 by virus, 3:404–405 E Early bunch stem necrosis of grape, 35:355–395 Easter lily, fertilization, 5:352–355 Eggplant: grafting, 28:103–104 history & iconography, 34:25–35 phytochemicals, 28:162–163 Elderberry, wild of Kazakhstan, 29: 349–350 Embryogenesis, see Asexual embryogenesis Endothia parasitica, 8:291–336 Energy efﬁciency, in greenhouses, 1: 141–171; 9:1–52 Environment: air pollution, 8:20–22 controlled for agriculture, 7:534–545 controlled for energy efﬁciency, 1: 141–171; 9:1–52 embryogenesis, 1:22, 43–44

CUMULATIVE SUBJECT INDEX fruit set, 1:411–412 ginseng, 9:211–226 greenhouse management, 9:32–38 navel orange, 8:138–140 nutrient ﬁlm technique, 5:13–26 Epipremnum, see Aroids, ornamental Eriobotrya japonica, see Loquat Erwinia: amylovora, 1:423–474 lathyri, 3:34 Essential elements: foliar nutrition, 6:287–355 pine bark media, 9:103–131 plant nutrition, 5:318–330 soil testing, 7:1–68 Ethylene: abscission, citrus, 15:158–161, 168–176 apple bioregulation, 10:366–369 avocado, 10:239–241 bloom delay, 15:107–111 CA storage, 1:317–319, 348 chilling injury, 15:80 citrus abscission, 15:158–161, 168–176 cut ﬂower storage, 10:44–46 dormancy, 7:277–279 ﬂower longevity, 3:66–75 ﬂowering, 15:295–296, 319 genetic regulation, 16:6–7, 14–15, 19–20 kiwifruit respiration, 6:47–48 mango fruit crop, 31:120–122 mechanical stress, 17:16–17 1-methylcyclopropene, 35:263–313 petal senescence, 11:16–19, 27–30 rose senescence, 9:65–66 Eucharis, 25:19–22 Eucrosia, 25:58 F Feed crops, cactus, 18:298–300 Feijoa, CA & MA, 22:148 Fertilization & fertilizer: anthurium, 5:334–335 azalea, 5:335–337 bedding plants, 5:337–341 blueberry, 10:183–227 carnation, 5:341–345 chrysanthemum, 5:345–352 controlled release, 1:79–139; 5:347–348 Easter lily, 5:352–355 Ericaceae, 10:183–227

503 foliage plants, 5:367–380 foliar, 6:287–355 geranium, 5:355–357 greenhouse crops, 5:317–403 lettuce, 2:175 nitrogen, 2:401–404 orchid, 5:357–358 poinsettia, 5:358–360 rose, 5:361–363 snapdragon, 5:363–364 soil testing, 7:1–68 trickle irrigation, 4:28–31 tulip, 5:364–366 Vaccinium, 10:183–227 zinc nutrition, 23:109–128 Fig: botany, horticulture, breeding, 34:113–195 industry, 12:409–490 ripening, 4:258–259 Filbert, in vitro culture, 9:313–314 Fire blight, 1:423–474 Flooding, fruit crops, 13:257–313 Floral scents, 24:31–53 Floricultural crops, see also individual crops: Amaryllidaceae, 25:1–70 Banksia, 22:1–25 China, protected culture, 30:141–148 daylily, 35:193–220 fertilization, 1:98–104 growth regulation, 7:399–481 heliconia, 14:1–55 Leucospermum, 22:27–90 postharvest physiology & senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43 Protea, 26:1–48 Florigen, 4:94–98 Flower & ﬂowering: Amaryllidaceae, 25:1–70 appleanatomy&morphology,10:277–283 apple bioregulation, 10:344–348 Arabidopsis, 27:1–39, 41–77 aroids, ornamental, 10:19–24 avocado, 8:257–289 Banksia, 22:1–25 blueberry development, 13:354–378 cactus, 18:325–335 citrus, 12:349–408

504 Flower & Flowering (Continued ) control, 4:159–160; 15:279–334 daylily, 35:193–220 development (postpollination), 19:1–58 ﬁg, 12:424–429 girdling, 20:1–26 grape anatomy & morphology, 13: 354–378 homeotic gene regulation, 27:41–77 honey bee pollination, 9:239–243 in vitro, 4:106–127 induction, 4:174–203, 254–256 initiation, 4:152–153 kiwifruit, 6:21–35; 12:316–318 Leucospermum, 22:27–90 lychee, 28:397–421 orchid, 5:297–300 pear bioregulation, 10:344–348 pecan, 8:217–255 perennial fruit crops, 12:223–264 phase change, 7:109–155 photoperiod, 4:66–105 pistachio, 3:378–387 postharvest physiology, 1:204–236; 3:59–143; 10:35–62; 11:15–43 postpollination development, 19:1–58 protea leaf blackening, 17:173–201 pruning, 8:359–362 raspberry, 11:187–188 regulation in ﬂoriculture, 7:416–424 rhododendron, 12:1–42 rose, 9:60–66 scents, 24:31–53 senescence, 1:204–236; 3:59–143; 10:35–62; 11:15–43;18:1–85 strawberry, 28:325–349 sugars, 4:114 thin cell layer morphogenesis, 14: 239–256 tulip, 5:57–59 water relations, 18:1–85 Fluid drilling, 3:1–58 Foliage plants: acclimatization, 6:119–154 fertilization, 1:102–103; 5:367–380 industry, 31:47–112 Foliar nutrition, 6:287–355 Freeze protection, see Frost protection Frost: apple fruit set, 1:407–408

CUMULATIVE SUBJECT INDEX citrus, 7:201–238 protection, 11:45–109 Fruit: abscission, 1:172–203 abscission, citrus, 15:145–182 apple anatomy & morphology, 10:283–297 apple bioregulation, 10:348–374 apple bitter pit, 11:289–355 apple crop load, 31:233–292 apple ﬂavor, 16:197–234 apple maturity indices, 13:407–432 apple ripening & quality, 10:361–374 apple scald, 27:227–267 apple weight loss, 25:197–234 avocado development & ripening, 10:229–271 bloom delay, 15:97–144 blueberry development, 13:378–390 CA storage & quality, 8:101–127 cactus physiology, 18:335–341 chilling injury, 15:63–95 coating physiology, 26:161–238 cracking, 19:217–262; 30:163–184 diseases in CA storage, 3:412–461 drop, apple ﬁg, 4:258–259; 12:409–490; 34:113–195 fresh cut, 30:185–251 functional phytochemicals, 27:269–315 grape, 35:355–395 growth measurement, 24:373–431 jujube, 32:229–298 kiwifruit, 6:35–48; 12:316–318 loquat, 23:233–276 lychee, 28:433–444 mango fruit drop, 31:113–155 maturity indices, 13:407–432 navel orange, 8:129–179 nectarine, postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 olive physiology, 31:157–231 olive processing, 25:235–260 pawpaw, 31:351–384 peach, postharvest, 11:413–452 pear, bioregulation, 10:348–374 pear, fruit disorders, 11:357–411 pear maturity indices, 13:407–432 pear ripening & quality, 10:361–374 pear scald, 27:227–267

CUMULATIVE SUBJECT INDEX pear volatiles, 28:237–324 pistachio, 3:382–391 phytochemicals, 28:125–185 plum, 23:179–231 pollination, 34:239–275 pomegranate, 35:127–191 quality & pruning, 8:365–367 red bayberry, 30: 83–113 ripening, 5:190–205 rose, wild of Kazakhstan, 29:353–360 set, 1:397–424; 4:153–154 set in navel oranges, 8:140–142 size & thinning, 1:293–294; 4:161 softening, 5:109–219; 10:107–152 splitting, 19:217–262 strawberry growth & ripening, 17:267–297 texture, 20:121–224 thinning, apple & pear, 10:353–359 tomato cracking, 30:163–184 tomato parthenocarpy, 6:65–84 tomato ripening, 13:67–103 volatiles, pear, 28:237–324 Fruit crops, see also Individual crop alternate bearing, 4:128–173 apple bitter pit, 11:289–355 apple crop load, 31:233–292 apple ﬂavor, 16:197–234 apple fruit splitting & cracking, 19: 217–262 apple germplasm, 29:1–61, 63–303 apple growth, 11:229–287 apple maturity indices, 13:407–432 apple scald, 27:227–267 apple, wild of Kazakhstan, 29:63–303, 305–315 apricot, origin & dissemination, 22:225–266 apricot, wild of Kazakhstan, 29–325–326 architecture, 32:1–61 avocado ﬂowering, 8:257–289 avocado rootstocks, 17:381–429 barberry, wild of Kazakhstan, 29:332–336 berry crop harvesting, 16:255–382 bilberry, wild of Kazakhstan, 29:347–348 blackberry, wild of Kazakhstan, 29:345 bloom delay, 15:97–144

505 blueberry developmental physiology, 13:339–405 blueberry harvesting, 16:257–282 blueberry nutrition, 10:183–227 bramble harvesting, 16:282–298 cactus, 18:302–309 CA & MA for tropicals, 22:123–183 CA storage, 1:301–336 CA storage diseases, 3:412–461 carbohydrate reserves, 10:403–430 cherry origin, 19:263–317 cherry, wild of Kazakhstan, 29:326–330 chilling injury, 15:145–182 chlorosis, 9:161–165 cider, 34:365–414 citrus abscission, 15:145–182 citrus cold hardiness, 7:201–238 citrus, culture of young trees, 24: 319–372 citrus dwarﬁng by viroids, 24:277–317 citrus ﬂowering, 12:349–408 citrus irrigation, 30:37–82 citrus nutrition diagnostics, 34:277–364 cotoneaster, wild of Kazakhstan, 29:316–317 cranberry, 21:215–249 cranberry harvesting, 16:298–311 cranberry, wild of Kazakhstan, 29:349 currant harvesting, 16:311–327 currant, wild of Kazakhstan, 29:341 deﬁcit irrigation, 21:105–131 dormancy release, 7:239–300 elderberry, wild of Kazakhstan, 29: 349–350 Ericaceae nutrition, 10:183–227 fertilization, 1:104–106 ﬁg, industry, 12:409–490; 34:113–195 ﬁreblight, 11:423–474 ﬂowering, 12:223–264 foliar nutrition, 6:287–355 frost control, 11:45–109 gooseberry, wild of Kazakhstan, 29: 341–342 grape ﬂower anatomy & morphology, 13:315–337 grape harvesting, 16:327–348 grape irrigation, 27:189–225 grape nitrogen metabolism, 14:407–452 grape physiological disorder, 35:355–395

506 Fruit crops (Continued ) grape pruning, 16:235–254, 336–340 grape root, 5:127–168 grape seedlessness, 11:164–176 grape, wild of Kazakhstan, 29:342–343 grapevine pruning, 16:235–254, 336–340 greenhouse, China, 30:149–158 honey bee pollination, 9:244–250, 254–256 jojoba, 17:233–266 jujube, 32:229–298 in vitro culture, 7:157–200; 9:273–349 irrigation, deﬁcit, 21:105–131 kiwifruit, 6:1–64; 12:307–347; 33:1–121 lingonberry, 27:79–123 lingonberry, wild of Kazakhstan, 29:348–349 longan, 16:143–196 loquat, 23:233–276 lychee, 16:143–196, 28:393–453 mango fruit drop, 113–155 mountain ash, wild of Kazakhstan, 29:322–324 mulberry, wild of Kazakhstan, 29:350–351 muscadine grape breeding, 14:357–405 navel orange, 8:129–179 nectarine postharvest, 11:413–452 nondestructive postharvest quality evaluation, 20:1–119 nutritional ranges, 2:143–164 oleaster, wild of Kazakhstan, 29:351–353 olive physiology, 31:157–231 olive salinity tolerance, 21:177–214 orange, navel, 8:129–179 orchard ﬂoor management, 9:377–430 pawpaw, 31:351–384 peach orchard systems, 32:63–109 peach origin, 17:331–379 peach postharvest, 11:413–452 peach thinning, 28:351–392 pear fruit disorders, 11:357–411; 27:227–267 pear maturity indices, 13:407–432 pear scald, 27:227–267 pear volatiles, 28:237–324 pear, wild of Kazakhstan, 29:315–316 pecan ﬂowering, 8:217–255

CUMULATIVE SUBJECT INDEX photosynthesis, 11:111–157 Phytophthora control, 17:299–330 plum origin, 23:179–231 plum, wild of Kazakhstan, 29:330–332 pollination, 34:239–275 pomegranate, 35:127–191 pruning, 8:339–380 rambutan, 16:143–196 raspberry, 11:185–228 raspberry, wild of Kazakhstan, 29:343–345 red bayberry, 30:83–113 roots, 2:453–457 sapindaceous fruits, 16:143–196 sea buckthorn, wild of Kazakhstan, 29:361 short life & replant problem, 2:1–116 strawberry fruit growth, 17:267–297 strawberry harvesting, 16:348–365 strawberry, wild of Kazakhstan, 29:347 summer pruning, 9:351–375 Vaccinium nutrition, 10:183–227 vacciniums, wild of Kazakhstan, 29:347–349 viburnam, wild of Kazakhstan, 29:361–362 virus elimination, 28:187–236 water status, 7:301–344 water stress, 32:111–165 Functional phytochemicals, fruit, 27:269–315 Fungi: ﬁg, 12:451–474 mushroom, 6:85–118 mycorrhiza, 3:172–213; 10:211–212 pathogens in postharvest storage, 3:412–461 trufﬂe cultivation, 16:71–107 Fungicide, & apple fruit set, 1:416 G Galanthus, 25:22–25 Gboma eggplant, history & iconography, 34:25 Garlic, 33:123–172 CA storage, 1:375 Genetic variation: alternate bearing, 4:146–150 kiwifruit, 33:1–121 photoperiodic response, 4:82

CUMULATIVE SUBJECT INDEX pollution injury, 8:16–19 temperature-photoperiod interaction, 17:73–123 wild apple, 29:63–303 Genetics & breeding: almond, 34:197–238 aroids (edible), 8:72–75; 12:169 aroids (ornamental), 10:18–25 bean, bacterial resistance, 3:28–58 bloom delay in fruits, 15:98–107 bulbs, ﬂowering, 18:119–123 cassava, 12:164 chestnut blight resistance, 8:313–321 citrus cold hardiness, 7:221–223 cranberry, 21:236–239 daylily, 35:207–214 embryogenesis, 1:23 ﬁg, 12:432–433; 34:165–170 ﬁre blight resistance, 1:435–436 ﬂower longevity, 1:208–209 ﬂowering, 15:287–290, 303–305, 306–309, 314–315; 27:1–39, 41–77 ginseng, 9:197–198 grafting use, 28:109–115 horseradish, 35:247–255 in vitro techniques, 9:318–324; 18:119–123 kiwifruit, 33:1–121 lettuce, 2:185–187 lingonberry, 27:108–111 loquat, 23:252–257 macadamia, 35:1–125 muscadine grapes, 14:357–405 mushroom, 6:100–111 navel orange, 8:150–156 nitrogen nutrition, 2:410–411 pineapple, 21:138–164 plant regeneration, 3:278–283 pollution insensitivity, 8:18–19 pomegranate, 35:172–175 potato tuberization, 14:121–124 rhododendron, 12:54–59 sweet potato, 12:175 sweet sorghum, 21:87–90 tomato parthenocarpy, 6:69–70 tomato ripening, 13:77–98 tree short life, 2:66–70 Vigna, 2:311–394 waxes, 23:50–53

507 woody legume tissue & cell culture, 14:311–314 yam (Dioscorea), 12:183 Geophyte, see bulb, tuber Geranium, fertilization, 5:355–357 Germination, seed, 2:117–141, 173–174; 24:229–275 Germplasm: acquisition, apple, 29:1–61 characterization, apple, 29:45–56 cryopreservation, 6:357–372 in vitro, 5:261–264; 9:324–325 macadamia, 35:1–125 pineapple, 21:133–175 pomegranate, 35:134–141 resources, apple, 29:1–61 Gibberellin: abscission, citrus, 15:166–167 bloom delay, 15:111–114 citrus, abscission, 15:166–167 cold hardiness, 11:63 dormancy, 7:270–271 ﬂoral promoter, 4:114 ﬂowering, 15:219–293, 315–318 genetic regulation, 16:15 grape root, 5:150–151 mango fruit drop, 31:113–155 mechanical stress, 17:19–20 Ginger postharvest physiology, 30: 297–299 Ginseng, 9:187–236 Girdling, 1;416–417; 4:251–252, 30:1–26 Glucosinolates, 19:99–215 Gooseberry, wild of Kazakhstan, 29: 341–342 Gourd, history, 25:71–171 Graft & grafting: herbaceous, 28:61–124 history, 35:437–493 incompatibility, 15:183–232 phase change, 7:136–137, 141–142 rose, 9:56–57 Grape: CA storage, 1:308 chlorosis, 9:165–166 ﬂower anatomy & morphology, 13:315–337 functional phytochemicals, 27:291–298 harvesting, 16:327–348 irrigation, 27:189–225

508 Grape (Continued ) muscadine breeding, 14:357–405 nitrogen metabolism, 14:407–452 physiological disorder, 35:355–395 pollen morphology, 13:331–332 pruning, 16:235–254, 336–340 root, 5:127–168 seedlessness, 11:159–187 sex determination, 13:329–331 wild of Kazakhstan, 29:342–343 Gravitropism, 15:233–278 Greenhouse & greenhouse crops: carbon dioxide, 7:357–360, 544–545 China protected cultivation, 30:115–162 energy efﬁciency, 1:141–171; 9:1–52 growth substances, 7:399–481 nutrition & fertilization, 5:317–403 pest management, 13:1–66 vegetables, 21:1–39 Growth regulators, see Growth substances Growth substances, 2:60–66; 24:55–138, see also Abscisic acid, Auxin, Cytokinins, Ethylene, Gibberellins abscission, citrus, 15:157–176 apple bioregulation, 10:309–401 apple dwarﬁng, 3:315–375 apple fruit set, 1:417 apple thinning, 1:270–300 aroids, ornamental, 10:14–18 avocado fruit development, 10:229–243 bloom delay, 15:107–119 CA storage in vegetables, 1:346–348 cell cultures, 3:214–314 chilling injury, 15:77–83 citrus abscission, 15:157–176 cold hardiness, 7:223–225; 11:58–66 dormancy, 7:270–279 embryogenesis, 1:41–43; 2:277–281 ﬂoriculture, 7:399–481 ﬂower induction, 4:190–195 ﬂower storage, 10:46–51 ﬂowering, 15:290–296 genetic regulation, 16:1–32 ginseng, 9:226 girdling, 20:1–26 grape seedlessness, 11:177–180 hormone reception, 26:49–84 in vitro ﬂowering, 4:112–115 mango fruit drop, 31:113–155 mechanical stress, 17:16–21

CUMULATIVE SUBJECT INDEX meristem & shoot-tip culture, 5:221–227 1-methylcyclopropene, 35:355–395 navel oranges, 8:146–147 pear bioregulation, 10:309–401 petal senescence, 3:76–78 phase change, 7:137–138, 142–143 raspberry, 11:196–197 regulation, 11:1–14 rose, 9:53–73 seedlessness in grape, 11:177–180 triazole, 10:63–105 H Haemanthus, 25:25–28 Halo blight of beans, 3:44–45 Hardiness, 4:250–251 Harvest: ﬂower stage, 1:211–212 index, 7:72–74 lettuce, 2:176–181 mechanical of berry crops, 16:255–382 Hawthorne, wild of Kazakhstan, 29:317–322 Hazelnut. See Filbert wild of Kazakhstan, 29:365–366 Health phytochemicals: fruit, 27:269–315 horseradish, 35:243–244 pomegranate, 175–177 vegetables, 28:125–185 Heat treatment (postharvest), 22:91–121 Heliconia, 14:1–55 Henbane, history & iconography, 34:10–14 Herbaceous plants, subzero stress, 6:373–417 Hippeastrum, 25:29–34 Histochemistry: ﬂower induction, 4:177–179 fruit abscission, 1:172–203 Histology, ﬂower induction, 4:179–184, see also Anatomy & Morphology History & iconography: alkekenge, 34:36–40 aubergine, see eggplant. belladonna, 34:14–19 capsicum pepper, 34:62–74 datura, 34:44–51 eggplant, 34:25–35. gboma eggplant, 34:25 grafting, 35:437–493

CUMULATIVE SUBJECT INDEX henbane, 34:10–14 husk tomato, 34:40–44 Lycium spp., 34:23 mandrake, 34:4–10 potato, 34:85–89 scarlet eggplant, 34:25 Scopolia spp., 34:20–23 Solanaceae, 34:1–111 Solanum dulcamara, 34:25 Solanum nigrum, 34:23–24 tobacco, 34:51–62 tomato, 34:75–85 Withania spp., 34:19–20 Honey bee, 9:237–272 Honeysuckle, wild of Kazakhstan, 29:350 Horseradish: botany, horticulture, breeding, 35:221–265 CA storage, 1:368 Husk tomato, history & iconography, 34:40–44 Hydrolases, 5:169–219 Hydroponic culture, 5:1–44; 7:483–558 Hymenocallis, 25:59 Hypovirulence, in Endothia parasitica, 8:299–310 I Ice, formation & spread in tissues, 13:215–255 Ice-nucleating bacteria, 7:210–212; 13:230–235 Iconography, see history. In vitro: abscission, 15:156–157 apple propagation, 10:325–326 aroids, ornamental, 10:13–14 artemisia, 19:342–345 bioreactor technology, 24:1–30 bulbs, ﬂowering, 18:87–169; 34:417–445 cassava propagation, 13:121–123; 26:99–119 cellular salinity tolerance, 16:33–69 cold acclimation, 6:382 cryopreservation, 6:357–372 embryogenesis, 1:1–78; 2:268–310; 7:157–200; 10:153–181 environmental control, 17:123–170 ﬂowering, 4:106–127 ﬂowering bulbs, 18:87–169; 34:417–445

509 geophytes, 34:417–445 pear propagation, 10:325–326 phase change, 7:144–145 propagation, 3:214–314; 5:221–277; 7:157– 200; 9:57–58, 273–349; 17:125–172 thin cell layer morphogenesis, 14:239–264 woody legume culture, 14:265–332 Industrial crops, cactus, 18:309–312 Insects & mites: aroids, 8:65–66 avocado pollination, 8:275–277 ﬁg, 12:442–447 hydroponic crops, 7:530–534 integrated pest management, 13:1–66 lettuce, 2:197–198 ornamental aroids, 10:18 particle ﬁlm control, 31:1–45 tree short life, 2:52 tulip, 5:63, 92 waxes, 23:1–68 Integrated pest management, greenhouse crops, 13:1–66 Invasive plants, 32:379–437 Iron: deﬁciency & toxicity symptoms in fruits & nuts, 2:150 deﬁciency chlorosis, 9:133–186 Ericaceae nutrition, 10:193–195 foliar application, 6:330 nutrition, 5:324–325 pine bark media, 9:123 Irrigation: citrus, 30:37–82 deﬁcit, deciduous orchards, 21: 105–131; 32:111–165 drip or trickle, 4:1–48 frost control, 11:76–82 fruit trees, 7:331–332 grape, 27:189–225 grape root growth, 5:140–141 lettuce industry, 2:175 navel orange, 8:161–162 root growth, 2:464–465 scheduling, 32:111–165 Ismene, 25:59 J Jojoba, 17:233–266 Jujube, 32:229–298

510 Juvenility, 4:111–112 pecan, 8:245–247 tulip, 5:62–63 woody plants, 7:109–155 K Kale, ﬂuid drilling of seed, 3:21 Kazakhstan, see Wild fruits & nuts Kiwifruit: botany, 6:1–64 genetic resources and breeding, 1–121 nutrition and vine growth, 12: 307–347 L Lamps, for plant growth, 2:514–531 Lanzon, CA & MA, 22:149 Leaves: apple morphology, 12:283–288 ﬂower induction, 4:188–189 Leek: CA storage, 1:375 fertilization, 1:118 Leguminosae, in vitro, 5:227–229; 14: 265–332 Lemon, rootstock, 1:244–246, see also Citrus Lettuce: CA storage, 1:369–371 classiﬁcation, 28:25–27 fertilization, 1:118 ﬂuid drilling of seed, 3:14–17 industry, 2:164–207 seed germination, 24:229–275 tipburn, 4:49–65 Leucadendron, 32:167–228 Leucojum, 25:34–39 Leucospermum, 22:27–90 Light: fertilization, greenhouse crops, 5: 330–331 ﬂowering, 15:282–287, 310–312 fruit set, 1:412–413 lamps, 2:514–531 nitrogen nutrition, 2:406–407 orchards, 2:208–267 ornamental aroids, 10:4–6 photoperiod, 4:66–105 photosynthesis, 11:117–121

CUMULATIVE SUBJECT INDEX plant growth, 2:491–537 tolerance, 18:215–246 Lingonberry, 27:79–123 wild of Kazakhstan, 29:348–349 Longan, see also Sapindaceous fruits CA & MA, 22:150 Loquat: botany & horticulture, 23:233–276 CA & MA, 22:149–150 Lychee, see also Sapindaceous fruits CA & MA, 22:150 ﬂowering, 28:397–421 fruit abscission, 28–437–443 fruit development, 28:433–436 pollination, 28:422–428 reproductive biology, 28:393–453 Lycium spp., history & iconography, 34:23 Lycoris, 25:39–43 M Macadamia, genetic resources & development, 35:1–125 Magnesium: container growing, 9:84–85 deﬁciency & toxicity symptoms in fruits & nuts, 2:148 Ericaceae nutrition, 10:196–198 foliar application, 6:331 nutrition, 5:323 pine bark media, 9:117–119 Magnetic resonance imaging, 20:78–86, 225–266 Male sterility, temperature-photoperiod induction, 17:103–106 Mandarin, rootstock, 1:250–252 Mandrake, history & iconography, 34:4–10 Manganese: deﬁciency & toxicity symptoms in fruits & nuts, 2:150–151 Ericaceae nutrition, 10:189–193 foliar application, 6:331 nutrition, 5:235–326 pine bark media, 9:123–124 Mango: alternate bearing, 4:145–146 asexual embryogenesis, 7:171–173 CA & MA, 22:151–157 CA storage, 1:313 fruit drop, 31:113–155 in vitro culture, 7:171–173

CUMULATIVE SUBJECT INDEX Mangosteen, CA & MA, 22:157 Master Gardener program, 33:393–420 Mechanical harvest, berry crops, 16:255– 382 Mechanical stress regulation, 17:1–42 Media: fertilization, greenhouse crops, 5:333 pine bark, 9:103–131 Medicinal crops: Artemisia, 19:319–371 poppy, 19:373–408 Taxus, 32:299–327 Melon grafting, 28:96–98 Meristem culture, 5:221–277 Metabolism: ﬂower, 1:219–223 nitrogen in citrus, 8:181–215 seed, 2:117–141 1-Methylcyclopropene, 35:263–313 Micronutrients: container growing, 9:85–87 pine bark media, 9:119–124 Micropropagation, see also In vitro; propagation: bulbs, ﬂowering, 18:89–113 environmental control, 17:125–172 nuts, 9:273–349 rose, 9:57–58 temperate fruits, 9:273–349 tropical fruits & palms, 7:157–200 Microtu, see Vole Modiﬁed atmosphere (MA) for tropical fruits, 22:123–183 Moisture & seed storage, 2:125–132 Molecular biology: cassava, 26:85–159 ﬂoral induction, 27:3–20 ﬂowering, 27:1–39;41–77 hormone reception, 26:49–84 Molybdenum nutrition, 5:328–329 Monocot, in vitro, 5:253–257 Monstera, see Aroids, ornamental Morphology: navel orange, 8:132–133 orchid, 5:283–286 pecan ﬂowering, 8:217–243 red bayberry, 30: 92–96 Moth bean, genetics, 2:373–374 Mountain ash, wild of Kazakhstan, 29:322–324

511 Mulberry, wild of Kazakhstan, 29:350–351 Multiple cropping, 30:355–500 Mung bean, genetics, 2:348–364 Mushroom: CA storage, 1:371–372 cultivation, 19:59–97 spawn, 6:85–118 Muskmelon, fertilization, 1:118–119 Mycoplasma-like organisms, tree short life, 2:50–51 Mycorrhizae: container growing, 9:93 Ericaceae, 10:211–212 fungi, 3:172–213 grape root, 5:145–146 Myrica, see Red bayberry N Narcissus, 25:43–48 Navel orange, 8:129–179 Nectarine: bloom delay, 15:105–106 CA storage, 1:309–310 postharvest physiology, 11:413–452 Nematodes: aroids, 8:66 ﬁg, 12:475–477 lettuce, 2:197–198 tree short life, 2:49–50 Nerine, 25:48–56 NFT (nutrient ﬁlm technique), 5:1–44 Nitrogen: CA storage, 8:116–117 container growing, 9:80–82 deﬁciency & toxicity symptoms in fruits & nuts, 2:146 Ericaceae nutrition, 10:198–202 ﬁxation in woody legumes, 14:322–323 foliar application, 6:332 in embryogenesis, 2:273–275 metabolism in apple, 4:204–246 metabolism in citrus, 8:181–215 metabolism in grapevine, 14:407–452 nutrition, 2:395, 423; 5:319–320 pine bark media, 9:108–112 trickle irrigation, 4:29–30 vegetable crops, 22:185–223 Nomenclature, 28:1–60 Nondestructive quality evaluation of fruits & vegetables, 20:1–119

512 Nursery crops: fertilization, 1:106–112 nutrition, 9:75–101 Nut crops, see also individual crop almond breeding, 34:197–238 almond postharvest technology & utilization, 20:267–311 almond, wild of Kazakhstan, 29:262–265 chestnut blight, 8:291–336 chestnut, botany & horticulture, 31:293–349 fertilization, 1:106 hazelnut, wild of Kazakhstan, 29:365–366 honey bee pollination, 9:250–251 in vitro culture, 9:273–349 macadamia, 35:1–125 nutritional ranges, 2:143–164 pine, wild of Kazakhstan, 29:368–369 pistachio culture, 3:376–396 pistachio, wild of Kazakhstan, 29:366–368 walnut, wild of Kazakhstan, 29:369–370 Nutrient: citrus diagnotics, 34:277–364 concentration in fruit & nut crops, 2:154–162 ﬁlm technique, 5:1–44 foliar-applied, 6:287–355 media, for asexual embryogenesis, 2:273–281 media, for organogenesis, 3:214–314 plant & tissue analysis, 7:30–56 solutions, 7:524–530 uptake, in trickle irrigation, 4:30–31 Nutrition (human): aroids, 8:79–84 CA storage, 8:101–127 phytochemicals in fruit, 27:269–315 phytochemicals in vegetables, 28:125–185 steroidal alkalois, 25:171–196 Nutrition (plant): air pollution, 8:22–23, 26 blueberry, 10:183–227 calcifuge, 10:183–227 citrus diagnostics, 34:277–364 cold hardiness, 3:144–171 container nursery crops, 9:75–101

CUMULATIVE SUBJECT INDEX cranberry, 21:234–235 ecologically based, 24:156–172 embryogenesis, 1:40–41 Ericaceae, 10:183–227 ﬁre blight, 1:438–441 foliar, 6:287–355 fruit & nut crops, 2:143–164 ginseng, 9:209–211 greenhouse crops, 5:317–403 kiwifruit, 12:325–332 mycorrhizal fungi, 3:185–191 navel orange, 8:162–166 nitrogen in apple, 4:204–246 nitrogen in vegetable crops, 22:185–223 nutrient ﬁlm techniques, 5:18–21, 31–53 ornamental aroids, 10:7–14 pine bark media, 9:103–131 raspberry, 11:194–195 slow-release fertilizers, 1:79–139 O Oil palm: asexual embryogenesis, 7:187–188 in vitro culture, 7:187–188 Okra: botany & horticulture, 21:41–72 CA storage, 1:372–373 Oleaster, wild of Kazakhstan, 29: 351–353 Olive: alternate bearing, 4:140–141 physiology, 31:147–231 pollination, 34:265–266 processing technology, 25:235–260 salinity tolerance, 21:177–214 Onion: CA storage, 1:373–375 ﬂuid drilling of seed, 3:17–18 Opium poppy, 19:373–408 Orange, see also Citrus alternate bearing, 4:143–144 sour, rootstock, 1:242–244 sweet, rootstock, 1:252–253 trifoliate, rootstock, 1:247–250 Orchard & orchard systems: ﬂoor management, 9:377–430 light, 2:208–267 root growth, 2:469–470 water, 7:301–344

CUMULATIVE SUBJECT INDEX Orchid: fertilization, 5:357–358 physiology, 5:279–315 pollination regulation of ﬂower development, 19:28–38 Organogenesis, 3:214–314, see also In vitro; tissue culture Ornamental plants, see also individual plant Amaryllidaceae Banksia, 22:1–25 cactus grafting, 28–106–109 chlorosis, 9:168–169 cotoneaster, wild of Kazakhstan, 29:316–317 fertilization, 1:98–104, 106–116 ﬂowering bulb roots, 14:57–88 ﬂowering bulbs in vitro, 18:87–169 foliage acclimatization, 6:119–154 foliage industry, 31:47–112 geophytes, in vitro, 34:417–445 heliconia, 14:1–55 honeysuckle, wild of Kazakhstan, 29:350 Leucadendron, 32:167–228 Leucospermum, 22:27–90 oleaster, wild of Kazakhstan, 29:351–353 orchid pollination regulation, 19:28–38 poppy, 19:373–408 protea leaf blackening, 17:173–201 rhododendron, 12:1–42 rose, wild of Kazakhstan, 29:353–360 Salix, 34:447–489 viburnam, wild of Kazakhstan, 29:361–362 Osier, see Salix P Paclobutrazol, see Triazole Papaya: asexual embryogenesis, 7:176–177 CA & MA, 22:157–160 CA storage, 1:314 in vitro culture, 7:175–178 Parasitic weeds, 33:267–349 Parsley: CA storage, 1:375 drilling of seed, 3:13–14 Parsnip, ﬂuid drilling of seed, 3:13–14 Parthenocarpy, tomato, 6:65–84 Particle ﬁlms, 31:1–45

513 Passion fruit: in vitro culture, 7:180–181 CA & MA, 22:160–161 Pathogen elimination, in vitro, 5:257–261 Pawpaw, 31:351–384 Peach: bloom delay, 15:105–106 CA storage, 1:309–310 orchard systems, 32:63–109 origin, 17:333–379 postharvest physiology, 11:413–452 short life, 2:4 summer pruning, 9:351–375 thinning, 28:351–392 wooliness, 20:198–199 Peach palm (Pejibaye): in vitro culture, 7:187–188 Pear: bioregulation, 10:309–401 bloom delay, 15:106–107 CA storage, 1:306–308 decline, 2:11 ﬁre blight control, 1:423–474 fruit disorders, 11:357–411; 27:227–267 fruit volatiles, 28:237–324 in vitro, 9:321 maturity indices, 13:407–432 root distribution, 2:456 scald, 27:227–267 short life, 2:6 wild of Kazakhstan, 29:315–316 Pecan: alternate bearing, 4:139–140 fertilization, 1:106 ﬂowering, 8:217–255 in vitro culture, 9:314–315 Pejibaye, in vitro culture, 7:189 Pepper (Capsicum): CA storage, 1:375–376 fertilization, 1:119 ﬂuid drilling in seed, 3:20 grafting, 28:104–105 phytochemicals, 28:161–162 Pepper (Piper), 33:173–266 Persimmon: CA storage, 1:314 quality, 4:259 Pest control: aroids (edible), 12:168–169 aroids (ornamental), 10:18

514 Pest control (Continued ) cassava, 12:163–164 cowpea, 12:210–213 ecologically based, 24:172–201 ﬁg, 12:442–477 ﬁre blight, 1:423–474 ginseng, 9:227–229 greenhouse management, 13:1–66 hydroponics, 7:530–534 parasitic weeds, 33:267–349 particle ﬁlms, 31:1–45 sweet potato, 12:173–175 vertebrate, 6:253–285 yam (Dioscorea), 12:181–183 Petal senescence, 11:15–43 pH: container growing, 9:87–88 fertilization greenhouse crops, 5:332–333 pine bark media, 9:114–117 soil testing, 7:8–12, 19–23 Phase change, 7:109–155 Phenology: apple, 11:231–237 raspberry, 11:186–190 Philodendron, see Aroids, ornamental Phosphonates, Phytophthora control, 17:299–330 Phosphorus: container growing, 9:82–84 deﬁciency & toxicity symptoms in fruits & nuts, 2:146–147 nutrition, 5:320–321 pine bark media, 9:112–113 trickle irrigation, 4:30 Photoautotrophic micropropagation, 17:125–172 Photoperiod, 4:66–105, 116–117; 17:73–123 ﬂowering, 15:282–284, 310–312 Photosynthesis: cassava, 13:112–114 efﬁciency, 7:71–72; 10:378 fruit crops, 11:111–157 ginseng, 9:223–226 light, 2:237–238 Physiology, see also Postharvest physiology Allium development, 32:329–378 apple crop load, 31:233–292

CUMULATIVE SUBJECT INDEX bitter pit, 11:289–355 blueberry development, 13:339–405 cactus reproductive biology, 18:321–346 calcium, 10:107–152 carbohydrate metabolism, 7:69–108 cassava, 13:105–129 citrus cold hardiness, 7:201–238 citrus irrigation, 30:55–67 conditioning 13:131–181 cut ﬂower, 1:204–236; 3:59–143; 10:35–62 desiccation tolerance, 18:171–213 disease resistance, 18:247–289 dormancy, 7:239–300 embryogenesis, 1:21–23; 2:268–310 ﬂoral scents, 24:31–53 ﬂower development, 19:1–58 ﬂowering, 4:106–127 fruit ripening, 13:67–103 fruit softening, 10:107–152 ginseng, 9:211–213 girdling, 30: 1–26 glucosinolates, 19:99–215 grafting, 28:78–84 heliconia, 14:5–13 hormone reception, 26:49–84 juvenility, 7:109–155 lettuce seed germination, 24:229–275 light tolerance, 18:215–246 loquat, 23:242–252 lychee reproduction, 28:393–453 male sterility, 17:103–106 mango fruit drop, 31:113–155 mechanical stress, 17:1–42 1-methylcyclopropene, 35:253–313 nitrogen metabolism in grapevine, 14:407–452 nutritional quality & CA storage, 8: 118–120 olive, 31:157–231 olive salinity tolerance, 21:177–214 orchid, 5:279–315 particle ﬁlms, 31–1–45 petal senescence, 11:15–43 photoperiodism, 17:73–123 pollution injury, 8:12–16 polyamines, 14:333–356 potato tuberization, 14:89–188 pruning, 8:339–380 raspberry, 11:190–199

CUMULATIVE SUBJECT INDEX red bayberry, 30:96–99 regulation, 11:1–14 root pruning, 6:158–171 roots of ﬂowering bulbs, 14:57–88 rose, 9:3–53 salinity hormone action, 16:1–32 salinity tolerance, 16:33–69 seed, 2:117–141 seed priming, 16:109–141 strawberry ﬂowering, 28:28:325–349 subzero stress, 6:373–417 summer pruning, 9:351–375 sweet potato, 23:277–338 thin cell layer morphogenesis, 14:239–264 tomato fruit ripening, 13:67–103 tomato parthenocarpy, 6:71–74 triazoles, 10:63–105; 24:55–138 tulip, 5:45–125 vernalization, 17:73–123 volatiles, 17:43–72 water relations cut ﬂowers, 18:1–85 watercore, 6:189–251 waxes, 23:1–68 Phytochemicals, functional: fruits, 27:269–315 vegetables, 28:125–185 Phytohormones, see Growth substances Phytophthora control, 17:299–330 Phytotoxins, 2:53–56 Pigmentation: ﬂower, 1:216–219 rose, 9:64–65 Pinching, by chemicals, 7:453–461 Pine, wild of Kazakhstan, 29:368–369 Pine bark, potting media, 9:103–131 Pineapple: CA & MA, 22:161–162 CA storage, 1:314 genetic resources, 21:138–141 in vitro culture, 7:181–182 Piper, see black pepper Pistachio: alternate bearing, 4:137–139 culture, 3:376–393 in vitro culture, 9:315 pollination, 34:264 wild of Kazakhstan, 29:366–368 Plant: architecture, 32:1–63 classiﬁcation, 28:1–60

515 protection, short life, 2:79–84 systematics, 28:1–60 Plantain: CA & MA, 22:141–146 in vitro culture, 7:178–180 Plastic cover, sod production, 27: 317–351 Plug transplant technology, 35:397–436 Plum: CA storage, 1:309 origin, 23:179–231 wild of Kazakhstan, 29:330–332 Poinsettia, fertilization, 1:103–104; 5: 358–360 Pollen, desiccation tolerance, 18:195 Pollination: apple, 1:402–404 artiﬁcial, 34:239–276 avocado, 8:272–283 cactus, 18:331–335 embryogenesis, 1:21–22 ﬁg, 12:426–429 ﬂoral scents, 24:31–53 ﬂower regulation, 19:1–58 fruit crops, 12:223–264 fruit set, 4:153–154 ginseng, 9:201–202 grape, 13:331–332 heliconia, 14:13–15 honey bee, 9:237–272 kiwifruit, 6:32–35 lychee, 28:422–428 navel orange, 8:145–146 orchid, 5:300–302 petal senescence, 11:33–35 protection, 7:463–464 rhododendron, 12:1–67 Pollution, 8:1–42 Polyamines: chilling injury, 15:80 in horticulture, 14:333–356 mango fruit drop, 31:125–127 Polygalacturonase, 13:67–103 Pomegranate, 35:127–191 Poppy, opium, 19:373–408 Postharvest physiology: almond, 20:267–311 apple bitter pit, 11:289–355 apple maturity indices, 13:407–432 apple scald, 27:227–257

516 Postharvest physiology (Continued ) apple weight loss, 25:197–234 aroids, 8:84–86 asparagus, 12:69–155 bitter melon, 35:343–344 CA for tropical fruit, 22:123–183 CA for storage & quality, 8:101–127 carrot storage: 30:284–288 cassava storage, 30:288–295 chlorophyll ﬂuorescence, 23:69–107 coated fruits & vegetables, 26:161–238 cucumber, 35:325–330 cucurbits, 35:315–354 cut ﬂower, 1:204–236; 3:59–143; 10: 35–62 ﬁg, 34:146–164 foliage plants, 6:119–154 fresh-cut fruits & vegetables, 30:85–255 fruit, 1:301–336 fruit softening, 10:107–152 ginger storage, 30:297–299 Jerusalem artichoke storage, 30:271–276 heat treatment, 22:91–121 lettuce, 2:181–185 low-temperature sweetening, 17: 203–231, 30:317–355 luffa, 35:344–345 MA for tropical fruit, 22:123–183 melon, 35:330–337 navel orange, 8:166–172 nectarine, 11:413–452 nondestructive quality evaluation, 20:1–119 pathogens, 3:412–461 peach, 11:413–452 pear disorders, 11:357–411; 27:227–267 pear maturity indices, 13:407–432 pear scald, 27:227–257 petal senescence, 11:15–43 potato low temperature sweetening, 30:317–355 potato storage, 30:259–271 protea leaf blackening, 17:173–201 pumpkin & squash, 35:337–341 quality evaluation, 20:1–119 scald, 27:227–267 seed, 2:117–141 sweet potato storage, 30:276–284 taro storage, 30:295–297;

CUMULATIVE SUBJECT INDEX texture in fresh fruit, 20:121–244 tomato fruit ripening, 13:67–103 tomato posthavest losses, 33:351–391 vegetables, 1:337–394 watercore, 6:189–251; 11:385–387 watermelon, 35:319–325 wax gourd, 35:342 Potassium: container growing, 9:84 deﬁciency & toxicity symptoms in fruits & nuts, 2:147–148 foliar application, 6:331–332 nutrition, 5:321–322 pine bark media, 9:113–114 trickle irrigation, 4:29 Potato: CA storage, 1:376–378 classiﬁcation, 28:23–26 fertilization, 1:120–121 history & iconography, 34:85–89 low temperature sweetening, 17:203– 231; 30:317–353 phytochemicals, 28:160–161 postharvest physiology & storage, 259–271 tuberization, 14:89–198 Processing, table olives, 25:235–260 Propagation, see also In vitro apple, 10:324–326; 12:288–295 aroids, ornamental, 10:12–13 bioreactor technology, 24:1–30 cassava, 13:120–123 ﬂoricultural crops, 7:461–462 foliage plants, 31:47–112 ginseng, 9:206–209 macadamia, 35:92–95 orchid, 5:291–297 pear, 10:324–326 rose, 9:54–58 tropical fruit, palms 7:157–200 woody legumes in vitro, 14:265–332 Protea ﬂoricultural crop, 26:1–48 leaf blackening, 17:173–201 Proteaceous ﬂower crop: Banksia, 22:1–25 Leucospermum, 22:27–90 Leukcadendron, 32:167–228 Protea, 17:173–201; 26:1–48

CUMULATIVE SUBJECT INDEX Protected crops, carbon dioxide, 7:345–398 Protoplast culture, woody species, 10:173–201 Pruning: alternate bearing, 4:161 apple, 9:351–375 apple training, 1:414 chemical, 7:453–461 cold hardiness, 11:56 ﬁre blight, 1:441–442 grapevines, 16:235–254 light interception, 2:250–251 peach, 9:351–375 phase change, 7:143–144 physiology, 8:339–380 plant architecture, 32:1–63 root, 6:155–188 Prunus, see also almond; cherry; nectarine; peach; plum in vitro, 5:243–244; 9:322 root distribution, 2:456 Pseudomonas: phaseolicola, 3:32–33, 39, 44–45 solanacearum, 3:33 syringae, 3:33, 40; 7:210–212 Pumpkin, history, 25:71–170 Q Quality evaluation: fruits & vegetables, 20:1–119, 121–224 nondestructive, 20:1–119 texture in fresh fruit, 20:121–224 R Rabbit, 6:275–276 Radish, fertilization, 1:121 Rambutan. see Sapindaceous fruits CA & MA, 22:163 Raspberry: harvesting, 16:282–298 productivity, 11:185–228 wild of Kazakhstan, 29:343–345 Red bayberry, 30:83–113 Rejuvenation: rose, 9:59–60 woody plants, 7:109–155 Replant problem, deciduous fruit trees, 2:1–116

517 Respiration: asparagus postharvest, 12:72–77 fruit in CA storage, 1:315–316 kiwifruit, 6:47–48 vegetables in CA storage, 1:341–346 Rhizobium, 3:34, 41 Rhododendron, 12:1–67 Rice bean, genetics, 2:375–376 Root: apple, 12:269–272 cactus, 18:297–298 diseases, 5:29–31 environment, nutrient ﬁlm technique, 5:13–26 Ericaceae, 10:202–209 grape, 5:127–168 kiwifruit, 12:310–313 physiology of bulbs, 14:57–88 pruning, 6:155–188 raspberry, 11:190 rose, 9:57 tree crops, 2:424–490 Root & tuber crops: Amaryllidaceae, 25:1–79 aroids, 8:43–99; 12:166–170 carrot postharvest physiology, 30:284–288 cassava crop physiology, 13:105–129 cassava molecular biology, 26:85–159 cassava multiple cropping, 30:355–500 cassava postharvest physiology, 30: 288–295 cassava root crop, 12:158–166 horseradish, 35:221–265 low-temperature sweetening, 17: 203–231, 30:317–355 minor crops, 12:184–188 potato low temperature sweetening, 30:317–355 potato tuberization, 14:89–188 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 sweet potato postharvest physiology, 30:276–284 taro postharvest physiology, 30:295–297 yam (Dioscorea), 12:177–184 Rootstocks: alternate bearing, 4:148 apple, 1:405–407; 12:295–297 avocado, 17:381–429

518 Rootstocks (Continued ) citrus, 1:237–269 clonal history, 35:475–478 cold hardiness, 11:57–58 ﬁre blight, 1:432–435 light interception, 2:249–250 macadamia, 35:92–95 navel orange, 8:156–161 root systems, 2:471–474 stress, 4:253–254 tree short life, 2:70–75 Rosaceae, in vitro, 5:239–248 Rose: fertilization, 1:104; 5:361–363 growth substances, 9:3–53 in vitro, 5:244–248 wild of Kazakhstan, 29:353–360 S Salinity: air pollution, 8:25–26 citrus irrigation, 30:37–83 olive, 21:177–214 soils, 4:22–27 tolerance, 16:33–69 Salix, botany & horticulture, 34:447–489 Sapindaceous fruits, 16:143–196 Sapodilla, CA & MA, 22:164 Scadoxus, 25:25–28 Scald, apple & pear, 27:227–265 Scarlet eggplant, history & iconography, 34:25 Scopolia spp., history & iconography, 34:20–23 Scoring & fruit set, 1:416–417 Sea buckthorn, wild of Kazakhstan, 29:361 Secondary metabolites, woody legumes, 14:314–322 Seed: abortion, 1:293–294 apple anatomy & morphology, 10: 285–286 conditioning, 13:131–181 desiccation tolerance, 18:196–203 environmental inﬂuences on size & composition, 13:183–213 ﬂower induction, 4:190–195 ﬂuid drilling, 3:1–58 grape seedlessness, 11:159–184 kiwifruit, 6:48–50

CUMULATIVE SUBJECT INDEX lettuce, 2:166–174 lettuce germination, 24:229–275 priming, 16:109–141 rose propagation, 9:54–55 vegetable, 3:1–58 viability & storage, 2:117–141 Senescence: chlorophyll senescence, 23:88–93 cut ﬂower, 1:204–236; 3:59–143; 10: 35–62; 18:1–85 petal, 11:15–43 pollination-induced, 19:4–25 rose, 9:65–66 whole plant, 15:335–370 Sensory quality: CA storage, 8:101–127 Shoot-tip culture, 5:221–277, see also Micropropagation Short life problem, fruit crops, 2:1–116 Signal transduction, 26:49–84 Small fruit, CA storage, 1:308 Snapdragon fertilization, 5:363–364 Sod production, 27:317–351 Sodium, deﬁciency & toxicity symptoms in fruits & nuts, 2:153–154 Soil: grape root growth, 5:141–144 management & root growth, 2:465–469 orchard ﬂoor management, 9:377–430 plant relations, trickle irrigation, 4: 18–21 stress, 4:151–152 testing, 7:1–68; 9:88–90 zinc, 23:109–178 Soilless culture, 5:1–44 Solanaceae: history and iconography, 34:1–111. in vitro, 5:229–232 steroidal alkaloids, 25:171–196 Solanum dulcamara, history & iconography, 34:25 Solanum nigrum, history & iconography, 34:23–24 Somatic embryogenesis. See Asexual embryogenesis Sorghum, sweet, 21:73–104 Spathiphyllum, see Aroids, ornamental Squash, history, 25:71–170 Stem, apple morphology, 12:272–283 Sternbergia, 25:59

CUMULATIVE SUBJECT INDEX Steroidal alkaloids, solanaceous, 25: 171–196 Storage, see also Postharvest physiology, Controlled-atmosphere (CA) storage carrot postharvest physiology, 30: 284–288 cassava postharvest physiology, 30: 288–295 cut ﬂower, 3:96–100; 10:35–62 ginger postharvest physiology, 30: 297–299 Jerusalem artichoke postharvest physiology, 30:259–271 low temperature sweetening, 17: 203–231; 30:317–353 potato low temperature sweetening, 30–317–353 potato postharvest physiology, 30: 259–271 root & tuber crops, 30:253–316 rose plants, 9:58–59 seed, 2:117–141 sweetpotato postharvest physiology, 30:295–297 taro postharvest physiology, 30:295–297 Strawberry: fertilization, 1:106 ﬂowering, 28:325–349 fruit growth & ripening, 17:267–297 functional phytonutrients, 27: 303–304 harvesting, 16:348–365 in vitro, 5:239–241 wild of Kazakhstan, 29:347 Stress: beneﬁts of, 4:247–271 chlorophyll ﬂuorescence, 23:69–107 climatic, 4:150–151 ﬂooding, 13:257–313 irrigation scheduling, 32:11–165 mechanical, 17:1–42 olive, 31:2207–217 petal, 11:32–33 plant, 2:34–37 protectants (triazoles), 24:55–138 protection, 7:463–466 salinity tolerance in olive, 21:177–214 subzero temperature, 6:373–417 waxes, 23:1–68

519 Sugar, see also Carbohydrate allocation, 7:74–94 ﬂowering, 4:114 Sugar apple, CA & MA, 22:164 Sugar beet, ﬂuid drilling of seed, 3:18–19 Sulfur: deﬁciency & toxicity symptoms in fruits & nuts, 2:154 nutrition, 5:323–324 Sweet potato: culture, 12:170–176 fertilization, 1:121 physiology, 23:277–338 postharvest physiology & storage, 30:276–284 Sweet sop, CA & MA, 22:164 Symptoms, deﬁciency & toxicity symptoms in fruits & nuts, 2:145–154 Syngonium, see Aroids, ornamental Systematics, 28:1–60 T Taro, see Aroids, edible postharvest physiology & storage, 30:276–284 Taxonomy, 28:1–60 Taxus, 32:299–327 Tea, botany & horticulture, 22:267–295 Temperature: apple fruit set, 1:408–411 bloom delay, 15:119–128 CA storage of vegetables, 1:340–341 chilling injury, 15:67–74 cryopreservation, 6:357–372 cut ﬂower storage, 10:40–43 fertilization, greenhouse crops, 5: 331–332 ﬁre blight forecasting, 1:456–459 ﬂowering, 15:284–287, 312–313 interaction with photoperiod, 4:80–81 low temperature sweetening, 17: 203–231 navel orange, 8:142 nutrient ﬁlm technique, 5:21–24 photoperiod interaction, 17:73–123 photosynthesis, 11:121–124 plant growth, 2:36–37 seed storage, 2:132–133 subzero stress, 6:373–417 Texture in fresh fruit, 20:121–224

520 Thinning: apple, 1:270–300 peach & Prunus, 28:351–392 Tipburn, in lettuce, 4:49–65 Tissue culture, see also In vitro culture, 1:1–78; 2:268–310; 3:214–314; 4:106–127; 5:221–277; 6:357–372; 7:157–200; 8:75–78; 9:273–349; 10:153–181, 24:1–30 bulb organ formation, 34:417–444 cassava, 26:85–159 dwarﬁng, 3:347–348 geophyte organ formation, 34:417–444 nutrient analysis, 7:52–56; 9:90 Tobacco, history & iconography, 34:51–62. Tomato: CA storage, 1:380–386 chilling injury, 20:199–200 classiﬁcation, 28:21–23 fertilization, 1:121–123 ﬂuid drilling of seed, 3:19–20 fruit cracking, 30:163–184 fruit ripening, 13:67–103 galacturonase, 13:67–103 grafting, 28:98–103 greenhouse quality, 26:239 history & iconography, 34:75–85 parthenocarpy, 6:65–84 phytochemicals, 28:160 postharvest losses, 33:351–391 Toxicity symptoms in fruit & nut crops, 2:145–154 Transport, cut ﬂowers, 3:100–104 Tree decline, 2:1–116 Triazoles, 10:63–105; 24:55–138 chilling injury, 15:79–80 Trickle irrigation, 4:1–48 Trufﬂe cultivation, 16:71–107 Tuber, potato, 14:89–188 Tuber & root crops. See Root & tuber crops Tulip, see also Bulb fertilization, 5:364–366 in vitro, 18:144–145 physiology, 5:45–125 Tunnel (cloche), 7:356–357 Turfgrass, fertilization, 1:112–117 Turnip, fertilization, 1:123–124 Turnip Mosaic Virus, 14:199–238

CUMULATIVE SUBJECT INDEX U Urd bean, genetics, 2:364–373 Urea, foliar application, 6:332 V Vaccinium, 10:185–187, see also Blueberry; Cranberry; Lingonberry functional phytonutrients, 27:303 wild of Kazakhstan, 29:347–349 Vase solutions, 3:82–95; 10:46–51 Vegetable crops, see also Speciﬁc crop Allium development, 329–378 Allium phytochemicals, 28:156–159 aroids, 8:43–99; 12:166–170 asparagus postharvest, 12:69–155 cactus, 18:300–302 carrot postharvest physiology & storage, 30:284–288 cassava crop physiology, 13:105–129 cassava molecular biology, 26:85–159 cassava multiple cropping, 30:355–500 cassava postharvest physiology & storage, 30:288–295 cassava root crop, 12:158–166 CA storage, 1:337–394 CA storage & quality, 8:101–127 CA storage diseases, 3:412–461 caper bush, 27:125–188 chilling injury, 15:63–95 coating physiology, 26:161–238 crucifer phytochemicals, 28:150–156 cucumber grafting, 28:91–96 cucurbit postharvest, 35:315–354 ecologically based, 24:139–228 eggplant grafting, 28:103–104 eggplant phytochemicals, 28:162–163 fertilization, 1:117–124 ﬂuid drilling of seeds, 3:1–58 fresh cut, 30:185–255 ginger postharvest physiology & storage, 30:297–299 gourd history, 25:71–170 grafting, 28:61–124 greenhouses in China, 30:126–141 greenhouse management, 21:1–39 greenhouse pest management, 13:1–66 honey bee pollination, 9:251–254 horseradish, 35:221–265 hydroponics, 7:483–558

CUMULATIVE SUBJECT INDEX Jerusalem artichoke postharvest physiology &; storage, 30:271–276 lettuce seed germination, 24:229–275 low-temperature sweetening, 17:203–231 melon grafting, 28:96–98 minor root & tubers, 12:184–188 mushroom cultivation, 19:59–97 mushroom spawn, 6:85–118 N nutrition, 22:185–223 nondestructive postharvest quality evaluation, 20:1–119 okra, 21:41–72 pepper phytochemicals, 28:161–162 phytochemicals, 28:125–185 plug industry & technology, 35:387–436 potato low temperature sweetening, 30:317–353 potato phytochemicals, 28:160–161 potato postharvest physiology & storage, 30:271–276 potato tuberization, 14:89–188 pumpkin history, 25:71–170 root & tuber postharvest & storage, 30: 295–297 seed conditioning, 13:131–181 seed priming, 16:109–141 squash history, 25:71–170 steroidal alkaloids, Solanaceae, 25: 171–196 sweet potato, 12:170–176 sweet potato physiology, 23:277–338 tomato fruit ripening, 13:67–103 tomato (greenhouse) fruit cracking, 30:163–184 tomato (greenhouse) quality: 26: 239–319 tomato parthenocarpy, 6:65–84 tomato phytochemicals, 28:160 tropical production, 24:139–228 trufﬂe cultivation, 16:71–107 watermelon grafting, 28:86–91 yam (Dioscorea), 12:177–184 Vegetative tissue, desiccation tolerance, 18:176–195 Vernalization, 4:117; 15:284–287; 17:73–123 Vertebrate pests, 6:253–285 Viburnam, wild of Kazakhstan, 29:361–362

521 Vigna, see also cowpea genetics, 2:311–394 U.S. production, 12:197–222 Viroid, dwarﬁng for citrus, 24:277–317 Virus: beneﬁts in horticulture, 3:394–411 dwarﬁng for citrus, 24:277–317 elimination, 7:157–200; 9:318; 18: 113–123; 28:187–236 ﬁg, 12:474–475 tree short life, 2:50–51 turnip mosaic, 14:199–238 Volatiles, 17:43–72; 24:31–53; 28:237–324 Vole, 6:254–274 W Walnut: in vitro culture, 9:312 wild of Kazakhstan, 29:369–370 Water relations: cut ﬂower, 3:61–66; 18:1–85 citrus, 30:37–83 deciduous orchards, 21:105–131 desiccation tolerance, 18:171–213 fertilization, grape & grapevine, 27:189–225 kiwifruit, 12:332–339 light in orchards, 2:248–249 photosynthesis, 11:124–131 trickle irrigation, 4:1–48 Watercore, 6:189–251 apple, 6:189–251 pear, 11:385–387 Watermelon: fertilization, 1:124 grafting, 28:86–91 Wax apple, CA & MA, 22:164 Waxes, 23:1–68 Weed control, ginseng, 9:228–229 Weeds: invasive, 32:379–437 lettuce research, 2:198 parasitic, 267–349 virus, 3:403 Wild fruit & nuts of Kazakhstan, 29:305–371 almond, 29:262–265 apple, 29:63–303, 305–315 apricot, 29:325–326 barberry, 29:332–336

522 Wild fruit (Continued ) bilberry, 29:347–348 blackberry, 29:345 cherry, 29:326–330 cotoneaster, 29:316–317 cranberry, 29:349 currant, 29:341 elderberry, 29:349–350 gooseberry, 29:341–342 grape, 29:342–343 hazelnut, 29:365–366 lingonberry, 29:348–349 mountain ash, 29:322–324 mulberry, 29:350–351 oleaster, 29:351–353 pear, 29:315–316 pine, 29:368–369 pistachio, 29:366–368 plum, 29:330–332 raspberry, 29:343–345 rose, 29:353–360 sea buckthorn, 29:361 strawberry, 29:347 vacciniums, 29:347–349 viburnam, 29:361–362 walnut, 29:369–370 Willow, see Salix Withania spp., history & iconography, 34:19–20

CUMULATIVE SUBJECT INDEX Woodchuck, 6:276–277 Woody species, somatic embryogenesis, 10:153–181 X Xanthomonas phaseoli, 3:29–32, 41, 45–46 Xanthophyll cycle, 18:226–239 Xanthosoma, 8:45–46, 56–57, see also Aroids Y Yam (Dioscorea), 12:177–184 Yield: determinants, 7:70–74; 97–99 limiting factors, 15:413–452 Z Zantedeschi, see Aroids, ornamental Zephyranthes, 25:60–61 Zinc: deﬁciency & toxicity symptoms in fruits & nuts, 2:151 foliar application, 6:332, 336 nutrition, 5:326; 23:109–178 pine bark media, 9:124 Zizipus, see Jujube

Cumulative Contributor Index (Volumes 1–35) Abbott, J.A., 20:1 Adams III, W.W., 18:215 Afek, U., 30:253 Albrigo, L.G. 34:277 Aldwinckle, H.S., 1:423; 15:xiii, 29:1 Alonso, J.M., 34:197 Aly, R., 33:267 Amarante, C., 28:161 Anderson, I.C., 21:73 Anderson, J.L., 15:97 Anderson, P.C., 13:257 Andrews, P.K., 15:183 Ascough, G.D., 34:417 Ashworth, E.N., 13:215; 23:1 Asokan, M.P., 8:43 Atkinson, D., 2:424 Aung, L.H., 5:45 Babadoost, M. 35:221 Bailey, W.G., 9:187 Baird, L.A.M., 1:172 Banks, N.H., 19:217; 25:197; 26:161 Barden, J.A., 9:351 Barker, A.V., 2:411 Bartz, J.A., 30:185; 33:351 Bar-Ya’akov, I. 35:127 Bass, L.N., 2:117 Bassett, C. L., 26:49 Becker, J.S., 18:247 Beer, S.V., 1:423 Behboudian, M.H., 21:105; 27:189 Ben-Jaacov, J., 32:167 Bennett, A.B., 13:67 Benschop, M., 5:45 Ben-Ya’acov, A., 17:381 Benzioni, A., 17:233

Bevington, K.B., 24:277 Bewley, J.D., 18:171 Bieleski, R.L. 35:xiii Binder, B.M., 35:263 Binzel, M.L., 16:33 Blanpied, G.D., 7:xi Blenkinsop, R.W., 30:317 Bliss, F.A., 16:xiii; 28:xi Boardman, K. 27 xi Borochov, A., 11:15 Bounous, G.; 31:293 Bower, J.P., 10:229 Bradley, G.A., 14:xiii Brandenburg, W., 28:1 Brecht, J.K., 30:185 Brennan, R., 16:255 Broadbent, P., 24:277 Broschat, T.K., 14:1 Brown, S. 15:xiii Buban, T., 4:174 Bukovac, M.J., 11:1 Burke, M.J., 11:xiii Buwalda, J.G., 12:307 Byers, R.E., 6:253; 28:351 Caldas, L.S., 2:568 Campbell, L.E., 2:524 Cantliffe, D.J., 16:109; 17:43; 24:229; 28:325; 35:397 Carter, G., 20:121 Carter, J., 35:193 Carter, J.V., 3:144 Cathey, H.M., 2:524 Chambers, R.J., 13:1 Chandler, C.K. 28:325 Charles, J., 34:447

Horticultural Reviews, Volume 35 Edited by Jules Janick Copyright & 2009 John Wiley & Sons, Inc. 523

524 Charron, C.S., 17:43 Chen, J., 31:47 Chen, K., 30:83 Chen, Z., 25:171 Chin, C.K., 5:221 Clarke, N.D., 21:1 Coetzee, J. H., 26:1 Cohen, M., 3:394 Collier, G.F., 4:49 Collins, G., 25:235 Collins, W.L., 7:483 Colmagro, S., 25:235 Compton, M.E., 14:239 Connor, D.J., 31:157 Conover, C.A., 5:317; 6:119 Coombs, B., 32:xi Coppens d’Eeckenbrugge, G., 21:133 Corelli-Grappadelli, L., 32:63 Costa, G. 28:351 Costes, E., 32:1 Coyne, D.P., 3:28 Crane, J.C., 3:376 Criley, R.A., 14:1; 22:27; 24:x Crowley, W., 15:1 Cuevas, J., 34:239 Cutting, J.G., 10:229 Daie, J., 7:69 Dale, A., 11:185; 16:255 Darnell, R.L., 13:339, 28:325 Daunay, M.-C., 34:1 Davenport, T.L., 8:257; 12:349; 31:113 Davies, F.S., 8:129; 24:319 Davies, P.J., 15:335 Davis, T.D., 10:63; 24:55 Decker, H.F., 27:317 DeEll, J.R., 23:69 DeGrandi-Hoffman, G., 9:237 De Hertogh, A.A., 5:45; 14:57; 18:87; 25:1 Deikman, J., 16:1 DellaPenna, D., 13:67 DeLong, J.M., 32:299 Demers, D.-A., 30:163 Demmig-Adams, B., 18:215 Dennis, F.G., Jr., 1:395 Dickson, E.E., 29:1 Dorais, M., 26:239; 30:163 Doud, S.L., 2:1 Dudareva, N., 24:31 Duke, S.O., 15:371

CUMULATIVE CONTRIBUTOR INDEX Dunavent, M.G., 9:103 Duval, M.-F., 21:133 Du¨zyaman, E., 21:41 Dyer, W.E., 15:371 Dzhangaliev, A.D., 29:63, 305 Early, J.D., 13:339 Eastman, K., 28:125 Eizenberg, H., 33:267 Ejeta, G., 33:267 Elfving, D.C., 4:1; 11:229 El-Goorani, M.A., 3:412 Erwin, J.E., 34:417 Esan, E.B., 1:1 Evans, D.A., 3:214 Ewing, E.E., 14:89 Faust, M., 2:vii, 142; 4:174; 6:287; 14:333; 17:331; 19:263; 22:225; 23:179 Felkey, K., 30:185 Fenner, M., 13:183 Fenwick, G.R., 19:99 Fereres, E., 31:157 Ferguson, A.R., 6:1; 33:1 Ferguson, I.B., 11:289; 30:83; 31:233 Ferguson, J.J., 24:277 Ferguson, L., 12:409 Ferree, D.C., 6:155; 31:xi Ferreira, J.F.S., 19:319 Fery, R.L., 2:311; 12:157 Fischer, R.L., 13:67 Flaishman, M.A., 34:113 Fletcher, R.A., 24:53 Flick, C.E., 3:214 Flore, J.A., 11:111 Forshey, C.G., 11:229 Forsline, P.L., 29:ix; 1 Franks, R. G., 27:41 Fujiwara, K., 17:125 Gazit, S., 28:393 Geisler, D., 6:155 Geneve, R.L., 14:265 George, K.J., 33:173 George, W.L., Jr., 6:25 Gerrath, J.M., 13:315 Gilley, A., 24:55 Giovannetti, G., 16:71 Giovannoni, J.J., 13:67

CUMULATIVE CONTRIBUTOR INDEX Glenn, G.M., 10:107; 31:1 Gofﬁnet, M.C., 20:ix Goldschmidt, E.E., 4:128; 30:1; 35:437 Goldy, R.G., 14:357 Goren, R., 15:145; 30:1 Gosselin, A., 26:239 Goszczynska, D.M., 10:35 Grace, S.C., 18:215 Gradziel, T.M., 30:xiii; 34:197 Graves, C.J., 5:1 Gray, D., 3:1 Grierson, W., 4:247 Griesbach, R.J., 35:193 Griffen, G.J., 8:291 Grodzinski, B., 7:345 Gucci, R., 21:177 Guest, D.I., 17:299 Guiltinan, M.J., 16:1 Gulia, S.K., 35:193 Hackett, W.P., 7:109 Halevy, A.H., 1:204; 3:59 Hallett, I.C., 20:121 Hammerschmidt, R., 18:247 Hanson, E.J., 16:255 Hardner, C.M., 35:1 Harker, F.R., 20:121 Hatib, K., 35:127 Heaney, R.K., 19:99 Heath, R.R., 17:43 Helzer, N.L., 13:1 Hendrix, J.W., 3:172 Henny, R.J., 10:1; 31:47 Hergert, G.B., 16:255 Hershenhorn, J., 33:267 Hess, F.D., 15:371 Hetterscheid, W.L.A., 28:1 Heywood, V., 15:1 Hjalmarsson, I., 27:79–123 Hogue, E.J., 9:377 Hokanson, S.C. 29:1 Holland, D., 35:127 Holt, J.S., 15:371 Huang, Hongwen, 33:1 Huber, D.J., 5:169 Huberman, M., 30:1 Hunter, E.L., 21:73 Hurst, S., 34:447

525 Hutchinson, J.F., 9:273 Hutton, R.J., 24:277 Indira, P., 23:277 Ingle, M. 27:227 Isenberg, F.M.R., 1:337 Iwakiri, B.T., 3:376 Jackson, J.E., 2:208 Janick, J., 1:ix; 8:xi; 17:xiii; 19:319; 21:xi; 23:233; 34:1. 35:437 Jarvis, W.R., 21:1 Jenks, M.A., 23:1 Jensen, M.H., 7:483 Jeong, B.R., 17:125 Jewett, T.J., 21:1 Jiang, W., 30:115 Joel, D.M., 33:267 Joiner, J.N., 5:317 Jones, H.G., 7:301 Jones, J.B., Jr., 7:1 Jones, R.B., 17:173 Kagan-Zur, V., 16:71 Kalt, W. 27:269; 28:125 Kamenetsky, R., 32:329; 33:123 Kang, S.-M., 4:204 Karp, A., 34:447 Kato, T., 8:181 Kawa, L., 14:57 Kawada, K., 4:247 Kays, S.J.. 30:253 Kelly, J.F., 10:ix; 22:xi Kester, D.E., 25:xii Khan, A.A., 13:131 Kierman, J., 3:172 Kim, K.-W., 18:87 Kinet, J.-M., 15:279 King, G.A., 11:413 Kingston, C.M., 13: 407–432 Kirschbaum, D.S. 28:325 Kliewer, W.M., 14:407 Knight, R.J., 19:xiii Knox, R.B., 12:1 Kodad, O., 34:197 Kofranek, A.M., 8:xi Korcak, R.F., 9:133; 10:183 Kozai, T., 17:125 Krezdorn, A.H., 1:vii

526 Kushad, M.M., 28:125 Kuzovkina, Y.A., 34:447 Labrecque, M. 34:447 Laimer, M., 28:187 Lakso, A.N., 7:301; 11:111 Lamb, R.C., 15:xiii Lang, G.A., 13:339 Larsen, R.P., 9:xi Larson, R.A., 7:399 Laterrot, H., 34:1 Lauri, P.E. 32:1 Layne, D.R., 31:351 Leal, F., 21:133 Ledbetter, C.A., 11:159 Lee, J.-M., 28:61 Levy, Y., 30:37 Li, P.H., 6:373 Lill, R.E., 11:413 Lin, S., 23:233 Liu, M., 32:229 Liu, Z., 27:41 Lipton, W.J., 12:69 Littlejohn, G.M., 26:1 Litz, R.E., 7:157 Lockard, R.G., 3:315 Loescher, W.H., 6:198 Lorenz, O.A., 1:79 Lowe, A.J. 35:1 Lu, R., 20:1 Luby, J.J., 29:1 Lurie, S., 22:91–121 Lyrene, P., 21:xi Maguire, K.M., 25:197 Mahovic, M.J., 33:351 Malik, A.U., 31:113 Manivel, L., 22:267 Maraffa, S.B., 2:268 Marangoni, A.G., 17:203; 30:317 Marini, R.P., 9:351; 32:63 Marinoni, D.T., 31:293 Marlow, G.C., 6:189 Maronek, D.M., 3:172 Martin, G.G., 13:339 Masiunas, J., 28:125 Mayak, S., 1:204; 3:59 Maynard, D.N., 1:79; 35:315 McConchie, R., 17:173

CUMULATIVE CONTRIBUTOR INDEX McConnell, D.B., 31:47 McIvor, I., 34:447 McNicol, R.J., 16:255 Merkle, S.A., 14:265 Merwin, I.A., 34:365 Meyer, M.H., 33:393 Michailides, T.J., 12:409 Michelson, E., 17:381 Mika, A., 8:339 Miller, A.R., 25:171 Miller, S.S., 10:309 Mills, H.A., 2:411; 9:103 Mills, T.M., 21:105 Mitchell, C.A., 17:1 Mizrahi, Y., 18:291, 321 Mohankumar, C.R., 30:355 Molnar, J.M., 9:1 Monk, G.J., 9:1 Monselise, S.P., 4:128 Moore, G.A., 7:157 Mor, Y., 9:53 Morris, J.R., 16:255 Mu, D., 30:115 Mudge, K. 35:437 Mulwa, R.M.S., 35:221 Murashige, T., 1:1 Mureinik, I., 34:xi Murr, D.P., 23:69 Murray, S.H., 20:121 Myers, P.N., 17:1 Nadeau, J.A., 19:1 Nair, R.R., 33:173 Naor, A., 32:111 Nascimento, W.M., 24:229 Neal, J., 35:1 Neilsen, G.H., 9:377 Nelson, P.V., 26:xi Nerd, A., 18:291, 321 Niemiera, A.X., 9:75; 32:379 Nobel, P.S., 18:291 Norman, D.J., 31:47 Norton, M.A., 35:221 NyujtŒ, F., 22:225 Oda, M., 28:61 O’Donoghue, E.M., 11:413 Ogden, R.J., 9:103 O’Hair, S.K., 8:43; 12:157

CUMULATIVE CONTRIBUTOR INDEX Oliveira, C.M., 10:403 Oliver, M.J., 18:171 O’Neill, S.D., 19:1 Opara, L.U., 19:217; 24:373; 25:197 Ormrod, D.P., 8:1 Ortiz, R., 27:79 Padilla-Zakour, O.I., 34:365 Palser, B.F., 12:1 Papadopoulos, A.P., 21:1; 26:239; 30:163 Pararajasingham, S., 21:1 Parera, C.A., 16:109 Paris, H.S., 25:71 Parthasarathy, V.A., 33:173 Peace, C., 35:1 Pegg, K.G., 17:299 Pellett, H.M., 3:144 Perkins-Veazil, P., 17:267 Phillips, G., 32:379 Pichersky, E., 24:31 Pickering, A.H., 35:355 Piechulla, B., 24:31 Pisanu, P., 35:1 Ploetz, R.C., 13:257 Pokorny, F.A., 9:103 Pomper, K.W., 31:351 Poole, R.T., 5:317; 6:119 Poovaiah, B.W., 10:107 Portas, C.A.M., 19:99 Porter, M.A., 7:345 Possingham, J.V., 16:235 Prange, R.K., 23:69; 32:299; 35:263 Pratt, C., 10:273; 12:265 Predieri, S., 28:237 Preece, J.E., 14:265 Priestley, C.A., 10:403 Proctor, J.T.A., 9:187 Puonti-Kaerlas, J., 26:85 Puterka, G.J., 31:1 Qu, D., 30:115 Quamme, H., 18:xiii Rabinowitch, H.D., 32:329 Raese, J.T., 11:357 Ramming, D.W., 11:159 Ransom, J.K., 33:267 Rapparini, F., 28:237

527 Ravi, V., 23:277; 30:355 Reddy, A.S.N., 10:107 Redgwell, R.J., 20:121 Regnard, J.L., 32:1 Reid, M., 12:xiii; 17:123 Reuveni, M., 16:33 Rich, P.J., 33:267 Richards, D., 5:127 Rieger, M., 11:45 Rodov, V., 34:113 Romero, M.A., 34:447 Roper, T.R., 21:215 Rosa, E.A.S., 19:99 Roth-Bejerano, N., 16:71 Roubelakis-Angelakis, K.A., 14:407 Rouse, J.L., 12:1 Royse, D.J., 19:59 Rubiales, D., 33:267 Rudnicki, R.M., 10:35 Ryder, E.J., 2:164; 3:vii Sachs, R., 12:xiii Sakai, A., 6:357 Salisbury, F.B., 4:66; 15:233 Salova, T. H., 29:305 Saltveit, M.E., 23:x; 23:185 San Antonio, J.P., 6:85 Sankhla, N., 10:63; 24:5 Sargent, S.A., 35:315 Sasikumar, B., 33:173 Sauerborn, J., 33:267 Saure, M.C., 7:239 Schaffer, B., 13:257 Schenk, M.K., 22:185 Schneider, G.W., 3:315 Schneider, K.R., 30:185; 33:351 Schotsmans, W.C., 35:263 Schuster, M.L., 3:28 Scoﬁeld, A. 35:437 Scorza, R., 4:106 Scott, J.W., 6:25 Sedgley, M., 12:223; 22:1; 25:235 Seeley, S.S., 15:97 Serrano Marquez, C., 15:183 Sharp, W.R., 2:268; 3:214 Sharpe, R.H., 23:233 Shattuck, V.I., 14:199 Shear, C.B., 2:142 Sheehan, T.J., 5:279 Shehata, A., 35:221

528 Shipp, J.L., 21:1 Shirra, M., 20:267 Shorey, H.H., 12:409 Silber, A., 32:167 Simon, J.E., 19:319 Singh, B.P., 35:193 Singh, N.B., 34:447 Singh, S.H., 34:277 Singh, Z. 27:189; 31:113 Skirvin, R., 35:221 Sklensky, D.E., 15:335 Smart, L.B., 34:447 Smith, A.H., Jr., 28:351 Smith, G.S., 12:307 Smith, M.A.L., 28:125 Smock, R.M., 1:301 Socias i Company, R., 34:197 Sommer, N.F., 3:412 Sondahl, M.R., 2:268 Sopp, P.I., 13:1 Soule, J., 4:247 Sozzi, G. O., 27:125 Sparks, D., 8:217 Splittstoesser, W.E., 6:25; 13:105 Spooner, D.M., 28:1 Srinivasan, C., 7:157 Srivastava, A.K., 34:277 Stang, E.J., 16:255 Steffens, G.L., 10:63 Stern, R.A., 28:393 Stevens, M.A., 4:vii Stoffella, P.J., 33:xi Stover, E., 34:113 Stroshine, R.L., 20:1 Struik, P.C., 14:89 Studman, C.J., 19:217 Stutte, G.W., 13:339 Styer, D.J., 5:221 Sunderland, K.D., 13:1 Sung, Y., 24:229 Sura´nyi, D., 19:263; 22:225; 23:179 Swanson, B., 12:xiii Swietlik, D., 6:287; 23:109 Syvertsen, J.P., 7:301, 30:37 Talcott, S.T., 30:185 Tattini, M., 21:177 Teodorescu, T.L., 34:447 ˜ tO ˜ nyi, P., 19:373 TO Theron, K.I., 25:1

CUMULATIVE CONTRIBUTOR INDEX Tibbitts, T.W., 4:49 Timon, B., 17:331 Tindall, H.D., 16:143 Tisserat, B., 1:1 Titus, J.S., 4:204 Trigiano, R.N., 14:265 Trybush, S., 34:447 Tunya, G.O., 13:105 Turekhanova, P.M., 29:305 Uchanski, M. 35:221 Upchurch, B.L., 20:1 Valenzuela, H.R., 24:139 Valois, S., 34:365 van den Berg, W.L.A., 28:1 van Doorn, W.G., 17:173; 18:1 Van Iepersen. W., 30: 163 van Kooten, O., 23:69 van Nocker, S. 27:1 van Staden, J., 34:417 Veilleux, R.E., 14:239 Vizzotto, G., 28: 351 Volk, T.A., 34:447 Vorsa, N., 21:215 Wallace, A., 15:413 Wallace, D.H., 17:73 Wallace, G.A., 15:413 Walters, S.A., 35:221 Wang, C.Y., 15:63 Wang, L., 30:115 Wang, S.Y., 14:333 Wann, S.R., 10:153 Warrington, I.J., 35:355 Watkins, C.B., 11:289 Watson, G.W., 15:1 Webster, B.D., 1:172; 13:xi Weichmann, J., 8:101 Weih, M., 34:447 Wetzstein, H.Y., 8:217 Whiley, A.W., 17:299 Whitaker, T.W., 2:164 White, J.W., 1:141 Williams, E.G., 12:1 Williams, M.W., 1:270 Wismer, W.V., 17:203 Wittwer, S.H., 6:xi Woodson, W.R., 11:15

CUMULATIVE CONTRIBUTOR INDEX Wooley, D.J., 35:355 Wright, R.D., 9:75 Wu¨nsche, J.N., 31:23 Wutscher, H.K., 1:237 Xu, C., 30:83. Yada, R.Y., 17:203; 30:317 Yadava, U.L., 2:1 Yahia, E.M., 16:197; 22:123

529 Yan, W., 17:73 Yarborough, D.E., 16:255 Yelenosky, G., 7:201 Zanini, E., 16:71 Zhang, B., 30:83 Zieslin, N., 9:53 Zimmerman, R.H., 5:vii; 9:273 Ziv, M., 24:1 Zucconi, F., 11:1

Plate 2.1. Pomegranate cultivars diversity and fruit development. A. Vase-shape ﬂower; B. Bell-shape ﬂower; C–E. Different stages of fruit color development in three cultivars: C. ‘C13’, D. ‘P.G.116-17’, E. ‘P.G.127-28’, 1. May, 2. June, 3. August, 4. October (NadlerHassar et al. unpublished); F. Fruit diversity of pomegranate cultivars grown in Israel.

Plate 2.2. Pomegranate cultivars. A. ‘Rosh Hapered’; B. ‘P.G.127-28’; C. ‘P.G.116-17’; D. ‘P.G.118-19’ (‘Hershkovich’); E. ‘Wonderful’; F. ‘Shani-Yonay’; G. ‘Kamel’; H. ‘P.G.128-29’ (‘Akko’); I. ‘Emek’.