Plant cell culture protocols

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M E T H 0 D S I N M 0 L E C U L A R B I 0 L 0 G Y'M John M. Walker, SERIES EDITOR 132 131 130 129 128 127 I 26 125 124 123 122 121 120 119 I I8 117 116 115 114 113 112

Biomformatics Methods and Protocols, ed1ted by Stephen M1sene1 and Stephen A Kwwetz, 1999 Flavoprotein Protocols, ed1ted by S K Chapman and G A Re1d /999 Transmption Factor Protocols, edited by Martm J T!•mm< /999 NMDA Protocols, edt ted by Mm L1, /999 Molecular Methods In Developmental Biology· Xenopus Developmental Biology Protocols, Vol. II, edited by Rocky S Tuan, /999 Developmental Biology Protocols, Vol. I, edited by Rody S Tuan, /999 Protein Kmase Protocols, ed1ted by Ala
Stephen flo Paddock, 1999 Natural Killer Ceil Protocols· Cel/ulm and Moleculm Method<, ed1ted by Keuv S Campbell and Ma1co Colonna, /999 Eicosanoid Protocols, ed1ted by Elias A Lwno< /999 Chromatin Protocols, edited by Peter B Bedel. /999

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90 89 88 87 86

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Protem L1pidation Protocols, edited by M1chae/ H Gelb, /999

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Calcium Signaling Protocols, edited by Da11d Lambeu /999 DNA Repair Protocols. Eukml'otlc S1
Protocols m DNA Topology and Topoisomerases, Pmt fl Oshe10jf. /999 Protocols in DNA Topology and Topoisomerases, Pm t f DNA

and Enzvmes,

Osheroff, 1999

edited by Man•-Ann BJOIIIItl and Ne1/

Protein Phosphatase Protocols, ed1ted by John W Lud/mv, /997 PCR

in Bioanaiysis, ed1ted by Slephen Meilw,

1997

Flow Cytometry Protocols, ed1ted by Mmk J Jmn�ze
Interactions. Methods Case S!udJe< and PIOF01, /997

Iocol< edued by Keuh R

Retm01d Protocols, ed1ted by Clulltophel RedfeJn, /997 Protein Targetmg Protocols, ed1ted by Roge> A Clegg

f99 7

Combinatonal Peptide L1brarv Protocols, ed1ted by Shmuel Cab1ll1,' /997 RNA Isolation and Characterizahon Protocols, edued by Ralph Rap/e�, /997

Differential Display Methods and Protocols, edited by Peng L1ang and Arthw B Pmdee, /997 Transmembrane Signaling Protocols, edited by Dafna Bm­ Sagl, /997 Receptor S1gnal TransductiOn Protocols, edited by R A J Chalilss /997

82 Arab1dop
ed1ted by Jose M Mm tmez-Zapatel

and Julw Sa/mas, /998

81 Plant Virology Protocols, edited by Gan· D Fo11e1 /998 80 lmmunochemJcai Pr ot ocols, IFUJYIJ IIJJTIIJI edited by John Pound, 1998

Lipoprotein Protocols, edited byJose M 01dovas, /998

79 Polyamine Protocols, edited by DaVId M L Mmgan, /998 78 Antibactenal Peptide Protocols, edned by W!l/wm M

Dool!tlle and Kmen Reue, /999

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Free Radical and Antioxidant Protocols, ed1ted by Donald A1 mst10ng, /998

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Elizabeth A Shephard, /998

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Receptor Binding Techniques, ed1ted by Man• Keen, /999 Phospholipid Signaling Protocols, edited by ian Bud /998 Mycoplasma Protocols, ed1ted by Roge1 J M1le< and Robm A J Nlclwlas, 1998

P1ch1a

Protocols, edited by DaVId R H1ggms and James

C1egg. /998

I 02 BIOluminescence Methods and Protocols, ed1ted by Rober/

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93 92 91

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Immunocytochemical Methods and Protocols (2nd ed.),

Thomson, /997

Adhesion Proteins Protocols, edited by Efl10hetta De,ana, /999

Topology

Electron Microscopy Methods and Protocols, ed1ted by Nmse1 HaJibagheu, 1999

I 07 Cytochrome P450 Protocols, ed1ted by fan R Ph1filps and

106 I05 I04

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RNA-Protem Interaction Protocols, ed1ted by Susan R Havnes, /999

edited by Lmette C Javo11, /999

DNA Profiling Protocols, ed1ted byJames M

Molecular Embryology: Methods and P10toco/s, edited by Paul T Shmpe and lvm Mawn /999

Enzl•molog� and D1ug<, ed1ted by Man··Ann 8jOJn
and Zebwjlsh, edited by Mall Gud/e, /999

I09 Lipase and Phospholipase Protocols, ed1ted by Mark H

I08

96 95

lntegrm Protocols, edited by Anthonv Howlett, /999

II I Plant Cell Culture Protocols, edited by Robell Hall /999

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98 97

A LaRossa, f998

Myobacteria Protocols, ed1ted by Tanya Pansh and Ne�l G Sloket, f998

I 00 Nitric Oude Protocols, edited by M A Tllheradge, f997 99 Human Cytokines and Cytokine Receptors, edited by Reno Debet<, /999

74 73 72 71 70 69 68

Shafer, /997

Protem SyntheSIS: Methods and P10tocol<, edited by Robm Marlin, 1998 Glycoanalysis Protocols, edited by Elizabeth F Houn<el f998 Basic Ceil Culture Protocols, ed1ted byJeffrev W Pollmd andJolm M Walke�, 1997 R1bozyme Protocols, ed1ted by Plullp C Twne�, /997 Neuropeptlde Protocols, ed1ted by Cmvel/ H Wdhmm, /997

Cr

Bw11

fnme

and

Neurotransmitter Methods, ed1ted by R1chmd C Rmne, /997 PRINS and Gosden, /997

In Sttu

PCR Protocols, ed1ted by John R

Sequence Data Analysis Guidebook, ed1ted by Smwn R Swmde/1, f997 eDNA Library Protocols, ed1ted by Jan G Cou ell and Ca10lme A Austm, /997 Gene Isolation and Mappmg Protocols, edited by Jacque/me Boultwood, /997

M E T H

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Plant Cell Culture Protocols

Edited by

Robert D. Hall CPRO-DLO, Wageningen, The Netherlands

Humana Press

* Totowa, New Jersey

© 1999 Humana Press Inc 999 R1verv1ew Dnve, SUite 208 Totowa, New Jersey 07512 All nghts reserved No part of th1s book may be reproduced, stored m a retneval system, or transmitted m any form or by any means, electromc, mechamcal, photocopymg, m1crofilmmg, recordmg, or otherwise Without wntten perm1sswn from the Publisher Methods

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Cover 11lustratton F1gure I B from Chapter 17, "Protoplast Isolation, Culture, and Plant RegeneratiOn from by Paul Anthony, Wagner Otom, J Bnan Power, Kenneth C Lowe, and M1chael R Davey

Pass1f/ora,"

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L1brary of Congress Catalogmg tn Publtcat1on Data Ma1n entry under tttle Methods m molecular b1ology'" Plant cell culture protocols I edtted by Robert D Hall p em -- (Methods tn molecular b1ology'" , v Il l ) Includes b1bliographtc references and mdex ISBN 0-89603-549-2 (alk paper) I Plant cell culture--Laboratory manuals I Hall, Robert D (Robert Davtd), 1958QK725 P5535 1999 571 6'382-
2 Plant tissue culture--Laboratory manuals

II Senes Methods m molecular b1ology (Totowa, NJ) , Il l 98-48664 CIP

Contents Preface

. v

Contributors .......

•

XI

PART I. INTRODUCTION

1

An Introduction to Plant-Cell Culture: Pomters to Success

Robert D. Hall PART II

2

........................................................................................

1

CELL CuLTURE AND PLANT REGENERATION

Callus Initiation, Maintenance, and Shoot Induction in Rice

Nigel W. Blackha/1, Joan P. Jotham, Kasimalai Azhakanandam, J. Brian Power, Kenneth C. Lowe, Edward C. Cocking, and Michael R. Davey . . . .. 19 .......................... ........... ....

3

............

..........

Callus Initiation, Maintenance, and Shoot Induction in Potato Momtormg of Spontaneous Genetic Variabtfity In Vitro and In VIVo

Rosario F. Curry and Alan C. Cassells

...........................................

4 Somatic Embryogenesis m Barley Suspension Cultures Makoto Kihara, Hideyuki Funatsuki, Kazutoshi Ito, and Paul A. Lazzeri

........................................................................

5

31

43

Somatic Embryogenesis in P1cea Suspension Cultures

Ulrika Egertsdotter

.

.

....

51

................................. .............................

61

.................................. ............................. .......

.

SPECIALIZED TECHNIQUES

6

Direct, Cyclic Somatic Embryogenesis of Cassava for Mass Production Purposes

Krlt J. J. M. Raemakers, Evert Jacobsen, and Richard G. F. Visser

.

7 Immature Inflorescence Culture of Cereals. A Highly Respons1ve System for Regeneration and Transformation

Sonriza Rasco-Gaunt and Pilar Barcelo

...........

8

Cryopreservation of Rice Tissue Cultures

9

Noncryogenic, Long-Term Germplasm Storage

Erica E. Benson and Paul T. Lynch .. .

Ali Golmirzaie and Judith Toledo

vii

..........

..........

.

.

..

.............................

.

....... ....................

.

.......

............. ..............................

71

83

95

viii

Contents

PART Ill. PLANT PROPAGATION IN VITRO

10

Micropropagation of Strawberry via Axillary Shoot Proliferation

11

Meristem- Tip Culture for Propagation and Virus Elimination

Philippe Boxus

..................................................................................

103

Brian W. W. Grout ............................................................................. 115 SPECIALIZED TECHNIQUES

12

Clonal Propagation of Orchids

13

In Vitro Propagation of Succulent Plants

14

Micropropagation of Flower Bulbs: Lily and Narcissus

15

Clonal Propagation of Woody Species

16

Spore-Derived Axenic Cultures of Ferns as a Method of Propagation

Brent Tisserat and Daniel Jones .................... ................... .......... 127 .

.

.

Jill Gratton and Michael F. Fay ....................................................... 135

Mere/ M. Langens-Gerrits and Geert-Jan M. De Klerk . ...... .. .. ... 141 .

.

.

lndra S. Harry and Trevor A. Thorpe .............................................. 149 Matthew V. Ford and Michael F. Fay .............. . .. .......................... 159 . .

.

PART IV. APPLICATIONS FOR PLANT PROTOPLASTS

17

18

Protoplast Isolation, Culture, and Plant Regeneration from Pass1flora

Paul Anthony, Wagner Otoni, J. Brian Power, Kenneth C. Lowe, and Michael R. Davey ..................... ............. 169 Isolation, Culture, and Plant Regeneration of Suspension-Derived Protoplasts of Loltum

Marianne Foiling and Annette Olesen ............ .............. ..... .......... 183 .

.

19

Protoplast Fus1on for Symmetnc Somatic Hybnd Production in Brassicaceae

20

Production of Cybrids in Rapeseed (Brassica napus)

Jan Fahleson and Kristina Glimelius ............ .......................... ...... 195 .

Stephen Yarrow ..................................... .......................... . ................ 211

SPECIALIZED T ECHNIQUES

21

Microprotoplast-Mediated Chromosome Transfer (MMCT ) for the Direct Production of Monosomic Addition Lines

Kamisetti S. Ramulu, Paul Dijkhuis, Jan Blaas, Frans A. Krens, and Harrie A. Verhoeven ...... ................ ......... 227 .

22

.

Guard Cell Protoplasts: Isolation, Culture, and Regeneration of Plants

Graham Boorse and Gary Tallman . ......................... ...................... 243 .

Contents 23

ix

In Vitro Fertilization with Isolated Single Gametes

Erhard Kranz PART v.

24

.....................................................................................

259

PROTOCOLS FOR GENOMIC MANIPULATION

Protocols for Anther and Microspore Culture of Barley

Alwine Jiiihne-Gartner and Horst Lorz

...........................................

25

Microspore Embryogenesis and In Vitro Pollen Maturation m Tobacco

26

Embryo Rescue Following Wide Crosses

Alisher Touraev and Erwin Heberle-Bors Hari C. Sharma

269

......................................

281

..................................................................................

293

SPECIALIZED TECHNIQUES

27

Mutagenesis and the Selection of Resistant Mutants

Philip J. Dix

.......................................................................................

309

28 The Generation of Plastid Mutants in Vitro

Philip J. Dix

.......................................................................................

319

PART VI. PROTOCOLS FOR THE INTRODUCTION OF SPECIFIC GENES

29

Agrobacterium Med1ated Transformation of Petuma Leaf Disks

30

Transformation of Rice via PEG-Mediated DNA Uptake into Protoplasts

31

Transformation of Wheat via Particle Bombardment

32

Plant Transformation via Protoplast Electroporation

-

Ingrid M. van der Meer

........................... ..........................................

Karabi Datta and Swapan K. Datta lndra K. Vasil and Vim/a Vasil .

349

...............................................................................

359

....

..

.

.

....... .................... ........

.

.......

.

335

...

..

George W. Bates

.................................................

327

SPECIALIZED TECHNIQUES

33

Transformation of Ma1ze via Tissue Electroporatlon

Kathleen D'Ha/luln, Els Bonne, Martien Bossut, and Rosita Le Page .

......................................... ...........................

34

Jim M. Dunwe/1 PART VII

35

367

Transformation of Maize Using Silicon Carbide Whiskers

.

...................................................... ...........................

375

SusPENSION CuLTURE INITIATION AND THE AccuMULATION OF METABOLITES

Directing Anthraquinone Accumulation via Manipulation of Morinda Suspens1on Cultures

Marc J. M. Hagendoorn, Dlaan C. L. Jamar, and Linus H. W. van der Plas

......................................................

383

Contents

X

36

Alkaloid Accumulation in Catharanthus roseus Suspension Cultures

37

Betalains Their Accumulation and Release In Vitro

Alan H. Scragg

..................................................................................

393

Christopher S. Hunter and Nigel J. Kilby ...................................... 403 Appendix

411

Index .

415

. ..

Contributors • Department of Life Sczences, Universzty of Nottmgham, University Park, Nottzngham, UK KASIMALAI AZHAKANANDAM Department of Life Sczences, Unzverszty of Nottingham, Universzty Park, Nottingham, UK PILAR BARCELO • Biochemistry and Physwlogy Department, IACR­ Rothamsted, Harpenden, Hertfordshzre, UK GEORGE W. BATES • Department of Biological Sciences, Florida State Unzverszty, Tallahassee, FL ERICA E. BENSON • School of Molecular and Life Sczences, Unzversity of Abertay Dundee, Dundee, Scotland NIGEL W. BLACKHALL • Department of Life Sciences, Umversity of Nottmgham, University Park, Nottmgham, UK JAN BLAAS • DLO-Centre for Plant Breeding and Reproductwn Research, CPRO-DLO, Wagen ingen, The Netherlands ELs BoNNE • Plant Genetic Systems, Gent, Belgium GRAHAM BooRSE • Department of Bwlogy, Wzllamette Universzty, Salem, OR MARTIEN BossuT • Plant Genetic Systems, Gent, Belg1um PHILLIPE Boxus • Biotechnology Department, Agricultural Research Centre, Gembloux, Belgium ALAN C. CASSELLS • Department of Plant Sczence, Umverszty College Cork, Cork, Ireland RosARIO F. CURRY • Department of Plant Sczence, Umversity College Cork, Cork, Ireland EowARD C. CocKING • Department of Life Sciences, Universzty of Nottingham, Umversity Park, Nottingham, UK MICHAEL R. DAVEY • Department of Life Sczences, University of Nottingham, University Park, Nottmgham UK KARABI DATTA • Plant Breeding, Genetics and Bzochemzstry Dzvzswn, IRRI, Manila, The Philippines SWAPAN K. DATTA • Plant Breeding, Geneflcs and Biochemzstry Dzviston, IRRI, Mamla, The Philzppines

PAUL ANTHONY

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xi

xii

Contributors

• Plant Genetic Systems, Gent, Belgzum Department of Developmental Biology, DLO-Centre for Plant Breeding and Reproductwn Research, CPRO-DLO, Wageningen, The Netherlands PHILIP J. D1x • Department of Biology, Saint Patrzck's College, Maynooth, Co. Kildare, Ireland JIM M. DuNWELL • Department of Agricultural Botany, University of Readmg, Whitekmghts, Reading, UK ULRIKA EGERTSDOTTER • Norwegian Forest Research Institute, As, Norway JAN FAHLESON • Department of Physwlogzcal Botany, Uppsala Umverszty, Uppsala, Sweden MICHAEL F. FAY • Royal Botanzc Gardens, Kew, Richmond, Surrey, UK MARIANNE PoLLING • Department of Agricultural Sciences, Plant Breedzng and Bwtechnology, Royal Veterinary and Agricultural Umversity, Copenhagen, Denmark MATTHEW V. FoRD • Royal Botanzc Gardens, Kew, Rzchmond, Surrey, UK HIDEYUKI FUNATSUKI • Plant Genetzc Resources Laboratory, Hokkaido National Agrzcultural Experiment Statzon, Shznsez, Memuro, Kasai, Hokkaido, Japan KRISTINA G LI MELI US • Department of Plant Breeding, Swedish Universzty of Agrzcultural Sciences, Uppsala, Sweden Au GoLDMIRZAIE • Genetic Resources Department, International Potato Centre (CIP), Lzma, Peru JILL GRATTON • Royal Botanzc Gardens, Kew, Richmond, Surrey, UK BRIAN W W. GROUT • Consumers Association, London, UK MARC J. M. HAGENDOORN • Department of Plant Physiology, Wageningen Agrzcultural University, Wageningen, The Netherlands RoBERT D. HALL • DLO-Centre for Plant Breeding and ReproductiOn Research, CPRO-DLO, Wageningen, The Netherlands lNDRA S. HARRY • Department of Biological Sciences, Unzversity of Calgary, Calgary, Alberta, Canada ERWIN HEBERLE-BORS • Institute of Microbiology and Genetics, Vienna Bwcenter, Unzversity of Vienna, Vienna, Austria CHRISTOPHER S. HUNTER • Faculty of Applied Sciences, University of the West of England, Frenchay, Bristol, UK KAZUTOSHI ITo • Plant Bioengzneerzng Research Laboratorzes, Sapporo Brewerzes Ltd , Nitta-Machi, Nztta-Gun, Gunma, Japan

KATHLEEN D'HALLUIN

PAUL DIJKHUIS

•

xiii

Contributors

• Department of Plant Breeding, Wageningen Agncultural University, Wageningen, The Netherlands ALWINE JAHNE-GARTNER • Angewandte Molekularbiologie der Pflanzen, Institut fuer Allgemeine Botanik, Hamburg, Germany DIAAN C. L. JAMAR • Department of Plant Physiology, Wageningen Agricultural University, Wageningen, The Netherlands DANIEL JoNES Fermentatzon Biochemistry, USDA, ARS, NCA UR, Peona, IL JoAN P. JoTHAM • Department of Life Scwnces, Umversity of Nottmgham, Universlfy Park, Nottmgham, UK MAKOTO KIHARA • Plant Bioengineering Research Laboratories, Sapporo Brewenes Ltd., Nitta-Machi, Nitta�Gun, Gunma, Japan NIGEL J. KILBY • Faculty of Applied Sciences, Universzty of West of England, Bristol, Bristol, UK GEERT-JAN M. DE KLERK • Centre for Plant Tissue Culture Research, Lzsse, The Netherlands ERHARD KRANZ Center for Applied Plant Molecular Bwlogy, Institute for General Botany, Umverszty of Hamburg, Hamburg, Germany FRANS A. KRENS • Department of Cell Bzology, DLO-Centre for Plant Breeding and Reproduction Research, CPRO-DLO, Wagenzngen, The Netherlands MEREL M. LANGENs�GERRITS Centre for Plant Tissue Culture Research, Lisse, The Netherlands PAUL A. LAZZERI • Biochemistry and Physwlogy Department, JACR­ Rothamsted, Harpenden, Hertfordshire, UK RosiTA LEPAGE • Plant Genet1c Systems, Gent, Belgium HoRST LoRz • Angewandte Molekularbiologie der Pflanzen, lnstztut fuer Allgemeine Botanik, Hamburg, Germany KENNETH C. L owE • Department of Life Sciences, Umversity of Nottingham, Universlfy Park, Nottingham, UK PAUL T. LYNCH • Diviszon of Biological Sciences, Universlfy of Derby, Derby, UK INGRID M. VAN DERMEER • Department of Cell Biology, DLO-Centre for Plant Breeding and Reproduction Research, CPRO-DLO, Wageningen, The Netherlands ANNETTE OLESEN • Department of Agricultural Sc1ences, Plant Breeding and Biotechnology, Royal Veterinary and Agricultural University, Copenhagen, Denmark WAGNER 0TONI • Department of Life Sciences, University of Nottingham, Unzverszty Park, Nottzngham, UK

EvERT JACOBSEN

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Contributors

XIV

Department of Plant Physiology, Wagenzngen Agricultural Unzversity, Wagenmgen, The Netherlands J. BRIAN PowER Department of Lzfe Sczences, University of Nottmgham, Universzty Park, Nottmgharn, UK KRIT J. J. M. RAEMAKERS Department of Plant Breeding, Wagenmgen Agncultural Unzversity, Wageningen, The Netherlands KAMISETTI S. RAMULU Department of Developmental Bwlogy, DLO-Centre for Plant Breedmg and Reproductwn Research, CPRO-DLO, Wagemngen, The Netherlands SoNRIZA RAsco-GAUNT • Bwchemistry and Physwlogy Department, IACR­ Rothamsted, Harpenden, Hertfordshzre, UK ALAN H. ScRAGG Department of Envzronmental Health and Science, Umverslty of the West of England, Frenchay, Bnstol, UK HARI C. SHARMA Department of Agronomy, Purdue Unzverszty, West Lafayette, IN GARY TALLMAN Department of Bwlogy, Wlllamette Unzverszty, Salem, OR TREVOR A. THORPE • Department of Biologzcal Sczences, Unzverszty of Calgary, Calgary, Alberta, Canada BRENT TISSERAT Fermentation Bwchemzstry, USDA, ARS, NCAUR, Peona, IL JuDITH TOLEDO Internatwnal Potato Centre (CIP), Genetzc Resources Department, Lima, Peru ALISHER TouRAEV Vienna Bwcentre, Institute of Microbiology and Genetics, Vzenna Unzverszty, Vienna, Austrza lNDRA K. VASIL • Laboratory of Plant Cell and Molecular Bwlogy, Department of Hortzcultural Sczences, Umverstty of Florida, Gamesville, FL VIMLA VASIL Department of Hortzcultural Sczences, University of Florida, Gamesvzlle, FL HARRIE A. VERHOEVEN • Department of Cell Bwlogy, DLO-Centre for Plant Breedmg and Reproductwn Research, CPRO-DLO, Wagenzngen, The Netherlands RICHARD G. F. VISSER • Department of Plant Breeding, Wagenzngen Agricultural University, Wagenzngen, The Netherlands STEPHEN YARROW Bwtechnology Strategies and Coordznation Office, Agriculture and Agri-Food Canada, Nepean, Ontarzo, Canada LINUS H. W. VAN DER PLAS

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__ __ __ __ __ __ __ __ __ ___________

INTRODUCTION

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_________________________________

An Introduction to Plant-Cell Cu lture Pointers to Success

Robert D. Hall

1. Introduction

With the continued expansion of in vitro technologies, plant-cell culture has become the general title for a very broad subject. Although in the beginnmg it was possible to culture plant cells either as established organs, such as roots, or as disorganized masses, it is now possible to culture plant cells m a variety of ways: individually (as single cells in microculture systems); collectively (as calluses or suspensions, on Petri dishes, in Erlenmeyer flasks, or m large-scale fermenters); or as organized units, whether this is shoots, roots, ovules, flow­ ers, fruits, and so forth. In the case of Arabidopsis, it is even possible to culture complete plants for generations from seed germination to seed set without hav­ ing to revert to an in vivo phase. In its most general definition, plant-cell culture covers all aspects of the cultivation and maintenance of plant material in vitro. The cultures produced are being put to an ever-increasing variety of uses. Initially, cultures were used exclusively as experimental tools for fundamental studies on plant cell divi­ sion, growth, differentiation, physiology, and biochemistry (1). Such systems were seen as ways to reduce the degree of complexity associated with whole plants, provtding additional exogenous control over endogenous processes, to enable more reliable conclusions to be made through simpler experimental designs. However, more recently, this technology has been increasingly exploited in a more applied context, and successes in a number of areas have resulted both in a major expansion in the number of people making use of these techniques and also m an enhancement of the degree of sophisticatiOn asso­ ciated with in vitro technology. Techniques for micropropagatwn and the From Methods m Molecular Biology, Vol 111 Plant Cell Culture Protocols Edtted by R D Hall © Humane Press Inc , Totowa, NJ

1

2

Hall

production of disease-free plant stocks have been defined and refined to such an extent that they have become standard practice for a range of (usually veg­ etatively propagated) horticultural and ornamental crop plants, such as ger­ beras, hhes, strawbernes, ferns, and so on, thus creating what is now a multimillion dollar mdustry. Nevertheless, the dtsctpline wtthm this technology that will eventually have the greatest impact on both fundamental and apphed plant science is that of genetic modification of plant cells. Although this methodology i s effectively still only in its infancy, tt is now already posstble, using a range of different techmques, to modtfy genetically virtually every plant spectes that has been tested so far, albeit with wtdely dtvergent degrees of efficiency (2) Without doubt, this technology provides us with the most powerful single tool wtth which to study all aspects of plant-cell physiology, metabohsm, and develop­ ment by allowing the molecular dtssection of individual components of the (sub)cellular organization of plants. In addttlon, the apphcat10n of genetic modification techniques has already enabled us to produce crop plants with altered phenotypes, concernmg e.g., herbicide resistance, insect resistance, and yield parameters (2). Many additional apphcattons are at the expenmental/pre­ commercial stage. In stmple terms, plant-cell culture can be considered to mvolve three phases : first, the isolatiOn of the plant (ttssue) from tts usual envuonment; second, the use of aseptic techmques to obtain clean matenal free of the usual bactenal, fungal, vtral, and even algal contammants, and thtrd, the culture and mainte­ nance of this matenal m vttro m a strictly controlled phystcal and chemical envtronment. The components of this environment are then in the hands of the researcher, who gams a considerable degree of external control over the subse­ quent fate of the plant matenal concerned. An extra, fourth phase may also be constdered where recovery of whole plants for rootmg and transfer to soil is the ultimate goal. The success of thts technology ts to a great extent, dependent on abtdmg by a number of fundamental rules and following a number of bastc protocols. For those who have no experience at all with in vitro technology, tt is strongly recommended, pnor to initiating a first research proJect, that some basic knowl­ edge be gained by visiting a working lab, preferably one doing similar work to that which is planned. This wtll not only save time, but also will help to avoid many of the pitfalls that could arise. Researchers can then also make direct contact with an experienced scientist who may later act as mentor. To proceed, a stratghtforward, well-tested protocol can be used to become acquamted wtth the manipulations reqmred to achieve a particular goal. Then, having gotten thts protocol to work, the researcher can begin with the modificatiOns needed to achieve the ongmal goal. The aim of the rest of this chapter ts to act as a refer-

Introduction to Plant-Cell Culture

3

ence giving some basic guidelines concerning how to initiate a research pro­ gram based on in vitro technology for plant tissues. The remaining chapters in this book will then describe individual protocols for spectfic techniques in detail.

2. Materials 2. 1. Plant Material

Probably the worst thing that any researcher can do when embarking on a new in vitro technique is to use material that is suboptimal. This not only means using the wrong species/variety/genotype, but also using the right material, but which has been grown under substandard conditions. Thus, choosmg m vivo­ grown material from plants that are diseased or too old, or have not been main­ tained in an active growth phase during their entire life should be avoided. With suboptimal material, problems can be encountered in obtaimng sterile cultures, excessive variability in in vitro response can result, and at worst, a complete failure of the experiment may occur. For most applications, explants from very young plants will respond best. For thts reason, m vitro germinated seedlings are a frequently favored choice. Seed is often also much more readily sterilized than softer plant tissues. This, therefore, maximizes the likelihood of obtaining explants that are not only healthy, but are also guaranteed to be free of undestrable contaminants. However, species producmg small seed can give nse to problems in obtaining sufficient experimental material. Furthermore, seed from outbreeders can also be genetically heterogenous, entailmg an unde­ sired variation in in vitro response that otherwise might be avoided by using explants from a single, larger greenhouse-grown individual. For specific applications, precise growth conditions may be essential, par­ ticularly with regard to the period directly before the plants are to be used. Similarly, even when plants are healthy and at the desired stage for use, it is often the case that only a specific part of these plants will glVe the best explants, e.g., a particular internode, the youngest fully expanded leaf, flower buds within a certain size range, and so forth. A good search of the literature and paying close attention to the recommendations of experienced researchers are always to be strongly recommended.

2.2. Equipment A plant-cell culture labOiatory does not differ greatly from most other botan­ ical laboratories in terms of layout or equipment. However, the requirement for sterility dominates. Plant cell cultures require rich media, but are relatively slow-growing. This places them in great danger of being lost, within days, through the accidental introduction of contaminating microorganisms. Plant-

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cell cultures also quickly exhaust their nutrient source, and therefore, stenle transfer to fresh media is a weekly to monthly reqmrement. A cell-culture laboratory should be kept tidy, and dust-free wtth clean work­ mg surfaces. Some type of stenle culture transfer facility is essential. A lami­ nar flow cabmet is preferable but a UV -stenlized transfer room or glove box, both of which are used solely for thts purpose, and whtch are UV madiated at all times when not m use, can also be employed effectively. Such facilities, when used for plant material, should never be used by colleagues for work on other orgamsms, such as yeast or Escherichw coli. It should also be held as a general rule that everything going into the sterile working area should already be sterile or, in the case of instruments, should be sterilized immediately on entry. Thts means also that m vivo grown plant material should only enter the transfer area after it has been submerged m the stenlizing solut10n. The other equally important piece of eqmpment is the autoclave whtch is needed to sterilize glassware, media, and so forth. This should be of a stze suffictent to cope with daily reqmrements. However, very large autoclaves should be avotded unless they are specifically designed for rapid heatmg and cooling before and after the high-pressure period to avoid long delays and also to prevent medta being severely "cooked" as well as being autoclaved. Although spectalized techniques have specific equipment requirements (noted in the relevant chapters), in addttion to the stenle transfer and autoclav­ ing facilities, the following are generally needed to perform baste cell-culture procedures: l . Tissue-culture-grade chemicals With appropriate storage space at room tempera­ ture, 4°C and -20°C. 2. Weighing and media preparation fac1lities: Balances to measure accurately mg to kg quantities should be avatlable. 3 A range of stenhzation facilities In addttlon to the autoclave, a hot-air stenlizmg oven is useful. Sterile filters (0.22-!lm) are required for sterilizing heat-lab1le compounds. If large volumes of stenle liquids are required, a penstalt1c or vacuum pump IS also to be strongly recommended 4 A source of (double) distilled water. 5 Stirnng facilities that allow a number of different media to be made simulta­ neously 6. A reliable pH meter with solutions of HCI and KOH (0.01 , 0 l , 1 .0, and 1 0 M) to adJust the pH accurately 7. Culture vessels e1ther of (preferably borosilicate) glass or disposable plastic, tubes, Erlenmeyer flasks, Jars, and so on. 8 Plastic disposables, e.g. Petri dishes (9, 6, 3 em), filter umts, syringes, and so forth, as well as plastic bottles of various sizes for freezing medta and stock solutions for long-term storage

Introduction to Plant-Cell Culture

5

9. Sealants, e.g., aluminium foil, Parafilm/Nescofilm, clingfilm/Saranwrap. 1 0. Basic glassware (measuring cylinders, volumetric flasks), dissection mstruments, hot plate/stirrer, gas, water, and electricity supply, microscopes, and so forth. 1 1 . Microwave: Although not essential, the ability to make solidified med1a in "bulk" and remelt it for pouring when required not only saves time, but also avmds the risk of undesired condensation building up in culture vessels (especially Petri dishes) on prolonged storage.

2.3. Washing Facilities The importance of cleaning glassware in a tissue-culture laboratory should never be underestimated. Furthermore, incorrect rinsing is equally as bad as incorrect washing. Traces of detergent or old media can cause devastation the next time the glassware is used. If not to be washed immediately, all glassware should be rinsed directiy after use and should not be allowed to dry out. There­ fore, keep a small amount of water in each vessel until it is cleaned. Certain media components (e.g., phytohormones), which are only poorly soluble in water when dried onto the inside of a flask, may not be removed by the normal washing procedures, but can redissolve the next time the vessel is autoclaved and contaminate the medium. For this reason, flasks used to make or store concentrated stocks of medium components should not be used for any other purpose. New automatic washing machines can be programmed to wash at tempera­ tures approaching 1 00°C, rinse extensively with warm and then cold water, and finally demineralized water before even blow-drying! However, if such equipment is not available, washing by hand is equally as good, if a little time­ consuming. In this case, glassware should be soaked overnight in a strong deter­ gent before being thoroughly scrubbed with a suitable bottle brush and then rinsed two to three times under running tap water and finally at least once with demineralized water. All glassware should then be dried upside down before being stored in a dust-free cupboard until reqmred. It is generally recommended that glassware be thoroughly washed in an acid bath on a regular basis.

2.4. Media There is a small number of standard culture media that are widely used with or without additional organic and inorganic supplements (see Appendix; 3-7). However, next to these, there is an almost unending list of media that have been reported to be appropriate for specific purposes (8). Protoplast culture media, for example, can have a wide variation in composition, reflective of the often critical conditions required by these highly sensitive and fully exposed cells. However, even these are to a large extent derived from one of the stan­ dard recipes. Plant-culture media generally consist of several inorganic salts, a

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(small) number of orgamc supplements (e.g., vttamins, phytohormones), and a carbon source. In addition to these standard components, the spectfic needs of particular species or tissues, or the precise conditwns required to initiate a desired m vitro response dictate which additional supplements are required. Today, with the wealth of knowledge concemmg a very divergent list of plant species that has been built up over the last 20 years and that ts readtly avatlable m the literature, the choice of medtum with whtch to begin for a particular plant should be made only after referrmg to prevtous publications on the same or related species. It can be seen, from the standard media recipes listed in the Appendix, that the mtcro- and macroelements and organic supplements can vary considerably. The species to be used will generally determine which medium to choose and, of course, the atm of the expenment (e.g., callus production, plant regenera­ tion, somatic embryogenests, anther culture, and so on) wtll determine which additional supplements are reqmred. This is especially so for the phytohor­ mones, whtch can play an extremely important role m determmg the response of plant cells/tissues in vttro. Indeed, m many cases, tt ts only the number, concentration, type, and balance of the phytohormones used that dtstmgmshes one experimental design from another. Of the macrocomponents, the source of nitrogen (N) is often constdered to be of particular mfluence. Most medta have N in the form of both mtrate and ammoma, but the ratio of one to the other can vary enormously to the extremes that one of the two sources ts absent. Alterna­ tively, both sources can be omttted and replaced by orgamc N sources in the form of ammo actds, as m the case of AA medmm (9). Although many medta are composed as a fine balance to promote and mamtam cell growth m vitro, temporary divergence from usmg the usual medta components IS often employed to dtrect growth and morphogenesis m particular dtrectwns. For example, by limitmg or removing the N or phosphate source, secondary meta­ bolite production can be sttmulated, and through the quahtative and quantita­ tive mampulatwn of the sugar supplement, organogenesis or embryogenesis may be mduced. Briefly, the Importance of the different media components can be given as follows: Inorganics a Macronutnents. Ca, K, Mg, N, P, and S are included m anton and/or cation form and are generally present at mM concentrattons All are essential for sustained growth in vitro b. Micronutrients: B, Co, Cu, Fe, I, Mo, Mn and Zn are generally mcluded at � concentrations Nt and Al may also be included, but the mtmscule amounts reqmred are posstbly already present as contammants in, e.g., agar.

Introduction to Plant-Cell Culture

7

2. Organics a. Vitamins: Generally, thiamine (vitamin B1), pyridoxme (vitamm B 6), nico­ timc acid (vitamin B3 ) and myoinositol are included, but only thiamine is considered to be essential. The others have growth-enhancmg properttes. The concentratiOns of each can vary significantly between the different medta compositiOns (see Appendix). b. Ammo acids: Some cultured plant cells can synthesize all amino actds, none are considered essenttal. However, some media do contain certain amino acids for their growth-enhancing properties, e.g., glycine in MS media (3). How­ ever, high concentrations of certain amino acids can prove toxic. Crude ammo acid preparations (e.g., casamino acids; 10) can also be used (e.g., for proto­ plast culture), but their undefined nature makes them less popular. c. Carbon source: Generally, most plant-cell cultures are nonautotrophtc and are therefore entirely dependent on an external source of carbon. In most cases, this is sucrose, but occasionally glucose (e.g., for cotton cultures) or maltose (e.g , for anther culture) is preferred. d. Phytohormones: The most commonly used phytohormones for plant-cell cul­ ture are the auxins and cytokinins. However, for spectfic applications wtth certain species, abscisic acid or gibberellic acid may be also used. Auxins induce/stimulate cell division in explants and can also stimulate root forma­ tion. Both natural (indole-3 -acetic acid, IAA) and synthetic (e.g., indole-3butyric acid, IBA; !-naphthalene acetic acid, NAA; 2,4-dtchlorophenoxyacetic acid, 2,4-D; p-chlorophenoxyacetic acid, pCPA) forms are used. Although the synthetic forms are relatively stable, IAA is considered to be rapidly inactivated by certam environmental factors (e g., light). In addition, auxin-like compounds, such as Dicamba and Ptcloram, can be used to the same effect. Cytokimns play an influential role in cel l dtvision, regeneration, and phytomorphogenesis, and are believed to be involved in tRNA and pro­ tein synthesis. Although the natural form, Zeatin (or Zeatin riboside) ts avatl­ able commercially and ts widely used for certain applications, the synthetic cytokinins (benzyladenine, BA, or 6-benzylaminopurme, BAP; kinetin, K; and tsopentyl adenine, 2-iP) are more generally used. Other compounds, such as Thidiazuron and phenylurea derivatives, also have cytokmm acttvtty wtth the former, for example, gaining increasing populanty for woody spectes. Gibberellin (usually GA3) is occasionally used to stimulate shoot elongation in cultures that contam meristems or stunted plantlets. Absctstc acid (ABA) is sporadically used, but its mode of action is unclear. In some cases, it is used for tts inhibitory and, in some cases, for its stimulatory effect on cell-culture growth and development. Altering the qualitative and quantitative balance of the phytohormones included in a culture medium, and especially m relation to the auxin/cyto­ kinin balance t s one of the most powerful tools available to the researcher to direct in vitro response. In many cases, making the correct choice, nght from culture mitiation, is all-determining.

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e. Others· In the past, a wide range of relatively mdefinable supplements have been used for plant-cell culture rangmg from protem hydrolysates to yeast extracts, fruit (e.g., banana) extracts, potato extracts, and coconut milk. How­ ever, the use of such components, through their unknown compositiOn com­ bmed w1th our Improved knowledge of cellular reqmrements m vitro, together w1th the mcreasing ava!lab1hty of components, such as zeatin, 1s now greatly reduced. Coconut milk, however, IS still widely used for protoplast culture and is now commercially available. 3 Antibiotics: Both synthetic and naturally occurrmg antibiotics can be used for plant­ cell culture. These play an essential role, for example, m ehmmatmg Agrobactenum species after coculttvatwn in transformation expenments or m providing selection pressure for stably transformed cells However, for standard practices, the use of antibiOtics IS usually avoided, smce these can have unknown physwlog1cal effects on cell development. Low levels are nevertheless often used m the more nsky/ expensive large-scale operatiOns, e g., m fermenters and m micropropagatwn programs. 4 Gelhng agent· It Is becommg mcreasmgly evident that not only the concentra­ tion, but also the type of agent used to make sohd media mfluences the m VItro response of cultured plant tissue Both natural products extracted from seaweeds (e g , agar, agarose, and alginate) and their more recently emerged substitutes (e.g., Gelrite, Phytagel), obtamed from microbial fermentatiOn, can be used. Each has 1ts advantages and disadvantages, and the chmce is usually determined by the species and the apphcatwn Agars and agaroses generally produce gels that are stable for prolonged penods and are considered not to bmd media components excessively. Products w1th vanous degrees of punty are ava1lable, and low-gel­ ling temperature types can even enable the embeddmg of sensitive cells, such as protoplasts. On the other hand, Gelrite/phytagel produces a rigid gel at much lower concentrations than agar or agarose. They are also almost transparent, which makes 1t easier, e.g , to identify contamination at an early stage These gels do, however, tend to liquify m long-term cultures owmg to pH changes or the depletiOn of salts necessary for crosslinking. Higher concentratiOns of antibiOtics (e g., kanamycin) may also be reqmred in Phytagel/Gelrite solid1fied media m comparison to those solidified w1th agar/agarose

In most countries, the most commonly used media are now commercially available (e.g., from S1gma, Duchefa) at competitive pnces, saving a lot of t1me and effort. Furthermore, when the exploratory work is completed and a specific modified medium has been designed for use, some compames (e.g., Duchefa, Haarlem, The Netherlands) will even make this medmm to order. 2.5. Culture Facilities It

to be strongly recommended that plant-cell cultures be incubated under stnctly controlled and defined environmental conditiOns. Although certain cul­ tures (e.g., shoot cultures) will have a set of opt1mum conditions for growth, they may contmue to surv1ve and grow under other, suboptimal conditions. IS

Introduction to Plant-Cell Culture

9

Other cultures, however, e.g., protoplast or microspore cultures require very precise treatments. DeviatiOns from this, by 1-2° in temperature can mean com­ plete experimental failure. Facilities are therefore required that ailow good and reliable regulation oflight quality and intensity, photoperiod, temperature (accu­ racy to ±1 °C), air circulation, and in certain countries, humidity. The space available should also be sufficient to allow the execution of experiments under uniform conditions. The choice of facility is often difficult. Several small incubators give flexibility, but generally increase variability in culture condi­ tions and can also prove expensive. A large walk-in growth room in which can be placed not only shelves, but also rotary shakers, bioreactors, and so on, reduces flexibility, but is generally more economical. The extra equip­ ment then no longer needs expensive stand-alone, controlled environment units. However, the failure (through an electrical fault, power cut, and so forth) of such a large growth room could be disastrous, and therefore, safety features should always be included, so that technical personnel can immedi­ ately be warned, 24 h/d, when the environmental conditions seriously deviate from the chosen settings. In incubators without lighting, obtaining uniform conditions is realtively easy. However, when light is introduced into a culture room, variation almost inevitably arises. Not every culture vessel can be placed at an equal distance from the light source. Limited space also often necessitates piling Petri dishes two or three deep. Furthermore, even with the best air circulation, local tem­ perature differences at culture/shelf level can be significant. Although little can be done about this, it is certainly important to be aware of these mequali­ ties. Consequently, it is recommended to carry out related experiments in the same place in the culture room if at all possible. The most uniform provision of light in a culture room is through fluorescent tubes placed above the shelves. However, since space usually has to be used efficiently, shelves are usually stacked above each other. This often results in significant localized mcreases in temperature on the upper shelves. This is not only undesirable, but also can result in the frequently occurring problem of Petri dish condensatiOn. This can be so extreme that the explants end up sitting m a pool of liquid, which can prove highly detrimental to culture development/survival. Insulating matenals placed above the hghts or channeled air flows along shelves can help to some extent, but the latter may increase the risk of contamination. The problem is immediately solved if the hghts are placed vertically on the walls behind the shelving, but this entails the disadvantage of a significant variation in light intensity across each shelf. The importance of these different factors to the plant material to be used and the nature of the work to be done determines which type of facility should be chosen and how it should be organized.

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3. Methods

3. 1. Sterilization of Equipment 1

Transfer facilities: On mstallahon, transfer areas (lammar flow cabmets, inocula­ tion rooms, glove boxes) should be thoroughly decontaminated usmg a SUitable d1smfectant and, then, 1fthe matenal allows, 70% ethanol (Note: any object made of perspex should never be exposed, however bnef, to alcohol, smce It will become bnttle and crazed). New flow cabmets should be left runmng overmght to clean the fi lters thoroughly before bemg brought mto circulatiOn Once m use, it should become standard practice for every user to spray down the transfer area with 70% alcohol both before and after use Furthermore, for transfer rooms and glove boxes, which are sterilized by UV light, an exposure of at least 1 5 mm between each user IS required to ensure complete decontammatwn. 2 Glassware Before stenlizmg open glassware (e g., beakers, Erlenmeyer flasks, and so on), these should be capped with a double layer of alummum fOil to ensure that sterility IS mamtamed after treatment Glassware with screw caps should always have these loosened half a turn before treatment to prevent high pressures bmldmg up, which can lead to the vessel explodmg. Glassware can routmely be autoclaved at 1 2 1 °C at a pressure of 1 5 ps1 for 1 5 mm Alternatively, dry heat can be used at l 60°C for 3 h The latter should, however, be avoided when plastic caps are used (e g., for closmg culture tubes), smce these cannot withstand the prolonged high temperatures Dry heat stenhzat10n IS also to be recommended for glassware destmed for use with protoplast media The osmolality of these media IS often very cntical, and even small amounts of condensatiOn, which can result from autoclavmg, can prove detnmental 3 Instruments: We routmely flame the lower parts of mstruments (e g., scalpels, forceps, and so on) m the lammar flow cabmet directly before use These are then always allowed to cool before bnngmg mto contact with plant tissue. Between mampulatwns, the mstruments are stored with their workmg surfaces submerged m 70% ethanol m a glass vessel (e g., a 1 00-mL measunng cylinder or beaker) kept m the transfer area for this purpose. The alcohol Is replaced at least once a day. Instruments and other metal objects can also be stenhzed usmg dry heat after first wrapping them m alummum fotl or heavy brown paper. Autoclaving I S to b e avOided, smce the combmatwn o f elevated temperatures and steam qmckly leads to corrosiOn 4 Heat-labile components. Certam plashes (e g., PVC, polystyrene) and other matenals may not tolerate the high temperatures generally reqmred for stenl­ tzatwn If It IS not known what matenal a component Is made of or If It IS unclear whether a known matenal is autoclavable, tt t s always unwise to gamble Check wtth a smgle ttem first tf posstble Otherwtse, use the alterna­ tive of a chemical method (e g., ImmersiOn for several mmutes m 70% ethanol or m one of the soluhons ltsted below for plant matenal) or UV rad1atton How­ ever, the latter ts only smtable if the UV rays can penetrate to all surfaces of the object concerned

Introduction to Plant-Cell Culture

11

3.2. Sterilization of Complete Media and Media Components 1 . Autoclavmg: The easiest and most widely used method to sterilize culture media is to autoclave for 1 5-20 min at 1 2 1 oc and a pressure of 1 5 psi. However, this is only possible if all the components in the medium are heat-stable. Longer times are to be avoided to prevent the risk of chemical modificatwn/decompositwn. For certain components, e.g., when glucose is used instead of sucrose, a lower temperature ( 1 1 0°C) is often recommended to avoid caramehzation of the carbon source. The autoclavmg time should be measured from the moment that the destred pressure is reached and not from the moment that the autoclave 1s switched on. To avoid excessively long periods before maximum pressure 1s reached, It is advisable never to overload the autoclave nor to autoclave large volumes m single flasks. Dividing the medium over a number of smaller flasks (preferably 500-mL flasks and only if absolutely necessary, 1 000-mL flasks) mcreases the surface area/volume ratio and, therefore, allows the medium to heat through quicker. This reduces the time needed to reach the destred steam pres­ sure. For this reason also, larger volumes need longer autoclaving times (e.g., 30 mm are recommended for volumes of 1 000 mL and 40 mm for 2000-mL vol). After autoclaving, the pressure should be allowed to fall relatively slowly to avo1d the media from boiling over in the flasks. In this regard, flasks should never be filled to more than 90% of their total volume 2. Filter sterilization: Med1a contaming heat-labile components should e1ther be fil­ ter-sterilized in their entirety, or the heat-labile components should be dissolved separately and added after autoclaving the other components. In the latter case, care must be taken to ensure that: The pH of the solution to be filter-sterilized is the same as that of the desired final pH of the medium. All components are fully dissolved before filtering. The temperature of the autoclaved fraction is as low as possible before adding the fi lter-sterilized components, i.e., room temperature for liqu1d media, 50°C for agar-based and 40°C for agarose-based media. If one or more of the components is poorly soluble, thus requiring a sig­ nificant volume to fully dissolve, the volume of the autoclaved components should be reduced accordingly in order to end up w1th the des1red final vol­ ume and concentration of all components. For example, it is standard practice when requiring solidified verswns of heat-labile media to make a double­ concentrated medtum stock for mtxing wtth an equal volume of double-con­ centrated agar/agarose stock in water, after the latter has been autoclaved and allowed to cool to the requtred temperature. Solutions for protoplast isolation and culture media should routinely be filter-sterilized. Autoclaving can result m a reduced pH, an altered osmolal­ ity, and undesirable chemical modifications, all ofwhich can prove detnmen­ tal to these very sensitive cells For filtration, various filters are now commercially available for filtering different types and volumes of media.

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The usual pore s1ze I S 0.22 Jlm, wh1ch IS appropnate for excluding microbial contaminants wh1le allowing the medtum to flow through relatively eas1ly Care should be taken to chose the right membrane type to use as certain solu­ tions, e.g , those containing proteins, DNA, or alcohol have special requtre­ ments. To this end, the manufacturer's recommendations should always be followed Disposable filter umts available from a wide range of sources are the ideal chmce, but a cheaper alternative 1s to buy autoclavable filter umts, which can be used With a w1de range of membrane mserts. The solutwn to be sterilized IS placed in a synnge that I S connected to the stenle filter unit in the sterile transfer area. The solution IS then forced through by hand and collected m an appropriate prestenhzed vessel Smce the pressures needed can some­ times be quite high, a syringe/filter combmation w1th a Luer-Lok system or a spring c lamp is to be recommended. For large volumes (>500 mL) peristaltiC or vacuum pump-based systems w11l save a lot of energy and frustratiOn

3.3. Sterilization of Plant Material

2.

Choice of explant· The first critical step m mit1atmg a new cell culture IS the obtamment of plant ttssue free of all contammatmg m1croorgamsms. For some species and explant types, this IS exteremly easy, whereas for others, 1t can be a desperately frustrating expenence. The ease w1th wh1ch plant matenal can be stenhzed 1s directly related to the way the plants prov1ding the explants have been grown Diseased plants or plants that have been attacked by bitmg or suck­ ing insects are likely to be contaminated both externally and internally, and 1t may prove imposs1ble to obtam microbe-free material without the continued/ prolonged use of antibiotics Seed IS a favored chmce, but even here, weather cond1t1ons before and dunng seed harvest can also prove to be greatly mfluential to the subsequent success of stenhzatwn. Choice of treatment: For material that is endogenously "clean," the usual proce­ dure 1s to apply an exogenous chemical treatment for a spec1fied penod. Differ­ ent sterilants are available for use at a range of different concentratiOns, and the choice of protocol 1s usually determined by the nature of the explant and the extent of external contaminatiOn A wettmg agent IS usually included (e.g , a drop of household detergent or Tween) to improve contact w1th the sterilant, smce the ease of wetting can be cnt1cal Very ha1ry or heavily waxed tissues can prove difficult to sterilize successfully. Ideally, a treatment should be chosen that is as mild as poss1ble while sttll guaranteemg decontammatton. For th1s reason, seed is often the matenal of choice, since these generally surv1ve stronger stenl­ Ization procedures better than softer tissues, such as leaves or stems A standard procedure is, for example, treatment with 70% ethanol for 3 0--60 s followed by a 1 5-min ImmersiOn m sodium hypochlorite ( l % available chlo­ rine). This IS then followed by three washes m stenle water to remove the ste­ rilant, which can prove to be toxic to the explants if it is carried over into culture. Vanations to th1s involve the use of different times of exposure to the sterilant, different concentratiOns of hypochlorite, or the use of one of the alter-

13

Introduction to Plant-Cell Culture

Table 1 A Summary of the Most Commonly Used Sterllants for Decontaminating Plant Material Sterilant

Typical concentrations

Ethanol

70%

Sodium hypochloritea Calctum hypochlorite Hydrogen peroxide Mercuric chloride

0.5-5% free chlorine 1-1 0% saturated soiution 1 .0-- 1 0% 0. 1-1 .0%

Silver nitrate Bromine water Combinations

1% 0.5-2%

Comments 70% has better wetting properties than 96% Most widely used Frequently used, prepare fresh Prepare fresh Very toxic, requires special handling and waste disposal Reqmres special waste disposal Shorter exposure ttmes (< 1 0 min) recommended Ethanol ts usually used as a short pretreatment in combi­ natiOn with one of the others; hydrogen peroxtde or mercunc chloride is sometimes used sequentially with sodmm hypo­ chlorite or calcium hypochlorite for particularly difficult material

0Household bleach (e.g., Domestos, Clorox) IS often used Chose one that does not mclude a thickening agent, and observe the stock concentration closely, because this varies among batches/ brands/countries.

native sterilants. The most widely used compounds and concentrations are listed in Table I . When working with material for the first time, the best approach is to choose seed or young, healthy plants and perform a small sterilization experiment where the hypochlorite concentration is varied (e.g., 0. 5-5% available chlorine) along with the exposure time (e.g., 5-30 min). The mildest conditions that give 1 00% sterile explants should then be used for all subsequent work.

3.4. Preparation of Media Plant-culture media are made either from commercially available powders or self-made concentrated stock solutions. All should be prepared using puri­ fied water that is de-ionized and distilled or the equivalent thereof, so that all pyrogens, organics, salts, and microorganisms have been removed. Commer­ cial media only need to be dissolved in an appropriate volume of water, and after the additional supplements have been added the pH is adjusted to the required value usmg HCI or KOH. For solid media, a gelling agent is required,

14

Hall

and this can be added either before or after the pH IS adJusted. No clear guide­ line can be gtven concernmg which is better, but it is important to be consis­ tent. However, the presence of gelhng agents wtll slowly decrease the efficiency of the pH electrode, whtch then reqmres more frequent cleamng m HCI. If a particular medmm IS not commercially available or if the relative con­ centrations of individual medmm components are to be changed, the most eco­ nomical option is to make stock solutions for repeated use. Generally, separate stocks of the different groups of components are made with the macronutrients being prepared as a x 1 0 or x20 stock, the mtcronutnents and orgamcs as a 1 OOx stock, and the phytohormones as x 1 000 stocks. These stocks are stored separately in the freezer m smtable ahquots. As a general rule, mdividual stock components should first be fully dissolved separately before addmg to the main solutiOn. Thts reduces the hkehhood of precipttatwn. It 1s also best to dtssolve the morganic N components first. Ifprecipatwn does occur, check that the pH t s close to 5.5, and try changmg the order in whtch the components are added. Owing to their very low concentratiOns, mtcronutnent stocks are usually pre­ pared from indlVldual stock solutwns, which are diluted and mixed to give the desued concentrations. These individual stocks may be stored frozen for many months Without detnmental effects. However, as wtth all stored stock solu­ tions, when a precipitate appears, the solutwn must be discarded and a new one prepared. Phytohormones are, m general, poorly soluble m water, and 1 000x stocks should be made by first dissolving m 1-5 mL of an appropnate solvent before making up to the final volume wtth distilled water and adjusting the pH to approx 5.5. HCl (0. 1 N), NaOH (0. 1 M), or ethanol can be used as solvent. Although most synthetic phytohormones can usually be autoclaved together wtth the rest of the medium, the naturally occurring forms (e.g., IAA, GA, ABA, Zeatin) are generally filter-sterilized. Prepared media stocks can usually be stored frozen for up to 6 mon without precipitation or reduced growth responses. Phytohormone stocks can similarly be stored frozen, but stocks for daily use can also be stored in the refngerator. However, these should be replaced monthly. Prepared medta m Petn dishes and jars should be used wtthm 2-3 wk of storage at room temperature. Storage at 4 °C is to be avoided, smce thts leads to excessive condensatiOn instde the vessels. 3.5. Preparation of Explants and Subculture Techniques Explants vary considerably in terms of size and shape in relation to the type of matenal and the aim of the experiment. Very small explants may not survive except on nch media or on medmm that has been "conditioned" (i.e., pre-

Introduction to Plant-Cell Culture

15

exposed i n some way to growing cultures). Initially grouping small explants can help. For culture initiation, new explants are prepared under aseptic condi­ tions from presterilized material. The outside edges are usually removed, smce these are likely to have been damaged by the stenlant. The tissue is cut mto pieces using a sharp (new) scalpel blade to give relatively uniform explants of an appropriate size (dimensions usually <1 em). The cut surfaces are important for nutrient uptake and for stimulating callus growth. Petn dishes or sterilized ceramic tiles make suitable cutting surfaces. On preparation, explants should be transferred to culture medium as quickly as possible to avoid desiccation. The orientation on the medium, in relation to orientation in the original plant, may be important (in relation to polar phytohormone transport, for example). In this regard, literature guidance should be sought, or otherwise all possibili­ ties should be tested in an imtial experiment to determine the best choice for a particular application. Once a culture has been established, regular transfer of all or part of the plant material to fresh medium is necessary. Before doing so, all cultures shouid be checked for sterility, and all contammated cultures dis­ carded. This is especially important for recently initiated cultures. A sharp scal­ pel blade should be used to remove that part that is to be transferred, smce a clean cut results in the least damage and gives the best growth response. The amount of matenal to be transferred is determined entirely by the growth rate of the culture concerned with the general rule being that the faster the growth, the less material needs to be transferred. Growth rate usually also determmes the subculture interval. Raptdly growing suspensions, for example, may need subculture every 4-7 d, whereas callus cultures may survive quite happily with transfers only every 1-2 mon. For established cultures, the risk of loss through contamination is of course greatest during subculture. Consequently, it is recommended always to keep a number of old cultures after each transfer, until 1t can be confirmed that the new cultures are sterile or, alternatively, transfers should be staggered so that not all cultures of a particular type are transferred at the same time. In this way, disastrous losses can be avoided. 3.6. Precautions and Hints to Success The following list represents a series of hints, precautions, and recommen­ dations that should help in avoiding some of the common pitfalls. 1 . Make culture medium several days before it is reqmred If the autoclave has been faulty, contammation will become evident before subculturing has taken place. 2. When preparing solid media, allow the temperature to fall to at least 50°C before pouring in order to prevent excessive condensation forming in the dishes and jars.

16 3

Hall When Initiating new cultures, always begm with healthy plant matenal

4. When prepanng culture media, stocks, and so forth, follow a strict routme regard­

5.

6.

7.

8

9

10

11

12.

13

14.

15

ing the way components are added, the order of addition, and so on, to avOid the risk of precipitation. When makmg media and stock solutions, work with checklists, and tick off each component as It Is added. This is particularly important when making stocks that are to be stored and used for a long time. A fault when making the stock can mean months of problems. To maintam maximum sterility m a culture lab, always autoclave contammated cultures before discarding Never try to "rescue" cultures mfected with a fungus that is already sporulatmg. The waste bin IS a frequent source of contammation m a lab. Clean It out, and sterilize It with ethanol regularly When autoclavmg media m bottles with screw caps always loosen these before­ hand, since this will avoid both the nsk of the bottles explodmg and also the creation of a vacuum after cooling, which can be so strong that It becomes Impos­ sible for the cap to be removed When remeltmg solidified media m a microwave, always loosen the cap and ensure that there IS sufficient air space to avoid the medium boiling out of the flask as It melts. Before using a new type of pen or marker, make sure that the ink I S not light­ sensitive (1f to be used for labeling cultures to be grown m the light for long penods) and that It does not disappear on autoclavmg When adding filter-sterilized ( 1 e., heat-labile) supplements to autoclaved medmm, always allow the latter to cool to approx l 0°C above gelling temperature, and then, after thorough mixing, pour the medium Immediately mto plates/jars to ensure that It cools as rapidly as possible When adding filter-sterilized supplements to autoclaved medmm, adjust the pH of the solutiOn to that of the medium before filtration This IS particularly impor­ tant with, for example, hormone stocks that are dissolved m HCl or KOH/NaOH solutions. If forced to use suboptimal culture conditions that result m considerable conden­ satiOn within culture vessels, allow the medium to solidify as a slope by placmg the dishes/j ars at a slight ( 1 0°) angle. ThiS allows the condensatiOn to collect at the lowest pomt, thus keepmg the explants h1gh and dry. When wishing to cut up tiny explants, e.g , meristems or embryos, even a stan­ dard scalpel blade IS often not fine enough. Use the sharpened point of a syringe needle, or alternatively, cut up p1eces of a double-edged razor blade w1th an old pair of scissors and fix them m an appropriate holder. Replace these regularly When usmg alcohol to store stenhzed mstruments between mampulatwns m a lammar flow cabmet, make sure that the vessel used has an even, flat top With no chips or pounng hps (as With beakers). Accidentally settmg the alcohol ahght (a not infrequent occurrence) can then Immediately be remedied by placmg any flat surface on top of the vessel, Immediately starvmg the flame of oxygen.

Introduction to Plant-Cell Culture

17

1 6. Since work in many tissue-culture labs is often now a combmation of cell biol­ ogy and molecular biology techniques, it is recommended to use separate glass­ ware for each type ofwork. This avoids the risk of suboptimal washmg practices resulting in the contamination of culture media with trace amounts of tox1c com­ pounds, such as SDS or ethidium bromide. 17. Even the richest labs often use cheap glassware (jam pots, honey pots, baby food jars) for mass culture purposes. These vessels are usually made of soda glass rather than the recommended borosilicate (Pyrex-type) glass. In such cases, 1t has been recommended (11) that these vessels be discarded after I yr of use or, alter­ natively, be coated with dichloro-silane for longer usage. 1 8 New glassware should always be thoroughly washed before using for the first time. Street (1) even recommends autoclaving new flasks filled w1th distilled water tw1ce before bnnging them mto CirculatiOn. 19. Since good suspensiOn cultures often take a long time to produce, keep a number of flasks on a second shaker, if space/expense allows, to avoid complete loss through motor failure. Backup cultures on solid medium are also to be recom­ mended. 20. When transferring cultures to a new medium to test, e.g , growth rate/regeneratiOn response, bear in mind that nutnent carryover can be significant. Th1s 1s espe­ cially true for synthetic hormones, such as 2,4-D. Consequently, when makmg assessments of a new medmm, subculture the tissues at least twice beforehand 2 1 . Medium pH s1gmficantly mfluences nutrient availability and culture response. Consequently, for certam applicatiOns, it should be borne m mind that the pH generally decreases 0.2-0.5 U on autoclaving. Furthermore, the stiffness of sohd media is also pH-dependent. If a low pH is desired, higher agar concentratiOns may be required to give the same gel strength and v1ce versa. References I . Street, H. E. (ed.) ( 1 974) Tzssue Culture and Plant Science. Academic, New York. 2. Christou, P. ( 1 995) Strategies for variety-independent genetic transformation of Important cereals, legumes and woody species utilizmg particle bombardment Euphytzca 85, 1 3-27 . 3. Murashige, T. and Skoog, F. ( 1 962) A revised medium for rapid growth and bw­ assays with tobacco tissue cultures. Physiol. Plant 1 5, 473-479 4. Linsmaier, E. M. and Skoog, F. ( 1 965) Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18, I 00-1 27. 5 . Gamborg, 0. L., M1ller, R. A., and Oj ima, K. ( 1 968) Nutnent requirements of suspension cultures of soybean root cells. Exp Cell. Res. 50, 1 5 1-1 57. 6. Schenk, R. U. and Hildebrandt, A. C. ( 1 972) Medium and techmques for induc­ tion and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Botany 50, 1 99-204. 7. Nitsch, J P. and Nitsch, C ( 1 969) Haploid plants from pollen. Science 1 63, 85-87 8. George, E. F., Puttock, D. J. M., and George, H. J (eds.) (1 987) Plant Culture Media I. Exeget1cs, Westbury, UK.

18

Hall

9. Tonyama, K , Hmata, K , and Sasaki, T. ( 1 986) Haploid and diplOid plant regen­ eration from protoplasts of anther callus m nee Theor. Appl Genet 73, 1 6-- 1 9 1 0 Kao, K. N and Michayluk, M R ( 1 975) Nutritional requirements for growth of Vzcw hajastana cells and protoplasts at a very low populatiOn density m liqmd media Planta 1 26, 1 05-1 1 0. 1 1 . de Fossard, R. A (ed.) ( 1 976) Tissue Culture for Plant Propagators Umverstty of New England Press, Armidale, Australia

2

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Callus Initiation, Maintenance, and Shoot Induction in Rice Nigel W. Blackhall, Joan P. Jotham, Kaslmalal Azhakanandam, J. Brian Power, Kenneth C. Lowe, Edward C. Cocking, and Michael A. Davey

1 . Introduction

Embryogenic suspension cultures provide the most wtdely employed source of totipotent cells for protoplast isolatiOn in rice (Oryza satlva L.), smce meso­ phyll-derived protoplasts of this tmportant cereal rarely undergo sustained mitotic division leading to the production of tissues capable of plant regenera­ tion. Cells from embryogenic suspensions provide an alternative to immature zygotic embryos for biolistic-mediated production of fertile transgenic rice plants (I) and are also amenable to transformation procedures employing agrobacteria (2). Currently, protocols are available for regenerating fertile plants from cell suspension-derived protoplasts of the three maJor subgroups of rice varieties, namely japonica (3), javanica (4) and indica (5) rices. Previous reports have stated that genotype and explant source are important parameters in determining the success of plant regeneration from cultured tis­ sues of rice (6-10). In japonica rices, callus cultures can be produced relatively easily from almost any part of the plant, including roots, shoots, leaves, leaf­ base meristems, mature and immature embryos, young inflorescences, pollen grains, ovaries, scutella, and endosperm. Such tissues can be induced to regen­ erate plants (11). Conversely, indica rices are more recalcitrant in culture (12, 13). In the procedures described in this chapter, scutella from mature seeds are used as the source of callus for both indica and japonica rices. The establishment and maintenance of embryogenic cell suspensions is gen­ erally difficult, with morphogenic competence of suspensions usually declin­ ing with successive subculture over prolonged periods (14) . However, From. Methods m Molecular Btology, Vol 1 1 1 Plant Ce/1 Culture Protocols Edited by R 0 Hall © Humana Press Inc , Totowa, NJ

19

20

B/ackha/1 et a/

followmg the development of reproducible protocols for cryopreservation of nee cell suspensiOns (15, 1 6), it has been poss1ble to dev1se strateg1es to over­ come difficulties associated w1th the loss of totipotency and the requirement to remitlate periodtcally new cultures capable of plant regeneratiOn. Samples of the cell suspensiOns should be cryopreserved as soon as posstble after mttla­ tton. The cultures should be resurrected from frozen stocks tmmed1ately tfthere are mdtcatwns of loss of embryogemc potential of the suspensiOns. Loss of totipotency can occur at any hme m the development and maintenance of cell suspensions. However, it anses most frequently 9-1 2 mon after mitlattan of the suspensiOn cultures. Frequently, 1t has been noted that the m1t1attan and growth of embryogemc callus requtre med1a contammg auxm (specifically 2,4-dlchlorophenoxyacetic acid 2,4-D), whereas for the development of embryos mto plants, auxms should be omitted from the culture medmm. The regeneratiOn of nee plants has been found to be enhanced by media lackmg auxm, but supplemented with reduced concentratiOns of cytokmms, such as 6-benzylammopurme (BAP) or kmetm (1 7, 18) . MicropropagatiOn provides a means of rapidly mult1plymg matenal of both cultivated and wild nces, as well as genetically modified plants (e.g., transgemcs, somatic hybrids, and cybrids) The abihty to multiply plants m vitro Is espectally Important for wild nces (Oryza spec1es other than 0. satzva), for whiCh only hmited supphes of seed may be available. Indeed, wild nces are an Important genetic resource, smce they possess resistances to biotic and abi­ otic stresses. These Oryzae can also be used to generate alloplasm1c hnes for the development of novel cytoplasmtc male stenlity systems.

2. Materials 2. 1. Initiation of Embryogenic Callus from Mature Seed Scutella 1 . Seeds of 0 sat1va cvs. Taipei 309 and Pusa Basmati 1 (obtained from the Inter­ natiOnal Rice Research Institute [IRRI], Los Bafios, Phihppmes) 2 "Domestos" bleach (Lever Jndustnal Ltd., Runcom, UK) or any commerctally avatlable bleach solutton contammg approx 5% available chlorine. 3. Sterile punfied water: water punfied by distillatiOn, reverse osmosis, or IOn­ exchange chromatography, wh1ch has been autoclaved ( 1 2 1 oc, 20 mm, saturated steam pressure) 4 MS basal medmm: based on the formulatiOn of Murashige and Skoog (19) This medmm can be purchased m powdered form lackmg growth regulators (Sigma, Poole, UK), to which 30 g/L sucrose is added The medmm is semisohdified by the addttion of 8 g/L SeaKem LE agarose (FMC BwProducts, Vallensbaek Strand, Denmark), pH 5.8 (see Table 1) Autoclave.

21

Callus Initiation in Rice Table 1 Formulation of Media-Macronutrients, Micronutrient&, Vitamins, and Other Supplements Concentration, mg/L Component Macronutrients CaCl2 KH2P04 MgS04 KCl KN03 NaH2P04·2H2 0 (NH4hS04 Micronutrients KI H3B03 MnS04 NaMo04·2H20 ZnS04·7H20 CuS04·5H20 CoCI2·6H20 FeS04 7H20 Na2 EDTA NaFeEDTA Vitamms Myo-inositol Nicotmic acid Pyridoxme HCl Thiamine HCI Glycine L-Glutamine L-Aspartic actd L-Arginine L-Proline Other supplements 2,4-Dichlorophenoxyacetic acid Gibberellic acid Kinetin Sucrose Maltose pH Sterilization

MS

LS2.5

AA2

3 32.2 1 70.0 1 80.7

332.2 1 70.0 1 80.7

332.2 1 70.0 1 80.7 2940.0

1 900.0

1 900 0

0.83 6.20 16.90 0.25 8.60 0.025 0.025 27.85 37.25

0.83 6 20 1 6.90 0.25 8.60 0.025 0.025 27.85 37.25

R2 147.0 1 70 0 1 20.4 4040.0 3 1 2.0 330.0

0.83 6.20 19 92 0.25 8.60 0.025 0.025 27.85 37.25

500 00 447.00 50.00 500.00 50.00

2.5 1 00.0 0.5 0.5 0. 1 2.0

1 00.0

1 .0

1 00 0 05 0. 1 0.5 75.0 877.0 266.0 228.0

1 .0

560.0 2.5

30000

30000

2.0 0. 1 0.2 20000

5.8 Autoclave

5.8 Autoclave

5.8 Filter

20

30000 5.8 Autoclave

Blackha/1 et a/.

22 5

LS2.5 medium based on the formulation of Linsmmer and Skoog (20) supple­ mented wtth 2 5 mg/L 2,4-D and semisolidified by the addttion o f 8 g/L SeaKem LE agarose, pH 5 8 (see Table 1) Autoclave 6. Nescofilm. Bando Chemtcal Ind. Ltd , Kobe, Japan

2.2. Micropropagation of Cultivated and Wild Rices I . Seeds of 0 sat1va cvs. Tat pet 309 and Pusa Basmati I , Oryza austrahens1s and

Oryza granulata (obtamed from IRRI). "Domestos" bleach: as m Subheading 2.1., item 2. Stenle punfied water: as in Subheading 2.1., item 3. MS basal medmm: as in Subheading 2.1., item 4. Autoclavable culture vessels, e g., 1 75-mL capacity screw-capped "Powder­ Round" glass Jars (Beatson Clark and Co Ltd , Rotherham, UK) 6 Micropropagatwn medmm MS medmm (as m Subheading 2.1., item 4) with 2 0 mg/L BAP and 50 g/L sucrose, semisohdified by the additiOn of 8 g/L SeaKem LE agarose, pH 5.8 Autoclave 7 Glazed white ceramic ttles ( 1 5 x 1 5 em), wrapped m alummum fml and autoclaved

2 3. 4 5

2.3. Initiation of Embryogenic Callus from Leaf Bases

of Micropropagated Plants l . LS2 . 5 medmm: as m Subheading 2.1., item 5. 2 Nescofilm: as m Subheading 2.1., item 6. 2.4. Shoot Regeneration from Callus

2 3.

4 5 6

Differentiation medmm MS medmm (as m Subheading 2. 1., item 4.) supple­ mented wtth 2 0 mg/L BAP and 30 g/L sucrose, semisohdtfied by the addition of 8 g/L SeaKem LE agarose, pH 5 8 Autoclave Nescofilm· as tn Subheading 2.1., item 6. Rootmg medmm: MS medmm (as m Subheading 2.1., item 4.), supplemented wtth 1 . 5 mg/L a-naphthaleneacetic aci d (NAA) and 30 g/L sucrose, semi­ solidified by the addttion of 8 g/L SeaKem LE agarose, pH 5 8 Autoclave Maxtcrop liquid fertthzer solutiOn Maxtcrop Plus Sequestered Iron, Maxtcrop Garden Products, Gr. Shelford, Cambridge, UK Inttiation compost a 1 2 · 1 (v v) mtxture of M3 sotl-less compost (Ftsons pic , lpswtch, UK) and Perlite (Stlvaperl Ltd., Gamsborough, UK) Mamtenance compost. a 6 l l (v·v) mtxture ofM3 sml-less compost, John Innes No 3 compost (J Bentley Ltd , Barton-on-Humber, UK) and Perlite

2.5. Initiation of Embryogenic Suspensions

1 . AA2 medmm: modtfied AA medmm (21) supplemented with 2 mg/L 2,4-D, pH 5.8 (see Table 1). Filter-stenhze. 2 R2 medmm: modtfied R2 medium (22) supplemented with l mg/L 2,4-D and 30 g/L maltose, pH 5.8 (see Table 1). Autoclave (see Note 1).

Callus Initiation in Rice

23

3. Disposable sterile plastic 1 0-mL ptpets with a wide orifice (e.g., Sterilin 47 1 1 0; Bibby Sterilin, Stone, UK}, or 1 0-mL glass sero1ogtcal pipets (e.g., Sterilin 7079- l ON) with the ends removed to produce a wider orifice. 3. Methods

3. 1. Initiation of Embryogenic Callus from Mature Seed Scutella of Cultivated Rices 1 . Debusk l 00 seeds each of 0 sativa cvs. Taipei 309 and Pusa Basmati 1 . 2 . Surface-sterilize the seeds by immersion i n a 30% (v/v) solution of "Domestos" bleach for 1 h at room temperature. 3. Remove the "Domestos" solution using five rinses with sterile purified water. 4. Germinate the seeds by laying on the surface of 20-mL aliquots of M S basal medium in 9-cm diameter Petri dishes (9 seeds/dish) Seal the dishes with Nescofilm and incubate in the dark at 28 ± 1 °C. 5. After 1 4 d, remove the coleoptiles and radicles, and transfer the explants to 20-mL aliquots of LS2 .5 medium in 9-cm diameter Petri dishes (9 explants/dtsh). Seal the dishes with Nescofilm, and incubate as in Subheading 3.1., step 4. 6. Subculture every 14-28 d (see Note 2) by selecting the most embryogenic callus, i.e., tissue with a dry, fnable appearance, and transferring 1-5 calli (each approx 5 mm in diameter) to 20-mL vol ofLS2.5 medium tn 9-cm diameter Petri dishes. 3.2. Micropropagation of Wild Rices I . Debusk 1 0 seeds each of 0 austraizensis and 0. granulata (the number of seeds can be varied depending on supply). 2. Surface-sterilize the seeds and germinate as in Subheading 3.1., steps 2--4. 3 . Subculture the seedlings after 1 4 d. Aseptically remove the seedlings and place onto the surface of a sterile white tile. Trim the roots to their base using a scalpel, and trim the leaves of the shoots to a length of 2 em 4 Transfer the shoot bases to screw-capped glass j ars each containing 50-mL aliquots of micropropagation medium (see Subheading 2.2., step 6.). Immerse the bases of the shoots 5 mm below the surface of the medium. 5. Subculture the shoots every 28 d. At each subculture, use forceps and a scalpel to separate the multiple shoots (tillers), which develop from each explant. Select healthy micropropagules (tillers), and trim the roots and stems as in Step 3. Trans­ fer to micropropagation medium as in Step 4. 3.3. Initiation of Embryogenic Callus from Leaf Bases of Micropropagated Shoots 1 . Use separate mtcropropagules of 0. australienszs and 0. granulata plants obtained as in Subheading 3.2., steps 3-5. Each micropropagule has hard white tissue at its base. Thts tissue ts embryogenic and is used for callus initiation. 2 Excise the tissue, and cut into 4-mm sections; culture the latter on 20-mL aliquots of LS2.5 medium in 9-cm diameter Petri dishes (eight tissue sections/dish). 2. Seal the Petri dishes with Nescofilm, and incubate in the dark at 28 ± I °C.

8/ackha/1 et a/.

24

Fig. 1 . Callus initiation from explants of 0. australiensis (A) and 0. granu/ata (B) after culture of the explants for 62 and 84 d, respectively (bars = I em). Embryogenic callus of 0. australiensis (C) and friable callus of 0. granulata (D), both suitable for the initiation of cell suspensions (bars = 0.5 em).

3. After 28 d, inspect the dishes for callus production by the explants. Select yel­ low-colored, rapidly dividing calli composed of small cell clusters, excise the tissue from the parent explants, and transfer the tissues to LS2.5 medium every 28 d (8 tissues/dish; see Note 3, Fig. lA-D). 3.4. Shoot Regeneration from Callus I. Transfer individual pieces of callus, obtained either from mature seed scutella or from leaf bases as in Subheading 3.3., step 1., to 9-cm diameter Petri dishes each containing 20-mL aliquots of differentiation medium (12 calli/dish, each approx 3 mm in diameter, Fig. 2A). Seal the dishes with Nescofilm and incubate

Callus Initiation in Rice

25

Fig. 2. Stages in plant regeneration from mature seed scutellum-derived callus of the indica rice cv. Pusa Basmati I . (A) Embryogenic callus 28 d after sub-cul­ ture on LS2.5 medium (bar = 0.5 mm). (B,C) Stages in shoot regeneration, 1 5 d after transfer of callus to differentiation medium in the dark (B), and 30 d after transfer to the light (C) (bars = 0.5 em). (D) Rooted shoots ready for transfer to compost (bar = 1 em).

Blackha/1 et a/.

26

2

3

4. 5. 6. 7 8. 9.

at 27 ± 2°C in the dark for 14 d, followed by transfer to the light ( 1 6-h photope­ 2 riod, 55 j.lmol/s/m , Daylight fluorescent tubes). Examine every 7 d for the regen­ eration of shoots and roots. When shoots appear, transfer each shoot, together with a 3-mm diameter piece of the adjacent parental callus, to rooting medmm ( 1-8 shoots/dish; Fig. 2B,C ; see Note 4). Seal the dishes with Nescofilm, and incubate at 27 ± 2°C in the light as m Subheading 3.4., step 1 . Transfer rooted shoots to initiation compost (see Subheading 2.4.5.) in 7.5-cm diameter plastic plant pots (Fig. 2D). Cover the regenerated plants with 20 x 20 em clear polythene bags (see Note 5). Maintain the potted plants in a glasshouse under natural daylight With maximum day and night temperatures of 28 ± 2°C and 24 ± 2°C, respectively. After 3 d, make five incisions with a pm mto the top of the bags. Four d later, remove, With scissors, one comer of each bag. After a further 4 d, remove the other comer of each bag. Every 2 d, cut off the top 1 em of each bag, until the topmost leaves of the potted plants are exposed. Remove each bag. Spray the plants daily with a 0. 1 % (v/v) aqueous solutiOn of Max1crop Transfer plants producing tillers and roots showing healthy, vigorous growth to 1 5-cm diameter pots containing maintenance compost.

3.5. Initiation of Embryogenic Suspensions When sufficient dry, fnable, callus has been obtamed (approx 5 wk for cultivated rices and up to 24 wk for wild nces, depending on the growth rate of the callus), initiate cell suspensiOn cultures by transferrmg 1 . 5 g fresh weight of embryo­ genic callus (see Notes 6 and 7) to a 75-mL Erlenmeyer flask containing 1 8 mL of either AA2 medmm (for 0. sativa cv. Taipei 309 and 0 granulata [see Note 8]) or R2 medium (for 0. sat1va cv. Pusa Basmati I and 0. australiensis). Incubate at 28 ± 2°C m the dark on a horizontal rotary shaker ( 1 20 rpm, 4-cm throw). 2 Subculture the suspensions every 3-4 d. Allow the cells to settle, remove 50% of the supernatant, and replace with an equal or slightly greater volume of medmm. Gradually i ncrease the volume of medium and the size of flasks m accordance with the rate of growth of the suspension cultures. 3. After 1 2- 1 5 wk, there should be sufficient quantity of small clusters of cells (see Note 9) to transfer 1 mL packed cell volume (PCV; see Note 10) of the small clusters to produce a "ptpetable" suspenston (see Note 9). Thts "ptpetable" cul­ ture is subcultured at 7-d mtervals by transferring 1 mL PCV, together with 9 mL of spent medium, to a 250-mL capacity Erlenmeyer flask containmg 42 mL of new medium (see Note 1 1). Mamtam the "stock" culture for a further 2 wk until the new "pipetable" suspensiOn has become established Repeat this procedure tf the "pipetable" culture fatls to become established 4. The growth characteristics of established cultures are determined by daily mea­ suring the settled cell volume (SCV). Decant the cell suspensiOn mto a graduated centnfuge tube, allow to sediment under gravity ( 1 0 mm), and note the SCV from the graduatiOns (see Note 12) 1.

Callus Initiation in Rice

27

4. Notes 1 . R2 medium should be autoclaved at 1 l 6°C for 30 min, since sterilization at 1 2 1 °C for 20 min causes excessive caramelization. 2. Regular subculture of scutellum-derived callus will produce fast-growing, globu­ lar tissues. Fourteen days are the optimal time between transfers, but the tissues can be left for 28 d before subculture. 3. Transfer of leaf base-derived calli to fresh LS2.5 medmm every 28 d is Impor­ tant, since compact embryogenic callus becomes more globular with time in cul­ ture and, therefore, more suitable for the initiation of finely divided cell suspensions. 4. More than 30% of the shoots regenerated from some callus cultures may be albmo. In such cases, the callus should be discarded, and new cultures initiated fro m seed. The regeneration of albinos appears to be cultivar specific. For example, it occurs in the Japonica rice cv. Taipei 3 09, but not in the indica cv. Pusa Basmati l . 5. Place polythene bags over the potted plants, and secure the bags by placing the pots inside other 7.5-cm pots. The bags are required to maintain high humidity around the plants and to reduce transpiration. The humidity Is reduced gradually by opening the bags as the plants become acclimatized. 6. In the case of 0. granulata, approx 2% of the stem bases produce fast-growing, fine yellow callus suitable for the initiation of cell suspension cultures. Transfer of this callus to new semisolidified medium results in rapid tissue proliferation, enabling initiation of cell suspensions after 8-1 2 wk of culture of the callus on semisolid medium. 7. For 0. australiensis, approx 90% of the stem bases produce embryogemc callus. Initially, the cell suspension cultures of 0. australiensis consist of large c lusters of callus (each in excess of 2 mm diameter), but after 8- 1 2 wk of subculture, the majority of the cell clusters are < I mm in diameter. 8. Maintenance of cell suspensions of 0. granulata is difficult because of the extreme viscosity of the cultures. It is necessary to shake the Erlenmeyer flasks vigorously immediately before subculture and to pay particular attention to the volume of cells (see Note 9) used for inoculation into new medium. 9 When a cell suspension is first initiated, the mitial or "stock" suspension consists of large cell clusters. As these clusters grow, they release smaller clumps of cells into the liquid medium. For experimental use, a "p1petable" culture is initiated from the small cell clusters. The "stock" culture is discarded once a "pipetable" culture has been established. 1 0 . PCV is measured by drawing a suspension of cells into a sterile 1 0-mL pipet (see Subheading 2.5., item 3.) The medium i s expelled while holding the pipet tip against the bottom of the flask. The cells will be retained in the tip of the pipet, allowmg the medium to escape. The PCV may be adjusted to 1 mL by drawing more suspension into the pipet or by releasing packed cells from the pipet tip. I I . The maintenance of cell suspension cultures requires careful attention to the sub­ culture procedure. Regular subculture, usually at 7-d intervals and use of the

28

B/ackha/1 et a/.

correct moculatwn volume ( I mL PCV) are vttal m maintaining totipotency. An excess of inoculum causes exhaustion of the nutrients early in the subculture cycle, resultmg m necrosis within the centers of the cell clusters Conversely, t f too small an moculum t s transferred, the growth rate o f the cells is reduced con­ siderably, smce the cells fall to reach the mimmum moculatwn denstty 1 2 . The doubhng ttme o f cultures varies, but 1 s generally 2--4 d

References

2

3. 4 5

6 7

8 9 1 0. 11

12.

13. 14

S1vamani, E , Shen, P , Opalka, N., Beachy R N , and Fauquet, C M ( 1 996) Selection of large quantities of embryogenic calli from md1ca nee seeds for pro­ ductiOn of fertlle transgenic plants usmg the bwlistic method. Plant Cell Rep 15, 322-327. Htei, Y , Ohta, S , Koman, T , and Kumashtro,T ( 1 994) Effictent transformatiOn of nee (Oryza sat1va L.) mediated by Agrobactenum and sequence analysis of the boundanes of the T-DNA Plant J 6, 27 1-282 Thompson, J A., Abdullah, R., and Cockmg, E. C ( 1 986) Protoplast culture of nee (Oryza sat1va L ) usmg media solidified wtth agarose Plant Sc1 47, 1 79-1 83 Suh, S. C , K1m, H 1., and Park, W ( 1 992) Plant regeneration from nee protoplasts culture m Javamca cv Texmont Res Rep Rural Dev Adm (Suweon) 34, 1-9 Jam, R. K., Khehra, G S , Lee, S-H , Blackhall, N W , Marchant, R , and Davey, M R. ( 1 995) An tmproved procedure for plant regeneratiOn from mdtca and J apomca nee protoplasts Plant Cell Rep 1 4, 5 1 5-5 1 9 La1, K L . and Lm, L F ( 1 982) Induction and plant regeneratiOn o f callus from tmmature embryos of nee plants (Oryza sat1va L.) Jpn J Crop Sc1 5 1 , 70-74 La1, K L. and Lm, L. F (1 986) Further studtes on the vanabthty of p lant regen­ eratiOn from young embryo callus cultures m nee plants (Oryza satzva L ) Jpn J Crop Sc1 55, 4 1--46 La1, K L and Lm, L F ( 1 988) Increased plant regeneration frequency m water­ stressed nee tissue cultures. Jpn J Crop Sc1. 57, 553-557 Abe, T. and Futsuhara, Y., ( 1 984) Vanetal difference of plant regeneratiOn from root callus tissues m rice. Jpn J Breed 34, 1 47-1 5 5 Abe, T , and Futsuhara, Y ( 1 986) Genotypic vanab1hty for callus formation and plant regeneratiOn m nee ( Oryza sat1va L ) Theor Appl Genet 72, 3-1 0 BaJ aJ , Y. P. S. ( 1 99 1 ) BIOtechnology in rice improvement, m Bzotechnology m Agnculture and Forestry, val 1 4, Rzce (BaJaJ , Y P S , ed ), Spnnger-Verlag, Heidelberg, pp 3-1 8. Kyozuka, J , Otoo, E., and Sh1mamoto, K ( 1 988) Plant regeneratiOn of proto­ plasts oflndica nee. genotyptc differences m culture response. Theor Appl Genet 76, 887-890. Lee, L . , Schroll, R. E., Gnmes, H. D., and Hodges, T. K. ( 1 989) Plant regenera­ tiOn from mdtca rice (Oryza satzva L.) protoplasts. Planta 1 78, 325-3 3 3 Abe, T and Futsuhara Y. ( 1 99 1 ) Regeneration o f nee plants from suspensiOn cultures, m Biotechnology m Forestry and Agnculture, vol 14, Rzce (BaJaJ, Y. P S., ed.), Springer-Verlag, Heidelberg, pp 38--46

Callus Initiation in Rice

29

1 5 . Anthony, P., Jelodar, N. B , Lowe, K. C., Power J. B., and Davey, M. R. ( 1 996) Pluronic F -68 increases the post-thaw growth of cryopreserved plant cells. Cryo­ biology 33, 508-5 14. 1 6. Lynch, P T., Benson, E. E., Jones, J., Cocking, E. C., Power, J. B., and Davey, M. R. (1 994) Rice cell cryopreservation: The mfluence of culture methods and the embryogenic potential of cell suspensions on post-thaw recovery. Plant Scz. 98, 1 85-1 92 1 7. Raghava-Ram, N. V. and Nabors, M. W. ( 1984) Cytokinm mediated long-term, high-frequency plant regeneration in rice tissue culture. Z fur Pflanzenphysiol 1 13, 3 1 5-32 3 . 1 8. Raghava-Ram, N . V. and Nabors, M . W. (1 985) Plant regeneratiOn from tissue cultures ofPokkali rice ( Oryza sativa) is promoted by optim1zing callus to medium volume ratio and by a medmm-conditwnmg factor produced by embryogemc cal­ lus Plant Cell, Tiss Org. Cult 4, 24 1-248 . 1 9. Murashige, T . and Skoog, F . ( 1962) A revised medmm for rapid growth and bio­ assays with tobacco tissue cultures. Physzol. Plant 15, 473-497 20. Linsmaier, E. M. and Skoog F. ( 1965) Organic growth factor reqmrements of tobacco tissue cultures Physwl Plant 18, 100-127 2 1 . Muller, A. J., and Grafe R. (1 978) Isolation and characterisation of cell lines of Nicotiana tabacum lacking nitrate reductase. Mol. Gen Genet. 1 61, 67-76. 22. Ohira, K., Ojima K., and Fujiwara A. ( 1 973) Studies on the nutritiOn of nee cell culture. I. A s1mple, defined medmm for rapid growth in suspension culture Plant Cell Physzol. 14, 1 1 1 3-1 1 2 1 .

3

__ _______________________________

Cal l us Initiation, Maintenance, and Shoot Induction in Potato Monitoring of Spontaneous Genetic Variability In Vitro and In Vivo Rosario F. Curry and Alan c. Cassells

1 . Introduction

Potato tissue culture has been widely researched both from the perspectives of mass clonal propagation, e.g., in potato seed certification schemes, and of genetic manipulation (1, 2). Potato micropropagation is central to most potato certification schemes (3), and genetic engineering has resulted in the release and sale to consumers in North America of transformed "Russet Burbank" pota­ toes (NatureMark Potatoes, Boise, ID). Early potato tissue-culture studies showed that the genome was unstable m callus formation and subculture, and gave rise to high-frequency spontaneous mutations ("somaclonal variation") in adventitiously regenerated plants (4). Various workers have sought to exploit somaclonal variation in potato improvement, but with limited success owing to the high level of useless vari­ ability produced, typical of mutation breeding, including polyploids, aneup­ loids, and unstable maturation mutants (5) . This problem of unwanted background variability is of concern to those involved m potato genetic trans­ formation (6). Here, direct and indirect adventitious pathways for potato regeneration are described (7). The use of flow cytometry is described for the detection of van­ ability (8) in explants used to initiate cultures, and in the callus and adventi­ tious shoots derived. This application of flow cytometry to monitor genetic drift is based on the high-frequency occurrence of polyploidy in potato tissues in vivo (9) and in callus and adventitious regenerants in vitro (10). Flow m Molecular Btology, Vol t t 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

From Methods

31

Curry and Cassells

32 Table 1 Constituents of Potato Culture Media8 Compound MS medmm Myo-mosttol Casem hydrolysate Sucrose Glucose Agar Manmtol NAA IAA t - Zeatin Kmetm 6 - BAP GA3 MES pH

Callus tmtlatwn Greenmg M2 Ml Full strength Full strength 1 00

30 g 7g 5

10 g 10 g 10 g 40 g 0.2 01

Shoot mductwn M3

Potato nodal medmm

Full strength 1 00 1 000 3g

Half strength

10 g 36 g 01 05 05

05 02 58

57

15 g 60g

0I 02

l .O g 57

58

aThe medmm (MS) used I S that ofMurashtge and Skoog (2 7) (see Appendix) ConcentratiOns expressed as mg/L, unless otherwise stated

cytometry also has potential apphcation m selectwn agamst polyplmds and aneuplmds in genetic transformatiOn and mutation breeding studies. Image analysis can be used to measure variability in the adventitious regenerant popu­ latiOns (11) using the "standard dev1ation assay" of De Klerk (12) , which ts based on the princ1ple that random mutation m a populatiOn w1ll increase char­ acter variability, e.g., variability in leaf shape, that 1s, in characters that are controlled by mmor genes. Image analysis can also be used to differentiate some nonmorphological mutants from the control population, but m this case, owing to large environment-genotype mterachon m potato, large clonal popu­ lations are reqmred for sampling (13)

2. Materials 1 . 1 40-mL polyvmylchloride food-grade tubs (see Note 1) sealed with "Parafilm M" (see Note 2) containmg 4 5 mL of potato nodal medmm (see Table 1); 9-cm Petn dishes (see Note 3) containing 25 mL of callus tnitiatwn medmm, callus greemng medium, or shoot mductwn medium, as appropnate (medta compost­ hons are given in Table 1) 2. Potato plants for explants: these should be grown from certtfied vtrus-free seed tubers m the glasshouse m insect-proof cages to exclude aphid vtrus vectors

Callus Initiation in Potato

3.

4. 5.

6.

33

These cages can be made from wooden frames covered wtth mushn. Alterna­ tively, the plants can be grown in plastic bags with msect-proof vents (Sunbags: Sigma Chemical Co., Poole, Dorset, UK). To introduce these plants from the glasshouse into culture in the growth room, nodes ( 1-1 . 5 em in length) are excised, surface-sterilized and used as explants. 80% (v/v) aqueous ethanol, 1 0% (v/v) commercial bleach (Domestos--active ingredient 0.5% sodium hypochlorite; Lever Industrial Ltd., Ireland), sterile dis­ tilled water. Potato miCroplants in culture are used as source explants for callus (see Note 4) A suitably equipped laboratory for plant tissue culture to include autoclavmg facilities; instruments and instrument-sterilizing equipment, and a transfer (lamt­ nar flow) cabinet (see Note 5). Suitable plant tissue-culture growth conditions include: a temperature maintained at 22 ± 3°C; a 1 6-h photoperiod of 35-50 f..lOlll Vm2/s, provided by "white" 65/80 W liteguard flourescent bulbs (Osram Ltd. , UK). Flow cytometer (see Note 6). Mungbean and parsley seeds as internal standards for flow cytometry.

7. 8. 9 Buffer for extraction of nuclei. It consists of 1 5 mM Tris (hydroxymethyl aminomethane), 2 mM Na2 EDTA, 0.5 mM spenmne, 80 mM KCI, 20 mM NaCl, 1 5 mM mercaptoethanol, and 0. 1% (v/v) Tnton X- 1 00, pH 7.5 (1 7). 1 0. 4', 6-diamidino-2-phenylindole (DAPI) stain· The solution of DAPI consists of 20 mg DAPI/ 1 mL of water. Ten millihters ofDAPI solution are added to 10 mL citrate buffer in a small flask with closure. The citrate buffer ts prepared by add­ ing Na2HP04 to citric acid until a pH of 4.0 is reached (ca. 6:5 ratio). The DAPI mixture ts covered wtth foil and stored in the refrigerator when not in use. 1 1 . Glass Petri dishes 1 2.

Razor blades.

1 3 . Image analysis system (see Notes 8 and 9).

3 . Methods

3. 1. Callus Initiation from In Vivo Material I . Internode and petiole explants are taken from the mid region of plants, 1 8-2 5 em

in height, growing in the glasshouse. 2. Surface-sterilize explants in 80% (v/v) aqueous ethanol for 30 s followed by immersion for 1 5 min in 1 0% (v/v) commerctal bleach. Rinse in sterile dtstilled water five times. 3. Trim back 3--5 mm from cut ends ofsterilized material to remove damaged matenal. 4. Cut explant longitudinally before placing both halves, and cut surface down on callus inittation medium (M 1 ) ( see Table 1).

3.2. Callus Initiation from In Vitro Material 1 . Choose healthy vigorous mternode explants (see Note 1 0) from 6-wk-old in vitro plantlets. Internodes should be l .0-2 0 em in length (7). 2. Place internodes on M l (see Table 1 ; Note 1 1) .

34

Curry and Cassells

3.3. Maintenance of Callus Mamtam callus under growth room conditions (see Subheading 2., item 6). Subculture every 4--5 wk onto fresh M l , by transferrmg single p1eces of callus approx 1 em in diameter. 3. Only subculture VIable, healthy calh (see Note 1 2).

1 2

3.4. Induction of Shoots

2. 3. 4. 5.

6

7.

Transfer callus from M 1 onto greenmg medmm (M2) for 4 wk (see Table 1 ; Note 13). Once callus has greened, transfer to shoot mduction medium (M3) (see Table 1) When shoots begm to grow, excise and transfer to potato nodal medmm (see Table 1; Note 14) (14, 15) . Seal tubs with parafilm (see Note 2), and mamtain under growth room condi­ tions When shoots have developed 5-6 nodes (approx 4 wk m the growth room) remove from contamer Wash off agar attached to roots wtth warm unsterile water (see Note 1 5) Ftll an unstenle plug tray wtth a mtxture of vermiCulite and pottmg compost ( I · 1 ratio) (see Note 16). Place one plantlet in each plug, and firm the compost at the crown. Mist gently wtth tepid water Place a translucent plastic cover over the tray to keep the relattve humidtty htgh. Weanmg is earned out for 3 wk m the glasshouse. Mtst the plantlets twice da1ly, and open atr vents m the covers one-third of the way each week. Remove the covers after 3 wk

3.5. DNA Quantitation by Flow Cytometry

3.5. 1 . Preparation of Nuclei Suspension Samples for DNA Measurement

2

3

4 5.

Mungbean and parsley seeds are surface-sterilized, Immersed m stenle distilled water for 24 h, and germmated on 2.2 g/L Murashtge and Skoog basal medium contammg 15 g/L sucrose and 8 g/L agar, pH 5 .8, m the growth room. Remove young leaf ttssue (see Notes 17 and 1 8) from the mtemal standard and I 0 mg of fresh tissue from test sample to be analyzed, and place on a clean Petn dtsh (see Note 19) Chop standard and sample wtth approx 200 clean strikes of a sharp blade m 0.4 mL of tce-cold nuclet extractton buffer (see Notes 20 and 2 1 ) (22) . Leave for 1 0-20 mm. Add 2.0 mL of DAPI stam mixture (see Note 7) Ftlter through a nylon mesh of 40 11m mto a sample tube

3.5.2. Use of Flow Cytometer 1.

Tum on machme, and allow lamp to stabthze for 30 mm before beginmng DNA measurements

Callus Initiation in Potato

35

2. Check that parameter settings are correct (see Notes 22 and 23). 3. Place the sample tube in the sample holder. 4. Adjust the gain and lower limits until all peaks are apparent on the x-ax1s (see Note 23). 5. Clear the sample measurement thus far once the flow rate stabilizes (see Note 24). All measurements taken after this point will be taken as the result for that sample. 6. Do not allow the volume of sample to decrease to the pomt where the electrode taking measurements emerges from the sample. If thts occurs, the sample data are cleared automatically. 7. Ensure Data Pool Application for Cytometry (DPAC) software is set to receive mformahon from the flow cytometer (see Note 25). 8. Cease measurement, and transfer data to computer software. 9. The next sample can now be analyzed. I 0. After all measurements are taken, clean the flow cuvet (see Note 26). 1 1 Switch off the flow cytometer, computer, printer, and UV lamp.

3.5.3. Use of DPAC Software 1 . Analyze data on the software once the information has been transferred and stored on disk (see Subheading 3.5.4.). 2. Name the sample. 3 . Analyze peaks. Try to align the reference line with the internal standard peak. This helps m the comparison of sample nuclei with internal standard nuclei. 4. If software does not identify all peaks, there is a facility to select peaks. 5. Print out graphs.

3.5.4. Interpretation of Results 1 . Examine DNA histograms. Ideally graphs should be clear with sharp peaks and low coefficients of variation (see Note 27). 2. Decide which peaks represent the fluorescence of internal standard nuclei. This can be done by referring to the peak number and index values printed underneath the graph (see Fig. 1 ., Notes 28 and 29). It may be necessary to calculate the DNA content of the mtemal standard peak to verify this (see Note 30). 3. Check if a second peak of the standard, the 4C amount, 1s also represented m the histogram. 4. The remaining peak(s) represents the fluorescence of potato sample nuclei (see Fig. 1., Notes 31 and 32). 5. To estimate the nuclear DNA content, calculate the ratto of the mode of the target nuclei to mternal standard nuclei. Multiply this by the nuclear DNA content m picograms of the standard. The result is the amount of DNA (pg) in that peak (see Note 30). 6. Analyze DNA content in picograms of each peak to determine ifpolyploid nuclei are present (see Notes 33 and 34).

Curry and Cassells

36 COlUtt

tc = J l O ·� tc

l1j

2fHI

"<�)

u

p 'ttec ID

Name Peak 1

=

n

SJSii

g ; � ;c; hn

n = �t.

1 so

l llll

=

j [J lW ..

u

5U

5250 callus Index

!UU

11= � n

-1

I

ISO

,A.

cUO

TC · 1 & mung Mode

2 71 38 2 3 85 54 :3 7 92 111 4 15 8 5 222 No c yc l e e valuated

Mean 38 55 111 22 1

IC

I

c5u

tntelt,tty

2 1 09 0

Area# 1 19

Area% 5 . 64

436 587 256

20 67 2 7 83 12 . 1 4

0 00% ) CV% 2 63

1 82 2 25 2 . 04

Fig l DNA histogram of DAPI-stamed nuclear preparatiOn of internal standard ( Vzgna radzata) and callus tissue of S tuberosum (see Note 32)

3.6. Image Analysis of Leaf Shape

3.6. 1 . Sampling of Plant Population l . Plants should be grown in the glasshouse in randomized blocks with at least 50 replicates for each treatment, under umform environmental conditiOns. Plants should be clearly coded/labeled 2. Termmal leaflets are taken from each plant m the batch and coded With respect to the batch (see Note 35) 3. Harvested leaflets should be placed m labeled plastic bags to avmd desiccatiOn durmg processmg

3 6.2. Image Analysis I . Tum on the hghtbox, focus the camera, and set a midrange aperture of approxi­

2

mately F-8 Open the system menu and chck on "set defaults " Set the parameter you want to measure, e g., leaf shape factor, and press "enter "

Callus Initiation in Potato

37

3 . Calibrate the equtpment (see Note 36) by selecting the "analysts menu" and "cali­ brate;" place a ruler in the measuring frame, and click the mouse cursor on the scale at the two ends. Key in the measured length, and enter the mimmum object stze. Enter units of measurement, e.g., em. 4. Place a sample leaflet on a hghtbox, and the raise or lower the video camera, such that the leaflet fits wtthin the camera frame. 5. Click on the "tmage menu" and select "acqutre image ." Place a sample leaflet on the lightbox, and set the threshold. Adjust the controls unttl the image ts fully thresholded with no extra light or dark highlighting outside the object Press "en­ ter" to acquire the image. Check the image has been acquired by placing your hand underneath the camera. If the image has been grabbed, the leaflet image wtll remam on the monitor when the camera is blocked. 6. Select "object" from the "analysis menu. " Press "enter," and "save;" label the image. 7 Press appropriate key to show shape factor values (see Note 37)

3. 6.3. Analysis and Interpretation of Image Data 1 Enter the indtvidual data points from above m a stattsttcal software spread sheet 2. Calculate the standard deviations for the control and test populations. The smaller the SD, the less the variabthty in the character chosen, t.e., m the respective popu­ lation (see Note 38) (12) .

4. Notes

I . All plantlets are grown and maintained in 1 40 mL clear stenle polyvinylchlonde tubs (cat. no. 1 50c, Wilsanco Plastics Ltd., Killyman Road, Industrial Estate, Dungannon, Co. Tyrone DT7 1 6LN, Northern Ireland). 2 Each tub is sealed with "Parafilm M" (American Can Co. ,CT). Parafilm has htgh gas and vapor barrier properties, and can prevent contamination from entering the culture vessel. There is enough gaseous exchange through the walls of the container to prevent ethylene buildup or oxygen deficiency mstde the vessel. 3. Each dish is wrapped m clear PVC "clmgfilm wrap." Thts acts as a barrier to contaminants, yet allows gaseous exchange (1 6). 4. Potato microp\ants are produced and maintained in vitro via nodal subculture. In sterile conditions, nodes are exctsed from microplants and placed on potato nodal medium. Subculturing takes place every 5-7 wk 5. All media, glassware, and instruments are sterilized at I I 0 kPa for 1 5 mm. It i s necessary to filter-sterilize certain hormones into sterilized media, since autoclaving them with other media components may result in loss of activity (see Table 2). Subculturing is performed under sterile conditions in a laminar flow cabinet (cat. no. KD2S, Brassaire, John Bass Ltd., Hampshtre, UK). Instruments are sterilized for 1 5-20 s at 250°C in a bead sterilizer placed m the flow cabinet. 6. In this study, the flow cytometer used is a Partee CA-lli (Partee GmbH, Otto­ Hahn-StraBe 32, D-48 1 6 1 , Munster, Germany) .

Curry and Cassells

38

Table 2 Plant Growth Regulators: Preparation and Storage of Stock Solutions Growth regulators Mol. wt solvent Diluent Lrquid storage Stenhzation

NAA 1 86.2 I N NaOH Water 2-8°C A

IAA

Zeatm

Kmetm

BAP

2 1 5 .2 1 75.2 2 1 9.2 225.3 ETOH/ I N NaOH l N NaOH I N NaOH l N NaOH Water Water Water Water < oc 2-8°C < 0°C < ooc AIF

AIF

AIF

AIF

GA3 346 4 ETOH Water 2-8°C AIF

NAA, ! -naphthalene acetic ac1d, IAA, mdole-3-acetlc ac1d, BAP, benzylademne; GA3, g1bberelhc ac1d

A, Autoclavable wrth other medra components. F, F!lter-stenhze A/F, Autoclavable, but may suffer some loss of actlVlty

7. The fluorescent dye DAPI has been demonstrated m the past to grve reproducible results in staining released nuclei, although some argue that the use of base preference fluorochromes, such as DAPI, can lead to errors tn calculations It 1s slow-fadmg and has greater specificity for double- stranded DNA, makmg It unnecessary to pretreat the sample with RNase. Other stains used frequently mclude· proptdium Iodide (mtercalatmg dye), Mithramycin (fluorescent antibi­ otic); Hoechst dyes (A-T specrfic stains) (18). 8. The rmage analysis system descnbed here is the DIAS system (Delta-T Instru­ ments, Cambridge, UK) 9 Any bas ic statistical software pack wrll be able to provtde values for the standard deviations of population means, e.g., Pnsm (GraphPad Software, San Dtego, CA) I 0. In vttro plantlets should be bactenal-mdexed routinely to ensure the health status of the matenal (1 9) 1 1 . When internode explants are first placed on M 1 (callus imtlatiOn medmm), 1t may take up to 8 wk before sigmficant callus forms at cut ends. After the first subculture, callus will grow raptdly and can be subcultured every 4 wk. 1 2 . If callus turns brown or a translucent-gray color, this means rt has senesced. Do not subculture senesced callus. It is tmportant to wrap plates m clingfilm wrap and not parafilm, so ethylene emitted from the cultures wrll not burld up msrde the Petri plate. Thus callus is mamtained m a less stressful environment, thereby decreasing the mctdence of senescence. 1 3 The "greemng" stage IS a necessary step m shoot mduct10n. It wrl l be observed that callus contmuously subcultured on M1 is mostly fnable. However, after 4 wk on greening medium, callus changes its texture to a hard, crusty mass.

Callus Initiation in Potato

39

1 4. It can take up to 6 wk for shoots to be induced. Once one shoot i s removed from a callus, many more shoots will be produced within days. Therefore, it is impor­ tant to excise the first shoot as soon as it appears. 1 5. It is important to wash agar off roots to prevent fungal contamination in the glass­ house. 1 6. Vermiculite has water-retention properties that prevents the compost from dry­ ing out in the glasshouse. 1 7. The amounts of standard used vary wtth each type of sample. It ts advisable to run a preliminary sample to dectde on the correct amqunt of test tissue and stan­ dard to use. At least 4000 nuclei are required. 1 8. Flow cytometry provides only relative values. Simultaneous measurement of the fluorescence of internal standard stained nuclei of known DNA content is neces­ sary to determine DNA content in picograms of the sample nuclei. Mungbean ( Vigna radzata cv. Berken, 2 C : I Opg ) or parsley (Petroselinum cnspum 2C:3.6 pg) nuclei are used as an internal standard (DNA amounts m plants are expressed m picograms, I pg = I 0-12 g). These were selected as standards be­ cause thetr peak positions do not coincide with either the GO + G l or the G2 peak of the potato sample (21) . 1 8. Flow cytometric analysis has been performed on fixed ttssues (20). Samples "fixed" wtth formaldehyde can be stored for weeks, without loss of resolution, before analysis. The number of released nuclet can be higher with fixed than w1th fresh tissues, so smaller samples can be used 20. Nuclei will be damaged if the blade is blunt, and this debris wtll lead to increased "background notse" on graphs (23). It may be difficult to chop callus in nuclet extraction buffer, because it tends to clump m the liquid. Incubation in the buffer and mechanical disruption of cells by syringmg may prove a more reliable method for isolation of nuclei. 2 1 . The sample must be kept at ice-cold temperatures after isolation of nuclei m order to decrease nuclease activity (24). 22. The speed of the flow of nuclei analyzed is best adjusted to as low a setting as possible, i.e , 20--40 nuclei/s. For greater accuracy, t.e., sharper peaks and w1th low coefficients of variatton, speed can be reduced even further, but analysis ts more time-consuming. 2 3 . The gain is reduced to shift the readmgs to the left, unttl all peaks on the x-axis are visible (Most machines have a screen that allows you to see the peaks form as each nucleus is being analyzed.) Adjusting the lower limits removes the back­ ground reading that arises from debris in the sample. 24. It is necessary to clear all previous measurements when the flow rate stabilizes to ensure accurate results. 25. Follow the manufacturer's guidelines to ensure the computer attached is ready to receive data from the flow cytometer. This may mvolve settmg the software to a transfer command. 26. The flow cuvet can be cleaned after each sample ts analyzed and when all the measurements have been taken. It allows water to flow through the inJection tubes to remove nuclei and debns.

Curry and Cassells

40

27. For accurate interpretation of results, coefficients of variatwn (CV) should be below 5%. Sample preparation IS very important m this respect, smce nuclei dam­ aged dunng mechamcal isolation wtll result m higher CV values (25) One could centnfuge each sample and discard debns before analysis, but this 1s time­ consummg Over- or understammg nuclei with DAPI can also result m larger coefficients of variatiOn and, therefore, more vanable results. In Fig. 1 ., CV val­ ues range from 2 63-1 82 Peak 1 representmg mungbean nuclei have the largest CV values 28. The mdex value of the mtemal standard does not always approximate to 1ts 2C DNA content, e.g., 1 06 pg m the case of mungbean (21), smce 1t depends on where the reference line IS positwned dunng the analysis. Th1s line should be aligned wtth the peak of the mtemal standard It IS found sometimes that the nuclei of the mternal standard tissue are dividmg, givmg a second peak, repre­ sentmg the 4C amount of DNA. 29 The C value refers to the DNA amount of the unreplicated haploid genome 2C IS the DNA amount of an unrephcated diploid genome 4C IS the DNA amount m nuclei with two replicated copies of a genome, i e , nuclei after d1vis10n and DNA synthests, m the G2 and M (mitottc) phase (21) . 30 CalculatiOn of nuclear DNA content: Mode of sample mo de o f refierence standard

x

DNA (pg) of reference standard

(1)

The "Mode" represents the most frequently occurring number in a sample 3 8 + 3 8 x 1 06 1 06 pg Peak 1 54 + 38 x I 06 I 50 pg Peak 2 1 1 1 38 x 1 06 3 09 pg Peak 3 222 -. 3 8 x 1 .06 6. 1 9 pg Peak 4 3 1 Exercise cautwn when determmmg the 2C DNA content of the sample A previ­ ous knowledge ofplmdy level of matenal is Important Solanum tuberosum 1s a tetraploid (2n 48) 4C IS the DNA amount of the unrephcated tetraploid genome (4C: 3 6 pg) (1 7) 3 2 . Th1s graph shows a mmor peak of 2C nuclei (peak I ) of mungbean cells Peak 2 ( l 50 pg) represents the unreplicated genome (4C DNA content) of potato nuclet m G011 phase Peak 3 (3 09 pg) represents the 8C DNA content of potato nuclei in G2 phase The nuclei have doubled the normal DNA content after d1v1sion and DNA synthes1s Peak 4 represents 6 1 9 pg of DNA Th1s 1s approximately twice the value of peak 3, md1catmg that some of these nucle1 have undergone diVISiOn. 3 3 . If the DNA amount is greater than twice the 2C DNA amount, polyploid nuclei may be present. For example, the DNA content of peak 3 ts shghtly htgher (3 09 pg) than twice the DNA content of peak 2 (3 .00 pg). One could hypothesize that the extra DNA 1s from aneuploid nuclei, or it could be the result of poor sample preparation It IS extremely difficult to determine aneuplo1dy (± chromosome differences) usmg flow cytometry. =

=

_,_

=

=

=

Callus Initiation in Potato

41

34. Accuracy of any result from flow cytometry is questionable because of the large portion of nuclear DNA m native chromatm that is unstained. The amount of DNA remaining unstained depends on chromatin structure (24). 3 5 . It is important that the leaves are fully expanded and are taken from eqmvalent leaf positions on the plants to reduce variability m the data. 3 6 The mstructions are for the MS-DOS verston o f the DlAS tmage analysis system (Delta-T Instruments, Cambridge, UK). The principles apply to other systems 37. Depending on the apparatus used, there wiii be a choice of system-generated parameters. It is recommended that a quantitative character is used, although for highly variable populations, this may not be essenttal. 38. The "standard deviation assay" (12) may be used to detect variability in a popu­ latiOn at the percentage level. To dtscriminate between clones, vanability m the data owmg to the genotype environment interactiOn reqmres that large sample stzes may be analyzed. (For analysts of Gausstan data see ref. 26)

References 1 . Bajaj, Y. P S., (ed.) ( 1 987) Potato, in Biotechnology in Agnculture and Forestry 3 Springer Verlag, Berlm 2. Salammi, F. and Motto, M. ( 1 993) The role of gene technology m plant breeding, in Plant Breedmg-Prmciples and Prospects (Hayward, M D., Bosemark, N 0 , and Romagosa, I. eds.) Chapman and Hall, London, pp 1 38-1 59. 3 Cassells, A. C. ( 1 997) Pathogen and microbial contammatton management, m micropropagation: an overview, in Pathogen and M1crobwl Contammatwn Man­ agement in Micropropagatwn, Kluwer, Dordrecht, pp. l-1 4. 4. Kumar, A. ( 1 994) Somaclonal variatton, in Potato Genet1cs (Bradshaw, J. E. and Mackay, G. R., eds.), CAB International, Wallingford, pp 1 97-2 1 2 5 . Cassells, A. C . and Sen, P . T . ( 1 996) In vitro productton of late bltght (Phyto­ phthora uifestans)-reststant potato plants, in Biotechnology 111 Agriculture and Forestry 36 (Bajaj, Y. P. S., ed.), Spnnger Verlag, Berltn, pp 1 07-1 1 8 . 6. Dale, P. J. and Hampson, K. K. ( 1 995) An assessment of morphologtcal and trans­ formation efficiency in a range of vanettes of potato (Solanum tuberosum L ), m Plant Genetic Manipulatwn m Plant Breed111g. Critena for DeciSion Makmg (Cassells, A. C . and Jones, P. W., eds.), Kluwer, Dordrecht, pp. 1 0 1-108. 7. Cassells, A. C., Austin, S., and Goetz, E. M. (1 987) Variatton in tubers m smgle cell-derived clones of potato in Ireland, in Bwtechnology m Agriculture and For­ estry 3 (Bajaj, Y . P . S., ed.), Springer-Verlag, Berlin, pp. 375-39 1 . 8 . Kubalakova, M., Dolezel, J. , and Lebeda, A. ( 1 966) Plotdy instability of embryo­ genic cucumber callus culture. Bioi Plant 38, 475-480. 9. D'Amato, F. ( 1 952) Polyploidy in the differentiation and function of tissues and cells in plants. Caryologw 4, 3 1 1-358. l 0. Karp, A. ( 1 990) Somaclonal vanation m potato, in Biotechnology 111 Agnculture and Forestry 1 1 (Baja.J, Y. P S., ed ,), Springer Verlag, Berlm, pp. 379-399 1 1 . Cassells, A. C., Walsh, C., and Periappuram, C ( 1 993 ) Diplonttc selectiOn as a positive factor in determining the fitness of mutants of Dianthus "Mystere" derived from x-irradiat10n of nodes in m wtro culture. Euphyt1ca 70, 1 67- 1 74.

42

Curry and Cassells

1 2. D e Klerk, G.-J. ( 1 990) How to measure somaclonal variation Acta Bot. Neerl 3 9, 1 29-1 44. 1 3 Cassells, A. C., Croke, J.T., and Doyle, B. M. ( 1 997) Evaluation of Image analy­ Sis, flow cytometry and RAPD analysis for the assessment of somaclonal vana­ tlon and induced mutation in tissue culture-derived pelargomum plants. Angewandte Bot. 7 1 , 1 25-1 30. 14. Ahloowaha, B . S. ( 1 982) Plant regeneration from callus culture m potato Euphytica 3 1 , 755-759. 1 5 . Sk1rvin, R. M., Lam, S . L., and Jamck, J. ( 1 975) Plantlet formation from potato callus zn vitro Hort. Science 10, 4 1 3 . 1 6 Cassells, A. C and Roche, T. ( 1 993) The influence of the gas permeability of the vessel ltd and growthroom light intensity on the characteristic of Dzanthus microplants m vitro and ex vitrum, in Physwlogy, Growth and Development of Plants and Cells zn Culture (Lumsden, P. J., Nicholas, J.R., and Davies, W. J , eds ), Kluwer, Dordrecht, pp.204-2 14. 17 Dolezel, J., Binarova, P., and Lucreth, S . ( 1 989) Analysis of nuclear DNA content m plant cells by flow cytometry. Bioi Plant 3 1 , 1 1 3-120. 1 8 Do1ezel, J., Sgorbati, S., and Lucretti, S. ( 1 992) Companson of three DNA fluoro­ chromes for flow cytometric estimation of nuclear DNA content m plants. Physzol Plant. 85, 625-63 1 1 9. Cassells, A. C . ( 1 998) Productton of pathogen-free plants, in Bwtechnology m Agriculture (Altman, A., ed ), Marcel Dekker, New York, pp 43-56 20. Sgorbati, S , Levi, M , Sparvoli, E., Trezz1, F., and Lucchmi, G ( 1 986) Cytometry and flow cytometry of 4', 6-diamidmo-2-phenylindole (DAPI)-stained suspen­ sions of nuclei released from fresh and fixed t1ssues of plants Physwl Plant 68, 47 1-476. 2 1 Bennett, M. D . and Leitch, I. J ( 1 995) Nuclear DNA amounts i n Angiosperms Ann. Bot 16, 1 1 3-1 76. 22 Galbraith, D. W., Harkins, K. R., Maddox, J. M., Ayres, N. M , Sharma, D. P., and Firoozabady, E. ( 1 983) Rapid flow cytometric analysis of the cell cycle m mtact plant tissues Sczence 220, I 049-105 1 23. Ulrich, I. and Ulnch, W. ( 1 99 1 ) H1gh-resolutton flow cytometry of nuclear DNA m higher plants Protoplasma 1 65, 2 1 2-2 1 5 24. Dolezel, J. ( 1 99 1 ) Flow cytometric analysis of nuclear DNA content m higher plants. Phytochem. Anal. 2, 1 43-1 54. 25. De Laat, A. M. M., Gohde, W., and Vogelzang, M. J. ( 1 987) Determmat10n of plo1dy of single plants and plant populations by flow cytometry. Plant Breeding 99, 3 03-3 07. 26. Motulsky, H. ( 1 995) Intu1tive Bwstatistics, Oxford University Press 27. Murash1ge, T. and Skoog, F. ( 1 962) A revised mediUm for rapid growth and bw­ assays with tobacco t1ssue cultures. Physiol Plant 15, 473-497

4

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Somatic Embryogenesis in Barley Suspension Cultures Makoto Kihara, Hideyuki Funatsuki, Kazutoshi Ito, and Pau l A. Lazzeri 1 . Introduction

Barley is an important crop both for brewmg and for animal feed. In addi­ tion to conventional breeding, in vitro culture is a useful technology for the improvement of barley quality. For example, a number of methods for barley anther and microspore culture have been published for the production of doubled haploid lines for use m plant breeding and in genetic analysts (1). Recently, novel technologies, such as protoplast fusion and DNA uptake by protoplasts, are being integrated into plant breeding programs (2) . For the uti­ lization of these novel technologies in the molecular breeding of cereals, embryogenic suspension cultures provide a valuable tool, because they are good sources of protoplasts. These allow mampulations at the single cell level, such as direct DNA uptake or somatic hybridization (3). In addition, such sus­ pensions are a suitable target for gene transfer by particle bombardment (4). The establishment of embryogenic suspension cultures has been reported for several graminaceous species. In rice, reliable and efficient protocols for the establishment of suspension cultures have been developed, and transgenic, somatic hybrid, and cybrid plants have been obtained by using protoplasts iso­ lated from embryogenic suspension cells (5, 6). In contrast to the significant progress in rice, barley has been classified as a difficult species for the estab­ lishment of embryogenic suspension cultures (2). In 199 1 , the first successful plant regeneration from barley protoplasts was reported (7). In this work, embryogenic suspension cells, as a source ofregenerable protoplasts, were ini­ tiated from anther-derived calluses. However, these cells lost their regenera­ tion capacity quickly, and the efficiency of plant regeneration was not h1gh From. Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

43

44

Kihara et a/.

Fig. 1 . Different stages in the initiation and establishment of embryogenic cell suspension cultures of barley. (A) Primary calluses on callus induction medium (approx 4 wk after callus initiation). (B) Suspension cells subcultured in modified AA (left) and L l (right) medium. (C) Shoot regeneration from embryogenic structures on regeneration medium. (D) Regenerated plantlet in Magenta box.

enough for genetic manipulation (8). In the last few years, however, with the combination of established transformation techniques (9) and improved sus­ pension culture protocols using immature embryo-derived calluses (I 0, 11; see Fig. 1 ), transgenic barley plants have been produced by direct gene transfer to suspension cell-derived protoplasts (12). Jmprovements in the efficiency of production of embryogenic barley sus­ pensions have been achieved by analysis and optimization of limiting factors at different steps in the overall process, as follows: l . Callus induction. a. Genotype and growth condition of donor plants. b. Developmental stage of explants. c. Callus induction medium. 2. Suspension establishment. a. Subculture technique. b. Culture vessel and subculture medium.

Barley Suspension Cultures

45

3. Plant regeneration. a. Plant regeneration medium. b. Analysis of regeneration ability of suspension cells and colonies derived from protoplasts isolated from suspension cells.

The following protocol has proven very successful in our lab for the estab­ lishment of embryogenic barley suspension cultures. 2. Materials

2. 1. Callus Induction

1. 2. 3. 4. 5 6. 7. 8

Immature embryos, cv. Ign (see Note 1). 70% Ethanol m water. Sodium hypochlorite solution ( 1 .0% active chlorine) in distilled water. Sterile distilled water. Sterile working surface, scalpel, forceps. Stereo mtcroscope. Modified L2 medium (see Table 1) in 60-mm Petri dishes. Culture room (25 °C, 1 6 h hght, 50-I 00 lx ).

2.2. Establishment of Suspension Cultures

1. 2. 3. 4 5 6.

Sterile 100-mL conical flasks (Iwaki Glass, Japan) capped with foil. Sterile macroplates with six wells (Falcon, NJ). Sterile 1 80-mL plastic vessels (Greiner, Germany). Modtfied AA or L 1 medium (see Table 1 ). Rotary shaker. Culture room (25°C, 1 6 h light, 5 0---- 1 00 lx).

2.3. Plant Regeneration

1 . Modified L3 medtum (see Table 1) in 90-mm Petn dishes. 2. Stereo microscope. 3. Hormone-free modified L3 medium in 90-mm Petrt dishes and Magenta boxes (Magenta Corp., IL). 4. Culture room (25°C, 16 h light, 50---- 1 00 lx and 2500----3 000 lx). 3 . Methods 3. 1. Callus Induction

1 . Sterilize immature seeds of donor plants (see Note 1 ) with 70% ethanol for 3 0 s and then with sodium hypochlorite containing 1 .0% active chlorine for 1-3 mm. Rinse these seeds three times with stenlized dtstilled water. 2 . Exctse immature embryos of length 0.5-1 .0 mm (see Note 2) from the seeds under a stereo microscope, and place on modtfied L2 medium (see Table 1 and Notes 3 and 4) in 60-mm Petn dtshes. Incubate these cultures at 25°C, under 1 6 h of dim light (50---- 1 00 lx).

Kihara et a/.

46 Table 1 Composition of Culture Media8

Modified L 1

Modified L2

Modified L3

700 1 750 200 350 450

1 500 1 750 200 350 450

200 1 750 200 350 450

M1cro Salts (mg/L) MnS04 4H20 H3B03 ZnS04 7H20 Kl Na2Mo04 2H20 CuS04 5H20 CoC12 6H20

15 5 1 3 .4 0.75 0 25 0.025 0 025

15 5 7.5 0.75 0 25 0 025 0 025

15 5 7.5 0.75 0 25 0 025 0 025

10 3 2 08 0 25 0.025 0 025

FeNaEDTA (mg/L) Na2EDTA FeS04 7H20

37 28

37 28

37 28

37 28

1 00 10

! 00 10

I

I

1

I

1 00 10 I 1

1 00 10 1 1

2

2

2

1 1 0.4 0.2 0 02 0 01

1 I 04 02 0 02 0 01

1 I 04 02 0 02 0 01

Macro salts (mg/L) NH4N03 KN03 KH2P04 MgS04 7H20 CaC12 2H20 NaH2P04 2H20 KCl •

•

V1tamms (mg/L) Myo-Inos1tol Thmmme HCl Pyndoxme HCl N1cotimc ac1d Ascorbic ac1d Ca pantothenate Cholme chlonde Folic ac1d Riboflavin p-Ammobenzmc acid Bwtm

Ammo ac1ds (mg/L)

Sugars (g/L) Hormone (mg/L)

252 1 50 1 50 2960

Gin 750 Pro 1 50 Asn 1 00

Gin 750 Pro ! 50 Asn 1 00

Gin 750 Pro 1 50 Asn 1 00

Gin 876 Asp 266 Arg HCI2 1 6 Gly 7 5

Maltose 50

Maltose 30

Maltose 30

Sucrose 30

2,4-D 2 0

2,4-D 2 5

BAP 1 .0

2,4-D 2.0

4

A "t

56

5 6

Agarose (g/L) pH

Mod1fied AA

5.6

5 8

aAll media are stenlized by ultrafiltratton For sohd cultures (modified L2 and modified L3 med1a), double-concentrated media are m1xed w1th double-concentrated type I-A (S1gma) agarose solutwn

47

Barley Suspension Cultures 3.2. Establishment of Suspension Cultures

After 3-4 wk of callus induction, transfer the calluses to I 00 mL conical flasks containing 5 mL of modified AA medium (see Table 1) or macroplates with stx wells containing 0.5 mL of modified AA medmm (see Note 5). Incubate the cul­ tures in comcal flasks and macroplates (25°C, under 1 6 h of dtm light) on a rotary shaker at 1 00-1 20 and 80-100 rpm, respectively (see Note 6). 2. Gradually increase the volume (0.3--0.5 mL in conical flasks and 0. 1--0.2 mL in macroplates) of the subculture medium each ttme. Maximum volume of medium is 12 and 2 mL in comcal flasks and macroplates, respectively (see Note 7). 3. In the case of macroplates, after 2-6 mon of subculture, transfer the suspension lines, which have released small cell aggregates, to plastic vessels containing 5 mL of modified AA medmm. Incubate the cultures (25°C, under 16 h of dim light) on a rotary shaker at 80-100 rpm, remove old medium, and replace with fresh medium at intervals of7-l 0 d. Gradually mcrease the volume (0.3--0.5 mL) of the subculture medtum each time Maximum volume of medium is 1 2 mL 4. At each subculture, remove elongated empty or brownish cells, and subculture only cytoplasmically rich and yellow or white cells (see Note 8) -

3.3. Plant Regeneration 1 . Directly plate suspension aggregates drained of suspension medium on 25 mL of modified L3 regeneration medium in 90-mm Petri dishes. 2. Transfer embryogenic structures, which are observed under a stereo miCroscope, to fresh regeneration medium 3-4 wk later, and culture at 25°C under dim hght

until distinct green shoots appear Incubate the green shoots (about 10 mm in length) at 25°C under hghtmg of 25 00-3 000 lx from cold white fluorescent tubes, 1 6 h day length. After 2-3 wk of exposure to tight, transfer the green shoots (20-30 mm length) to hormone­ free modified L3 medium m 90 mm Petri dishes for the induction of roots. 4. Transfer shoots more than ca. 40 mm length to 25 mL hormone-free modified L3 medium in Magenta boxes. 5. After visible root induction, pot the plantlets in sol i (see Note 9). 6. For the detailed analysis of regeneration ability of suspension cultures, isolate protoplasts from suspension cells (see Note 10) and culture (see Note 1 1 ). After about 1 mon of protoplast culture, check the regeneration ability of protoplast­ denved colonies as described above (see Note 1 2). 3

-

4. Notes 1 . Growth conditions of donor plants have a major mfluence on the efficiency of cell suspensiOn establishment and their regeneration ability. In our study, after 2-3 wk from germmation, donor plants (cv. Ign) are vernalized for 6-8 wk at 5 ± 2°C under 2000 lx from cold white fluorescent tubes ( 1 6 h day length), and then grown m a growth chamber ( 1 2-l 6°C, 20,000-30,000 lx from high-intensity discharge lamps, 1 6 h day length). These plants are ready for removal of the tmmature

seeds after 2-3 mon after vernalization These conditions provide much better

48

2.

3.

4.

5

6

7

8.

9.

1 0.

Kihara et a!. results than usmg greenhouse-grown plants (10, 11). However, growth conditions must be optimtzed for each genotype used m the expenments, because the same tendency was not found for another cultivar (cv Dtssa) (11) The developmental stage of immature embryos affects the effic1ency of callus induction and cell suspenswn estabhshment (1 1) Optimal embryo size depends on the genotype, and in Igri, optimal length is 0 5--1 0 mm (I 0-20 d after anthesis) One to three immature embryos should be plated scutellum-side up on 1 0 mL callus induction medmm m 60-mm Petri dtshes (Falcon) All Petn dishes used m this protocol are sealed With Nesco film (Nippon ShOJI, Japan) The embryos with a length of more than 1 .0 mm tend to germmate. The removal of root and shoot meristems from embryos prior to culture prevents germmatwn and pro­ motes callus mductwn Higher concentratiOns ( 1 2 5 and 5 0 mg/L) of 2,4-D m callus mductwn medmm suppress the prohferatwn of callus, resultmg m a lower efficiency of cell suspen­ sion establi shment m cv Ign (11). Modified L 1 medium (see Table 1 ) IS also suitable for suspensiOn establishment In the experiments performed m the years 1 994--1995, 22, 43, and 3 1 cell hues showed consistent proliferation by using modified AA medium m comcal flasks, modified AA mediUm m macroplates and modified L 1 medium m macroplates, respectively Among them, 2, 8, and 4 cell hnes showed green shoot regeneratiOn ability from protoplasts In the case of comcal flasks, mtervals of first and second subculture are approx 14 d. After the second subculture, mtervals are 9-1 1 d. In the case ofmacroplates, mtervals of subculture are 7-1 0 d After 1 mon of transfer to liqmd medmm, suspenswn cel ls consist of elongated empty cells and cytoplasmically rich cells. After a mmimum time of 2 mon and max1mum t1me of6 mon, cytoplasmically nch cells release small cell aggregates Until the release of small cell aggregates, two-thirds to three-quarters of the old medium IS removed and replaced with fresh subculture medium, and suspensiOn cultures are maintamed at a high callus:medmm ratio After the release of small cell aggregates, cultures are gradually brought to a lower density The cell den­ Sity IS finally 4--6 g cells/ 1 0-1 2 mL liqmd medmm SuspensiOn cells subcultured m modified L l medium tend to grow as homog­ enous, yellowish small cell aggregates In contrast, suspenswn cells subcul­ tured in modified AA medmm tend to be heterogeneous and whitish with larger aggregates. Immediately after transfer to pots, regenerated plants are often damaged by con­ ditiOns of low humidity, so these plants are grown m a growth chamber con­ trolled at 1 8°C (20,000 lx from cold white fluorescent tubes, 1 6 h day length) and 80% humidity for 2-3 wk Flowering plants are obtained by the same vernaliza­ tion and growth conditiOns as descnbed for donor plants For protoplast IsolatiOn, incubate approx 2 g of suspension cells (at day 3--6 after subculture) in 20 mL filter-stenlized enzyme solutiOn for 3-4 h at 25°C. The enzyme solutiOn compnses 1 % Cellulase Onozuka RS (Yakult, Japan) and 0 l %

Barley Suspension Cultures

49

Pectolyase Y-23 (Se1shin, Japan) dissolved in LW solution (9) . LW solution con­ tains the macro- and microsalts and amino acids of modified L l medmm and 0.6 M mannitol. 1 1 . Wash the isolated protoplasts three times with LW solutiOn by centnfugation After the last centrifugation, suspend the protoplast pellets in modified L l medium containmg 0.4 M maltose and 1 .8% sea plaque agarose (FMC, ME). Culture these protoplasts with nurse cells (10) Fast-growing suspension cells should be used as the nurse cells. 1 2. The methods for the analys1s of regeneration abihty of protoplast-denved colo­ mes are the same as described for those of suspension cells. These colomes show a rapid response on regeneration medium, and after about 2 wk of transfer, somatic embryogenesis or shoot regeneration can be observed.

References I . P1ckering, A. R. and Devaux, P ( 1 992) HaplOid production: Approaches and

2. 3. 4.

5. 6. 7.

8. 9.

1 0.

11.

12.

use in plant breedmg, in Barley Genetics, Biochemistry, Molecular Biology and Biotechnology (Shewry, P. R., ed.), CAB International, Wallingfold, UK, pp. 5 1 9--547. Vasil, I. K. ( 1994) Molecular improvement of cereals. Plant Mol Bioi. 25, 925-937 Krautwig, B. and Lorz, H ( 1 995) Cereal protoplasts Plant Sc1 1 1 1 , 1-10. Fromm, M. E., Morrish, F., Armstrong, C., Williams, R., Thomas, J., and Klem, T. M. ( 1 990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8, 833-839 Shimamoto, K., Terada, R , Izawa, T., and Fujimoto, H. ( 1 989) Fertile transgemc rice plants regenerated from transformed protoplasts. Nature 338, 274-276. Yang, Z. Q., Shikanai, T., Mori, K., and Yamada, Y. ( 1 989) Plant regeneratiOn from cytoplasmic hybnds of rice (Oryza sativa L.). Theor. Appl. Genet 77, 305-3 1 0. Jahne, A., Lazzeri, P. A., and Lbrz H. ( 1 99 1 ) Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensiOns of barley (Hordeum vulgare L.). Plant Cell Rep 10, 1-6. Lorz, H. and Lazzeri, P. A ( 1 992) In v1tro regeneratiOn and genetic transforma­ tion of barley. Barley Genet VI II, 807-8 1 5. Lazzeri, P. A., Brettschneider, R., Luhrs, R , and Lorz, H. ( 1 991 ) Stable transfor­ mation of barley via PEG-induced d1rect DNA uptake into protoplasts. Theor Appl. Genet. 81, 437-444. Kihara, M. and Funatsuki, H. ( 1 994) Fertile plant regeneration from barley (Hor­ deum vulgare L.) protoplasts isolated from long-term suspension culture. Breed­ ing Sci. 44, 1 57-160. Funatsuk1, H. and Kihara, M. ( 1 994) Influence of primary callus induction condi­ tions on the establishment of barley cell suspensions y1elding regenerable proto­ plasts. Plant Cell Rep 13, 55 1-555 Funatsuki, H., Kuroda, H., K1hara, M., Lazzeri, P. A., Muller, E., and Lbrz, H ( 1 995) Fertile transgenic barley generated by direct gene transfer to protoplasts. Theor. Appl. Genet. 91, 707-7 1 2 .

5

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Somatic Em bryogenesis in Picea Suspension Cu ltu res Ulrika Egertsdotter 1 . Introduction

Somatic embryogenesis is a nonsexual propagation process where somatic cells differentiate into somatic embryos (1). In gymnosperms, somatic embryo­ genesis was first reported in Picea abies (2,3), but has now been induced in a variety of cycads and comfers (4). The development of somatic embryos 1s similar in different coniferous species, and in pnnciple, it resembles their zygo­ tic counterparts during development. The technique of multiplying conifers vta somatic embryos offers many advantages, both in the mass propagation of selected genotypes and as a part of breeding programs. Some of the advantages of somatic embryogenesis are: the somatic embryos can be propagated on a large scale in bioreactors, a high yieid of plants can be obtained in a short time, the embryos already have a tap root, the embryos can be encapsulated and treated like seeds, true rejuvenation can be obtained even if the somatic embryos are regenerated from mature trees, and the somatic embryos can be cryopreserved (5). By using somatic embryos in breeding programs, it is possible to keep simultaneously all genotypes cryopreserved until valuable clones have been identified in field tests. The cryopreserved material can then be mass propagated. Somatic embryos from conifers have been stably transformed using micro­ projectile bombardment (6-8) . Also, several Agrobacterium strains are capable of infecting conifers and forming tumors and roots, but the regeneration of plantlets from transformed embryos has been less successful. The fi rst transgenic conifer (Picea glauca) was obtained after the bombardment of mature somatic embryos. Embryogenic cultures were subsequently established and plants regenerated (9). From· Methods In Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

51

52

Egertsdotter

The regeneration of plants from somatic embryos m P1cea can be divided mto four main steps: Imtiation of somatic embryos from the imtlal explant, proliferation of the embryogemc cultures, maturation ofthe somatic embryos, and regeneration of plants from the somatic embryos. Embryogemc cultures have been successfully mitiated and proliferated in several different P1cea spe­ cies (4). However, the maturation and regeneration steps still contain many difficulties, and are at present areas of mtensive research. Initiat10n: Embryogenic cultures can be mduced from seedlings, and both immature and mature zygotic embryos. It has also been imphed that embryo­ genic cultures can be imtiated from more mature tissue (10). The different explants have been successfully used within different species (4) . The initia­ tiOn frequency iS genotype-dependent, and 1t dechnes with the mcreasing age of the primary explant. The cells in the pnmary explant are stimulated to form somatic embryos on a medium contaming auxin and cytokimn. The LP medmm (II; Table l) has been successfully used for the imtiatwn and subsequent proliferation of sev­ eral Picea species (Table 2). Proliferation: The somattc embryos formed on the mitiatwn medmm con­ tmue to proliferate when transferred to fresh medmm. The embryos can then, after a few subculture mtervals, be transferred to growth in hqmd medium At the prohferat10n stage, embryos withm different cell hnes reach different developmental stages, which IS reflected in the number of cells, both m the embryonic and suspensor regions, and by the degree of orgamzation of the cells mto a polarized structure. The embryogenic cell lines appear to be of two mam types, A and B. Embryos in type A cell lines are composed of densely packed, large embryomc regions and many suspensor cells, whereas in type B, they are composed of small, loosely packed embryonic reg10ns and few sus­ pensor cells (12; Fig. 1). In general, the embryos in suspension cultures have a less polanzed morphology than embryos grown on sohd medmm. The embryo morphology IS parttally dependent on the hormonal balance present m the medium (13) . Also, different proteins are secreted mto the medium in suspension cultures of types A and B, which influence the develop­ mental stage of the embryos under proliferatiOn (14). Changmg the hormonal balance, or addmg condit10ned medmm does not change the original type of embryo in the cell line . However, embryo morphology can be strongly Improved by adding extracts of mature seed (15). The seed extracts stimulate the formation of larger embryomc regwns, more suspensor cells, and a more polanzed embryo. The size of the embryonic reg10n and how polanzed the embryo ts are both important characters for the subsequent maturation process Maturatwn· In the maturatiOn process, the somatiC embryos stop proliferat­ mg, mcrease in size, and start to accumulate storage matenals, includmg starch,

53

Picea Suspension

Table 1 Components of 1/2 LP Mediums for Initiation and Proliferation of Plcea

Component 1 . KN03 2. NH4N03 3 . MgS04 · 7 H20 4. KH2P04 5 CaCI2 2 H20 6. Micronutrientsh 7. Amino acidsc 8 Fe-EDTA 9. Pyridoxine-HC1 1 0. Nicotinic acid I I . Glycine 1 2. 2, 4-Dd 1 3 . BN 1 4. Thiamine-HC1 1 5 Myo-inosito1 1 6 o-G1ucose 1 7 . o-Xylose 1 8 . L-Arabinose 1 9 Glutamine 20. Sucrose 2 1 . Ge1ntef •

Concentration m stock, g/L 95 60 37 34 44

0.07342 0.0 1 0 0.020 0.020 0 10 0. 1 0

Volume stock, mL, for 1 L of final medium 10 5 5 5 5 0.5 10 1 25 0.5 05 0.5 10 5

Concentration m final medmm, mg/L 950 3 00 1 85 1 70 220

6.95 0.5 1 .0 I 0 1

0.5 25 50 90 75 75 500 1 0 X 1 03 3 4 X 10

acu1ture medium 112 LP: M1x all components m water, adjust to pH 5 8, and autoclave Stock solutions nos. 1-1 1 can be stored at +4°C for several months, hormonal stocks are stored at -20°C and kept at +4°C for a maximum of 4 wk. Prepared medmm is stored at +4°C for a maxi­ mum of4 wk h1 000X Micronutrient stock· Zn-EDTA (4. 7 1 6 g/L), MnS04 2 H20 (2 23 g/L), H3B03 (0 63 g/L), Na2Mo04 2 H20 (0 025 giL), CuS04 5 H20 (0 0025 g/L), CoC12 6 H20 (0 0025 g/L), KI (0 7 5 g/L) '50X Ammo acid stock. L-glutamme (0.02 g/L), L-a1amne (0 0025 g/L), L-cysteme HC1 (0.00 I g/L), L-argmine (0 0005 g/L), L-leucine (0.0005 g/L), L-phenylalanine (0 0005 g/L), L-tyrosme (0 0005 g/L) d2, 4-Dichlorophenoxyacetic acid. "N6-benzyladenine fGelnte IS a trademark of Merck and Co , Inc. Rahway, NJ, Kelco DIVISion USA Gelnte IS used in mit1ation medium or in sohd proliferation medmm For standard purposes, 25 mL of gelrite medmm IS poured/1 0-cm Petri plate.

proteins, and lipids (16). The ability of the somatic embryo to mature varies significantly among dtfferent cell lines, and this appears to be closely corre­ lated to embryo morphology during the proliferatiOn stage. In general , type A

Egertsdotter

54 Table 2 Reported Variations in Culture Requirements Within Picea Species

P abzes

P engelmannz P glauca

P glauca-engelmanm P glehnz P Jezoenszs P manana

P P P P

omonka pungens rubens sztchensis

lmtial explant0

M ed mm , InitiatiOn and proliferatiOn

IE

1 12 LP

ME

1 /2 LP

s

1 12 LP

IE IE MEe s IE MP ME ME IE ME s s ME ME IE ME

LP LP LP LP LP LP Lepmvreh Lepmvre LP 1 /2 LMC

LP LP LP 1 /2 MSd 1 /2 LP

0lE, munature embryos, ME, mature embryos, S, seedling matenal "Lepmvre medmm (24). 'LJtvays medmm (25). dMurash1ge-Skoog medmm (26). ermtJatwn m light

cell lines produce many mature embryos, whereas type B cell lines seldom produce mature embryos. Aberrant mature embryos are common among most cell lines, and these embryos often fail to form a tap root. The embryos are stimulated to mature by abscisic acid (ABA) (1 7) and a raised osmotic potential (18) . The medmm used for maturatiOn IS Similar to that used for proliferation, but With different growth regulators, e.g., ABA, a raised osmotic potential and, in a few cases, auxm and/or cytokmm. Embryo­ gemc cultures of comfers have until now been most commonly grown on a sohdlfied medmm, with the maturatiOn treatments, as reported for different species, generally bemg developed for solid grown cultures. However, meth-

55

Picea Suspension

A

B

Fig l . Schematic drawmg of the mam types of somatic embryos that appear in

suspension cultures of Picea. (A) Type A somatic embryo, (B) Type B somatic embryo

ods for successful maturation and regeneration from suspension cultures have been reported for P. glauca (18, 1 9), P. abies and Picea sztchensis (20,21) , Picea mariana (22), and Picea g/auca-enge/manni (23). Basically, the suspen­ sion is successively transferred to a prematuratwn medium lacking auxm and cytokmin. The embryos are then collected and transferred on a filter paper, to a medium containing ABA and a raised osmotic potential. The concentration of ABA reqmred for maturation to start differs depending on the species and cell lines bemg used. Sucrose and/or polyethylene glycol (PEG) are commonly used to raise the osmotic potential. To improve further development, the mature embryos can then be partially desiccated. In P. abies, between 50 and 400 mature embryos can be producedlg of embryogenic tissue. Regeneration: Plantlet regeneration from mature embryos ts routme in many laboratones, although for a limtted number of cell hnes. A common problem ts that the mature somatic embryos can only form cotyledons and a hypocotyl, but not a root. The mature embryos are initially imbibed on a solidtfied medmm under low light intensity. As the root develops, the plantlets can be transferred m an upright position to a vermiculite medium, then to nonsterile conditions m growth chambers, then to greenhouses, and finally to the field. Regenerated plants have been extensively compared to seed-derived plants. In field tests, P. abies plants derived from somatic embryos and from seeds flowered at the same time after 1 0 growth seasons. Suspension cultures offer many advantages over solid-grown cultures, both in pure and applied research. Studies on the developmental regulation of pro­ liferating somatic embryos provide information on embryo development m general and are also necessary to increase the number of genotypes that can be used for regeneration. The response of the somatic embryos to different envi­ ronmental stimuli are best studied in suspension cultures as all embryos are

56

Egertsdotter

exposed to the same condthons. Studies on thetr response to dtfferent com­ pounds are easily undertaken, and samples removed from the suspension should represent the whole culture. For the apphcation of somattc embryogenesis to forestry, suspenston cultures usmg bioreactors ts a prereqUisite for future, large­ scale plantlet production. The following descriptions on tmtiation and proliferatiOn are based on pro­ tocols optimized for P abzes. There are at present no general methods for matu­ ration and regeneration that are likely to be successful m all Pzcea spectes and genotypes. Therefore, it is advisable to find current publicatiOns on the spectes of mterest, whtch wtll then allow the development of smtable methods for maturatiOn and regeneratiOn of whtchever cell hnes are bemg used. The time schedule for different steps involved may vary dependmg on the species and cell hne. It ts therefore important to follow carefully the growth and development of the embryos at the different stages. Imttation and proliferation of embryogenic tissue. a Stenhze seeds b. Imbibe seeds. c Isolate zygotic embryos, and tmtlate embryogemc tissue: 4--8 wk. d. Isolate embryogenic tissue from tmtial explant and subculture onto fresh medmm. e Proliferate on sohd medmm 4-8 wk 2 Start of suspension culture· a. Subculture mto small aliquots and proliferate: 2-4 wk b Transfer to, and establish growth in, liquid medmm 4-1 2 wk

2. Materials I . Commercial bleach, Tween, and sterile water. Flow cabinet, forceps, and scalpel 3. Seeds (see Note 1). 4 Dissectmg microscope 5. Petn plates containing proliferatiOn medmm (Table 1). 6 Erlenmeyer flasks (see Note 2) contammg proliferation medmm (Table 1 ) 7 Wide-mouthed pipets (see Note 3) 2

3. Methods

3. 1. Initiation of Embryogenic Tissue 1 . Stenhze the seeds by soakmg them m 1 5 % commercial bleach (6% [v/v] sodmm hypochlonte) with a few drops of Tween for 1 5 min with agitatiOn Remove the bleach solution, and rinse the seeds with plenty of stenle water (see Note 4) 2 Imbibe seeds overnight m distilled water at +4°C in darkness.

3. Isolate the zygotic embryos wtth a scalpel and forceps under a dissectmg micro­ scope. Put the embryos gently onto the proliferatiOn medium, making sure that they are m good contact with the medmm Seal the plates with stnps of cling film.

Picea Suspension

57

4. Leave the plates at room temperature under darkness. Subculture embryos onto fresh medium at least once a month. Continuously check for the formatwn of embryogenic tissue (see Note 5). When the somatic embryos have reached a size of 5 mm, the embryogenic ttssue can be isolated and transferred onto fresh medium with sterile forceps. Avoid transferring the mttial explant. 5. Subculture onto fresh medium at 2-4 wk intervals. 6. Take samples of the somatic embryos, and examine under a microscope to deter­ mine the morphology of the proliferating embryos (Fig. 1).

3.2. Establishing Suspension Cultures 1 . Two to 4 wk after the last subculturing, when new embryogenic tissue has been formed, carefully remove small (3-mm) pteces of embryogenic tissue from the outer part of the embryogenic mass (see Note 6). Place I 0 pieces on single Petri plates containing 25 mL of fresh, solid proliferatiOn medium. Subculture onto fresh medium every 2 wk until new embryogenic tissue has formed. 2. Remove the embryogenic tissue masses from the Petri plate, and place them m 250-mL Erlenmeyer flasks containmg 20 mL of hquid proliferation medium. Incubate on a rotary shaker at 50 rpm m darkness. 3 . After two weeks, add 5 mL of fresh medium. (see Note 7). 4. After another two weeks, let the embryos settle out, use a wtde-mouthed pipet to move the loosened embryos to a 250-mL Erlenmeyer flask containing 25 mL of proliferation medium, and incubate on a rotary shaker at I 00 rpm. A void trans­ ferring the initial embryogemc tissue mass. 5. Subculture the embryos to fresh medmm every week (see Note 8). Transfer 1 0 mL of the embryos to 50 mL of freshly made medium as described above. If growth and embryo morphology (see Note 9) are unsatisfactory, add mature seed extract (see Note 10).

4. Notes 1 . Mature seeds are generally the most accessible and therefore the most commonly used as initial explant. However, the initiation frequency from immature seed ts generally higher. 2 Glass or plastic Erlenmeyer flasks can be used for suspension cultures. When establishing suspension cultures, baffleflasks can be used to facilitate loosening of the embryos from the initial masses. All flasks should be capped wtth cotton plugs (optional) and alummum foil. 3 . Measuring pipets with wide mouths allow the transferring of the embryos wtth less damage, and they can be necessary to enable the transfer of larger embryos. 4 The seeds can be sterilized and rinsed m a Petri plate. If sterility is a problem, the seeds can be stenlized in a presterile Erlenmeyer flask. The seeds can then be poured onto a sterile nylon mesh, and rinsed while on the mesh. 5. The embryogenic tissue is white to translucent m appearance Under the dissecting microscope, the embryos can be seen to branch out from the ini­ tial explant.

Egertsdotter

58

6 The embryogemc tissue is growing most actively on the penphery. Only transfer pieces of tissue from this area; rapidly proliferating embryos are more easy to establish in liqmd medium. 7 The formation of new embryos m the suspensiOn culture can stop tf the suspen­ sion IS diluted too much. However, a too high density will cause brownmg of the culture and eventually death of embryos. The optimal cell density vanes between cell hnes, and depends on the proliferatiOn rate and the type of embryo To become fam1har with cell line reqmrements, it IS advisable to view samples of the suspension under the microscope to determme the v1abihty of the embryos present. 8. Dependmg on the type of embryos m the cell line, different subcultunng mter­ vals should be employed. Generally, type A reqmres subculturing weekly, whereas type B IS insensitive to prolonged subculturing mtervals and can be left for months in the same medium without loss of viability 9 Cell lmes composed of large, well-developed embryos (type A) can be difficult to establish as suspension cultures, whereas cell lmes composed of small, less developed embryos (type B) are easy to establish. Embryos cultured m liqmd medium generally develop fewer suspensor cells and tend to loose their polanzed appearance. 10. Seed extracts can be prepared from mature seed by grindmg 1 0 g of seed m a food mixer together with 1 00 mL of proliferation medium Larger debns IS removed by filtering through cheesecloth or M1racloth. The extract IS filter-ster­ Ilized mto the suspensiOn cultures to a concentration eqmvalent to 50-I 00 mg of seeds/mL of medmm. The treatment can be repeated over a few subculturing mtervals, but this seems to be detnmental If applied for longer periods The seed extract stimulates the growth and development of somatic embryos of both type A and B. Additionally, the frequency of the formatton of mature embryos 1s increased dramatically m all cell hnes tested.

References

2 3

4 5.

Bajaj , Y. P S ( 1 995) Bzotechnology zn Agrzculture and Forestry 30 Somatic Embryogenesis and Synthetic Seed I Sprmger Verlag, Berlm Hakman, I. and von Arnold, S. ( 1 985) Plantlet regeneration through somatic embryogenesis m P1cea ab1es (Norway spruce). J Plant Physzol 1 2 1 , 149-1 58 Chalupa, V. ( 1 985) Somatic embryogenesis and plantlet regeneratiOn from cul­ tured Immature and mature embryos of Pzcea ab1es (L ) Karst. Commun Inst For. 14, 57-Q3. Jain, S., Gupta, P , and Newton, R ( 1 995) Somatic EmbryogeneSIS m Woody Plants Kluwer Academtc Publishers, Dordrecht, The Netherlands. Norgaard, J , Duran, V., Johnsen, 0., Krogstrup, P , Ba1durson, S . , and von Arnold, S. ( 1993) Vanatwns m cryotolerance of embryogemc P1cea ab1es cell hnes and the assoctatwns to genetic, morphologtcal and physwlogical factors Can J For Res 23, 2560--2 567.

Picea Suspension

59

6 Robertson, D., Weissmger, A. K., Ackley, R , Glover, S., and Sederoff, R. R ( 1 992) Genetic transformatiOn of Norway spruce (Picea abzes [L.] Karst) using somatic embryo explants by mtcroprojecttle bombardment. Plant Mol Bzol 19, 925-93 5. 7 . B ercetche, J., Dinant, M., Matagne, R. F., and Paques, M. ( 1 994) Selection of transformed embryogenic tissues of Picea abies (L.) Karst after microprojectile bombardment, in Abstracts Vlllth International Congress ofPlant Tissue and Cell Culture, Firenze, June 1 2-17, 1 994. 8 Charest, P. J., Lachance, D., Devantier, Y , and Klimaszewska, K. ( 1 993) Tran­ sient gene expression and stable genetic transformation in Picea mariana (black spruce) and Larix laricines (tamarack), in: Abstracts Fifth International Work­ shop, IUFRO Working Party 82.04-{)7, Spain 9. Ellis, D. D., McCabe, D E., Mcinnes, S., Ramachandran, R., Russell, D. R., and Wallace, K. M. ( 1 993 ) Stable transformation of Ptcea glauca by particle accelera­ tion. Bio/Technology 1 1 , 84-89. 1 0. Westcott, R. J. ( 1 994) Production of embryogenic callus from nonembryogenic explants of Norway spruce (Picea abies [L.] Karst). Plant Cell Rep. 1 4, 47-49 I I von Arnold, S and Eriksson, T ( 1 98 1 ) In vitro studies of adventitious shoot for­ mation in Pinus contorta Can J Bot 59, 870-874. 1 2. Egertsdotter, U. ( 1 996) Regulation of somatic embryo development in Norway spruce (Pzcea abies). Doctoral thesis, Swedish Umversity of Agncultural Sci­ ences, Uppsala. 1 3 . Bellarosa, R , Mo, L. H., and von Arnold, S ( 1 992) The mfluence of auxm and cytokinin on proliferation and morphology of somatic embryos of Picea abzes (L.) Karst. Ann. Bot 10, 1 99-206 1 4. Egertsdotter, U., Mo, L H., and von Arnold, S. ( 1 993) Extracellular protems m embryogenic suspension cultures ofNorway spruce (Pzcea abies). Physwl Plant 88, 3 I 5-3 2 l . 1 5 . Egertsdotter, U and von Arnold, S. ( 1 995) Importance of arabmogalactan pro­ teins for the development of somatic embryos of Norway spruce (Pzcea abzes) Physiol. Plant 93, 334-345 1 6. Hakman, I. ( 1 99 3 ) Embryology in Norway spruce (Pzcea abzes) An analysts of the composition of seed storage protems and deposition of storage reserves during seed development and somatic embryogenes is. Physwl Plant 81, 1 48- 1 5 9 . 1 7 von Arnold, S . and Hakman, I. ( 1 988) Regulat10n o f somattc embryo develop­ ment m Picea abies by absctstc ac1d (ABA). J Plant Physzol. 132, 1 64-1 69. 1 8 Attree, S. M., Pomeroy, M. K., and Fowke, L. C ( 1 995) Development of whtte spruce (Pzcea glauca [Moench.] Voss) somatic embryos during culture wtth abscisic acid and osmoticum, and thetr tolerance to drymg and frozen storage. J Exp. Bot. 285, 433-439 I 9. Attree, S. M., Pomeroy, M. K., and Fowke, L C. ( 1 992) ManipulatiOns of condi­ ttOns for the culture of somatic embryos of whtte spruce for 1mproved tnacy­ lglycerol bwsynthests and des1ccat10n tolerance. Planta 187, 395-404

60

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20. Krogstrup, P ( 1 990) Effect of cellular densities on cell proliferation and regen­ eration from embryogemc cell suspensions of Pzcea abies. Plant Scz 72, 1 1 5-123 2 1 . Krogstrup, P., Enksen, E. N., M0ller, J. D., and Rou1und, H. ( 1 988) Somatic em­ bryogenesis m s1tka spruce (Pzcea sztchenszs [Bong ] Carr.). Plant Cell Rep. 7, 594--597. 22 Tautorus, T. E., Lulsdorf, M. M., Kikc10, S. I , and Dunstan, D. I. ( 1 992) Bw­ reactor culture of Pzcea manana Mill (black spruce) and the spectes complex Pzcea glauca-engelmannu (intenor spruce) somatic embryos Growth parameters. Appl M1crobwl Biotechnol 38, 46--5 1 23. Tautorus, T. E., Lulsdorf, M M , Ktkcw, S. I., and Dunstan, D. I. ( 1 994) Nutnent utilization dunng bwreactor culture, and maturation of somatic embryo culture of Pzcea manana and Pzcea glauca-engelmannu. In Vitro Cell Dev Bwl 30, 58-65. 24. Aitken-Christte, J. and Thorpe, T A. (1 984) Clonal propagatiOn. gymnosperms m Cell Culture and Somat1c Cell Genet1cs ofPlants (Vas1l, I. K., ed.), Academic, London, pp 82-95 . 25. L1tvay, J. D , Johnson, M. A., Verma, D , Emspahr, D., and Weyrauch, K ( 1 98 1 ) Conifer suspensiOn culture medmm development usmg analytical data from developmg seeds. !PC Techmcal Paper Serzes 1 15, 1-1 7. 26 Murash1ge, T. and Skoog, F. ( 1 962) A rev1sed medmm for rap1d growth and bto­ assays with tobacco t1ssue cultures Physwl Plant. 15, 473--497.

6

------

Direct Cyclic Somatic Embryogenesis of Cassava for Mass Production Pu rposes Krit J. J. M. Raemakers, Evert Jacobsen, and Richard G. F. Visser 1 . Introduction

Cassava (Manihot esculenta Crantz) is a perennial shrub of the same family, the Euphorbiaceae, as castor bean (Ricinus communis) and rubber tree (Hevea brasiliensis). It is grown for its starch-containing tuberized roots, which are used for human consumptiOn, as animal feed, and as raw material for the starch industry. The centers of diversity are in central and North eastern Brazil, south­ western Mexico, and eastern Bolivia (1). Cassava is also grown on the African and Asian continents (2). The average yield in the world is 8.8 tonnes/hectare (3), which is only a fraction of the potential y1eld of90 tonnes/hectare (4). One of the reasons for this low yield is the use of cuttmgs infected with diseases and pests as the starting material. Meristem culture (5, 6) 1s used to produce healthy planting material (see Note 1 ), and multiple shoot culture (7) 1s used to produce large numbers of healthy plants. However, this multiple-shoot culture technique requires several tissue­ culture steps (mechanical isolation of cuttings, rooting of cuttings, and harden­ mg of plants), which makes the procedure labor-intensive. Somatic embryos have the developmental program to grow into complete plants wtthout mechan­ ical isolation and separate shooting and rooting steps (8). Therefore, the multi­ plication of planting material by somatic embryogenesis would reduce labor input significantly. In cassava, both a direct and indirect form of somatic embryogenesis exists. In the direct form, the somatic embryos develop directly into mature somatic embryos with large green cotyledons (Fig. lA-D). In the indirect form of somatic embryogenesis, also called friable embryogenic callus, the embryogenic propaFrom Methods m Molecular Biology, Vol 111 Plant Cell Culture Protocols Edited by A D Hall © Human a Press Inc , Totowa, NJ

61

Raemakers, Jacobsen, and Visser

62

gules do not develop beyond the preglobular stage, but mstead break up mto new embryogemc propagules (see Note 2). Both types of somatic embryogenesis start wtth the culture ofleafexplants on Murashige and Skoog (9) medmm supplemented wtth auxms (10-1 7). Clumps of globular embryos (Fig. lA) are formed after 1 0-- 1 4 d of culture. Transfer of these clumps wtth globular embryos to a Greshoffand Doy medmm (18) supple­ mented wtth auxms mitiates fnable embryogenic callus (16), and transfer of these clumps to a Murash1ge and Skoog (9) medmm without auxins allows the globu­ lar embryos to develop mto mature somatic embryos (Fig. lB; 10-15, 1 7) Somatic embryogenesis can only be used for plant multtplicatlon tf there IS an efficient system of plant regeneration. In the dtrect form of somatic embryo­ genesis, mature embryos germinate effictently into plants (Fig. lE; 12, 19,20). In the indtrect form, the fnable embryogemc callus should first produce mature somatic embryos (maturatiOn) before they can be germmated. However, because maturation occurs at very low frequencies (16, 20), friable embryo­ gemc callus formatiOn cannot be used for efficient plant multiplication. Another advantage of direct somatic embryogenesis m cassava Is that mature somatic embryos are tdeal explants for direct secondary/cyclic somatic embryo­ genesis, thus allowmg the production ofmtlhons of propagules (11, 19,21-25) . For thts, mature somatic embryos are chopped mto small pteces and are sub­ cultured m a medmm supplemented with auxms. Both hqmd and sohd medmm can be used. Dependmg on the auxm used, after 2-4 wk, new mature embryos are formed, which can then be used for a new cycle of secondary somatic embryogenesis (see Note 3). This chapter descnbes methods to tmtiate somatic embryogenesis in ehte genotypes of cassava, the subsequent multiplicatiOn of the embryogemc tissue by secondary/cyclic somatic embryogenesis, the germination of somatic embryos into plants, and finally, the transfer to the greenhouse. An overvtew of the whole procedure IS given m Table 1 . 2. Materials 2. 1. General Requirements 1

Rotary shaker ( 1 20 rpm)

2 Sterile 300-mL Erlenmeyer flasks, capped wtth alummum fOil 3 . Stenle tubes and Petn plates 4 Bottles (0 5 , I L) that can be autoclaved 5 Microwave oven to dtssolve sohd medmm. 6 pH meter. 7. 1 M KOH and 0. 1 M HCl for setting pH of the medium 8 Pipet ( 100---1 000 1-1L) w1th stenle tips to add stock solutiOns of growth regulators to the medmm

Fig. 1. Somatic embryogenesis in cassava (M. esculenta Crantz): (A) formation of globular somatic embryos on leaf explant cultured for 10 d on solid MS2 + 8 mg!L 2,4-D medium, (B) formation of mature somatic embryos on leaf explant cultured for 21 d on solid MS2 + 8 mg/L 2,4-D medium followed by 10 days on solid MS2 + 0.1 mg/L BAP medium, (C) scanning electron microscopy of the formation of secondary somatic embryos formed on primary somatic embryo cultured for 15 d on solid MS2 + 8 mg/L 2,4-D medium, (D) mature cyclic somatic embryos maintained in liquid MS2 + 10 mg/L NAA medium, (E) germinated cyclic somatic embryos (induced in liquid NAA suppl� mented medium and cultured after desiccation on solid MS2 + 0,1 mg/L BAP medium).

Raemakers, Jacobsen, and Visser

64

Table 1 Characteristics of the Culture Procedures Used in Somatic Embryogenesis in Cassava Transfer penod, wk

Medtum

Step Transfer of plants to m vitro Mul ttph cation of plants

Genotype dependencya 28/28

3-25

MS2

28/28

IsolatiOn of leaf explants Pnmary somatic embryogenesis Secondary/cyclic somatic embryogenesis establishment mamtenance

14,27 MS2 + 8 mg/L 2,4-Dh MS2+0 1 mg/L BAh

3 2-3

pt MS2 2nd. MS2 pt MS2 2"d MS2

or

+ 8 mg/L 2,4-Dh + 0 1 mg/L BAh + 8 mg/L 2,4-D' + 0 I mg/L BA'

MS2 + 10 mg/L NAN

Germmatwn

Refs

MS2 + 0 1-1 0 mg/L BAh

24/28

15,25

1 -2 3 1-2

1 9/20

15,25

1 9/20

15,25

2

1 3/20

19

717

19,20

0Genotype dependency: number of successful genotypes/number of tested genotypes bQnly sohd medmm can be used 'Both sohd and hquid med1a can be used

9. 1 0. 11. 12. 13

14. 1 5. 16 17 18 1 9. 20.

1 00-mL volumetric flask, for preparation of stock solutiOns of the growth regulators 0. 2-!lM filter units for stenlizat1on of stock solutiOns Dimethyl sulfoxide for dissolving the growth regulator ptcloram. 1 M NaOH for dissolvmg the growth regulators NAA, 2,4-D, and BAP. Sodmm hypochlonte solut10n ( 1 % free chlonne) and stenle water for stenhzatlon of plant matenal Scalpel and forceps Bmocular microscope for Isolation of explants. Sterile transfer facllttles. Refngerator for storage of stock solutions Growth chamber temperature 30°C, day length of 1 2 h, h ght mtens1 ty of 2 40 11mo l lm /s. Rock wool plugs. Sterile sml and an mcubator for transfer of plants from m vttro to the greenhouse.

65

Somatic Embryogenesis Table 2 Preparation of Stock Solutions of Growth Regulators

Desired concentration Powder for 1 00 mL Solvent Storage of stocks

BAP

NAA

Picloram

2,4-D

I mg/mL I OO mg I M NaOH 4°C

10 mg/mL 1 000 mg I M NaOH -20°C

10 mg/mL 1 000 mg DMSO -20°C

8 mg/mL 800 mg I M NaOH -20°C

2.2. Plant Materials and Culture Media I . Cassava plants grown in the greenhouse. 2. Distilled water. 3 . Preparation of stock solutions of the growth regulators (details for each growth regulator are gtVen m Table 2): Add growth regulator to a 1 00-mL volumetnc flask, dissolve the powder in a 0.2-25 mL of solvent, fill volumetric flask to 100 mL with disti lled water, sterihze solution by filtering it through a double 0.2-f.!M fi lter unit, dispense sterile solution in 1 -mL Eppendorf tubes and store Eppendorf tubes. 4. Preparation ofMS2-medium: Add 800 mL to 1 -L bottle, and dissolve Murash1ge and Skoog (9) basal salts and vitamins, and 20 g sucrose Set pH to 5. 7 by addmg droplets of I M KOH or 0. 1 M HCI, fill bottle to 1 L, autoclave it for 20 min at 2 1 2 ! °C and 1 .2 kg/cm • In the case of solid medium, 8 g/L agar IS added after setting the pH . After sterilization, the bottles are stored m a dark cabmet. Before use in tissue culture, the solid medium IS melted in a microwave oven, and growth regulators are added. The volume of the stock solution added to the medium can be calculated using the following fonnula:

[Concentration of growth regulator in medium (mg/L) x volume of the medium (L)]/concentration of stock solution (mg/mL)

=

volume of stock solution required for medmm (mL)

(l)

The concentration of stock solutions is chosen, in such a way that in most cases, I mL is needed for I L of MS2 medmm. An exception is the MS2 + 6 mg/L NAA and 6 mg/L Picloram medmm used for primary somatic e mbryogenesis by Tay­ lor (personal communication): 0.6 mL of both 1 0 mg/mL NAA and 1 0 mgimL Picloram are added to 1 L of MS2 medium. 3. Methods

3. 1. Transfer of Plants to In Vitro and Multiplication 1 . Isolate the upper single nodes ( 1-3 em) from healthy and vigorously growing plants, preferably on a warm and sunny day.

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2. Transfer each node to one tube wtth a 1% solution of sodium hypochlorite (20 min). Wash each node three times wtth sterilized, distilled water. 3 Remove discolored parts of the stem and transfer nodes to tubes with solid MS-2 medium. 4. Multiply plant matenal m-vitro every 3 wk by smgle-node cuttmgs until there are about 30 smgle plants.

3.2. Isolation of Leaf Explants and Culture for Primary Somatic Embryogenesis Transfer single node cuttmgs to IIqmd MS2 medium The plants are kept upnght using rock wool plugs. After 12 d of growth, add 2,4-D to the liqutd medium to a final concentration of 8 mg/L The shoot tips are harvested 2 d later 2. Under a bmocular microscope, remove leaves larger than 4 mm from the shoot tips. Isolate smgle leaves smaller than 4 mm (tf posstble, dtvtde them mto smgle leaf lobes) and the remams of the shoot ttp and culture these m the dark on sohd MS2 + 8 mg/L 2,4-D medmm (see Notes 4 and 5). 3 After 1 4-2 1 d transfer the explants to solid MS2 + 0 1 mg/L BA medium m the light, and subculture every 2-3 wk. 4. Cut offwtth a scalpel mature pnmary somatic embryos. Mature somatic embryos possess large green cotyledons (Fig. 1 8-D) 1

3.3. Establishment and Maintenance of Secondary/Cyclic Somatic Embryogenic Cultures 2 I . Cut with a scalpel the mature pnmary somatic embryos mto fragments of 1 -6 mm , and culture on solid MS2 + 8 mg/L 2,4-D medmm for 20 d. Then transfer to soltd MS2 + 0 I mg/L BA medtum 2. Harvest agam the mature somattc embryos, cut mto small fragments, and culture 0.2 g m 300-mL Erlenmeyer flasks contammg 50 mL of liquid MS2 + 8 mg/L 2,4-D or 1 0 mg/L NAA medtum The Erlenmeyer flasks are cultured m the light on a shaker at 1 20 rpm. 3 After 2 1 d, transfer the matenal cultured m 2,4-D-supplemented medtum to IIq­ md MS2 + 0. 1 mg/L BA medtum (see Notes 6 and 7) 4 Every 1 4 d (NAA-supplemented medmm) or 30 d (2,4-D-supplemented medium) mature somattc embryos can be Isolated, and the procedure descnbed m steps 1 , 2, and 3 can be repeated 3.4. Germination of Cyclic Somatic Embryos

1 . Mature somattc embryos mamtamed m NAA-supplemented medmm can be used directly for germmatton (see, for example, Fig. 1 D). Mature embryos mamtamed m 2,4-D-supplemented medmm must be multtphed first for at least I mon m NAA-supplemented medmm (see Note 8) 2 Dry the mature embryos first on sterile filter paper in the flow hood. After that, transfer them to an empty stenle Petn dtsh for desiccatiOn m the growth cabinet. The hd of the Petn dtsh ts changed every other day to remove condensed water.

Somatic Embryogenesis

67

3. After 4-8 d, well-desiccated mature embryos are cultured in the light on solid MS2 medium supplemented with 0. 1-1 mg/L BA (see Note 9). 4. The mature somatic embryos will first form roots and then shoots. Depending on the quality of the roots, transfer the plants directly to the greenhouse (for example, plants shown in Fig. lE) or after one round of multiplication on solid MS2 medium.

3.5. Transfer of Plants to the Greenhouse Plantlets, 2-4 em tall, can be transferred to the greenhouse. Remove the plantlets carefully from the tubes or containers, and wash away the agar from the roots thoroughly. 2. Grow plants in pots ( l :3 sterilized mixture of soil with fine sand), and keep at high humidity in an incubator. After 1 wk, the plants can be acclimatized to the greenhouse conditions by gradual reduction of the humidity 4. Notes

1 . Healthy plants can also be obtained by growing them m vitro under a day/mght temperature of 40/35°C and subculturing the shoot tips every 1 2 d (26). Another option is to combine meristem culture with primary somatic embryogenesis by using meristems as explants. Different methods are available to screen the plants for diseases (for details, see 2 7) . 2. Friable embryogenic callus has been shown to be an excellent source o f tissue to obtain transgenic plants (28-30) and to obtain regenerable protoplasts (31), which can also be used to obtain transgemc plants by electroporatwn (Raemakers et a!., unpublished results) 3. In a well established culture, one mature somatic embryo multiplied m liquid medium via cyclic somatic embryogenesis will yteld more than 20 new mature somatic embryos in 2-4 wk, resulting in 201 3-2026 mature somatic embryos a year. 4. Leaf explants from greenhouse-grown plants can also be used as the starting material for primary somatic embryogenesis However, it has been shown that the embryogenic response of these leaf explants depends on the growth condi­ tiOns in the greenhouse (25). 5. Other auxins, such as Dicamba and Picloram, can also be used to induce primary somatic embryogenesis (13) However, in our laboratory, these media were not as efficient as the procedure described in Subheading 3.2. Another option (Tay­ lor, personal communication) is to culture leaf explants (harvested from donor plants grown on MS2 medium) on solid MS2 + 6 mg/L NAA + 6 mg/L Picloram medium to induce primary somatic embryogenesis. In our laboratory this method proved also to be very efficient 6. The developmental range at which somatic embryos can be used to initiate new cyclic somatic embryogenesis varies from globular to young mature somatic embryos (light green cotyledons) for NAA-supplemented medium and from globular to mature somatic embryos (with dark green cotyledons) for 2,4-D­ supplemented medium (19) .

68

Raemakers, Jacobsen, and Visser

7. In our laboratory, Jt JS found that in about 70% of the genotypes, NAA-supple­ mented medmm can be used to mamtain cyclic embryogemc cultures. In the other 3 0%, prolonged culture m NAA-supplemented medmm resulted m root forma­ tion (19) In the first instance, cultures w1l l contam somatic embryos w1th tap­ roots, but later this w1ll shift to the formation of adventitious roots Such cultures can be mduced to be embryogenic agam by isolatmg the remammg embryogemc tissue and cultunng this for cyclic/secondary somatic embryogenesis m MS2 medium + 8 mg/L 2,4-D medmm 8 Des1ccatwn has no posttlve effect on germmation of cyclic somatic embryos, whtch are mttlated m hqutd MS2 medium + 8 mg/L 2,4-D Desiccatwn only stimulates root formatiOn (19, 20). However, Mathews et al. (12) showed that secondary somatic embryos that were mamtamed on solid MS2 medmm + 4 mg/L 2,4D germmated properly after des1ccation 9. The desiccatiOn procedure descnbed m Subheading 3.4. 1s used m our labora­ tory. Each laboratory has to find the optimal conditiOns for their own matenal Furthermore, the procedure descnbed m Subheading 3.4., can only be used for small amounts of somatic embryos The method has to be adjusted for large-scale desiccatiOn. In our hands, mature somatic embryos, which had lost 80% of their fresh weight, germmated at the highest frequencies (20) These optimally desic­ cated mature secondary somatic embryos reqUire a lower level ofBA (0 I mg/L) for germmatwn than subopt1mally destccated ones. Higher BA levels (> 1 mg/L) stimulate the formatiOn of thick roots and highly branched plantlets With short internodes Because of these charactenstics, such plantlets cannot be transferred directly to the greenhouse. It has also been found that germinatiOn m the dark decreases the level of BA needed for normal germmatwn (19,20)

References 1 . Nasser, N. M A. ( 1 978) ConservatiOn of genetic resources of cassava (Mamhot esculenta): determmatJon of wild species locahzatwn with emphasis on possible ongm. Econ Bot. 32, 3 1 1-320 2. Byrne, D. ( 1 984) Breedmg cassava, in Plant Breedzng Revzews, vol . 2 (Jamck, J , ed.), A VI, Westport CT, pp 73- 1 34 3. FAO ( 1 993) Yearbook 1992 Rome, pp 1 0 1-102. 4 Cock, J. H (ed.) ( 1 985) Cassava New Potentzalfor a Neglected Crop. Wetvtew Press, Boulder and London. 5 Kartha, K K. and Gamborg, 0 L. ( 1 975) Ehminatwn of cassava mosmc vtrus dtsease by menstems Plant Scz Lett 2, 1 07-1 1 3 6 Roca, W. M . ( 1 984) Cassava, m Handbook of Plant Cell Culture, vol. 2 (Sharp, W. R., Evans, D. A., Ammirato, P V., and Yamada, Y., eds.), Macmillan, New York, pp. 269-3 0 1 . 7 Smith, M K , Biggs, B J , and Scott, K. J ( 1 9 86) In vitro propagatton of cassava (Manzhot esculenta Crantz). Plant Cell Tiss Org. Cult. 6, 22 1-229 8 Parrot, W A , Merkle, S A , and Wtlliams, E G ( 1 99 1 ) Somatic embryogenesis potential for use m propagatiOn and gene transfer systems, m Advanced Methods

Somatic Embryogenesis

9. 1 0. 1 1. 12.

13.

14.

1 5. 16.

1 7.

1 8. 1 9.

20. 21 22. 23.

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in Plant Breeding and Biotechnology (Murray, D. R., ed.), CAB Int. Wallingford, Oxon UK, pp. 1 58-200. Murashige, T. and Skoog, F. ( 1 962) A revised medium for rapid growth and bio­ assay with tobacco cultures. Physiol Plant. 15, 473-497. Stamp, J. A. and Henshaw, G. G. ( 1 987) Somattc embryogenesis from clonal leaf tissue of cassava. Ann. Bot. 59, 445-450. Szabados, L., Hoyos, R., and Roca, W. ( 1 987) In vztro somatic embryogenesis and plant regeneration of cassava. Plant Cell Rep 6, 248-25 1 . Mathews, H., Schopke, C., Carcamo, R , Chavarnaga, P , Fauquet, C., and Beachy, R. N. ( 1 993) Improvement of somatic embryogenesis and plant regeneratiOn m cassava. Plant Cell Rep 12, 328-333. Sudarmonowati, E. and Henshaw, G. G. ( 1 993) The induction of somatic embryo­ genesis of recalcitrant cassava cultivars using Ptcloram and D tcamba, m Proceed­ ings of the First Internatwnal Sczentzfic Meetzng of the Cassava Biotechnology Network (Roca, W. M. and Thro, A. M., eds.), Centro International de Agricultura Tropical, Cartagena de Indias, Columbia, pp. 1 28-1 34. Raemakers, C. J. J. M., Bessembinder, J., Staritsky, G., Jacobsen, E., and Vtsser, R G F. ( 1 993) Induction, germination and shoot development of somatic em­ bryos in cassava. Plant Cell Tiss Org. Cult 33, 1 5 1-1 56. Sofiari, E. ( 1 996) Regeneration and transformation of cassava. PhD thests, Wageningen Agricultural University, Netherlands. Taylor, N. J., Edwards, M. Kiernan, R. J. , Davey, C. D. M., Blakesley, D., and Henshaw, G. G. ( 1 996) Development of friable embryogemc callus and embryo­ genic suspension culture systems in cassava (Mamhot escu/enta Crantz) Nature Biotechnol. 14, 726-730. Taylor, N. J., and Henshaw, G. G., ( 1 993) The induction of somatic embryogen­ esis in 1 5 African and one South American cassava cu1tivars, in Proceedzngs of the First International Scientific Meeting of the Cassava Biotechnology Network (Roca, W . M. and Thro, A. M., eds.), Centro International de Agricultura Tropt­ cal, Cartagena de Indias, Columbia, pp. 229-240 Gresshoff, P. M. and Doy, C. H. ( 1 974) Development and dtfferenttation of hap­ loid Lycopersicon escu/entum (tomato). Planta 1 07, 1 6 1-1 70. Sofiari, E., Raemakers, C. J. J. M., Kanju, E., Danso, K., van Lammeren, A. M., and Jacobsen, E. ( 1 997), Companson ofNAA and 2,4-D mduced somatic embryo­ genesis in cassava. Plant Cell Tzss. Org. Cult, in press. Raemakers, C. J. J. M., Rozenboom, M. G. M., Danso, K., Jacobsen, E., and Visser, R. G. F. ( 1 997) Regeneration of plants from somatic embryos and friable embryogenic callus of cassava (Manzhot esculenta Crantz). Afr Crop Scz J , in press. Stamp, J. A . and Henshaw, G. G. ( 1 98 7) Secondary somatic embryogenesis and plant regeneration in cassava. Plant Cell Tzss Org Cult. 1 0, 227-233. Raemakers, C. J. J. M., Arnatt, M., Stantsky, G., Jacobsen, E., and Visser, R. G. F. ( 1 993) Cyclic somatic embryogenesis in cassava. Ann Bot. 71, 289-294 Raemakers, C. J. J. M., Schavemaker, C. M , Jacobsen, E., and Vtsser, R. G F. ( 1 993) Improvements of cyclic somatic embryogenesis of cassava (Manihot escu/enta Crantz). Plant Cell Rep 1 2, 226-229

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24 Li, H Q , Huang, Y. W , Liang, C Y , and Guo, J Y. ( 1 995) Improvement of plant regeneratiOn from secondary somatic embryos of cassava, m Proceedmgs of Second Internatzonal Meetmg of Cassava Biotechnology Network, Bogor, Jndo­ nesza, August 22-26, Centro Intemacwnal de Agncultura Tropical, Cali, Colum­ bia, pp. 289-302 . 25. Raemakers, C J. J M ( 1 993) Pnmary and cyclic somatic embryogenesis m cas­ sava PhD thesis Wagenmgen Agncultural UmvefS!ty, Netherlands. 26. VIllegas, L. and Bravato, M. ( 1 990) ConservatiOn m vitro of cassava germplasm, m In Vztro methodsfor Conservatzon ofPlant Genetic Resources (Dodds, J , ed ), Chapman and Hall, London, pp. 1 1 1- 1 2 1 27 Raemakers, C J J M., Jacobsen, E., and Visser, R. G . F ( 1 997) Micropropagatwn of Manzhot esculenta Crantz (cassava), in Bzotechnology m Agriculture and For­ estry, vol 3 9 (Bajaj, Y. P. S , ed ), Sprmger Verlag, Berlm, pp 77- 1 03 28. Schbpke, C., Taylor, N., Carcamo, R , Konan, N K , Marmey, P , and Henshaw, G ( 1 996) RegeneratiOn of transgemc cassava plants (Mamhot esculenta Crantz) from microbombarded embryogemc suspensiOn cultures Nature Bzotechnol 14, 73 1-735 29 Raemakers, C J. J. M., Sofian, E., Taylor, N., Henshaw, G. G., Jacobsen, E , and VIsser, R G. F ( 1 996) ProductiOn of transgemc cassava (Mamhot esculenta Crantz) plants by particle bombardment usmg luciferase activity as selectiOn marker. Mol Breedmg 2, 339-349. 30 Snepvangers, S . C. H. J., Raemakers, C J J M , Jacobsen, E , and VIsser, R. G F. ( 1 997) Optimization of chemical selection of transgemc friable embryogemc cal­ lus of cassava using the luciferase reporter gene system. Afr Crop Scz J. , m press 3 1 Sofian, E , Raemakers, C. J. J. M., Bergervoet, J E. M., Jacobsen, E , and Visser, R. G. F. ( 1 997) Plant regeneratiOn from protoplasts Isolated from friable embryo­ genic callus of cassava. Plant Cell Rep. , m press

' -----I mmature Inflorescence Culture of Cereals A Highly Responsive System for Regeneration and Transformation

Sonriza Rasco-Gaunt and Pilar Barcelo 1 . Introduction

Success in obtaining transgenic cereals depends largely on efficient plant regen­ eration protocols. It is not trivial to establish regenerable ttssue cultures m cereals, since only immature tissues of a limited age range can be induced to regenerate efficiently. Generally, the tissue-culture factors that determine the success of regen­ eration and transformation in cereals are: genotype, choice of explant, physiologtcal state of the explants/donor plants, culture conditions, and medium composttton. Although scutellar tissues are now conventionally used for cereal transfor­ mation, immature inflorescences also provide a practicable source of young tissues for somatic embryogenesis and plant regeneration. Inflorescence cul­ tures of rice (1, 2) , maize (3), wheat (4-8), sorghum (9, 10), barley (11), millet (12,13), rye (6, 14,15), and Triticeae sp. (tritordeum [1 6]) are documented. However, cereal transformation using inflorescence ttssues is novel and has, so far, only been demonstrated by Barcelo et al. (1 7). The advantages of usmg inflorescence explants for ttssue culture and trans­ formation over the use of scutellar tissues are: I . Their isolation IS easier and quicker, particularly if the embryo ax1s must be exctsed to prevent precocious germinatiOn in scutellum cultures. 2. Their physwlogtcal state seems to be relatively stable in response to plant growth conditions. Therefore, variability in regeneration 1s generally less than is observed With scutellum cultures. 3 . It is quicker to grow donor plants for mflorescence culture, resultmg in a more effictent use of greenhouse/growth space, and there IS less time for pest and patho­ gen problems to develop. From Methods m Molecular Brology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc Totowa, NJ

,

71

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Plants from genotypes that normally require vernalization may not require ver­ nalization when regenerated from mflorescences (18) 5 In certam genotypes, mflorescence tissues may be more amenable for culture and/or transformation than scutellar tissues 6 Inflorescence tissues seem to withstand better the damage mcurred by gene delivery via particle bombardment. 4

The hmttatton of mflorescence culture ts that only a narrow range of cereal genotypes are responsive etther to both tissue culture and transformation or to the latter alone. This chapter presents procedures for the tissue culture (embryogenic callus mductwn and regeneration) and transformatiOn vta particle bombardment of tritordeum and wheat using Immature inflorescence tissues It includes recent findings and modificatiOns to the authors' ongmal protocol (1 7). 2. Materials 2. 1. Donor Plant Materials and Growth Conditions To produce mflorescences, grow plants oftntordeum (a ferttle cereal amphi­ ploid obtained from crosses between Hordeum chliense and durum wheat cul­ ttvars, contaming the genome H CH H CH AABB {1 9]) and wheat ( Triticum aestzvum, e.g., vanety Baldus [see Note 1]) m growth rooms under a 1 6-h pho­ topenod, a1r temperatures of 1 8-20°C durmg the day and l 4°C at mght, and at 50-70% relative humidity. If using winter wheat vaneties, vernalize 1mb1bed seeds (sown m verm1cuhte) for a penod of 8 wk at 5-6.5°C pri o r to transfer­ ring to the above condttions (see Note 2). 2.2. Tissue-Culture Media Components and Other Solutions

2.2. 1 . Culture Media Components 1 . L7 Macrosalts ( l OX stock). 2 5 g NH4N03, 1 5 0 g KN03, 2.0 g KH 2P04 , 3.5 g MgS04 · 7H 20, 4.5 g CaC12 · 2H20 Dissolve salts separately m a small volume of distilled water, mix together, and make up to 1 L Autoclave and store at 4°C (see Note 3). 2. L Mtcrosalts ( l OOOX stock). 1 5 0 g MnS04, 5 0 g H3B03 , 7 5 g ZnS04·7H20, 0 75 g Kl, 0 2 5 g Na2Mo04 2H20, 0.025 g CuS04·5H20, 0.025 g CoC12 6H20 Make up to 1 L With distilled water Fi lter-stenhze through a 0 2-J..L m medmm filter (e g , MediaKap, Laguna Hills, CA) and store at 4°C 3 Ferrous sulfate/chelate ( 1 00X stock, S1gma F-05 1 8 , UK) 4 L V1tamms/mositol (200X stock) 40.0 g myomos1tol, 2 0 g th1amine HCl, 0 20 g pyndoxme HCI, 0 20 g mcotinic ac1d, 0 20 g Ca-pantothenate, 0.20 g ascorbic ac1d Make up to 1 L w1th distilled water Store solution at -20°C m 10 mL aliquots

Culture of Cereals

73

5. Ammo acids, 3AA (25X stock): 1 8 .75 g L-glutamme, 3 . 7 5 g L-prohne, 2 . 5 0 g L-asparagine. Make up to I L with distilled water. Store solutions at -20°C in 40 mL aliquots. 6. Gelling agent: Agargel-make ( 1 0 g/L or 2X concentrated stock, S1gma A-330 I , UK), agarose--make (8 g/L or 2X stock): Prepare stock solutions of Agargel and agarose. Autoclave and store at room temperature (see Note 4) 7. Silver Nitrate (20 mg/mL stock): Prepare AgN03 solutiOn and filter-sterilize through a 0.2-IJlll syringe filter. Store solutiOns at -20°C in I mL aliquots. A lter­ natively, silver thiosulfate (STS) may be used in place of AgN03 (see Note 5). 8. Zeatin Mixed Isomers (I 0 mg/mL stock): Prepare solution by d1 ssolvmg zeatin in a few drops of 1 M HCI. Make up to concentratiOn with distilled water. F1lter-stenlize through a 0.2-IJlll syringe filter. Store solutions in 1 -mL aliquots at -20°C. 9. 2,4-Dichlorophenoxyacetic acid, 2,4-D ( 1 mg/mL stock): Prepare solutiOn by dis­ solving 2,4-D in a few drops of I M NaOH. Make up to concentration with dis­ tilled water. Filter-sterilize through a 0.2-j.Ul1 syringe filter. Store solutions at -20°C in 2-mL aliquots (see Note 6). 1 0 Ptcloram ( I mg/mL stock): Prepare solutton by dissolving m a few drops o f 1 M NaOH. Make up to concentration w1th distilled water. Filter-stenlize through a 0.2-IJlll syringe filter. Store solutiOns in 2-mL ahquots at -20°C (see Note 6)

2.2.2. Culture Media I . L 7 -V liquid medium (2X concentrated stock). 200 mL L 7 macrosalts ( I OX), 2 mL L microsalts ( l OOOX), 20 mL ferrous sulfate/chelate (Sigma F-05 1 8, I OOX), 0.4 g myoinositol, 80 mL 3AA (25X), 60 or 1 80 g maltose (see Note 7). Make up to 1 L with distilled water. Adjust pH to 5 7 Filter-stenlize through a 0.2- IJlll medium filter and store at 4°C 2. R liqmd medium (2X concentrated stock): 200 mL L7 macrosalts ( I OX), 2 mL L microsalts ( 1 OOOX), 20 mL ferrous sulfate/chelate (Sigma F-05 1 8 , I OOX), 1 0 mL vitamins/inositol (200X), 60 g maltose. Make up to l L w1th distilled water Adjust pH to 5.7 Filter-sterilize through a 0.2-IJlll medmm filter, and store at 4°C. 3 Callus induction medium: To prepare 800 mL, m1x 400 mL L7-V liquid medium (2X), 0.4- 1 6 mL of 1 mg/mL 2,4-D stock solutiOn for wheat culture or 3.2 mL of 1 mg/mL Picloram stock solution for tritordeum culture, 0.4 mL 20 mg/mL AgN03 stock, and 400 mL Agargel ( 1 0 g/L) or 400 mL agarose (8 g/L) Pour medmm into sterile tissue-culture Petn dishes (see Notes 8-10). 4. RegeneratiOn medium (RZ): To prepare 800 mL, mix 400 mL R hquid medium (2X), 80 J.1L 2,4-D stock, 0 4 mL zeatm stock, 0.4 mL AgN03 stock, 400 mL Agargel, or agarose. Pour medmm mto sterile Petri dishes or into Magenta boxes (see Notes 9 and 10) . 5. RegeneratiOn medium (RO): To prepare 800 mL, m1x 400 m L R liquid medmm (2X) and 400 mL Agargel or agarose. Pour medium into sterile Petri dishes or mto Magenta boxes (see Note 10).

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2. 2. 3. Solutions for DNA Delivery and Selection 1 . Gold particle mixture: Suspend 40 mg gold powder (see Note 1 1 ) m 1 mL of absolute ethanol, and somcate for 2 mm until particles are clearly dispersed. Cen­ trifuge bnefly (5 s), and discard supernatant Repeat ethanol wash twice Resus­ pend gold in I mL of sterile distilled water, and sonicate for 2 mm Centnfuge for 5 s, and discard supernatant Repeat the process once, and resuspend finally m 1 mL stenle distilled water. Aliquot m 50-J.!L vol/Eppendorftube, mixmg between aliquots to ensure an equal distnbution of particles. Store at -20°C 2. Calcmm chlonde (2 . 5 M stock)· Prepare solutiOn and filter-stenhze through a 0.2-J.!m synnge filter. Store at 4°C in 5 5-J.!L aliquots. 3 Spermidme solution (0. 1 M stock)· Spermidme is supplied as a 0.92 g/mL den­ Sity solutiOn Take 15 8 f..lL of solutiOn, and add sterile distilled water to give a total volume of 1 mL Store at -20°C m 25-f..l L ahquots Do not reuse thawed aliquots (see Note 12) 4. Plasmid DNA (1 f..lg/f..lL stock) Prepare plasmid DNA usmg a Qiagen punficatwn kit Resuspend DNA pellet m stenle distilled water or TE buffer ( I 0 mMTris and 1 mM EDTA, pH 8 0), and adJust concentration as appropnate (see Note 13) 5 SelectiOn agents: Glufosinate ammomum (10 mglmL stock), Bialaphos ( 1 0 mg!mL stock), genettcin dtsulfate (G4 1 8) (50 mg/mL stock), paromomyc in sulfate ( 1 00 mg/mL stock). Prepare solutiOns, and filter-sterilize through a 0.2-f..lm syringe filter. Store solutions at -20°C m ahquots (see Note 6) 2. 2. 4 . Histochemical GUS Assay Solutions 1 . Sodium-phosphate buffer, pH 7.0 (0.5 M stock)· Prepare 1 M Na2HP04 and 1 M NaH2P04 Mtx 58 mL 1 M Na2HP04 and 42 mL I M NaH2P04. Make up to 200 mL with distilled water Store solution at -20°C m 20-mL aliquots. 2 Potassmm ferncyanide (50-mM stock): Prepare solution, and store at -20°C m 1 0-mL ahquots 3 . Potassium ferrocyanide (50-mM stock): Prepare solution, and store at -20°C m 1 0-mL aliquots. 4. X-Giuc buffer· To prepare 1 00 mL, dissolve 50 mg X-Gluc (5 -bromo-4-chioro3-indolyl-�-o-glucuronide, cyclohexylammomum salt) in I mL methyl cellusolve or dimethylformamtde. Add the followmg soluttons: 20 mL 0 5 M sodiUm-phos­ phate buffer, pH 7 .0, I mL each of 50 mM potassmm ferncyamde and potassiUm ferrocyamde and 77 mL distilled water. Ftlter-sterilize through a 0.2-f,.lm synnge filter and store at -20°C in 2-mL aliquots.

2.3. Additional Requirements 1. 2. 3 4. 5.

1 M and 5 M NaOH or KOH and HCl. 70% Absolute ethanol. 1 0% Domestos commercial bleach (see Note 1 5). Sterile water Culture room conditions: 1 6-h photoperiod, 25 ± 1 °C.

Culture of Cereals

75

Fig. I . Wheat inflorescences ranging in length from <0.5-1 .0 em ready for dissection.

3. Methods 3. 1. Collection and Dissection of Inflorescences 1. Harvest tillers containing inflorescences ranging in length from 0.5-1.0 em (see Note 14 and Fig. 1) by cutting below the inflorescence-bearing node {the last node of the tiller). Trim the tillers to approx 8- to 10-cm length, and seal the upper end with Nescofilm. 2. Sterilize the tiller sections by rinsing in 70% ethanol for 5 min, then 30 min in 100/o Domestos (see Note 15) with gentle agitation, and rinse twice with sterile distilled water. 3. Dissect inflorescences under sterile conditions by cutting away the whorled leaves surrounding the inflorescence with a scalpel blade. Cut inflorescence into transverse sections of approx 1 -mm length (see Note 16).

3.2. Culture of Inflorescences

3.2. 1. Culture of Inflorescence Explants for Somatic Embryogenesis and Plant Regeneration 1. Plate the pieces of inflorescence ( 1 -mrn explants) on callus induction medium. Culture approx 10-15 explants/9-cm Petri dish. 2. Keep cultures in the dark at 25 ± I °C for 3-4 wk.

Rasco-Gaunt and Barcelo

76

Fig. 2. Regenerating callus from inflorescence

3.

Transfer cultures to regeneration medium 25

4.

after 3 wk in regeneration medium.

(RZ) and culture further for 3-4 wk at

± 1 °C under 16 h photoperiod (see Note 17 and Fig. 2).

On sur.ceeding rounds of subculture, small plantlets are transferred to regenera­ tion medium (RO) for further development prior to transfer to soil (see

Note 18).

Incubate cultures under the same conditions specified above. 5. Transfer plantlets to 2 : 1 soil-perlite mixture once a good root system is estab­ lished, and keep initially in a propagator for acclimation for 1-2 wk. Grow the plants in a controlled environment chamber or under greenhouse conditions as outlined in Subheading

2.1.

Repot the plants when necessary.

3.2.2. Culture of Inflorescence Explants for Particle Bombardment 1 . To prepare for particle bombardment, place up to

30 explants at the center of a

Petri dish containing callus induction medium, covering a target area of2.5 em in diameter. 2. Preculture the explants in the dark at 25

± l °C for 1-6 d prior to bombardment

(see Note 19). 3.3. Particle Bombardment

3.3. 1. DNA-Gold Precipitation I.

Sonicate a 50-�L aliquot of the gold solution for

I

min for deagglomeration of

particles 2. Add 5

(see Note 20). �L (5 �g) of plasmid DNA,

and mix gently. If using more than one plas­

mid, i.e., for cotransformation, the amounts ofDNA are added in equimolar quan­ tities (for bombardment of controls, see

Note 21)

Culture of Cereals

77

Place 50 ).IL of CaC12 and 20 ).IL of spermidme into an Eppendorf tube hd, and mix well. Close the lid, and tap the solution down into the gold-DNA mixture (see Note 22). Pulse centrifuge for 5 s to pellet DNA-coated particles. Dtscard supernatant. 4 Add I SO � of absolute ethanol to wash the particles (see Note 23). Pulse centn­ fuge for 5 s to pellet particles. Discard the supernatant and resuspend particles in 85 J.LL of absolute ethanol (see Note 24). 3

3.3.2. DNA Delivery 1 . Gently mtx the coated particles, and Immediately take a 5-J.LL aliquot. Drop the mixture onto the center of a macrocarrier, and allow to dry (see Note 25). 2 Place target cultures (prepared as exp1amed in Subheadings 3.1. and 3.2.) into the chamber. 3 . Operate the particle gun according to the instructions supphed with the B10-Rad PDS 1 000/He gun using the followmg conditiOns: He acceleration pressure 650-1 1 00 psi, vacuum 28 m. Hg, 2.5 em gap distance (distance between rupture disk and macrocarrier), 5.5 em target distance (di stance between stoppmg screen and target plate), and 0 8 em stopping plate aperture (see Note 26)

3.3.3. Transient GUS Expression Assay Two days after bombardment, randomly select a few pieces of tissues for a hts­ tochemtcal GUS assay Immerse the tissues m X-Gluc buffer wtth 0. 1 % v/v Tri­ ton X- 1 00. 2. Incubate overnight at 37 °C and then for 2 d at 26°C pnor to assessment. Assess blue staining (see Note 27) 3 . After initial incubation, explants may be stored in 70% ethanol or m 1 M TE buffer with a pH higher than 7.

3. 3. 4. Selection of Cultures for Recovery of Transgenic Plants 1 . After bombardment (see Subheading 3.3.2.), spread the explants evenly over the surface of the culture medium m 2-3 9-cm Petri dishes (not exceeding 1 0- 1 5 explants/plate). 2. Culture explants on callus inductiOn medium for 3-4 wk as explained in Sub­ heading 3.2.1 . 3 . Transfer exp1ants to regeneratiOn medmm (RZ) contaimng the appropnate selec­ tion agent, and incubate as usual for 3-4 wk (see Note 28). 4. Transfer cultures to RO medium containing selective agent, and culture for a further 3-4 wk until survivmg plantlets show signs of resi stance 5. Transfer plantlets to pots, and grow them as explamed m Subheading 3.2. 1 . 4. Notes

1 . The followmg elite wheat cultivars are also responsive, Consort, Hereward, Rta1to, Riband Sots sons, Cadenza, Imp, Avans, Canon

78

Rasco-Gaunt and Barcelo

2 We also grow donor plant matenals under greenhouse condltwns, but these plants are subJected to seasonal changes. Our conditions for growmg tritordeum and wheat plants are. a. Sot! composition· 75% L&P fine-grade peat, 1 2% screened stenhzed loam, 1 0% 6 mm screened, hme free gnt, 3% medtum-grade vermtcuhte, 3 . 5 kg 3 Osmocote/m sotl (slow-release fertilizer, 1 5 - 1 1 - 1 3 NPK plus mtcronutn­ 3 ents), 0 5 kg PG mtx/m soil ( 1 4- 1 6- 1 8 NPK granular fertthzer plus mtcro­ nutnents). b Ltght: growth rooms provtded wtth HQI lamps 400W (Osram) and glass­ houses with natural hght supplemented wtth sodmm lamps 400 W (Son-T) c. Pest control: sulfur (Thiovit) spray every 4-5 wk and bwlogtcal control usmg Amblyse1us callgmosus for the control of thnps 3 Autoclave settmgs are 1 2 1 °C, 1 5 psi for 20 mm. 4 Agargel and agarose are used at end concentratiOns of 0.5 and 0 4%, respec­ tively Shake solutions very well before and just after autoclaving to avoid lumps Agarose ts only used m medta supplemented wtth the selectiOn agent paromomy­ cm, which prectpttates out of so Iutton m Agargel-sohdtfied medta. 5 We prepare STS by m1Xlng 0. 1 M AgN03 and 0 1 M Na2S2 03 at 1 4 rat10 Add AgN03 stock mto Na2 S2 03 slowly to avmd prectpttatwn STS ts used freshly made at a final concentration of 30 !J.M (eqmvalent to 1 0 mg/L) AgN03 and Na2S 203 solutions are atr- and photo-senstttve. Store filter-sterilized solutwns m the dark at 4 oc 6 Ptcloram, 2,4-D, glufosmate ammonium, Btalaphos, G4 1 8, and paromomycm are toxtc substances Check chemtcal punttes of glufosinate ammonium (Grey­ hound, UK) and Btalaphos (Metji Seika Kaisha LTD, Tokyo) after every pur­ chase Calculations to achteve accurate concentrations may need to be adjusted accordmgly 7 Use 60 and 1 80 g maltose in the callus mduction medium for tritordeum and wheat cultures, respectively We find that 9% maltose m the callus mduct10n medmm ts opttmal for regeneratiOn in wheat 8 The callus inductton medmm ts supplemented wtth 0 5-2 mg/L 2,4-D and 4 mg/L Ptcloram for wheat and tritordeum culture, respectively The optimal 2,4-D con­ centration vanes for dtfferent wheat genotypes 9 Silver mtrate is not essenttal for tntordeum, but does not have any negative effect The addition of AgN03 m the mductwn medmm has been seen to be beneficial m most wheat cultures, although tt has to be tested for each new genotype 1 0 The gellmg agent stock solution is melted m a mtcrowave oven Mtx all the medta components, and finally add the gelhng agent. To mmimtze condensatiOn m the plates, do not pour the medium until tt has cooled to approximately 50°C Pour approx 40 mL of medmrn/9-cm tissue-culture Petri dtsh or 80 mL/Magenta box Therefore, 800 mL medmm wtll make approx 20 plates or 1 0 Magenta boxes 1 1 . The stzes and sources of gold parttcles we use are. 0 6-!J.m particles {Bto-Rad, CA) and 0 4 to 1 2-IJ.m parttcles (Heraeus, Karlsruhe, Germany) Bto-Rad par­ ticles are used for wheat transformatiOn, but etther type ts used for tntordeum

Culture of Cereals

79

1 2. Spenmdme solution ts very hygroscoptc and oxtdizable. We suggest storing the original spermidine solution m 1 5 .8-JJL aliquots at -80°C. Each aliquot will be used to make up 1 mL of 0. 1 M spermidine stock. 1 3 . We use a number of combinatiOns of scorable and selectable marker genes cobombarded with genes of interest. Generally, we use the uidA gene as scorable marker, and bar and neo genes as selectable markers (see also Note 28). 1 4. Inflorescences are located above the last node of a tiller. Therefore, by cutting under this node, the part of the tiller harvested will contain the inflorescence. The upper end of the tiller is sealed to prevent the penetration of sterilizing agent mto the mner region where the explant is located. Inflorescences of 0.5- 1 .0 em are generally most responsive, but there is genotypic vanation. It takes an element of familiarity with the plant material to be able to harvest tillers wtth the right age/ size of inflorescence. 1 5 . Domestos is a chlorme-based commercial bleach containing 4.5 JJgiL free chlorine. While sterilizing, continuously agitate the tillers by placing on a shaker. 1 6. Regeneratton is inhibited in wheat if explants are < I mm size 1 7 . Silver nitrate is not essential in the regeneration medium for tritordeum cultures. 1 8. It takes about three to four rounds of subculture (9-12 wk) on regeneration medium before plantlets are of sufficient size to be potted out Wheat plants may need more than one round of regeneration on RZ medium. 1 9 Preculture allows the tissues to recover from tsolatwn before bemg subjected to bombardment. This should also allow for any contamination to be detected pnor to bombardment. In wheat, the preculture period condttions the tissues for bom­ bardment, since the htgh-maltose concentratiOn m the medium (9%) partially plasmolyses the cells. Partial plasmolysis ts apparently tmportant for the tissues to withstand particle bombardment. Preculture of explants for 1-() d prior to bom­ bardment increases the number of transgenic plants produced in tntordeum. Preculture of wheat explants for l d appears optimal. 20 There is evidence that oversonicatlon may result in partiCle aggregatiOn. 2 1 . For bombardment controls, t.e., particle bombardment without DNA, 5 1JL of sterile distilled water are added in place of plasmtd DNA 2 22. Precipitation onto the gold particles occurs very rapidly. The Ca + wns from CaCl2 act to bind the DNA onto the gold particles Therefore, for a uniform pre­ cipitation, DNA-gold and CaCirspermidtne mixture should be mixed well before they are combined. 2 3 . Resuspend as many of the parttcles as possible by scraping them off the stde of the tube with a pipet tip and breakup any aggregatiOn. It is important to make sure that the gold particles are fully resuspended at this stage, smce aggregations are more difficult to deal with later 24. Seal Eppendorftube hds wtth Nescofilm to mimmtze evaporatiOn of ethanol, and keep on ice until required However, tt ts advtsable to use the gold-DNA prepa­ ration immediately. 25. Allow the particles to dry slowly on a nonvibratmg surface in a 6-cm Petn dish Only a few macrocarriers should be loaded with gold at any one time, so that they

Rasco-Gaunt and Barcelo

80

are used when recently dried Examme macrocarners microscopically pnor to bombardment to determme the umformity and spread of partiCles, discarding any that have agglomerated A "sand-dune" appearance of particles IS observed as a consequence of too rapid ethanol evaporatiOn. 26. We use acceleratiOn pressures of 650 or 900 psi for wheat and 1 1 00 for tritordeum. Wheat tissues are more sensitive to high bombardment pressures. 27 The same procedure may be used to assay GUS expression m tissues of puta­ tively transformed plants, such as leaves, roots, and flowers. For GUS assays on green tissues, chlorophyll should be removed after staming by incubating the tissues in absolute ethanol The length of the incubatiOn time depends on the age of the leaf and should last until the chlorophyll has been totally extracted It may be necessary to replace the ethanol several t1mes 28 We generally use the selectable marker genes bar and neo for transformatiOn The bar gene confers resistance to the herbicides Basta (glufosmate ammomum/ PPT) and B ialaphos The neo gene confers resistance to the antibiotics G4 1 8 and paromomycm We use the followmg concentrations for selectiOn' 2-4 mg/L gluphosmate ammonium, 3-5 mg/L Bialaphos, 25-50 mg/L G4 1 8, and 50 mg/L paromomycin References

2

3

4

5.

6. 7

8

Chen, T. H., Lam L., and Chen, S C. ( 1 985) Somatic embryogenesis and plant regeneratiOn from cultunng young mflorescences of Oryza satzva L (nee). Plant Cell Ttssue Organ Cult 4, 5 1-54. Rout, J. R and Lucas, W J. ( 1 996) Charactenzatwn and mampulatlon of embryo­ gemc response from in-vitro-cultured Immature mflorescences of rice ( Oryza satlva L.). Planta 198, 1 27-1 38. Pareddy, D. R and Petolmo, J F. ( 1 990) Somatic embryogenesis and plant regen­ eration from immature inflorescences of several elite mbreds of maize. Plant Set 67, 2 1 1-2 1 9 Ozias-Akms, P . and Vasil, I K ( 1 982) Plant regeneration from cultured Imma­ ture embryos and mflorescences of Trztzcum aesttvum L (wheat) Evtdence for somatiC embryogenesis Protoplasma 1 10, 95-1 05 Maddock, S. E , Lancaster, V A , RtsiOtt, R , and Franklin, J ( 1 983) Plant regen­ eration from cultured Immature embryos and mflorescences of 25 cultivars of wheat ( Trztzcum aestzvum). J Exp Bot 34, 9 1 5-926 Eapen, S. and Rao, P S. ( 1 985) Plant regeneratiOn from immature mflorescence callus cultures of wheat, rye and triticale. Euphytzca 34, 1 53-1 59. Redway, F A , Vasil, V., Lu, D , and Vast!, I K ( 1 990) IdentificatiOn of callus types for long-term mamtenance and regeneratiOn from commerctal cultivars of wheat ( Trztzcum aestzvum L ) Theor Appl Genet 79, 609-6 1 7 Sharma, V K , Rao, A , Varshney, A., and Kothari, S L ( 1 995) Companson of developmental stages of mflorescence for high requency plant regeneratiOn m Trztzcum aestlvum L. and T durum Desf. Plant Cell Rep. 1 5, 227-23 1 .

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9 . George, L . , Eapen, S . , and Rao, P . S ( 1 989) High frequency somatic embryogen­ esis and plant regeneration from immature inflorescence cultures of two Indian cultivars of sorghum (Sorghum bicolor L. Moench). Proc. Indzan A cad. Sci. 99, 405-4 1 0. 1 0. Wen, F. S., Sorensen, E . L., Barnett, F L., and Liang, G. H. ( 1 99 1 ) Callus induc­ tion and plant regeneration from anther and inflorescence culture of Sorghum. Euphytzca 52, 1 77-1 8 1 . 1 1 . Thomas, M . R. and Scott, K . J . ( 1 985) Plant regeneration by somatic embryogen­ esis from callus initiated from Immature embryos and Immature inflorescences of Hordeum vulgare. J. Plant Physwl. 121, 1 59-1 69. 12. Nagarathna, K. C., Shetty, S A , Harinarayana, G , and Shetty, H. S. ( 1 993) Selec­ tion for downy mildew resistance from the regenerants of pearl millet. Plant Scz 90, 53-6 1 . 1 3 . Vishnoi, R. K . and Kothari, S . L . ( 1 996) Somatic embryogenesis and efficient plant regeneration in immature inflroescence culture of Setana ztallca (L.) Beauv Cereal Res. Commun. 24, 29 1-297. 1 4 . Xu, Z. H., Wang, D. Y., Yang, L. J., and Wei, Z M ( 1 984) Somatic embryogen­ esis and plant regeneration in cultured immature inflorescences of Setana itahca Plant Cell. Rep. 3, 1 49-1 50. 1 5. Lmacero, R. and Vazquez, A M. ( 1 990) Somatic embryogenesis from immature inflorescences of rye. Plant Sci. 72, 253-258. 1 6. Barcelo, P., Vazquez, A., and Martin, A. ( 1 989) Somat1c embryogenesis and plant regeneration from tritordeum. Plant Breeding 103, 23 5-240. 1 7. Barcelo, P., Hagel, C., Becker, D., Martin, A., and Lorz, H. ( 1 994) Transgemc cereal (tritordeum) plants obtamed at high effictency by m1croproject1le bombard­ ment of inflorescence tissue. Plant J 5, 5 83-592 1 8. Marcinska, 1., Dubert, F., and Biesaga-Koscielmak, J. ( 1995) Transfer of the abil­ ity to flower in winter wheat via callus tissue regenerated from immature Inflores­ cences Plant Cell Tissue Organ Cult. 41, 285-288. 1 9. Martin, A. and Sanchez-Mange, E ( 1 982) Cytology and morphology of the amphiploid Hordeum chzlense X Tntzcum turgzdum conv. durum. Euphytzca 3 1 , 26 1 -267.

8

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Cryo preservation of Rice Tissue Cu ltures Erica E. Benson and Paul T. Lynch 1 . Introduction

The biotechnological improvement of rice is largely dependent on the mam­ tenance of dedtfferentiated cultures as either callus and/or suspension cultures. For example, the production of transgenic plants of rice (Oryza sativa L.) etther by direct DNA uptake into protoplasts or by particle bombardment IS depen­ dent on embryogemc callus or cell suspension cultures from whtch fertile plants can be regenerated (1,2). However, over time the morphogenic competence of dedifferentiated nee cultures declines (3). Therefore, new cultures have to be regularly mitmted and characterized in order to maintain a constant supply of embryogenic cells. This approach is highly problematic, particularly with Indica, Varietal Group 1 (4) , nee varieties (5) . Cryopreservation of embryo­ genic cells provides a more efficient means of ensuring a constant supply of competent cells for genetic mampulation. The recovery of embryogenic nee cultures after cryogenic storage that were capable of plant regeneration has been reported by several groups (6-9). Embryogenic callus, and more com­ monly, suspension cultures from a range of different rice varieties have been cryopreserved , mcluding Indtca (Varietal Group 1 ) varieties (9), Japomca (Varietal Group 6) varieties (7. 10) . Transgenic nee suspension cultures have also been successfully recovered from cryogenic storage (6). Suspension cultures of rice have been cryopreserved using controlled-rate cooling (6, 7) and vitnfication procedures (11,12) . Protocols based on the con­ trolled-rate freezing of cells normally requires preculture in culture medium containing osmotically active compounds (for example, mannitol) prior to cryopreservatwn. Preculture can sigmficantly enhance the postthaw recovery of plant cells (13). This effect ts not solely owmg to dehydratiOn effects (14), but can also mvolve the activation of genes coding for factors that protect plant From Methods m Molecular Biology, Vol 1 1 1 . Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

83

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Benson and Lynch

cells from environmental stresses (1�1 7). After preculture, the cells are treated with cryoprotectants (total concentration 1-2 M), which results m moderate cell dehydration (18). Further dehydration occurs dunng slow freezing. Ice crystal for­ mation m the external medium leads to an increase m solute concentratiOn and, therefore, the osmotic removal of water from the cells. On further ultrarapid cool­ ing m liquid nitrogen, the cell contents vitnfy (19). Although plant cells can toler­ ate extracellular Ice crystal formation, such ice formation can be damagmg, for example, owing to the mechanical stress to cells caused by the growth of extracel­ lular Ice crystals (20). The more recently developed vitrification procedures (21,22) involve cryopreservation strategies, which prevent Ice nucleatiOn and the subse­ quent growth of ice crystals. They are based on a combmatwn of cryoprotective mechamsms, includmg severe cell dehydratiOn at nonfreezmg temperatures as a result of exposure to concentrated nonpenetratmg cryoprotectants (total concentra­ tion 5-8 M), followed by rapid freezmg leadmg to vttnficatwn (23). The applica­ tiOn of high concentratiOns of penetratmg cryoprotectants, for example, dimethy1 sulfoxide (DMSO), mcreases the VIscosity of the mtracellular solutes, and as a result, ice nucleatmg events are mhibited. The avoidance of Ice crystal formatiOn m vitrificatiOn procedures and the lack of a need for a controlled-rate freezer are gen­ erally considered to be advantagous (23). However, this must be counterbalanced by the need to use high concentratiOns of potentially toxic chemicals m the VItrifi­ cation solutions. Glasses formed during vitnfication are not stable and can desta­ bilize on rewarming, dunng which Ice nucleation can occur. It IS thus cntlcal to ensue rapid rewarmmg. Therefore, in vitrification, It IS the rewarming rate that IS cntical rather than coolmg parameters. This chapter presents a series of protocols based on published methods used to cryopreserve nee suspension and callus cultures. In the case of suspensiOn cultures, controlled-rate freezing and vitnfication procedures are descnbed. Factors influencing postthaw regrowth and methods of determmmg postthaw cell viability are also presented in the context of protocol development. 2. Materials To maintain stenhty ofthe nee cultures, all appropnate mampulatwns should be performed in a lammar flow bench, usmg aseptic techniques and sterile materials. 2. 1. General Requirements 1 . Liquid nitrogen and Ice. 2 Small bench-top dewar of 2- to 5-L capacity. 3 . Long-term storage dewar with an appropriate drawer or cane inventory system 4 Polypropylene cryovials, 2 mL With graduatiOn marks and label, or 1 - to 0.5-mL polypropylene straws. Cryovials can be obtamed, for example, from Stgma and straws (type 1 0 1 , cattle, nonstenlized) from Instruments de Medtcme Veterinaire, I' Atgle, France

Rice Tissue Cultures

85

Small spatula, sieves with 45-J..Lm nylon mesh, alummum cryocanes, Pasteur pipets, forceps, ice bucket, I 00- and 25 0-mL conical flasks. 6. A water bath set at 45°C (see Note 1). 7 Rice callus and suspension cultures grown on appropnate culture medmm, e g , AA2 (24) or modified R2 (9) 8 . Safety eqmpment· mcludmg, cryogloves, face shield. 5

2.2. Controlled-Rate Freezing Requirements (Cell Suspensions) Controlled-rate freezer able to cool at a rate of - 1 °C/min However, alternattve cooling methods may suffice (see Note 2) 2. Liquid preculture medmm. rice culture medmm, e.g., AA2 (24) or modtfied R2 (9), both contammg 60 g/L manmtol (see Note 3). 3 Cryoprotectant solution. glycerol, 46 g/L, DMSO, 39 g/L, sucrose 342 3 g/L, and prolme 1 0 0 g/L made up m hqmd standard culture medmm (see Note 4). 4. Postthaw recovery medmm: rice culture medium contammg 4.0 g/L agarose (see Notes 3 and 5) in 9 0-cm Petri dishes (25 mL of medta/dish) The medta onto which the cells will be placed Immediately after thawing should be overlatd with two filter paper dtsks (Whatman No. 1 , 5 5-cm diameter), one on top of the other

2.3. Controlled-Rate Freezing Requirements (Callus Cultures) 1 . Controlled-rate freezer able to cool at a rate of -1 °C/min (see Note 2) 2. Full-strength cryoprotectant solution· glycerol, 46 g/L, DMSO, 39 g/L, sucrose 342.3 g/L, and prolme 3 3 g/L made up m liquid standard culture medmm, such as modified R2 medmm (8) (see Note 4). 3 Half-strength cryoprotectant solutton, prepared m full-strength culture medmm, but at half the above concentratiOns 4 Water bath set at 30°C (see Note 1 ) 5 Ammomum-ion-free liqutd rice culture medium 6 Postthaw culture media semtsohd rice culture medmm (see Notes 3 and 5) m Petn dishes.

2.4. Vitrification Requirements

2.4. 1 . Method A, Based on Huang et a/. (1 2) 1 . Preculture medmm A-liquid nee culture medmm (e.g., AA2) contammg 60 g/L sucrose 2. Preculture medmm B--l tqutd nee culture medium contammg 72.9 g/L sorbttol 3 PVS2 VItnfication solutwn (25,26) comprising rice preculture medium B to which ts added: 30% (v/v) glycerol, 1 5 % (v/v) ethylene glycol, and 1 5 % (v/v) DMSO (see Note 4). 4. A 25% (v/v) solutwn of PVS2 dtluted with preculture medtum B 5 . Washing solutiOn comprising I 2 M sorbitol made up m hqmd nee culture medmm 6. Semisolid nee culture medmm (e.g., AA2 medmm contammg 0 8% w/v agar) containing 40% (w/v) soluble starch 7. Semisolid nee culture medmm

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2.4.2. Method B, Based on Watanabe and Steponkus (1 1 ) Loading solution 1 26.3 g/L DMSO in hquid rice culture medium, e g , Lmsmater and Skoog medmm, LS (2 7) , held at 20°C. 2. Vitrification solution, 445 giL DMSO and 1 87 giL sorbitol in rice culture medmm, held at 0°C (see Note 4) 3 Unloadmg solutwn, 9 1 . 1 g/L sorbttol m hqmd nee culture medmm 4. Semtsolid rice culture medium (see Note 5).

2.5. Selection of Tissue for Freezing For the successful cryopreservation of rice tissue cultures, It is vital to select the most appropnate tissue. Generally, an actively growmg area of callus cul­ tures and cell suspens10ns m the exponential phase of growth should be selected. Factors including the embryogenic potential of the cells (7), cell aggregation (10), and cultivar (9), have been shown to influence postthaw rice cell regrowth significantly. 2.6. Requirements for the Assessment of Postthaw Recovery

2.6. 1 . Fluorescein Diacetate (FDA) Stock solution of FDA 0 1 % (w/v) m acetone, stored m the dark at 4°C 2 Pasteur ptpets, test tube, shdes and cover shps 3. Liquid standard culture medium. 4. UV mtcroscope wtth 490-nm excitatwn and 530-nm barrier filters

2. 6.2. Triphenyl Tetrazolium Chloride (TTC)

2 3. 4. 5 6

Reagent mtxture. 0.6% (w/v) TTC m 0.5 M Na2HP04/KH2P04 buffer, pH 7 4, supplemented with 0.05% (v/v) Tween 80 The buffer can be stored m a refng­ erator for several months Do not use tf the solution appears cloudy The reagent mtxture should be prepared tmmedtately pnor to use Dtstllled water. 95% (v/v) Ethanol. Pipets ( 1 0-mL ), Pasteur ptpets, cuvets, test tubes Boiling water bath Spectrophotometer capable of measunng absorbance at 490 nm.

3 . Methods

3. 1. Controlled-Rate Freezing (Cell Suspensions}, Based on Lynch et a/. (7) 1 . At the standard subculture time, transfer the cells to be cryopreserved to preculture medmm usmg the standard culture protocol 2. Three to 4 d after subculture harvest the cells on 45-f..lm nylon mesh filters and transfer to cryovials (0 75 mL/vial)

Rice Tissue Cultures

87

3. Prelabel the vials, and to each vial add 0.75 mL of filter-sterilized, chilled (on Ice) cryoprotectant mixture. Immediately seal the vial, and shake to ensure the cells and the cryoprotectant mixture are well mixed and place on ice 4. Mount the vials onto labeled aluminium cryocanes, and incubate on Ice for 1 h 5 . Shake the Ice and water off the canes and vials. If necessary, dry With tissue paper and transfer to the controlled-rate freezer (e.g., a Planer Kryo I 0 Senes III; Planer BioMed, Sunbury, Middlesex, UK) precooled to ooc Place the contro lled­ rate freezer's sample chamber temperature probe mto a vial contammg I 5 mL of chilled cryoprotectant mixture. This provides a temperature profile of a "sample" durmg freezing. Start the freezing program (see Note 6). 6. Immediately after the controlled-rate freezer programme is completed remove the vials from the freezer sample chamber and plunge them mto a dewar of hqmd nitrogen. This should be located next to the freezer. After 5- 10 mm, the vials can be transferred to a long-term storage dewar, and their position logged mto an appropriate inventory system (see Note 7). 7 After storage, plunge the vials mto a water bath at 45°C (see Note 1). Complete thawmg normally takes approx 2 min Remove the vials from the water bath as soon as all the ice has melted, and transfer to a lammar flow bench (see Notes 7 and 8). 8. Dry the vials with tissue paper. 9. Aspirate the cryoprotectants with a Pasteur p1pet and place the cells on the top of a double layer of filter paper (Whatman no I , 5 5-cm diameter) overlaymg the postthaw recovery medium (see Note 5). 1 0 Seal the dishes with Nescofilm (Nippon ShOJI Kaisha Ltd., Osaka, Japan), and incubate in the dark at 28°C, or as appropriate for the rice culture being used. I I After 3 d, transfer the cells and filter paper to a fresh dish of postthaw recovery medmm (see Note 5). 1 2. Postthaw cell regrowth IS normally visible to the naked eye within 5 d of thaw­ ing Sufficient cell regrowth to allow reinitiation of suspension cultures normally occurs withm 25 d of thawing. 1 3 Suspension cultures can be reinit1ated using the same techniques used to imtiate new nonfrozen cultures. It has been noted that cultures Initiated from frozen cells often develop more quickly than new, nonfrozen cultures and can be m a state suitable for protoplast isolatiOn Within 28 d after mitiation (7) 3.2. Controlled-Rate Freezing (Callus Cultures),

Based on Cornejo et al. (8) Select callus with a compact nodular appearance from cultures 4- to 8-mon old, which have been subcultured every 4 wk 2. Place individual calli mto vials containing 1 mL of 1 12 strength chil led cryoprotectant solutiOn, and mcubate on ice for 1 5 mm. 3. Remove the cryoprotectant solution, replace with an equal volume of ful l strength chilled cryoprotectant solutiOn and mcubate on ice for a further 1 5 mm.

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4. Mount vials on aluminum canes, and transfer to a controlled-rate freezer wtth tts sample chamber at 4oc Start the freezing program, cool to -25°C at - 1 °C/m10, and hold at -25°C for 30 mm. 5 Immedtately after the controlled-rate freezer program ts completed, remove the vtals from the freezer sample chamber, and plunge them 10to a dewar of hquid mtrogen. After 5-1 0 m10, the vtals can be transferred m to a long-term storage dewar and appropnate 10ventory system (see Note 7) 6 After storage, plunge the vtals mto a water bath at 30°C (see Note 1) Complete thawing normally takes approx 2 min. Remove the vtals from the water bath as soon as all the ice has melted, transfer to a lammar flow bench, and place on tee (see Notes 7 and 8) 7 Gradually dilute the cryoprotectant solutwn wtth tee-cold ammomum ton-free culture medmm. 8 Remove the thawed calh from the vtals and transfer to semtsohd culture medmm (9) . Seal the Petn dtshes wtth Nescofilm, and mcubate m the dark at 27°C, or as appropnate for the rice culture bemg used. 9. Transfer to fresh culture medmm every 3 wk. 1 0. One to 2 moo after thawmg, remove the regrowmg callus from nongrowmg sec­ twos placed on fresh culture medium, and mamtam for a further 3-6 wk unttl actively growmg callus hoes have been estabhshed.

3.3. Vitrification

3 3 1 . Method A, based on Huang et a/. ( 1 2) 1 . Using standard culture procedures, l l d pnor to freezing, transfer the suspenswn cells mto preculture medium A, and mcubate under normal conditwns 2 Usmg standard culture procedures, 1 d prior to freezmg, transfer the suspension cells into preculture medium B, and mcubate under normal condttions 3 Transfer 3 mL of cell suspension culture into a 1 0-mL centnfuge tube, add 6 mL of 25% (v/v) PVS2 solution, and mcubate at 20°C for 1 0 mm 4. Remove the 25% (v/v) PVS2 solution, resuspend the cells m 3 mL of 1 00% (v/v) chtlled PVS2 solutton, and mcubate on tee for 7.5 mm 5. Usmg a wtde-bore Pasture ptpet, transfer 0.75-mL ahquots of the suspended cells (at a final packed-cell volume of 40%) into polypropylene straws (straws surface stenhzed wtth ethanol). 6 Qmckly seal each straw by drummmg seal10g powder ( obta10ed from Instruments de Medtcme Vetennatre) mto the open end. Ensure the powder ts wetted by the suspensiOn 10 the straw. AlternatiVely, the straws can be sealed with a hot pair of forceps or a seahng machme. 7. Plunge straws mto hqmd mtrogen After 5-1 0 mm, the samples can be trans­ ferred into a long-term storage dewar and appropnate inventory system (see Note 7). 8. Rapidly thaw the cells by placmg the straws 10 a water bath at 37°C 9 Introduce 1 rnL of washing solution into each warmed straw, and mcubate for 2 m10. 1 0. Remove the supernatant, replace with 1 mL of washing solution, and mcubaie the cells for 25 mm at 25°C.

89

Rice Tissue Cultures

1 1 . Drain the cells from the cut end of each straw onto semt-sohd nee culture medmm contaming 40% (w/v) starch. Incubate at 26°C for 2d. 1 2. Transfer the cells onto semtsolid AA2 medium, and incubate at 26°C Cell growth can be observed wtthin 4 d after thawmg.

3. 3. 2. Method 8, Based on Watanabe and Steponkus (1 1 ) Allow the cells to settle m cultures 3-4 d after subculture, and drain off the cul­ ture medium. 2 To 500 J.!L of settled cells add 2 5 mL of loading solut10n, and incubate at 20°C for 20 mm. 3 Remove the loadmg solutiOn, and place the cells on tee. Resuspend the cells m 2.5 mL of chtlled vttnficatton solution 4 Transfer 500-J.!L aliquots of the cell suspenston m to polypropylene straws, and plunge m to hquid nitrogen. The total ttme the cells were exposed to vitrificatton solutton prior to freezmg ts 4 mm. After 5-1 0 min, the vtals can be transferred to a long-term storage dewar with an appropnate mventory system (see Note 7) 5 Store straws in hqutd nitrogen 6 After storage, thaw the cells by warmmg the straws m the air for 1 0 s followed by 1 0 s in an ethyl alcohol bath at 20°C (see Notes 1 , 7, and 8). 7. Immediately after thawmg, transfer the straw to a lammar flow bench, cut both ends and expel the contents mto 8 mL of unloadmg solution Allow the cells to settle, remove the supernatant, and replace With 2 mL of fresh unloading solu­ tion. Maintam at 20°C for 30 mm 8. Transfer 1 00 J.!L of settled cells onto 4 mL of semisolid culture medmm m a 3 . 5-cm Petri dish. Seal the dishes with Nescofilm Incubate cultures at 25°C under fluorescent light (2000 lx) or m the dark at a temperature appropnate for the nee cell line used. 9 Withm 3-4 wk of thawmg, sufficient cell regrowth should have occurred to allow the reimtiation of suspension cultures.

3.4. Assessment of Postthaw Reco very The presence of postthaw cell regrowth can be determmed by regular (at 7-d intervals) examination of the thawed cells under a binocular microscope. Such examinations can be combmed with an assessment of morphological features, such as color and wetness, of the thawed cells, which can be Important mdica­ tors of cryomjury

(3).

Growth can be quantified by fresh and/or dry weight

determinations, and the growth expressed in terms of % change after thawmg or % difference as compared with nonfrozen controls. In combmation wtth such observations it is also useful to assess the viabihty of the thawed cells usmg techniques ,such as FDA (28) staining or TTC reduction (29) as described below. VIability determinatiOns can provtde a useful mdication of potential postthaw growth and can be used to ascertain the effects of dtfferent postthaw culture conditions

(3).

However, such determinations should not be made m

90

Benson and Lynch

tsolatwn, since apparently htgh cell vtability ( determmed for example by TTC reduction) soon after thawing ts not always a prelude to postthaw cell growth (3) .

3.4. 1 . FDA, Based on Widholm (28) Place 1-2 drops of the FDA stock solutwn into 1 0 mL of hqmd culture medmm and mix well 2. Add 1-2 drops of the di luted stain to the cell sample mounted on a mtcroscope shde. 3. Leave for a few mmutes before placing the cover slip over the sample. 4 Count the number of cells m a field ofvtew under bnght field, and then count the number of fluorescent cells in the same field of vtew with UV tlluminatton % Vtabtlity = (number of fluorescent cells/total number of cells) x 1 00

(1 )

Determme the viabthty in at least five fields of view to calculate an overall sample mean vtabihty.

3. 4.2. TTC, Based on Steponkus and Lamphear (29) I . Take 1 00 mg fresh wetght cell samples, place

2 3. 4 5. 6 7

8

9.

m a test tube, and add 3.0 mL of TTC reagent solutiOn Infiltrate reagent solution under vacuum for I 0 mm. Incubate samples in the dark ovemtght at 25°C. Remove the TTC reagent and wash the cells by resuspendmg m distilled water Remove the dtsttlled water Resuspended the cells in 7 mL of 95% ethanol Extract the formazm color complex by placmg the samples m bothng water unttl all the ethanol had been dnven off To avotd the butldup of ethanol fumes m the laboratory, thts stage should be performed tn a fume cupboard. Resuspend cooled samples m l 0 mL of ethanol, vortex, and allow the cells to settle out. Settling of the cells can be assisted by centrifuging the samples at 80g for 5 mm. Measure the absorbance of the supernatant at 490 nm , against an ethanol blank. Vtabihty can be expressed as absorbance at 490 nm/ 1 00 mg or /g fresh weight, or as a percentage of an unfrozen control. Sample rephcatton ts advtsable to take into account vanatwn between cell sampling procedures.

4. Notes

1 . To avmd the cryovials movmg around m the Circulation of the water bath, and to reduce the potential for microbial contamination, place jars of stenle water m the bath, which should be allowed to equilibrate to the temperature of the water bath To thaw, the cryovtals are plunged mto the stenle water in the jars. 2. Controlled-rate freezing has generally been achteved using computer program­ mable cooling systems manufactured, for example, by Planer Products Ltd. and CryoMed. These machmes provtde a very accurate and reproducible means of freezing nee cultures However, controlled-rate freezers are expensive (usually

Rice Tissue Cultures

3.

4

5

6.

7.

91

over $ 1 2,000). Less sophisticated alternatives using standard laboratory freezers have been successfully used to cryopreserve rice cultures (9). Specifically, v1als are incubated for 2 h at -25 °C pnor to plungmg into liqmd nitrogen, or mcubated for I h at -25°C followed by 1 h at -70°C prior to plunging mto liqmd nitrogen. AA2-based culture medium must be filter-sterilized. To prepare semisolid AA2 based medmm, m1x equal quantities of double-strength AA2 medmm (filter-ster­ Ilized), w1th melted double-strength agarose (Sigma Type 1 ) 8.0 g/L in water (autoclaved), and immediately pour into Petn dishes. Cryoprotectants must be of a high purity or, m the case of DMSO, spectroscopi­ cally pure. After preparing the cryoprotectant mixture, adjust the pH to 5 8. Cryoprotectants should be prepared immediately prior to use, chilled on Iced water, and filter-stenlized Owing to the high viscosity of v1tnfication solutiOns, autoclaving is the only practical method of sterilization. Postthaw recovery medium tends to be based on the media the nee cells are nor­ mally maintained in, for example, AA2 (7), modified R2 (9), or LS (10). How­ ever, the presence of ammonium Ions in the culture medmm Immediately after thawing may be detrimental to postthaw cell growth (7,30) , as is the presence of fructose as the carbon source in the culture medmm (3) . The addition of the sur­ factant Pluronic F -68 (0 0 l % w/v) to postthaw culture medmm has been shown to promote postthaw rice cell growth (31). Supplementmg the culture medium with activated charcoal for the first 3 d postthaw can enhance postthaw cell growth (18) . Modifications o f the pre growth and recovery medium by the addition o f the iron chelating agent desfernoxamine can enhance the recovery of cryopreserved rice cell cultures (32). Thus, short-term applications of the drug (3 d exposure) before and after cryopreservation, at concentrations withm the range of 0.5- 1 0 mg/L can improve recovery potential. This i s further enhanced if catiOns are removed from the culture medium during the period of application Desfern­ oxamme has been marketed as the drug Desferral by C1ba Geigy and can be obtamed from Ciba Laboratories, Horsham UK. Its mode of actiOn involves the reduction of oxidative stress v1a the removal ofcations, which enhance the Fenton reaction and hydroxyl radical production. RICe cell cultures are known to un­ dergo oxidative damage dunng cryopreservation (33) . Where controlled-rate freezers have been employed to freeze nee cultures, the freezing programmes most widely employed are based on that of Withers and King (34) . This specifically involves a hold for 10 mm at 0°C, cooling at- 1 °C/mm to -35 °C, and holding at -3 5°C for 3 5 min. Samples can be stored either under liquid mtrogen or m liquid nitrogen vapor, with the level of liqmd nitrogen maintained below the lowest vials. Under the latter conditions, no liquid mtrogen enters the vials, reducmg the chances of vials exploding when thawed. Losses ofliquid mtrogen by evaporation are less in these systems. However, significantly the temperature of the uppermost Vials may tend to oscillate and rise above - 1 1 0°C. Thts can cause devitrificatwn and Ice crystal growth in the cells, which Will result in the loss of cell v1abihty.

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8. Care should be taken when thawmg vials or straws, smce any hqmd nitrogen m them w1ll rapidly evaporate, and this can lead to etther vials or straws ruptunng explosively. Therefore, appropnate safety precautions should be observed, includmg weanng a face shteld. 9. To reduce photo-ox1dation damage to the cells (35) , all mampulations dunng the recovery phase are earned out m a nomlluminated laminar flow bench.

References 1 . Kothan, S. L., Davey, M R., Lynch, P. T., Finch, P. T., Fmch, R. P., and Cockmg, E. C ( 1 99 1 ) Transgenic rice, m Transgemc Plants, vol. 2 (Kung, S D. and Wu, R., eds ), Butterworths, London, pp 3-20 2. Cao, Duan, J , X., McElroy, D., and Wu, R ( 1 992) RegeneratiOn of herbicide res1stant transgemc nee plants followmg miCroprojectlle-medtated transforma­ tiOn of suspensiOn culture cells Plant Cell Rep 1 1, 586--5 9 1 . 3 Lynch, P. T. and Benson, E. E. ( 1 99 1 ) Cryopreservatwn, a method for mamtam­ mg the plant regeneratiOn capac tty of nee cell suspensiOn cultures, in Proceed­ mgs of the Second Rzce Genetzc Symposzum, IRRI, Los Banos, pp. 32 1-332 4 Glaszmann, J C. ( 1 987) Isozymes and classificatiOn of Astan nee vaneties Theor Appl Genet 14, 2 1-30 5. Hodges, T K., Peng, Lyzmk, J., L A., and Koetje, D. S. ( 1 99 1 ) TransformatiOn and regeneratwn of nee protoplasts, in Rzce Biotechnology (Khush, G. S and Toenniessen, G H , eds.), CAB InternatiOnal, Wallingford, pp 1 5 7-1 74 6 MeiJer, E. G M , van Iren, F , SchnJnemakers, E , Hensgens, L A. M., van Zijderveld, M., and Schilperoort, R. A. ( 1 990) Retention of the capactty to pro­ duce plants from protoplasts m cryopreserved hne of rice (Oryza satzva L ). Plant Cell Rep 1 0, 1 7 1- 1 74. 7 Lynch, P. T., Benson, E. E., Jones, J , Cockmg, E C , Power, J B , and Davey M R. ( 1 994) Rtce cell cryopreservatwn: the mfluence of culture methods and the embryogemc potential of cell suspensions on post-thaw recovery. Plant Scz 98, 1 85-1 92 8 Cornejo, M-J, Wong, V. L , and Blechi, A. E ( 1 995) Cryopreserved callus - A source of protop lasts for rice transformation. Plant Cell Rep 14, 2 1 0--2 1 4 9 Jam, S., Jain, R. K , and Wu, R ( 1 996) A s1mple and effic1ent procedure for cryopreservatwn of embryogenic cells of aromatic Indtca nee vanet1es. Plant Cell Rep 15, 7 1 2-7 1 7. 10. Watanabe, K., Kawai, F., and Kanamorii, M. ( 1 995) Factors affectmg cryopro­ tectabthty of cultured nee (Oryza satlva L ) cells--cell wall and cell aggregate size. Cryo Lett 16, 1 47-1 56. 1 1 . Watanabe, K and Steponkus, P. L. ( 1 995) The vttnfication of Oryza satzva cells, L . Cryo Lett 16, 25 5-262 1 2 Huang, C. N., Wang, J. H., Yan, Q. S., Zhang, X Q., and Yan, Q. F. ( 1 995) Plant regeneration from nee (Oryza satlva L ) embryogemc suspensiOn cells, cryopre­ served by v1tnfication Plant Cell Rep 14, 730--734.

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1 3 . Ribeno, R. C. S., Jekkel, Z., Mulligan, B. J., Cockmg, E. C., Power, J. B., and Davey, M. R. ( 1 996) Regeneration of fertile plants from cryopreserved cell sus­ pensiOns of A rabidopszs thaliana (L.) Heynh. Plant Sci. 1 1 5, 1 1 5-1 2 1 . 1 4 . Chen, T . H . H., Kartha, K . K., Constabel, F., and Gusta, L V . ( 1 984) Cryopre­ servatwn of alkaloid-producing cell-cultures ofpenwmkle (Catharanthus roseus). Plant Physiol. 75, 726-73 1 1 5 . Skriver, K. and Mundy, J ( 1 990) Gene expressiOn in response to abscistc actd and osmotic stress. Plant Ce// 2, 503-5 1 2 . 1 6. Watanabe, K . , Sato, F , Yamada, Y . , Kawai, F . , Kanamori, M . , and Mttsuda, H ( 1 992) Characten sation of polypeptides in cultured rice cells differing in cryoprotectability J Plant Physzol. 136, 443--447 1 7. Chandler, P. M. and Robertson, M. ( 1 994) Gene expression regulated by absctstc acid and tts relation to stress tolerance Ann. Rev Plant Physzol Plant Mol Bzol 45, 1 1 3-1 4 1 . 1 8 Scrijnemakers, E. W . M . and van Iren, F. ( 1 995) A two-step or eqmhbrium freez­ ing procedure for the cryopreservation of plant cell suspensiOns, in Cryopre­ servatwn and Freeze-Dryzng Protocols (Day, D J and McLellan, M R , eds.), Humana Press, Totowa, NJ, pp. 1 03-1 1 1 1 9 Meryman , H. T. and Williams, R. J ( 1 985) Basic pnnciples of freezmg InJUry 10 cells; natural tolerance and approaches to cryopreservation, 10 Cryopreservatzon of Plant Cells and Organs (Kartha K. K., ed.), CRC, Boca Raton, pp. 1 3--48 . 20 Grout, B W. W . ( 1 995) IntroductiOn to the z n vztro preservatiOn o f plant cells, tissues and organs, in Genetic Preservatzon ofPlant Cells In Vztro (Grout, B. W W., ed.), Springer-Verlag, Berlm, pp 1-1 2 . 2 1 . Uragami, A., Sakai, A., Nagai, M . , and Takahashi, T ( 1 989) Survival of cultured cell and somatic embryos of Asparagus officznalis cryopreserved by vitnficatwn. Plant Cell Rep 8, 4 1 8--42 1 . 22. Langis, R., Schnabel, B , Earle, E D . and Steponkus, P. L ( \ 989) Cryopre­ servatwn of Brasszca campestris L. cell suspensions by vitrification Cryo Lett 10, 42 1--428. 23. Reinhoud, P J., Uragami, A., Sakai, A. and Van Iren, F ( 1 995) VItrificatton of plant cell suspensions, m Cryopreservatzon and Freeze-Drying Protocols (Day, D.J and McLellan, M R., eds.), Humana Press, Totowa, NJ, pp. 1 1 3-1 20. 24. Abdullah, R., Cockmg, E . C , and Thompson, J. A. ( 1 986) Efficient plant regen­ eration from nee protoplasts through somatic embryogenesis. Rio/Technology 4, 1 087-1 093 . 25. Uragami, U. ( 1 99 1 ) Cryopreservatwn of asparagus (Asparagus officznalzs L.) cul­ tured zn vztro. Res. Bul l. Hooatdo Nat Agric. Expt. Stat. 1 56, 1 3 7 . 26. Sakai, A., Kobayashi, S., and Oiyama, I. ( 1 990) Cryopreservatwn ofnucellar cells of navel orange (Citrus sznenszs Osb var. brasz/zenszs Tanaka) by vitnficatwn Plant Cell Rep. 9, 3 0-3 3 27 Linsmaier, E. M. and Skoog, F. ( 1 965) Organic growth factor reqmrements of tobacco tissue cultures Physzol Plant 8, 100-1 27 -

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28. Widholm, J M. ( 1 972) The use of florescein dtacetate and phenosafranine for determming viability of cultured plant cells. Stam Techno/. 47, 1 89. 29 Steponkus, P. L. and Lamphear, F. 0. ( 1 967) Refinement of the tnphenyl tetrazo­ hum chloride method of determining cold injury. Plant Physwl. 42, 1 423-1426. 30. Kuriyama, A., Watanabe, K., Ueno, S., and Mttsuda, H. ( 1 989) Inhibitory effect of ammonium ions on recovery of cryopreserved rice cells. Plant Scz. 64, 23 1 -23 5 3 1 Anthony, P., Jelodar, N. B., Lowe, K. C., Power, J. B. and Davey, M . R. ( 1 996) Pluromc F -68 mcreases the post-thaw growth of cryopreserved plant cells. Cryo­ biology 33, 508-5 14. 32 Benson, E. E , Lynch, P. T , and Jones, J. ( 1 995) The use of the tron chelatmg agent desferrioxamine m rice cell cryopreservatwn· a novel approach for tmprov­ mg recovery. Plant Scz 1 10, 249-258 3 3 . Benson, E. E., Lynch, P. T., and Jones, J ( 1 992) The detectiOn of hpid peroxt­ dation products m cryoprotected and frozen nee cell, consequences for post freeze recovery Plant Scz. 85, 1 07-1 14. 34 Withers, L. A and King, P. J. ( 1 980) A stmple freezing umt and cryopreservatwn method for plant cell suspenstons. Cryo Lett. 1, 2 1 3-220. 35 Benson, E E and Noronha-Dutra, A. A ( 1 988) Chemtlummescence m cryopre­ served plant tissue cultures. the possible involvement of smglet oxygen m cryoinJury. Cryo Lett. 9, 120-- 1 3 1

9

-------

Noncryogenic, Long-Term Germ plasm Storage Ali Golmirzaie and Judith Toledo

1.

Introduction Germplasm conservation is an important goal in the preservation of genetic

diverstty. Conventtonal methods, such as field and seed conservation, are used for most crops, although nonconventional methods, such as tissue culture and cryopreservation, have been used to support those methods. Tissue culture can be used to maintain p lant material, thus safeguarding 1t against losses caused by adverse environmental changes, pests, and diseases durmg growth under field condtttons. For in v1tro establishment, the choiCe of a particular medmm depends on the plant species, but m general, the medium should contain minerals, a carbon source, vitamins, and low concentratiOns of growth regulators

(Table 1).

In this medium, plants can grow m vitro for

2-3 mon, when it is then necessary to make subcultures to fresh media. Thts procedure increases the amount of labor required, especially when workmg with a large collection. In vttro maintenance of plants for longer periods of time, without subcul­ ture, is done through growth rate reduction, in which modifications to environ­ mental conditions or changes to some media components are made. Examples are reducing growth temperature, reducing hght mtensity, usmg growth regu­ lators, hmiting mineral supply, addmg osmotic stressants, or combming any of these procedures.

1. 1. Growth Temperature Temperature ranges established for culture rooms are similar to field tem­ peratures for a given crop. To minimize the growth rate of temperate species, we can reduce room temperature to near zero; for tropical crops, a moderate reductiOn in normal growth temperature is applicable (1,2) . From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

95

96

Golmirzaie and Taledo

Table 1 Components of Culture Media of Potato Included with the Murashige and Skoog Mineral Salts Mixture and Vitamins ( 1 1, and see Appendix) (International Potato Center, 1 997) Ingredients, mg/mL Gibberellic acid

Sucrose% Sorbitol% Benzyl ammo punne Chlorocholine chloride Agar% Phytagel% pH

PropagatiOn medium 01 2.5

ConservatiOn medmm 2.5 40

Tubenzat10n medmm 80 50 500

3 5 56

80 3.5 5.6

5.6

1.2. Light Intensity In VItro plants use sugars as a carbon source through heterotrophic absorp­ tion in the culture vessel. Although these plants still mamtam their photosyn­ thetic ability (autotrophic absorption), this can be restncted by low C02 concentrations. By using low hght intensity, we can reduce the support of car­ bon obtamed autotrophically, which then results m delayed growth (3). For in vitro conservation of potato plantlets, low light intensity is used along with other stressants (4). 1.3. Growth Regulators Abscisic acid (ABA) reduces the overall growth rate ofin vitro plants. Although it is used for several crops, physiOlogiCal changes or mutations can appear, which can threaten germplasm genetic stability (2-5) . 1.4. Nutrient Supply Limiting the nutrient supply can delay plant growth by decreasmg the amount of specific minerals, such as nitrogen or magnesium. This IS done for vanous crops (4, 6). However, symptoms of nutrient deficiency can appear, which lead to severe stress, thus reducmg plantlet viability. 1.5. Osmotic Stressants Minerals dissolved in water are mtroduced into cells through differences in osmotic pressures (inside and outside the plant cell). The inclusiOn of sugar m media increases the osmotic potential, thus reducing the uptake of minerals by cells. As a consequence, plant growth is delayed. Sugar alcohols (as mannitol

97

Noncryogenic, Long- Term Germplasm Storage Table 2 Number of Accessions of Solanum Species Maintained Under Long-Term Conditions from 1-5 Years (Unpublished Data) Solanum species

1

2

3

4

5

Total

S andzgena S stenotomum S tuberosum S. phureja S chaucha S gonzocalyx S JUzepczuku S a]anhuzrz Others

I 0 0 0 0 0 0 0 0

569 57 38 37 23 IS 6 6 1 84

666 54 24 37 23 6 3 3 1 25

1 55 5 I 29 68 66 61 3I 8 8 280

70 5 8 9

286I 245 1 38 1 49 1 08 53 17 I7 607

I 0 0 18

and sorbitol) are used extensively in germplasm conservation. Osmoticums enter the cell slowly and produce osmotic effects without being metabolized (1,2, 7). A combinatiOn of osmotic stressants and low temperatures increases the length of time between subcultures. For the in vitro conservation of potato, good results have been obtamed by combming osmotic stressants, low temperatures, and low hght intensity (8). Most potato accessions can be maintained for up to 4 yr With­ out subculture under these conditions (Table 2) (Fig. 1 ) .

1.6. Long-Term Maintenance Through In Vitro Tubers Microtuber production I S an alternative method for clonal multiplicatiOn m tuberous species. Microtubers of potato have been developed at CIP for a wtde range of genotypes for the purpose of international germplasm distribution, potato seed production, and as an alternative to germplasm conservatiOn. Microtubers are produced in 2-3 mon and can be stored at 1 ooc for 1 0 mo (9) after harvest. AdditiOnally, tuber dormancy can be controlled by environmen­ tal changes (9, 1 0), or sprout growth can be retarded by storage of sprouted tubers embedded in conservation medium, thus permitting another means of long-term conservatiOn. 2. Materials 1 Potato plantlets growmg on propagation medium 2. Culture media (Table 1) contam Murashige and Skoog salts (11; see Appendix) (Gibco I 0632--0 1 6) and Murashige and Skoog vitamms (11; see Appendix) (Sigma M7 1 50); growth regulators are mcluded in the media before autoclavmg, agar or phytagel is mcluded after adjusting the pH The media are autoclaved at I 2 I oc for 20 mm at I S ps1 before use

98

Golmirzaie and Toledo

3 months

4 years

Fig. I . Potato plantlets after 4 yr of storage at 6-8°C in conservation media. 3. Growth regulators: Gibberellic acid (Sigma G7645), 6-benzylaminopurine (Sigma 85898), chlorocholine chloride (Sigma C4049). 4. Sucrose grade II (Sigma S5391). 5. Phytagel (Sigma P8169) or agar (Sigma Al296). 6. Surgical knife handle # 1 1 with blade #15. 7. Stainless-steel forceps, 1 8 mm. 8. 25 x ISO mrn pyrex test tubes, containing 1 2 mL ofculture media. 9. lOOX 15-mm Petri dishes containing two sheets Whatman no. I filter paper. I 0. Bacti-incinerator for instruments. l l . Laminar flow cabinet for aseptic manipulation. 12. l 8-22°C culture room (air conditioner with thermostat control), 3000 lx, 1 6 h light. 13. 6-8°C culture room (air conditioner with thermostat control}, l 000 Ix, 16 h light. 3. Methods

3.1. In Vitro Propagation by Single Nodes I . Under aseptic conditions, take out in vitro plantlets obtained from buds growing

on propagation media from the culture tube, and place on a Petri dish (see Notes 1 and 2).

Noncryogenic, Long- Term Germplasm Storage

99

2 Slice the plantlets mto pieces or nodes (miCropropagatwn) Transfer each node mto a glass tube 1 8 x I SO mm wtth propagation medium (Table I), 3 nodes/ tube (11) 3 Transfer to incubatiOn room. After 2-3 mo, plants w1th more than l O nodes w1ll be obtamed. IncubatiOn room conditions are 1 8-22°C, 16 hr hght, 3000 lx.

3.2. Transfer to Slow-Growth Conditions

2.

3. 4.

5

Cut plants growing on propagatiOn medium at 1 8-22°C into single nodes, and place m tubes of 25 x 1 50 mm contaimng conservat10n medmm (Table I), approx 5 nodes/tube and 5 tubes/accession (see Note 3) Grow plantlets for 2-3 wk at 1 8-22°C (untll roots and the first two nodes are obtamed), after which, transfer to a cool room at 6°C, 1 6 hr light, I 000 lx (see Note 4). Make evaluatiOns of stress response weekly for the first few months, and then make monthly evaluatwns (see Notes 5, 6, and 7). When plantlets show glassmess, or become yellowish or browmsh, replace with new nodes. Sometimes some explants produce this effect, whereas other nodes grow normally. When plants begm to dte and produce symptoms, such as stems turmng yellow at the bottom, media show empty bubbles, or when plants reach the top of the tube, they must be subcultured. Under this evaluation, most accessiOns of the potato collectiOn were maintamed for 4 yr (Table 2, see Note 8)

3.3. Subculture After Slow Maintenance 1 . Transfer plants to propagation medium when the mamtenance period IS fimshed m order to recover normal growth. During the first month, nodes will show slow growth However, normal growth will be recovered dunng the next few months. IncubatiOn room conditwns for this period are the same as for the propagatiOn room. If stress symptoms persist, a second micropropagation (Subheading 3.1.) 1s necessary (see Notes 1 and 3) 2. Place the micropropagated plants obtamed on conservatiOn medmm (Subhead­ ing 3.2.).

3.4. Long-Term Maintenance Through in Vitro Tubers l . Propagate in VItro plantlets In magenta boxes using propagatiOn medium. 2 After 3 wk of growth, add the hqmd tuberization medmm over the plants grow­ mg in the magenta boxes, and place in a darkroom for 2-4 mo 3 . M1crotubers appear from the second month and continue growing for the next few months 4. When the microtubers have taken on a rounded form and the connectiOn With the stolon is tight, they are ready for harvesting 5 Store microtubers either in Petri dishes w1th a wet filter paper or embedded in conservatiOn medium

1 00

Golmirzaie and Toledo

4. Notes

2

3.

4

5.

6

7. 8

Plantlets for slow-growth conservation must come from young cultures that have good phystologtcal status m order to obtam the best effect of osmotic stress and therefore a longer penod of conservatiOn Mamtenance of large collections mvolves the use of numbers or names to tden­ tify accesstons. Databases (Foxpro for Windows, MS Access quenes, or MS SQL-Server) wtth spectfic informatton can be useful for management, because they provtde a source of labels to pnnt when necessary When plantlets grown m conservatiOn medium are subcultured onto new conser­ vation medmm after thetr growth penod, vtabt hty m nodes ts reduced and plant­ lets have low vtgor. In thts case, we recommend a source ofplantlets growmg on propagatiOn medmm The culture growth room for longer penods of plantlet conservatiOn has to be mamtained in the best aseptic conditiOns as posstble by avoiding sources of con­ tammatwn (dust, dtrt, or contammated matenal) and cleanmg the room frequently (using bactenctdes, fungicides, and acanctdes) At the same time, a restncted area has to be constdered to prevent trespassmg. To ensure the stabthty of culture room growth condttlons, we recommend usmg an electromc alarm system con­ nected to a control panel m a secure room. Durmg growth, the lateral buds are stimulated to grow and produce several branches that dunng the first year, look hke a bush at the bottom of the tube After the first year, each branch contmues growmg toward the top of the tube Aenal roots are normally present and develop profusely over time. Leaves are normally very small, and stems are thick. Slow growth can be affected by a reduction m osmotic stress produced when plants take up manmtol, thus accumulating the substance m tissues or using it as a source of carbon Thts has been reported m wheat (embryo culture), rape (seed­ hng culture), and potato (stem culture) (12) There ts no dtfference between dtrty (vtrus-mfected) and clean (vtrus-free) plant­ lets growmg on conservation medmm The genetic stabthty of plantlets growing in slow-growth condttwns has to be monitored; there are some mdtcattons that low temperatures (5-l 0°C) mtght cause biochemtcal leswns dunng seed storage and cryopreservatwn. In thts case, morphological comparisons and molecular techmques should be developed to compare field and m vitro accessiOns

References Dodds, J. H. and Roberts, L. W ( 1 985) Expenments m Plant Tissue Culture, 2nd ed. Cambndge Umversity Press, Cambndge, NY 2. George, E. F. and Shernngton, P. D. ( 1 984) Plant Propagatwn by Tissue Culture Exegetics Ltd , Edmgton, Wetsbury, Wilts, England 3. Hughes, K W. ( 1 98 1 ) In v1tro ecology. exogenous factors affectmg growth and morphogenesis in plant culture system. Envzron Exp Botany 2 1 , 28 1-288.

Noncryogenic, Long- Term Germplasm Storage

101

4 . Lizarraga, R., Huaman, Z , and Dodds, J. H . ( 1 989) I n vitro conservation ofpotato germp1asm at the InternatiOnal Potato Center. Am. Potato J 66, 253-276. 5. Wescott, R. J. ( 1 98 1 ) Ttssue culture storage of potato germplasm. 2 Use of growth retardants Potato Res 24, 343-352 6 Wescott, R J ( 1 98 1 ) Tissue culture storage of potato germplasm. 1 Mtmmal growth storage Potato Res 24, 33 1 -342. 7. Thompson, M. R , Douglas, T. J., Obata-Sasamoto, H., and Thorpe, T. A. ( 1 986) Manmto1 metabolism m cultured plant cells Physwl Plant. 67, 365-369 8. Espinoza, N., Ltzarraga, R., Stguefias, C., Buitron, F., Bryan, J , and Dodds, J H. ( 1 992) Tzssue Culture: Mzcropropagatwn, Conservatwn, and Export of Potato Germplasm CIP Research Guide 1 . International Potato Center, Ltma, Peru 9 Estrada, R., Tovar, P , and Dodds, J H. ( 1 986) Induction of in vttro tubers m a broad range of potato genotypes. Plant Cell Tzss. Org Cult 7, 3- 10. 1 0 Tovar, P., Estrada, R., Schtlde-Rentschler, L , and Dodds, J. H ( 1 985). Inductton of m vitro potato tubers. CIP Circular 13 (4): 1-4. InternatiOnal Potato Center, Lima, Peru 1 1 . Murashtge, T and Skoog, F ( 1 962) A revtsed medmm for rapid growth and bto­ assays with tobacco ttssue culture Physzol Plant 15, 473-497 1 2 Ltpavska, H and Vreugdenhtl, D. ( 1 996) Uptake of manmtol from the medta by m vitro grown plants. Plant Cell, Tzss Org. Cult 45, 1 03-1 07

10

__ __ __ __ __ __ __ __ __ __ __ __ __ __

Micropropagation of Strawberry via Axi l lary Shoot Prol iferation Philippe Boxus

1 . Introduction Elite strawberry plants can only be multiphed by a vegetative method. This explains the great disseminatiOn of parasites, such as viruses, mycoplasmas, nematodes, soil fungi, and tarsonems, which are transmitted directly or mdi­ rectly to the descendants. The tmpossibihty of efficiently fightmg agamst such parasites, the introductiOn of nonresistant culttvars, and mtemational trade have rendered the problem more and more acute. However, most strawberry­ producmg countries have drawn up a program of certificatiOn that takes these vanous parasites into account. After heat therapy or meristem culture, healthy plants are Isolated m screened cages, far from any source of contaminatiOn. The sotl is dismfected by fumigation. Every mother plant Is indexed, and daughter plants are then planted in the open air m well-isolated and dismfected plots. They remam under very strict phytosanitary observatiOn. The plants produced m this way consti­ tute registered stock, which IS considered virus-free, though It ts tmposstble to give absolute evidence of thts. This traditional method of multtplying healthy plants requires a minimum delay of 3 yr before a healthy cultivar is at the producer' s disposal. Massive production of strawberry plants in vitro, introduced in 1 974 (1), offered an interesting improvement to this very strict and very slow scheme. Therefore, within five years, European growers had adopted this method. The technical process IS very simple. The presence of I mg/L benzylammopunne (BAP), an active cytokmm, m the basal culture medium mduces an exten­ sive development of axillary dormant buds. This axillary branchmg contmues From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

1 03

1 04

Boxus

from one subculture to the next, but as soon as the transfer ts made onto a BAP-free basal medium, the branchmg stops, and rooted strawberry plantlets develop. The first industnal apphcatwn of strawberry micropropagation was reported m Germany (2). About 1 0 years later, the annual productiOn ofmicropropagated strawberry plants reached 7.5 million. Thts high-quality matenal, used as stock mother plants, produces a large portion of the btllion conventional strawberry plants annually produced m the world (CEE, Cost 87, January 1 98 8). In the US alone, m 1 985, 350,000 plants were ratsed m vitro, whereas 700 million plants were produced m the field (3) Numerous field tnals, made to compare frmt production of healthy plants orgmating from tissue culture with standard material, dtd not show any statts­ tical differences (4-7). However, some sporadic occurrences of abnormal frutt setting were observed on specific cultivars. A hyperflowermg habit entailed malformations and small fruits, although this trouble was only present on spe­ cific cultlvars after numerous subcultures. Therefore, it was decided m several countnes to limit to 1 0 the number of subcultures for each mericlone (6). More­ over, legislation in some countries rejected or limited the use of this new meth­ odology. In The Netherlands, for mstance, only the everbearing cultivars could be propagated in vitro. Although the limitation of subculture number was very effective in control­ ling the frutt quality of strawberry microplants, we were obliged to wait for J emmali et al. ' s researchs to understand the reasons for this hyperflowering behavior (8) . Indeed, they observed a new kind of bud during the proliferation phase. Named "stipular buds," they appeared at a specific position on the sttpule (Fig. 1). After isolation and transfer onto a proliferation medium, they showed a very high propagation rate in VItro, and a hyperflowermg habit in production fields. Jemmali et al. identtfied a htgher free BAP content in these buds (9). It is possible to limit the occurrence of these sttpular buds by decreasing the BAP concentration from 1 mg/L to 0.5 or 0.25 mg/L. Moreover, the number of subcultures has to be stnctly limited to 1 0. Under these conditions, we have a very htgh-quality stock material, which ts well adapted to the new mtensive crop system. These observatiOns underline the nsks introduced by adventitious regenera­ tion in a mass propagation system. However, on the contrary, the axlllary branches developed from preformed axillary buds are genetically and pheno­ typically stable, as was already underlined (I 0) and agam confirmed in our very recent tnals on strawberry plants (11).

105

Micropropagation of Strawberry

Fig. I . Neoformation of stipular buds on cv 'Gorella' subcultured on a proliferation medium containing I mg/L BAP. (A) The buds present a fasciation, paral lel to the petiole axis. (B) The stipular bud develops just on the middle of the two stipule tips. LS: left stipule, RS: right stipule, SB: stipular bud, AB: axillary bud, PA: petiole axis.

Table 1 Stock Solutions for Strawberry Culture Medium (Knop Macronutrients) (12) Ca (N03h

I 00 giL

KN03

100 giL

Mg S04 KH2PO4

·

?H20

I 00 giL 100 giL

2. Materials

I . Plant material must be selected in a field production. From the selected mother plants, collect preferentially the youngest runners (see Notes 1 and 2). 2. Reagents for plant material sterilization: alcoholic solution 96%, calcium hypo­ chlorite 5% (w/v, see Note 3). 3. Binocular microscope and dissecting tools, razor blades and handles, scalpels with disposable blades, and fine forceps adapted for meristem excision ( 12 em long).

4. Stock mother solutions: prepared with chemicals, quality pro analysis (see Note 4) a. Macronutrients: the four salts are stored at room temperature in liquid solu­ tion (100 giL) (Table 1).

1 06

Boxus

Table 2 Stock Solution for (1 000X) Murashige and Skoog Micronutrients (13) MS solution, mg/L end concentratwn

Salts

MnS04 H 20 ZnS04 7H2 0 H3B03 KI Na2Mo04 2 H2 0 CuS04 5H20 CoC12 6H2 0 •

•

•

•

•

16 8.6 6.2 0.83 0.25 0.025 0.025

1 L MS: stock solutiOn l ,OOOX, mg/L

1 6,000 8,600 6,200 830 250 25 25

Table 3 Stock Solution for (1 000 x) Murashige and Skoog Vitamins and Amino Acids (12) Vitamms or ammo acid Nicotinic acida Pyndoxme, HCl Glycine Thiamine, HCl Meso-mositol

MS solution, mg/L end concentration

1 L MS. stock solution l ,OOOX, mg/L

05 05 20 0 1 1 00.0

5 00 500 2000 1 00 1 00,000

"Nicotmic acid must be dissolved by gentle heatmg

b. Mtcronutnents: strength l OOOX, stored at +4°C (Table 2) c Vttamms strength l OOOX , stored at +4°C (Table 3) d. Growth substances· 1 Cytokmm BAP strength l OOOX, dissolved m a few drops HCl 32%, then make up to volume with distilled water, stored at +4°C m dark bottle. 11 Auxm, mdolyl butync acid (IBA), strength I OOOX, dissolved m 1 M KOH, stored at +4°C m dark bottle. iii. Gibberellic acid, GA3, strength l OOOX, dissolved m water, stored at +4°C 5 Agar and glucose of good quality (see Note 5) 6. Glassware: a Tissue-culture tubes· 1 6 x 75 mm with screw cap for menstem culture, and 25 x 1 50 mm with loose-fittmg caps (kaputs, Belco) for the first subcultures. b . Tissue-culture Jars, 0 5 L, diameter 1 0 em, With a glass hds (see Note 6) c Precut food polythene film (23 x 23 em) to seal the hds of the J ars.

107

Micropropagation of Strawberry Table 4 Composition of Strawberry Culture Media (1)

Macronutrients Micronutrients Vitamin mixture Hormonal substances (mg/L) BAP IBA GA3 Glucose (g/L) Agar (giL) Activated charcoal (g/L) pH

Meristem culture medium

Propagation medium

Rooting medium

Knop Knop MS

Knop Knop MS

Knop Knop MS

0. 1 1 .0 01 40 0 5.2

05 1 0 0. 1 40.0 5.2

5.6

56

1 0 40 0 52 0.5 5.6

M S Murash1ge and Skoog, BAP benzylammopunne; IBA' indo! butync aCid, GA3• gibberellic ac1d

7. Honzontal lammar air flow hood for stenle transfers 8. Sterile disposable Petri dishes (90-1 00 x 1 5 mm), fine but long forceps (25 em) to divide clusters of axillary shoots. 9 Draft-free growth room w1th a1r conditioning, temperature between 22 and 25°C. The photoperiod IS fixed at 1 6-8 h/d and mght, With a li ght intensity of 2 3 0-50 f..1.mol/m /s, diffused by dayltght TL lamps. The control of relative humidity is only necessary m very humid countnes The growth room shelves (0 75- or I 50-m Width) are metal m order to prevent an excessive heat mside the Jars resultmg from the greenhouse effect during the light penod (see Note 7). 1 0 . Greenhouse facilities or plastic tunnels must be avmlable dunng the five or SIX weaning weeks. The strawberry plantlets are normally very resilient Therefore, heating or coohng eqmpment IS not obligatory.

3. Methods After media preparatton and the stenlization of the plant material, all subse­ quent steps should be earned out in a flow cabmet under aseptic conditions. 3. 1. Preparation of Culture Medium 1 . Prepare the culture media from mother stock solutions and distilled water. Except for the hormonal balance, the media have the same compositions for the three propagatiOn stages (Table 4). 2. Prepare culture media in a gauged flask, and before adding the agar and the glu­ cose, adjust the pH to 5.6 with I M KOH or 1 N HC1 solutions 3. After bmhng, the medmm is distnbuted, 2.5 mL in the smali test tubes, 1 5 mL m the large ones and 1 40 mL m the 0.5-L J ar s . Finally the matenal 1 s

1 08

Boxus

sten l l zed by autoclavmg at l l 0°C for 2 0-3 0 min, depending on the size of your autoclave. 4. After autoclaving, the jars or test tubes are kept m a clean room. They are ready to use 1 d later (see Note 8) 3.2. Initiation Phase or Meristem Tip Culture

1 . The position of the meristem t1p on the mother plant does not influence the plant propagation ability However, excisiOn IS easier from young runner tips than from well-developed plants (see Note 9) 2 . D1smfect the samples by washmg with water and weak detergent Then, d1p for 2 mm m an absolute alcohol solutiOn. Afterward, dip the samples for 1 0-20 mm m a 5% calcium hypochloride solution Fmally, the samples are transferred to a washing bath filled with stenle water (see Note 10) 3 . The samples are excised one by one usmg fine forceps and pieces of razor blade The explant size should be <0.3 mm to guarantee a maximum samtary state of the produced plants. They are aseptically placed m test tubes ( 1 6 x 75 mm) contam­ mg 2 . 5 mL menstem culture medmm 4 The cultures are placed on the shelves m the growth room 5 About 1 mo later, the excised meristem t1ps have developed into a rosette of leaflets. They must be transferred to a propagation medium to start the men clonal propagatiOn (see Note 1 1 ) 3.3. Mericlonal Propagation

l . As soon as the first rosette of leaflets has developed to a size of a few millimeters, transfer to a 25 x 1 50 culture tube sealed by kaputa (loose-fitting caps), contam­ ing 1 5 mL propagatiOn medmm Each culture tube IS labeled to keep the mericlones separate 2 The racks with the culture tubes are placed m the light on the shelves m the growth room for 6-8 wk 3 Within 3 or 4 wk after this transfer, two or three new buds will appear at the base of the petioles of the oldest leaves. These young axillary buds grow very qmckly and, m tum, produce new axillary buds. After 6-8 wk m the presence of cytoki­ nm (BAP 0.5 mg/L), the axillary buds mvade the entire culture tube The 1mtial plantlet is transformed mto a cluster of buds, often Without any callus These clusters may contam 1 5-25 small buds Each has several short petioles, ended by a very small unifohate leaf No roots are present on these buds. 4 At this stage, the clusters of buds may be aseptically separated and transferred onto a fresh medmm to contmue the clonal propagation. Every 6-8 wk, one Iso­ lated bud wtll produce 1 5-25 new axillary buds (see Note 1 2) 5 . Durmg the second diVISion of the clusters, only a small fractiOn of the 1solated buds IS transferred to proli feratiOn medmm to contmue the propagatiOn. The remamder IS placed on rooting medium to be stored at +2°C as mother stock plants ("germplasm") or to produce rooted plantlets to be used for virus mdexing (see Subheading 3.4.).

Micropropagat10n of Strawberry

1 09

6. Economtcally, the dtvis ion withm isolated buds is not very easy and takes a lot of time Therefore, after one or two transfers on prohferatwn medium, the c lusters are now dtvided more roughly into four to ftve small tufts of buds These are transferred onto fresh proliferatiOn medium, 7 explants/0. 5 L J ar (see Note 1 2) 7 The J ars are placed on the shelves m the light. About 4 wk later m the presence of 0.5 mg/L cytokimn, new large clusters are formed and may be divtded further (see Note 13) 8 To maintain a high proliferation rate, the transfers must be done tmmediately after the appearance of the first roots In thts way, the prohferatton rate ts about four- to fivefold per month, t.e , several milhon from one menstem m only 1 yr (see Note 14)

3.4. Rooting Phase

2 3.

4

5.

To obtain rooted plantlets, the clusters are dtvided into small tufts of three well­ developed buds. These explants are transferred to a BAP-free medtum contam­ mg 0.5 g/L acttvated charcoal. Each 0.5-L Jar contams 1 5 explants (see Note 1 5). Placed on the lighted shelves in the growth room, the first roots appear 7-1 0 d later, and the first true leaves (tnfoliate leaves) develop (see Note 16) Wtthm 5-6 wk, well-rooted plantlets occupy the whole Jar These have devel­ oped 4- to 8-cm high leaves and 3- to 7-cm long roots They are now ready for weanmg (see Note 1 7) Under Subheading 3.3., step 5, the possibility of creating a germplasm wtth a part of the clusters obtamed from the second or thtrd subculture was constdered. Toward thts atm, tsolated buds are placed m 25 x ! 50 mm test tubes, closed wtth kaputs and sealed wtth food polythene film After 4-5 wk m the ltght, the test tubes contaming well-developed, rooted plantlets can be stored m complete dark­ ness m a cold room at +2°C These can then be stored in this way for 2 or 3 yr These cold-stored plantlets could then be used to start a new propagatiOn !me They wtll replace the matenal coming from meristem culture. Of course, in thts case, tt wtll be necessary to take the total number of subcultures into account to av01d the nsk of dev1ant plants emergmg (see Subheading 1 .) Under Subheading 3.3., step 5, 1t was also recommended to root, dunng the first steps of the propagatton process, a mm1mum of two plants, which must serve for vtrus mdexmg. As the propagatiOn line ts pursued during the mdexing tests, tt ts ev1dent that all the productiOn should be destroyed m the case of v1rus contami­ nation (see Note 18).

3.5. Weaning Phase I . Rooted plantlets are removed from the Jars coming directly etther from the growth room or the cold room 2. Wash under tap water to ehmmate a maxtmum of gelled agar from the roots 3. The small tufts of clean rooted plantlets are separated mto m smgie plantlets wtth the help of fine forceps (see Note 1 9)

1 10

Boxus

Table 5 Field Performance, Runner Production in Italy, Year 1 979 (4) Culttvar

Propagat10n°

'Gorella'

TC

' Belrubt '

standard TC standard

' Ahso'

TC

standard

Number of runners 62 0 39.3 26 2 10 8 1 46.3 89.5

% runners >8

mm

7 1 .7 79.9 88.6 83 .4 54 2 6 1 .5

0TC mtcropropagatwn m tissue culture

4

5

6

7

8 9.

After grading and countmg, the wetted smgle plantlets are stored m small plastic boxes and placed at +2°C until the transfer to the soil (see Note 20) The rooted plantlets are acchmat1zed m trays filled w1th a peat m1xture substrate w1th a separatwn of 5 x 5 em. They are covered w1th a plastic sheet. The opttmum ambient temperature I S 22°C/1 5°C day and mght Strawberry plant\ets can sur­ vive more drastic temperatures wtthout damage if they are covered by a plastic sheet. However, long penods of low temperature (±5°C) or high temperature (±32 °C) must be avo1ded Covenng wtth a mtlky white plastic sheet can protect the plantlets from dtrect mtense solar radiation Immediately after transplantation, the plantlets are watered w1th a solution con­ tammg a broad spectrum fungtctde to avmd damage from fungt (see Note 21) After the watenng wtth the fungictde, the plastic sheet IS firmly sealed Every 2 or 3 d, the state of the plants must be controlled As soon as there is need for a fungicide treatment, repeat. After 2 or 3 wk, when the plants develop new leaves, the plastic sheet can pro­ gressively be hfted up Generally, 4 wk after soil transfer, the first runners come out. Then young plants can be planted in the nursenes for the productiOn of runners (see Note 22)

3. 6. Field Behavior Runner production: The mam characteristics of micropropagated strawberry plants m the field are a flatter habit of the nonflowenng plants and a very large number of stolon imtiatlons. At the begmmng of the summer, the stolons are shorter and finer than those formed on standard plants At the end of the growmg season, the runner plants obtamed from micropropagated plants are identical to the conventional ones Only the number of runners per mother plant can dtfferen­ ttate the two ongms (Table 5). Thts ability to produce larger quantities of run­ ners disappears dunng the second year of production This mcreased runner production ts very i mportant for the everbeanng culttvars, whtch are generally poor producers of runners

Micropropagation of Strawberry

111

Table 6 Field Performance: Fruit Production in France, cv. "Gorella" (Mean of 3 Years, 1 984-1 986) (6) Southeast

Standard New clones0 Old clones6

Southwest

Commercial grade, g/plant

Mean weight, g/fruit

Commercial grade, g/plant

Mean we1ght, g/fru1t

837 892 700

1 8.2 18 0 1 6. 1

510 602 418

17 5 17 6 1 4. 8

0New clones 'Gorella' clones w1th < 1 0 subcultures m v1tro hOld clones 'Gorella' clones w1th >30 subcultures m v1tro

Table 7 Fruit Production: Year 1 988, cv. "Rapella," Wilhelminadorp and Horst (The Netherlands) (7) Productwn kg/acre, class I and I I F1rst runner progeny o f T C plants Second runner progeny of TC plants Standard plants

278 A0 281 A 244 B

0Dlfferent letters mdJCate statlsllcal differences at p = 0 05 2. Fruit productwn: The comparisons made in several countnes, w1th different van­

eties, do not show any significant difference between the fru1t productwn from micropropagated or standard strawberry plants, providing that mericlones w1th an high number of subcultures have been avmded (Tables 6 and 7)

4. Notes

1 . To be sure to start the clonal propagation with the true cultivar, 1t 1s better to select the material in a field production. Ask the help of a good farmer, a special­ ist of the chosen variety, to select the best mother plants in the field. Some labo­ ratories keep the mother plants in screenhouses for several years, and always use the same plants for the menstem excision. Th1s method seems not as reliable as the previous one. 2. If possible, collect ±20 young runner tips, removed from several mother plants. Give a number to each mother plant, and keep the runner t1ps of each mother plant in separate bags. As soon as possible, place the bags in the vegetable compartment of a refrigerator. The samples can be stored easily for 2 or 3 wk before exciswn 3 . Calcium hypochlorite is very hygroscopiC and must be stored under dry condi­ tions. The 5% calcium hypochlorite solution must be prepared fresh before use This solutwn can be used only for 2 or 3 d

1 12

Boxus

4 All the mother stock soluttons are prepared from proanalySIS chemtcal reagents dissolved m distilled water Preparation of large quantities is possible In this case, distnbute the mother stock solution into different containers Sterilize the mineral mother stock solutions by autoclavmg, freeze the mother stock solutions of vttamm, and growth substances Before use, always control that there IS no precipitatwn in the concentrated mother solutwns. 5. Use a microbtological-grade agar and sugar 6. Some laboratones use other glass containers, named "Meh" j ars. These have a polythene hd and a content of 3 50 mL 7. The control of relative humidtty IS important in countnes wtth very humid and hot seasons, monsoon seasons for mstance If not controlled, the nsks of mould and bactenal contammatwns are too high. In spite of the mr regulatiOn m the room, a temperature gradient exists m most growth rooms Generally, the upper shelves are warmer than the lower ones Too high-temperature m the Jars results from a high ambtent temperature, but also from the greenhouse effect, which can cause an mcrease of 6-8°C in the Jars m companson With the ambient air There­ fore, it is important to place the jars Immediately on a metal shelf. This has the ambtent temperature and wtll eliminate the excesstve temperature from the Jars. 8 After autoclaving, cool the Jars as early as possible to avoid hydrolysis of the culture medmm. This can occur m summer dunng hot days, especially when the media contam httle agar In very hot countries, tt will be necessary to mcrease the agar concentration 9. To excise the menstems, the very young runner tips are the best plant matenal Thts matenal IS collected in a fruit productiOn field m June, before the runners have formed leaves and roots. Thts very young matenal has fewer halTS surround­ mg the menstemattc zone and results m httle contaminatiOn. When meristems are removed from dormant plants or from cold stored plants, a higher percentage of the cultures will be contammated. When only crowns of adult plants are avail­ able, avOid deep crowns or those from cold stored plants, because these are fre­ quently contaminated by bactena I 0. After stenhzatwn wtth calcium or sodium hypochlonde, 1t is not necessary to rinse the explants several times With stenle water, because chlonde tons are not toxic m the culture medium If after 1 mo the excised meristem has not developed a rosette of leaflets, this explant can be subcultured for another month onto the same menstem culture medium. 1 1 At each transfer, carefully clean the explants by removal of all necrotic parts, callus, and roots to avoid oxtdation or diffuston of phenohc compounds mto the fresh culture medmm. 1 2 . Dip the explants deeply into the medium to assure a good contact between the dormant axillary buds and the medium. This Will enlarge the number of axillary shoots developed dunng the subculture. There IS no risk of asphyxia provided that only a portion of the explant IS out the medtum. 1 3 The vaneta! sensitivity to the temperature 1 s variable. The most resthent cultiVars should be placed on the upper shelves. A general yellowmg of the plantlets dur-

Micropropagation of Strawberry

1 4.

15.

16

17.

1 8.

1 9.

20.

21.

1 13

mg the proliferatiOn phase 1s charactenstic of too htgh a temperature. In th1s case, change the level of the shelves or decrease the air temperature, but take care, because at a temperature that is too low, the plants will not grow fast enough. At each transfer, take the number of subcultures into account to be sure to stop at the l Oth subculture Dip the explants correctly mto the medium Take care not to have too hard a medium. Control that the mother jar, which must be divided, is not contaminated by fungus or bacteria. For these latter infectiOns, 1t is necessary to inspect v1sually the jars by transparency. If there 1s any doubt, e.g., presence of whtte cloud at the base of the explants or some turbidity in the condensation water at the surface of the medmm, discard this mother jar. Some bacteria are very diffi­ cult to eliminate, even by flaming! Finally, always transfer before or just at the appearance of the first roots. If this IS not posstble, e.g., no medta or no manpower available, immediately place the Jars m the cold room at +2°C to stop development. This last point is very important to maintam the high rate of multiplicatiOn The introductiOn of activated charcoal in the rooting medium tmproves the growth of the roots a little, but also, the black color of the medium is an excellent Identi­ fication label Some laboratories prefer MS medium (12) to Knop medmm (13) as macronutrients during the rooting phase Indeed, on this medium, the plantlets will grow faster. However, these MS plantlets are softer and also more suscep­ tible during the weaning phase Plantlets i n rootmg phase are more sensitive to an excess temperature. I n thts case, the roots stop their growth and develop a black tip. As soon as the jars are placed at a lower temperature, the roots start to grow agam To extend the penod of work m the laboratory, it is posstble to root strawberry plantlets some weeks before the normal weanmg ttme Rooted plantlets can be stored at +2°C for 4-6 mo without any damage, but in the case of long storage periods, place the rooted plantlets as early as posstble m the cold room to avoid oxidations or phenol productiOn. Very often, the propagation of new menclones ts pursued dunng the ttme reqmred for virus indexing. However, until now, the percentage of virus eliminatiOn has been about 1 00%, so the risk of being obliged to destroy all the productiOn for sanitary reasons is very low. In general, each 0.5-L jar produces 45 to 60 rooted plantlets, 3-7 em high. Some cultivars with very fine buds can give, per jar, higher numbers of rooted plantlets However, they are so thin that it is better to keep 2 or 3 buds together to facilitate their acclimatization. The little rooted plantlets are stored in 1 2 x 8 x 4 em plastic boxes covered by a see-through lid. These plantlets are motst when they are mtroduced m these boxes Also, after a few days, check the condensation water inside and eliminate the excess water to keep the matenal in good condition for 1 mo. Thirarn (TMTD) 0.5-1 .0% is a very effective fungtcide, without any phytotoxic­ ity for control of Botrytzs and Pythzum, the most dangerous fungt during the acclimatization phase. Avoid use of very freshly stenlized substrates, which are too easily mvaded by unfavorable germs.

1 14

Boxus

22. Transfer the plants to the nursery field as soon as possible to avOid a halt m the development of the young runners. Severe stress, mduced by a poor transfer from the greenhouse to the field, can even mduce flowers under long day length and, consecutively, can stop runner production for several weeks.

References

2 3.

4.

5 6.

7 8 9.

1 0.

II

12 13

Boxus, Ph ( 1 974) The production of strawberry plants by m vitro micropropa­ gation J Hart Sc1 49, 209-2 1 0 Westphalen, H. J. and Bill en, W . ( 1 976) Erzugung von Erdbeerpflanzen m grossen Mengen durch Sprosspitzenkultur. Erwerbsobstbau 18, 49, 50. Swartz, H. J and Lmdstrom, J. T. ( 1 986) Small frutt and grape tissue culture from 1 980 to 1 985 Commerciahzation of the techmque, m T1ssue Culture as a Plant Productwn System for Horticultural Crops (Zimmerman, R H. , Gnesbach, R. J , Hammerschlag, F. A , and Lawson, R H, eds ), Martinus NIJhoff, Dordrecht, pp. 20 1-220. Damiano, C. ( 1 980) Strawberry micropropagatiOn, m Proceedmgs ofthe Confer­ ence on Nursery Productwn of Fru1t Plant Through T1ssue Culture Apphcatwns and Feas1b1hty. USDA-SEA, Agncultural Research Results, ARR-NE. Beltsville, MD, pp l l-22. Boxus, Ph. ( 1 985) Comportement du fraiSier Issu de micropropagatton m v1tro . Fru1t Beige 4 10, 1 06-- 1 1 0. Navatel, J. C., Varachaud, G., Roudeillac, P., and Bardet, A. ( 1 986) Multiphca­ tion m v1tro· comportement agronomique de plants de fraiSiers 1ssus de pteds­ meres prodmts par micropropagatwn par rapport au materiel class1que. Synthese de trms annees d'expenmentatton. lnfos-CT!FL 21, 2-8 . DIJkstra, J ( 1 9 8 8 ) Samenvatting onderzoekresultaten vollegrondsaardbe1en ( 1 987) Internal Report (personal commumcatwn) Jemmah, A , Boxus, Ph., and Kinet, J M ( 1 992) Are strawberry ansmg from adventitious stipule buds also true to type? Acta Hart. 319, 1 7 1-1 76. Jemmah, A , Boxus, Ph., Kevers, Cl , and Gaspar, Th ( 1 995) Carry-over of mor­ phological and biochemical charactenstics associated With hyperflowenng of m1cropropagated strawbernes J Plant Phys1ol 147, 43 5-440 d ' Amato, F ( 1 977 ) Cytogenetics of differentiatiOn m tissue and cell cultures, m Applled and Fundamental Aspects of Plant Cell, T1ssue and Organ Culture (Remert, J and BaJaJ , Y. P. S., eds ), Spnnger Verlag, Berlin, pp. 344-3 57. Jemmah, A , Robbe, A., and Boxus, Ph ( 1 997) Histochemical events and other fundamental differences between axillary and adventitiOus stipular shoots m m1cropropagated strawberry Third Intern Strawberry SymposiUm, Veldhoven (The Netherlands) Acta Hort 439, 34 1-345 Knop, W. ( 1 965) Quantitative Untersuchungen uber d1e Emahrungsprozesse der Pflanzen Landwzrstsch Vers Stn 7, 93-1 07 Murashige, T and Skoog, F. ( 1 962) A revised medmm for raptd growth and bw­ assays With tobacco tissue culture. Physiol Plant 1 5, 473-497

ll

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Meristem-Tip Cu lture for Propagation and Virus Elimi nation Brian W. W. G rout 1. Introduction

The essence of meristem-tip culture is the excision of the organized apex of the shoot from a selected donor plant for subsequent in vitro culture. The conditions of culture are regulated to allow only for organized outgrowth of the apex directly mto a shoot, Without the mtervention of any adventitious organs (1-3) . The excised meristem tip is typically small (often < 1 mm in length) and is removed by sterile dissection under the microscope, as in the potato example detailed in this chapter (Fig. 1 ) . The explant compnses the apical dome and a limited number of the youngest leaf primordia, and excludes any differentiated provascular or vascular tissues. A major advan­ tage of working with such a small explant is the potential that th1s holds for excluding pathogenic orgamsms that may have been present in the donor plants from the in vitro culture (see below). A second advantage ts the gene­ tic stability inherent m the techmque, since plantlet productiOn Is from an already differentiated apical meristem and propagation from adventitious meristems can be avmded (3-9) . Shoot development directly from the mer­ istem avoids callus tissue formation and adventitious organogenesis, ensur­ mg that genetic instability and somaclonal variation are minimized. If there i s no requirement for vtrus eliminatiOn, then the less demandmg, related tech­ nique of shoot-tip culture may be more expedient for plant propagatiOn. In thi s related procedure the explant is still a dissected shoot apex, but a much larger one that is easter to remove and contains a relatively large number of deve loping leaf primordta. Typtcally, this explant is between 3 and 20 mm in length, and development in vitro can still be regulated to allow for direct outgrowth of the organized apex. From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

1 15

116

Grout

Fig. 1 . A freshly excised meristem tip from an axillary bud of the potato Solanum tuberosum. The two smallest emergent leaf primordia are present. Scale bar repre­ sents

50

f..IM.

The axillary buds of in vitro plantlets derived from meristem-tip culture may also be used as a secondary propagule. When the in vitro plantlet has devel­ oped expanded internodes, it may be divided into segments, each containing a small leafand an even smaller axillary bud (Fig. 2). When these nodal explants are placed on fresh culture medium, the axillary bud will grow directly into a new plantlet, at which time the process can be repeated. This technique adds a high propagation rate to the original meristem-tip culture technique, and together the techniques form the basis of micropropagation, which is so important to the horticulture industry (1-4; see Table 1). It is possible, however, that callus tissue may develop on certain portions of the growing explant, particularly at the surface damaged by excision (Fig. 3). The only acceptable situation under such circumstances is where the callus is slow-growing and localized, and the callus mass and any organized develop­ ment on it can be excised at the first available opportunity. Studies from the author's laboratory on cauliflower plants regenerated from wound callus and floral meristems in vitro showed normal phenotypes for all the plants produced by

Meristem-Tip Culture

1 17

Fig. 2. An axillary bud from a freshly excised nodal segment, taken from a mer­ istem-derived potato plantlet in vitro. A number of small leaf primordia are apparent, emphasizing the effectively normal structure of this very small axillary organ. Scale bar represents 50 jJM.

Table 1 The Propagation Potential Inherent In the Merlstem-Tip Culture TechniqueTime from culture initiation, mo

No of nodes availableb for further subculture, equivalent to plantlet number

3

5

4 5

25 125

6 7

625 3125

8

1 5,625

"The data are for potato species, Solanum curtilobum. and were calculated from propagation rates achieved in the author's laboratory. bAssumes 5 nodes availablelplantlet.

1 18

Grout

Fig. 3. A meristem-tip culture of potato 6 d after initiation. A distinct swelling can be seen at the wound surface, and larger callus cells are beginning to proliferate from it. Scale bar represents 500 �·

meristem culture, whereas those of callus origin included rosette and stunted forms, and both glossy and serrated leaves as examples of phenotypic deviance. These abnormal plants, derived from callus tissue, and normal plants from meristem culture showed the same DNA levels, as measured by scanning microdensitometry, but showed considerable differences when the isozymes of acid phosphatase was examined by gel electrophoresis. This suggests a range of unacceptable variation at the gene level, introduced by the adventitious ori­ gin of the shoots. A further advantage of meristem culture is that the technique preserves the precise arrangement of cell layers necessary if a chimeral genetic structure is to be maintained. In a typical chimera, the surface layers of the developing meristem are of differing genetic background, and it is their contribution in a particular arrangement to the plant organs that produces the desired character­ istics, e.g., the flower color in some African violets. As long as the integrity of the meristem remains intact and development is normal in vitro, then the chimeral pattern will be preserved. If, however, callus tissues are allowed to

1 19

Meristem-Tip Culture Table 2 The Relationship of Explant Size to Effective Virus (WCMV) Elimination in Clover8 mm

No. explants taken mto culture

No of plants estabhshed in soil

No. virus-free plants

<0.6 0.6-I .2 1 .3-1 .8 1 .9-2.4 2.5--3 .0

90 1 13 1 90 158 I 74

I8 45 102 88 92

18 19 25 II I1

Explant size,

aoata from (15)

form and shoot proliferation, subsequently, is from adventitious origins, then there will be a risk that the chimera! layers of the original explant may not all be represented in the specially required form in the adventitious shoots. The techmque of menstem culture may be explmted m situations where the donor plant is infected with vual, bacterial, or fungal pathogens, whether or not symptoms of the infection are expressed The basis of eradicatiOn is that the terminal region of the shoot meristem, above the zone of vascular differentation, is unlikely to contain pathogemc particles. If a sufficiently small explant can be taken from an mfected donor and raised in vitro, then there is a real possibility of the derived culture being pathogen-free. Such cultures, once screened and certified, can form the basis of a guaranteed disease-free stock for further propagation (9-11) . The meristem-tlp technique can be hnked with heat therapy to improve the efficacy of disease elimination, or antiviral, chemo­ therapeutic agents may be investigated (9-14). Whatever variants of techmque are employed for virus eradication, the key to success is undoubtably the s1ze of the explant. The smallest explants are those that typically, will be the least successful during in vitro culture, but will produce the highest proportion of virus-free material when entire plants are reared in the glasshouse or field. This is clearly lilustrated by attempts at the elimination of WCMV from Trifolium pratense (Table 2 ; 15). If meristem culture alone is not successful m producing any virus-free plants, then temperature-stress treatment of donor plants and/or the use of antiviral agents will have to be considered. There are no prescriptive, global methodolo­ gies for these treatments, and an empmcal study will be required. Heat therapy relies on the growth of the donor plants at elevated temperatures, typically 30-37°C and may involve a treatment of several weeks. Where in vitro cul­ tures provide the donor material, It IS possible to heat treat the cultures also. A number of factors may contribute to the absence of virus particles from the

Grout

120 Table 3 The Effect of Extended Low·Temperature (2-4°C) Treatment on the Elimination of Hop Latent Viroid from Hop Plants8

Vanety 1 Vanety 2

Duratwn of cold treatment, mo

Propagation of vtrotd-free plants

6 17 21 9

0/4 4/23 1114 3/ 1 2

a

Adapted from (14).

meristems of treated plants, mcludmg reduced movement of the particles to the apical regwns, a thermally induced block on viral RNA synthesis, and mactl­ vation of vtrus particles. It IS useful to note that extended, low-temperature treatment of donor matenal may also be effective (14). In attempts to ehmmate hop latent vtroid, the low-temperature (2-4°C) treatment of parent plants was only effective If extended beyond 6 mo (Table 3). Particularly when subJect­ ing donor plants to temperature stresses, It is extremely important to under­ stand the physiology and developmental behavior of the plants concerned, so that maximum stress can be apphed while maintaming an acceptable pattern of growth. A good knowledge of the taxonomy of the plant under investigation Is also valuable when selectmg likely treatments from the hterature, since close, or distant, relatives can be identified. Antiviral chemicals can be used as additives in the culture medmm (11, 12), and one of the most widely used is nbavann, also known as vtrazole. This compound ts a guanosme analog with broad-spectrum activity against animal viruses and appears also to be active against plant vtrus replicatiOn m whole plants. Increasmg concentrations of ribavirin and increasing length of culture mcubation m the presence of the compound typically mcrease the effective­ ness of virus elimmation, but slowed growth and phytotoxity may be evident at high concentrations. The maJor advantages of menstem culture are that It provides: 1 clonal propagation m vitro wtth maxtmal genetic stabthty; 2 the potential for removal of viral, bactenal, and fungal pathogens from donor plants; 3. the menstem ttp as a practtcal propagule for cryopreservation and other tech­ mques of culture storage, 4. a technique for accurate mtcropropagation of chtmeric material; and 5. cultures that are often acceptable for mtematwnal transport wtth respect to quar­ antine regulations.

Meristem-Tip Culture

121

Fig. 4. A meristem-tip culture of potato l 0 d after initiation. When excised, this explant had only two obvious leaf primordia, and has both developed and extended considerably during the culture period. Scale bar represents 500 J-IM.

The technique ofmeristem culture, with optional procedures for virus elimi­ nation, is detailed below for potato species (see Fig. 4). The steps described are broadly applicable to a wide range of plant subjects, although formulations of media and reagents may need to be altered. 2. Materials I . Glasshouse-grown potato plants provide the stem sections with axillary buds to be dissected. These are raised in an insect-prooffacility where possible, and main­ tained on capillary matting on raised benches so that overhead watering is avoided. The donor plants can be subjected to heat treatments for virus eradica­ tion, involving growth at 33°C for 4-6 wk.

2. Wide-necked test tubes make suitable culture vessels for incubating meristem cultures, capped with purpose-bought tops, or aluminum foil, and sealed after inoculation with a Parafilm strip.

3. The culture vessels contain 2.5 mL ofMurashige and Skoog medium (16 and see Appendix) solidified with 0.8-1.0% w/v agar and supplemented with 0.2 mg/L

Grout

122

8

c

A Ftg. 5 . Propagation from menstem-ttp denved plantlets by the techmque of nodal culture (A) A plantlet showing extenston growth m vttro. The nodal segment to be excised is indicated (NS). (B) The excised nodal segment as tt ts transferred onto fresh culture medium, showing the axtllary bud (AX) that will be responsible for subse­ quent growth. (C) The pattern of development of a successful nodal segment culture, showmg extenswn growth of the new plantlet ! -naphthalene acettc actd and 0 5 mg/L gibberelhc actd, pH 5.8 (see also 1 7-20) Alternatively, hqu1d med1a may be used m conJunction w1th paper bndges or fiber supports m the culture vessel. lf reqmred for virus elimmatton, nbovarin at 200 j.tM is filter-sterihzed and added to the medium before soltdtficatton. 4. Nodal cultures, denved from m vitro plantlets, are exctsed as detatled in Fig. 5 and cultured m identical vessels to the exc1sed menstems. The growth medium 1s the same as that for menstem culture without growth hormone supplements. 5. Prestenhzatwn of the exctsed stem segments IS carried out using an absolute ethanol dtp for 30 s wtth a subsequent sten ltzatwn using 5% v/v sodium hypo­ chlorite with a drop of Tween 80 (added as a wettmg agent) Thts ts parttcularly tmportant when the ttssue surfaces are waxy or coated w1th epidermal hatrS. Ster­ ile distilled water for rmsmg ts also reqmred

Meristem- Tip Culture

123

4. A dissectiOn microscope with a magnificatiOn of at least 1 5 x and mounted in a laminar flow cabinet IS required A ptece of expanded polystyrene covered 10 white plastic film and taped to the m1croscope stage 1s tdeal for hold10g stenltzed stem segments in place for dissection, usmg stenlized pms. The tips of 1 2-gage hypodermic needles are used to carry out the dissectiOn 5. A growth room is needed that provides a controlled environment 10 which to incubate the cultures. L1ght10g at 4000 lx at culture level wtth a 1 6-h photo­ period, provided by warm white fluorescent tubes, and a constant temperature of 25 ± 1 °C provides opttmal culture condttlons

3. Methods

2.

3.

4. 5.

6. 7. 8. 9. 1 0. 11

Select a su1table donor plant, m this case, any of the Solanum tuberosum ssp tuberosum types (see Note 1 ), following any desired temperature pretreatments Excise stem segments contaming at least one node from the donor plant. Remove mature and expandmg fo hage to expose the term10al and ax11Iary buds. Cut donor segments to 4-cm lengths, and presteri lize by immersion in absolute ethanol for 30 s. Sterilize by immersing the donor tissues in the sodium hypochlorite solution, with added detergent, for 8 min (see Note 2). Following surface stenlization, nnse the tissues three times m sterile d1stilled water Mount the stem segment on the stage of the d1ssection mtcroscope, and use the tips of hypodermtc needles to dtssect away progressively smaller, developing leaves to expose the apical menstem of the bud, with the few youngest of the leaf pnmordia (see Note 3). Excise the explant ttssue that should compnse the apical dome and the required number of the youngest leaf primordia (see Note 4). After excision, the explant ts transferred di rectly onto the selected growth medtum, and the culture vessel ts closed. Transfer the completed menstem-ttp culture to the growth room. If the explant is vtable, then enlargement, development of chlorophyll, and some elongation will be visible wtthin 7-14 d (Fig. 4; see Note 5). Maintain the developmg plantlet in vitro until the mternodes are suffictently elon­ gated to allow dissectiOn mto nodal explants. To prepare nodal explants, remove the plantlets from the culture vessels under sterile condttlons, and separate into nodal segments (Fig. 5) Each of these 1s transferred directly onto fresh growth medium to allow ax 1llary bud outgrowth. ExtensiOn of this bud should be evtdent wtthm 7-1 4 d of culture initiation (see Note 6)

4. Notes

1 . Donor plants should be selected for general health and vigor. Donor tissues should be taken from young, acttvely growmg stems to ensure the best chance of suc­ cess. However, if donor plants have been subjected to temperature stresses as a pretreatment, then allowances must be made for apparent foliar injury and slowed

124

2. 3

4 5

6.

Grout growth Usmg donor plants that have been glasshouse-raised and only watered from below will help mimm1ze mfectwn problems Be careful. It is often easy to oversterilize dunng surface sterilizatiOn and lethally dam­ age tissues and, therefore, the menstems m an attempt to eradicate surface pathogens It is only w1th practice that reproducible, high levels of culture success will be achieved, smce early attempts at dissection will often result m damaged tissue. Hypodermic needles used as dissecting tools often need to be discarded after two to three meristem exclSlons It seems that the presence of leaf pnmord1a IS essential for successful culture growth, so removal of too small an explant may restnct success A consequence of d1ssect1on may be the productiOn m the tissue of tox1c, oxi­ dized polyphenohc compounds (3) These are more prevalent in cultures of woody species. Their effects may be mimm1zed by rap1d senal transfer of the excised menstem tip to fresh medmm as soon as s1gmficant brownmg of the medmm occurs, or by reducmg light available to the culture m the early stages of development Attempts can be made to reduce oxidatiOn of the secreted polyphe­ nohc compounds by mcorporatmg antioxidants mto the growth medium These might include ascorbic acid, d1thwthreitol, and polyvmyl pyrrolidone Media are commonly species-specific With regard to the formulatiOn of the required growth regulators and their concentratiOns, and may also be specific for the other organic and morgamc constituents. The specificity may even extend to the cultJVar/race level with respect to optimal growth. For potato, a range of suc­ cessful media for different vaneties and species, at a range of ploidy levels, have been pubhshed (1 7-20) Reference sources can provide an appropnate growth medmm to begm an empirical study, usmg the formulatiOn for the closest taxo­ nomic relative 1f no medmm is detailed for the particular plant under mvestlgatlon

References 1 . Murash1ge, T. ( 1 978) The impact of plant tissue culture on agriculture, in Fron­ tzers of Plant Tzssue Culture (Thorpe, T A , ed ), IAPTC, Calgary, pp 1 5-26 2 Murashige, T. ( 1 978) Pnnc1ples of rapid propagation, m Propagation of Hzgher Plants Through Tzssue Culture (Hughes, K W , Henke. R., and Constantm., eds.), US Department of Energy CONF-7804 1 1 1 , US Technical Information Center, Washington, DC, pp 1 4-24 . 3 Hu, C. Y . and Wang, P. J ( 1 984) Menstem, shoot-tip and bud culture, i n Hand­ book of Plant Cell Culture (Evans, D. A., Sharp, W R , Ammtrato, P V , and Yamada, Y., eds ), Macmillan, New York, pp. 1 77-277 4. Debergh, P. C. and Zimmerman, R H. (eds.) ( 1 99 1 ) M1cropropagatwn Technol­ ogy and Applzcatzon Kluwer Academic Pubhshers, p 484. 5. Murashige. T. ( 1 974) Plant propagation through tissue culture. Ann. Rev Plant Physzol. 25, 1 3 5-1 66 6 Ancora, G., Belli-Donini, M. L., and Cozzo, L ( 1 98 1 ) Globe artichoke plants obtained from shoot aptces through rapid m VItro micropropagatwn. Scz Hort 14, 207-2 1 3.

Meristem- Tip Culture

125

7 Reisch, B. ( 1 984) Genetic vanab1lity m regenerated plants, m Handbook of Plant Cell Culture, vol. I , (Evans, D A., Sharp, W R , Ammirato, P. V., and Yamada, Y., eds.), Macmillan, New York, pp 748-769 8. Scowcroft, W. R., Bretell, I. R S., Ryal, S. A., Davies, P , and Pallotta, M. ( 1 987) Somaclonal variatiOn and genom1x flux, m Plant Tzssue and Cell Culture (Green, C. F., Somers, D. A., Hackett, W. P., and Bisboev, D. D., eds.), A. L1ss, pp. 275-288. 9. Vanzaayen, A , Vane1jk, C , and Versluijs, J. ( 1 992) Production of high quahty, healthy ornamental crops through meristem culture. Acta Bot Neerl. 41, 425-433 1 0. Walkey, D. G. A. ( 1 980) Production of virus-free plants by tissue culture, m Tis­ sue Culture Methods for Plant Pathologists (Ingram, D S. and Helgeson, J. P., eds.), B lackwell Scientific, UK, pp I 09-1 19 1 1 . Kartha, K. K. ( 1 986) Production and mdexmg of disease-free plants, m Plant Tis­ sue Culture and Its Agricultural Applzcatwns (Withers, L A. and Alderson, P. G., eds ), Butterworths, UK, pp 2 1 9-238 12. Long, R. D. and Cassells, A C ( 1 986) Elimination of viruses from tissue cultures in the presence of antiviral chemicals, m Plant T1ssue Culture and Its Agncultura/ Applications (Withers, L. A. and Alderson, P G , eds ), Butterworths, UK, pp 239-248 1 3 . Lim, S., Wong, S., and Goh, C ( 1 993) Elimination of Cymbidium mosaic v1rus and Odontoglossum Ringspot VIrus from orchids by menstem culture and thm section culture with chemotherapy Ann. Appl Bwl 122, 289-297 1 4. Adams, A. N., Barbara, D J , Morton, A., and Darby, P. ( 1 996) The expenmental transmission of hop latent v1r01d and Its ehmmatwn by low temperature treatment and meristem culture. Ann Appl Bw l 128, 37-44 1 5 . Dale, P. J. and Cheyne, V. A ( 1 993) The ehmmatwn of clover diseases by shoot tip culture. A nn Appl Bwl 123, 25-32. 1 6. Murashige, T. and Skoog, F. ( 1 962) A revised medmm for rapid growth and bio­ assay using tobacco tissue cultures. Physwl Plant 15, 473-497. 1 7 . Westcott, R. J., Henshaw, G. G , Grout, B. W W. and Roca, W. M. ( 1 977) Tissue culture methods and germplasm storage in v1tro Acta Hart 18, 45-49. 1 8 . Miller, S. A. and Lipschutz, L ( 1 984) Potato, m Handbook ofPlant Cell Culture, vol. 3 (Ammirato, P. V , Evans, D. A., Sharp, W R., and Yamada, Y., eds.), Macmillan, pp. 29 1-326 1 9 . Henshaw, G. G., O' Hara, J. F and Westcott, R J. ( 1 980) Storage of potato germplasm, in Tissue Culture Methods for Plant Pathologzsts (Ingram, D. S. and Helgeson, J. P., eds ), Blackwell, pp 7 1-78 20. George, R. A. T. (ed.) ( 1 986) Technical Guzdelme or Seed Potato Mzcro­ propagation and Mult1p/zcatwn Food and Agnculture OrganizatiOn of the United Nations, Rome, Italy.

12

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Clonal Propagation of Orchids Brent Tisserat and Daniel Jones

1 . Introduction Methods for clonal propagation of the two major morphological groups of orchids, i.e., sympodials and monopodials, are presented. The first group, sympodials, includes such genera as Cymbidium, Cattleya, Dendrobium, and Oncidium. They are characterized by a multibranching rhizome that can supply an abundance of axillary shoots for use as explants. They were among the first orchids to be successfully propagated, and techmques for their in vitro mitiation (i.e., establishment) and subsequent proliferation are well established (1-8). The second group, monopodtals, include Phalaenopsis and Vanda, and are character­ ized by a single, unbranched axis that possesses few available axillary shoots for use as explants. Significantly different m their morphologies, these two groups require different approaches to explant selection and subsequent culturing. The successful large-scale micropropagation of monopodials is, in fact, a relatively recent achievement (9) and is the culmmahon of a wide variety of studies using different media compositions and supplements (10-10). Since the introduction of orchtd meristem culture in 1 960, a divergence in orchid micropropagation techmques developed based on using two distinctly different culture schemes. In one method, the imtial explant is induced into (and is subsequently maintamed in) an undifferentiated callus or protocorm­ like body (PLB) state. PLBs are spherical tissue masses that resemble an early stage of orchid embryo development. Proliferation occurs via PLB multiplica­ tion, and differentiation mto plantlets is permitted only after the desired vol­ ume of callus is achieved. The alternate method minimizes the role of callus, and encourages the differentiation of cultures into plantlets early in the proce­ dure. Consequently, proliferation is accomplished by the induction of axillary shoots from plantlets derived from the original explant. From. Methods m Molecular 81ology, Vol 111 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

127

128

Tisserat and Jones

This chapter describes four methods of propagatiOn: one callus and one axil­ lary shoot techmque for each of the two morphological groups of orchids. Advantages and disadvantages associated with these two methods will be dts­ cussed and possible variation pointed out (see Subheading 4.). 2. Materials

2

3.

4 5. 6

Dtsmfesting solutwn· Use a solutiOn of 1% sodmm hypochlorite (household bleach diluted with distilled water) With 0. 1 % Tween 80 emulsifier. Prepare fresh solution for each treatment, and use wtthin the hour Postdisinfestation holding solution . Prepare a I 00-mM phosphate buffer with 8. 7 6 mM sucrose Adjust the pH to 5 8, and autoclave for 1 5 mm at I 05 kg/cm2 and 1 2 1 oc. Culture media: Use Murash1ge and Skoog morgamc salts (see Appendix) With 8. 76 mM sucrose, 0 003 mM thiamme-HCl, 0.005 mM pyndoxme-HCl, 0 008 mM mcotinic acid, 0 5 5 5 mM myo-mosJtol, and I 00 mg/L casem hydrolysate This baste formulation ts supplemented as indicated in Table 1. An mitiation medium (IM) and a differing proliferation medmm (PM) are reqmred for each method The proliferatiOn steps of Subheadings 3.3. and 3.4. necessitate the preparatiOn of a senes of media (Table 1) to determme optimum yields. The pH of all solu­ tions ts adJ usted to 5 . 7 with 1 . 0 N HCl or NaOH before bemg dispensed mto 25 x 1 50 mm culture tubes (for agar media) or 250-mL flasks (for liqmd media) m 25 -mL ahquots. Culture tubes and flasks, capped With appropnate closures, are autoclaved for 1 5 mm at I 05 kg · cm-2 and 1 2 1 oc. Culture conditiOns: Cultures receive 1 5-25 )lmol/m2/s from wide-spectrum fluo­ rescent tubes for 16 h daily. Temperature is mamtamed at 27°C. Sympodial orchid explants (see Table 2). Developmg shoots (< lh their mature size) are severed from the rhizome (Fig. 1 ) Monopodia! orchid ex plants (see Table 2): Inflorescences (preferably with flow­ ers still intact) should be severed near their point of emergence from leaf bases

3. Methods 3. 1. Clonal Propagation of Sympodials by Callus Production Leaves surroundmg developmg shoots are removed, exposmg underlymg lateral buds and shoot Ups. Though the form of the leaves may vary from one sympodtal genus to another, the basic structure of the developmg shoot IS as depicted m Fig. lA. 2. Excised shoots are sterilized for 20 min in d1smfestmg solutiOn 3 Replace disinfesting solution With buffered holding solution Tissues may remam in this solution while they await final trimmmg 4. Exctse explants for cultunng by maneuvermg the blade of a scalpel behmd the lateral buds. Remove the buds by cuttmg approx I mm mto the underlymg stem tissue. The shoot tip is removed by cutting JUSt below the base of the elliptical leaf pnmordta. All dissectmg operations are performed while the tissues are Immersed m the holding solutiOn Fmal trimming of ex plants mvolves the removal

129

Orchid Clonal Propagation Table 1 Supplements to the Basal Medium Employed In the Four Orchid Micropropagation Methods8 Supplements Methods 3.1. 3.2. 3.3.

Media

BA, 1-fM

NAA, 11M

IM PM IM PM IM PM

7 83 0 78 7.83 I I 75 1 9.58 0.39 0.39 0.39 3 92 3 .92 3 .92 19 58 7 84 1 5 .67 3 1 .34 7 84 1 5 .67 3 1 . 34

l 07 1 3 .43 1 .07 2 .68 5 37 2 68 5.37 8.05 2.68 5.37 8.05 5 . 37 0 54 0.54 0.54 5 37 5.37 5.37

3.4.

IM PM

3.1.-3.4.

RM

Agar, mg/Lb 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000 6000

0lM, mitiation mediUm, PM, prohferahon mediUm; RM, rootmg mediUm; BA, benzyladenme, NAA, naphthaleneacetic ac1d hAgar Type. Bacto-agar, D1fco Laboratone�. DetrOit, MI

Table 2 Some Commonly Encountered Monopodia! and Sympodial Orchid Genera Monopodia is Aerzdes Aerangzs A ngraecum Ascocentrum Sympodials Brassavola Brassza Broughtoma Cattleya Cochlwda

Cyrtorchzs Dorllis Neoflnetia Phalaenopsis

Renanthera Rhynchosty/is Vanda Vandopszs

Cymbzdium Dendrobium Encvclw Epidendrum Laelza

Masdevalia Mzltonia Odontog/ossum Oncidium Sophromtzs

Tisserat and Jones

1 30 A Pseudobulb

Leaves Surrounding , ' Developing Shoot

�:

tip

�,., ..

,

Fig. 1 . Orchid explant selecttons. (A) Shoot explant selection for sympodial orchtds. Dotted line indicates point of removal of developmg shoot (left) Exctsed shoot show­ ing exposed lateral buds and shoot tip used for culturmg (right). (B) Inflorescence stem explant selectton for monopodtal orchtds. Explant wtth ongmal bract mtact (left) Dotted line indicates location where bract was formerly attached to the stem (nght) of one or two of the tumcate leaf scales of the lateral buds or leaf pnmordta sur­ rounding the shoot tips Fmally, carefully remove any fibrous stem tissue that may have adhered to the lateral buds or shoot tips when dissected from the stem 5 Place explants m flasks contammg hquid IM (Table 1), and agitate on a gyrotory shaker at 1 00 rpm. 6. Wtthm a few days, explants should turn green and begm to swell After 4--6 wk, the explants should be large and plump. At thts time, remove the explants from culture, hghtly scar wtth two or three superfictal scalpel mctstons, and place on PM (Table 1). If, after the tmttal 4-6 wk, any cultures appear to have undergone httle or no growth, reculture to fresh IM, and observe for suitability for transfer to PM in another few weeks In addttion, if at any time the medmm of an imtla­ tton culture begms to discolor (I.e , darken because of phenolic compound accu­ mulation), tt should be repiaced wtth fresh medium tmmediately (see Note 1 ). 7. On transfer to PM, explants begm to produce callus or PLBs on thetr surface and from the incistons made before transfer Large PLB clusters should be broken up and subcultured to fresh PM to enhance multiplication. Continue secttonmg and subcultunng callus masses until the destred volume of PLBs ts achteved.

Orchid Clonal Propagation

131

8 To mduce differentiatiOn mto plantlets With roots, transfer PLBs to the rooting medmm (RM) m Table 1

3.2. Clonal Propagation of Sympodials by Axillary Shoot Formation I . Follow steps 1-5 as descnbed m Subheading 3.1. Step 6 IS executed usmg Sub­ heading 3.2. PM mstead of Subheading 3.1. PM (Table 1). 2. When transferred to PM, cultures will start to produce well-differentiated axil­ lary shoots and/or callus masses, which quickly differentiate into shoots. From this pomt onward, most shoots should g1ve nse to additiOnal axillary shoots With­ out passmg through a callus stage Shoot clumps should be separated to enhance proliferatiOn, and mdividual shoots subcultured to fresh PM This step IS repeated until the desired number of plants IS obtamed (see Note 2) 3. To establish a root system, shoots are transferred to RM (Table I )

3.3. Clonal Propagation of MonopodiaIs by Callus Production I . Cut the inflorescence mto sectwns, each pwce possessmg one dormant node w1th

approx 2 em of the stem attached above and below the node (see Note 3) 2 With fine-tipped forceps, remove the bracts surrounding the dormant buds (Fig. 1 B) All traces of the bracts, including any old, papery, or scared tissues that may be present at their bases, should be removed before dismfestatwn. 3. Wash nodal sectiOns by agitation m dilute household detergent for 5 mm. 4 Stenhze m dismfesting solutiOn for 20 min Agitate frequently. 5 Replace dismfestmg solutiOn w1th buffered holdmg solutiOn. Agitate vigorously to ensure deactivation of the remammg stenlant 6 With a scalpel, recut the two ends of the nodal sections, makmg sure that each fresh cut IS at least 5 mm below the margins created by the bleached (damaged) areas at each end of the sectiOn 7 Place nodal sections on Subheading 3.3. IM so that the bud IS just above the medium surface. Within a week, buds turn green and begin to swell After 1 2-16 wk, explants possess multiple shoots with well-developed leaves. OccasiOnally, this shoot cluster may also possess peripheral callus masses that contain newly devel­ oping plantlets m various stages of development (Fig. IB) 8 If callus masses described in step 7 are present on the explant, they should be severed from the rest of the node culture, sectiOned mto smaller pieces, and trans­ ferred to the PM senes of Subheading 3.3. (Table 1) 9. Cultures not possessmg visible callus are treated as follows· Sever the nodal plantlets from the stem, bemg sure to leave at least a 3-5-mm stub of the nodal base still attached to the stem Severed shoots may be used for Subheading 3.4. (Fig. 2). Reculture the stem sectiOn, devoid of Its nodal shoots, on fresh IM. The cut surface and periphery of the nodal stub will produce callus, which can be handled as descnbed m step 8 (Fig. 2). 1 0. The PM (from Table 1) deemed optimum for a particular callus should be employed for further subcultunng (see Notes 4--{;) Contmue sectiOning callus clusters and transferring to fresh PM until the desired volume of callus IS achieved.

Tisserat and Jones

132

� A

-......

. -�, :1

D

B E

�

) Fig 2 . Steps m the micropropagation of monopodtal orchids. (A) Newly mtttated inflorescence node culture (B) Multiple Shoots developmg from a node. (C) An mdi­ vtdual shoot separated from the shoot cluster derived from either B or D. (D) Multiple shoots, with accompanymg callus, developmg from node. (E) Recultured inflores­ cence node with shoots removed to mduce callus from the remainmg stub. (F) Isolated callus and shoots obtained from culture D 11

To mduce dtfferentiation into plantlets w ith roots, callus or PLBs should be trans­ ferred to RM (Table 1).

3.4. Clonal Propagation of Monopodia Is

by Axi!!ary Shoot Formation 1 . Follow steps 1-7 as described for Subheading 3.3. 2 Multiple nodal shoots are separated, so that each plantlet has a sufficient base to support Its leaves above the medmm surface (Fig. 2) Culture plantlets on the entire PM series m this sectwn (Table 1).

Orchid Clonal Propagatton

133

3 The medmm gtving the highest axillary shoot yteld for a particular plant is used for all subsequent proliferatiOn. Contmue separating shoot clusters into indlVldual plantlets and subcultunng on fresh PM until the desired number of plantlets is achteved (see Note 7). 4 To establish a root system, shoots are transferred to RM (Table 1 ). 4. Notes

1 . Exudation of phytotoxic phenolics from wounded orchtd tissue poses a senous problem for m vitro culture. The most effective way of overcommg thts problem in orchids IS frequent media replenishment. Inclusion of activated charcoal into the media may also be beneficial. It is important that culture vigor not be com­ promised by phenolic accumulation, because once attenuated, the productivity of a culture is dtfficult to restore. 2 It should be apparent that the IM formulatiOns of all four methods are closely allied to the PM formulatiOns of Subheadings 3.2. and 3.4. The primary reason for i ndirectly approachmg even the callus proliferation techniques by first employing a shoot proliferation analog is to achieve greater mitml explant sur­ VIval. The presence of cytokmms in the IM improves the survival rate of explants, probably owing to the antisenescence properties of the cytokinms 3. In monopodia! propagation, it is Important that mflorescences be young and healthy. Ideally, flowers at the apex should still be fresh. Older mflorescences, or ones from which the flowers have faded, will gtve adequate, but not optimum results. 4. In monopodml callus propagation, loss of chlorophyll, and an accompanymg decline in growth, may be observed m some cultures. In such instances, transfer of the callus to PM lacking sucrose can often restore color and growth to cultures All subsequent proliferatiOn steps should, therefore, employ sucrose-free media. When differentiation mto plantlets is desired, use sucrose-containmg RM (Table 1) 5. Generally, callus propagation tends to be swifter than axillary shoot propagatiOn However, cultunng orchids for extended periods in an undifferentiated callus state can result m sigmficant occurrences of mutation Genetic aberratiOn has long been accepted as a nsk in callus proliferatiOn. However, as the demand for orchtd clones increases, the problem of mutation m callus IS only hkely to worsen. Conversely, axillary shoot proliferatiOn IS very resistant to genetic aberratiOn, and its development was largely m response to mcreasmg concern over the occur­ rence of mutatiOn (3, 5) . 6. Though NAA and BA perform sattsfactonly, experiments t o substitute other growth regulators for them should be benefic mi. In particular, cytokmins, such as tsopentenyladenine and zeatin riboside (the latter bemg very expenstve), enhance orchtd miCropropagatlon. Also, numerous other growth regulators, such as tht diazuron, have also been employed that show promtse m orchtd micro­ propagatiOn (19). 7 Both axillary shoot methods presented are capable of producmg approx 1 000 mdtvtdual plants m the first year of culture, and as many as I 00,000 by the end of the second year The two callus methods descnbed are capable of producmg 1 00,000 or more plants wtthm the first year of culture

1 34

Tisserat and Jones

References 1 Churchill, M. E ( 1 973) Tissue culture of orchids New Phytol. 72, 1 6 1- 1 66 2 Lmdennann, E G P. ( 1970) Menstem culture of Cattleya Am. Orchzd Soc Bull 39, 1 002-1 004. 3 Morel, G. ( 1 960) Producing virus free Cymbzdzums Am Orchzd Soc Bull 29, 495--497 4. Morel, G ( 1 964) A new means of clonal propagatiOn of orch1ds Am Orchzd Soc Bull 33, 4 73--478 5 Sagawa, Y ( 1 966) Clonal propagation of Cymbzdzums through shoot menstem culture. Am Orchid Soc Bull 35, 1 88-1 92 6 Muj ib, A and Jana, B. K. ( 1 994) Clonal propagation of Dendrobium madame pompa­ dour through apical menstem. Adv Plant Scz 7, 340--346 7 Scully, R ( 1 967) Aspects of menstem culture m the Cattleya alliance Am Orchtd Soc Bull 36, 1 03-1 08 8. W1mber, D. ( 1 963) Clonal multiplication of Cymbtdtums through tissue culture of the shoot menstem Am Orchtd Soc Bull. 32, 1 05- 1 07. 9 Kuhn, L ( 1 982) Orchid propagatiOn m the EYMC Marze Selby Bot Gard Bull 9, 32-34 1 0. Gnesbach, R. J. ( 1 983) The use of indoleacetylammo acids m the m v1tro propa­ gation of Phalaenopsts orchids Sci Hart 19, 363-366. 1 1 . Intuwong, 0. ( 1 974) Clonal propagatton of Phalaenopsts by shoot ttp culture. Am Orchtd Soc Bull 43, 893-895 1 2 . Huang, L C ( 1 984) Alternative media and method for Cattleya propagatwn by t1ssue culture. Am Orchzd Soc Bull 53, 1 67-1 70 1 3 Rotor, G. ( 1 949) a method of vegetative propagatwn of Phalaenopsts species and hybnds Am Orchzd Soc Bull 1 8, 738-739 14 Sagawa, Y ( 1 96 1 ) Vegetative propagatiOn of Phalaenopszs stem cuttmgs. Am Orchzd Soc Bull 30, 803-809 1 5 Scully, R ( 1 966) Stem propagatwn of Phalaenopsts Am Orchtd Soc Bull 35, 40--42 1 6 Lay, M and Fan, F. ( 1 978) Studtes on the tissue culture of orchtds Orchzd Rev Oct, 308-3 1 0 1 7 Tanaka, M . ( 1 977) Clonal propagatwn of Phalaenopszs by leaf tissue culture. Am Orchzd Soc Bull 46, 733-737 18 Zimmer, K and Pieper, W ( 1 978) Clonal propagation of Phalaenopszs by excised buds. Orchzd Rev July, 223-227 1 9 Ernst, R ( 1 994) Effects of thtdtazuron on m vttro propagatiOn of Phalaenopszs and Dorztaenopszs (Orch1daceae). Plant Cell Tzssue Organ Cult. 39, 273-275 20 Tokuhara, K and Mu, M ( 1 993) Micropropagahon of Phalaenopsts and Don ­ taenopszs by cultunng shoot tips of flower stalk buds Plant Cell Re 1 3, 7-1 1

13

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In Vitro Propagation of Succu lent Plants Jill Gratton and Michael F. Fay

1 . Introduction Maintenance of collecttons of succulent plants can be problematic, because many of these spectes are very susceptible to rots caused by bacteria and fungi. Rootmg and establishment of cuttmgs can also be difficult. Tissue-culture tech­ mques have been applied to a w1de range of succulents, and the relevant litera­ ture has recently been reviewed (1 , 2). Here we descnbe methods for the micropropagation of cacil and other succulents that have been developed at Kew for overcoming the problems mentioned above. The techniques have been used most successfully with members ofAsclepza­ daceae, healthy plants havmg been produced from small pteces of unhealthy tissue in the genera Cara/luma, Huernia, Stapeha, and Ceropegia. Some other genera, notably Hoodza, have, however, proven intractable using the methods described here. Succulent species of A izoaceae and Crassulaceae have also been propagated, but the methods will not be descnbed here, since they are closely based on those used for Asclepiadaceae. Cactt have proven more difficult. This is probably largely owmg to the struc­ ture of the plants, with the menstematic areas bemg embedded m the areole tissue. Removal of woody spines can damage this tissue or allow it to be dam­ aged by the sterilant. Successes have, however, been achieved, and include species of Opuntia and Mammillaria. Succulent Euphorbza spp. are also diffi­ cult, but several species have been micropropagated. Species with woody spines seem to be particularly problematic. Micropropagation can be of particular value in the propagation of rare and endangered species, especially where viable seeds are not available. Bulk propagation and subsequent distribution of those species that are endangered by overcollection may alleviate pressure on the wild populations. From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

135

1 36

Gratton and Fay

2. Materials 1 . Media: Murashrge and Skoog (MS) mediUm (3 and see Appendrx) IS obtamed as a ready-mtxed powder, without sucrose, growth regulators, or agar It rs made up at the standard concentration wtth 30 g/L sucrose and a range of growth regulators, which are added before autoclaving. These are deta1led m the Subheading 3. Adjust the pH of the medium to 5.6-5.8 using dtlute NaOH or HCl as appro­ priate Dissolve 9 g/L of agar m the medmm usmg a mterowave oven (2 mm for each 2 50 mL of medmm m a 1 000-W mtcrowave oven), and stir thoroughly This method was developed usmg Sigma punfied agar (Product No A7002). D1spense the medmm into the required vessels, and stenhze m the autoclave for 1 5 mm at 2 1 2 l °C and 1 .05 kg/cm • If the medmm is not to be used rmmed1ately, store the vessels m plastic bags at 4°C m the dark 2 Sterilizatwn· An ethanol (95%) dip t s used as a pretreatment ( 1 0-30 s) when the tissues are mfested wtth pests. Sodium hypochlorite (BDH, 1 0-14% avail­ able chlonne) IS used as the sten lant, di luted to 3-I O% v/v m deiomzed water (0 . 3-1 .4% available chlorme), with the addttion of a few drops of Tween 80/L 3 Compost Open, free-draining composts are used, since these have been shown to promote rootmg and to decrease losses from rottmg off during the weaning stage. The most frequently used compost is composed of three parts fine loam, two parts cmr, one part sharp gnt, s1x parts montmorillomte (particle s1ze up to 4 mm), and s1x parts Perlite (3- to 6-mm particle stze). To thts compost, slow­ release fertilizer ts added as reqmred. Calcmed montmonllonite possesses good absorption properties. The montmorillonite and Perlite make the compost con­ siderably more open, thus aiding establishment 3. Methods

The techniques descnbed here were developed at Kew in an attempt to make the best possible use of what httle material is often available. The basic prin­ ciple on which the method is based is the induction of shoot formation from dormant menstems in the areole of each or from axrllary buds m other succu­ lents, using media containing cytokinms, alone or m combination with low concentratiOns of auxms. 3. 1. Initial Preparation of Tissue 1 . Excise and dtscard dead or rotting material.

If the matenal ts mfested wtth pests, dtp 1t in 95% ethanol for 1 0-30 s, and then wtpe It with a soft tissue or paintbrush. 3. Remove any soil adhering to the plant matenal by washmg under running water or agitating m water containing a few drops of Tween 80. A fine paintbrush can be useful for d1 slodgmg debns trapped by baiTS or spines 4. Remove any damaged ttssue, takmg care not to damage the underlymg tissues

2

Succulent Plants

137

5 . Woody spines and halTS found m cacti and succulent Euphorbza spp. are difficult to surface-sterihze effectively They are therefore removed, usmg watchmaker' s forceps, fine-pointed scalpel blades, o r hypodermic needles (see Note 1 ) . Great care must be taken not to damage the tissue underneath the spme, since the mer­ istematic area often hes very close to the base of the spines Where the spines are less woody, they can be cut back as close as posstble to the base, rather than bemg totally removed.

3.2. Surface Sterilization The pieces of plant tissue should be left as large as posstble for surface ster­ Ilization, to minimize damage to healthy tissue by the stenlant. Where pos­ sible, a range of stenlizatwn times and stenlant concentratiOns are used, but this depends on the amount of tissue available. Sodtum hypochlonte (0.3- 1 4% available chlorine i n deionized water) i s used as the stenlant, with a few drops of Tween 80/L. Stenh zation times range from 5 to 1 5 mm The most com­ monly used regime ts a 5% dilution of BDH sodium hypochlorite (0 5-0 7% final avatlable chlonne) plus Tween 80 for 10 or 15 mm Surface stenhzatwn is improved if the beaker is put onto a magnetic stirrer set at a speed high enough to keep the plant tissue submerged, but not so high that it wtll damage the plant tissue 2 Rinse the matenal three times m sterile deionized water

3.3. Preparation of the Surface Sterile Material for Culture The size of the explant used for culture vanes greatly, depending on the structure of the plant and the extent to which any rot present has advanced. The general procedures for each of the groups of plants are described below .

3.3. 1 . Asclepiadaceae In asclepiads, axillary buds are found in the axils of the vestigtal leaves and are often vistble to the naked eye. Remove these buds w1th some surrounding tissue, using a scalpel and forceps. The first cut should be made directly below the vestigial leaf and the second as far above the bud, smce the structure of the plant will allow. A verti cal cut made behind these cuts will separate the required explant from the main body of the plant (Fig. 1 ) . In species where the stems are very ridged and the vestigial leaves are very close together, make vertical cuts between the ndges as far away from the buds as possible. In these spec1es, we have found it better to use pieces of tissue with several buds for Initiating cultures, rather than trymg to separate out the indt­ vidual buds. In Ceropegia spp., nodal sections are taken. Where there are two buds present at the node, make a vertical cut through the node, and culture the buds separately.

138

Gratton and Fay

Fig. 1 . Caralluma micropropagation. A shoot ready for explant excision (left, approx x I .5), a prepared explant with two donnant buds (middle, approx x4), and an established proliferating shoot culture (right, approx x3).

3.3.2. Cactaceae In cacti where the areoles are widely spaced, remove them with 3-5 mm of the surrounding tissue. In Mammillaria and other genera with tubercles, remove the areoles with as much of the tubercle tissue as possible. Where the areoles are close together, make vertical incisions into the tissue, taking care not to cut right through, and excise small pieces consisting of several areoles.

3.3.3.

Euphorbiaceae

These are cut up into explants similar to those from asclepiads, after removal of spines. 3.4. Culture I . Place the individual explants in boiling tubes containing 10-15 mL of medium, with the cut surface of the explant in contact with the medium, and the bud or areole facing upward. Where sufficient tissue is available, explants are cultured on a range of semisolid media based on MS with added growth regulators. The most commonly used growth regulator combinations are 1 or 2 mg/L benzyl­ aminopurine (BAP) + 0. I mg/L naphthylacetic acid (NAA) (see Note 2). 2. Maintain the cultures in a culture room at 22-25°C with a 16-h photoperiod (light intensity 1000-4200 lx) (see Note 3).

Succulent Plants 3

4

5.

6. 7

8.

139

Examine the cultures after a few days for signs of contammation. Where no more material is readily available, It has proven possible in some cases to carry out a second surface sterilization if contammation has occurred. SolutiOns of sodmm hypochlorite (0. 5- 1 .0% avmlable chlorine) have been used for 5-30 mm, depend­ ing on the type of matenal, but it IS dtfficult to gtve more specific mstructwns. After a variable length of time on the media described above, axtllary buds on some of the ex plants will begm to grow out (see Note 4) When these have grown sufficiently (to 2-3 em in ascleptads), cut them mto sections, each wtth one or a few buds, and place them onto fresh medmm m l f2. or l -Ib honey Jars These sectwns and the stump on the original explant should contmue to pro­ duce lateral shoots, thus establishing a proliferating system (Fig. 1 ) Continue subcultunng m the same way at 1 - to 2-mo mtervals until sufficient shoots have been obtamed When this stage is reached, rootmg m vttro is attempted Place excised shoots onto a range of semisolid medm based on MS ± growth regulators The most commonly used media for rootmg are MS wtth· (a) no growth regulators; or (b) 0.0 1 , 0. 1 , 0 25, 0.5, or 1 0 mg/L NAA. Alternative media are detailed m Note 5. When the plantlets are well rooted, tease them out of the medium gently, and wash them carefully under running water to remove traces of agar. Pot the plantlets m 2-in square pots wtth 1 -4 plants/pot, using the compost descnbed in Subheading 2. (see Note 6). Place the pots on a bed of sand wtth under heating (tfavailable) in a dry intermediate glasshouse (2 1 oc day, 1 6°C night). Where rotting off is a problem, fungtctde drenches have proven useful. Plants can take up to 6 mo to become fully establtshed and begm to grow vtgorously.

4. Notes

1 . In cacti, the removal of spmes can result m damage to the dormant buds located in the areole. In particularly sensitive species, stenlization wtth 0 1 % mercunc chlonde for 2-1 0 mm can obvtate the necessity to remove spines pnor to stenl­ izatlon. As a result, the sensitive bud tissue is not exposed to damage dunng the subsequent treatment wtth sodmm hypochlonte Mauseth and Halperin (4) recommended smgemg the spmes on each instead of removing them. Thts has been tned wtth little success A major problem with removmg spines can be that the newly exposed tissues are very susceptible to damage by the stenlant. Spmes in some species of Euphor­ bta can be removed after stenlization. Where there ts very little tissue avatlable, the cut ends can be sealed wtth paraffin wax, thus mtmmtzing the damage. 2 Wtth some plants, tt has proven necessary to use medta wtth higher cytokimn concentrations to imtlate proliferatmg cultures. The most frequently used con­ centrations are 5 or 10 mg/L BAP with 0 1 or 0 5 mg/L NAA. 3. Dunng the tmtiat10n process wtth Asclepzadaceae, compact callus sometimes forms at the base of explants Remove this at each subculture . If the callus threat­ ens to take over the culture, transfer the tissue to a medmm wtth lower growth regulator concentratiOns

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Gratton and Fay

4 The pathway for cacti descnbed in the Subheading 3. Is the tdeal, but m certam species of cactt (e.g., some Mammzllana spp.), a callus ts sometimes formed before shoots are regenerated Vttrificatwn can also occur; techmques for over­ commg thts that can be tned mclude: a IncorporatiOn of 1-3 g/L activated charcoal b. Use of media of lower ionic strength, e.g , half-strength MS ( 'hMS). c Use of medta wtth htgher agar concentratiOn (e g , 1 4%) d Allowmg the medmm to dehydrate parttally before transfernng the cultures 5. Where rootmg will not take place on any of the medta hsted above, the followmg variations have been used wtth some success. a Indoleacetic actd (IAA) can be used at the same concentratiOns m place of NAA b NAA can be used at higher concentrations (5-20 mg/L) c. lf2MS ± NAA or IAA can be used. 6 Where dehydratiOn IS a problem dunng weanmg, steps must be taken to stop the plantlets from dymg Keep the plants under a clear propagator hd until they have become acclimatized to the lower humtdity Gradually lower the humidity by openmg the vents m the hd, and eventually remove the hd altogether. The use of open, free-drammg composts ts very important at this stage. With particularly dtfficult spectes, we have had some success usmg an mter­ medmte stage between agar and pot culture Stenhze the standard compost m honey jars, and then add stenle water or mineral salt solutiOns (e.g , 1 /z MS with­ out sucrose or agar) Then "pot" the plantlets in this, and seal the vessels as nor­ mal. When the plantlets appear to have become established, transfer the Jars to the glasshouse, gradually loosen the tops over a few days, and then pot the plant­ lets normally

References Fay, M F. and Gratton, J. ( 1 992) Ttssue culture of cacti and other succulents-a literature review and report ofmtcropropagauon at Kew Brad/eya 10, 3 3--48 2 Fay, M F., Gratton, J and Atkmson P J. ( 1 995) Tissue culture of succulent plants-an annotated btbhography. Bradleya 13, 38--42. 3 Murashtge, T and Skoog, F. ( 1 962) A revised medmm for rapid growth and bw­ assays wtth tobacco t1ssue cultures. Physzol Plant 1 5, 473--497 4 Mauseth, J. D and Halperin, W ( 1 975) Hormonal control of organogenesis m Opuntza polyacantha (Cactaceae). J Am Bot 62, 869-877 ,

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Micropropagation of Flower Bul bs Lily and Narcissus

Merel M. Langens-Gerrlts and Geert-Jan M. De Klerk

1 . Introduction For most bulbous crops, artificial (vegetative) propagation methods have been developed, such as scalmg (lily), scooping (hyacinth), and chipping (nar­ cissus). Because the speed of these methods is often low, introduction of newly bred cultivars (either produced by conventional breedmg or by genetic modifi­ catiOn) or of pathogen-free bulbs (produced by meristem culture) requires a long period of time. In tulip, for which no artificial propagation method exists, this can even take 20-25 yr. Micropropagation considerably shortens this period. Furthermore, because of the large number of propagatiOn cycles in the field, conventionally produced bulbs may become easily mfected. Micro­ propagation produces starting material that is completely or predominantly pathogen-free. The mam difference between micropropagation of bulbs and other crops is that bulblets and not microcuttings are produced. In contrast to microcuttings, bulb lets mostly do not reqmre a rooting treatment and acclimatization, but they do reqmre breaking of dormancy. It should be noted that m almost all proto­ cols, at least one step involves adventitious regeneration of a shoot menstem. Many If not all sports (mutated plants) are chimeras, and smce the chimeric structure is lost in adventitious regeneration, the characteristic for which a sport has been selected may very well get lost when these protocols are used. The protocols for micropropagation of flower bulbs consist of six stages: 1 . Mother plant preparation: Storage conditions of the bulbs may determine the abil­ ity to regenerate. To avoid contamination, healthy undamaged bulbs are used Imttal contamination can be strongly reduced by a hot water treatment· 1 h at 54°C m narcissus (1) and l h at 43°C in lily (2) From Methods m Molecular 81o/ogy, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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2 lmtlatwn· Usually, explants are exc1sed from scales or flower stems. From these explants, adventitious shoots/bulb lets regenerate m vitro. Ap1cal and ax1llary buds may also be used and may be the preferable explant, since they give better results m virus ehmmatwn and also have better propagatwn rates than adventitious buds In iris, bulblets ongmating from aptcal or axillary buds show better performance for a number of years after planting (M van Schadewtjk, personal communicatiOn) 3. Propagation. Both shoots and bulblets can be used for further advent1t10us or ax11lary propagation. Shoots have htgher propagation rates than bulblets In hya­ cinth, e g., 2-3 new bulb lets regenerate/bulblet, whereas 1 0--40 new shoots regen­ erate/shoot 4 Bulb growth· In the final propagatwn cycle, bulb lets should be produced Bulb­ lets are firm structures and can be handled eas1ly Shoots planted m sOil some­ times do not form a bulb or form aberrant bulbs (3) ConditiOns st1mulatmg bulb growth Include h1gh sucrose concentratiOn, absence or low concentratiOns of growth regulators, darkness, charcoal, and moderate temperatures Bulblets should be sufficiently large (about 1 00 mg) to achieve good growth after plant­ mg. Large bulb lets of hly grow faster than small ones (4) 5 Dormancy breaking For fast and umform sproutmg, and for optimal growth, bulblets need a temperature treatment to break dormancy (5, 6) This requires either several weeks at low temperatures (2-9°C) or in some species, e g., ms, a temperature treatment of 4 wk at 30°C (3) . The optimal temperature and duration for tissue-culture-derived bulblets are the same as for conventwnally produced ones. Some tissue-culture cond1ttons, in particular, temperature, sugar concen­ tratiOn, and duratiOn of the culture cycle, affect the dormancy level and leaf for­ mation (7-9) Dormancy development i s prevented by addition of flundone, an mhtbttor of abscistc actd synthesis (10) . 6 . Plantmg: For plantmg, usually the same steamed pottmg composts can be used as for conventionally propagated bulbs. Dunng the first year, growth may be m a glass house. Bulb growth is considerably enhanced by planting directly m sotl, probably because of the lower sml temperature (hyacinth and ms, A. F L M Derks, personal commumcatwn) Growth m an aphid-proof glass house or gauze house IS essential when virus mfectton has to be avoided

The protocols developed for propagation in vitro are often stmtlar to con­ ventiOnal propagatiOn protocols. For the five mam bulbous crops, tulip, lily, narcissus, hyacmth and ms, satisfactory mtcropropagation protocols exist, except for tuhp (11, 12) . In thts chapter, protocols for lily (straightforward) and narctssus (more complicated) will be descnbed. A protocol for ms can be found m ref. (3) and for h y a c inth in ref. (1 3) . 2. Materials 2. 1. Lily Clean, healthy bulbs (see Note 1). 2. 70% (v/v) Alcohol, stenle water

Micropropagation of Flower Bulbs

1 43

3 Sodium hypochlorite solutiOn ( 1 % available chlorine) + 0.03 + Tween 20 4 Containers with medium: the medium consists ofMS salts (14), 3% sucrose (w/v), I 00 mg/L myo-inositol, 0.4 mg/L thiamine-HCl, 0.27 J.IM naphthaleneacetic acid (NAA), 0 6% (w/v) agar, and a pH of 6.0 before autoclaving. This medium can be used both for initiation and propagation (see Note 2). Bulb growth m the final step can be on the same medium, but with 6% sucrose.

2.2. Narcissus 1. 2 3 4.

Clean, healthy bulbs (see Note 3). 70% (v/v) Alcohol, sterile water. Sodium hypochlorite solution ( 1 % available chlonne) + 0.03 + Tween 20 Containers with medium: basal medium consists of MS salts (14), 3% sucrose (w/v), 1 00 mg/L myoinositol, 0.4 mg/L thiamine-HCl, 0.54 J.IM NAA, 4 4 )1M benzyladenine (BAP), 0.6% (w/v) agar, and a pH of 6.0 before autoclavmg The basal medium can be used for initiation and propagation. Bulb growth can be induced on the same medium, but with 9% sucrose, without BAP and with 5 g/L activated charcoal. Rooting takes place on basal medium without BAP

3. Methods 3. 1 . Lily

1 . Remove roots. 2. Wash bulbs thoroughly with water. 3. Remove brown or damaged outer scales (see Note 4). 4 . Peel off clean and healthy-looking scales. Remove brown tips Smce regeneration IS best at the basal stde of the scale, take care not to damage this part 5. Rmse scales for about 30 s with 70% alcohol 6. Sterilize scales for 30 min m sodmm hypochlonte. 7. Rmse three times with stenle water. 8. Cut explants of about 7 x 7 mm (see Note 5; Fig. 1). 9 . Place explants with abaxial side on medium (Fig. 1) 1 0 Explants can b e cultured at 20---2 5°C. In the dark, larger bulblets are obtamed than in the hght ( 1 6 hid, 30 j..lmol/m-2/s-1 ). l l After about l 0 wk, regenerated bulblets can be used for further propagation Indi­ VIdual scales of the bulblets are used as explant, large scales can be cut m two or three parts. This step can be repeated every 1 0 wk. 1 2 . After the final propagation cycle, larger bulblets can be obtained by subcultunng single bulblets on medmm wtth high- (6%) sucrose concentration for a number of weeks or months. This treatment is often used to "store" the bulblets until plant­ ing time in the spring. 1 3 . Before planting, bulblets receive a cold treatment of several (6-1 2) wk at 5°C in the dark (see Note 6). 1 4. Bulb lets are excised from the explant, planted m soil, and grown in a gauze house (at l 7°C, most bulblets wtll sprout within 2-4 wk).

Langens-Gerrits and De Klerk

144

Fig. l. Preparation of a lily explant.

3.2. Narcissus 1. 2. 3. 4. 5. 6.

7. 8. 9.

10.

II.

Remove roots and dry tunic. Submerge bulbs completely in hot water of 54°C for 1 h (hot water treatment). Dry bulbs on filter paper at room temperature for 1 d. Remove outer scale and 'h of the upper part of the bulbs. Halve the bulbs longitudinally. Sterilize bulbs for 30 min in sodium hypochlorite. Rinse three times with sterile water. Cut "twin scales": two bulb scale segments 1 em wide and 1.5-2 cm Jong,joined by a part of the basal plate (Fig. 2). Culture explants in upright position with the basal plate on medium, in the dark at 15-20°C. After about 14 wk, the bulb scale segments are excised from the explant. Shoot clumps attached to the basal plate are propagated by splitting them into smaller clumps. Removal of large shoots stimulates formation of new shoots. This cycle can be repeated about every 5-6 wk. The propagation rate decreases every cycle, but is restored by an intermediate bulb formation phase after every three to four propagation cycles: single shoots are placed on bulbing medium for about 6 wk {20°C, dark), so the shoot base swells to form a bulblet. From the bulblets, "minitwin scales" are cut (ca. 5 mm wide and 5-10 mm high). Depending on the size of the bulblet, two to four minitwin scales can be cut (see Note 7). Culture minitwin scales on basal medium for I 0-12 wk. Outgrowth ofnew shoots is stimulated by removal of large shoots and leaf)' structures after about 6 wk.

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Fig. 2. A twin-scale explant of narcissus with regenerating shoots. 12. Shoot clusters regenerated from the minitwin scales are propagated again by splitting. 1 3 . Culture single shoots on bulb growth medium at 20°C in the dark for about 6 wk. 14. Culture bulblets on rooting medium at l5°C in the dark for 7-8 wk. 15. Rooted bulblets are planted in soil (see Note. 8). 4. Notes 1 . Before storage, the bulbs are given a cold treatment (2°C for 6-8 wk) after har­ vest, and then packed in moistened peat and stored at -1 °C. 2. The optimal NAA concentration for regeneration (number ofbulblets) decreases with duration of storage (15) . 3. To obtain regeneration after a hot water treatment, bulbs should be stored dry at 30°C immediately after harvest. 4. At this point, a hot water treatment ( 1 h at 43°C) can be given to decrease endog­ enous contamination. Let the bulbs dry for 1 d at room temperature before cutting explants. After a hot water treatment, culture tubes should not be closed air-tight, since released gases may reduce viability of the explant. Some genotypes (e.g., Lilium speciosum "Rubrum No.lO") are more sensitive to this than others.

1 46

Langens-Gerrits and De Klerk

5. RegeneratiOn ts highest in explants cut from the base of a scale Some genotypes have a very strong regeneration gradient (only explants cut from the base of a scale show regeneratiOn). In others, the whole scale can be used. The size of an explant strongly affects the s1ze of the regeneratmg bulblets: from a large explant, large bulblets regenerate (4). 6 The cold treatment can also be grven to bulblets excised from the explant Store the bulb lets on mOistened filter paper. 7. Chow et al (16) also developed a propagatiOn method for narcissus. In th1s method, shoot clumps are subjected every other cycle to "severe cuttmg" (down to the basal plate region) to restore the propagation rate. 8. Rooting ts essential for optimal growth after planting. Without roots, leaves remam small (ca 0.5 em) and dte a few weeks later. Rooted bulblets do not need a cold treatment to break dormancy (1 7).

References 1 . Hoi, G. M G M. and Van Der Lmde, P. C. G. ( 1 992) Reduction of contaminatiOn m bulb-explant cultures of Narcissus by a hot-water treatment of parent bulbs. Plant Cell Ttss. Org. Cult 3 1 , 75-79. 2 Langens-Gerrits, M , Albers, M., and De Klerk, G J ( 1 998) Hot-water treatment before tissue culture reduces mitial contammatwn in Lzlzum and Acer. Plant Cell Tzss Org Cult 52, 75-77 3 Van Der Linde, P. C. G. and Schtpper, J. A. ( 1 992) M1cropropagation of ms with special reference to Ins x hollandzca Tub., m Bzotechnology zn Agrzculture and Forestry, vol. 20 (BajaJ, Y P S , ed ), Springer-Verlag, Berhn, pp 1 73-1 97. 4. Langens-Gernts, M., M1ller, W B , Lthen-K1pms H , Kolloffel, C . , Croes, A F and De Klerk G. J ( 1 997) Bulb growth in lily regenerated m VItro. Acta Hortzc. 430, 267-27 3 . 5 Gerrits, M . , Kim, K S . , and D e Klerk, G. J . ( 1 992) Hormonal control o f dormancy m bulblets of Lzlium specwsum cultured in vitro Acta Hortzc 325, 52 1-527 6. Langens-Gerrits, M., Hoi, T., M11ler W. B., Hardin, B., Croes, A. F., and De Klerk, G J. ( 1 997) Dormancy breakmg m lily bulb lets regenerated m vitro. effects on growth after plantmg Acta Hortzc 430, 429--436 7. Aguettaz, P , Paffen, A., Delvallee, I , Van Der Linde, P., and De Klerk, G. J. (1 990) The development of dormancy m bulblets ofLzlzum specwsum generated in vitro I The effects of culture conditwns. Plant Cell Tzss Org Cult 22, 1 67-1 72 8 Delvallee, 1., Paffen, A., and De Klerk, G. J. ( 1 990). The development of dor­ mancy in bulb lets of Lilium specwsum generated in vitro. II. The effect of tem­ perature. Physwl. Plant 80, 43 1--436 9. Gerrits, M . and De Klerk, G J. ( 1 992) Dry-matter partttioning between bulbs and leaves m plantlets of Lilzum speciosum regenerated i n vttro Acta Bot. Neerl. 4 1 , 46 1--468. 1 0. Ktm, K S., Davelaar, E , and De Klerk, G. J. ( 1 994) Absctsic acid controls dor­ mancy development and bulb formatiOn m hly plantlets regenerated in v1tro Physwl Plant 90, 463--469

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1 1 Le Nard, M , Ducommun, C , Weber, G , Don on, N , and Bigot, G ( 1 987) Obser­ vatiOns sur Ia mu1tiphcatwn in vitro de Ia tuhpe (Tulipa gesneriana L ) a partu de hampes florales prelevees chez des bulbes en cours de conservation. Agronomze 7, 32 1-329. 12. Hulscher, M., Krijgsheld, H. T., and Van Der Llnde, P. C. G. ( 1 992) Propagatwn of shoots and bulb growth of tultp in vitro. Acta Hortzc 325, 441-446 1 3 . Paek, K. Y. and Thorpe, T A ( 1 990) Hyacmth, in Handbook of Plant Cell Cul­ ture, vol. 5, Ornamental Speczes (Amm1rato, P. V., Evans, D R., Sharp, W R., and BaJaJ, Y. P. S . , eds.), McGraw-Hill, New York, pp. 479-508. 14 Murashige, T. and Skoog, F ( 1962) A revised medium for rapid growth and bio­ assays w1th tobacco tissue cultures. Physiol Plant 15, 473-497 1 5 . Van Aartnjk, J. and Blom-Bamhoom, G. J ( 1 98 1 ) Growth regulator requirements for adventitious regeneratiOn from L1lium bulb-scale tissue m vitro, m relatwn to duration of bulb storage and cult1Var. Sci Hort 14, 261-268 1 6 Chow, Y. N , Selby, C., and Harvey, B M R ( 1 992) A s1mple method for mam­ taining high multiplicatiOn of Narc1ssus shoot cultures in vttro. Plant Cell T!ss Org Cult. 30, 227-230 1 7. Langens-Gerrits, M and Nashtmoto, S ( 1 997) Improved protocol for the propa­ gation of Narc1ssus m v1tro Acta Hort1c 430, 3 1 1-3 1 3

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Clonal Propagation of Woody Species lndra S. Harry and Trevor A. Thorpe 1 . Introduction

Tissue-culture technology is Widely used for the vegetative propagation of selected plants in agnculture and horticulture and, to a lesser extent, in for­ estry. The objective is to produce large numbers of plants with uniform qual­ ity. Historically, commercial applications of this technology were restricted to herbaceous plants. However, for the last two decades, considerable success has been obtained with woody plants (1,2). These include both gymnosperms and angiosperms, i.e., softwoods and hardwoods, and both trees and shrubs. Eco­ nomically, these trees are extremely important for wood products, includmg lumber, pulp and paper, forestry plantations, and reforestation. Large-scale clonal systems can be an asset for selected high-performance trees, and reh­ able protocols are necessary for further genetic manipulatiOn. However, a maJor problem in the propagation of woody plants Is that most success ts achieved with juvenile tissue and not from proven mature trees (3,4). Traditional methods for propagation include rooted cuttings, grafting and layering. For in vitro propagation, the methods generally used are first, the mductwn of adventitious buds or axillary bud breaking, both of whtch produce shoots that are subsequently elongated and rooted, and second, somattc embryogenesis mainly from Juvenile tissue. Only propagation vta the first two methods are dtscussed here. To obtain axillary shoots, shoot tips, lateral buds, and small nodal cuttings are used as explants (2). Axillary shoot bud prolifera­ tion IS mduced from preformed meristems, and m general, true-to-type and genetically stable plants are produced (3). Cytokinins with or without an auxm are used for the development of these menstems. Adventitious buds are either induced directly on explants or on callus derived from primary explants. Again, cytokinins are used pnmanly for dtrect organogenesis, whereas a combmatwn From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Edited by A D Hall © Hu mana Press Inc , Totowa, NJ

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of cytokmins and auxms are used for the latter. In additwn, the methods used should be rel atively simpl e, have htgh mult!phcatwn rates, be repro­ ductble, and allow differentiation of shoots without mtervenmg callus. Also, shoots should be rootable, and plantlets should survtve transfer to ex vttro condttions (5-7). In general, for gymnosperms, whole embryos or excised seedling parts are used as explants. In contrast, a major advantage of mampulatmg angiosperm trees m vttro, ts that a wide range of matenal can be used as explants. These include seeds, seedlmg and floral parts, leaves, and shoots and buds from both JUVemle or mature trees. Thts extensive source of explants IS Important, smce the vtable penod of seeds from many hardwood trees is quite short. Although the desirable charactenstics m tree spectes are obvtous at matunty, propaga­ tion using mature tissue is sttll problematic. Other dtfficulties encountered wtth woody spectes mclude systemic mfecttons, episodic growth, the productiOn of polyphenols, tannms and volatile substances, and hyperdric1ty (vitrificatiOn) of cultures (3). To Illustrate the tissue-culture procedures commonly used for these two groups of plants, Thuja occtdentalls or eastern white cedar and Populus tremulozdes or aspen are used. For both species, a multistage process is used (5) . For cedar, this includes adventitious bud mductiOn on the explant, bud elongatiOn, axillary budding, and rootmg and hardening of plantlets before transfer to greenhouse conditions (8, 9). For poplar, this process mvolves axil­ lary bud breakmg and shoot imtiatJon, shoot mult1phcation and elongatiOn, followed by rooting and acchmattzatwn (6, 10, 1 1)

2. Materials Stocks of vanous salt formulatiOns, both for mductwn and elongation, e.g., QP (12; see Table 1) for cedar and MS (13; see Appendix) for poplar, and phytohor­ mones includmg Jii-benzylademne (BA), zeatin, and mdole-3-butync ac1d (IBA) (see Note 1 ) BA and zeatin are d1ssolved m l MNaOH, and stored at 4°C. BA 1s added to medmm before autoclaving. However, the pH of the zeatm stock IS adJusted to 5 7-5.8 and 1s filter-sterilized, and the reqmred amount IS added to autoclaved med1um before the plates are poured IBA solutions are made up when needed, the pH is adJusted to 5 0, and are filter-stenlized before use. The med1a needed for Thuja are a. lf2 QP, I j.lM BA, 3% sucrose. b. l f2 QP, l 0 11M zeat m , 2% sucrose c. l f2 QP, 3% sucrose. d. 1/z QP, 2% sucrose and 0.05% activated charcoal Those for poplar are: a. MS, 2 11M BA, 3% sucrose. b MS, I 11M BA, 3% sucrose

151

Clonal Propagation of Woody Species Table 1 Ouoirin and Lepoivre Nutrient Medium (12) as Modified in Ref. 14 mg/L MaJor salts KN03 NH4N03 Ca(N03)z · 4H20 KH2P04 MgS04 4H20 Mmor salts MnS04 4H20 H3 B03 ZnS04 7H20 KI CuS04 5 H20 Na2Mo04 2H20 CoCI2 6H20 Vttamins (15) Ntcotmic ac1d Pyridoxine HCI Th1amme HCI Iron (13) Na2EDTA · H20 FeS04 7H20 Asparagme Myo-mos1tol •

•

•

•

2 3. 4.

5. 6. 7.

1800 400 1 200 270 360 1 .0 6.2 8.6 0.08 0 025 0 25 0 025 5 .0 0.5 5.0 74 5 55 7 I 00 I 00

c. lf2 MS (half-strength maJor salts only), 3% sucrose. For all culture med1a, the p H 1s adJusted to 5.7-5.8 before adding 0 8 % (w/v) Dtfco Bacto-agar for autoclavmg. Petn d1shes (25 mL medium) and glass Jars ( 1 00 mL medmm) are prepared. 9-cm Petri plates with 25 mL induction medium Autoclavable glass jars w1th I 00 mL of elongation, propagatiOn, and rooting medta. Relatively clean seeds and, preferably, greenhouse-grown plant matenal for bud explants. Stenlants, mcludmg commerc1al bleach, e.g., Javex ® (6% NaOCI),H202, HgCI2, 70% ethanol and sterile water, and wettmg agent, e.g., Tween 20®. Parafilm® or Stretch 'n Seal® for sealing plates and Jars. Lammar flow hood for aseptic work.

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8. Instruments mcluding scalpels and # I 0 and # 1 1 blades, long forceps, fine-tip dtssectiOn forceps, and a dtssecting microscope. 9 Growth cabmets mamtamed at 2�26°C, and 16-h photoperiod at about 80 1Jll10I/m2/s from wtde-spectrum fluorescent tubes (see Note 2).

3. Methods: Softwoods 3. 1. Softwoods

3. 1 . 1 . Explant Sterilization and Induction 1 . Imbibe cedar (T occzdentalzs) seeds ovemtght under runnmg tap water. 2 Dismfest seeds for 20 min m 30% Javex® bleach (6% NaOCI) wtth 3-4 drops of Tween 20®/ 1 00 mL Rmse three times with sterile distilled water Treat seeds for 5 mm with 1 0% H202 Rmse several times wtth stenle water (see Note 3). 3 For T occtdentalts, 1hQP ( l h-strength maJor salts only) With I 1-JM BA for 2 1 d was the most effecttve mductton treatment (see Notes 4 and 5). Excise embryos from dtsinfested seeds, and plate on this medmm about 1 2 explants/plate 4. After phytohormone exposure, transfer explants to the same medmm wtthout BA for 3 wk (see Note 6).

3. 1.2. Bud Development Transfer explants to l f2QP, 2% sucrose, and 0 05% comfer-denved activated charcoal for shoot elongation. 2. Subculture every 3-4 wk. As shoots elongate, transfer to glass Jars wtth elonga­ tiOn medmm and seal wtth Parafilm® 3. Divtde explants into smaller pteces for faster shoot elongatiOn; separate mdt­ vidual shoots after two passages and when shoots are >5 mm in hetght. Maintam shoots on shoot elongation medmm

3. 1 . 3. Multiplication via Axillary Buds l . Use 8-10 mm adventitious shoots (see Note 7) 2. Prepare medta in glass Jars wtth phytohormone(s) to activate the axillary mer­ Istems. For cedar, culture shoots on l f2 QP, 2% sucrose with 1 0 1-JM zeatin for 4 wk, followed by culture on llzQP, 2% sucrose and 0.05% activated charcoal for axillary shoot elongatton. 3. Separate axillary shoots when 5-1 0 mm, and culture on the same medmm.

3. 1 .4. Rooting and Acclimatization I . Select shoots > 1 0 mm m height for rootmg (see Note 8) 2. Prepare rootmg medmm or substrate, substrate can be a smlless commerctal mtx, or a 1 : 1 mtxture of peat and vermtcuhte mmstened with dt1uted (1f2QP, 1% sucrose) medmm and autoclaved Stir mtxture after autoclaving to aerate and redistnbute peat/vermiculite 3 Treat shoots for 3-4 h m an auxm solution, 1 e , immerse shoot bases or ends m IBA ( I mM), pH 5 .0; transfer treated shoots to substrate of chotce. Seai Jars wtth Parafilm® . Roots appear after � wk

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4. Harden rooted shoots at 20°C for 1-2 wk before transfer to greenhouse condi­ tions, usmg an appropriate sotl mix for conifers.

3.2. Hardwoods 3.2. 1 . Explant: Sterilization and Shoot Initiation 1 . Select dormant twtgs (e.g., P tremuloides) wtth nodal or axtllary buds, preferably

from greenhouse matenal, since tt will be less contammated than field material. 2. Cut twigs into small sections (±7 mm) each with an intact bud 3. Surface-sterilize by dipping in 70% ethanol for I mm and rinsing three times wtth sterile dtstilled water; then, soak sections in 20% bleach (6% NaOCJ) with 2-3 drops Tween® for 1 5 mm. Rinse several times with sterile distilled water. 4. Separate buds, and remove the outermost scales under dissecting mteroscope, removal of these scales reduces the amount of phenohc exudates and enhances bud breakmg (see Note 9). 5 Plate on MS medium with 2 )1M BA, 3% sucrose, and 0.8% agar (see Note 1 0) 6. Seal plates with ParafiJm® or Stretch n' Seal ® (see Note 1 1 ) , and mcubate at 24--25°C under 1 6 h hght for 2-4 wk. 7. Subculture at 2- to 3-wk mtervals or shoots wi ll begm to tum brown. Always remove the lower end of the shoot (±2-4 mm), thus providmg a freshly cut sur­ face for nutrient uptake

3. 2.2. Shoot Elongation and Multiplication 1 . To enhance elongatton, transfer shoots at 2-wk mtervals. 2. For multtplicatton, select shoots ± 1 0 mm, and subculture these on MS medium wtth I )1M BA, 3% sucrose, and 0.8% agar m glass jars Seal Jars with Parafilm® and subculture at 3- to 4-wk intervals (see Notes 12 and 13). 3 . Observe shoots carefully for culture contaminants (see Note 14) .

3.2.3. Rooting and Acclimatization l . After 8-1 2 wk on multtphcation medium, transfer indtvtdual shoots to l f2MS medium without phytohormones m glass Jars (see Note 1 4). 2. Transfer at 3-wk intervals until root primordta develop. 3 Remove plantlets from agar medmm when they have well-developed root sys­ tems, and transfer to plastic boxes or enclosed seedhng tr�ys wtth stenle ver­ miculite. Maintam these under high light and humidity. Mist plantlets With stenle water every 1 2 h to maintain high humidity Harden plants for 3-4 wk before transfernng to greenhouse condtttons (see Note 1 5). 4 . Notes 1 . For prepanng stock solutions and mformatton on commonly used media see refs (5-7) 2. For more information on the above eqmpment, tmplements, and so on, see refs

(7-20) 3 . Some softwood seeds require much more elaborate pretreatments, including stratificatton before excision and explantatton (see 21). Strattficatton procedures

1 54

4.

5

6

7

8.

9

I0

II

1 2. 13. 14

Harry and Thorpe earned out at temperatures of 3-5°C for periods of days or weeks are useful for breaking seed dormancy. Salt formulatiOns generally used for comfers mclude von Arnold and Enksson's AE or LP (22), Bornman 's MCM (23) , and Schenk and Hildebrandt (24) . These can be used at full- or half-strength In general, for mduction and elongation, variOus salt formulations should be tested at both full- and half-strength and somettmes at lower concentratiOns Select medium based on overall expl ant appearance, percent explant response, number of adventitiOus shoots, and elon­ gation rates (4). Thus, both quantitative and qualitattve factors must be constdered For each spectes, the optimum BA concentratiOn, exposure ttme, and sucrose concentration have to be determmed Other cytokmms, like kmetm, zeatm, and 2tP used alone or m combinatiOn wtth BA can sometimes 1mprove organogen­ esis, and the effects of these have to be determmed (4). For species w1th larger seeds and embryos, e.g., rad1ata pme, 1t 1s necessary to germmate the whole seed in a m01 stened, stenle, soilless mtx (25) , or conversely, the exc1sed embryo 1s germmated on 1% sucrose-agar for 3-4 d, e.g , Canary lsland pme (26) [ndJVJdual cotyledons can then be exctsed and used as explants Multtplicatwn ts a most Important step m developmg a mtcropropagatton proto­ col, smce th1s step allows for the production of large numbers of shoots, e1ther via ax1llary buddmg (preferably) or by adventitious buddmg (wh1ch may mvolve bud formation m callus) For common approaches used, see refs (2-4) . W1thout adequate rates ofmultiphcation, the developed method w1ll probably not be eco­ nomtcal (7) Act1vated charcoal is often needed. For some softwoods, only com­ fer-denved charcoal was effective (2 7) For some comfers, rootmg and acchmat1zat10n may be earned out concurrently, but for most, two dtstmct sets of operations are needed. In such cases, rootmg ts often achteved or at least mitiated under m vttro sten le condtttons, and may reqUJre separate steps of root mductwn and root development (see 2,6,28) The productiOn of toxic secrettons, mcludmg polyphenols and tannms, IS a maJor problem that often has to be overcome on mtroducmg hardwoods mto culture (3) Several approaches have been developed to deal w1th th1s problem (29), but m some cases, the only successful method J S the very labor-mtensive process of frequent transfer of the explants onto fresh medtum (3) . Although 2 J!M BA ts recommended for th1s spectes, a range of2-5 J.lM can be used effectively. MS 1s used extensively for hardwoods, but other formulations like Gamborg 's B5 (30) and McCown's Woody Plant Medtum (31) are also effecttve Compansons were made w1th Parafilm® (permeable) and Stretch 'n Seal ® (Imper­ meable), but no stgmficant dtfferences were observed. The add1t1on of 0 .05% activated charcoal d1d not s1gmficantly Improve shoot imttatJOn or elongatiOn in our experiments Frequent transfer was the best optton In compansons wtth d1fferent types of culture jars, we found that poplar shoots grew slower m Magenta Jars when compared to glass jars. Low denstty of shoots in the culture vessel and regular transfers Will help m the detectiOn of contammants and mamtenance of asepttc cultures. However, when

Clonal Propagation of Woody Species

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contaminatiOn occurs, precautions must be taken to prevent the spread of the contaminant whether tt ts mtroduced or systemic to the plant matenal 1 5 In-vttro grown plantlets cannot be transferred directly to ex vttro conditiOns These plantlets have to be gradually acclimatized to higher hght mtenstttes and lower humtdity under greenhouse or field condttwns They are particularly sus­ ceptible to ex vitro stresses because of reduced epicuticular wax, and have mt­ ttally, a slow stomatal response to water stress and ltmtted capac tty for photosynthesis, smce they were cultured on sucrose-contammg medta (see 32) In addttion, for hardwoods, leaves must be fully developed, smce poor leaf for­ matiOn results m shoot deterioratiOn (33).

References I Ztmmerman, R H. ( 1 986) Regeneration m woody ornamentals and frutt trees, m Cell Culture and Somat1c Cell Genet1cs of Plants, vol. 3 , (Vastl, I K., ed ), Aca­ demic, New York, pp. 243-258 2 Thorpe, T. A , Harry, I S , and Kumar P. P { 1 99 1 ) Apphcatton of mtcropropa­ gatton to forestry, m Micropropagation (Debergh, P. C. and Ztmmerman, R. H , eds ), Kluwer Academtc, Dordrecht, pp. 3 1 1-3 36 3 Harry, I S. and Thorpe, T A ( 1 990) Spec tal problems and prospects m the propa­ gatiOn of woody spectes, m Plant Agmg Bas1c and Applzed Approaches, (Rodnguez, R., Tames, R S , and Durzan, D. J., eds ), Plenum, New York, pp 67-74. 4 Dunstan, D. I. and Thorpe, T. A. { 1 986) RegeneratiOn m forest trees, m Cell Cul­ ture and Somatic Cell Genetics zn Plants, vol. 3 (Vasil, I K , ed.), Academtc, New York, pp. 233-24 1 5. Thorpe, T. A . and Patel, K R ( 1 984) Clonal propagation· Adventitious buds, m Cell Culture and Somatic Cell Genetzcs of Plants, vol . I (Vast!, I. K , ed.), Aca­ demic, New York, pp. 49--60. 6 Ahuja, M R. ( 1 987) In VItro propagation of poplar and aspen, in Cell and T1ssue Culture m Forestry, vol 3 (Bonga, J. M. and Durzan, D J., eds.), Martmus Nijhoff, Dordrecht, pp 207-223 . 7. Harry, I S. and Thorpe, T. A. ( 1 994) In vztro methods for forest trees, m Plant Cell and T1ssue Culture (Vasil, I. K. and Thorpe, T. A., eds.), Kluwer Academtc, Dordrecht, pp. 539-560. 8. Harry, I S., Thompson, M. R., Lu, C -Y , and Thorpe, T. A. ( 1 987) In vrtro plant­ let fonnatwn from embryonic explants of eastern white cedar (ThuJa occ1dentalzs L). Tree Physiol 3, 273-283 . 9. Nour, K. A and Thorpe, T. A. ( 1 993) In vztro shoot multipltcation of eastern whtte cedar. In V1tro Cell Dev Bzol. 29, 65-7 1 . I 0. Lubrano, L ( 1 992) Mt cropropagation of Poplars (Populus spp ) , m Biotechnol­ ogy zn Agrrculture and Forestry-Hzgh- Tech and M1cropropagatzon !!, vol 1 8 (Bajaj , Y P S., ed ), Springer-Verlag, Berhn, pp 1 5 1 -1 76. I I Thorpe, T A (1 995) Aspen Mtcropropagation Part of a report Aspen Decay and Stain and Genetic Fmgerpnntmg of Ciones, Report A50 1 2 , Canadian Forest Ser­ vtce, and Land and Forest Servtces, Edmonton, Alberta

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1 2 Quotrin, M. and Lepotvre, P. ( 1 977) Etude de miheux adaptes aux cultures in vztro de Prunus Acta Hortzc. 78, 437-442. 1 3 Murash1ge, T and Skoog, F . ( 1 962) A rev1sed medium for rap1d growth and blo­ assays w1th tobacco t1ssue cultures Physwl Plant 15, 473-497 14 A1tken-Chnst1e, J and Thorpe, T. A. ( 1 984) Clonal propagatiOn. Gymnosperms, in Cell Culture and Somatic Cell Genetzcs of Plants, vol I , Laboratory Proce­ dures and The1r Applications (Vastl, I K , ed ), Academ1c, New York, pp 82-95 1 5 Gamborg, 0. L., Murash1ge, T , Thorpe, T A., and Vas1l, I K, ( 1 976) Plant T1ssue Culture Media. In VItro 1 2, 473-478 1 6 Chandler, S. F. and Thorpe, T A ( 1 985) Culture of plant cells· techmques and growth medium, in Cell Bwlogy, vol C l , Techmques m Settmg Up and Mamie­ nance of Tzssue and Cell Cultures, C l l 2 (Kurstak, E., ed.), Elsevier, Ireland, pp. l-2 1 1 7 Gamborg, 0 L and Ph1lhps, G C (eds.) ( 1 995) Plant Cell, Tzssue and Organ Culture-Fundamental Methods, Sectton I Sprmger, Berhn, pp 3-42 1 8. Btondt, S. and Thorpe, T. A. ( 1 98 1 ) Requirements for a tissue culture fac 1hty, m Plant Tissue Culture Methods and Apphcatwns m Agnculture (Thorpe, T A , ed.), Academic, New York, pp l-20 1 9 Bhojwani, S S. and Razdan, M K ( 1 983) Developments m Crop Sc1ence, vol 5, Plant T1ssue Culture Theory and Practice Elsev1er, Amsterdam, p 502 20. Brown, D C W. and Thorpe, T. A. ( 1 984) Organization of a plant ttssue culture labora­ tory, m Cell Culture and Somatic Cell GenetiCS ofPlants, vol I , Laboratory Procedures and Thezr Applzcatwns (Vasil, I K , ed ), Academic, New York, pp l-12 2 1 . Thorpe, T A and Harry, I . S ( 1 99 1 ) Clonal propagation ofcomfers, m Plant Tzssue Culture Manual C3 (Lmdsey, K., ed.), Kluwer Academic, Dordrecht, pp. l-16 22 von Arnold, S and Eriksson, T ( 1 98 1 ) In v1tro studtes of adventitious shoot for­ mation in Pmus contorta Can J. Botany 59, 870--8 74 23. Bornman, C H. ( 1 983) Possibilities and constramts m the regeneratiOn of trees from cotyledonary needles of P1cea ab1es in vitro Physwl Plant 80, 534--5 40 24. Schenk, R U and H1ldebrandt, A. C ( 1 972) Medmm and techmques for induc­ tion and growth ofmonocotyledonous and dicotyledonous plant cell cultures Can J Botany 50, 1 99-204. 25 Biondi, S. and Thorpe, T A ( 1 982) Growth regulator effects, metabohc changes, and resp1rat10n durmg short 1nt t1atwns m cultured cotyledon explants of Pmus radwta Bot Gaz 143, 20--2 5 26 Martmez Pultdo C, Harry, I S , and Thorpe, T. A. ( 1 990) In vllro regeneration of plantlets of Canary Island pme (Pmus canarzens1s) Can J Forest Res 20, 1 200-- 1 2 1 1 27. Rumary, C. and Thorpe, T. A ( 1 984) Plantlet formation m black and whtte spruce I . In vztro techniques. Can. J. Forest Res 14, 1 0-- 1 6 . 28 Mohammed, G. H . and Vidaver, W E ( 1 988) Root productton and plantlet devel­ opment m tissue-cultured comfers. Plant Cell T1ss Org. Cult 14, 1 3 7-1 60. 29. Pienk, R L M ( 1 987) In Vttro Culture of H1gher Plants Martmus Nlj hoff, Dordrecht.

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30. Gamborg, 0 L , Miller, R A., and Ojima, K. ( 1 968) Nutnent requirements of suspension cultures of soybean root cells. Exp Cell Res 50, 1 5 1-158 3 1 Lloyd, G. and McCown, B . ( 1 980) Commercially feasible micropropagatwn of mountain laurel, Kalmza latifolia, by use of shoot-tip culture. Jnt Plant Propaga­ tors Soczety Combzned Proceedzngs 30, 42 1 -427 32. Chalupa, V. ( 1 987) European Hardwoods, m Cell and Tzssue Culture zn Forestry, vol 3 (Bonga, I M and Durzan, D J eds ), Martmus NIJhoff, Dordrecht, pp. 224-246. 3 3 . McCown, D. D. and McCown, B. H ( 1 987) North Amencan hardwoods, m Cell and Tzssue Culture in Forestry, vol. 3 (Bonga, J. M. and Durzan, D. J., eds ), Martinus Nijhoff, Dordrecht, pp. 247-260 ,

16

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Spore-Derived Axenic Cultures of Ferns as a Method of Propagation Matthew V. Ford and Michael F. Fay 1 . Introduction

A method by whtch many fern species can be successfully grown from spores in axenic culture ts described. Unllke the conventional method of sow­ mg the spores on compost, thts method allows spore populations free from contammatiOn by spores of other spectes to be sown. The method can be used for the production of mature sporophytes or to provide a controllable system for biosystematic studtes of, or expenmentation with, fern gametophytes (1, 2). For many years, It has been known that spores of most ferns require expo­ sure to hght for germination and growth (1,3,4). However, dark germmatiOn has been recorded in Pteridium aquilinum (5, 6), Blechnum spzcant (7), and Onoclea sensibilis (8, 9), among others. This abihty to germinate m the dark may vary wtth specimen age, exposure to different temperatures, and plant hormone treatment (1,3, 10). In general, the percentage of dark germinatiOn ts low, and subsequent growth is abnormal or retarded. Many workers have mvestigated the relative effectiveness of dtfferent spec­ tral regions of light on spore germmatiOn, and have obtained results indicating the involvement of phytochrome (11-13). In general, exposure to red light induces germination (14, 15). Thts ts reversed by exposure to far-red light and blocked by blue light (16). Light quality also influences prothallial growth (1 7-20). Spectral filters are not necessary for germination of the majority of ferns usmg the method we describe here, since white light provides an ade­ quate spectral balance. Light mtensity has also been reported to have a marked influence on germinatiOn and subsequent prothallial growth (21). This phe­ nomenon can be observed m nature in the various habitat preferences of ferns. From Methods m Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols Edtted by R D Hall © Humana Press Inc , Totowa, NJ

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Spore sowing density can prove to be cntical at its extremes (22), wtth slower germmation at lower densities and reduced germmation at higher den­ sities. This appears to be the result of volatile substances, posstbly mcludmg ethylene, whtch are produced by germmatmg spores. Overcrowding may also lead to aberrations in prothallial growth. Competition for light and mmeral resources may also cause problems. The productiOn of large amounts of antheridogens, often found m high-denstty gametophyte populations, can also prove problematic, smce they mduce a predominance of maleness m the cul­ tures (23), hence preventing fertilization and sporophyte formatiOn. It can be seen from the bnef resume gtven above that much work has been camed out on the physiological aspects of spore germmatton and gametophyte growth. The followmg method takes mto account much of the available knowl­ edge, but has been designed to be simple and effective. 2. Materials Media: Murashtge and Skoog (MS) medium (24 and see Appendix) 1s obtamed as a ready-mixed powder, wtthout sucrose, growth regulators, or agar. The tntttal medmm is made up at half the recommended strength ( 1 hMS), together wtth 1 5 g sucrose dtssolved m 1 L of punfied water (Mtllt-Q, dtsttlled or equtvalent) For subsequent transfers, the medium ts supplemented wtth I g/L activated charcoal (25). Adjust the pH to 5.6 usmg dtlute HCI or NaOH as appropnate. Dtssolve 9 g of agar in the medmm using a microwave oven (2 mm for each 250 mL of me­ dtum in a 1 000-W oven), and stir thoroughly. This method was developed usmg Stgma purified agar (Product No. A 7002). Dtspense the medmm into the demed vessels (we normally use ' h-- and l -Ib honey Jars), and stenltze m the autoclave for 1 5 min at 1 2 1 oc and 1 05 kg/cm2 For sowmg, we use dtsposab1e sterile Petn dtshes, and in thts case, the medmm is autoclaved in conical flasks and then dts­ pensed mto the Petn dtshes m a 1ammar atrflow bench If the medmm ts not to be used immediately, store the vessels at 4°C m the dark 2. Stenltzation· Sodmm hypochlonte (BDH, I 0--1 4% available chlonne) ts used as the stenlant, dt luted to 3-1 0% v/v m detomzed water (0.3-1 .4% available chlo­ nne ), wtth the addttton of a few drops of Tween 80/L Spores are stenltzed m filter paper packets made from a 4 25-cm ctrcle of filter paper folded m from four stdes, as shown m Fig. 1 . 3 . Compost: Some ferns, such as Cyathea and Angwpterzs, are fatrly sturdy when they are brought out of culture These can be weaned in the first compost gtven below Others (Adtantum, Chetlanthes, Nephrolep1s, and so forth) are qutte delt­ cate when first out of culture, and thus requtre a much finer compost, such as the second one gtven below In all cases, good dramage must be provtded. For the more robust ferns, use: 2 parts leaf mold; 2 parts cotr; I part bark wtth added fertthzer, e.g., 2 g/L Vttax UN2. For more dehcate ferns, the same compost ts used after stevmg through a 5 mm steve.

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I + I I

3

� I

spores

Fig. 1 Ft lter paper packet used for fern spore sterilizatiOn The spores are placed on the sheet of filter paper (4 25-cm diameter), whtch ts then folded as mdtcated to form a packet This ts then sealed wtth a staple.

3. Methods 3. 1. Spore Collection and Storage I . For each spectes a frond, or ptece of frond, wtth mature sporangia ts collected and kept warm and dry m a folded white card, or paper, awaiting dehiscence (3) Depending on the matunty of the sporangia collected, this usually takes place withm 48 h. More than enough spores can be obtained from a smgle frond It has been calculated that the approximate spore content of an individual frond can range from 7 50,000 to 7 50,000,000 dependmg on the spectes (10) . Spore vmbil­ tty declines with age and depends on storage conditiOns, but thts also vanes between species. Ferns wtth chlorophyllous spores (those in Osmundaceae, Gletchemaceae, Grammttidaceae, and Hymenophyllaceae) should be sown as soon as possible, because they lose their vtabthty after only a few days (26, 10). Nonchlorophyllous spores, m general, retam theu vtabthty for substantial pen­ ods when kept at room temperature, but there are exceptiOns (e g., spores from the tree fern family, Cyatheaceae, lose vtabthty rapidly after only a few weeks) (1 0) If storage of the spores ts unavotdable, they are best kept m sealed packets m a destccator, or m sealed tubes at 4°C.

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3.2. Sterilization

2.

3 4

5.

6

Ensure that the spore sample 1s reasonably free from chaff and other debns. Th1s can be done e1ther manually with fine forceps, for the larger p1eces, or by g1vmg the wh1te card a short, sharp, but gentle tap Th1s should separate chaff from spore. Place some spores m the filter paper packet, and seal w1th a metal staple (remem­ benng that too small or too great a quant1ty may result m dens1ty problems). Soak the spore packets for 5-1 0 mm in stenle detomzed water, and carefully expel any a1r w1thm them by gently squeezmg w1th forceps. Remove the packets from the water, put them m a plastic beaker, and place on a magnetic stmer Sodium hypochlonte (BDH, I 0-- 1 4% avatlable chlonne) is used as the sterilant, diluted to I 0% v/v in deiomzed water ( 1-1 4% available chlo­ nne), w1th the add1tton of a few drops of Tween 80/L Add enough NaOCI solu­ tton to more than cover the packets, and set the speed on the stmer to prov1de gentle ag1tat1on Leave to ag1tate for I 0 mm M ove the beaker mto a lammar a1rflow bench. Standard asept1c techmques should now be followed Remove each packet from the solut1on, and nnse m separate tubes of stenle deton­ lzed water three t1mes

3.3. Sowing Carefully take the packet from the water with large forceps, and squeeze gently to expel any excess water 2 Remove the staple end of the packet w1th sc1ssors. 3 W1th the Petn d1sh open, usmg two pairS of forceps, unfold the packet, and w1pe the spores m a sw1rling motton over the surface of the medmm 4 If any obvious clumpmg of spores 1s observed, gently spread them out w1th the flat end of a spatula 5 Seal the Petn d1sh With Parafi lm or Nescofilm, and place m the growth room (temperature 22 ± 2°C, light mtensity 400-4200 lx, photopenod 16 h) or m an mcubator The hght mtens1ty used depends on the hab1tat type of the spec1es bemg cultured

3.4. Germination and Gametophyte Growth Normally a penod of 3-96 h m the dark after sowmg ts reqUired for tmbtbttton before the spores become hght-recepttve (2 7) Thts premductwn phase of germi­ natiOn is not directly accommodated m th1s method because the photopenod pro­ VIded m the growth room has proven adequate m supplying the necessary environment. If, however, synchronous or um form germmatton IS des1red, then a dark penod before exposure to hght IS advisable 2. The inductton phase of germinatton w1ll go largely unnot1ced. It is followed by the postmductton phase, where dark processes are triggered, resultmg m the pro­ trusiOn of a rh1zotd and the protonema (28). The t1me from sowmg to th1s stage vanes greatly between spec1es and can range from a few days to many months

Spore-Derived Axenic Cultures

163

Fig. 2. A mass of gametophytes ofActiniopteris semiflabellata (left) forming a few sporophytes around the edge and a later stage (right) with well-developed sporophytes ready for separation from the gametophytes (approx x3). Some spores will inevitably be killed by the surface sterilization process, but a good percentage of germination should still result.

3. Normally after a few weeks, the characteristic thalloid gametophyte form will be observed (see Fig. 2). From this stage, the rate of growth varies between species and with the light conditions to which the gametophyte has been subjected. If growth to the mature gametophyte appears particularly retarded, try moving some dishes to higher and lower Jjght intensities.

4. During growth, the following problems may occur: a. The population is too dense. b. The medium begins to dry and split. c. The gametoyphytes begin to grow brown around their bases and discolor the medium. d. Mass sporophyte formation begins. The solution to problem (a) is to ease the gametophytes gently away from the medium and move them to fresh medium in either jars or dishes, depending on the size of the plants. A spatula with a flattened end bent to a 90° angle is useful for this operation. By gently pulling the latter across the surface ofthe media, the rhizoids (vital for the uptake of nutrients and moisture and anchorage to the sub­ strate) are eased out of the media with minimal physical damage. Remove any media adhering to the plant during transfer. If problem (b) occurs, movement to fresh media is required as soon as pos­ sible. Gametophytes have been shown to be sensitive to drying (29). This can lead to a rapid death of the population if left too long.

Ford and Fay

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If problem (c) happens, then e1ther (i) the medium IS not su1table for th1s spe­ Cies and It needs to be transferred to another medmm (see Note 1 ), or (11) the nutnents are bemg rapidly exhausted, and more frequent transfer to fresh medmm 1s necessary. Situation (d) qmte often occurs where the level of moisture m the environment is sufficient to facilitate fertihzatwn. Th1s becomes a problem If the contamer 1s too small or the sporophytes arise too close to their neighbors. Th1s can lead to a high degree of competitiOn and stunted growth. It 1s therefore necessary to trans­ fer the plants to a fresh medmm, ensunng adequate space I S provided for unre­ stncted growth If the gametophytes form clumps, then gently tease them apart and move to fresh medmm

3.5. Sporophyte Formation and Potting I . Many ferns are homosporous, producing spores that germmate to form b1sexual, haplo1d gametophytes These have both anthend1a (male) and archegoma (female) parts, and produce gametes that fuse and form the dtploid sporophyte (30) Other ferns are heterosporous, producmg micro- and megaspores that ger­ minate to form mterogametophytes (male) and megagametophytes (female), respectively. These spores must be sown together 1f the ultimate aim ts to pro­ duce sporophytes (e.g , in Marsilea and Platyzoma) 2 Some species w1ll produce sporophytes read1ly (Fig. 2), and others wtll do so only after several transfers to fresh medium A few reqmre supplementary mois­ ture. Thts can be provtded s1mply m one of two ways. a. Rmsmg in stenle dewmzed water between transfers b. Addmg a small amount of stenle deionized water to the Jar, swtrhng gently, and pounng off the excess (care has to be taken here not to add so much water that the structure of the medium is damaged). Do not attempt th1s 1fthe game­ tophytes are not firmly anchored to the medmm In the early stages, the sporophyte ts dependent on the gametophyte for motsture and nutnents, which are transferred to 1t v1a a foot 3 . Subsequently roots will be formed, and when these are large enough, the sporo­ phytes may be pncked out onto fresh medium and cultured separately. It ts often advtsable to leave the parent gametophyte attached to the sporophyte, reducmg the nsk of physical damage. The gametophytes may then senesce and d1e, or contmue growmg, m which case they can then be removed and cultured sepa­ rately, if so desued. 4. As the sporophyte grows, more frequent transfers to larger vessels may prove necessary. When a reasonable number of roots have been formed, the plant IS ready for pottmg. Thts can be done usmg one of two methods: a Duect pottmg of the fern m the compost descnbed m the Subheading 2. Adhering med1a are carefully removed by nnsmg in tepid de1onized water before pottmg Good dramage must be prov1ded, and a covenng layer of gravel helps to conserve water and prevents the growth of algae The potted

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Spore-Derived Axenic Cultures

ferns are grown m shaded frames in a glass house at temperatures between 20 and 30°C and high relative humidity (90-98%). They can then be gradually acclimatized to the recommended growth conditiOns for the spec1es m questiOn. b. Transfernng to sealed Jars of stenle compost. Some ferns are slow to produce or produce only small amounts of root. Th1s mtermediate step can help to encourage root growth and reduce the potential damage to roots when the p lant is finally potted as m (a).

4. Notes l . Some ferns may prefer a different medium to that given here, e g Angwptens boivmu will grow on 'hMS, but grows better on Knudson's C medmm (29) I f frequent transfers are made. Useful media vanations for experimentatiOn are . Knudson 's C and Knudson 's C modified (with l mL microsalt solutwn/L) media, or Moore 's medmm (personal communicatiOn, Moore, London Umvers1ty). 1 .0 g/L NH4N03 0 2 g/L KH2P04 0 2 g/L MgS04 7H20 CaCI 2 2H 2 0 0. 1 g/L D1ssolve each component separately. Then combme, and add l mLIL ofsolutwn (a) and 2 mLIL of solution (b). a Microsalt stock solution. 6 20 g/L B PJ H2 0 0 25 g/L Na2 Mo04 2H20 8 60 g/L ZnS04 · 7H2 0 KI 0.83 g/L CuS04 · 5H2 0 0.025 g/L CoCl2 6H20 0 025 g/L (Dissolve separately and then combme; store in the dark at 0°C.) b. Ferric citrate stock solutwn: C6 H507Fe · 5H20, I g/L. (Store m the dark at 0°C). After combmmg all components, adjust pH to 5 .6, and add 9 g/L agar 2. The time period and the strength of the stenlant can be varied Increasmg the time or concentration will 1mprove the sterilizatiOn of those specimens that are repeat­ edly contammated. Reducmg the time or concentration will decrease the percent­ age of spore fatality in especmlly delicate samples 3. For some spec1es with short spore viab1hty, manual dissection of the sporangia may be required 1f dehiscence has not occurred withm 48 h. Th1s 1s earned out m a draft-free environment usmg a clean blade and fine forceps. 4. If fungal contammatwn 1s severe, the culture must be thrown away. If m1ld, sec­ tiOns of the populatiOn may be saved by movmg the uncontammated parts to a sugar-free medmm (such as Moore 's) or to 1hMS supplemented w1th a fungi­ cide, e g , l % Benlate. Growth on the former may be slow and pale, but the plants should reach matunty. Transfer back to a sugar-based medmm should be pos­ Sible at a later stage, but the fungus may mamfest itself agam, after lymg dormant m the absence of sugar ,

•

•

•

1 66

Ford and Fay

5 . Abnormal growth may occur at etther the gametophyte or sporophyte stage a Elongatton of the proth allus may occur. This is hkely to be a denstty problem and should be treated as such. b. Sporophytes are sometimes formed without the fuston of gametes takmg place, 1 e., apogamously This sometimes occurs m old cultures of gameto­ phytes or in cultures of spectes that are parttcularly susceptible to thts prob­ lem Ethylene has been tmphcated as a promoter of thts process (31) . The apogamous (haplotd) sporophyte may anse from the prothalltal cushion, apt­ cal regton, or on the end of an aptcal protuberance or podmm (32) c The sporophytes grow down mto the medta as well as upward Th ts t s geotrophtc confuston, posstbly brought on by etther ethylene or too much hght below the culture Thts can be averted by thmnmg out the population and blockmg hght from below. 6 The pH used m thts method was chosen because tt sutts a range of ferns (33) However, for obligate calcicoles or calctfuges, the pH may have to be adJusted If lower pH values are destred, then a htgher concentration of agar ts needed to mamtain a suffictent gel strength ( 1 2 g/L or more) I f pH values >7.0 are reqmred, tt ts better to use Ca(OH)2 rather than NaOH, smce htgh concentrations ofsodmm can prove toxtc 7 Wtthermg of fronds durmg transfer occurs m particularly senstttve ferns or tfthe transfer takes a long time Thts ts the result of destccatton caused by the stream of atr m the flow bench. A screen, usmg one or more stenle dtshes set up m the bench, helps to prevent this problem

Acknowledgments We would hke to thank L . Goss and N. Rothwell for their advtce on compost and glass house conditions. References

2. 3

4. 5.

6

Miller, J H. ( 1 968) Fern gametophytes as expenmenta1 matenal. Bot Rev 34, 362--441 Windham, M D and Haufler, C H ( 1 986) Btosystematlc uses of fern gameto­ phytes denved from herbanum spectmens. Am Fern J 16, 1 1 4-1 28 Dyer, A. F. ( 1 979) The culture of fern gametophytes for expenmental mvestlga­ tlon, m The Expenmental Bwlogy ofFerns (Dyer, A F , ed ), Academtc, London, pp 254-3 05 Wtll tams, S. ( 1 9 3 8 ) Expenmental morphology, m Manual of Ptendology (Verdoorn, Fr , ed ), Martmus Ntjhoff, The Hague, pp 1 05- 1 40. Ragavan, V. R. ( 1 970) Germmatton of bracken fern spores. Regulatton of protem and RNA synthests dunng tmtlation and growth of the rhtzotd. Exp Cell Res 63, 341-352 Wemberg, E. S and Voeller, B R. ( 1 969) Inductton of fern spore germmatton. Proc Nat/ Acad Sc1 USA 64, 83 5-842

Spore-Derived Axenic Cultures

1 67

7. Orth, R ( 1 93 7) Zur Ketmungsphyswlogte der Farnsporen m versch � edenen Spektralvemken J Wis Bot 84, 358-426 8. Miller, J. H. and Greany, R. H. ( 1 974) DeterminatiOn of rhtzotd onentatwn by hght and darkness tn germmating spores of Onoc/ea sens1blils Am J Bot 5 1 , 329-334. 9. Mtller, J. H. and Miller, P M. ( 1 970) Unusual dark-growth and antheridial dtffer­ entiatwn in some gametophytes of the fern Onoclea Am J Bot 57, 1 245-1 248 1 0. Page, C N. ( 1 979) Expenmental aspects of fern ecology, in The Expenmental Bwlogy of Ferns (Dyer, A. F , ed.), Academtc, London, pp 552-589 I I Raga van, V R . ( 1 97 1 ) Phytochrome control of germmatwn ofthe spores ofAsple­ mum mdus Plant Physwl 48, 1 00-- 1 02 1 2 Ragavan, V R ( 1 973) Blue hght mterference in the phytochrome-controlled ger­ mmatton of the spores of Chellanthes farmosa Plant Physwl 5 1 , 306--3 I I 1 3 . Sugai, M., Take no, K., and Furuya, M ( 1 977) Diverse responses of spores m the hght-dependent germination of LygodiumJaponicum PI Sci. Lett 8, 333-3 38 1 4 Towtll, L R. and Ikuma, H. ( 1 973) Photocontrol of the germmatwn of Onoclea spores I. Actwn spectrum. Plant Physwl 5 1 , 973-978. 15. Towill, L R and lkuma, H ( 1 975) Photocontrol of the germinatiOn of Onoclea spores II. Analysts of germmatton processes by means of anaerobtosts Plant Physwl 55, 1 50--1 54 16 Furuya, M ( 1 985) Photocontrol of spore germmatt on and elementary processes of development tn fern gametophytes, in Biology of Ptendophytes (Dyer, A F and Page, C N , eds. ), Proc Royal Soc Edin. 868, 1 3-1 9 1 7. Greany, R H and Mtller, J. H. ( 1 976) An Interpretation of dose-response curves for hght mduced cell elongatiOn m the fern protonemata Am J Bot 63, 1 03 1-1037. 1 8 Howland, G P and Edwards, M. E. ( 1 979) Photomorphogenests of fern gameto­ phytes, m The Expenmental Bwlogy ofFerns (Dyer, A F , ed ), Academic, Lon­ don, pp 394-434. 1 9 Mtller, J. H. and Mtl ler, P M ( 1 967) Actwn spectra for hght mduced elongatiOn m fern protonemata. Physwlog1a Pl. 20, 1 28-1 38 20. Miller, J. H. and Miller, P. M ( 1 974) InteractiOn of photomorphogenetic pig­ ments m fern gametophytes Phytochrome and a yellow-hght absorbmg pigment. Pl. Cell Physwl. Tokyo 8, 765-769. 2 1 Ford, M . V. ( 1 984) Growth responses o f selected fern prothalh to various hght regtmes. Unpublished 22 Smtth, D. L and Robinson, P. M. ( 1 97 1 ) Growth factors produced by germmatmg spores of PolypodlUm vulgare (L.) New Phytol. 70, 1 043- 1 052 23 . Bell, P R ( 1 979) The contnbution of the ferns to an understandmg of the ltfe cycles of vascular plants. IV Sexuality m gametophyttc growth, tn The Experz­ mental Bwlogy of Ferns (Dyer, A F , ed.), Academic, London, pp 64, 65 24. Murashige, T. and Skoog, F. ( 1 962) A revtsed medmm for raptd growth and bto­ assays wtth tobacco ttssue cultures. Physwl. Plant 1 5, 473-497. 25. Ford, M. V. ( 1 992) Growing ferns from spores in stenle culture, in Fern Horticul­ ture, Past, Present and Future Perspectives (Ide, J M , Jermy, A. C , and Paul, A. M., eds.), Intercept, Andover, pp 295-297

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26 Lloyd, R. M. and Klekowski, E. J., Jr ( 1 970) Spore germinatiOn and vtability m the Pteridophyta: EvolutiOnary significance of chlorophyllous spores. Bwtropica 2, 1 29-1 3 7 2 7 Jarvts, S . J and Wtlkms, M. B. ( 1 973) Photoresponses o f Matteuccza struthwpterzs (L ) Todaro J. Exp Bot 24, 1 1 49-1 1 57. 28 Brandes, H . ( 1 973) Spore Germmatton Ann Rev Plant Physwl 24, 1 1 5- 1 28 29 Knudson, L. ( 1 946) A new nutnent solutton for the germinatton of orch1d seed A m Orchzd Soc Bull 1 5, 2 1 4-2 1 7 30. Hyde, H . A., Wade, A . E., and Harrison, S G . ( 1 978) Welsh Ferns, Clubmosses, Quzllworts, and Horsetazls, Nattonal Museum of Wales, Cardiff 3 1 Elmore, H W and Wh1tt1er, D P. ( 1 973) The role of ethylene m the inductton of apogamous buds m Pterzdzum gametophytes. Planta 1 1 1, 85-90. 32 Walker, T. G ( 1 985) Some aspects of agamospory m ferns-the Braithwaite Sys­ tem, m Bwlogy of Pterzdophytes (Dyer, A. F. and Page, C N . , eds ). Proc Royal Soc Edm 86B, 59-66. 33 Otto, E. A., Crow, J. H., and Ktrby, E. G ( 1 984) Effects of ac1d1c growth conditions on spore germmation and reproductive development m Dryopterzs margznalzs (L ) A nn Bot. 53, 439--442

17

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Proto plast Iso lation, Culture, and Plant Regeneration from Passiflora Paul Anthony, Wagner Oton i, J. Brian Power, Kenneth C. Lowe, and Michael R. Davey

1 . Introduction The famtly Passifloraceae contams over 580 woody or herbaceous spectes (1) , the maJonty of spectes wtthin the genus Passiflora being found m tropical South Amenca. Passiflora edulzs fv. tlavtcarpa is considered to be the most important spectes (2) because of tts value in the frutt JUICe mdustry. Addttton­ ally, thts spectes is resistant to the soil-borne pathogen Fusarium oxysporum and, consequently, is frequently used as a rootstock onto which is grafted P edulzs S tms. Interspeci fic sexual hybridization has been attempted in Passzjlora breedmg programs, using wild-type germplasms to transfer dtsease reststance and other potentially demable trmts into culttvated spectes. However, fertile hybnds have been difficult to obtam (3). Somatic hybridization provtdes a means of circumventmg such sexual incompattbthttes. Indeed, novel ferttle somatic hybnds have been produced between P. edulzs fv . tlavicarpa and Passiflora incarnata (4). Smce the latter species can survive winter tempera­ tures of -1 6°C, such transfer of cold tolerance to the commercial crop would permit cultivatiOn m more temperate climates (5) . Somatic hybnd plants have also been produced between P edulis fv. tlavtcarpa and Passiflora amethystina, Passiflora cmcinnata, Passiflora gibertz, and Passiflora alata, respectively (6). A prerequisite to any somatic hybndizahon program ts the successful tsola­ twn and culture of protoplasts (wall-less cells) of at least one of the parental spectes, leading to the effictent regeneration of normal, ferttle plants. Proto­ plast tsolatwn ts mtluenced by several factors, mcludmg the choice of plant species and/or cultivar, together wtth the source tissue (e.g., leaves, cell sus­ pensiOns, cotyledons, roots, pollen tetrads) and tts physwlogtcal status. The From Methods m Molecular Biology, Vol 111 Plant Cell Culture Protocols Edtted by R D Hall © Humana Press Inc , Totowa, NJ

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nature of the cell wall of the source tlssue(s) IS an Important parameter, smce It determines the composition and concentration of the enzyme mixture reqmred for efficient wall degradatiOn and protoplast release High protoplast viability is crucial immediately after isolation, and also dunng subsequent culture. To date, plant regeneratiOn from Passzjlora protoplasts has been reported for P. edulis fv. flav1carpa (7,8), P amethystina, and P. czncinnata (8). The followmg protocol Is an excellent starting point for those interested m dicot protoplast culture, smce It concerns a well-tested and highly responsive system. 2. Materials 2. 1. Glasshouse-Grown Seedlings of P. edulis and P. giberti 1 . Seeds of P edulzs fv flavicarpa and P gzbertz (J C Oliveira and C Ruggiero, Departmento de Fttotecma, FCA VJ-Umvendade Estadual Pauhsta [UNESP], Jaboticabal, Sao Paulo, Brazil). 2. Levington M3 sotlless compost (Fisons, lpswtch, UK) and John Innes no 3 com­ post (J Bentley Ltd , Barrow-on-Humber, UK). 3. "Vac-trays" (H Smith Plastics Ltd., Wtckford, Essex, UK)

2.2. Isolation of Protoplasts Directly from Seedling Leaves of P. edulis I . "Domestos" bleach (Lever Industnal Ltd , Runcorn, UK), or any commerctally available bleach solutiOn contammg approx 5% available chlonne. 2 Enzyme 1 solutiOn (7) " 2 g/L Macerozyme R I 0 (Yakult Honsha Co Ltd. , Ntshmomiya Hyogo, Japan), 1 0 giL Cellulase R I O (Yakult Honsha Co Ltd.), 1 gi'L Dnselase (Kyowa Hakko Co. Ltd , Tokyo, Japan), 1 1 g/L 2-[N-morpholmo]ethane­ sulfonic actd (MES), 250 mg/L polyvmylpyrrolidone (PVP- 1 0, Stgma), 250 mg/L cefotaxime ("Ciaforan", Roussel Laboratones, Uxbndge, UK) with CPW salts (9), pH 5.8 Filter-stenhze 3 Modified CPW salts solution (9) 27 2 mg/L KH2P04, 1 0 1 mg/L KN03, 246 mg/L MgS04 7H20, 0 1 6 mg/L KI, 0.025 mg/L CuS04 5H20, 1 480 mg/L CaC12 2H2 0, pH 5 8 4. CPW 1 3 M solutiOn: modified CPW salts solutiOn With 1 3 0 g/L manmtol (item 3), pH 5 8 . Autoclave ( 1 5 mm, 1 2 1 °C, steam pressure) 5 Seaplaque agarose (FMC Btoproducts, Rockwell, USA). Autoclave.

2.3. Culture of Leaf Protop/asts ofP. edulis KM8P med mm . based on the formulatiOn of Kao and Michayluk (10) with modi­ ficatiOns (1 1) (Table 1) and supplemented wtth 250 mg/L cefotaxime. For semt­ solid KM8P medtum, m1x double-strength KM8P medium with an equal volume of molten (40-60°C) 1 .6% (w/v) aqueous Seaplaque agarose, the latter prepared (as with all med1a) with reverse-osmosis water Add filter-stenhzed cefotax1me to the molten medtum at 40°C

1 71

lsolatton, Culture, and Regeneration Table 1 Formulation of Media Macronutrients, Micronutrients, Vitamins, and Other Supplements ConcentratiOn, mg/L Component Macronutnents NH4N03 KN03 CaC12 2H2 0 MgS04 7H2 0 KH2 P04 Sequestrene 330 Fe M1cronutnents KI H3B03 MnS04 4H2 0 ZnS04 7H2 0 NaMo04 2 H2 0 CuS04 5H 2 0 CoC1 2 6H2 0 V1tamms Myo-mos1to1 Nicotmamide Pyndoxme HCI Thiamme HCI D-Ca Pantothenate Folic acid Absc1sic ac1d Bwtm Cholme chlonde R1boflavm Ascorbic acid VItamm A Vitamm D3 VItamm B 1 2 Na pyruvate C1tnc ac1d Mahc ac1d Fumaric acid Other supplements Fructose Ribose •

•

•

KM8P 600 1 900 600 300 1 70 28 0.75 30 1 0.0 20 0 25 0 025 0 025

KM8 600 1 900 600 300 300 28 0.75 30 10 0 20 0 25 0 025 0 025

KPR 600 1 900 600 300 300 28

K8 600 1 900 600 300 300 28

0 75 30 10 0 20 0.25 0.025 0 025

0 75 30 10 0 20 0 25 0 025 0 025

1 00 I 0 1 0 1 .0 1 .0 0.4 0 02 0 01 I 0 02 20 0 01 0 01 0 02 20 40 40 40

1 00 1 .0 1 0 1 .0 1 0 04 0 02 0.01 1 .0 0.2 20 0.0 1 0 01 0 02 20 40 40 40

1 00 10 10 1 0 05 02 0 01 0 005 05 0I 1 .0 0 005 0 005 0.01 10 20 20 20

1 00 I 0 1 0 I 0 05 02 0 01 0 005 05 01 1 .0 0 005 0 005 0.0 1 10 20 20 20

250 250

250 250

1 25 1 25

125 1 25 (contmued)

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Anthony et a/.

Table 1 (continued) ConcentratiOn, mg/L Component

KM8P

KM8

KPR

K8

Other supplements 250 i 25 250 125 Xylose 250 1 25 250 1 25 Mannose 1 25 250 1 25 250 Rhamnose 250 250 1 25 1 25 Cellobiose 250 1 25 250 125 Sorbitol 250 1 25 125 250 Manmtol 250 125 250 1 25 V1tamm-free casammo acids 20 mLIL 20 mLIL 1 0 mLIL 1 0 mLIL Coconut milk 0 I 05 0 I 02 2,4-Dichlorophenoxyacetic acid 02 02 05 05 Zeatm I 0 I 0 I 0 I 0 a-Naphthaleneacetlc acid 250 20,000 20,000 250 Sucrose 1 00,000 1 0,000 1 0,000 1 00,000 Glucose 58 58 5.8 5.8 pH 2 Fluorescem dmcetate (FDA). 3 mg/mL stock solutiOn m acetone (12) Store m the dark at 4°C 3 KM8 medmm based on the formulation of Kao and M1chayluk (10) with modi­ fications (1 1) (Table I) Filter-stenhze.

2.4. Plant Regeneration from Protoplast-Derived Tissues of P. edulis I . MSRI medmm: based on the formulatiOn ofMurash1ge and Skoog (13, see Appen­ dix) with 5.0 mg!L a-naphthaleneacet1c acid (NAA), 0 25 mg!L 6-benzylammopunne (BAP), 50 mg/L cysteme, 50 mg/L glutamme, 50 mg/L glutamic acid, 0 5 mg/L biotin, 0 5 mg/L folic acid, 30 g/L sucrose, and 8 g/L agar, pH 5 8 Autoclave the medmm, and add filter-stenhzed solutiOns of the ammo acids and v1tamms when the medmm has cooled to about 40°C 2 MSR2 medmm. MS-based medmm with 1 .0 mg/L BAP, 30 g/L sucrose, and 8 g/L agar, pH 5 8 Autoclave 3. MSR3 . half-strength MS-based medmm contaimng 3 . 0 mg/L mdole-3-butync acid (IBA), 0.5 mg/L NAA, 30 g/L sucrose, and 8 g/L agar, pH 5 8 Autoclave

2.5. Initiation of Cell Suspensions of P. giberti 1 "Domestos" bleach· as m Subheading 2.2, item 1. 2 MS 1 medmm based on the formulation of Murash1ge and Skoog (1 3) , supple­ mented with 7.2 mg/L 4-ammo-3 ,5,6-tnchloropicolmic acid (Picloram, Aldnch Chemical Co , Mi lwaukee, WI) and 8 g/L agar (Sigma, Poole, UK) Autoclave 3 Nescofilm· Bando ChemiCal Ind Ltd. (Kobe, Japan)

Isolation, Culture, and Regeneration

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Table 2 Formulation of Modified AA2 Medium Macronutrients, Micronutrients, Vitamins, and Other Supplements Component Macronutnents CaCI2 2H20 KH2P04 MgS04 7H20 KCI Micronutnents KI H3B03 MnS04 4H2 0 NaMo04 · 2 H 20 ZnS04 · 7H20 CuS04 5H20 CoCI2 6H 20 FeS04 7H20 Na2EDTA VI tamms Myo-mositol Ntcotmic acid Pyridoxine HCI Thiamine HCI Glycine L-Giutamme L-Aspartic acid L-Arginme Other supplements 2,4-Dichlorophenoxyacetic acid Gibberellic acid Kinetin Sucrose pH •

•

ConcentratiOn, mg/L 440 1 70 370 2940 0 83 62 22 3 0 25 86 0 025 0 025 27 85 37 25 1 00 05 0. 1 05 75 877 266 228 2.0 0 1 0.2 20,000 58

4. Modified AA2 medium: based on the formulatwn of Muller and Grafe (14) (Table 2) Filter-stenhze

2.6. Isolation of Protoplasts from Cell Suspensions of P. giberti Enzyme solution 1 0 1 (15) : 1 0 g/L Cellulase RS (Yakult Honsha Co Ltd.), 1 g/L Pectolyase Y23 (Seishim Pharmaceutical, Tokyo, Japan), 1 l g/L MES m CPW 1 3 M solution (9), pH 5.8. Filter-sterilize. 2. CPW I 3 M solution: as in Subheading 2.2, item 1.

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Anthony et a/.

2. 7. Culture of Cell Suspension-Derived Protoplasts of P. giberti KPR medmm (16) : (Table 1) Ftlter-stenhze. 2 FDA solutwn: as m Subheading 2.3, item 2. 3 . K8 medium: based on the formulatwn of Kao and MJChayluk (10) wtth modifica­ tiOns (11) (Table 1) Ftlter-stenhze

2.8. Plant Regeneration from Protoplast-Derived Tissues of P. giberti MSE l medmm· half-strength MS-based medmm (13) containmg 20 g/L sucrose, 4 8 mg/L Picloram, and semtsohdtfied wtth 2 g/L Phytagel {Stgma), pH 5 8 Autoclave 2 MSE2 medium· half-strength MS-based medmm (13) contammg 20 g/L sucrose, 0 5 mg/L gtbberelhc actd (GA3), and semtsohdtfied wtth 2 g/L Phytagel, pH 5 8 Autoclave 3 MSE3 medmm. half-strength MS-based medmm (13) contaming 20 g/L sucrose and semtsohdified wtth 2 g/L Phytagel, pH 5 8. Autoclave.

3 . Methods 3. 1. Glasshouse-Grown Seedlings of P. edu lis and P. giberti Prepare a 1 : 1 (v.v) mtxture of Levmgton M3 compost and John Innes No 3 com­ post, and fill the mdtv1dual compartments of plastic propagator trays 2. Place 2-3 seeds (approx 1 em deep) mto each compartment of the plastic propa­ gator trays and trngate from above (see Note 1) 3 Mamtam propagators at a max1mum day temperature of28 ± 2°C and a mmtmum mght temperature of 1 8 ± 2°C m the glasshouse under natural dayhght supple­ mented w1th a 1 6 -h photopenod provided by Cool Wh1te fluorescent tubes ( 1 80 f..J.mol/m2/s)

3.2. Isolation of Protoplasts Directly from Seedling Leaves of P. edu lis Surface-sterilize fully expanded, young leaves exc1sed from 45- to 60-d-old plants (Fig. 1A), m 7% (v/v) "Domestos" bleach solutwn for 20 mm Wash thoroughly w1th stenle, reverse osmos1s water (three changes) 2 Cut the leaves transversely mto 1 -mm stnps (see Note 2) and mcubate (30 mm) approx l g f wt portwns of matenal m 20-mL ahquots of CPW 1 3M solutwn (see Subheading 2.4., item 4.) contamed m 9-cm Petn dtshes Seal the d1shes w1th Nescofilm 3 Remove the CPW 1 3M solutiOn, and replace w1th Enzyme 1 solutwn (see Note 3), mcubatmg I g f wt of tissue m I 0-mL enzyme solutwn Seal the dtshes w1th Nescofilm, and mcubate on a slow shaker (40 rpm) at 25 ± 2°C for 1 6 h m the dark 4. F1lter the enzyme-protoplast mixture through a nylon steve of 64-f..J.m pore SIZe (see Note 4) Place the filtrate m 1 6-mL capactty screw-capped centnfuge tubes

Isolation, Culture, and Regeneration

1 75

Fig. I. Isolation, culture and plant regeneration from leafprotoplasts ofP. edulis fv. flavicarpa. (A) Forty-five-d-old glasshouse-grown seedlings suitable for the isolation ofleafprotoplasts (bar = 2.6 em). (B) Freshly isolated leaf protoplasts (bar= I 00 J..lm). (C, D, E) Protoplasts undergoing mitotic division after 8, 10, and 1 2 d of culture, respectively (bars = 75 J..lm). (F) A protoplast-derived colony after 15 d of culture (bar = 75 f..I)ID . (G) Protoplast-derived tissues (bar = 5 mm). (H) Protoplast-derived callus undergoing shoot regeneration (bar= 1.5 mm). (I) A rooted, protoplast-derived plant (bar = 1.5 em). 5. Centrifuge (80g; 7 min). Discard the supernatants, and resuspend the protoplast pellets in CPW 13M solution. Centrifuge and wash the protoplasts twice by resuspension and centrifugation in CPW13M solution.

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Anthony et at.

6. Resuspend the protoplasts m a known volume (e.g., 1 0-mL) of CPW 1 3M solu­ tion (Fig. 1B). Remove an aliquot of the suspension ( e g , 0. 1 -mL ), and transfer to a haemocytometer. Count the protoplasts, yields of ± 1 2 x 1 06/g f. wt can be expected. 7. Assess protoplast vmbihty usmg FDA (see Note 5) Add 1 00-J.tL of FDA stock solutiOn to 1 6-mL of CPW 1 3M solutiOn M1x one drop of this solutiOn, by Pas­ teur ptpet, w1th an equal volume of protoplast suspenston. Observe the proto­ plasts under UV illummatiOn (e g., usmg a Ntkon Dtaphot TMD mverted miCroscope with high-pressure mercury vapor lamp HBO I 00 W/2, a B I FITC exciter filter IF 420-485 nm, dtchromic mirror DM5 I 0, and eyepiece absorptiOn filter 570). VIable protoplasts fluoresce yellow-green (see Note 6) Protoplast vtabthty IS usually ±90%. 3. 3. Culture of Leaf Protoplasts of P. edulis I 2 3.

4. 5

Centrifuge the protoplast suspensiOn from Subheading 3.2., step 6 at 80g for 7 mm Di scard the supernatant Resuspend protoplasts in agarose-sohdtfied KM8P medmm (see Note 7) at a den­ sity of I 5 x 1 05 /mL (see Note 8). Dispense 40-J.tL droplets of KM8P medium containing suspended protoplasts m 5-cm Petri dishes (25 droplets/dish). Allow the droplets to solidify (see Note 9), and bathe the droplets in each dish in 2-rnL vol of liqmd KM8P medmm containing cefotaxime Seal the dishes w1th Nescofilm, and mcubate at 25 ± 2°C m the dark Replace the KM8P bathing medmm every 5 d, w1th KM8P medmm mixed with KM8 medmm, m the ratiOs of 3 : 1 , 2. 1 , 1 . 1 , and 0 : 1 (v·v). Assess the protoplast platmg efficiency (number of diVIdmg [mitotic] protop lasts expressed as a percentage of the number of viable [FDA assessed] protoplasts ongmally plated) after 6-1 0 d of culture (Fig. 1C,D). The platmg efficiency should be ±40%.

3.4. Plant Regeneration from Protoplast-Derived Tissues of P. edulis Transfer protoplast-derived colomes (Fig. 1 E,F) (see Note 10), 20 d after Isola­ tiOn ofprotoplasts, to agar-solidified MSR 1 medmm (50 colomes/20-mL ahquots of medmm in 9-cm Petn dishes). Seal the dtshes With Nescofilm and mcubate at 25°C ± 2°C m the light under a 1 6-h photopenod (25 J.tmollm2 /s; Daylight fluo­ rescent tubes). 2 After 25 d, transfer protoplast-denved tissues (Fig. 1 G) to agar-solidified MSR2 medium (5 calli/45-mL aliquots of medmm m 1 75-mL glass J ars) Incubate as m step 1. 3 Subculture protoplast-denved tissues every 30 d to MSR2 shoot regeneration medmm as m step 2. Shoots should appear progressively w1thm a further 60 d of culture (Fig. 1H). 4. Excise developmg shoots when 5-6 em in height. Root the shoots by transfer to agar-solidified MSR3 medmm for 7 d (Fig. 1 1), followed by transfer to MS-

Isolation, Culture, and Regeneration

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based medtum, lackmg growth regulators ( 1-3 shoots/50 mL medium for both stages in 1 75-mL jars). Maintam the shoots under growth conditiOns as in step 1 .

3.5. 1nitiation of Cell Suspensions of P. giberti 1 . Excise and sterilize leaves as in Subheading 3.2., step 1. 2 Place the leaves With their adaxial surfaces in contact with 20-mL ahquots of agar-solidified MS 1 medium (see step 2) using 10 leaves/9-cm Petn dtsh. Seal the dishes with Nescofilm. 3. Incubate the cultures m the dark at 28 ± 2°C. After 28 d, transfer the cultures to the light under a 1 6-h photopenod (25 J.lmollm2 /s, Daylight fluorescent tubes) and incubate for a further 40 d 4. Transfer 2-3 g f. wt of embryogenic callus (see Note 1 1 ) mto 40-mL ahquots of hquid AA2 medium m 250-mL Erlenmeyer flasks. Incubate on a honzontal rotary shaker (90 rpm) in the dark at 28 ± 2°C 5. Subculture 5-8 mL packed volume of cells (see Note 12) to 40-mL of fresh AA2 medium every 4 d, for the first 4 passages. Thereafter, subculture cell suspen­ SIOns every 7 d (Fig. 2A).

3.6. Isolation of Protoplasts from Cell Suspensions of P. giberti 1 . Harvest cells 4 d after subculture (Passage 5 onward; see Subheading 3.5., step 5) of suspensions usmg a nylon sieve of 1 00-J.lm pore size (see Note 4). 2. Transfer cells to 9-cm Petri dishes contammg Enzyme solutiOn 1 0 1 ( 1 g f. wt of tissue m 20-mL enzyme solutiOn) Seal dishes with Nescofilm, and mcubate on a slow shaker (40 rpm) for 1 6 h at 25 ± 2°C in the dark. 3 Filter the enzyme-protoplast mixture through a nylon sieve of 45-J.lm pore size (see Note 4). Place the filtrate m 1 6-mL capac1ty screw-capped centnfuge tubes. 4 Follow stages as in Subheading 3.2., steps 5-7

3. 7. Culture of Protoplasts of P. giberti Resuspend protoplasts m hqmd KPR medmm (Fig. 2B) at a density of 1 0 x 1 05/mL, and dispense as 4-mL aliquots m 5-cm Petri d1shes. 2 Seal the d1shes with Nescofilm, and mcubate at 25 ± 2°C m the dark. 3 Remove 0.5-mL ahquots of KPR medtum at 5 and 1 0 d (see Note 13); replace with a s1mllar volume of hqmd K8 medmm 4 Assess the protoplast platmg effic1ency after 6-- 1 0 d of culture (Fig. 2C,D) as m Subheading 3.3, step 4 Thts should be ± 1 0%.

3.8. Plant Regeneration from Protoplast-derived Tissues of P. g i berti 1

Transfer protoplast-denved colomes (Fig. 2E) (see Note 10), 40 d after 1solatwn, to MSE l medtum (5 colomes/ 1 0-mL medmm m 5-cm Petn d1shes) Seal the dishes with Nescofilm, and mcubate at 27 ± 2°C in the light under a 1 2 h photo­ penod (25 J.lmol/m2/s; Daylight fluorescent tubes).

1 78

Anthony et a/.

Fig. 2. Isolation, culture, and plant regeneration from cell suspension protoplasts of P.

giberti.(A) Embryogenic cell suspension suitable for protoplast isolation (bar = I

cm).(B) Freshly isolated cell suspension protoplasts (bar = 50 ,.un).(C) Protoplasts after 5 d in culture. Note the change from a spherical to a more oval shape as the protoplasts enter mitotic division (bar = 50 ,.un).(D) A protoplast that has undergone mitotic division to form two daughter cells (bar = 50 J.lm). (E) A protoplast-derived cell colony after 28 d of culture (bar = 50 J.lm). (F, G) Somatic embryos developing from protoplast-derived callus (bars = 5 nun). (H) Somatic embryo-derived shoots (bar = 1.5 mm). (I) A rooted protoplast-derived plant (bar = t .5 em). 2. After 20 d, transfer protoplast-derived colonies to MSE2 medium, as in step 1. Incu­ bate at 27 ± 2°C in the light under a 16 h photoperiod (25 J.lmoVm2/s; Daylight fluo­ rescent tubes) to induce shoot regeneration by somatic embryogenesis (Fig. 2F,G).

Isolation, Culture, and Regeneration

1 79

3 Excise developmg shoots when I em in hetght (Fig. 2H). Root the shoots by transfer to MSE3 medmm ( 1 -3 shoots/50-mL medmm in 1 75-mL J ars) (Fig. 21) Incubate the shoots as m step 2.

3.9. Characterization of Regenerated Plants Morphologically, regenerated plants should be scored within 2-3 mo fol­ lowmg transfer to the glasshouse, since the juvenile characteristics of leaf shape and pigmentation in vitro may be different from those of seed-derived plants raised directly in the glasshouse. Furthermore, this will enable the floral char­ acteristics, fertility, and fruitmg properties to be compared with those features of seed-denved (control) plants. The somatic chromosome complement can be assessed using root tip preparations and any shtft in ploidy wtthm populations of regenerated plants raptdly confirmed by flow cytometnc analysts of isolated nuclei (1 7) . Further DNA molecular studtes may be performed on those protoplast­ derived plants that exhibtt stable phenotypic, fertility, and cytologtcal charac­ ters, whtch dtffer from those of seed-derived plants. These techmques mclude the use of AFLP (18), RAPD (19), RFLP (20), and simple sequence repeats (microsatellites) (21) . 4 . Notes 1 . Store Passiflora seeds at 4°C Sow Pass1jlora seeds m excess of the number of seedhngs reqmred, since germinatiOn within the genus Passzflora can be slow and erratic . A mtmmum of 1 0 seedlings wtll be reqmred for each protoplast iso­ latiOn Leaves should be harvested before seedlmgs produce tendnls 2. Repeatedly use a new scapel blade to ensure precise cuttmg, rather than tearmg and bruismg, of the leaf material 3 . The enzyme solutiOns should be prefiltered, using 0 2-�-tm pore stze mtrocellu­ lose 47-mm dtameter membrane filters (Whatman) to remove insoluble tmpun­ ties. Thts prevents premature blockage of the stenle microb1al filters (0.2-).lm pore stze, 30-mm dtameter; Mtmsart NML, Sartonus AG, Gottmgen, Germany) dunng subsequent stenltzatlon of the enzyme solutwns 4 Inexpenstve, nylon steves m a range of pore stzes may be obtamed from Wtlson Steves, Common Lane, Hucknall, Nottmgham, UK 5 . Prepare the FDA dilution in CPW 1 3 M solution, immediately pnor to vtabihty assessments, smce even short-term storage wtll result m cleavage of FDA to fluo­ rescein, espectally tf the so IuttOn is exposed to strong illummation 6 FDA taken up into protoplasts is acted on enzymatically by esterases, to release fluorescem This molecule ts excited under UV t llummatton and fluoresces yel­ low-green. Only vtable protoplasts wtth mtact plasma membranes that retam fluo­ rescem wtll fluoresce. 7.

The agarose-sohdtfied KM8P medmm must be prepared as: (a) aqueous double­ strength agarose and (b) hqmd, double-strength medmm components. The agar-

1 80

8. 9.

10 11 12.

13

Anthony et at. ose ts sterilized by autoclavmg (20 min, 1 2 1 °C, saturated steam pressure); the hquid component IS filter-stenhzed The double-strength liquid component IS mtxed wtth the molten agarose component at 50--60°C, Immediately pnor to use. Ensure that the agarose-sohdtfied KM8P medmm ts at 3 5--40°C, pnor to mtxmg wtth, and resuspenswn of, the protoplast pellets Allow the KM8P droplets, contammg protoplasts, to sohdtfy for at ieast 1 h (at room temperature) before the addtttOn of the hqmd KM8P bathmg medium Thts permtts the droplets to adhere to the bottom of the Petn dtshes Transfer mdtvtdual colomes usmg fine, Jeweler's forceps, flame-stenhze, and cool the forceps immediately pnor to use. Embryogenic callus is yellow m color, dry, friable, and translucent m appearance Use a 1 0-mL graduated pipet, from which the end has been removed to increase the Size of the orifice to transfer the cells The packed cell volume can be deter­ mmed by removing 1 0-mL ahquots of liquid medium, holding the pipet verti­ cally, and allowing the cells to settle ( 1 0 mm) Angle the Petn dtsh, and allow the protoplasts to settle (5 mm) to the bottom of the Petn dish before removal of the overlymg hqmd culture medmm.

References 1 . Ohvtera, J. C ( 1 987) Melhoramento genettco in Cultura do maracuJazeiro, vol 1 (Ruggtero, C , ed.), Edttora Legts Summa, Ribeirao Petro, pp 2 1 8-246. 2 Vanderplank, J. ( 199 1 ) Passwn Flowers, Cassel Publishers, London. 3 Payan, F. R , and Martm, F. W. ( 1 975) Earners to the hybndtzatwn of Passiflora species Euphyt!ca 24, 709-7 16 4 Otom, W C., Blackhall, N W , d' Utra Vaz, F. B . , Casah, V . W., Power, J. B., and Davey, M. R. ( 1 995) Somatic hybndtzat10n of the Passiflora species, Passiflora edulis fv flavicarpa Degener and P mcarnata L J Exp Bot 46, 777-785 5 . Dozier, W. A , Jr., Rodriguez-Kahana, R., Caylor, A W , Htmelnck, D. G , McDaniel, N R , and McGuire, J. A. ( 1 99 1 ) Ethephon hastens matunty of pass10nfruit grown as an annual in a temperate zone Hort Scz 26, 146-1 47. 6. Dornelas, M C , Tavares, F. C A., Oliviera, J C., and Vieira, M L C ( 1 995) Plant regeneratiOn from protoplast fuswn m Passiflora spp. Plant Cell Rep 15, 1 06-1 10 7. d ' Utra Vaz, F B , dos Santos, A V P , Manders, G , Cocking, E. C , Davey, M R., and Power, J. B ( 1 993 ) Plant regeneration from leaf mesophyll protop lasts of the tropical woody plant, pass10nfrmt (Passiflora edulzs fv flavicarpa Degener ) the Importance of the antibiOtic cefotaxime in the culture medmm. Plant Cell Rep. 1 2, 220-225 8. Domelas, M. C. and Vtetra, M. L. C. ( 1 993) Plant regeneration from piOtoplast cultures of Passiflora edulzs var jlavzcarpa Deg., P amethystzna Mtkan. and P czncznnata Mast Plant Cell Rep 13, 1 03-1 06 9 Frearson, E. M., Power, J B., and Cocking, E C ( 1 973) The IsolatiOn, culture and regeneration of Petunza protoplasts Dev. Bwl. 33, 1 30-1 37.

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1 0. Kao, K . N . and Michayluk, M . R . ( 1 975) Nutntwnal reqmrements for growth of Vzcza hajastana cells and protoplasts at a very low populatiOn density m hqmd media Planta 126, 1 05-1 1 0. 1 1 . Gilmour, D M , Golds, T. J., and Davey, M. R. ( 1 989) Medzcago protoplasts. fusion, culture and plant regeneration, in Bwtechnology zn Forestry and Agrzcul­ ture, vol 8, Plant Protoplasts and Genetzc Engzneerzng I (BajaJ, Y. P. S , ed ), Springer-Verlag, Heidelberg, pp. 370--3 88. 12. Widholm, J. ( 1 972) The use ofFDA and phenosafranine for determining viability of cultured plant cells. Stain Techno! 47, 1 86-- 1 94 1 3 Murashige, T. and Skoog, F ( 1 962) A revised medtum for rapid growth and bw­ assays w1th tobacco tissue cultures. Physwl. Plant 56, 4 73-497 1 4 Muller, A. J. and Grafe, R. ( 1 978) IsolatiOn and characterisatiOn of cell hnes of Nzcotzana tabacum lackmg mtrate reductase. Mol Gen Genet 161, 67-76. 1 5 Jam, R. K., Khehra, G S , Lee, S-H., Blackhall, N. W., Marchant, R , Davey, M R , et a!. ( 1 995) An Improved procedure for plant regeneration from mdica and J aponica rice protoplasts. Plant Cell Rep. 14, 5 1 5-5 1 9. 1 6 Thompson, J. A., Abdullah, R., and Cocking, E C. ( 1 986) Protoplast culture of nce (Oryza satzva L.) usmg media solidified with agarose Plant Scz 47, 1 79-1 83 1 7. Hammatt, N., Blackhall, N W , and Davey, M R ( 1 99 1 ) Vanatwn m the DNA content of Glycine species. J Exp Bot. 42, 659-665 1 8 Vos, P , Hogers, R , Bleeker, M., Reijans, M., van de Lee, T , Homes, M , et al ( 1 995) AFLP: a new techmque for DNA fingerpnntmg Nuclezc Acids Res 23, 4407-441 4 1 9 Wllhams, 1 G K , Kubehk, A . R., Livark, K. J., Rafalski, J . A., and Fingey, S V. ( 1 990) DNA polymorph1sms amplified by arbitrary pnmers are useful as genetic markers Nuclezc Aczds Res 18, 653 1-6535 20. Vaccmo, P., Accerb1, M , and Carbelhm, M. ( 1 993) Cultivar Identification m Tntz­ cum aestzvum using highly polymorphic RFLP probes Theor Appl Genet 86, 833-836. 2 1 . Powell, W., Machray, G. C., and Provan, 1. ( 1 996) Polymorphism revealed by simple sequence repeats. Trends Plant Scz 1, 2 1 5-222.

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Isolation, Cultu re, and Plant Regeneration of Suspension- Derived Protoplasts of Lolium Marianne Foiling and An nette Olesen

1 . Introduction An efficient protoplast regeneratiOn system makes somatic hybridizatiOn and direct gene transfer attractive tools in the breeding of perennial rye grass, one of the most important forage grass species in temperate regions. Despite many efforts durmg the last decade, regeneration from protoplasts of Lolium perenne L. has generally been low and further complicated by the frequent occurrence of albino plantlets (1-3). Recently, however, the use of nurse culture (4, 5) and condittoned medium (5,6) has considerably tmproved platmg effictencies and reproducibility. Protoplasts of grasses are usually obtained by treating cell colonies from embryogenic suspension cultures with enzymes that digest cell-wall compo­ nents. Smce protoplasts cannot normally perform better than the donor suspen­ stan, the regeneration behavior of these donor suspenswns is very important for success with the protoplast culture system. For ryegrass suspensiOn cul­ tures, regeneration capactty usually decreases and albino frequency mcreases wtth culture age (7). Thts often leads to total loss of abthty to regenerate green plantlets within 25 wk after culture imttation (1,2). The genotype and subcul­ turing regime strongly affect regeneration behavtor of suspenston cultures (7), but responstve genotypes can be found wtthm most vanettes (8) . Based on correlation studies, we found regeneration ability in suspension and anther cul­ ture to be controlled to some extent by the same genes (7) . Old suspension cultures can adapt to i n vitro culture condtttons, and protoplasts isolated from such cultures may dtvtde m fairly stmple media. From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

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However, protoplasts tsolated from younger morphogenic suspension cul­ tures are usually fragile and require more complex media (6, 9) . Cocultivation of protoplasts with nurse cells or conditioned medium is often necessary for obtammg d1vts10n m such protoplasts, but wtll usually also tmprove plating effic1ency and the proportiOn of green plants from protoplasts that are able to dtvtde wtthout mcluswn of nurse or conditioned medium (5) . In our labora­ tory, the use of nurse culture, in combinatiOn with the use of protoplasts from highly regenerable and well estabhshed suspensiOns has proven very suc­ cessful (see Fig. 1 ) . By usmg of nurse cells, we obtamed average platmg effictenctes up to 1 2% from the best culture, 40-50% regeneration frequency, about 80% green plants, and a total yield of almost 60,000 green protoplast­ derived plants (wtthout clonmg) per mL suspensiOns cells. Wtthout nurse cells or condtttoned medmm, the same culture gave about 1 200 green plants/mL suspensiOn cells, and wtth condtttoned medmm, the yteld of plants was approx 26,000/mL (5) . The mam effect of nurse culture ts tmprovement of mtttal cell d1Vlsion and microcolony formation. After thts stage, nurse cells are removed, smce pro­ longed cocultivatton may result in competition for nutrients and accumulatiOn oftoxtc waste products excreted from the nurse cells. Attempts have been made to charactenze and tdenttfy the condtttoning factor(s) (10-13). However, sev­ eral compounds may be involved (9. 14. 15), and the mdtcatton that some are unstable (5) makes thetr 1dent1ficat10n difficult. Thts mstabt hty may explain why the nurse system generally ts more effictent than condtttonmg media (5). Interactions between protoplasts and sources of condtttonmg or nurse cells may complicate thetr use (9, 15) and should be taken into consideration. We found that nurse cells and condtttoned medium obtamed from the protoplast donor suspensiOn had the best effect on the plating effictency, mtcrocolony growth, and frequency of green plants (5). Here, we describe a protocol for the tsolation, culture and plant regeneratiOn of Lolium suspenstan-denved protoplasts mvolvmg nurse cells or conditioned medium It includes the following steps Preparation of suspenston cultures pn o r to protoplast culture (5 d). 2 Protoplast tsolatwn and purificatiOn 3. Protoplast culture wtth nurse or condtttoned medmm (2 wk). 4. Culture of microcolonies embedded m agarose drops in liquid protoplast colony platmg medmm (2 wk) 5 Macrocolony culture on solid protoplast colony platmg medmm. Thts step mcludes one subculture of mdiv1dual colonies (2-6 wk dependmg on colony growth rate + 4 wk). 6. Plant regeneratiOn (3-4 wk).

c

Fig. 1. A-D. Plant regeneration from cell suspension protoplasts of L. perenne L. (A) Freshly isolated protoplasts. {B) Colonies formed in agarose drops after 2 wk with nurse culture and l wk in liquid protoplast colony plating medium. (C) Colony con­ sisting of small dense cells with a regular shape. This type of colony will often regen­ erate into plantlets. (D) Colony with loose irregular growth. This type of colony will usually not be able to regenerate plants.

Fig. 1 . E-G. (E) Agarose drop (40 ¢-) containing protoplast-derived colonies 4 wk after protoplast isolation. (F) Regenerating calli 21 h mo after protoplast isolation. (G) Shoots of L. perenne 3 mo after protoplast isolation.

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1 87

2. Materials All media are made with distilled deionized water and based on Murashige and Skoog's medium (MS) (16; and see Appendix). This is commercially avail­ able from, e.g., Flow Laboratories Inc. Hormone stocks described below can be stored at 4 oc for up to 2 mo. 1 . 2,4-DichlorophenoxyacetJc acid (2,4-D), 0. 1 mg/mL stock. dissolve I 00 mg 2,4-D in a small volume of 96% ethanol, and adjust volume to l 000 mL with water 2. 6-benzylamino purine (BAP), 0. 1 mg/mL stock: dissolve l 00 mg BAP m a small volume of 0.5 N HCI, and adJust volume to l 000 mL with water 3 Kinetm, 0 l mg/mL stock. dtssolve 1 00 mg kmetm m a small volume of O 5 N HCI and adjust volume to l 000 mL wtth water

2. 1. Suspension Culture Medium SuspensiOn maintenance medium (SM) consists of MS salts and vitamins supplemented with 3% (w/v) sucrose and 3 mg/L 2,4-D. Adjust pH to 6.2 and autoclave. 2.2. Protoplast Isolation Solutions l . Enzyme solution I % (w/v) Cellulase Onozuka RS (Yakult Pharmaceuttcal Indus­ try Co Ltd ), 1% (w/v) Metcelase (MetJ i Setka Katsha Ltd.), 0 3% (w/v) Macerozyme R- l 0 (Yakult Pharmaceuttcal Industry Co. Ltd ), and 0 l% (w/v) Pectolyase Y-23 (Setshm Pharmaceutical Industry Ltd.) dtssolved m MS medmm supplemented wtth 1 0 mM CaCI2, 3% (w/v) sucrose, 8% (w/v) manmtol , and 2 mg/L 2,4-D Stir solutton at low rotatton for l-2 h, and pellet undissolved particles by centrifugation at l 25g for 20 mm at 4°C. Adjust pH to 6 0 and filter­ sterilize supernatant (0 22-!lffi membrane filter) Store m l 0-mL ahquots at -20°C for up to l yr 2 Protoplast washmg medium: PC medium I X (see Subheading 2.3., step 1).

2.3. Protoplast Culture Media I . Liqmd protoplast culture medium (PC l X)· MS salts and vitamms supplemented with l 0 mM CaCI2 , I I % (w/v) glucose, 0. 1 mg/L 2,4-D, and 0. 1 mg/L 6-benzyl­ ammo purine (BAP) Adjust pH to 6 0 and filter-stenhze (0.22-!IDl membrane filter). Medmm can be kept at room temperature for up to l -2 mo. 2. Sohd protoplast culture medium (agarose platmg medium) Prepare medmm descnbed above, but wtth a twofold concentration of all constituents (PC 2X) and filter-sterilize. Prepare an agarose solution wtth 2.4% (w/v) Sea-Plaque aga­ rose (FMC) m dtsttlled water and autoclave. Whtle sttll hot and fluid, mtx agar­ ose solution and PC 2X medmm m the ratio l : l . Cool the medmm to approx 32°C m water bath (see Note 1) 3 . Conditioned medmm: Harvest spent suspensiOn culture medmm from the proto­ plast-donor suspenston 5 d after last subculture (see Note 2). Remove cells

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by fi ltermg the medium through a 22 5-IJlll nyl on steve Adjust osmolanty to 800 mOsM/kg wtth a saturated glucose solutiOn, and adjust pH to 6.0 (see Note 3) Ftlter-stenhze the medmm (0 22-IJlll membrane filter), use immedtately, or store at 4°C until next day

2.4. Colony Plating and Plant Regeneration Media Protoplast colony platmg medmm (PCP I X) MS salts and vttamms supplemented wtth 6. 84% (w/v) glucose, 0. 1 mg/L 2,4-D, and 0. 1 mg/L BAP. Adjust pH to 6 0 and filter-stenhze (0 22-!illl membrane filter) For sohd platmg medta, prepare medium at a twofold concentration (PCP 2X) and filter-sterilize (0.22-!illl mem­ brane filter). Prepare a 0.6% (w/v) Gelnte (Kelco) solutiOn m dtstiiied water and autoclave While still at 90-1 00°C, mtx Gelrite solution wtth PCP 2X in the ratto 1 1 (final concentration, PCP I X wtth 0 3% [w/v] Gelnte), and pour mto 9-cm plastic Petn dtshes (approx 50 mL!dish). 2. Plant regeneratiOn medmm (PR). MS salts and vttamms supplemented wtth 3% (w/v) sucrose, 0.2 mg/L kmetm, and 0 8% (w/v) agar (Dtfco) Adjust pH to 6 2 and autoclave Pour medmm mto 9-cm Petri dtshes (approx 50 mL!dtsh)

3. Methods 3. 1. Maintenance of Suspension Cultures

SuspensiOns may be mitmted from embryos or menstems (1, 7) . Success wtth suspension culture of Lo/ium depends primanly on the donor genotype, an opti­ mal suspension mamtenance scheme, and personal expenence (see Note 4) Suspenston cultures are cultivated m 250-mL Erlenmeyer flasks, and main­ tamed by weekly subcu1tunng. Do thts by ttltmg the flask to allow the cells to sedtment, pour off 90% of the used medmm, and add fresh SM mediUm to a total volume of80- 1 00 mL Culttvate at 25°C in drm hght (7 llmol!m2/s) on a gyratory shaker at 1 50 rpm. Seal flasks wrth two layers of polyethylene (PE) film (see N ote 5) 2. SuspensiOns are renewed at 2 1 - or 28-d intervals by transferrmg usmg a wtde­ bore pipette (5-mm opening) 1-2 mL of small compact cell colomes (see Note 6) together wtth approx I 0 mL spent suspensiOn mediUm to new 250-mL flasks contaming 50-70 mL of fresh SM medmm (see Note 7).

3.2. Preparation of Suspension Cultures Prior to Protoplast Culture (see Note 8) Cultures for protoplast Jsolatwn: 5 d pnor to protoplast Isolation and 7 d after last renewal by ptpet subculture (see Note 9), transfer approx I mL of small compact cell colonies with a wtde-bore p1pet to a 250-mL Erlenmeyer flask. 2 Cultures for nurse culture· 5 d prior to protoplast isolatton and 7 d after last subcul­ ture transfer approx 1 .5-2 mL of small compact suspensiOn colomes from the protoplast donor suspensiOn culture (see Note 10) to a 2 50-mL Erlenmeyer flask

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3 . To each flask, add 80 mL of fresh SM medmm, and seal the flasks wtth two layers of PE film. Culttvate at 25°C m dtm light (7 IJlllOI/m2 /s) on a gyratory shaker at 1 50 rpm for 5 d.

3.3. Protoplast Isolation and Purification 1 . Transfer approx 1 mL of small compact suspension colomes with a wtde-bore ptpet to a 9-cm Petri dtsh, and remove excess SM media. Add 1 0 mL of enzyme solutiOn, and seal the dish with PE film. 2. Incubate dtshes for 5 h m the dark at 25°C on a gyratory shaker at 30 rpm (see Note 1 1 ). 3 Fi lter the protopl ast/enzyme solution through 9 1 - and 22 5-IJlll nylon steves (see Note 1 2) Tilt the steves with the ttp of a ptpet, allowmg protoplasts and medmm to pass through the sieves, and wash wtth PC I X medmm (up to 1 0 mL). Carefully transfer the protoplast solutton to a 3 0-mL container (Bibby Sterihn Ltd.) by use of a wide-bore pipet. Add additiOnal PC I X medtum to a final volume of 30 mL. Centrifuge at 5 0g for 5 mm at room temperature Dtscard supernatant 4. Resuspend protoplasts tmmedtately m 20 mL of PC IX medmm (see Note 1 3), and centnfugate agam. 5. Resuspend protoplast pellet m 1 0 mL of PC I X medium, and store protoplasts overnight at 4°C or proceed immediately with next step 6 Centrifuge protoplast suspenston, and resuspend the protoplast pellet m 1 0-20 mL of PC l X medium. Measure the protoplast dens1ty with a hemocytometer (Fuch­ Rosenthal), and calculate total protoplast yield.

3.4. Protoplast Culture I . Dispense aliquots of the protoplast suspension into centrifuge tubes, and centn­ fuge at 5 0g for 5 mm at room temperature. Discard the supernatant, and resus­ pend the protoplast pellet in agarose platmg medmm to a final density of 2 x I 05 protopl asts/mL (see Note 14) 2 Place, usmg a Pasteur pipet, one agarose drop (approx 24 drops/mL) m the middle of each well of a 24-we ll multidish (Nunclon ® 1 5 5 mm, NUNC), and allow agarose to solid ify m darkness for 1 h at room temperature. The proto­ plasts can e1ther be cultivated in conditioned medmm or cocultivated w1th nurse cells. 3. Use of conditioned medmm: Dispense 0.5-mL aliquots of undiluted condttioned medmm into each well. F11l the mner space between the wells w1th stenle water to reduce evaporation, seal with PE film, and mcubate d1shes in plastic bags at 25°C in darkness. 4. Use of nurse culture: D1spense 0.5-mL ahquots of hquid PC IX medmm to each well. Add approx 30 mg of small, dense suspens10n colomes (without suspens10n culture medium) to each well, fill the inner space between the wells wtth stenle water, seal, and mcubate the dishes as descnbed in step 3

1 90

Foiling and Olesen

3. 5. Microcolony Culture After 14 d of culture when the microcolomes consist of 6-8 cells (see Note 15), determine platmg efficiency (number of microcolonies/total number of cultured protoplasts) by countmg m1crocolonies using an mverted microscope. For proto­ plasts cultured w1th nurse cells, wash agarose drops twice with hquid PCP I X medmm (see Note 1 6) These washes ensure removal of all nurse cells. No wash­ mg is needed for protoplast agarose drops cultured w1th conditioned medmm 2 Transfer agarose drops to new multidishes, and add 0.5 mL hqUJd PCP I X medmm Culture for further 2 wk 3. 6. Culture of Micro- and Macrocolonies I . Transfer one or two agarose drops w1th protoplast-denved colomes to sohd PCP IX mediUm (see Note 17) Chop agarose-drops lightly w1th a blunt scalpel (see Note 18), and spread the agarose p1eces on the surface Seal the dishes w1th PE 2 film and incubate at 22°C m contmuous white fluorescent hght (70 j..llllOI/m /s) 2. After 2 wk of culture, transfer the most rapidly growmg colonies ( 1 S-2 mm) Individually to fresh PCP (sohd) dishes Incubate at same temperature and under the same hght conditions as descnbed above Leave the remammg small colomes on ongmal PCP d1shes to contmue their growth by spreadmg them over the entire surface. Repeat transfernng colomes from the ongmal dishes until all colomes have been moved mdiVIdually to fresh PCP medmm (4--6 wk) 3. 7. Plant Regeneration I . After 4 wk of culture of mdiv1dual colomes on PCP medium, transfer calluses to PR medium Seal dishes w1th PE film and mcubate at 20°C m contmuous white fluorescent light (70 !Jillol/m 2/s) for 4 wk 2. Count the number of calluses regeneratmg at least one plantlet, and transfer plantlets to vessels contammg PR medium Cultivate under h1gh hght intensities (70 j..lolll l/m2/s) With a 1 6/8 h day/mght hght regime 3 Transfer rooted plants to peat/soil, and continue growth m greenhouse

4. Notes 1 . Alternatively, stored sohd agarose solution may be reheated m a water bath or microwave oven and mixed I I With stored PC 2X To avoid damage to proto­ plasts, medium should never be warmer than 32°C when used for platmg 2. Do not harvest spent medmm from cultures more than 3 wk after latest pipet subculturing (see Subheading 3.1., step 2). Spent medmm harvested later than this or conditioned medium from other cultures than the protoplast-donor sus­ pensiOn may have a detnmental effect on protoplast diVISIOn The best condi­ tioned medium IS obtamed from the protoplast-donor suspensiOn taken 12 d (7 + S d) after pipet subculturing. 3 . The osmolanty ofSM medmm must be raised to a value Similar to PC medium to avoid burstmg of the protoplasts The pH of spent SM medium iS usually reduced

Protoplasts of Lol i u m

4

5

6.

7

8.

9. I0

1 I.

191

to below 5.6 as a consequence of suspension growth, although 1t may occasion­ ally be higher than 6 0. Suspenstons should be tested for regeneration capacity every 4th week. Do this by pipeting l-2 mL of small dense suspension colomes to solid PCP 1 X Culti­ vate for 4 wk with light and temperature conditions as described in Subheading 3.6., step 1, and proceed with steps described in Su bheading 3.7. Type of sealing is very important for suspension growth. Foil sealing will mcrease dramatically the rate of suspension growth because of better aeratton. However, this will often reduce regeneratiOn longevity of the suspensions We use house­ hold cling film (PE, l 0 �). Tilt the flask and allow the cells to sediment. Then slowly rotate the flask in one hand so that the small dense cell colomes concentrate in the top layer of the sed1mented cells for a few seconds. Transfer small compact cell colonies to new flasks The handling of suspenston cultures is a critical step for protoplast culture. Preferably only one person should handle the cultures and thereby obtain expe­ rience for a correct subculturing scheme for each culture. For most cultures, pipet subculturing every 4th week is adequate, whereas others benefit from being subcultured by pipet every 2-3 wk. This 1s the case 1f suspensions turn darkish yellow before the 4th week after pipet subcultunng. If the culture eas­ ily turns brown (espec1ally older cell colonies), pipet small light-colored cell colonies to new flasks (avoid all brown cell colontes), and add 30-50 mL of fresh suspensiOn medmm. Repeat this With periodic mtervals (each 2-3 wk) unttl brownmg has stopped For fast-growmg suspenstons (usually relevant for nonembryogenic suspensions only), it may be necessary to discard some cells every week. Do this by spmnmg the flask, and immediately pour off some medium with cells before they sed1ment For slow-growmg suspensions, be careful not to dilute to much. The dilution of cell colomes 5 d pnor to protoplast ISolation w1ll usually improve yield and VIability of protoplasts as well as quahty of nurse cells. Use of suspension cultures more than 14 d after latest pipet subcultunng may reduce protoplast yield and viability The best nursing effect ts obtamed with cell colonies from the protoplast donor suspension. However, embryogentc suspension cultures often grow slowly, and for large experiments, It may be necessary to take nurse cells from a fast-growmg nonembryogentc suspenswn so that adequate amounts of nurse cells can be obtamed. In this case, screen several Lolium suspensiOns for their ability to sup­ port the protoplast culture. After 5 h of enzyme treatment, some undigested cell colomes w1ll remam, the amount dependmg on the suspensiOn culture. However, prolonged digestion wtll reduce protoplast viability. For time-consuming transformation or fusion expen­ ments, it may be advantageous to 1solate protoplasts overnight in half-strength enzyme. Dishes wtth cells for overmght dtgestwn are placed at very low rotatton (almost no movement of cells).

Follmg and Olesen

1 92

12

13

14

15

1 6. 17.

18

This Is easiest done by placmg the 9 ! -IJlll sieve mside the 2 2 5-J.Ull sieve in a Petn dish. We use homemade sieves that touch the bottom of the d1sh, smce this Im­ proves flow through the meshes and ts more gentle to the protoplasts These are constructed by securing preces of nylon filters of appropnate meshes to crrcular glass rings with diameters of 4 em (9 1 -IJlll Steves) and 5 5 em (22.5-J.Ull s1eves) Keep m mind that protoplasts are very fragile, and take great care m steps, such as pipeting and the resuspending of protoplast pellets Protop1asts may form aggregates that are difficult to drspense rf resuspending rs delayed Some suspensiOn cultures are known to grve nse to protoplast cultures with hrgh plating efficrency (more than 1 0%) when cocultivated with a nurse cu lture Reducmg platmg density to I x I 05 protoplasts/mL for such cultures may mcrease platmg efficiency further (5) For very slow-developmg protoplast cultures, 2 wk with a nurse culture may be too short a penod for reachmg the srx- to eight-cell stage, and cocultunng for a further I to 2 wk rs then beneficial This IS most easily done by usmg a small blunt scalpel to hft agarose drops from the wells and transfer them to a 9-cm Petn dish with liqUid PCP 1 X medmm Culture on PCP medmm matures the colomes and Improves their ab1hty to regen­ erate into plantlets. The number of agarose drops per plate depends on the platmg efficiency When platmg efficiency Is low (below 1 %), proliferatiOn of micro­ colomes may be stimu lated by mcreasmg the number of agarose drops per plate, smce mrcrocolomes at this stage still depend on the nursmg effect of other microcolomes Protoplast-denved microcolonies have a very compact structure, and choppmg the agarose drops with a blunt scalpel does not damage or break up tndlVldual colomes

Acknowledgments The authors wish to thank DLF-TRIFOLIUM Ltd. and the Damsh Academy of Techmcal Sctence (ATV) for financml supports References I . Dalton, S. J ( 1 988) Plant regeneration from cell suspensiOn protoplasts of Festuca arundmacea Schreb. (tall fescue) and Latium perenne L. (perenmal ryegrass). J Plant Physwl. 132, 1 70---1 75 2. Creemers-Molenaar, J., Van der Valk, P., Loeffen, J P. M , and Zaal, M A. C M. ( 1 989) Plant regeneratiOn from suspensiOn cultures and protoplasts of Loll urn perenne L. Plant Set 63, 1 67-1 76. 3 Zaghmout, 0 M -F and Torello, W. A ( 1992) Plant regeneratiOn from callus and protoplasts of perennial ryegrass (La !tum perenne L. ). J Plant Physwl 140, I 0 1-1 05 4 Wang, Z Y., Nagel, J., Potrykus, I and Spangenberg, G ( 1 993) Plants from suspension-derived protoplasts in Loltum species. Plant Set 94, 1 79-1 93 ,

Protoplasts of Lol i u m

193

5. Follmg, M , Madsen, S , and Olesen, A ( 1 995) Effect of nurse culture and condi­ tioned medium on colony formation and plant regeneratiOn from ryegrass proto­ plasts Plant Scz 108, 229-239. 6. Creemers-Molenaar, J , van Eeuwijk, F. A , and Krens, F A ( 1 992) Culture opti­ mization for perennial ryegrass protoplasts. J. Plant Physzol. 139, 303-308. 7 Olesen, A., Storgaard, M., Madsen, S., and Andersen, S B. ( 1 995) Somatic m vztro culture response of Lolzum perenne L . : genetic effects and correlations with anther culture response Euphytzca 86, 1 99-209 8 Olesen, A., Storgaard, M , and Madsen, S ( 1996) Suspension culture performance m commercial vanettes of perenmal ryegrass Euphytzca 88, 1 5 1 -1 57 9 Funatsuki, H , Lorz, H , and Lazzen, P A ( 1 992) Use of feeder cells to Improve barley protoplast culture and regeneration. Plant Scz 85, 1 79- 187 I 0. Stuart, R and Street, H E ( 1 97 1 ) Studies on the growth m culture of plant cells J Exp Bot 22, 96-1 06 1 1 . Somers, D. A., Birnberg, P R., Petersen, W. L., and Brenner, M. L. ( 1 987) The effect of conditioned medmm on colony formation from "Black Mexican Sweet" corn protoplasts. Plant Scz 53, 249-256 12 Btrnberg, P R Somers, D A , and Brenner, M L ( 1 988) Charactenzation of condittonmg factors that increase colony formatton from "Black Mexican Sweet Corn" protoplasts. J Plant Physzol 132, 3 1 6-32 1 1 3 . Schroder, R., Gartner F , Stembrenner, B., Knoop, B , and Beiderbeck, R ( 1 989) VIability factors in plant suspensiOn cultures-some properttes. J Plant Physwl 135, 422-427 1 4 Bellmcampi, D. and Morpurgo, G ( 1 989) Evidence for the presence of a second condtttonmg factor m plant cell cultures. Plant Scz 65, 125- 130 15. J0rgensen, R B , Andersen, B , and Andersen, J M ( 1 992) Effects and character­ Ization of the conditiOmng medmm that increase colony formation from barley (Hordeum vulgare L ) protoplasts J Plant Physwl 140, 328-3 3 3 1 6. Murashige, T and Skoog, F ( 1 962) A revised medmm for rapid growth and bw­ assays with tobacco tissue cultures Physzol Plant 15, 473-497 ,

19

__ __ __ __ __ __ __ __ __ __ __

Protoplast Fusion for Symmetric Somatic Hybrid Production in B rassicaceae Jan Fahleson and Kristina Glimelius

1 . Introduction This chapter will focus on somatic hybridization m Brassicaceae. The results from a vast number of investigations have clearly shown that somatic hybrid­ Ization m this family can overcome sexual barriers, and that the nuclear, mito­ chondrial, and chloroplast genomes from different sexual ly mcompatible species can be combined. Reproducible protocols for protoplast culture and regeneration to plants have been established for a large number of crop species within this family, such as oilseed rape, turnip rape, cabbage, and Indian mus­ tard (1). This has laid the foundation for the production of somatic hybnds (2, 3) . A substantial number of somatic hybnds have been produced between species within the genus Brasszca, combinmg both diploid and allopolyploid Brassica species mto hybrid plants. For example, the resynthesis of rapeseed (4-7) , but also a combinatiOn of all three Brasszca genomes (A, B, and C) have been performed (8, 9) . To widen the gene pool of rapeseed, several mtergenenc somatic hybnds combming Brassica napus with species from the genera Eruca (10), Sznapzs (1 1, 12), Raphanus (13), Moncandza (14, 15), and Dip/otaxzs (16, 1 7) have been made. Furthermore, It has even been possible to combme species from different tnbes, by obtaming somatic hybnds, e.g., between rape­ seed and A rabzdopsis thaltana (18-21), rapeseed and Thlaspi perfoliatum (22), as well as rapeseed and Lesquerellafendleri (23). In general, It can be stated that the process of somatic hybndization has widened the gene pool of crops within the Brassicaceae family. In addition to this, successful results regarding somatic hybridizatiOn wtth the purpose of improvmg Brasszca oleracea (24, 25) and Brassica JUncea have been made (26-30) . It must be recognized, however, that even If sexual barners are From Methods m Molecular BIOlogy, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

195

1 96

Fahfeson and Gfimefius

circumvented by th1s method, interspecific incompatibility barners can be present at other stages of plant development, mhtbtting the production of fer­ tile hybrid plants (31). Furthermore, except m a few cases (32,33) the chloro­ plast genomes seem unable to recombme, whereas in the m1tochondnal genomes, a considerable degree of recombmation has been detected (34-36). The method for protoplast fuswn descnbed m th1s chapter 1s the mass-fuswn method. H1gh numbers of protoplasts from the parental species are m1xed to­ gether and mduced to fuse vm a polyethylene glycol (PEG)/CaCl2 treatment. The mass-fusion method ts m ttself random, since homofus10ns (fus10ns of protoplasts wtthm the same species) as well as heterofuswns (fus10ns of proto­ plasts between the two parental spec1es) are obtamed. In addition, the number ofprotoplasts participating m a fuswn event cannot be controlled by the mass­ fusiOn method. The mass-fuswn method has to be combined with an efficient system for selectwn of hybnd cells. Different approaches have been adopted, such as the use of metabolic mutants (1 1) or selectiOn with iodacetamide (lOA) m combi­ nation w1th madmtwn/medm selectiOn (3 7,38). The selectwn system used m our laboratory IS flow cytometry and cell sorting. Th1s is a general system and relies on differential fluorescence from the parental protoplasts, obtamed through staining of the protoplasts and/or chlorophyll autofluorescence from one of the parental protoplasts (39) . The hybnd cells will produce a double­ fluorescent signal and can thus be selected. Irrespective of the selection method used, a procedure for identification of hybrid md1viduals has to be developed. The 1denttficatton procedure can be based on b1ochem1cal methods (40), morphological features (14) or genetic markers (2 1, 22, 31). Genet1c markers based on DNA sequences, such as restnct10n-length polymorph1sms (RFLPs) or spec1es-spec1 fic repetitive DNA sequences, are generally robust markers and are not mfluenced by changes m cell metabolism or environment. Thts chapter covers the first steps m the producttOn of somatic hybrids w1thm Brass1caceae, and descnbes Isolatton of protoplasts, protoplast fuswn, prepa­ ratiOn for flow cytometry and cell sortmg, cultivatiOn and regeneration. As a procedure to identify hybnds, we have also mcluded a section covenng Isola­ tion of spectes-speclfic DNA sequences. This last section mcludes some stan­ dard procedures that are not descnbed m detail. 2. Materials 2. 1. Plant Material and Culture Conditions I Etwlated hypocotyls, grown for 4-5 d m vitro, m darkness at 25°C 2 Green leaves from m vitro grown plants, approximately 3-wk-old, grown at a light mtensity of 1 2 W/m2 and a day/mght regime of 1 6/8 h at 25°C (See Note 1 )

Hybrid Production in Brassicaceae

197

2.2. Equipment All materials must be sterilized prior to use. 2. 3 4. 5 6 7. 8 9. 10 II. I2 13 14 15 1 6. 17. 18 1 9. 20. 21 22 23

Protoplast filter ( 1 00-IJill nylon mesh). Forceps. Scalpel Spatula. Pipets, 1 0-, 5-, and 1 -mL. Pasteur ptpets. Petn dtshes, 3 5-, 5-, and 9-cm. Glass Jars (baby food Jar-type) Flasks, 1 00-, 250-, and 500-mL. Cylinders, 50-, 1 00-, and 500-mL Graded 1 5-mL glass centrifuge tubes. Mu1ttboxes, 48-wells (Costar®) 1 5-mL Falcon tubes wtth rounded bottom. Hemocytometer (Burker chamber) Flow hood. Mtcroscope, mverted, eqwpped wtth a UV lamp. Fluorescent activated cell sorter (e.g., a Becton Dtckmson instrument or equtvalent) X-ray film. Cassettes for X-ray fi lm Shaker. Membranes for colony hybndizatton. Toothpicks 3 Ohgolabelmg ktt for 2P labelmg of DNA.

2.3. Media and Chemicals l 2 3 4.

5. 6 7. 8 9. 1 0. II 1 2. 1 3.

Calcium hypochlonte (7.5% w/v). Tween 20. Ethanol, 70 and 96%. Cell -wall-degrading enzymes ( cellu\ysm and macerase, Ca\bwchem) I % cellu1ysm, 0 I % macerase (w/v) dtssolved m the culture medmm K 3 (see Table 1 and Note 2). Manmtol, 0.4 M, dtssolved tn the culture medium 8p (see Table 1 ). Agarose, 0 1 3 % and 0.4% m K3 medtum SeaP!aque agarose 1 .47% (FMC ®) in detontzed water, autoclaved. CPW 1 6 (41 , see Table 2). Stenhzed by filtration. TVL (see Table 2), autoclaved W5 (42; see Table 2), autoclaved KB (see Table 1), autoclaved. 5(6)-Carboxyfluorescein dtacetate, 0.22 M, dtssolved m W5 (CFDA, Molecular Probes; see Note 3). PEG solution (see Table 2 and Note 4).

Fahleson and Glimelius

1 98

Table 1 Media Compositions for Cultivation of Protoplasts Medm

Components

MS

A 8

pH 5 . 7-5 . 8

Amount 20 14

c

10

D E MS II Fe-EDTA Glycme

10 10 10 5

I

Nicotinic acid

K3 pH

5 . 8--6. 0

Vitamm 86 Vitamm 8 1 Edamm (casem hydrolysate) Inositol Sucrose Gellan gum A 8 c

8p pH 5.6

E K K3 F Fe-EDTA Zn-EDTA V1tamm 8 1 V1tamm 86 N1cotimc acid Xylose Inositol A 8 c

D E Kao's micro Fe-EDTA

I I 1 00 10 3 26 2 7 7 10

5 10 5

I 10 2 2 250 1 00 20 3 . 75

8. 1 10 1 3.6 10 5

mLIL mL/L mLIL mL/L mLIL mL/L mLIL mL/L mL!L mLIL mL/L g/L mg/L g/L g/L mLIL mL!L mL/L mL/L mL/L mL/L mL/L mL/L mL/L mg/L mL/L mL/L mg/L mg/L mL/L mL/L mLIL mLIL mL/L mL/L mL/L

(contznued)

Hybrid Production in Brassicaceae

1 99

Table 1 (continued) Medta

Components

Ntcotinic actd Vitamin B6 Vitamm B 1 Ascorbic actd Na pyruvate Cltnc acid Mahc actd Fumanc acid Fructose Rtbose Xylose Mannose Rhamnose Cellobiose Sorbitol Manmtol Inosttol Casammo actds Coconut water Sucrose Glucose

Amount 2 2 10 2 20 40 40 40 250 250 250 250 250

250 250 250 1 00 250 20 250 72. 1

mLIL mL/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mLIL mg/L g/L

1 4 CaC12 PEG 2 · 1 (see Table 2): The CaC12 solutiOn t s autoclaved. 1 5 CaCI2:PEG 5 1 (see Table 2) 1 6 . Stock soluttons ofmedta chemtcals (see Table 3)· Kept at +4°C to prevent fungal and/or bactenal growth 1 7 MS medmm, autoclaved (43: see Table 1 ) 1 8 K3 medium, filter-stenhzed (42; wtth modtficatwns accordmg to 44, 45, see Table 1) 1 9 8p medium, filter-stenlized ( 46; with modtfications accordmg to 47, 48; see Table I ) 20 Restnctwn enzyme Sa u3A 2 1 . Restriction enzyme buffer I OX (provided wtth the restnctwn enzyme). 22. lsopropylthio-�-o-galactoside (IPTG)· 2 g of lPTG to 8 mL dtstilled H20. Adj ust the volume to I 0 mL, and stenlize by filtratiOn through a 0 22- IJ.lll dtsposable filter. Store m 1 -mL ahquots at -20°C. 23. 5-Bromo-4-chloro-3-mdolyl-�-o-galactostde (X-gal). Dtssolve X-gal m dtmethyl­ formamJde (DMF), 20 mg of X-gal/mL DMF Use polypropylene tubes, put m a box to prevent from hght. Store at -20°C (See Note 5.) 24 Phenol chloroform mtxed 1: I (Ins-saturated phenol).

200

Fahfeson and Gfimefius Table 2 Solutions for Protoplast Isolation and Fusion Solution

Components

CPW 1 6 pH 5 . 5-5 8

KH2P04 KN03 CaCI2 2H20 MgS04 7H20 KI CuS04 5H20 Sucrose •

25 26. 27

28

29.

30 31 32

Amount 27 2 101 1480 246 0 16 0 025 1 60

mg/L mg/L mg/L mg/L mg/L mg/L g/L

TVL pH 5 6-5 .8

Sorbitol CaCI2 2H20

54.654 7 35

g/L g/L

ws

CaCI2 2H20 NaCI Glucose KCI

18 4 9 I 08

g/L g/L g/L g/L

KB pH 7. 2

KCl CaCI2 2H20 Tns

1 1 18 I 47 13.1

g/L g/L g/L

PEG pH 7 0

PEG 1 5 00 Glucose CaCI2 2H20

400 54 7.35

giL giL g/L

CaCI2 pH 7 0

CaCI2 2H20 Sorbitol

14 7 1 8 .2

g/L g/L

·

•

0.5 M, disodmm etylendiammtetraacetate (EDTA) Dtssolve 1 86. 1 g/L with mtenstve stirnng, heating and adJustmg pH to 8.0 IS also reqmred. NaAc, 3 M (sodmm acetate 3H20): Add 408 . 1 g to 800 mL H20. AdJ USt pH to 5 2 wtth glactal acettc actd, and dtlute to I L Autoclave TE buffer, I O rnM Tris-HCI (pH 7.5), I rnM EDTA (pH 8 0): To 988 mL of H20, add 1 0 mL I M Tns-HCI + 2 mL of O 5 M EDTA Adenosme tnphosphate (ATP), 0 I M: Dissolve 60 mg m 0 . 8 mL H20 AdJust pH to 7.0 wtth NaOH, O . I N Adj ust volume to I mL, and store m altquots at -20°C Dithiothreitol (DTT), 0. 1 M: Dissolve 3.09 g in 20 mL ofO.Ol M sodmm acetate (pH 5 2). Steriltze by filtration Store m 1 -mL ahquots at -20°C PEG 50%: PEG 8000 dtssolved m deiOnized water T4 DNA ltgase Ltgatwn buffer ( l OX, provided with T4 DNA hgase) ·

20 1

Hybrid Production in Brassicaceae Table 3 Stock Solutions of Media Chemicals Amount

Component A B C D. E F

KN03 NH4N03 MgS04 7H20 KH2P04 CaCI2 2H20 MnS04 H20 Na2Mo04 · 2H20 CuS04 5 H 20 CoC1 2 6H20 H3B03 G KI H (NH4}zS04 K. NaH2P04 H20 K3 (NH4}zS04 L MgCI2 6H20 ZnS04 7H20 Zn-EDTA ·

·

·

·

·

Fe-EDTA Fe S04 7H20 Na2EDTA ·

Glycine N1Cotm1c ac1d Thiamine (B1) Pyndoxine (B6) 2,4 Dichlorophenoxy acetic ac1d (2,4-D) 1 -N aphtylacetic acid (NAA) 6-Benzy!amino punne (BAP) Zeatm

g/L 95 1 20 g/L 3 g/L 17 g/L 44 giL g/L I 69 0.025 g/L 0.0025 g/L 0.0025 g/L 0.620 g/L 10 g/L 1 50 g/L 15 g/L g/L 26 8 35 g/L 0 86 mg/mL 15 mg/mL

Component Kao's microelements KI MnS04 4H20 H3B03 ZnS04 · 7H20 Na2 Mo04 2H20 CuS04 5H20 CoC12 6H20

Amount (22) 75 1 000 300 200 25 25 25

mg/L mg/L mg/L mg/L mg/L mg/L mg/L

MS II (m1croelements) KI 83 2230 MnS04 4H20 620 H3B03 1 234 ZnS04 7H20 Na2Mo04 2H20 25 25 CuS04 5H20 CoC12 6H20 25

mg/L mg/L mg/L mg/L mg/L mg/L mg/L

·

5 57 mg/mL 7.45 mg/mL 2 05 0. 1 0.5

mg/mL mg/mL mg/mL mg/mL

0. 1

mg/mL

0. 1

mg/mL

0. 1 0. 1

mg/mL mg/mL

3 3 . Competent Escherichw coli XL 1 , prepared according to standard methods 34. Luria-Bertam medmm (LB medmm) To 950 mL of deiomzed H20 add 10 g Bacto-tryptone, 5 g Bacto-yeast extract, and 20 g NaCI Dissolve under stirring, adjust pH to 7.0 with 5 N NaOH, and dilute to l L Autoclave. 3 5 . Amp i cilli n stock solution, 1 00 mg/mL Dilute to a final concentratiOn of 50-- 1 00 ).lg/mL when used tn media.

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3. Methods 3. 1. Sterilization and Cultivation of Plant Material Stenlize approx 5 00 seeds of one parent for I h w1th continuous shakmg m 50-I 00 mL of a 7 5% calcmm hypochlonte solution to whtch a few drops of Tween 20 have been added (See Note 6 ) 2. Rmse seeds for I mm m 70% ethanol, and thereafter wash three times for 2 mm each m stenle water 3 Germmate seeds m 9-cm Petri dtshes containmg 20 mL MS medmm supple­ mented with 3% (w/v) sucrose In case of etwlated hypocotyls, seeds are placed in a row at one end of a 9-cm Petri dtsh (about 20 seeds/dish) and then placed slightly tilted m the dark for 4--5 d at 25°C 4 Green leaves of the other parent are obtained from 3 -wk-old plants, grown in glass 2 jars at a hght mtenstty of 1 2 W/m · and a day/mght regtme of 1 6/8 h at 25 °C

3.2. Pretreatment of Plant Material I . W1th a forceps and scalpel, carefully cut the etiOlated hypocotyls into 0.5- to 1 -mm

segments The leaves are gently cut mto small pteces For a typical experiment, 25 Petn dishes (about 500 seeds) and leaves from 2-3 m vitro plants are needed 2 Place each matenal m a separate TVL solutwn to plasmolyze for I h 3 . Withdraw the solutiOn with a p1pet and replace tt With enzyme solutiOn ( I % cellulysm, 0 I % macerase m K3 medmm) 4 Place the dtshes m darkness, w1th the leaf dtshes statwnary and the hypocotyl dishes on a shaker, about 1 5 rpm, overmght

3.3. Isolation of Protoplasts

2 3 4 5. 6 7

8 9 I 0. 11

Check the protoplast suspenston under an mverted mtcroscope to confirm the absence of contammatton by bactena or fungi Ftlter the protoplast suspenswns through a 1 00-f.illl nylon mesh Add approximately one-third the volume of CPW 1 6 Transfer the suspenswns to centnfuge tubes and centnfuge for 5 mm at I OOg Remove the floatmg protoplasts wtth a Pasteur ptpet and add approxtmately five ttmes the volume ofWS (See Note 7 ) Centnfuge as before Remove the supernatant, and resuspend the hypocotyl protoplast pellets m a small volume of W5 (about I 5 mL!centrifuge tube) The leaf protoplasts are resus­ pended m a small volume of TVL.KB 1 · 1 . Stam the hypocotyl protoplasts wtth 250-500 !J.L of 0 22 M 5(6)-carboxy-fluo­ rescein dtacetate (CFDA) for 1 0 mm. Add W5 (8.5 mL!centnfuge tube), and centnfuge the hypocotyl protoplasts as before Resuspend the hypocotyl protoplasts m a small volume ofTVL KB 1 · 1 , and cal­ culate the protoplast density m the haemocytometer Dilute the hypocotyl protoplasts to 9 x 1 05 protoplasts/mL and the leaf proto­ plasts to 9 x I 0 5 protop1asts/mL

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3.4. Fusion of Protoplasts 1 . Mix the protoplasts 1 . 1 wtth a Pasteur ptpet and place approx 20 separate drops (about 0 4 mL/droplet) m a 9-cm Petn dtsh 2. Turn the flowhood off, and let the protoplasts settle for 5 min 3 Add 2 droplets ofthe PEG solutiOn to each droplet ofprotoplasts Leave for 3-5 mm. (See Note 8.) 4 Tilt the Petn dtsh, and remove the PEG so Iutton wtth a ptpet. Wtth another ptpet, add 2-3 mL of the CaCI2· PEG 2 : 1 solution for 5 min to wash the protoplasts (which now should be slightly attached to the bottom of the Petn dtsh). 5 Remove the CaCJ2·PEG 2 : 1 solution, and replace tt wtth CaCI2 ·PEG 5 I solutiOn for 5 m m (See Note 9 ) 6. Wash the protoplasts wtth 4 mL 8p medmm (without hormones) After thi s point, selectiOn of hybnd cells may be performed in several ways The followmg sec­ tion describes how cells are prepared for flow cytometry and cell sortmg How­ ever, alternative selection methods are indtcated elsewhere (see Note 10)

3.5. Preparation of Protoplasts for Flow Cytometry and Cell Sorting I . Store protoplasts for at least 2 h at +8°C. Remove 8p medium, and cover the protoplasts with 7-8 mL of 8p medmm containing 0.4 M mannitol and mtxed wtth W5 ( 1 : 1 v/v) Leave at +8°C for at least 2 h. (See Note 1 1 .) 2. Usmg a Pasteur ptpet, carefully detach the protoplasts from the bottom of the Petn dtsh, and resuspend them in W5 Add about the same amount of W5 as m step 5, Subheading 3.3. (See Note 12 ) 3. Centnfuge m I 5-mL Falcon tubes at 75g for 5 mm Dtscard supernatant 4. Resuspend the protoplasts in a small volume of 8p medmm wtth 0.4 M manmtol to gtve a protoplast concentratiOn of about I x I 0 6 protoplasts/mL 5 Sort the protoplasts m the flow cytometer (this machme is usually operated by a spectalist and is adjusted to sort out protoplasts exhibiting a clear stgnal of double fluorescence). (See Note 13 ) The protoplasts are sorted mto separate wells of a multivtal dtsh, Costar®, 48 wells, each well contammg 1 00 � of 8p medmm supplemented wtth 4 5 � 2,4-dtchlorophenoxyacettc actd (2,4-D), 0 5 � NAA, and 2 2 � BAP

3.6. Culture of Hybrid Cells and Regeneration of Plants 2 I . The hybrid cells are cultured m a culture room wtth a ltght mtensity of I 2 W1m and a day/mght regtme of 1 6/8 h at 25°C . The medmm used ts the same as men­ tioned m Subheading 3.5. 2 When the first divtstons are observed (usually after 4-7 d), the cells are dtluted with fresh culture medmm, contammg 0 55% Sea Plaque agarose , w/v (FMC) with­ out hormones, to four ttmes the ongmal volume (See Note 14.) 3 When the agarose has soltdtfied, use a spatula, and move the beads to 2 5-cm Petn dishes With 3 mL 8p medmm, contammg the same hormone concentratiOn

as m the beads ( 1 /4 of the ongmal hormone concentration m the multtvtal dtsh)

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4. At weekly mtervals, replace the medtum wtth fresh medmm. 5 When small callt ( 1-2 mm m diameter) are observed, usually after 2-4 wk, they are removed from the beads and plated on 0. 1 3 % agarose (w/v) in K3 medmm with 3 4% sucrose (w/v) and the same hormone concentratiOns as m the beads 6 After about 2 wk the calli are mduced to dtfferentiate mto plants by transfernng them to K3 medium supplemented with 0 5% sucrose, 0 4% agarose (w/v), 0 6 J1M mdole-3-acettc actd, 2.2 J1M BAP, and 2 3 J1M zeatm 7 Transfer calli to fresh dtfferentJatJon medmm every 3 wk 8 When shoots emerge from a callus and have reached a stze of about 1 em m height, transfer the shoot to a baby food Jar wtth MS medtum, I % sucrose (w/v) without hormones

3. 7. Isolation of Species-Specific Repetitive Sequences

2

3. 4 5 6

7

8

9 1 0.

Isolate DNA from parental spectes accordmg to standard methods There are a vanety of DNA IsolatiOn methods One commonly used m our laboratory IS described m ref. (35) To a clean and autoclaved mtcrofuge tube, add the fo llowing· 0 2 � DNA 0.5 )lL Sau3A I )lL restnctwn enzyme buffer ( I OX) Distilled water to a final volume of I 0 ).JL. For each species, set up five reactiOns, and dtgest DNA for 2, 4, 6, 8, and 1 0 mm at room temperature Stop dtgestwn by adding 2 J..LL of 0 5 M EDTA Check the different digests on a 0.6% agarose gel Determme the reactiOn that produces fragments m the range of 500-- 1 000 bp. Set up five reactiOns, and dtgest for the amount of ttme determmed m step 4. Pool the 5 samples, extract once wtth phenol chloroform I · I , and prectpttate wtth 3X the volume of cold absolute ethanol together with 1 1 1 0 of the volume of 3 M NaAc. Put m -70°C freezer for 1-2 h Spm for 1 5 mm at maximum speed m a microcentnfuge. Discard supernatant and wash pellet m 70% ethanol Spm for 1 0 mm, dry, and dtssolve pellet m 25 )lL of TE buffer Set up hgatton reactiOn. Add the followmg components to a c lean mtcrofuge tube 25 )lL dtgested DNA 6 )lL hgatwn buffer ( l OX, supphed with the DNA hgase) I J..LL dephosphorylated pUC 1 8 (Pharmacia) 6 )lL 0 I M ATP 6 J..LL 0 I M DTT 6 J..LL 50% PEG. 1 )lL T4 DNA ligase 9 )lL distilled water Ligate overnight at l 6°C Heat-mactivate the reaction at 65°C for I 0 mm

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I I . Take 20 !JL of the hgatwn mtx, and transform competent E coil XL I Thaw competent cells slowly on ice. Add 80 !JL of TE buffer and 200 !JL of competent cells 1 2. Place on ice for 30 mm. 1 3 . Acttvate membranes for 2 min at 42°C. Place on ice for 15 mm. 1 4 Add I mL L B wtthout ampicillin. Culture i n 1 5-mL Falcon tubes with rounded bottom on a shaker (about 200 rpm) at 37°C for l h. 1 5 . Prepare LB plates plus ampiCillin (50--1 00 J..tg/mL). Spread X-gal and IPTG on the plate (40 !JL of each). 1 6 Spread bactenal suspensiOn over the plate, and culture overmght at 37°C 1 7 Ptck whtte colomes over t o gndded membranes (810-Rad) The membranes are laid on an LB plate Use a toothptck, and produce two identical membranes and a master plate This means that each colony will be represented three ttmes at the same posttwn on each plate Pick 500 colonies from each transformatiOn (m total l 000 colonies since DNA from two species are used). Usually I 00 colonies/mem­ brane or plate ts a smtable denstty. 1 8 Culture the colomes to a stze of about 1 mm m diameter. Wash the filters accord­ ing to manufacturer' s mstructwns 2 1 9 Label 25-50 ng of undigested parental DNA with 3 P usmg oligolabehng ktt (Pharmacia) Set up two reactions, one for each DNA of the parental spectes. 20. Hybridtze and wash filters accordmg to manufacturer's mstructwns Expose on X-ray film (7-8 h or overmght), tdentlfy those colomes that gtve a strong stgnal to one of the labeled DNAs, but not to the other. The selected colomes are restreaked on a fresh LB plate and then grown in a larger volume of hqmd LB medium ( 1 00--500 mL) for plasmtd tsolatton (See Note 15 )

4 . Notes I . The best parents to start wtth are some of the cultivated Brass1ca species, e.g., Brasszca campestrzs, B napus, and B oleracea Usually there is a vanatwn m the yield of hypocotyl protoplasts among different spectes and even vanetles, whereas It is easier to obtain enough material from green leaves. In our courses for undergraduate students, we have used B. napus cv "Hanna" as a source for hypocotyl protoplasts 2. Dtssolve the Enzyme powder under intensive stirring and store at +4°C over­ night. Ftlter-stenlize. 3. Boll the suspensiOn heavtly, let cool, and stenlize by filtration. 4. Melt PEG by gentle heatmg. Dissolve glucose and CaCI2 m distilled water m 50% of final volume. Mtx wtth melted PEG. Cool, adJust pH, and dtlute to final volume. Stenlize by filtration. 5. DMF is a highly toxic compound 6. The percentage of contaminated seed should be very low. Normally, 3-4 Petn dishes (out of 25) have to be dtscarded owing to fungal growth. If fungal growth is dtscovered in a Petn dtsh, the whole dtsh has to be dtscarded. 7. In CPW 1 6, only the clean v1able protoplasts w1ll float. The debns w1ll form a pellet to be discarded. The addition of W5 will pellet the protoplasts

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8 To Illustrate what IS happenmg when PEG is added, place a few drops of mixed protoplasts in a Petri dtsh under the inverted microscope, and add the PEG solu­ tton It can be seen that the protoplasts shrink heavily, owmg to the mcrease m the osmotic pressure within the cells 9 The dtlutton of the PEG solutton m two steps will help to recover the round shape of the protoplasts m a gentle way 1 0 Selectton o f hybnd cells can be done m several ways. The procedure descnbed here, used in our lab, involves the preparatton of cells for flow cytometry and cell sorting (39, 49) . Other alternative selectiOn methods could be micromanipulatiOn (40), or use of iOA treatment m combmahon With madtatwn (3 7-38) Irradiation or treatment with lOA has to be done at some pomt pnor to fusiOn See specific papers for detai ls I I . The storage of protoplasts m cold will help to recover the protoplasts from the rough PEG treatment. Also, the passage through the flow cytometer ts somewhat rough and freshly fused protoplasts mtght break. 1 2 Be very gentle when releasing the pro toplasts from the bottom of the Petn dtsh Do not flush directly on the cells. 1 3 The double fluorescence from a hybnd cell ts obtained because the CFDA stam­ mg of the hypocotyl protoplasts (green fluorescence) and the red autofluoresence from the chloroplasts m the leaf protoplasts. The typical filter eqmpment m the flow cytometer was as follows. fluorescence emission IS first filtered With a 5 1 0-nm htgh-pass filter and split mto red and green channels by a half-silvered mmor The green channel has a selection filter centered at 530 nm, band wtdth 250 nm Correspondmg data for the red channel Is 588 nm, and band width 20 nm 1 4 Make a 1 .47% (w/v) of a SeaPlaque solution Dissolve in dewmzed water by heat­ ing and then autoclavmg. Mix wtth double-concentrated 8p mediUm (stenlized by filtration), cool to about 45-50°C, and add 300 flL of this solutton to each well 1 5 Before plasmtd tsolatwn, make glycerol stocks of the selected colomes

References Vamling, K and Glimelius, K ( 1 990) Regeneratton of plants from protoplasts of oilseed Brass1ca crops, m Legumes and Ozlseed Crops, vol I (BaJaJ, Y. P S , ed ), Spnnger-Verlag, Berlin, pp 385-4 1 7. 2. Glimelius, K., Fahleson, J., Landgren, M., Sjodm, C , and Sundberg, E. ( 1 99 1 ) Gene transfer vta somattc hybndtzatton m plants TzBtech 9, 24--30. 3 Waara, S and Glimelius, K. ( 1 995) The potenttal of somattc hybndtzatton for crop breeding Euphytica 85, 2 1 7-23 3 . 4 Schenck, H R and Robbe len, G ( 1 982) Somatic hybnds b y fusiOn o f protoplasts from Brasszca oleracea and B campestris Z Pflanzenz , 89, 278-288 5 Sundberg, E , Landgren, M , and Glimelius, K ( 1 987) Fertility and chromosome stability in Brasszca napus resynthestzed by protoplast fusiOn. Theor Appl Genet 75, 96-1 04. 6 Heath, D. W. and Earle, E. D. ( 1 996) Resynthests of rapeseed (Brasszca napus L )· A comparison of sexual versus somattc hybndtzation Plant Breed 1 15, 395-40 I .

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7. Terada, R , Yamashtta, Y , Ntshibayasht, S., and Shimamoto, K. ( 1 987) Somattc hybrids between Brasszca oleracea and B. campestris selection by the use of iodoacetamide inactivatiOn and regeneratiOn abihty Theor. Appl Genet. 73, 3 79-3 84. 8 Sj odm, C. and Glimelms, K ( 1 989) Brasszca naponigra, a somatic hybrid resis­ tant to Phoma lingam Theor. Appl Genet 77, 65 I--656 9 Sacnstan, M. D., Gerdemann, K. M., and Schieder, 0 ( 1 989) Incorporation of hygromycm resistance in Brassica nigra and 1ts transfer to B napus through asym­ metric protoplast fusion. Theor Appl Genet. 78, 1 94-200. I 0 Fahleson, J , RAhlen, L., and Ghmelius K. ( 1 988) Analysis of plants regenerated from protoplast fuswns between Brassica napus and Eruca sativa Theor Appl Genet 16, 507-5 1 2 . I I . Toriyama, K . , Kameya, T., and H i nata, K . ( 1 987) SelectiOn of a umversal hybrid­ Izer in Smapis turgzda Del and regeneration of plantlets from somatic hybrids wtth Brasszca spec1es. Planta 1 70, 308-3 1 3 1 2 . Chevre, A. M., Eber, F , Margale, E., Kerlan, M. C , Pnmard, C., Vedel, F , and Delseny, M. ( 1 994) Companson of somatic and sexual Brasszca napus-smapzs alba hybnds and the1r progeny by cytogenetiC stud1es and molecular charactenza­ tion Genome 37, 367-374. 1 3 . Lehvelt, C. and Krens, F. A ( 1 992) Transfer of reststance to the beet cyst nema­ tode (Heterodera schachtu Schm.) mto the Brasszca napus L gene pool through intergeneric somatic hybnd1zation w1th Raphanus satzvus L. Theor Appl Genet 83, 887-894 14 Tonyama, K , Hmata, K , and Kameya, T ( 1 987) ProductiOn of somat1c hybnd plants "Brasszcomoncandza" through protoplast fusion between Moncandza arvens1s and Brasszca oleracea Plant Scz 48, 12 3- 128 15 O'Ne1ll, C M . , Murata, T , Morgan, C. L , and Mathias, R J ( 1 996) Expres­ sion of the CrC4 mtermediate character tn somatic hybnds between Brasszca napus and the CrC4 species Moricandia arvensis. Theor Appl Genet. 93, 1 2 34- 1 24 1 . 1 6 McLellan, M S , Olesen, P , and Power, J. B ( 1 987) Towards the introduction of cytoplasmtc male sterility (CMS) mto Brassica napus through protoplast fuswn, in Progress in Plant Protoplast Research (Puite, K. J., Dons, J. J. M., Huizmg, H J , Kool, A J , Koomneef, M and Krens, F A , eds ), Wagenmgen, The Nether­ lands, pp. 1 87-1 8 8. I 7 Khmaszewska, K and Keller, W ( 1 988) RegeneratiOn and charactenzation of soma­ he hybrids between Brasszca napus and Dzplotaxzs harra Plant Sc1 58, 2 1 1-222 1 8. Gleba, Y Y. and Hoffmann, F. ( 1 979) "Arabidobrassica"· plant-genome engi­ neering by protoplast fuston Naturwzssenschaften 66, 547-5 54. 1 9. Gleba, Y. Y. and Hoffmann, F. ( 1 980) "Arabidobrass1ca" a novel plant obtamed by protoplast fuswn. Planta 149, 1 1 2-1 1 7 . 20. Bauer-Weston, W B , Keller, W , Webb, J., and Gledd1e, S ( 1 993) Productwn and charactenzatton of asymmetnc somatic hybnds between Arabzdopszs thallana and Brassica napus Theor. Appl Genet. 86, 1 50--1 58

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2 1 . Forsberg, J , Landgren, M , and Ghmehus, K ( 1 994) Fert1 le somatic hybnds between Brass1ca napus and Arab1dops1s thahana. Plant Sc1. 95, 2 1 3-223. 22 Fahleson, J., Enksson, 1., Landgren, M , Styrnne, S., and Ghmehus, K. ( 1 994) Inter­ tribal somatic hybrids between Brass1ca napus and Thlasp1 perfollatum with h1gh content ofthe T. perfollatum speclfic nervomc ac1d Theor Appl Genet 87, 795-804. 23 Skarzhinskaya, M., Landgren, M., and Ghmehus, K ( 1 996) Production of inter­ tnbal somatic hybnds between Brass1ca napus L and Lesquerellafendlen (Gray) Wats. Theor Appl Genet 93, 1 242-1250 24. Tonyama, K., Hmata, K., and Kameya, T. ( 1 987) ProductiOn of somatic hybnd plants, "Brass1comon candia," through protoplast fusiOn between Moncandw arvens1s and Brass1ca oleracea. Plant SCI 48, 1 23-1 28 2 5 . Hag1mon, M , Nagaoka, M., Kato, N., and Yosh1kawa, H ( 1 992) ProductiOn and characterizatiOn of somatic hybnds between the Japanese rad1sh and cauliflower Theor Appl Genet 82, 8 1 9-824 26 Sikdar, S R , Chatterjee, G , Das, S , and Sen, S K ( 1 990) "Eruss1ca," the mter­ genenc fertile somatiC hybrid developed through protoplast fuswn between Eruca sativa Lam and BrasslCa ]uncea (L ) Czem Theor Appl Genet 79, 56 1-567 27 Chatterj ee, G , S1kdar, S R Das, S , and Sen, S K ( 1 988) lntergeneric somatic hybnd productiOn through protoplast fusion between Brass1ca JUncea and D1plotax1s muralls Theor Appl Genet 76, 9 1 5-922 28 K1rt1, P. B., Naras1mhulu, S B , Prakash, S., and Chopra, V L ( 1 992) Somatic hybrid1zat10n between Brass1ca JUncea and Moncandw arvens1s by protoplast fusion. Plant Cell Rep 1 1, 3 1 8-3 2 1 2 9 . K1rt1, P B , Mohapatra, T , Khanna, H , Prakash, S . , and Chopra, V L. ( 1 995) D1plotax1s cathohca + Brasszca JUncea somatic hybnds. molecular and cytoge­ netic charactenzatwn Plant Cell Rep 14, 593-5 97 30 Kirti, P. B . , Narasimhulu, S B , Prakash, S , and Chopra, V L ( 1 992) ProductiOn and charactenzatwn of mtergenenc somatic hybnds of Trachystoma ballll and BrasslcaJuncea. Plant Cell Rep 1 1 , 90-92 3 1 . Fahleson, J., Eriksson, I . , and Ghmehus, K ( 1 994) Intertnbal somatic hybrids between Brass1ca napus and Barbarea vulgans. Plant Cell Rep 13, 4 1 1-4 1 6 3 2 . Medgyesy, P . , FeJes, E , and Maltga, P. ( 1 985) Interspecific chloroplast recombi­ nation m a Nzcotwna somatic hybnd Proc Nat! A cad Sc1 USA 82, 6960-6964 33 Thanh, N D and Medgyesy, P ( 1 989) Limited choroplast gene transfer via recombmatton overcomes plastome-genome mcompat1b1ltty between N1cotzana tabacum and Solanum tuberosum Plant Mol Bzol. 12, 87-93 34. Pellet1er, R . G. ( 1 986) Plant organelle genetics through somatic hybndtzatlon Oxford Survey Plant Mol Cell Bzol 3, 97-1 2 1 . 3 5 . Landgren, M . and Gltmehus, K. ( 1 990) Analysis of chloroplast and m1tochondnal segregatiOn m three d1fferent combmatwns of somat1c hybnds produced w1thm Brasszcaceae Theor Appl. Genet 80, 776-784. 36. Landgren, M. and Ghmehus, K ( 1 994) A high frequency of mtergenomtc mlto­ chondnal recombmatwn and an overall biased segregatiOn of B campestns or -

,

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37.

38

39

40.

41

42

43 44. 45 46

47 48 49.

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recombmed B campestris mttochondna were found in somatic hybrids withtn Brasszcaceae. Theor Appl Genet 87, 854-862. Yamashtta, Y., Terada, R , Nishtbayasht, K., and Shimamoto, K ( 1 989) Asym­ metnc somattc hybnds of Brasszca. partial transfer of B campestns genome mto B oleracea by cell fusion Theor. Appl Genet. 77, 1 89-1 94. Terada, R., Yamashtta, Y , Ntshibayashi, S., and Shtmamoto, K. ( 1 987) Somatic hybnds between Brasszca oleracea: selectiOn by the use of wdoacetamtde mactt­ vatwn and regeneration abtltty Theor. Appl Genet 73, 379-384 Glimehus, K , Djupsjobacka, M., and Fellner-Feldegg, H ( 1 986) SelectiOn and ennchment of plant protoplast heterokaryons of Brasszcaceae by flow sortmg Plant Sci 45, 1 33-1 4 1 Sundberg, E . and Gl tmehus, K. ( 1 9 86) A method for productiOn o f mterspectfic hybrids wtthin Brasszcaceae vta somatic hybridtzatwn, usmg resynthests of Bras­ SICa napus as a model. Plant Sc1 45, I 55- I 62. Banks, M. S and Evans, P. K ( 1 976) A companson o f the tsolatwn and culture of mesophyll protoplasts from several Nicotzana species and thetr hybnds Plant Sc1 Lett 7, 409-4 1 6 Menczel, L , Nagy, F , Kiss, Z s R , and Maltga, P ( 1 98 1 ) Streptomycm resistant and sensitive hybnds of N1cotzana tabacum + Nzcotzana knzghtzana correlatiOn of resistance to N tabacum plasttds Theor Appl Genet 59, 1 9 1- 1 9 5 . Murashtge, T and Skoog, F ( 1 962) A revised medmm for raptd growth and bio­ assays wtth tobacco ttssue cultures. Phystol Plant 15, 4 73-497 Kao, K. N , Constabel, F . , Mtchayluk, M R., and Gamborg, 0. L ( 1 974) Plant protoplast fuston and growth of mtergenenc hybnd cells Planta 120, 2 1 5-227 Nagy, J. I. and Mahga, P ( 1 976) Callus induction and plant regeneration from mesophyll protoplasts of N1cotzana sylvestns Z Pflanzenphyswl 78, 453-455 Kao, K. N and Mtchayluk, M R. ( 1 975) NutnttOnal requtrements for growth of Vzcza hajstana cells and protoplasts at a very low populatiOn denstty m hqutd medm Planta 1 26, 1 05-1 1 0 Ghmeltus, K. ( 1 984) Htgh growth rate and regeneration capacity o f hypocotyl protoplasts m some Brasstcaceae. Physwl Plant 61, 3 8-44 Gam borg, 0 L., Mtller, R A., and Oj ima, K. ( 1 968) Nutnent requtrements of suspenston cultures of soybean root cells. Exp Cell Res 50, 1 5 1- 1 5 8 Sundberg, E., Lagercrantz, U . , and Gltmehus, K . ( 1 99 1 ) Effects of cell type used for fusion on chromosome ehmmatwn and chloroplast segregatiOn m Brass1ca oleracea (+) Brass1ca napus hybrids Plant Scz 78, 89-98

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Prod uction of Cybrids in Rapeseed (Brassica napus) Stephen Yarrow

1 . Introduction Protoplast fusion produces cells that contain a mixture of the DNA-contain­ mg organelles (nuclei, chloroplasts, and mitochondria) from both fusiOn par­ ents. This chapter describes the production of rapeseed (Brassica napus) fuswn-product cells where one of the nuclei IS ehmmated. In the absence of selection pressure, the remaining chloroplast/mitochondrial mixture segregates randomly as the cell divides, eventually creating different nuc lear/chloroplast/ mitochondnal combmatlons. These novel nucleo-cytoplasmic combmations are called cytoplasmic hybrids or "cybnds." As defined here, a cybrid can include cases where the chloroplast/mitochon­ drial combination is novel, as well as alloplasmic substitutions, where the cyto­ plasm of one parent is substituted with another. The existence of new types of mitochondria or chloroplasts derived from the recombination of organelle DNA also constitutes a cybrid. With few exceptions, novel chloroplast/mitochondrial combmations are unobtainable from conventional hybridization of plants, since the male gamete rarely contributes functional cytoplasm during fertilization. The identification of useful and important traits that are conferred by the organelles of certain plants has made the production of cybrids commercially important. Cytoplasmic male sterility (CMS) is one such trait of great agronomic importance for crop species. By preventing self-fertilization, CMS can allow the economic production of single-cross F 1 hybrids. The productiOn of cybrids as a means to incorporate or transfer CMS, conferred by the mitochondria, has been demonstrated in several crop species, including rapeseed (B. napus) (1), particularly noteworthy smce this relatively recent technology is progressing From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edtted by R D Hall © Humana Press Inc , Totowa, NJ

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in this species to the commercial stage (2); broccoli (Brassica oleracea) (3), rice (Oryza sativa) (4); carrot (Daucus carota L.) (5); Lolium perenne (6); and, potato (Solanum tuberosum) (7). In some cases, cybnd technology has been used further to improve CMS hnes, as demonstrated by K1rti et al. (8), whereby the chloroplasts associated w1th CMS-mitochondna prevwusly transferred from Raphanus sativus to Brassicajuncea, were causmg chlorosis. These were substituted via "cybndizatwn" with non-chlorosis causmg chloroplasts from Brassica JUncea. Cybnd technology has been used to transfer other traits, includmg tolerance to the herbicide triazine in rapeseed (B napus) (9, 10), and resistance to the antibiotic streptomycin in potato (11). In addit10n, the technology has been used to study organelle mteractions m Citrus (12), tomato (13), and tobacco/ petuma (14). A variety of methods of producing and selectmg for cybnds has been reported. For a detailed review on cybnd research, see Pelletier and Romagosa et al. (15), and Kumar and Cocking (16). This chapter describes the productwn of rapeseed (B nap us) cybnds usmg a "donor-recipient" aided selection scheme first descnbed for Nicotiana by Zelcer et al. (1 7) . The nuclei of the "donor" parent are inactivated prior to fusiOn, so that effectively It only donates cytoplasm to the other "recipient" nuclear-bearing parent. Nuclear mactivation can be by y or X-irradiation (10, 18-20). The select10n IS complemented by the treatment of the "recipient" parent with an inhibitor, iodoacetic acid (lOA), that kills the cells by metabolic machvatton of the cytoplasm (10,19,20) . Only fusiOn products survive due to complementation. This selection is only for fusion products and not for spe­ cific cybrids; these are formed after organelle segregation during cell division and plant regeneration (see Fig. 1). The scheme described here has been successfully used to produce important cybrids in both rice (4) and rapeseed (B napus) (1 0, 21). The cybrid example described is the combination ofCMS and cytoplasmic triazine tolerance (CTT) traits. In rapeseed, CTT is conferred by the chloroplast genome (22) and CMS, by the chondriome (9, 10,23, 24) For commercial production of single-cross­ hybnd CTT canola, as proposed by Beversdorf et al. (1), both CMS and CTT traits must be present in the female parent. However, since these are generally only transmitted through the female hne, It IS not possible to combme these traits by conventional breedmg approaches (see Notes 1 and 2). Protoplast fusion, however, allows the biparental transmiSSion of both CMS and CTT traits (9,10,23). In addition, mcorporating CTT into rapeseed hybrids can allow the crop to be grown in weed-infested areas. The weeds are eliminated with applications of triazme (25).

213

Production of Cybrids "Recipieat" Puental

"Doaor" l'arental

Liu A Protoplast � dria : � "1 ���· 0

0

Cl ol oroplut (�p-AJ

Line B Protoplast

Nuleu B

Nadeu A

.

� (J Protoplast Fusion

,.·

lOA Metabolic.

0 V

labibltlou

Cbloropl.-1 [t.p-BJ

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Nudear luacttvalion by Gamma or X-lrradlallon

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laacrlvated Nucleua B Biparental Fu1lon Product

�

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and Random Segregation

/ / \�

A l•t.Bit.p-BJ

A [mi-Bitp-AJ

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Fig. 1 . Protoplast fusion and possible organelle segregation.

The procedures below describe how mesophyll protoplasts of the CIT rape­ seed "donor" parent are fused with "recipient" CMS rapeseed hypocotyl proto­ plasts. Fusion is by a modified combination of polyethylene glycol (PEG) (26,2 7) and ca+2 ions at high pH (28). Protoplasts are cultured following a protocol that was devised for rapeseed (29), with the addition oftobacco nurse cells (9), that augment fusion product survival. 2. Materials

2. 1. Protoplast Source Material l . Mesophyll: CIT plants are grown from seed and established in ''Metro-.mix" pot­ ting compost, fertilized daily with "Peters" 20-10-20 fertilizer (both from W.R.

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214

2. 3. 4. 5 6.

6

Grace and Co, Canada), maintained within growth chambers under the regimen of 2 a 1 2-h photopenod of 1 60 J.lmol/m /s of photosynthetically active radiatiOn (pro­ vided by 40 W "cool white" fluorescent tubes), at a constant temperature (23°C) TypiCally, four to s1x CTT plants are needed for each md1vidual fusion operatiOn. Hypocotyl : CMS seedlings are grown from surface-sterilized seed. 1 0% (v/v) SolutiOn of 6% commercial preparation of hypochlonte Paper towels (wrapped in aluminum fml and autoclaved). Forceps, stenlized by flammg. MSS medmm. basal salts of Murash1ge and Skoog 's med1a (30 and see Appen­ dix), containing 1 0 g/L sucrose, solidified with 4 g/L agarose (Sigma Type 1), pH adJusted to 5 7, poured mto 1 0-cm Petn plates (70 mLid1sh) Sieves, pore s1ze <0.5 mm (autoclaved).

2.2. Protoplast Isolation 1 . Soft nylon pamt brush, stenhzed by 1mmerswn m 70% ethanol for 5 mm and dned on stenle paper towels 2. Scalpel, with # 1 1 blade, stenhzed by flammg. 3 Stenle 250-mL Erlenmeyer flasks, capped with fml 4 Sterile fi ltermg funnels (double-layer cheesecloth hnmg a glass funnel). 5. Stenle Babcock bottles, capped with foil. 6. Stenle 230-mm cotton-plugged Pasteur pipets 7 Porcelain tile (wrapped in fml and autoclaved). 8 Low-speed bench-type centrifuge 9. Soak solutwn (for 1 L). 475 mg KN03 1 1 0 mg CaC12 2H20 92 5 mg MgS04 7H20 42. 5 mg KH2P04 0 1 25 mg Thiamine HC1 G1ycme 0.5 mg 1 25 mg Nicotinic acid 0 . 1 25 mg Pyndoxme HCl 0 . 1 2 5 mg Fohc ac1d 0 0 1 25 mg B iotin 25.0 mg Casein hydrolysate 6-Benzylamino purine 0 5 mg 1 .0 mg 2,4-Dichlorophenoxyacetic acid pH 5.6, autoclaved. The soak solutiOn can be stored at 4°C for several weeks 1 0. Enzyme solution for 1 00 mL. 0 I g Macerozyme R- I O* 1 0g Cellulase R- 1 0* 0. 1 g 2 (N-morpholine) ethanesulfonic acid (MES)* •

Production of Cybrids

215

1 .0 g Sucrose 1 2.0 g p H 5 .6, fi lter-sterilized through 0.2-!Jm Nalgene filter The enzyme solution can be stored frozen for up to 6 mo. Mesophyll protoplast isolation reqmres a l / 1 0 strength enzyme solutiOn, prepared by dtlution With nnse solution (see below). For the tsolation of hypocotyl protoplasts, full-strength enzyme solution IS used *Yakult Honsha Co , Ltd , Tokyo, Japan 1 1 . Rinse solutiOn (for 1 L). 1 20 g Sucrose MES 0.5 g pH 5 6, autoclaved. Stored at room temperature in sealed bottles for several months

Polyvmylpyrrolidone

2.3. Protoplast Inactivation 1 . Iodoacetic acid (lOA) solution: The lOA solution for protoplast inactivation IS prepared from a 30 mM stock, diluted wlth rinse solutiOn, pH adJUSted to 5 . 6, and filter-sterilized with a 0 2-!Jm Nalgene filter. The final concentratiOn of iOA necessary for protoplast inactivation is determmed pnor to fuswn. 2 30 mM stock: 0.279 g lOA in 50 mL H20. This stock solution can be stored at 4 °C, m the dark for up to 3-4 mo, wtth no noticeable loss of acttv1ty 3. X ray or y-radiatwn source.

2.4. Protoplast Fusion PEG solutiOn (for 1 00 mL). 25.0 g PEG 8000 (Sigma) 0. 147 g CaCl2 2H20 4.0 g Sucrose Autoclaved in fml wrapped vials. Can be stored at 4°C for up to 6-8 wk 2 2 . Ca + solution (for 1 00 mL): 0.735 g CaC12 2H20 Glycine 0.375 g 1 2.0 g Sucrose pH 1 0.5, filter-sterilized with 0.2-!Jm Nalgene filter. Solution must be prepared on the day of fusion •

•

2.5. Protoplast Culture 1 . PreparatiOn of med1a: a. Cell-layer (Cl) medium (31 and see Table 1). Prepared on the day of fusion, in two forms: 1. "Liquid," agarose-free---fi lter-sterilized with 0.2-!Jm Nalgene filter. ii. "Semisolidified," with double-strength agarose. Made up by mixing equal volumes of double strength "liqutd" Cl (filter-stenlized wtth 0 .2-!Jm Nalgene filter) w1th molten quadruple-strength solutiOn of the appropri-

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216 Table 1 Protoplast Media Cl0

Medium MaJor elements, mg/L KN03 CaC12 2H20 MgS04 7H20 KH2P04 Iron and minor elements, mg/L Na2 · EDTA FeS04 7H20 H3B03 MnC12 4H20 ZnS04 7H20 KI Na2Mo04 2H20 CuS04 5 H20 CoS04 7H20 Orgamcs, mg/L Thiamme HCI Glycme Nicotmtc acid Pyndoxme HCI Folic acid Btotin Casem hydrolysate Osmoticum, g/L Sucrose Myo-mositol o-Mannitol Sorbttol Xylttol Hormones, mg/L NAA b BAPb 2,4-Db •

•

·

•

•

•

·

Kmetm Agarose (g/L) Type VII (Stgma)

7600 1 760 1 480 680 1 8.5 1 3.9 3 l 99 4.6 0.42 0. 1 3 0.0 1 3 0.0 1 5

Ra 1 900 440 370 1 70 18 5 18 5 3.1 99 4.6 0.42 0 13 0 013 O.o i 5

DMa 1 900 440 370 1 70 1 8.5 1 8 .5 3 l 99 4.6 0.42 0. 1 3 0 013 0.0 1 5

0.5 2.0 5.0 05 05 0 05 5 0.0

05 2.0 5.0 05 0.5 0 05 1 00

0.5 2.0 5.0 05 05 0 05 1 00

85.6 5.7 5.7 5.7 4.7

34 25 01 18 25

34 25 0. 1 1 8 .25

0l 0.4 1 .0

0. 1 1 .0

1 .0 0 1

0.6

4.5

0.6

aFrom Shepard (31) modtfied table repnnted by permtsston from The Umverstty of Mmnesota Press bAbbrevJatwns. NAA-1 -napthaleneacetlc actd, BAP----
Production of Cybrids

c.

d.

e f.

2.

3.

217

ate agarose (sterilized by autoclaving at 1 20°C for 20 min)-final agar­ ose strength of 1 .2 g/L. Maintained at 40°C b Reservoir (R) and differentiation (DM) media (31 and see Table 1 ) Proliferation medmm (PM): MS medium (30 and see Appendix) contammg 10 g/L sucrose, 1 . 0 mg/L 2,4-Dt, 0 . 1 mg/L k metin, 4.0 g/L agarose (Sigma Type 1). RegeneratiOn medmm (RM 1 ) : MS medmm (30 and see Appendix) contammg 2.0 g/L sucrose, 2.0 mg/L zeatm, 2.0 mg/L kmetm, 0. 1 mg/L IAAt, 4 0 g/L aga­ rose (S1gma Type I) Regeneration medium (RM2)· same as RM 1 medmm, except the sucrose is reduced to 1 .0 g/L, and the zeatm and IAA t are each mcreased to 3 .0 mg/L SP (shoot proliferation) medmm: Gamborg's B5 medium (32 and see Appendix) contammg 2.0 g/L sucrose, 0.03 mg/L GA3 t and 4.0 g/L agarose (Stgma Type 1). With the exception of the Cl medium, all media are made up by m1xmg equal volumes of. a. Double-strength components, without agarose, filter-sterilized w1th 0 2-f!m Nalgene filter with b. Molten double-strength agarose, stenhzed by autoclavmg at 1 20°C for 20 mm. All media are adjusted to pH 5.6. Preparation ofR plates: Sterile deep Petn plates (I 00 x 25 mm) are filled With 70 mL of molten R medium On settmg, an X IS cut mto the medmm, reachmg nght to the edges of the plate. Two diametrically opposite quadrants are then removed with a sterile spatula to complete the R plate preparation. Twenty or so R plates should be prepared in advance Unused plates can be stored at 4°C m the dark, for up to 4 wk. PreparatiOn of RM 1 , RM2, and SP media plates: RM l , RM2, and SP media are dispensed mto sterile deep Petn plates ( 1 00 x 25 mm), 70 mL/plate Plates can be stored at 4°C m the dark for several weeks.

2.6. Triazine Tolerance Test Triazine paste: A paste of the triazine herbicide Atrazine WP (80%; Ctba Giegy) ts prepared by mixing 1 .47 g of Atrazme in 1 00 mL water. 3. Methods All procedures must be performed aseptically, wtthin a lammar flow sterile atr cabinet. 3. 1. Production of Etiolated Hypocotyls 1 . Seed of CMS-rapeseed are surface-sterilized by immersion in 1 0% (v/v) commer­ cial hypochlorite solution (0.6% free chlorine) for 1 0 min and then nnsed for 5 min tAbbreviations: 2,4-D- 2,4-dtchlorophenoxyacetic actd; IAA- 3-mdoleacettc actd; GA3-g tb ­ berelhc acid

218

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m stenle water. These procedures are best performed by placmg the seed m pre­ sterilized sieves, for ease of handling and immersion mto the different solutions 2 . For some batches of seed, this procedure may be ineffective. In these cases, seeds are surface-sterilized by 1mmerswn m a solution of0.05% mercunc chlonde (tak­ ing particular care in the handlmg of this substance) and 0. 1 % sodmm dodecyl sulfate, for 1 0 min, and then rinsed s1x times m stenle water 3 . Seeds are transferred to paper towels to remove excess moisture and then onto MSS medmm (approx 20 seeds/Petn plate) 4. Plates are incubated in the dark at constant temperature ( 2 5°C) for germmatwn Typically, 20-30 plates of CMS-rapeseed seed are reqUired/fusion operation

3.2. Isolation of Protoplasts

3.2. 1 . Mesophy/1 Protoplasts l . Leaves from 3-wk-old CTT plants are surface-stenlized by immersmg m 1 0% (v/v) commerctal hypochlonte solution for 3 mm, and nnsed once m stenle water and once again, for 30 s, m 70% ethanol 2 The leaves are placed onto paper towels to remove excess mOisture, care bemg taken not to dry the leaves completely to the point of wiltmg 3 The lower epidermis of the leaves are abraded by gentle strokmg With the pamt­ brush until bnstle marks are JUSt visible. Excessive brushmg should be avoided to prevent damage to the subepidermal cells that could reduce protoplast ytelds 4 Leaves are cut into 1 -cm wide stnps and transferred to the soak solution. Approx­ Imately 3.0 g of tissue (around 6-9 leaves) are Immersed m each flask of soak (1 00 mL of soak/flask). Typically, two flasks of material are prepared for each mdtvtdual fuswn operation 5 The flasks of leafmaterial are then incubated for 2--4 h m the dark, at 4°C. Longer mcubations, of up to 1 2 h, are possible without undue harm . 6. Following cold mcubatwn, the soak solutwn 1s carefully poured off and replaced with l/10 strength enzyme solutiOn Typically, leaf material is prepared early in the afternoon and the soak solutiOn substituted wtth enzyme solution in the early evening. 7 . Flasks are incubated for 1 5-1 8 h (1 e., overnight) at 25°C in the dark, on a rotary shaker, set to gtve a very gentle agitation. Successful enzyme digestion is evtdent when the leaf pteces are floatmg and have a translucent appearance. However, leaves that have sunk and/or appear undigested can still yteld protoplasts, but the numbers are generally reduced. From this stage, all manipulations must be performed particularly carefully to avoid damaging the fragile protoplasts. 8. The contents of each flask IS poured through a filtering funnel, into two babcock bottles A retort stand is used to hold the funnel. Gentle agitatiOn with a Pasteur pipet will assist the liberation of protoplasts. 9 . The babcock bottles are then topped up wtth nnse solutiOn and recapped wtth sterile foil.

Production of Cybrids

219

1 0 . Protoplasts will float to the top of the bottle following centrifugation at 500 rpm (350g) for 1 0 min (in a bench model centnfuge With babcock bottle-size swmg­ out buckets). 1 1 . Protop lasts are "rinsed" free of enzyme solution by transferring them with a Pas­ teur pipet to a babcock bottle previously half filled with rinse solution, thereby combmmg mto one the protoplasts from two babcocks. 12. The bottle is then filled completely with rinse solution, resuspending the proto­ plasts, recapped, and centnfuged once again, as described before.

3.2.2. Hypocotyl Protoplasts 1 . Hypocotyls excised from etiolated CMS rapeseed seedlings (3-5 d after sowmg) are chopped on a porcelain tile into 2-5 mm transverse segments. These are Immediately placed mto full-strength enzyme solutiOn Generally, 200--3 00 chopped hypocotyls are added to each flask filled w1th 1 00 mL of enzyme solutiOn. 2. The hypocotyl material is mcubated overnight (25°C m the dark, on a rotary shaker, set to give a very gentle agitation), and protoplasts Isolated as descnbed for mesophyll CTT material (see Note 3). 3. 3. Prelusion Treatment of Protoplasts

3.3. 1 . Donor Parent Nuclear mact1vation of mesophyll CTT protoplasts can be by either y-ray or X-ray irradiatiOn. Whichever is used, 1t IS necessary to establish the dosage and check the protoplast response in control cultures. Irradiation should produce protoplasts that do not divide, but remam "dormant" for several days, under normal culture conditions, before eventually dymg. However, a low frequency of first divisions is acceptable, provided there is no colony formatiOn. Irradia­ tiOn is performed Immediately after protoplast isolation in the babcock bottle (see Note 4). 3.3.2. Recipient Parent The effective dosage level ofiOA must be established prior to fusion experi­ ments. The lOA concentration should be suffic1ent to kill the protoplasts, under normal culture conditions, withm 24 h. For B. napus rapeseed, 3--4 mM lOA are typical. 1 . Following isolation, CMS protoplasts are carefully transferred to a sterile centri­ fuge tube, of 1 0- to 25-mL capacity. 2. The tube is then filled with the diluted lOA solution, resuspending the protoplasts 3 Following I 0 mm of mcubat10n at room temperature, the tube ts centnfuged at 350g for 1 0 min. 4. The lOA solution is removed by two consecutive centnfugattons, each time trans­ ferrmg the floatmg protoplasts to separate babcock bottles filled with rmse solu­ tion. It IS typ1cal for about 25% of the protoplasts to be lost as a result of the centrifugation steps.

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3.4. Protoplast Fusion I . Protoplasts of both donor and rectptent parents are carefully combmed together in a stenle round-bottom tube ( 1 3 x l 00 mm). Actual numbers of protoplasts from each parent will vary widely wtth each fusion owing to yteld variations of dtfferent tissue samples and through varying degrees of cell death caused by cen­ trifugatiOn. Ratios of the CTT and CMS parents should be in the l : l-1 ·4 range. Some adjustments may be necessary. 2. Pnor to mitlatmg the fuston process, approx Ifs of the mtxed protoplast suspen­ sion is removed and transferred to a separate tube These will constitute the "unfused mixed control" (UFMC) to be cultured separately (see below). 3 The volume ofthe remammg protoplast suspension should be adJusted to 0.5--l 0 mL by the addition of extra rinse solution, or by centrifugmg the tube and removmg excess nnse solutiOn from underneath the floatmg protoplasts. 4 To this is added an equal volume (0 5-1 0 mL) of PEG solution, followed by the gentle resuspension of the protoplasts by careful rotations of the tube for approx 30 s 5 After 1 0 mm of mcubatton at room temperature, 2 mL ofCa2 + solutiOn are gradu­ ally added, further resuspendmg the protoplasts. 6 The tube ts then fi l led wtth further additiOns of 2 mL of Ca2+ solutiOn, with careful mixm g each ttme, until the tube ts full, and centnfuged at 400 rpm for 1 0 m m . 7. Floating protoplasts are carefully removed and resuspended i n nnse solution con­ tamed m a babcock bottle. 8. After a final centrifugatiOn at 350g for 1 0 mm, the fuswn process ts complete.

3.5. Protoplast Culture Fusion protoplasts and the UFMC are plated in nurse culture plates prepared 24 h in advance.

3.5. 1. Nurse Culture Preparatton I . Nicotzana tabacum (tobacco) plants are grown from seed under the same condi­ tiOns as described Subheading 2.1. Conditions for the preparation of the leaf mate­ rial and the isolation ofprotoplasts are the same as described m Subheading 3.2.1. Protoplasts are mitotically inactivated by irradiatiOn as descnbed m Subheading 3.3.1. (see Note 5). 2. Irradiated tobacco protoplasts are then transferred from the rinse solution to sev­ eral milliliters of "liquid" CL medium and resuspended. 3 . With a hemocytometer, the number of cells in the suspensiOn ts calculated from a small sample. 4. Usmg further volumes of "hqutd" CL medmm, the denstty of the protoplast sus­ pension is adjusted to 2 x 1 05 protoplasts/mL 5 . Next, the protoplast suspension in "liquid" Cl medium is gently mixed wtth equal volume of molten "semisolid" Cl medium (containing double-strength agarose, at 40°C). Four mtlhliters of the adjusted protoplast suspension (in the "semt-

Production of Cybrids

22 1

solid" Cl medmm now with smgle strength agarose) is transferred to each empty quadrant in the R medmm plates. Ten to 1 2 tobacco-filled nurse plates should be sufficient for each fusion and its corresponding UFMC. 6. Completed nurse plates are mcubated at 25°C in the dark.

3. 5.2. Culture of Fused and UFMC Protoplasts 1 . Protoplasts from fusion and UFMC samples are separately resuspended m a few milliliters of "hqmd" CL medmm. 2 With the aid of a hemocytometer, the number of cells in the suspension IS calcu­ lated and adjusted with further volumes of "hqmd" CL medmm to 1 05 proto­ plasts/mL. 3. One milliliter of the post-fusion suspensiOn IS then gently mixed into each nurse 4 quadrant, givmg a final Brasszca protoplast density of 2 x 1 0 protoplasts/mL. The UFMC suspension is dispensed similarly. 4. FusiOn and UFMC plates are then sealed with parafilm and mcubated at 25°C m the dark 5. One week to 1 0 d after platmg, suspensions are transferred to regular Petri plates (contents of one quadrant/ 100 x 15 mm plate) and diluted With an equal volume of molten DM medium precooled to 40°C. The combmed suspensiOn and DM medium should be spread evenly over the bottom of the Petn plates to form a thm layer for maximum colony formatiOn.

Subsequent culture cond1t1ons are changed to 25°C with a 1 6-h photopenod of 1 50-200 �ol!m2 /s of photosynthetically active rad1at10n. 3. 6. Callus Proliferation and Plant Regeneration From 1 0-1 4 d after dilution, colonies in the fusion plates will have grown to approx 0.5-1 .0 mm in size. Ideally, there should be no colony development in the UFMC plates, since the irradiation and lOA treatments should have inacti­ vated or killed the unfused protoplasts (see Note 5, re. nurse controls). How­ ever, it is hke1y that some colonies will develop from the irradiated population, since the madiation treatment is often not 1 00% effective. If the number of colonies in the UFMC plates approaches the colony count in the fusion plates, then the fusion must be discarded and the inactivatiOn treatment checked and re-established. 1 . Assuming there is no colony formatiOn m the UFMC plates or at least consider­ ably less than the fusion plates, then colonies of0.5-l .O-mm size from the fusion plates are individually transferred to the surface of PM medium, 35-40 colonies/ plate, for further proliferatiOn. 2. Colonies that are 1 .0- to 2. 0-mm in diameter (usually after 1 0-14 d at wh1ch t1me they should be colorless or very pale green) are transferred to RMl medmm, 20-25 colomes/plate, to mduce differentiatiOn.

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3. W1thm 2-3 wk, it is likely that some of the colonies w1ll have regenerated small shoots. These colomes must then be transferred to SP medmm to induce shoot proliferatiOn and growout 4. The remammg colomes are transferred to RM2 medmm to further mduce differ­ entiatiOn 5 Any shoots developmg on the RM2 medmm should also be transferred to SP medmm. It IS unlikely that more than 1-5% of the colomes will respond on the differentiatiOn medmm, smce the vanous treatments and fusiOn processes typi­ cally reduce the regeneratiOn response considerabiy. 6. Shoots that have clearly differentiated menstems are transferred to peat pellets ("Jiffy 7," Jiffy Products [N B ] Ltd , Shippegan, Canada; sterilized by autoclav­ mg) to induce rootformation 7. Pellets are placed in stenle glass Jars to mamtain a high humidity. Peat pellets should be mOistened with filter-stenhzed fertilizer (see Subheading 2.1., item 1) To prevent premature flowering, the photopenod IS reduced to l 0 h hght/14 h dark 8 When several roots are protrudmg from the sides of the peat pellets, the plantlets are transferred to pottmg compost for further plant development. 9 Regenerated plants are maintained at 23°C with a 1 6-h photopenod of 1 60 !J.moll m2/s of photosynthetically active radiatiOn to mduce flowenng.

3. 7. Identification of Cybrids I . On flowermg, the flower morphology of the regenerants IS exammed for male sterility (absence of pollen production), which md1cates the presence of CMS mitochondria 2. Plants are then tested for their sensitivity to tnazine herbicides Two to three mature leaves still attached to each plant are painted with the atrazme paste. Those plants carrymg CTT chloroplasts should remam undamaged. The other plants will suffer yellowmg of the leaves and may be dead withm 7-14 d.

Those plants that are male stenle and are tolerant to atrazme are the destred cybrids (see Note 6). Male fertile, atrazine-susceptlble plants are also cybnds, but with the opposite organelle combination, so-called reverse-cybrids. In addi­ tion, male ferttle, atrazine tolerant plants can also be constdered cybrids, smce by alloplasmic substitution, they possess the nucleus of the onginal CMS par­ ent. However, if the original parents had identical nuclei, e.g., if they were both versions of the same rapeseed variety, then these plants cannot be constdered as cybrids. Male sterility sometimes appears m regenerants that have aneuploid chro­ mosome complements, independent of the mitochondria. The genotype of the mitochondrial population can be distinguished by "restnctwn fragment-length polymorphism" (RFLP) analysis of the mitochondrial DNA. Plastomes can be stmtlarly identified. The techmques involved are beyond the scope ofthts chap­ ter, and the reader 1s referred to Kemble (33).

Production of Cybrids

223

4. Notes I . The described procedure can produce cybrids m 8-1 2 mo from sowing the seed of the protoplast source material to identifymg the cybnds m the whole-plant regenerants. This time penod predommantly depends on the rapidity of shoot differentiation. This in turn depends on the careful timing of each transfer onto the different culture media. 2. These procedures can be utilized w1th different parents to produce rapeseed cybrids w1th other desired organelle combmations For example, similar tech­ niques have transferred the CMS trait from spring-planted rapeseed to wmter forms, producing cybrids that can be useful to single-cross-hybnd winter rape­ seed productiOn program (34). In th1s case, conventional crossmg also could have produced CMS winter lines, but this would have taken up to 3 yr. Fusion accom­ plished the same goal m just I yr, a sigmficant saving of tlme. 3 In some circumstances, it may be necessary to use hypocotyl protoplasts as the "donor" parent when attempting to produce a particular rapeseed cybnd. Hypo­ cotyl protoplasts will require h1gher dosages of irradiation for mactivatwn, since their nuclei are more resistant to radiation damage than their mesophyll counterparts. 4. When madiating donor parent protoplasts pnor to fusiOn, the exposure period should not be longer than 1-2 h to prevent undue cell-wall regeneration that can mhibit fuswn. If the exposure penod has to be longer, owmg to the nature of the madiatwn equipment, then the problem can be circumvented by directly madi­ ating the leaves mstead, prior to preparations for protoplast isolation Indeed, th1s may be more convenient, both for inactivatmg the donor parent and the nurse matenal. 5 . The effectiveness of the mitotic inactivation may vary from time to time, both for the Brassica donor parent and for the tobacco nurse cultures. Therefore, nurse control cultures should be prepared, in addition to the UFMC controls. Instead of adding I mL of fusion or UFMC protoplasts, add I mL of protoplast-free "liq­ uid" Cl medium to each nurse cell quadrant. If colonies appear in the nurse con­ trols, then the fuswn must be discarded, and the mactivatwn treatment checked and re-established. 6 Figure 1 suggests that one-quarter of the regenerants will be the desired cybnds, wh1ch indeed there would be if there was random segregatiOn of the organelles followmg cell d1visions of the fuswn product cells. However, the proport10ns of the different segregants is, m practice, usually not equal because of differences m protoplast tissue sources, differential responses in culture, and adverse effects of the cell inactivation processes References I . Beversdorf, W. D., Enckson, L R., and Grant, I ( 1 985) HybridizatiOn process utilizing a combinatiOn of cytoplasmic male sterility and herbicide tolerance US Patent No. 45 1 7763 . 2 Renard, M . , Delourme, R., Pelletier, G., Pnmard, C , Mesquida, J , Darrozes, G., and Morice, J. ( 1 99 1 ) Un systeme de productwn de semences hybrides F1 utlhsant

224

3.

4

5.

6.

7.

8

9.

10

II

12

13 14.

1 5.

Yarrow

une sterilite male cytoplasmique. Comptes Rendus de l 'A cademze d'Agrzculture de France 77, 49-5 8. Yarrow, S A , Burnett, L., Wildeman, R. P., and Kemble, R J ( 1 990). The trans­ fer of "Pohma" Cytoplasmic Male Sterility from otlseed rape (Brasszca napus) to broccoli (B. oleracea) by protoplast fusiOn. Plant Cell Rep 9, 1 8 5-1 88. Akagt, H , Tagucht, T , and Fujimura, T ( 1 995) Stable mhentance and expressiOn of the CMS tratts introduced by asymmetnc protoplast fusiOn Theor Appl. Genet 91, 563-567 Suenaga, L. ( 1 99 1 ) Basic studtes on transfer of cytoplasmic male steriltty by means of cytoplasmiC hybridizatiOn m carrot (Daucus carota L.). J. Faculty Agn­ culture Hokkazdo Umv Japan 65, 62- 1 1 8. Creemers-Molenaar, J., Hall, R. D., and Krens, F . A ( 1 992) Asymmetnc proto­ plast fusiOn atmed at mtraspecific transfer of cytoplasmtc male sterihty (CMS) m Lohum perenne L Theor. Appl Genet 84, 763-770 Ga1un, E , AviV, D , and Perl, A ( 1 99 1 ) Protoplast fus1on for potato-cybnd for­ mation. Report of the plannmg conference on application of molecular techmques to potato germp1asm enhancement, InternatiOnal Potato Center, pp. 1 05-1 1 1 K1rt1, P. B . , Banga, S. S , Prakash, S., and Chopra, V. L. ( 1 995) Transfer of Ogu cytoplasmic male sterility to BrasszcaJuncea and Improvement of the male stenle hne through somatic cell fusiOn Theor. Appl. Genet 91, 5 1 7-52 1 Yarrow, S. A., Wu, S C., Barsby, T. L., Kemble, R. J., and Shepard, J. F. ( 1 986) The introductiOn of CMS mitochondna to triazine tolerant Brassz ca napus L., var "Regent," by micromanipulation of mdtvidual heterokaryons Plant Cell Rep. 5, 4 1 5-4 1 8. Barsby, T. L., Chuong, P. V., Yarrow, S. A., Wu, S. C , Coumans, M , and Kemble, R. J. ( 1 987) The combmauon of Pohma ems and cytoplasmic tnazme resistance m Brasszca napus Theor Appl Genet 73, 809-8 1 4 S tdorov, V. A , Yevtushenko, D P , Shakhovsky, A M , Gleba, Y Y , and Gleba, Yu-Yu ( 1 994) Cybnd production based on mutagemc macttvatton of protop1asts and rescuing of mutant plastids m fusiOn products: potato with a p1astome from S bulbocastanum and S. pznnatisectum Theor. Appl Genet. 88, 525-529. Grosser, J W., Gmttter, F G , Jr., Tusa, N , Recupero, G R , and Cucmotta, P ( 1 996) Further evidence of a cybndtzatwn requirement for plant regeneration from cttrus leafprotoplasts following somatic fusion. Plant Cell Rep 1 5, 672-676 Bonnema, A . B., Melzer, J M , and O'Connell, M A. ( 1 99 1 ) Tomato cybnds w1th mitochondnal DNA from Lycoperszcon pennellu. Theor Appl Genet. 81, 339-348 DJurberg, I , Kofer, W , Hakansson, G., and Glimelius, K. ( 1 99 1 ) Exchange of chloroplasts in a Nzcotiana tabacum cybnd with Petunza chloroplasts in order to establish that a vanegated and abnormal development of the cybnd 1s caused by a mttochondnal mutatiOn. Physiol Plant. 82, A28 Pelletier, G and Romagosa, I. ( 1 993) Somatic hybndtzation, m P/ant Breedzng Prznczp/es and Prospects Plant Breeding Senes 1 (Hayward, M D and Bosemark, N 0., eds.), Chapman and Hall, London, pp. 93- 1 06

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1 6. Kumar, A. and Cocking, E. C. ( 1 987) Protoplast fusiOn: A novel approach to or­ ganelle genetics in higher plants. Am. J. Bot. 74, 1 289-1 303 1 7. Ze1cer, A., Aviv, D., and Galun, E. ( 1 978) Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts of normal Nicotiona sylvestrzs and X-ray Irra­ diated protoplasts of male stenle N. tabacum. Z. Pjlanzenphyswl 90, 397-407. 1 8 . Aviv, D , Arzee-Gonen, P., BleiChman, S , and Galun, E. ( 1 984) Novel alloplasmic plants by "donor-recipient" protoplast fusion: cybnds havmg N tabacum or N. sylvestris nuclear genomes and either or both plastomes and chondriomes from alien species Mol. Gen Genet 1 96, 244-253 19 Sidorov, V. A., Menczel, L , Nagy, F., and Maliga, P ( 1 98 1 ) Chloroplast transfer m Nzcotzana based on metabolic complementatiOn between irradiated and iodoacetate treated protoplasts. Planta 1 52, 34 1-345. 20. Ichikawa, H., Tanno-Suenaga, L., and Imamura, J. ( 1 987) Selectton of Daucus cybnds based on metabolic complementation between X-madiated D capillifolius and wdoacetamide-treated D carota by somatic cell fusion Theor Appl. Genet 74, 746-752 2 1 . Sakai,T., Liu, H J , Iwabuchi, M., Kohno-Murase, J , and Imamura, J. ( 1 996) IntroductiOn of a gene from fertility restored radtsh (Raphan us satzvus) mto Bras­ szca napus by fuston of X-madiated protoplasts from a radish restorer hne and todacetoamide-treated protoplasts from a cytoplasmic male-sterile cybnd of B napus Theor Appl. Genet 93, 373-3 79. 22 Retth, M. and Straus, N. A. ( 1 987) Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola. Theor. Appl Genet 73, 357-3 63 23. Pelletier, G., Pnmard, C., Vedel, F , Chetrit, P., Remy, R., Rouselle, P., and Renard, M. ( 1 983) Intergenenc cytoplasmic hybridization in Cruciferae by proto­ plast fusion. Mol. Gen Genet 191, 244-250. 24. Chetrit, P., Mathieu, C., Vedel, F., Pelletier, G., and Primard, C ( 1 985) Mito­ chondrial DNA polymorphism induced by protoplast fusion in Cruciferae. Theor Appl Genet. 69, 36 1-366. 25. Beversdorf, W. D., Weiss-Lerman, J., Erickson, L. R., and Souza-Machado, Z ( 1 980) Transfer of cytoplasmic mherited tnazme resistance from bird's rape to cultivated oilseed rape (Brasszca campestrzs and B napus) Can J Genet Cytol 22, 1 67-1 72. 26. Kao, K. N and Michayluk, M. R. ( 1 974) A method for high frequency mterge­ neric fusiOn of plant protoplasts. P/anta 1 1 5, 355-3 67 27. Wallin, A. K., Ghmehus, K., and Eriksson, T. ( 1 974) The induction of aggrega­ tion and fusion of Daucus carota protoplasts by polyethylene glycol. Z Pjlanzen­ physiol. 74, 64-80. 28. Keller, W A. and Melchers, G. ( 1 973) The effect of high pH and calciUm on Tobacco leaf protoplast fusiOn. Z Naturforsch. 28c, 73 7-74 1 . 29. Barsby, T L., Yarrow, S A., and Shepard, J . F . (1 986) A rapid and efficient alternative procedure for the regeneration of plants from hypocotyl protoplasts of Brassica napus. Plant Cell Rep. 5, 1 0 1-1 03

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3 0 Murashige, T. and Skoog, F . ( 1 962) A revised medium for rapid growth and bw­ assays wtth tobacco ttssue cultures. Physzol Plant 15, 473-497 3 1 . Shepard, J. F. ( 1 980) Mutant selectiOn and plant regeneration from Potato meso­ phyll protoplasts, m Genetic Improvement of Crops · Emergent Techmques (Rubenstem, 1 ., Gengenbach, B., Phthps, R. L. , and Green, C. E., eds.), Umver­ stty Mmnesota Press, pp. 1 85-2 1 9. 32. Gamborg, 0. L., Mtller, R. A., and Ojima, K. ( 1 968) Nutrient requtrements of suspension cultures of soybean root cells. Exp Cell Res. 50, 1 5 1-158. 3 3 Kemble, R . J . ( 1 987) A raptd, smgle leaf, nucleic actd assay for determmmg the cytoplasmic organelle complement of rapeseed and related Brass1ca spectes Theor. Appl Genet 73, 364-370 34. Barsby, T L , Yarrow, S A , Kemble, R. J., and Grant, I. ( 1 987) The transfer of cytoplasmtc male stenlity to wmter-type oilseed rape (Brasszca napus L.) by pro­ toplast fusion. Plant Sci 53, 243-248.

21

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Microprotoplast-Med iated Chromosome Tra nsfer (MMCT) for the Direct Production of Monosomic Add ition Li nes Kamisetti S. Ramulu, Paul Dijkhuis, Jan Blaas, Frans A. Krens, and Harrie A. Verhoeven

1 . I ntroduction The transfer of smgle chromosomes carrying important genes between related, but sexually mcongruent species, and the productiOn of monosomic addition plants, can speed up gene introgress10n through homoelogous recom­ bination or other mechamsms of gene transfer (1,2). Monosomic addition lines form the most Important material for the transfer of desirable alien genes from a wild donor species to a cultivated species. Because of sexual incongruity between the wtld spectes and the cultivated species, the demands of breeders for the transfer of desirable traits, such as disease or stress resistance and apo­ mixis, are insuffictently met by conventiOnal breeding methods. DNA trans­ formatiOn using the tsolated, cloned genes makes it feasible to transfer genes across sexual barners or taxonomic boundanes. However, several of the agro ­ nomically important traits are encoded by polygenes, which are clustered within blocks on specific chromosomes or scattered throughout the genome, and therefore they are not yet amenable to this technique. In addition to sexual mcongruity, and the possible gene clustermg for a locus (e.g., controllmg apomictic reproduction in matze-Tripsacum backcross progeny), the occur­ rence of male sterility, poor seed set, and the low frequency of demed traits hmder the transfer of economically important genes. Also, by backcrossmg, It ts often difficult to eliminate the recombined undestrable donor genes and pre­ vent linkage drag "hitchhiking" genes (1). On the other hand, distant hybnds that cannot be produced by conventional sexual crosses, can be obtained by symmetric somatic hybndization (3). Thus far, the hybrids obtamed after From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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asymmetric hybndization were genetically complex, with several donor chro­ mosomes and unwanted genes, and were often sterile (4-7). Recently, we found that m1croprotoplast-mediated chromosome transfer (MMCT) is an efficient techmque for transferring smgle chromosomes from one species to another (8, 9) . The establishment of procedures for the isolatiOn of small subd1plmd microprotoplasts and the fusiOn of these with recipient protoplasts enabled the development of MMCT technology (10, 11). Usmg the MMCT techmque, we have transferred smgle chromosomes of potato carrying the selectable marker gene for kanamycm resistance (npt/1) and the reporter gene P- glucuronidase (uidA) to tomato (Lycopersiconperuvzanum) and tobacco. Several monosomic addition hybrid plants could be regenerated withm a short period of 3-4 mo after polyethylene glycol-mduced mass fusion between donor potato micro­ protoplasts contaming one or a few chromosomes and normal recipient L. peruvzanum or tobacco protoplasts. These plants contamed one potato chro­ mosome carrying a smgle copy of uidA and one or two copies of the neomycm phosphotransferase (nptll) gene conferrmg kanamycm resistance, together with the complete set of chromosomes of the rec1p1ent tomato or tobacco (8, 9) . The monosomic additiOn plants were phenotypically normal, resemblmg the recipient lines. Genomic m sztu hybridizatiOn (GISH) and RFLP analysis usmg chromosome-specific markers revealed that the monosomic addition plants contained potato chromosome 3 or 5, harboring the npt/1 and uidA genes (12) . The alien genes nptll and uidA were stably expressed in both the tobacco and tomato backgrounds. RFLP analysis and GISH confirmed sexual transmissiOn of the potato chromosome to the BC 1 progeny plants A few BC 1 plants con­ tained the nptll and uidA genes m the absence of the additional potato chromo­ some, indtcatmg that the marker genes had been mtegrated mto the tomato genome. The dtrect productiOn of monosomic additions and the occurrence of donor DNA integration achteved through MMCT m sexually nonhybridtzmg, but related spectes, will be potentially useful for the transfer of economically important traits, introgresstve breedmg and for analyzing plant genome evolu­ tiOn (1, 2, 13, 14) . The mtergenomtc monosomic additions are useful tools for the constructiOn of chromosome-spectfic DNA libraries (15), and for investi­ gatmg the regulation of gene expression related to chromosome-gene domams (14, 16). In v1ew of these perspectives, we have described below in detail the essential reqmrements of the MMCT technique, as established m our investigations: 1. 2. 3 4. 5. 6.

InductiOn of miCronuclei at high frequencies m donor species IsolatiOn of miCronucleate protoplasts m donor species. Isolation and enrichment of small subdiplmd microprotoplasts m donor species Efficient fusiOn of the donor m1croprotoplasts With normal recipient protoplasts Selection of fusiOn products P lant regeneratiOn from fusion products and the production of monosomic addi­ tion hybnds

MMCT for Production of Monosomic Lines

229

2. Materials I . Donor species: We use a suspensiOn cell culture of transformed potato (Solanum tuberosum, line 4 1 3 , 2 n 3x 36) carrying the nptll and uzdA genes (1 7, 18) (see Note 1 ) 2 Recipient plant species, e.g., shoot cultures ofthe wild tomato species L peruvianum (Hygromycm-resistant !me PI 1 28650, 2n 2x 24) (19) or tobacco (Nzcotzana tabacum, cv. Petit Havana, streptomycm-resistant !me SR 1 , 2n 4x 48) (20). 3 . Spmdle toxms· 32 j.!M amiprophos-methyl (APM) (Tokunol M: 0-methyl-0-0[4methyl-6-nitrophenyl]-N-isopropyl-phosphoro thioam1date, Bayer Mobay Corpo­ ration, Agricultural Chemical Division, Kansas Ctty, MO) IS applied to the cell suspension culture from a stock solution (20 mg/mL) m water-free DMSO, and 7.5 )..IM cremart (CR) (Butamtphos, 0-ethyl-0-[3-methyl-6-mtrophenyl]N-sec­ butylphosphorothioamidate, Sum1tomo Chemical Company, Osaka, Japan) from a stock solution ( 1 0 mg/mL) in water-free dimethyl sulfoxide (DMSO) (21,22) . 4. Inhibitors of DNA synthesis: I 0 mM hydroxy urea (HU) is applied to the cell sus­ pension culture as a concentrated, freshly prepared solution in the culture medium (40 mg/mL), and 1 5 )..IM aphidtcolm (APH) from a stock solution (20 mg/mL) m (DMSO) (both from Sigma Chemical Company, St Louis, MO) (10,21). 5. Cell-wall-dtgestmg enzyme mixture· cellulase Onozuka-RI O ( I %), macerozyme Onozuka-R 10 (0 2%) (both Yakult Honsha Co., Tokyo, Japan), half-strength V-KM medium (see Table 1; 23) wtth 0 2 M glucose and 0 2 M manmtol, but no hor­ mones (11) Adjust the osmolality to 500. m/sM/kg and the pH to 5 6 Centnfuge the mixture for 3 mm at 3000 rpm and filter-stenhze The mtxture can be stored for several months at -20°C 6. Cytochalasm-B (CB) (St gma Chemtcal Company) ts applted at 20 )..IM from a stock solutwn (20 mg/mL) m DMSO 7. Chnst Omega ultracentrifuge with swmg out rotor for tubes of about 6 mL, capable of producmg at least 1 05 x g 8. Percoll solutwn (Sigma) 9. Nylon sieves of decreasing pore size 48, 20, 1 5 , 10, and 5 )..lm (Nybott; Swiss Silk Bolting Cloth Mfg Co , Zunch, Switzerland). 1 0. W5 medmm (see Table 2) ts used for mtxmg ofmtcroprotoplasts wtth the recip­ Ient protoplasts for PEG mass fuswn. 1 1 . PEG stock solution for mass fusion is prepared as follows PEG stock solutwn. 30% w/v PEG 4000, 0.3 M glucose, 66 mM CaC12 2H20 1 2. High-pH buffer is prepared from two stock solutions, stock A and stock B stock A. 0.4 M glucose, 66 mM CaC12 2H 20, 1 0% DMSO, stock B: 0 3 M glyc me, pH I 0.5. High-pH buffer Mix stock A and stock B in the ratio of 9: 1 to prepare the htgh-pH buffer solution just before use. 1 3 . Callus mduction (CI) medta· TM-2 for tomato (24,25) and H-460M for tobacco (26) (see Tables 3 and 4). 14. CI liquid media for selectlng fusion products: TM-2 medmm (Table 3) supple­ mented with 50 mg/L kanamycin (Kan 50) prepared from a stock solutton (50 mg/L) in H20 + 25 mg/L hygromycin (Hyg 25) prepared from a stock solution (25 mg/L) in H 20 is used for tomato (18). In the case of tobacco, H-460M medmm =

=

=

=

=

•

•

=

230

Ramulu et a/.

Table 1 Composition of V-KM Medium (23)

Quantity (mg/L)

Component Macroelements KN03 MgS04 ?H20 KH2P04 FeS04 7H20 Na2 EDTA 2H20 CaC12 2H20 Microelements H3B03 MnS04 H20 ZnS04 7H20 Na2Mo04 2H20 CuS04 5H20 CoCI2 6H20 KI Orgamc acids Pyruvic acid Fumanc ac1d Citnc ac1d Mahc ac1d Vitamins d-Ca-panthotenate Cholme

740 0 492.0 34.0 14 0 19 0 368 0

•

•

·

1 .5 5.0 1 .0 0. 1 2 0.0 1 2 0.0 1 2 0 38

·

•

·

·

1 0.0 20.0 20 0 20 0 05 05

Component Vitamms (cont) Ascorbic ac1d p-Ammobenzoic ac1d Nicotinic acid Pyndoxme-HCI Th1amme-HCI Folic ac1d Biotin Vitamm A Vitamin D3 V1tamm B 1 2 Sugars Mannitol Sorbitol Sucrose Fructose Ribose Xylose Mannose Rhamnose Cellobiose Myo-mos1tol pH

Table 2 Composition of WS Medium

Component NaCI CaCI2 2H20

KCI Glucose pH

Quantity, g/L 9

18 3 0.37 0.90 58

Quantity (mg/L) 1 0 0.0 1 0.5 05 50 0 2

0.005 0.005 0.005 O.Q l 1 25 0 1 25 0 1 25 .0 1 25 0 125 0 1 25 0 1 25 0 1 25.0 1 25 0 50 0 56

MMCT for Production of Monosomic Lmes

23 1

Table 3 Composition of the Media Used for Tomato Component KH2P04 CaCI 2 2H2 0 KN03 NH4N03 NH4H2P04 (NH4hS04 MgS04 7H20 KI H3B03 MnS04 4H 20 ZnS04 7H 20 Na2 Mo04 2H2 0 CuS04 5H20 CoCI 2 6H20 FeS04 7H20 Na2EDTA Nicotimc acid Th1amine-HCI Pyndoxme-HCI Biotm o-Ca-pantothenate Chohne chloride Glycme Casem hydrolysate L-Cysteme Malic actd Ascorbic ac1d Adenine sulfate L-Glutamine Myo-mositol R1boflavine Sucrose Manmtol Xyhtol Sorb1tol MES Agar NAA Zeatin 2,4-D BAP GA3 pH •

·

•

•

•

TM-2, mg/L, (24,25)

TM-3, mg/L, (24,25)

1 70 0 440.0 1 500.0

1 70 0 440 0 1 500 0

370.0 0 83 6.20 22 30 8 60 0 25 0 025 0.025 1 3 90 1 8 50 2 50 10 0 I 0 0.05 0 50 0 10 0.50 1 50 0 1 .0 10 0 0 50 40.0 1 00 0 4600 0 0.25 7% w/v 4560.0 3800 0 4560.0 97.60 1 0 1 .0

5.6

370.0 0 83 6 20 22.30 8.60 0 25 0 025 0.025 1 3 90 1 8 50 50 50 0 50 0 05 0 10 2 50 1 00.0

TM-4, mg/L, (24,25) 1 50 1 900.0 320 0 230 0 1 34.0 247.0 0.83 6.20 22.30 8 60 0 25 0 025 0.025 1 3 90 18 50 5.0 50 0.50 0 05 0 10 2 50

40.0 100.0 1 00.0

1 00 0

5% w/v

3% w/v

97 60 7% w/v

97.60 7% w/v

0.2 0.5 58

20 0.2 58

Table 4 Composition of the Media Used for Tobacco H-460M mg/L (26)

Component NaH2P04 H20 Ca(H2P04)2 H20 CaCl2 2Hp KN03 NH4N03 (NH4hS04 MgS04 7H20 KH2P04 (NH4h succinate H3B03 MnS04 H20 ZnS04 7H20 KI Na2Mo04 2H20 CuS04 SH20 CoCl2, 6H20 Na2EDTA FeS04 7H20 Ca-panthotenate Myo-inosttol Ntcottmc actd Pyridoxme Thtamine-HCl Biotin Casem hydrolysate Fructose Xylose Sorbttol Mannitol Glucose H20 Sucrose NAA BAP 2,4-D Agar pH ·

·

·

·

·

1 70.0 50.0 900.0 2500.0 250.0 1 34 0 250 0

1 24 3 .38 2 12 0 17 0 05 0 005 0 005 37.30 27.80 1 .0 1 00.0 1 0 1 0 5 .0 0 01 1 25 .0 250.0 250 0 250.0 250 0 80 g 250.0 l 0 0.2 0 l 5.8

232

AG mg/L (1 8)

MSR- 1 mg/L (18)

440.0 1 000.0 800 0

440.0 1 900.0 1 650.0

740 0 1 36.0 1 00 0 0 62 1 .7 1 06 0 08 0 03 0 003 0.003 37.30 27.80 1 .0 100.0 1 0 1 0 5.0 0 01

370 0 1 70.0 62 1 7.0 10 6 08 0.25 0.025 0.025 37 30 27.80 1 .0 1 00 0 1 0 1 0 50 0 01

50 g 30 g 0l 1 .0

30 g

8g 5.7

8g 5.8

0.25

MMCT for Production of Monosomic Lines

233

Table 5 Composition of Rooting Medium for Tomato : MS Medium Supplemented with 2% Sucrose Component Macroelements CaCI2 KH2P04 KN03 MgS04 NH4N03 M1croelements CoCl2 6H20 CuS04 5H2 0 FeNaEDTA H3B03 KI MnS04 H20 Na2Mo04 2H20 ZnS04 7H20 ·

•

Quantity mg/L 332.02 1 70.00 1 900.00 1 80 54 1 650 00 0.025 0.025 36 70 6.20 0.83 1 6.90 0.25 8.60

Quant1ty Component Vitamins Glycine Myo-mos1tol Nicotmic acid Pyndoxine HCl Thiamme HCI Sugars Sucrose pH

mg/L 2 00 1 000.00 0.50 0.50 0 10 V1tamms 20 g 58

supplemented with 50 mg/L kanamycm (Kan 50) prepared from a stock solut10n (50 mg/L) in H20 is used (18) . 1 5 Sohd media, i.e., callus growth med1a (CG) for selecting fusion products TM-3 medium (Table 3) supplemented with I 00 mg/L kanamycin (Kan 1 00) prepared from a stock solution (50 mg/mL) in H20 + 50 mg/ L hygromycin (HYG 50) pre­ pared from a stock solution (25 mg/mL) m H20 IS used for tomato (18). In the case of tobacco, AG medium (Table 4) supplemented with 1 00 mg/L kanamycin (Kan 100) prepared from a stock solution of (50 mg/mL) m H20 IS used (18). 1 6 RegeneratiOn media· TM-4 medmm is used for tomato (Table 3), and MSR- 1 for tobacco (Table 4). 1 7 . Rooting med1a: MS supplemented with 2% sucrose for potato m1croprotoplast (+) tomato protoplast fus10ns (Table 5), and MS supplemented with 2% sucrose and indole butyric ac1d (0.25 mg/L) for potato (+) tobacco fus10ns. The compositiOn of rooting medium for shoots denved from potato (+) tobacco fusions 1s the same as above, i.e., MS medmm, but 1s supplemented with mdole butyric acid (0.25 mg/L).

3. Methods 3. 1. Induction of Micronuclei 1 . For species/genotypes, which respond to synchronization, e g . , Nzcotzana plumbagmifo/za, treat early log-phase suspensiOn cells at day l after subculture w1th the mh1bltors of DNA synthesis HU at 1 0 mM or APH at 1 5 J..LM for 24 h.

234

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2 Wash the suspension cells free of HU or APH w1th culture medium, and treat w1th APM at 32 � for 48 h or CR at 3 7 or 7.5 J..LM for 48 h. Figure 1 A shows the various steps and processes mvolved m micronucleation (see Notes 2 and 3). 3 For species/genotypes in wh1ch spontaneous cell synchrony occurs (e g , potato line 4 1 3), treat early log-phase suspension cells at day 1 after subculture w1th CR at 3 7 or 7.5 J..LM or with 3 2 � APM 4 Incubate the APM- or CR-treated suspenswn cells m the cell-wall-d1gestmg enzyme mixture, supplemented w1th CB (20 J.!M) and e1ther APH (32 J.!M) or CR (7.5 J.!M) for 16 h in 9-cm Petn d1shes contammg 1 . 5-mL packed cell vol­ ume and 1 5-mL enzyme solution on a gyratory shaker (30 rpm) at 28°C (see Notes 4 and 5) 5 Filter the suspension of protop lasts through 297- and 88-J.!m nylon meshes, and wash w1th an equal volume of half-strength V-KM medmm, contammg only macro- and m1croelements (Table 1 ) and 0.24 MNaCI (pH 5 .6) Punfy the proto­ plasts by repeated washmg and centnfugatwn three to four times (see Notes 6 and 7) Resuspend the pellet m sucrose solution (0 43 M), and load onto an already prepared Percoll grad1ent (see below)

3.2. Isolation of Microprotoplasts I

Prepare in advance the Percoll gradient by addmg 7.2% (w/v) manmtol to the Percoll solution (S1gma), filter-stenhze, and centnfuge for 1 h at 1 05g to produce a contmuous iso-osmotic grad1ent of Percoll Afterwards, remove the top layer of about 5 mm 2 Load the punfied dense suspenston of protoplasts (mono- and m1cronucleated) onto the continuous !So-osmotic gradient of Percoll (Sigma), and centnfuge at h1gh speed, I 0 5g for 2 h m a Chnst Omega Ultracentnfuge, using a swmg-out rotor. Several bands contaimng a m1xture of evacuolated protoplasts, microproto­ plasts of various s1zes, and cytoplasts form at vanous distances from the top of the centrifuge tube. Figure 18 g1ves a schematiC representation of vanous steps for the isolatiOn of m1croprotoplasts (see Note 5)

Fig. 1 . (opposite page) (A) Vanous steps and processes mvolved in mlcronucle­ atton Inductton of micronuclei in donor suspenston cells after treatment With the mhib1tors of DNA synthesiS, HU (I 0 mM, 24 h) or APH ( 1 5 �. 24 h) followed by treatment with the spmdle toxms APM (32 J.!M, 48 h) or CR (3 7 or 7 5 J..LM, 48 h) Afterwards, m1cronucleated cells are mcubated m a cell-wall-d1gestmg m1xture, which also contains CB (20 11M) and APH (32 J,!M) or CR (7 5 J.!M) for 1 6 h to Isolate micronucleated protoplasts. (B) High-speed centnfugatwn ( I 05 x g) of a suspen­ siOn of micronucleated protoplasts and mononucleate protoplasts for the Isolation of md1vidual m1croprotoplasts (MPPS) and protoplasts (PPS), and ennchment of MPPS by sequential filtratiOn through nylon s1eves of decreasmg pore size to Isolate smaller subdipl01d microprotoplasts (reproduced with permissiOn, 18)

235

MMCT for Production of Monosomic Lines

A Donor cell

B Micronucleated protoplast

protoplast

t

Treatment with HU or APH

t

Treatment with APM or CR

High-speed centrifugation (100,000 X g)

t

P-.,. --*- .

Band contalna 1.5

MPPSC 2 ·12

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3.0

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101)

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Chromosome decondensatlon

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Enrichment of MPPS through aequenUal flltraUon

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20

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Mlcronucleated protoplast

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236

Ramulu et a/.

3.3. Isolation and Enrichment of Small Subdiploid Microprotoplasts

2 3

Collect the bands, and filter them sequentially through nylon steves of decreasmg pore size (48, 20, I 5, l 0, and 5 !lffi) (see Notes 6 and 7). Rmse wtth manmtol (0 4 M) Collect the filtered fracttons m mannitol ( 0.4 M), and centrifuge at 80g for l 0 mm Recentrifuge the supernatant at I 60g for l O mm Resuspend the pellets m manmtol, and analyze the samples by light and UV microscopy to esttmate the number of FDA-stamed microprotoplasts and by microdensitometry and flow cytometry to determine the nuclear DNA content of the mtcroprotoplasts

3.4. Fusion of Donor Microprotoplasts with Recipient Protoplasts

2 3 4.

5

Mix the donor microprotoplasts and recipient protoplasts m a ratio of 2 I (e.g , 3 0 x 1 05 mtcroprotoplasts of potato 1 .5 x 1 05 protoplasts of tomato or tobacco) In 6-cm Falcon Petn dishes, place 5 separate 60-).lL drops of of protoplasts sus­ pended m W5 medmm (18, 24) to give a final platmg denstty of I x 1 0 6 /mL for PEG-based mass fusiOn. After 20 mm, add PEG (PEG 4000) to give a final concentration of 8-1 0%. After 7 mm, carefully remove the PEG and W5 medmm usmg a Pasteur pipet, and slowly add 20 ).lL high-pH buffer solutiOn to each group of cells Twenty minutes later, when the microprotoplast-protoplast mixture has agam settled, rinse the dish gently with 3 mL of H460 M medmm for fuswn samples of potato (+) tobacco and With TM2 medmm for fuswns of potato (+) tomato As a control for microprotoplast (+) protoplast fuswns, carry out symmetnc fuswns between the donor cell suspenswn protoplasts and the rectpient leaf pro­ toplasts under the same conditions and at a similar platmg density

3.5. Selection of Fusion Products 1

Isolate the hybrid calli usmg selectiOn pressure w1th antibiotics, e.g , kanamycm and hygromycm, as m the case of fustons of potato microprotoplasts (+) tomato protoplasts, or only kanamycm, as m the case of potato microprotoplasts (+) tobacco protoplasts dtlutwn and selectiOn m CI hqmd medmm (18,24-26) on days 12 and 1 9, and selection on sohd callus growth (CG) medmm (l8, 24-26) on day 2 5 (see Note 8). 2 Transfer the resistant calli (when turned green) to regeneratiOn medmm contam­ ing no kanamycm or hygromycin 3 Transfer the regenerated shoots to rootmg medmm, e.g., MS supplemented With 2% sucrose m the case of potato microprotoplasts ( +) tomato protoplast fustons (Table 5), and MS supplemented With 2% sucrose and mdole butyric acid (0 025 mg/L) m the case of potato-tobacco fusions

3. 6. Production of Monosomic Addition Hybrid Plants I . Analyse the regenerated plants for the expressiOn of donor marker genes, e g., kanamycm reststance and GUS, as m potato (+) tomato fuswns, or only kanamy­ cm reststance as m potato (+) tobacco fuswns to tdenttfy the hybrid plants (8, 9) .

MMCT for Production of Monosomic Lines

237

2. Determine the chromosome compositiOn of the hybnd plants by GISH to select the monosomic additiOn plants contaimng a smgle donor chromosome carrying the marker genes, and a complete chromosome set of the recipient parent, e g., 1 potato chromosome and 24 tomato chromosomes, as m potato ( +) tomato fusiOns (8, 9, 12) (see Notes 9 and 1 0) .

4. Notes l . N. plumbagmifolia VIviani (Doba hne 1- 1 25 - 1 line resistant to kanamycin) has been used as a model plant species in our investigations to establish optimum conditions, together with the donor potato line 4 1 3 for the production of mono­ somic additions in tomato and tobacco backgrounds Cremart at 7 5 !JM for S tuberosum and APM at 32 !J.M for N plumbagmifolia gave the highest frequency of micronucleated cells. 2 Micronucleatwn in suspensiOn cells/protoplasts occurs through modificatiOn of mitosis under the influence of the spmdle toxms APM or CR The metaphase­ blocked cells/protoplasts with single or groups of chromosomes show no diVI­ sion of centromere or chromatid separatiOn (no anaphase), but decondense and form micronuclei. Thus, the higher the frequency of metaphases, the greater the yield of micronuclei Will be 3 . The yield of micronuclei depends not only on the frequency of micronucleated protoplasts, but also on the frequency ofmicronuclei/micronucleated protoplast The sequential treatment with HU or APH, followed by APM or CR treatment, results m a synchronous progression of cells through S-phase to G2/M, enhanc­ mg the frequency of micronuclei 4. Further, the presence of CB m the enzyme mixture can positively influence the micronucleatton of protoplasts. In potato and N. plumbagmifoha, the frequency of miCronuclei could be mcreased two- to threefold by incubatmg CR- or APM­ treated suspension cells m an enzyme mixture containing CB and CR/APM CB disrupts the microfilaments, whereas CR avmds the reformation of miCrofila­ ments in micronucleated protoplasts Therefore, miCronuclei formed pnor to enzyme incubation can be maintained m a stable state without undergomg fuswn and restitution. Additionally, the chromosomes m the remaming metaphases and prophases from the late dividmg cells decondense and form micronuclei, thus mcreasing the percentage of micronucleated protoplasts. 5 The yield of microprotoplasts strongly depends on an efficient fragmentation of micronucleated protoplasts and the presence of CB and APM/CR during ultra­ centrifugation. To Isolate mtcroprotoplasts from micronucleated protoplasts, the protoplasts have to be fragmented, whtle maintaining the integnty of the plasma membrane. The presence of mtcrofilaments opposes changes to the shape of pro­ toplasts under the influence of g forces during ultracentnfugation, thus resulting in a lower yteld of subprotoplasts Therefore, the miCrofilaments have to be dis­ rupted by mcubation with CB, combined with APM/CR to avmd reformation of miCrotubules m miCronucleated protoplasts. Also, high-speed centrifugatiOn at I 05 x g IS required for a longer duratiOn (2 h) to obtam a better fragmentatiOn and separation of mtcroprotoplasts.

Ramulu et at.

238

(+)

RECIPIENT PROTOPLAST

!

G2 (4C=24 chromosomes)

D ONOR MICROPROTOPLAST COne chromosome

with two Intact chromatids)

CD

FUSION PRODUCT (G2)

Enters Into mitosis without extra DNA synthesis MONOSOMIC ADDITION UNE

24

' +,

Fig. 2 Production of a monosomic additiOn I me With a complete chromosome set (24 chromosomes) and one donor chromosome after fuswn of a recipient protoplast with a donor m1croprotoplast ( e g , potato) (reproduced w1th permisswn; 18)

6. In addition to the yield of m1croprotoplasts, the frequency of smaller subd1pl01d miCroprotoplasts containing single chromosomes IS critiCal for the successful pro­ duction of monosomiC additiOn hybrids after fusiOn. Therefore, sequential filtra­ tion w1th nylon sieves of decreasmg pore s1ze IS essential to separate the small m1croprotoplasts from the larger ones 7. A repeated washmg of micronucleated protoplasts with a washmg mediUm con­ tainmg manmtol (0 4 M) is important to avOid clogging of sieves dunng the pun­ ficatwn step, thus mcreasmg the total yield of m1cronucleated protoplasts. 8. The populatiOn of small subdiplOid m1croprotoplasts with a partial genome (con­ taimng one to four chromosomes) ennched after sequential filtration remams mtact and viable (FDA-positive) for several days, but should not regenerate cell walls or undergo cell divlSlon (as shown for N. plumbagznifoila or potato miCroprotoplasts) The lack of cell diVISion activity of the donor m1croprotoplasts with a part1al genome can be an additiOnal advantage m fus1on expenments, because it avOids contammatwn of the donor partner wh1le selectmg for the fuswn products

239

MMCT for Productton of Monosomic Lines

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DONOR MICROPROTOPLAST (One chromosome

RECIPIENT PROTOPLAST (G1)

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FUSION PRODUCT Earlier progression of recipient genome to M (G1 - S - G2 - M)

I l

Progression of donor G1 chromosome to S phase (G1 - S)

,.

Donor chromosome/DNA breakage due to pre1 chromosome condensation (PCC) f lnduced by recipient chromosomes

© ...

DNA INTEGRATION

Fig. 3 . Donor DNA mtegration in rectptent chromosomes after fusiOn of a rectptent protoplast with a donor mtcroprotoplast (reproduced with penmssion; 18)

9. By microprotoplast fuston, the regeneratton abtlity of the rectpient hoe remains unchanged as compared to that after protoplast-protoplast fusion (symmetric fusion). Since the donor protoplast contains a single chromosome m mterphase and a small amount of cytoplasm surrounded by an intact plasma membrane, tts mtegratwn mto the reciptent genome ts stable, thus producing monosomic addi­ tion hybrids with one donor chromosome and a complete chromosome set of the recepient species (Fig. 2). 1 0. From potato microprotoplast (+) tomato protoplast fusiOn, several monosomtc addition plants (2n 24 + l ) were regenerated withm a short penod of 3-4 mo Also, donor DNA integratiOn can occur in mtcroprotoplast hybrid plants. A delayed replication of the addition chromosome and mttottc failure mtght lead to chromosome/DNA breakage and integration of donor DNA into the reciptent genome (Fig. 3) The mtegratton of donor DNA occurred 10 the progeny after =

240

Ramulu et at. backcrossing of monosomic addition hybnds with the rec1p1ent species (I.e., m potato microprotoplasts [+] tomato protop lasts) (9, 12), showing sexual transmis­ siOn of the ahen gene. The progeny with the integrated donor DNA can be Iden­ tified by usmg the marker genes (e.g , the reporter mdA gene or the selectable nptll gene). In generatively produced addition hnes, the mtegrat10n of donor DNA/chromosome segments apparently occur by similar mechamsms· double­ strand breaks of the donor chromosome dunng Its dismtegratwn at some stage in meiosis/mitOSIS, followed by repair-induced mvasion and mtegrat10n of donor DNA mto the host chromosomes (1) Thus, the loss or disintegration of the addi­ tiOnal donor chromosome can occur after the productiOn of monosomic additiOn plants Consequently, the frequency of the monosomic addition plants as well as the stability and mamtamance of the added donor chromosome m subsequent somatic/generative cycles might depend on vanous factors, 1 e , chromosome type, ploidy level of the recipient, genotypic background, and so forth (26) The fre­ quency of donor DNA mtegrat10n can be determmed by usmg appropnate markers

References Sybenga, J ( 1 992) Cytogenetics m plant breeding. Monographs on Theoretzcal and Applied Genetzcs, vol 1 7 . Springer-Verlag, Heidelberg 2 Fedak, G ( 1 992) Perspectives on wide crosses m barley Barley Genetzcs News­ letter, vol 6 Department of Agronomy, Colorado State Umvers1ty, Fort Colhns, CP, pp 683-689. 3 . Jacobsen, E., de Jong, J H., Kamstra, S. A., van den Berg, P M M. M , and Ramanna, M S. ( 1 994) The first and second backcross progeny of the mterge­ nenc fusiOn hybnds of potato and tomato after crosses with potato Theor Appl Genet. 88, 1 8 1-1 86. 4. Famelaer, I , Gleba, Y Y , S1derov, V A., Kaleda, V A , Parokonny, A S , and Boryshuk, N V ( 1 989) Intrageneric asymmetnc hybnds between Nzcotzana plumbaginifolza and Nzcotzana sylvestrzs obtamed by "gamma-fuswn." Plant Scz 61, 1 05-1 1 7 5 WIJbrandi, J , Zabel, P., and Koomneef, M ( 1 990) Restnction fragment length polymorphism analysis of somatic hybrids between Lycoperszcon esculentum and madiated L peruvzanum evidence for hm1ted donor genome eliminatiOn and extensive chromosome rearrangements Mol Gen Genet 222, 270--2 77 6 Pmte, K J and Schaart, J G ( 1 993) Nuclear genomiC compositiOn of asymmetnc fusion products between Irradiated transgemc Solanum brevzdens and S tuberosum

1

limited elimination of donor chromosomes and polyploidizatwn of the recipient genome Theor Appl Genet 86, 23 7-244 7. Wolters, A. M. A , Jacobsen, E , O'Connel, M , Bonnema, G , Ramulu, K. S., and de Jong, J H ( 1 994) Somatic hybnd1zauon as a tool for tomato breedmg Euphytzca 79, 265-277 8. Ramulu, K. S , DIJkhuis, P., Rutgers, E., Blaas, J., Krens, F. A., and Verbeek, W. H. J. ( 1 996) lntergenenc transfer of partial genome and direct productiOn of monosomic addition plants by m1croprotoplast fuswn. Theor Appl Genet 92, 3 1 6--3 25.

MMCT for Production of Monosomic Lmes

24 1

9. Ramulu, K. S., Dijkhms, P., Rutgers, E., Blaas, J., Krens, F. A , and Dons, J. J. M. ( 1 996) Microprotoplast mediated transfer of single chromosomes between sexually mcompatible plants Genome 39, 92 1-933 10. Verhoeven, H A. and Ramulu, K. S . ( 1 99 1 ) Isolation and chractenzation of mtcroprotoplasts from APM-treated suspenston cells of Nzcollana plumbagmifolza Theor. Appl Genet 82, 346-352. I I Ramulu, K. S., Dijkhms, P , Famelaer, 1 . , Cardt, T , and Verhoeven, H. A. ( 1 993) Isolation of subdtplmd mtcroprotoplasts for parttal genome transfer m plants: enhancement of mtcronucleatwn and ennchment of microprotoplasts wtth one or a few chromosomes Planta 190, 1 90-- 1 98. 1 2. Rutgers, E , Ramulu, K. S., Dtjkhuis, P., Blaas, J., Krens, F A., and Verhoeven, H. A.( l 997) IdentificatiOn and molecular analysis of transgemc potato chromo­ somes transferred to tomato through microprotoplast fusion. Theor. Appl Genet. 94, 1 053-1059 1 3 Sybenga, J. ( 1 989) Genetic mampulation. generative vs somatic, m Biotechnol­ ogy m Agnculture and Forestry 9 Plant Protoplasts and GenetiC Engeneenng II (Bajaj, Y P S., ed ), Spnnger-Verlag, Hetdelberg, pp. 26-53. 1 4 Heslop-Harrison, J S., Lettch, A. R , and Schwarzacher, T ( 1 993) The physiCal orgamzatwn of mterphase nuclet, m The Chromosome (Heslop-Hamson, J. S and Flavell, R B eds ), Bws, Oxford. pp. 22 1-232. 1 5. Schondelmater, J , Martin, R , Jahoor, J , Houben, J , Graner, J , and Koop, H U ( 1 993) Mtcrodissectwn and mtcroclonmg of the barley (Hordeum vulgare L ) chromosome 1HS Theor Appl Genet 86, 629-636 16. B ennett, M D ( 1 988) Parental genome separatiOn m F 1 hybrids between grass spectes, in Proceedmgs ofKew Chromosome Conference III (Brandham, P.B, ed ), Her MaJesty's Stationary Office, London, pp 1 95-208 1 7 . Gtlissen, L J., Ramulu, K S., Fhpse, E , Memen, E , and Stlekema, W J ( 1 99 1 ) Transformation of dtplotd potato genotypes through Agrobacterzum vectors and expressiOn of T-DNA markers m root clones, regenerated plants and suspenswn cells. Acta Bot Neerl 40, 53-6 1 . 1 8 Ramulu, K. S , Dtjkhms, P., Rutgers, E., Blaas, J., Verbeek, W. H. J , Verhoeven, H. A., and Cohjn-Hooymans, C M ( 1 995) Microprotoplast fuswn techmque. a new tool for gene transfer between sexually mcongruent plant spectes. Euphyt1ca 85, 255-268. 1 9. Koomneef, M., Hanhart, C G , and Martmelh, L. ( 1 987) A genetic analysts of cell culture tratts m tomato Theor Appl Genet 74, 63 3-64 1 20 Mahga, P., Sz.-Brenovits, A., Maarten, L., and Joo, F. ( 1 975) Non mendehan streptomycin resistant tobacco mutant with altered chloroplasts and mttochon­ dna. Nature 255, 40 1-402 2 1 . Ramulu, K. S., Dijkhms, P , Famelaer, 1., Cardt, T., and Verhoeven, H. A. ( 1 994) Cremart: a new chemtcal for effictent mductwn of mtcronuclei in cells and proto­ plasts for parttal genome transfer Plant Cell Rep 13, 687-69 1 . 22. Verhoeven, H. A , Ramulu, K S., Gihssen, L J W., Famel aer, 1., Dtjkhms, P., and Blaas J ( 1 99 1 ) Partial genome transfer through micronuclei in plants Acta Bot. Neerl 40, 97-1 1 3 .

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23 Bokelmann, G S. and Roest, S. ( 1 983) Plant regeneration from protoplasts of potato (Solanum tuberosum cv. B intje) Z Pflanzenphysiol 109, 259-265 24 Shahm, E. A ( 1 985) Totipotency of tomato protoplasts Theor. Appl Genet 69, 23 5-240. 25. Derks, F. H. M., Hakkert, J C., Verbeek, W. H. J., and Cohjn-Hooymans, C. M. ( 1 992) Genome composition of asymmetric hybnds m relation to the phyloge­ netic distance of the parents Nucleus-chloroplast interactiOn. Theor. Appl Genet 84, 930-940. 26 Nagy, J I and Mahga, P. ( 1 976) Callus inductwn and plant regeneration from mesophyll-protoplasts of Nicotzana sylvestrls Z. Pflanzenphyswl 78, 453-455 27 Smg, R.G ( 1 993 ) Plant Cytogenetzcs CRC, Boca Raton, FL

22

__ __ __ __ __ __ __ __ __ __ __

Guard Cell Protoplasts Isolation, Culture, and Regeneration of Plants

Graham Boorse and Gary Tal lman 1 . Introduction

The guard cells that flank stomata undergo environmentally induced, tur­ gor-dnven cellular movements that regulate stomatal dimensions. Under many environmental conditions, changes in stomatal dtmenswns regulate rates of transpiration and photosynthetic carbon fixation (1). Among the environmental signals that guard cells receive and transduce are light quality, light intensity, intercellular concentrations ofleafcarbon dioxide, and apoplastic concentratiOns of abscisic acid (ABA) (2). How guard cells mtegrate the variety of Signals present in the environment and activate the appropriate signal transduction mechamsms to adjust stomatal dimensions for prevailing environmental condi­ tions is the subject of intense investigation (3) . Many fundamental studies of stomata have been performed with detached leaf epidermis, but for many types of experiments, epidermis is not an adequate material. Guard cells are among the smallest and least numerous of cell types in the leaf, and the presence of even a very small number of contammatmg cells in epidermal preparatiOns (e.g., epidermal cells that neighbor guard cells and mesophyll cells) can result m significant expenmental artifacts (4). This is particularly true of experiments that involve guard cell biochemistry or mole­ cular biology and/or experiments in which guard cell metabolism is measured. Furthermore, the presence of the relatively thick guard cell wall precludes cer­ tain types of studies (e.g., electrophysiology). Good methods for makmg large (::::: I x 10 6 cells), highly punfied prepara­ tions (<0.0 1 % contamination with other cell types) of guard cell protoplasts (GCP) were first reported about 1 5 years ago (4-7). The availability of these From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R 0 Hall © Humana Press Inc , Totowa, NJ

243

Boorse and Tallman

244

methods (8) contributed sigmficantly to a resurgence of interest in the baste cell biOlogy of guard cells. GCP have been used to demonstrate that guard cells possess: 1. 2. 3. 4. 5

Both PSI and PSII acttvtty (4) A functional photosynthetic carbon reduction pathway (9) A blue light-activated plasma membrane H+ -translocating ATPase (1 0-12) Voltage-gated, mward rectifymg plasma membrane K+ channels (13, 14). Outward-rectifymg plasma membrane anion channels (see 15 for a revtew of wn channel studies usmg GCP).

GCP have been used to mvestigate: 1

Regulation of K+ channel activity m the guard cell pl asma membrane (15, 1 6) , 2 2 ElevatiOn of cytosolic free Ca + by movement of extracellular Ca + through AHA­ activated, Ca2 +-permeable channels (15), and 3. Ca2 +-dependent and Ca2 +-mdependent ABA stgnal transductiOn pathways (15)

2

Recently, two partial-length cDNAs codmg for dtfferent plasma membrane H+ -ATPase isoforms have been isolated from GCP of View faba L. and have been used to locahze expression of these genes (1 7) Most studies using GCP have been short-term experiments of a few hours ' duratiOn. Until recently, no in vitro experimental systems existed that would enable the study of responses of GCP to envtronmental stgnals admmistered over longer penods. However, GCP have now been established and main­ tained in culture (18-21) . As with any culture system, an advantage of cultur­ mg GCP is that culture condttlons can be rigorously defined and carefully controlled. An additional advantage of cultunng GCP is that the homogeneity of such preparations confers on them a umformity of response that ts seldom observed m cultures of mixed-cell types (20, 21). In the latter, Identification of the cell type responding to a change m culture condition may be difficult. If the responding cell type is Identified, the basts for any particular response to a change m a culture condition (e.g. , an mcrease or decrease m synthesis of a particular protein or transcription of a particular gene) may still not be identifi­ able, because the condition can evoke from each umque cell type a response that affects the response(s) of each and/or every other cell type. Monocultures of GCP hold promise for studies of the stgnal transduction mechamsms underlymg: 1 . Cellular dtfferentiatton. 2 Chloroplast senescence 3 . RegulatiOn of the cell cycle m plants, processes that are only acttvated by longer penods of exposure to environmental stgnals.

Guard Cell Protoplasts

245

Each of these processes can be activated and directed ill cultures of GCP by manipulating concentrations and/or ratios of plant growth regulators (auxill, cytokinins, or ABA [18,20, 22]) and/or temperature (20) . For example, when GCP of Nicotiana glauca are cultured at temperatures <34°C, they dedifferen­ tiate and divide to form a callus from which plants can be regenerated (22). In the process of dedifferentiatiOn, their chloroplasts senesce (20). When they are cultured at temperatures >34°C in media containing 0. 1 � ABA, they remain differentiated. GCP cultured at temperatures >34°C for 1 wk do not dtvtde (20), but when they are transferred to temperatures <34°C, at least a small percentage redtfferentiate and divide (unpublished). These data indicate that ill monocultures of GCP, alteration of only one or two culture conditwns (±ABA, temperature) is sufficient to: I . Maintam cultured GCP m or trigger entry of cultured GCP into certain stages of the cell cycle. 2 Determine whether GCP remain differentiated or dedtfferenttate. 3 Determine whether or not chloroplast senescence I S activated

Cultured GCP of N. glauca (tree tobacco) and Beta vulgaris (sugar beet) have been used to produce fnable, embryogenic callus from which plants have been regenerated, demonstrating that GCP are totipotent (22-24). Regenera­ tion of plants from cultured GCP of B. vulgaris is of major commerctal tmpor­ tance, because callus denved from other cell types, tissues, and organs of this plant are recalcttrant to regeneration (24) . Cultured GCP of B vulgarts have already been used to produce transgemc plants with enhanced reststance to herbicides by a relatively raptd protocol (25). GCP are isolated usmg a two-step procedure. To remove contammatmg mesophyll and epidermal cells, detached eptdermis ts treated with a mix­ ture of cellulase and pectmase dissolved m a hypotomc solutiOn. Because the cell walls of contammating epidermal and mesophyll cells are thinner and are of a different chemical composttion than those of guard cells, they are digested more quickly than those of guard cells. As protoplasts of con­ tammatmg epidermal cells and mesophyll cells are released into the hypo­ tome medium, they swell and burst. After sufficient time has passed to destroy contaminatmg cells, the remaming cutlcle contaming guard cells ts collected on a nylon net, rinsed, and transferred to a solutiOn of cellulase and pectinase in a solution that ts slightly hypertonic to guard cells. After a few hours of dtgestion, GCP are released into the medmm. The cells are collected by filtration and centrifugation, washed, and suspended in cul ture media (Fig. 1 ) .

246

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Fig. I. Guard cell protoplasts isolated from leaves of N. glauca (Graham), tree tobacco. Differential interference contrast optics; large organelles in protoplasts are chloroplasts. Average diamter is ::::. 1 5 jlm.

2. Materials

2.1. Plants 1 . Seeds ofNicotiana glauca (Graham), tree tobacco (see Note 1). 2. Pots (0.16-, 2.0,- 10-L), potting soil, and sand. We have used Supersoil, Chino, CA, or Pro-Mix "HP" High-Porosity Growing Medium, Premier-Western US, Laguna Niguel, CA. Sand is 30 grade. Plants grow best in well-drained, porous media. 3. Environmental growth chamber: Conviron Model E7 (Conviron, Pembina, ND) 4. Fluorescent light bank: Lamps are model FT72Tl2.CW.l 500, General Electric, Cleveland, OH, or equivalent. 5. Modified Hoagland's nutrient solution (Table 1). 2.2. Day Before Culture I . 2-L Erlenmeryer flasks with cotton stoppers covered with cheesecloth (4), paper

towels, aluminum foil, and autoclave tape. 2. Pyrex casserole dish: 1 1 x 7 x 1.5 in. (1). 3. Pyrex casserole dish: 11 x 7 x 1.5 in. (1) containing: fine-point forceps wrapped individually in foil (1), glass plate: 4.5 x 4.5 x 0.13 in. (1), 6-in. cotton swab (1), single-edge razor blade (1), Petri dish (glass, bottom only}, 15 x 60 rnm (1). 4. Plastic beakers: 2 L (1), 1 L (1).

24 7

Guard Cell Protoplasts Table 1 Modified Hoagland's Nutrient Solution (26)

Constituent

I. 2. 3. 4. 5.

NH4H2P04 KN03 Ca(N03)z 4H20 MgS04 7H20 H3B03 MnS04 H20 ZnS04 7H20 CuS04 5H20 Mo03 NaCI 6. Iron solution FirSt dissolve: Na2 EDTA Then add: FeS04 7H20 7 KOH ·

•

•

•

•

To prepare nutnent

g/L m stock solution

Fmal concentratiOn in nutnent solution after dllutwn of stock, mM

2 1 .0 1 09.0 1 70.0 43.6 0.56 0.308 0.042 0.0 1 8 0 01 1 1 .045

1 .0 6.0 40 2.0 0.4 10 0 08 04 0.4 100 0

60 4.5 4.0

0 09 0 09 7 1 .3

solutwn from stock solutwns, add 1 0 0 mL each of solutwns

50 mL of solutiOn 7 to a 20-L carboy Add water to 1 8 L, shake to m1x

1-6

and

5. Plastic beaker: 2 L (1) containmg. plastic, disposable Erlenmeyer flasks With screw caps-125 mL (3; loosen caps and cover with foil), plastic funnels with top dmm­ eter = 3.5 in., stem diameter = 0.5 in., (2; one funnel is lined with 220 x 220 !liD mesh nylon net; the other is lined with a 30 x 30 !!ill mesh nylon net; nets are secured to rims of funnels with autoclave tape; wrap funnels in foil Nylon nets are N1tex® from TETKO®, Inc., I l l Calumet St., Depew, NY 14043).

2.3. Isolation of Guard Cell Protoplasts I . Plastic bag for holding leaf in moist paper towels. 2. Gyratory shaking water bath, pH meter, balance. 3 . SolutiOns-make fresh daily; filter-sterilize. All chemicals are reagent-grade. Powdered enzymes are stored refngerated. Cellulase "Onozuka" RS from Yakult Pharmaceutical Ind Co., Ltd., 1 - 1 - 1 9, Higashi-Shmbashi, Minato-Ku, Tokyo, 1 05 Japan. Pectolyase Y-23 from Seishin PharmaceutiCal Co., Ltd , 4- 1 3 , Koami­ cho, Nihonbashi, Chuo-ku, Tokyo 1 03, Japan Media contammg agar are steril­ Ized by autoclavmg at 1 2 1 °C, 1 5 psi for 20 min. A: 0.5% polyvmylpyrrolidone 40 (PVP 40) and 0.05% ascorbic acid, pH 6 5, Dissolve 2.5 g of PVP 40 and 0.25 g of ascorbic acid m 450 mL of dewmzed water; adjust pH to 6 5 with NaOH, bring to final volume of 500 mL with deion­ ized water, and mix.

Boorse and Tallman

248

4.

B . In a small beaker combine 2.533 g sucrose, 0.0054 g CaCI2, 0.4 g Cellulase Onozuka RS, 0 003 g Pectolyase Y-23, 0. 1 85 g PVP 40, and 0.075 g bovine serum albumm (BSA) Add 35 mL of dewmzed water and stir until components are d1ssolved The pH of the enzyme solutiOn 1s adJusted 1mtially to 3.4 w1th stmmg for 7 mm, and then raised to 5.5 before filter-stenhzatwn (see Note 2). Fmal concentratiOn of sucrose is ca. 0 2 M; final concentratiOn of CaCI2 IS ca 0 7 mM C· 0 2 M sucrose, I mM CaC12 Dissolve 1 3 692 g of sucrose and 0 0294 g of CaCI2 m 1 90 mL of dewmzed water; adjust pH to 5 5 with HCI and/or NaOH Bring final volume to 200 mL with deionized water and m1x D· In a small beaker combine 2. 1 40 g sucrose, 0 0037 g CaC1 2, 0 4 g Cellulase Onozuka RS, 0 003 g Pectolyase Y-23, 0. 1 25 g PVP 40, and 0 075 g BSA Add 23 mL of dewmzed water, and stir until components are dissolved Adjust pH to 5 5 (see B and Note 2) Final concentratiOn of sucrose 1s ca 0 25 M, final concen­ tratiOn of CaCI2 1s ca I rnM. Stenle 0 45 !-LID cellulose mtrate fi lters m disposable f1lter umts 1 1 5 mL (4) , 250 mL (1), 500 mL (1) Enzyme solutiOns and culture media lackmg agar are sterilized by filtratiOn through 0.45 -�-tm ce llulose mtrate filters m disposable filter units ( 1 1 5 mL = model 1 25 -0045, 2 5 0 mL 1 2 6-0045 , 500 mL = 450-0045 , Nalgene Co , Rochester, NY) To prevent particulates from plugging filters, a 50-mm diameter prefilter (Gelman Sc1ences Type A/E Glass F1ber Filter, PIN 6/632, Gelman, Ann Arbor, MI) 1s used when enzyme solu­ tions are sterilized Lammar flow cabmet Stenle latex gloves (see Note 3) D1smfectant (see Note 4) 5.25% sodmm hypochlorite = Clorox bleach, Oakland, CA. Free chlorme con­ centration = 5 .25%. 95% Ethanol. Centrifuge tubes· stenle, disposable, plast1c comcal w1th caps; 15 mL (3) , 50 mL (3) Synnge: plastic, 60 mL (1), disposable 0.45-�-tm membrane synnge filter (1; Com­ ing disposable syringe filter 0 45 !-LID, 25-mm d1ameter cellulose acetate mem­ brane m acrylic holder, model # 2 1 053-25 ; Coming, Inc , Commg, NY). Glass pipets· sterile, disposable, glass, 1 0 mL (5), 1 mL (2) Chmcal centnfuge. Hemocytometer, m1croscope, hand tally counter Incomplete medmm I (Table 2). =

5 6 7 8. 9. 1 0. 11

12 13 14 15.

2.4. Primary Cultures and Colony Formation Plastic eight-well m1crochamber culture slides, Lab-Tek Chamber Slide™, Model 1 77402, Nunc, Inc., Naperville, IL 2. Petn d1sh {I; 2.5 em deep x 1 5 em d1ameter), Parafilm (Amencan Natwnal Can Co , Greenwich, CT) 3 . Hormone stock solutwn (see Subheading 3.3., step 27). 4. Incubator (lightmg optwnal)

Guard Cell Protoplasts

249

Table 2 Media Used to Culture and Regenerate Plants from Guard Cell Protoplasts of N. glauca (Graham), Tree Tobacco Medmm I, mg/L

Constituent Salts Ca(N03h 4H20 NH4N03 KN03 CaCl2 • 2H20 CaCl2 MgS04 7H20 MgS04 Na2S04 KH2P04 NaH2P04 HzO KCl Na2 EDTA FeS04 7H20 Fez(S04)3 KI H3B03 MnS04 H20 MnCl2 • 4H20 ZnS04 7H20 Na2Mo04 2H20 CuS04 5H2 0 CoS04 7H20 CoC12 6H20 FeNa EDTA ·

·

·

·

·

·

·

·

1 80.0 82 5 1 67.0 84 .7 333.0 447 .3 1 8 1 .0 68.0 14.9 88.3 3.7 2.8 23 0 76 2.0 3 1 2.0 2.3 0.03 0 003 0.003

Medium II, mg/L

825.0 950 0 220 0

1 650 0 1 900.0 1 1 0.0

1 223 0

61 1 5

680 0

1 80.0 1 70 0

37.3 27.8 0.83 62 19 8 92 0 25 0.025 0 03

·

Root medtum, mg/L

4 1 2. 5 475 .0

340 0

18 7 13 9 .

0 83 6.2 1 6.9 8.6 0.25 0.025

0.42 3.1 9.9 46 0 13 0.0 1 3 0.0 1 5

0 025 36 7

·

Orgamcs 39.7 i-Inositol 0.19 Thiamme HCl l 35 Glycine 0.45 N1acm 0. 1 Pyridoxine HCl Nicotmic acid Casein Hydrolysate 976.0 MES 95,840.0 Sucrose Kinetm

Tobacco shoot medmm, mg/L

1 00.0 1 .0

1 00.0 04 2.0 .

50 0 05

0.5 05 1 000.0

·

976.0 79,4 1 9.0

863 .25 1 .0 (contrnued)

Boorse and Tallman

250 Table 2 (continued) Medium I, mg/L

Constituent Orgamcs 1 -NAA 6-BAP Agarose Agar MediUm

03 0.075

Medtum II, mg/L

To bacco shoot medtum, mg/L

Root medmm, mg/L

03 0 075 5000.0 8000 0

I,

mediUm

II, and root mediUm are mod1ficatwns

of those descnbed by Shepard

and Totten (2 7) All salts and orgamcs through casem hydrolysate are made as l OOOX stocks, except CaC12 2H20 (260X) and KC l (663X) Na2EDTA, FeS04 7H20, and Fe2(S04h are mixed m a smgle stock solutiOn m that order Stocks are stored frozen at -20°C, any matenal precipitated by freezmg IS resolubhzed after thawmg. An mcomplete mediUm all components through

I

conta1mng

2(N-Morpholmo) ethanesulfomc ac1d [MES] IS prepared m 1 -L

batches usmg stock solutiOns and sohd MES The mcomplete medmm IS stored frozen m 1 00-mL ahquots at -20°C until the day GCP are to be Isolated Sucrose and hormones are added to complete wash and culture media on the day of protoplast IsolatiOn as descnbed m

Subheading 3.3., step

II,

27

The final sucrose concentratiOn m mediUm

I IS 0

2 8 M, m medmm

0 .2 3 M, and m shoot differentiatiOn medmm, 0 . 025 M. Concentration of MES m media

and

II

I

IS 5 mM. Tobacco shoot medmm IS commerc ially available from Carolma Bwlog1cal

Supply, Gladstone,

OR

2.5. Primary Callus 2

Medtum II in Petri dishes ( 1 5 em deep x 1 0 em diameter; ca 20 mL/dish; Table 2) Lighted mcubator.

2.6. Secondary Callus 1 . Tobacco shoot medmm (Table 2). 2 Lighted incubator.

2. 7. Plant Regeneration 1 . Tobacco shoot medium (Table 2). 2 Root medmm (Table 2) m Magenta vessels (Sigma Chemical Co , St Louis, MO) , 75 mL!vessel. 3. Lighted mcubator. 4. 0. 1 6-L small pots, pottmg soiL

3. Method 3. 1. Plants Germmate seeds of N. glauca at high density on the surface of mOistened, auto­ pottmg sOil m small pots (0 1 6 L; see Note 1 )

elaved

Guard Cell Protoplasts

25 1

2 . Germinate seeds and mamtain seedltngs on a 1 6-h light/8-h dark cycle in an envi­ ronmental chamber. Mean {± SE) temperature dunng the light cycle is 28 ±2°C; mean temperature durmg the dark cycle is 2 1 ±2°C. Relative humidity m the chamber is 65-75% Water seedlings With tap water daily. The photosynthetic photon flux density (PPFD) at seedling height is 50-70 fJmollm2/s of photons of photosynthetically active radiation (PAR). 3 . After 4---{) wk of growth, transfer plants to 2-L plastic pots contammg an auto­ claved mixture of 60% soil/40% sand (v·v). 4. After another 4-6 wk of growth, cull plants to 2 plants/pot and allow to grow to a height of 0.2 to 0.3 m. 5 . Transfer p lants to I 0-L plastic pots containmg the same soil/sand mtx. Grow on a table under htgh-intensity fluorescent lights. The PPFD at the top of the canopy is 800-900 j.tmol/m2/s of PAR. 6 . Water plants three times daily with tap water at 6 h mtervals for 4 min and every other day with Hoagland 's nutnent solution {Table 1) Mean temperature I s 27 ±2°C during the 1 6 h light cycle and 2 3 ±2°C durmg the 8 h dark cycle. Relative humidity m the room IS 45-65%

3.2. Day Before Culture l . Autoclave at 1 2 1 °C, 1 5 psi for 20 min; exhaust on dry cycle: Four, 2-L Erlenmeyer flasks each containing 1 .4 L of deiomzed water Stopper flasks wtth cotton wrapped in cheesecloth. Place a paper towel over each stop­ per and secure on each side with autoclave tape. Place a double layer of alumi­ num foi l on top of each stopper, and secure to the flask wtth autoclave tape One casserole dish, empty: Cover the dish with a single layer of foil and secure with autoclave tape. One casserole dish containing: two pairs of fine-pomt forceps wrapped individually in foil, one 6-in. cotton swab, one glass plate, one 1 5 x 60 mm glass Petri dish bottom, and one single-edge razor blade Cover the dish with a smgle layer of foil, and secure with autoclave tape Two, 1 -L plastic Nalgene beakers, empty. Cover beakers with a double layer of foil and secure with autoclave tape. One, 2-L plastic beaker, empty; one, 2-L plastlc beaker contammg three 1 25-mL, screw-cap disposable plastic Erlenmeyer flasks, (caps secured loosely and covered with foil) and two plastic funnels, one lined wtth 220 x 220 j.lm mesh nylon netting and the other with 30 x 30 j.lm mesh nylon netting. Secure nettmg to rim of each funnel with autoclave tape; wrap funnels in foil. Cover beakers with a double layer of foi l secured with autoclave tape.

3.3. Isolation of Guard Cell Protoplasts I Tum on shaking water bath, check water level, bring temperature to 28°C 2 . Thaw 1 00 mL of incomplete medium I {Table 2). 3 For each experiment, harvest one flat leaffrom Insertion level 4 or 5 from the top of the plant with a blade length of O. l l-0.2 m and With a relatively thtck cuticle. Harvest 0.5 to 1 .5 h pnor to the onset of the hght cycle (see Note 5). Store leaves

252

4 5. 6. 7. 8

9 I 0.

I I.

1 2. 13

14

15. 16

1 7. 1 8. 1 9. 20.

Boorse and Tallman in moist paper towels in a plastic bag m darkness until detachment of epidermis is imtiated Weigh, mix, and adjust pH of solutions A, B, C, and D; filter-sterilize. Tum on laminar flow hood; purge for I 0--30 mm. Cover hands with stenle, latex gloves (see Note 3) Wipe the sides and bottom of the lammar flow cabmet with full-strength dlSln­ fectant (see Note 4). Transfer solutions, glassware, and mstruments to the lammar flow cabmet. Back row: solutions, middle row, L to R. 2-L beaker, 2-L beaker, 1 -L beaker, 1 -L beaker; front row L: casserole dish With Implements; front row R. empty casse­ role dish. Remove foil and contents from beakers. Pour 2 L of sodium hypochlo­ nte mto 2 L beaker at left Fill the second 2-L beaker with 2 L ofstenle, deionized water; fill each of the two I L-beakers with I L of stenle, deionized water Remove fml from both casserole dishes Spray leaf with 95% ethanol, and then lightly buffw1th tissues to remove some of the wax. Immerse nght hand m sodmm hypochlonte for 3-5 s. Using the nght hand, immerse the leaf m the sodmm hypochlonte solution for 3-5 s D1p left hand by the same method. Immerse leaf in each beaker of water for 3-5 s, starting w1th the 2-L beaker. Lay the leaf m the empty casserole dish, top (adaxial) side up Transfer a few milhhters of solutiOn A to the small Petri dish in the other casse­ role dish; pour the remainder over the leaf. Keeping the leaf beneath the solution and startmg at the base of the leaf, break (tear) the leaf near the midnb. Bend the tom leaf sectiOn toward you at angles of 1 20--1 60° to the leaf surface, and "peel" the adax1al ep1dermis away from the mesophyll (See Note 5; It may be necessary to repeat this procedure three to five t1mes to get most of the ep1derm1s from one-half of the leaf ) Transfer each sheet o f epiderm1s, and any attached leafmatenal to the glass plate in the neighbormg casserole dish. Spread the epidermis on the plate with the side that normally faces the mesophyll upward Brush the epidermis gently w1th the cotton swab to remove adhering mesophyll. Usmg the single-edge razor blade, slice the epidermis honzontally mto stnps, and then vertically to small p1eces (ca. 5 x 5 mm). Usmg fine forceps, transfer pieces to the small Petn dish. Repeat steps 13 and 14 until most of the epidermis has been peeled from the leaf Decant the solutiOn i n the Petri dish mto the casserole dish. Gather epidermal fragments in the Petri d1sh mto a ball With forceps, and drop them down the m1ddle of the neck of a 125-mL Erlenmeyer flask. Add solution B to the flask, nnsmg any epidermis adhering to the sides down mto the flask With the solutiOn. Cap Incubate for 1 5 mm at 28°C in a shaking water bath at 1 7 5 rpm. Depending on leaf age, digestiOn may take more or less tlme Remove used materials from the hood. Leave 2-L beaker of sodium hypochlorite and 2-L beaker of water. After 1 0 min of mcubation, stenhze gloved hands as above.

Guard Cell Protoplasts

253

2 1 . In hood, posttion funnel wtth 220 x 220 �m mesh nylon net over mouth of second 1 2 5-mL Erlenmeyer flask. 22. After 1 5 min, remove the incubating flask from the water bath. In the laminar flow cab met, remove cap, and pour contents of flask over the 220 x 220 �m mesh nylon net to collect "cleaned" epidermis. 23. Rinse epidermis on net with 1 00 mL of solutwn C 24. Open second set of stenle forceps; collect epidermis in a ball, and transfer to third 1 25-mL Erlenmeyer. Rmse stdes of flask dunng additiOn of solution D. Cap. 25. Incubate for 3 h and 1 5 min at 28°C with shaking at 25 excurswns/min 26 Remove used matenal from lammar flow cabmet. 27. Dunng incubation, prepare wash and culture media. Dtssolve 9.584 g of sucrose m a total volume of 1 00 mL usmg mcomplete medium I (Table 2). Dtvide 60 and 40 mL into separate beakers Adjust pH of 60-mL aliquot to 6. 1 ; adjust pH of 40-mL aliquot to pH 6.8. Dissolve 0 0 1 2 g of 1 -napthalene acetic actd (NAA) and 0 003 g of 6-benzylammopurine (BAP) m 1 0 mL of 95% ethanol to make a hormone stock of 1 .2 mg/L NAA and 0.3 mg!L BAP. Add 1 0 �L of hormone stock to 1 0 mL of medium at pH 6 1 In laminar flow cabinet, usmg a 60-mL synnge, filter medium containing hormones through a disposable syringe filter mto a 1 5 -mL sterile, plastic conical centrifuge tube Cap 28. Filter remaining media at pH 6. 1 and 6.8 through separate 0 45-�m d1sposable filter units; place in laminar flow cabinet along with two 1 5- and three 50-mL stenle plastic comcal centnfuge tubes w1th caps. Uncap two 50-mL centnfuge tubes. Transfer pH 6.8 medmm from filter unit to one 50-mL tube and cap. Trans­ fer pH 6. 1 medmm to a second 50 mL tube and cap 29 In hood, unwrap and positwn funnel w1th 30 x 30 �m mesh nylon net over mouth of an open 50-mL com cal centnfuge tube 3 0. After 3 h and 1 5 min, remove the mcubatmg suspension of ep1derm1s from the water bath and swirl. In the laminar flow cabinet, remove cap from mcubatwn flask, and pour contents over the nylon net. 3 1 . With a 10-mL sterile, disposable pipet, div1de filtrate equally between two 1 5 mL stenle plastic comcal centrifuge tubes; cap. 32. Collect GCP by centrifuging the filtrate at 40g for 6-7 mm 3 3 . After centrifugation, remove supernatant with 1 0-mL p1pette and d1scard m waste container in hood 34. With a fresh, sterile 1 0-mL pipet, add 8 mL of medmm at pH 6.8 to each tube. Resuspend GCP in wash medmm by rolling centrifuge tubes gently between the palms of the hands Cap tubes, and centrifuge at 40g for 6-7 mm 35 Repeat steps 33 and 34 two more t1mes, with medmm of pH 6 1 wtthout hormones. After the second centri fugation m th is med1 um, asp1 rate the supernatant in each tube to 0.5 mL. Use a 1 -mL sterile p1pet to resuspend GCP in one of the tubes and transfer them into the other (combmed total volume of 1 mL). 36. Usmg a hemocytometer, count GCP to estimate cell dens1ty and dilute accord­ mgly w1th medium (pH 6 I ) to gtve a final value of I 25 x 1 05 cells/mL.

254

Boorse and Tallman

3.4. Primary Cultures and Colony Formation I . Initiate liquid cultures by pipeting 0.3 mL of the cell suspension to wells of eight­ well mJCrochamber culture shdes Add to each chamber 0 1 mL of the medmm contaming NAA and BAP. 2 Incubate chamber slides m stenle plastic Petri dtshes (2.5 em deep x 1 5 em diam­ eter) contaimng moist paper towels; seal edges of dishes tightly with Parafiim® 3 . Incubate cultures at 25-32°C (see Note 6) in darkness or under red light ( 1 5-20 !lffiOI/m2/s of photons of PAR) on a 1 2-h hght/ 1 2-h dark cycle. For the latter expenments, red light can be provided by filtering the fluorescent hght of the growth chamber descnbed above through a red Plextglas® filter

3.5. Primary Callus I . After s-I 0 wk of culture m m.Jcrochamber shdes, transfer cultured cells to medmm II 2. Seal dishes With Parafiim®, and mcubate at 25°C under contmuous whtte fluo­ rescent hght (21-27 I.J.mollm2/s of photons of PAR)

3.6. Secondary Callus I . After 8-I 0 wk, transfer green callus tissue (Fig. 2A) to a commerctal N tabacum shoot differentiatiOn medmm (Fig. 28, Table 2) 2 2 Incubate at 25°C under contmuous whtte fluorescent hght ( 1 4-23 I.J.mol/m /s of photons ofPAR)

3. 7. Plant Regeneration After 8-I 0 wk of growth on shoot medium, transfer callus to fresh shoot differ­ entiatiOn medium. 2. When shoots are 0.5-1 em m height (Fig. 2C), transfer them to root medmm (Table 2) m Magenta vessels, and mcubate at 25°C under contmuous whtte fluo­ rescent light (30 I.J.mol/m 2 /s of photons of PAR). 3 When roots are sufficiently developed (6--8 wk), transplant plants to small pots, and grow under the conditions described above for seedlings.

4. Notes Seeds may be obtamed from Lena Dugal, Exotic Seeds from around the World, 1 8 14 N.E Schuyler, Portland, OR 972 1 2 ; URL http·//www.rdrop.com/users/ idaho/tobacco.htm; phone and FAX 503-282-1 235 Seeds are sprinkled on the top of the soil and misted daily with an atomizer to prevent flushmg seeds deep into soil. Once seedlings are 5- 10 mm m height they are watered directly 2. Treatment at low pH appears to precipitate some mhtbttory, msoluble matenal that does not return to solution when the pH IS raised to 5 5. Each new lot of enzyme should be tested, and the concentration adjusted for any differences m activity from the previOus lot. Pectolyase activity IS increased dramatically with an increase of only 1 or 2°C in temperature. The time required for the first enzyme dtgestion may vary depending on leaf age, with younger leaves requiring less

255

Guard Cell Protoplasts

A

c

Fig. 2. Callus and shoots derived from cultured guard cell protoplasts of N. glauca (Graham), tree tobacco. (A) Primary callus, (B) secondary callus, and (C) shoots derived from secondary callus.

3.

4. 5.

6.

time. If leaves are too young, yields will be low, and peanut-shaped remnants of cells will appear in preparations. Gloves are not powdered; they are air-tested for pinholes. Gloves are pulled over lab coat sleeves to cover any area of the arm or wrist that might be exposed. We use Lysol Pine Action Cleaner, Household Products Division, Reckitt & Colman, Inc. (Montvale, NJ). Plants are not used after they reached a height of l m. Leaf should be fully expanded with a well-developed waxy cuticle. Leaves are removed from dark­ ness so that all stomata are closed. Osmotic potentials of guard cells of leaves with open stomata will not be uniform, and thus, solution D may not be hyper­ tonic to all guard cells, reducing yields. Older leaves may give lower yields of GCP. Higher yields ofepidermis from older (larger) leaves may saturate enzymes and reduce the number of GCP released over the 3-h, 15-min digestion period. Temperature has a dramatic effect on survival; highest survival is at 32°C (20).

256

Boorse and Tallman

References 1 . Cowan, I. R. ( 1982) Regulation of water use m relatiOn to carbon gain m htgher plants, in Encyclopedia of Plant Physiology, voi. 128 (Lange, 0. L., Nobel, P. S , Osmond, C B., and Ztegler, H , eds.), Spnnger, Heidelberg, pp. 589-6 1 3 2. Zeiger, E ( 1 983) The biology of stomatal guard cells. Annu. Rev Plant Physwl 34, 44 1--475. 3 Assmann, S M. ( 1 993) Stgnal transduction m guard cells. Annu Rev Cell Bwl 9, 345-375 4 Outlaw, W H., Jr , Mayne, B C , Zenger, V. E., and Manchester, J. ( 1 98 1 ) Presence ofboth photosystems in guard cells of Vzczafaba L tmphcations for envtronmental signal processing Plant Physiol 61, 12-16. 5. Shimazaki, K , Gotow, K , and Kondo, N. ( 1982) Photosynthetic properties of guard cell protoplasts from Vzcza faba L Plant Cell Physzol 23, 87 1-879 6 Gotow, K., Kondo, N , and Syono, K. ( 1 982) Effect of C0 2 on volume change of guard cell protoplasts from Vzczafaba L. Plant Cell Physzol 23, 1 063-1 070 7. Gotow, K., Shtmazakt, K., Kondo, N., and Syono, K ( 1 984) Photosynthesis­ dependent volume regulatton m guard cell protoplasts from Vzcza faba L Plant Cell Physwl 25, 67 1 -675 8. Weyers, J. D. B., Fttzsimmons, P. J., Mansey, G. M., and Martin, E S. ( 1 983) Guard cell protoplasts--aspects of work with an important new research tool

Physiol Plant 58, 33 1-339. 9. Gotow, K., Taylor, S., and Zeiger, E. ( 1 988) Photosynthetic carbon fixation m guard cell protoplasts of Vicia faba L.--evidence from radiolabel experiments Plant Physwl 86, 700--705 1 0 Assmann, S. M , Stmoncmi, L , and Schroeder, J. I ( 1 985) Blue light activates elec­ trogemc Ion pumpmg in guard cell protoplasts of Vzczafaba Nature 3 18, 285-287 I I . Shimazakt, K., Imo, M , and Zetger, E. ( 1 986) Blue ltght-dependent proton extru­ ston by guard-cell protoplasts of Viczafaba. Nature 3 19, 324-326. 1 2 Sh1mazakt, K. and Kondo, N. ( 1 987) Plasma membrane H+-ATPase m guard-cell protop1asts from Vzczafaba L Plant Cell Physzol 28, 893-900 1 3 . Schroeder, J. 1., Hedrich, R., and Fernandez, J. M. ( 1 984) Potassmm-selectlve smgle channels m guard cell protoplasts of Vzczafaba Nature 312, 36 1-362. 14. Schroeder, J. 1., Raschke, K., and Neher, E. ( 1 987) Voltage dependence of K+ channels in guard cell protoplasts Proc Nat/. Acad Scz USA 84, 4 1 08--4 1 1 2 1 5 . Ward, J M., Pe1, J-M , and Schroeder, J I ( 1 995) Roles of 10n channels m mttia­ tiOn of signal transductiOn in htgher plants Plant Cell 1, 833-844. 1 6 Fairley-Grenot, K and Assmann, S. M. ( 1 99 1 ) Evtdence for G-protem regulation of mward K+ channel current m guard cells of fava bean Plant Cell 3, 1 037- 1 044 1 7 Hentzen, A. E., Smart, L. B , W1mmers, L. E , Fang, H. H , Schroeder, J. I., and Bennett, A. B ( 1 996) Two plasma membrane W-ATPase genes expressed m guard cells of Vzcza faba are also expressed throughout the plant. Plant Cell Physzol 37, 650-659. 1 8 Cupples, W , Lee, J ., and Tallman, G. ( 1 99 1 ) DIVIsiOn of guard cell protoplasts of Nzcotzana glauca (Graham) in hqUid cultures Plant Cell Environ 14, 69 1-697

Guard Cell Protoplasts

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19. Herscovich, S , Tallman, G., and Zeiger, E. ( 1 992) Long-term survival of Vicia guard cell protoplasts m cell culture. Plant Scz 81, 23 7-244. 20. Roberts, C., Sahgal, P., Merritt, F , Perlman, B., and Tallman, G ( l 995) Tem­ perature and abscisic acid can be used to regulate survival, growth, and differen­ tiation of cultured guard cell protoplasts of tree tobacco. Plant Physwl 1 09,

1 4 1 1-1420. 2 1 . Hall, R. D., Riksen-Brumsma, T., Weyens, G., Lefebvre, M , Dunwell, J M , and van Tunen, A ( 1 997) Sugar beet guard cell protoplasts demonstrate a remarkable

22.

23

24.

25.

26.

capacity for cell divisiOn enabhng applications m stomatal phys wlogy and molecular breeding. J Exp Bot 48, 255-263 . Sahgal, P , Martinez, G , Roberts, C , and Tallman, G ( 1 994) Regeneration of plants from cultured guard cell protoplasts of Nzcotiana glauca (Graham) Plant Sci 97, 199-208 Hall, R D., Pedersen, C , and Krens, F A ( 1 994) RegeneratiOn of plants from protoplasts of sugarbeet (Beta vulgarzs L.), in Plant Protoplasts and Genetzc Engmeenng, vol. V (BaJaJ, Y. P. S , ed.), Spnnger-Verlag, Berlin, pp 1 6-37 Hall, R D , Verhoeven, H A., and Krens, F A ( 1 995) Computer-assisted Identi­ ficatiOn of protoplasts responsible for rare diVISIOn events reveal guard-cell toti­ potency. Plant Physwl 107, 1 3 79-1 3 86 Hall, R. D., Riksen-Brumsma, T., Weyens, G. J , Rosqum, I J., Denys, P. N., and Evans, I. J. ( 1 996) A high efficiency technique for the generatiOn of transgemc sugar beets from stomatal guard cells Nature Biotechnol 14, 1 1 33-1 1 38 Hoagland. D. R. and Amon, D I. ( 1 938) The water culture method for growmg plants without soil. Circular of the Ca/iforma Agrzcultural Expenment Statzon,

No. 347 27 Shepard, J F. and Totten, R E. ( 1 975) Isolation and regeneration of tobacco meso­ phyll cell protoplasts under low osmotic conditions. Plant Physzol 55, 689-694.

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I n Vitro Fertil ization with Isolated Single Gametes Erhard Kranz 1 . Introduction

An experimental system has been estabhshed to isolate, handle, and fuse smgle gametes, which enables studies of gamete interaction, of gametic hybndtzatwn, and of events that occur immediately after gamete fusion. Also, startmg with gamete fusion, the development of a single zygote into an embryo and finally into a plant can be followed m vttro. With experimental access to smgle gametes, to gamete fusion, and to events after fertilization, the consequences and sigmfi­ cance of such early events on plant formation can be studied. In contrast to animal and lower plant systems, which use naturally free-livmg gametes, m vitro fertilization wtth higher plants presupposes the isolation of gametes. Sperm cells have to be isolated from pollen grains or tubes, egg and central cells from an embryo sac, which also normally contams two synergtds, and some antipodal cells. Moreover, the embryo sac ts generally embedded in the nucellar tissue of the ovule. Double fertilization ts the fus10n of one sperm with the egg to create the embryo and the fusion of the other sperm wtth the central cell to form the endosperm (1). In vivo this occurs deep within ovule tissues m the embryo sac and normally with the help of one of the two syner­ gtds. In vitro, these fusions are performed without any surrounding cells. Also, by use of a microculture system, zygote, embryo, and plant development take place in the absence of mother tissue, as is the case with endosperm formation. Therefore, in vttro fertilization with tsolated, higher plant gametes is different from that in vivo. In vitro fertilization includes the combination of three basic microtechniques: l. The Isolation and selectiOn of male and female gametes. 2. The fusion of pairs of gametes. 3 Smgle cell culture From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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Cells of the embryo sac are usually isolated using mixtures of cell-wall­ degrading enzymes m combinatiOn wtth a manual Isolation procedure (for review, see 2). It is also possible to tsolate female gametes of, for example barley, wheat, and rapeseed using only mechanical means (3-5) . The same can be achieved m mmze (6), but tt is more effective to treat the matze ovule tissue with cell-wall-degrading enzymes for a short penod pnor to the manual tsola­ tion step in order to soften the nucellar ttssue. Sperm cells are obtained from pollen grains (tricellular pollen) or tubes (bicellular pollen) mainly by osmotic bursting or by squashing or grindmg of the matenal (for review, see 2). Egg cells, central cells, and synergtds can be tsolated only m small numbers. How­ ever, the posstbtlity of selection, mdivtdual transfer, and handhng of smgle­ gametes enables studies at the single cell level, for example, on cell phystology, adhesiOn, and putative recogmtton events wtth one egg and one sperm. Fur­ thermore, with microtechniques onginally developed for somatic protoplasts (7, 8), defined gamete fusion is posstble (9). Isolated gametes, generative cells, nongametic cells of the embryo sac, and somatic cells have been fused electrically (6, 1{}-12) and chemiCally, by cal­ cium (13, 14) and by polyethylene glycol (15). Fus10genic media, for example, those mcluding calcmm, might be used to determme condttions, whtch pro­ mote in vtvo membrane fusiOn. The fusion method usmg electncal pulses for cell fusion is described here in detatl, because it ts well established and an effictent part of the m vttro fertilization procedure. Early steps m zygote devel­ opment can be analyzed in mtcrodroplet culture under defined conditions with­ out feeder cells (6, 1 6, 1 7). However, to achieve sustained growth of in vitro zygotes, coculttvation with feeder cells is necessary (3, 10, 12). Embryogenesis and plant regeneration from isolated male and female gametes fused m vttro have, so far, been achieved exclusively usmg nurse culture and electrofus10n techmques (18). Moreover, because of the effictent productiOn of in vttro zygotes by the latter method, the numbers of such cells allow molecular analy­ ses to be performed at the single-cell level for gene expressiOn studies ofknown genes mvolved in events dunng fertthzation and zygote formation (19) . Also by use of reverse transcriptase-polymerase cham reaction (RT-PCR) methods, eDNA-libraries have been generated from a small, but suffictent number of egg cells (20) and m vttro zygotes (21) to tsolate egg-cell-spectfic and fertthza­ tion-mduced genes. The potential of in vitro fertthzation techniques for studies on ferttltzation mechanisms and development have been revtewed (for example, 22,23). Sktll and experience m the handling of mdivtdual cells are necessary. Unless otherwtse mentioned, the procedures are described for matze (Zea mays).

Isolated Single Gametes

26 1

2. Materials 2. 1. General Materials l . Plant matenal grown in the greenhouse under standard condttions (see Note 1 ) 2 3-cm plastic dtshes 3. Cover shps, siliconized at the edges wtth Repel-Silane (Merck, Darmstadt, Ger­ many, Pharmacia BIOtech, Uppsala, Sweden) and UV-stenhzed. 4. Mmeral oil, autoclaved (paraffin Iiqmd for spectroscopy, Merck, Darmstadt, Germany). 5. Manmtol solutwn adjusted to 570 mosM/kg H2 0 before autoclavmg (concentra­ tion about 530 mM), after autoclavmg 600 mosM/kg H20 6. Mixture of cell-wall-degrading enzymes containing 1 . 5% pectmase (Serva, Heidelberg), 0 5% pectolyase Y23 (Seishm, Tokyo), l 0% cellulase Onozuka RS (Yakult Honsha, Tokyo), and 1 .0% hemicellulase (Stgma) dissolved m btdtsttlled water, adjusted to 570 mosM/kg H20 with manmtol at pH 5 0, and filter-stenl­ ized before freezmg Store I 0-mL aliquots at -20°C 7. Glass needles wtth fine tips, preferably prepared usmg a mtcroforge 8 CaptIIanes, tip openmgs I 00-300 f..lm (drawn by hand) and 20 f..lm (drawn by a puller). 9. Electrodes (platinum wtre, dtameter 50 �-tm) fixed to an electrode support, whtch IS mounted under the condenser of the microscope 10. Feeder: maize suspension cells (see Note 2). 1 1 . Medmm for in vttro zygotes and feeder cells (ZMS). MS medmm (24) wtth the modifications NH4N03 ( 1 65 mg/L) and orgamc constituents. These are. mco­ timc actd ( 1 . 0 mg/L), thtamme-HCI ( 1 0.0 mg/L), pyridoxine-HCI ( 1 . 0 mg/L), L-glutamme (750 mg/L), prolme ( 1 50 mg/L), asparagme ( 1 00 mg/L), myo-mosi­ tol ( 1 00 mg/L) (25), and 2,4-D (2 0 mg/L), adjusted to 600 mosM/kg H20 with glucose, pH 5.5, and fi lter-stenltzed (18) 12. "Mtllicell-CM" mserts (diameter 1 2 mm), Milhpore, Bedford, MA I 3 RegeneratiOn media (RMS) Ftlter-stenltzed MS medta (24) sohdtfied wtth 4 g/L agarose (type I-A, Stgma) and the modtficatwns. RMS J · medmm w1thout hor­ mones and supplemented w1th 60 g/L sucrose; RMS2 same medium as RM S I , but contaming 40 g/L sucrose, RMS3: medmm wtthout hormones and supple­ mented with I 0 g/L sucrose, macro- and mtcrosalts half-concentratiOn (1 8)

2.2. Equipment I 2. 3 4.

Lammar flow box. Inverted microscope. Shding stage for the msertion ofa cover slip and a plastic dish, self-made (see Fig. I). Mtcropump. computer-controlled dtspenser/dilutor (Mtcrolab-M, Hamilton, Darmstadt, Germany). 5. Electrofuswn apparatus (CFA 500, Kruss, Hamburg, Germany). 6 Electrode support, self-made. 7. Posttionmg system ( optwnal), especially useful for gently movmg the electrodes along the z-axts by a step motor (type MCL, Lang, Huettenberg, Germany)

262

Kranz I s o la t i o n

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Fig. 1 Setup for Isolation, mdividual selectiOn, and transfer of maize gametes Ear sptkelets are cut as mdtcated (dotted lmes). After removing mteguments, cells of the embryo sac are Isolated from nucellar ttssue pieces m the IsolatiOn chamber (plastic dish) manually wtth needles followmg transfer with a capillary mto microdroplets on a cover shp. Subsequently, sperm cells are selected m the isolatiOn chamber after release from the pollen grams by osmotic shock and transferred mto microdroplets Manipulations are performed in these microdroplets under microscopic observatiOn physwlogical and biochemical studies, cell fusiOn, and micromJection for further analysis and culture

262

Isolated Single Gametes

263

3. Methods 3. 1. Isolation of Gametes I . Collect pollen from freshly dehisced anthers Pollen can be stored for several hours at room temperature in plastic dtshes contaimng a wet filter paper fixed to the ltd to provide a moistened atmosphere. 2. Collect ears after silk emergence, and sterihze the outer leaves with ethanol (70%) 3 Dissect 20--3 0 nucellar tissue pieces from the ovules under a dtssectmg mtcro­ scope (see Fig. 1). The embryo sac should be vistble in the tissue pteces Collect the tissue pieces m l mL mannitol solution (600 mosM/kg H 2 0) in 3-cm plastic dishes, and add 0.5 mL enzyme mtxture (see Notes 3 and 7) Incubate the mtxture at room temperature for 30 min wtthout shaking. After treatment, the dishes can be stored m the refrigerator at 6°C (see Note 4). 4. Overlay the stlicomzed and UV-sterihzed cover slip wtth 3 00 11L autoclaved mmeral oil, and inject 2 11L mannitol droplets (600 mosM/kg H 2 0) m three rows, each wtth I 0 droplets usmg a microcaptllary and a micropump Take care that the droplets spread over the glass surface 5 Iso late egg cells and the other cells of the embryo sac dtrectly m the mcuba­ tion dish wtth glass needles under mtcroscoptc observation, and transfer the cells by a microcapillary (tip opening I 00--200 11m for egg cells and 3 00 11m for central cells) into the microdroplets using the micropump (see Notes 1 and 4, and Fig. 1). 6. Overlay about 1 000 pollen grains tn a 3. 5-cm diameter plasttc dish wtth I 5 mL mannitol solutiOn (600 mosM/kg/H 2 0). After gram burstmg, transfer the sperm cells {tip openmg of the capillary 20 11m) by use of the micropump mto the microdroplets containmg egg cells (see Notes 5 and 6, and Fig. 1 )

3.2. Fusion of Gametes 1 . Ftx two electrodes (S0-11m diameter platmum wtre) to an electrode support. Before use, stenlize the ends of the electrodes in a weak flame . Mount the support under the condenser of the microscope. Adjust the electrodes to the crosshairs positiOn and lower these onto the cover slip and into one droplet 2. Align and ftx the two gametes at one electrode. Prepare and adJ ust the elec­ trodes carefully as demonstrated in Fig. 1 . By movmg the mtcroscope stage, first move one egg cell toward the electrode. Fmally, the egg cell is ftxed to the e lectrode by dielectrophoresis ( 1 MHz, 70 V x cm-1). Using the same procedure, the sperm cell is fixed to the egg cell. Now the fmal distance of the electrodes is adjusted to be approximately twice the sum of the dtameters of the cells. 3. Egg-sperm fusion is induced by applymg a smgle, or a maxtmum of three nega­ tive De-pulses (50 11s; 0.9-1 .0 kV x cm-1) (see Note 7) 4. The fusion products are removed from the electrode by gently movmg the shdmg stage. Move the electrodes out of the droplet (see Notes 8 and 9)

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3.3. Zygote and Embryo Culture� Plant Regeneration 1 . In a "Millicell-CM" insert, place 1 00 f..lL ZMS medmm, and insert 1t mto a 3 . 5-cm plastic dtsh, contammg 1 .5 mL of a feeder suspensiOn (see Notes 2 and 9). Trans­ fer the fusiOn products mto the insert usmg a mJCrocapillary. During the next day, the dish can be placed on a rotary shaker (5 0--70 rpm). Thts may be advantageous when suspensiOn feeder cells are used. Culture conditiOns are 26 ± I 0°C, a hght/ dark cycle of 1 6/8 h, and a light mtensity of about 50 f..lmol/m2/s 2 About 1 0--14 d after gamete fusiOn in liqmd culture, transfer embryos (mm1mal stze of0.4-mm length) by use of a Pasteur pipet onto sohd regeneratiOn medmm without hormones and supplemented with 60 giL sucrose ( 1 .5 mL RMS I medmm) m a 3 . 5-cm plastic dish for a first passage of2 wk (see Note 1 0). When a coleop­ tile and roots are formed, transfer the structures for another 1-2 wk onto 1 . 5 mL of RMS2 medmm contaming 40 g/L sucrose Transfer plantlets mto a glass Jar contammg 50 mL ofRMS3 medmm ( 1 0 g/L sucrose, macro- and microsalts half­ concentration, solidified w1th 4 g/L agarose) After about another 2 wk, transfer the ma1ze plants (leaf lengths about 1 5-20 em) to soil.

4 . Notes To obtain a h1gh yield of egg cells, use only healthy and well-growing donor plants. Using maize, routinely 20--40 egg cells can be isolated/expenmenter/d, and the same number of fusion products can be obtained. Under optimal condi­ tions, up to 60 zygotes can be created by I person/d. The procedure IS hne-mde­ pendent 2. Feeder cells should grow actiVely It can be useful to subculture suspension cells twice a week before use. M1crospore cultures also can be used as a feeder system (3). Although the feeding effect can also be ach1eved w1th nurse cells of a differ­ ent species than the zygote, make sure that culture cond1ttons meet the reqmre­ ments of both the zygotes and the feeder cells For example, when suspensiOn cells are used for feeding, these have to be adapted (usually several passages before use) to a htgher osmolality than normally used for these cells to meet the reqUirement for egg protoplasts and zygotes 3 Dependmg on the quahty ofthe material, the use of mannitol solutiOn (650 mosM!kg H2 0 after autoclavmg) in this step can be advantageous m Improving cell fusion. 4 When ovule ttssue 1s treated with cell-wall-degradmg enzymes, the concentra­ tion of the enzymes should be kept low, and treatment should be short to avo1d spontaneous fusion of the cells of the embryo sac. 5. Sperm cells are short-hved after tsolatwn. Therefore, It IS important to optimize conditions that guarantee the maintenance of good quality of these cells dunng a sUitable time necessary to perform cell fuston. For example, whereas sperm cells of ma1ze can be used for fusiOn w1thin 30 mm after ISolation (10, 1 1), sperm cells of wheat are useful for fusion for only few mmutes after isolatiOn (6, 12). If pos­ sible, use oval- or spmdle-shaped sperm cells for fuston, because these fuse more efficiently with egg cells than round ones (6; and see Fig. 2). Occasionally, the

Isolated Single Gametes

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o o cr 2

1

3

4

5

Fig. 2. Influence of turgor and shape of protoplasts on the effectiveness and speed of cell fusion. 1 , Protoplasts (pairS of two somatic, two egg or one somatic and one egg protoplast) with high turgor are attached only locally at one point resulting in h1gh frequency of fuswn. 2, Same protoplast paiTS, but with low turgor are attached at a broader area creating space between the protoplasts, which results in only a low fre­ quency or no fusiOn. 3, Alignment of an egg and sperm protoplast each w1th a h1gh turgor, results in high frequency of fusiOn. The speed and effectiveness of fusion is lower with low turgor Speed of fuswn and effectiveness is h1gh using oval-shaped sperm cells (4) or after alignment of the pomted end of one of the tail-like extensions of the spindle-shaped sperm cell on the egg membrane (5).

two sperm cells spontaneously fuse after release from the pollen gram. To avoid the use of such a fusiOn product, select sperm cells only when two are VISible m close proximity to each other and separated from others 6. For efficient cell fusion, the turgor of the cells should be h1gh enough (see Fig. 2) Av01d shifts from low to higher osmolalities between Isolation and fuswn media. Use the same medium preparation. Take care that the mJCrodroplets always are covered by 01l. Otherwise, the osmolality of the mediUm can dramatically mcrease with deleterious effects to the cells. 7. With well-prepared electrodes, the fusion frequency wJil be nearly 1 00% When no cell fusion occurs, lower the distance of the two electrodes and pulse again Sometimes some remnant cell-wall matenal is attached to the egg membrane, which prevents fusion This can be overcome by rotation of the egg cell to estab­ lish a new fusion site. Another reason for nonfusion can be that the turgor of the protoplasts 1s too low (see Fig. 2). If this is the case, transfer the protoplasts mto a fusion medium w1th a slightly lower osmolality. When a lot ofprotoplasts burst after the applied pulse, the osmolahty of the fusion medium 1s too low or the electrodes are not well adjusted.

266

Kranz

For the fusion, combmat1on of cells of the same s1ze, for example, egg + egg or egg + somatic protoplast, use d1electnc alignment conditwns of 1 MHz, 30 V/cm and oc pulses of0.3 kV x cm-1, 50 J.!S. 8 There are only two reports descnbmg calcmm-mediated egg-sperm fusion (13, 14). It has to be seen, if th1 s time-consummg method can be used effi­ ciently for routine work. The ahgnment of the two gametes can be performed using a glass needle tn a m1crodroplet. When adhesiOn has occurred, egg and sperm cells can be fused m manmtol solutton (about 400-500 mosM/kg H 2 0) contammg 1-50 mM calcmm (CaC12 ) As w1th electncal fuswn, the turgor of the protoplasts IS Important Thus, determme the opt1mum osmola­ lity of the fusiOn medmm 9 Single egg cells and zygotes can be encapsuled m droplets of agarose-contammg manmtol solutiOn (ultralow gellmg temperature agarose, type IX, Stgma, I %) After cell transfer, the droplets are sohd1fied for I 0 mm at 6°C. The droplets can be fixed on small plastic pieces, wh1ch can be useful, for example, for fixatwn, electron m1croscop1cal stud1es, and transportatiOn of smgle cells to another labo­ ratory for further analyses (16, 1 7). To follow development at a fixed posttJOn, zygotes can be embedded m agarose-containmg medmm, prevwusly filled mto the "Mtlhcell-CM" mserts ( I 00 J.!L, ultralow gellmg temperature agarose, type IX, S1gma, I %) I 0 To obtam secondary embryogenesis, transfer embryos from hqmd culture medmm (ZMS) onto solid RMS I medium supplemented w1th 2 ,4-D (2 mg/L) for two to three passages

References I Goldberg, R B., de Patva, G., and Yadegari, R. ( 1 994) Plant embryogenesis. Zygote to seed. Sczence 266, 605--6 14 2. Theums, C H., Pierson, E. S., and Crestt, M ( 1 99 1 ) IsolatiOn of male and female gametes m higher plants. Sex Plant Reprod. 4, 145-154 3. Holm, P. B., Knudsen, S , Mountzen, P , Negn, D , Olsen, F. L., and Roue, C ( 1 994) Regeneratton of ferttle barley plants from mechamcally tsolated proto­ plasts of the ferttltzed egg cell. Plant Cel/ 6, 53 1-543 4. Kovacs, M., Barnabas, B , and Kranz, E ( 1 994) The Isolation of vtable egg cells of wheat (Trztlcum aestzvum L.). Sex Plant Reprod 1, 3 1 1-3 1 2 5. Katoh, N., Lorz, H . , and Kranz, E . IsolatiOn of vtable egg cells o f rape (Brasstea napus L.) Zygote 5, 3 1-3 3 . 6. Kranz, E , von W1egen, P., and Lorz, H . ( 1 995) Early cytologtcal events after mduction of cell dtvtswn m egg cells and zygote development followmg m vttro fertthzation w1th angiOsperm gametes Plant J 8, 9-23 7. Koop, H.-U and Schweiger, H.-G. ( 1 985) Regeneration of plants after electro­ fusiOn of selected pairS of protoplasts Eur J Cell Bwl 39, 46--49 8. Spangenberg, G and Koop, H -U. ( 1 992) Low density cultures· mtcrodroplets and single cell nurse cultures. Plant Tissue Culture Manual A l O (Lindsey, K , ed.), Kluwer Academ1c Publishers, Dordrecht, pp. I-28.

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9. Kranz, E. ( 1 992) In v1tro fertJhzatwn ofmmze mediated by e1ectrofuswn ofsmg1e gametes Plant T1ssue Culture Manual, EI (Lindsey, K , ed.), K1uwer Academic Publishers, Dordrecht, pp 1-12. 10. Kranz, E., Bautor, J., and Lorz, H. ( 1 99 1 ) In v1tro fertihzatwn of smgle, Isolated gametes of matze mediated by electrofuston Sex Plant Reprod 4, 1 2-16 1 1 . Kranz, E , Bautor, J., and Lorz, H ( 1 99 1 ) Electrofusion-mediated transmission of cytoplasmic organelles through the m vitro fertilization process, fuswn of sperm cells With synergids and central cells, and cell reconstitutiOn m maize Sex Plant Reprod 4, 1 7-2 1 . 1 2 . Kovacs, M., Barnabas, B., and Kranz, E. ( 1 995) Electro-fused isolated wheat (Triticum aest1vum L ) gametes develop into multicellular structures. Plant Cell Rep 1 5, 1 78-1 80 1 3 Kranz, E. and Lorz, H ( 1 994) In vztro fertthsation of maize by smgle egg and sperm cell protoplast fusion medtated by high calcium and htgh pH Zygote 2, 1 25-12 8. 1 4. Faure, J.-E., Digonnet, C , and Dumas, C ( 1 994) An m v1tro system for adhesiOn and fusiOn of ma1ze gametes. Science 263, 1 5 98- 1 600 1 5 Sun, M., Yang, H., Zhou, C., and Koop, H.-U ( 1 995) Smgle-patr fusion of variOus combinatiOns between female gametoplasts and other protoplasts m N!cotwna tabacum. Acta Botamca S1mca 37, 1--Q. 1 6 . Faure, J -E., Mogensen, H. L , Dumas, C , Lorz, H , and Kranz, E ( 1 993) Karyo­ gamy after electrofuswn of single egg and sperm cell protoplasts from matze· Cytological evtdence and time course Plant Cell S, 747-7 55 1 7 Ttrlapur, U K., Kranz, E., and Crestt, M ( 1 995) Charactenzatton of tsolated egg cells, in v1tro fuston products and zygotes of Zea mays L usmg the techmque of 1mage analysts and confocal laser scanning mtcroscopy. Zygote 3, 57--{)4 1 8 . Kranz, E and Lorz, H ( 1 993) In vitro fertthzation with ISolated, smgle gametes results in zygotic embryogenesis and fertile matze plants. Plant Cell S, 739-746. 1 9. Richert, J., Kranz, E., Lorz, H., and Dresselhaus, T. ( 1 996) A reverse transcnptase­ polymerase cham reaction assay for gene expression studies at the smgle cell level Plant Sci 1 1 4, 93-99. 20. Dresselhaus, T , Lorz, H , and Kranz, E ( 1 994) Representative eDNA libraries from few plant cells. Plant J 5, 605-6 1 0 . 2 1 Dresselhaus, T., Hagel, C . , Lorz, H . , and Kranz, E ( 1 996) Isolatton o f a full­ length eDNA encodmg calreticulin from a PCR library of m v1tro zygotes of matze Plant Mol Bioi 31, 23-34 22. Kranz, E. and Dresselhaus, T. ( 1 996) In vitro ferttlizatwn wtth Isolated htgher plant gametes Trends Plant Sc1 1, 82-89. 23. Rougier, M., Antoine, A. F., Aldon, D., and Dumas, C. ( 1 996) New lights in early steps of in v1tro fertilization m plants. Sex Plant Reprod 9, 3 24-329 24. Murashtge, T and Skoog, F ( 1962) A revtsed medium for raptd growth and bio­ assays with tobacco tissue cultures . Physzol Plant 1 5, 473--497 25. Olsen, F L. ( 1 987). Inductwn of microspore embryogenesis m cultured anthers of Hordeum vulgare The effects of ammonium mtrate, glutamme and asparagme as nitrogen sources Carlsberg Res Commun 52, 393-404.

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Protocols for Anther and Microspore Cu lture of Barley Alwine Jahne-Gartner and Horst Lorz

1 . Introduction The establishment of true breedmg lines is a critical step for variety devel­ opment in most crop plants. TraditiOnally, plant breeders have achteved homo­ zygosity for all genes by using the time- and labor-intens1ve methods of self-fertilization or backcrossing. The phenomenon of parthenogenesis allows the use of androgenetic in vitro methods in order to shorten the t1me needed for the production of homozygous plants. It IS possible to sw1tch m1crospore development from the normal gametophyttc pathway into the sporophytic path­ way. The process ofm!Crospore embryogenesis makes it posstble to regenerate homozygous plants originating from smgle cells. In barley, there is a high per­ centage--up to 90% dependent on the genotype--of dihaploid regenerants owing to a smgle autoendoreduplication of the genome durmg the first d1vts10n of the m1crospore. Accordmgly, there is no need for an applicatiOn of agents, such as, e.g., colchtcme, to mduce chromosome doubltng. The most common androgenetic m vitro method ts the culture of mtact anthers, which is a relatively simple and fast method, and requires only mmi­ mal facilities. Anther culture ts widely used for the production of homozygous plants. Research on barley anther culture was initiated by Clapham (1), and since then, thts technique has been optimized extensively. Many efforts have been made in order to develop efficient and reliable culture systems. Several important modtfications have enabled plant breeders to use anther culture very successfully as a routme method in breedmg programs for the productiOn of pure lines. The development of effictent anther culture methods and the1r appli­ cations have been the subJect of several revtews (2-5). From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edited by A D Hall © Humana Press Inc , Totowa, NJ

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Alternatively, microspores can be mechanically Isolated and cultured mde­ pendent of the anther. They represent a unique experimental matenal, since 1t is possible to isolate large numbers of single, almost synchronously develop­ ing cells, which w1ll eventually undergo embryogenesis. Therefore, 1solated microspores are considered as ideal target cells for in vitro selection and trans­ formation. Progress in the development of barley micros pore culture has been very rapid, espectally in the last few years. There are now several methods for the isolatiOn and culture ofbarley m1crospores that have been reviewed in detml (5, 6) . MeanwhJie, fertile, transgemc barley plants have been produced by par­ ticle bombardment of isolated m1crospores (7). There are a huge number ofpublicatwns on the opt1m1zation of methods for barley anther and microspore culture. A review of the hterature shows that 1t 1s obviously necessary to refine, m each laboratory, the expenmental parameters dependent on the local conditions It can be frequently observed that the most declSlve step m barley anther and microspore culture IS the growth of the donor plants. Only m1crospores of supenor quality are a smtable startmg matenal for successful culture expen­ ments. The number of m1crospores capable of diVISIOn and regeneratiOn can vary widely withm one vanety because of the envuonmental cond1t10ns m wh1ch the plants are grown (8). The vigor of donor plants is mfluenced by several parameters, the most important of which are: photoperiod, light inten­ sity and spectrum, temperature, and nutritwn (6). For barley, high hght mten­ s ity (20,000 lx) and low temperatures are favorable for the support of androgenesis (9). Plant growth takes longer under low temperatures. There­ fore, the development of m1crospores is more homogeneous and it is easier to harvest the spikes at the Ideal stage. Furthermore, 1t has been shown that the culture response is optimal when spikes are harvested at the begmning of the flowering period and dechnes w1th mcreasmg plant age (1 0, 11). For establish­ ment of a rehable cuiture system, the donor plants should be grown under con­ trolled conditions, either m a greenhouse or in a phytotron. Optimal donor plants should be free of diseases and pests. However, pest control procedures should be avoided, since they reduce explant quahty and subsequent culture response (12, 13). Here the growth conditions are described that could be real­ Ized with our facilities in the institute. For successful microspore culture experiments, 1t is Important to determme a smtable developmental stage for the m1crospores to be used. In barley, most scientists use the mid- to late unmucleate stage (Fig. 1 ) . The hab1t of the tiller can--to a certam extent--be correlated to the developmental stage of the micro­ spores. Usually, sp1kes can be preselected on the basis of the interligule length between the flag leaf and the second leaf, and on the thtckness of the tiller

Anther and Microspore Culture of Barley

271

Fi g. l . Developmental stage ofmicrospores, which is suitable for barley anther and microspore culture. Miduninucleate stage, the nucleus is close to the germ pore.

For switching the genetic program of barley pollen from the gametophytic to the sporophytic pathway, a signal is necessary. In barley, two procedures are being used routinely: cold pretreatment and starvation. The storage of spikes for 3-5 wk at 4°C, at relatively high humidity in the dark (14) is a common and simple method. Another, more labor-intensive procedure is the incubation ofisolated anthers for 4 d on a medium containing 0.3 M mannitol instead of a metabolizable sugar at 25°C (15). Each of the respective meth­ ods has been reported to be more effective than the other (16-18), so that it can be recommended to compare both kinds of pretreatments. ln our labora­ tory, the cold pretreatment has proven to be more successful and, further­ more, much easier to perform. It is therefore the method that is described in this protocol. The optimization of culture media for barley anther and microspore culture has been the subject of many investigations (5). Major improvements have been achieved through modification ofthe nitrogen (N) supply (19) and the carbo­ hydrate source (20). The medium that is presented here comprises all maj or 'improvements and has been found to be suitable for anther as well as micro­ spore culture.

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2. Materials 2. 1. Donor Plants 1 . The model genotype m barley anther and microspore culture is a two-rowed winter­ type barley (Hordeum vulgare L. cv. lgri). 2. Controlled greenhouse or phytotron ( 1 4/ l 2°C day/mght, 16 h hght, 1 0,0001 6 ,000 lx, lamp type F96T l 2/CWNHO 1 500, 2 1 5 W, Philtps, relative humidity 70-90%) 3 . Vemahzation chamber (2° C, 9 h light, 4000 lx, lamp type VIALOX NAV-T, 400 W, Osram, relative humidity 95%).

2.2. Pretreatment For cold pretreatment, two-compartment Petri dtshes (Gremer, Germany) are recommended. 2.3. Culture Media The culture medium described in Table 1 (see Note 1) IS suitable for anther as well as for microspore culture. The pH of media IS adjusted to 5 . 6---5- .8, and solutions are sterilized by filtration. For solid culture medium, solutions are prepared double-concentrated and mixed with an equal volume of autoclaved 0.4% GelRite (Roth, Germany) m water. For anther culture, a final concentra­ tion of 20% Ftcoll 400 (Stgma, Germany) can also be included to increase the buoyancy of the medmm. Soltd medmm used for 1solated m1crospore culture is identtcal to liquid medium, but the L-glutamine 1s omitted. For regeneratiOn, the carbohydrate content of the medium is reduced to 30 g/L, and 6-BAP and L-glutamine are om1tted. Medmm 1 : Medtum 2 : Medium 3 . Medium 4· Medmm 5 :

solid medtum (anther culture). liquid medium supplemented With 20% Ftcoll (anther culture) liquid medium (microspore culture). solid medmm without L-glutamme (microspore culture) solid medium With reduced maltose (30 g/L) and without 6-BAP and L-glutamme

2.4. Anther and Microspore Isolation The use of fine-tipped forceps, e.g., Aesculap BD32 1 , ts recommended for the removal of awns prior to cold pretreatment and for the preparatiOn of mtact anthers. For determmation of the m1crospore stage and the exammatwn ofmtcro­ spore cultures, a hght mtcroscope is needed. For the tsolation of mtcrospores, the following matenals are needed: 1 2

Forceps and scalpel. Waring Mtcro Blendor (Eberbach Corporation, US) (Blender Cup autoclaved)

273

Anther and Microspore Culture of Barley Table 1 Composition of the Medium Used for Barley Anther and Mlcrospore Culture (19,21) Macroelements (mg/L) NH4Cl KN03 CaCl2 2H20

76 1 300 450

MgS04 7H20

350

KH2P04

200

Na2EDTA FeS04·7H20

37 28

Microelements (mg/L) H3B03 MnS04·4Hp

5 00 25 00

ZnS04·H20

7.50

KI

0.75

Na2Mo04 2H20

0.25

CuS04·5H20

0.025

CoCI2·6H20

0 025

Vttamins (mg/L) Myomositol Nicotmic actd Pyndoxine HCI Thtamine HCI

1 00 1 I 10

Amino actds (mg/L) L-Glutamme

420

Sugars (g/L) L-Maltose

50

Growth regulator (mg/L) 6-BAP

3. Centrifuge (required speed: 7 1 4 rpm, e.g., Sigma 3 K l 2). 4.

l 00-j.Ul1 nylon steve (autoclaved).

5

I 0- and 50-mL sterile conical polystyrene tubes

6. Hemocytometer

7. 0.3 M mannitol (autoclaved). 8 1 9% Maltose (autoclaved) 3. Methods 3. 1. Growth of Donor Plants 1 . Germinate seeds in a peat-soil mix in growth chamber. 2. After 2-3 wk, vernahze the Ign seedlings for a period of 8- 1 0 wk.

Jahne-Gartner and Lorz

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Add fert1 hzer to the plants startmg 8 wk after vernalizatiOn (0 7% Wuxal , Schermg, N.P.K 1 2 4.6) With each watenng. 4. Two to 4 wk before the first sp1kes are harvested, transfer the plants to a con­ trolled greenhouse ( 1 8/ l 4°C day/mght, 1 6 h light, at least 1 8,000-20,000 lx, lamp type SON-T Agro, Phihps, relative hum1d1ty 70-90%). 5. After transfer, add one teaspoon of N1trophoska blue (N P:K:Mg 1 2 1 2. 1 7 2), and increase fertilizing to 1 % Wuxal The applicatiOn of pestiCides should be avoided as far as possible, and t1llenng plants should not be treated at all 6 Select tillers on the basis of the thickness of the sp1ke, the mterligule length between the flag leaf, and the second leaf and emergence of the awns (see Note 2) In the case of lgn, the appropnate morphologJcal stage for harvestmg sp1kes occurs when the awns have emerged about 0 3-0 5 em from the flag leaf The distance between the penultimate leaf and the flag leaf I S usually about 3-6 em

3

=

=

3.2. Pretreatment

1 Surface-sterilize freshly harvested sp1kes With an aerosol of 70% ethanol 2. Remove the ensheathmg leaves under sterile conditiOns 3 . Stage the m1crospores by removmg an anther from the floret in the central part of the spike 4. Squash the anther m a drop of water (see Note 3). 5 Examme the m1crospores microscop!cally Only those spikes w1th m1crospores at the mid- to late umnucleate stage should be used for cold pretreatment 6 Remove awns from the spikes under sterile conditiOns in a lammar flow bench About five sp1kes can be transferred to one-half of a two-compartment Petn dtsh A drop of water 1s placed m the other half m order to mamtam a relatively h1gh humidity 7 Perform the cold pretreatment by wrappmg the d1shes m alummum foil, and store them for 2-5 wk at 4-6°C 3.3. Anther Culture After cold pretreatment, anthers can be prepared from the spikes. Dunng cold pretreatment, the anthers usually change their color from green to yellow. Anthers from the base and the top of the spike should not be used, since they are generally not at the appropnate stage. 1 2

3 4 5

Isolate anthers with fine ti pp ed forceps, and avoid damage to the anthers Place all anthers from one spike on solid medium (medmm 1) or on Ficoll-con­ taming medmm (medmm 2) (Fig. 2A). Incubate the dishes m the dark for 4-5 wk at 26°C After 4 wk, assess the anther culture response by countmg the number of respond­ ing anthers, I.e., the anthers that gave nse to embryogenic callus (Fig. 2B) Transfer callus carefully to regeneratiOn medmm (medmm 5), and mcubate for about 3-4 wk in the light ( 1 6 h) at 24°C. -

Anther and Microspore Culture of Barley

275

Fig. 2. Barley anther culture. (A) Freshly isolated anthers on solid (left dish) and on Ficoll medium (right dish). (B) Anther-derived callus after 3 wk on solid (left dish) and on Ficoll medium (right dish) .

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6. Transfer plantlets to soil, and determme the number of regenerants 7. Grow regenerants initially m soil under glass or plastic boxes m order to acclima­ tize the plantlets 8 Vernalize vigorous plants, and grow to matunty.

3.4. Microspore Culture Up to 30 cold-pretreated spikes can be used for one IsolatiOn procedure (1 9). 1. Cut the spikes with a scalpel mto 1-cm segments, and discard the top and the bottom segment. 2. Transfer the segments with a forceps into a Waring Microblender, and add 20 mL of 0.3 M mannitol. 3. Isolate microspores by blendmg twice for 5 s at low speed. 4 Filter the crude miCros pore preparatiOn through a 1 00-J..lm sieve, and retransfer the material remammg on the sieve back mto the blender. 5. Repeat the whole procedure three times w1th 1 0 mL of 0.3 M mannitol. 6 Transfer the whole extract into a 50-mL tube, and centrifuge for 8 mm at 85g (20°C) 7. Remove the supernatant with a pipet Do not pour off, smce the micros pore pellet IS soft and resuspends quickly. 8. Resuspend the pellet m 8 mL of 19% maltose, and transfer the suspensiOn to a 1 0-mL tube. 9. Carefully place a 1 -mL layer of 0 3 M manmtol on top. 1 0 Centrifuge for 1 0 min as described above. The fraction of viable microspores (Fig. 3B) is located m a band at the manmtol/maltose mterphase (Fig. 3A). l l Collect the band carefully w1th a 1 -mL pipet, and transfer 1t to a 50-mL tube. 1 2 . Add 20-30 mL of 0.3 M mannitol. 1 3 Determine the total number o f m1crospores With a hemocytometer. Addi­ tionally, the viabil ity of m1crospores can be determined with fluorescem d1 acetate (22) . 1 4. Centrifuge the m1crospore suspension as before, remove the supernatant care­ fully, and add liquid culture medium (medium 3 ) to final density of 2-5 x J 05 microspores/mL. 1 5 . Culture 1 -mL ahquots in Petn dishes (3-cm 0) or in 6-well plates. 1 6 . Keep cultures m the dark at 26°C, and add after 1-2 wk, 1 mL of fresh liqmd culture medmm (medium 3). 1 7 . Determine the percentage of prohferatmg microspores (Fig. 3C) after 2 wk. 1 8 After 3-4 wk, transfer the m1cospore-denved aggregates onto solid medmm With­ out L-glutamine (medmm 4) and culture at 26°C in the dark. 1 9 . Transfer cultures after 1 wk to the light for regeneratiOn ( 1 6 h, 24°C) 20. Subculture at intervals of 2 wk, and transfer shoots to regeneratiOn medmm (medmm 5). 2 1 . Transfer regenerated plants to soil as described above.

Anther and Microspore Culture of Barley

277

Fig. 3. Culture of isolated barley microspores. (A) Viable microspores collected in a band after centrifugation. (B) Freshly isolated microspores (C) Microspore-derived aggregates after 2 wk in culture.

Jahne-Gartner and Lorz

278 4 . Notes

2

3

Standard stock solutions can be used for media preparatiOn. Stock solutions of vi tamms should be kept at -20°C For preparatiOn of a 6-BAP stock solution ( 1 mg/mL), the hormone should first be dissolved m 1 mL 1 M KOH and then made up to volume With water. These morphological parameters can give an mdication on the micros pore stage However, these parameters depend on the actual growth conditions and the geno­ type. Therefore, mtcroscopic examination of squashed anthers is strongly recom­ mended in order to find a correlation between the morphological habtt and the mtcrospore stage In barley, staimng with acetocarmine is usually not necessary, the nucleus can be recognized Without staining.

References

2 3 4

5 6 7. 8 9

1 0. 11 12

13

Clapham, D. ( 1 973) Haplmd Hordeum plants from anthers m vitro Z Pjlanzenz 69, 142-1 55. Dunwell, J M. ( 1 985) Anther and ovary culture, m Cereral Tzssue and Cell Cul­ ture (Junk, W., ed.), Martinus NIJhoff, Dordrecht, The Netherlands, pp. 1--44 Luckett, D J. and Davey, N L. ( 1 992) Utilisation ofm1crospore culture m wheat and barley Improvement. Aust J. Bot 40, 807-828 Pickenng, R A and Devaux, P ( 1 992) Haploid productton: approaches and use m plant breedmg, m Bwtechnology m Agnculture No 5 Barley Genetzcs, Bw­ chemzstry, Molecular Biology and Bwtechnology(Shewry, P R., ed ), C A B InternatiOnal, Wallingford, pp. 5 1 9-547 Jahne-Gartner, A and Lorz, H Anther and m1crospore culture of barley Submitted Jahne, A. and Lorz, H. ( 1995) Cereal microspore culture. Review article Plant Scz 1 09, 1-1 2. Jahne, A., Becker, D., Brettschneider, R., and Lcirz, H ( 1 994) RegeneratiOn of transgemc, miCrospore-derived, fertile barley. Theor Appl Genet 89, 525-533 Heberle-Bors, E. ( 1 989) Isolated pollen culture in tobacco. plant reproductive development in a nutshell Sex Plant Reprod. 2, 1-10 Forough1-Wehr, B. and Mix, G ( l 979) ln vztro response of Hordeum vulgare L anthers cultured from plants grown under different envuonments. Env Exp Bot 19, 303-309. Gaul, H , Mix, B Foroughi-Wehr, B., and Okamoto, M. ( 1 976) Pollen gram development of Hordeum vulgare Z Pjlanzenz. 16, 77-80. Wheatley, W. G , Marsolais, A. A., and Kasha, K. J. (1 986) MICrospore growth and anther staging in barley anther culture. Plant Cell Rep 5, 47--49. Kasha K. J., Ziauddm, A , and Cho, U H . ( 1990) Haploids m cereal Improve­ ment anther and m1crospore culture, in Gene Manipulatwn zn Plant Improve­ ment II (Gustafson, J. P., ed.), Plenum, New York, pp. 2 1 3-235 Jahne A., Lazzen, P.A, Jager-Gussen, M , and Lorz, H ( 199 1 ) Plant regeneration from embryogenic cell suspensiOns derived from anther cultures of barley (Hor­ deum vulgare L ) Theor Appl Genet 82, 74-80

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1 4 Huang, B . and Sunderland, N . ( 1 982) Temperature-stress m barley anther culture Ann Bot. 49, 77-88. 1 5 . Wei, Z. M., Kyo, M., and Harada, H. ( 1 986) Callus formation and plant regenera­ tion through direct culture of isolated pollen of Hordeum vulgare cv. "Sabarhs." Theor. Appl Genet. 72, 252-255. 1 6 Olsen, F . L. ( 1 99 1 ) Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.) Heredztas 1 1 5, 255-266. 1 7 . Roberts-Oehlschlager, S. L. and Dunwell, J. M. ( 1 990) Barley anther culture. pretreatment on manmtol stimulates production of microspore-denved embryos. Plant Cell Tzss. Org Cult 20, 23 5-240. 1 8 . Hoekstra, S., van Zijderveld, M. H., van Bergen, S., van der Mark, F , and Heidekamp, F. ( 1 994) Genetic modificatiOn of barley for end use quality, m Improvement of Cereal Qualzty by Genetic Engmeerzng (Henry, R. J and Ronalds, J. A , eds.), Plenum, New York, pp. 1 3 9-- 1 44 1 9. Mordhorst A. P and Lorz, H ( 1 993) Embryogenesis and development of iso­ lated barley (Hordeum vulgare L ) microspores are mfluenced by the amount and composition ofmtrogen sources in culture medta J Plant Physwl. 142, 484--492 20. Hunter, C. P ( 1 987) Plant generation method European patent application No 87200773 . 7 2 1 . Jahne, A . , Lazzeri, P. A . , Jager-Gussen, M and Lorz, H. ( 1 99 1 ) Plant regenera­ tion from embryogenic cell suspenswns derived from anther cultures of barley (Hordeum vulgare L.). Theor Appl. Genet 82, 74--80 22. Widhalm, J. M. ( 1 972) The use of fluoresceine diacetate and phenosafranine for determining viability of cultured plant cells. Stain Techno! 47, 189-194.

25

__ __ __ __ __ __ __ __ __ __ __ __ ____

Microspore Embryog enesis and In Vitro Pollen Maturation in Tobacco Alisher Touraev and Erwin Heberle-Bors

1 . Introduction MICrospores have two developmental options when they are Isolated from anthers and cultured in vitro. In a nch medmm, they can develop mto mature pollen grains that are fertile on pollination in vivo (1, 2). Their development closely resembles pollen formed m VIVO, whereas the m vitro culture condt­ tions simulate the changing envtronment a microspore/pollen grain experiences inside the anther. The microspore divides by an asymmetrical division, and the daughter cells differentiate into mature pollen grains, with theu characteristic cell-cycle arrest (vegetative cell m G 1 , generatiVe cells m 02). This simple pathway makes m vitro pollen maturatiOn an excellent system to study devel­ opmental processes, such as cell fate determination and cellular dtfferentiation (3). In addition, in vitro pollen maturation has also been used for pollen selec­ tion (4) and plant transformation (5). Another potential applicatiOn IS the res­ cue of sterile or self-incompatible pollen for "self'-polhnatwns. When microspores or young pollen grains are stressed, e.g., by starvatiOn or heat, gametophyttc development stops, and a novel type of microspore/pollen grain IS formed, which on transfer to nonstress conditions (rich medium with­ out growth factors at ambtent temperature) divides continuously and eventu­ ally develops into an embryo, i.e., a sporophyte (6, 7). S tarvatiOn mduces microspore embryogenesis in tobacco (6), wheat (8), barley (9), and nee (10), whereas heat shock works in the Brasszcas (11), tobacco (6), and wheat (8). On colchicine treatment of these haploid embryos, the resultmg doubled hap­ loids are completely homozygous, and a populatiOn of doubled haploids repre­ sents the genetic variability of male meiosis in a hybrid from whtch the microspores are taken. Such doubled-haploid "recombmant inbreds" are valuFrom Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

28 1

282

Touraev and Heberle-Bors

able tools for gene mapping, and they allow us to speed up breedmg pro­ cesses (12, 13). In molecular breedmg, they help to transfer rapidly and fix transgenes from a easily transformable genotype mto a culttvar, whereas the delivery of DNA into embryogemc microspores further advances genetic improvement of crop plants by producing homozygous transgemcs m one step (14, 15). At present, tobacco is the only system m which both the gametophytic and sporophytic pathway are well established. Both are highly effictent and can be eastly reproduced m any lab. 2. Materials 2. 1. Equipment I . Lammar air flow. 2 Autoclave. 3. Two mcubators or growth rooms (25 and 33°C) 4. Growth room, 16 h hght, 25°C 5. Mtcrowave oven 6 Chmcal centnfuges and centnfuge tubes. 7. Growth chambers for donor plants (optional) 8 UV/hght microscope. 9 Inverted microscope. 1 0 Domestic fridge and freezer 1 1 . Balance. 12 pH meter 1 3 . Millipore (or Commg) filtration units (0 2-J..lm membrane) 14. Magnettc sttrrer.

2.2. Glassware, Culture Vessels, and Miscellaneous Items 1 . Glass beakers (for 1 00--1 000 mL), flasks ( 1 00--500 mL), funnels ( 1 0--1 000 mL) 2. 6-, 1 2-, and 24- well plasttc Petn dtshes {ttssue-culture tested from Falcon or Corning). 3. Glass vials wtth cap ( 1 7-mL, 26-mm dtameter, Merck), and 1 2-mL conical ster­ tle plastic centnfuge tubes (Kabe Labortechnik, Numberg, Germany) 4 Magnetic rod ( 1 8 mm in length), which moves freely on bottom of the glass vtal. 5. Metal steve (60-J..lm pore stze, 3 0-mm dtameter, S igma) fittmg to the top of a 1 00-mL Erlenmeyer flask wtth wtde neck 6. Parafilm (American NatiOnal Can ) 7. Pasteur pipet (plastic, disposable from local producer) 8. Gilson pipetman (for 20, 200, and 1 000 J..lL). 9 Plastic Petn dishes (tissue-culture-treated, Falcon or Cormng) I 00 x 1 5 mm, 1 00 x 20 mm, 60 x 1 5 mm, 35 x 1 0 mm. 1 0. Paper bags to prevent cross pollination 1 1 . Labels with thread, pen.

Microspore Embryogenesis

283

2.3. Chemicals 2.3. 1 . General 1. 2 3. 4. 5.

Agar (Sigma, US). Act1 vated charcoal (S1gma, US) Ethanol (70%). DAPI stam (Partee solution or S1gma [powder] ). Chemicals used m tissue culture media (Stgma, US, see below)

2.3.2. Media Compositions Used in Thts Chapter B medmm (1 6) Molanty mg/L Chemicals 20 mM 1 490 KCI l mM 147 CaCI2 2H20 l mM 250 MgS04 7H20 300 mM 54,700 Manmtol l mM Phosphate buffer Adjust pH to 7.0 by addition of KOH Filter-sterilize 2 AT3 medmm (unpublished) Molanty mg/L Chemicals 1 9 mM 1 950 KN03 2 mM 277 (NH4)2S04 2 9 mM 400 KH2P04 1 1 mM 1 66 CaCI2 2H20 185 0.7 mM MgS04 7H20 0. 1 mM 10 mL from a 3 .67 g/L stock solutiOn Fe-EDTA 1 mL from 1 OOOX stock so Iutton B5 vitamms (see Appendix) 1 mL from 1 OOOX stock solutwn MS microsalts (see Appendix) I O mM 1 950 MES 8 5 rnM 1 256 Glutamme 250 mM 90,000 Maltose Adjust pH to 6.2 by addition of KOH. Filter-sterilize. 3 T l medium (11)· mg/L Molanty Chemicals lO rnM 1 00 1 KN03 l mM 236 Ca(N03h 4H20 1 mM 247 Mg(S04) 7H20 0. 1 6 mM 10 H3B03 1 mM Uridine 244 1 27 0 5 mM Cytldme 3 mM 438 Glutamme Lactalbumine hydrolysate 1 0,000 ·

284

4.

5.

Touraev and Heberle-Bors 500 mM 1 7 1 , 1 50 Sucrose 1 mM Phosphate buffer Adjust pH to 7 0 by additiOn of KOH Filter-stenltze P medium (1 7) mg/L Molanty Chemicals 1 17 M 400 5 Sucrose Prolme 1 1 .5 1 00 mM Adjust pH to 7.0 by additiOn of KOH Filter-stenltze PEG8000 medmm (18) Molanty mg/L Chemicals 1 mM 111 CaCl2 75 1 mM KCI MgS04 7H20 0.8 mM 200 H3B03 l 6 mM 1 00 Trace amount 30 !J.M CuS04 300 Casem hydrolysate 1 25,000 PEG 8000 1 46 mM 50,000 Sucrose 1 5 mM 2900 MES Adjust pH to 5 . 9 by additiOn of KOH. Autoclave. GK medmm (19): Chemtcals mg/L Molanty 200 0 8 mM MgS04 7H20 200 3 2 mM H3B03 Trace amounts CuS04 30 !J.M 50,000 Sucrose 1 46 mM Adjust pH to 5 8 by additwn of KOH. Autoclave. MS medmm· Murashige and Skoog medium (20 and see Appendix) supplemented With 1 % activated charcoal, I % sucrose, and 0.8 % agar The pH IS adJusted to 5 8 before autoclaving. •

6

•

7.

3. Methods 3. 1. Preparations Glassware and distilled water are stenlized by autoclavmg at 1 20°C, 1 5 pst for 20 mm. The concentrated medta stocks contammg salts and orgamc supple­ ments can be prepared in advance and stored m a freezer for several months. Media for washmg and culture of mtcrospores must be filter stenlized 3.2. Growth of Donor Plants Many tobacco genotypes and spectes, mcludmg Nzcotzana tabacum L. and Nicotiana rustzca L., have been shown to be good donor plants for microspore cultures. Emphasis should be on conditions for profuse flower formatiOn and htgh male fertility (good-quality pollen). Here we give a protocol established

Microspore Embryogenesis

285

m our lab for N. tabacum L. cv. Petit Havana S R l (21). Donor plants are grown in a growth chamber (25°C, 1 6 h light) with regular supply of fertilizers and routine watering. Continuous flowering can be achieved by regular harvest of open flowers. Thus, one generation of tobacco plants can be used for 4-6 mo without significant decrease in miCrospore culture response. 3.3. Flower Bud Sterilization Flower buds with an approximate length of 1 0--1 1 mm, representing unicel­ lular microspores (which can be confirmed by DAPI staining; see Note 1, and Fig. lA,B) are exctsed. The buds are sterilized by dipping them m 70% ethanol for a maximum of 5 min (see Note 2). 3.4. Microspore Isolation

2 3.

4. 5. 6. 7.

Under sterile conditions, squeeze the anthers out of the flower buds mto a glass vial ( 1 7 mL), contaming approx 3 mL of medium B (16; see Note 3). Anthers from 20-30 buds can be squeezed/vial. Remove the medium, and replace with 6 mL of fresh medmm B. Put a sterile magnetic bar into the vial, and stir for 2-3 min at maximum speed until the mtxture of microspores/anther debris and medmm becomes mtlky (see Note 4). Collect the suspension of released microspores and anther debris wtth a Pasteur pipet, and filter the suspension through a 60-f.lM metal sieve (see Note 5) Centnfuge the filtrate for 2-3 min at 1 200 rpm (about 200g; see Note 6). Remove the top green layer of the two-layered pellet, wh1ch contains anther wall debns by using a 200- or 1 000-f.lL Gilson pipette tip (see Note 7). Resuspend the whitish pellet composed of m icrospores in 2-1 0 mL of medium B and centnfuge again Repeat this procedure at least two to three times until there is no greenish layer above the whitish pellet. The micro­ spores can be used for embryogenesis (see Subheading 3.5.) or pollen matura­ tion (see Subheading 3.6.).

3.5. Induction of Microspore Embryogenesis and Regeneration of Haploid Tobacco Plants I . Resuspend the final whitish pellet in medium B (microspores from one flower bud in 2 mL of medium B give the optimal density for a good response), and pour into small Petri dishes (35 x 10 mm, Falcon or Commg) wtth 1 .5 mL of suspen­ sion/dish (see Note 8). 2. Seal Petn dishes with Parafilm, and incubate at 3 3 °C for 5-6 d in the dark (see Note 9, see Fig. I C). 3. Collect the suspension from the Petri dishes in 1 2-mL test tubes, and pellet by centrifugation at 1 000 rpm ( 1 75g) for 5 min (see Note 10). 4. Di scard the supernatant, resuspend the pellet m AT3 medmm, and pour back mto the original Petri dish with 1 . 0 mL/Petri dish.

286

Touraev and Heberle-Bors

Fig.

I.

Microspore Embryogenesis 5

287

Seal the Petn dish wtth Parafilm, and mcubate for 1 . 5-2 mo at 25°C in the dark

(see Fig. 1 E,G). 6 . Transfer seedlings onto agar soltdtfied MS medmm (20; and see Appendix) wtth I % activated charcoal and I % sucrose, and move to ltght.

3.6. Maturation of Tobacco Microspores In Vitro Resuspend the final whtttsh pellet in medmm T l (1 7; microspores from one bud m I mL of medmm give optimal maturation) and pour into small Petn dtshes (35 x 10 mm, Falcon or Coming) with 1 mL suspension/dish 2. Seal Petri dishes with Parafilm, and incubate at 25°C for 4 d in the dark (see Note 1 1 and Fig. lD) 3. Dilute cultures with I mL of medmm P, and mcubate for I d (day 5; see Note 12) 4. Dilute cultures wtth another 1 mL of medium P, and mcubate for one more day (day 6, see Note 13) Fully matured pollen grams (see Fig. I F) can be used for m vttro germinatiOn (see Subheading 3.7. and Fig. 1H) or for m situ pollmation (see Subheading 3.8.).

3. 7. Germination of In Vitro Matured Tobacco Pollen Collect the m vitro matured pollen grains m 1 2-cm test tubes. Centrifuge the suspension at 1 5 00 rpm (275g) for l-2 mm. Discard the supernatant, and resuspend the pellet m a volume ofPEG8000 medmm to give a density of 1 0,000 pollen grains/mL (18; see Note 14). 4. Pour the suspenswn into 6- or I 0-cm Petri dtshes (cultures should be diluted by a factor of 2-3 in order to make observation of germinatiOn easier) 5. Seal Petn dtshes wtth Parafilm, and incubate at 25°C m the dark 6 Observe and evaluate germmation frequency and tube length after 5-l 0 h or longer (see Note 1 5) 1 2 3

3.8. In Situ Pollination with In Vitro Matured Tobacco Pollen Collect in vitro matured pollen grains in 1 2-cm test tubes 2. Centrifuge suspension at 1 500 rpm (275g) for 1-2 mm.

Ftg. 1. (prevzous page) Embryogenesis and maturatiOn from Isolated tobacco miCrospores m vttro (A) and (B) freshly Isolated unmucleate tobacco mtcrospores stamed wtth DAPI (A) and FDA (B), (C,E,G) m vttro mterospore embryogenesis (C) mtcrospores after 6 d starvatiOn m sucrose- and mtrogen-free medium B; {E) globular embryos after 30 d of culture of embryogemc mtcrospores in medmm AT3; {G) tor­ pedo and cotyledonary embryos after 1 . 5 mo culture of microspores in AT3. (D,E,F) m vitro mtcrospore maturation : (D) starch formation in microspores after 2 d of mcu­ batton in maturation medium T l ; (E) ful ly mature pollen after 6 d of culture of mt­ crospores in vitro , (H) germinatiOn of in vttro matured pollen after 5 h mcubatwn m medmm PEG8000

288

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3. Dtscard the supernatant, and resuspend the pellet m l-2 mL of medtum GK (see Note 16). 4. Centrifuge the suspensiOn at 1 5 00 rpm (275g) for another 1-2 mm. 5 Dtscard the supernatant, and resuspend the pellet m 0.5 mL of medmm GK 6 Transfer the suspensiOn into small PCR Eppendorf tubes (e g., 500-�-tL tubes). 7 Centrifuge the suspension at 1 500 rpm (275g) for l-2 mm 8 Dtscard the supernatant, and resuspend the pellet m a volume of medmm GK to give a density of 1 .250 pollen grains/j..t L (see Note 17). 9. Take 4 J,tL of pollen suspensiOn using a 1 0- or 20-J,tL Gtlson ptpet, and apply as a drop carefully onto the stigma of a flower, whtch has been emasculated I d before (see Note 1 8). 1 0. Label the pollinated flowers, and bag them 5--6 h after polhnat10n when the drop of pollen suspensiOn on the stigma has dried. 1 1 Harvest seed pods, and count seeds after about 1 mo.

4 . Notes I . Diamidmo-2-phenylmdole (DAPI) IS a specific fluorescent stam to visualize DNA It can be purchased as a ready-to-use solution from Partee (Germany) or as a powder from Stgma (US). The powder IS dtssolved m buffer (22) or m 50% ethanol. The nuclei of tobacco mtcrospores are stamed well in both solutiOns, and can be observed with a range of UV filter sets under a fluorescent micro­ scope. AlternatJVely, stagmg can be performed by acetocarmine or acetoorcein stain, but the sensitivity of these stams IS less than that of the DAPI stain. 2 In general, surface stenlizatwn of buds wtth 70% ethanol is sufficient to obtain sterile cultures, but sometimes one can also use sodmm or calcium hypochlonte as stenhzatwn agents after the ethanol treatment. 3 . Medmm B is stmple, cheap, osmotically well balanced, and used m our lab for all tobacco mtcrospore tsolatwn procedures. Dependmg on skill and the IsolatiOn procedure used, the VIability of Isolated microspores should be 90--95% in th1s medmm. 4 The duration and time of stunng depend on the vial used and on the type of stirrer, and can easily be optimtzed emptrically The magnetic bar should move mstde the glass vtal freely. In case of small-scale isolations (<5 buds), anther maceration wtth a glass rod can be used successfully (23), and m the case of large-scale isolattons (more than 50 buds), a Wanng blendor IS the best chotce (24). 5. Umcellular and early btcellular tobacco mJCrospores have average diameters of 20--23 j..tM, and the pore stze of the steve used for filtratiOn may vary from 40 to 60 f.lm. 6. M icrospores can resist centnfugation speeds even htgher than 600g, but htgh speed may affect subsequent vtabthty of the microspores 7 The mtcrospores pellet much faster than the anther debris, and therefore, one can see two clear pellet layers (one greenish, one whitish) after centrifugation. The greenish layer can be removed easily after some practice.

Microspore Embryogenesis

289

8 . Microspore dens1ty 1s an important parameter for opt1mal induction of embryo­ genesis. The calculation of optimal density for tobacco microspores ts g1ven m refs. (1 7) and (25), and has been found to be: mtcrospores from one bud to 2--4 mL of medmm. 9. This is the key step to block the gametophytic pathway of development and d1vert mtcrospores into the sporophytic pathway. Embryogenic m1crospores at the end ofthe starvation treatment should have mcreased m size and should show a "star"­ like phenotype under the inverted microscope with the nucleus being m a more central positiOn as compared to the starting stage and cytoplasmic strands radiat­ ing out from it through the vacuole (see Fig. I C). 1 0 The embryogenic microspores formed are very sensitive to damage, and low speed must be used to pellet them gently. 1 1 . Depending on the developmental stage and synchrony of the startmg population (ratio of unicellular to early bicellular microspores), the mcubation time m medium T l can be shortened to 3 d (1 7). Starch accumulatiOn is seen in Immature pollen grains after 2-3 d of culture in medium T l , which is a good criteria for optimal and synchronous maturation of microspores (see Fig. lD). 1 2 This step and the following step are simulatmg the dehydratiOn o f maturmg pollen. I 3 A mature pollen grain ts 30-32 !liD in size and has a slightly rectangular shape, whereas earlier stages are fully spherical. The exine should be fully mtact. No protrusiOns from the germ pores should be seen The number and s1ze of starch grains are smaller when compared to a slightly earlier stage. I 4 The germination medium based o n the use o f PEG8000 (12) giVes a very htgh frequency of germinatiOn of m v1tro matured tobacco pollen (see Fig. lH) as compared to medmm GK (19) or GV (1 7). 1 5 . In v1tro germination usually starts 4--5 h after mcubatton, but the best time to evaluate germmation frequency 1s after 9-- I O h I 6. Do not use the PEG8000 medium for m situ pollination experiments The m vitro matured pollen when suspended in PEG8000 medmm and loaded onto a sttgma, germinate rapidly, but do not penetrate the style and form a wh1te cover on the surface of the stigma. I 7. It has been shown (5, 1 9) that the minimum number of m vttro matured pollen grains applied onto one stigma should not be < 1 000 m order to obtain any seed set. A mmimum of 5000 1s needed to obtain optimum seed set 1 8 The receptivity o f the stigma o f an emasculated flower bud IS a maJor hmit­ ing factor in obtammg seed set. The woundmg, wh1ch accompames emascu­ latiOn 1 d before zn situ poll ination, induces the formatwn of flavonols m the stigma and style, wh1ch are Important growth factors for the germmattng pol­ len grains (1 9, 26) The stigmatic surface should be wet, and a drop of stig­ matic diffusate should be vis1ble at the moment of polhnation. A drop of up to 6 1-1L can be apphed onto a stigma, but usually 4 !lL drops of pollen suspen­ siOn are used (5) .

290

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References Bemto Moreno, R. M., Macke, F , AI wen, A., and Heberle-Bors, E. ( 1 988) ln-sztu seed production after pollination with zn-vztro-matured, Isolated polien Planta

1 76, 145-1 48 2. Stauffer, C., Bemto Moreno, R. M., and Heberle-Bors, E ( 1 99 1 ) In sttu pollina­ tion with m vitro matured pollen of Trztzcum aestzvum Theor Appl Genet 81, 576-5 80. 3 McCormick, S. ( 1 993) Male gametophyte development Plant Cell S, 1 265-1 275 4. Touraev, A., Fink, Ch., Stoger, E., and Heberle-Bors, E ( 1 995). Pollen selectton: a transgemc reconstruction approach Proc Nat/ Acad Scz USA 92, 1 2 , 1 65-1 2 , 1 69 5. Touraev, A., Stoger, E., Voronm, V., and Heberle-Bors, E. ( 1 998) Plant male germ Ime transformatiOn. Plant J 12(4), 949-956 6 Touraev, A , Ilham, A , Vicente, 0 , and Heberle-Bors, E ( 1 996) Stress mduced

mtcrospore embryogenesis from tobacco microspores: an optlmtzed system for molecular studtes Plant Cell Rep 1 5, 561-565. 7 Touraev, A , Vtcente, 0 , and Heberle-Bors, E. ( 1 997) Inittation of mtcrospore embryogenests by stress. Trends Plant Scz 2, 297-302 8. Touraev, A., Indnanto, A., Wratscko, 1., Vtcente, 0., and Heberle-Bors, E. ( 1 996) Effictent mtcrospore embryogenesis in wheat (Trztzcum aestzvum L ) mduced by starvatiOn at htgh temperatures. Sex Plant Reprod 9, 209-2 1 5 9 . Hoekstra, S., van Ztjderveld, M H , Louwerse, J. D , Hetdekamp, F , and van der Mark, F. ( 1 992) Anther and mtcrospore culture of Hordeum vulgare L cv Ign.

Plant Scz 86, 89-96 1 0. Ogawa T., Fukuoka, H , and Ohkawa, Y ( 1 994) Inductton of cell dtvtsion of tso­ lated pollen grams by sugar starvatton m nee Breedmg Sci (Jpn ) 44, 75-77 1 1 . Pechan, P. M. and Keller, W A ( 1 988) Identtficatton of potenttally embryogemc mtcrospores m Brasszca napus Physiol Plant 74, 377-384. 1 2 Mornson, R A and Evans, D A ( 1 988) Haplotd plants from tissue culture: new plant varieties m a shortened ttme frame. Bw!Technology 6, 684--690 1 3 Ferne, A M. R , Palmer, C E., and Keller, W A ( 1 995). Haplotd embryogen­ esis, m In Vztro Plant Embryogenesis (Thorpe, T A , ed.), Kluwer, Dordrecht, pp 309-344 1 4. Jahne, A., Becker, D., Brettschneider, R , and Lorz, A. ( 1 994) RegeneratiOn of transgemc, microspore-derived, fertile barley Theor App/ Genet 89, 525-533 15 Stoger, E , Fmk, C., pfosser, M., and Heberle-Bors, E. ( 1 995) Plant transformation by particle bombardment of embryogemc pollen Plant Cell Rep 14, 27 3-278 . 1 6 Kyo, M. and Harada, H. ( 1 986) Control o f the developmental pathway of tobacco pollen zn vitro Planta 168, 427--43 2. 1 7 . Tupy, J , R' hova, L., and Zarsky, V ( 1 99 1 ) Production of fertile tobacco pol len from microspores m suspensiOn cultures and tts storage for m sztu pollinatiOn Sex Plant Reprod 4, 284--2 87 1 8 Read, S. M., Clarke, A. E., and Bactc, A ( 1 993) Stimulation of growth of cultured Nicotzana tabacum W38 pollen tubes by poly (ethylene glycol) and Cu(lll salts Protoplasma 1 77, 1-14

Microspore Embryogenesis

29 1

1 9 Ylstra, B . , Touraev, A., Bemto Moreno, R. M, Stoger, E., van Tunen, A J., Vicente, 0., Mol, J N. M , et al ( 1 992). Flavonols stimulate development, germi­ nation, and tube growth of tobacco pollen. Plant Physwl. 100, 902-907 20. Murashige, T. and Skoog, F ( 1 962) A revtsed medmm for rapid growth and bio­ assays with tobacco tissue cultures. Physwl Plant 1 5, 473-497 2 1 . Mabga, P , Breznovits, A , and Marton, L ( 1 973) Streptomycin-reststant plants from callus culture of haploid tobacco Nature 244, 29-30. 22. Vergne, P., Delvallee, I., and Dumas, C. ( 1 987) Rapid assessment of microspore and pollen development stage in wheat and maize usmg DAPI and membrane permeabilizat10n. Stazn Techno/. 62, 299--3 04. 23. Benito Moreno, R. M., Macke, F., Hauser, M.-T , Alwen, A., and Heberle-Bors, E ( 1 988) Sporophytes and male gametophytes from m vztro cultured, Immature tobacco pollen, in Sexual Reproduction m Hzgher Plants (Crest!, M , Gon, P and Pacmi, E., eds.), Sprmger, Heidelberg, NY, pp 1 3 7-142 24. Swanson, E B , Coumans, M. P., Wu, S. C., Barsby, T L., and Beversdorf, W. D ( 1 987) Efficient Isolatton of microspores and the production of m�erospore-de­ rived embryos from Brasszca napus. Plant Cell Rep 6, 94-97 25. Garrido, D., Charvat, B , Benito Moreno, R. M , Alwen, A , VIcente, 0 , and Heberle- Bors, E. ( 1 99 1 ) Pollen culture for haplmd plant production in tobacco, m A Laboratory Guzde for Cellular and Molecular Plant Biology (Negrutiu, I. and Ghartl-Chhetn, G , eds ), Birkhlmser, Basel, pp 59---() 9 . 26 Vogt, T., Pollak, P., Tarlyn, N., and Taylor, L. P. ( 1 994) Pollination- or wound­ induced kaempferol accumulat10n in petuma stigmas enhances seed production Plant Cell 6, 1 1-23

26

__ __ __ __ __ __ __ __ __ __ __ __ ____

Embryo Rescue Following Wide Crosses Hari C. Sharma

1 . Introduction Crosses between species of the same or different genera are called wide crosses (WCs). They are useful in plant improvement for gene transfer and haploid production, and in general biology, for genome mapping, study of chro­ mosome behavior, and phylogenetic relatiOnships (1-8) . Broadening the genetic base of modem crops is necessary to prevent genetic vulnerability. If genes are not present in crop species, we resort to mutagenesis, transformation, protoplast fusion, and wide crosses. The first two technologies are not routme for a number of reasons (9, 10). The utility of protoplast fusion may he m trans­ formation or production of hybrids, which are sexually impossible (1 1 , 12). Nevertheless, wide crossmg w1ll remain an important method of genome ma­ nipulation for mtroducmg unique vanatton to crop species. In recent years, It has become Imperative that genetic material from WCs be explmted m breed­ ing (10, 13) . In the mampulatwn, exploitatiOn, and study of alien genomes through WCs, the first required step is to make a hybrid. The ahen genome can then be stud­ Ied and utilized to various degrees from the whole genome and single chromo­ somes to specific gene transfer by manipulating the hybnd genome through either natural recombinatiOn or specialized techmques dependmg on the genomic relationship of the two species (14). However, crossability barners often make hybridization difficult. These barners, caused by gene action, genome incompatibility, ploidy levels of species, and the environment, lead to no or poor seed set on crossmg, retard or stop hybnd seed development, or lead to stenlity of the hybrid plant (15, 1 6) . However, reproductive barners are not always absolute (1 7). Molecular analysis shows that genomic changes in wide hybnds only partially comcide with those of speciation (18), and methods to From Methods m Molecular BIOlogy, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R 0 Hall © Humana Press Inc , Totowa, NJ

293

294

Sharma

Fig. I . 14-d-old normal seed of wheat (left) and Agropyron junceum (right), and aborting seed ofwheat x A.junceum wide cross wfth only watery endosperm (middle),

overcome crossability barriers have been devised (7,19-21). 1n nature, hybrid­ ization is suppressed owing to less favorable conditions for fertilization, hybrid seed development, and survival of .F 1 sterile plants, but under artificial condi­ tions, genotypic variation for crossability, multiple pollinations, appl ication of growth regulators, and in vitro techniques can be exploited to make the WC possible. In making the hybrid, seed abortion is the major barrier, largely caused by the failure of proper endosperm development and its inability to nourish the embryo (Fig. 1 ). The technique of rearing the embryo from an aborting seed into a plant in vitro is called embryo rescue (ER) or embryo culture (EC). Thus, in ER following WCs, synthetic medium supplies the life support for the hybrid embryo. ER following WCs is one of the most success­ ful and beneficial plant cell/tissue-culture techniques (7,20,22,23). The tech­ nique has been used routinely, but procedures and modifications are rarely or incompletely described in publications. The few protocols that are available deal with EC in general, and not ER in WCs (22-25). Kaltsikes et al. (26) described a method for the production of wheat x rye hybrids by EC. Collins and Grasser (27) reviewed interspecific hybridization and EC in Trifolium, Mok et al. (28) in Phaseolus, and Williams (29) in forages. There are different ways of ER following WCs: direct ER where the seed is dissected, the embryo excised and cultured to germinate into a plant (30) ; ovule culture, followed (31) or not followed (32), by EC; ovary culture followed by ovule culture or EC (22); segmenting the cultured embryo for further culturing

Embryo Rescue

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(33); inducmg callus and plantlet regeneration from embryos (34); culturing embryos onto nurse endosperm on medium (35) ; and spike culture followed by EC (36). Compared to direct ER, other vanations are more cumbersome and require transfers to different media, and are useful tf duect ER IS unsuccessful. In some cases where comparative studies have been made, duect ER was more successful than some of these variations (3 7,38) . Ovule culture has worked largely in cotton (39), but is not useful when maternal tissue or endosperm is inhibitory to the embryo (40, 41). It has utility where embryo abortwn occurs early or when dissectwn ts not possible. Postpollination application of growth regulators can prolong seed development on the mother plant. Nurse culture may be useful when no suitable medmm is found. Direct ER followmg WC IS a much simpler technique A survey of literature from 1 984 to 1 996 by the author, based on Center for Agn. and Bwsci. (CAB), has shown that direct ER has been used far more frequently than these vanations. However, detatled, stepwtse procedures that can be followed and per­ formed by a new researcher are lacking. This chapter provides an updated overview and complete step-by-step pro to co l of ER from seeds that result from WCs. 2. Materials l.

2 3

4

5. 6. 7. 8.

9

1 0.

11.

Seeds of parental species of wheat (Trztzcum aestivum) and wheatgrass (Agropy­ ron camnum) , greenhouse, growth chamber or field facilities, pots, and s01l to grow plants. Polhnatmg bags, surgica l sctssors, forceps, tags, paper clips, pencil, and com envelops to make we General laboratory glassware, mcludmg flasks, beakers, graduated cylinders, pipets, pipet rubber bulbs, stir bars for makmg solutions, and medta. Refngerator, lammar flow cabmet, dtsttlled water umt, autoclave, analytJCal bal­ ance to wetgh from 0. 1 mg up to a few grams, pH meter, stirrer wtth hot plate, and aluminum foil Chemicals listed m Table 1 plus mositol, sucrose, agar, and hormone(s) as needed for media. Acid (0.5 N HC!) and alkah (0 5 M NaOH) solutions to adjust pH of the media Clorox bleach and cheesecloth to surface-stenlize WC seeds. Gas flame or a burner, spray bottles With 95 and 70% ethanol, and Coplin Jar containmg 70% ethanol Dissectmg microscope or stereomiCroscope wtth a ltght source, glass Petn dishes with moistened filter papers, sharp surgtcal forceps, scalpel, narrow spatula, needle with handle for dissecting seeds, and isolatmg embryos. Culture tubes for medium, and slanting test tube racks ( 1 50 x 25 mm glass tubes wtth slip-on caps [Fig. 2], avatlable from Carolina Biologtcal Supply Co or Sigma Chemical Co.). Incubator wtth temperature and light controls.

Sharma

296 Table 1 Composition of Stock Solutions and Final Concentrations of Ingredients in Media Component

Stock sr. no.

Inorgamc

Stock vol., mL 500

2

500

Stock amount, Fmal cone., g mg/L

Ingredient NH4 N03 KN03 MnS04 4H20 ZnS04 7Hz0 CuS04 5H20 MgS04 7H2 0 CaC12 2H20 •

•

•

3

500

•

KI

CoCI2 6H2 0 KH2P04 H3B03 Na2Mo04 2H 20 FeS04 7H20 Na2 EDTA Thiamine-HCI Glycme L-Argmme-HCI L-Tyrosme •

4

500

5

500

a b c

500 250 250 250

·

Vi tamms Ammo acids

·

82.5 95 0 1 1 15 0 43 0 00 1 2 5 18 5 22.0 0 04 1 5

0 00 1 25 85 0.3 1 0.0 1 25 I 39 I 865 0 02 0 25 0 25 0.25

1 650 1 900 22.3 86 0 025 370 440 0.83 0 025 1 70 62 0.25 27 8 37 3 04 10 10 10

3 . Methods 1 . Germinate the seeds of the parental species (e.g., wheat and wheatgrass) to be crossed and grow plants to flowering. Stagger two to three plantmgs to increase the chances of simultaneous flowenng of the two parental species and makmg the cross (see Note 1) 2 Emasculate the florets of the plants to be used as females when the anthers are yellowish green and have not dehtsced. Pollmate usmg pollen of the other spe­ cies when the stigma becomes feathery (receptive). Enclose the sptkes m polh­ natmg bags usmg paper clips (Fig. 3) Pollinate twtce over 2 d to mcrease seed set. Make rectprocal crosses to enhance chances of success. Write mformatton about the cross, includmg date of pollmation, on a tag, and tte it to the tiller (see Note 2) 3. Score the number of florets po11tnated and seed set after 4-5 d. Remove the pol­ hnatmg bags, and cover the spikes bearmg seed(s) with com envelopes to block ltght and faci lttate seed development. 4 At about day 1 0 after pollination, inspect the growmg caryopses by openmg the bracts With forceps, and tentattvely decide when the embryos are to be excised for culture Plan to prepare stock solutions and ER media accordmgly (see Note 3)

Embryo Rescue

Fig.

297

2. Slip-on-type embryo culture tubes in a slanting test tube rack with germinat­

ing embryos on media demonstrating the embryo culture system.

Fig.

3.

Wide crossing between wheat and Agropyron

intermedium.

5. Turn on the laminar flow hood. Wipe it thoroughly with cloth well soaked in 95% ethanol (see Note 4). 6. Sterilize all glassware and water in the autoclave ( 121oc and 15 psi, 20 min). Sterilize the various dissecting tools listed in the Subheading 2. in the autoclave

298

7

8

9

1 0.

Sharma or m 95% ethanol and keep m the sterile hood m Coplin jar with 70% ethanol with their points downward. Sterilize glass Petn d1shes contammg moistened filter papers. Wrap the glassware, tools, and Petri d1shes in alummum foil before puttmg in the autoclave Bring all the autoclaved glassware and tools to the lami­ nar flow hood. Spray with 95% ethanol as you put them in the hood. Prepare stock solutwns for modified MS media (42) followmg Table 1 (see Notes 5 and 6). Prepare inorgamc and VItamin stocks at strengths SOX and the ammo actds stocks at 25X the final concentratiOns needed for the actual media To pre­ pare each morgamc stock, dtssolve each salt m a mimmum volume of dtsttlled water in a beaker and then pool m the order shown to prevent prectpitatwn. Make volume up to 500 mL by addmg water. For Fe· EDTA complex, weigh 1 . 865 g of Na2 'EDTA, and put it into a beaker wtth 1 00 mL dtsttlled water. St1r and apply gentle heat or give a longer time on a stirrer to dissolve Dissolve 1 .39 g of FeS04·7H2 0 separately m the same way. Pool the two solutions, and bring vol­ ume to 500 mL With dtsttlled water. Sttr for 1 h to ensure complete mixing of the complex. Bring pH to 5.8 Store all stock solutiOns at 4°C (see Note 7). From the above stock solutiOns, prepare the actual media w1th supplements To prepare I L media, put 500 mL of distilled water m a I L beaker, and stir at moderate speed. Usmg separate ptpets for each stock, add 1 0 mL each of mar­ game stocks 1-5 (Table 1) m ascendmg order to avOid precipttatton Mtx well before addmg each solutiOn Add I 0 mL ofvitamm stock and then 1 0 mL each of the three amino actd stocks (a--<:) Weigh and add 1 00 mg of inosito1 Add 30-50 g of sucrose Add 0. 1-1 0 mg of kmetin (see Note 8) Make volume up to 800 mL Adjust pH to 5.8 by addmg dropwise, etther 0 5 N HCl or 0 5 M NaOH Make volume up to 1 000 mL. Add 7 g of agar. Pour the media mto a 1 5- or 2-L flask and close the flask mouth with alummum foil. Autoclave for 3 mm or bot! on burner to melt agar (see Note 9). Distribute 20-25 mL/tube Close the tubes With hds, and wnte name and date Put the tubes in a metallic or any autoclavable contamer, and autoclave for 20 min to stenlize the media (see Note 10) After stenlizatwn, close the hds well onto the culture tubes, since they may have hfted somewhat durmg autoclavmg. Put the tubes on the slanting test tube rack to solid­ t fy Wipe or spray with ethanol as you bnng them into the lam mar flow hood (see Note 1 1). Harvest the seed by cuttmg the spike from the mother plant With a patr of scts­ sors Put the spikes in a beaker contaming water and bnng to the laboratory Remove the seed with a pau of forceps and put m water to avoid drymg out (see Note 12) Record the number of seeds harvested Prepare 1 5% (v/v) Clorox bleach (= 0.8% sodmm hypochlonte = 0 37% chlo­ nne) solution using distilled water, and fill a small stenle beaker. Put the seeds m the solutiOn. If the beaker has been exposed outside the hood while pounng or preparing the solutton, wtpe the outer walls, and top of the beaker wtth ethanol after placing in the hood Allow 20 min for surface-sterilization of the seeds m the solutton. If the seeds float, keep dtppmg them under the hqmd wtth forceps (see Notes 13 and 14). Clean the working area Wipe hands wtth ethanol . Spray

Embryo Rescue

I I.

1 2.

1 3.

1 4. 15

1 6.

299

and wipe the whole hood once again wtth ethanol during this time After 20 mm, decant the solutwn, blockmg the seeds with forceps. Rinse three times wtth auto­ claved water, shaking, and dtscardmg the water each time Fmally, add some sterile water to keep seeds motst, and store, if necessary, at 5-8°C m darkness. Organize the working area mside the larnmar bench: Spray agam the dissectmg mstruments (forceps, scalpel, needle and spatula) wtth ethanol and leave dipped m the Coplin Jar containing 70% ethanol. Before every use, flame dry tools to bum off the alcohol and allow to cool. Wtpe/spray the dtssectmg miCroscope thoroughly wtth ethanol paymg particular attention to the stage and the surround­ ing area. Tum on the gas flame or burner Place an autoclaved Petri d1sh contam­ mg a moistened filter paper on the dissecting microscope stage. Transfer a seed from the beaker onto the dish (see Note 1 5). Usmg the seed as the object, adjust the d1ssectmg microscope at a magmficatwn comfortable to your eyes (see Note 16) View the seed and locate the basal end contammg the embryo (see Note 17). Hold the seed in the mtddle wtth a patr of flamed and cooled forceps Make a shallow mcision (slit) on the pericarp next to the holding pomt of the basal half of the seed with a scalpel. Using sharp forceps, open (peel) the pencarp dtrectly above the embryo, exposing the embryo (Fig. 4) If dunng the dtssectmg opera­ tiOn, the filter paper in the Petn dtsh dnes out, add a little water, to avmd dehy­ dratiOn of the embryo, by dtpping the scalpel or forceps m the water beaker contammg the stenle seeds, and then touching the filter paper. Working m a dry environment IS otherwise difficult as the starch of the endosperm st1cks to the forceps and hinders the operation (see Note 18). Wtpe the outside of a medta tube with ethanol, espectally tts upper portion, close to the lid, and allow to evaporate Open the medta tube over the flame Flame and cool the spatula, motsten tt by dtppmg in water, and lift the embryo Place the embryo onto the medmm m the tube with flat stde of scutellum m contact wtth the medium, t .e., embryomc axts upward (see Note 1 7) Close the tube w1th the lid after passmg the mner stde of the lid over the flame. Label the tube with mformatwn about the cross, embryo age, and date of culture. Put the tube back m the rack in the same posttion (see Fig. 2). Flame all the tools before starting on the next seed. Culture each embryo in a separate tube (see Notes 19 and 20) Incubate the embryos at low temperature by placing the test tube rack(s) m the vernalizatiOn chamber or refngerator (5-8°C) m the dark for 4-5 d and then trans­ fer to the incubator at 24°C, 1 2 h photoperiod, and htgh humidity (see Note 21 ) During gerrnmation within the first week, if any embryo becomes contammated, resterilize for 2-5 min m disinfectant as for seeds. After 2-3 wk, when the plantlet is well rooted, have two to three leaves, and its tip has reached the top of the culture tube, transplant i t in a pot contammg stan­ dard greenhouse sotl wtth fertthzers and organic matter Tease or invert and gen­ tly pull out the seedhng from the medium, and wash carefully under running water to remove traces of agar (see Note 22). Place the plants m a growth chamber or greenhouse and protect from dtrect sun­ light. Cover the seedlings w1th a beaker or a clear plasttc bag for a few days to

300

Sharma

c

Fig. 4. Dissecting a wide cross-seed, and isolating and culturing the embryo on medium: Take the seed (A), place in the dissecting dish below dissecting scope and hold with one hand using forceps, and operate with the other (B). Incise with a scalpel and peel the pericarp with forceps (C). Lift the naked embryo (D), and place the embryo on the media in the tube (E).

provide enough humidity. Uncover the plant after about a week when new leaves stan appearing. When growth is resumed (Fig. 5), you have the hybrid(s) at hand (see Notes 23 and 24).

Embryo Rescue

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Fig. 5. Embryo rescued A. caninum x wheat F1 hybrid seedling (middle) in soil after the plastic cover has been removed and good growth has started. On the sides are A. caninum (left) and wheat parent seedlings.

4. Notes

The example ofER following WC's in wheat (T. aestivum) is used for the proto­ col. Wheat is one of the crop species in which ER following WCs has proven most valuable. Seedlings of winter wheat and wheatgrasses (e.g., Agropyron, Elymus species) require vernalization (cold treatment, 5-8°C) for 8-10 wk to induce flowering. Grow the plants under optimum conditions in the greenhouse or growth chamber. Conditions in the field may be better, but unpredictable. A greenhouse environment with 2 1 °C day, l 8°C night temperatures and lO-h pho­ toperiod for tillering, and 25°C day, 2 1 °C night temperatures and 1 8-h photope­ riod for flowering and crossing is good for wheat x wheatgrass crosses. 2. Since there is variation in the WC seed and embryo development in a spike, some or several of the seeds when dissected may not have embryos, not every embryo will be placed in the best appropriate position on the most appropriate media or grown under the most appropriate conditions, and some or several embryos may not grow. The researcher's first goal must therefore be to produce as many hybrid seeds as possible. Pollinating 1000 florets/cross is recommended. To facilitate fertilization and caryopsis growth, you may douse the floret after pollination with 50 ppm (50 mg!L) gibberellic acid (GA3) solution. ln several wheat x wbeatgrass crosses, seed set is possible or higher when the alien species having the lower chromosome number is used as female. 3. Monitor the seed development very closely on a daily basis starting 1 wk after pollination. Delay the removal of seed from the mother plant as long as possible. I.

302

4.

5

Sharma In wheat x wheatgrass crosses, usually the caryopses will begin to turn brown (drying) and are ready for ER, 1 2 d after pollination, and the embryo can be observed in the shriveling seed. The hood should be turned on well before use to minimize chances of contamma­ tion. Beware: 95 % ethanol catches fire. Use only 70% ethanol when the burner or the gas flame is on Buy plant cell culture or analytical-reagent-grade chemicals, and store m a dark and dry place followmg label mstructwns Should you have different formula­ tiOns of any salt, recalculate the amount accordmgly For example, m MS salts, one can use 370 mg/L MgS04 7H20 or 1 8 1 mg/L MgS04 because their formula weights are 246.5 and 1 20.4, respectively. Instead of prepanng stocks, media can be obtained m prepared powder form, e.g , S1gma Chemical Co. 's product no. M55 1 9, which I S MS-based medmm We use this medmm at 2.22 g/L with 50 g/L sucrose and 7 g/L agar, pH = 5.8 for ER followmg wheat x maize WCs (data unpublished) At 4°C, the stock solutiOns should remam usable for a few months, but prepare fresh 1f microbial growth is seen. Unless you need a large quantity of media, prepare regularly small quantities of stocks. The hormone(s) reqUirement will depend on the cross, growth, and age of hybrid seed, and If embryos do not germmate without hormonal stimulatiOn Media con­ tainmg 1 mg kmetin/L for certam wheat x wheatgrass crosses facilitate embryo germmatwn, but root primordia fail to elongate and lead to seedling starvatiOn (43) . Using a more labile form or a lower concentration of hormone(s) m such situations might solve the problem Alternatively, transfer p\antlets to hormone­ free medium. If agar IS to be melted on a burner, keep shaking the flask constantly, or the agar Will stick to the bottom of the container and will bum. A lternatively, autoclave the media m bulk m the flask and dispense mto autoclaved tubes m the lammar hood after it has cooled to around 50°C. All mgredients m the medmm are heat-stable. If a heat-labile compound has to be added, then autoclave the rest of the medium in a 1 . 5- to 2-L flask, filter­ sterilize the stock solutiOn, and add to precooled (50°C) medium, and then dis­ tnbute mto stenle tubes The stock solutiOn concentratiOn should be such that addition of no more than 5 mL will give the desired end concentration in the medmm If the media Is to be used w1thm 1 wk, It IS okay to keep It m the hood at room temperature For a longer duration, store at 4°C to prevent desiccatiOn There may be some accidental self-pollinated seed D1stmgmsh It by Its normal and more advanced development, and discard. On the other hand, some of the shriveling hybrid seeds may be small, so open the bracts well to ensure that you do not overlook them. Alternatively, wrap the seeds in cheesecloth, and 1mmerse in the disinfectant w1th periodic agitatiOn manually or by magnetic stirring to disperse a1r bubbles and facilitate d1stnbutwn of the d1smfectant. ·

6.

7

8

9.

10.

1 1. 1 2.

13.

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Fig. 6 Drawing of a seed showmg main structures encountered m ER

Ftg 7 Drawmg of an embryo showmg tts components.

14. If the seeds are from rectprocal matmgs or from more than one cross, then har­ vest, label, and surface-stenlize the seeds separately You may surface-stenltze a few tmmature seeds from the parent plants for practtce m excising embryos 1 5 . You can dtssect the seed on the stereomtcroscope stage ttself, but thts ts not rec­ ommended. There wtll be dehydratton of embryo wtthout water and glare to eyes wtth water, and scratching of the stereomtcroscope stage can result 1 6. A lower magnification provtding sharper and whole-seed vtew may be most destr­ able for mampulatwn 1 7 . To begin wtth, a new worker may want to practice wtth parental seeds The cereal seed, such as wheat x wheatgrass hybnd seed, ts composed of pencarp, endo­ sperm, and embryo (Fig. 6). The pericarp ts the seed-covenng over the embryo and endosperm (44), and forms a hairy tip at the apex of the seed. The embryo is at the end oppostte to the hairy ttp. The scutellum forms the largest part of the embryo It is attached to the embryonic axis (bulgmg side), and tts lower s1de ts flat (Fig. 7). Wtth expenence, JUSt two patrS of forceps w1ll do, and no scalpel ts needed for incision, an opening can be made with the forceps used to peel off the pencarp. If the embryo, after one attempt, IS uncovered only partly, a little more pericarp can be peeled away. It IS a simple questiOn of breakmg the seed and gettmg the embryo out. Any vanatwn m the operation is fine as long as the embryo ts not mJured. Sometimes, the naked embryo may not stay on the seed and may fall off 1 8. It is useful to record embryo type (torpedo, globular, or heart shape), any charac­ tensttcs of endosperm (starchy, watery, no endosperm), measure embryo stze, or

304

19

20. 21 22

23.

24

Sharma take photograph(s) For measurements, the stereom1croscope should have an ocu­ lar micrometer, and for photography, it should be eqmpped with a camera Also make a note If no embryo is found L1ftmg the embryo i s easy I f the spatula i s mmst. A somewhat dehydrated embryo will stick to the surface mOisture of the spatula Be careful not to inJure the embryomc ax is or push the embryo into the medmm If the embryo is too small to handle with the spatula, the scalpel or a d1ssectmg needle With Its tip moistened w1th culture medmm or stenle water can be used to piCk up the embryo. If proper placmg of the embryo becomes a concern, then use media m Petri dishes rather than tubes. The media dish can be placed under the dissect­ mg microscope while placmg the embryo. Petn dishes can be sealed with parafi lm. Petn dishes may be more economical, smce they need less media and one can culture more than one embryo per dish However, Petn dishes do not provide enough space for the seedling to grow, and the leaves may spread over the medmm. Culturing more than one embryo per dish may be nsky m that if one is contammated, then It may contaminate the others Furthermore, one seedling may grow too b1g, but cannot be transplanted because the others are still too young. There may also be some depletion of media components lead­ ing to weak seedlmgs. After d1ssectmg 5-1 0 seeds, replace the d1ssectmg d1sh with a new one to avOid the possibility of contamination or losmg the embryo m the torn filter paper. Place a pan of water at the bottom of mcubator If it does not have a humidity regulator To count chromosomes for an early confirmation of hybnd nature of the seed­ ling, harvest 1-2 root tips, pretreat m 2% bromonaphthalene for 6-h overnight at 4°C, and fix m glacial acetic acid for 20 min at 4°C Hydrolyze the root tips in I N HCI at 60°C for I 0 min, stain in Feulgen stam for I 0-1 5 min, and then squash in 1% acetocarmine (45) If the parents were wmter wheat and a wheatgrass, vernalization would be needed for flowering and backcrossmg, or for studying inflorescence or pollen mother cells of the hybnds The number of embryos found and cultured, number of seedlings produced, and behavwr of the remammg cultured embryos may be recorded. From the vanous data recorded, calculate % seed set, embryo recovery, and ER efficiency This and other information will be useful for further actwns

Acknowledgments Support of USAID (Grant No. HRN-5600-G-00--2 032-00, PSTC, o/o Agri. and Food Secunty, Center for Economic Growth, Bureau for Global Programs, Field Support and Research), and Public Varieties of Indiana is gratefully acknowledged. This work IS dedicated to my family, gurus, colleagues, and relatives who Impacted my hfe. Purdue Umv. Agn. Res. Programs contnbu­ tton no. 1 5408.

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References 1 . Chandler, J., Jan, C., and Beard, B. ( 1 986) Chromosomal di fferentiatiOn among the annual Helianthus species. Syst. Bot 11, 354-37 1 . 2 . Friebe, B . , Mukai, Y., Dhaliwal, H., Martin, T., and Gill, B . ( 1 9 9 1 ) Identification of alien chromatin in wheat gennplasm by C-bandmg and m sztu hybnd1zatwn. Theor Appl. Genet. 81, 3 8 1-3 89. 3. Riera-Lizarazu, 0., Rmes, H., and Phillips, R. ( 1 993) Retention of maize chromo­ somes in haplOid oat plants from oat x maize crosses. Agron. Abstract 84, 1 1 2 4. Jiang, J., Friebe, B., and Gill, B. ( 1 994) Recent advances m alien gene transfer m wheat. Euphytzca 73, 1 99-2 1 2. 5 . Ozminkowski, R. and Jourdan, P ( 1 994) Comparing the resynthesis of Brasszca napus by interspecific hybridization J. Am Soc Hort Sci 1 19, 808-8 1 5 . 6 Bonhomme, A . , Gale, M., Koebner, R., NIColas, P., Jahier, J., and Bernard, M ( 1 995) RFLP analysis of an Aegilops ventrzcosa chromosome that carnes a gene conferring resistance to leaf rust when transferred to hexaploid wheat. Theor Appl Genet. 90, 1 042-1 048. 7. Sharma, H. ( 1 995) How wide can a wide cross be? Euphytica 82, 43--M. 8. Anderson, A., Crasta, 0., Francki, M., Bucholtz, D., Sharma, H., and Ohm, H. ( 1 997) Molecular and cytogenetic analysis of Thmopyrum intermedium translo­ catwns. Plant Genome 5, 90. 9. Hayward, M., Bosemark, N., and Romagosa, I. ( 1 993) Plant Breedmg, Prmczples and Prospects Chapman and Hill, London. 1 0 Jones, S , Murray, T., and Allen, R. ( 1 995) Use of alien genes for disease resis­ tance m wheat A nnu Rev. Phytopathol 33, 429-443 1 1 . Evans, D., Flick, C., and Jensen, R ( 1 98 1 ) Disease resistance: mcorporatwn mto sexually incompatible somatic hybnds ofthe genus N!cotzana Science 213, 907-909 1 2. Hansen, L. and Earle, E. ( 1 995) Transfer of resistance to Xanthomonas campestrzs into B. oleracea by protoplast fusion. Theor Appl Genet. 91, 1 2 93-1300. 1 3 . Shanna, H., Ohm, H., Goulart, L., Lister, R., Appels, R , and Benlhabib, 0 ( 1 995) Introgression and charactenzation ofBYD resistance from Thinopyrum mtermedzum in wheat. Genome 38, 406--4 1 3 . 1 4 Clark, M and Wall, W . ( 1 996) Chromosomes. Alden, Oxford, UK. p . 345. 1 5 . Stebbins, G. ( 1 958) The inviability and sterility of interspecific hybrids. Adv. Genet 16, 63 7-655 . 1 6. Linskens, H . ( 1 972) The reaction o f inhibition during incompatible pollinatiOn and its elimination. Sovzet Pl. Physiol. 20, 1 56-1 66 1 7 Grant, V ( 1 98 1 ) Plant Speciation. Columbia University Press, p 1 83 1 8 . Vershnin, A., Salma, E., and Svitashev, S. ( 1 992) Is there a connection between genomic changes and wide hybridization? Hereditas 1 1 6, 2 1 3-2 1 7 . 1 9. Stalker, H. ( 1980) Utilization of wild species for crop improvement. Adv Agron. 33, 1 1 1-147 . 2 0 . Collins, G . , Taylor, N . , and DeVema, J . ( 1 984) In vztro approaches to interspe­ cific hybridizatiOn and chromosome manipulation in crop plants. Stadler Genet Symp. 16, 3 23-3 8 3 .

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2 1 Baum, M , Laguda, E , and Appels, R. ( 1 992) Wide crosses in cereals A nnu. Rev PI Physiol. Mol Bwl 43, 1 1 7-143 22 Bridgen, M. ( 1 994) A rev1ew of plant embryo culture. Hort Sci 29, 1 243-1 246 23 Hu, C., Helena, M., and Zanettm1, B. ( 1 995) Embryo cu lture and embryo res­ cue for wide cross hybnds, m Plant Cell, Tissue and Organ Culture Funda­ mental Methods (Gamborg, 0. and Phillips, G eds ), Springer-Veriag, Berlin, pp 1 29-1 4 1 . 24. Raghavan, V. ( 1 980) Embryo culture Int Rev. Cytol Suppl l l B, 209-240 25 Monmer, M. ( 1 990) Culture of zygotic embryos of h1gher plants, m Methods zn Molecular Bwlogy, Plant Cell and T1ssue Culture (Pollard, J., and Walker, J., eds.), Humana Press, Clifton, NJ, pp. 1 29-1 39. 26 Kaltsikes, P. J., Gustafson, J., and Kalts1kes, P I. ( 1 986) Tntlcale· productiOn through embryo culture, in Bwtechnology zn Agriculture and Forestry (BajaJ, Y P S , ed.), Springer-Verlag, Berlin, pp 523-529. 27 Collms, G. and Grosser, J ( 1 984) Embryo culture, m Cell Culture and Somat1c Cell Genet1cs of Plants, Laboratory Techmques (Vasil, I , ed.), Academic, New York, pp. 242-257 28 Mok, D., Mok, M , Rabakoanhanta, A , and Shu, C ( 1 986) Phaseolus w1de hybridizatiOn through embryo culture, m Bwtech zn Agrz. and Forestry (BaJ aJ, Y., ed.) Spnnger-Verlag, Berlm, pp. 309-3 1 8 29. Williams, E. ( 1 987) Interspecific hybnd1zatwn m pasture legumes. PI Breed Rev 5, 237-305. 30 Sharma, H. and Ohm, H ( 1 990) Crossability and embryo rescue enhancement m w1de crosses between wheat and three Agropyron spec1es. Euphyt1ca 42, 209-2 14 3 1 . McCoy, T. and Sm1th, L ( 1 986) Interspecific hybnd1zahon of perenmal Med1cago spec1es using ovule-embryo culture Theor Appl Genet 71, 772-783. 32 Shubhada, T., ParanJpe, S , Khuspe, S., and Mascarenhas, A. ( 1 986) Hybndiza­ tJOn of Gossypzum species through zn ovulo culture. PI Cell, T1ssue Organ Cult 6, 209-2 1 9. 33. Guan, G., LI, P., and Wu, D. ( 1 988) A study of backcrossmg the interspecific F l hybnd of Lycopers1con esculentum x L peruvzanum to L. esculentum Acta Hort S1mca 15, 39-44. 34. Chen, Q., Jahier, J., and Cauderon, Y ( 1 992) ProductiOn of embryo callus-regen­ erated hybnds between Trzt1cum aestivum x Agropyron crzstatum With I B chro­ mosome Agronom1e 1, 5 5 1 -555 35. Kruse, A . ( 1 974) An zn vitro/zn VIVO embryo culture techmque. Hereditas 11, 2 1 9-224 36. Laune, D. and Bennet, M. ( 1 988) Cytological evidence of fertihzatwn m hexap­ lmd wheat x sorghum. Plant. Breed 1 00, 73-82 3 7. Hossam, M., Inden, H., and Asahtra, T. ( 1 989) Interspecific hybnds between Bras­ Sica campestrls and B oleracea by embryo and ovary culture. Memo1rs College Agrz Kyoto Univ. 135, 2 1-30. 38. Sirkka, A and Immonen, T. ( 1 993) Companson of callus culture at different t1mes of embryo rescue. Euphyt1ca 10, 1 85-1 90.

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39. Beasley, C., Tmg, I , Linkins, A., Binbaum, E., and Delmer, D. ( 1 974) Cotton ovule culture: progress and preview of potential, m Tissue Culture and Plant Sci­ ence (Street, H., ed.), , Academic, New York, pp. 1 69-1 92. 40. Sharma, D . , Chowdhury, J . , Ahuja, U . , and Dhankhar, B ( 1 980) Interspecific hybndizatwn m the genus Solanum Z Pflanzenzuchtng 85, 248-253 4 1 . Hong, K., Om, Y , and Park, H . ( 1 994) Interspecific hybndizat10n between Cucurblta pepo and C. moschata through ovule culture. J Korean Soc Hort Sc1 35, 43 8-448. 42. Murashtge, T. and Skoog, F ( 1 962) A revised medium for rapid growth and bw­ assays with tobacco t1ssue cultures. Physiol Plant 15, 473-497 43 Sharma, H. and Baenziger, P. ( 1 986) ProductiOn, morphology and cytogenetic analys1s of Agropyron camnum (Elymus canmus) x Trztzcum aest1vum hybnds and backcross- I derivatives Theor. Appl Genet 71, 750-756 44. Briggle, L. ( 1 967) Morphology, m Wheat and Wheat Improvement (QUisenberry, K., ed.), Am. Soc. Agronomy Pub , Madison, WI, pp. 92-1 1 5 45 Sharma, H. ( 1 982) A techmque for somatic counts from root-tips of cereal seed­ lings raised by embryo culture Curr Sc1 51, 1 43-144

27

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Mutagenesis and the Selection of Resista nt Mutants Philip J. Dix

1 . Introduction The prospect of utihzmg plant tissue cultures as a means of generating and Identifying novel genetic vanants IS one that has sparked the mterest of researchers for many years. It has resulted in large numbers of research papers describmg the selectiOn and characterization of mutants, and several exhaus­ tive reviews (e.g., 1,2), mcludmg a treatise givmg detailed coverage of the whole field (3) . By far the largest number of articles relate to the selection of resistant mutants, which are the most straightforward category to select m vttro, and whtch are the focus of the present chapter. One thmg that is immediately apparent on superficially scannmg the literature on these mutants i s a high level of repetition. This is because the concept is simple: one challenges a large population of cells with a selective agent, and identifies "survivors" on the basis of their capacity to divide to form cell masses, or organized structures. Thus, the procedures used to select mutants resistant to a range of different antlmetabolites have a certain uniformity. By the same token, selectiOn for the same type of mutant (e.g., salt tolerant) in a range of species, involves a broadly similar strategy. On closer scrutiny of the same literature, however, it becomes apparent that far from there being a simple formula to obtain "results" for se­ lection of every phenotype in every species, a great deal of refinement, some­ times major, sometimes subtle, has been necessary in each case. This is owmg to the diversity of detail, both in the tissue-culture procedures available for each crop and m the cellular consequences of exposure to different antime­ tabolites. Awareness of this is important in appreciating the limitations of any set protocol for selection of mutants in vitro. In choosing species, culture sys­ tems, mutagenesis treatment, and selective agent for this chapter, the author IS providmg a specific workable system, which should lead to the selection of From Methods m Molecular Btology, Val 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc , Totowa, NJ

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mutants. Inevitably, however, variations in any of these will require refine­ ments to the procedures. It is hoped that these protocols will provide a sound baselme on which those modifications can be based. The procedures utilize Nicotzana plumbaginifo/ia, an amenable species wtth whtch the author has considerable experience, and two culture systems, cell suspenston cultures and mesophyll protoplasts. They should be appli­ cable without modification to related species, such as Nicotiana tabacum, tobacco, and be adaptable for use with other species with smtable cell sus­ pension and/or protoplast culture systems. The author' s laboratory has a spe­ cial mterest in the role of prolme m stress tolerance, and therefore , m hydroxy-proline-resistant lines, which accumulate prohne. It should be pos­ sible to substitute other antimetabolites subject to modtficattons to the proce­ dures. The mutagenesis treatments described here relate to exposure to low concentratiOns of the two most commonly used nitroso-ureas, but short-term exposure to higher concentratiOns, and to ethyl methane sulfonate (EMS) is discussed m Note 9, whereas UV irradiation is covered in Note 10. For advice on other approaches and procedures for in vitro mutagenesis, the reader is referred to earlier articles (4, 5). 2 . Materials 1 . MS plant salt mtx (see Appendix) 2 Vttamins and plant growth hormones m aqueous stock solutions as follows a Thiamme-HCI, I mg/mL b. Meso-mositol· no stock, used as powder. c. 2,4-Dichlorophenoxyacetic acid (2,4-D): 0. 1 mg/mL d 6-Furfurylammopurine (kinetin)· 0.05 mg/mL. e Benzylammopurme (BA) : 0.5 mg/mL. f Naphthalene acetic acid (NAA): I mg/mL. g. Gtbberellic actd (GA3): 0.5 mg/mL 2,4-D, kinetm, NAA, and GA3 will dissolve more readily if a small amount of I M NaOH is added dropwtse to the solution until the hormone dtssolves, whereas BA ts most easily dtssolved m 0. 1 M HCI 3 All components of K3 medium and W5 salts (6; Table 1) 4. 4-Hydroxy-L-prolme. 1 M. 5 . N-Nitroso-N-ethyl urea (NEU) or N-mtroso-N-methyl urea (NMU): 1 0 rnM. 6 20% (v/v) Domestos, or other proprietary bleach, givmg a final concentratiOn of I % sodium hypochlonte. 7 RM medmm: 4.6 g MS plant salt mix, 20 g sucrose, and 6.5 g Difco Bacto-agar (or equivalent)IL Thts and all other media are autoclaved for 20 mm at 1 . 1 kg/cm2 8. RMP medium. as RM, but with I 00 mg meso-mosttol, I mg thiamine-HCI, O. I mg 2,4-D, and 0. 1 mg kinetm/L. 9. RMOP medmm: as RM, but wtth I OO mg meso-inosttoi, I mg th tamme-HCI, i mg BA, and 0 I mg NAA/L

31 1

Resistant Mutants Table 1 Components of Modified K3 Medium Used for Isolation and Culture of N. plumbagin/folia Protoplasts8 mg/L

Ingredient

900 2400 1 20 240 1 30 0.025 0.025 27.85 37 3 3.0

CaC12 · 2H20 KN03 NaH2P04 · 2H20 NH4N03 (NH4hS04 CoC12 · 6H20 CuS04 7H20 FeS04 7H20 Na2EDTA H3B03 •

•

asugar

IS

Ingredient

mg/L

KI MnS04 · 4H20 Na2Mo04 · 2H20 ZnS04 · 7H20 meso-inositwl Nicottmc acid Pyndoxine · HCl Thiamme· HCl Xylose pH

0 75 67 0 24 2.3 1 00 1 .0 1 .0 10 250 5.6

not included m the above table Sucrose or glucose 1s added as descnbed m the hst

under Subheading 2.

It

IS

convement to make up 5 L of the medmm double-strength, om1ttmg

only the sucrose or glucose, and stormg at -20°C It I S generally recommended to filter-stenlize the medtum, but for N medmm.

p/umbagmifoha protoplasts, good results can be obtamed w1th autoclaved

1 0. N. plumbaginifolia seeds. 1 1 . Culture room facihttes: Preferred conditions: 25°C, 1 500 lx, 1 6-h d. Cell suspen­ sion cultures are mamtained on a rotary shaker at 1 00 rpm in the culture room.

3. Methods These are dealt with under the appropriate subheadmgs below. They include the sterihzation and germination of seeds and the initiation of shoot, callus, and cell suspension cultures; the determination of the appropriate selective level of hydroxyproline; mutagenesis and the selection of resistant colonies; and the regeneration of plants from them. 3. 1. Initiation of Shoot, Callus, and Cell Suspension Cultures 1 Place a few (ca. 1 00-200) tobacco seeds in an Eppendorf tube. 2. Drop a few drops of 70% ethanol m the tube for a few seconds, and then add 20% Domestos (without removmg the alcohol) for 10 min 3. Spin for 30 s at top speed m a microfuge, pour off the Domestos, and replace with sterile dtstilled water. 4. Repeat step 3, providing a second wash in sterile dtstilled water. 5. Pour the seeds out on to three to four 9-cm Petri dtshes, each contaimng 20 mL RM medmm. Seal wtth Parafilm and mcubate m the culture room until the seeds germinate (see Note 1). 6. Thin out seedlings at 0.5-1 em m height, transferrmg to RM medium in sterile tubs or Jars (e.g., Plantcon containers-Flow Laboratories). At a height of 4-5 em,

Dix

312

7.

8

9

10

11. 12.

transfer to mdtvtdual contamers. When domg so, remove the roots and the basal 2 mm of the stem with a single clean cut, and insert the cut surface into the fresh medium Thts encourages vigorous growth of adventitious roots When the seedling approaches the top of the container, establish shoot cultures by dtssecting out nodes (with 0 5- 1 -cm internode remaining above and below) and transferring to RM medmm in fresh containers. Axillary buds will grow, and the process can be repeated at 4- to 8-wk intervals To mitiate callus cultures, remove leaves and cut mto stnps (3- to 4-mm wtde) with stenle scalpel and forceps. Transfer to the surface of 20 mL RMP medium in 9-cm Petn dishes, 5 explants/dish, wtth the lower eptdermts downward. Seal with Parafi lm, and incubate at 25°C in culture room or an incubator (illumination not necessary) When sufficient callus has developed from cut surfaces of the explants (4--6 wk), exctse small pteces (0.3- to 0.5-cm dtameter), transfer to fresh RMP medmm, and culture as before. In thts way, callus stocks can be maintamed mdefimtely by subculturing at monthly mtervals. To mitiate cell suspension cultures, transfer about I g of callus to 5 0 mL of RMP medium (wtthout agar) m a 250-mL Erlenmeyer flask. Development of finely dtspersed cell suspensions may be accelerated by cutting the callus mto pteces first Incubate the cultures at 25°C on an rotary shaker at 1 00 rpm Subculture the newly formed suspensiOns after 3 wk by swtrhng each flask and pouring at 3-4x dilution into flasks of fresh RMP medmm. Subculture the cell suspensiOn cultures in the same way but at about SX dilutiOn, at 1 0- to 14-d intervals. After several such culture passages, the suspensiOns should become more finely dispersed and raptdly dtvtding At thts stage, the culture pas­ sage can be shortened to 7 d to ehmmate lag and statiOnary phases of the growth curve. (For further comments on the suspensiOn culture procedure, see Note 2.)

3.2. Determination of Selective Level of the Antimetabolite ( See Also Note 3) 1 . Prepare RMP medta containing the followmg concentratiOns of hydroxyprolme (mol wt = 1 3 1 ): 0, 0. 1 , 0.3, I , 3, 1 0, 30, and 1 00 mM (see Note 4). The medta are supplemented with 0.65% Difco Bacto-agar and dispensed, 20 mL!dtsh, into 9-cm Petn dtshes. Hydroxyprolme, from freshly prepared filter-sterilized stock solu­ tiOns (prepared in hquid RMP medium), ts added to flasks of autoclaved medium held at about 45°C Smce add1t10n of stock wtll m some cases result m substan­ ttal dilution of the agar m the medium, calculated adjustments m the imtial vol­ ume of liqu1d medmm m such flasks may be reqmred. Two stock solutiOns are recommended: 300 mM (39.3 mg/mL) for 1 00- and 3 0-mM final concentratiOns, and 1 00 mM ( 1 3 . 1 mg/mL) for the remainder. The volumes of med1a and stock solution required to gtve final volumes of 250 mL (sufficient for 1 2 dtshes) are summanzed in Table 2. 2. Place small (20- to 30-mg) pieces of healthy callus, taken from culture about 3 wk after subculture, on the surface of the medta described above. Five mocula should

Resistant Mutants

313

Table 2 Preparation of Media Containing a Range of Hydroxyproline Concentrations Fmal concentration,

Stock solution,

mM

mM

Volume autoclaved mediuma, mL

Volume autoclaved solution added, mL

1 00 30 10 3

300 300 1 00 1 00 1 00 1 00 1 00

1 67 225 225 242.5 250 250 250 250

83 25 25 7.5 2.5 0 75 0 25 0

1

0.3 0. 1 0

aEach flask contams agar for 0 65% concentration in a final volume of 250 mL (1 e , I 63 g)

be evenly spaced on each dish. Use 1 0 dishes/hydroxyproline concentratiOn to give a total of 50 calli/treatment. Seal dishes with Parafilm, and incubate at 25°C 3. After 4-6 wk, weigh the calli, determine the mean and standard deviation for each treatment, and after subtracting the initial inoculum weight, calculate the percentage of fresh weight mcrease compared to the control. Plot these values against hydroxyproline concentratiOn (log scale) 4. Select the lowest concentration givmg complete inhibition of growth (likely to be 1 0 or 30 mM) as the selective concentration for mutant isolation.

3.3. Mutagenesis and Selection of Resistant Cell Lines The following protocol commences With cell suspension cultures. I f pro to­ plasts are preferred, see Note 5. 1 . To 1 0 flasks of freshly subcultured cell suspension cultures, on a 7-d passage cycle, add filter-sterilized NEU or NMU to give a final concentratiOn of 0. 1 mM (important: see Note 6). 2. Culture the treated cell suspensions and also nonmutagenized controls in the usual way for 7 d. 3 . For each suspensiOn culture, prepare RMP medium· levels of all components and agar for 250 mL, and selective levels of hydroxyproline (again refer to Table 1). In this case, however, make each flask up to a final volume of200 rnL, not 250 rnL (omit 50 mL distilled water when preparing the medium). Place autoclaved flasks of medmm in a water bath at 45°C, to keep agar molten, for 30--60 mm prior to plating the cells. 4. F1lter the contents of each flask of cells through two layers of stenle mushn to remove large aggregates (this step may be omitted i f the suspensiOn is very fine), and pour the suspensiOn mto one of the flasks of molten medium. M1x by swirl-

Dix

314

Freshly Isolated protoplasts cultured at 1 OS /ml 1n 5 em Petn d1shes, 5 mUd1sh 1n K3 liqUid med1um w1th 0 4 M glucose and 0 1 mM NEU

t

7- 1 0 days

Contents of each dish transferred to a 9 em d1sh and diluted With K3 med1um w1th 0 4 M glucose to a f1nal volume of 1 0 ml

t

7- 1 0 days

Dilut1on With an equal volume of K3 w1th 0 3 M glucose Transferred to fresh 9 em dishes at 10 mUd1sh

t

7- 1 0 days

K3 w1th 0.2 M 9 em d1shes at

Dilution w1th an equal volume of glucose transferred to fresh 1 0 mUd1sh

t

7- 1 0 days

Contents of 5 dishes p1petted 1nto RMP w1th 0 2 M glucose, the selective level of hydroxyproline, and agar, to g1ve total volume of 250 ml Poured 1nto 1 2 x 9 em dishes

I

t

5-8 weeks

Remove resistant colomes

I

Fig 1 . A flow d1agram for 1solation of hydroxyproline-resistant colonies from meso­ phyll protoplasts of N plumbagmifolla. ing and pour mto 1 2 sterile 9-cm Petri dishes. Over 1 00 dishes contaimng the selective level of hydroxyproline and 1 2 control dishes without hydroxyproline should be obtamed. Cell aggregates arising from mesophyll protoplasts (see Note 5 and Fig. 1) are plated m the same way, except that filtration through muslin IS not necessary, and the platmg medmm mcludes 0.2 M glucose or sucrose. In thts case, ptpet (usmg a sterile Pasteur pipet) the contents of five dishes of protoplast-denved colonies mto each flask of molten medmm, sw1rl, and pour as above. The number of dtshes of protoplast-derived colonies and, therefore, the number of flasks of medium needed, will depend on the tmtial yield and viability of protoplasts, whtch ts difficult to standardize.

Resistant Mutants

315

5 . Allow the agar to set, seal each dish w1th a strip of Parafilm, and mcubate at 25°C in the dark. 6 Examine the dishes at weekly intervals. After 5-8 wk, remove any growmg colo­ mes from selective plates, and transfer mdividually to dishes of fresh RMP medium supplemented With the selective level of hydroxyproline. 7. After 4-7 wk, transfer small pieces (20-30 mg) of callus, from lines contmumg to grow on medium contammg hydroxyproline to RMOP medium in Petri dishes. Seal with parafilm, and incubate in the culture room at moderate (I 000-2000 lx) illumination. 8. After 5-8 wk, excise any adventitious shoots w1th normal leaves, and transfer to RM medium. Incubate for a further 4-6 wk, until a vigorous root system is estab­ lished. Transfer calluses that did not produce adventitious shoots on RMOP medium to fresh RMB medmm. Shoots may be regenerated on RMB, or after several altematmg culture passages on RMB and RMOP (see also Note 7). 9. Shoot cultures can either be mamtained m vitro by transferring nodal cuttings to fresh medmm as already described, or transferred to the greenhouse for growth to maturity and genetic analysis. In the latter case, gently remove agar from the roots, and plant individually in 1 2-cm pots 213 full of pottmg compost To avmd desiccation, cover tightly with chng film for 1-2 wk, until the plantlets are estab­ lished. When growing vigorously, transfer them to larger pots, where they should flower within 2-3 mo. 10. Check resistance of selected plants by imtiatmg callus from shoot cultures and testing for growth on medium containing selective levels of hydroxyprohne as already descnbed (see Note 8).

4 . Notes 1 . In the event of poor germmation, a gibberellic ac1d treatment can be mcluded. Steri lized seeds are placed m an aqueous solution of GA3 (0. 5 mg/mL) for 1 h before transferring to Petri dishes. 2. The object of th1s protocol is to provide a straightforward set of instructiOns that will give a good chance of obtaining the desired mutants. For this reason, detailed monitoring of the growth of the suspension cultures has been excluded in favor of a simple empirical approach that has proven effective in our hands If quanti­ tative data on mutatiOn frequency or d1rect compansons between a number of experiments are required, it may be desirable to momtor the growth of the sus­ pensiOns to ensure that comparable cultures are always used, and to perform cell 4 counts prior to platmg to ensure that a uniform cell density (about 1 0 cells/mL) is used in all experiments. Such measurements can pose their own problems in highly aggregated suspension cultures, but some useful guidelines and proce­ dures can be found in ref (7) and (8). 3 . Callus is the most convement material for producmg a dosage-response curve, as described in the protocol. In the case of hydroxyproline, there is a good correla­ tion between the sensitivity of callus and that of plated cells or protoplasts. For some chemtcals, thts correlation can be less precise, and it may be Wise, 1f suffi-

316

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c1ent material is available, to use as add1ttonal selective levels concentratiOns above and below that determmed by the callus test A more ngorous alternative is to measure dose-response by plating cells or protoplasts at the complete range of anttmetabohte concentrations, usmg exactly the same procedure descnbed m Sub heading 3.3. In this case, mstead of fresh weight, colony number after a smtable period (4--6 wk for plated cell suspenswns, 6-8 wk for plated proto­ plasts) should be plotted against antimetabolite concentratiOn. 4. Storage t1mes of culture medium: All media descnbed here, mcluding those con­ tammg hydroxyproline, can be stored for 3-4 wk, preferabiy in the cold room w1thout serious detenoratwn. Medmm m Petri dishes, however, IS subJect to con­ centratiOn by evaporation and should be used wtthm 2 wk of preparatiOn Even for this penod, it is important that they are sealed m bags (e g., those m wh1ch the sterile dishes are obtained from supphers) and kept m the cold room NMU and NEU should be added to medmm only 1mmed1ately before use. 5. If mesophyll protoplasts are the deSlfed starting matenal, the protocol for !Sola­ tion, mutagenesis, and culture of the protoplasts is exactly as described for the isolation of chloroplast mutants in Chapter 28, th1s volume. When the colonies are at the stage where they are ready to be incorporated mto solid medmm, they can be handled m exactly the same way as mutagen-treated cell suspenswn cul­ tures in the current protocol, except that 0.2 M glucose or sucrose should be mcluded m the RMP medmm used for plating. The steps in the isolation of pro­ lme res1stant mutants from mesophyll protoplasts are outhned m the flow dia­ gram (Fig. 1 ). 6. Both NMU and NEU, which are closely related, are dangerous carcinogens, and must be handled wtth extreme care We recommend the use of a respirator, and protective gloves and apron during all mampulatwns mvolving mutagens. It is important to avoid skm contact All working surfaces, balances, and the hke, where sp1llage m1ght have occurred, should be washed down 1mmed1ately after use Placing large sheets of absorbent paper backed w1th fo1l on the lammar flow work surface helps to contam any sp11lage It IS a good 1dea to exclude other workers from the work area while manipulatiOns with mutagen are m progress In the event of skm contact, and routmely after use, wash hands m soap and water gently, avo1dmg excessive rubbing of the skin. Do not use NMU in alkalme solu­ tions, because 1t IS very unstable After filter-sterihzing mutagen solutiOns, do not remove the syringe from the millipore unit Immediately, since the pressure that has bmlt up in the synnge can result in the release of an aerosol ofthe muta­ gen. Mutagen solutwns should be inactivated before d1sposal. Add an excess of 5% NaOH in the fume hood, and leave open overn1ght, before pourmg down the smk and chasmg w1th a large volume of tap water Contammated apparatus should also be treated wtth 5% NaOH, but in this case, after an overnight soakmg, a second wash (> 1 h) IS recommended, followed by a thorough nnse m running tap water. Both mutagens are unstable in aqueous solution, with the advantage that the low concentratiOns used here do not need to be washed from the cells. The rate of breakdown is highly pH-dependent, and it IS important that mutagen solu-

Resistant Mutants

7

8. 9.

10.

317

tions should be prepared using culture medium at tts normal p H (5.6). Further information about these mutagens can be found in ref. (9). It ts also posstble to select spontaneous mutants without mutagenic treatment, but a larger number of cultures may be requtred. Shoot regeneration from the resistant cell hnes ts a critical phase of the proce­ dure. The efficiency with which regeneration can be mduced by mampulating the hormonal composition of the medium (i.e., usmg RMOP or RMB) ts variable, and in the case of cell suspension cultures, may be quite low. Regeneration can often be stimulated by the mcluswn in the medmm of silver ions, m the form of silver nitrate at I 0 or 50 mg/L (10) . Resistance to the antimetabolite can also b e checked i n seedling progeny and callus derived from them. Mutagens can also be supplied at higher concentrations for short time penods to freshly isolated protoplasts or cell suspension cultures. The method ts fairly gen­ eral for a range of mutagens, mcluding NEU, NMU, and EMS In all cases, pre­ liminary expenments are needed to establish a suitable concentration and duration of treatment, one that gives a reduction to 1 0-50% colony formatwn m plated protoplasts, or cell suspension cultures compared to untreated controls. Recom­ mended concentration ranges to investigate are 0.3-1 0 rnM for NEU or NMU, or 0. 1-3% v/v EMS The duratiOn of treatment should be about 60 mm, after whtch the cells should be washed twice with fresh culture medium, before contmumg to culture and plating m the usual way. EMS is a volatile Iiqmd that needs to be dispensed into the liquid medmm m a fume hood. Prior to dtsposal, solutions contammg EMS should be inactivated by gradual addttion to a large excess of 3 M KOH in 95% ethanol, heated under reflux, repeatedly stirred for 2 h before dis­ posal down the drain, chased by a large volume of tap water UV mutagenesis t s best appl ied to protoplasts 24 h after tsol ation. D tshes o f protoplasts are placed under the UV source and the l ids removed before it ts turned on. A dose givmg 1 0-5 0% subsequent colony formatiOn com­ pared to nonmutagemzed contro ls should be used (typ tcally in the range 2 200-2000 erg/mm ).

References 1 . Dix, P. J. ( 1 986) Cell hne selection, in Plant Cell Culture Technology (Yeoman, M. M , ed.), B lackwell Scientific, Oxford, pp. 143-20 1 2. Dix, P. J. ( 1 994) IsolatiOn and charactensation of mutant cell lines, in Plant Tis­ sue Culture-Theory and Applications (Vasil, I . K. and Thorpe, T. A., eds.), Kluwer Academic, Dordrecht, pp. 1 19-138. 3 Dix, P. J. (ed.) ( 1 990) Plant Cell Line Selection. VCH, Weinhetm. 4. Negrutm I. (1 990) In vitro mutagenesis, in Plant Cell Line Selection (Dix, P. J , ed), VCH, Weinhetm, pp. 1 9-3 8. 5. Dix, P. J. ( 1 993) Use of chemical and physical mutagens zn vitro, m Plant Tissue Culture Manual · Fundamentals and Applicatwns (Lmdsey, K., ed. ), Kluwer Aca­ demtc, Dordrecht, F 1 , pp. 1-1 7.

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6. Maliga, P. ( 1 984) Cell culture procedures for mutant selection and characterisation

7

8.

9.

10

m Nzcotzana plumbaginifo/za, in Cell Culture and Somatzc Cell Genetzcs ofPlants, vol. 1 , Laboratory Procedures and Thezr Applzcations (Vasil, I. K., ed.), Aca­ demic, New York, pp. 552-5562 Kmg, P. J. ( 1 9 84) InductiOn and mamtenance of cell suspensiOn cultures, m Cell Culture and Somatic Cell Genetzcs ofPlants, vol. 1 , Laboratory Procedures and Thezr applzcatzons (Vasil, I. K , ed ), Academic, New York, pp 1 30-1 3 8 Gihs sen, L. W J., Hamsch ten Cate, C. H , and Keen, B . ( 1 983) A rapid method of determinmg growth characteristics of plant cell populations in batch suspen­ sion culture. Plant Cell Rep 2, 232-23 5. Hagemann, R. ( 1 982) Induction ofplastome mutatiOns by nitrosourea compounds, in Methods zn Chloroplast Molecular Bwlogy (Edelman, M., Hallick, R. B . , and Chua, N H., eds ), Elsevier/North Holland, BIOmedical, Amsterdam, pp. 1 1 9-- 1 27 Pumhauser, L., Medgyesy, P., Czako, M., Dix, P. J., and Marton, L. ( 1 987) Sttmu­ latwn of shoot regeneration m Trztzcum aestzvum and Nzcotzana plumbagznifolza VIv tissue cultures usmg the ethylene mhibitor AgN03 • Plant Cell Rep. 6, 1 -4

28

__ __ __ __ __ __ __ __ __ __ __

The Ge neration of Plastid Mutants In Vitro Philip J . Dix

1 . Introduction The plastid genome (or "plastome") encodes a number of proteins associ­ ated with the structure and function of chloroplasts, as well as tRNAs and rRNAs associated wtth the plastid translational machinery (1,2). Although there have been numerous studtes on the genetics of algal chloroplasts, similar stud­ Ies with higher plants have been hampered by the uniparental (maternal) pat­ tern of transmissiOn of chloroplasts observed in most species and also the shortage of suitable genetic markers (3). Two developments have added impetus to studies on htgher plant plastid genetics. First, procedures have been developed (4, 5) for the more efficient generation of plastome mutations. Second, breakthroughs m the genetic trans­ formation of plastids (6, 7) have led to an appreciation of the great biotechno­ logical potential of expressing foreign genes in the plastids (8). DNA delivery mto plastids has been achieved through biolistics or PEG-mediated uptake mto protoplasts, and selection of transformants IS based on one of two alternate strategies (9). Of these, the more attractive IS the use of plastid mutations con­ fernng msensitivity to antibiotics (10). As well as rapidly generatmg homo­ plasmic transplastomic hnes, these avoid the controversial use of bactenal antibiOtic resistance genes (9) and indeed can restrict the foreign DNA intro­ duced to solely the gene of interest. Thus, plastid mutations are now of interest both for the fundamental infor­ mation they can provtde on plastid function and as a means for selecting gene­ tically transformed plastJds. The protocols given here describe two reliable procedures to select for mutations m plastid ribosomal RNA or protem genes conferring insensitivity to several antibiOtics (streptomycm, spectinomycin, lmcomycin), m Solanaceous species. They use the mutagen mtroso-methylurea From Methods m Molecular Btology, Vol 111 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

319

320

Dix

Table 1 Components of Modified K3 Medium Used for Isolation and Culture of N. plumbaginifolia Protoplasts8 Ingredient CaC12 2H20 KN03 NaH2P04 2H20 NH4N03 (NH4hS04 CoC12 6H20 CuS04 7H2 0 FeS04 7H20 Na2 EDTA H3B03 •

·

•

•

mg/L 900 2400 1 20 240 1 30 0.025 0.025 27.85 37.3 3.0

Ingredient

mg/L

KI MnS04 4H20 Na2 Mo04 2H20 ZnS04 7H2 0 myo-mosJtwl Nicotinic acid Pyridoxine HCl Thiamine HCl Xylose pH

0.75 6.7 0.24 2.3 1 00 1 .0 1 .0 10 250 5.6

·

·

"Sugar 1s not mcluded m the above table Sucrose or glucose 1s added as descnbed m the hst under Subheading 2. It 1s convement to make up 5 L of the mediUm double-strength, om1ttmg only the sucrose or glucose, and stormg at -20°C. It is generally recommended to filter-stenhze the mediUm, but for N plumbagmifolla protoplasts, good results can be obtamed w1th autoclaved mediUm

(NMU), which has been shown to be efficient in targeting the plastome (11). The procedure usmg protoplasts IS effective for Nlcotwna plumbagmifolza (4) and with modifications (see Note 4) can also be used to obtam herbicide resis­ tant mutants. The leaf stnp procedure (5) has been used successfully for sev­ eral species (N1cotiana tabacum, N. plumbaginifolia, Nicotiana sylvestns, Solanum mgrum, and Lycopers1con peruvwnum) and can also generate chloro­ phyll-deficient mutants on nonselective plates. 2 . Materials I . Shoot cultures of the species to be used (see above)· The procedures for obtain­ ing and maintaining these cultures, starting from seed, are as descnbed m Chap­ ter 27, this volume. 2. Protopl ast enzyme solutiOn. Modified K3 medmm {12; Table 1) containing 0.4 M sucrose and 0.5% Dnselase (Kyowa Hakko Kogyo Co., Tokyo, Japan) (see Note 1). K3 can be filter-sterilized or autoclaved m a pressure cooker, and stored for up to 4 wk, preferably m the cold room Driselase must be added immediately before use, and the resultmg enzyme solutiOn filter-stenhzed. 3. Protoplast wash solution, W5 (12): 1 5 0 rnM NaCl, 1 25 rnM CaC12, 5 mM KCi, 5 mM glucose, pH 5.6 W5 may be autoclaved and stored m the cold room for up to 2 mo 4 Protoplast culture medmm Modified K3 supplemented w1th 0 4 M glucose, 0 1 mg/L 2,4-dlchlorophenoxyacetic ac1d (2,4-D, from a 0. 1 mg/mL stock pre-

Plasmid Mutants

5.

6.

7. 8 9. l 0. 11. 12

13. 14. 15. 16. 1 7. 1 8.

32 1

pared w ith the dropw ise addition of 0 . 1 M NaOH to dissolve the hormone), 0.2 mg/L 6-benzylammopunne (BA, from a 0.5 mg/mL stock, prepared in 0. 1 M HCl), and 1 mg/L naphthaleneacetlc acid (NAA, from a l mg/mL stock prepared as for 2,4-D, above). Also, the same medmm w1th glucose levels reduced to 0.3 and 0.2 M Regeneration medmm (RMOP): Contams (per liter)· 4 6 g MS salts (see Appen­ dix), 20 g sucrose, 1 00 mg mesoinositol, 1 mg th1amine-HCl, 1 mg BA, 0 1 mg NAA (see item 4 above for stock solutions of hormones), pH 5.6, solidified with 0.65% D1fco Bacto-agar. Leaf strip medium for L. peruvzanum: Contains (per litre): 4.6 g MS salts, 20 g sucrose, l 00 mg mesoinositol, l mg thiamme-HCl, 0 5 mg mcotimc acid, 0.5 mg pyridoxine-HCl, 0.2 mg mdoleacetic ac1d (IAA), and 2 mg zeatin (Sigma) Stock solutiOns ( l mg/mL) of these hormones should be freshly prepared m 0. 1 M NaOH (IAA) or HCl (zeatm), filter-sterilized, and added to molten, autoclaved medium (pH 5 6, 0.65% D1fco Bacto-agar) before pounng Leaf stnp medmm for S mgrum: As for L. peruvzanum, except zeatm is replaced by l mg/L BA RM medium: Contains (per liter)· 4 6 mg MS salts and 20 g sucrose, pH 5.6, solidified with 0.65% Difco Bacto-agar. RM solution: as for RM medmm, but Without the agar. 60-Jlm mesh nylon boltmg cloth. Mutagen: N-mtroso-N-methylurea (NMU) (see Note 2). Protective clothmg. Plastic or rubber apron and disposable gloves, mdustnal organic vapor cartndge respirator with cartndges, and prefilters (obtamable from "Sa-fir," East Hoathly, East Sussex, England) Large sheets of absorbent paper backed w1th fml . 5 M NaOH. 5% (w/v) Streptomycm sulfate in H20, filter-stenhzed. 5% (w/v) Lmcomycm hydrochloride in H20, filter-sterilized. Culture room facilities: Preferred conditiOns: 25° C, 1 500 lx, 1 6-h d. 0.4 M glucose.

3. Methods Mutant isolation is described below, using two different starting materials, leaf strips and mesophyll protoplasts. The use of leaf strips is technically more straightforward and is described first. Healthy leaves from shoot cultures of any of the species mentioned in Subheading 1 . can be used in exactly the same way, the only differences being the culture media required. In our experience, S. nigrum gives substantially higher yields of streptomycin-, spectmomycin-, or lincomycin-insensitive mutants than the other species. Albmo mutants can readily be obtained in the same experiments, as described in Note 3 . The rest of this sectiOn deals wtth the isolatiOn of streptomycin- and hnco­ mycin-insensitive mutants of N. plumbagznifolia from cultures initiated from

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mesophyll protoplasts. ModificatiOns of this procedure, necessary to obtam mutants resistant to terbutryn and other photosynthetic herbicides, are described in Note 4 . 3. 1 . Isolation o f Antibiotic-Insensitive Mutants from Leaf Strips

2

3 4 5.

6.

7

8.

Prepare mutagen solutwns after carefully readmg Note 2 Prepare a stock solu­ tion contammg 80 mg NMU m 20 mL of RM solutiOn, and filter-stenlize. For a 1 -mM solutwn, add 2.6 mL of stock to 97.4 mL of autoclaved RM solutiOn m a wide-necked 250-mL Erlenmeyer flask For a 5-rnM solutiOn, add 1 2 9 mL-8 7 1 mL of RM solutton. It ts Important that the RM solutiOns used have pH values of 5 5---{) 0 to enhance the stability of the mutagen Remove leaves from shoot cultures and cut into small stnps (2- to 3-mm wtde by 5- to 1 5-mm long, depending on leaf size). Add 2 5 0 stnps to 1 00 mL of each ( l and 5 mM) NMU solution and to 1 00 mL of RM solution (nonmutagenized control). Incubate the flasks on a rotary shaker (about 50 rpm) at 25°C for 90 min Decant mutagen solutiOn, and wash the leaf stnps four to five times m a large excess of RM solution or sterile distilled water (pH adjusted to 5 5-6 .0) For each treatment, place 200 of the stnps on the surface of selective medium m 40 plastic Petn dtshes (5 stnps/dish) The selective medta are RMOP for Nzcotz­ ana spectes, and the media descnbed in Subheading 2. for S mgrum and L peruvwnum, m each case supplemented with 500 mg/L streptomycm sulfate ( 1 000 mg/L can also be used for Nzcotzana spp and S mgrum ), I 00 mg/L spectmomycm, or lmcomycm hydrochlonde. The medmm is prepared by addt­ tton of a small aliquot from the concentrated, filter-stenhzed stock solutiOn of antibiotic to the autoclaved culture medmm, cooled to about 50°C pnor to pour­ mg mto dtshes. Place the remammg 50 stnps on the same medmm Without strep­ tomycin. Seal all dishes with parafilm, and mcubate m the culture room After about 40 d, the leaf strips on control dishes wtll show prolific shoot regen­ eratiOn, whereas those on antibiotic-containing medium are bleached and show little morphogenesis Green nodules wtll appear at the edges of some of the bleached leaf strips (from one or both mutagen treatments), and most of these will develop mto shoots. When shoots have at least two leaves that are normal, not "vitreous" (glassy and translucent) m appearance, remove cleanly with a scal­ pel, and transfer to RM medium (embedding cut stem m the medium) for rootmg Contmue to mcubate m the culture room After 4-8 wk, p lantlets with vtgorous roots are obtamed. Remove a smgle leaf, cut mto stnps, and test for msensttiVIty to the antJbtotlc on the same medium as that tmttally used for mutant selectiOn. Typically, msenstttvtty ts expressed m the mutants by the retentiOn of chlorophyll and the appearance of numerous green advent1t1ous shoots Rooted plants can either be propagated m vitro by nodal cuttmgs, or transferred to sotl for growth to matunty and genetic analysts. Both these procedures are descnbed m Chapter 28, thts volume.

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3.2. Isolation of Streptomycin or Lincomycin-Insensitive Mutants from Protoplast Cultures of N. plumbagin ifolia I . Remove healthy, fully expanded leaves from shoot cultures, sltce finely usmg a sterile razor blade (m a holder) or scalpel and forceps, and transfer to stenle protoplast enzyme solutiOn m 9-cm Petn dishes. Typically, 10 mL of solutiOn m a dish should be sufficient for three to four leaves, and three such dishes should provide enough protoplasts for one expenment. Protoplast yields are vanable, however, so It may be necessary to start with more material 2 Incubate overnight ( 1 2-1 6 h) at 25°C m the dark 3 . Swul the dishes several times to liberate protoplasts from leaf debns, remove the solutiOn with a Pasteur pipet, and filter through 60-�.tm nylon mesh mto a 1 00-mL Erlenmeyer flask. 4. Transfer the protoplast preparation to 1 0-mL capped glass centnfuge tubes (ster­ Ile), and spin at about 300g for 3 min. 5 Intact protoplasts float and form a tight green band at the surface of the medmm Remove this carefully with a Pasteur pipet, and transfer to a clean tube (not more than 1 mL/tube) Fill the tubes with W5 solutiOn, cap, invert to ensure thorough m1xmg, and spm at about 50g for 2 mm 6. Pipet off the supernatant, and resuspend the protoplast pellet m a small volume of protoplast culture mediUm (K3) supplemented with 0.4 M glucose Mix the con­ tents of the tube, count the mtact (spherical, with an umnterrupted plasma mem­ brane) protoplasts, and dilute to 1 05 /mL with the culture medmm 7 Transfer to 5 em Petn dishes, 5 mL/dish, usmg either previOusly calibrated Pas­ teur pipets or automatic pipets with wide-bore tips 8 To individual dishes, add (to a final concentration of 0. 1 or 0.3 mM ) NMU from a concentrated stock prepared m the culture medium and filter-stenltzed. (Impor­ tant: read Note 2 carefully before usmg the mutagen ) Wrap all dishes With parafilm and mcubate in the culture room at low light mtensity ( ca I 00 lx) 9 After 7-1 0 d, providmg divisions can be observed in the protoplasts, dilute the protoplasts 2x with fresh K3 mediUm with 0 4 M glucose. To do this, pipet the contents of each dish mto a 9-cm dish, and add 5 mL of medmm Seal and incu­ bate as before There is no need to wash out the mutagen, smce It IS unstable and breaks down within 48 h at the pH used 1 0. Monitor the development of the protoplast-derived cell aggregates, and make dilutions at suitable mtervals (see Note 5) In a good preparatiOn, these intervals should be of 7-1 0 d Each dilutiOn should be 2x, and lead to a doubling of the number of dishes contammg 1 0 mL culture. For the first dilutiOn, use K3 medmm with the glucose reduced to 0.3 M, and for the second, glucose reduced to 0 2 M W1thm 1 0--1 4 d of the latter di lutiOn, numerous microcolomes (about 1 -mm diameter) should be visible, and the cultures are then ready for platmg mto soltd medium 1 1 . For every four (more If there is a low colony density) dishes, prepare 500 mL of RMOP mediUm w1th 0.2 M glucose (instead of 2% sucrose), 0.65% agar, and 1 000 mg/L streptomycm sulfate or lincomycm hydrochlonde The antibiOtics are

Dix

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1 2.

13

14.

1 5.

added from the concentrated filter-stenhzed stock solutwn to autoclaved medmm, held at 45°C m a water bath. Usmg a fine-tipped Pasteur pipet, remove the excess K3 medmm from the cul­ tures to be plated. This Is best done by tilting the dishes slightly and allowing the microcolonies to settle, so that the medtum can be removed from above them Then, using a broad-tipped pipet, wash the contents of four dishes mto the 500-mL molten medmm by repeatedly transferrmg small amounts of the medium to the dishes and suckmg up agam, together w1th the colomes. Mix well, and pour mto 9 e m Petri dishes (about 2 0 mLidish) Allow the agar to set, wrap the dishes with parafilm, and incubate in the culture room at I 000- to 1 500-lx dlummatwn Numerous white colomes should appear after 1-2 mo. Streptomycm- or hncomycm­ msensitive colomes are green and are easily selected visually. P1ck them off when large enough and transfer to the same medmm, but With sucrose reduced to 0. I M Resistant colonies will contmue to grow and remam green. Subculture them onto RMOP medmm, Without the antibiOtic, for shoot regeneratiOn Regenerated shoots are handled as described for mutants isolated from leaf stnps.

4 . Notes 1 . Dnselase powder 1s a crude preparation containmg much msoluble material that can quickly block the milhpore filter. This can be prevented by either spmning for a few minutes m a bench centnfuge, or filtenng, to obtam a clean solutiOn pnor to filter sterilization. 2 NMU Is a dangerous carcmogen and must be used wtth great care We recom­ mend the use of a respirator, and protective gloves and apron during all mampu­ lations mvolving the mutagen. It 1s Important to avmd skm contact. All working surfaces, balances, and the hke, where spillage m1ght have occurred, should be washed down immedtate1y after use. Placing large sheets of absorbent paper backed w1th fm1 on the Iammar flow work surface helps to contam any spillage. It IS a good Idea to exclude other workers from the work area wh1le manipula­ tiOns with mutagen are m progress In the event of skm contact, and routmely after use, wash hands m soap and water gently, avmdmg excess we rubbmg of the skin. Do not use NMU m alkahne solutiOns because 1t is very unstable. After filter-steriltzmg mutagen solutions, do not remove the syrmge from the mtlltpore umt tmmedtately, smce the pressure that has butlt up m the synnge can result m the release of an aerosol of the mutagen Mutagen solutwns should be macttvated before disposal. Add an excess of 5% NaOH in the fume hood, and leave open overnight, before pouring down the smk and chasmg with a large volume of tap water. Contammated apparatus should also be treated wtth 5% NaOH, but in th1s case, after an overmght soakmg, a second wash (> I h) is recommended, followed by a thorough nnse m runnmg tap water. Additional advtce on the use of these mutagens ts given m ref (11). 3 . For all the species mentiOned, albmo mutants have also been obtained from leaf stnps. These anse m response to the mutagenesis treatment on the dishes from

Plasmid Mutants

325

which the selective agent (antibiOtic or herbicide) has been excluded. Among the nonnal green shoots differentiatmg from the leaf strips, some albino or vane­ gated shoots are frequently obtamed. Albmo shoots can be rooted and mamtained on RM medium m the same way as other shoot cultures. Albmo shoots can be obtained from vanegated ones by dissecting out white sectors and cultunng on the appropriate regeneration medium. 4 The procedure for Isolating antibwtic-res1stant mutants from protoplast cultures of N. plumbagzmfolia can be applted to the selectwn of mutants res1stant to her­ bicides that inh1b1t photosynthesis, providing a selective medtum perm1ttmg photomixotrophic growth IS used. Th1s is achieved by lowermg to 0.3% the glu­ cose level m the RMOP medium m which the m1crocolomes are plated. In order to reduce the osmotic stress resulting from plating m such a low-sugar medmm, an additional dilution step (with K3 medmm plus 0. 1 M glucose) 1s introduced into the protoplast culture procedure. For tnazme herb1c1des (e.g., terbutryn, atra­ 4 zine, stmazine ), a suttable selective level 1s I 0- M, and selection ts based on the greenmg of colomes, exactly as m the case of antlbtottc-resistant mutants. After retesting for resistance on selective medium, shoots are regenerated by transfer of small callus pieces to RMOP medium wtthout the herb1c1de Mutants reststant to metobromuron and bromoxynil have also been obtamed m thts way 5 The instructions for the gradual dilutiOn of protoplast cultures w1th fresh medium of decreasmg glucose concentration are given as accurately as poss1ble. The 7- to 1 0-d mterva1 should work, but careful monitonng of the cultures ts desirable If the growth rate of the colomes seems to be slow, a longer interval must be used On the other hand, raptd growth rates, especially tf accompamed by brownmg or the appearance of dead cells, mdtcates a requtrement for more raptd dtlutwn

References 1 . Dyer, T. A ( 1 985) The chloroplast genome and its products, in Oxford Surveys of Plant Molecular and Cell Bwlogy, vol 2 (Mifhn, B. J., ed.), Oxford Umverstty Press, New York, pp 147-1 77. 2. Borner, T and Sears, B. B. ( 1 986) Plastome mutants. Plant Mol Bzol 4, 69--92. 3. Medgyesy, P. ( 1 990) Selection and analysis of cytoplasmic hybrids, in Plant Cell Lzne Selection (Dtx, P J., ed ), VCH, Weinhetm, pp. 287-3 1 6 4. Cseplo, A and Ma!iga, P ( 1 984) Large scale isolation of maternally inhented hncomycin rest stance mutatwns m dtplotd Nzcotzana plumbagznzfolza protoplast cultures Mol Gen Genet 196, 407-4 1 2 5 McCabe, P F , Ttmmons, A. M., and Dtx, P. J. ( 1 989) A stmple procedure for the tsolatwn of streptomycm resistant plants in Solanaceae. Mol Gen Genet 216, 1 32-1 3 7. 6. Svab, Z., HaJduklewitz, P , and Maltga, P. ( 1 990) Stable transformation of plas­ tids in htgher plants Proc Nat!. Acad. Sci USA 87, 8526-8530 7. O'Neill, C M., Horvath, G. V , Horvath, E , Dtx, P J., and Medgyesy, P ( 1 993) Chloroplast transfonnatwn m plants: polyethylene glycol (PEG) treatment of pro­

toplasts IS an alternative to bwltsttc deltvery systems. Plant J. 3, 729-738

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8. McBnde, K. E., Svab, Z., Schaaf, D J., Hogan, P S. Stalker, D. M., and Maliga, P ( 1995) Ampltfication of a chtmenc Baczllus gene m chloroplasts leads to an extra­ ordinary level of an msecttctdal protein m tobacco. Rio/Technology 13, 362-365. 9. Dtx, P J. and Kavanagh, T A. ( 1 995) Transformmg the plastome genettc mark­ ers and DNA dehvery systems. Euphytzca 85, 29-34. 1 0. Kavanagh, T A., O'Driscoll, K. M , McCabe, P F., and Dix, P. J ( 1 994) Muta­ tions confernng hncomycm, spectmomycin, and streptomycin resistance m Solanum nigrum are located m three dtfferent chloroplast genes Mol Gen Genet 242, 675-680 1 1 . Hagemann, R ( 1 982) Induction of plastome mutations by nttroso-urea-com­ pounds, m Methods zn Chloroplast Molecular Bzology (Edelman, M., Hallick, R B., and Chua, N. H , eds.), Elsevter Btomedtcal, Amsterdam, pp. 1 1 9-1 27. 12. Maltga, P. ( 1 984) Cell culture procedures for mutant selection and charactenza­ twn in Nicotzana plumbagmifolza, m Cell Culture and Somatzc Cell Genetzcs of Plants, vol 1 (Vasil, I. K., ed.), Academic, New York, pp. 552-562.

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Agrobacterium-Med iated Tra nsformation of Petunia Leaf Disks Ingrid M. van der Meer

1 . Introduction Agrobacterzum-medtated transformation of plants is now applicable to many dicotyledonous and also several monocotyledonous plant species. It can be used to transform many dtfferent species based on various factors: the broad host range of Agrobacterium (1), the regeneration responsiveness of many dif­ ferent explant tissues (2), and the utility of a wide range of selectable marker genes (3) . In addition to tobacco, one of the first species that was routinely transformed usmg this method was Petunia hybrida. P. hybrida is a very good model plant for the analysis of gene function and promoter activity. It 1s readily transformed, the culture conditions are easy ful­ filled, generation time is 3--4 mo and one can grow up to 1 00 plants/m2 . Fur­ thermore, its genetic map is well developed, and it contams active transposable elements (4). The protocol presented here 1s a simplified vers10n of that of Horsch et al. (5) . The basic protocol involves the inoculation of surface-sterilized leaf disks with the appropriate disarmed strain of Agrobacterium tumefaciens carrying the vector of choice, which in this protocol confers kanamycin resistance. The plant tissue and Agrobacterzum are then coculttvated on regenerat10n medmm for a period of 2 or 3 d. During this tlme, the virulence genes m the bacteria are induced, the bacteria bind to the plant cells around the wounded edge of the explant, and the gene-transfer process occurs (6). Using a nurse culture of tobacco or Petunia cells during the coculture period may increase the transfor­ mation frequency. This is probably owing to a more efficient induction of the vtrulence genes. After the cocultivat10n period, the growth of the bactenal population is inhibited by bacteriostatic antib10tics ( cefotaxim or carbenicilFrom· Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by A D Hall © Humana Press Inc , Totowa, NJ

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lm), and the leaf ttssue IS mduced to regenerate. The induction and develop­ ment of shoots on leaf explants occur m the presence of a selective agent agamst untransformed plant cells, usually kanamycin. Dunng the next 2-3 wk, the transformed cells grow mto callus or differentiate into shoots vta organogen­ esis. After 4-6 wk, the shoots have developed enough to be removed from the explant and induce rooting m preparation for transfer to sml. To speed up the rooting period, the shoots can be rooted without selection on kanamycin. In total, it takes about 2 mo, after inoculation of the leaf disks with Agrobacterium to obtain rooted plantlets that can be transferred to soil. 2. Materials 2. 1. Bacteria Media I . For the growth of Agrobactenum, use Luna broth (LB) medmm· I % Bacto-peptone (Dtfco, DetrOit, Ml). 0.5% Bacto-yeast extract (D1fco) 1 % NaCI. Autoclave, and cool medmm to at least 60°C Add appropnate ant1b10t1cs to select for plasm1ds (50 mg/L kanamycin for pBin l 9 {7}) 2. LB agar: LB medmm w1th 1 5 g/L agar (Difco) Autoclave, and cool medium to at least 60°C Add appropnate antibiotics to select for plasm1ds (50 mg/L kanamy­ cm for pBm l 9). Pour mto stenle 20-mm Petn d1shes 3 Agrobactenum inoculatiOn dliutton medmm Murashige and Skoog (8, and see Appendix) salts and v1tamms (4.4 g/L) (S1gma, Amsterdam). Autoclave.

2.2. Stock Solutions For convemence, most stock solutiOns are prepared at 1 000 times the con­ centration needed for the final media. The antibiotics are added to the medta after autoclaving when the temperature of the medta has cooled to 60°C. Cefotax1me. 250 mg/mL (Duchefa, Haarlem, The Netherlands, or S1gma), filter­ stenhze, keep at -20°C 2 Kanamycm: 250 mg/mL (Duchefa or Sigma), filter-stenhze, keep at -20°C 3 6-Benzylammopunne (6-BAP), (Sigma)· 2 mg/mL Dissolve 200 mg BAP in 4 mL 0 5 N HCl Add, while stirring, drop by drop H20 at 80-90°C and make up to I 00 mL Fi lter-sterihze. 4 1 -Naphtalene acetic acid (NAA) (Sigma)· I mg/mL, dissolved m DMSO No need to stenhze. Keep at -20°C DMSO should be handled under a fume hood

2.3. Plant Culture Media I . Cocult1vatton medmm. Murashtge and Skoog (8, and see Appendtx) salts and v1tamms (4.4 g/L) (Stgma), 30 g/L sucrose, 2 mg/L 6-BAP, 0 0 1 mg/L NAA, adjust pH to 5 . 8 w1th I M KOH, add 8 g/L agar (Bacto Difco) and autoclave. Pour mto stenle plastic dishes that are 20 mm h1gh (Greiner, Kremsmunster, Austna)

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2. Regeneration and selection medium: Murashige and Skoog salts and vttamins (4.4 g/L) (Sigma), 30 g/L sucrose, 2 mg/L 6-BAP, 0.0 1 mg/L NAA, adJUSt pH to 5.8 with 1 M KOH, add 8 g/L agar (Bacto Difco ). Autoclave and cool medta to 60°C, add 250 mg/L cefotaxtme to kill off Agrobactenum, and the appropnate selective agent to select for transformed cells depending on the vector used ( 1 00 mg/L kanamycin for pBin 1 9) . Pour 25 mL mto each sterile 20-mm htgh Petri dish (Gremer) 3 . Rooting medium: Murashige and Skoog salts and vttamms (4.4 g/L) ( Stgma), 30 g/L sucrose, adjust pH to 5 . 8 with 1 M KOH, and add 7 g/L agar (Bacto Difco) Autoclave and cool media to 60°C, add 250 mg/L cefotaxtme to ktll off Agrobactenum, and add the appropnate selective agent to select for transformed shoots dependmg on the vector used ( 1 00 mg/L kanamycm for pBm 1 9). To speed up the rootmg process, kanamycin may be omttted from the rootmg medmm Pour m Magenta GA7 boxes (S tgma, 80 mL per box).

2.4. Plant Material, Sterilization, and Transformation P hybnda c.v W 1 1 5 (Mitchell), grown under standard greenhouse conditions 2. A tumejac1ens stram LBA 4404 and A tumefaczens LBA 4404 contaming pBm 1 9 (m which the gene of interest t s mserted) 3. 1 0% So Iutton of household bleach contammg 0. 1 % Tween or other surfactant 4 Stenle H20 5 Stenle filter paper (Whatman) and sterile round filters (Whatman, dtameter 90 mm)

2.5. General Equipment Stenle transfer factlities

2. Rotary shaker at 28°C 3 . Cork borer (or a paper punch) 4 Magenta GA 7 boxes (Stgma) and 20-mm htgh Petn dtshes (Gremer). 3. Methods 3. 1. Plant Material Young leaves are used as the explant source for transformation. These explants can be obtained from aseptically germinated seedlings or mtcro­ propagated shoots, but m this protocol, they are obtamed from greenhouse­ grown material. The genotype of the source material is important in order to obtain high transformation rates. P. hybnda cv.W l 1 5 gtves the best results and is most often used (see Notes 1 and 2). To obtain plant maten al suitable for transformation, seedlmgs should be germmated 4-6 wk pnor to transformation. Sow P. hybrzda (W 1 1 5) seeds in sotl 4--6 wk pnor to transformatiOn, and grow under standard greenhouse conditions in 1 0 x 1 0 x 1 0 em plastic pots. Use commerctally avatlable nutnttve solutiOn for house plants.

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3.2. Leaf Disk Inoculation I . Grow A tumefaczens culture overntght in LB at 28°C on a rotary shaker ( 1 3 0 rpm) with appropnate antibiotics to select for the vector (50 mg/L of kana­ mycm for pBin 1 9) (see Note 3). The Agrobacterzum hqutd culture should be started by inoculating 2 mL of liqutd LB with several bacterial colomes obtamed from an Agrobactenum streak culture grown on an LB agar plate at 28°C for 2-3 d. The streaked plate 1tself can be moculated from the ongmal -80°C frozen stock of the Agrobacterium stram (see Note 4). Th1s stock IS composed of a bac­ terial solutwn made from a 1 . 1 mixture of stenle glycerol (99%) and an over­ night LB culture of the Agrobacterzum 2 Prepare the culture for moculation of explants by taking 0.5 mL of the overmght culture and dilutmg 1 to 200 with Murashige and Skoog salts and v1tamms medmm (4 4 g/L) (Sigma) to a final volume of 1 00 mL The A tumefaciens mocu­ lum should be vortexed well pnor to use Pour the Agrobacterzum inoculum into four Petn d1shes 3. When the plants are I 0- 1 5 em h1gh, harvest the top leaves to prov1de ex plants (leaves 3-8 from the top) Lower leaves and leaves from flowermg plants should not be used, smce they usually have a lower transformatiOn and regeneratiOn response (see Note 1 ) 4 . Prepare harvested leaves for moculatwn by surface stenhzatwn for 1 5 m m m 1 0% solutiOn of household bleach contammg 0 I% Tween or other surfactant Wash the leaves thoroughly three t1mes with stenle H20 Keep them m stenle H20 until needed (see Note 5) All procedures followmg the bleach treatment are conducted m a stenle transfer hood to maintam t1ssue stenhty 5. Punch out leaf d1sks w1th a stenle ( 1 -cm dmmeter) cork borer (or cut mto small squares to produce a wounded edge) in one of the Petn d1shes contaimng the Agrobacterium moculum (20-25 disks/Petri d1sh) (see Note 6) Avmd the m1dnb of the leaf or any necrotic areas Cut 80-100 d1sks/construct (see Note 7). 6. Leave the d1sks m the inoculum for 20 mm After moculatwn, the explants are gently sandw1ched between two layers of stenle filter paper (Whatman) to remove excess moculum 7 (Optional) Prepare nurse culture plates by addmg 3 mL of cell suspenswn culture (e g , P. hybrzda cv Coomanche or Nzcotzana tabacum cv SR I ) to Petn d1shes contammg cocult1vatwn medmm Sw1rl the suspensiOn to spread the cells over the surface of the medium and cover w1th a stenle Whatman filter paper (diam­ eter 90 mm) (see Note 8). 8 Place 20 explants w1th the adax1al surface downward on each plate w1th coculttvation medmm (either with or without nurse cells), and mcubate for 2-3 d (see Note 9). Seal the plates w1th Nescofilm The culture cond1t10ns for the leaf d1sks are as follows a temperature of 25°C and a photopenod of 14 h hght (hght mtens1ty· 25--40 11ffi OI!m2/s)l l O h dark The controls to check the transformatiOn protocol are Leaf d1sks maculated with "empty" Agrobacterzum (w1thout pBm 1 9 vector) on regeneratiOn medmm w1thout selective agent (to check the regeneratwn).

Petunia Leaf Disks

33 1

Leaf disks inoculated With "empty" Agrobacterzum (without pBin 1 9 vector) on regeneration medium with selective agent (to check the efficiency of antibiotic selection) 9. After cocultlvation, transfer the disks to regeneration and selection medmm (seal the plates with Nescofilm), and continue incubation until shoots regenerate Transfer the explants every 2-3 wk to fresh regeneration and selection medium (see Note 1 0).

3.3. Recovery of Transformed Shoots 1 . After 2-3 wk, the first shoots will develop Cut off the shoots cleanly from the explant/ callus when they are 1-1 .5 em long, and place them upright in rootmg medium in Magenta boxes The shoots should be excised at the base without taking any callus tissue (see Note 1 1) . Take only one shoot from each callus on the explant to ensure no siblmgs are propagated representmg the same transfor­ mation event (see Note 12). Shoots from distinctly different calli on the same explant are, however, likely to be derived from different transformatiOn events and should be transferred separately. Give each shoot a code that allows it to be traced back to specific explants Cefotaxime is kept in the medtum to avoid Agrobacterzum regrowth. The anti­ biotic used to select for the transgenic shoots can be added to the medmm to select against escapes, although the rooting process is sped up when it is omitted. 2. (Optional) Before removing rooted shoots from sterile culture, transfer a leaf to selection medium to test for resistance to kanamycin Ifthe leaf 1s obtamed from a tranformed plantlet 1t should stay green and form callus on selectiOn med1um. If It ongmates from an untransformed plantlet, 1t should become brown/white and die within a few weeks. 3. After 3-4 wk the shoots will have formed roots. Remove plantlets, wash agar from the base under a running tap, plant the transformants m soli, and transfer them to the greenhouse (see Note 13). To retain humidity, cover the pots w1th Magenta boxes or place them m a plastic propagatiOn dome. The plants should then be allowed to come to ambient humidity slowly by gradually openmg the dome or Magenta box over a penod of 7 d (see Note 14). 4. Fertilize and grow under standard plant growth conditiOns.

3.4. Analysis of Transformants Tissue culture can be used to confirm that the putative transgenic shoots produced are expressmg the selectable marker gene (see Subheading 3.3., step 2). However, DNA analysis using Southern blotting or PCR w il l con­ firm whether regenerants have integrated the antibiOtic resistance gene (and also a gene of mterest if this was linked to it withm the T-DNA). For DNA analysis using Southern blotting (9), leaf DNA can be isolated accord m g to the protocol described by Dellaporte et al. (1 0) . A more rapid method can be used to Isolate genomic DNA as descnbed by Wang et al. (1 1) when PCR Is

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going to be used to analyze the presence of foretgn DNA in the transformed plants (see Note 1 5) Usually, one to five coptes of the foretgn DNA is mtegrated m the plant genome usi n g the Agrobacterzum-mediated transformation method. However, position effects may silence the expression of the introduced gene (see Note 7) 4 . Notes

2.

3

4 5.

6.

7.

8

9. 10

Thts transformatiOn protocol works very well for the often-used P hybnda vane­ ties W l l 5 (Mitchell) and V26 However, some Petuma ltnes show poor regen­ eration from leaf dtsk explants, and consequently, few or no trans formants can be obtained from these plants. Umform, clean, and young plants will perform best It is important not to take leaf matenal from old, flowenng plants Instead of greenhouse matenal, aseptically germmated seedlings or micro­ propagated shoots could also be used as explant source. Then, of course, there IS no need to surface-stenhze the leaves. The Agrobactenum stram that ts most often used is LBA4404 (Clontech {12}). Also the more Virulent strams C58 or AGLO (13) can be used, but these can be more dtfficult to ehmmate after cocultlvatwn. Dunng growth, the Agrobacterzum culture will aggregate. The streaked plate can be reused for approx 3 wk if kept at 4 oc after growth Be very gentle wtth the plant matenal dunng stenhzatwn, smce the bleach will easily damage the leaves, especially wounded or weak, etiolated leaves Damaged tissue should not be used. Dtsks provide a very uniform explant and are conveniently generated with a cork borer or paper punch However, square explants or strips can also be used Av01d excessive woundmg during the process. The cork borer should be allowed to cool before use after flammg. Thts transformatiOn protocol will yteld approx 20 transgenic plants from I 00 tmttal explants The expressiOn level of the construct of interest can be greatly mfluenced by a positiOn effect owmg to Its stte of mtegratton mto the host plant genome. Thts stlencmg of expression owing to position effects can occur in 2�0% ofthe transgenic plants, especially tf weak promoters are used. Therefore, at least 20 transformants should be generated/construct The nurse culture ts not essential for transformatiOn, but can facilitate the process by increasing frequency and reducmg damage to the explant by the bactenum Any healthy suspension of tobacco or Petuma should work The suspensiOn cul­ tures can be maintamed by weekly transfer of 1 0 mL mto 50 mL of fresh suspen­ siOn culture medtum The cocultivation time may have to be optimized for different Agrobactenum strains carrying different vectors I f Agrobacterium contmues to grow on the regeneratiOn and selection medtum (formmg shmy gray-whtte bactenal colonies), 1 5 0 mg/L ofvancomycm can also be added to the medium already contammg cefotaxtme

Petunia Leaf Disks 11 1 2.

13.

14

15.

333

Care should be taken that only the stem and none of the associated callus IS moved to the rootmg medium Otherwise, no roots will develop. It is common for multiple shoots to arise from a single transformed cell. There­ fore, it is Important to separate mdependent transformatiOn events carefully so that siblmg shoots are not excised It is important to wash away all of the agar medium from the roots and to trans­ plant before the roots become too long When there is still agar left, Jt could enhance fungal growth. Gradual reduction m the humidity I S necessary to harden off the plantlets m soil. The roots must grow mto the sml, and the leaves must develop a protective wax cuticle. If the plantlets start to turn yellow and die from fungal contammatwn, the hd should be opened faster If the plantlets begm to wilt, the hd should be opened slower Confirmation of transformatiOn by PCR may not always be reliable, owmg to possible carryover of the Agrobacterium mto the whole plant. DNA analysis usmg Southern blot hybnd1zation Is a better way to confirm whether regenerants are true trans formants Furthermore, the number of mserts can be determmed at the same time using thts method

References I . Richie, S W. and Hodeges, T. K. ( 1 993) Cell culture and regeneratiOn of trans­ gemc plants, m Transgenzc Plants, vol I (Kung, S. and Wu, R., eds.), Academic, San D1ego, pp. 147-1 7 8 2 Jenes, B., Morre, H , Cao, J . , Zhang, W., and Wu, R (1 993). Techmques for gene transfer, m Transgenzc Plants, vo1 1 (Kung, S and Wu, R., eds) , Academic, San Diego, pp. 1 25-146 3 Bowen, B. A. ( 1 993) Markers for gene transfer, m Transgenzc Plants, vol l (Kung, S. and Wu, R., eds.), Academic, San Diego, pp. 89-124 4 Gerats, A G M , Hmts, H , VriJlandt, E , Marana, C , Souer, E , and Beld, M ( 1 990) Molecular characterization of a nonautonomous transposable element (dTph l ) of petuma Plant Cel/ 2, 1 1 2 1-1 1 2 8 5 Horsch, R B , Fry, J E., Hoffman, N. L., Eichho1tz, D , Rogers, S C , and Fraley, R. T ( 1 985) A simple and general method for transfernng genes mto plants Sczence 227, 1 229-! 23 ! 6. Hooykaas, P J. J. ( 1 989) Transformation of plant cells via Agrobactenum. Plant Mol Bwl 13, 327-3 36 7. Bevan, M. ( 1 984) Bmary Agrobactenum vectors for plant transformatiOn. Nuclezc Aczds Res 12, 87 1 1-872 1 8. Murash1ge, T. and Skoog, F. ( 1 962) A revised medmm for rapid growth and bw­ assays with tobacco tissue cultures. Plant Physwl 15, 473-497. 9 Mamahs, T , Fritsch, E. F , and Sambrook, J ( 1 982) Molecular clonzng A Labo­ ratory Manual Cold Spring Harbor Laboratory Press, Cold Spnng Harbor, NY 1 0. Dellaporte, S. L., Wood, J., and Hicks, J. B. ( 1 983) A plant DNA mm1preparat10n: versiOn II. Plant Mol. Bwl Rep. 1, 1 9--2 1

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1 1 . Wang, H., Qi, M , and Cutler, A J ( 1 993) A simple method of preparing plant samples for PCR. Nucleic Aczds Res 21, 4 1 53-41 54 12 Hoekema, A., HirSch, P. R , Hooykaas, P J 1., and Schilperoort, R. A ( 1 983) A bmary plant vector strategy based on separation of vzr and T region of the Agrobactenum tumefaczens TI-plasmid Nature 303, 1 79-180 13. Lazo, G R., Stein, P. A , and Ludwig, R. A. ( 1 99 1 ) A DNA transformation-compe­ tent Arabidopsts genomic hbrary m Agrobacterzum Bzo/Technology 9, 963-967

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Transformation of Rice via PEG-Medi ated DNA Uptake i nto Protoplasts Karabi Datta and Swapan K. Datta

1 . Introduction For stable transformation of cereals through PEG-mediated DNA uptake into protoplasts, the two most critical requisites are the ability to Isolate and culture protoplasts m large numbers, and the development of an efficient and rehable system for routine plant regeneration from protoplasts (1-3). Based on early suc­ cess with mesophyll protoplasts of some dicotyledonous species, extensive efforts were made to induce sustained divisiOn m protoplasts Isolated from leaves or young shoots of different cereal plants. However, there is still no convmcmg evtdence of sustained dtvtsions in protoplasts isolated from leaves or shoots of any cereal In contrast, protoplasts isolated from embryogenic suspension cul­ tures could be induced to divide m culture (4). Obtainmg a fast-growmg and highly embryogemc suspensiOn culture is the most important factor for cereal plant regeneration from protoplasts (4-8). Microspore cultures, and immature or mature embryos may be used to obtain embryogenic call i which can eventual ly be used to establish embryogenic cell suspensiOns ECS (2, 7) . PEG-mediated gene transfer to rice protoplasts appears to be the most effi­ cient, reliable, inexpensive, and simplest method, when it works (2, 8, 9). In this system, from a suspension culture, a large population of protoplasts can be readily obtamed for transformation enabhng many chances of obtaimng inde­ pendent transformation events. RegeneratiOn of transgenic plants is possible under suitable in vitro conditiOns through selectiOn at an early stage of develop­ ment. However, the tissue-culture response may vary dependmg on the plant genotype, handling, and the condttion of the suspension cells. We have success­ fully used the procedure descnbed below for gene transfer to rice for at least I 0 Indica and several Japomca cultivars. From Methods

Edited

m

Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols by R D Hall © Humana Press Inc , Totowa, NJ

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336 2 . Materials 2. 1. Suspension Culture Establishment

2. 1 . 1 Plant Material Plant material is Oryza sativa-Indtca-type rice cultivar, Chinsurah Boro II, IR 72, Japomca type rice culttvar-Yamabiko: Immature mflorescence (panicles collected pnor to the emergence from the flag leaf sheath), immature embryos (pamcles collected 7-1 0 d after anthesis) and healthy mature seeds from the mentioned cultlvars.

2. 1 .2. General Equipment Laminar flow cabmet, mverted mtcroscope, hght microscope, mcubator, gyrat­ mg shaker, autoclave, and disttlled water plant 2 Nescofilm, flasks, beaker, measunng cylmder, medta bottles, forceps, scalpel, sctssors, sptrit lamp, and ptpet bulb 3 Plastic Petn dtshes (50 mm), 24-well COST A plates, disposable ptpets, 2- and I 0-mL capacity. 4 Greenhouse facihttes

2. 1 . 3. Sterilizing Solutions I . 70% Ethanol. 2 Sodium hypochlonte solut10n (e.g , 1 00 mL 1 .8% sodmm hypochlonte w1th two drops Tween 20) 3. Sterile dtsttlled water.

2. 1 .4. Media Mtcrospore culture medmm (R I ) Quanttty ( mg/L) Component NH4N03 1 650 KN03 1 900 CaCI2 2H20 440 MgS04 7H20 370 KH2P04 1 70 KI 0.83 6.3 H3 B03 MnS04 4H20 22.3 ZnS04 7H20 86 0 25 Na2Mo04 2H20 0 025 CuS04 5H20 CoCI2 6H20 0.025 37 3 Na2EDTA 27.8 FeS04 7H20 •

•

•

•

Transformation of Rice 10 Thiamine HCI 500 Glutamme 300 Casem hydrolysate 1 00 Myo-mosttol NAA 2 1 2,4-D Sucrose 6% w/v Fico II 400 1 0% w/v pH should be 5.6. Based on MS medium (10) . Steri lize by filtration 2. AA medmm (11) Quanttty (mg/L) Component 1 50 CaC1 2 2H20 250 MgS04 7H20 1 50 NaHzP04 H20 2950 KCI 0 75 KI 3.0 H3B03 10 0 MnS04 H20 ZnS04 7Hz0 2.0 0.25 Na2Mo04 2H20 0.025 CuS04 5H20 CoCI2 6H20 0 025 I 0 Ntcotme actd 1 .0 Pyndoxme-HCI 10 0 Thtamme-HCl 1 00 Inosttol 37.3 Na2EDTA 27 8 FeS04 7H20 876 L-G1utamine 266 Aspartic acid 1 74 Arginine Glycine 75 2,4-D 1 .0 Kinetin 0.2 GA3 0 1 20 g/L Sucrose/maltose pH should be 5 6 Sterilize by filterat10n. 3. Modtfied MS medtum Quanttty (mg/L) Component 1 650 NH4N03 1 900 KN03 440 CaCI2 2H20 370 MgS04 7H20 ·

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•

•

•

•

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1 70 KH2P04 KI 0.83 H3B03 6.3 22 3 MnS04 4H20 8.6 ZnS04 7H20 Na2Mo04, 2H20 0 25 CuS04 5 H20 0 025 CoCI2 6H20 0 025 27.8 FeS04 7H20 37.3 Na2 EDTA 05 Nicotmtc actd Pyridoxtne-HCl 0.5 1 0 Thmmine-HCl Glycine 20 300 Casein hydrolysate 1 00 Myo-mosttol 1 5 2 ,4-D Kmetm 0.5 Sucrose/maltose 30 g/L Agar 8 g/L pH should be 5 . 8 . This is based on MS medium (10) Steriltze by autoclavmg 4. R2 medmm (11) Quantity (mg/L) Component NaH2P04 2H20 240 KN03 4040 330 (NH4)2S04 247 MgS04 7H20 1 47 CaC12 2H20 0.50 MnS04 H20 ZnS04 7H20 0 50 H3B03 0 50 CuS04 5H20 0 05 0 05 Na2 Mo04 37.3 Na2 EDTA 27 8 FeS04 7Hz0 0.5 Ntcottmc actd 0.5 Pyndoxine-HCI 1 Thtamme-HCI 2 Glycine 1 00 Inositol 1 2,4-D 20 g Sucrose/maltose pH should be 5 8. S teriltze by autoclaving. •

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Transformation of Rice

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2.2. Protoplast Isolation, Transformation, and Regeneration

2.2. 1. General Equipment I. 2 3 4 5 6.

Centrifuge. Temperature-controlled shaker 1 2-mL round-bottom screw-cap centnfuge tubes. Petn dishes, 1 0 (deep model) and 3 5 em (Falcon) Nylon Sieves-50 and 25 J..U1l (Saulas, F93 1 00 Montreutl, France) Counting chamber (hemocytometer)

2.2.2. Solutions and Medium 1 . Enzyme so Iutton. 4% w/v cellulase onozuka RS, I % w/v macerozyme R l 0 (both Yakult Honsha Co., Japan), 0.02% pectolyase-Y23 (Se1shin Pharmaceu­ tical Co , Japan), 0.4 M manmtol, 6.8 mM CaCI2, pH 5 . 6; filter-sten hze, and store at -20°C 2 Wash solution 0 4 M mannitol, 0 1 6 M CaC12; autoclave 3. MaMg solutiOn (transformatiOn buffer). 0 4 M manmtol, 15 rnM MgCI 2 , I% (w/v) MES, pH 5.8, autoclave. 4. PEG solutiOn (40% w/v): Dt ssolve 80 g PEG 6000 (Merck, Art 1 2 033, 1 000) in 1 00 mL distilled water contammg 4 72 g Ca (N03h · 2H20 and 14.57 g manmtol. The PEG is dissolved by heatmg carefully m a microwave. Make the total volume 200 mL w1th distilled water. Divide the solution into two parts. In one part, the pH is adJusted to 1 0 .0 using I M KOH and left over­ night to stabilize Then, adJUSt the final pH to 8.2 usmg the second part of the solution Filter-stenlize the solutiOn and store as 5-mL ahquots at -20°C (see Note 1 ). 5 P I medmm· R2 medmm w1th 2 mg/L 2,4-D and 0 4 M maltose, pH 5 6, filter­ stenlize (Table 1). 6 Agarose-protoplast medtum 600 mg (dry autoclaved) Sea plaque agarose melted in 30 mL P I medmm 7. P2 medium. Soft agarose N 6 medium (13) with 6% maltose or sucrose, 2 mg/L 2,4-D, and 0.3 g Sea plaque agarose; autoclave (Table 1 ). 8. DNA for transformation - Plasmid DNA is used, 1 0 �Jg/sample of protoplast sus­ pensiOn ( 1 .5 x 1 06 protoplasts) is used for transformation. D1ssolve 1 0 mg calf thymus DNA (used as earner DNA) in 5 mL distilled water. Shear by passage through an I S -gauge needle to give an average fragment s1ze of 5- 1 0 kb (check by running gel) Carner and plasmid DNA is sterihzed by precipitation and washing in 96% ethanol (also possible with 70% ethanol), and dried m a laminar flow hood. DNA 1s dissolved m sterile double-distilled water (2 j..ig/fll) and stored at 4°C (or at -20°C for longer period). 9. Nurse cells--OC cell hne derived from seedlmgs of 0. sativa L. C5924, pro­ Vided by K. Syono of Umversity of Tokyo (see Note 2).

Datta and Datta

340 Table 1 Composition of the Media Used Component (NH4)2 S04 KH2 P04 KN03 NH4N03 CaCI2 2H2 0 MgS04 7H2 0 Na2 EDTA FeSO 7H2 0 NaH2P04 2H2 0 2H2 0 MnS04 4H20 H3B03 ZnS04 H 2 0 Kl CoC12 6H2 0 CuS04 5 H2 0 Na2 Mo04 2H 20 Thmmine-HCl Nicotinic acid Pyridox me-HCI Glycme Myo-mositol Kinetin NAA 2,4-D Sucrose g/U Maltose g/U Agar g/L Agarose g/L ·

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·

P 1 mg/L

P2 mg/L

330 0

463 0 400 0 2830 0

4044 0 1 47.0 247 0 37 3 27.8 240 240 05 0.5 05

0.05 0 05 I 0 0.5 05 2.0 1 00 0

1 66 0 I 85 0 37.3 27 8

44 1 6 1 .5 08

I 0 05 05 20 1 00.0

P3 mg/L 1 70 0 1 900 0 1 650 0 440 0 370 0 37.3 27 8

22.3 6.3 86 0 83 0.025 0.025 0 25 10 05 0.5 2.0 I OO.O

2.0 1 .0

20 1 3 6.92 144. 1 2

20 60.0 60.0

30.0 8.0

pH 5 6 F i Iter-sterilized

3 or 6 pH 5 8 Autoclaved

pH 5 8 Autoclaved

0Either sucrose or maltose to be used

3. Method 3. 1. Growth of Donor Plants I . Break the dormancy of clean and pure seeds of denved lines m the oven at 50°C for 3-5 d. 2. Sow the seeds in seed boxes and water sufficiently to wet the soil

Transformation of Rice

34 1

3 . At 2 1 d after sowing, transplant the seedlings to 6-m. diameter pots (one seedling per pot) contammg soil and fertilizer (2.5 g [NH4h S04, 1 .25 g P205 and 0. 75 g K2 0/pot). 4. Grow the plants m the glass house, and keep them well watered. The glass house should have full sunshme and sufficient ventilation to mamtam daytime tempera­ tures of 27-29°C With a 90% humidity level.

3.2. Establishment of Embryogenic Suspension Culture

3.2. 1 . From Microspores I . Collect the tillers from the donor plants when most of the microspores are at the m1duninucleate stage In all cereals mvestigated, the early or midunmucleate stage of microspore development was found to give the best results (14). 2. Stenlize the selected spikes wtth 70% ethanol for 30 s, and then treat wtth sodmm hypochlorite solution for 7 min followed by washing three times with sterile deiomzed water (15) . 3. Float 30 anthers, each containing microspores a t the miduninucleate stage on the sur­ face of 10 mL of liquid microspore culture media (R 1) m 50-mm sterile Petri dishes. 4. Culture in the dark at 25°C. These floating anthers shed microspores mto the medium within 3-7 d. 5. After 4-6 wk, transfer the developing embryogemc calli (about 0 5 g) to 7 mL of AA medium (11) to establish the embryogemc cell suspension (Fig. lA). At th1s stage, subculturing t s necessary at least twice a week until a fine cell suspenswn IS obtamed. This cell suspension can then be subcultured every 7 d for long-time maintenance (at least 1 yr).

3.2.2. From Immature or Mature Embryos 1 . Stenlize dehulled Immature ( 1 2 d after anthesis) or mature seeds m 70% ethanol for 1 min, and then sodium hypochlorite ( 1 .8%) solution for 30 min followed by washing three times with sterile dewnized water. 2. Isolate the embryos from the stenle seeds, and place mdividually in 24-well COSTA plates with each well contammg 1 .5 mL of modified MS medmm. The scutellar tissue should be upward, since callus growth occurs only With th 1s onentatwn. 3. Incubate the embryos in the dark at 25°C After 3-4 d, cut off the emerging shoots and roots, and subculture the remaining tissues onto fresh medium. At least two more subcultures are necessary at 2-wk intervals until yellowish-white soft embryogenic calli develop on the surfaces of the scutella 4. Transfer this callus to 6 mL of liquid AA medium (11) m 50-mm Petri dishes, and 2 incubate on a gyratmg shaker at low speed (80 rpm) m diffuse light (3 ,.unol/m /s) at 25°C. 5. Subculture the callus every 7 d with continued manual selectiOn of small and densely cytoplasmic cells, which are transferred to 20 mL ofR2 medium in 100-mL Erlenmeyer flasks (see Note 3) These suspensiOns can be maintained for a long

Datta and Datta

342

Fig.

t.

Transformation of Rice

343

time by subcultunng every 7 d, and incubatmg with gyratory shakmg (80 rpm) at 25°C in dark or diffuse light.

3.3. Protoplast Isolation I . Transfer (approx) 3-5 g of suspension cells 3-4 d after subculture mto a deep 1 0-

2. 3. 4. 5 6.

cm Falcon Petri dish. Allow the suspension cells to settle and remove the medium by pipetmg off (see Note 4). Add 20 mL of enzyme solutwn, seal with Nescofilm, and mcubate Without shakmg at 30°C in the dark for 3-4 h (dependmg on protoplast release) (see Note 5) Add an equal volume of wash solutiOn to each Petn dish contammg the proto­ plast enzyme mixture Remove the protoplast suspension with a 1 0-mL pipet, and pour through 50- and 25-fllll steves into a sterile glass beaker ( I OO mL) Transfer the filtrate to l 0-mL round-bottom screw-cap centrifuge tubes, and cen­ trifuge for 1 0 min at 70g to separate off the enzyme solutiOn Di scard the supernatant, and resuspend the protoplast pellet m washmg solutiOn. Centrifuge and repeat once. Resuspend the pellet m l 0 mL of washing solutiOn. Count the density of protoplasts/mL usmg a hemocytometer Freshly Isolated protoplasts from ECS should appear densely cytoplasmic (Fig. lB; see Note 6)

3.4. Direct Gene Transfer to Protoplasts Using PEG I . Centnfuge the protoplasts

2. 3

4.

5. 6

m wash solutiOn at 70g for I 0 mm (I.e., third washing). Remove the supernatant, and resuspend the protoplast pellet m transformation buffer (i.e., MaMg solution). Adjust the protoplast density to 1 .5-2 0 x I 06/0.4 mL Distribute the protoplast suspension (0.4 mL contaimng 1 .5 x 1 0 6 protoplasts) into different centrifuge tubes using a 2 mL-pipet. Add 6-1 0 j.Ig of stenle plasmid DNA (preferably hneanzed) and 20 j.Ig of calf thymus carrier DNA (transformation can be performed without calf thymus) to each 0.4-mL ahquot of the protoplast suspension (see Note 7) Add 0.5 mL of the PEG solution dropw1se, and mix gently. Incubate at room temperature (or preferably at zooq for 1 0 mm. Add slowly 1 0 mL of wash solutiOn, mix, and centrifuge at 70g for 10 mm to remove the PEG. Discard the supernatant. Resuspend the protoplast pellet in 0.4 mL P 1 medium

Fig. I . (prevwus page) (A) Fertile transgenic nee plants from transformed proto­ plasts obtained usmg the PEG method. ECS developed from microspore culture (B) Freshly 1solated, densely cytoplasmic, protoplasts from ECS. (C) Early div1s1on in protoplast culture. (D) Protoplasts m selection medium (left: Petn dish nontransformed cell, right: Petri dtsh wtth transformed calli developing from transformed protoplasts). (E) Protoplast-derived calli m bead-type culture without selectiOn. (F) Putative transgenic rice plants in a plastic tray containing Yosh1da solutiOn after transfer to the transgenic greenhouse. (G) Transgenic fertile rice plants in the greenhouse.

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3.5. Culture of Protoplasts I . Melt agarose-protoplast medium m a microwave oven, and cool to 40°C 2 M1x 0.6 mL of the agarose-protoplast medmm with 0.4 mL of P I medmm con­ tammg protoplasts, and transfer mto a 3.5-cm Petn d1sh. Incubate at 20°C for I h The final density of the protoplasts should be around 1 5 x I 0 6/mL 3. Cut the solidified agarose gel mto four segments, and transfer each to a 5-cm Petn dish contaming 5 mL of P I medium (bead-type culture) (16) with or With­ out adding nurse culture (6,17) Incubate cultures in the dark at 28 5°C with slow shaking (40 rpm) 4. After 7 d, replace 2 mL medium w1th fresh P l medmm 5. At the l Oth d after protoplast isolation, remove (all) the nurse cells by transfer­ ring the segments to a fresh Petn d1sh con taming 5 mL of P 1 medi urn. Keep m dark at 28.5°C w1th slow shaking (30 rpm)

3. 6. Selection 1 . FirSt selectiOn. 1 4 d after transformatiOn, add the selective agent to the medmm (e.g., hygromycm B 25 mg/L, or kanamycm 50 mg/mL, G-4 1 8 25 mg/L, or phosphinotricm 20 mg/L, depending on the selectable marker gene used) Incu­ bate as above, but wtthout shakmg Early dtviswns of protoplasts are already present at this stage (Fig. 1 C). 2 Second selection· after 2 wk, the selection pressure should be mamtained in the same way by replacing 4 mL of medium with fresh medmm supplemented w1th the same concentratiOn of selective agent. Culture for another 2 wk Putatively transformed, protoplast-derived colomes are vts1ble at thts stage (Fig. 1 D) Nontransformed colonies without selectiOn are shown in Fig. IE. 3. Replace 4 mL of P l medmm with 2 mL of fresh P l medium and 2 mL of suspen­ sion culture medium (R2 medium) Without selective agent Culture for 2 wk. 4 Th1rd selectiOn: Transfer the visible calli (putative transformed colomes) onto soft agarose medium (P2 medmm) contaming 0.3% agarose and the same con­ centration of selective agent. Incubate m the dark at 25°C for 2 wk (see Note 8) 3. 7. Regeneration I . Transfer the vtsJble colonies to P2 medmm contaming 0.6% Sea plaque agarose without the selecttve agent (Table 1 ) Culture for another 2 wk under the same condttions 2. Transfer the selected embryogemc colonies with a size of ca. 1 .5--3 .0 mm d1ameter with developing somatic embryos to P3 medium (Table 1). Incubate in darkness at 25°C until the development ofembryos or embryogemc calh 1s observed (see Note 9). 3. Transfer the developmg embryos onto the same medium, and culture m the hght 2 (24 f..UTIOI!m /s), with a 1 6-h photoperiod at 25°C to obtain plantlets 4 Transfer the plantlets to MS medmm wtthout hormones unt1l a well-developed root system IS obtained. These plants can be transferred to the transgemc green­ house tf mamtamed under h1gh hum1dity for the first 2 wk (see Note 10) Alter­ natively, plants can be transferred to a culture solutton (18) m a plastic trays,

Transformation of Rice

345

which may be placed m a phytotron or greenhouse {wtth 29°C light penod, 2 1 °C dark period), 1 4 h/day, hght 1 60 ).lll10l/m2 /s, daylight supplemented with Philips HPL N400W mercury lamps if necessary and 70--95% relative humtdity (Fig. lF; Note 1 1 ). The plants when transferred to soil (Fig. lG) set seed after 3-4 mo.

4 . Notes 1 . For each transformation expenment ( 1 0 samples), use a new tube (5 mL) of PEG from the -20°C freezer to avoid posstble toxic effects and changes of pH 2. For nurse cells: any actively dividing nee cell suspension, even the same suspen­ sion culture used for protoplast isolation can be used. In our case, we use an OC cell line that Is not regenerable, but still actively dtvides 3 . Some suspension hnes (IR72) grow better m N 6LP medium, I.e., N6/P l basal medmm (13) supplemented with 1 g/L prolme, 860 mg/L glutamme, 1 . 5 mg/L 2,4-D, and 3% w/v sucrose or maltose (1, 5) 4 Regenerable embryogemc cell suspensiOn cultures (ECS) should be used for protoplast transformation. ECS can be obtamed for all genotypes of japonica, mdiCa, and IRRI-New plant-type nee. Usually, a 3- to 6-mo old suspensiOn culture shows a better effictency for protoplast tsolatwn, transformatiOn, and sub­ sequent regeneratiOn (1 9). 5 . Enzymatic digestion of suspension cells should not exceed 5 h; if sufficient pro­ toplasts are not released, the experiment for that day should be terminated. 6. The handling of protoplasts should be very gentle at all stages of the protocol 7. For the selectiOn, we use the selectable marker genes, such as the hygromycin resistance gene, kanamycm resistance gene, or a herbicide resistance gene Some­ times a gene of interest may be linked to the selectable marker gene. For cotransformabon, we add 1 0 J,tg ofplasm1d DNA contammg the gene of mterest, l 0 J.tg of selectable marker gene, and 20 J.tg of calf thymus DNA Supercotled plasmtd DNA (50 J,tg/mL/1 0 x 1 06 protoplasts even Without calf thymus DNA) is suitable for transient gene expression, as examined 24 h after transformation. 8. A total period of selectiOn pressure of <2 wk may produce escapes SelectiOn pres­ sure for longer than 8 wk may affect the further growth and fertility of the plants, and may produce abnormal plants Keep the selection pressure to the mmtmum t1me requtred to obtain the transformants. It IS always better to remove the culture from the selection pressure to avoid later abnormality and sterility of the plants 9. Take care of those embryogenic calli developmg faster and separate them from the rest. Transfer them earlier for regeneratiOn to plants. Faster-developmg calh produce healthy (normal) plants It is recommended to place a few (four to s1x) embryogemc calli together in a Petri d1shlflask for regeneration. 1 0. Regenerated transgemc plants should be transferred to sml within 6 wk to mtm­ mize the tissue-culture effect. 1 1 In some countries, e g , m Philippines, nee plants grow very well in the glass house/greenhouse conditions throughout the year without any art1fictal hght 1 2 . Molecular analysis (Southern and Western analysis) should confirm the integra­ tion and expression of the transgene in the nee genome

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Referen ces 1. Datta, K , Potrykus, I., and Datta, S. ( 1 992) Effictent fertile plant regeneration from protoplasts of the Indica rice breedmg line IR 72 ( Oryza sativa L ) Plant Cell Rep 1 1, 229-233. 2. Datta, S. K., Peterhans, A , Datta, K., and Potrykus, I. ( 1 990) Genetically engi­ neered fertile Indica-rice plants recovered from protoplasts. Bio/Technology 8, 73 6--740. 3 . Peterhans, A , Datta, S. K., Datta, K , Goodall, G. J., Potrykus, I., and Paszkowski, J ( 1 990) Recogmtton efficiency of Dzcotyledoneae-specific promoter and RNA processing signals m nee. Mol. Gen Genet 222, 36 1-368. 4. Vasil, I . K. ( 1 987) Developing cell and tissue culture systems for the Improve­ ment of cereal and grass crops. J Plant Physiol 128, 1 93-2 18 5. Datta, S. K., Datta, K., Soltamfar, N , Donn, G., and Potrykus, I. ( 1 992) Herbicide­ resistant Indica nee plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts Plant Mol B10l 20, 6 1 9-629. 6 Kyozuka, J., Hayashi, Y., and Shimamoto, K. ( 1 987) High frequency plant regen­ eration form nee protoplasts by novel nurse culture methods. Mol Gen Genet 206, 408-4 1 3 7 . Datta, S . K , Datta, K., and Potrykus, I ( 1 990) Ferttle Indica nee plants regener­ ated from protoplasts tsolated from mtcrospore derived cell suspensions. Plant Cell Rep 9, 253-256. 8. Datta, S. K. ( 1 995) Polyethylene-glycol-mediated direct gene transfer to mdica rice protoplasts and regeneration of transgenic plants, m Gene Transfer to Plants (Potrykus, I . and Spangenberg, G , eds ) , Spnnger-Verlag, New York, pp 66--7 4. 9. Datta, S. K. ( 1 996) Genetic transformatiOn of rice from protoplasts of haploid on­ gm, m In Vttro Haplozd ProductiOn m Hzgher Plants, 2 (Jam, S. M., Sopory, S K , and Veilleux, R. E , eds.), Kluwer Academic, The Netherlands, pp. 41 1-423. l O Murashige, T. and Skoog, F. ( 1 962) A revtsed medium for rapid growth and bto­ assays with tobacco tissue cultures Physiol Plant. 15, 473-497. 1 1 Muller, A. J and Graffe, R. ( 1978) Isolatwn and characterization of cell hnes of Nicotzana tabacum lacking nitrate reductase. Mol Gen Genet. 1 6 1 , 67-76. 1 2. Ohira, K., OJima, K., and Fuj iwara, A ( 1 973) Studies on the nutrition of rice cell culture. I . A simple, defined medmm for rapid growth m the suspenston culture Plant Cell Physzol 14, 1 1 1 3-1 1 2 1 . 1 3 . Chu, C C., Wang, C . C , Sun, C. S., Hsu, C., Ym, K. C , and Chu, C . Y { 1 975) Establishment of an efficient medmm for anther culture of nee through compara­ tiVe expenments on the mtrogen sources. Scz. Sm 18, 659-668 14. Datta, S. K., Datta, K., and Potrykus, I. ( 1 990) Embryogenesis and plant regen­ eration from microspores of both "mdica" and "Japomca" nee (Oryza satzva) Plant Scz. 67, 83-88. 15 Datta, S K. and Wenzel, G. ( 1 987) Isolated microspore denved plant formation vta embryogenesis m Trittcum aestivum Plant Scz 48, 49-54.

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1 6. Shillito, R D , Paszkowski, J., and Potrykus I ( 1 983) Agarose platmg and a bead type culture techmque enable and stimulate development of protoplast-denved colonies m a number of p1ant species. Plant Cell Rep. 2, 244--247 1 7. Smith, J. A., Green, C. E., and Grengenbach, B. G. ( 1 984) Feeder layer support of low density populatiOns of Zea mays suspension cells. Plant Sc1 Lett 36, 67-72 1 8 . Yoshida, S., Forno, D. A., Cock, J. H., Gomez, K. A. ( 1 976) Routme procedure for growing rice plant in culture solution. Laboratory Manual for Physwlogical Studies ofR1ce The International Rice Research Institute Los Banos, Philippines. pp 6 1-66 1 9 A1am, M. F., Datta, K., Vasquez, A. R., Oliva, N., Khush, G. S., and Datta, S K ( 1 996) Transformation of new plant type lmes through biolistic and protoplast methods R1ce Genet Newsletter 13, 1 39-1 4 1

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Tra nsformation of Wheat via Particle Bombard ment lndra K. Vasil and Vimla Vasil

1 . Introduction Wheat (Triticum aestzvum L.) is the number-one food crop in the world based on acreage under cultivation and total production. It is also the most widely cultivated crop as a result of breeding for adaptation to a wide range of eco­ logical conditions Wheat was first domesticated nearly 1 0,000 years ago m the Fertile Crescent of the Ttgns-Euphrates basm m southwestern Asia. As a major commodity in international agriculture, and an important source of nutri­ tion and protein in the human diet, wheat has long played a central role m world food secunty. Dunng 1 965-1 990, introductiOn of the Green RevolutiOn high-mput and high-yieldmg varieties led to a nearly threefold mcrease m world wheat production. However, increases m the productivity of wheat and other major food crops attamed through breedmg and selectiOn have begun to decline. This is happenmg at a time when nearly a third (34%) of the wheat crop IS lost to pests, pathogens, and weeds (1), in addition to postharvest losses during storage. Introduction of single genes through genetic transformation into crops such as maize, soybean, potato, canola, and cotton, has shown that such losses cannot only be greatly reduced or even ehmmated, but can also result m reduced use of pesticides and herbicides. Wheat was the last of the major crops to be transformed (2, 3), because of technical difficulties and the long time needed to establish reliable protocols for high-efficiency regeneration and transformation. Owing to the inherent dif­ ficulties m the establishment of embryogemc suspensiOn cultures and regen­ eration of plants from protoplasts, fertile transgenic wheat plants have so far been obtained only by the direct delivery of DNA mto regenerable tissue explants by accelerated microproj ectiles (3-6) . The followmg protocol From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Edtted by R D Hall © Humana Press Inc , Totowa, NJ

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descnbes the genetic transformatiOn of wheat by particle bombardment of tmmature embryos usmg the PDS- 1 000/He (B 10-Rad) system (3, 4, 6). Although genes for resistance to hygromycin (7), kanamycm (8), and glyphosate (9) have been used as selectable markers m wheat transformation, the best results have been obtained with the bar gene (phosphinothncm acetyltransferase, PAT, 3-6, 8, 1 0, 1 1), which confers res1stance to DL-phosphmothncm (PPT) or glufosinate, the active mgredient m the commercial herbicides Herbtace, Basta, and Btalaphos (12). The method descnbed here 1s based on the use of the plas­ mid pAHC25 (4, 13), contaming the uidA (p-glucuromdase) and bar as reporter and selectable marker genes, respectively (4, 6, 14). Both genes are under the control of the matze ubtqmtm promoter (4, 13), which has been shown to pro­ vide h igh levels of gene expression in gramineous species (15). T h e stabi lity of integration and express10n of the transgenes has been shown for several gen­ eratiOns (14, 16, 1 7). 2. Materials 2. 1. Culture of Immature Embryos

2

3

4. 5.

Plant matenal: Wheat (T. aestivum L., cv Bobwhtte) plants are grown either m the field, greenhouse, or in growth chambers (first 40 d at l 5°C/ l 2°C day/mght 2 temperature and 1 0-h photopenod at 1 50 !1ffiOl/m /s, followed by mamtenance at 20°C/1 6°C temperature and 1 6-h photopenod; see Note 1). Medium for culture of immature Embryos· Murashige and Skoog ' s (18) formula­ tiOn was used as follows (MS + medium) 4.3 g MS salts (Sigma #M5524). 20 0 g Sucrose 1 00.0 mg Myo-mosttol 500.0 mg Glutamme. 1 00.0 mg Casem hydrolysate 2.0 mL 2,4-dichlorophenoxyacetic acid (2,4-D) from stock (see item 4 below). 2.5 g Gelrite. Bnng volume to 1 L with dtsttlled water, and adJust pH to 5.8 with 4 N NaOH After autoclavmg, add 1 mL filter-stenhzed vitamin stock (see item 5 below) Pour medium in Petri dishes ( 1 00 x 1 5 mm, Fisher). The stenle medta can be stored for 1 mo at room temperature Medmm for osmotic treatment of embryos. Add 36 44 g sorbitol (0 2 M) and 36 44 g mannitol (0 2 M) to make up l L of MS+ medium. Pour in Petn dishes (60 x 1 5 mm) after autoclaving. 2,4-D stock: Dissolve I 00 mg 2,4-D in 40 mL 95% ethanol, and make up the volume to 1 00 mL with distilled water. Store m refngerator. VItamm stock: Dissolve 1 0 mg thiamin, 50 mg nicotinic acid, 50 mg pyndoxme HCI, and 200 mg glycme m dtsttlled water, and make up volume to I 00 mL. Store at -20°C

Particle Bombardment

35 1

2.2. Preparation and Delivery of DNA-Coated Gold Particles 1 . Stenle distilled water 2 Gold particle stock 1 s prepared essentially accordmg to protocol provided by Bw­ Rad with PDS/ 1 000 He. Suspend 60-mg gold particles (ca. l - J.l111 diameter) in 1 mL 1 00% ethanol for 2 mm in 1 .5-mL Eppendorf tube, w1th occaswnal vortexmg. Centrifuge for 1 mm. Discard supernatant, and resuspend in 1 mL stenle distilled water by vortexing. Wash two more times, and finally resuspend m 1 mL sterile water Ahquot 25 or 50 J.IL in 0. 5-mL Eppendorf tubes, wtth vortexing between aliquots to ensure proper mixing Store at -20°C 3. CaC12 (2.5 M, filter-stenhzed) m 1 -mL ahquots, stored at -20°C. 4. Spermidine-free base (0 I M, filter-sterilized) in 1 -mL aliquots, stored at -20°C 5. Ethanol ( 1 00%). 6. pAHC25 DNA at 1 ).lg/J.IL tn TE ( 1 0 mM Tns-HCl, 1 mM EDTA, pH 8). 7. Hehum gas cylinder. 8. Sonicator (optional)

2.3. Culture, Selection, and Regeneration

2. 3.

4

5. 6.

Medmm for selectiOn. MS+ medmm Without glutamme and casem hydrolysate, with 3 mg/L b1alaphos (4 mg/mL stock) added after autoclaving. Pour in Petn dishes ( 1 00 x 1 5 mm). Medmm for regeneration· Selection medium without 2 ,4-D, and supplemented with l-10 mg/L zeatin, added after autoclavmg. Medium for shoot elongatiOn: MS salts (2. 1 5 g), sucrose ( 1 5 g), myo-mos1tol (50 mg), and 2.5 g gelrite. Make volume to I L with distilled water, pH 5 . 8. After autoc1aving, add 0 5 mL vitamm stock and 5 mg/L bialaphos. Pour m Petn dishes ( 1 00 x 20 mm). Medmm for root elongatwn: Shoot elongation medium ( 1 0 mL) w1th 4-5 mg/L bialaphos in culture tubes ( 1 00 x 25 mm). All med1a with b1alaphos can be stored for 2 wk at room temperature. Zeatin stock solutwn: Dissolve 40 mg zeatm in few drops of I M NaOH, and make volume up to 1 0 mL in distilled water. F ilter-sterilize. Bialaphos stock solution: Dissolve 40 mg of bialaphos in 5 mL d1sttlled water, and make up volume to 1 0 mL Filter-sterilize and store at -20°C.

2.4. Analysis of Transient and Stable Expression

2.4. 1 Assay for GUS Activtty X-gluc ( 1 5-bromo-4-chloro-3-indolyl-b-glucuronide)· 1 00 mL; 1 00 mg X-gluc dissolved in 40 mL distilled water (2 mM). 50 mL 0 2 M phosphate buffer, pH 7.0 ( 1 00 mM). 1 mL 0.5 M potassium ferricyanide (5 mM). 1 mL 0.5 M potassium ferrocyanide (5 mM) . I mL 1 M EDTA, pH 7.0 ( 1 0 mM).

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Bnng volume to I 00 mL and sttr unttl dissolved. Add I 00 � Tnton X- I 00, filter-stenlize (22 J.lll)l , and store m small ahquots at -20°C.

2.4.2. PA T Assay l . PAT extraction buffer (EB buffer) 1 00 mL 5 mL 1 M Tns-HCI, pH 7.5 (50 mM). 0.4 mL 0.5 M EDTA (2 mM) 1 5 mg leupeptine (0. 1 5 mg/mL) 1 5 mg phenylmethylsulphonyl fluonde (PMSF, Stgma) (0 1 5 mg/mL). 30 mg bovme albumm (BSA) (0 3 mg/mL) 30 mg dtth!Othrettol (DTT) (0 3 mg/mL) St1r to dtssolve, filter-stenhze, and store at -20°C 2 AB buffer 1 00 mL 5 mL 1 M Tns-HCI, pH 7 5 (50 mM) 0.4 mL 0 5 M EDTA (2 mM) 100 mg BSA (0. 1 %) Make up volume to 1 00 mL with dtstilled water, and store m refngerator 3 Saturated ammomum sulfate. 400 g (NH4hS04 in 400 mL distilled water, pH 7.8 Autoclave and store at room temperature Can be used for at least 3 mo. 4. Thm-layer chromatography (TLC) m1grat10n solvent 500 mL· 300 mL !-propanol 1 7 8.6 mL Ammonmm hydroxide Make up volume to 500 mL w1th water (saturate the chromatography tank over­ mght with 3-mm paper) Htghly pungent; wear mask 4 5 1 C-labeled acetyl-Co-A Wear gloves and work m area designated for radiOactive work. 6 TLC plates. 7. Chromatography tank. 8 Kodak (X-Omat) AR X-ray film 9 Bulk reactiOn m1xture for PAT assay Prepare the necessary volume for two reac­ tiOns more than the total number of reactions to allow for p1petmg error For one 4 react10n. 0 6 � PPT ( l mg/mL) , I � 1 C acetyl Co-A (0 0 5 llCi/�), 1 .4 11L distilled water. 3. Methods

3. 1. Culture of Immature Embryos Collect spikes ( 1 1-14 d post-anthesis }, and wrap m moist paper towels Sptkes can be used fresh or stored for up to 5 d, wtth cut ends Immersed m water, m a refngerator (see Note 2). 2 Remove caryopses from the middle half of each spike, and surface-stenlize wtth 70% ethano l for 2 m m , followed by slow stt rnng for 1 5-20 mm m 20% chlorox solutiOn ( I 05% sodmm hypochlonte) contaimng 0 l % Tween 20 Remove chlorox by washmg with four changes of stenle distilled water at 5-mm mtervals (see Notes 3 and 4).

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Aseptically remove the Immature embryos (0.8-1 5 mm) under a stereomJCroscope, and place in Petri dishes containing MS+ medium, with the scutellum exposed and the embryo axis m contact with the medium (see Note 5) Twenty to 25 embryos can be cultured m one Petn dish Seal the culture dish With Parafilm, and mcubate m the dark at 27°C to induce proliferation of scutellar cells (see Note 6) 4 Transfer embryos 4-6 h pnor to bombardment (4-6 d after culture, when cell proliferation IS visible at the edges of the scutellum) to Petri dishes contammg the medmm for osmotic treatment. Arrange 30--40 embryos, With scutellum facmg up, m a 2-cm diameter circle in the center of the d1sh (see Note 7). 3

3.2. Bombardment of Embryos I.

2 3. 4. 5.

6. 7 8. 9.

10 II

12. 13.

14. 15

Stenhze the bombardment chamber of PDS I 000/He and the gas acceleratiOn cylinder with 70% ethanol, and allow to dry. Stenhze the rupture and macrocamer disks by soakmg m 1 00% ethanol for 5 mm and a1r-drymg m lammar flow hood Stenlize steel screens by autoclaving Add to two, 25-J.tL ahquots of washed sterile gold particles, an equal volume of stenle distilled water, and vortex for 5 s Add 5 IJL plasmid DNA or 5 1JL/construct for cotransformatwn to one tube of gold particles (the other tube of gold particles serves as control for bombardments) Vortex Immediately for 30 s to ensure good mixing of DNA and gold partiCles Add, in qmck successiOn, 50 IlL of CaC1 2 and 20 IlL of sperm1dme, and vortex for 2 mm Hold on 1ce for 5- 1 0 mm. Centnfuge for I 0 s, discard supernatant, and resuspend m 200 IlL I 00% ethanol (can somcate bnefly to disperse) Centrifuge for I 0 s, d1scard supernatant, and resuspend the pellet in 1 50-250 IJ.L of 1 00% ethanol. Leave on 1ce, but use w1thm 2 h. Somcate and quickly spread 5 1-!L of gold from control Eppendorf tube (w1thout DNA) or from gold-DNA tube to the center of a macrocarner disk placed m the holder (gold concentration 30-50 �J.g/shot) A1r-dry At least two disks should be prepared for control bombardments, and the reqmred amount w1th gold-DNA based on the number of samples m the expenment F1ve macrocarner disks can be prepared at one t1me from each suspensiOn (see Note 8) With the helium tank on, set the delivery pressure to 1 300 ps1 (200 ps1 above the desired rupture disk value) Turn on the vacuum pump. Place a rupture d1sk ( 1 1 00 psi) in the holder, and screw tightly m place (see Note 9) W1th the steel mesh m place, transfer the coated macrocarner disk (coated side facing down) to the holder, and place the macrocarner assembly unit m the cham­ ber at level 2 from top. Place a Petri dish contammg the cultured embryos on the sample holder at level 4 from top, and close the door. Follow manufacturer's directiOns for bombardment, and follow all md1cated safety procedures.

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3.3. Culture, Selection, and Regeneration 1 . Sixteen to 20 h after bombardment, separate and transfer the embryos to Petn dishes contaimng either MS+ medium without glutamme and casem hydrolysate (delayed selection) or selection medium for callus proliferation (early selectiOn), for up to 2 wk m the dark Place 1 6-20 embryos (from each bombarded d1sh) m two dishes to avoid overcrowdmg and crossfeeding (see Note 10). 2 Transfer the embryogemc calli from callus proliferation medium to regeneratiOn 2 medium for shoot formation under 1 6-h photoperiod (40 j..U110l!m /s) for 8-1 0 d At the end of this penod, green areas mdicat1ve of shoot formation are VISible to the naked eye. 3 . Transfer the green shoots, along w1th the callus, as a umt to shoot elongatiOn medmm and culture m the hght for 1-2 cycles of 2 wk each. Keep each callus­ shoot piece well separated from others (see Note 11). 4 Transfer green shoots (>2 em in length) to root elongation medium in tubes. The smaller (< 1 em) shoots can be subcultured for one more cycle of shoot elongatiOn m Petn dishes. Calh w1th no shoots or shoots smaller than 1 em after two cycles on shoot elongation medium can be discarded (see Note 12). 5 Plants reachmg to the top of the culture tubes and With well-developed roots can be tested for PAT activity and transferred to sml in 56-66 d (see Fig. 1 ) They are grown to maturity m growth chambers under conditions similar to those used for donor plants.

3.4. Analysis of Transgene Expression

3.4. 1. Histochemical GUS Assay for Trans1ent Express1on Remove a sample of embryos (2--4 from 4 dishes each of control and +DNA) 2 d after bombardment, and soak separately m X-gluc solution (50--100 !JL!well of a mJCrotJter plate) 2 Seal the plate w1th Parafilm, and mcubate overnight at 37°C 3 Examme embryos under a stereo d1ssectmg miCroscope to v1suahze the blue, GUS-expressing umts

3.4.2. PA T Assay This assay is based on the detection of 1 4C-labeled acetylated PPT (nonra­ dioactive PPT used as substrate) after separation by TLC. Collect leaf samples on Ice m 1 5-mL Eppendorftubes (pnor to transferring puta­ tive trans formants to soil). Be sure to include ± control samples (optimum sample we1ght 20--3 0 mg; freeze m liqmd mtrogen and store at -70°C 1f not to be used the same day) 2. Add about 2 mg Po1yvmyi-Pyrrolidone (Sigma PVP40), some Silicone powder and 1 00 !JL of EB buffer to each tube Grind for 30 s with a polytron and keep samples on 1ce. 3. Centnfuge 1 0 m at 4°C, and collect supernatant m a fresh tube Repeat centnfugatwn

Particle Bombardment

355 Plant Seeds - 60 d

,j,

Anthes1s - 12 d

,j,

Embryo Culture (dark) 4-6 d Callus lmt1at1on

,j,

Prebombardment Treatment 4-6 h Osmot1curr.

,j,

Bombardment

,j,

Postbombardment Treatment 1 6-20 h Osmot1cum � Culture 1n the Dark No Selection or 3 mg/1 B1alaphos 14 d Callus Formation � Culture 1n the Light 3 mg/1 B1alaphos B-1 0 d Formation of Shoots

,j,

Culture 1n the L1ght 5 mg/1 81alaphos 14 d Elongation of Shoots 1n Petn dishes � Culture 1n the Light 4-5 mg/1 81alaphos 1 4-20 d Elongation of Shoots 1n Tubes � Transfer of Plants to Soil - 60 d � Anthes1s - 30 d � Mature Seeds

Ftg. 1 Time frame for the production of transgenic wheat (cv Bobwhite) plants Times shown are averages for experiments performed in 1 995, those destgnated by ­ are approximate, and vary etther with the batch of donor plants or the individual callus I me or plant. Transgenic plants were transferred to soil 56-66 d after the mit1at10n of cultures (6)

4. Measure protem concentratiOn in the crude leaf extracts followmg the Bw-Rad protem assay relative to BSA as standard. 5. Adjust protein concentratiOn to 2.5 (�Jg/f!L for each sample wtth EB buffer Assay can be stopped here overnight (store at -20°C). The background acttvtty can be ehmmated by prectpttating the extracts wtth saturated (NH4hS04• 6. Use I 0 flL of extract for each sample for assay. 7. Aliquot 3 � ofbulk reactwn mtxture to 1 0 � of each sample extract. Mix well, and mcubate at 37°C for 1 h. Stop reactiOn on 1ce.

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Spot 1 3 � of each sample on a TLC plate (can be done by first spottmg 6.5 �. drymg wtth a han dryer, and spotting the rest to avmd spreading of mtxture). Up to 1 5 samples can be spotted on one plate. 9 Transfer the TLC plate (after all the sample extracts have dned) to a saturated chromatography tank for 1-1 5 h. 1 0. Remove plate and let dry completely (at least 1 5 mm) 4 I I Vtsuahze the 1 C-labeled acetylated PPT by overnight exposure to X-ray film 1 2 . Plants testing posthve for PAT are regarded as transformed (as the assay tests for expresston of the enzyme encoded by the transgene bar) and can be transferred to sotl. Characterizatwn of the trans genes can be accomphshed by DNA isolatwn and Southern hybridizatiOn using standard protocols 8

4. Notes Because the quality of donor plants directly affects the capac tty of the tmmature embryos to produce embryogemc callus, tt ts tmportant that the plants be grown under opttmal conditwns. Pestictde applicatiOn from pollination to harvest ttme should be avoided 2 For optimum response (espectally when using field-grown matenal, which is often contaminated), use within 2 d after collection 3 The stze of the caryopsts ts variable along the length of the sptke Therefore, a more umform sample ts obtained by collectmg caryopses only from the mtddle half of each sptke 4 The duration of sterilization treatment depends on the quality of the donor plants. Caryopses from growth chamber grown plants tend to be clean and can be stenl­ ized effectively m 1 0-- 1 5 mm. Matenal from field-grown plants needs a longer (30-mm) Clorox treatment 5 Although embryos rangmg m stze from 0.5-1 .5 mm are capable of producmg embryogemc callus, the best postbombardment response ts obtamed from 1 - to 1 2-mm embryos. Embryos at thts stage of development are the eastest to dtssect, since younger embryos are nearly transparent and difficult to locate, whereas the older ones are whtte owmg to stored starch m the scutellum Holdmg the cary­ opsts with forceps and making an mcision at the base easily exposes the embryo (the outline of the embryo is vtstble before dtssectwn) 6. Fifty to 60 tmmature embryos can be cultured in the same dtsh t f the donor plants appear healthy and free of infectton Nonetheless, tt ts better to culture an average of 20 embryos/dtsh from field matenal owmg to the probabthty of infectwn. The cultures should be exammed on a datly basis to detect early stgns of contammatwn. The contammated embryos should be removed along wtth the medmm around them to avotd infectmg the adjacent embryos. 7 The htghest frequenctes of transformatiOn and regeneration are obtamed wtth 4-6 h of pre- and 1 6 h of postosmottc treatment. 8. The concentration of gold particles and DNA m gold/DNA mixture can vary. We recommend 30--5 0 J.tg gold/shot for a uniform spread on the macrocarner disk, to obtam a more even and finer stze of blue GUS umts, and a conststent effictency of transformatiOn

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9. Rupture disks in a range of 650-1 550 are used, with the 1 1 00 psi dtsk bemg most common. 1 0. Among Basta, btalaphos, and PPT, btalaphos was found to be most reliable for selectiOn of wheat transformants in the accelerated protocol (6) . The time when selectiOn 1s imposed, the number of explants/d1sh, and the concentration of the selective agent are critical variables that depend on the quality of the embryos cultured. Generally, early selection is recommended for htgh-quahty embryos, and delayed selectiOn for inferior embryos. TransformatiOn frequencies of 0. 1-2.5% were obtamed with the protocol shown m Fig. 1 . 1 1 Cultures should be examined frequently to assess the concentration of the selec­ tive agent reqmred for the next step. For example, with high zeatin m the regen­ eratiOn medium ( 1 0 mg/L), too many shoots are formed. With such cultures, It is safe to use 5 or 6 mg/L b1alaphos during shoot elongation. With low ( 1 mg/L) or no zeatin, when only one or two green areas are seen in each explant, 4 or 5 mg/L bialaphos are sufficient 12. During the penod the explants and the regenerants are m Petn dtshes, bmlaphos can be used at 5 or 6 mg/L, since cross-protectiOn IS provided by the adJacent explants. However, tf very few shoots emerge after the first cycle of shoot elon­ gatwn, then 4-5 mg/L bmlaphos are sufficient, because only a smgle explant IS present and there is no crossprotectwn.

References 1 Oerke, E-C., Dehne, H-W , Schonbeck, F., and Weber, A ( 1 994) Crop Produc­ twn and Crop Protectwn Estzmated Losses m Ma;or Food and Cash Crops Elsevier, Amsterdam 2 Bialy, H. ( 1 992) Transgemc wheat finally produced Bw/Technology 10, 675 . 3 . Vast!, V , Castillo, A. M., Fromm, M E , and Vast!, I K ( 1 992) Herbtctde rests­ taut ferttle transgemc wheat plants obtamed by microproJecttle bombardment of regenerable embryogemc callus. Bw/Technology 10, 667-674 4. Vasil, V., Snvastava, V., Castillo, A. M., Fromm, M. E., and Vasil, I. K ( 1 993) Rapid productwn of transgenic wheat plants by direct bombardment of cultured tmmature embryos Rio/Technology 1 1 , 1 5 53-1 558. 5 Weeks, J. T., Anderson 0 D , and Blechl, A. E ( 1 993) Raptd productiOn of mul­ tiple mdependent lines of ferttle transgenic wheat (Trztzcum aestzvum) Plant Physiol 1 02, 1 077-1084 6. Altpeter, F., Vasil, V., Srivastava, V., Stoger, E., and Vast!, I K. ( 1 996) Acceler­ ated production of transgemc wheat ( Tntzcum aestivum L.) plants. Plant Cell Rep 16, 1 2-1 7 7. Ortiz, J P A , Regg1ardo, M I , Ravizzm1, R A., Altabe, S. G., Cervtgm, G. D. L , Spitteler, M. A., et a!. ( 1 996) Hygromycin rest stance as an effictent selectable marker for wheat stable transformation Plant Cell Rep 15, 877-8 8 1 8. Nehra, N. S , Chibbar, R. N., Leung, N . , Caswell, K., Mallard, C., Stemhauer, L , et a! ( 1 994) Self-fertile transgenic wheat plants regenerated from Isolated scutellar t1ssues followmg m1croprojectile bombardment w1th two dtstmct gene constructs. Plant J 5, 285-297

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9 Zhou, H., Arrowsmtth, J W., Fromm, M. E., Htronaka, C M., Taylor, M. L , Rodriguez, D., et al ( 1 995) Glyphosate-tolerant CP4 and GOX genes as a select­ able marker m wheat transformation Plant Cell Rep. 15, 1 59-1 63 1 0 . Becker, D., Brettschneider, R., and Lorz, H. ( 1 994) Ferti le transgenic wheat from microproJ ecttle bombardment of scutellar tissue. Plant J. 5, 299-307. 1 1 Takumt, S and Shimada, T ( 1 996) Production of transgenic wheat through par­ ticle bombardment of scutellar tissues: frequency ts mfluenced by culture dura­ tion J Plant Physwl 14 9, 4 1 8--423 1 2 Vasil, I. K. ( 1 996) Phosphmothncm-resistant crops, tn Herb1c1de-ReS1Stant Crops (Duke, S 0 , ed. ), Lewis Publishers, Boca Raton, FL, pp. 85-9 1 1 3 . Christensen, A. H. and Quat!, P. H. ( 1 996) UbtqUtttn promoter-based vectors for high-level expresston of selectable and/or screenable marker genes in monocoty­ ledonous plants. Transg Res 5, 2 1 3-2 1 8. 1 4. Altpeter, F . , Vast!, V , Srtvastava, V., and Vast!, I. K. ( 1 996) Integratton and expression of the htgh-molecular-wetght glutenin subumt l Ax 1 gene tnto wheat Nature Biotechno/ 14, 1 1 5 5-1 1 5 9 1 5 Taylor, M. G , Vast!, V., and Vast!, I K. ( 1 993) Enhanced GUS gene expresswn tn cereal/grass cell suspensions and immature embryos usng the matze ubtquittn­ based plasmtd pAHC25 Plant Cell Rep 1 2, 49 1--495 16 Snvastava, V., Vast!, V , and Vast!, I K. ( 1 996) Molecular charactenzation of the fate of transgenes tn transformed wheat ( Tnt1cum aest1vum L ) Theor Appl Genet. 92, 1 03 1-1 037. 1 7. Blechl, A. E. and Anderson, 0 D ( 1 996) ExpressiOn of a novel htgh-molecular­ wetght glutemn subumt gene in transgemc wheat Nature Bwtechnol 14, 875-879 1 8 . Murashige, T. and Skoog, F. ( 1 962) A revised medmm for rapid growth and bio­ assays wtth tobacco ttssue cultures. Physwl Plant 15, 4 73--497.

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Pl ant Transformation via Protoplast Electropo ration Georg e W. Bate s

1 . Introduction In electroporatwn, cells are permeabihzed by the application of very short, high-voltage electric pulses. Molecules ranging in size from small orgamc metabolites and reporter dyes to large macromolecules-including antibodies and plasmids--can be mtroduced into cells by electroporation. Electroporation Is effective on virtually any type of cell, and ts now the method of chOice for the genetic transformation of bactena and certain animal cell hnes. The pn­ mary application of electroporatiOn to plants has been for DNA uptake for studies of transient gene expressiOn and for stable transformation. However, electroporation has also been used to mtroduce RNAs (1,2), antibodies (3), and small molecules (4) into plant cells and Isolated organelles (5). Because the thick plant cell wall restricts macromolecule movement, most work on plant cell electroporation utilizes protoplasts. This has limited the use of electroporation for stable transformation to species whose protoplasts are regenerable. As protoplast regeneration systems are Improved, reports of new species of plants transformed by electroporat10n contmue to appear (for example, ref. 6). One advantage of electroporation over particle bombardment for stable transformation IS that electroporation results predominantly m single­ copy plasmid msertions (Bates, unpublished observations), whereas particle bombardment tends to introduce large plasmid concatemers. However, the main use of protoplast electroporation 1s in transient express1on assays for studies of transcriptional regulation (for example, 7, 8). These stud1es do not require protoplast regeneration. A growing number of recent reports indicate that electroporation can be used to introduce DNA into walled plant cells and plant tissues (9, 10). Tissue electroporation does not work in all cases, and the parameters for successful plant tissue electroporation are not yet clear. From Methods Ed1ted

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Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols

by R D Hall © Humana Press Inc , Totowa, NJ

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However, the success of tissue electroporatton in crops, such as maize and soybean, reopens the use of electroporation for stable transformation in major crops. This chapter provides a protocol for protoplast electroporation, a protocol for the selection of stable, kanamycin-resistant transformants, and notes on how to optimize these protocols for both stable transformation and transient expresswn. These protocols have been used for many years in the author's laboratory for the transformation of tobacco protoplasts, but they can be readily modified for use with protoplasts of other species and for the uptake of mole­ cules other than DNA. 2. Materials 2. 1. Protoplast Electroporation InstrumentatiOn· Electroporatwn equipment IS available from a variety of com­ mereta! manufacturers and can also be homemade Lists of commercial manufac­ turers and instrument specificatiOns, as well as a general dtscusston of homemade eqmpment can be found in Chassy et a!. (1 1). Two types of oc electrical pulses, square-wave pulses and capacitive dts­ charges, may be used for electroporahon. However, because the eqUipment IS less expenstve, most iaboratories use capaclilve-dtscharge electroporation sys­ tems The equipment presently used m the author' s laboratory ts the Cell-Porator® Electroporatwn System I manufactured by Gtbco BRL Life Technologies Inc. (Gaithersburg, MD). Thts capacttlve-dtscharge mstrument allows the pulse volt­ age to be adJUSted from 0 to 400 V, pulse length can be varied by selectmg one of etght dtfferent-stzed capacitors (from 1 0-1 980 )lF) (see Note 1) The Gtbco BRL Celi-Porator utilizes presterilized, disposable electroporatwn chambers to hold the cells during electroporatiOn For work wtth plant protoplasts, electroporation chambers should be selected that have a 0.4-cm electrode gap Chambers with 0 1 -cm electrode gaps can also be purchased, but are designed for electroporation of bacteria 2. Protoplasts: Protoplasts Isolated by standard procedures are suitable for electro­ poratwn. Protoplasts from a wtde range of spectes, organs, and cell cultures have been successfully electroporated However, tt ts important that the protoplasts be ofhtgh quality. Even m htgh-quahty protoplast preparatiOns, electroporatwn ktlls a substantial fractwn of the protoplasts PreparatiOns of margmal or low-quality protoplasts are likely to be completely killed by the electric shocks 3 DNA" The plasmid DNA used m electroporatwn does not have to be highly pun­ fied, but should be free of RNA and protems (such as RNase and restnctwn enzymes) that m1ght affect the vtabthty of the protoplasts The author's labora­ tory routmely uses plasmids isolated by alkaline lysts (12). RNA is removed from the plasmtd preparatiOns by RNaseA dtgestton followed by phenol extractiOn and ethanol precipitatiOn (see Note 2) Before electroporatton, the DNA must be sierihzed. DNA that has been etha­ nol-prectpttated and redtssolved m autoclaved water or TE ts probably suffi-

Protoplast Electroporatlon

36 1

ciently stenle. However, to be certam the DNA is sterile it can be passed through a 0.2-f..Ull pore, low-bindmg, cellulose acetate syrmge filter (e.g., Nalgene # 1 902520). It is convenient to fi lter-sterilize the DNA after d1lutmg 1t mto electro­ poration medium at the start of an experiment For transient expressiOn studies, linear and superc01led plasm1ds are equally effective (13). However, for stable transformation, lmear DNA IS 3- to 1 0-fold more efficient than superc01led (14). 4. Electroporation medmm: Hepes-buffered sahne (HBS): 1 50 mM KCI, 4 mM CaCI 2, I 0 mM HE PES (pH 7 .2), and enough mannitol to balance osmotically the protoplasts For tobacco mesophyll protoplasts, 0 2 1 M mannitol is used. The HBS can be prepared in advance, stenlized by autoclavmg, and stored at 4°C.

2.2. Selection of Stable Transformants 1 . Media for protoplast culture and selection of stable transformants: Tobacco mesophyll protoplasts may be cultured m K3G medium (K3 salts, v1tamms, and hormones {15] containing 0.4 M glucose as the carbon source and osmotic stabi­ lizer). K3G may be sterilized by autoclaving, and stored at 4°C 2 Callus medmm (CM)· Murash1ge and Skoog salts and v1tamms (1 6) plus 1 00 mg/L inositol, 3% sucrose, 1 mg/L benzyladenme, and I mg/L a-naph­ thaleneacetic acid For selection of kanamycm-res1stant stable trans formants, CM is amended by addition of vanous amounts of mannitol, Sea plaque agarose (FMC Corp , Rockland, ME), and kanamycin as descnbed below in the Subheading 3. CM and CM amended w1th mannitol and Sea plaque agarose can be prepared m advance, sterilized by autoclaving, and stored at 4°C Kana­ mycm should not be autoclaved. A I OOOX stock of kanamycin can be prepared by d1ssolvmg 1 00 mg/mL kanamycin sulfate m water; the stock should be fil­ ter-sterilized and stored at -20°C.

3. Methods The following procedures must be carried out under sterile conditiOns, prefer­ ably in a lammar flow hood. All solutions should be at room temperature (see Note 3). 3. 1. Protoplast Electroporation Freshly isolated protoplasts should be used for electroporation: After washing the protoplasts free of the enzymes used for cell-wall d1gestwn, pellet the proto­ plasts and resuspend them in 1 0 mL HBS + manmtol (see Note 4). 2 Determine the number of protoplasts/mL usmg a hemacytometer. 3 . Place aliquots of I x I 0 6 protoplasts into 1 5-mL conical centrifuge tubes, and pellet the protoplasts by centrifugation (50g for 5 mm). 4. Discard the supernatant. Usmg a disposable plastic transfer p1pet, gently resus­ pend each protoplast sample in 0.5 mL HBS + manmtol + DNA. The plasmid DNA concentration m the electroporatwn medmm should be 1 0- 1 00 �Jg/mL (see Note 5).

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Bates Use a dtsposable plasttc transfer ptpet to transfer the protoplast samples to electroporation chambers Let stand for 5 mm. Resuspend the protoplasts by gentle agttatwn of the electroporatwn chamber, and then Immediately apply a smgle electric pulse (325 1!f, 300 V; see Note 6) [f you are using the Gtbco BRL Ceii-Porator, make sure the mstrument is on the low .n setting. Watt 2-3 mm, and then resuspend the protoplasts by agttation of the electro­ poratwn chamber. Transfer the protoplasts to a conical centrifuge containing 5 mL of K3G Rinse the electroporatwn chamber wtth 0. 5 mL HBS + manmtol, and combme the rinse wtth the protoplast sample (see Note 7) When all the protoplast samples have been electroporated, pellet the protoplasts, and resuspend each sample in 5 mL of culture medtum. Transfer the protoplast samples to a 60 x 1 5 mm Petn dish, and culture the proto­ plasts at 27°C. Protop lasts may be sampled after 24 h for transtent gene expresswn or roam­ tamed in culture for later selectiOn of stable trans formants

3.2. Selection of Stable Transformants The following procedure for selectiOn of stable transformants ts based on the agarose-bead culture technique of Shill ito et al. (1 7) . In this procedure, the protoplast culture is diluted and solidified by additiOn of low-melting-pomt agarose. When properly diluted, the embedded protoplasts are well separated from each other and grow into mdividual calh. Selection for transformants I S carried out after embeddmg the cells. This procedure permits calculation of plating efficiency and transformation efficiency, and allows isolation of indi­ vidual transformed clones. The method outlined here describes selection of kanamycm-resistant clones, but it can easily be adapted for use with other selectable markers or reporter genes. Dunng selectwn, the culture is progres­ sively diluted and the osmotic strength of the medium ts reduced. The sequence of medium changes described here has been optimized for work with proto­ plasts of Nicotiana tabacum, but can be modified for work With other species. l . Choose a plasmtd containmg a functiOnal neomycm phosphotransferase II gene such as pMON200 (18) or pBI 1 2 l (Clonetech, Palo Alto, CA). Lineanze the plasmtd by digestion wtth a suitable restnctwn enzyme. It is convenient to cut enough of the plasmid for several expenments. Then phenol-extract and ethanol­ precipitate the DNA, and resuspend tt m water at a concentration of l �JgiJ..!L . 2 Electroporate the protoplasts m the presence of 1 0-100 J..!g/mL of lmeanzed DNA, and culture them m 60 x 1 5 mm Petri plates m 5 mL of K3G (see Note 8) 3. After l wk of culture, the protoplasts wi ll have grown mto small-cell clusters (see Note 9) At this pomt, the protoplast-denved colomes are tmmoblltzed by a l : l dilution of the culture wtth medium containmg agarose Prepare in advance CM medtum + 0.23 M manmtol + 2 4% Sea plaque agarose, and stenhze It by

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autoclavmg. Just before the transfer, melt this medium, and let it cool to JUSt above its gellmg temperature. While the agarose Is cooling, scrape any adhering protoplast colonies off of the Petn plate with a micropipet tip Transfer half of the culture (2.5 mL) to a new 60 x 1 5 mm Petn plate Now add 2 5 mL of the agarose-contaming medium to both Petn plates of protoplasts Mix thoroughly by swirling the plates. Place the Petn plates in the refngerator for 1 5 min to solidify the agarose. Culture the protoplasts at 27°C At the end of the second week of culture, divide the solidified culture mto wedges using a spatula, transfer the wedges to 100 x 1 5 mm Petn plates, and add 5 mL of liquid CM + 0. 1 3 M mannitol supplemented with 1 00 j.J.g/mL kanamycm (see Note 10). Culture at 27°C. At the end of the third week, add 5 mL CM supplemented with 1 00 j.J.g/mL kana­ mycm to each Petri plate. Thereafter, at weekly mterval s, remove 5 mL of hqmd from each Petn plate, and replace It With 5 mL of fresh CM supplemented With 1 00 j.J.g/mL kanamycm Transformed colomes should be visible after 4-5 wk of culture. When the transformed colomes are 2-3 mm in diameter (4-6 wk of culture), they can be picked out of the agarose using a spatula, and cultured on CM + 0.8% agar + 1 00 j.J.g/mL kanamycm. After another 2 wk of growth, they should be large enough to be transferred to regeneration medmm.

4 . Notes 1 . Pulse voltage, or more precisely electric field strength, and pulse length are two critical parameters m electroporati on, and must be optimized for each species and cell type Field strength depends on the voltage applied to the electroporatton chamber and the distance between the electrodes in the chamber. The appropnate units for field strength are V/em. Application of a 1 00 V pulse to a chamber with a 0.4-cm electrode gap results in a field strength of 250 V/em. Pulse length is determined by the size of the capacitor and the resistance of the electroporation medium. Di scharging a capacitor produces an exponentially decaymg pulse. The length of such pulses is best described by their RC time constant, which is the time reqmred for the pulse voltage to drop to 3 7% of its Initial value 2. Both RNA and protems present in a crude plasmid preparatton can be introduced mto the protoplasts by electroporation along with the DNA and may affect the experimental outcome. For example, in some early experiments in the author ' s laboratory, protoplasts were electroporated in the presence of a plasmid that had been treated with RNase, but not phenol-extracted. No transient gene expressiOn was observed until the RNase was removed from the plasmid preparations 3. Some electroporatlon protocols cail for chilling the cells m an ice bath during and Immediately after electroporation. The pores that form in the plasma membrane owing to electnc shocks have been shown to stay open longer if the cells are maintained at a temperature below the membrane's phase transition temperature. Chilling the cells would be expected to improve the efficiency of transformatiOn

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Bates by electroporatwn, because tt would allow more time for DNA uptake How­ ever, several studtes of transient and stable gene expressiOn have shown that chilling does not Improve electroporation effictency It turns out that the uptake of DNA dunng electroporatwn is electrophoretic (and not diffustve) and occurs dunng the electric pulse itself Thus, chillmg the cells ts unneces­ sary tf transformatiOn ts the goal of electroporation Chilhng may be useful, however, when el ectroporatwn ts used to mduce the uptake of molecules other than nuc letc acids. Because HBS is a nonphyswlogtcal, htgh-salt medmm, it ts advtsabie to hmlt the exposure of the protoplasts to thts medmm to 30 min. If a large number of samples are gomg to be electroporated, divide the protoplast preparation mto two batches. Leave one batch in the protoplast wash or in culture medmm, while the other batch ts resuspended m HBS, divided mto samples, and electroporated. Then when the first batch of protoplasts has been electroporated and dtluted mto cul­ ture medium (Subheading 3.1., step 7), pellet the second batch of protoplasts resuspend them m HBS and process them for electroporat10n. The effictency of both transtent expressiOn and stable transformation mcreases l inearly with DNA concentration from l 0 to l OO f.!g/mL. Transient expressiOn and stable transformatiOn are also increased by addition of "carner DNA," such as salmon sperm DNA. For transient gene expressiOn, the author's laboratory uses 1 0 f.!glmL supercoiled plasmid DNA + 50 f.!g/mL salmon sperm DNA (sheared by sonication). Because the earner DNA has been found to integrate along with the plasmtd, carrier DNA should be avotded for stable transformatiOn When the goal t s stable transformatiOn, thts laboratory uses 50 f.lg/mL of linear­ tzed plasmid DNA and no earner The efficiency of electroporation depends on pulse length and voltage A 325-!lf capactttve dtscharge mto 0.5 mL of HBS gtves a pulse of about l 0 ms (RC ttme constant) For tobacco mesophyll protoplasts, a l O-ms pulse of 300 V (750 V/em field strength m the electroporatton chamber) usually gives optimal results. The optimal setting can vary wtth species and cell type, and should be determmed empirically m prelimmary expenments. A quick way to begin to look for effec­ tive electroporat10n parameters ts to find pulse settings that result in 50% proto­ plast death by 24 h after the shocks Electroporatwn effictency can also vary between batches ofprotoplasts because ofbatch-to-batch dtfferences m protoplast viabthty. Batch-to-batch vanabthty com­ phcates transtent expressiOn studtes. To handle thts vanability, rephcates and appropnate controls must be mcluded m every expenment. Some transient expres­ sion studtes also mclude an mternal control. The approach t s to add a second plas­ mid carrying a reporter gene to each sample before electroporatwn as an internal control. ExpressiOn of the second plasmtd can be used to normalize both batch-to­ batch and sample-to-sample differences in transient gene expression Manufacturers of electroporation eqmpment mtend for the electroporatwn cham­ bers to be dtscarded after each use. However, the author finds that the chambers can be reused two or three times wtthout affectmg electroporation efficiency or

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protoplast viability. Rmsmg the chamber after electroporatwn with 0.5 mL HBS + manmtol not only helps remove all the electroporated protoplasts from the chamber, but also readies the chamber for the next sample. 8. High initial protoplast densities improve surviVal of the electric shocks. Up to 500 transformed clones can be recovered from a sample of I x I 0 6 protoplasts This would give an absolute transformation efficiency of 1 transformant for every 2000 electroporated protoplasts. However, the transformatiOn efficiency IS actu­ ally severalfold higher, because 50% of the protoplasts are killed directly by the electnc shock and no more than half of the surviVing protoplasts grow into calli This laboratory uses 50 J.tg/mL hnear DNA for stable transformatiOn South­ em blotting shows that 50-75% of the transformants have a single copy of the plasmid integrated. 9. The timing of the media changes described here should not be adhered too rigidly, but should be modified depending on how fast the protoplasts grow after electroporation. For tobacco protoplasts, the first dilution of the cultures IS done when the protoplasts have grown into m1crocolomes of 5- l 0 cells, which IS sually after 6-7 d of culture This first dilution may have to be delayed if the protoplasts are growmg more slowly-as will happen if electroporatwn kills more than about 75% of the protoplasts Too rapid a dilutiOn of the culture results m death of the protoplast-derived colomes within 24 h of the dilution I 0. As descnbed here, the selectiOn pressure starts out at 50 llg/mL of kanamycin and mcreases to about 1 00 llg/mL over a penod of a few weeks. Selection can be started earlier. For example, It can be convemently started I wk after e lectroporation by adding kanamycin at the first dilution of the culture This lab obtains the highest number of trans formants when addition of kanamycm IS delayed until the end of the second week. This may be because piating efficiency depends on cell density, so startmg selection early reduces the cell density in the culture, and this in turn, inhibits the growth of some trans­ formed clones SelectiOn for kanamycm resistance is very clean in tobacco. The author never observes untransformed clones growmg m the presence of kanamycin.

References I . Bailey-Serres, J. and Dawe, R. K. ( 1 996) Both 5' and 3' sequences of maize adh l mRNA are reqmred for enhanced translatiOn under low-oxygen conditiOns. Plant Physwl. 1 12, 685--695 2. Wintz, H. and Dietrich, A. ( 1996) Electroporation of small RNAs into plant proto­ plasts. mitochondrial uptake of transfer RNAs. Bwchem Bwphys Res Commun 223, 204--2 1 0. 3 Maccarrone, M., Veldmk, G A., Finazzi Agro, A., and Vhegenthart, J. F. ( 1 995) Lentil root protoplasts. a transient expression system suitable for coelectroporatwn of monocolonal ant1bodtes and plasmid molecules Bwchzm Bwphys. Acta 1 243, 1 3 6-142. 4. Jang, J.-C. and Sheen, J. ( 1 994) Sugar sensmg in higher plants. Plant Cell 6, 1 665--1679.

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5 To, K. Y , Cheng, M C , Chen, L F., and Chen, S C (1 996) Introductton and expressiOn of foretgn DNA m t solated spmach chloroplasts by electroporation Plant J 10, 737-743 . 6 Lee, L , Larmore, C. L., Day, P. R , and Turner, N. E ( 1 996) Transformation and regeneratiOn of creeping bentgrass (Agrostzs palustrzs Huds) protoplasts. Crop Scz 36, 40 1-406 7. Kao, C. Y., Cocciolone, S. M , Vastl, I. K., and McCarty, D. R. ( 1 996) Localiza­ tion and interactiOn of the cts-actmg elements for absctsic acid VIVIPAROUS 1 , and !tght achvahon o f the C 1 gene of matze. Plant Cel/ 8, i i 7 1-i 1 79. 8 Snowden, K. C., Buchholz, W. G., and Hall, T. C. ( 1996) Intron position affects expressiOn from the tpt promoter m nee. Plant Mol Bwl 3 1 , 689-692 9 Laursen, C M., Krzyzek, R. A , Fhck, C E., Anderson, P. C , and Spencer, T M ( 1 994) Productton of ferttle transgemc matze by electroporatwn of suspension culture cells Plant Mol Bwl. 24, 5 1-6 1 . 10. Chownra, G . M , Akella, V., Fuerst, P. E., and Lurqum, P F ( 1 996) Transgemc gram legumes obtamed by m planta electroporatton-medtated gene transfer. Mol Bwtechnol. 5, 85-96. 1 1 Chassy, B. M., Saunders, J A., and Sowers, A. E. (1 992) Pulse generators for electrofusion and electroporation, in Guzde to Electroporatwn and Electrofuswn (Chang, D. C , Chassy, B. M., Suanders J A , and Sowers, A. E., eds.), Academic, San Diego, pp. 555-569. 12 Sambrook, J , Fntsch, E F , and Mamatis, T . ( 1 989) Molecular Clonmg A Labora­ tory Manual, 2d ed. Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, NY 1 3 . Bates, G W , Carle, S A., and Piastuch, W C ( 1 990) Lmear DNA mtroduced into carrot protoplasts by electroporatton undergoes ltgatwn and recirculanzatton Plant Mol. Bioi 14, 899-908 1 4. Bates, G. W ( 1 994) Genetic transformatiOn of plants by protoplast electro­ poratwn. Mol Bwtechnol. 2, 1 3 5-145. 15. Nagy, J. I. and Maitga, P ( 1 976) Callus mductton and plant regeneration from mesophyll protoplasts of Nzcotzana sylvestrzs. Z Pflanzenphyswl 78, 453-455 16 Murashtge, T and Skoog, F ( 1962) Revtsed medium for raptd growth and bioas­ says wtth tobacco tissue cultures Physwl. Plant 15, 4 73-497 1 7 Shilhto, R D , Paszkowskt, J , and Potrykus, I ( 1 983) Agarose platmg and a bead type culture technique enable and stimulate development of protoplast-denved colonies m a number of plant spectes Plant Cell Rep 2, 244-247. 18 Rogers, S G , Horsch, R. B., and Fraley, R. T ( 1 986) Gene transfer m plants· production of transformed plants usmg T1 plasmid vectors Methods Enzymol 1 18, 627-64 1 .

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Transformation of Maize via Tissue Electroporation Kathleen D 'Halluin, Els Bon ne, Martl e n Bossut, and Rosita Le Page

1 . Introduction Introduction of DNA into plant protoplasts via electroporation is a well­ known procedure. By givmg electrical impulses of high field strength, the cell membrane is reversibly permeabihzed, so that DNA molecules can be mtro­ duced into the cell {1) . Originally, maize transformatiOn efforts focused on electroporation of proto­ plasts (2). However, transient transformation by tissue electroporation was first demonstrated by Dekeyser et al. (3). Leaf bases of several monocot species, mclud­ ing maize, were shown to be amenable to electroporatiOn-mediated DNA uptake based on the transient expression of reporter genes. Stable transformants via tissue electroporation of matze have been described for enzymatically treated or mecham­ cally wounded immature embryos and type I callus (4), for mechanically wounded immature zygotic embryos (5), for enzymatically treated embryogenic maize sus­ pension cells (6), and for type II callus (7). Furthermore, transient expressiOn was observed after electroporation of intact immature maize embryos (8). Transient and stable electrotransformations were obtamed after electroporation of preplas­ molyzed, intact black Mexican sweet maize suspension cells (9). The followmg schedule outlines the steps for the stable transformation of enzymatically treated, Immature embryos and mechamcally wounded type I callus of maize. 2. Materials 2. 1. Electroporation 1 . Embryo culture medmm, Mah l VII· N6 basic medium (10) supplemented with

1 00 mg/L casem hydrolysate, 6 rnM L-prohne, 0.5 g/L MES, and 1 mg/L 2,4-D sohddied wtth 2.5 g/L Gelrite (Duchefa), pH 5 . 8 (see Notes 1 and 3). From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Edited by R D Hall © Humana Press Inc Totowa, NJ

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5 6. 7.

Mah l VII substrate. M: N6 basic medium; a: 1 00 mg/L casem hydrolysate and 6 mM L-proline; h: 0.5 giL MES; I · I mg/L 2,4-D; VII· 2.5 g/L Gelnte (Duchefa) Enzyme solution 0.3% macerozyme (Kmki Yakult, Nishinomiya, Japan) in CPW salts (11) supplemented with 1 0% manmtol and 5 mM MES, pH 5.6, filter-steril­ Ized (see Note 2) Washing medmm: N6 salt solution supplemented w1th 6 mM asparagme, 12 mM L-proline, I mg/L thiamin-HCl, 0 5 mg/L nicotinic ac1d, 1 00 mg/L casein hydroly­ sate, 1 00 mg/L mos1tol, 30 g/L sucrose, and 54 g/L manmtol, filter-stenhzed EPM buffer. 80 mM KCI, 5 mM CaCI2 , i 0 mM HEPES, and 0 425 M manmtol, pH 7 2 The same buffer without KCI 1s also needed. Agarose substrate· water with 0 45% BRL agarose Electroporatwn apparatus, electrodes, and cuvets (see Notes 4 and 6). DNA ofan appropriate plasmid beanng the bar selectable marker gene (see Note 5)

2.2. Selection and Regeneration of Transformants I . SelectiOn medmm Mah1 l VII Mah I VII substrate supplemented With 0 2 M man­ mtol and 2-5 mg/L filter-stenhzed phosphmothricm (PPT) (see Notes l and 3) Mahi l VII substrate. M. N6 basic medmm, a. 1 00 mg/L casem hydrolysate and 6 mM L-prohne; h. 0.5 g/L MES, , . 0 2 M manmtol, l . l mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) 2 SelectiOn medmm Mhi l VII: Mah l VII substrate from which casem hydrolysate and L-proline are omitted, and supplemented With 0 2 M manmtol and 2-5 mg/L filter-stenlized PPT Mh! l VII substrate M N6 basic medmm, h 0.5 g/L MES; , . 0 2 M manmtol, I 1 mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) (see Notes 1 and 3). 3 SelectiOn medmm Mh 1 VII Mah 1 VII substrate from which casem hydrolysate and L-prolme are omitted, and supplemented With 2-5 mg/L filter-stenhzed PPT (see Notes l and 3) Mh l VII substrate M: N6 basic medmm, h· 0 5 g/L MES; I . I mg/L 2,4-D, VII: 2.5 g/L Gelnte (Duchefa) 4 Selection medium Ahi i .5VII · correspondmg substrate, ofMh1 l VII substrate but MS (12, and see Appendix) basic medmm ("A") mstead of N6 basic medium ("M") supplemented with 0 2 M mannitol, 0 5 giL MES, I 5 mg/L 2 4-D, and 5 mg/L filter­ stenhzed PPT, solidified with 2.5 g/L Gelnte (Duchefa), pH 5.8 (see Notes l and 3) Ahi l .5 VII substrate A MS medmm; h· 0 5 g/L MES; 1 . 0 2 M manmtol, 1 5 1 5 mg/L 2,4-D, VII 2 5 g/L Gelnte (Duchefa) 5 RegeneratiOn medmm· MS medmm supplemented with 5 mg/L 6-benzylammo­ punne and 2 mg/L filter-stenhzed PPT (see Notes 1 and 3). 6. Shoot development media (see Notes 1 and 3): MS6%: MS medmm with 6% sucrose and supplemented With 2 mg/L fi lter­ stenhzed PPT MS1h: half-concentratiOn of MS medmm 7 Basta 0 5%· 0 5% (v/v) of a Basta stock solutiOn contaming 200 g/L giufosinate­ ammomum

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3. Methods 3. 1. Electroporation

3. 1 . 1 . Immature Embryos

2 3. 4

5 6. 7. 8

9.

Maize plants of the public inbred line H99 are grown in the greenhouse tn 20-L pots containing slow-release fertilizer. Growth conditions are at 25°C and 1 6-h hght of -20,000 lx (daylight supplemented by sodium vapor and mercury halide lamps); at night, temperature is reduced to 1 5-20°C. Kernels from ears 9-1 4 d after pollination are surface-sterilized m 3. 6% sodium hypochlorite for 1 5 mm. Immature zygotic embryos of the mbred lme H99 ( 1 -1 5 mm m length) are exc1sed and plated on Mah l VII substrate (see Note 1) Freshly isolated Immature embryos are collected from the Mah 1 VII substrate and enzymatically treated for 1-3 mm at room temperature with the enzyme solution (see Note 2) The embryos are then carefully, washed with filter-stenhzed washmg medmm (see Note 3). After washmg, approx 50 embryos are transferred mto a dtsposable mtcrocuvet contammg I 00 !JL matze electroporation buffer (EPM) (see Note 4). Ten m1crograms of hneanzed plasm1d DNA are added per cuvet and coincubated with the enzyme-treated embryos (see Note 5). After 1 h, the electroporatwn is carried out by dischargmg one pulse w1th a field strength of 375 V/cm from a 900-� capac1tor (see Note 6). The onginally descnbed (4) pre- or postelectroporat1on incubatiOn on ice can be omitted After a few minutes, the embryos are transferred onto culture substrate.

3. 1 .2. Type I Callus I . Immature embryos ( 1-1 5 mm m length) of the backcross (Pa9 1 x H99) x H99 are exc1sed from ears 9-1 2 d after pollination and plated w1th the1r embryonic axts m contact wtth the Mah l VII substrate (see Note 7). 2. Type I callus 1s imttated from tmmature embryos m the dark at 25°C 3 . Yellow, not too far differentiated, embryogenic t1ssue IS microscopically dts­ sected from developing, type I callus that has been cultured on Mah 1 VII sub­ strate for a period of - 1 .5--6 mo with subculture intervals of approx 3--4 wk Tissues older than 6 mo are discarded, smce the quahty of the callus declmes 4. The embryogenic tissue is cut through the menstems, using a mtcroscope, mto pteces of approx 1-1 5 mm and collected m EPM buffer without KCI. 5 The finely chopped tissue is washed several times wtth EPM buffer wtthout KCI to remove nucleases. 6. After 3 h of preplasmolysis in th1s buffer, the callus pieces are transferred to cuvets containmg 1 00 ,L of EPM supplemented with 80 mM KCl. 7. Ten mtcrograms of hnearized plasmid DNA are added per cuvet, and subsequent conditiOns are as for electroporatwn of Immature embryos

D'Halluin et a/

370 3.2. Selection and Regeneration of Transformants

3.2. 1 . Immature Embryos I

2 3. 4.

5 6 7. 8

The embryos are transferred either Immediately after electroporatwn to selective substrate MahJ I VII or to nonselective substrate for a few days (3-7 d) before transferring to selective substrate Mh1 l VII (see Note 8). The quality of the embryos IS the determmmg factor for the duect or nondirect transfer to selective substrate immediately after transformatiOn. The omisswn of L-proline and casein hydroly­ sate from the selective substrate makes the selectton more stnngent. Good-qual­ ity embryos from maize plants grown under more optimal conditions are plated Immediately on selective Mhi 1 VII substrate. After 2-3 wk, the embryos are transferred to selectiVe Mh l VII substrate. After 3-4 wk, the embryogemc tissue is selected and subcultured for another 3-4 wk on selective Mh l VII substrate w1th 2-5 mg/L PPT. Afterwards, the developmg embryogemc tissue IS Isolated and transferred to regeneratiOn medmm at 2 5 °C w1th a daylength of 1 6 h. Fluorescent lamps ("lumJiux wh1te" and "natural"; Osram, Mumch, Germany) are used with a hght mtensity of 2000 lx After 2 wk, the embryogenic tissue 1s subcultured onto fresh substrate with the same composition. Developmg shoots are transferred to MS6% medium With 2 mg/L PPT from which hormones are omitted Further developing shoots are transferred to nonselective MSI!z medium for fur­ ther development mto plantlets Plantlets are transferred to soil and grown to maturity m the greenhouse.

3.2.2. Type I Callus 1 . Immediately after electroporation, the callus p1eces are transferred to seiect1ve Ahi l .5VII substrate with 5 mg/L PPT. 2. After 3-4 wk, the prohferatmg callus pteces are subcultured onto Mh 1 VII sub­ strate with 5 mg/L PPT for a period of approx 2 mo with a subculture mterval of approx 3-4 wk. 3 Subsequent conditiOns for regeneratiOn are as for immature embryos of H99, except that 5 mg/L zeatm are used mstead of 6-benzylaminopurine for the mduc­ tlon of regeneratiOn from the selected embryogenic tissue.

3.3. Analysis of Putative Transgenic Plants

3.3 1 Phosphinothricin Acetyltransferase (PA T) Activity PAT activtty is detected by an enzymatic assay usmg 14C-Iabeled CoA. 1 4C-labeled acetylated PPT IS detected after separation by thin-layer chroma­ tography (13).

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3.3.2. Dot Assay or Basta Spraying Leaves of in vivo-grown plants are brushed With a 0.5% Basta® solution or 2 sprayed with 1 00 mL/m of a 0.5% Basta solution. Plants are assessed 8 d after Basta application. Plants expressing the bar gene have no symptoms, whereas leaves of nontransformed plants show necrosis.

3. 3. 3. Inheritance of the bar Gene Most plants should develop normally and form a normal tassel and ear. Seed set is obtained by selfing or crosspollination. Transgenic lines can be used either as male or female parent m the crossing or as both. The segregation of the Basta resistance gene is then determined by using a Basta dot assay or by a Basta spraying, and scored for segregation of resistant and sensitive pheno­ types. The maJority of the hnes obtained via electroporation show a Mendelian mhentance pattern for the bar gene locus (see Note 9). 4 . Notes

2. 3

4

5

6. 7.

All solid substrates are prepared m the following way· the sugar solutiOn and the salt solution with MES and the solidifying agent are autoclaved separately All other components are filter-sterilized and added after autoclavmg. The filter-sterilized enzyme solution is stored in the freezer at -20°C All liquid substrates and buffers are filter-stenlized and stored at room temperature in the dark, solutwns of hormones and L-prohne are stored m the freezer at -20°C, and v1tamm and casein hydrolysate solutions are stored m the refrigerator Microcuvets ( 1 93 8 PS; Kartell, Binasco, Italy) are stenlized m 70% ethanol for several minutes. Afterward microcuvets are filled with 1 mL agarose substrate (water with 0 45% BRL agarose), and after solidification I 00 J.!L of the electroporatwn buffer (EPM) IS p1peted on top of th1s agarose substrate. Parallel stainless-steel electrodes, 30 mm long, 2 mm thick, and 6 mm apart are mserted in such a way that by pushing the electrodes on the agarose substrate, the plant tissue I S collected between the electrodes in the EPM buffer. Plasmid DNA was purified on Qiagen (Q1agen Inc.) columns and resuspended m 1 0 rnM Tns-HCI, pH 7.9, and 0. 1 rnM EDTA at a concentration of I mg/mL. The plasmid DNA, carrying a chimeric cauliflower mosaic virus (CaMV) 35 S-bar3'nos gene, was linearized pnor to electroporation, usmg an appropriate enzyme A homemade electroporation umt consisting of a power supplier connected with an array ofcapacitors arranged m a circuit, as descnbed by Fromm et a!. (1), was used Since we observed a higher transformatiOn frequency from backcross matenal (Pa9 1 x H99) x H99 compared with the inbred lines H99 and Pa9 1 , and since It was easier, under our climatic conditions, to produce good donor material from the backcross compared with the inbred lmes, we continue to use the backcross m transformatiOn expenments

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8. The manmtol IS added m the first cu lture substrate immediately after electro­ poratJon to prevent an osmotic shock m the tissue that has been enzymatically treated or preplasmolyzed for 3 h in the EPM buffer with 0 425 M mannitol. 9 The maJority of the lines obtained with electroporatJon show a Mendelian inheri­ tance pattern for the bar gene locus m contrast with lines produced by particle bombardment. TransformatiOn of maize based on electroporation or micropro­ Jectile bombardment of type I callus and immature embryos have been further compared The level of transient expressiOn as duected by anthocyanin bwsyn­ thetJc regulatory genes revealed a sigmficantiy higher number of red spots on particle bombardment compared with electroporatwn. B oth transformatiOn procedures lead to the Iso latiOn of stab le trans­ formants denved from Immature embryos and type I callus as targets for transformatiOn usmg the bar gene as selectable marker Upon e lectroporahon, a clear selection was observed m ti ssue culture, but Basta-resistant pl ants could only be regenerated from a mmor fraction of the selected calli. With particle bombardment, a sigmfJcantly higher number of regenerants express­ ing the bar gene (up to ca. 40-fold) was obtamed However, segregatiOn analysis of progemes from transgemc lmes obtained from both transforma­ tiOn procedures revealed that m the maJ ority of the electroporatwn-denved hnes, the gene is inherited m normal Mendelian fashwn, whereas a lower fractiOn (about 50%) of the bar lines obtamed by microproJectile bombard­ ment show a normal Mendelian segregatiOn About 30% of the hnes produced by particle bombardment do not show an inhen tance of the Basta resi stance phenotype at all and yield only sensitive progemes. In reciprocal crosses of transformants produced by particle bombardment, segregation could be w1thm the expected range m one directiOn and abnormal, even all-sensitive, m the other duectwn Transformants should correctly express the mtroduced genes, 1f they are to be of practical value A drawback of d1rect gene delivery IS the unpredictable and often complex pattern of mtegration On ei ectroporation, about half of the transformants have a rather Simple IntegratiOn pattern, whereas the other half have a more complex mtegrahon pattern. With bwhstJc transformatiOn, the majority of the transformants have mtegrated multiple copies of the transgene, often fragmented and rearranged. This could explam the high proportion oflines obtamed with biol istics havmg an aberrant segre­ gation, since the presence of multiple copies of transgenes can lead to msta­ bil ity of their expression (14). In conclusiOn, stably transformed maize hnes using type I callus and Imma­ ture embryos can be produced by both direct gene transfer methods. The effi­ Ciency of transformatiOn IS significantly higher with particle bombardment compared with electroporatwn. However, qualitatively better transformants are produced by e l ectroporation, since a sig nific ant lower number of transformants have to be generated by elec troporatwn compared W ith biolistJcs m order to obtam a transgenic line d1splaymg a simple and "cor­ rect'' mtegration pattern

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References I . Fromm, M. E., Mornsh, F , Annstrong, C. W1lhams, R , Thomas, J , and Klein, T M. ( 1 990) Inheritance and expression of chimenc genes m the progeny of transgenic maize plants. Rio/Technology 8, 833-839 2. Rhodes, C. A., P1erce, D A , Mettler, I. J., Mascarenhas, D., and Detmer, J. J ( 1 988) Genetically transfonned maize plants from protoplasts Scrence 240, 204-207. 3 Dekeyser, R A , Claes, B., De RIJCke, R M U , Habets, M. E., Van Montagu, M C., and Caplan, A. B. ( 1 990) Transient gene expression m mtact and orgamzed rice tissues. Plant Cel/ 2, 59 1--{;02. 4. D ' Hallum, K., Bonne, E., Bossut, M , De Beuckeleer, M. and Leemans, J ( 1 992) Transgenic ma1ze plants by tissue electroporatwn Plant Cell 4, 1 495-1 505 5. Xiay1, K., Xmwen, Z., Heping, S., and BaoJian, L. ( 1 996) Electroporatwn of immature maize zygotic embryos and regeneration of transgemc plants. Transgemc Res. 5, 2 1 9-22 1 . 6. Laursen, C M , Krzyzek, R. A , Flick, C E , Anderson, P C , and Spencer, T M . ( 1 994) ProductiOn of fertile transgemc maize by electroporation o f suspensiOn culture cells Plant Mol Bzol 24, 5 1-{i I 7 Pescitelli, S. M. and Sukhapmda, K ( 1 995) Stable transformatiOn v1a electro­ poration mto maize Type II callus and regeneratiOn of ferti le transgemc plants. Plant Cell Rep. 14, 7 1 2-7 1 6 8 . Songstad, D . D., Halaka, F . G . , DeBoer, D . L , Annstrong, C. L., Hinchee, M. A. W., Ford-Santmo, C. G., et a! ( 1 993) Transient expressiOn of GUS and anthocya­ nin constructs in intact maize Immature embryos followmg electroporation. Plant Cell, Tissue Organ Cult 33, 1 95-20 1 . 9. Sabn, N., Pelissier, B , and TeJSSJe, J. ( 1 996) Transient and stable electrotrans­ formations of mtact black Mexican sweet maize cells are obtamed after preplasmolysJs Plant Cell Rep 15, 924-928. 10 Chu, C. C., Wang, C C , Sun, C. S., Hsu, C., Ym, K C., Chu, C. Y , et a! ( 1 975) Establishment of an efficient medmm for anther culture of rice through compara­ tive expenments on the mtrogen sources Scz Szn 18, 659-ti68 1 1 . Frearson, E. M., Power, J B., and Cocking, E. C. ( 1 973) The Isolation, culture and regeneration of Petuma leafprotoplasts Dev Bzol 33, 1 30-1 37. 1 2. Murash1ge, T. and Skoog, F ( 1 962) A revised medmm for rapid growth and bw­ assays with tobacco tissue cultures. Physzol Plant 15, 473-497 1 3 . De Block, M., Bottennan, J , Vandew�ele, M., Dockx, J , Thoen, C , Gosse1e, V., et a!. ( 1 987) Engmeenng herbiCide resi stance m plants by expressiOn of a detoxi­ fying enzyme. EMBO J 6, 25 1 3-25 1 8 14. Matzke, M A and Matzke, A J M ( 1 995) How and why do plants machvate homologous (trans)genes? Plant Physzol 107, 679--{;85

34

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Transformation of Maize Using Silicon Carbide Whiskers Jim M. Dunwell 1 . Introduction

As the most commercially valuable cereal grown worldwide and the best­ characterized in genetic terms, maize was predictably the first target for transfor­ mation among the important crops. Indeed, the first attempt at transformatiOn of any plant was conducted on maize (1). These early efforts, however, were inevi­ tably unsuccessful, since at that time, there were no rehable methods to permit the introduction of DNA into a cell, the expression of that DNA, and the Iden­ tification of progeny derived from such a "transgenic" cell (2). Almost 20 years later, these technologies were finally combined, and the first transgemc cereals were produced. In the last few years, methods have become mcreasingly effi­ cient, and transgenic maize has now been produced from protoplasts as well as from Agrobacterium-mediated or "Biolistic" delivery to embryogenic tissue (for a general comparison of methods used for maize, the reader is referred to a recent review-ref. 3). The present chapter will describe probably the simplest of the available procedures, namely the delivery of DNA to the recipient cells by vortexing them in the presence of silicon carbide (SiC) whiskers (this name will be used in preference to the term "fiber," smce it more correctly descnbes the single crystal nature of the material). This needle-shaped matenal was selected as a means of perforating cells (thereby allowing the entry of DNA), since it was known to be one of the hard­ est of"man-made" products; it is used commercially as an abrasive and a com­ ponent of saw blades. In fact, it is not a completely synthetic material, but is produced by carbonizmg (at high temperature in carbon monoxide) the s1licate found within the cells of rice husks. The chemically transformed product of one cereal is therefore being used to transform another cereal genetically. From Methods 1n Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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Dunwell

Fig. l . (A) Scanning electronmicrograph of silicon carbide whiskers of the type used for transformation. (B) Group of maize cells after vortexing with silicon car­ bide whiskers, showing dark staining of a single-cell expressing the introduced GUS gene. In essence. the transfonnation method described below involves the agita­ tion ofsuspension-culture cells, with SiC whiskers and plasmid DNA

(Fig. 1).

The resultant transgenic cells are selected by growth on bialaphos and plants subsequently regenerated. Details of the procedure as developed for maize can

Silicon Carbide Whiskers

377

be found in a series of recent papers and reviews (4-8). The scope of the method, however, is not restricted to matze, or indeed to cells of htgher plants, as shown by successful dehvery of DNA to cells of various algae (9), mcluding Chlamydomonas (10), tobacco (11, 12), Agrostis (13), and Lolium (Dalton, per­ sonal communication). Most recently, transient expression of the GUS marker was detected when dry wheat embryos were vortexed with DNA and whiskers for 1 0--30 min (14). It was also demonstrated that callus ttssue induced from the treated embryos contamed GUS-expressing sectors more than 1 mo after treatment. This very promising finding suggests that cells in culture are not necessanly the only possible target. AddttJOnally, recent results obtamed from node slices of Ameri can chestnut showed that treatment with whiskers increased the proportion of wounded tissue and thereby enhanced subsequent Agro ba cterium mediated gene delivery (15). It should be noted that the method as developed for the production of transgemc maize is the subject of a granted US patent (16) and that other pat­ ents are pending. These do not of course prevent the use of the technique for research purposes. -

2. Materials 2. 1. Source of Whiskers This ts one of the most important factors in the process. The preferred type of whiskers is that designated "Silar SC-9" and obtained from Advanced Com­ posites ( 1 525 South Buncombe Road, Greer, SC 2965 1 -9208). Other available types are TWS I OO (Tokat Carbon Company, Tokyo, Japan) (13) and Alfa Aesar (Johnson Mathey, Ward Hill, MA). Most whisker preparations are highly heterogeneous, rangmg m length from 5-500 J..U11 They can also dtffer in diameter (usual mean of 1 J..U11) and m the degree of hydrophobicity, both fac­ tors that are hkely to affect their efficiency (8). SiC whiskers are known to be a respiratory hazard (1 7, 18) and should therefore be treated with caution at all times, but especially when bemg weighed m the dry state. They should be dis­ posed of as hazardous waste. 2.2. Donor Plants Glass house-grown plants of the matze hybrid A l 88 x B73 are used as a source of embryos (see Note 1). 2.3. Callus Maintenance Medium For 1 L of N6 medmm (19), dissolve in distilled water: 4 g powdered N6 salts (Sigma), 30 g sucrose, 1 00 mg myo-inositol, 2 mg glycine, I mg thia­ mine, 0.5 mg pyridoxine HCl, 0.5 mg nicotmic acid, and 2 mg 2,4-D (from

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stock solutiOn [ 1 mglmL] made by dissolving 2,4-D m dilute KOH). Adjust to pH 6.0 with 1 M KOH, add 3 g Gelnte, and autoclave. 2.4. Callus Initiation Medium Medium N61 is N6 (20) modified by adding 0.69 g/L proline (6 mM) and reducmg the sucrose content to 20 g/L. Adjust pH to 6.0, add gelling agent as above, and autoclave. 2.5. Suspension Initiation Medium For 1 L MS medium (21), dissolve in distilled water: 4.3 g powdered salts (Sigma), 2 mg glycine, 0.5 mg thiamme, 0.5 mg pyridoxine HCl, 0.05 mg nico­ tinic acid, and 0 . 0 3 73 mg N a2EDTA. Adjust the pH to 6 .0 and autoclave . 2.6. Suspension Maintenance Medium For 1 L ofH9CP+, add to liquid MS· 30 g sucrose, 1 00 mg myo-inositol, 2 mg 2,4-D, 2 mg NAA, 0.69 g proline, 200 mg casein hydrolysate (both phytohor­ mones added from predissolved stocks in dilute KOH). Adjust to pH 6.0, and autoclave. (Add 5% sterile coconut water [Gibco] before subculture.) 2. 7. Pretreatment of Cells Medium N6(S/M) used for osmotic treatment ts liqmd N6 wtth 45 giL o-sorbitol, 45 giL o-mannitol, and 30 giL sucrose. Adjust pH to 6.0 and autoclave. 2.8. Selection of Transgenic Cells 1 . Bialaphos is obtamed by defonnulatiOn of the herbicide Herbiace (Metj i Seika, Japan). 2. Prepare N6( 1 B) from N6 and 1 mg/L btalaphos and N6(0.5B) with 0 5 mg/L bialaphos Solidify with 0.3% gelnte, or for embedding, with 0.6% Sea plaque agarose (FMC Bioproducts). Add bialaphos by filter-stenhzation after autoclav­ ing the media

2.9. Regeneration Media 1 . Regen 1 medium 1s MS with (per L) 60 g sucrose, 1 mg NAA, and 1 g myo-inosi­ tol Solidify with 0 3% Gelrite, pH 6.0 2. Regen 2 medium Is as above with 0.25 mg NAA and 30 g sucrose. 3 Regen 3 Is half-strength MS with 30 g sucrose, sohd1fied, and w1th pH as above.

2. 10. DNA Preparation Use a QIAGEN plasmid Maxi kit to purify plasmid DNA, and resuspend the DNA pellet in sterile Millipore-purified water at a final concentration of 1 f..lg/)!L.

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2. 1 1. Additional Requirements Bleach for sterilization, sterile water, forceps (curved tip), scalpel, Erlenm­ eyer flasks (250 mL), disposable pipets ( 1 0-mL w1de-bore), and 20-mesh stain­ less-steel s1eve are needed. 3. Methods 3. 1. Establishment of Suspension Cultures 1 . 1 0- 1 2 d after pollinatiOn, sterilize husked ears wtth 50% commercial bleach (final concentration 2.6% available chlorine), rinse three ttmes m water, remove Imma­ ture zygotic embryos under aseptic conditions, and place scutellum surface upward on N6I medmm 2 Add 3 g of proliferatmg type II callus from a selected smgle embryo to 20 mL of H9CP+ medium 3. Mamtain m 1 25-mL Erlenmeyer flask at 28°C in darkness on a rotary shaker ( 1 25 rpm) 4. Subculture every 3 . 5 d by addmg 3 mL packed cell volume (see Subheading 3.2.) and 7 mL old culture medmm to 20 mL fresh medmm. 5. If reqmred, cell suspensions can be cryopreserved (22). To thaw, submerge tubes in a water bath at 45°C, and immediately use a 5-mL dtsposable p1pet to plate cells onto a sterile filter paper m a 60 x 20 mm Petn dtsh. Remove excess cryoprotectant by repeated blotting wtth filter paper, place cells on solid N6 medtum, and subculture weekly for 3 wk, after which the suspensiOn can be reimtiated as above.

3.2. Pretreatment of Cells Prior to Transfo;mation l 2 3. 4. 5. 6.

7

One day after subculture, sieve suspensiOn cells through a 20-mesh stainless steve, and return to ongmal Erlenmeyer flask In used medmm. Measure packed cell volume (PCV) by using 1 0 mL wtde-bore dtsposable ptpet and allowmg cells to settle in lower half of ptpet. Replace H9CP+ medium with liqUid N6(S/M) at rate of3 mL to every I mL PCV. Return flasks to rotary shaker for 45 min. Resuspend cells evenly by pipetmg cells and medmm up and down repeatedly Usmg 1 0-mL disposable pipet, dispense 1 -mL aliquots mto 1 . 5-mL Eppendorf tubes. When cells have settled to bottom of tube, remove 0.5 mL medmm, leaving 1 · 1 proportions of PCV:medium.

3.3. Preparation of Whiskers I . Transfer about I 0-50 mg dry whiskers to preweighed 1 .5-mL Eppendorftubes in a fume hood. 2. Rewetg h tubes, and calculate the weight of whiskers. 3 Pterce the lid to avoid tts bemg displaced during heating, cover tt in aluminium fml, and autoclave the tubes.

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4. Store at room temperature prior to use Whiskers should be prepared fresh m an aqueous suspenston (5% w/v m stertle water) pnor to use (8).

3.4. Transformation Procedure I . Add 40 � of 5% whisker suspenston and 2 5 � plasmid DNA ( I j..tg/�) to the pretreated cells m an Eppendorf; gently premtx by tapping. 2. Place tube in multtsample holder of Vortex Geme II, and mtx at full speed for 60 s, or m the holder of a Mtxomat dental amalgam mtxer shaken at fixed speed for 1 s (See Notes 2, 3, and 4)

3.5. Selection of Transgenic Tissue 1 Add 0.5 mL fresh N6(S/M) medmm to the mtxture m the tube 2. Use a 200-� wtde-bore Ptpetman tip to dtspense the dtluted contents onto a 55-mm Whatman no. 4 filter paper overlaymg solid N6 medmm m a 60 x 20 mm plastic Petri dish. 3. Wrap dtshes m gas-permeable Urgopore tape (Stenlco, Brussels), and incubate at 28°C in darkness. 4. After 7 d, transfer the filter paper and cel ls to the surface of N6(1B) selectwn medium. Repeat after a further 7 d. (see Note 5) 5 Use a spatula to transfer cells from the filter paper mto 5 mL N6(0.5B) medium contammg 0 6% Sea plaque low-gellmg-pomt agarose m a stenle test tube kept at 3 7°C. 6. 01Vide the suspension into two altquots, and p late each evenly over 20 mL N6(0.5B) medmm soltdtfied with 0 3% Gelrite m a 1 00 x 25 mm Petri dtsh. 7 Seal with Nescofilm, and incubate at 28°C. 8 After 4--5 wk ptck offpotenttally transformed cal It, and transfer to the surface of fresh N6(1B) medmm 9. Subculture onto same medium every 2 wk.

3.6. Regeneration of Plants 1 . Transfer type II callus onto Regen I medium, and incubate at 2 5°C m darkness. 2. After 2-3 wk, transfer opaque, mature somatic embryos onto Regen 2 medmm. 2 Incubate in light ( -200 J..lO lll l/m /s with 1 6-h photoperiod) 3 . After a further 2-3 wk, shoots and roots emerge, and small plantlets can subse­ quently be transferred to glass tubes contaimng 1 5 mL Regen 2 medium. 4. After 7 d, transfer to sot! m a glass house for analysts (see Note 6). Grow to maturity in 3-gallon pots contammg equal parts of peat:perltte:s01l. 4. Notes 1.

The genotype recommended m this study produces a form of dispersed suspen­ sion very suitable for the method descnbed. Any other genotype with this charac­ teristic Is likely to be equally suttable. As ment10ned m Subheading 1., mature embryos have also been used successfully m wheat (14) .

Silicon Carbide Whiskers

38 1

2 Although these results were obtained usmg two particular forms of apparatus for mixmg, it can be assumed that any other eqUivalent form of mixmg device would give similar results. For example, the results on wheat (14) were achieved wtth a Micro tube mixer MT-360 (Tomy Seiko Co., Ltd., Japan) 3. To date, tnals on a range of material have gtven the best results with StC whis­ kers. There may, however, be other, as yet untested crystalline material of equal efficiency 4. As an alternative to the use of a random-mixing process for cells and whiskers, a fixed array of microneedles has been developed (23) 5 . It can be expected that other selective agents could be used in place of bialaphos 6. In an interesting comparative study (24) , It has been shown with Chlamydomo­ nas reinhardtu that transformants produced by a glass bead method (25) gtve a htgher frequency of random to homologous ( 1 000: 1 ) mtegratton events than those produced by using particle bombardment (24 1 ) (ref. 26). It may be that the form of the DNA (degree of condensatton) is an Important determmant of the type of integration pattern. If so, then the SiC method, m which the DNA IS not precipi­ tated, may be advantageous m those systems where random patterns are reqmred.

References 1 Coe, E H. and Sarkar, K. R ( 1 966) Preparatton of nucletc actds and a genetic transformatton attempt m matze. Crop Scz 6, 432-435 2. Dunwell, J M. ( 1 995) Transgenic cereals Chern Ind Sept 1 8 , 730-73 3 3 . Wilson, H M . , Bullock, W P., Dunwell, J M., Ellis, J. R , Frame, B , Register, J., et a!. ( 1 995) Maize, m Transformation ofPlants and Sml Mzcroorgamsms (Wang, K., Herrera-Estrella, A., and Van Montagu, M , eds.), Cambridge Umverstty Press, pp. 65-80 4. Frame, B. R., Drayton, P. R., Bagnell, S. V., Lewnau, C. J., Bullock, W. P., Wtlson, H M., et a!. ( 1 994) ProductiOn of ferttle transgemc matze plants by si licon carbtde whtsker-mediated transformatiOn. Plant J 6, 94 1-94 8. 5. Kaeppler, H. F. and Somers, D. A. ( 1 994) DNA dehvery to maize cell cultures using st hcon carbide fibers, m The Mmze Handbook (Freeltng, M., and Walbot, V , eds. ), Springer-Verlag, New York, pp 6 1 0--6 1 3 6. Thompson, J. A., Drayton, P. R., Frame, B . R., Wang, K , and Dunwell, J . M. ( 1 995) Maize transformatiOn utilizmg silicon carbide whiskers: a review. Euphytzca 85, 75-80. 7. Wang, K., Frame, B . R , Drayton, P. R , and Thompson, J. A. ( 1 995) Silicon­ carbide whtsker-mediated transformation: regeneration oftransgemc matze plants, in Gene Transfer to Plants (Potrykus, I. and Spangenberg, G., eds.), Springer, Berlm, pp 1 86-1 92. 8. Wang, K., Drayton, P. R., Dunwell, J. M., and Thompson, J. T ( 1 995) Whtsker­ medtated plant transformatiOn' an alternative technology. In Vztro Cell Dev Bzol 31, 10 1-104 9. Dunahay, T. G . , Adler, S A , and Jarvik, J W ( 1 997) Transformation of microalgae using sthcon carbide whiskers. Methods Mol Bzol. 62, 503-509

Dun well

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1 0 Dunahay, T. G. ( 1 993) Transformation of Chlamydomonas reznhardtii with sib­ con carbide wh1skers. BwTechmques 15, 452--460. 1 1 Kaeppler, H F Gu, W . , Somers, D A , Rmes, H W , and Cockburn, A. F ( 1 990) Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Rep 9, 4 1 5--4 1 8. 1 2 . Kaepp1er, H. F , Somers, D. A., Rmes, H. W., and Cockburn, A F. ( 1 992) Silicon carbide fiber-mediated stable transformation of plant cells Theor Appl Genet 84, 560--566. 1 3 Asano, Y., Otsuki, Y , and Ugaki, M ( 1 99 1 ) Electroporation-medtated and sib­ con carbide whisker-medtated DNA dehvery m Agrostzs alba L. (Redtop) Plant Sci 9, 247-252. 14. Senk, 0 , Amur, I., Murat, K., Tetsuo, M , and Masaki, I. ( 1 996) Sihcon carb1de fiber-mediated DNA deli very mto cells of wheat (Tntzcum aestzvum L.) mature embryos. Plant Cell Rep 1 6, 1 3 3-1 36 Zmg, Z., Powell, W. A., and Maynard, C. A. ( 1 997) Usmg stlicon carbtde fibers 15 to enhance Agrobacterium-mediated transformatiOn of Amencan chestnut In Vztro Cell Dev Bwl 33, 63A 16 US Patent 5 302523, TransformatiOn of Plant Cells. April 1 2, 1 994 1 7 Vaughan, G L., Jordan, J , and Karr, S ( 1 99 1 ) The toxiCity, in vitro, of sthcon carbide whiskers. Envzron Res 56, 57-67. 1 8 Vaughan, G. L., Trendy, S A., and Wtlson, R B. ( 1 993) Pulmonary response, m vzvo, to sihcon carbtde whiskers. Envzron. Res 63, 1 9 1-201 19 Chu, C C., Wang, C. C., Sun, C S., Hsu, C., Yin, K C., Chu, C Y , et a!. ( 1 975) Establishment of an efficient mediUm for anther culture of rice through compara­ tive experiments on the mtrogen sources. Scz Sm 1 8, 659-668 20 Armstrong, C L. and Green, C. E ( 1 985) Estabhshment and maintenance of fnab1e, embryogemc matze callus and the involvement of L-proline. Planta 1 64, 207-2 1 4. 2 1 . Murashige, T. and Skoog, F. ( 1 962) A revised medium for rapid growth and bio­ assays wtth tobacco t1ssue cultures Physiol Plant 15, 473--497. 22 Shill ito, R. D., Carswell, G. K., Johnson, C. M., D1 Maio, J J., and Harms, C. T. ( 1 989) RegeneratiOn of ferti le plants from protop1asts of ehte mbred maize. Bzol Technology 7, 58 1-587 23 US Patent 545704 1 , Needle Array and Method of Introducmg Btolog1cal Sub­ stances mto Ltving Cells usmg the Needle Array. October 1 0, 1 995 24. Sodeinde, 0. A. and Kindle, K. L ( 1 993) Homologous recombmatwn in the nuclear genome of Chlamydomonas remhardtu. Proc. Nat/ Acad Scz USA 90, 9 1 99-9203. 25. Kindle, K L ( 1 990) Htgh-frequency nuclear transformatiOn of Chlamydomonas reznhardtu. Proc. Nat/. Acad. Scz USA 87, 1 228- 1232. 26 Kindle, K. L., Schnell, R. A., Fernandez, E., and Lefebvre, P. A. ( 1 990) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for mtrate reductase. J Cell Bwl 109, 2589-260 1 ,

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Directing Anthraq ui none Accumulat ion via Man ipulation of Morinda Suspension Cultures Marc J. M. Hagendoorn, Diaan C. L. Jamar, and Linus H. W. van der Plas

1 . Introduction Secondary metabolite accumulation in cell suspensions is the result of an interplay between primary metabolism, supplying the machinery, the energy, and the precursors, and the secondary metabolic pathways. For this reason, a successful strategy for producing considerable amounts of secondary plant products can hardly be obtained without the knowledge of the interactiOns between primary and secondary metabolism. These interactions are best studied m cell suspensions that produce significant amounts of secondary metabolites. In Morinda citrifolia cell suspensions, secondary metabolic pathways can easily be switched on, resulting m a considerable accumulatiOn (more than 1 0% of the dry weight) of anthraquinones (AQs, [1}). Anthraqumones are dyes that can be treated to give a variety of colors, e.g., yellow, red, purple, brown, or black (2). Rubiaceae AQs are all derivatives from the basic structure as shown in Fig. 1. M citrifolia is a tropical tree, inhabiting the islands on the edge of the Pacific Ocean (2). In M. Cltrifolia plants, AQs are only accumulated m the roots, and then mostly in the bark. Most of the AQs of M. citrifolia cell suspensions are glycosylated, especially as 0-glucosylxylosyl (therefore called primverosides [3}) and subsequently are stored in the vacuole. In the Rubiaceae, anthraquinones are produced via the shiktmate-mevalonate pathway. Intermediates from glycolysis and the pentose phosphate pathway are combmed to form shikimate and subsequently chorismate. This compound can, via several steps, be converted into phenylalanine, tyrosine, tryptophan, and via isochorismate, into anthraquinones. Thus, the b10synthes1s of anthraquinones From Methods

m

Molecular Brology, Vol 1 1 1 Plant Cell Culture Protocols Hall © Humana Press Inc , Totowa, NJ

Edited by R. D.

383

384

Hagendoorn, Jamar, and van der Plas

Ftg. I Baste structure of anthraqumones found m the Rubtaceae. SubstJtuents mclude methyl, hydroxyl, and methoxyl groups. Numbered atoms ( 1-14) are C-atoms Os indtcate the posttlon of the 2-oxygen atoms.

shows mteractions with protein synthesis, other secondary pathways (phenyl­ propanmd pathway via phenylalanine), and hormone synthesis (auxm synthe­ SIS via tryptophan). We have stud1ed the mfluence of several auxm analogs on anthraqumone production. For 2,4-dlchlorophenoxy acetic ac1d (2,4-D) and ! -naphthalene acetic ac1d (NAA), we also have determmed the effects on cell d1vision. It appears that auxin analogs that are able to maintam cell d1vision have a nega­ tive influence on AQ production (4) . The auxin analog 2,4-D already stimu­ lates cell division at a much lower concentration than NAA. In Morinda cells growing in the presence of 4.5 � NAA, the AQ production can be Immedi­ ately blocked by the addition of 2,4-D (final concentration 4.5 � (5) . There­ fore, Mormda cell suspensions can be used as a model system, m wh1ch AQ production can be easily switched on and offby changing the auxm concentrations. Although there often is a negative correlatiOn between cell division and anthraquinone accumulation (see Fig. 2), affecting specifically the cell divi­ sion rate, th1s does not result in a change in AQ productwn. Poss1bly two dis­ tinct programs can be sw1tched on under regulation of auxins· a program focused on fast cell div1s1on and one in which productiOn prevails (4). These separate programs are also reflected m the cell compositiOn. The slowly diVid­ ing cells show a lower respiration rate, a h1gher soluble sugar content, and contam less protein (5, 6) . M. citrifolza cell suspenswns can also be grown m fermenters, as so-called contmuous cultures. After several weeks, a steady state is reached in wh1ch growth rate, cell composttton and AQ content (4, 7) are constant. Therefore, interactions between primary and secondary metabolism can be studied quan­ titatively over prolonged periods. Another advantage of the Morinda system is that changes m AQ accumula­ tion can easily be followed visually. A simple extraction and subsequent spec-

Directing Anthraquinone Accumulation

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Fig. 2. Correlation between final cell number and AQ content in M citrifolia cell suspensiOns at the end of a batch culture ( 14 d after moculation) Data are used from several mdependent expenments. Each pomt is based on three AQ determmations and on 1 6 cell number determmations. troscopic determination can give an indication of the AQ content. Methods are described for the growth and maintenance of batch and continuous Monnda cultures and for the measurement of the different specific anthraquinones (gly­ cosldes and/or aglucons). 2. Materials 2. 1 . Growth and Maintenance of Morinda Citrifolia Cultures Batch cultures: 1 . 250-mL Erlenmeyer flasks for the maintenance of the M cltrifolla L cell hoe (1) 2. Growth medium· Gamborgs B5 medium (8, see Appendtx), supplemented with sucrose (40 g/L), 0.2 mg/L kinetm (Sigma), and NAA or 2,4-D {Stgma) as auxm (analog), (see also Note 1) 3 . Stock solutions of NAA and 2,4-D ( 1 g/L): dissolve m 1 M KOH (p.a.) and store at -20°C.

Continuous cultures 1 . A standard 2-L btoreactor with round bottom vessel, stirred with an ADI 1 0 1 2 motor controller (Applikon) 2. 0.2-� filters {Mtlhpore) for aeration. 3. Gamborg 's B5 medium supplemented wtth 5 or 10 g/L sucrose, 0.2 mg/L kmetm, and auxin (concentration and type depending on the expenment).

386

Hagendoorn, Jamar, and van der Plas

2.2. Measurement of Growth Parameters I 2 3. 4 5.

A Fuchs-Rosenthal hemocytometer for cell countmg. Buchner funnel, filter papers (e g , Schleicher and Schull). Vacuum/water pump 60°C drymg oven FDA solutiOn. prepare a stock solutiOn by d1ssolvmg FDA m acetone to giVe a concentration of 5 g/L. Dilute I 00 ttmes m growth medmm before use Make fresh each time 6. Standard UV/vJsJble microscope with FITC filter

2.3. Anthraquinone Determinations 1 2 3

Ethanol (p.a ) was used. AIIzarm (BDH) as a standard (see also Note 2 ) Silica gel TLC (0 2 mm, Merck) plates The mob1le phase consists of Isopro­ panol water: ethyl ether toluol (50· 1 2 5· 25: 1 0 , v/v/v/v) 4. Thymol-sulfunc acid is 0 5 g thymol m 95 mL ethanol, to wh1ch 5 mL 1 8 N sulfunc acid IS carefully added

3. Methods 3. 1. Growth and Maintenance of M. Citrifolia Cultures To start a new cell hne, Morinda seeds are used. 1 . Surface-stenhze seeds m a solution of 10 g/L Na hypochlonte and 50 mg/L Tween-20 for 1 0 mm. 2. Rmse with stenle water (three times). Germinate the seeds m Petn dishes con­ taining water-based agar. 3 When the first true leaves have appeared, transfer the obtamed seedlings to hqmd growth medium (see Subheading 2.) supplemented w1th 4 5 1-iM 2,4-D. 4 Loose cells will detach from the seedlings w1thm a few weeks Use these cells as an moculum, and mitiate a new cell lme. 5. Scaling up of such a newly formed Ime can be done by usmg the whole batch as moculum for a new culture m a larger (6x) volume

For culture maintenance, cells are grown m the presence of 4.5 � 2,4-D. Batch cultures should be subcultured every 1 4 d. Subculturmg and/or the start of an experiment are as follows. 1 . In a laminar flow cabinet, add 10 mL of a 1 4-d old suspensiOn culture to 50 mL of fresh medmm in a 25 0-mL Erlenmeyer flask 2. Close the flasks with two layers of alummum foil After each handling, the fml should be replaced by a new, stenle one 3 Shake the flasks on an orbttal shaker ( I 00 rpm), and culture at 25°C with a 1 6-h day 2 period and an 8-h night period; mad�ance 1 0-20 W/m (Ph1hps Fluorescent no. 84) 4. By repeatmg steps 1-3 cultures can be mamtamed indefimtely.

387

Directing Anthraquinone Accumulation

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Fig. 3. Schematic drawing of the fermenter cultures. Fresh medium IS pumped mto the culture vessei at a constant rate. The overflow/sample IS removed by a vacuum pump (C) through an enlarged tube (A) that IS placed at a certam height to keep the culture volume constant (B). The overflow IS collected m a sample bottle (D). The mcoming a1r is mOistened by leadmg 1t through a bottle filled w1th sterile water (E).

3. 1. 1 . Continuous Culture Using the configuration as shown in Fig. 3, Morinda cells can be grown under sucrose or phosphate hm1tation. When cells are grown under phosphate limita­ tion, 0.2 mM phosphate is used in combination with an otherwise standard Gamborg' s B5 medium, supplemented with l 0 g/L sucrose. For sucrose limita­ tiOn, only 5 g/L sucrose is added to Gamborg's B5 medium. Air is contmuously supplied at a rate of 20 L/h. The fermenter is stirred at 1 00 rpm. Cells are grown at a dilution rate of 0. 1/d (i.e., 1 0% of the culture ts replaced with fresh medium every day). Auxm type and concentration depend on the experiment.

388

Hagendoorn, Jamar, and van der Plas

Inoculation procedure: 1 . Sterilize the entire apparatus m an autoclave for 1 5 mm at l 20°C and 1 .4 bar. 2. In a laminar flow cabmet, add 300 mL cells from a 14-d old batch culture and 1 . 5-L fresh medium to a bottle that IS subsequently connected to the sample port 3 . Pour the content of the bottle into the fermenter. The empty bottle can then be used for the first overflow/sample. 4. Connect the stmer, the pH electrode, the thermostated water bath, and the medium pump, and the culture 1s started by the contmuous addition of fresh mediUm. 5 The overflow 1s discontmuous; sample and overflow procedures are descnbed m the legend to Fig. 3 (see also Note 3).

3. 1.2. Measurement of Growth Parameters I

Cell number determmat1on: dilute the cells m B5 mediUm (final number of cells per square should be between 10 and 30). Count two samples in a hemocytom­ eter The cells in 16 squares should easily be counted (see also Note 4). 2. Fresh weight and dry weight: collect a sample of the culture (up to 10 mL) with a sterile pipet (Gremer, wide-mouthed) Remove the mediUm usmg a paper filter on a BUchner funnel. Remove the cells from the paper filter to obtain the fresh we1ght (see also Note 5) To determme dry we1ght, cells are dned at 60°C for 24 h 3 . V�ability of the cells: Mix I vol of cell suspension w1th I vol of 50 mg!L fluorescem d1acetate solution. Using a hght microscope, count the number of cells that show green fluorescence under UV Illumination. Divide by the total number of cells (vis­ ible under white light). Multiply by I 00 to give the percentage of viable cells

3.2. Anthraquinone Determinations

3.2. 1. Quantitative Estimation Cells are harvested as described above. Cells are either directly used or stored at -20°C. I . Add approx 0.0�.2 g cells (fresh wt) to 5 mL 80% aqueous ethanol m a 1 5-mL centrifuge tube (see Note 6). 2. Heat the tubes in a water bath at 80°C for 1 0 mm 3 . Collect the ethanolic extract by centrifugmg the tubes at 1 500 g for 5 mm. 4. Add another 5 mL of 80% ethanol to the pellet, and repeat steps 2 and 3 . 5 . Pool the two supernatants, and make up to 1 0 mL w1th 80% ethanol 6. Determme the absorptiOn at 434 nm. When necessary, the extracts can be diluted to obtain an absorptiOn between 0. 1 and 1 .0. 7 . Calculate the AQ content by using a milhmolar extmctwn coefficient of 5.5 and a mean mol-wt value of 580 (see Notes 2 and 7).

3.2.2. Qualitative Determination To acquire a first impression of the anthraquinone composition, the extract described above can be analyzed using thin-layer chromatography (TLC) (see

389

Directing Anthraquinone Accumulation

also Note 9). In this manner, the glycosides and the aglucons can be separated and the RF 's compared with literature data (e.g., 9). 1 . Apply a sample of the extract to the TLC plate, 1 .5 em from the bottom The amount of extract that can be loaded onto a TLC plate depends on the concentra­ tion of the dissolved compounds. Spots should be clearly visible. 2. Develop the plate until the front of the mobile phase 1s I em from the top of the plate in order to be able to determine the RFs (see Note 8). 3 . Anthraquinone spots are readily v isible The glycosides can be identified after spraying with thymol -sulfuric ac1d and subsequent bak mg of the plate for 1 5-20 min at 1 20°C. In this way, the sugartails are colored dark pmk.

3.3. Manipulation of Anthraquinone production

3.3. 1 . Upmanipulation by Auxin Deprivation Growth in the presence of 2,4-D, down to a concentration of ca. 5 does not result in significant AQ accumulation (4).

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1 o-S M,

I . Use cultures that have been grown in the presence of 5 x w--8 M2,4-D for several subcultures. 2. On d 0, transfer cells to auxm-free medium or to medmm containing 4.5 1-JM NAA 3 . Subculture every 14 d to auxm-free medium or to medium contaming 4 5 1-JMNAA

Withm a few days, AQ accumulation can be seen. When using NAA, the cells contmue dividing, and the effects of AQ accumulation on cell growth can also be studied. After about four to five subcultures (ca. 60 d {6)), the AQ content becomes toxic and the cells eventually die. In an auxin-free medium, Mormda cells produce AQ more rapidly, but they stop dividing and die withm 2 wk.

3.3.2. Downmanipulation by Auxin Addition Use cultures that have been grown in the presence of 4 5 1-JM NAA. To study the kmetlcs of the inhibition of the AQ accumulation, cells should be used that con­ tain ca 50 mg AQ/g dry wt at the start of the batch culture. 2. Add 2,4-D (final concentration 4.5 �: the AQ accumulation IS stopped w1thin a day. When effects on growth are stud1ed, 2,4-D can best be added in the early loganthmic growth phase (normally ca. 4-8 d after inoculatiOn). 3 . Effects on AQ accumulation can be measured after 1 d. 1

4 . Notes 1 . Culture medmm can be prepared and stored m advance. Caution should be taken

that some growth regulators are heat-labile (e.g., IAA) and that autoclavmg at too high a temperature can lead to caramelization of the sucrose, the avOidance of wh1ch is especially important with medium for continuous cultures (prepared in larger quantities, 3 L)

390

Hagendoorn, Jamar, and van der Plas

2. The mixture of AQs extracted from M. citrifolia suspension cells shows a simtlar spectrum (400-600 nm) to the alizarin absorptton spectrum. For quantitative determmattons, a millimolar extmctton coefficient of 5.5 should be used, also based on alizann 3. Dunng contmuous culture of plant cell suspenstons, tt ts very important to have an efficient overflow and to be able to take representative samples Because the dtlutton rate of plant cell contmuous cultures ts very slow, a contmuous overflow would specifically remove only medmm, because the upward movement of the culture through the overflow tube would take place at a lower velocity than the downward movement of the cells caused by grav1ty. Therefore, every few hours the excess of culture is removed at a h1gh speed, with a vacuum pump (see Fig. 3) The same holds for sample takmg. The diameter of the overflow/sample tube i s rather large to prevent blockage b y cell clumps. 4. Many plant cell suspenston cultures contam clumps of cells, whtch makes count­ mg the cells rather problematic. In these cell suspensiOns, the cell clumps have to be macerated m order to obtam a correct est1matton of the cell number In M Cltrifolza suspenstons, however, the cells are etther loose or are attached to each other m chams. Both types of cells are eas1ly counted In order to avotd overesti­ mation of the cell number, cells that cross the lmes m a hemocytometer are only counted 1f they cross 1t on the upper or the left s1de of the square 5 An exact esttmatton of the fresh wetght content IS hampered by two thmgs. F1rst, the layer of cells harvested on the paper filter w1ll lose water Therefore, one has to use a stnct, reproducible ttme schedule. Second, m the course of growth of a batch culture, more cells are harvested and, therefore, the cell layer on the paper filter w1ll become thicker, therefore, more Intercellular water will be mcl uded m the FW determmatton at the later growth phases One has to adJust the size of the sample to avotd this 6 In 2,4-D cultures, 0.2 g FW 1s used because of the very low AQ content. So-called NAA cultures can produce so much AQ that the sample size must be reduced to 0 05 g FW or even less, m order to ensure that all AQs can be Isolated from the cells. A good md1cation of a proper anthraqumone extractiOn 1s the color of the pellet after AQs have been extracted tw1ce The pellet should look whitish 7 The mean molecular weight of the anthraqumones that occur m M cztrifoha IS calculated, supposmg that 95% occurs as glycos1des and 5% as aglucons (1, 9). 8 At least 30 mm before the plates can be developed, the chromatography tank should be equilibrated To do thts, pour a 1 -cm layer of mobile phase mto the tank and close the lid 9. To get more insight mto the exact structure of the anthraqumones, other more elaborate methods have to be used such as HPLC, GCMS, and so forth (9) .

References Zenk, M H , El Shagi, H., and Schulte, U. ( 1 975) Anthraqumone production by cell suspensiOn cultures of Mormda cztrifolza Planta Med. Suppl. 79-1 0 1 . 2 . Morton, J . F ( 1 992) The ocean-gomg Noni or Indian Mulberry (Mormda cltrifolza, Rubiaceae) and some of tts "colorful" relatives Econ Bot 46, 24 1-256

Directing Anthraqwnone Accumulation

39 1

3 Inoue, K., Nayesh1ro, H., Inouye, H., and Zenk, M. H. ( 1 98 1 ) Anthraquinones m cell suspension cultures of Morinda citrzfolia. Phytochemzstry 20, 1 693-1 700. 4. Hagendoorn, M. J M., Jamar, D. C. L., Meykamp, B., and Van der Plas, L H W ( 1 996) Cell division versus secondary metabolite production in Morznda citrzfolza cell suspensions J. Plant Physwl. 1 50, 3 25-330. 5 . Hagendoorn, M J M , van der Plas, L H W , and Segers G J ( 1 994) Accumu­ latiOn of anthraqumones m Morinda cztrzfolza cell suspenswns A model system for the study of the mteraction between secondary and pnmary metabolism. Plant Cell Tzss. Org. Cult 38, 227-234. 6. Van der Plas, L. H W , EIJkelboom, C , and Hagendoorn, M J M. ( 1 995) Rela­ tion between pnmary and secondary metabolism m plant cell suspensions. com­ petitiOn between secondary metabolite production and growth in a model system (Morznda cztrifolza). Plant Cell Tiss Organ Cult 43, 1 1 1-1 1 6. 7. De Gucht, L. P E and Van der Plas, L H W. ( 1 995) DeterminatiOn of expen­ mental values for growth and maintenance parameters Bzotechnol Bzoeng 47, 42-52 8 Gamborg, 0 L , Miller, R A , and OJima, V ( 1 968) Nutnent reqmrements of suspensiOn cultures of soybean root cells. Exp Cell. Res 50, 1 5 1 -1 58. 9. WiJnsma, R and Verpoorte, R ( 1 986) Anthraqumones m the Rub1aceae. Progress Chern Org Nat Prod 49, 79-149

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Alkaloid Accumu lation in Catharanthus roseus Suspension Cultures Alan H . Scragg

1 . Introduction Plants have traditionally been the source of pharmaceuticals and fine chemi­ cals, and at this time, plant material is still bemg screened for new compounds (1) . Initially, plant cell and tissue culture was developed m order to study the biochemistry and physiology of p l ant s with out th e comp l i c at i on of the whole plant. However, plant cell or tissue cultures were also found to accumu­ late many of the compounds characteristic of the ongmal plant (2). Many plants are the source of plant-derived products, which mclude drugs, fragrances, col­ ors, oils, and pesticides (3). Most of these valuable plant products are defined as secondary metabohtes, which are often complex in structure, the end prod­ uct of long biochemical pathways, not essentml for growth, but do often confer some advantage to the plant, such as reduction of predation (3). A number of these secondary products are very valuable, such as taxol (4), and plant cell culture may provide an additional source of supply if· •

• •

•

There ts a high demand for the product. The product is affected by disease. Supply is affected by the climate Only low levels accumulate in plants.

It may also provide an alternative supply if: •

•

•

The plant is rare. The plant species is protected. No chemical synthesis IS available

From Methods m Molecular Btology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

393

394

Scragg

These reasons, and in particular, the high values of some secondary prod­ ucts (2) have been the driving force in the attempts at producing them using plant cell cultures. Although high levels of some secondary products have been obtained, those of commercial value have remamed stubbornly low. Therefore, numerous approaches have been taken m order to mcrease the yield of second­ ary products m plant cell cultures. These were: •

•

•

•

•

SelectiOn of high-yteldmg hnes. Changes m medium composition· carbon source quantity and quahty, mtrogen, phosphate, growth regulators Changes m culture condtttons pH, temperature, light Cultivation strategtes. tmmobthzatwn, organ culture, hatry roots, two-stage cul­ tivation. Spectahzed techmques ehcttatwn, product removal.

Much of this research has been earned out usmg cultures of Catharan thus roseus (L) G.Don, the Madagascan periwmkle. This plant has been extensively studied, smce It contains the Important antileukemics vincnstme and vmblas­ tine and the antihypertensives ajmalicine and serpentme (5, 6) . Despite considerable efforts, the two dimeric alkaloids vincristme and vm­ blastine have not been detected in C. roseus cultures, even though a number of other alkalmds have been detected (6). Although none of the approaches described above have developed a stable, high-yteldmg culture of C. roseus, the use of a two-stage process has proven to be the most successful. Secondary products are often accumulated after growth has ceased and a change in cultural conditions, whtch slows or stops growth, will often encourage the accumulatiOn of secondary products. Thts led to the development of two-stage processes for the accumulatiOn of ajmalicine and serpentme in C. roseus cultures (7,8) . In the first stage, growth ts encouraged, and in the second stage, secondary product accumulatiOn is stimulated. In one C roseus system, the change to the productton medium was the replacement of the growth regulator 2,4-D by NAA (7, 9) (see Note 1). An alternative two­ stage system that has been used by other groups ts based on L&S medmm (1 0) , and the production medmm lacks mtrate, ammomum, and phosphate, combmed with an mcrease m glucose from 20--80 g/L (see Note 2) (8,1 1) . However, an alternattve single-stage C. roseus system has been developed where the growth and production medta have been combmed and secondary product and growth are partially linked (12, 13). All three C. roseus systems are capable of alkaloid accumulation desptte their differences in cultural condt­ ttOns, such as hght/dark, use of sucrose or glucose, and growth regulators. Thus, tt would appear that there are no specific condttlons for secondary product accumulation, and these vary wtth the mdividual cell hnes developed. Table 1

395

Alkaloid Accumulation

Table 1 Growth and Alkaloid Accumulation by C. roseus Suspension Cultures8

Medmm B5 zb

8%C Growthe (L&S) Production APMg APM M3h

Alkal01d accumulatiOn mg/g dry wt

Growth rate

Growth regulators

Carbon source

mg/L

g/L

)ll
1 .0 2,4-D 0. 1 kinetm 0 17 IAA 1 . 1 3 BAP Zero 2.0 NAA 0.2 kinetin

20

0 21

0 1

0

50

0 12

30

I 2

80 30d

0 01

1 2 0

0 0

0 1 7 IAA 0 1 7 IAA I O NAA

god 20 50 20

0 25 0 18 0 18

Serpentme

0 0.023 0 68 56

AJmahcme

12 0 00 1 0 34 I 5

0Usmg production media In smgle and two-stage processes. hProductlon medmm based on L&S (7) 'The medmm contams only water and sucrose (8) dThe carbon source was glucose, whereas all other media used sucrose (11) eorowth and productwn medmm bases on L&S (11) fL&S mediUm mmus mtrate, ammomum, and phosphate (11) gBased on B5 medmm, smgle-stage culture (9) hBased on M&S mediUm, smgle-stage culture (12)

gives details of the conditiOns encouragmg alkalOid accumulation m C. roseus usmg both one- and two-stage processes. The last system (M3) IS described m this chapter along w1th the extractiOn, separation, and identificatiOn of the alka­ loids aJmalicine and serpentme. 2. Materials 2. 1. Suspension Cultures and Growth Analysis

2.

3.

4 5

Suspens ton cultures of C. roseus can be developed from ex plants VIa callus usmg standard techmques (5, 6) . We use a selected !me C roseus IDI (14) Mamtenance medmm M3 IS based on Murash1ge and Skoog salts (1 5, and see Appendix) (w1th the om1sswn of KH2P04) and contains 20 g/L sucrose, 0. 1 mg/L kinetin, and 1 .0 mg/L naphthaleneacetlc ac1d (NAA). 250-mL conical flasks contammg 1 00 mL M3 medium. The flasks are closed with a double layer of aluminum foil. Orbital shaker operatmg at 150 rpm A culture room at 25°C, and constant light at 50 Jlmol/m21s

396

Scragg

TOP SECTION

MESH

Fig I A stamless-steei filter umt used for wet and dry weight determmation. The filter is supported by the mesh on the lower half, and the weight of the unit holds the filter in place and the filter on the Buchner flask. 6. 7 8. 9

Whatman no. 1 filter paper disks (2.5-cm). Oven at 60°C Stamiess-steel filter bed (see Fig. 1). 2 50-mL B uchner flask.

2.2. Alkaloid Extraction and Analysis

2 3 4 5. 6. 7. 8. 9 10. 1I 12 13. 14.

TLC plates· 20 x 20 em, 0.25 mm sihca 601254 fluorescence (E Merck, Darmstadt); activate by heatmg to I oooc for I h 9-cm Hartley funnel . Miracloth (Calbiochem-Behrmg, La Jolla, CA) 1 0 x 50 mm Whatman cellulose thimbles. SEP-PAK C 1 8 cartndges (Waters Associates) Rotary evaporator (Rotavapor R I I O, BuchiLabs). Somcator bath (Decon Frequency Sweep bath, Decon Ultrasomc Ltd., Hove, UK) Iodoplatinate stam. 5% w/v platinum chlonde ( 1 0 mL), cone. HCI (5 mL), 2% w/v KI (240 mL) Ceric ammomum sulfate stam: cenc ammonium sulfate ( I g) m I 00 mL of 85% a-phosphonc acid 0.8 x 1 0 em J.ll11 Bondpak C I 8 HPLC column HPLC-grade methanol N-heptane sulfonate. Acetomtnie. TLC tank suitable for 20 x 20 em plates.

397

Alkaloid Accumulation

14

12_

_

12

0 �----0

2

3

4

5

6

7

8

9

10

11

12

13

TIME (Days)

Fig 2 The accumulation of serpentine in a smgle-stage system usmg C grown m M3 medium (0) Dry weight g/L, (D) serpentme mg/g dry wt

roseus

!D I

3 . Methods 3. 1. Growth and Growth Analysis An example of the growth and alkaloid accumulation by a culture of C ID l cultured m M3 medium ts shown m Fig. 2 . The accumulation of serpentine under these conditiOns can be mcreased by raising the concentration of sucrose m the medium (see Table 2). To avotd osmotically stressmg the cells by addmg htgh concentrations of sucrose at the start of the culture, the sucrose can be apphed m a number of small aliquots, m a fed-batch process.

roseus

I. Subculture the suspensiOn every 2 wk by adding 20 mL of culture to I 00 mL of fresh medium m a 250 mL flask. 2. To measure wet and dry weight, place a supply of filter paper disks m the oven for at least 24 h before use. 3. Wetgh the dned filterdtsk after removal from the oven usmg ! /sample and 3 samples/ measurement 4. Place the fi lter paper on the ft lter untt seated on a bored rubber stopper on a Buchner fl ask under vacuum. The ft lter used ts a stamless-steel untt as

398

Scragg

Table 2 Improvements in the Alkaloid Accumulation in the One-Stage Process Using 101 on M3 Medium Sucrose % 2 6 6 (fed batch)a

Growth rate

Bwmass g/L d- 1

Serpentine mg/g dry wt

ProductiOn mg/Lid

0 46 0.33 0 36

8.3 23.9 26.6

2.55 9.65 5.07

I 32 8.24 5 62

0Sucrose added m 3

x

2% batches

durmg the growth of the culture

shown m Fig. 1 . Other ftlter umts such as those supplied by Mtllepore can also be used. 5 Wet the filter w1th about 3 mL d1st1lled water and retam under vacuum for a fixed ttme period (e.g., l O s) Rewe1gh Immediately to avoid loss owing to evaporatiOn 6 Replace the filter m the filter umt, and filter a known volume of culture (e.g , 3-5 mL) The culture should be well mixed before samplmg to ensure a represen­ tative sample (see Note 3). When the cells appear "dry," contmue the vacuum for the same fixed time penod as above and weigh immediately. 7 Place the filter m a labeled Petn dish, and dry for at least 24 h in the oven before wetghing the dry filter and cells 8. The wet and dry we1ghts of the cells are calculated as followed Wet wt (g/L) Dry wt (g/L)

=

=

[wt of wet d1sk + cells (g) - wt of wet d1sk (g)/ sample volume (mL)] x 1000

(I)

[wt of dry dtsk + cells (g) - wt of dry disk (g)/ sample vol (mL)] x 1 000

(2)

3.2. Alkaloid Extraction and Analysis I . Samples of between 50 and 1 00 mg dry weight are required for alkalmd extrac­ tiOn Thus, the sample w1ll need to be of 0 5-1 .0 g wet wt. For a fully grown culture, this w11l represent a volume of 5-20 mL culture (wet weights are 1 00-200 g/L) (see Note 4) 2 . The samples are filtered using a 9-cm Hartley funnel fitted w1th a M1racloth fil­ ter The cells are washed w1th I 00 mL d1sttlled water, placed m a small plastic bag, and rap1dly frozen usmg hqUtd mtrogen (see Note 5) The frozen cells can be stored at -20° C, but are normally freeze-dned. Freeze-drymg appears to diS­ rupt the cell membrane and allows extraction of the alkalmds without usmg any other form of cell d1sruptton (see Note 6) 3 Freeze-dried cells (0. 05--0 I ) are p laced m the extraction thimble and plugged w1th nonadsorbent cotton wool The actual we1ght to be extracted can be deter­ mmed by wetghmg the th1mble before and after addmg the cells.

399

Alkaloid Accumulation

CONDENSER

SAMPLE IN TIDMBLE

SYPHON

SOLVENT

Ftg. 3. A Soxhlet apparatus used to extract alkalotds from dried cells. The flask at the bottom contains 1 00 mL methanol, which is immersed in a water bath at 85°C The methanol that botls off ts condensed at the top, and the warm methanol bathes the sample m the thimble. Once the thimble umt is filled, the syphon drams the chamber and the process ts repeated. 4. The thimble is placed in a small Soxhlet apparatus (Fig. 3 ) and refluxed with 1 00 mL methanol for 2 h at 85°C, The extract (in the round-bottomed flask) ts reduced to dryness by rotary evaporatiOn at 60°C under vacuum.

400 5

6.

7.

8.

9

10 1 1. 12. 13 14. 15. 16

Scragg The restdue ts resuspended m 1 mL methanol usmg a 1 -mm exposure m a somca­ tor bath to help resuspens10n. Two further 1 -mL methanol washes are used, and these are combmed and stored m a sealed bottle at 4°C. Alkalotd accumulation m the medium can be estimated by collectmg 1 00 mL medium after filtration and extractmg this with 50 mL chloroform. A more con­ venient method mvolves solid--liqmd separation usmg a SEP-PAK C 1 8 cartndge. The cartridges are preactivated with 4 mL of acetonitrile, followed by 4 mL of 95% methanol The medium sample ts centrifuged ( 1 O,OOOg for 1 0 min) or fil­ tered to remove any cell debris, and 20-- 1 00 mL apphed to the column. The alka­ loids are eluted with 4 mL methanol and can be then identified by HPLC, and so forth For tdenttfication of the alkaloids in a routine system, HPLC ts preferred, and t f mformation o n all alkaloids I S reqmred, UV detectiOn at two wavelengths will give Indications of peak punty A diOde array detection system will often resolve any purity problems. For the identificatiOn of aj mahcme and serpentme, the followmg HPLC system can be used. The column ts a 0 8 x 1 0 em Jll1l Bondpak C 1 8 reverse phase, run isocrattcally at 55:45 : 5 methanol·watern-heptane sulfonate (nHS) for 14 mm followed by linear gradient to 70:30:5 methanol: water·nHS The sample volume was 1 0--3 0 �. with a flow rate of 1 5-2 0 mL!min, and the wavelengths of 2 54 and 280 nm are used for detection. The standard should be run every 6-1 0 runs, and the retention times updated, smce these will vary owmg to changes m run condtttons, such as temperature. Although d10de array detectiOn can give details of peak punty, a second method should be used to confirm the Identity of a peak. Th is has been achieved b y TLC (16, 1 7), MS, or NMR (12) . There are a number of methods that can be used for the analysts and detectiOn of alkalotds by TLC, and the followmg are smtable for serpentine and aJmalicme Solvent system 1 . chloroform, acetone, diethylamme 5 :4: 1 Solvent system 2: ethylacetate, absolute alcohol, NH40H 3· 1 . 1 The plates are run m these solvents m normal TLC tanks for 45-50 mm and dned at 60°C. The dried plates can be exammed using the followmg techmques. Under UV hght at 2 54 nm, the alkalotds aJmalicme and serpentme fluoresce so that their Rr values can be compared with standards. At a wavelength of 366 nm, the alkaloids will quench and thus form dark spots When sprayed wtth 10doplatinate, serpentine wtll give a purple color and the other alkaloids a red-purple or pink color. When sprayed With cenc ammomum sulfate (CAS), ajmahcme gtves a gray color and serpentine has no reaction

4 . Notes 1 . The cell line C. roseus MCR 1 7 (9) was maintained on Gamborgs B5 medmm (18) containmg 1 .0 mg/L 2,4-D and 20 g/L sucrose The pH was adjusted to 5 5 before sterilization The cultures were subcultured every week by adding 20 mL

Alkaloid Accumulation

2.

3.

4.

5

6

401

culture to 1 80 mL fresh medium in a 500-mL flask. The culture was mcubated at 28°C under constant illuminatiOn and shaken at 1 5 0 rpm The cell I me of C. roseus B I X was tmtiated from seed (1 1) and was maintained on L&S mediUm (10), containing 2.0 mg/L NAA, 0.2 mg/L kmetm, and 30 g/L glucose. The pH was adjusted to 5.8 prior to autoclaving. The cultures were sub­ cultured every 2 wk by adding 35-1 65 mL fresh medium in a I 000-mL flask The cultures were incubated at 25°C in the dark and shaken at 1 00 rpm Plant cell suspensions are generally aggregated, and thus settle rapidly and clog narrow pipet ttps. A convenient method of avoiding problems wtth these aggre­ gated cultures is to use a 5-mL automatic pipet fitted wtth ttp, which has had the end 5 mm removed to give a very wtde bore. These tips can be sterilized by autoc1aving in beakers sealed with alummum foil. One to 3 samples can be removed from a single 120-mL culture in a 250-mL shake flask before the reductton in volume significantly alters condtttons m the flask. However, if alkalotd accumulatiOn is to be followed early m the culture when the cell density is low and duplicate samples are needed for accuracy, large­ volume samples are needed (50--100 mL). Therefore, a large number of flasks will need to be initiated from a single inoculum, and the contents of a whole flask used m the early stages. The number of flasks used will depend on the frequency of sampling and the rate of growth of the culture The use of small plasttc bags allows a large number of samples to be freeze-dned at one time using a large metal container and avoids the use of glass flasks. It is difficult to extract all the sample from a glass flask Most of the alkaloids can be exacted from plant matenal with orgamc solvents at alkaline pH. Thus, dtchloromethane (19), ethyl acetate (20), and methanol have been used to extract alkalmds. The crude extract can be cleaned up and concen­ trated by using solid-phase extractwn. In this case, the extract ts passed through a cartridge of C 1 8 reversed-phase ion-exchange groups as described for the analy­ sis of the medtum.

References I . Cox, P. A. and Balick, M. J. ( 1 994) The ethnobotamcal approach to drug discov­ ery. Sci. Am. June, 60--65 . 2 Verpoorte, R., Van der Heij den, R., and Schrtpesma, J ( 1 993) Plant cell technol­ ogy for the production of alkaloids: present status and prospects. J Nat Prod. 56, 1 86--207. 3. Balandrin, M. F. and Klocke, J. A. ( 1 988) Medicinal, aromatic and industnal materials from plants, in Bwtechnology 1n Agriculture and Forestry, 4, Medicmal and Aromatic Plants I ( Bajaj, Y. P. S., ed.), Springer-Verlag, Berlin, pp 3-36. 4. Zhong, J.-J. ( 1 995) Recent advances in cell cultures of Taxus spp. for production of the natural anttcancer drug taxol. Plant Tissue Biotechnol 1, 75-80. 5 . Van der Hijden, R., Verpoorte, R , and Ten Hoopen, H. J G ( 1 989) Cell and tissue cultures of Catharanthus roseus (L) G.Don. a hterature survey. Plant Cell T1ss Organ Cult 18, 23 1-280.

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6 Moreno, P. R. H , Van der Htjden, R , and Verpoorte, R ( 1 995) Cell and tissue culture of Catharanthus roseus· a literature survey II updatmg from 1 988 to 1 993. Plant Cell Tzss Organ Cult 42, 1-25 7. Zenk, M. H., El-Shagi, H., Arens, H., Stocktgt, J , Wetler, E W., and Deus, B. ( 1 977) FormatiOn of the indole alkaloids serpentine and ajmahcme m cel l suspen­ sion cultures of Catharanthus roseus, m Plant Tzssue Cultures and Its Bwtechno­ logzcal Applzcatwn (Barz, W , Remhard, E., and Zenk, M H , eds ), Spnnger Verlag, Berhn, pp. 27-44 8 Knobloch, K. H., Hansen, B , and Berhn, J. ( 1 98 1 ) Medmm-mduced formation of mdole alkaloids and concomttant changes of interrelated enzyme activtttes in cell suspension cultures of Catharanthus roseus Z. Naturforsch 36c, 40--43 . 9 Tom, R , Jardm, B., Chavarie, C , and Archambault, J ( 1 99 1 ) Effect of culture process on alkaloid production by Catharanthus roseus J Bwtechnol 21, 1-20 1 0 L msmai er , E. M and Skoog, F. ( 1965) Orgamc growth factor requuements of tobacco tissue cultures Physzol. Plant 1 8, 1 00-1 27. 1 1 . Moreno, P. R H , Schlatmann, J E , Van der Hetjden, R., Van Guhk, W M , Ten Hoopen, H. J. G , Verpoorte, R., et a! ( 1 993) Induction of aJmahcme formation and related enzyme actJvies m Catharanthus roseus cells· effect ofmcoulum den­ sity Appl Mzcrobwl Bwtechnol 39, 42-47 12 Moms, P. ( 1 985) RegulatiOn of product synthests m cell cultures of Catharanthus roseus II Companson of production media. Planta Medica 52, 1 2 1-1 26 1 3 Scragg, A. H (1 993) The problems associated wtth htgh biomass levels m plant cell suspenswns. Plant Cell Tzss Org. Cult 43, 1 63-1 70. 14 Cresswell, R C ( 1 986) Selectwn studtes on Catharanthus roseus, m Secondary Metabolzsm zn Plant Cell Cultures (Moms, P., Scragg, A H., Stafford, A., and Fowler, M. W , eds.), Cambridge Universtty Press, Cambndge, pp. 23 1-236 1 5. Murashige, T. and Skoog, F ( 1 962) A revtsed medium for rapid growth and bw­ assays wtth tobacco tissue cultures. Physwl. Plant 15, 473-497 1 6 Baerhetm Svendsen, A. and Verpoorte, R. ( 1 983) Chromatography of alkalotds, part A. thin layer chromataography J Chromatogr Library 23a, Elsevter, Amsterdam. 1 7. Verpoorte, R. and Baerheim Svendsen, A. ( 1 984) Chromatography of alkalOids, part B· gas-Iiqmd chromatagraphy and high-performance hquid chromatography J. Chromatogr Ltbrary 23b, Elsevier, Amsterdam. 1 8 Gamborg, 0. L., Mtller, R A., and Oj tma, K. ( 1 968) Nutntional reqUirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 1 5 1-1 58. 1 9. Dos Santos, R. I., Scnpsema, J., and Verpoorte, R. ( 1 994) AJ mahcme metabohsm in Catharanthus roseus cell cultures. Phytochemzstry 35, 677---()8 1 20 Facchini, P. J. and DtCosmo, F. ( 1 99 1 ) Secondary metabolite biosynthesis m cul­ tured cells of Catharanthus roseus (L) G Don tmmobthzed by adheswn to glass fibres. Appl Microbwl. Bwtechnol 35, 3 82-392.

37

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Betalains Their Accumulation and Release In Vitro

Christopher S. Hunter and Nigel J. Kilby

1 . Introduction Since the mid- 1 980s the diverse group of compounds known as plant sec­ ondary metabolites, or secondary products, have assumed even greater s igmfi­ cance than previOusly as compounds of commercial (e.g., taxol, rosmarinic acid) and soctal (e.g., "natural" fragrances and flavors) sigmficance. The com­ mercial production of these secondary metabolites remamed field-based until the production of shikonin from plant cells in vitro (1). With few exceptiOns, the main world supply for most plant secondary metabolites remains the har­ vesting of field-grown plants followed by the extractiOn and/or direct utiliza­ tion of the active prmciple(s). The proportion of the plant that constitutes the compound of mterest is mvariably only a few percent of the total plant dry weight. For example, the maximum quinme content of Cmchona bark IS 6-1 4%, and the bark IS only a small portion of the entire tree (2) . Some plants produc­ ing secondary metabolites are annuals or biennials, but many require long­ term field-growth (Cinchona, 7-1 6 yr, Lzthospermum erythrorhizon, 5-7 yr) before economic harvest. Field-grown crops are also subject to the vaganes of weather, pests, diseases, and nutrient availability. These, w1th other biotic and abiotic factors, continue to stimulate evaluation of alternative productwn sys­ tems for these compounds. Recent developments in gene manipulation have given nse to transgemc organisms able to synthesize secondary products characteristic of the transgemc parent (3). Although ethical and safety concerns dominate the statutory licens­ ing and commercial development of such transgenic products, it is unlikely that their productwn in VIVO will be superseded. Whether or not m vitro From Methods m Molecular Biology, Vol 1 1 1 Plant Cell Culture Protocols Ed1ted by R D Hall © Humana Press Inc , Totowa, NJ

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Hunter and Kilby

production from nontransformed cell lines will be subject to comparable legis­ lation remains unresolved, but is potentially less controversial The m vitro productiOn systems mvestigated mclude Agrobactenum-trans­ forrned "hairy roots" (4) , the growth of free cells and aggregates (5, 6) sus­ pended m stirred-tank chemostats and turbtdostats, m atr-lift btoreactors, m vanous tmmobthzed systems, and as calluses, both m flmdtzed-bed btoreactors (7) and on agar-based media in flasks. To enhance product yield, medta have often been espectally developed. Dependmg on whether or not the product yield is substantially proportional to the biomass or whether the product is produced pnncipally dunng statiOnary phase (that IS, the period after whtch culture growth has essentially stopped), media may be designed to maximize cell growth or reduce potential cell growth, but enhance product y1eld For shikonm productiOn m Japan (MitsUI Petroleum Co.), cells were grown is stirred tanks First, growth of mostly colorless cells was maximized for 9 d in medmm MG-5. Then, cells were transferred to a medmm (M9) for a further 14 d durmg which the red napthoqumone compound, shikonin, was produced. The harvest ofboth shikonm, taxol/taxane and most other products of large­ scale culture of plant cells depends on the destructive extraction of the biom­ ass. With a cell doubling time rangmg between 1 6 h and several days, the productiOn of cells and secondary products IS an expensive process (8) . To produce secondary metabolites m a commercially sound way, It has been van­ ously estimated that the compound of mterest must have an end-user value >(US)$7500/kg. This figure IS J UStified on the basts of the provision of aseptic culture conditions for several weeks, the (generally) low percentage yteld of the secondary metabolite, and the costs of laboratory "overheads" and down­ stream processmg. The long-term apphcabihty of production m vitro thus depends on either product demand that can sustam the high price of the product on the market or the reductiOn of costs m production. ProductiOn costs can be reduced by a range of optiOns, for example, mcreasmg yields through the addi­ tiOn of elicitors (9), selecting higher-yieldmg clones, continumg to develop media that stimulate metabolite production (10), or permeabiltzmg the cells (II) to enable harvest of the secondary metabohte m a nondestructive way, thereby allowing the cells to produce second and subsequent metabolite crops (12, 13) . A range o f expenmental techmques has been applied to cells to release the metabolites of interest. Permeabilizatwn has three critical requirements: I Mamtenance of asepsts 2 Survival of a sufficient cell populatiOn to grow and produce subsequent crops 3 Reversibility Perrneabtl izatwn usmg dtmethyl sulfoxide, glycerol, medta of low pH value, and other treatments has been ofhmtted value only, pnnctpally because there has been either li mited release of the metabolite, or cell death/lysis has occurred

Betalams

405

Permeabilizatton by either ultrasonication or electropermeabihzatwn offers a robust system that satisfies the three critical requirements. Both techniques are amenable to small-scale operatiOn m the laboratory, and ultrasonication has potential for use m pilot or large-scale production. This chapter provides detatls of a laboratory ultrasomc technique. The ultrasomc process subJects cells to somcatton for relattvely short ttme periods; thts ultrasonication is followed by short-term incubation of the cells, durmg whtch there is the gradual release of a proportiOn ofthe vacuole-located secondary metabolite into the medmm during approx 30 mm postsonicatton. 1. 1. Red Beetroot: A Model System Although of low financtal value, the red pigment from Beta vulgaris (red beetroot) ts a commercmlly Important food colorant (E 1 62) used m such prod­ ucts as salamt and soups. Betalam-producing B. vulgaris has been used as a "model" system for studies on the production of secondary products in vitro Its particular merit lies m the obvtous color and the consequent stmpltctty of colonmetnc quantification, m companson wtth colorless compounds that are usually assayed by gas chromatography or HPLC. The prmctpal ptgments m beetroot are two red betacyanins (betamn and prebetanm) and two yellow betaxanthins (vulgaxanthin I and ll) (14, 15). In most of the recent studtes on pigment productiOn by B. vulgaris in vitro, attentiOn has focused on cultures selected for theu ability to produce the dominant red pigment betanin (16). The beetroot system has been used m hairy root (1 7), immobilization (18), and permeabilization (19) mvesttgations. The following protocol enables: •

•

•

•

Axenic beetroot cultures to be mitiated. Callus to be grown on agar-based media Suspension cultures to be initiated and subcultured Pigment to be extracted usmg both destructive (whole-cell disruptton) and non­ destructive (ultrasomcatton) methods.

2. Materials I . B. vulgaris cv. Boltardy. Seed t s widely available from seed merchants or dtrect from Suttons Seeds pic, Torquay, Devon, UK. Surface-stenlants. 70% (v/v) etha­ nol, sodium hypochlorite { I or 2% available chlonne), sterile distilled water. 2. Tissue-culture media: All medta are based on that of Gamborg 's BS (20; and see Appendix). The basic medium without plant growth regulators, sucrose or agar can be purchased from Stgma, Poole, UK. 3. Suspension culture (S l ) medium: 3 . 1 9 g Gamborg' s BS medium, 20 g sucrose, 0 I mg kinetin (6-furfurylaminopurine), 0.02 mg 2,4-dichlorophenoxyactetic acid (2,4-D) (both hormones are taken from I mg/mL stocks dtssolved in dilute KOH).

Hunter and Kilby

406

Styrene Jar

SonrcatJon medJum Cells for sonication Couplmg gel

Transducer

Fig 1 . Ultrasound transducer attached to the styrene Jar pnor to somcat1on.

4 5 6.

7.

8.

9.

10

Medium 1s made up to 1 L with single dtstilled water and then the pH adJusted to 5.5 prior to autoclaving at 1 06 kPa at 1 2 1 oc for 1 5 mm. Agar-based medium (A2) for callus growth: as for S 1 , except that 6 g/L agar are added prior to pH adjustment. Callus induction medmm (A I ): as for A2, except that the 2,4-D concentratiOn ts 1 mg/L. Seed germmatwn medium (A3) . as for A2, except that tt contains neither kinetm nor 2,4-D. Erlenmeyer flasks ( 1 00 mL and/or 250 mL), w1th the necks closed by a double layer of alummum fml and oven-sterilized (dry) at l 60°C for 90 min, are used for seed germmation, callus m1tiatwn, growth, and suspenswn cultures. Stenle sty­ rene screw-capped J ars (60 mL) (Sterilin, UK, type l 25AP) are used to contain the cells and medmm dunng the sonication process The ultrasound is produced from an Impulsaphon ultrasound therapy unit, type M55 w1th a 3 5-cm plane dtsk ceram1c transducer (Ultrashall-Gerateban, 2 Kronberg, Germany) The power output of the transducer 1s 3 W/cm The fre­ quency of the ultrasound is \ .02 MHz The styrene Jar ts connected to the trans­ ducer by a few drops of a sound-conducting couphng gel (Electromed1cal Supplies, Wantage, UK) (see Fig. 1 ) General equ1pment. wide-bore pipets, glass mtcrofiber filter paper (type GF/C, Whatman, Maidstone, UK), spectrophotometer. Culture mcubation. all cultures are grown at 25°C m 1 6-h day length i n wh1te fluorescent light (30 wm-2, but the mtensity and spectral conditions are not cnttcally Important) . Suspension cultures are mcubated on an orbttal shaker at 1 00 rpm.

Betalains

407

3. Methods 3. 1. Callus Initiation Callus may be mttiated from a range of sources-from seed, from petioles, or from flowering stems.

3. 1 . 1 . From Seed Soak seeds in ethanol for 30 s. Then transfer them to a stenle flask contaming an aqueous solution of sodium hypochlorite (2% available chlorine) plus 1-2 drops of Tween 20 (see Note 1). 2. Wash the seeds (4x) in sterile distilled water (SOW), and transfer them asepti­ cally to the surface of A3 medmm, use 5 seeds/250 mL flask (see Note 2 ) 3 . When the seeds have germinated and seedlings grown t o produce the second true leaf, aseptically remove mdividual seedlings, dissect petiOles and hypo­ cotyls mto sections approx 1 5 mm long, and implant onto the surface of A 1 medium. 4. Withm 2 1 d, callus will have tmtiated and should grow into structures several mtlltmeters m dtameter. Exctse these calluses, and transfer them to A2 medmm.

3. 1.2. From the Flowering Stems and Petioles of Mature Plants Remove the flowering stem and/or the petioles, trim off any leaf lamma or flow­ ers, and then surface-stenltze them in sodium hypochlorite ( I % available chlo­ nne + 1 drop Tween 20) for 20 min 2 Wash the stems (4 x) in SOW, and then cut aseptically mto 20-mm lengths 3. Bisect each 20-mm length longitudinally and place, cut sides down, onto A I medium. 4. Incubate the flasks: within 21 d, callus should have grown and should be treated as described above for the seed-denved callus and subcultured to A2 medium.

3.2. Callus and Suspension Culture When the calluses have grown to an approximate diameter of20 mm, they should be subcultured by transfer wtth a sterile loop to fresh A2 medium The quantity transferred at subculture IS not critical, but a ptece 5-1 0 mm in diameter IS rec­ ommended A fast-growmg callus will require subculture at 3 -wk intervals. 2 Transfer approx 5 g of callus to 50 mL of S 1 medium, and incubate on the orbital shaker. 3. Subculture the suspensiOn at 7-d intervals to fresh S l medmm (20% v/v mocu­ lum volume). Incubate on the orbital shaker at 1 00 rpm.

3.3. Pigment Production Maximization and Analysis Most calluses and suspension cultures of beetroot contam a mixture of cells that can visually be classified as "colorless, pink, or red." For the production of betanin, It is clear that at least in the short term, one must select for "red" cells.

408

Hunter and Kilby - zero son1cat1on - 10 -

sec son1cn

20 sec son1cn

- 30 sec son1cn _,....

40 sec son1cn SOsec son1cn

- 60 sec somcn

2

4

5

6

7

Post-sonication incubation period (mmutes)

8

F1g 2 Effect of duration of somcation pigment release

To select for red cells, prepare several flasks of A2 medium, and onto the surface of each, pour approx 2 mL of suspensiOn culture, sw1rl the suspensiOn over the A2 surface to distnbute the cells (smgle cells and aggregates), and incubate the flasks. 2. In each flask, many calluses w1ll develop that can be assessed VIsually for the1r color, selected and subcultured to fresh A2 and later, after sufficient growth, to S l medmm for suspensiOn culture It 1s our expenence that selected cell hnes will reqmre reselection after many subcultures. Also, we have found that 1t 1s prudent to have a stock of "backup" callus cultures in reserve to overcome the seemingly mev1table problems of a shaker power-supply failure or other such disaster! 3. To quantify the relative amount of betanin in a cell extract (see Note 3) or the medium surrounding cells postsonicatwn, take a standard volume/extract dilu­ twn, filter 1t through glass microfiber paper, check and adJust the pH to 5.5, and measure the absorbance of the filtrate at 537 nm (betanm "-max). W1thin the absor­ bance range 0-1 0, Beer's Law 1s observed Thus, the quantity of betamn IS pro­ portwnal to the absorbance.

3.4. Ultrasonic Release of Pigments (see Fig. 2) I . The suspensiOn culture is removed from the shaker, and the cells allowed to settle. Usmg a 1 0-mL w1de-bore p1pet, mod1fied by cuttmg off and d1scardmg the tapered end, remove 4 mL of settled cells. Dispense the cells to the styrene Jar, add 35 mL S I medium, screw on the plastic lid, and place the styrene jar onto the transducer, usmg the couplmg gel between the Jar and the transducer 2 Tum on the ultrasonicator for the desired period (see Notes 4, 5, and 6). After somcatwn, transfer the entue contents of the J ar to a 250-mL Erlenmeyer flask containing 60 mL fresh S I medmm, and incubate on the orbital shaker. During the next 30-40 min the pigments will diffuse through the permeab!lized mem­ branes into the incubatmg medium: thereafter, no further p1gment efflux 1s likely

Betalains

409

Remove samples for spectrophotometnc analys1s If 1t IS des1red to contmue to grow the cells for a further "crop" of pigment, ensure that all handlmg has been asept1c and that the cells are incubated with the appropnate volume of S 1 medium. 4. Notes

2. 3.

4. 5.

6

The preparation of aseptic cuitures from seed or from in vivo plants 1s the most difficult part of the entire protocol. The seed is megular and rough-textured, often the plants in v1vo appear to be contaminated with endophytic microorgan­ I sms. If external contammation IS a problem, mcrease the NaOCl concentratiOn for seed up to 5% available chlorine: endophytes will not be killed by surface sterilizatiOn, so d1 fferent plant sources of beetroot should be used. Normal aseptic techniques must be adopted for work with the cultures. To harvest destructively all the pigments from a sample of cells, homogenize the cells in S l medium at ca. 4°C, centrifuge the supernatant from the cells, retam the supernatant pigmented medium, and re-extract the cells in S 1 medmm Repeat the process until no further pigment is extracted. Pool the supernatant solutions, adjust to pH 5.5, measure absorbance at 537 nm for betanin, or scan the extract between 350 and 700 nm to plot the absorptiOn spectrum of the combmed p1gments. Investigate a range of times of ultrasomc treatment times from I 0 s to 5 min. To observe the effect of dissolved gasses in the S 1 medium on the ultrasomc release of pigments, try degassing the medium by bubbling helium mto the medmm for 5 min pnor to sonicatiOn� Also try gassing the medium by bubbling laboratory air into the S 1 medium prior to sonicatiOn In both cases, a suitable air filter should be used to maintain the stenhty of the cultures To observe the effect of temperature on the pigment efflux, cool or heat the me­ dmm (range 4-42°C) pnor to sonication and dunng the postsomcat10n mcuba­ tion penod.

References l . Fuj ita, Y., Tabata, M., Nish1, A., and Yamada, Y. ( 1 982) New medium and pro­ duction of secondary compounds with the two-staged culture method, in Proc 5th Inti Cong. Plant Tzssue and Cell Culture (Fujiwara, A., ed ), Maruzen Co , Tokyo, pp. 399---4 00. 2 McHale, D. ( 1 986) The cinchona tree Bwlog1st 33(1), 45-53. 3 . Taya, M , Mine, K., Kmo-Ora, M., Tone, S., and Ich1, T. ( 1 992) ProductiOn and release ofpigments by culture of transformed hairy root of red beet J Fermenta­ tiOn Bweng 73(1), 3 1-36 4. Flores, H E. and Medina-Bolivar, F. ( 1 995) Root cultures and plant natural prod­ ucts · "unearthing" the hidden half of plant metabolism. Plant Tissue Cult. Bzotechnol 1(2), 59-74. 5. Scragg, A. H. ( 1 995) The problems associated with h1gh biomass levels m plant cell suspensiOns Plant Cell Tiss Org Cult 43, 1 63-1 70 . 6 . Alfermann, A W . and Peterson, M . ( 1 995) Natural product formation b y plant cell biotechnology. Plant Cell TISS Org Cult 43, 1 99-205.

410

Hunter and Kilby

7. Khlebnikov, A , Dubms, B., Kut, 0. M., and Prenosil, J. E ( 1 995) Growth and productivity of Beta vulgarzs cell culture m flmdized bed reactors Bwprocess Eng 14, 5 1-56. 8. Ten Hoopen, H. J G., van Gulik, W M., Me1jer, J. J , and Verpoorte, R ( 1994) Econom1c feas1bihty of industrial plant cell biotechnology: the effect of various process options, in Plant Cell, Tissue and Organ Cultures m L1qu1d Medza (Macek, T. and Vanek, T., eds ), Abstracts ofsymposmm, July 8-1 1 , Prague, CZ. pp. 22-30 9. Sankawa, U., Hakamatsuka, T., Shmkai, K., Yosh1da, M., Park, H-H , and Ebizuka, Y. ( 1 995) Changes of secondary metabolism by ehcitor treatment m Puerarza lobatqa cell cultures, in Current Issues m Plant Molecular and Cellular Bwlogy (Terzi, M., Cella, R., and Falavigna, A , eds ), Kluwer Academ1c, Dordrecht, NL, pp. 595--604. 1 0. Taya, M , Yakura, K., Kmo-Oka, M , and Tone, S ( 1 994) Influence of medium constituents on enhancement of pigment production by batch culture of red beet hairy roots. J Fermentatwn Bweng 77, 2 1 5-2 1 7 . I I D1Iono, A A., Weathers, P. J., and Cheetham, R. D ( 1 993) Non-lethal secondary product release from transformed root cultures of Beta vulgarzs Appl. Mzcrobiol Bwtechnol 39, 1 74- 1 80 1 2. Ktlby, N. J. and Hunter, C S. ( 1 990) Towards opt1m1sation of the use of 1 .02-MHz ultrasound to harvest vacuole-located secondary product from m VItro grown plant cells Appl Mzcrobwl Bwtechnol 34, 478-480. 1 3 Kilby, N. J. and Hunter, C . S . ( 1 990) Repeated harvest of vacuole-located second­ ary product from zn vitro grown plant cells using 1 .02 MHz ultrasound. Appl M1crob1ol B1otechnol 33, 448-45 1 . 1 4. Leathers, R. R., Davm, C , and Zryd, J. P. ( 1 992) Betalam producmg cell cultures of Beta vulgarzs L var. Biokores Monogerm (red beet). In Vztro Cell Dev. Bzol 28P, 39-45 1 5 . Bohm, H. and Rink, E. ( 1 988) Betalams m, Cell Culture and Somat1c Cell Genet­ ICS ofPlants, vol. 5 (Vas1l, I. K , ed.), Academic, pp 449--463 . 1 6 K1lby, N J. ( 1 987) An mvestlgatwn of metabohte release from plant cells in v1tro to the1r surroundmg medium PhD thes1s, Bristol Polytechmc, UK 1 7. Hamill, J. D., Parr, A. J., Robms, R. J., and Rhodes, M. J C. ( 1 986) Secondary product formatiOn by cultures of Beta vulgaris and Nzcot1ana rust1ca transformed with Agrobacterium rhizogenes Plant Cell Rep 5, 1 1 1-1 1 4. 1 8 Rhodes, M. J C., Smith, J. I , and Robms, R. J. ( 1 987) Factors affectmg the unmo­ b!llzatwn of plant cells on reticulated polyurethane foam part1cles. Appl Mzcrobiol Biotechnol 26, 28-35 1 9. Kilby, N. J. and Hunter, C. S. ( 1 986) Ultrasomc st1mulat10n of betamn release from Beta vulgans cells m vttro. a non-thennal, cavJtatwn-medJated effect, m Abstracts of VI International Congress ofPlant Cell and Tissue Culture (Somers, D A., Gengenback, B. G., Biesboer, D D., Hackett, W. P , and Green, C. E., eds.), Umvers1ty of Minnesota, MN, p. 352. 20. Gamborg, 0. ( 1 970) The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physzol 45, 372-3 75

APPENDIX

_ _ _ _ _ _ _ _ _ _ _ _ _

Widely Used Plant Cel l Cu ltu re Med ia The following represent the most Widely used media for plant cell culture. Recipes for additional media used for specific applications are to be found in the relevant chapters and are hsted in the mdex.

Macroelements (mg/L) CaCI2 KH2P04 KN03 MgS04 NH4N03 Microe1ements (mg/L) CoCI2 · 6H20 CuS04 · 5H20 FeNaEDTN H 3B03 KI MnS04 ·H20 Na2Mo04·2H20 ZnS04 · 7H20 Organics (mg/L) Glycine Myo-inos1to1 Nicotmic acid Pyndoxine-HCI Thiamine-HC1

Murashige and Skoog complete medium (1)

Murashige and Skoog plant salt m1xture (1)

332.020a 1 70.000 1 900.000 1 80.5406 1 650 000

3 3 2 020a 1 70.000 1 900.000 1 80.5406 1 650 000

332 02oa 1 70 000 1 900 000 1 80 5406 1 650 000

0.025 0.025 36.700 6.200 0.830 1 6.900 0.250 8.600

0 025 0.025 36.700 6.200 0 830 1 6.900 0.250 8.600

0.025 0.025 36.700 6.200 0 830 1 6 900 0.250 8.600

2.000 1 00.000 0.500 0.500 0. 1 00

Linsmaier and Skoog medtum (2)

1 00 000

0.400

0440 mg/L CaCI2·2H20

h370 mg/L MgS04·7H20

congmal rec1pe uses

5 mLIL of a stock' 5.57 gFeS04·7H20, 7.45 g Na2EDTA dissolved m

From Methods m Molecular B1ology, Vol 1 1 1 Plant Cell Culture Protocols Edited by A 0 Hall © Humana Press Inc • Totowa, NJ

41 1

I L

Hall

412 Gamborg's B 5 medmm (3) Macroelements (mg/L) CaCI2 KH2P04 KN03 MgS04 NH4N03 NaH2P04 (NH4)2 S04 (NH4)H2P04 Mtcroelements (mg/L) CoC\2 6H20 CuS04 5H20 FeNaEDTA H3B03 KI MnS04 H20 Na2Mo04 2H20 ZnS04 7H20 Organics (mg/L) B wtin Fohc acid Glycme Myo-mositol Nicotmtc acid Pyndoxme-HCI Thtamine-HCI

Schenk and Htldebrandt medmm (4)

1 1 3 .230a

1 5 1 ooob

2500.000 1 2 1 .560d

2500 000 1 95.050e

Nitsch's medmm (5)

\ 66 oooc 68 000 950 000 90.271 720.000

1 30.440 1 34 000 300 000 0 025 0.025 36 700 3 000 0.750 10 000 0.250 2 000

1 00.000 1 .000 1 .000 1 0 .000

0 010 0 020 1 9 800 5 000 1 .000 1 0 000 0. 1 00 1 000

1 000 000 5.000 0 500 5.000

0 025 36 700 1 0 000 1 8 940 0.250 1 0 000 0 050 0.500 2.000 1 00.000 5 000 0 500 0.500

0 1 50 mg/L CaCI2 2H20 h200 mg/L CaCI2 2 H20 '220 mg/L CaCI2 2H20 d250 mg/L MgS04 7H20 e400 mg/L MgS04 ?H20 .

fl 85 mg/L MgS04 7H20.

References Murashige, T. and Skoog, F . ( 1 962) A revised medmm for raptd growth and b!O­ assays with tobacco ttssue cultures. Physwl Plant 15, 473-479. 2. Lmsmaier, E. M. and Skoog, F. ( 1 965) Orgamc growth factor reqmrements of tobacco ttssue cultures. Physwl Plant 18, 1 00-1 27 3 Gamborg, 0. L , Miller, R A , and Ojima, K. ( 1 968) Nutnent reqmrements of suspenston cultures of soybean root cells Exp. Cell. Res 50, 1 5 1-1 57. l.

Appendix

413

4 . Schenk, R. U . and Hildebrandt, A. C ( 1 972) Medmm and techmques for mductwn and growth of monocotyledonous and dicotyledonous plant cell cultures Can J Bot 50, 1 99-2 04. 5 Nitsch, J. P. and Nitsch, C . ( 1 969) Haploid plants from pollen Sc1ence 163, 85-87

Index A Abscistc acid, 96, 243 Activated charcoal, 9 1 , 1 09, 1 33, 1 50, 1 54, 1 60 Agarose embedding, for protoplast culture, 1 79, 203, 220, 22 1 , 266, 344, 363 Agrobacterium, hatry roots m Beta, 404 transformation of Petunia, 3 27-334 Albino regenerants, 1 83 Alkalmds, 393--402 analysis, 398--400 Amiprophos-methyl (APM), 229 Ampicillm, 2 0 1 Anther culture, Hordeum, 269-279 Anthraquinones, 383-3 9 1 analysis, 3 88, 3 89 Antlbwtics, 8, 1 70, 20 1 , 229,230, 233, 3 2 1 , 322, 328, 344, 350, 3 6 1 Aphtdicohn (APH), 229 APM, see Amtprophos-methyl Asc 1epiadaceae, micropropagation of, 1 3 7, I 38 Auxin, see 2 ,4 D, IBA, IAA, NAA Axillary shoot culture, orchids, 1 3 1-1 33 strawberry, 1 03-1 1 4 -

B

bar gene, 80, 350, 368 Barley, see Hordeum vulgare BAP, see 6-Benzylamino purine Batch culture,

Morinda suspensiOns, 383-3 9 1 6-Benzylamino purme, 38, 65, 96, 1 03, 1 04, 1 06, 1 39, 1 50, 1 72, 1 87, 278, 328 Beta vulgaris, suspensiOns cultures, 403--4 1 0 Beta1ams, 403--4 1 0 B ialaphos, 35 1 Brasszca, protoplasts, 1 95-2 1 0, 2 1 1-226 Bulb scale culture, 143 c

Cactaceae, micropropagation of, 1 3 5-140 Calf thymus camer DNA, 345 Callus culture, Beta vulgarzs, 407 Hordeum vulgare, 274 Nzcotzana, 254, 3 1 2 orchids, 1 28-- 1 3 3 Oryza satzva, 1 9-29 Zea mays, 369, 377, 378 Cassava, see Manmhot esculenta Catharanthus roseus, suspension culture 3 03--402 Cefotaxtme, 1 70, 328 Cell sorting, 203 Ceric ammonmm sulfate stam, 396 Charcoal, see Acttvated charcoal Chlamydomonas, 38 I Chlorocholine chloride, 96 Chromosome countmg, 304 Clover, vtrus ehmination, 1 1 9

4 15

Index

416 CMS, 2 1 1-226 Cond1t10ned medmm, 1 84, 1 87 Continuous cultures, 387, 3 8 8 Controlled rate freezmg, 87, 88 Co-transformation, 345 CPW salts, 1 70 Cybrid production, 2 1 1-226 Cychc somatic embryogenesis, 6 1-70 Cytochalasm-B, 229 Cytokmin, see 6 BAP, kmetm, zeatm Cryopreservatwn, 83-94 Cryoprotection solutiOn, 85 -

D 2 ,4-D, see 2,4-Dichlorophenoxyacetic acid DAPI stammg, 3 3 , 288 Dedi fferentiatwn, 245 DesJccauon of somatic embryos, 66-68 DICamba, 67 2,4-DJchlorophenoxyacetJc ac1d, 65, 73 , 1 8 7, 2 1 7, 3 5 0, 3 8 5 D1rect DNA transfer, cereals v1a bombardment, 76, 77 Nicotlana, v1a electroporation, 359-366 Oryza v1a PEG, 343 Trztzcum v1a bombardment, 349-35 8 Zea mays v i a S I carb1de wh1skers, 375-3 82 Dithwthreitol, 200 DMSO, 84 DNA, analysis of transformants, 33 1 , 332 Isolation, from plants, 204, 205 plasmid preparation, 74, 3 3 9, 360, 36 1 , 378 synthesis mhibitors, 229 Dormancy breaking, bulbs, 1 42 nee grams, 340

OTT, see Dithwthreitol

E Electroporation, of protoplasts, 359-366 of tlssues, 367-373 Embryo culture, 264 for bombardment, 350-354 for electroporatwn, 367-3 73 Embryo rescue, 293-307 Embryogenic hoes, rice, 83-94, 34 1-343 see also somatic embryogenesis Enzyme mixtures, for protoplast IsolatiOn, Brassica, 1 97, 2 1 4, 2 1 5 Lycoperszcon, 229 N1cotiana, 229, 247, 320 Oryza, 339 Passij7ora, 1 70, 1 73 Solanum, 229 Zea gametes, 26 1 Equtpment, for plant cell culture, 3-5 Euphorbiaceae, micropropagatwn, 1 3 5-1 40 F FCM, see Flow Cytometry FDA, see Fluorescem diacetate Feeder cells, see Nurse cultures Fermentor culture, Mormda suspen stons, 383-3 9 1 Ferns, spore culture o f 1 59-1 68 F ertihzation, m vitro via gametic fuswn, 259-267 Ficoll, 272 Flow cytometry, 3 1 , 203 Fluorescein d1acetate, 86, 90, 1 76, 202, 287, 386 Fungicide use, during transplantatiOn, 1 1 3, 1 65

Index G

G-4 1 8, 344 GA3, see Gibberelhc actd Gametic fusion, 259-267 Gelling agents, 8 Genettc modification, of cereals, 7 1-83 of Nzcotwna, 359-366 of Oryza , 3 3 5-347 of Petuma, 327-334 of Tritzcum, 349-358 of Zea mays, 367-373, 375-382 Genetic variatiOn, analysts of, 3 1-42 Germp1asm storage, noncryogemc, 95-1 0 l vta cryopreservation, 83-94 Gibberelltc acid, 38, 96, 1 06, 1 2 1 , 1 74, 2 1 7, 3 0 1 Glycerol, 330 Greenhouse, transfer of regenerants, of cereals, 76 of ferns, 1 64, 1 65 of Lzlium, 1 43 of Manihot, 67 of Oryza, 344 of strawberry, 1 09, 1 1 0 of succulents, 1 39 of Thuja, 1 52 Growth analysts, of suspension cultures, Mormda, 3 88 Catharanthus, 395, 396 Guard cells, 243-257 GUS assay, 74, 35 1-352 Gymnosperms, mteropropagation of, 149-157 H Haploid plants, 285-286 HEPES, 3 6 1 , 368

417 Herbicides, Basta, 350 bromoxyml, 325 tnazine, 2 1 7, 325 metobromuron, 325 phosphinotricin, 344, 3 50 Hordeum vulgare, anther/mtcrospore culture, 269-279 suspenston culture, 43-50 Hybrids, genettc identification of, 204, 222 Hydroxyproline as antimetabolite, 3 1 2 Hydroxy urea, 229 Hygromycin, 230, 344 Hyperflowering in strawberry, 1 04 I

IAA, see Indole acettc actd IBA , see Indole Butyric actd Image analysts, 36-37 Indole acetic actd, 38, 1 72, 2 1 7, 32 1 Indole butync actd, 106 Inflorescence culture, 7 1 Iodoacetamide, for cybrid production, 1 96, 206, 2 1 5, 2 1 9 Iodoplatinate stain, 396 IrradiatiOn, pretreatment, for cybrid productton, 2 1 2-2 1 3, 2 1 9 UV mutagenesis, 3 I 7 K

Kanamycin, 229-230, 328, 344, 3 6 1 Kinetin, 1 87 , 2 1 6 L

LB medmm, 20 1 , 3 28 Lihum, micropropagatton of, 1 4 1-148

Index

418 Lincomycin, 32 1 Lohum, suspension cell protoplast culture, 1 83-1 94 Lycopers1con, protoplast fusion, 227-242 mutagenesiS, 320 M

Maize, see Zea mays Manzhot esculenta, somatlc embryogenesis, 6 1-70 Mature seed extract, 58 Media, for bacteria culture, LB, 201 , 328 for plant cell culture, components and preparatiOn, 5-8 AA, 2 1 , 46, 337 AE, 1 54 AT3 , 283 Gamborg ' s 85, 2 1 7, 385, 405, 4 1 2 Knudson ' s C, 1 65 L 1 /L2/L3 , 4 6 LP, 5 3 Lmsmater & Skoog (LS), 2 1 , 86, 4 1 1 MCM, 1 54 Murashige & Skoog (MS), 4 1 1 McCowans woody plant medmm, 1 54 Moore ' s fern, 1 65 Nitsch, 4 1 2 N6, 3 7 7 R, 2 1 , R l , 336---3 37 R2 , 3 3 8 Quoirm & Lepo1vre, 1 5 1 Schenk & Hildebrandt, 1 54, 4 1 2 TM, 23 1 for protoplast culture, KM8p, 1 7 1- 1 72

K3, 1 98, 3 1 1 for protoplast isolatiOn, CPW salts, 1 70 W5 salts, 230, 320 Meristem culture, Manihot esculenta, 6 1 orchids, 1 27-1 3 5 strawberry, 1 03 , I 0 8 Solanum, 1 1 5-125 MES, 1 70, 2 1 4, 250, 283, 367 Microdroplet culture, 260 Mtcronuclet, induction, 23 3-234 Micropropagatwn, of bulbs, 14 1-148 of cactl, 1 3 5-140 of Lllium, 14 1-148 of Manihot esculenta, 65-67 of Narczssus, 1 4 1-148 of orchids, 1 27-1 34 of Oryza satzva, 20 of Populus, 149-1 57 of Solanum, 3 1---42, 98-99 of strawberry, 1 03-1 1 5 of succulents, 1 3 5-140 of Thuja, 1 49- 1 5 7 Microprotoplast production/fusion, 227-242 Mtcrospore culture, of Hordeum vulgare, 269-279 of Nzcotzana, 28 1-29 1 of Oryza satzva, 341-343 M1crotubers, of potato, 97 Mormda, cell suspensions, 383-3 9 1 Mutagenesis, and antimetabolite res1stance, 309-3 1 8 of plastids, 3 1 9-326 N NAA, see napthalene acetic ac1d

Index Napthalene acetic acid, 38, 65, 1 2 1 , 1 39, 1 72, 328, 3 85 Narcissus, micropropagation of, 1 4 1-148 Nzcotiana, genetic modificatiOn, 359-366 guard cell protoplasts of, 243-257 leaf protoplasts of, 229, 3 1 9-326 microspore culture, 28 1-292 mutagenesis, 3 09--3 1 8, 3 1 9-326 Nitroso-N-ethyl urea (NEU), 3 10 Nitroso-N-methy1 urea (NMU), 3 10, 321 npt II gene, for selectiOn, 223, 3 62 Nuclei extraction buffer, 33 Nurse cultures, 49, 1 84, 264, 330, 3 3 9 0

Orchids, micropropagation of, 127-1 34 Organelle transfer, 2 1 1-226 Oryza sativa, callus initiation and shoot induction, 1 9-29 cryopreservation embryogemc cultures, 83-94 genetic modificatiOn, 33 5-347 Osmotic stressants, 96-97, 1 00 p

Packed Cell Volume, 26, 1 77, 379 Particle bombardment, 76-77, 349--3 58 Passzjlora, protoplast culture, 1 69-1 82 PAT, see Phosphinotrycm acetyl transferase PCV, see Packed Cell Volume PEG, see Polyethylene glycol Permeabihzatwn of suspensiOn cells, 405 Petunia, genetic modification, 327-3 34

419 Phosphinotricin, 344, 368 Phosphinotrycm acetyl transferase assay, 252, 3 70--3 7 1 Phytohormones, as medmm supplement, 7-8 preparatwn of stock solutiOns, 3 8, 65, 1 87 see also ABA, 6-BAP, 2,4-D, GA3 , IAA, IBA, Kinetin, NAA, Picloram, Zeatm Picea, embryogemc suspenswns, 5 1--61 Picloram, 65, 73, 1 72, 173 Pigments, in cell culture, 3 83-3 9 1 , 403--4 1 0 Plastid mutants, 3 1 9-326 Pollen maturatwn, m VItro, 281-292 Pollination, With zn vztro matured pollen, 287 Polyethylene glycol, for protoplast fuswn, 200, 203 -mediated DNA transfer, 3 3 5-347 Polyphenols, productiOn m cultures, 1 24, 1 3 3 , 1 50, 1 53 Polyvinylpyrrohdone, 1 70, 2 1 5 , 247 Potato, see Solanum Protoplast fuswn, see SomatiC hybndization Protoplasts from, Hordeum vulgare suspenswns, 43, 48 Brasszca, 1 95-2 1 0, 2 1 1-226 Lolzum suspenswns, 1 83-1 94 Nicotzana glauca guard cells, 243-257 Nzcotiana tabacum, 3 1 6, 3 1 9-326, 359-366 Oryza suspensiOns, 3 35--3 47 Pass zjlora leaf, 1 69-- 1 82 viabtlity of, 176 PVP, see Polyvinylpyrrohdone

Index

420 R

DNA transfer, 375-3 82 Silver mtrate, 73 Silver thiosulfate, 78 Solanum, callus 1mtlatwn and shoot mductwn, 3 1-42 long-term culture storage, 95-1 0 1 mersJtem tip culture, 1 1 5- 1 2 5 rnlcroprotoplasts, 227-242 VIruS elimmatwn, 1 1 5-125 Somaclonal variatwn, in Passiflora, 1 79 in Solanum, 3 1 -42, 1 1 8 strawberry hyperflowering, 1 04 Somatic embryogenesis, m Hordeum vulgare, 43-50, 269-279 m Manrhot esculenta, 6 1 -70 in Oryza sativa, 1 9-29, 83-94 m Pzcea, 5 1--6 1 in Zea mays, 369-3 70 Sil tcon carbide whJsker-medJated

Rice, see Oryza sativa Rootmg m vitro,

Brasszca, 222 fern gametophytes, 1 64 Hordeum vulgare, 47 Nzcotwna, 254, 3 1 5 orchtds, 1 3 1 , 1 3 2 Oryza satzva, 22, 344 Passiflora, 1 76-1 77 Petuma, 33 1 Populus, 1 52 Solanum, 3 1 -42 strawberry, I 09 succulents, 1 39-140 Thu;a, 1 53 Zea mays, 380

s

SCV, see Settled cell volume Secondary metabolism, in Beta suspensions, 403-4 1 0 in Catharanthus suspensions, 393-402 m Mormda suspensions, 383-3 9 1 SelectiOn agents, for genetic modification,

Basta, 80, 368 b1a1aphos, 74, 3 5 1 , 3 7 8 G4 1 8 (geneticin sulfate), 74, 344 g1ufosinate ammomum, 74 hygromycm, 344 kanamycin, 328, 344, 362 paromomycin sulfate, 74 phosphinotrycm, 344, 368 Settled cell volume, 26 Shikonm, 404 Shoot mductwn, Hordeum vulgare, 43-50 Oryza satzva, 1 9-2 9 Solanum, 3 1-42 Tntlcum aestivum, 7 1-82

Somatic hybnd1zation, for Brasszca,

symmetnc hybnds, 1 9 5-2 1 0 asymmetnc hybnds, 2 1 1-226 m Passiflora, 1 69 of Solanum x Lycoperszcon protoplasts 227-242 of Zea gametes, 259-267 Spectinomycm, 3 22 Spermidme, 74, 794 Spores, ferns, collection/ storage, 1 6 1-1 62 germinatiOn, 1 62-1 64 Stenhzatwn agents, 1 3 Sterihzatwn procedures, 1 0-1 3 Strawberry mtcropropagatwn, 1 03-1 1 5 Streptomycm, 32 1 SuspensiOn cultures, of Beta vulgaris, 403-4 1 0 of Catharanthus, 393-402

Index of Hordeum vulgare, embryogemc, 43-50 of Lolium, embryogenic, 1 83-1 94 of Monnda, 383-39 1 o f Nzcotwna, 3 1 2 of Oryza satzva, embryogemc, 26, 335-347 of Passijlora, 1 77-1 79 of Picea, embryogemc, 5 1--6 1 of Zea mays, 379 T TE buffer, 200 Thm Layer Chromatography, alkalords, 3 96--400 anthraqumones, 388-389 Tissue electroporatwn, 367-373 TLC, see Thm Layer Chromatography Tobacco, see Nzcotwna Tomato, see Lycoperstcon Transformation, see Genetic modificatiOn Tnazme tolerance test, 2 1 7, 325 Tnphenyl Tetrazohum Chlonde, 86, 90 Trzticum aestivum,

mflorescence culture and regeneration, 7 1-82 embryo rescue, 293-307 genetic modificatiOn, 349-3 58, 3 8 1 TTC, see Tnphenyl Tetrazohum Chloride

42 1 u

uzdA (GUS) gene, 228, 350 Ultrasonic cell permeabihzatwn, 408--409 UV mutagenesis, 3 1 7 v

Vancomycm, 332 VernalizatiOn, of Hordeum vulgare, 47, 273 of Tntzcum aestzvum, 72 Viability testmg, FDA, 86, 90, 1 72, 202, 287, 388 TTC, 86, 90 Virus ehminatwn, 1 1 5-1 25, 142 antiviral chemtcals, 1 20 temperature treatments, 1 1 9-1 20 VItnficatton, for cryopreservatwn, 88-89 in tissue cultures, 1 40, 1 50 w

Wheat, see Tntzcum aestlvum X

X-gal, 1 99 z

Zea mays, m vitro ferttlizatwn, 259-267 genetic modification, 367-3 73, 375-3 82 Zeatin, 38, 73, 1 50, 2 1 7, 3 2 1 , 3 5 1

METHODS I N MOLECULAR B I OLOCY"'

•

111

Series Editor: john M. Walker

Plant Cell Culture Protocols Edited by Robert D. Hall CPRO-OLO, Wageningen, The Netherlands

In Plant Cell Culture Protocols, Robert Hall and a panel of expert researchers present a comprehensive collection ofthe most frequently used and broadly applicable techniques for plant cell and tissue culture. Readily reproducible and extensively annotated, the methods cover culture initiation, maintenance, manipulation, application, and long-term storage, with emphasis on techniquesfor genetic modification and micropropagation. Many of these protocols are currently used in major projects designed to produce improved varieties of important crop plants. In addition, a number of specialized protocols have been included to illustrate the diversity of the techniques available and their widespread applicability. Plant Cell Culture Protocols is aimed at scientists involved in all aspects of plant biotechnological research, as well as thoseworking in other areas ofagriculture and horticulture who are interested in expanding their technical repertoire to include in vitro methodology. lts state-of-the-art techniques are certain to make the book today's reference of choice, an indispensable tool in the development of new transgenic plants and full­ scale commercial applications. FEATURES • Includes clear step-by-step instructions that are

readily reproducible

• Uses examples easily modified to suit the

reader's own plant materials and aims

• Provides extensive tips on pitfalls to avoid and

on handling unexpected problems CONTENTS PART I. INTIOOUCTION. An Introduction to Plant Cell Culture: Pointers to Success. PART II. CELL CULTURE AND PLANT REGENERATION. Callus Initiation, Maintenance, and Shoot Induction in Rice. Callus Initiation, Maintenance, and Shoot Induction in Potato: Monitoring ofSpontaneous Genetic Vari­ ability In Vitro and In Vivo. Somatic Embryogenesis in Barley Suspension Cultures. Somatic Embryogenesis in Picea Suspension Cultures. SPECIALIZED TECHNIQUES. Direct, Cyclic Somatic Embryogenesis of Cassava for Mass Production Pur­ poses. Immature Inflorescence Culture of Cereals: A Highly Responsive System of Regeneration and Transformation. Cryopreservation of Rice Tissue Cultures. Noncryogenic, Long­ Term Germplasm Storage. PART Ill. PlANT PROPAGATION IN VrrRo. Micropropagation of Strawberry Via Axillary Shoot Prolifera­ tion. Meristem-Tip Culture for Propagation and Virus Elimination. SPECIALIZED TECHNIQUES. Clonal Propagation of Orchids. In Vitro Propagation of Succulent Plants. Micro­ propagation of Flower Bulbs: Lily and Narcissus. Clonal Propagation of Woody Species. Spore-Derived Axenic Cul­ tures of Ferns as a Method of Propagation. PART IV. APPLICATIONS Foa PLANT PROTOPLASTS. Protoplast Isolation, Culture, and Plant Regeneration from Passif/ora. lsolation, Culture and Plant Re­ generation of Suspension-Derived Protoplasts of Lolium. Protoplast Fusion for Symmetric Somatic Hybrid Production in Brassicaceae. Production of Cybrids in Rapeseed (Brassica napus). SPECIALIZED TECHNIQUES. Microprotoplast-Mediated Chromosome Transfer (MMCD for the Direct Production of Monosomic Addition Lines. Guard Cell Protoplasts: Isolation,

Culture, and Regeneration ofPlants. In Vitro Fertilization With Isolated Single Gametes. PART V. PROTOCOLS foa GENOMIC MANIPULATION. Protocols for Anther and Microspore Culture of Barley. Microspore Embryogenesis and In Vitro Pollen Matu­ ration in Tobacco. Embryo Rescue Following Wide Crosses. SPECIALIZED TECHNIQUES. Mutagenesis and the Selection of Resistant Mutants. The Generation of Plastid Mutants In Vitro. PART VI. PROTOCOLS FOR THE INTROOUCTION Of SPECIFIC GENES. Agrobacterium-Mediated Transformation of Petunia Leaf Disks. Transformation of Rice Via PEG-Mediated DNA Up­ take into Protoplasts. Transformation of Wheat Via Particle Bombardment. Plant Transformation Via Protoplast Electroporation. SPECIALIZED TECHNIQUES. Transformation of Maize via Tissue Electroporation. Transformation of Maize Using Silicon Carbide Whiskers. PART VII. SUSPENSION CULTURE INITIATION AND THE ACCUMULATION Of METABOLITES.

Directing Anth raqui none Accumulation via Manipulation of Morinda Suspension Cultures. Alkaloid Accumulation in Catharanthus roseus Suspension Cultures. Betalains: Their Accumulation and Release In Vitro. PART VII. APPENDIX.

ISBN 0-89603-549-2 9 D O 0 0>

Methods in Molecular BiologrM • 1 1 1 PLANT CELL CULTURE PROTOCOLS ISBN: 0-89603-549-2

9