Table Of ContentPlant Cell Culture
MonoclonalAntibodies
PCR
AnalyzingChromosomes
SeparatingCells
Biological Centrifugation
Plant Cell Culture
Forthcoming titles
AnalysingGene Expression
Animal Cell Culture andTechnology, Second Edition
Gene Mapping
ReconstructingEvolutionaryTrees
Plant Cell Culture
D. E. Evans
Oxford Brookes University, Oxford, UK
J.O.D. Coleman
Oxford Brookes University, Oxford, UK
and
A. Kearns
Oxford Brookes University, Oxford, UK
@
Taylor&Francis
~
Taylor&FrancisGroup
LONDONANDNEWYORK
©Taylor & Francis, 2003
First published 2003
All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any
electronic, mechanical, or other means, now known or hereafter invented, including photocopying and
recording, or in any information storage or retrieval system, without permission in writing from the
publishers.
A CIP catalogue record for this book is available from the British Library.
ISBN 1 85996 320 X
Published by Taylor & Francis
2 Park Square, Milton Park, Abingdon, Oxon, 0Xl4 4RN
52 Vanderbilt Avenue, New York, NY 10017
Transferred to Digital Printing 2007
Typeset by J&L Composition, N Yorks, UK
Publisher's Note
The publisher has gone to great lengths to ensure the quality of this
reprint but points out that some imperfections in the original
may be apparent
Contents
Abbreviations xi
Preface xiii
Chapter I An introduction to plant cell and
tissue culture
1. Plantcellandtissue culture 1
2. Industrial-scaleplantcellculture 3
3. Planttissue culture, plantbreedingand crop improvement 3
4. Planttissue cultureandplantgenetic engineering 4
Chapter 2 Basic plant biology for cell culture 7
1. Tissuesandorgans 7
2. Planttissues 7
3. Meristems 8
4. Plantreproductivetissues 9
5. The embryoandembryogenesis 10
6. Developmentoftissues 11
7. Protoplasts 12
8. Media, nutrientsandrequirementsforgrowth 12
Chapter 3 Tissue culture in agriculture,
horticulture and forestry I9
1. Introduction 19
2. Micropropagation 20
3. Plantbreeding 23
4. Biodiversityand conservationofgermplasm 26
Chapter 4 Tissue culture in genetic
engineering and biotechnology 3I
1. Introduction 31
2. Genetic engineering 31
3. Applicationsofplantgenetic engineering 39
VI Contents
Chapter 5 Culture facilities, sterile technique
and media preparation 43
1. Introduction 43
2. Thebasic laboratorylayoutandequipment 43
3. Sterilization 47
4. Media preparation 50
5. Contamination 53
6. Disposingofcontaminatedwaste 53
7. Safetyin the laboratory 54
Protocol5.1: Hygiene in the tissue culturelaboratory 55
Protocol5.2: Preparingbasal mediumfrom commercial
formulations orfrom individualcomponents 57
Protocol5.3: Preparationofhormoneadditionsto basal media 60
Protocol5.4: Preparationofcoconutwater supplementfor
basal media 62
Chapter 6 Callus cultures 63
1. Introduction 63
2. Originofcallus 63
3. Typesofcallus 63
4. Roleofcallus in embryogenesis, organogenesisand cellculture 64
5. Initiationandestablishmentofcallus cultures 64
6. Monitoringthegrowthofcallus 67
7. Genetictransformationofcallus 68
Protocol 6.1: Preparationofplantmaterialandexplants 70
Protocol 6.2: Callus from adicotroot: Arabidopsis thaliana and
Nicotiana tabacum 73
Protocol 6.3: Callus from dicotshoot/leaf- Arabidopsisthaliana
andNicotiana tabacum 74
Protocol 6.4: Callus from amonocot(e.g.maize, rice) 75
Protocol 6.5: Callus from gymnosperms (Norwayspruce) 76
Protocol 6.6: Transformingmaizecallus byparticlebombardment 77
Protocol 6.7: PreparationofatransgenicArabidopsisthalianacallus
usingAgrobacterium 79
Chapter 7 Cell suspension cultures 81
1. Introduction 81
2. Initiationofcellsuspensioncultures 81
3. Maintenanceofcellsuspensioncultures 82
4. Growthcharacteristicsofcellsuspensioncultures 83
5. Usesofcellsuspensioncultures 87
Protocol 7.1: Ageneric protocolfor initiatingasuspensionculture 88
Protocol 7.2: Monitoringthe growthofsuspensioncultures 90
Protocol 7.3: Measurementofcellviability 92
Protocol 7.4: Genetictransformationofsuspensionculturedcells 94
Contents vii
Chapter 8 Protoplast culture 95
1. Introduction 95
2. Isolationofprotoplasts 95
3. Protoplastculture 97
4. Usesofprotoplasts 98
Protocol8.1: Preparationofprotoplastsfrom suspensioncultures
(e.g.maize) usingalongincubation 101
Protocol8.2: Isolationofprotoplastsfrom carrotsuspension
culturesusingarapid incubation 102
Protocol8.3: Rapid preparationofmesophyllprotoplastsfrom
maizeleaves 103
Protocol8.4: Preparationofmesophyllprotoplastsfrom tobacco
leavesandpurificationofprotoplastson adensity
gradient 104
Protocol8.5: Rapidpreparationofprotoplastsfrom maizeroots 106
Protocol8.6: Preparationofprotoplastsfrom roots (arabidopsis)
withpartialpurificationofprotoplastsbyflotation 108
Protocol 8.7: The cultureofprotoplastsusingnurseculture 110
Protocol8.8: The cultureofprotoplastsusingthe hangingdrop
method 112
Protocol8.9: Detectingregenerationofcellwall in isolated
protoplasts 114
Protocol8.10: Protoplastfusion inducedbypolyethyleneglycol
(PEG) 115
Protocol 8.11: Electricalfusion ofprotoplasts 116
Protocol 8.12: Protoplasttransformationbyelectroporation 118
Protocol8.13: Transformationofprotoplastsmediatedby
polyethyleneglycol(PEG) 119
Chapter 9 Haploid cultures 121
1. Introduction 121
2. Antherculture 121
3. Microsporeculture 122
4. Anthercultureversus microsporeculture 122
5. Intergenericcrosses andembryorescue 123
6. Proceduresfor the inductionofandrogenesis 124
Protocol 9.1: Anthercultureofwheat (Triticum aestivum L.) 125
Protocol 9.2: Wheatmicrosporeculture 127
Chapter I0 Organ and embryo culture 129
1. Introduction 129
2. Hairy roots 129
3. Embryo culture 129
4. Minitubersandmicrotubers 131
VIII Contents
Protocol 10.1: Isolationandcultureofthe primaryseedlingroot
ofdicots 133
Protocol 10.2: Isolationandcultureofrootsofmonocots
(e.g.maize) 134
Protocol 10.3: Hairy rootcultures (planttransformationwith
Agrobacteriumrhizogenes) 135
Protocol 10.4: Sterilecultureofthe ear ofacereal (e.g.maize) 137
Protocol 10.5: Embryo rescue ofadicot- Phaseolus vulgaris 138
Chapter II Regeneration ofplants and
micropropagation 141
1. Introduction 141
2. Regenerationvia somaticembryogenesis 141
3. Control ofembryogenesis 144
4. Regenerationvia organogenesis 144
5. Protocols 145
Protocol 11.1: Embryogenesisfrom callus in adicot(e.g.carrot) or
monocot(cereals, rice) 148
Protocol 11.2: Plantregenerationbyorganogenesis 149
Protocol 11.3: SomaticembryogenesisofNorway spruce usinga
suspensionculturestep 151
Chapter 12 Somaclonal variation IS3
1. Introduction 153
2. Originsandmechanismsofsomaclonalvariability 153
3. The importanceofsomaclonalvariation 155
4. Methodsofassessing somaclonalvariationin regeneratedplants 155
5. Somaclonalvariationasatechniqueforcrop improvement 158
Chapter 13 Bacterial culture in the plant cell
culture laboratory IS9
1. Introduction 159
2. Facilitiesfor bacterialculture 159
Protocol 13.1: TocultureE.coliin Luriabroth (LB) 161
Protocol 13.2: Calciumchloride-mediatedtransformationofE.coli 162
Protocol 13.3: Calciumchloride-mediatedtransformationof
Agrobacterium 164
Protocol 13.4: CultureofAgrobacterium tumefaciensin culture
medium,YEB 166
Protocol 13.5: CultureofAgrobacteriumrhizogenes in culture
medium,YMA 167
Contents ix
Chapter 14 Industrial usesofplant cell culture 169
1. Introduction 169
2. Plantcellsin culture 169
3. Apparatusfor large-scaleplantcultures 170
4. Examples ofindustrial-scaleproductionusing culturedcells 172
Chapter I5 Prospects and future challenges 175
1. Recentdevelopments 175
2. Thefuture 175
Suppliers ofchemicals, apparatus and cell
culture products 181
Glossary 185
Index 189
