PLANT TISSUE CULTURE (THEORY AND PRACTICALS)

2nd Edition

PLANT TISSUE CULTURE (THEORY AND PRACTICALS)

2nd Edition

T. Pullaiah Department of Botany, Sri Krishnadevaraya University, Anantapur - 515003, Andhra Pradesh (India)

M.V. Subba Rao Department of Botany, Andhra University, Visakhapatnam - 530003, Andhra Pradesh (India)

and

E. Sreedevi Department of Botany, Government Degree College, Anantapur - 515001, Andhra Pradesh (India)

Published by SCIENTIFIC PUBLISHERS (INDIA)

Jodhpur –

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© 2017, Authors

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ISBN: 978-93-86347-35-0 (PB) 978-93-86347-34-3 (HB)

eISBN: 978-93-87869-80-6

Visit the Scientific Publishers (India) website at http://www.scientificpub.com

Printed in India

PREFACE

Since the publication of the first edition in 2009, several new and exciting developments have taken place in the field of plant tissue culture which pursues us to attempt to provide current information and examples. From this book, faculty in various fields of plant sciences, graduate students, and undergraduates may gain basic and advanced knowledge about application of various aspects of Plant Cell and Tissue Culture. The revised edition presents updated information and will prove to be an excellent text and reference book for theoretical, practical and applied aspects of plant tissue culture with hands on training approach. Each chapter as before, is written in lucid language which has now been thoroughly revised for recent developments to include relevant media protocols, and is profusely illustrated with self-explanatory diagrams. This book includes new additions in every chapter starting from tissue culture history to haploid production, suspension cultures, nucellus culture, protoplast fusion and hybridization and secondary metabolite production and enables to make the readers to know about recent developments in Plant tissue culture. This revised edition includes detailed drawings that complement both knowledge of theory and experiments.

T. Pullaiah M.V. Subba Rao

E. Sreedevi

LIST OF ABBREVIATIONS

% Per cent

2iP 2-Isopentenyl adenine

ABA Abscisic Acid

AC Activated Charcoal

AIDS Acquired Immuno Deficiency Syndrome

AR Analytical Reagent

ATP Adenosine Triphosphate

BB5 Gamborg’s Medium

BA 6-Benzyladenine

BAP 6-Benzyl-Aminopurine

BM Basal Medium

BuDR BromoDeoxyuridine

CCC Cycocel

cDNA complementary DNA

CH Casein Hydrolysate

CM Coconut Milk

CMS Cytoplasmic Male Sterility

CPPU N-2-chloro-4puridyl-N-phenyl Urea

CPW Cell-protoplast Washing

DES Diethyl Sulphate

DFR Dihydroflavonol-4-reductase

DH Diploid Homozygous

DMSO Dimethyl Sulphoxide

DNA Deoxyribonucleic Acid

vi Plant Tissue Culture (Theory and Practicals)

DPU N,N’-diphenyl Urea

DRT Decimal Reduction Time

ECs Embryogenic Clumps

EDTA EthylenediamineTetraacetic acid

ELISA Enzyme-linked Immunosorbent Assay

EMS Ethyl Methane Sulphonate

F3’5’H Flavanoid 3’, 5’- hydroxylase

FDA Fluorescein Diacetate

FudR 5-Fluorodeoxyuridine

GA3 Gibberellic Acid

GCMS Gas Chromatography Mass Spectrometer

GR Guaranteed Reagent

H2O2 Hydrogen peroxide

HEPA High Efficiency Particulate Air filter

HgCl2 Mercuric chloride

HPLC High Performance Liquid Chromatography

HU Hydroxy Urea

IAA Indole-3-Acetic Acid

IBA Indole-3-Butyric Acid

IBPGR International Board for Plant Genetic Resources

IEDCs Induced Embryogenic Determined Cells

JA Jasmonic Acid

JAMe methyl Jasmonate

KN Kinetin

LN Liquid Nitrogen

LOS Low-oxygen Storage

LPS Low-pressure Storage

LS medium Linsmaier and Skoog medium

MCO Mitotic Crossing Over

MCPA 2-methyl-4-chlorophenoxyacetic acid

mRNA messenger RNA

List of Abbreviations vii

MS Murashige and Skoog’s Medium

MSO Methionine Sulphoximide

NAA Naphthalene Acetic Acid

NOA Naphthoxyacetic Acid

NR Nitrate Reductase oC Degree Celsius

OD Optical Density

ODAP β-oxalyl-L-α,β-diaminopropionic acid

PCPA 4-Chlorophenoxyacetic acid

PCR Polymerase Chain Reaction

PCV Packed Cell Volume

PE Plating Efficiency

PEDCs Pre-embryogenic Determined Cells

PEG Polyethylene Glycol

PGRs Plant Growth Regulators

PLRV Potato Leaf-Roll Virus

ppm Parts per million

Psi Pounds per square inch

PVP Polyvinylpyrrolidone

RBC Red Blood Cell

RFLP Restriction Fragment Length Polymorphism

RO Reverse Osmosis

rRNA Ribosomal Ribonucleic Acid

T-DNA Transfer Deoxyribonucleic Acid

TDP Thermal Death Point

TDT Thermal Death Time

TDZ Thiadiazuron

TMV Tobacco Mosaic Virus

UV Ultraviolet

WBC White Blood Cell

WM White’s Medium

viii Plant Tissue Culture (Theory and Practicals)

WPM Woody Plants Medium

TTC 2,3,5-Triphenyltetrazolium Chloride

2,4,5-T 2,4,5-Trichloro phenoxy acetic acid

Picloram 4-Amino, 3,5,6-Trichloropicolinic acid

B-9 Succinic acid 2, 2-dimethyl hydrazide

2,4-D 2,4-Dichloro phenoxy acetic acid

CONTENTS

1. HISTORY OF PLANT TISSUE CULTURE 1 1.1. Introduction 1 1.2. Totipotency of the Cells 3 1.3. Morphogenesis 4 1.4. History of Plant Tissue Culture 9 1.5. Applications of Plant Tissue Culture 19 1.6. Terminology 20 1.7. Books for Further Reading 24

2. LABORATORY REQUIREMENTS FOR PLANT TISSUE CULTURE

25

2.1. Laboratory Organization 25

3. TISSUE CULTURE MEDIA 34

3.1. Introduction 34 3.2. Media Composition 34 3.3. Media Preparation 48 3.4. Commonly used Concentrations in Tissue Culture 50

4. METHODS OF STERILIZATION 52

4.1. Introduction 52 4.2. Sterilization by Heat 53 4.3. Filter Sterilization 55 4.4. Chemicals 55 4.5. Infrared Radiation 56 4.6. Surfactants 56 4.7. Antibiotics for Control of Bacterial and Fungal Contaminants 57 4.8. Maintenance of Aseptic Conditions 58

x Plant Tissue Culture (Theory and Practicals)

4.9. Surface Sterilization of Explant 58

4.10. Transfer of An Explant 59

5. CELL AND SUSPENSION CULTURE 62

5.1. Introduction 62 5.2. Procedure for Cell and Suspension Culture 62 5.3. Establishment of Cell Suspension Culture 62 5.4. Cell Suspension Culture and Cell Growth 64 5.5. Formation of Somatic Embryos and Plant Regeneration 64 5.6. Types of Suspension Cultures 64 5.7. Growth Patterns in Suspension Culture 66 5.8. Measurement of Cell Growth in Suspension Cultures 67 5.9. Synchronization of Suspension Cell Cultures 69 5.10. Technique for Single Cell Culture 69 5.11. Tests for Viability of Cultured Cells 71 5.12. Advantages of Cell and Suspension Culture 72 5.13. Plant Cell Reactors 72 5.14. Immobilized Plant Cell Reactors 77 5.15. Culture of Isolated Single Cells 78

6. SOMATIC EMBRYOGENESIS 80

6.1. Introduction 80 6.2. Some Examples of Somatic Embryogenesis 82 6.3. Theories on Embryogenesis 83 6.4. Factors Affecting Somatic Embryogenesis 85 6.5. Anatomy and Cytology to Somatic Embryo Development 88

7. SYNTHETIC SEEDS 93

7.1. Introduction 93

7.2. Encapsulation 93

7.3. Production of Artificial Seeds 95

7.4. Applications 97

7.5. Limitations 98

7.6. Uses of Artificial Seeds 98

7.7. Reference 99

Contents xi

8. MICROPROPAGATION OF HIGHER PLANTS 100

8.1. Introduction 100 8.2. Somatic Cell Embryogenesis 102 8.3. Production of Adventitious Shoots and Related Organs 103 8.4. Enhanced Axillary Branching 103 8.5. Steps Involved in Plant Multiplication 105 8.6. Buds Culture 107 8.7. Factors Affecting Micropropagation 109 8.8. Micropropagation of Ornamental Plants 112 8.9. Advantages of Micropropagation 113

9. HAPLOID PRODUCTION 115

9.1. Introduction 115 9.2. Anther Culture 115 9.3. Pollen Culture 122 9.4. Comparison between Anther and Pollen Cultures 123 9.5. Significance of Uses of Haploids 124 9.6. Limitations 126 9.7. Gynogenesis 127 9.8. Bulbosum Technique 133

10. NUCELLUS CULTURE, OVULE CULTURE, OVARY CULTURE AND FLOWER CULTURE

134

10.1. Nucellus Culture 134 10.2. Ovule Culture 138 10.3. Ovary Culture 140 10.4. Flower Culture 142

11. EMBRYO AND ENDOSPERM CULTURE 146

11.1. Embryo Culture 146 11.2. Endosperm Culture 152

12. PROTOPLAST CULTURE AND SOMATIC HYBRIDIZATION

156

12.1. Introduction 156 12.2. Sources of Protoplasts 157 12.3. Isolation of Protoplasts 158

xii Plant Tissue Culture (Theory and Practicals)

12.4. Culture of Protoplasts 163 12.5. Applications 166 12.6. Protoplast Fusion and Somatic Hybridization 166 12.7. Cybrids 173 12.8. Applications 175

13. SOMACLONAL VARIATIONS 180

13.1. Introduction 180 13.2. Origin of Somaclonal Variation 180 13.3. Isolation of Variants 186 13.4. Gametoclonal Variation 190

14. GERMPLASM CONSERVATION 191

14.1. Introduction 191 14.2. Determination of Cell Survival 198 14.3. Action of Cryoprotectants 198 14.4. Factors Affecting Revival of Frozen Cells 199 14.5. Applications 200

15. PRODUCTION OF SECONDARY METABOLITES THROUGH TISSUE CULTURE

203

15.1. Introduction 203 15.2. Types of Cultures 204 15.3. Bioreactors 209 15.4. Cell Immobilization 209 15.5. Improving the Product Quality and Quantity 211 15.6. Commercial Production 217 15.7. Characteristics of Plant Cells in Suspension Culture 218 15.8. Factors Affecting Secondary Metabolite Production in Cell

Suspension Cultures 218

15.9. Ways to Increase Product Formation 219

16. ISOLATION OF MUTANTS FROM CELL CULTURE 220

16.1. Introduction 220

17. PRACTICALS 224