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ova, E. Zazimalova and E.F. George 6 Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists 205 J. van Staden, E. Zazimalova and E.F. George 7 Plant Growth Regulators III: Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds 227 I.E. Moshkov, G.V. Novikova, M.A. Hall and E.F. George 8 Developmental Biology 283 D. Chriqui 9 Somatic Embryogenesis 335 S. Von Arnold 10 Adventitious Regeneration 355 P.B. Gahan and E.F. George 11 Stock Plant Physiological Factors Affecting Growth and Morphogenesis 403 J. Preece 12 Effects of the Phyova, E. Zazimalova and E.F. George 6 Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists 205 J. van Staden, E. Zazimalova and E.F. George 7 Plant Growth Regulators III: Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds 227 I.E. Moshkov, G.V. Novikova, M.A. Hall and E.F. George 8 Developmental Biology 283 D. Chriqui 9 Somatic Embryogenesis 335 S. Von Arnold 10 Adventitious Regeneration 355 P.B. Gahan and E.F. George 11 Stock Plant Physiological Factors Affecting Growth and Morphogenesis 403 J. Preece 12 Effects of the Physical Environment 423 E.F. George and W. Davies 13 The Anatomy and Morphology of Tissue Culturedova, E. Zazimalova and E.F. George 6 Plant Growth Regulators II: Cytokinins, their Analogues and Antagonists 205 J. van Staden, E. Zazimalova and E.F. George 7 Plant Growth Regulators III: Gibberellins, Ethylene, Abscisic Acid, their Analogues and Inhibitors; Miscellaneous Compounds 227 I.E. Moshkov, G.V. Novikova, M.A. Hall and E.F. George 8 Developmental Biology 283 D. Chriqui 9 Somatic Embryogenesis 335 S. Von Arnold 10 Adventitious Regeneration 355 P.B. Gahan and E.F. George 11 Stock Plant Physiological Factors Affecting Growth and Morphogenesis 403 J. Preece 12 Effects of the Physical Environment 423 E.F. George and W. Davies 13 The Anatomy and Morphology of Tissue Culturedsical Environment 423 E.F. George and W. Davies 13 The Anatomy and Morphology of Tissue Cultured
Sant Saran Bhojwani Prem Kumar Dantu Plant Tissue Culture: An Introductory Text Plant Tissue Culture: An Introductory Text Sant Saran Bhojwani Prem Kumar Dantu Plant Tissue Culture: An Introductory Text 123 Sant Saran Bhojwani Prem Kumar Dantu Department of Botany Dayalbagh Educational Institute Agra, Uttar Pradesh India ISBN 978-81-322-1025-2 DOI 10.1007/978-81-322-1026-9 ISBN 978-81-322-1026-9 (eBook) Springer New Delhi Heidelberg New York Dordrecht London Library of Congress Control Number: 2012954643 Ó Springer India 2013 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Dedicated to the most Revered Dr M B Lal Sahab (1907–2002) D.Sc (Lucknow), D.Sc (Edinburgh), the visionary Founder Director of the Dayalbagh Educational Institute, for the inspiration and strength to undertake and complete the task of writing this book Preface Plant tissue culture (PTC) broadly refers to cultivation of plant cells, tissues, organs, and plantlets on artificial medium under aseptic and controlled environmental conditions PTC is as much an art as a science It is the art of growing experimental plants, selecting a suitable plant organ or tissue to initiate cultures, cleaning, sterilization and trimming it to a suitable size, and planting it on a culture medium in right orientation while maintaining complete asepsis It also requires an experienced and vigilant eye to select healthy and normal tissues for subculture PTC involves a scientific approach to systematically optimize physical (nature of the substrate, pH, light, temperature and humidity), chemical (composition of the culture medium, particularly nutrients and growth regulators), biological (source, physiological status and size of the explant), and environmental (gaseous environment inside the culture vial) parameters to achieve the desired growth rate, cellular metabolism, and differentiation The most important contribution made through PTC is the demonstration of the unique capacity of plant cells to regenerate full plants, via organogenesis or embryogenesis, irrespective of their source (root, leaf, stem, floral parts, pollen, endosperm) and ploidy level (haploid, diploid, triploid) PTC is also the best technique to exploit the cellular totipotency of plant cells for numerous practical applications, and offers technologies for crop improvement (haploid and triploid production, in vitro fertilization, hybrid embryo rescue, variant selection), clonal propagation (Micropropagation), virus elimination (shoot tip culture), germplasm conservation, production of industrial phytochemicals, and regeneration of plants from genetically manipulated cells by recombinant DNA technology (genetic engineering) or cell fusion (somatic hybridization) PTC has been extensively employed for basic studies related to plant physiology (photosynthesis, nutrition of plant cells, and embryos), biochemistry, cellular metabolism, morphogenesis (organogenesis, embryogenesis), phytopathology (plant microbe interaction), histology (cytodifferentiation), cytology (cell cycle), etc Indeed the discovery of first cytokinin is based on PTC studies Thus, PTC is an exciting area of basic and applied sciences with considerable scope for further research Considerable work is being done to understand the physiology and genetics of embryogenesis and vii viii Preface organogenesis using PTC systems, especially Arabidopsis and carrot, which are likely to enhance the efficiency of in vitro regeneration protocols Therefore, PTC forms a part of most of the courses on plant sciences (Developmental Botany, Embryology, Physiology, Genetics, Plant Breeding, Horticulture, Sylviculture, Phytopathology, etc.) and is an essential component of Plant Biotechnology After the first book on ‘‘Plant Tissue Culture’’ by Prof P R White in 1943, several volumes describing different aspects of PTC have been published Most of these are compilations of invited articles by different experts or proceedings of conferences More recently, a number of books describing the methods and protocols for one or more techniques of PTC have been published which should serve as useful laboratory manuals The impetus for writing this book was to make available an up-to-date text covering all theoretical and practical aspects of PTC for the students and early career researchers of plant sciences and agricultural biotechnology The book includes 19 chapters profusely illustrated with half-tone pictures and self-explanatory diagrams Most of the chapters include relevant media compositions and protocols that should be helpful in conducting laboratory exercises For those who are interested in further details, Suggested Further Reading are given at the end of each chapter We hope that the readers will find it useful Suggestions for further improvement of the book are most welcome During the past two decades or so research in the area of plant biotechnology has become a closed door activity because many renowned scientists have moved from public research laboratories in universities and institutions to the private industry Consequently, detailed information on many recent developments is not readily available We would like to thank many scientists who provided illustrations from their works and those who have helped us in completing this mammoth task The help of Mr Jai Bhargava and Mr Atul Haseja in preparing some of the illustrations is gratefully acknowledged October 2012 Sant Saran Bhojwani Prem Kumar Dantu Contents Historical Sketch 1.1 Landmarks/Milestones Suggested Further Reading 10 General Requirements and Techniques 2.1 Introduction 2.2 Requirements 2.2.1 Structure and Utilities 2.2.2 Washing Room 2.2.3 Media Room 2.2.4 Glassware/Plasticware 2.2.5 Transfer Room 2.2.6 Growth Room 2.2.7 Cold Storage 2.2.8 Greenhouse 2.3 Techniques 2.3.1 Glassware and Plasticware Washing 2.3.2 Sterilization 2.4 Appendix I 2.5 Appendix II Suggested Further Reading 11 11 11 11 12 13 14 14 15 16 16 16 17 17 22 23 25 Culture Media 3.1 Introduction 3.2 Media Constituents 3.2.1 Inorganic Nutrients 3.2.2 Organic Nutrients 3.2.3 Plant Growth Regulators 3.2.4 Other Supplements 3.2.5 Undefined Supplements 3.2.6 Gelling Agents 3.3 pH of the Medium 3.4 Media Preparation 3.4.1 Steps in the Preparation of Culture Medium 3.4.2 Use of Commercial Pre-Mixes Suggested Further Reading 27 27 27 29 29 31 33 33 34 34 35 35 36 36 ix x Contents and Cell Culture Introduction Callus Cultures Suspension Cultures 4.3.1 Batch Cultures 4.3.2 Continuous Cultures 4.3.3 Medium for Suspension Cultures 4.3.4 Synchronous Cell Suspension Cultures 4.3.5 Determination of Growth in Suspension Cultures 4.3.6 Tests for Viability of Cultured Cells 4.4 Large Scale Cell Culture 4.5 Single Cell Culture 4.5.1 Isolation of Single Cells 4.5.2 Culture of Single cells 4.5.3 Factors Affecting Single Cell Culture 4.6 Concluding Remarks 4.7 Appendix Suggested Further Reading Tissue 4.1 4.2 4.3 Cytodifferentiation 5.1 Introduction 5.2 Experimental Systems 5.2.1 Tracheary Element Differentiation In Vitro 5.2.2 Phloem Differentiation In Vitro 5.3 Factors Affecting Vascular Tissue Differentiation 5.3.1 Growth Regulators 5.3.2 Other Factors 5.4 Cell Cycle and Tracheary Element Differentiation 5.5 Changes Associated with Tracheary Element Differentiation 5.6 Process of TE Differentiation 5.7 Concluding Remarks 5.8 Appendix Suggested Further Reading Cellular Totipotency 6.1 Introduction 6.2 Factors Affecting Shoot Bud Differentiation 6.2.1 Culture Medium 6.2.2 Genotype 6.2.3 Explant 6.2.4 Electrical and Ultrasound Stimulation of Shoot Differentiation 6.3 Thin Cell Layer Culture 6.4 Totipotency of Crown Gall Tumor Cells 6.5 Ontogeny of Shoots 6.6 Induction of Organogenic Differentiation 39 39 39 40 41 41 43 43 43 44 45 46 46 46 49 49 49 50 51 51 52 52 53 53 53 55 55 56 58 59 60 60 63 63 64 64 67 67 68 68 69 69 70 Contents xi 6.7 Concluding Remarks Suggested Further Reading 73 74 Somatic Embryogenesis 7.1 Introduction 7.2 Factors Affecting Somatic Embryogenesis 7.2.1 Explant 7.2.2 Genotype 7.2.3 Medium 7.2.4 Growth Regulators 7.2.5 Selective Subculture 7.2.6 Electrical Stimulation 7.2.7 Other Factors 7.3 Induction and Development 7.3.1 Induction 7.3.2 Development 7.3.3 Single Cell Origin of Somatic Embryos 7.4 Synchronization of Somatic Embryo Development 7.5 Physiological and Biochemical Aspects of Somatic Embryogenesis 7.6 Molecular Markers and Somatic Embryogenesis 7.7 Maturation and Conversion of Somatic Embryos 7.8 Somatic Embryos Versus Zygotic Embryo 7.9 Large Scale Production of Somatic Embryos 7.10 Synthetic Seeds 7.11 Practical Applications of Somatic Embryogenesis 7.12 Concluding Remarks 7.13 Appendix Suggested Further Reading 75 75 76 77 77 78 78 79 79 80 80 81 81 82 82 83 84 85 86 86 89 90 90 91 92 Androgenesis 8.1 Introduction 8.2 Androgenesis 8.2.1 Techniques 8.3 Factors Effecting In Vitro Androgenesis 8.3.1 Genetic Potential 8.3.2 Physiological Status of the Donor Plants 8.3.3 Stage of Pollen Development 8.3.4 Pretreatments 8.3.5 Culture Medium 8.4 Origin of Androgenic Plants 8.4.1 Induction 8.4.2 Early Segmentation of Microspores 8.4.3 Regeneration of Plants 8.5 Diploidization 8.6 Applications 8.7 Concluding Remarks 8.8 Appendix Suggested Further Reading 93 93 93 93 95 95 98 98 98 100 100 101 102 103 104 105 106 107 110 .. .Plant Tissue Culture: An Introductory Text Sant Saran Bhojwani Prem Kumar Dantu Plant Tissue Culture: An Introductory Text 123 Sant Saran Bhojwani Prem Kumar Dantu Department of Botany Dayalbagh... inspiration and strength to undertake and complete the task of writing this book Preface Plant tissue culture (PTC) broadly refers to cultivation of plant cells, tissues, organs, and plantlets on... quantities (Chap 15) These, in brief, are some of the milestones in the history of plant tissue culture Like any other area of science, plant tissue culture started as an academic exercise to answer