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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 SantSaran Bhojwani PremKumar Dantu Department of Botany Dayalbagh Educational Institute Agra,Uttar Pradesh India ISBN 978-81-322-1025-2 ISBN 978-81-322-1026-9 (eBook) DOI 10.1007/978-81-322-1026-9 SpringerNewDelhiHeidelbergNewYorkDordrechtLondon LibraryofCongressControlNumber:2012954643 (cid:2)SpringerIndia2013 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeor part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway, andtransmissionorinformationstorageandretrieval,electronicadaptation,computersoftware, orbysimilarordissimilarmethodologynowknownorhereafterdeveloped.Exemptedfromthis legalreservationarebriefexcerptsinconnectionwithreviewsorscholarlyanalysisormaterial suppliedspecificallyforthepurposeofbeingenteredandexecutedonacomputersystem,for exclusiveusebythepurchaserofthework.Duplicationofthispublicationorpartsthereofis permitted only under the provisions of the Copyright Law of the Publisher’s location, in its currentversion,andpermissionforusemustalwaysbeobtainedfromSpringer.Permissionsfor use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liabletoprosecutionundertherespectiveCopyrightLaw. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthis publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesare exemptfromtherelevantprotectivelawsandregulationsandthereforefreeforgeneraluse. Whiletheadviceandinformationinthisbookarebelievedtobetrueandaccurateatthedateof publication, neither the authors nor the editors nor the publisher can accept any legal responsibilityforanyerrorsoromissionsthatmaybemade.Thepublishermakesnowarranty, expressorimplied,withrespecttothematerialcontainedherein. Printedonacid-freepaper SpringerispartofSpringerScience+BusinessMedia(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 controlledenvironmentalconditions.PTCisasmuchanartasascience. It is the art of growing experimental plants, selecting a suitable plant organortissuetoinitiatecultures,cleaning,sterilizationandtrimmingit to a suitable size, and planting it on a culture medium in right orien- tation while maintaining complete asepsis. It also requires an experi- enced and vigilant eye to select healthy and normal tissues for subculture. PTC involves a scientific approach to systematically opti- mize physical (nature of the substrate, pH, light, temperature and humidity), chemical (composition of the culture medium, particularly nutrientsandgrowthregulators),biological(source,physiologicalstatus andsizeoftheexplant),andenvironmental(gaseousenvironmentinside the culture vial) parameters to achieve the desired growth rate, cellular metabolism, and differentiation. The most important contribution made through PTC is the demon- strationoftheuniquecapacityofplantcellstoregeneratefullplants,via organogenesis or embryogenesis, irrespective of their source (root, leaf, stem,floralparts,pollen,endosperm)andploidylevel(haploid,diploid, triploid). PTC is also the best technique to exploit the cellular totipo- tency of plant cells for numerous practical applications, and offers technologiesforcropimprovement(haploidandtriploidproduction,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 recombi- nant DNA technology (genetic engineering) or cell fusion (somatic hybridization). PTC has been extensively employed for basic studies relatedtoplantphysiology(photosynthesis,nutritionofplantcells,and embryos), biochemistry, cellular metabolism, morphogenesis (organo- genesis, embryogenesis), phytopathology (plant microbe interaction), histology (cytodifferentiation), cytology (cell cycle), etc. Indeed the discovery offirst 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 pro- tocols. 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. Afterthefirstbookon‘‘PlantTissueCulture’’byProf.P.R.Whitein 1943, several volumes describing different aspects of PTC have been published.Mostofthesearecompilationsofinvitedarticlesbydifferent experts or proceedings of conferences. More recently, a number of booksdescribingthemethodsandprotocolsforoneormoretechniques 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-datetextcoveringalltheoreticalandpractical aspectsofPTCfor the students and early career researchers of plant sciences and agricul- turalbiotechnology.Thebookincludes19chaptersprofuselyillustrated with half-tone pictures and self-explanatory diagrams. Most of the chaptersincluderelevantmediacompositionsandprotocolsthatshould be helpful in conducting laboratory exercises. For those who are inter- ested in furtherdetails, SuggestedFurther Readingare given atthe end ofeachchapter.Wehopethatthereaderswillfindituseful.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 1 Historical Sketch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Landmarks/Milestones. . . . . . . . . . . . . . . . . . . . . . . 8 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 10 2 General Requirements and Techniques. . . . . . . . . . . . . . . 11 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.2 Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.2.1 Structure and Utilities . . . . . . . . . . . . . . . . 11 2.2.2 Washing Room. . . . . . . . . . . . . . . . . . . . . 12 2.2.3 Media Room . . . . . . . . . . . . . . . . . . . . . . 13 2.2.4 Glassware/Plasticware . . . . . . . . . . . . . . . . 14 2.2.5 Transfer Room . . . . . . . . . . . . . . . . . . . . . 14 2.2.6 Growth Room. . . . . . . . . . . . . . . . . . . . . . 15 2.2.7 Cold Storage . . . . . . . . . . . . . . . . . . . . . . 16 2.2.8 Greenhouse . . . . . . . . . . . . . . . . . . . . . . . 16 2.3 Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.1 Glassware and Plasticware Washing . . . . . . 17 2.3.2 Sterilization . . . . . . . . . . . . . . . . . . . . . . . 17 2.4 Appendix I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.5 Appendix II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 25 3 Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.2 Media Constituents. . . . . . . . . . . . . . . . . . . . . . . . . 27 3.2.1 Inorganic Nutrients . . . . . . . . . . . . . . . . . . 29 3.2.2 Organic Nutrients . . . . . . . . . . . . . . . . . . . 29 3.2.3 Plant Growth Regulators . . . . . . . . . . . . . . 31 3.2.4 Other Supplements . . . . . . . . . . . . . . . . . . 33 3.2.5 Undefined Supplements. . . . . . . . . . . . . . . 33 3.2.6 Gelling Agents . . . . . . . . . . . . . . . . . . . . . 34 3.3 pH of the Medium . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.4 Media Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.4.1 Steps in the Preparation of Culture Medium. . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.4.2 Use of Commercial Pre-Mixes . . . . . . . . . . 36 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 36 ix x Contents 4 Tissue and Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4.2 Callus Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4.3 Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . 40 4.3.1 Batch Cultures . . . . . . . . . . . . . . . . . . . . . 41 4.3.2 Continuous Cultures . . . . . . . . . . . . . . . . . 41 4.3.3 Medium for Suspension Cultures . . . . . . . . 43 4.3.4 Synchronous Cell Suspension Cultures . . . . 43 4.3.5 Determination of Growth in Suspension Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . 43 4.3.6 Tests for Viability of Cultured Cells . . . . . . 44 4.4 Large Scale Cell Culture. . . . . . . . . . . . . . . . . . . . . 45 4.5 Single Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . . 46 4.5.1 Isolation of Single Cells. . . . . . . . . . . . . . . 46 4.5.2 Culture of Single cells. . . . . . . . . . . . . . . . 46 4.5.3 Factors Affecting Single Cell Culture . . . . . 49 4.6 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . 49 4.7 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 50 5 Cytodifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5.2 Experimental Systems. . . . . . . . . . . . . . . . . . . . . . . 52 5.2.1 Tracheary Element Differentiation In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . 52 5.2.2 Phloem Differentiation In Vitro . . . . . . . . . 53 5.3 Factors Affecting Vascular Tissue Differentiation. . . . 53 5.3.1 Growth Regulators . . . . . . . . . . . . . . . . . . 53 5.3.2 Other Factors . . . . . . . . . . . . . . . . . . . . . . 55 5.4 Cell Cycle and Tracheary Element Differentiation . . . 55 5.5 Changes Associated with Tracheary Element Differentiation. . . . . . . . . . . . . . . . . . . . . . 56 5.6 Process of TE Differentiation. . . . . . . . . . . . . . . . . . 58 5.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . 59 5.8 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 60 6 Cellular Totipotency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 6.2 Factors Affecting Shoot Bud Differentiation . . . . . . . 64 6.2.1 Culture Medium . . . . . . . . . . . . . . . . . . . . 64 6.2.2 Genotype. . . . . . . . . . . . . . . . . . . . . . . . . 67 6.2.3 Explant . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6.2.4 Electrical and Ultrasound Stimulation of Shoot Differentiation. . . . . . . . . . . . . . . 68 6.3 Thin Cell Layer Culture . . . . . . . . . . . . . . . . . . . . . 68 6.4 Totipotency of Crown Gall Tumor Cells . . . . . . . . . . 69 6.5 Ontogeny of Shoots . . . . . . . . . . . . . . . . . . . . . . . . 69 6.6 Induction of Organogenic Differentiation. . . . . . . . . . 70 Contents xi 6.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . 73 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 74 7 Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . 75 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 7.2 Factors Affecting Somatic Embryogenesis. . . . . . . . . 76 7.2.1 Explant . . . . . . . . . . . . . . . . . . . . . . . . . . 77 7.2.2 Genotype. . . . . . . . . . . . . . . . . . . . . . . . . 77 7.2.3 Medium. . . . . . . . . . . . . . . . . . . . . . . . . . 78 7.2.4 Growth Regulators . . . . . . . . . . . . . . . . . . 78 7.2.5 Selective Subculture . . . . . . . . . . . . . . . . . 79 7.2.6 Electrical Stimulation . . . . . . . . . . . . . . . . 79 7.2.7 Other Factors . . . . . . . . . . . . . . . . . . . . . . 80 7.3 Induction and Development. . . . . . . . . . . . . . . . . . . 80 7.3.1 Induction . . . . . . . . . . . . . . . . . . . . . . . . . 81 7.3.2 Development . . . . . . . . . . . . . . . . . . . . . . 81 7.3.3 Single Cell Origin of Somatic Embryos. . . . 82 7.4 Synchronization of Somatic Embryo Development . . . 82 7.5 Physiological and Biochemical Aspects of Somatic Embryogenesis. . . . . . . . . . . . . . . . . . . . 83 7.6 Molecular Markers and Somatic Embryogenesis. . . . . 84 7.7 Maturation and Conversion of Somatic Embryos . . . . 85 7.8 Somatic Embryos Versus Zygotic Embryo. . . . . . . . . 86 7.9 Large Scale Production of Somatic Embryos . . . . . . . 86 7.10 Synthetic Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 7.11 Practical Applications of Somatic Embryogenesis. . . . 90 7.12 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . 90 7.13 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 92 8 Androgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 8.2 Androgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 8.2.1 Techniques. . . . . . . . . . . . . . . . . . . . . . . . 93 8.3 Factors Effecting In Vitro Androgenesis . . . . . . . . . . 95 8.3.1 Genetic Potential. . . . . . . . . . . . . . . . . . . . 95 8.3.2 Physiological Status of the Donor Plants . . . 98 8.3.3 Stage of Pollen Development. . . . . . . . . . . 98 8.3.4 Pretreatments . . . . . . . . . . . . . . . . . . . . . . 98 8.3.5 Culture Medium . . . . . . . . . . . . . . . . . . . . 100 8.4 Origin of Androgenic Plants. . . . . . . . . . . . . . . . . . . 100 8.4.1 Induction . . . . . . . . . . . . . . . . . . . . . . . . . 101 8.4.2 Early Segmentation of Microspores. . . . . . . 102 8.4.3 Regeneration of Plants. . . . . . . . . . . . . . . . 103 8.5 Diploidization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 8.6 Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 8.7 Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . 106 8.8 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 110

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