Sant Saran Bhojwani Prem Kumar Dantu

318 Pages·2013·9.52 MB·Indonesian

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
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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