TISSUE CULTURE STUDIES in TAMARIND (Tamarindus Indica L.), a LEGUMINOUS TREE SPECIES
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TISSUE CULTURE STUDIES IN TAMARIND (Tamarindus indica L.), A LEGUMINOUS TREE SPECIES URMIL J. MEHTA NOVEMBER, 2001 TISSUE CULTURE STUDIES IN TAMARIND (Tamarindus indica L.), A LEGUMINOUS TREE SPECIES A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY URMIL J. MEHTA PLANT TISSUE CULTURE DIVISION NATIONAL CHEMICAL LABORATORY PUNE – 411 008, INDIA NOVEMBER, 2001 Dedicated to my husband, Dr. J.K. Mehta and children, Akshay, Shikha, Shilpa and Shruti CONTENTS Page No. Acknowledgements iii Certificate iv Key to abbreviations v Conversion table of Molarity concentrations vi of plant growth regulators used Abstract vi i CHAPTER 1: GENERAL INTRODUCTION 1-29 CHAPTER 2 : MATERIALS AND METHODS 30-38 CHAPTER 3: CLONAL PROPAGATION 39-87 A Clonal propagation from seedling explants 3.1A Introduction 43 3.2A Experimental Protocol 44 3.3A Results and Discussion 47 3.4A Conclusion 63 B Clonal propagation from mature explants 3.1B Introduction 63 3.2B Experimental Protocol 64 3.3B Results and Discussion 67 3.4B Conclusions 78 C. Micrografting 3.1C Introduction 78 3.2C Experimental Protocol 79 3.3C Results and Discussion 81 3.4C Conclusions 84 D. Variation in germination and seedling growth among seeds 3.1D Introduction 85 3.2D Experimental Protocol 85 3.3D Results and Discussion 86 3.4D Conclusions 87 CHAPTER 4: DE NOVO ORGANOGENESIS 88-108 4.1 Introduction 88 4.2 Experimental Protocol 94 4.3 Results and Discussion 97 4.4 Conclusions 107 CHAPTER 5: INDUCTION OF SOMATIC EMBRYOGENESIS 109–145 5.1 Introduction 109 5.2 Experimental Protocol 119 5.3 Results and Discussion 123 5.4 Conclusions 144 CHAPTER 6 : MORPHOGENETIC RESPONSE IN SEEDLINGS 146–160 GERMINATED IN PRESENCE OF THIDIAZURON 6.1 Introduction 146 6.2 Experimental Protocol 149 6.3 Results and Discussion 151 6.4 Conclusions 160 SUMMARY 161-163 BIBLIOGRAPHY 164-190 AUTHOR’S PUBLICATION ACKNOWLEDGEMENT I would like to place on record my deep sense of gratitude to my research guide, Dr. (Mrs.) Sulekha Hazra, of Plant Tissue Culture Division, National Chemical Laboratory. I am indebted to her for the constant supervision, support and encouragement through advice and constructive criticism throughout my research work. Her positive attitude and motivation abilities goaded me into working harder and putting in my best. I am also grateful to Dr. K.V. Krishnamurthy, Head, Plant Tissue Culture Division, National Chemical Laboratory for his constant support during my research period. I am especially thankful to my senior colleagues Dr. B. G. Hazra, Dr. A. Sarkar, Dr. (Mrs) N.A. Sahastrabudhe for their valuable and timely help which has a major contribution in the progress of this work. I am specially thankful to my friends Vandana Mhaske, K. Chengalrayan, Madhumita Joshi, Abhay Sagare, M.L. Mohan, Anjan Banerjee, Satish Nalawade, K. Sujatha, Shaila Maria Barretto, Neelima Renukdas and Dinesh Chauk whose timely help needs special mention. I wish to offer my thanks to the members of the PTC Division who whole -heartedly co-operated during the course of this work. Last but not the least, it is difficult to word my gratitude towards my family members, without whose encouragement and unstinted moral support this endeavor would have never been possible. I take this opportunity to thank Dr. A.F. Mascarenhas, former Head, Plant Tissue Culture and Prof. S.B. David, my teacher who initiated me to the field of Plant Tissue Culture. Finally I would like to thank Dr. R.A. Mashelkar, former Director and Paul Ratnasamy, present Director, National Chemical Laboratory, for allowing me to submit my work in the form of a thesis. ( Mrs. Urmil J. Mehta ) Date : Place: Pune CERTIFICATE This is to certify that the work incorporated in the thesis entitle “ Tissue Culture Studies in Tamarind (Tamarindus indica L.), a leguminous tree species ”submitted by Mrs. Urmil J. Mehta was carried out by the candidate under my supervision at the Plant Tissue Culture Division, National Chemical Laboratory, Pune. Such material as has been obtained from other sources has been duly acknowledged in the thesis. (Dr. Mrs. Sulekha Hazra) Guide Pune Date: Key to abbreviations °C Degree Celsius cv. Cultivars v/v Volume/volume (concentration) w/v Weight/ volume (concentration) ANOVA Analysis of variance s.d. Standard deviation HgCl2 Mercuric Chloride MS Murashige and Skoog medium (1962) PGR Plant Growth Regulator KN Kinetin (6-furfuryl amino purine) BAP 6-Benzyl amino purine Z Zeatin 2iP 6-g,g-dimethylallylaminopurine TDZ Thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea) NAA a-Naphthaleneacetic acid GA3 Gibberellic acid PVP Polyvinyl pyrrolidone Dicamba 3,6-dichloro-o-anisic acid 2,4 -D 2,4-di chlorophenoxy acetic acid 2,4,5-T 2,4,5-trichlorophenoxy acetic acid Picloram 4-amino-3,5,6-trichloro picolinic acid IAA Indole acetic acid IBA Indole butyric acid IPA Indole propionic acid ABA Abscissic acid TBA Tertiary butyl alcohol (2-methyl propan-2-ol) DPX-4 mountant [189-(2-chloro-N-(4-methoxy-1,3,5-triazin-2-yl amino carbonyl) benzene sulphanamide)] MOLARITY CONCENTRATIONS OF PLANT GROWTH REGULATORS USED NAME Mol.Wt. Mg.l1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 2.0 5.0 10.0 20.0 2-iP 203.25 ìM 0.49 0.98 1.48 1.97 2.46 2.95 3.44 3.94 4.43 4.92 9.84 24.60 49.20 98.40 BAP 225.26 ìM 0.44 0.89 1.33 1.78 2.22 2.66 3.11 3.55 4.00 4.44 8.88 22.20 44.39 88.79 KN 215.22 ìM 0.46 0.93 1.39 1.86 2.32 2.79 3.25 3.72 4.18 4.65 9.29 23.23 46.46 92.93 Z 219.25 ìM 0.46 0.91 1.37 1.82 2.28 2.74 3.19 3.65 4.10 4.56 9.12 22.81 45.61 91.22 TDZ 220.20 ìM 0.45 0.91 1.36 1.82 2.27 2.72 3.18 3.63 4.09 4.54 9.08 22.71 45.41 90.83 NAA 186.21 ìM 0.54 1.07 1.61 2.15 2.69 3.22 3.76 4.30 4.83 5.37 10.74 26.85 53.70 107.41 IAA 175.20 ìM 0.57 1.14 1.71 2.28 2.85 3.42 4.00 4.57 5.14 5.71 11.42 28.54 57.08 114.16 2,4-D 221.04 ìM 0.45 0.90 1.36 1.81 2.26 2.71 3.17 3.62 4.07 4.52 9.05 22.62 45.24 90.48 2,4,5-T 255.49 ìM 0.39 0.78 1.17 1.57 1.96 2.35 2.74 3.13 3.52 3.91 7.83 19.57 39.14 78.28 PIC 241.46 ìM 0.41 0.83 1.24 1.66 2.07 2.48 2.90 3.31 3.73 4.14 8.28 20.71 41.41 82.83 DICAMBA 221.00 ìM 0.45 0.90 1.36 1.81 2.26 2.71 3.17 3.62 4.07 4.52 9.05 22.62 45.25 90.50 IBA 203.24 ìM 0.49 0.98 1.48 1.97 2.46 2.95 3.44 3.94 4.43 4.92 9.84 24.60 49.20 98.41 GA3 346.40 ìM 0.29 0.58 0.87 1.15 1.44 1.73 2.02 2.31 2.60 2.89 5.77 14.43 28.87 57.74 ABA 264.32 ìM 0.38 0.76 1.13 1.51 1.89 2.27 2.65 3.03 3.40 3.78 7.57 18.92 37.83 75.67 ABSTRACT Trees are complex biological systems in themselves and they are part of an even more complex system – the forest ecosystem. As trees grow and reproduce, they interact in many ways with other biological systems and with the environment. The importance of trees in maintaining our ecosystem and their importance in the world economy, need no emphasis. The global demand for wood products and wood as source of energy and raw material for various industries are increasing with the ascent in world population. With industrialization of the developing countries like India, the natural forests are disappearing rapidly. To increase the forest cover major efforts are directed towards afforestation of the denuded and barren lands, commonly known as wastelands. The role of biotechnology and tissue culture lies in enhancing the production of superior planting material. Tree biotechnology has to be an integral part of an overall forest genetics and tree-breeding program. One of the most important things that biotechnology can offer is to cut the long periods required for tree improvement using conventional technology. The trees thus grown through organized program will not only support the ecosystem but also serve as the source of raw material for industries and renewable resource of energy. Leguminous tree species are well known for their utility to mankind. They are of special interest due to their economic and ecological importance. These trees are particularly considered in afforestation and soil erosion control programs due to their ability to fix atmospheric nitrogen. Survey of existing literature reveals that although research in tree tissue culture was initiated earlier, research in the area of tree legumes started only two decades ago.