TISSUE CULTURE STUDIES AND MOLECULAR CHARACTERIZATION OF PONGAMIA (Pongamia pinnata (L.) Pierre), AN OIL PRODUCING TREE LEGUME A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY K. SUJATHA PLANT TISSUE CULTURE DIVISION NATIONAL CHEMICAL LABORATORY PUNE – 411 008, INDIA. MAY 2007 Dedicated to my beloved mother-in-law, husband, daughter and my parents CONTENTS Page No. ACKNOWLEDGEMENTS i CERTIFICATE iii DECLARATION iv KEY TO ABBREVIATIONS v ABSTRACT vi CHAPTER 1: GENERAL INTRODUCTION 1-26 CHAPTER 2: MATERIALS AND METHODS 27-37 CHAPTER 3: CLONAL PROPAGATION USING JUVENILE TISSUES 3.1 Introduction 3.2 Experimental Protocol 38-62 3.3 Results and Discussion 3.4 Conclusion CHAPTER 4: CLONAL PROPAGATION USING MATURE NODAL BUDS 4.1 Introduction 4.2 Experimental Protocol 63-87 4.3 Results and Discussion 4.4 Conclusions CHAPTER 5: DE NOVO MORPHOGENESIS 5.1 Introduction 88-110 5.2 Experimental Protocol 5.3 Results and Discussion 5.4 Conclusions CHAPTER 6: MOLECULAR CHARACTERIZATION 6.1 Introduction 111-136 6.2 Experimental Protocol 6.3 Results and Discussion 6.4 Conclusions SUMMARY 137-140 BIBLIOGRAPHY 141-162 AUTHOR’S PUBLICATION 163-164 ACKNOWLEDGEMENT I would like to place on record my deep sense of gratitude to my research guide, Dr. 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 overly enthusiasm and integral view on research and her mission for providing 'only high-quality work’, has made a deep impression on me. I owe her lots of gratitude for having shown me the way of research. Her positive attitude and motivation abilities guided me to work harder & harder and put in my best. I am also grateful to Dr. S.K. Rawal, Head, Plant Tissue Culture Division, National Chemical Laboratory for his constant support, encouragement and constructive criticism throughout my research period. My heartfelt thanks go to Dr. Vidya S. Gupta, for providing me ISSR primers and permitting me to perform few molecular techniques in her lab. Her warm personality, encouragement, support and patience in handling my shortcomings, helped me immensely towards developing my writng skills. I am especially thankful to Dr. Urmil J. Mehta, who comforted me with her caring and taught me the essence of life and research. My special thanks to Dr. Subhada Thengane, Dr. D.K. Kulkarni, Mr. Dhage, Dr. D.C. Agrawal, Dr. B.M. Khan, Dr. M.M. Jana for their timely help during my research work. My special thanks to Dr. Niu Chen, who offered timely help in preparation of the chapter of my thesis. Its my pleasure to offer my thanks to my friends viz, Shweta, Sunil, Bhuban, Raju, Shaila, Nookaraju, Harini, Bharti, Kiran, Neelima, Anish, Arohi and Ramchander whose timely help needs special mention. My special thanks to Ashwini Rajwade, who helped me in screening of ISSR primers and trained me in basic molecular biology techniques. I owe my thanks to all the members of Plant molecular biology group especially to Dr. Ashok Giri, Ilangovan, Soumiya, Gayatri, Radhika, and others for their timely help during my research. I wish to offer my thanks to all the students of Plant Tissue Culture Division, Biochemical Division and the members of the PTC Division who whole-heartedly co- operated during the course of this work. Finally, I would like to thank all technical staff for their help, friendship and making my stay at NCL enjoyable. I take this opportunity to thank Prof. R.J.Thengane, Department of Botany, University of Pune, who directed me to the field of Plant Tissue Culture in NCL. This research has been supported by grant from The Council of Scientific and Industrial Research (CSIR), New Delhi who awarded me Junior and Senior Research Fellowship for this work. The financial support from CSIR is thus gratefully i acknowledged. I would like to thank Dr. Sivaram, Director, National Chemical Laboratory, for allowing me to submit my work in the form of a thesis. Last but not the least, it is difficult to express my gratitude towards my family members, without whose encouragement and unstinted moral support, this endeavor would have never been possible. I would like to thank to my mother in-law Smt. Padma Rangarajan, who have been very patiently waiting for me to get this thesis done and made me indulgent from household responsibilities. Special thanks to my husband and best pal, R. Raman, for his heartfelt love and support towards me over the years. I hereby place on record, the kind and incessant cooperation of my daughter, Harini, throughout my research period. Over and above, I would like to state that, the Blessings of my beloved parents, Krishnaswamy and Lakshmi was also instrumental in the completion of my thesis and obtaining the degree of Philosophy. ( Mrs. K. Sujatha ) Date : Place: Pune ii CERTIFICATE OF THE GUIDE CERTIFIED that the work incorporated in the thesis entitled “Tissue Culture Studies and molecular characterization of Pongamia (Pongamia pinnata (L.) Pierre), an oil producing tree legume” submitted by Mrs. K. Sujatha 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: iii DECLARATION BY THE CANDIDATE I declare that the thesis entitled “Tissue Culture Studies and molecular characterization of Pongamia (Pongamia pinnata (L.) Pierre), an oil producing tree legume” Submitted by me for the degree of Doctor of Philosophy is the record of work carried out by me under the guidance of Dr. (Mrs.) Sulekha Hazra and has not formed the basis for the award of any degree, diploma, associateship, fellowship, titles in this or any other university or other institute of Higher learning. I further declare that the material obtained from other sources has been duly acknowledged in the thesis. May 2007 Mrs. K. Sujatha. iv Key to abbreviations °C Degree Celsius v/v Volume/volume (concentration) w/v Weight/ volume (concentration) ANOVA Analysis of variance sd Standard deviation HgCl2 Mercuric Chloride MS Murashige and Skoog medium (1962) GR Plant Growth Regulator KN Kinetin (6-furfuryl amino purine) BA 6-Benzyl amino purine Z Zeatin TDZ Thidiazuron (N-phenyl-N'-1,2,3-thidiazol-5-ylurea) NAA α-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 AdS Adenine Sulphate Taxim Cefotaxime (antibiotic) PPM Plant Preservative Mixture TBA Tertiary butyl alcohol (2-methyl propan-2-ol) DPX-4 [189-(2-chloro-N-(4-methoxy-1,3,5-triazin-2-yl mountant amino carbonyl) benzene sulphanamide)] v ABSTRACT Pongamia pinnata (L) Pierre (Syn: Pongamia glabra. Vent), an oil producing tree legume, popularly known as Karanja, is the most widely available non-edible oil tree in India. This multipurpose tree species is well adapted for various agro climatic conditions and is one of the most extensively chemically investigated plants. Potential of the seed derived oil of Pongamia as a substitute for diesel is recognized and often identified as "Bio-diesel". A large number of bioactive compounds have been isolated and characterized from various parts of this tree (flavonoids, furanoflavonoids), which has many industrial and medicinal uses. Pongam oil resembles groundnut oil in its fatty acid composition. However, due to presence of substances like Karanjin and Pongamol, the oil is non-edible. Biotechnological approaches offer the potential to alter qualitative and quantitative improvement in the products obtained from trees. By subjecting to these approaches, Pongamia can serve as a source of high quality fuel and raw material for industries. Raising plantations of clonally propagated, high seed yielding trees will contribute to increased seed production. These plantations will also serve the purpose of seed orchard for production of superior quality seeds. Pongamia tree can also be exploited as an alternate source of edible oil by silencing the related genes, which cause production of undesirable substances. From the existing literature it is apparent that there is a lot of scope for genetic modification of Pongamia for which there is need to develop a reproducible in vitro regeneration system. There is no literature on in vitro regeneration or tissue culture studies in this species. Keeping in view, the recalcitrant nature of leguminous tree species, standardization of protocols for in vitro morphogenesis remains a challenging proposition. To date, there is no literature available regarding the molecular scaffold of this tree. There is a need to undertake a preliminary study to assess the variability/ similarity among the individual tree species as well as the fidelity of the tissue culture raised plantlets. vi The present research work entitled “Tissue Culture Studies and Molecular Characterization of Pongamia (Pongamia pinnata (L.) Pierre), an oil producing tree legume” is designed to address some of the limitations. 1. Optimization of conditions for propagation using juvenile tissues. 2. Optimization of conditions for clonal propagation using mature nodal buds. 3. Optimization of protocol for de novo morphogenesis (organogenesis and somatic embryogenesis). 4. Assessment of variability/similarity among the trees and fidelity of the in vitro raised clones using ISSR markers. The thesis is divided into six chapters followed by summary and list of references. CHAPTER 1: General Introduction This chapter covers the literature on the nature of Pongamia, chemical constituents and its industrial and chemical applications and importance of Pongamia. It also includes the literature on the current status of research on in vitro morphogenesis in tree legumes.