STUDIES ON SEED GERMINATION AND MICRO PROPAGATION OF TREMA ORIENTALIS (L.) BLUME AND MACARANGA PELT AT A (ROXB.) MULL. ARG. AND THEIR USE IN REVEGETATION OF MINE WASTELANDS A THESIS SUBMITTED TO GOA UNIVERSITY FOR THE AWARD OF THE DEGREE OF 5 8 1 • - # 7 DOCTOR OF PHILOSOPHY tfOM/SfU IN /O V)CX BOTANY 6* tfc FACULTY OF LIFE SCIENCE ^- i PUi> BY CASSIE R. RODRIGUES V* Research Guide PROF. B. F. RODRIGUES UGC-SAP DEPARTMENT OF BOTANY ) GOA UNIVERSITY December 2013 T — S 2.2- T - 62 - 2- <Dedicated to, M y Parents Mrs. Adetina g. (Rpdrignes Mr. A- Bosco <Rgdrigues M y g u id e Prof. (Bernard <F. (Rgdrigues DECLARATION I hereby declare that the matter embodied in this thesis entitled, “Studies on seed germination and micro propagation of Trema orientalis (L.) Blume and Macaranga peltata (Roxb.) Mull. Arg. and their use in revegetation of mine wastelands.” is the result of investigations carried out by me, under the supervision of Prof. B. F Rodrigues and it has not previously formed the basis for the award of any degree, diploma, associate ship, fellowship or the other such similar title. Goa University Ms. Cassie R. Rodrigues December 2013 (Candidate) CERTIFICATE This is to certify that the work incorporated in this thesis entitled, “Studies on seed germination and micro propagation of Trema orientalis (L.) Blume and Macaranga peltata (Roxb.) Mull. Arg. and their use in revegetation of mine wastelands.” submitted by Ms. Cassie R. Rodrigues, constitutes her independent work and the same has not been previously submitted for the award of any other degree, diploma, associate ship, fellowship or the other such title. Goa University December 2013 (Research Guide) Department of Botany Goa University ACKNOWLEDGEMENT I thank God for blessing me with strength and courage throughout the completion of this work and surrounding me with all wonderful people listed here. This Ph.D. thesis is an output of not only my passion for science but the valuable support extended to me by my guide Prof. B. F. Rodrigues and my parents. I am privileged to be given an opportunity to work under the guidance of Prof. B. F. Rodrigues, initially as a project research fellow and later as a Ph. D scholar. I am greatly indebted to my guide for seeing me though the challenging process of designing experiments, implementation, data collection, analyses and the synthesis of scientific research. His knowledge, scientific advice and persistent suggestions have helped me throughout the study period, as well as to put it all together in this thesis. He is my primary resource for getting my scientific questions answered. Most of all 1 wish thank Sir for the professional skills he instilled in me. His hardworking and tireless efforts balancing both routine academics to everyday problem solving are so inspiring. It’s been quite a learning experience for the past five years. I am also grateful to the Vice Chancellors’ nominee Dr. S. Ghadi, Department of Biotechnology, Goa University, for his encouraging comments and support throughout the study period. The road to my Ph. D started with the financial support extended by M/s Fomento Mines Pvt. Ltd. for the initial two years and I wish to expressly thank them for the funding. Also, I would like to thank UGC-MANF for the fellowship awarded, due to which this thesis has come to its completion. Some of the results described in this thesis would not have been obtained without collaboration with a few laboratories. I owe a great deal of appreciation and gratitude to Dr. Rahul Mohan, Scientist, NCAOR, for SEM imaging, Dr. Manish Tiwari, Scientist, NCAOR, for CHN analyses and the Director of National Centre for Antarctic and Ocean Research, Vasco, Goa for extending help through these facilities. I also wish to thank Mr. Vishwas Shirodkar, Soil testing laboratory, Goa Agriculture Department, Old Goa and Mr. Swapnil Suktankar, Soil testing laboratory, Goa Agriculture Department, Margao, for assisting with AAS analysis. Special thanks to Dr. Nandakumar Mekhot, HOD and Dean, Department of Management Studies, Goa University, for guiding me with data analysis using SPSS and Dr. Y. S. Valaulikar Head, Department of Mathematics, Goa University, for helping me with the statistical analysis. I would like to thank Prof. D. J. Bhat former HOD, Department of Botany, Goa University, for his constant encouragement and help throughout my work. I also wish to thank Prof. M. K. Janarthanam, Head Department of Botany, Goa University, for providing all facilities for the smooth conduct of my work. My sincere thanks to the other teaching staff of the Botany Department, Dr. P. K Sharma, Professor; Dr. V. Kerkar, Associate Professor; Dr. S. Krishnan, Associate Professor and Dr. N. M. Kamat, Assistant Professor for their help. A good support system is important for surviving and staying sane and to overcome all odds during research. 1 was blessed to be a part of this amazing, funny and cheerful group in our lab. M y special thanks to my colleagues from the lab Dr. Radhika Nair, Dr. James D’ Souza, Dr. Jyoti Vaingankar, Mr. Andy Willis, Ms. Seema Desai and Ms. Kim Rodrigues for their valuable support. My colleagues from the department have also been a great help. I thank Mr. M. Baskaran, Mr. Siddesh Naik, Mr. Anup Deshpande, Mr. Ravikiran Pagare, Ms. Sonashia Velho- Periera and Ms. Vera Maria Da costa. I am grateful to all my friends from other departments, especially Ms. Reshma Raut Desai from the Department of Physics, for her help and support throughout my work. I also thank Dr. Emilia Mascarenhas, Dr. Ashish Prabhugaokar and Dr. Pratibha Zalmi for their support. I wish to thank all non-teaching staff of the Department of Botany, Goa University and the University administrative staff for all their help. My family has contributed immensely towards the completion of my work in countless ways. Lastly, I take this opportunity to thank my parents, my brother and sister for being there throughout. Without their patience, encouragement and understanding, it would not have been possible. Cassie R. Rodrigues CONTENTS SR. CONTENTS PAGE NO. NO. 1. CHAPTER 1: Introduction 1-9 2. CHAPTER 2: Review of literature 10-37 3. CHAPTER 3: Study of seed dormancy and germination dynamics in Trema orientalis (L.) Blume and 38-55 Macaranga peltata (Roxb.) Miill. Arg. Introduction 38-40 Material and methods 40-46 Results 46-50 Discussion 50-55 CHAPTER 4: To develop and standardize efficient protocol for in vitro regeneration, multiplication and 4. 56-72 acclimatization for T. orientalis and Af. peltata. Introduction 56-57 M aterial and m ethods 57-62 Results 62-67 Discussion 68-73 CHAPTER 5: To study comparative growth and nutrient uptake in plants grown separately in mine reject and 5. 74-92 garden soil. Introduction 74-76 M aterial and m ethods 76-80 Results 81-86 Discussion 86-93 6. Summary 94-96 7. Bibliography 97-135 8. Synopsis 136-150 9. Appendices 151-159 LIST OF TABLES TABLE TITLE AFTER NO. PAGE NO. 1 Classes of seed dormancy 30 2 Characteristics of physiological seed dormancy-deep, 30 intermediate and non-deep 3 Logistic regression analysis predicting the effect of 46 different parameters on seed germination probability in T. orientalis. 4 Effect of chemical treatments on seed germination, 46 seedling length, vigour and imbibition time in T. orientalis. 5 Effect of storage time on germination in treated 48 seeds (cone. H2SO4 ; 15 min.) of T. orientalis. 6 Effect of pre-sowing treatments on seed 49 germination of M. peltata. 7 Logistic regression analysis predicting the effect of 50 different parameters on seed germination probability in M. peltata. 8 Effect of different concentrations of Kin, BA and TDZ in 63 combination with NAA on shoot regeneration from nodal explants of T. orientalis after 6 weeks of culture. 9 Effect of BA in combination with NAA, adenine sulphate 65 (AS) and coconut water (CW) on shoot regeneration from leaf calli of T. orientalis after 6 weeks of culture. 10 Effect of NAA on root development in T. orientalis shoots 65 after 3 weeks of culture on half strength MS medium. 11 Effect of growth regulators on shoot proliferation after 6 66 weeks of culturing M. peltata nodal explants on WPM medium. 12 Effect of different concentrations of BA with and without 67 2, 4-D on shoot regeneration from leaf calli of M. peltata after 8 weeks of culture. 13 Effect of IBA at different concentrations and AC on root 67 development in M. peltata shoots after 2 weeks of culture on half strength WPM medium. 14 Physico-chemical properties of mine spoil with 81 various amendments at the beginning of the experiment 15 Seedling growth in M. peltata in amended mine spoils 81 recorded after one year. 16 Nutrient status of M. peltata plants growing in amended 81 mine spoil. 17 Seedling growth in T. orientalis in garden soil and mine 84 spoil with and without amendments. 18 Nutrient status of T. orientalis plants growing in pure and 85 amended mine spoil and garden soil. LIST OF FIGURES FIG. TITLE AFTER NO. PAGE NO. 1 Map of Goa showing the mining belt adJoining forest area 1 2 Time course of maJor events associated with germination 28 and subsequent post germination growth. 3 Hormonal interactions during the regulation of seed 31 dormancy release and germination of (a) Nicotiana and (b) Brassica model species. 4 Germination curve depicting cumulative germination 47 percentage of T. orientalis seeds for different treatments. 5 Effect of storage time on seed viability of M. peltata. 49 6 Germination curve depicting cumulative germination 49 percentage of M. peltata seeds upon treatment with concentrated sulphuric acid for different time durations.