MICROPROPAGATION, BIOCHEMICAL AND MOLECULAR STUDIES OF ORCHIDS OF WESTERN GHATS OF GOA AND ITS ENVIRONS Thesis submitted to the Goa University for the Award of Degree of DOCTOR OF PHILOSOPHY IN t BOTANY P“-- ■ Po F-1 ,1 47 k By Govind V. Parab eV-6 1..6 s-ers Ce,4, coVre.1-4.--As s 4i1 6,424e.r- Department of Botany 4‘'`"\ Goa University, Goa - 403206 be. C. S'AreiZso VOttkos-rt.. India -r k))stvb-,e„,") May 2009 cp, s. o4-1 2-1 o 9 (CALick.) STATEMENT As required by the University Ordinance 0.19.8(ii), I state that the present thesis "Micropropagation, Biochemical and Molecular Studies of Orchids of Western Ghats of Goa and its Environs", is my original contribution and the same has not been submitted on any occasion for any other degree or diploma of this University or any other University/ Institute. To the best of my knowledge, the present study is the first comprehensive work of its kind from the area mentioned. The literature related to the problem investigated has been cited. Due acknowledgements have been made wherever facilities and suggestions have been availed of. Place: Goa (Govm V31- ra Date: 2 I o 420'05 Candidate CERTIFICATE As required by the University Ordinance 0.19.8(IV), this is to certify that the thesis entitled "Micropropagation, Biochemical and Molecular Studies of Orchids of Western Ghats of Goa and its Environs", submitted by Mr Govind V. Parab for the award of the degree of Doctor of Philosophy in Botany, is based on his original and independent work carried out by him during the period of study, under our supervision. The thesis or any part thereof has not been previously submitted for any other degree or diploma in any University or Institute. riltopsyv t2TeriTO (S. Krishnan) Research Guide Place: Goa Date: IA.105-12_00,3 ACKNOWLEDGEMENTS This endeavor of mine would not have been accomplished without the profound help and co-operation of many people. I take this opportunity to remember all of them with sincere pleasure and gratitude. I express my deep and sincere thanks to my guide Dr. S. Krishnan, Sr. Lecturer, Department of Botany, Goa University, for extending his valuable guidance throughout my work. His constant encouragement and help has enabled to complete this work. No word can fulfill his sincere commitment towards the completion of the research work. I would also like to express my gratitude to Prof. D. J. Bhat, Head, Department of Botany, Goa University, for providing me the facilities to carry out my research work. My sincere thanks to my co guide, Prof. M. K. Janarthanam for his help and valuable suggestions during this study. My sincere thank to subject experts Prof. P. K. Sharma and Prof. B. F. Rodrigues, for their valuable comments, suggestions and encouragement. I am also thankful to Dr. Vijaya U. Kerkar and Dr. Nandkumar Kamat, for their encouragement and support. I also thank to Prof. P. V. Desai, Dean, Faculty of Life Sciences & Environment and Chairman of FRC for his valuable suggestions, comments and support during this study. I am indebted to Dr. Ajay Parida, Director, Division of Biotechnology and Dr. K. R. Sivaprakash, Research Scholar, M. S. Swaminathan Research Foundation, Chennai, for providing laboratory facilities and help during study. I thank Dr. C. Sathish Kumar, Scientist, Tropical Botanical Garden and Research Institute, Pacha Palode, Trivandrum, for the confirmation of orchid identification. I am also thankful to Mr. S. S. P. Tendulkar, Director, Department of Agriculture, Govt. of Goa, for utilizing greenhouse facility available at Zonal Agriculture Office, Ela- Goa. Also it is my duty to thank Mr. S. Gandhi, Assistant Agriculture Officer, Zonal Agriculture Office, Ela-Goa for giving valuable suggestions during the research work. I thank The Western Ghat Secretariat, Planning Commission, New Delhi and UGC Special Assistance Programme (SAP) for their financial support to carry out the research work. I thank Mr. D. G. Tripati, Director, Tulip Dignostic Pvt. Ltd. Bambolim-Goa (TDPL) for his cooperation for completion of the research work. I also take this opportunity to thank Mr. R. B. Mane, Production Manager, (TDPL) for giving constant inspiration during completion of this work. My sincere thanks to Mr. Sarvesh P. Timblo, Director, Panduronga Timblo Industrias (PTI), Margoa-Goa for providing leave facility to complete this work. I also thank Mr. R. K. Singh, Sr. Manager (PTI) for his constant encouragement and inspiration for the completion of this work. I am grateful to Mr. M. K. Reddy, Deputy General Manager and Mr. Sushil Kukade, Assistant Manager, Dempo Mining Corporation Pvt. Ltd (DMC), Bicholim, Goa, for providing leave facility and their valuable support during writing of the thesis. I also thank Mr. S. N. Shanbhag (Manager, Personnal) Mr. S. C. Mandrekar, Mr. S. D. Dewari, Mr. Rishikumar, Mr. Dharmanand Pednekar, Mr. Daveedu Shaik (DMC) for their help for completion of writing of the thesis. I express my sincere thank to Mr. Ramchandra Gawas, Goa University Library, Mr. Vithal Naik and Mr. Gajanan P. Tari, Department of Botany, Goa University, for their constant assistance and support towards the completion of this work. I thank M. Baskaran, Shilpa J. Bhonsle, Sidhesh S. Naik research scholars and Anand Girap (M. Sc., student), Department of Botany, Goa University, for their help and support during this study. My special thanks to non-teaching and administrative staffs Mr. Suresh Fondekar, Mr. Krishna Velip, Ms. Anna D'souza and Ms. Gracy Godinho, Department of Botany, Goa University, for their kind co-operation and help. I thank Mr. Dhillan Velip, Research Assistant, Department of Botany, Goa University, for his help during the field trips. I would like to thank Harshala Gad, Radhika Nair, Jyosna Desai, Rupali Bhandari, Puja Gawas, Ashish Prabhugaonkar, Pratibha Jalmi, Research Scholars, for their help and support during this work. My special thanks to my cousin Mr. Pandarinath N. Parab, brother in law Mr. Jaidatt B. Gad and Mr. Atmaram K. Gaonkar for their constant inspiration and support during this study. I offer my thanks to my friends Mr. Rajesh J. Parab, Mr. Anant S. Gawas, Mr. Bhanudus R. Parab, Mr. Venkatesh M. Sinari, Mr. Babaji P. Sinari, Mr. Uday S. Sinari and Mr. Ganapati L. Parab, Residents of Amona, for giving encouragement, support and help during the period of research work. Last but not least, I express my thanks to my loving parents and relatives (Smt. Premavati K. Govenkar, Mr. Suryakant K. Govenkar, Mr. Gurudas K. Govenkar, Mr. Sameer D. Gawas) for their constant moral support, encouragement and help during the entire period of study. My whole hearted thanks to my wife (Nutan) and son (Omkar), who have been the source of encouragement and constant support for the successful completion of this research work. PAnnimnninnnntImpninmnnpm ,g6 Dedicated to the Memories of My eloved F ther Shri Vasitnt G. Parab mrAnnvpme4r. 11,* " ;00!' CONTENTS CHAPTERS PAGE NO 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 6 3. MATERIALS AND METHODS 29 4. RESULTS 46 5. DISCUSSION 86 6. CONCLUSIONS 105 7. SUMMARY 106 REFERENCES 109 APPENDIX ABBREVIATIONS Percent Plus or minus °C Degree centigrade Micro mole m Micrometer 111 Microliter BA 6-benzylamino purine bp Base pair About cv Cultivar CW Coconut water 2, 4-D 2, 4-Dichlorophenoxyacetic acid EDTA Ethylenediaminetetraacetic acid Fl Flower Grams Hour HC1 Hydrochloric acid IAA Indole-3-acetic acid ISSR Inter simple sequence repeat KIN Kinetin Km Kilometer kb Kilo base pair 1 Litre m Meter M Molar mA Milli ampere mg Milligram MI Mitra et al., medium min Minutes ml Milliliter mm Millimeter mM Millimolar mg L-1 Miligram per liter MS Murashige and Skoog medium N Normal NAA 1-Naphthaleneacetic acid NaOH Sodium hydroxide ng Nanogram nm Nano meter OD Optical density PGRs Plant growth regulators PLBs Protocorm- like bodies ppm Part per million psi Pounds per square inch RAPD Random amplified polymorphism DNA rPm Revolutions per minute RT Room temperature SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Sq. Km Square kilometer sec Seconds Var Variety UV Ultra violet Vern Vernacular v/v Volume by volume VW Vacin and Went medium w/v Weight by volume WAP Weeks after pollination LIST OF FIGURES Fig. 1. Map of Goa showing collection sites of orchid species. Fig. 2. Map of Goa showing collection sites of orchid populations for molecular and biochemical studies. Fig. 3. Response of coconut water on callus induction from immature seeds of Aerides maculosa. Fig 4. Response of cytokinin combinations and auxin and cytokinin combinations on PLB formation in Aerides maculosa on Vacin and Went medium supplemented with 15% coconut water. Fig. 5. Percentage survival of plantlets of Aerides maculosa. Fig. 6. Response of coconut water on callus induction from immature seeds of Rhynchostylis retusa. Fig. 7. Response of cytokinin combinations and auxin and cytokinin combinations on PLB formation in Rhynchostylis retusa on MS medium supplemented with 15% coconut water. Fig. 8. Percentage survival of plantlets of Rhynchostylis retusa. Fig. 9. Amplified and polymorphic bands in RAPD analysis of Aerides maculosa. Fig. 10. Percentage polymorphism in RAPD analysis of Aerides maculosa. Fig. 11. Dendrogram of Nei's genetic identities between the populations of Aerides maculosa based on RAPD data. Fig. 12. Amplified and polymorphic bands in ISSR analysis of Aerides maculosa. Fig. 13. Percentage polymorphism in ISSR analysis of Aerides maculosa. Fig. 14. Dendrogram of Nei's genetic identities between the populations of Aerides maculosa based on ISSR data. Fig.15. Detected and polymorphic band in SDS-PAGE analysis of Aerides maculosa. Fig.16. Percentage polymorphism in SDS-PAGE analysis of Aerides maculosa. Fig.17. Dendrogram of Nei's genetic identities between the populations of Aerides maculosa based on SDS-PAGE data.