MICROPROPAGATION OF SOME EDIBLE BAMBOO SPECIES AND MOLECULAR CHARACTERIZATION OF THE REGENERATED PLANTS A thesis submitted in fulfillment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY in BIOTECHNOLOGY By JASMINE BRAR (REGD. NO: 90700005) Department of Biotechnology Thapar University Patiala-147004, Punjab, India April, 2014 CANDIDATE’S DECLARATION I hereby declare that the work presented in the thesis entitled “Micropropagation of some edible bamboo species and molecular characterization of the regenerated plants” in fulfillment of the requirement for the award of the Degree of Doctor of Philosophy at the Department of Biotechnology, Thapar University, Patiala, is an authentic record of my own work during the period from July 2007 to April 2014, under the supervision of Dr. Manju Anand, Assistant Professor, Department of Biotechnology, Thapar University, Patiala and Dr. Anil Sood, Chief Scientist and Head, Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur (HP). The report has not been submitted for the award of any other degree or certificate in this or any other University. Place: Patiala Jasmine Brar Date:14.04.14 Dedicated to my Parents for their endless Love, Support and Encouragement ACKNOWLEDGEMENT This thesis is the completion of one stage of my pursuit of knowledge and has been kept on track with the support and encouragement of my well wishers, friends, colleagues and various institutions. I would like to express my deep sense of gratitude to my esteemed supervisor, Dr. Manju Anand, Assistant Professor, Department of Biotechnology, Thapar University for her valuable guidance, keen interest and constructive criticism rendered during the course of this work. I am thankful to her for editing the thesis meticulously offering valuable suggestions and giving it a technical perfection. I am indebted to her for making my dreams come true with her perceptiveness, able guidance, constant enthusiasm, valuable insight and unconditional support at all stages of work. I had a valuable and enriching experience with her which taught me a lot and will be cherished throughout the life. I wish to place on record my profound sense of gratitude to my co-supervisor, Dr. Anil Sood, Chief Scientist and Head, Biotechnology Division, CSIR-IHBT, Palampur for conceptualizing and developing the theme, for providing me the best laboratory facilities and for his support in all the stages of work. I am grateful for his patience, systematic guidance, editing of thesis and the efforts he made in training me in the scientific field. He nurtured my determination to find and realize my potential and always supported me at those times when i felt almost defeated by the circumstances. Just saying thank you will never repay his kindness. I am indebted to Dr. P.S. Ahuja, Director, CSIR-IHBT, Palampur for providing me the platform to grow in life and explore the scientific field. He has always been a guiding force with his visionary ideas and his help in various ways during the tenure of my work at IHBT, Palampur. I am thankful to Dr. Ram Kumar Sharma, Senior Scientist, CSIR-IHBT for providing me guidance on molecular biology work and gel doc studies. I am extremely thankful to Dr R.D. Singh, Scientist and Head Biodiversity Division, CSIR-IHBT, Palampur for his valuable guidance in statistical analysis without which many of the aspects of thesis would not have been completed. I express my regards to Dr. Madhu Sharma and Dr. Amita Bhattacharya, Principal Scientist, CSIR-IHBT, Palampur for their worthy and moral boosting suggestions from time to time. My thanks to Mr. Pavitra, Photographer for helping me in photography work. I also want to thank Mr. Om Prakash, Senior Technical Officer for his help in maintaining the plants in green house and fields. I am grateful to Librarian Mr. Mukthiar Singh and other Library staff including Mr. Sarvan and Mr. Dogra for always helping me in accessing the knowledge base available in the library. I express my gratitude to Dr. Prakash Gopalan, Director, Thapar University, Patiala for providing necessary support. My earnest thanks to Dr. Dinesh Goyal, Head of the Department and member of the Doctoral Committee for his constant encouragement and valuable suggestions. I express my thanks to Dr. Anil Kumar, Assistant Professor, DBT and member of the Doctoral Committee for his suggestions during presentations of progress reports. My special thanks to Pankaj Bhardwaj, Amrina Shafi, and Awadhesh Kumar Pal for their care, understanding, encouragement and suggestions in planning some experiments which further strengthened my resolve to complete this work. Words cannot express my thanks to all my friends especially Shammi, Reenu, Vanita, Mrigaya and Ankush whose friendship has made my life a wonderful and memorable experience. The kind co-operation of the technical staff is duly acknowledged. I am indebted to my parents for their love and support throughout my life. I thank them both for giving me courage to pursue my aim and held me strongly during the bad phases of my life. I always look for the last opinion from them without whom I think I don’t stand anywhere. My younger sister, Rosen and brother, Jiveshjit deserve my thanks as well for their care and prayers for me. I thank almighty for blessing me in the path of obtaining higher knowledge. Jasmine Brar ABSTRACT The present study was conducted on three important edible bamboo species namely Dendrocalamus membranaceus, Bambusa balcooa and B. bambos which have been listed as priority species by International Union for Conservation of Nature (IUCN) with an aim to establish an efficient and reproducible protocol for their large scale multiplication under in vitro conditions. The other objective was to assess the genetic fidelity of in vitro raised plants by molecular markers and to study their field performance. A comparative evaluation of various physiological and biochemical parameters was carried out to compare in vitro plants with hardened ex vitro plants. Different explants like nodal explants, apical buds and leaves were taken from precocious branches of field grown, healthy plants for initiating aseptic cultures in all the three species. Among the various explants tested, unexpanded buds from secondary and tertiary branches were found to be the best for raising cultures as they responded favourably to different media combinations besides being easier to handle. The collection time of explant for culture initiation greatly influenced the frequency of bud break and number of shoots produced. Explants collected during spring in the months of February to April gave best response in terms of increased bud break (> 90%), early shoot initiation and decreased contamination. Rainy season (June to August) depicted more than 80% bud sprouting but was not a preferred season as it had a direct influence on contamination rate (95%) and survival percentage of explants. Likewise, larger explants (more than 25mm) had a direct effect on culture initiation and took least time for bud sprouting. Bacterial contamination was the major problem encountered during the initiation of aseptic cultures which was solved by treating explants with streptomycin sulphate and tetracycline (0.02% each) in addition to sodium hypochlorite (15%) and mercuric chloride (0.1%) while carrying out disinfection. An efficient procedure for multiple shoot proliferation from axillary buds was achieved on cytokinins supplemented media. In D. membranaceus and B. bambos, best response for multiple shoots induction was achieved on Murashige and Skoog’s (1962) medium containing 4.4 µM of 6-Benzylaminopurine (BAP) alongwith 1.16 µM of Kinetin (Kn) forming 13.40 ± 1.5 and 21.70 ± 2.40 shoots respectively. In B. balcooa, highest shoot proliferation (19.8 ± 1.4 shoots) was obtained when 4.4 µM of BAP was used in conjunction with 0.53µM of Naphthalene Acetic Acid (NAA). Once the clusters of shoots were formed, small clumps of 3-4 shoots were excised and transferred onto fresh multiplication medium every 4 weeks for continuous shoot proliferation. Rooting is a major bottleneck while carrying out in vitro multiplication of bamboos. For root induction, in vitro raised shoots were divided into clumps of 3-4 shoots and transferred onto MS medium containing different auxins. In D. membranaceus, half strength MS medium containing 5.37 µM of NAA in conjunction with 4.4 µM of BAP gave the best rooting percentage of 65%. In B. balcooa, 76.6% rooting was observed on full MS medium supplemented with 16.11 µM of NAA. In the present investigation on B. bambos, rooting was difficult to achieve and has been a major problem to be worked out. Best rooting response of 60% was achieved on half strength MS medium supplemented with 9.80 µM Indole-3 Butyric Acid (IBA). An initial pulse treatment of coumarin (9 mg/l) for 10 days and then shifting the cultures to coumarin free medium improved the rooting percentage by 15 to 20%. In the present study, IBA and NAA proved effective for providing initial stimulus for in vitro rooting but advanced root growth was noticed only on transfer to auxin free medium. Propagule size used for recurrent multiplication of shoots as well as in vitro rooting was a crucial factor during present study. Shoot clumps each having three shoots rather than a single shoot were found to be most effective for shoot proliferation and in vitro rooting. In vitro raised plants of D. membranaceus, B. balcooa and B. bambos were carefully rescued from the vessels and were initially transferred to the plastic pots containing moist riverbed sand and covered with perforated plastic jars/covers to maintain high internal humidity and were kept for a period of 14 days in the growth room. Thereafter, they were transferred onto potting mixture containing sand: soil: farmyard manure (1:1:1) and shifted to green house under controlled conditions of low radiance and high RH where they depicted 67% survivability in D.