In vitro studies on morphogenesis and conservation of some threatened medicinal plants THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF DOCTOR Of PhilOsOPhy IN BOTANY BY SHIWALI SHARMA Department of Botany Aligarh Muslim University Aligarh (INDIA) 2011 Plant Biotechnology Lab., Dr. Anwar Shahzad Ph.D. (AMU) Department of Botany, Assistant Professor Aligarh Muslim University DST Young Scientist Aligarh-202002, UP, India Member Academic Council, [email protected] AMU [email protected] Dated: ………………. CertifiCate This is to certify that the thesis entitled “In vitro studies on morphogenesis and conservation of some threatened medicinal plants” submitted for the award of the degree of Doctor of Philosophy, embodies the original research work carried out in the Plant Biotechnology Laboratory at the Department of Botany, Aligarh Muslim University, Aligarh by Ms. Shiwali Sharma under my guidance and supervision and has not been submitted in part or full for the award of any other degree or diploma of this or any other university. (DR. ANWAR SHAHZAD) (Research Supervisor) Contact no. +91- 9837061683 Acknowledgement Begins with the name of GOD who is our creator and sustainer my all praise goes to almighty GOD, the most beneficent and merciful for bestowing and blessing me during the completion of this work. I consider it my right to communicate profound and overwhelmed instinct of gratitude and indebtedness to my esteemed Supervisor Dr. Anwar Shahzad, Assistant Professor, Plant Biotechnology Laboratory, Department of Botany for his insight sagacity, insinuation of belief, compassion, commiseration, critical suggestions, persuasion and continued interest during the present work. Despite his busy schedule, he gave me liberty to encroach his valuable time. It is immense pleasure for me to express my thanks to Prof. M. Anis, the Chairman and Head of Plant Biotechnology Laboratory, Department of Botany for his indispensible guidance and providing all necessary and possible facilities required to conduct the experiments. He has been a constant source of strength and inspiration to me throughout the execution of this study. My special “Vote of Thanks” goes to Prof. Sayeed A. Siddiqui, founder of Plant Tissue Culture & Experimental Embryology Laboratory in our Department for his best wishes from beginning to the completion of this thesis. I wholeheartedly acknowledge my seniors, Dr. Naseem Ahmad (Young Scientist, DST- Fast-Track), Dr. Iram Siddique (RA-CSIR), Ankita Varshney and Anushi A. Jahan for their help and support during the crucial stages of my research work. Continuous encouragement and valuable suggestion I received from my friends, Aastha Sahai (SRF-CSIR), Shahina Parveen (SRF-MANF UGC), Neelu Singh, Kavita Parihar, Nigar Fatima, Ruphi Naaz and Shahnaz Parveen. They always stimulated me to work hard. My colleagues, Taiba Saeed (RA-UPCST) and Arjumend Shaheen (PF-UGC) have striven hard to make the thesis error-free as possible. I am most thankful to both of them for their untiring help and support. In true sense, it will be unjustified if I would not acknowledge my friend, Pratush Raj Singh (B. Tech. & M.Tech., IIT-Delhi) for boosting my moral, strength and being a source of i never ending inspiration. Without his valuable cooperation at each stride during writing, this task could not have been possible. My sincere and heartiest thanks go to my Parents, brothers, Sachin & Arjun and nephew, Abhishek whose moral support, inspiration and affection sustained me during this work. I sincerely acknowledge various authors and publishers both inside and outside the country, to whom I have referred to in the text. Thus, my sincere gratitude goes to many personalities who inspired me to shape this thesis. Finally, financial support in the form of Research Fellowship in Science under the scheme of UGC, New Delhi for Meritorious student is greatly acknowledged. Dated: …….. (Shiwali Sharma) ii ABBREVIATIONS ADS : Adenine sulphate ABA : Abscissic acid + NH4 : Ammonium ion BA : 6-Benzyladenine ++ : Ca2 Calcium ion CA : Chlorengenic acid CaCl2·2H2O : Calcium chloride (dihydrated) CAT : Catalase CH : Casein hydrolysate Chl : Chlorophyll CCC : Chlorocholine chloride CKOx : Cytokinin oxidase CPPU : 1-(2-chloro-4-pyridyl)-3-phenylurea cm : Centimeter °C : Degree centigrade 2, 4-D : 2, 4-Dichlorophenoxyacetic acid DDW : Double distilled water DPU : Diphenylurea EDTA : Ethylene diamine tetra acetic acid FAA : Formalin acetic acid B5 : Gamborg’s medium (1968) GA : Gibberellic acid g : Gram Glu : Glutamine g l-1 : Gram per litre h : Hour Ha : Hectare H+ : Hydrogen ion HCl : Hydrochloric acid OH- : Hydroxyl ion IAA : Indole-3-acetic acid IBA : Indole-3-butyric acid iii IRGA : Infra Red Gas Analyzer 2-iP : (∆2-Isopentenyl) adenine Km : Kilometer Kn : Kinetin LS : Linsmaier and Skoog’s medium (1965) HgCl2 : Mercuric chloride µg : Microgram µl : Microlitre µm : Micrometer µM : Micromolar mM : Millimolar mg : Milligram ml : Millilitre mm : Millimeter mg dm-3 : Milligram per decimeter cube mg g-1 : Milligram per gram mg l-1 : Milligram per litre min : Minute M : Molarity MS : Murashige and Skoog’s medium (1962) NAA : α-Naphthalene acetic acid PN : Net photosynthetic rate NN : Nitsch and Nitsch medium (1969) - NO3 : Nitrate ion N : Normality % : Percentage PAA : Phenyl acetic acid PG : Phloroglucinol PGR : Plant growth regulator PPM : Plant preservative medium Polyox : Polyoxyethyelene glycol PLBs : Protocorm-like bodies RH : Relative humidity SA : Salicyclic acid iv SH : Schenk and Hilderbrandt’s medium (1972) NaCl : Sodium chloride AgNO3 : Silver nitrate 2, 4, 5-T : 2, 4, 5-Trichlorophenoxyacetic acid TDZ : Thidiazuron [N-phenyl-(1, 2, 3- thidiazol)-5-ylurea] TRIA : Triacontanol UV : Ultraviolet V : Volt v/v : Volume by volume W : Watt w/v : Weight by volume WPM : Woody plant medium (Lloyd and McCown 1980) YE : Yeast extract v CONTENT Page ACKNOWLEDGEMENT i-ii ABBREVIATIONS iii-v CHAPTER 1 INTRODUCTION 1-20 1.1 Biodiversity loss: crisis facing medicinal plants 1.2 Conservation strategies 1.3 Plant tissue culture 1.4 Synseed technology 1.5 Spilanthes acmella (L.) Murr. 1.5.1 Habit 1.5.2 Habitat 1.5.3 Botanical description 1.5.4 Chemical constituents 1.5.5 Medicinal importance 1.5.5.1 Traditional uses 1.5.5.2 Larvicidal and insecticidal activities 1.5.5.3 Antiobesity activity 1.5.5.4 Antifungal activity 1.5.5.5 Transmucosal behavior 1.5.5.6 Antiaging activity 1.5.5.7 Antiinflammatory and analgesic activities 1.5.6 Causes of its extinction and need of micropropagation 1.5.7 Work conducted and lacuna for further research 1.6 Spilanthes mauritiana DC. 1.6.1 Habit 1.6.2 Habitat 1.6.3 Botanical description 1.6.4 Chemical constituents 1.6.5 Medicinal importance 1.6.5.1 Traditional uses 1.6.5.2 Larvicidal and insecticidal activities 1.6.5.3 Antifungal activity 1.6.6 Causes of its extinction and need of micropropagation 1.6.7 Work conducted and lacuna for further research 1.7 Decalepis hamiltonii Wight and Arn. 1.7.1 Habit 1.7.2 Habitat 1.7.3 Botanical description 1.7.4 Chemical constituents 1.7.5 Medicinal importance 1.7.5.1 Traditional uses 1.7.5.2 Antimicrobial activity 1.7.5.3 Insecticidal activity 1.7.5.4 Other properties 1.7.6 Causes of its extinction and need of micropropagation 1.7.7 Work conducted and lacuna for further research 1.8 Objectives CHAPTER 2 REVIEW OF LITERATURE 21-51 2.1 Organogenesis 2.1.1 Meristem, shoot tip and nodal segment culture 2.1.1.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.1.2 Effect of urea-based cytokinins on shoot regeneration 2.1.2 Leaf culture 2.1.2.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.2.2 Effect of urea-based cytokinins on shoot regeneration 2.1.3 Cotyledon culture 2.1.3.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.3.2 Effect of urea-based cytokinins on shoot regeneration 2.2 Indirect organogenesis 2.3 Other factors influencing regeneration 2.3.1 Effect of different culture media on shoot regeneration 2.3.2 Effect of different carbon sources on shoot regeneration 2.3.3 Effect of different pH on shoot regeneration 2.4 In vitro rooting of microshoots 2.5 Synseed production 2.6 Acclimatization of plantlets CHAPTER 3 MATERIALS AND METHODS 52-62 3.1 Plant material and explant source 3.2 Culture media 3.3 Preparation of culture medium 3.3.1 Preparation of stock solutions 3.3.2 Plant growth regulators (PGRs) 3.3.3 Carbon and energy sources 3.3.4 pH adjustment and gelling of the medium 3.3.5 Medium filling 3.4 Sterilization 3.4.1 Sterilization of the medium 3.4.2 Sterilization of glass-wares, DDW and instruments 3.4.3 Sterilization of laminar airflow hood 3.4.4 Sterilization of seeds 3.5 Inoculation of sterilized seeds and germination 3.6 Culture establishment and shoot regeneration 3.7 Sub-culturing and shoot proliferation 3.8 In vitro rooting 3.9 Culture room conditions 3.10 Synseed production 3.10.1 Plant material 3.10.2 Encapsulation matrix 3.10.3 Encapsulation and in vitro germination 3.10.4 Low temperature storage 3.10.5 Direct or ex vitro sowing 3.11 Acclimatization of plantlets 3.12 Physiological study 3.12.1 Chlorophyll (a, b and total) and carotenoids content estimation 3.12.1.1 Procedure 3.12.1.2 Estimation 3.12.2 Net photosynthetic rate (PN) estimation 3.13 Histological study 3.13.1 Fixation and storage 3.13.2 Embedding and sectioning 3.13.3 Staining 3.14 Chemicals and glass-wares used 3.15 Statistical analysis CHAPTER 4 OBSERVATIONS AND RESULTS 63-115 4.1 Spilanthes acmella 4.1.1 Seed germination and collection of explants 4.1.2 Nodal segment culture