In vitro studies on growth and morphogenesis in selected medicinally important Cassia spp. THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF DOCTOR of Philosophy IN BOTANY BY SHAHINA PARVEEN Department of Botany Aligarh Muslim University Aligarh, UP (INDIA) 2012 Dedicated To My Parents Plant Biotechnology Dr. Anwar Shahzad Ph.D. (AMU) Laboratory, Department of Assistant Professor Botany, Aligarh Muslim DST Young Scientist University , Aligarh-202002 Member Academic Council, UP (India) AMU [email protected] [email protected] Dated: ………………. Certificate This is to certify that the thesis entitled “In vitro studies on growth and morphogenesis in selected medicinally important Cassia spp.” 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. Shahina Parveen under my guidance and supervision and has not been submitted in part or full for the award of any other degree of this or any other university. (DR. ANWAR SHAHZAD) (Supervisor) Contact no. +91- 9837061683 ACKNOWLEDGEMENTS Begins with the name of Almighty Allah the most beneficent and merciful. My all praises are for Almighty Allah, for giving me this glorious opportunity to share my efforts with you in the form of thesis and blessing me with the strength to accomplish this goal. At this moment of accomplishment, I am deeply appreciative and indeed indebted to my supervisor, Dr. Anwar Shahzad, Assistant Professor, Department of Botany, A.M.U. Aligarh, for his valuable suggestions, constant vigilance, persistent patience and timely help which has made this work possible. It is no exaggeration to say without his generous sharing of insight, experiences and encouragements this thesis could not have been in its present form. I sincerely express my deepest sense of gratitude to Prof. Mohammad Anis, Chairman, Department of Botany, A.M.U. Aligarh, for his indispensable guidance, encouragement and providing all the necessary facilities to complete the present work efficiently. It’s my immense pleasure to acknowledge Prof. Sayeed A. Siddiqui, former Chairman, Department of Botany, A.M.U. Aligarh, for their motivation and concern throughout the experimental work. I am delighted to express my thanks to my seniors Dr. Mohammad Faisal (Assistant Professor, KSU Riyadh), Dr. Naseem Ahmad (Young Scientist, DST-Fast Track) and Dr. Iram Siddique (Assistant Professor, KSU Riyadh) for their critical suggestions, guidance and co-operation throughout the work. I am highly thankful to my colleagues Dr. Aastha Sahai, Dr. Shiwali Sharma, Ms. Arjumend Shaheen (Project Fellow - UGC) and Ms. Taiba Saeed (Research Assistant - UPCST) for their untiring help, co-operation, support and providing conducive environment to sort out the intricacies and complications of my work. i My special thanks to my friends Ms. Ankita Varshney, Dr. Anushi Arjumend Jahan, Dr. Zeba Khan, Mrs. Honey Gupta, Mrs. Razzaquia Khan, Mrs. Seemab Mukhtar, Dr. Hamid Iqbal, Mr. Abid Rafiq, Dr. Faheem Ahmad and Mr. Hilal Ahmad for all their support, encouragement, understanding and for being with me through thick and thin. My heart-felt thanks to all my labmates Ms. Nigar Fatima, Ms. Ruphi Naaz, Ms. Shahnaz Perveen, Ms. Afshan Naaz, Mr. Imran Khan, Mr. Rafique Ahmad and Mr. Saad Bin Javed. Finally with deep adoration, I acknowledge my Parents, elder brothers (Mr. Mohammad Saleem, Mr. Mohammad Sadiq, Mr. Abdul Majid, Mr. Mohammad Sajid) and younger sister Ms. Shazia Hasan for their love, affection, inspiration, blessings and moral support without which this work would have been impossible. I take this opportunity to sincerely acknowledge the University Grants Commission (UGC) for providing financial assistance under the scheme of NON-NET Fellowship and Maulana Azad National Fellowship (MANF) in the form of Senior Research Fellowship (SRF). Dated: (Shahina Parveen) ii ABBREVIATIONS ABA : Abscisic acid AdS : Adenine sulphate AgNO3 : Silver nitrate B5 : Gamborg et al. medium BA : 6-Benzyladenine CA : Chlorengenic acid CW : Coconut water ++ : Ca2 Calcium ion CaCl2∙2H2O : Calcium chloride (dihydrated) CH : Casein hydrolysate Chl : Chlorophyll cm : Centimeter °C : Degree centigrade DDW : Double distilled water 2,4-D : 2, 4-Dichlorophenoxyacetic acid DPU : Diphenylurea EDTA : Ethylene diamine tetra acetic acid FAA : Formalin acetic acid g : Gram GA 3 : Gibberellic acid h : Hour HCl : Hydrochloric acid HgCl2 : Mercuric chloride 2iP : 2-Isopentenyl adenine IAA : Indole-3-acetic acid IBA : Indole-3-butyric acid IRGA : Infra-Red Gas Analyzer Kn : Kinetin L2 : Philips and Collins medium µg : Microgram µl : Microlitre iii µm : Micrometer µM : Micromolar M : Molarity mg : Milligram mg/g : Milligram per gram mg/l : Milligram per litre min : Minute ml : Millilitre mm : Millimeter mM : Millimolar MS : Murashige and Skoog medium N : Normality % : Percentage NAA : α-Naphthalene acetic acid NOA : Naphthoxy acetic acid NaCl : Sodium chloride + NH4 : Ammonium ion nM : Nanomolar - NO3 : Nitrate ion - OH : Hydroxyl ion PG : Phloroglucinol PGR : Plant growth regulator PN : Net photosynthetic rate RH : Relative humidity s : Second TDZ : Thidiazuron 2,4,5-T : 2,4,5-Trichlorophenoxyacetic acid UV : Ultraviolet V : Volt v/v : Volume by volume W : Watt w/v : Weight by volume WPM : Woody plant medium YE : Yeast extract iv CONTENTS Page no. ACKNOWLEDGEMENTS i-ii ABBREVIATIONS iii-iv CHAPTER 1 INTRODUCTION 1-14 1.1 India: A mega biodiversity centre 1.2 Plants in traditional medicine 1.3 Threat to medicinal plants 1.4 Conservation of plants through in vitro techniques 1.5 Plant description 1.5.1 Cassia angustifolia Vahl. 1.5.1.1 Distribution 1.5.1.2 Morphological characters 1.5.1.3 Medicinal uses 1.5.1.4 Active compounds 1.5.1.5 Commercial products 1.5.1.6 Other uses 1.5.1.7 Propagation 1.5.2 Cassia sophera Linn. 1.5.2.1 Distribution 1.5.2.2 Morphological characters 1.5.2.3 Medicinal uses 1.5.2.4 Propagation 1.6 Plant tissue culture studies on C. angustifolia and C. sophera till date 1.7 Objectives CHAPTER 2 REVIEW OF LITERATURE 15-65 2.1 A brief history of plant tissue culture 2.2 Micropropagation or in vitro propagation 2.3 Tissue culture studies in some medicinally important Cassia spp. 2.3.1 Shoot induction and multiplication 2.3.1.1 C. angustifolia Vahl. 2.3.1.2 C. auriculata L. 2.3.1.3 C. obtusifolia L. 2.3.1.4 C. alata L. 2.3.1.5 C. siamea Lam. 2.3.1.6 C. sophera Linn. 2.3.1.7 C. tora L. 2.3.2 Rooting and acclimatization 2.4 Different strategies of micropropagation in other plant species 2.4.1 Direct regeneration 2.4.1.1 Apical meristem/axillary bud proliferation 2.4.1.1.1 Effect of cytokinins and auxins on shoot multiplication 2.4.1.1.2 Effect of TDZ on shoot multiplication 2.4.1.1.3 Effect of subculture passages 2.4.1.2 Adventitious shoot regeneration 2.4.2 Indirect organogenesis 2.4.3 Somatic embryogenesis 2.5 Rooting in microshoots 2.5.1 In vitro rooting 2.5.2 Ex vitro rooting 2.6 Synthetic seeds 2.7 Acclimatization 2.8 Physiological studies 2.9 Different factors affecting in vitro regeneration 2.9.1 Nature of explant: source, type and age of the explant 2.9.2 Media composition 2.9.3 Sources of carbohydrate and their concentration 2.9.4 pH of the medium CHAPTER 3 MATERIALS AND METHODS 66-79 3.1 Plant material and explant source 3.2 Culture media 3.2.1 Inorganic nutrients 3.2.2 Organic supplements 3.2.3 Preparation of stock solutions of different media 3.3 Plant Growth Regulators (PGRs) or plant hormones 3.3.1 Preparation of stocks of different PGRs 3.4 Carbon and energy source 3.5 Adjustment of pH and gelling of the medium 3.6 Filling of the medium 3.7 Sterilization 3.7.1 Sterilization of the medium 3.7.2 Sterilization of glasswares and instruments 3.7.3 Sterilization of plant material (seeds) 3.7.4 Sterilization of laminar air flow hood 3.8 Inoculation of seeds and establishment of aseptic seedlings 3.9 Collection of explants and establishment of cultures 3.10 Rooting in microshoots 3.11 Hardening and acclimatization 3.12 Synthetic seeds 3.12.1 Explant source 3.12.2 Encapsulation matrix 3.12.2.1 Encapsulation of explants 3.12.3 Planting media and culture conditions 3.12.4 Low temperature storage 3.12.5 Ex vitro conversion of synthetic seeds into plantlets 3.13 Physiological studies 3.13.1 Chlorophyll and carotenoids estimation 3.13.2 Leaf gas exchange measurements 3.14 Histological studies 3.14.1 Fixation and storage 3.14.2 Embedding, sectioning and staining 3.15 Chemicals and glasswares used 3.16 Statistical analysis CHAPTER 4 OBSERVATIONS AND RESULTS 80-142 4.1 C. angustifolia Vahl. 4.1.1 In vitro seed germination 4.1.2 Direct shoot regeneration 4.1.2.1 Cotyledonary node (CN) explant 4.1.2.1.1 Effect of explant age on multiple shoot regeneration 4.1.2.1.2 Effect of cytokinins on multiple shoot regeneration 4.1.2.1.3 Effect of cytokinin-auxin combinations 4.1.2.1.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.1.2.1.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.1.2.1.5 Effect of different media 4.1.2.1.6 Effect of pH 4.1.2.1.7 Effect of sucrose concentrations 4.1.2.2 Nodal segment (NS) explant 4.1.2.2.1 Effect of explant age on multiple shoot regeneration 4.1.2.2.2 Effect of cytokinins on multiple shoot regeneration 4.1.2.2.3 Effect of cytokinin-auxin combinations 4.1.2.2.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.1.2.2.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot