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Conservation and Assessment of Physico-chemical and Molecular diversity of the genetic resources of the genus Aloe occurring in Uttaranchal, India (Kumaon and Garhwal region) A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY RIJWAN NAJMODDIN PINJARI UNDER THE GUIDANCE OF Dr. (Mrs.) SHUBHADA R. THENGANE PLANT TISSUE CULTURE DIVISION NATIONAL CHEMICAL LABORATORY PUNE – 411 008, INDIA JULY 2010 I INTER-INSTITUTIONAL RESEARCH RESEARCH EMBODIES CARRIED OUT AT Herbal Medicine Division, Defence Agricultural Research Laboratory, Defence Research and Development Organisation, Pithoragarh, Uttaranchal. INDIA AND Plant Tissue Culture Division, National Chemical Laboratory (NCL) Pune, Maharashtra. INDIA. Research Guide Dr. (Mrs.) Shubhada R. Thengane Senior Scientist Plant Tissue Culture Division, National Chemical Laboratory, (N.C.L) Pune, INDIA II DEDICATED TO MY WIFE AND well wishers III CONTENTS Page Number ACKNOWLEDGEMENTS IX CERTIFICATE XI DECLARATION XII KEY TO ABBREVIATIONS XIII LIST OF TABLES XV LIST OF FIG URES XVII ABSTRACT 1- 4 Chapter 1: Introduction 5- 45 1.1 Introduction 1.2 Biodiversity: The Indian Scenario 1.2.1 Mega- Biodiversity hotspots 1.2.1 Trade of herbal drugs in India 1.3 The Himalayas 1.3.1 Western Himalaya 1.4 Uttaranchal (Uttarakhand) 1.5 The genus Aloe 1.6 Aloe barbadensis (Mill.) 1.6.1 Systematic position of Aloe barbadensis (Mill.) or Aloe vera (L.) 1.6.2 Chemical composition of Aloe barbadensis (Mill.) 1.7 Uses of Aloe species 1.8 Biotechnology of medicinal plants 1.8.1 Future frontiers 1.9 Genesis of thesis IV Chapter 2: Collection, Conservation, Authentication and Maintenance. 2.1 Introduction 46- 82 2.2 Materials and Methods 2.2.1 Collection 2.2.2 Conservation 2.2.3 Authentication 2.2.4 Development of Agro technology 2.2.5 Morphological parameters 2.2.6 Implementation of agro technology for mass cultivation at higher altitude 2.3 Results 2.3.1 Collection 2.3.2 Authentication 2.3.3 Morphological parameters 2.3.4 High altitude cultivation 2.4 Discussion 2.5 Conclusion 2.6 Applications Chapter 3: Nutraceutical assessment 83- 127 3.1 Introduction 3.2 Materials 3.3 Methods A). Preparation of sample B). Moisture content E). Biochemical parameters C). Biochemical parameters D). Minerals E). Aloin estimation Extraction of aloin V 1). Mechanical process 2). Chemical process F.) Solvent extracts preparation from whole leaf dried material Concentration of extracts Drying of extract G). Extraction from fresh leaves H). Processing and preservation of pulp Preservation of pulp or gel filet 1. Chemical preservation 2. Preservation of gel by precipitation 3. Preservation by lyophilizing filet 3.4 Results 3.5 Discussion 3.6 Conclusion and Application Chapter 4: In vitro evaluation of prebiotic and synbiotic potential 4.1 Introduction 128- 157 Probiotics, Prebiotics and Synbiotic Perspectives of Aloe 4.2 Materials 4.3 Methods Bacterial strains and growth conditions Basal media Maintenance of stock Inoculum Isolation of Bifidobacterium bifidum Preparation of gel solution Optimization of gel concentration Prebiotics assay Statistical analysis 4.4 Results VI 4.5 Discussion 4.6 Conclusion 4.7 Application Chapter 5: Assessment for antimicrobial activity of the extracts 5.1 Introduction 158- 171 5.2 Materials 5.3 Methods Samples Test organisms Inoculum Preparation of extract Determination MIC Antibacterial activity Statistical analysis 5.4 Results 5.5 Discussion 5.6 Conclusion 5.7 Application Chapter 6: Genetic diversity analysis by ISSR 172- 202 6.1 Introduction 6.1.1 Molecular markers and PCR analysis 1. Hybridization based (non-PCR) techniques 2. Arbitrarily primed PCR and other PCR based multi-locus profiling techniques PCR analysis for genetic diversity 6.1.2 ISSR markers 6.2 Materials 6.3 Methods VII A. DNA isolation protocol B. Quantification of DNA C. PCR protocol using ISSR primers Data analysis 6.4 Results Conservation implications 6.5 Discussion 6.6 Conclusion 6.7 Application Chapter No. 7 Summary and Future directions 203-209 7.1 Summary and Conclusion 7.2 Future Directions BIBLIOGRAPHY 210- 246 VIII ACKNOWLEDGEMENT It has been a great moment to place on record the sense of gratitude and support from bottom of my heart towards my Research Guide Dr. (Mrs.) Shubhada Thengane, Plant Tissue Culture Division, National Chemical Laboratory, Pune. Her crystal clear guidance, decision making power, enthusiasm and support for any inventive and quality work made this task easy and enjoyable. She didn’t leave any stone unturned for this work to be completed. My sincere thanks Dr. Puran Singh Rawat (DARL, Pithoragarh) and Dr. Narendra Kumar (Directorate of Life Sciences, DRDO, Delhi for encouraging me to initiate this work and constant support and motivation. I am thankful to Dr. Sivaram, Director and Dr. B.D. Kulkarni Dy. Director NCL, Pune for permitting me to register and perform the part of work here with world-class facilities. I am thankful to Dr. B. M. Khan (Head, PTC Div. NCL) and Dr. S. K. Rawal (Former Head, PTC Div.) for their timely help and guidance. Guidance and academic support by Prof. Dr. R. J. Thangane is also thankfully acknowledged hereby. It’s a time to offer gratitude to Late Dr. S. G. Patil, Dr. S. L. Shinde, Dr. B. C. Behera (ARI, Pune) for their valuable suggestions, untired efforts and standing by me in every task and situation. I am also thankful to Dr. D. K. Kulkarni and Mrs. V.A. Parashrami for their timely support. It has been an appropriate time to thank all my colleagues and lab mates in DARL, Shree Gaurishankar Verma, Shree Deendayal Thakur, Dr. Hemantkumar Pandey, Govind, Deepak and others who made my stay one of the most memorable and very admirable event in my life. I would like to place on record help and cooperation for DNA extraction facility by Dr. S. M. Gupta, DIBER, Haldwani and Dr. S. L. Laware, Head Dept. of Biotechnology, Fergusson College, Pune. IX I am very much thankful to Dr. Ravindra Patil, ARI and Dr. M. V. Kulkarni (University of Pune) for checking my thesis and help during the delicate phase of life and research. Valuable suggestion over thesis and support by them is also acknowledged. I would like specially thanks to my dear friends Mahesh, Girish, Raju, Bhuban, Rakesh, Vrushali, Dr. Nitasha, Dr. Kiran and Bharti for their restless efforts to come to a proper end during the work. Help and support from my colleagues Swapna, Varsha, Shweta, Reeta, Kanchan is hereby also acknowledged. My sincere thanks to Dr. M. M. Jana, Dr. D. C. Agrawal, Mr. A. B. Dhage, Mr. R Suryaprasad and all the staff of Plant Tissue Culture Division. It is difficult to imagine writing a thesis without the full support and understanding of the family. I express my heartfelt gratitude to my parents Mr. N.A Pinjari and Mrs. Raziya Pinjari and in laws Mr. B.S. Yadav and Mrs. Mithilesh Yadav for their constant support and encouragement. I express my warmest feelings for the understanding, courage, patience and for believing by my wife Nutan during completion of my thesis without which it would have been a dream only. Finally, I thank all my teachers, friends and well wishers, whose names may not have mentioned here, but whose good wishes and support have enabled me to complete this work. X CERTIFICATE This is to certify that the work incorporated in the thesis entitled “Conservation and Assessment of Physico-chemical and Molecular diversity of the genetic resources of the genus Aloe occurring in Uttaranchal, India (Kumaon and Garhwal region)” submitted by Mr. Rijwan N. Pinjari was carried out by the candidate under my supervision at the Plant Tissue Culture Division, National Chemical Laboratory, Pune. Any material obtained from other sources has been duly acknowledged in the thesis. (Dr. Mrs. S. R. Thengane) Guide Pune Date: XI DECLARATION BY THE CANDIDATE I hereby declare that the thesis entitled “Conservation and Assessment of Physico-chemical and Molecular diversity of the genetic resources of the genus Aloe occurring in Uttaranchal, India (Kumaon and Garhwal region)” submitted for Ph. D. degree to the University of Pune has not been submitted by me to any other university for a degree or diploma. Pinjari Rijwan Najmoddin, Plant Tissue Culture Division, National Chemical Laboratory, Pune- 411 008. Pune Date : XII KEY TO ABBREVIATIONS 0C- Degree Celsius. 2-D TLC – 2 Dimensional Thin Layer Chromatography. AAA – Amino Acid Analysis AOAC- Association of Official Analytical Chemists. CAM – Crassulecean Acid Metabolism cm- Centi meter DARL – Defenec Agricultural Research Laboratory DRDO – Defence Research and Development Organisation EDTA – Ethylene Diamine Tetra Acetic acid. FACE- Fluorophore-Assisted Carbohydrate Electrophoresis FPLC – Fats Performance/ Protein Liquid chromatography. GAP- Good Agricultural Practise. GC- MS – Gas Chrpmatography Mass Specroscopy GLC – Gas Liquid Chromatography. ICP/AES – Inductively Coupled Plasma Atomic Emission Spectrphotometry. IEF – Iso- Electric Focussing IR – Infra Red Mb- Milibar Min – Minutes MORA- Mandibular Orthopedic Repositioning Appliance. MP – Morse Potential MSL – Main Sea Level. NCIM- National Collection of Industrial Microorganisms. NMR – Nuclear Megnetic Resonance. OD – Optical Density ORAC – Oxygen Radical Absorbance Capacity. PAGE – Polyacrylaminde Gel Electrophoresis. PVP- Polyvinyl pyrrolidone RAPD- Rapid Amplified Polymorphic DNA. XIII RNase- Ribonuclease. Rpm- Rotations Per Minute. SCF Supercritical Fluid Extraction SDS- Sodium Dodesyl Sulphate SOD – Sulphur Oxide Dismnutase TBA – Tertiary Butyl Alcohol. TBE – Tris Borate EDTA TE- Tris EDTA TLC- Thin Layer Chromatography µ mole – Micro mole USD- United States Dollar UV- Ultra violet. WHO- World Health Organisation λ- Wavelength XIV LIST OF TABLES Table. No. Title Page No. Table No. 1/1 Climatic zones and altitudes of Western Himalayas 11 Table No. 1/2 Chemical composition of whole leaf of Aloe barbadensis 21 Table No. 1/3 Chemical composition of gel of Aloe barbadensis 22 Table No.
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