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Molecular Cloning of Korrigan and Sucrose Synthase Genes and Genetic Transformation of Eucalyptus for Cellulose Enhancement

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Molecular Cloning of Korrigan and Sucrose Synthase Genes and Genetic Transformation of Eucalyptus for Cellulose Enhancement Molecular cloning of korrigan and sucrose synthase genes and genetic transformation of Eucalyptus for cellulose enhancement A Thesis Submitted in fulfillment of the requirements for the award of degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY DIWAKAR AGGARWAL Regn. No. 90700006 Department of Biotechnology and Environmental Sciences THAPAR UNIVERSITY PATIALA-147004, INDIA AUGUST 2013 DECLARATION I hereby declare that the work which is being presented in this thesis “Molecular cloning of korrigan and sucrose synthase genes and genetic transformation of Eucalyptus for cellulose enhancement” submitted by me for the award of the degree of Doctor of Philosophy in the Department of Biotechnology and Environmental Sciences , Thapar University, Patiala, is true and original record of my own independent and original research work carried out under the supervision of Dr. Anil Kumar, Assistant Professor, Department of Biotechnology and Environmental Sciences , Thapar University, Patiala, India. The matter embodied in this thesis has not been submitted in part or full to any other university or institute for the award of any degree in India or Abroad. (Diwakar Aggarwal) ACKNOWLEDGEMENT In pursuit of this academic endeavor, I feel that I have been singularly fortunate because inspiration, guidance, direction, cooperation, love and care - all came in my way in abundance and it seems almost an impossible task for me to acknowledge the same in adequate term. Yes, I shall be failing in my duty if I do not record my profound sense of indebtedness and heart felt gratitude to my supervisor, Dr. Anil Kumar , Assistant Professor, Department of Biotechnology and Environmental Sciences (DBTES), Thapar University (TU), Patiala, who guided and inspired me in pursuance of this work. He nurtured my personality to adopt a socio-scientific temperament which enabled the metamorphosis of my raw skills into scientific expertise. It was his able supervision, advice, and guidance from the very early stage of this research as well as giving me extraordinary experiences through out the work which has resulted in fruitful outcome. Above all and the most needed, he provided me unflinching encouragement and support in various ways. His truly scientist intuition has made him as a constant oasis of ideas and passions in science, which exceptionally inspire and enrich my growth as a student and a researcher. I feel bereft of words to acknowledge his contribution to shape my academic perceptivity. I wish to express my deep sense of gratitude with reverence towards my project supervisor and revered teacher, Prof. M. Sudhakara Reddy , Professor and Head, Department of Biotechnology and Environmental Sciences, Thapar University (TU), Patiala, for providing inspiration, motivation, encouragement and financial support for the present work. I am highly indebted to him for his support and thought provoking suggestions during many exigent circumstances. His systematic approach and unconditional co-operation made me work hard. Indeed, I shall remain grateful to him forever. I am very thankful to Dr. N. Das , Associate Professor and Ex-Head, DBTES, TU, Patiala for providing suggestions, for thoughts and taking interest in the progress of this work since inception. I express my gratitude to Director as well as Dean (Research and Sponsored Projects), Thapar University, Patiala, for his encouragement and support during my research work in the Institute. ii Constructive suggestions and critical remarks of all the faculty members from DBTES towards amelioration of this work have been of immense help in the progress of this work. I am highly obliged to them for their support in carrying out present research work. I am thankful to Department of Biotechnology and Environmental Sciences (DBTES) and TIFAC-CORE at Thapar University, Patiala for providing necessary infrastructural support, assistance and co-operation. Here the time for the acknowledging friends comes. I was not sure to write this page as I didn’t want to end up with a long list of `cliché (which is, anyhow, exactly what I am going to do!). However, I decided to do it because there are plenty of people I want to thank and because I do think that most of the people, like me, enjoy reading their name in the acknowledgements. The cooperation received through my colleagues namely Pankaj Krishna, Santosh Karn, Achal, Giri, Balwant, Anshu, Raghu and other research scholars are thankfully acknowledged. A special word of thanks to all the members of tissue culture unit (Mr. Rajesh, Mr. Khem Raj, Mr. Sandeep, Mr. Baljeet and Mr. Bhupinder) of TIFAC- CORE, Thapar University, Patiala. I would like to convey my thanks to Mr. Manmeet Singh, Mr. Harish Madan and Mr. Ashwani for their cooperation. I also owe a word of thanks to Mr. Babban (Ram Naval) and Lallan Yadav for their help. I also thank those who could not find a separate name but helped me directly or indirectly. Financial Support in the form of SRF and Teaching Associateship from CSIR, New Dehli and Thapar University, Patiala is also duly acknowledged. Above all it’s my family members, who encouraged me to complete my Ph.D besides lots of odds and troughs in my life. I owe my personal reckoning of gratitude and benevolence to them for love, care, patience, inspiration, encouragement; motivation and constant driving force which has enabled me to complete my task. Date : 08.0813 Place : Patiala Diwakar Aggarwal iii DEDICATION I dedicate this thesis to my parents whom I have a deep respect, admiration and love for, and who taught me to be strong, to embrace life with enthusiasm, and to be generous and forgiving; to my wife who makes me a better person, gives me strength, and supports me all the way with his unselfish sacrifices and enduring love; to my son Krish who brings so much joy to my life. Thank you all! iv The following publications are the outcome of the present research work: Aggarwal D, Kumar A and Reddy MS (2010): Shoot Organogenesis from elite plants of Eucalyptus tereticornis . Plant Cell Tissue and Organ Culture 102:45-52 (IF: 3.63) Aggarwal D , Kumar A and Reddy MS (2011): Agrobacterium tumefaciens mediated genetic transformation of selected elite clones of Eucalyptus tereticornis. Acta Physiologiae Plantarum 33:1603–1611 (IF: 1.30) Aggarwal D , Kumar A, Sharma J and Reddy MS (2012): Factors affecting micropropagation and acclimatization of elite clone of Eucalyptus tereticornis Sm. In Vitro Cellular and Developmental Biology-Plant 48:521-529 (IF:1.13) CONFERENCE PRESENTATIONS Aggarwal D , Kumar A and Reddy MS (2010): Studies on shoot regeneration and genetic transformation in elite clones of Eucalyptus. National symposium on “Plant Cell Tissue and Organ Culture: The Present Scenario.” Centre of advanced study, Department of Botany, University of Calcutta, Kolkata. 3-5 March, 2010 Aggarwal D , Kumar A and Reddy MS (2012): Micropropagation of commercially important elite clone of E. tereticornis Sm . National symposium on “Emerging Trends in Biotechnology ” Department of Biotechnology, MM Modi College, Patiala.24th Feb., 2012 Aggarwal D, Kumar A and Reddy MS (2012) : Agrobacterium tumefaciens mediated genetic transformation of E tereticornis Sm . National seminar on “Plant Cell Tissue and Organ Culture: Emerging Trends.” Department of Botany, Aligarh Muslim University, Aligarh. 10-11 March, 2012 Aggarwal D , Kumar A and Reddy MS (2013): Biotechnological Approaches for the improvement of Eucalyptus. National Seminar on Plant Biotechnology “Plant Sciences: New Technologies, Conservation and Environment, Department of Botany, Aligarh Muslim University, Aligarh.23- 24 th February 2013. v Abbreviations % Percent 2,4-D 2,4-Dichlorophenoxy acetic acid B5 Gamborgs medium (Gamborgs et al. 1968) Abs Absorbance AFLP Amplified fragment length polymorphism BA 6-Benzyl adenine bp Base pair CaMV Cauliflower Mosaic Virus cDNA Complementary deoxyribonucleic acid CFL Cool Fluorescent Light cm Centimeter CTAB Cetyl Trimethyl Ammonium Bromide Dia Diameter DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid dNTPs deoxynucleotide Triphosphates EDTA Ethylene Diamine Tetraacetic Acid EGases Endo- 1,4- β -glucanases FAA Formalin–Acetic acid–Alcohol solution g Gram g Gravitational constant GUS β-Glucuronidase GA 3 Gibberellic acid h Hour ht Height IAA Indole-3-acetic acid IBA Indole-3-butyric acid IPA Indole-3-propionic acid IPTG Isopropyl- β –thiogalactoside ISSR Inter-simple sequence repeat kb Kilobase Kn Kinetin (N6-furfuryladenine) l Liter M Molar m Meter mg Milligram min Minute ml Mililitre mM Milli molar mm Millimeter MMLV Moloney murine leukemia virus MOPS 3-(N-morpholino) propanesulfonic acid MPa Megapascals mRNA Messenger RNA vi MS Murashige and Skoog medium 1962 NAA α-Naphthalene Acetic Acid ng Nanogram nm Nanometer nmol Nanomole nos Nopaline synthase nptII Neomycin phosphotransferase ºC Degree Celsius OD Optical density ORF Open reading frame PAR Photosynthetically Active Radiation PCR Polymerase chain reaction PGRs Plant growth regulator (s) pI Isoeletric point qRT-PCR Quantitative Reverse Transcription Polymerase Chain Reaction RAPD Random amplification of polymorphic DNA RFLP Restriction fragment length polymorphism RNA Ribonucleic acid rpm Rotation per minute rRNA Ribosomal ribonucleic acid RT Room Temperature RT-PCR Reverse Transcriptase Polymerase chain Reaction s Second S Svedberg SDS Sodium Dodecyl Sulphate TAE Tris-Acetate-EDTA TBE Tris-Borate-EDTA TDZ Thidiazuron TE Tris-EDTA Tris Tris-(hydroxymethyl)- aminomethane U Unit UV Ultra Violet V Volt v/v volume by volume w/v weight by volume WPM Woody Plant Medium (Lloyd & McCown 1980) X-gal 5-Bromo-4-chloro-3-indolyl-β-D-galactoside X-gluc 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid YEP Yeast Extract Peptone µg Microgram µl Microlitre µm Micrometer µmol Micrmole µM Micromolar vii TABLE OF CONTENTS Chapters Page No. 1. Introduction 1 2. Review of literature 12 3. Materials and methods 36 Results & Discussion 4. Micropropagation and shoot organogenesis of selected clones of Eucalyptus tereticornis 75 5. Development of Agrobacterium mediated genetic transformation protocol for Eucalyptus tereticorni s 103 6. Molecular cloning of korrigan and sucrose synthase genes from Populus deltoides 122 7.
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