(Solanum Lycopersicum Mill.) by SABIR HUSSAIN
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GENETIC TRANSFORMATION AND EXPRESSION ANALYSIS OF COLD TOLERANT GENE IN TOMATO (Solanum lycopersicum Mill.) BY SABIR HUSSAIN SHAH A dissertation submitted to The University of Agriculture, Peshawar in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (PLANT GENOMICS & BIOTECHNOLOGY) DEPARTMENT OF PLANT GENOMICS & BIOTECHNOLOGY PARC INSTITUTE OF ADVANCED STUDIES IN AGRICULTURE THE UNIVERSITY OF AGRICULTURE PESHAWAR, PAKISTAN AUGUST, 2014 TABLE OF CONTENTS CHAPTER NO. CONTENTS PAGE NO. ACKNOWLEDGEMENTS i ABBREVIATIONS iii LIST OF TABLES v LIST OF FIGURES viii LIST OF APPENDICES x ABSTRACT xiii I INTRODUCTION 1 II REVIEW OF LITERATURE 7 III MATERIALS AND METHODS 16 IV RESULTS 47 V DISCUSSION 131 VI SUMMARY, CONCLUSIONS AND 181 RECOMMENDATIONS LITERATURE CITED 186 APPENDICES 233 ACKNOWLEDGEMENTS The words become meaningless to express my deepest sense of gratitude to Almighty Allah, the most merciful, the most beneficent, Who created the universe, knows whatever is there in it, hidden or evident and Who bestowed upon me the intellectual ability and wisdom to search for the secrets. I must bow my head before Allah Almighty Who is compassionate and merciful and Who enabled me to complete this work, which marks an important turning point in my life. Cordial gratitude to the Holy Prophet Muhammad (Peace be upon him), who is forever a torch of guidance and knowledge for humanity. The words are inadequate to express my deepest sense of appreciation and devotion to my worthy supervisor Dr. Shaukat Ali, PSO/Associate Professor, NIGAB, NARC Islamabad. I am extremely grateful to his scholastic and sympathetic attitude, inspiring guidance, generous assistance, constructive criticism, timely advice and enlightened supervision in the accomplishment of this manuscript. I deem it utmost pleasure to avail this opportunity to express the heartiest gratitude and deep sense of obligation to Dr. Ghulam Muhammad Ali, CSO/Professor, Chairman Department of Plant Genomics & Biotechnology, PIASA and director NIGAB, NARC Islamabad for his obligation, well wishes, constructive criticism and encouragement during the course of my research studies. I am highly indebted to Dr. Muhammad Ramzan Khan PSO/Associate Professor NIGAB, NARC Islamabad for his sincere cooperation in my research work. I offer my earnest gratitude to Dr. Jalal-Ud-Din PSO/Associate Professor, Plant Physiology Program, Crop Sciences Institute, NARC Islamabad for his help, keen interest, support, vibrant supervision and moral encouragement during my research work. I acknowledge the profound gratitude of Dr. Sami Ullah Khan Assistant Professor Department of Agriculture, University of Haripur for his valuable consideration, enthusiastic guidance and keen concern in solving the problems during the course of my work. I am really thankful to Prof. Dr. Nadeem Akhtar Abbasi Chairman Department of Horticulture, Prof. Dr. Ishfaq Ahmed Hafiz and Abdul Ahad Qureshi Lecturer; i Department of Horticulture, PMAS Arid Agriculture University, Rawalpindi for providing the facilities for evaluation of cold tolerance in tomato transgenic lines. I wish to extend my thanks to Prof. Dr. Peter Stephen Baenziger, Institute of Agriculture and Natural Resources, Department of Agronomy and Horticulture University of Nebraska Lincoln, USA for his help, keen interest and support in the accomplishment of this manuscript. I would like to thanks to all of my teachers Dr. Armghan Shahzad, Dr. Aish Muhammad, Dr. Iqbal Hussain, Dr. Muhammad Iqbal, Dr. Iftikhar Ahmad and Dr. Umer Rashid for their help and support throughout my study. I would also like to thanks to my friends; Muhammad Ali Faqeer, Arshad Iqbal, Muhammad Amir Zia, Nadeem Ahmad, Zahid Hussain, Sohaib Roomi, Izhar Muhammad, Sohail Ahmad Jan, Aamir Rana, Ali Bahadur, Shafqat Ullah Khan Parova D. I. Khan, Muhammad Shahzaman and all of my colleagues for their special interest and encouragement during my research work. Among them, I am really grateful to Arshad Iqbal for his intellectual guidance, moral support and all-round help in writing this manuscript. I take as an opportunity to thank Muzaffar Ali, Equipment Technician and Lab Attendant, NIGAB for his miscellaneous help and excellent working environment in the lab. How can I overlook to articulate my admiration and unfathomable sagacity of appreciation from the core of my heart to my loving parents, brothers and sisters who always remembered me in their prayers and bestowed me with this blessing of higher education. May Allah bestow strength and contentment to all these splendid celebrities. I would feel incomplete without thanking to my maternal uncle Syed Ghulam Asghar Shah who filled me the love for seeking knowledge by teaching me in earlier classes; it is the fruit of his hard work, that I became able to get higher studies. I will remain grateful to him throughout my life. At the end, I would like to express my gratitude for officials of Higher Education Commission; Indigenous 5000 PhD Fellowship Program, Govt. of Pakistan for providing me financial assistance (vide letter no. 117-7583(Av7-118)/HEC/Sch/2011). This aid helped me to accomplish my research work in time with tangible achievements pubished in international journals. (Sabir Hussain Shah) ii ABBREVIATIONS % Percent µl Micro litre 1 O2 Singlet oxygen 2, 4-D 2, 4-dichlorophenoxyacetic acid ABA Abscisic acid AgNO3 Silver nitrate ANOVA Analysis of variance APX Ascorbate peroxidase AS Acetosyringone AsA Ascorbic acid BA 6-Benzyl adenine BAP 6-benzylaminopurine CaMV 35S Cauliflower mosaic virus 35 S CaMV Cauliflower mosaic virus CAT Catalase CBF3 Cold responsive-element binding factor 3 CBFs C-repeat binding factors CCM Co-cultivation medium Cf Cefotaxime CIM Callus induction medium CoCl2 Cobalt chloride CRD Completely randomized design CS Carbon sources CTAB Cetyl trimethyl ammonium bromide Cvs Cultivars DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DRE/CRT Dehydration responsive element/C-repeat DREB1A Dehydration responsive element binding factor 1A EC Electrical conductivity EDTA Ethylenediaminetetraacetic acid g Gram GA3 Gibberellic acid GB Glycinebetaine GM Germination medium GUS β-glucuronidase gene H2O2 Hydrogen peroxide HCl Hydrochloric acid hpt Hygromycin phosphotransferase IAA Indole-3-acetic acid IBA Indole-3-butyric acid IM Inoculation medium Kin Kinetin LB Luria broth iii Lip Lipase LSD Least significant difference MDA Malondialdehyde mg/l Milli gram per litre mM Milli mole MS Murashige and Skoog (basal medium) NAA Naphthalene acetic acid NADP+ Nicotinamide adenine dinucleotide phosphate NaOCl Sodium hypochlorite NOS Nopaline synthase NPT II Neomycin phosphotransferase gene NT Non-transgenic - O2 Superoxide radical OD Optical density OH Hydroxyl radical PCM Pre-culture medium PCR Polymerase chain reaction PEG Polyethylene glycol PGRs Plant growth regulators pH -log H+ POD Peroxidase PPO Polyphenol oxidase PSM Pre-selection medium RIM Root induction medium RM Regeneration medium ROS Reactive oxygen species RPM Revolutions per minute RT-PCR Reverse transcriptase polymerase chain reaction RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase RWC Relative water contents SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Shoot elongation medium SIM Shoot induction medium SM Selection medium SOD Superoxide dismutase Taq Thermus aquaticus T-DNA Transfer DNA TE Transformation efficiency T-G7 Terminator of gene 7 of Agrobacterium Ti-plasmid Tumour inducing plasmid TSS Total soluble sugars Vir-gene Virulence gene WM Washing medium YEP Yeast extract peptone ZEA Zeatin iv LIST OF TABLES Table Description Page No. No. 3.1 Type I callus induction media used for calli proliferation 19 3.2 Type II callus induction media used for calli proliferation 19 3.3 Type III callus induction media used for calli proliferation 19 3.4 Type I shoot induction media used for in vitro shoot regeneration 20 3.5 Type II shoot induction media used for in vitro shoot regeneration 20 3.6 Type III shoot induction media used for in vitro shoot regeneration 21 3.7 Type IV shoot induction media used for in vitro shoot regeneration 21 3.8 Type V shoot induction media used for in vitro shoot regeneration 21 3.9 Type VI shoot induction media used for in vitro shoot regeneration 26 3.10 Type VII shoot induction media used for in vitro shoot regeneration 26 3.11 Different types of culture media used for regeneration and transformation 33 4.1 Assessment of different concentrations of clorox on contamination 49 4.2 Assessment of clorox (40 and 50%) on germination frequency 49 4.3 Assessment of various explants on the basis of their regeneration 50 frequency and number of primordial shoots per explant 4.4 Assessment of various combinations of PGRs on callus induction 53 4.5 Assessment of AgNO3 in combination with various PGRs on callus 53 induction 4.6 Assessment of CoCl2 in combination with various PGRs on callus 57 induction 4.7 Assessment of various combinations of PGRs on in vitro shoot 57 regeneration 4.8 Assessment of AgNO3 in combination with various PGRs on in vitro 58 shoot regeneration 4.9 Assessment of CoCl2 in combination with various PGRs on in vitro shoot 63 regeneration 4.10 Assessment of synergistic effect of sucrose and sorbitol in MS basal 68 media without PGRs on in vitro shoot regeneration 4.11 Assessment of synergistic effect of sucrose and sorbitol in N6 basal 68 media without PGRs on in vitro shoot regeneration v 4.12 Assessment of synergistic effect of sucrose and sorbitol in MS basal 70 media enriched