EFFECT OF RADIATION ON VARIETAL IMPROVEMENT OF REGENERATED PLANTS OF LILIUM AND THEIR CHARACTERIZATION THROUGH MOLECULAR MARKERS _______________________________________________________________ A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY By FARAH ASLAM DEPARTMENT OF BOTANY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE, PAKISTAN 2016 CERTIFICATE This is to certify that the research work described in this thesis submitted by Ms. Farah Aslam to Department of Botany, Lahore College for Women University has been carried out under my direct supervision. I have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. I further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in complete fulfillment of the award of any other degree from any other institution or home or abroad. I also certify that the enclosed manuscript has been prepared under my supervision and I endorse its evaluation for the award of PhD degree through the official procedure of University. ________________ Name: Prof. Dr. Shagufta Naz Supervisor Date: Verified By ________________ Name: Prof. Dr. Farah Khan Chairperson Department of Botany Stamp _________________ Controller of Examination Stamp Date: ___________ Dedicated To Holy Prophet Hazrat (ﷺ) Muhammad My loving mother Uncle Khalid Pervaiz & my niece Maira ACKNOWLEDGMENTS “For instance, there is a tradition that one who treads a path in search of knowledge has his way paved to paradise by God as a reward for this noble deed” (Bukhari, Muslim) In the name of Allah, most compassionate, Merciful. All praise and love be to Allah, Lord of world’s most kind and merciful, who is the entire source of knowledge delivered to mankind. My all efforts are nothing without blessing of Almighty Allah to complete my research work. My all love and respect for Holy prophet, Hazrat Mohammad (PBUH), who is forever a model of guidance and knowledge for all mankind eternally. I offer indebted thanks to acting Vice Chancellor, Prof. Dr. Uzma Qurashi for providing me excellent research facilities and support to continue my research. I am also very grateful to Prof. Dr. Farah Khan, Head of Botany department, Lahore College for Women University, for her valuable guidance and kind behavior during my PhD tenure. I would like to pay my regards and gratitude to my most respected teacher and research supervisor Prof. Dr. Shagufta Naz, Head of Biotechnology department, Lahore College for Women University, for her enthusiastic guidance, keen attention, continued interest, analytical ideas, critical suggestions, polite and forbidding behavior (where and when necessary) during my research work. Her advice and counsel were always a valuable addition to any decision making process and her encouragement always succeeded in helping me to reach the next milestone. Also, special thanks to my senior teachers Dr. Sumera Javed, Dr. Kiran Shahzadi, Dr. Saiqa Ilyas and Mrs. Amina Tariq for their kind support and technical help. I would like to appreciate my friends especially, Ambreen, Rukhama and also to Intiaz, Ayesha, Sannia and Noureen Ashraf, Research assistant, for her moral support, generous advices and cooperation. Words are inadequate to express my profound and warmest gratitude to my respected Ammi, Abu sisters and brothers Saba, Hafsa, Ali and Kashif bhai the love and inspiration of whom encouraged me at each step of life and their prayers made it possible for me to carry out my work progressively. No words to express heartfelt gratitude to my family. I would also like to express my gratitude to my little love, Maira, who give me hope in the life. I am also thankful to Iqra for her help during my research work. Thanks are extended to Abdul-Rehman, Atif, Ansar and Fouzia attendants of Plant Biotechnology lab and plant tissue culture laboratory, Bagh-e-Jinnah, Lahore. Last but not the least; I would like to show my greatest appreciation and sincere regards for whole team of Higher Education Commission, Govt. of Pakistan, for providing me scholarship as well as for their efforts, encouragement and guidance. Thanks Farah Aslam CONTENTS TitleTitles Page No. List of Tables i List of Figures v List of Abbreviations No No. xiv Abstract xvi Chapter 1 : Introduction 1 1.1: Aims and Objectives 5 Chapter 2: Review of Literature 6 2.1: Family Liliaceae 6 2.1.1: Lilium Genus 6 2.1.2: Habitat 6 2.1.3: Habit and Taxonomy 7 2.2: Economic importance 7 2.2.1: Used as ornamental plant 7 2.2.2: Medicinal importance 7 2.2.3: Nutritive value 8 2.2.4 Other uses 9 2.3: Plant tissue culture 9 2.3.1: Why plant tissue culture techniques 9 2.4: Induced mutagenesis 12 2.4.1: Why mutagenesis 12 2.4.2: Gamma irradiation 13 2.4.3: In vitro mutagenesis 14 2.6: Why molecular characterization 15 2.6.1: RAPD analysis 16 2.7.1: SSR analysis 18 Chapter 3: Materials and Methods 20 3.1: In vitro propagation by using plant tissue culture 20 technique 3.1.1: Plant material 20 3.1.2: Sterilization 21 3.1.3: Surface sterilization of explants 21 3.1.4: Glassware sterilization 21 3.1.5: Instruments sterilization 22 3.1.6: Laminar air flow cabinet sterilization 22 3.1.7: Sterilization of culture room and inoculation room 22 3.18: MS medium preparation and sterilization 22 3.1.9: Preparation of stock solutions of plant hormones 23 3.1.10: Explant inoculation 23 3.1.11: Study of parameters 23 3.1.11.1: Micropropagation 23 3.1.11.1.1: Shoot and root formation 23 3.1.11.1.2: Acclimatization of plantlets 24 3.1.11.1.3: Data recorded 24 3.2: Induced mutagenesis 24 3.2.1: In vivo mutagenesis 24 3.2.1.1: Gamma irradiation treatment 24 3.2.1.2: Maintenance of experiment under in vivo 24 conditions 3.2.1.3: Data analysis for morphological study 25 3.2.1.3.1: Morphological characters 25 3.2.1.3.2: Germination response, percentage 25 and survival rate 3.2.1.3.3: Number of leaves 25 3.2.1.3.4: Leaf length and leaf width 25 3.2.1.3.5: Shoot length (cm) 25 3.2.2: In vitro mutagenesis 26 3.2.2.1: Data analysis 26 3.2.2.1.1: Multiple shoot formation 26 3.2.2.1.2: Morphological study of regenerated 26 plantlets in green house 3.3: Molecular characterization of Lilium (Control and 26 irradiated genotypes) 3.3.1: DNA extraction 26 3.3.1.1: Determination of genomic DNA 27 3.3.2: RAPD analysis 27 3.3.2.1: Polymerase Chain Reaction (PCR) for RAPD 27 analysis 3.3.2.1.1: Data analysis for RAPD 28 3.3.3: SSR analysis 28 3.3.3.1: Polymerase Chain Reaction (PCR) for SSR 28 analysis 3.3.3.1.1: Data analysis for SSR 29 3.4: Statistical analysis 29 30 Chapter 4: Results 4.1: Micropropagation of different Lilium Cultivars 30 4.1.1: Oriental (O) group 31 4.1.1.1: In vitro propagation of Oriental cultivars 31 4.1.2: Oriental x Trumpet (OT) group 37 4.1.2.1: In vitro propagation of Oriental x Trumpet 37 cultivars 4.1.3: Longiflorum x Oriental (LO) group 43 4.1.3.1: In vitro propagation of Longiflorum x 43 Oriental cultivar 4.1.4: Longiflorum (L) group 46 4.1.4.1: In vitro propagation of Longiflorum cultivar 46 4.1.5: Root initiation 49 4.1.6: Acclimatization of mericlones 52 4.2: Induced mutagenesis 55 4.2.1: Oriental (O) group 56 4.2.1.1: In vivo mutagenesis 56 4.2.1.1.1: Morphological study of Oriental 56 cultivars 4.2.1.2: In vitro mutagenesis 67 4.2.1.2.1: Shoot multiplication 67 4.2.1.2.2: Studies on morphology of M1V4 and 76 M1V5 generation of in vitro derived mutants 4.2.2: Oriental x Trumpet (OT) group 85 4.2.2.1: In vivo mutagenesis 85 4.2.2.1.1: Morphological study of Oriental x 85 Trumpet cultivars 4.2.2.2: In vitro mutagenesis 95 4.2.2.2.1: Shoot multiplication 95 4.2.2.2.2: Studies on morphology of M1V4 and 103 M V generation of in vitro derived 1 5 mutants 4.2.3: Longiflorum x Oriental (LO) group 112 4.2.3.1: In vivo mutagenesis 112 4.2.3.1.1: Morphological study of Longiflorum 112 x Oriental cultivar 4.2.3.2: In vitro mutagenesis 116 4.2.3.2.1: Shoot multiplication 116 4.2.3.2.2: Studies on morphology of M1V4 and 121 M1V5 generation of in vitro derived mutants 4.2.4: Longiflorum (L) group 126 4.2.4.1: In vivo mutagenesis 126 4.2.4.1.1: Morphological study of 126 Longiflorum cultivar 4.2.4.2: In vitro mutagenesis 130 4.2.4.2.1: Shoot multiplication 130 4.2.4.2.2: Studies on morphology of 135 M1V4 and M1V5 generation of in vitro derived mutants 4.3: Molecular characterization by using random 140 amplified polymorphic DNA (RAPD) markers 4.3.1: Oriental (O) group 141 4.3.1.1: Molecular characterization of mutants by 141 using RAPD markers 4.3.1.2: Phylogenetic relationship among mutants 149 4.3.2: Oriental x Trumpet (OT) group 151 4.3.2.1: Molecular characterization of mutants using 151 RAPD markers 4.3.2.2: Phylogenetic relationship among mutants 159 4.3.3: Longiflorum x Oriental (LO) 161 4.3.3.1: Molecular characterization of mutants by 161 using RAPD markers 4.3.3.2: Phylogenetic relationship among mutants 164 4.3.4: Longiflorum (L) group 165 4.3.4.1: Molecular characterization of mutants by 165 using RAPD markers 4.3.4.2: Phylogenetic relationship among mutants 168 4.4: Molecular characterization by simple sequence 169 repeats (SSR) markers 4.4.1: Oriental (O) group 170 4.4.1.1: Molecular characterization of mutants by 170 using SSR markers 4.4.1.2: Phylogenetic relationship among mutants 176 4.4.2: Oriental x Trumpet (OT) group 178 4.4.2.1: Molecular characterization of mutants using 178 SSR markers 4.4.2.2: Phylogenetic relationship among mutants 184 4.4.3: Lilium Longiflorum