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And Ex Vitro- Propagated Blueberry Plants At CHARACTERISTICS OF IN VITRO- AND EX VITRO- PROPAGATED BLUEBERRY PLANTS AT MORPHOLOGICAL, CHEMICAL AND MOLECULAR LEVELS By © JURAN CHANDRA GOYALI B. Sc. Ag., M. Sc. A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland January 2018 St. John’s Newfoundland and Labrador Canada This work is dedicated to my beloved wife Shikha Roy ii ABSTRACT The lowbush blueberry (Vaccinium angustifolium Ait.), a commercially important fruit crop in Canada and USA, is one of the richest sources of antioxidant metabolites which have highly potential to reduce the incidence of several degenerative diseases. The aim of this study is to investigate the effect of propagation on the morphological, chemical and molecular characteristics of blueberries. The study evaluated the genetic and epigenetic variation in micropropagated plants. A lowbush wild clone ‘QB9C’ and the cultivar ‘Fundy’ were studied after being propagated by conventional softwood cutting (SC), and by tissue culture (TC) using nodal explants. The antioxidant metabolites in leaves and fruits of both genotypes were investigated in different maturity stages. The TC-regenerated plants were grown more vigorously and produced higher number of stems, branches, and larger leaves compared to SC plants. However, TC plants of both genotypes produced less flowers and fruits compared with SC counterparts. Micropropagation influenced the synthesis of phenolic and flavonoid compounds, and their antioxidant activities in blueberry which were genotype specific. ‘QB9C’ plants were highly influenced by micropropagation for their phytochemical content and antioxidant capacity. Leaves contained substantially higher levels of polyphenolics, flavonoids and proanthocyanidins than berries. The total soluble phenolic and flavonoid content and reducing power of ferric ion were boosted in fruits of the micropropagated plants, whereas the levels of these metabolites and total antioxidant activity were decreased in the leaves of TC plants. Red leaves had higher phenolic and flavonoid content and antioxidant potential than the green leaves, and green fruits had higher levels of bioactive phytochemicals than semi-ripe and full ripe berries. In iii contrary, anthocyanin content increased with the advancement of fruit maturity. Molecular marker analysis with expressed sequence tag (EST)-simple sequence repeat (SSR) and EST-polymerase chain reaction (PCR) makers detected the identical monomorphic amplification profiles within the TC plants of each genotypes which confirmed their genetic integrity. Methylation sensitivity amplification polymorphism (MSAP) analysis demonstrated that TC plants of both genotypes had higher DNA methylation compared to SC plants. Discrete methylation polymorphism was observed among the tissue culture regenerated plants. These results indicate that although in vitro derived plants maintained trueness-to-type genetic makeup, tissue culture induces DNA methylation alterations and the possibility of involvement of these DNA fragments in the dynamic processes regulating plant growth and development under prevailing growth conditions. iv ACKNOWLEDGEMENTS Foremost I would like to extend my sincere and hearted appreciation to my supervisor, Dr. Samir C. Debnath for providing me with invaluable guidance and financial support throughout my Ph. D. programme. Through his guidance, vision and encouragement as a supervisor, I have had the opportunity to develop my ability as a graduate student, researcher and author. His insights were key in developing a direction for this research thesis and for my future career path. I am also grateful to my co-supervisor, Dr. Andrei Igamberdiev for his positive criticism and encouragement. He is an excellent advisor and a source of inspiration on my academic grounds. I would like to thank other members of my supervisory committee Dr. Natalia Bykova and Dr. Dawn Marshall for their ever- present support and insightful comments. I will forever be thankful to my mother whose blessing, kindness, and warm heart inspire me every day. I am always grateful to my brothers and sister for their moral support and blessings. I express my deepest appreciation to my wife Shikha Roy, my daughters Joyeeta and Sreejita who are my constant source of encouragement, and have understood the time taken from them in my devotion to this work. I am highly grateful to Dr. Bipul Hawlader and Krishna Roy who assisted me to put my step in such an excellent academic and professional environment in Canada. Their continuous encouragement and support have made my academic life enjoyable. I am very grateful to my parents-in-law for their inspiration and blessing to complete this programme successfully. I always thankful to Sima, Anup and Oishi for their encouraging support every day. v My sincere appreciation to the School of Graduate Studies for a fellowship throughout my programme. I also wish to extend my thanks to the Department of Biology and St. John’s Research and Development Centre (SJRDC) previous name Atlantic Cool Crop Research Centre for allowing me to use the resources for this study. Special thanks to Dr. Brian Staveley for helping me in DNA methylation project. I am also grateful to Dr. David Schneider for his help in using Minitab software. My sincere thanks to Neel Chandrasekara, Jay Shah and Poorva Vyas for sharing their knowledge and expertise. I am indebted to the wonderful research team at SJRDC who always made time to assist me with my research, despite having their busy schedule. Special thanks are owed to Sandy Todd and Cherry Dooley for their administrative support. I am grateful to Glen Chubbs, Darryl Martin and Sarah Leonard who helped me in maintaining plants in greenhouse and in performing laboratory analysis. Special thanks must be given to Carolyn Parsons for her expert photography. Last but not the least, my thanks go to my lab-mates specially Dong, Sapan, Dhrumit, Amrita and Devin for their valuable help and consultation. vi TABLE OF CONTENTS Abstract .............................................................................................................................. iii Acknowledgements ............................................................................................................. v Table of contents ............................................................................................................... vii List of tables ..................................................................................................................... xvi List of figures ................................................................................................................. xxiii List of abbreviations .................................................................................................... xxxiii CHAPTER 1 ....................................................................................................................... 1 Introduction and Overview.............................................................................................. 1 1.1 Introduction ........................................................................................................... 1 1.2 Overview ............................................................................................................... 4 1.2.1 Taxonomy of blueberry.................................................................................. 4 1.2.3 Blueberry types .............................................................................................. 4 1.2.3 Biology of blueberry ...................................................................................... 6 1.2.4 Health benefits of blueberry ........................................................................... 8 vii 1.2.5 Propagation of blueberry.............................................................................. 11 1.2.5.1 Sexual propagation................................................................................ 11 1.2.5.2 Asexual propagation ............................................................................. 12 1.2.5.2.1 Propagation by stem cutting........................................................... 12 1.2.5.2.2 In vitro propagation or micropropagation ...................................... 14 1.2.5.2.2.1 Propagation via axillary shoot proliferation ............................. 23 1.2.5.2.2.2 Adventitious shoot regeneration ............................................... 24 1.2.5.2.2.3 Somatic embryogenesis ............................................................ 26 1.2.5.2.3 Advantages of micropropagation ................................................... 26 1.2.5.2.4 Disadvantages of micropropagation .............................................. 27 1.2.6 Micropropagation and somaclonal variation in berry crop improvement ... 28 1.2.7 Morphological characteristics of blueberry plants propagated by different methods ................................................................................................................. 30 1.2.7.1 Characteristics of bush or plant stature ................................................. 30 1.2.7.2 Characteristics of leaf ........................................................................... 31 viii 1.2.7.3 Characteristics of flower and fruit ........................................................ 32 1.2.8 Plant phenolics ............................................................................................
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