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200916697 Apr2017.Pdf A metabolomics and transcriptomics comparison of Narcissus pseudonarcissus cv. Carlton field and in vitro tissues in relation to alkaloid production Aleya Ferdausi The University of Liverpool April 2017 Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Aleya Ferdausi Acknowledgements First, I would like to express my profound gratitude to my supervisor Dr Meriel Jones for her continuous support, motivation, suggestions, and guidelines to continue my project work smoothly. I am also grateful to her for giving critical evaluation on my thesis. I would also like to thank my secondary supervisor Professor Anthony Hall for his support and guidelines to lead me on the bioinformatics discovery. I would like to thank my PhD assessors Professor Martin Mortimer and Dr James Hartwell for their valuable suggestions and feedback on my annual project progress. My sincere acknowledgements also goes to Dr Xianmin Chang for his entire help throughout the major part of my research such as providing Narcissus bulbs, tissue culture and alkaloid analysis method development, calculations and data interpretations. Mark Preston, Centre of Proteome analysis for helping with GC-MS analysis and Dr Phelan Marie, NMR Centre, University of Liverpool for helping with NMR analysis. Dr Ryan Joynson, for his helps regarding transcript annotation. Centre of Genomic Research, University of Liverpool, for RNA-sequencing. Dr Jane Pulman for her helps to learn basic molecular biology techniques. My sincere thanks are given to Jean Wood, Senior Technician, Lab G, Institute of Integrative Biology. I am also grateful to all other staff, research groups, post-graduate students and all members of the Biosciences building, University of Liverpool for their friendly and humble attitude, which made me feel my work place like home. I also wish to express special thanks to Commonwealth Scholarship Commission, UK, and University of Liverpool, UK, for funding this PhD project. I am also thankful to Bangladesh Agriculture University, Bangladesh and Government, Peoples Republic of Bangladesh for allowing me the 4-years study-leave for pursuing my PhD. I would like to thank my friends Wazeera, Majida and Nirja. I am also grateful to my lovely pet birds Red, Blue and Chris. Special thanks to go Shams Khan for the daffodil plant drawing. Finally, those people who are everything to me, my parents, my two little sisters, Sharmin and Azmiri, my nephew, Saifan and my brother, Shuvo; without their support and love I would not be what I am today. With love, I dedicate my PhD thesis to my parents. i Table of contents: Page No. LIST OF FIGURES ............................................................................................................. ix LIST OF TABLES .......................................................................................................... .... xi ABSTRACT ..................................................................................................................... .... xii Abbreviations .................................................................................................................. .... xiii 1 Chapter One: Background .............................................................................. 1 1.1 The genus Narcissus ................................................................................................... .... 1 1.1.1 Growth cycle of Narcissus .................................................................................... .... 2 1.1.2 Narcissus morphology and development .............................................................. .... 3 1.2 Plant tissue culture ...................................................................................................... .... 3 1.2.1 Aspects of plant cell or tissue culture .................................................................... .... 4 1.2.2 Growth requirements for plant tissue culture ........................................................ .... 4 1.3 Plant secondary metabolism ........................................................................................ .... 5 1.3.1 Secondary metabolites........................................................................................... .... 5 1.3.2 Role of secondary metabolism in plants ................................................................ .... 6 1.3.3 Types of secondary metabolites ............................................................................ .... 6 1.3.3.1 Terpenes ............................................................................................................ 6 1.3.3.2 Phenolic compounds ......................................................................................... 7 1.3.3.3 Alkaloids ........................................................................................................... 8 1.3.4 Production of secondary metabolites in plant cell or tissue culture ........................... 8 1.3.5 Plant and plant tissue culture as a source of commercially important secondary metabolites ................................................................................................. 8 1.3.5.1 Pharmaceuticals ................................................................................................. 9 1.3.5.2 Commercial alkaloids ........................................................................................ 9 1.3.5.3 Other commercial products ............................................................................... 10 1.3.6 Problems associated with secondary metabolism in plant tissue culture ................... 10 1.3.7 Strategies for enhanced production of secondary metabolites ................................... 11 1.3.7.1 Organ culture ..................................................................................................... 11 1.3.7.2 Manipulation of nutrient or culture media......................................................... 12 1.3.7.3 Metabolic engineering ....................................................................................... 12 1.3.8 Molecular basis of plant secondary metabolism ........................................................ 13 1.4 Amaryllidaceae alkaloids ................................................................................................. 14 1.4.1 Types of alkaloids ...................................................................................................... 14 1.4.2 Pharmaceutical properties .......................................................................................... 15 1.4.3 Biochemical understanding of biosynthesis of Amaryllidaceae alkaloids ................. 16 1.4.4 Molecular biological understanding of biosynthesis of Amaryllidaceae alkaloids .... 17 1.4.5 Alkaloids in Narcissus ............................................................................................... 18 1.4.6 Galanthamine accumulation in plants ........................................................................ 19 1.4.6.1 In vivo (field) production .................................................................................. 19 1.4.6.2 In vitro (tissue culture) production ................................................................... 20 1.5 Metabolomics in plant secondary metabolism ................................................................. 21 1.5.1 Metabolomics ............................................................................................................. 21 1.5.2 Plant metabolomics .................................................................................................... 21 1.5.3 Plant metabolomic platforms ..................................................................................... 22 1.6 Transcriptomic study in the biosynthesis of plant secondary metabolites ....................... 23 1.6.1 Transcriptomic methodology ..................................................................................... 23 1.6.2 Transcriptomics in non-model plants ......................................................................... 23 1.6.3 Transcriptomic studies of Amaryllidaceae ................................................................. 24 1.7 Aims of the project ........................................................................................................... 25 ii Chapter Two: Tissue culture of N. pseudonarcissus cv. Carlton .................... 26 2.1 Introduction .................................................................................................................... 26 2.1.1 Difficulties with Narcissus propagation ..................................................................... 26 2.1.2 Plant parts (explants) for Narcissus tissue culture ..................................................... 27 2.1.2.1 Leaf explant culture .......................................................................................... 27 2.1.2.2 Scape (flower stem) culture .............................................................................. 27 2.1.2.3 Anther and ovary culture .................................................................................. 27 2.1.2.4 Chipping ........................................................................................................... 27 2.1.2.5 Twin-scaling ..................................................................................................... 28 2.1.3 Pre-culture treatments to break dormancy and reduce contamination....................... 28 2.1.3.1
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