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University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/77863 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. Delayed Bolting in Rocket for Improved Quality and Greater Sustainability Jemma Louise Taylor PhD – Plant and Environmental Sciences School of Life Sciences, University of Warwick September 2015 i Table of Contents A list of tables and figures .......................................................................................................vii Acknowledgements...................................................................................................................xi Declaration...............................................................................................................................xii Summary................................................................................................................................ xiii Abbreviations..........................................................................................................................xiv Chapter 1. Introduction..........................................................................................................1 1.1. Project background......................................................................................................1 1.2. Overall aim..................................................................................................................2 1.3. Objectives....................................................................................................................2 1.4. Introduction to rocket..................................................................................................2 1.4.1. Overview..............................................................................................................2 1.4.2. Phylogeny ............................................................................................................3 1.4.3. Cultivation............................................................................................................4 1.4.4. Taste and health ...................................................................................................5 1.4.5. Breeding...............................................................................................................6 1.4.6. Bolting..................................................................................................................6 1.5. Introduction to flowering ............................................................................................7 1.5.1. Overview..............................................................................................................7 1.5.2. Photoperiodic pathway.........................................................................................8 1.5.3. Autonomous pathway ........................................................................................10 1.5.4. Vernalization pathway .......................................................................................10 1.5.5. Age dependant pathway.....................................................................................11 1.5.6. Ambient temperature pathway...........................................................................12 1.5.7. Gibberellic acid pathway ...................................................................................12 1.5.8. Role of flowering pathway genes in crop species..............................................13 1.6. Project strategy..........................................................................................................14 1.6.1. Understanding the flowering pathway in rocket................................................14 1.6.2. Use of mutagenesis to delay the onset of flowering in rocket ...........................16 1.7. Elsoms Seeds Ltd and CASE award .........................................................................17 Chapter 2. Materials and Methods.......................................................................................19 2.1. Materials....................................................................................................................19 2.1.1. Arabidopsis ........................................................................................................19 2.1.2. Rocket (Diplotaxis tenuifolia)............................................................................19 2.1.3. Scoring plants for bolting...................................................................................20 2.1.4. Rocket pollination techniques............................................................................21 2.1.5. Seed threshing....................................................................................................23 ii 2.1.6. Rocket ethyl methanesulphonate (EMS) mutagenesis.......................................23 2.1.7. Growth conditions..............................................................................................26 2.2. Standard laboratory methods.....................................................................................28 2.2.1. DNA extraction..................................................................................................28 2.2.2. RNA extraction and cDNA synthesis ................................................................28 2.2.3. Primer design .....................................................................................................29 2.2.4. Polymerase Chain Reaction (PCR)....................................................................29 2.2.5. PCR product visualisation and purification .......................................................29 2.2.6. DNA sequencing................................................................................................30 2.2.7. GeneRacer™......................................................................................................30 2.2.8. Molecular cloning ..............................................................................................30 2.2.9. Functional complementation of Arabidopsis mutants .......................................32 2.2.10. Real time PCR gene expression analysis .......................................................33 Chapter 3. Flowering Time Gene Isolation .........................................................................35 3.1. Introduction ...............................................................................................................35 3.1.1. CONSTANS ......................................................................................................35 3.1.2. GIGANTEA.......................................................................................................36 3.1.3. FLOWERING LOCUS C ..................................................................................36 3.1.4. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 .........................37 3.1.5. FLOWERING LOCUS T ..................................................................................37 3.2. Methods.....................................................................................................................38 3.2.1. Gene isolation ....................................................................................................38 3.2.2. Functional complementation of Arabidopsis mutants .......................................39 3.2.3. Diurnal time course experiment.........................................................................40 3.2.4. Developmental time course................................................................................41 3.2.5. Gene expression analysis using real time PCR..................................................41 3.3. Results .......................................................................................................................41 3.3.1. CONSTANS (CO) .............................................................................................41 3.3.2. GIGANTEA (GI)...............................................................................................49 3.3.3. FLOWERING LOCUS C (FLC) .......................................................................52 3.3.4. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)............60 3.3.5. FLOWERING LOCUS T (FT) ..........................................................................64 3.4. Discussion .................................................................................................................86 3.4.1. CONSTANS ......................................................................................................86 3.4.2. GIGANTEA.......................................................................................................87 3.4.3. FLOWERING LOCUS C ..................................................................................89 3.4.4. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 .........................90 iii 3.4.5. FLOWERING LOCUS T and FT-like genes.....................................................91 Chapter 4. Investigating the Photoperiodic and Vernalization Responses in Rocket .........94 4.1. Introduction ...............................................................................................................94