CHAPTER 1 INTRODUCTION 1.1 Fruit 1.1.1 General introduction 1.1.2 Tomato 1.2 Fruit ripening 1.2.1 Colour, flavour and texture changes in fruit ripening 1.2.2 Ethylene in fruit ripening 1.3 Plant cell wall 1.3.1 General introduction 1.3.2 Cell wall components 1.3.3 Cell wall structure 1.4 Cell wall enzymes 1.4.1 Polygalacturonase (PG) 1.4.2 Pectate lyases 1.4.3 β-Galactosidase 1.4.4 Expansin 1.5 Pectinmethylesterase 1.5.1 PE proteins 1.5.2 PE modes of action and regulation 1.5.3 PE genes and isoforms 1 5.4 Tomato PE antisense lines CHAPTER 2 MATERIALS AND METHODS 2.1 Plant material 2.1.1 Tomato growth and maintenance 2.1.2 Crossing tomato 2.2 Chemicals 2.3 Fruit texture analysis 2.4 Biochemical methods 2.4.1 Extraction of crude protein from tomato fruits 2.4.2 Bio-Rad (Bradford) protein assay 2.4.3 Pectinesterase assay 2.4.4 Heparin affinity chromatography 2.4.5 PE profile assay 2.4.6 Concentration of pooled PE isoforms 2.4.7 Total leaf protein extraction for SDS PAGE analysis. 2.4.8 Making acetone insoluble solid (AIS) 2.4.9 Determination of degree of esterification (DE) of the pectin by titration 2.4.10 Extraction of CDTA Soluble Pectin 2.4.11 Determination of Neutral Sugars 2.4.12 Determination of uronic acid 2.5 Molecular biological methods 2.5.1 DNA extraction from tomato leaves 2.5.2 DNA measurement 2.5.3 Polymerase chain reaction (PCR) 2.5.4 Restriction enzyme digestion 2.5.5 Agarose gel electrophoresis 2.5.6 DNA sequence (MWG value read) 2.5.7 PCR product purification by Perfectprep Gel Cleanup Kit (Eppendorf): 2.6 Immunochemistry and immunolabelling 2.6.1 Immunodot assay
2.6.2 Immunolocalization 2.7 Statistics 2.8 Capillary Electrophoresis (CE) 2.8.1 Enzyme digests 2.8.2 CE CHAPTER 3 GENERATION OF DOUBLE ANTISENSE PLANTS 3.1 Plant generation 3.2 Generation of the double antisense plants 3.3 PE Isoform profilers of double antisense plants 3.3.1 PE extraction method optimisation 3.3.2 PE profiles in tomato fruit 3.4 Summary CHAPTER 4 CHARACTERIZATION OF PE SINGLE AND DOUBLE ANTISENSE PLANTS 4.1 Fruit PE activity,and morphology analysis 4.1.1 Generation of wild-type controls 4.1.2 Fruit PE activities 4.1.3 Fruit morphology and softening 4.2 Cell wall component analysis 4.2.1 UA in acetone insoluble solids (AIS) and CDTA soluble pectin 4.2.2 DE of pectin in AIS 4.2.3 Neutral sugar analysis by GC 4.3 Capillary electrophoresis (CE) of pectin 4.3.1 DE of CDTA soluble pectin 4.3.2 CE profiles of PG digested CDTA soluble pectin 4.4 Characterization of antisense lines using monodonal antibodies 4.4.1 Immuno-dot blot analysis of CDTA soluble pectin 4.4.2 Immunolocalization: Fruit pericarp 4.4.3 Immunolocalization: Stem 4.5 Summary 4.5.1 Biochemical and morphological study 4.5.2 Immunological study CHAPTER 5 USE OF ANTISENSE LINES FOR PE ISOFORMS ANALYSIS 5.1 Introduction 5.2 Effect of pH on PE activity 5.3 PE isoform profiling 5.3.1 Fruit 5.3.2 Stem 5.3.3 Leaf 5.4 Summary CHAPTER 6 OVERALL DISCUSSION 6.1 Role of PE during fruit ripening 6.2 PE isoforms in vegetative tissues 6.3 Unstable PE1 expression in double antisense lines. 6.4 Future work REFERENCES
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