Agrobacterium Tumefaciens-mediated Transformation of Tobacco (nicotiana Tabacum L.) Leaf Disks: Evaluation of the Co-cultivation Conditions to Increase Β-glucuronidase Gene Activity

………………………………………………………………………………….…...vii Chapter 1. Introduction .…………………………………………………………….……….....1 Chapter 2. Review of Literature ...…………………………………………………….………. 3 2.1 Molecular Mechanism of Agrobacterium-mediated DNA Transfer ……...........…3 2.2 Adoption for Plant Molecular Biology ………….……………………………...10 2.3 Agrobacterium-mediated Plant Transformation Protocol Development ….…...12 Chapter 3. Materials and Methods ……………………………….………………...........20 3.1 Materials ………………………………………………………………….……..20 3.2 Methods ………….……………………………………………………………...22 Chapter 4. Results ……………………….……………………………………………..........27 4.1 Effect of Temperature During the Co-cultivation ……………..……...…….....27 4.2 Comparison of Two β-glucuronidase Genes ….……………...…....………........28 4.3 Effect of Surfactant, Vacuum Pressure Strength and Duration …………….....32 4.4 Effect of Co-cultivation Durations and Bacteria Concentrations …..……...........39 4.5 Effect of Bacterial Pre-culture Conditions and Acetosyringone Concentrations .39 4.6 Effect of Wounding and Bacterial Removal ……………………...….…………44 4.7 Timecourse Experiment …….……………………………………..……………46 4.8 Evaluation of Antibiotic Selection Agents …………………...….…......……….46 Chapter 5. Discussion .……………………………………………………..............................49 References .………………………………………………………………….………...............52 Appendix. Phaseolin Promoter and Terminator Construction Scheme ……………..………...60 Vita .………………………………………………………………………………...…………67