by Lam Ying Hoo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 145-149). / Acknowledgements --- p.i / Abstract --- p.ii / Contents --- p.iv / Abbreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Trichosanthin --- p.1 / Chapter 1.2. --- Anti-plant viral and fungal properties of RIPs --- p.3 / Chapter 1.3. --- Agrobacterium-mediated transformation --- p.5 / Chapter 1.3.1. --- Ti (tumor inducing) plasmid --- p.6 / Chapter 1.3.2. --- Role of vir proteins in T-DNA transfer --- p.6 / Chapter 1.3.3. --- Integration of T-DNA into plant genome --- p.11 / Chapter 1.3.4. --- Use of Agrobacterium plasmid as transformation vectors --- p.13 / Chapter 1.4. --- Objective and strategy of producing transgenic plants that express TCS --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- Bacterial Strains used --- p.19 / Chapter 2.2. --- General Techniques --- p.20 / Chapter 2.2.1. --- Growth of bacterial strains --- p.20 / Chapter 2.2.2. --- Restriction Enzyme Digestion of DNA --- p.21 / Chapter 2.2.3. --- Agarose Gel Electrophoresis of DNA --- p.21 / Chapter 2.2.4. --- Purification of DNA fragments from Agarose Gel using GeneClean II® ( BIO 101 Inc.) kit --- p.22 / Chapter 2.2.5. --- Purification of DNA fragments by Phenol/Chloroform Extraction --- p.23 / Chapter 2.2.6. --- Ligation of DNA fragments --- p.24 / Chapter 2.2.7. --- Preparation and Transformation of Escherichia coli Competent Cells --- p.24 / Chapter 2.2.8. --- Minipreparation of Plasmid DNA --- p.26 / Chapter 2.2.9. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.27 / Chapter 2.2.10. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.29 / Chapter 2.2.11. --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.30 / Chapter 2.2.12. --- Western Blot detection of TCS --- p.33 / Chapter 2.2.13. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.3. --- Construction of Plant Transformation Vectors --- p.36 / Chapter 2.3.1. --- Construction of pSLJ58210 --- p.36 / Chapter 2.3.2. --- Construction of pSLJ TCS1 and pSLJ TCS2 --- p.38 / Chapter 2.3.3. --- Conjugation of pSLJ TCS1 and pSLJ TCS2 into A. tumefaciens by Triparental Mating --- p.41 / Chapter 2.4. --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens --- p.43 / Chapter 2.4.1. --- Growth of A. tumefaciens LBA4404 (pSLJ TCS1) --- p.43 / Chapter 2.4.2. --- Surface Sterilization of tobacco leaves --- p.43 / Chapter 2.4.3. --- Inoculation of tobacco leaf explants with A. tumefaciens LBA4404 (pSLJ TCS1) --- p.44 / Chapter 2.4.4. --- Regeneration of shoots from Transformed explants --- p.45 / Chapter 2.4.5. --- Rooting of Transformed shoots --- p.45 / Chapter 2.4.6. --- Re-establishment of cultured Plantlets in soil --- p.45 / Chapter 2.5. --- Analysis of the Regenerated Transgenic Tobacco --- p.46 / Chapter 2.5.1. --- Isolation of plant leaf protein --- p.46 / Chapter 2.5.2. --- SDS-PAGE and Western blot detection of TCS --- p.48 / Chapter 2.5.3. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.48 / Chapter 2.6. --- Bioassay of Inhibitory activity of TCS protein against TuMV --- p.49 / Chapter 2.6.1. --- Preparation of biologically active TCS protein --- p.49 / Chapter 2.6.2. --- Purification of TuMV from infected plant leaves --- p.51 / Chapter 2.6.3. --- Mechanical Inoculation of virus onto host plant --- p.53 / Chapter 2.6.4. --- Anti-viral assay on Local Lesion host --- p.54 / Chapter 2.6.5. --- Anti-viral assay on Systemic host --- p.55 / Chapter 2.7 --- Establishment of the plant culture medium for efficient Regeneration from tissue explants of Brassica parachinensis --- p.56 / Chapter 2.7.1. --- Preparation and Sterilization of culture medium --- p.57 / Chapter 2.7.2. --- Preparation of Sterile seedlings of B. parachinensis --- p.57 / Chapter 2.7.3. --- Regeneration from Cotyledon petiole and Hypocotyl segment explants --- p.58 / Chapter 2.7.4. --- Regeneration from Internode stem segment explants of shoot culture --- p.60 / Chapter 2.8. --- Reagents and Buffers --- p.61 / Chapter 2.8.1. --- Media for Bacterial culture --- p.61 / Chapter 2.8.2. --- Media for Plant tissue culture --- p.64 / Chapter 2.8.3. --- Restriction Enzymes --- p.66 / Chapter 2.8.4. --- Buffers for Agarose Gel Electrophoresis --- p.66 / Chapter 2.8.5. --- DNA ligation Buffer --- p.67 / Chapter 2.8.6. --- Reagents for preparation of E.coli competent cells --- p.67 / Chapter 2.8.7. --- Reagents for preparation of Plasmid DNA --- p.68 / Chapter 2.8.8. --- Reagents for Qiagen-pack 100 Cartridge --- p.69 / Chapter 2.8.9. --- Reagents for SDS-PAGE --- p.70 / Chapter 2.8.10. --- Reagents for Western Blotting --- p.71 / Chapter Chapter 3 --- Construction of Plant Transformation Vectors / Chapter 3.1. --- Introduction --- p.73 / Chapter 3.2. --- Results --- p.74 / Chapter 3.2.1. --- Construction of pSLJ58210 --- p.74 / Chapter 3.2.2. --- Construction of the recombinant binary vectors pSLJ TCSl and pSLJ TCS --- p.78 / Chapter 3.2.3. --- Conjugation ofpSLJ TCS 1 and pSLJ TCS 2 into Agrobacterium tumefaciens via Triparental Mating --- p.82 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Regeneration of leaf explants after transformation --- p.95 / Chapter 4.2.2. --- The level of expression of TCS in transgenic tobacco leaf --- p.100 / Chapter 4.3. --- Discussion --- p.104 / Chapter 4.3.1. --- Regeneration of transgenic tobacco plants --- p.104 / Chapter 4.3.2. --- Expression of TCS in transgenic tobacco plants --- p.108 / Chapter Chapter 5 --- Two approaches to study the Inhibitory effect of TCS on TuMV / Chapter 5.1. --- Introduction --- p.112 / Chapter 5.2. --- Results --- p.113 / Chapter 5.2.1. --- Expression and purification of recombinant TCS --- p.113 / Chapter 5.2.2. --- Purification of TuMV --- p.119 / Chapter 5.2.3. --- Anti-viral assay on local lesion host --- p.119 / Chapter 5.2.4. --- Anti-viral assay on Systemic host --- p.124 / Chapter 5.2.5. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.126 / Chapter 5.3. --- Discussion --- p.129 / Chapter Chapter 6 --- Establishment of plant culture conditions for efficient shoot regeneration from tissue explants of B.parachinensis / Chapter 6.1. --- Introduction --- p.133 / Chapter 6.2. --- Results --- p.133 / Chapter 6.3. --- Discussion --- p.137 / Chapter Chapter 7 --- Conclusion / Appendix / Chapter A.1. --- Size of molecular weight markers --- p.143 / Chapter A.2. --- References --- p.145