Genetic Transformation Of Wine Grape Cultivars And Construction Of Plant Bivalent-expression Vectors Containing Chi./Rs-AFP2 Or Chi./Glu. Genes

Plant gene engineering techniques offer new and efficientmethods for breeding cultivars resistant to disease. In recent years,great achievements have been obtained in genetically engineeringplants resistant to disease and pests. Several genes for resistance tofungal disease have been cloned and demonstrated, leading to manydisease resistant plants with stable heredity.Most cultivars used in viticulture are Vitis vinifera L., productiveand high-quality but with poor disease resistance. In China especially,most areas suitable for cultivation have high rainfall, which results infungal diseases such as powdery mildew and downy mildew affectingboth the productivity and quality of wine grapes. So, it is veryimportant to grow wine grape cultivars of high-quality and resistanceto disease.This paper, describes a propagation and regeneration system forCabernet Sauvignon, Pinot Noir and Chardonnay; regeneration ofembryogenic callus of Cabernet Sauvignon and Merlot; selection oftransgenic embryogenic callus of Merlot and the application of GFP;construction of bivalent-expression vectors containing Chitinase (Chi.)/ Raphnus sativus - antifungal protein (Rs-AFPs) or Chitinase (Chi.) /β-1,3-Glucanase (Glu.).The results were as follows:1. For Cabernet Sauvignon, Pinot Noir and Chardonnay shoot tippropagation, the best propagation culture medium was: MS + BA(1.0~1.5mg/L) + NAA (0.02mg/L). The best rooting culturemedium was: ?MS + IBA (0.5~1.0mg/L).2. For the first time in China, adventitious buds were obtained ofCabernet Sauvignon, Pinot Noir and Chardonnay by usingcytokinin-containing TDZ. The following culture media were viisuccessful: leaf regeneration of Cabernet Sauvignon andChardonnay: MS + TDZ (4.0mg/L); leaf regeneration of Pinot Noir:MS + TDZ (6.0mg/L) + NAA (0.05mg/L); Cabernet Sauvignonpetiole regeneration: MS + TDZ (4.0mg/L) + NAA (0.01mg/L);rooting medium for Cabernet Sauvignon leaf regeneration buds:?MS + IBA (1.0mg/L).3. For the first time in China, using embryogenic callus of CabernetSauvignon and Merlot, somatic embryos were obtained. Themedium was: CP + NOA (1.0mg/L) + BA (0.2mg/L), and theregeneration rates were respectively 41.59% and 47.50%.4. In this study, higher kanamycin (Kan. 100 mg/L) was used to selectembryogenic callus of transgenic Merlot: 22.2% plantlets weretransformed with Agrobacterium-mediated OSM-GUS gene bySouthern Blot analysis. A practicable Agrobacterium-mediatedtransgenic system was developed.5. For the first time, transgenic cells from particle bombardment-mediated 35S-EGFP were obtained using EGFP as the reportergene. Transformed callus showed fluorescence under GFP2. Itwas found that using pressures of 93 bar (1,350 psi) improvedtransformation rates of embryogenic callus. A practicable particlebombardment-mediated transgenic system was developed.6. The bivalent-expression vectors containing Chitinase (Chi.) /Raphnus sativus-antifingal protein (Rs-AFPs), Chitinase (Chi.) / β-1,3-Glucanase (Glu.) were constructed and transferred intoAgrobacterium tumefaciens EHA105 through direct transformationby gene engineering techniques.This work will help us to: research genetic transformation ofgrapevines; select transgenic cells using antibiotics; develop new winegrape cultivars of high quality and with resistance to disease.