Construction Of Trivalent Expression Vectors Containing Chi./Glu./Rs-AFP2 Genes And Organogenesis Regenerating System Of Winegrape 'Merlot'

Plant gene engineering techniques offer new and efficient methods for breeding cultivars resistant to disease. In recent years, great achievements have been obtained in genetically engineering plants resistant to disease and pests. Several genes for resistance to fungal disease have been cloned and demonstrated, leading to many disease resistant plants with stable heredity.Most cultivars used in viticulture are Vitis vinifera L., productive and high-quality but with poor disease resistance. In China especially, most areas suitable for cultivation have high rainfall, which results in fungal diseases such as powdery mildew and downy mildew affecting both the productivity and quality of wine grapes. So, it is very important to grow wine grape cultivars of high-quality and resistance to disease.This paper describes construction of trivalent-expression vectors containing Chitinase (Chi.), β-1,3-Glucanase (Glu.) and Raphnus sativus - antifungal protein (Rs-AFPs) by gene engineering techniques. By manipulating hormone levels, light intensities and temperature, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir. The results were as follows:By manipulating hormone levels, light intensities and temperatures, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir, For Merlot Poir, The best rooting culture medium was: ?MS + IBA (0.1mg/L). The optimum medium for adventitious bud regeneration from petioles was MS+TDZ2.0mg/L, and the germination rate was 62.42%. The optimum medium for adventitious buds regeneration from germination of somatic embryos of Merlot Noir was MS+TDZ(4.0mg/L), and the germination rate was 52.25%. The best adventitious bud propagation culture medium was MS + BA (0.5mg/L).In this study, TDZ was best for inducing adventitious bud from petioles in vitro, and it was easy to regenerate adventitious bud from the younger petiole. Maintaining cultures in dark had an important effect on regenerated adventitious bud, 30 days is best.The trivalent-expression vectors containing Chitinase(Chi.)/β-1, 3-Glucanase (Glu.)/Raphnus sativus-antifingal protein (Rs-AFPs), were constructed and transferred into Agrobacterium tumefaciens EHA105 through direct transformation by gene engineering techniques.This work will help us to: research genetic transformation of grapevines; select transgenic cells using antibiotics; develop new wine grape cultivars of high quality and with resistance to disease.