葡萄器官离体再生和遗传转化体系的建立

以‘森田尼无核’为试材，研究了生长调节物质、外植体类型、抑菌素等主要因素对葡萄离体不定芽再生和转化效率的影响，建立和优化了葡萄的再生和转化体系。结果表明：相对于BA和KT，细胞分裂素TDZ无论单独使用还是与生长素配合使用均可使葡萄不定芽再生频率达到最高；在MS + TDZ 2.0 mg·L^-1 、MS + TDZ 4.0 mg·L^-1 或MS + TDZ 4.0 mg·L^-1 + IAA0.1 mg·L^-1培养基上均可以较好地诱导森田尼无核葡萄再生不定芽，再生频率分别为37.4%,40.2%和50.0%；叶盘、茎段和叶柄都可以诱导再生不定芽，在同一种培养上再生频率有差异，但差异不大；采用根癌农杆菌介导的叶盘法转化葡萄时，卡那霉素抗性筛选浓度为20 mg·L^-1；抑菌抗生素使用头胞霉素适宜浓度为400 mg·L^-1，而使用羧苄青霉素时以300 mg·L^-1最佳；获得转化芽45个，部分株系用PCR和Southern 杂交证实，外源基因已整合在葡萄基因组DNA上。

Studies on Adventitious Shoots Regeneration and Transformation System of Grape in Vitro

Establishing an efficient regeneration and transformation system is a fundamental role to the transgenic study. For Centennial Seedless, the effects of growth regulators, explants and ablastin to the generation of adventitious shoots were studied, based on which the optimal condition for regeneration and transformation was determined. Our experiments showed that, when being used alone or accompanied with auxins, the cytokinin TDZ is always better than BA and KT in inducing adventitious shoots. The adventitious shoots of Centennial Seedless could be efficiently induced in the media of MS + TDZ 2.0 mg·L^-1, MS + TDZ 4.0 mg·L^-1 or MS + TDZ 4.0 mg·L^-1 + IAA0.1 mg·L^-1and adventitious shoot regeneration rates were 37.4%,40.2% and 50.0%, respectively. For fixed medium, adventitious shoots can be generated with minor difference in efficiency from leaf disc, stem and petiole. When this regeneration system was used in genetic transformation mediated by Agrobaterium tumefaciens, the optimal concentration of screening resistance medium was 20 mg·L^-1 for kanamycin, and for the control of agrobacterium growth, the best concentration was 400 mg·L^-1 for cephaloridine or 300 mg·L^-1 for carbencillin . Transformants were obtained 45. PCR and Southern blotting confirmed that foreign gene was integrated genome of grape.