葡萄叶盘法基因转化中抗生素种类和浓度的筛选  

The Selection of Antibiotics and Their Concentration during Gene Transformation via Leaf Disc Regeneration System in Grapes

作　　者：王彦立[1] 贺柱[2] 师校欣[2] 杜国强[2] 

机构地区：[1]河北农业大学农村发展学院,河北保定071001 [2]河北农业大学园艺学院,河北保定071001

出　　处：《中国农学通报》2009年第21期59-63,共5页Chinese Agricultural Science Bulletin

基　　金：河北省自然科学基金资助项目"不同继代次数组培苗种质保存特性研究"(C2008000287)资助;国家葡萄现代产业技术体系建设项目

摘　　要：以优良无核葡萄品种莫利莎葡萄试管苗为试材,研究了卡那霉素(Kan)、潮霉素(Hyg)对葡萄继代苗生长和离体叶片再生的影响,以确定叶盘法基因转化的选择压和转化体的筛选浓度;同时研究了不同浓度的头孢霉素(Cef)和羧苄青霉素(Carb)对葡萄离体叶片再生的影响,以确定侵菌共培养后合适的抑菌抗生素种类和浓度。试验结果表明葡萄继代苗对Hyg的敏感性强于Kan,葡萄遗传转化更适合使用潮霉素磷酸转移酶基因作为选择标记。Hyg3.0mg/L即达到继代苗致死浓度,可作为转化后抗性苗的筛选浓度,Hyg0.8mg/L完全抑制叶片不定芽的分化,可作为叶盘法转化时抗性芽的选择压。葡萄叶盘法基因转化中抑制农杆菌生长的抗生素可选用Cef300mg/L或Carb400mg/L,但对不定芽的分化有抑制作用。It was studied that the effect of Kanamycin and Hygromycin on the growth of subcultured explants and the efficiency of adventitious bud regeneration from leaves in vitro by using seedless grape cultivar of Melissa cultured in vitro, in order to determine the optimal concentration for the adventitious bud regeneration and trangnic plants screening; and the effect of the concentration of Cefotaxime and Carbenicillin on the efficiency of adventitious bud regeneration, in order to determine the optimal antibiotics and their concentration used after co-culture process. The results showed that the subculture plantlets were more sensitive to Hygromycin than to Kanamycin. The Hygromycin was considered to be more useful than Kanamycin as a selecting marker during gene transformation in grapes. The supplying of Hygromycin 3.0 mg/L in the media could led the subculture plantlets to death and was suggested to be used as the optimal concentration for transgenic lines screening. The supplying of Hygromycin 3.0 mg/L in the media could fully restrict the adventitious bud regeneration and could be used as a selecting pressure for the regeneration of adventitious buds with antibiotic-resistant. The supplying of Cefotaxime 300 mg/L or Carbenicillin 400 mg/L could eliminated the growth of Agrobacteria eventually, but it could limit the adventitious bud regeneration from leave as well.

关 键 词：葡萄 基因转化 农杆菌 抗生素 

分 类 号：S663.1[农业科学—果树学]