根癌农杆菌介导的葡萄遗传转化体系的优化

Optimization of Agrobacterium-Mediated Transformation System of Grape

以梅鹿辄葡萄离体叶片为转化材料,对根癌农杆菌介导的遗传转化过程中涉及的几个主要影响因素：抗菌素羧苄青霉素（Carb）的浓度、乙酰丁香酮（AS）的浓度、农杆菌菌液的浓度及卡那霉素筛选压进行优化。结果表明,抗菌素羧苄青霉素在浓度300mg·L^-1下即可有效去除农杆菌,且对胚性愈伤组织的分化的影响不大;乙酰丁香酮（AS）的最适浓度为50μm·L^-1;农杆菌菌液的浓度控制在OD6000.5～0.7时转化率最高;卡那霉素的筛选浓度控制在100mg·L^-1。利用优化的农杆菌介导法进行转化,对再生苗进行PCR检测,结果显示,外源基因已导入了葡萄基因组中。本研究优化了农杆菌介导的葡萄遗传转化影响因素,并获得了转基因植株。

In this paper,using the leaves of grape cultivar ‘Merlot＇as transformation material,many influence factors including the concentration of carbenicillin, the concentration of Acetosyringone（ AS）, the concentration of Agrobacterium,the concentration of kanamycin were studied to screen the optimal system. The results show that,the300mg·L^-1 carbenicillin could effectively remove the Agrobacterium and with rational differentiation of embryogenic callus,the optimal acetosyringone（ AS） concentration was 50μm·L^-1 ,the concentration of Agrobacterium at 0. 5 ～0. 7 of OD600 had the highest transformation rate,and the optimal kanamycin concentration was 100mg·L^-1 . By using the optimal agrobacterium-mediated transformation system,the regeneration plants were detected by PCR,and the results indicated that the exogenous gene was transformed into the grape genome. In this study,the factors affecting to Agrobacteriummediated transfermation of grape were optimized,and the transgenic plants were obtained.