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基于体细胞胚胎发生技术平台,利用携带pSuper1300+质粒,以潮霉素为筛选标记基因的农杆菌GV3101介导日本落叶松遗传转化,对植物受体材料生理状态、农杆菌浓度和浸染时间以及共培养时间等影响因素进行了研究、分析和讨论。结果表明:综合优化各影响因素,生长旺盛的日本落叶松胚性细胞,经浓度为0.4(OD600)的农杆菌浸染10min,共培养2d,再用含400mg/L的头孢霉素的液体培养基清洗脱菌,然后在含400mg/L的头孢霉素固体培养基上恢复培养,并置于含5mg/L潮霉素的固体培养基上多次筛选,最终共获得54个抗性细胞系,转化率平均为0.94个/g。PCR检测鉴定,所有抗性细胞系均为阳性转化体,并排除了农杆菌污染导致的假阳性。研究建立并优化了农杆菌介导的日本落叶松遗传转化技术,为进行遗传改良和基因功能鉴定提供有利平台。
Based on the technology platform of somatic embryogenesis, Agrobacterium tumefaciens GV3101 harboring pSuper1300 + plasmid and hygromycin as selectable marker gene was used to mediate the genetic transformation of Japanese larch. Physiological status of Agrobacterium tumefaciens, Agrobacterium tumefaciens concentration and time of infection, Time and other influencing factors were studied, analyzed and discussed. The results showed that the optimum culture conditions were as follows: embryogenic cells of Larix principis-rupprechtii growing at a concentration of 0.4 (OD600) for 10 min were co-cultured for 2 days and then treated with 400 mg / L of cefotaxime liquid medium After washing, the cells were resuspended in solid medium containing 400 mg / L cefotaxime and were screened on solid medium containing 5 mg / L hygromycin for a total of 54 resistant cell lines. The average conversion was 0.94 pcs / g. PCR detection and identification, all resistant cell lines were positive transformants, and ruled out the false positives caused by Agrobacterium contamination. The research established and optimized Agrobacterium-mediated genetic transformation of Japanese larch, which provided a favorable platform for genetic improvement and identification of gene function.