【摘要】 对影响花生遗传转化效率的PPT筛选压、根癌农杆菌的侵染浓度、侵染及共培养时间等因素进行研究,采用农杆菌转化花生叶片,成功地将抗虫基因豇豆蛋白酶抑制基因（CpTI）和耐除草剂基因（Bar）导入花生。实验结果表明,外植体在 MS+3mg/L BA+0.8mg/L NAA+2mg/L AgNO₃+6mg/L Gln培养基上预培养 3d,于 OD₆₀₀为0.3 的农杆菌菌液中浸泡 5～7min后培养 2d,再转移至含有 3mg/L BA+0.8mg/L NAA+2mg/L AgNO₃+6mg/LGln+500mg/L Cb+300mg/L Cef+0.25mg/L PPT 的MS培养基诱导筛选培养,3个月后得到 24个再生株系。经PCR初步检测,有7个株系显示了310bp的CpTI基因核酸片段,Southern杂交证实了5个株系有外源基因的整合。更多还原

【Abstract】 Genetic transformation of peanut using Agrobactierium -- mediaed approach is still technically chal-lenging. The factors thecting the transformation, such as the selective pressure of PPT, the concentration of theAgrobacteria, the timing of infection and co -- cultivation were examined. The optimized procedure was as follow-ing. After pre -- cultured for 3 days on MS medium containing 3mg/L BA, 0.8mg/L NAA, 2mg/L AgNO₃ and6mg/L Gln, the leanets were dipped into an Agrobacterium suspension (OD₆₀₀=0.3) for 5～7min. The leafletswere then co -- cultured with the Agrobacteria on a shoot induction medium in the dark. Two days later, the ex-plants were transferred to above pre -- culture media with 500mg/L Cb, 300mg/L Cef, and 0.25mg/L PPT. Thisoptimized protocol was successfully used to transfer the CpTI and Bar genes into peanut （Arachis hypogaea L. cv.Shanyou 523）. A total of 24 regeneration lines were produced in 3 months. Out of these, seven lines were provento contain a characteristic 310bp DNA segment of the CpTI gene by PCR analysis. The integration of foreign DNAinto the peanut genome was further confirmed by Southern blot analysis.更多还原