影响根癌农杆菌介导的丰香草莓遗传转化因素分析

将构建的携带NPTⅡ和AP-D基因的植物表达载体pBI121导入根癌农杆菌EH105中后,用其对草莓主栽品种丰香叶盘进行遗传转化,探讨了卡那霉素(Kan)的浓度、菌液浓度、侵染时间、预培养时间、共培养时间和筛选方式等因素对转化率的影响。结果表明:丰香草莓适宜的转化条件是Kan筛选浓度为20 mg/L,预培养时间为2~3 d,菌液浓度OD6000.3~0.5,侵染时间10~20 min;共培养2~3 d后将外植体接到含有5 mg/L Kan和500 mg/L Carb的诱导培养基上进行培养,以后每次继代逐步提高Kan选择压力至终浓度20 mg/L(每次增加5 mg/L);或共培养2~3 d后将外植体接到含有500 mg/L Carb的诱导培养基上,培养2周后继代到含20 mg/L Kan和400 mg/L Carb的培养基上进行选择培养。根据PCR检测结果,抗性愈伤组织转化率高达18.5%。抗性愈伤组织经进一步诱导,最后获得了17株抗性植株。本文探讨了影响丰香草莓转化的多个因素,以期建立高效的遗传转化体系,为草莓属植物种质的遗传转化奠定基础。

FACTORS INFLUENCING THE EFFICIENCY OF AGROBACTERIUM-MEDIATED TRANSFORMATION IN STRAWBERRY CULTIVAR TOYONOKA

Various aspects of transformation were examined in efforts to improve the transformation frequency of strawberry(Fragaria ananassa Duch.) using Agrobacterium-mediated method based on constructing a plant expression vector pBI121 containing the NPT Ⅱ gene and the antibacterial peptide-D gene(APD).The results indicated that 20 mg/L kanamycin(Kan) was suitable for selection of transformation.The optimal pre-culture time was 2-3 d and then inoculated 10-20 min with OD_(600) 0.3-0.5 Agrobacterium,and co-cultured for 2-3 d in succession.The co-cultured leaf disks were transferred to the inducing medium containing 5 mg/L Kan plus 500 mg/L Carbenicillin(Carb) and Kan concentration was gradually increased to 20 mg/L(5 mg/L Kan at a time),or to the inducing medium containing 500 mg/L Carb for two weeks and transferred to the inducing medium supplemented with 20 mg/L Kan plus 400 mg/L Carb.Inoculated leaf explants produced transgenic calli at a frequency of 18.5% according to PCR analysis.Seventeen transgenic lines were obtained and propagated in the greenhouse for further analysis.