影响西瓜农杆菌介导的高效遗传转化效率的主要因子

Establishment of efficient transgenic system mediated by Agrobacterium tumefaciens in watermelon

以西瓜子叶为外植体材料,利用GUS染色瞬时表达方法研究农杆菌浓度,预培养、共培养时间,预培养条件以及添加乙酰丁酸酮(AS)对西瓜转化效率的影响,优化西瓜遗传转化参数,初步建立以MS+BAP 2.0mg/L+IAA 0.2 mg/L+Hyg 10 mg/L+Cef 300 mg/L为分化培养基,黑暗条件下预培养2 d,使用OD为0.3的菌液侵染10 min后,黑暗条件下共培养3 d,然后在25℃,光周期为16 h的条件下筛选抗性芽的遗传转化体系。

An efficient watermelon cotyledon explant transgenic system mediated by Agrobacterium tumefaciens was established through the exploration of the factors affecting transformation efficiency of Agrobacterium tumefaciens. The parameters were optimized by measuring the efficiency of hi stochemical GUS transsient expression including preculture period, co-cultivation period, addition of AS and the composition of regeneration media. The watermelon cotyledon explants were pre-cultured for 2 d in the darkness, then inoculated with 0.3 of OD600 Agrobacterium tumefaciens for 10 min. Inoculated explants were co-culutred in the dark at 25℃ for 3 d, then transferred to the MS medium contained BAP （2.0 mg/L） and IAA （0.2 mg/L） plus 10 mg/L Hygromycin B and 300 mg/L cefalothin for the selection of transgenic shoots in the photoperiod of 16 h light/8 h dark at 25℃.