萝卜遗传转化体系的建立

系统研究了影响根癌农杆菌介导的萝卜(Raphanus sativus L.)遗传转化的若干因素,建立了萝卜遗传转化体系:即切取萝卜带柄子叶外植体,先进行2 d预培养,用OD600为0.3~0.5的EHA105农杆菌菌液侵染5~7 min后,再进行5 d共培养,然后将外植体转接到MS+6-BA 6 mg.L-1+NAA 0.05 mg.L-1+Cef 500 mg.L-1的培养基上进行7 d延迟筛选,再将外植体转接到MS+6-BA 6 mg.L-1+NAA 0.05 mg.L-1+Cef 500 mg.L-1+Hyg 5 mg.L-1的培养基上进行10 d的抗性筛选。结果共获得126个抗性芽,其中8个抗性芽经PCR检测扩增出目的基因的启动子BcA9,约850bp,阳性率为6.3%,初步验证目的基因已经转入萝卜再生芽中。

Establishment of Genetic Transformation System in Radish

The factors for Agrobacterium tumefaciens-mediated genetic transformation in Radish(Raphanus sativus L.)were studied.A radish genetic transformation system is established.Cotyledons with petiole were cultured in MS + 6-BA 6 mg·L-1 + NAA 0.05 mg·L-1 for 2 days as pre-culture.They were then infected for 5-7 min by the EHA105 Agrobacterium at OD600 = 0.3-0.5,and co-cultured in same medium for 5 days.The explants were then transferred to MS + 6-BA 6 mg·L-1 + NAA 0.05 mg·L-1 + 500 mg·L-1 Cef medium for 7 days for pre-screening.The explants were later cultured no MS + 6-BA 6 mg·L-1 + NAA 0.05 mg·L-1 + 500 mg·L-1 Cef + 5 mg·L-1 Hyg medium for 10 days for selecting transgenic events.We got 126 resistant shoots,and 8 of them were amplified the target gene BcA9 with size about 850 bp.The preliminary validation showed that the target gene has been transferred into radish.