细胞转化获得转白细胞介素-4基因胡萝卜(Daucus carrot)

Transferred Interleukin-4 Gene in Carrot (Daucus carota) by Cell Transfor- mation

本研究以胡萝卜‘开拓者’无菌苗胚轴诱导的愈伤组织经振荡培养获得悬浮培养细胞为受体材料,以卡那霉素抗性基因为选择标记基因,对农杆菌介导的胡萝卜细胞遗传转化体系进行了优化。结果表明：悬浮细胞预培养1-3d,胡萝卜细胞与农杆菌细胞比例为1：10或1：100,共培养时间为1-3d时,转化效率较高;但以悬浮细胞预培养1d,胡萝卜细胞与农杆菌细胞比例为1：10,共培养时间为1d时,可得到较多的转化苗。共获得抗性苗290株,经PCR检测66株扩增出特异性条带;部分阳性植株经PCR-Southern检测,初步证明目的基因已转入到胡萝卜中,并收获了转基因胡萝卜。

In this research, we optimized an Agrobacterium tumfaciens-mediated genetic transformation system of carot ＇Kaituozhe＇, which suspension cultured cells from the callus induced by disinfectant hypocotyls were used as the explants for genetic transformation and the Kanamycin resistant gene as selective marker. The results showed that the transformation frequency is higher with the procedures of the suspension cells pre-cultured 1-3 d, carrot cells with Agrobacterium cell ratio of 1：10 or 1：100, co-culture time is 1-3 d; and the transferred carrot＇s plantlets are higher with the procedures of the suspension cells pre-cultured 1 d, carrot cells with Agrobacterium cell ratio of 1：10, co-culture time is 1 d. Finally, 290 resistant plants were obtained, 66 out of those amplified specific bands by PCR assay. Some positive plants detected by PCR-Southern, preliminary evidence proved that the target gene has been integrated into the carrotgenome, and finally the transgenic carrots were harvested in this researchs.