根癌农杆菌介导观赏羽衣甘蓝遗传转化体系优化

Agrobacterium Tumefaciens-mediated Genetic Transformation System in Brassica oleracea var. acephala

为建立稳定的羽衣甘蓝遗传转化体系,以羽衣甘蓝自交系’Mark 10’,’W02-7’和’Curly 6’为试材,通过单因子试验,比较羽衣甘蓝子叶、子叶柄、子叶片、下胚轴4种外植体的再生能力,筛选培养基中的适宜激素浓度,优化了羽衣甘蓝不同外植体的再生体系。将播种后6d的子叶于最适再生培养基上进行黑暗预培养,以根瘤农杆菌为介导侵染子叶后,于黑暗条件下进行农杆菌与植物材料的共培养。随后转入含有头孢霉素的抑菌培养基中进行抑菌培养,再将子叶移入含有卡那霉素浓度的筛选培养基中进行抗性芽选择,并提取植物基因组DNA进行目的基因的PCR验证,从而建立高效的羽衣甘蓝遗传转化体系。结果表明:最佳的外植体是羽衣甘蓝的子叶,在培养基MS+6-BA 1.0mg·L^-1+NAA 0.1mg·L^-1上,不定芽分化率可达100%;子叶经2~3d预培养,将OD600值为0.3~0.5的携带DFR基因表达载体的农杆菌菌液离心,以50mL MS重悬液重悬,侵染外植体5min,共培养2d;采用头孢霉素300mg·L^-1,抑菌培养3d后,采用10mg·L^-1卡那霉素进行抗性芽选择。在播种后30~40d共获得了24个抗性芽,以抗性芽的基因组DNA为模板进行PCR扩增,9株呈DFR阳性,显示外源基因已经成功整合到羽衣甘蓝基因组中,平均转化率为37.5%。本研究结果可以为羽衣甘蓝重要性状基因的遗传转化提供依据。

In order to establish a stable kale genetic transformation system, kale inbred lines ’Mark 10’,’W02-7’ and ’Curly 6’were employed as test materials using single-factor test to compare the regenerative capacity of explants cotyledons, cotyledonary petiole, cotyledonary blade and hypocotyl, and to select the suitable hormone concentration, and optimize the regeneration system of different explants of ornamental kale. In order to establish an efficient genetic transformation system in kale, we pre-cultured6-day cotyledons on the optimal regeneration medium, then the cotyledons were infected by Agrobacterium tumefaciens. The coculture of plant material and Agrobacterium was carried out under dark conditions. Subsequently, the infected cotyledons were transferred to an antibacterial medium containing cephalosporin. Then the cotyledons were transferred into a screening medium containing kanamycin for selection of resistant buds. Genomic DNA of transgenic plant was extracted for target gene verification by polymerase chain reaction. The results showed that the best explants were cotyledons of kale, and the adventitious bud differentiation rate was 100% in the medium MS+6-BA 1.0 mg·L^-1+ NAA 0.1 mg·L^-1;cotyledons were pre-cultured for 2-3 days, and bacteriostatic culture containing DFR gene expression vector was cultured to OD600.3-0.5 and resuspended in 50 mL of MS and used to infect the explants for 5 min, culturing for 2 days;after 3 days of bacteriostatic culture with cephalosporin300 mg·L^-1, 10 mg·L^-1 kanamycin was used for selection of resistant buds. In this experiment, 24 resistant buds were obtained from 30-40 d after sowing, and the genomic DNA of resistant buds was used as a template for PCR amplification, and 9 strains were positive for DFR, indicating that the foreign gene has been successfully integrated into the kale genome. The average transformation rate is 37.5%. These results can provide basic information for the genetic transformation of kale-like genes.