根癌农杆菌介导ACS反义基因转化香蕉的研究

Transformation of Antisense ACS Gene to Banana Mediated by Agrobacterium tumefaciens

以香蕉（Musa spp．）品种‘天宝蕉’（Musa spp．,AAA类群）为试材，对以根癌农杆菌介导法的香蕉遗传转化体系进行较全面的研究，并以该系统进行了ACS反义基因转化香蕉的研究。结果表明：不经预培养的香蕉茎尖横切薄片，侵染前用附加0．1mg／L甘露醇的高渗固体培养基前处理4h，农杆菌重悬液浓度为0D。在1．0左右，重悬液中含100g／L蔗糖，接菌时间为10～15min，于26℃黑暗条件下共培养4d，共培养培养基pH值为5．8是较为适合的转化条件：采用附加100mg／L卡那霉素、2mg／LAgNO，筛选培养基对共培养后的香蕉横切薄片进行筛选，共获得5个转ACS反义基因的抗性芽系；经GUS组织化学法及PCR检测，gus基因已整合进香蕉基因组．

A high efficiency and systematic transgenic procedure mediated by Agrobacterium was developed in Musa spp. cv. Tianbaojiao （AAA group）. The introduction of antisense A CS gone to banana was conducted, and the optimized parameters used in Agrobacterium-mediated transformation were obtained. The best transient expression of GUS was obtained under the conditions as follows： the thin cross-sections which were not precultured but were pretreated with 0.1 mol/L mannitol for 4 h, and the Agrobacterium suspension was with OD600 value of 1.0 and supplemented with 100 g/L sucrose, the samples were infected for 10-15 mins, and then they were transferred onto the non-selective medium （pH5.8） supplemented with 0.1 mg/L NAA and 1.0 mg/L BA for co-culture for 4 days at 26 ℃ in the dark. After co-culture, the thin cross-sections were selected on MS medium supplemented with 100 mg/L kanamycin and 2 mg/L AgNO3, which could be used to inhibit Agrobacterium contamination and be good for bud differentiation. At last, 5 resistant bud lines were maintained for further studies. The GUS histochemical and PCR assays proved that the gus gene had integrated into the genome of the 5 resistant bud lines.