摘要
为了建立橡胶树胚性悬浮细胞遗传转化体系,用根癌农杆菌EHA105(携带pCAMBIA1301,含有gusA和hptⅡ)侵染胚性悬浮细胞团,用GUS检测的方法分析了共培养时间和菌液浓度对转化效率的影响;同时,测定了胚性细胞团对潮霉素敏感性。结果表明,适宜的共培养时间为3天,适宜的菌液浓度为OD600=0.4～0.6。潮霉素的致死剂量为15mg/L。使用大约1g鲜重的胚性细胞团,经过转化和选择培养,获得53块抗性愈伤组织,经诱导获得7个体细胞胚。GUS检测和PCR鉴定证实了gusA基因已经整合到橡胶树基因组中。本研究为根癌农杆菌介导的橡胶树胚性悬浮细胞遗传转化体系建立奠定了基础。
In order to establish transformation system based on embryogenic suspension cultures in rubber tree（Hevea brasiliensis Mu ll.Arg）,cell aggregates from embryogenic suspension cultures were co-cultivated with the A.tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hpt II genes.Effect of co-cultivation time and A.tumefaciens concentration on transient expression of gusA was examined by GUS assay.The sensitivity of cell aggregates to hygromycin（Hyg） added to the medium was also tested in order to determine the optimal concentration of this antibiotic in the selective medium.The results suggested that 3 days was an optimal co-cultivation time,a desired OD 600 of strain EHA105 was 0.4-0.6,and 15 mg/L concentration of Hyg was found to be enough to inhibit the growth of cell aggregates.A total of 53 Hyg-resistant embryogenic calli were obtained after transformation and co-cultivation.At last,7 somatic embryos were further formed.GUS assay and PCR analysis indicated that gusA had been integrated into the genome of rubber tree.In all,this study laid the groundwork for efficient Agrobacterium tumefaciens mediated transformation using embryogenic suspension cultures in rubber tree.
出处
《中国农学通报》
CSCD
2012年第31期15-20,共6页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金项目"橡胶树胚性悬浮细胞遗传转化体系的建立和焦磷酸酶基因在乳管中的高效表达"(30800881)
关键词
橡胶树
根癌农杆菌
遗传转化
胚性悬浮细胞
rubber tree
Agrobacterium tumefaciens
transformation
embryogenic suspension cultures
