根癌农杆菌介导的木薯遗传转化条件的优化  

Optimization of the Conditions for Agrobacterium-mediated Cassava Genetic Transformation

作　　者：方佳敏[1,2] 刘佳[2] 唐克轩[1] 张鹏[2] 

机构地区：[1]上海交通大学农业与生物学院,上海200240 [2]中国科学院上海生命科学研究院植物生理生态研究所植物分子遗传国家重点实验室,上海200032

出　　处：《热带作物学报》2011年第9期1697-1703,共7页Chinese Journal of Tropical Crops

基　　金：973计划项目(No.2010CB126605);国家木薯产业技术体系项目(No.nycytx-17)

摘　　要：对中国及非洲11个主栽木薯品种(KU50、NZ188、NZ199、SC5、SC6、SC8、SC124、TME7、TME12、Ebwanateraka、TMS60444)的离体培养研究结果表明,不同品种之间的体细胞胚胎发生和芽器官发生能力因基因型不同而差异显著。多数品种体细胞胚胎发生频率在60%以上,有几个可达到80%以上,如TMS60444和Ebwanateraka;而NZ199、SC124和TME2的诱导频率较低,分别只有25%、43%和36%。次生胚状体形成的子叶胚切块可诱导芽器官发生,诱导频率在15.8%～75.8%之间,其中TME7和TMS60444诱导效果最好。与TMS60444相似,KU50、SC5、SC8及Ebwana的次级体细胞胚经过多次继代诱导后,观察到脆性胚性愈伤组织的形成,但发生频率较低。对不同根癌农杆菌侵染外植体及抗生素浓度的优化结果表明,用改良型的农杆菌LBA4404进行侵染,在培养基中添加200μmol/L乙酰丁香酮,共培养温度22℃,培养4 d时,转化效果最好。共培养后,使用含有500 mg/L羧苄霉素及15 mg/L潮霉素的培养基来筛选体细胞胚胎发生是较为理想的。As cassava genetic transformation is a complex process, it is pivotal to optimize relative conditions including explant culturing, infection and cocultivation by Agrobacterium, selection and plant regeneration of transformants. Among 11 farmer-preferred cassava varieties（KU50, NZ188, NZ199, SC5, SC6, SC8, SC124, TME7, TME12, Ebwanateraka and TMS60444）from Africa and China, different capacities for shoot organogenesis and somatic embryogenesis were noticed during the tissue culture processes. Most varieties could have a frequency of somatic embryogenesis above 60% with several more than 80%, such as TMS60444 and Ebwanateraka. Cuhivars NZ199, SC124 and TME2 only gave very low frequencies, 25%, 43% and 36%, respectively. Shoot organogenesis could be induced from the cutting pieces of somatic cotyledons. After 2 weeks, somatic cotyledons produced shoot primordia and adventitious buds with a range of 15.8% to 75.8%. Among the tested cuhivars, TME7 and TMS6Oa, a.a. performed the best. Similar to TMS60444, secondary somatic embryos of KU50, Ebwanateraka, SC5 and SC8 could form friable embryogenic callus after cyclic subcuhuring. Nevertheless, induction rates were very low in these cuhivars in comparison with TMS60444. According to the results of cocuhivation with different Agrobacterium tumefaciens strains and antibiotic dose responses, optimized conditions were established for the best transformation efficiency in cassava transformation. These conditions included infection with the modified Agrobacterium strain LBA4404/CS, the use of 200 ~m AS, cultivation temperature at 22 ~C and cocultivation duration of 4 days. After cocultivation, antibiotic resistant somatic embryos should be regenerated under the selection containing 500 mg/L Carbenicillin and 15 mg/L of hygromycin. This study provides a protocol for condition optimization in cassava genetic transformation and will be useful for guiding the production of transgenic cassava using farmer-preferred cultivars.

关 键 词：木薯 体细胞胚胎发生 芽器官发生 遗传转化 条件优化 

分 类 号：S533[农业科学—作物学]