根癌土壤杆菌介导转化诸葛菜子叶获得转基因植株  

Agrobacterium-mediated transformation of Orychophragmus Violaceus Cotyledon and Regeneration of Transgenic Plants

作　　者：周冀明[1] 卫志明[1] 许智宏[1] 刘世贵[2] 罗鹏[3] 

机构地区：[1]中国科学院上海植物生理研究所植物分子遗传国家重点实验室 [2]四川大学生物工程系 [3]四川大学生物系,成都610064

出　　处：《生物工程学报》1996年第1期34-39,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金

摘　　要：采用诸葛菜子叶为材料,在MS+BA3mg/L+NAA0.2mg/L培养基上诱导芽的再生,1/2MS+IBA0.03mg/L上诱导生根,获得完整再生植株,建立了诸葛菜组织培养高频再生系统,其再生率可达100％。采用土壤杆菌介导转化诸葛菜子叶,在附加一定量的氨苄青霉素、头孢霉素和卡那霉素的相应培养基上进行筛选,进而培养出再生成苗。获得完整植株,经GUS和NPTⅡ酶活测定,以及Southern blot分析,证实为转基因植株。转化子叶的芽再生率为51％,获得完整转基因植株的转化率为5.53％。在国内外首次建立了以根癌土壤杆菌(Agrobacterium tumefaciens)为载体,诸葛菜子叶为受体的遗传操作系统。Excised cotyledons of Orychophragmus violaceus, were used as explants for tissue culture. They were cultured on MS medium supplemented with BA 3 mg/L and NAA 0. 2mg/L. When the regenerated buds were 2cm long, they were excised and transferred onto 1/2MS medium with IBA 0. 03mg/L, then the whole planticts were regenerated . The frequency of plant regenration was 100%. Then we began to study the genetic transformation of O. violaceus, After 2-3 days of cocultivation with Agrobacteri-um tumefaciens strain A208se (pTiT37 pROA93), the cotyledons were transferred onto the selection medium containing 25mg/L Km and 250mg/L Ap. After shoots emerged, they were excised and transferred onto the root medium containing 25mg/L Km and 100mg/L Cef, the roots were formed within 4-5 weeks. The whole plants were transplanted into pots and grew well. The frequency of plant regeneration was about 51%. The regenerated plants showed high enzymatic activities of B-glucuronidase and neomycine phosphotransferase I . Southern blot analysis confirmed that NPT I gene had been stably integrated into the chomosomal genome of O. violaceus. The transformation frequency was 5. 6%. In this paper, the first transgenic plant of O. violaceus is being reported.

关 键 词：诸葛菜 子叶 转基因植株 根癌土壤杆菌 

分 类 号：Q949.748.3[生物学—植物学] S188[农业科学—农业基础科学]