粳型恢复系C418转基因再生体系的建立

Establishment of Transgenic Regeneration System for Japonica Restorer Line C418

为水稻转基因育种和突变体库的建立创造有利条件,以粳稻C418成熟胚为外植体,通过植物组织培养技术,考察不同基础培养基(N6、MS、MSN、NMB、NB、MB和NBD)和生长素2,4-D浓度(1 mg/L、2 mg/L和3 mg/L)对外植体愈伤组织诱导效果;将继代培养获得的优良愈伤组织采用农杆菌介导法进行遗传转化,并考察适宜的共培养时间以及不同抗生素(头孢霉素、氨苄霉素和m潮霉素)对愈伤组织的分化效果,以建立北方骨干粳型恢复系C418转基因再生体系。结果表明:在愈伤组织诱导过程中,基础培养基NBD+3 mg/L 2,4-D处理粳稻C418成熟胚愈伤组织诱导率最高,达90.38%,愈伤组织生长状态最佳;水稻愈伤组织共培养2 d获得的抗性愈伤组织最多,筛选培养基中降低头孢霉素浓度并添加氨苄霉素可提高水稻愈伤组织分化率。通过培养基类型、激素浓度、共培养时间及抗生素种类的最佳组合,成功建立北方骨干粳型恢复系C418转基因再生体系,即水稻外植体经次氯酸钠处理后接种于诱导培养基NBD+3 mg/L 2,4-D,获得愈伤组织,再用农杆菌悬浮液(含目的基因)侵染愈伤组织,最后获得含目的基因的转基因植株。

The mature embryos from japonica restorer line C418 were cultured on different media of N6,MS,MSN,NMB,NB,MB and NBD)with different 2,4-D concentration(1 mg/L,2 mg/L and 3 mg/L)to investigate the effect of different media on callus induction rate and the excellent calli obtained from subculture were genetically transformed by agrobacterium-mediated method to study the effect of different co-culture time and different antibiotics(cephalosporin,ampicillin and hygromycin)on callus differentiation and to build the transgenic regeneration system of japonica restorer line C418 for creating favorable conditions of rice transgenic breeding and mutant library establishment.Result:The callus induction rate of mature embryos from japonica restorer line C418 cultured on NBD medium with 3 mg/L 2,4-D reaches 90.38%and the growth status of the induced calli is the best.The maximum resistance calli are obtained after 2 d co-culture.The screening medium with low cephalosporin concentration and adding ampicillin can increase differentiation of calli.The transgenic regeneration system of japonica restorer line C418 includes sodium hypochlorite for explant sterilization,NBD medium with 3 mg/L 2,4-D for callus induction,agrobacterium suspension liquid for callus infection.