羽衣甘蓝下胚轴农杆菌介导遗传转化体系的优化  

Optimization of Agrobacterium-mediated Transformation System for Brassica oleracea var. acephala with Hypocotyls as Explants

作　　者：高航[1] 高玉亮[2] 李葵花[1] 

机构地区：[1]延边大学农学院,延吉133002 [2]延边朝鲜族自治州农业科学院,龙井133400

出　　处：《生物技术通报》2015年第6期111-115,共5页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31260470);延边大学启动金项目(2011800-601010018)

摘　　要：以红鸥羽衣甘蓝下胚轴为试材,首先优化了不定芽分化体系,其次探讨了农杆菌介导遗传转化过程中农杆菌侵染浓度、侵染时间对不定芽分化率的影响及不定芽分化和生根过程中潮霉素B筛选压力,最后对抗性植株进行了潮霉素B筛选基因的PCR检测。结果表明,在MS+6-BA 5.0 mg/L+Ag NO3 9.0 mg/L培养基中不定芽分化率最高,为84.17%;农杆菌侵染浓度OD600值为0.5、侵染5 min时利于遗传转化,分化率为69.17%;不定芽分化和生根过程中潮霉素B筛选压力分别为4.0 mg/L和30.0 mg/L;潮霉素B筛选基因的PCR鉴定结果,在预期的602 bp处出现了条带,初步证明潮霉素B筛选基因已整合到羽衣甘蓝基因组中。An efficient transformation method for kale（Brassica oleracea var.acephala）cultivar Hongouwasdeveloped using hypocotyls as explant tissues with the following 3 steps：firstly optimizing the differentiation system of adventitious buds;secondly investigating the effects ofAgrobacterium infection concentration and time on differentiation rate and the selective concentration ofHygromicin B（Hyg） gene during differentiation and rooting of adventitious buds;finally verifying the selective geneHyg in the resistant plants by PCR. The highest differentiation rate（84.17%）of adventitious buds was found on MS ＋ 6-BA 5.0 mg/L ＋ AgNO3 9.0 mg/Lmedium. An infection concentration OD600 of Agrobacterium as 0.5 and infection time for 5 min were favorable for genetic transformation, and the differentiation rate was 69.17%. The selective concentration ofHyg during the differentiation and rooting of adventitious buds were 4.0 mg/Land 30.0 mg/L, respectively. Moreover, the amplified specific geneHyg bands were observed at the expected 602 bp by PCR analysis, which preliminarily demonstrated that the screened geneHyg was integrated into theB. oleraceavar.acephalagenome.

关 键 词：羽衣甘蓝 农杆菌侵染效率 潮霉素筛选压力浓度 

分 类 号：S635.9[农业科学—蔬菜学]