摘要
采用正交旋转设计方法对影响芥蓝遗传转化体系的因素进行了优化研究,结果表明:影响芥蓝KanR苗率的最主要因素是Kan浓度,而预培养时间和共培养时间是芥蓝遗传转化的主要影响因素。最利于芥蓝遗传转化的操作程序为:将芥蓝无菌苗下胚轴在预培养基上预培养2天后,在LBA4404菌液中感染8min,置于共培养培养基上培养2天,随后把外植体转入含Kan的选择分化培养基上诱导不定芽,28天转瓶一次,当抗性幼苗长至2～3cm时,齐愈伤组织处切下幼苗在生根培养基上诱导不定根,25天左右后等不定根长成即可开瓶炼苗,继而移栽至营养土中,正常管理至开花结果;经PCR、Southern印迹检测,证明CYP86MF基因已经整合至转基因植株染色体中。
By the orthogonalty design, the factors which influenced the transformation of Brassica oleracea L. vat. alboglabra have been studied, the results showed that the major factor for KanR frequency is Kan concentration, and the major factor for Chinese kale transformation are pre-culture time and co-culture time. The best procedure for transformation is： Hypocotyl explants from 7-day-old germinated seedlings were pre-cultured for 2 d on preculture medium and co-cultivated for 2 d. The explants were then transferred to selection medium. Vigorous green shoot were transferred to rooted medium for 25 d when they grow at 2-3 cm. The vigorous seedlings were transplanted to vermiculite medium after exercised. In order to the results of PCR, Southern blot analyses, the gene CYP86MF have been transformated into the Chinese kale Kan^R plantlets.
出处
《细胞生物学杂志》
CAS
CSCD
2007年第1期147-152,共6页
Chinese Journal of Cell Biology
基金
国家自然科学基金(No.30671426)
浙江省重大科技项目(No.2005C12019-02)资助~~
关键词
芥蓝
遗传转化体系
正交旋转组合设计
Brassica oleracea L.var. alboglabra
Chinese kale
transformation
orthogonalty design
