葡萄胚性培养物的起始与转化

Initiation and transformation of grapevine embryogenic cultures.

作者信息

Dhekney Sadanand A, Li Zhijian T, Dutt Manjul, Gray Dennis J

机构信息

Mid-Florida Research and Education Center, University of Florida/IFAS, Apopka, FL, USA.

出版信息

Methods Mol Biol. 2012;847:215-25. doi: 10.1007/978-1-61779-558-9_18.

DOI:10.1007/978-1-61779-558-9_18

PMID:22351011

Abstract

Protocols for the production and transformation of grapevine embryogenic cultures are described. Embryogenic cultures are initiated from leaves or stamens and pistils and transformed with Agrobacterium containing an enhanced green fluorescent protein/neomycin phosphotransferase II (egfp/nptII) fusion gene. Cultures are transferred to induction medium in the dark for callus formation and proliferation. Resulting cultures are transferred to somatic embryo development medium to induce secondary embryogenesis and formation of transgenic somatic embryos. Transgenic embryos identified on the basis on GFP fluorescence and kanamycin resistance are transferred to germination medium to regenerate transgenic plants. The presence of transgenes in independent plant lines is confirmed by PCR.

摘要

本文描述了葡萄胚性培养物的生产和转化方案。胚性培养物从叶片、雄蕊和雌蕊起始，并用含有增强型绿色荧光蛋白/新霉素磷酸转移酶II（egfp/nptII）融合基因的农杆菌进行转化。培养物在黑暗中转移至诱导培养基以形成愈伤组织并增殖。所得培养物转移至体细胞胚发育培养基以诱导次生胚发生和转基因体细胞胚的形成。基于绿色荧光蛋白（GFP）荧光和卡那霉素抗性鉴定出的转基因胚转移至萌发培养基以再生转基因植株。通过聚合酶链反应（PCR）确认独立植株系中转基因的存在。