Sangwan R S, Bourgeois Y, Sangwan-Norreel B S
Laboratoire Androgenèse et Biotechnologie, Université de Picardie, Faculté des Sciences, Amiens, France.
Mol Gen Genet. 1991 Dec;230(3):475-85. doi: 10.1007/BF00280305.
An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15-20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4-5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and beta-glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7-8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.
已开发出一种高效的农杆菌介导的转化方法，用于转化来自三种不同拟南芥生态型的合子胚。该方法对C24生态型的平均转化率为76%，对Landsberg-erecta和哥伦比亚生态型的转化率为15%-20%。实现最佳转化的关键步骤是将胚在含植物激素的培养基上进行预培养。光学显微镜和电子显微镜研究表明，在预培养期间，胚的原形成层细胞对农杆菌感染变得高度敏感。转化细胞在培养4-5周内形成愈伤组织并再生出芽。总共再生出1500株可育的转基因植株。通过基因组Southern印迹分析、报告基因（新霉素磷酸转移酶II和β-葡萄糖醛酸酶）的酶活性测定以及遗传分离试验，验证了再生植株中插入DNA的存在。通过形态学和细胞学标准确定，45个随机选择的转化株系和150个独立再生株的R1后代未表现出任何体细胞克隆变异。短周期（7-8周）、高效率、可重复性和低体细胞克隆变异频率使得合子胚转化特别适合于T-DNA标签诱变。
