Institute of Plant Sciences, Federal Institute of Technology, 8092, Zurich, Switzerland.
Plant Cell Rep. 1993 Oct;12(12):671-5. doi: 10.1007/BF00233417.
Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-μF capacitor and with 100 μg DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.
将基因转入完整细胞是通过对未经任何特殊预处理的合子小麦胚胎进行电穿孔来实现的。电穿孔在组织上是特异性的，因为发现盾片细胞比胚胎的其他细胞类型更容易接受基因转移。胚胎在电穿孔室中的取向也影响了转化的盾片细胞的数量；在电穿孔过程中，与电泳一样，带负电荷的质粒 DNA 分子似乎朝着正极移动。因此，将胚胎排列成使盾片朝向负极。使用携带两个嵌合花青素调控基因或一个嵌合 gusA 基因的质粒，允许在盾片中清楚地识别转化细胞。在一些胚胎上，用 960μF 电容器放电的 275V/cm 的单个电脉冲进行电穿孔，并使用 100μg/ml 的电穿孔缓冲液进行电穿孔后，发现超过 100 个转化的盾片细胞。使用花青素标记系统，可见转化的细胞生长产生红色区域。
