Study on gene transfer into plant cells by electroinjection

我们已经发现，通过使用电场脉冲，我们可以用质粒DNA转化完整的酵母细胞，用病毒RNA（电注射）去除烟草的游离叶肉细胞。本课题研究了电注射法将外源基因导入完整培养细胞和组织的可行性。研究了细胞壁对电场介导外源基因进入原生质体细胞及其表达的影响通过质粒DNA、病毒RNA和病毒颗粒的瞬时表达测定、扫描电镜和Southern杂交分析，我们已经揭示了在原生质体培养的初始阶段再生的细胞壁不抑制质粒DNA和病毒RNA的摄取和表达，但是完整细胞的细胞壁抑制这些DNA和RNA的摄取。在所研究的条件下，通过使用电脉冲将这些遗传物质直接转移到完整的培养细胞和组织中是不成功的。这些结果表明，需要增加完整细胞壁的孔隙度（通过酶或机械处理），以通过电脉冲将遗传物质引入细胞。

We have found that by use of electric field pulses we can transform intact yeast cells with plasmid DNA and transfect free mesophyll cells of tobacco with virus RNA (ELECTROINJECTION). In this project we studied the feasibility of the electro-injection to transfer foreign genes into intact cultured cells and tissues. We also studied the effects of cell walls on the electric field-mediated entry of foreign genes into and expression of the genes in the protoplast-derived cells (the cells that were obtained by the culture of protoplasts).Based on transient expression assay of plasmid DNA and virus RNA and particles, scanning electron microscopy and Southern hybridizaiton analysis, we have revealed that the regenerated cell walls at the initial stages of protoplast culture did not inhibit uptake and expression of plasmid DNA and virus RNA but that cell walls of intact cells inhibit uptake of these DNA and RNA. Direct transfer of these genetic materials into intact cultured cells and tissues by use of electric pulses was not successful under the conditions studied. These results indicate that increase of the porosity of intact cell walls (by enzymatic or mechanical treatment) is needed to introduce genetic materials into the cells by electric pulses.