Guerche P, Bellini C, Le Moullec J M, Caboche M
Laboratoire de Biologie Cellulaire, INRA, Versailles, France.
Biochimie. 1987 Jun-Jul;69(6-7):621-8. doi: 10.1016/0300-9084(87)90181-7.
Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250-300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat-shock (45 degrees C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.
通过电穿孔法将质粒DNA转染到烟草叶肉原生质体中。使用基于氯霉素转乙酰酶活性测量的瞬时表达测定法或通过对表达对与卡那霉素相关的氨基糖苷类药物巴龙霉素抗性的菌落进行评分来估计转染效率。在电穿孔后细胞存活率高于50%的条件下，发现瞬时表达信号和转化效率成正比。研究了影响转化效率的因素。检测到一个明确的最佳电压（250 - 300 V/cm）。在测试的各种盐中，氯化钾是最佳电解质。在电穿孔前进行热休克（45℃/5分钟）处理或在电穿孔介质中加入聚乙二醇均未提高电穿孔效率。用作原生质体来源的植物的生理状态显著影响所得原生质体的转染能力。对这些结果进行了讨论，并与先前发表的方法进行了比较。
