Saunders J A, Lin C H, Hou B H, Cheng J, Tsengwa N, Lin J J, Smith C R, McIntosh M S, Van Wert S
SARL, USDA/ARS, Beltsville, MD 20705, USA.
Mol Biotechnol. 1995 Jun;3(3):181-90. doi: 10.1007/BF02789328.
The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.
在直接基因转移实验中，优化电穿孔条件以实现DNA的最大摄取对于在转化的植物细胞中实现高水平的基因表达至关重要。使用不同的方波和指数波电穿孔装置，已应用台盼蓝和荧光素二乙酸酯这两种染料来优化三种植物细胞类型的电穿孔条件。不同的细胞类型包括烟草原生质体、具有完整细胞壁的来自大豆的稳定混合营养悬浮细胞培养物以及来自苜蓿和烟草的萌发花粉。即使存在完整的细胞壁，当针对电穿孔脉冲优化条件时，这些细胞类型中的每一种都成功实现了电穿孔。这些细胞中每一种的最佳场强都不同，原生质体的最佳脉冲场强最低，其次是悬浮细胞，最后是萌发花粉，需要最强的电穿孔脉冲。描述了一种使用来自不同植物来源的不同类型细胞优化电穿孔参数的快速方法。
