RESEARC H Rapid Optimization of Electroporation Condition s for Plant Cells, Protoplasts

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfe r 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 cel l 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 cel l walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell type s was obtained, even in the presence of an intact cell wall when conditions were optimized for the electropora-tion pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optima l pulse field strength, followed by suspension cells and finally germinating pollen requiring the stronges t electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using differ-ent types of cells from different plant sources