Electroporation-Mediated DNA Transfer to Tobacco Protoplasts for Transient Gene Expression Assays

Abstract

This chapter focuses on the electroporation-mediated DNA transfer to tobacco protoplasts for transient gene expression assays. Transient gene expression assays in protoplasts are widely used because of two important advantages: (1) gene expression can be measured very shortly after DNA uptake, and (2) because most of the introduced plasmid DNA remains extrachromosomal during the period of the assay, gene activity measurements are not biased by position effects, that is, the influence exerted by DNA sequences surrounding genes that are integrated into chromosomes. The application of short electric pulses of high field strength transiently permeabilizes cell membranes led to the development of electroporation-mediated gene transfer techniques for mammalian cells and plant cell protoplasts. Electroporation is currently the preferred technique for direct DNA transfer to plant protoplasts. Protoplasts of other plant species can be electroporated with similar procedures as described in the chapter for tobacco protoplasts. Pulse length and field strength should, however, be optimized for each species and genotype. Optimal DNA uptake usually requires conditions that are lethal for part of the protoplasts.