A simplified and efficient Agrobacterium tumefaciens electroporation method

Evelin Kámán-Tóth, Miklós Pogány, Tamás Dankó, Ágnes Szatmári, Zoltán Bozsó

Agrobacterium tumefaciens is a widely used microbial tool in plant molecular biology to transfer DNA into plant cells and produce, e.g., stable or transient transformants or induce gene silencing. In our study, we present a simplified version of electrocompetent cell preparation that is not only time and cost efficient, but it requires minimal handling of bacterial cells. Liquid cultures are normally used to prepare competentAgrobacteriumcells. To overcome the difficulties of working with liquid cultures, we propose suspending bacterial cells directly from overnight agar plate cultures. In addition, we optimized several parameters to simplify the procedure and maximize the number of transformants (e.g.,Agrobacteriumstrains, number of washing steps, amount of required plasmid DNA, electroporation parameters, type of incubation media, or incubation time). This optimized, simple, and fast protocol has proved to be efficient enough to obtain transformed colonies with low amounts (as little as 1 ng) of plasmid DNA. In addition, it also enabled us to introduce ligated plasmids directly intoAgrobacteriumomitting theE. colitransformation step and accelerating the cloning procedure further.