A simplified and efficient Agrobacterium tumefaciens electroporation method
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 competent Agrobacterium cells. 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., Agrobacterium strains, 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 into Agrobacterium omitting the E. coli transformation step and accelerating the cloning procedure further.
KeywordsAgrobacterium Electroporation Plate method Direct transformation
Research in the authors’ laboratories has been funded by the Hungarian Scientific Research Fund (OTKA K 104730, OTKA PD 109050), the Bolyai Scholarship (BO 609 12, BO 00348/09), and the Széchenyi 2020 Programme, the European Regional Development Fund and the Hungarian Government (GINOP-2.3.2-15-2016-00061) which are gratefully acknowledged.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest in the publication.
- Agrios GN (1997) Bacterial galls. Plant Pathology. Academic Press, San Diego, pp 438–441Google Scholar
- De la Riva G, Van Montagu M, Inzé D, Dhaese P (1991) High-efficiency transformation of Agrobacterium tumefaciens with plasmid DNA by electroporation. Biotecnol Aplicada 8:345–351Google Scholar
- den Dulk-Ras A, Hooykaas PJ (1995) Electroporation of Agrobacterium tumefaciens. Methods Mol Biol 55:63–72Google Scholar
- Gonzales MF, Brooks T, Pukatzki SU, Provenzano D (2013) Rapid protocol for preparation of electrocompetent Escherichia coli and Vibrio cholerae. J Vis Exp 80:e50684Google Scholar
- Lin JJ (1995) Electrotransformation of Agrobacterium. Methods Mol Biol 47:171–178Google Scholar
- Main GD, Reynolds S, Gartland JS (1995) Electroporation protocols for Agrobacterium. Methods Mol Biol 44:405–412Google Scholar