Electroporation of Agrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens is a soil bacterium that causes tumors on dicotyledonous plants. Virulent strains harbor a large plasmid, the Ti (tumor-inducing) plasmid, which is involved in tumorigenesis. A small segment of this plasmid, the T-DNA, is transferred to the plant cell and becomes integrated into one of the chromosomes in the nucleus. The T-DNA contains genes for the production of phytohormones viz an auxin and a cytokinin. Therefore, expression of the T-DNA in the plant cell leads to tumor formation (for review, see ref. 1). Deletion of the onc genes within the T region of the Ti plasmid results in nononcogenic strains. However, if the 24-bp border repeat, which surrounds the T region in the Ti plasmid, is kept intact, the mutated T-DNA is still delivered to plant cells by Agrobacterium. This natural plant vector system is used for the genetic engineering of plants (for review, see ref. 2). If genes are added to the T region of the Ti plasmid, these are cotransferred to the plant cell. An important finding was that separating the T region from the remaining part of the Ti plasmid did not prevent transfer of the T-DNA to the plant cell (3). On the basis of this principle, the binary vector system was developed. Binary vectors are wide host-range plasmids that are maintained by both E. coli and A. tumefaciens and contain an artificial T region into which genes of interest can be cloned. Traditionally, cloning with binary vectors is done in E. coli. The resulting vector is then introduced into an A. tumefaciens helper strain for delivery of the T-DNA to plant cells.