Electroporation of Agrobacterium tumefaciens

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.