Agrobacterium Transformation
Agrobacterium tumefaciens is a soil bacterium that naturally causes crown gall disease by transferring a segment of its own DNA (T-DNA) into the plant nuclear genome. Researchers have repurposed this natural transformation machinery to deliver any DNA sequence into plant chromosomes — removing the tumor-inducing genes and replacing them with the construct of interest.
The T-DNA System
The Ti (tumor-inducing) plasmid carries the T-DNA flanked by 25-bp border sequences. During infection, the virulence (vir) genes encode machinery that excises the T-DNA, coats it in VirD2 protein, and shuttles it through the plant cell wall and nuclear envelope. The T-DNA integrates semi-randomly into the plant genome via illegitimate recombination.
Modern binary vector systems separate the vir genes (on a helper plasmid) from the T-DNA (on a smaller, easier-to-manipulate binary plasmid). The construct, flanked by left and right border sequences, can be any size up to ~150 kb.
Transformation Protocols
The most common method is floral dip (for Arabidopsis) or leaf disk co-cultivation (for most crops). Leaf disks or callus tissue are co-cultivated with Agrobacterium, the bacteria are removed with antibiotics, and the tissue is transferred to selective medium where only transformed cells survive to regenerate. Scoville Splice delivers Agrobacterium transformation guides with every standard construct.