Several essential features are required for Agrobacterium-mediated transformation of plants:
- vir genes. Approximately 35 vir genes map outside the T-DNA (transferred DNA) region and encode products required for excision, transfer, and integration of T-DNA into a plant genome. vir genes act in trans, meaning they do not need to be physically attached to the T-DNA to cause integration into the plant genome.
- T-DNA border sequences. These are sequences of 25 bp imperfect repeats that flank the T-DNA and are required for its transfer. Border sequences encompass the recognition sites for a site-specific endonuclease, which is encoded by the vir D operon, part of the vir genes. The endonuclease cleaves the lower DNA strand of the T-DNA marking the starting point of the transfer.
Apart from the T-DNA border sequences, most of the genes of the T-DNA can be replaced by genes of interest to be transferred into the plant. Such engineered T-DNA is generally referred to as mutant T-DNA, engineered T-DNA or disarmed T-DNA.
However, the exact definition of the terms in a patent application is often provided by the inventor.
- cis-regulatory regions. These include the right and left T-DNA borders, which are physically attached to the genes to be transferred into the plant genome. Other cis -regulatory regions include promoters and terminators that flank the transgenes and regulate their expression. Commonly used promoters and terminators are the nopaline synthesis gene (NOS) and Cauliflower Mosaic Virus (CaMV) 35S promoter.
- Selectable marker genes. In plants, they allow the identification and selection of cells with the gene of interest incorporated in their genome. Bacterial selectable markers permit the identification of bacteria transformed with a vector carrying the marker. Examples of plant and bacterial selectable markers are hygromycin phosphotransferase and kanamycin, respectively.
The above-mentioned elements are incorporated in two basic types of vectors used to transform a wide range of plants via Agrobacterium:
- Binary vectors. In this system, the T-DNA and the vir region reside in separate plasmids within the same Agrobacterium strain. The vir genes are located in a disarmed (without tumor genes) Ti plasmid and the T-DNA with the gene of interest is located in a small vector molecule.
- Co-integrated vectors. These vectors result from the recombination of a small vector plasmid, for example an E. coli vector, and a Ti plasmid harbored in A. tumefaciens. The recombination takes place through a homologous region present in both of the plasmids. An engineered T-DNA containing the gene of interest can be in either one of the plasmids.