Background
The gram negative soil bacterium Agrobacterium tumefaciens causes crown gall disease in plants. This disease is characterized by the formation of tumors on plants after infection of wound sites by the bacterium. The crown gall cells gain two genetic properties not possessed by normal plant cells. These are the ability to grow in culture in the absence of externally added hormones, and the production of unusual compounds called opines. These opines are used by the bacterium as sources of carbon and nitrogen. The bacterium therefore creates a niche in the crown gall which is favorable for growth.
The molecular basis for this disease was identified in 1974 with the discovery of a 200 kb plasmid called the Ti plasmid (tumor inducing). Removal of the plasmid leads to avirulence, and re introduction restores virulence. The significant finding was that a 13 kb piece of the Ti plasmid is transferred into the genome of the recipient cell. This piece of transferred DNA is called the T DNA. The transformation of cells with this T DNA results in the development of the neoplastic growth called the crown gall tumor. This is accomplished by the products of three genes, tms 1, tms 2, and tmr. Tms 1 and 2 codes for steps in the synthesis of auxin and tmr code for the synthesis of cytokinin, the two phytohormones involved in cell growth and development. The increased levels of auxin and cytokinin directed by the T DNA leads to the tumorous morphology in transformed plant tissues.
The transfer of the T DNA into the plant genome is determined by a series of virulence (vir) genes located elsewhere on the Ti plasmid, and a short 25 base pair regions located at each end of the T DNA. Through the interaction of several vir gene products and the T DNA border sequences, the T DNA is transferred into the host genome. The transfer of this T DNA to plant cells is the key step in using Agrobacterium tumefaciens as an agent for directed transformation and genetic modification of higher plants. Vectors for successful transformation of plants with foreign genes are easily constructed by disarming (removal of the hormone genes) the Ti plasmid, and by inserting any gene(s) to be transferred within the border sequences. In the presence of the vir genes either on the same plasmid or another plasmid in the same bacterial cell, the genes are transferred to the plant genome. A growing number of plant species have been successfully transformed with foreign genes using such artificially constructed plasmid vectors. These foreign genes include selectable antibiotic resistance markers, easily scorable bacterial genes, and genes for useful agronomic traits such as herbicide tolerance, virus resistance, and insect resistance.
