PLASMID (Ti AND Ri) MEDIATED GENE TRANSFER
Many plants have received attention for DNA cloning and expression of foreign DNA in plant cells. Major work is done on soil-borne bacteria such as Agrobacterium tumefaciens causing crown gall disease and Agrobacteirum rhizogenes causing hairy root disease on the stems of numerous plants. Agrobacterium plasmids (Ti and Ri) have been used for introduction of genes of desirable traits into plants. Explants are commonly used for gene transfer. These explants are co-cultured with Agrobacterium containing vector with modified foreign gene.
The transformed colonies are selected and used for regeneration of whole plant. These plants are tested for the transfer of gene with the help of screenable markers (reporter genes). The reporter genes are also transferred along with desired genes.
Agrobacterium tumefaciens contains plasmid which induces tumour in plants, these plasmid is called as Ti-plasmid (180 to 250 Kb). Ti-plasmid contains T-DNA region of about 23 to 25 Kb which is transferred into plant cells. Agrobacterium tumefaciens mainly present in soil and cause infection to dicotyledonous plants.
Bacterial species secrete lipopolysaccharide which help in attachment with polygalacturonic acid fractions of plant cell wall, Phenolic compound (acetosyringone) is secreted from the wounded cells walls of plants which induces the vir genes of Ti-plasmids. Vir genes encode an enzyme which nick the double stranded T-DNA on the same strand at two points. It produces single stranded DNA molecules and are carried into plant cells, A desired DNA fragment can be cloned in the region of Ti plasmid which will introduce the DNA fragment into plant genome. Agrobacterium rhizogenes causes formation of adventitious roots and this rhizogenicity has been correlated with the presence of a large plasmid (Ri-plasmid). Plasmid of this species also contains a T-DNA which causes the development of hairy roots. Ri-plasmid of Agrobacterium rhizogenes is useful for production of secondary root systems for ability to resist anoxia from flooding of the soil and better anchorage of plants.