Gene transfer in higher plants for the development of genetically modified crops (gm crops)

Plant transformation using Ti-plasmid of Agrobacterium tumefaciens has become the most powerful method of foreign gene introduction into plant cells and subsequent regeneration of transgenic plants. The genetic engineering of plants actually started with the discovery of a pathogenic bacterium, Agrobacterium tumefaciens, causing a disease known as crown gall (tumor). A. tumefaciens, a rod shaped spore-forming Gram negative pathogenic soil bacterium. The bacterium is considered as natural genetic engineer in higher plants. The property of natural tendency of gene transfer to plants comes from the genes carried by Ti-plasmid (> 200 kbp) of Agrobacterium, which catalyzes the transfer of T-DNA to plant genome naturally. This feature is exploited in plant transformation, where a foreign gene inserted into T-DNA region (~23 kbp) is naturally transferred to plant. Thus bacterial Ti-plasmid is designated as key component in the gene transfer mechanisms. Natural Ti-plasmid is not suitable for gene transfer because of its large size and virulence genes content, which may create disease not desirable in transgenic system. Disarmed Ti-plasmid is used without any virulence genes on itself. Virulence genes are necessary for gene transfer processes but supplied by other helper plasmid. Only the T-DNA region flanking (25 bp direct repeat) with left and right border is transferred into the plant nucleus where it integrate with the genome for expression. Natural T-DNA mainly carries plant hormone genes (auxin and cytokinin) to develop tumoregenic growth (crown gall) by misbalancing the hormonal balance of the plant. T-DNA region also carries some special kinds of metabolizing gene for the synthesis of opines (amino acid derivatives- nopaline/octopine). These compounds serve as sources of carbon and/or nitrogen and energy and also stimulate plasmid conjugative processes as well as virulence genes (vir genes) expression. The genes encoding enzymes for opine catabolism is located outside the T-DNA. Natural Ti-plasmids are used in vector construction are as follows -pTiAch5 (octopine type) and pTiC58 (nopaline type). It was the findings that Agrobacterium can infect only dicots but nowadays routinely used in gene transfer in monocots including cereals crops. A number of plants have been transformed using this Agrobacterium-mediated gene transfer technique. Most prominent vectors such as co-integrate vectors (pGV2260) and binary vectors (pBIN19 and helper vector pAL4404) are used in foreign gene introduction into plant. The recombinant vectors with foreign gene are then used for transformation of plant explants (protoplast, tissues, leaf disks). Co-cultivation is widely used for transformation of dicot as well as monocot species. Ri-plasmid is another type plasmid present in Agrobacterium rhizogenes, which is responsible for root induction in plants and has been used in gene transfer system.