Agrobacterium Mediated Gene Transfer
In the soil, a 'natural genetic engineer' is present and is a bacterium called Agrobacterium tumefaciems. From the time immemorial, this little bacterium transfer a gene fragment called T-DNA (transfer DNA) that lies in Ti (tumor inducing) plasmid into the plant genome. Bacteria transfers a specific gene segment to the plant genome without any outside intervention. We are exploiting bacteria's ability by placing our gene of interest in the place of bacterial Transfer DNA.
What is Agrobacterium tumefaciens?
Agrobacterium tumefaciens is a Gram negative, rod shaped soil bacterium that invade many dicot plants and causes crown gall disease by a naturally evolved 'genetic engineering' process.
The causative agent of crown gall disease is a plasmid called as tumor inducing plasmid or Ti plasmid.A part of the plasmid called as transfer DNA (T-DNA) is transferred from the bacterium to the plant cells and later gets integrated into the plant genome.The T DNA has genes for auxin, cytokinnin and opines. Expression of auxin and cytokinnin induces cell division leading to crown gall or tumor formation in the site.
Why bacteria transfers its T-DNA?
The T-DNA of Ti plasmid has genes for auxin, cytokinin and opine. Opine is a growth promoting nutrient for the infecting bacterium. As cytokinin and auxin promotes cell division and tumor formation, bacterium utilizes these cells as a factory for the production of opines.
The mechanism of gene transfer by Agrobacterium
The Ti plamid has
- a transfer DNA (T-DNA): The region that is transferred and integrated into the plant genome
- a 24 bp left and right repeats or (T-DNA borders): essential sequence for gene transfer
- an origin of replication,
- a virulence region (vir region): has genes that mediated gene transfer
- an opine catabolism region: has genes for utilization of opines synthesised by the plant cells
Essential requirements for T-DNA transfer
- T-DNA borders are essential for gene transfer
- vir region mediated transfer of T-DNA into the plant genome.
How we exploit the gene transfer capability of Agrobacterium?
The major fact that allowed the use of Ti plasmid as our gene transfer carrier is
'Any gene fragment of suitable size if placed between T-DNA borders will be transferred and integrated into the plant genome. The only requirement is vir region and T-DNA borders'.
So we can replace the T-DNA with our gene of interest. If T-DNA borders and vir region are present it will be transferred and integrated into the plant genome irrespective of the gene sequence. The process of replacing T-DNA (tumor causing gene) with our gene of interest is called as 'disarming'. The plasmid with our gene of interest in the place of T-DNA is called as 'disarmed plasmids'.
This video will help you a lot in getting into the details of Agrobacterium mediated gene transfer in plants.
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