Agrobacterium mediated gene transfer is one of the most widely used methods of gene transfer. Agrobacterium tumefaciens is a bacterial plant pathogen found in the soil which causes crown gall disease in plants.
The basic mechanism that works behind it is, transfer of a segment or small piece of bacterial DNA to the plant genome. This bacterial DNA segment gets incorporated in the plant genome, stabilizes there and transcribes or expresses itself.
The bacterial DNA causing disease is situated on a plasmid, Ti plasmid (Tumour inducing plasmid, 200kb) instead of on a bacterial chromosome.
The segment which is actually transferred to plant genome is situated on T-DNA (transferred DNA, 23kb) of this plasmid.
In the expression of disease symptom, two major sets of genes are involved
- Genes on T-DNA (total 15 genes)
- Virulence genes i.e. vir genes
Both of the above sets of gene can be present on the same plasmid (co-integrative vectors) or separate plasmid (binary vector).
Genes on T-DNA
The T-DNA contains two groups / types of genes, which possess the ability to express in plants.
The two types of gene are
Oncogenes – Encode for synthesis of auxins and cytokinins (phytohormones). The overproduction of phytohormones leads to proliferation of callus or tumour formation.
Opine synthesizing genes – Encode for synthesis of opines (a product from amino acids + sugars, which are produced and excreted by the crown gall infected cells and consumed by A. tumefaciens as carbon and nitrogen sources this means opines act as source of nutrient for bacteria.
Virulence Genes
These are located on vir region (30 kb) of Ti plasmid.
These genes are not transferred to the plant genome, just help in the process; organized in six operons that are essential for the transfer (virA, virB, virD, and virG) or increase transfer efficiency (virC and virE) of T-DNA.
They are involved in activities like recognition of host plant, the chemotaxis, attachment to the plant cell, transfer of DNA in plant cell nucleus and integration of inserted DNA into the plant chromosome.
Procedure
For transformation oncogenic T-DNA is removed and replaced by another desired DNA segment with border sequence.
While working binary vectors the vir genes are removed and the desired DNA is integrated between the borders.
The vir genes necessary for transfer to the plant genome are arranged on a second plasmid with T-DNA and both borders removed.
Such binary vectors restrict the transferred Ti plasmid from spreading uncontrollably as genes responsible for that are do not get transferred.
Advantages of Agrobacterium mediated gene transfer
Some of the merits of the method are listed below
- Simple and comparatively less expensive
- High transformation efficiency
- Transgenic crop obtained have better fertility percentage
- Today, protocols for both monocotyledons and dicotyledons are available
- Relatively large length chromosomal segment can be transferred with little arrangement
Disadvantages of Agrobacterium mediated gene transfer
Some of the demerits of the method are listed below
- Time consuming
- Not all kind of cells can be treated by this method
- Can not transform organelles
- Sometimes leads to false positive results