Agrobacterium mediated gene transformation .

 WHAT IS AGROBACTERIUM ..........? 

Agrobacterium is a genus of Gram-negative bacteria that use horizontal gene transfer to cause tumors in plant .

                        Agrobacterium is well known for its ability to  transfer DNA between itself and plant ,and for this reason it has become an important tool for genetic engineering .

Agrobacterium is rod shaped plant pathogenic soil bacteria having two strain.....

1) A. tumefaciens  

2) A. rhizogenes 

A.tumefaciens  cause crown gall ( tumor ) and A.rhizogenes  hairy root disease in dicot plant by infecting through wounds on root or stem at the soil surface.

the bacterium contains Ti ( Tumor inducing ) and Ri ( root inducing ) plasmids .Both of these plasmid can transfer part of their DNA ( T- DNA) into plant cell chromosome by which plant cells become transformed by expression of T-DNA gene which induce disease.

Method of Agrobacterium mediated gene transformation -

1) Co- culture with tissue explants 

2) In plant transformation 

1) Co-culture with tissue explant :- 

-For transformation explant like protoplast ,callus, tissue slices, leaf disc , stem or floral tissue can be co cultured with genetically engineered Agrobacterium with recombinant vector for about 2 days.

- During co culture acetosyringone ( phenolic signal molecule ) released wound ( cut leaf disc) induce the Vir gene which bring about the transfer of recombinant T-DNA into many of the the plant cell .

-The explant can them be transferred to shoot inducing ( regeneration medium) containing  Kanamycin and carbenicillin.

- Kanamycin allows only transformed plant cell to divide and regenerate shoot in about 3-4 week ,while carbenicillin kills agrobacterium cells .

-The shoot are separated and transferred to root inducing medium and finally after few weeks are transferred to root inducing medium and finally after few weeks are transferred to soil.

-By this co- culture method only dicot plant can be transformed ,agrobacterium mediated transformation of monocot plant cell can be achieved by adding acetosyringone in the medium during co-culture.

2) In plant transformation -

- Transformation can be achieved by imbibition of seed in fresh culture of agrobacterium .T-DNA becomes integrated into plant genome .

- It appears that agrobacterium cells enter the seedling during germination ,are retained within the plants, when flower developed ,the zygote or cell become transformed .

Transgene can be introduced into plants by various direct and indirect method.

Advantages :- 

- simple and comparatively less expensive 

- High transformation efficiency 

- Transgenic crop obtained have better fertility percentage.

- Protocols for both dicotyledon and monocotyledon are available 

- Relatively large length DNA segment can be transferred .

Disadvantages :-

- Time consuming 

- Not all kind of cell can be treated by this method 

- Sometimes lead to false positive result.