Gene transfer in plants, agro bacterium mediated gene transfer. Crown gall disease, the tumour inducing principle and Ti plasmid, incorporation of T- DNA into plant cells

Indirect Gene Transfer:- When stable transformation is achieved by incorporating genes into the genome of plant cells with the help of a biological factor, it is called indirect gene transfer. Agrobacterium bacterium is used in this.

Agrobacterium mediated Gene Transfer:-

•  Agrobacterium is a gram -ve bacterium found in soil around plant roots.

•  Two species of Agrobacterium bacteria cause diseases in dicot plants:-

i. Agrobacterium tumefaciens

ii. Agrobacterium rhizogenes

i. Agrobacterium tumefaciens:- It causes Crown gall disease in angiosperm plants. Ti-plasmid is found in it.

ii. Agrobacterium rhizogenes:- It cause hairy root disease in angiosperm plants. Ri - plasmid is found in it.

•  Recombinant DNA is made by incorporating the desired gene into the T - DNA portion of the Ti plasmid. Which are transferred to Agrobacterium cell.

•  Now cut round pieces of sterilized leaf.

•  Now incubate these pieces with Agrobacterium overnight.

•  Now kept these pieces in Shooting medium for 2 days.

•  Now add Kanamycin and Carbenicillin in this shooting medium and cultured for 3-4 weeks.

•  Now transfer this to the rooting medium, add Kanamycin and Carbenicilin, culture it for 3-4 weeks.

•  Now establish this plant in a pot in green house.

•  After a few days, establish the plant in the soil of the field.

Mechanism of Gene Transfer:-

•  The T - DNA portion of the Ti - plasmid has the property that it can integrate into the plant's genome.

•  Leaf pieces release the Aceto - Syringine chemical that activates the Vir - Operon of T - DNA.

•  Once activated, the T-DNA portion separates from the plasmid and enters into many plant cells.

•  Now this T-DNA integrates into the genome by going into the nucleus.

•  This results in stable transformation of the plant.