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.