Question

You have identified a useful gene in bacteria. Make a flow chart of the steps that you would follow to transfer this gene to a plant.

Answer 1

Isolation of DNA:

• The bacteria which contains the gene of interest is grown and harvested.
• The bacterial cells were digested by lysozyme enzymes.
• The DNA is concentrated by appropriate treatments and the purified DNA is precipitated by adding chilled ethanol.
• The Ti plasmid (vector) is also isolated from Agrobacterium.

Fragmentation of DNA by restriction enzymes :

• Digestion of both the DNA and the vector (Ti plasmid) is performed by incubating the purified DNA and vector separately with restriction enzymes.
• Both the vector and DNA with the gene of interest are digested with the same restriction enzyme.

Separation and isolation of desired DNA fragment :

• After restriction enzyme digestion, separation of DNA fragments is carried out by gel electrophoresis. The fragments are separated based on their size.
• Separated bands of DNA on the gel plate, are extracted by elution.

Amplification of gene of interest using PCR :

• After elution the DNA fragments are subjected to Polymerase Chain Reaction (PCR).
• The desired gene of interest is amplified to millions of copies.
• It takes place in three steps like denaturation, annealing and extension. The primer, Taq polymerase and deoxynucleotides are used for this procedure.

Ligation of the DNA fragment into the vector :

• The amplified gene is ligated with the Ti plasmid in the T - DNA region.
• Thus the gene of interest inactivates the tumour inducing capability of Ti plasmid.

Transferring the recombinant DNA into the host :

• The Ti plasmid is injected into the Agrobacterium host by transformation. Then the Agrobacterium is made to infect the plant cell and the gene of interest present in the T-DNA region is incorporated into the plant genome.

Summary:

The following steps are required to transfer a useful gene from bacteria into a plant :