This plasmid is isolated and is cleaved open at a site in the T-DNA segment with the help of a specific restriction endonuclease. Now the isolated Nif gene is spliced into the opening with the help of the enzyme ligase. Thus the Nif gene is incorporated into the Ti plasmid. This Nif gene, integrated with the plasmid is called recombinant DNA.

3. Cloning the Nif gene

Cloning is a process by which hundreds of copies of Nif genes are produced. For this, the recombinant plasmid is introduced into the Agrobacterium. As these cells divide the plasmid and the Nif genes are also replicated. Thus thousands of copies of Nif genes are produced.

4. Introduction into the plant genome

Now the bacterium containing the recombinant plasmid is introduced into the plant cell. In the cell, the T-DNA containing the Nif gene of the plasmid gets separated and is transferred to the plant-cell genome. It gets incorporated with the plant genome and replicates along with the plant genome. Thus the plant genome gets the property of nitrogen fixation.