last step

5. Culture of plant cells into trees
The plant cell is cultured by tissue culture methods and is converted into a tree. All the cells of this tree contain the Nif gene and they can fix free nitrogen.

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.

1. Isolation of Nif gene

Nif genes are present in the nitrogen fixing bacteria Rhizobium. The bacterial cell is opened by the enzyme Lysozyme. The DNA is treated with a specific restriction endonuclease.
It produces DNA fragments with sticky ends. These fragments carry Nif genes.

2. Integration of Nif gene in the vector

steps

1. Isolation of Nif gene

Nif genes are present in the nitrogen fixing bacteria Rhizobium. The bacterial cell is opened by the enzyme Lysozyme. The DNA is treated with a specific restriction endonuclease.
It produces DNA fragments with sticky ends. These fragments carry Nif genes.

2. Integration of Nif gene in the vector

Mechanism of genetic engineering

Mechanism of genetic engineering
The mechanism of genetic engineering can be studied by transferring a Nif gene (nitrogen fixing gene) from a bacterium to a dicotyledonous plant. The Nif genes help the bacteria to convert free N2 into nitrates. But dicotyledonous plants do not contain Nif genes; so they cannot use free N2. When Nif genes are transferred from bacteria to plants, the plants get the ability to use free N2. So the plant need not be given artificial fertilizers. This saves money labour and increases the yield.

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