Question

(i) Insertional inactivation is a method to detect the recombinant DNA. Explain this method.

(ii) Name the commonly used vector for transferring genes in plants from bacteria.

(iii) How is this able to transform a normal plant cell into a tumour ?

Hint:

Insert the gene inside lacZ so recombinants stay colourless, the vector is the Ti plasmid of Agrobacterium, and T-DNA oncogenes cause the tumour.

Answer 1

Step 1: The principle of insertional inactivation:
A good vector carries a marker gene. A popular marker is lacZ, which codes for the enzyme \(\beta\)-galactosidase.
The gene of interest is cloned inside a restriction site that lies within this marker gene. This breaks the marker gene, so the enzyme is no longer made. This is called insertional inactivation.

Step 2: How the recombinants are detected:
The host bacteria are grown on a medium with a chromogenic substrate.
Cells with a non-recombinant plasmid make active \(\beta\)-galactosidase. They turn the substrate into a blue colour, so the colonies are blue.
Cells with a recombinant plasmid cannot make the enzyme, so their colonies stay white. The white colonies are picked as recombinants.

Step 3: Part (ii): the vector:
The commonly used vector is the Ti plasmid (tumour-inducing plasmid) of Agrobacterium tumefaciens.

Step 4: Part (iii): how it makes a tumour:
Agrobacterium tumefaciens is a soil pathogen that infects dicot plants.
Its Ti plasmid carries a T-DNA segment. This T-DNA moves into the plant cell and joins the plant genome.
T-DNA has oncogenes. They make the cell divide without control, and the normal cell turns into a tumour (crown gall). Scientists remove the oncogenes (disarmed Ti plasmid) and use the plasmid to carry useful genes.

Final Answer:
(i) Insertional inactivation: insert in the marker gene, so recombinants lose its activity (white colonies). (ii) Ti plasmid. (iii) T-DNA oncogenes transform the cell.