Read the given paragraph carefully and answer the following questions:

Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). There are some basic steps. in genetically modifying an organism-identification of DNA with desirable genes: (i) introduction of the identified DNA into the host; (iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny. Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as 'T-DNA' to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen. Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation.

Which one gives blue colored colonies if the plasmid in the bacteria does not have an insert?

The presence of chromogenic substrate

The correct answer is Option (3) → The presence of chromogenic substrate   

The method used to differentiate between recombinants and non-recombinants is insertional inactivation. In this technique, a selectable marker, such as an antibiotic resistance gene, is replaced by the foreign DNA (insert) within the coding sequence of an enzyme called β-galactosidase.

This insertion inactivates the gene responsible for the synthesis of β-galactosidase, resulting in the inability of the recombinant bacteria to produce this enzyme.

To identify recombinant colonies, a chromogenic substrate is added to the medium. Bacteria carrying non-recombinant plasmids (without the insert) will produce functional β-galactosidase and produce blue-colored colonies when exposed to the substrate. On the other hand, bacteria with recombinant plasmids (with the insert) will have the β-galactosidase gene inactivated, and these colonies will not produce any color. Thus, the lack of color indicates the presence of recombinant colonies. This method simplifies the identification of recombinant colonies without the need for simultaneous plating on two different antibiotic plates, making the process more efficient and less cumbersome.