However, even after transformation, it is essential to confirm the integration of genes, for the success of transgenesis.
The incorporation can be confirmed through
(i) The use of markers,
(ii) Southern blotting,
(iii) Northern blotting, and
(iv) Polymerase chain reaction.
(i) Marker and Reporter Gene:
Transformation in eukaryotic system is a very low frequency event. It is, therefore, important that there should be some means for selecting the transformed plant tissue. There are certain selectable markers genes present in vectors that facilitate the selection process. In transformed cells the selectable marker genes are introduced through vector.
For each marker gene there is one substrate which is a toxic substance like an antibiotic or any herbicide. The transformed cells are cultured in medium containing the toxic substance or antibiotic which facilitates the selection of transformed tissue to grow as the marker gene confers the resistance property to the transformed cell only.
The most widely used selectable marker gene is the npt II (neomycin phosphotransferase) gene that confers resistance to kanamycin, G418, neomycin. Other antibiotic resistance genes against bleomycin (We), spectinomycin and hygromycin (hpt) are used quite widely. The resistance genes against some herbicides like chlorosulphuron (als) and bialaphos (bar) are also in use (Table 24.1).
In addition there is an alternative procedure where there will be no selection pressure imposed on cells to grow further. The samples from grown tissue are tested for expression of the marker genes which are described as reporter gene or scoreable gene or screenable gene. Reporter genes are used in plant transformation vectors, both as a means of assessing gene expression by promoter analysis and as easily scored indicators of transformation.
Ideally, reporter genes should be easy to assay, preferably with a nondestructive assay system and there should be little or no endogenous activity in the plant to be transformed.
At present only a small number of reporter genes are in widespread use in plant transformation vectors, these being β-glucuronidase (gus)—causing blue colour (Fig. 24.8), green fluorescent protein (gfp), luciferase genes (lux and luc)—causing luminescence and to a lesser degree (although it is widely used in animal systems) the chloramphenicol acetyl transferase gene (cat).
(ii) Southern Blotting or Southern Hybridization:
It is a molecular technique which combines electrophoretic separation of DNA fragments with nucleic acid hybridization. In this method, the desired DNA to be tested is cut by restriction enzymes; gel electrophoresed and denatured gel fragments are transferred to nitrocellulose filter sheets.
The denatured DNA probe—the alien DNA, is tagged with radioisotope and added under renaturing condition (lower temperature) to the filter containing target DNA. The hybridized target DNA probe sequence can be detected by radioactive spots or auto-radiography.
(iii) Northern Hybridization:
It is an analogous method of Southern hybridization where the recipient or target sequence is the mRNA (messenger RNA) and not DNA. Northern hybridization becomes necessary to check that the introduced DNA in the chromosome is able to function or express and thus undergoes transcription to produce the desired messenger. Northern Blot confirms the presence of desired messenger RNA- complementary to the alien DNA, which is used as probe.
(iv) Polymerase Chain Reaction (PCR):
The genomic DNA is isolated from the suspected transformed plant tissue; designing the appropriate primers for the transgene, the PCR technique is used for gene amplification. Amplified transgene can be easily detected through gel electrophoresis only.
This technique is now easier and quicker method for detection of transgene as it does not require much purified plant DNA and within a very little period of time many samples can be screened for the presence of transgene.