**2.4 Confirmation of transgene integration by molecular techniques**

Confirmation of transgene integration has to be done through an appropriate method based on the transgene constructs, selectable marker, and reporter gene used. The antibiotic resistance genes are screened by the addition of herbicides or antibiotics to the growing media to distinguish transformed plant cells from the non-transformed plant cells. However, this method requires a large quantity of antibiotics and herbicides that are expensive and worsen by the risk of horizontal gene transfer to other bacteria.

An alternative screening method such as polymerase chain reaction (PCR) and reporter gene expression are used for the identification of transgenic plants. Some reporter genes such as the GFP, GUS, CAT, LacZ and Luc are used for the expression of these genes could be observed visually or directly under microscopy [12]. The GUS expression can also be detected through histochemical assay in which the localization of the transgene can be observed. Southern blotting is a molecular method which is generally used to identify the number of transgenes (copy number) inserted into the host genome as well as for the detection of transgene integrity and transgene rearrangement [13]. The polymerase chain reaction (PCR) method is one of the most sensitive and easiest methods among all the molecular techniques employed for the verification of the transgene. The PCR is generally done with primers specific to the site of plasmid constructs and gene of interest used for development of the transgenic plants. Successful amplification of the DNA fragment with expected band indicates the possible presence of transgene, and this DNA fragment is further confirmed through DNA sequencing. A real-time PCR provides fast, sensitive, and high-throughput molecular PCR–based analysis compared to the traditional Southern blot analysis especially in the area of transgene copy number and zygosity detection in transgenic plants [14]. In recent years, the availability of NGS tools and bioinformatics resources facilitates the study of genome and molecular characterization of complex traits. Hence, NGS approach provides an alternative high-resolution analysis tool for transgenes insertion in GM crops [15].
