To demonstrate that the transgene was expressed, the deoxyribonucleic acid (DNA) was isolated from the suspected transgenic plant and examine by southern hybridization technique. When a combination of transformed and untransformed callus is located on an antibiotic selection medium, only the transformed callus with T-DNA holding antibiotic resistant marker is capable to survive and grow.
Normally, only a single copy of the T-DNA region is introduced into a certain plant genome where it separates at meiosis like a common Mendelian allele. The existence of the insert can be confirmed by screening the transgenic tissue for transgenic markers or by screening purified DNA with a T-DNA probe in southern hybridization technique.
Southern blot verifies incorporation of T-DNA into the genomic DNA of the target organism and also gives insertion copy number. In this technique, the genomic DNA is first digested with restriction enzyme EcoRI. Then run the digested DNA on agarose gel by applying electric field to separate fragmented DNA by size and transfer the fragments to nylon membrane. On the nylon membrane, hybridize with radioactive DNA probe, which is designed to be complimentary to the gene of interest. The probe that is complimentary will only bind to the concern gene and results in the fluorescent bands.