PCR based detection of genetically modified tomato with AVP1D gene employing seed sampling strategy

Sampling is an essential requirement for developing precise and sensitive analytical methods for genetically modified (GM) crops to maintain seed purity in the seed and grain supply chains and to detect adventitious presence or unintentional mixing of GM seeds in seed lots. GM tomato line XAVP1D-2, overexpressing a mutant of Arabidopsis vacuolar H⁺-pyrophosphatase (AVP1D) gene, is highly tolerant to salinity and drought. Being a widely consumed vegetable crop, it is imperative to develop diagnostic protocols for GM tomato in order to effectively address the consumer's concerns. In the present study, a seed sampling strategy based on the International Rules of Seed Testing adopted by International Seed Testing Association (ISTA) has been reported to detect AVP1D gene and desired regions of transgene construct in GM tomato using PCR assays. The limit of detection (LOD) of the simplex PCR for AVP1D gene and transgene construct was up to 0.1%. The multiplex PCR assays were also developed for simultaneous amplification of AVP1D transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, neomycin phosphotransferase (nptII) selectable marker gene and nopaline synthase (nos) terminator along with β-fructosidase gene, an endogenous gene of Solanaceae family. The developed PCR assays can be effectively used for routine detection and monitoring of trait stability.