Simple, Efficient and High-Throughput Method for Transgenic Confirmation

Abstract

The present study reports a simple, efficient and high-throughput method for confirmation of neomycin phosphotransferase transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby) plants. For confirmation at seedlings and maturity stage, transgenic (T7) and wild type plants were sprayed with various Kanamycin concentrations (100–400 mg/l). Only the wild type plants sprayed with concentrations ≥300 mg/l developed typical chlorotic symptoms after 2 days, thus differentiating from the transgenic. For transgenic confirmation at earlier stage, transgenic and wild type seeds were germinated on filter paper soaked with Kanamycin solutions (100–500 mg/l). Opening of cotyledonary leaves and growth of wild type seedlings reduced considerably in Kanamycin concentrations ≥200 mg/l, than the transgenic, thereby differentiating from the transgenic. The transgenic confirmation experiments were repeated in subsequent T8, T9 and T10 generations with the same results. Thus, the Kanamycin resistance based transgenic confirmation methods are of wide applications for research community, as well as for enforcing bio-safety regulations, especially during the containment and confined field trials.