A rapid method for the analysis of zygosity in transgenic plants

Abstract Original ( T 0 ) transgenic plants are usually heterozygous for the transgene. In routine analysis, homozygous transgenic plants are sought among first generation ( T 1 ) plants, the descendants of each independent transgenic plant, based on segregation analysis of the second-generation ( T 2 ) seedlings. This procedure requires the growth of T 1 plants until seed production, which is not only time-consuming and laborious, but is often accompanied by the waste of the T 1 generation. Here we suggest an easy, rapid method to accurately identify homozygous and heterozygous transgenic plants at the seedling stage of T 1 plants. DNA of T 1 tomato seedlings was extracted by a fast extraction method and used as a template in duplex quantitative real-time PCR reactions. An endogenous single copy gene served to normalize the differences in the initial DNA levels. The parents, T 0 transgenic plants, were used to neutralize transgene copy number and to bypass the need of calibration with known heterozygous and homozygous plants. Using this assay, the zygosity of tens of T 1 seedlings was rapidly identified with 100% fidelity. This is the first report showing the profitability of this method in determining zygosity in plants.