Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction

Abstract

Quantitative real-time polymerase chain reaction (PCR) assays were designed that enabled the zygosity of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) to be determined. The two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers, were both accurate and reproducible in the determination of the number of transgenes present in a cell line. In the first assay, in which TaqMan assays were performed on increasing amounts of a plasmid containing the transgene of interest, a linear relationship between the level of fluorescence and the template amount was produced. Using the resultant linear relationships as standard curves, we were able to determine the zygosity of both soybeans segregating for the cry1Ac transgene and that of a T₁ peanut segregating for the hph transgene. In the second assay, a relative determination of copy number (referred to as comparative Ct) was performed on transgenic soybeans by comparing the amplification efficiency of the transgene of interest to that of an endogenous gene in a multiplexed PCR reaction. Both methods proved to be sufficiently sensitive to differentiate between homozygotes and hemizygotes. These assays have numerous potential applications in plant genetic engineering and tissue culture, including the hastening of the identification of transgenic tissue, selecting transformation events with a low number of transgenes and the monitoring of the transmission of transgenes in subsequent crosses.