Development of real time PCR assays for detection and quantification of transgene DNA of a Bacillus thuringiensis (Bt) corn hybrid in soil samples

Abstract

Real time PCR assays were developed to detect and quantify the transgene DNA of a commercially released Bacillus thuringiensis (Bt) corn (Zea mays L.) hybrid (DKC42-23), which was derived from the event MON863 and also carried a neomycin phosphotransferase gene (the nptII gene). We applied the real time PCR assays to investigate the persistence of the transgene DNA in a field trial grown with DKC42-23 over 3 years, in combination with bacterial natural transformation. The results showed that under continuous cultivation of DKC42-23, its transgene DNA was detectable in the field plots all year around. Meanwhile, when soil DNA extracts from DKC42-23 plots were used as donor in bacterial natural transformation, successful recovery of kanamycin resistant (Km(R)) transformants indicated that the nptII gene carried by DKC42-23 could be taken up and integrated into naturally competent Pseudomonas stutzeri pMR7 cells, leading to the restoration of the antibiotic resistance of P. stutzeri pMR7. However, after the cultivation of a soybean line in the same plots for the subsequent growing season, the presence of transgene DNA of DKC42-23 was reduced to undetectable levels at the end of that growing season. Therefore, existing corn-soybean crop rotation practices reduce the availability of transgene DNA in soil and thus minimize the risks that might be attributable to horizontal gene transfer. The real time PCR assays are useful for investigating the persistence of transgene DNA derived from the MON863 event in soil environments.

Soil microbial properties in Bt (Bacillus thuringiensis) corn cropping systems

“…In Bt (Cry3Bb1) corn grown in monoculture for three years in Ontario, Canada, Zhu et al (2010) detected the neomycin phosphotranferase (nptII) gene, an antibiotic-resistance gene commonly used as a marker in GM plants, in soil throughout the year. However, there was no evidence of transfer of this gene to soil bacteria .…”

“…Lawhorn et al, 2009) although some negative effects were reported after four years of Bt cotton (Gossypium hirsutum L.) monoculture (Chen et al, 2011). The neomycin phosphotranferase (nptII) gene, an antibiotic-resistance gene commonly used as a marker in GM plants, was detected in soil throughout the season in Bt (Cry3Bb1) corn grown in monoculture for three years (Zhu et al, 2010). However, there was no evidence of transfer of this gene to soil bacteria .…”