CP4-EPSPS protein expression levels
To facilitate the biosafety assessment of transgene escape to populations of wild relative species, it is important to conduct scientific research to properly estimate the expression levels of transgenes in wild individuals as well as the inheritance of the transgenes in wild populations (Zhu et al. 2004; Lu and Snow 2005; Lu 2008). In the present study, ELISA was used to detect CP4-EPSPS protein expression levels at different soybean growth stages, while EPSPS protein was detectable in F1-, F2- and F3-resistant plants. This observation was consistent with the findings of Kubo et al. (2013), who also showed that the gene was stably expressed and produced functional protein. Our study and the studies mentioned previously (Kubo et al. 2013) suggest that the transgene will be able to confer tolerance to glyphosate in the new host wild population. In addition, it is worth noting that a significant decline in the total protein content in both hybrids was observed compared to that in GM soybean. Since tolerance to glyphosate is very dependent on EPSPS protein expression levels in plant tissues, a reduction in the amount of endotoxin proteins in hybrids may contribute to the variability in tolerance. Consistent with our results, a similar conclusion was reported by Zhu et al. (2004), who also reported a decrease in Bt protein content in transgenic Brassica rapa and crop-weed hybrids. This difference may be associated with a weedy genetic background, positional effects, and the number of transgenes inserted per event (Moon et al. 2007).
Our study confirmed that the CP4-EPSPS protein was stably expressed in the hybrid soybean line, endowing these hybrid soybeans with herbicide tolerance, and the RR, RS and SS of F2 or F3 populations had similar seed germination, aboveground biomass, pod and seed number per plant and 100-seed weight, which indicated that the presence and absence of EPSPS or the copy number of the EPSPS gene were not significantly correlated with vegetative growth and fecundity. In contrast, heterosis between GM and wild soybean raises new competitive advantages for hybrids, allowing hybrids to obtain some similar growth characteristics as female wild soybean, such as seed dormancy, a higher stable grain weight, and greater pod and seed numbers per plant; these growth characteristics could increase the possibility of dispersal of transgenes through seed systems and may adversely affect genetic and species diversity of wild soybean. Thus, it is critical to build effective risk management and control measures for the gene flow of transgenes from GM soybean to wild soybean before commercial planting of GM soybean in China.