Plant materials
Glyphosate-resistant (GR) transgenic soybean 40-3-2 (labeled GM) and wild soybean Jiang pu were used in this study. The GM soybean expressing synthetic CP4-EPSPS gene confer tolerance against glyphosate herbicide, and wild soybean accessions used in these crosses were obtained in Jiang pu (32.05°N, 118.62°E, Nanjing, China). The experiment was carried out over four consecutive years, 2017-2020, in a greenhouse at the Key Laboratory on Biosafety of Nanjing Institute of Environmental Sciences. In 2017, using GM plants as the male parent (the pollen donor) and wild soybean as the female parent (the pollen recipient), crosses were performed in July by artificial pollination, and 54 F1hybrid seeds were collected in mid-October. The next year (2018), 23 out of 54 F1 seeds germinated and were subsequently transplanted, and the hybridity of F1 plants was double checked by spraying with glyphosate (14.4 g L-1) and PCR analysis as reported by previous studies (Sumarji and Suparno 2017). Among these 23 F1 individuals, 11 plants were glyphosate resistant and were grown in a greenhouse with their parental lines to examine their characteristics and produce second filial generation (F2) seeds by self-pollination. F2hybrids were examined for their characteristics and harvested separately to obtain third filial generation F3 seeds by self-pollination in 2019, and F3 individuals were planted to evaluate their traits in 2020.