Fig. 1

T-DNA region of the binary construct pTGH-1"

Table 1

Primer sequences used in this study"

Fig. 2

PCR identification of positive transgenic plants of the I. variabilis EPSPS gene A: PCR identification of the target gene I. variabilis EPSPS by primer pair EPSPS-F/EPSPS-R; B: PCR identification of the CaMV 35S promoter by primer pair 35SP-1/35SP-2; C: PCR identification of the CaMV 35S terminator by primer pair 35ST-1/35ST-2. M: 2 kb DNA marker; P: the positive control of pTGH-1plasmid; N: the negative control of J9707; 1-12: the T0 transgenic plants from EPS-1 to EPS-12."

Fig. 3

Determination of the I. variabilis EPSPS transgenic copy number by Southern blotting analysis in the T0-positive transgenic plants A: detection of Southern blot analysis. The hybrid probe is on the I. variabilis EPSPS gene; the genomic DNA was digested with Hind Ш before electrophoresis. M: DNA marker; 1-31: the positive transgenic plants; 1: EPS-1; 3: EPS-2; 12: EPS-7; 29: EPS-3; 31: EPS-6; N: J9707 (negative control). B: summary of the copy number of transgenic plants."

Fig. 4

Determination of the I. variabilis EPSPS transgenic copy number by Southern blotting analysis in the T2 and T3 positive transgenic plants A: detection of Southern blot with the hybrid probe of CaMV35S promoter and EcoR I restriction enzyme; B: detection of Southern blot with the hybrid probe of CaMV35S promoter and Hind Ш restriction enzyme; C: detection of Southern blot with the hybrid probe of EPSPS gene and EcoR I restriction enzyme; D: detection of Southern blot with the hybrid probe of EPSPS gene and Hind Ш restriction enzyme. M: DNA marker; P: pTGH-1 plasmid (positive control); N: J9707 (negative control); 1-9: the positive transgenic plants, which are a T2 plant of EPS-1, a T2 plant of EPS-2, a T2 plant of EPS-3, a T2 plant of EPS-4, a T2 plant of EPS-5, a T2 plant of EPS-6, a T3 plant of EPS-6, a T2 plant of EPS-7, and a T3 plant of EPS-7, respectively."

Table 2

Glyphosate-resistant segregation analysis of transgenic lines with single T-DNA insertion"

Fig. 5

Insertion location information of T-DNA of EPS-2, EPS-6, and EPS-7 in the transgenic lines"

Fig. 6

PCR confirmation of the T-DNA insertion in the transgenic lines of EPS-2, EPS-6, and EPS-7 with specific primers A: PCR confirmation of the T-DNA insertion in the transgenic line of EPS-2 with specific primer pair EPSPS2 F/P5. B: PCR confirmation of the T-DNA insertion in the transgenic line of EPSPS6 with specific primer pair EPSPS6 F/P5. C: PCR confirmation of the T-DNA insertion in the transgenic line of EPS-7 with specific primer pair EPSPS7 F/P5. M: 2 kb DNA marker; P: pTGH-1 plasmid (positive control); N: J9707 (negative control); 1: T0-transgenic plant; 2-4: T1-transgenic plant; 5-7: T2-transgenic plant; 8-10: T3-transgenic plant."

Fig. 7

Expression analysis of the transgenic lines of I. variabilis EPSPS A: the RNA expression levels of the target gene in the leaf of T0-positive transgenic lines detected by RT-PCR. M: 3 kb DNA marker; 1-5: the positive transgenic lines of EPS-1, EPS-6, EPS-7, EPS-2, and EPS-5, respectively; N: J9707 (negative control); P: pTGH-1 (positive control). B: the protein expression level of the target gene in the leaves of the T0-positive transgenic lines detected by Western blot. N: J9707 (negative control); 1-6: the positive transgenic lines of EPS-1, EPS-6, EPS-36, EPS-7, EPS-2, and EPS-5, respectively. C: the relative expression of the target gene in different tissues of the T3-positive transgenic lines detected by qRT-PCR. Actin2 (AF111812) in rape is an internal reference gene."

Fig. 8

Phenotypes of T1 transgenic plants of I. variabilis EPSPS under the treatments of glyphosate"

Fig. 9

Phenotypes of T3 transgenic plants of I. variabilis EPSPS under the treatments with different concentrations of glyphosate at seedling stage"