FIGURE 5.

Confirmation of the stable integration of the pANIC12A transgene in S. viridis transformants via Southern blot hybridisation analysis. (A) PCR analyses confirmed the integration of two pANIC12A transgene-derived sequences in 5 T₁ transformant lines, including the HYGROMYCIN PHOSPHOTRANSFERASE II [HPTII at 845 base pair (bp)] selectable marker gene and the RED FLUORESCENT PROTEIN (RFP at 360 bp) visual reporter gene. In addition, the S. viridis gene, ELONGATION FACTOR 1 (EF1 at 192 bp), was used as an internal control for PCR screening of S. viridis plant lines. (B) Southern blot hybridisation analysis revealed that transformant lines 1, 3, 4, and 5 harboured a single copy of the HPTII gene, as indicated by a single distinct band. However, two distinct bands were visualised for S. viridis transformant line 2 to indicate that this transformant line harboured multiple pANIC12A integration events. WT: the non-transformed S. viridis wild-type plant (negative PCR control). P: pANIC12A plasmid DNA (positive PCR control). Lines 1 to 5: S. viridis transformant line 1 to 5.