Figure 6

Kanamycin selection and regeneration of transgenic sugar beet guard cells transformed with the pSTM17::GAPc2ox1 constructs containing the neomycin gene driven by the mouse PGK promoter. A: Transgenic sugar beet callus grown from transformed sugar beet guard cell protoplasts cultured on medium supplemented with kanamycin at 100 μg ml^-1, clearly showing a difference between the healthy (green) callus and the dead (brown) non-transgenic callus. B: Shoots regenerated from transgenic sugar beet callus as above. C: Resultant transgenic seedlings in compost. D: Agarose gel showing the results of neomycin gene-specific PCR conducted on genomic DNA template extracted from sugar beets generated from two independent transformation events with pSTM17::GAPc2ox1 including the kanamycin driven by the mouse PGK promoter (arrows) compared with individuals from lines transformed with constructs containing a kanamycin cassette driven the CaMV35S promoter (the remaining PCR product bands). Sequences from other regions of the pSTM17::GAPc2ox1 construct were also detected by PCR and Southern blot hybridisation (results not shown). +ve = DNA plasmid template control, -ve = DNA template-free control; M = New England Biolabs 100 bp ladder. Diagnostic PCR for the neomycin gene sequences was performed using the primers Neo-For 5' CAG GAT GAT CTG GAC GAA GA 3' (Tm = 57.3 °C) and Neo-Rev 5' AAG AAG GCG ATA GAA GGC GA 3' (Tm = 57.3°C). The reactions (Qiagen Master Mix) contained 10 μM of each primer, and 50–100 ng genomic DNA template and were incubated at 94°C for 3 min, followed by 30 cycles of 94°C for 15 sec.; 55°C for 30 sec.; 72°C for 1 min and one cycle of 72 °C for 2 min.