Evaluation of Bxb1 functionality in switchgrass. Schematic of (a) the pMBXS638 T-DNA containing the bxb1 recombinase gene, (b) the pMBXS640 T-DNA carrying a non-coding DNA fragment (stuffer), and (c) the pMBXS640 predicted T-DNA structures after stuffer excision. PCR primers are shown as r1&r2, g1&g2, e1&e2 and s1&s2, att sites as grey arrows and hybridization probes as grey rectangles. (d) Southern blot with probes for the bxb1 gene (left panel) and the gusA gene (right panel). Lanes: P1, pMBXS638 DNA; P2, pMBXS640 DNA; WT, wild-type plant; 1–4, individual co-transformed plants from the same callus line; 5, co-transformed plant regenerated from another callus line; 6, plant transformed with the vector pMBXS640. (e) PCR for detection of Bxb1-mediated excision of the stuffer: e, PCR reactions for the stuffer construct using primers annealing to sequences outside the region flanked by the att sites (primers e1&e2); s, PCR reactions for the stuffer fragment (primers s1&s2); Lanes: P1, pMBXS638 DNA; P2, pMBXS640 DNA; the plant samples are in the same order as in (d). (f) Sequence of a PCR product (obtained with primers e1&e2) representing the attL footprint. (g) RT-PCR detection of bxb1 (with primers r1&r2 in 5 ng of total RNA), gusA (g1&g2, 200 ng RNA), and actin (ActA and ActB, 0.5 ng RNA) as a loading control. Lanes: 1–6, transgenic plants in the same order as in (d) and (e); Abbreviations: PZmUbi1, promoter and first intron of the maize ubiquitin1 gene; bxb1, Bxb1 recombinase gene; P35S, CaMV35S promoter; bar, marker gene conferring resistance to bialaphos; POsActin1, promoter and first intron of the rice actin1 gene; gusAeGFP, β-glucuronidase-enhanced Green Fluorescent Protein fusion; attB and attP, Bxb1 recombinase recognition sites; attL and attR, sequences formed after excision; T, transcription terminator; LB, left border sequence; RB, right border sequence; N, NcoI site.