Selection of T1 transgenic lines. Transgenic T1 individuals are selected for inherited antibiotic resistance separately at the gametophyte (a–e) and sporophyte (f–i) stages. Example shown is 35S::GUS-HygR. a–e Gametophyte selection. Spores are sown on two C-fern 1% agar (pH 6.0) plates: one containing antibiotic (selective plate), the other not (control plate). By 11 days after sowing, WT (a) and T1 (b) gametophyte populations have developed on the control plates. Under antibiotic selection, WT gametophytes have not developed (c) but some resistant T1 gametophytes have developed successfully (d). Inset shows resistant gametophyte developing on the plate (arrowhead). Resistant T1 gametophytes can then be analysed for transgene expression in addition to antibiotic resistance (e). f–i Sporophyte selection. T1 sporophytes are induced from the control gametophyte plate by repeated application of sterile water. Sporophytes typically become visible on the plate within 2 weeks (f). Individual sporophytes are then transferred to C-fern 1% agar (pH 6.0) plates containing antibiotic to screen for antibiotic resistance. Selection typically requires 7–14 days. Antibiotic selection kills WT (g) and non-transgenic T1 sporophytes (h), whilst transgenic T1 sporophytes within the population survive and continue to grow (h). After antibiotic selection resistant T1 sporophytes can be transplanted to soil, or analysed for transgene expression in addition to antibiotic resistance (i).