Figure 3
Callus bombardment and T0 sporophyte regeneration. Genetic transformation of C. richardii callus is through particle bombardment (a–d). Example shown is transformation of 35S::GUS-HygR. Callus is assembled for bombardment on a 50 mm plate containing 1× MS 2% sucrose 0.7% agar media (pH 5.8) containing 5 μM KT (MS-KT), creating a dense 25–30 mm diameter target for bombardment. After bombardment callus is transferred onto MS-KT plates containing antibiotic (b) and selection is maintained for 8 weeks, transferring to fresh selective media every 2 weeks. KT treatment is removed after 2 weeks to allow tissue differentiation. Untransformed callus is killed and turns dark brown without further growth (c). Transformed callus cells survive and divide, regenerating into differentiated sporophytes (d) suitable for transplanting to soil. Multiple sporophytes can regenerate from the same piece of callus. Transgene expression within regenerated sporophytes is variable (e–f), with some demonstrating full transformation (e), others apparently chimeric expression (f) and some demonstrating no expression despite antibiotic resistance (g–i).