Production of transgenic gentian plants by particle bombardment of suspension-culture cells

Cell suspension cultures were established from leaf explants of gentian (Ge ntiana triflora x G. scabra) for the generation of transgenic plants by par ticle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expres sion of a gene for beta-glucuronidase driven by the cauliflower mosaic viru s (CaMV) 35S promoter. We found that prior culture of suspension culture ce lls for 5 days on solid medium was optimum for successful particle bombardm ent. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30mg l(-1) hyg romycin in liquid MS medium that contained 10 mg l(-1)N-phenyl-N'-1,2,3-thi adiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l(-1) sucrose a nd then on solid medium prepared from the same liquid medium plus 2g l(-1) gellan gum. After 12 weeks of selection on solid medium that contained 30 m g l(-1) hygromycin, two transgenic gentian plants were regenerated from eac h selected callus. Analysis by the polymerase chain reaction and Southern b lotting revealed the stable integration of transferred DNA.