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

Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora x G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for beta-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30mg l(-1) hygromycin in liquid MS medium that contained 10 mg l(-1)N-phenyl-N'-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l(-1) sucrose and 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 mg l(-1) hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA.