Gene transfer by particle bombardment to embryogenic cultures of Picea abies and the production of transgenic plantlets

A particle inflow gun enabled efficient production of transgenic plantlets of Picea abies from embryogenic suspension cultures. In transient assays, the Zea ubiquitin promoter was 12-16 times as active as the 35S promoter. For stable transformation, the plasmid pAHC25 contained the bar gene and the gusA gene, both driven by the Zea ubiquitin promoter. Cells were maintained from 1 to 3 h before bombardment on proliferation medium supplemented with 0.25 M myoinositol and, from day 8, supplemented with Basta as selective agent. Embryogenic colonies resistant to Basta appeared from two months after bombardment. Of over 100 independent Basta-resistant sublines tested, 65% expressed the co-transformed reporter gene, even when it was not linked to the selectable marker. Over 80% of the sublines retained their embryogenic potential. Of 11 transformants analyzed, 4 contained transgenes in low copy number (1-3), the rest contained transgenes in up to 15-20 copies. Over 200 Basta-resistant sublines from four cell lines have been established, of which 138 are confirmed as transformed. Plantlets have been regenerated and grown on in pots.