RECOVERY OF TRANSGENIC TREES AFTER ELECTROPORATION OF POPLAR PROTOPLASTS

Protoplasts from leaflets of in vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying the nptII gene for resistance to neomycin; PGH1, carrying a mutant acetolactate synthase gene, als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10(5) to 10(4) protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 mu M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS of Arabidopsis and 25 mu M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated. Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.