The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb.)

Protoplasts of Festuca arundinacea (Schreb.) were transformed with plasmids containing a hygromycin resistance gene, and plants were regenerated under a variety of selection regimes. Plant regeneration was highest under discontinuous selection, but resulted in the production of significant numbers of escapes. Continuous selection at low concentrations of hygromycin produced the highest numbers of transgenic plants without escapes. Transgene copy numbers for 83 plants were estimated and related to the selection regime under which the plants were regenerated. Discontinuous application of hygromycin produced a high proportion of plants containing two or fewer inserted copies of the transgene (87%), as did low levels (50 mg·l−1) of continuous selection on hygromycin (88%). Continuous selection at high concentrations of hygromycin biased selection in favour of multiple-copy containing plants. Using a plasmid encoding a hygromycin phosphotransferase enzyme of higher specific activity (pROB5) resulted in the production of significantly more transgenic plants.