Use of paromomycin as a selective agent for oat transformation

Summary

Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R₁ seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.