Production of transgenic rice homozygous lines with enhanced resistance to the rice brown planthopper

Mature seed-derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was co-transformed with two plasmids, pWRG1515 and pRSSGNA1, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin-containing medium, resistant callus was transferred to hygromycin-containing regeneration medium for plant regeneration. Twenty-six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy-three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.