Particle-bombardment-mediated co-transformation of elite Chinese rice cultivars with genes conferring resistance to bacterial blight and sap-sucking insect pests

Abstract

Transgenic rice plants were generated using particle bombardment to simultaneously introduce the rice Xa21 gene eAective against bacterial blight and the Galanthus nivalis agglutinin (snowdrop lectin; gna) gene eAective against sap-sucking insect pests, specifically the brown plant hopper. Using three plasmids, we co- transformed 5- to 10-d-old, mature seed-derived rice (Oryza sativa L.) callus of two elite Chinese rice cultivars, Eyi 105 and Ewan 5. The plasmids carried a total of four genes. The gna and Xa21 genes were carried on separate plasmids. The selectable marker hygromycin phosphotransferase (hpt) and the reporter gene b-glucu- ronidase (gusA) were linked on the same, co-integrate vector. We recovered over 160 independently derived transgenic rice plants. Over 70% of the transgenic plants carried all four genes, as confirmed by polymerase chain reaction and/or Southern blot analysis. Furthermore, 70% of transgenic plants carrying all four genes also co- expressed all four genes, as confirmed by growth on selection media (hpt), GUS histochemical assays (gusA), western blotting (gna) and reverse transcriptase-poly- merase chain reaction (Xa21) analysis. The co-expres- sion eAciency reported for the four transgenes in our study is the highest ever found in any transgenic plant population generated through co-transformation. The linked genes (hpt and gusA) co-integrated with a frequency of near 100%, and we observed a co-integra- tion frequency greater than 70% for the genes carried on separate plasmids. We observed no preferential integra- tion of any particular gene(s). Genetic analysis con- firmed Mendelian segregation of the transgenes in subsequent generations. We report, for the first time, generation and analysis of transgenic rice lines carrying genes eAective against more than one taxa of pathogen or pest, substantiating that particle bombardment rep- resents an eAective way to introduce unlinked complex multiple traits into plants.