Large-scale production of enhancer trapping lines for rice functional genomics

Abstract

Using a rapid and efficient Agrobacterium tumefaciens mediated rice transformation protocol, we had got about 100,000 independent enhancer trapping lines of japonica rice variety Nipponbare. T 1 seeds had already been obtained from 60,000 lines. Regeneration of transgenic plants could be as short as 3 months starting from callus induction, thus the occurrence of somaclonal variation was reduced to a minimum level. The frequencies of hygromycin-resistant callus induction and plant regeneration were over 90 and 80%, respectively. PCR and Southern blot analysis showed that about 95% hygromycin resistant plants were transgenic. Southern blot and segregation analysis of T 1 seeds showed that about 40% transgenic plants contained single copy insertion, and the average copy number was 1.7. Histochemical assay of T 0 plants revealed expression patterns in root, leaf, embryo and endosperm of the reporter gene β-glucurodinase (GUS). This large-scale enhancer trapping pool provides a valuable tool for functional analysis of rice genome.