Abstract:

RNA silencing is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA (dsRNA). In an attempt to generate dsRNA-mediated transgenic maize plants resistant to SCMV (sugarcane mosaic virus), we cloned SCMV NIb gene-specific sequences and inserted it into the binary vector p3301 in the sense and antisense orientations (named SCMVirNIb), which could produce RNAs capable of duplex formation in plant cells. Maize immature embryos were co-cultured with Agrobacterium carrying two vectors, one marker-free vector harboring the SCMVirNIb and one vector harboring bar gene as the selective marker. Resistant calli were recovered by selection on medium containing Biolaphos. Among the regenerated plantlets from resistant calli, 14 plants have been certified to contain SCMVirNIb by PCR amplification and DNA dot blot. T₁ plants derived from the 14 plants were challenged in greenhouse with SCMV inoculums and the percentages of resistant plants in 11 T₁ lines were higher than 60%. One plant in the T1 lines was found to carry SCMVirNIb without bar gene by PCR assay.
T₂ plants derived from T₁ SCMV resistant transgenic plants were challenged with SCMV inoculums in field. The percentages of resistant plants from 3 lines, including the line derived from the marker-free transgenic plant, were higher than 85%. And non-transgenic control plants were all susceptive. Further molecular analysis confirmed that the resistant plants from the marker-free transgenic line contained SCMVirNIb but bar gene.

Key words: Inverted-repeat transgene, Marker-free, Maize (Zea mays), SCMV