SCMV-resistant Transgenic Maize Obtained By Using Different Strategies

Maize Dwarf Mosaic (MDM) disease in China was mainly caused by Sugarcane mosaic virus (SCMV). In an attempt to generate dsRNA-mediated transgenic maize plants resistant to SCMV, we inserted SCMV NIb gene-specific sequences 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 not bar gene.Both cp gene and crylacm gene were inserted into same plasmid and a double-gene expression vector was constructed. Maize immature embryos were cultivated with Agrobacterium harbouring the vector and regenerated seedlings were obtained by selection on medium containing Biolaphos. Moreover, leaf painting of herbicide and PCR amplification of both cp gene and crylacm gene were applied for detecting the transgenic status and 7 transgenic plants were selected for propagating progeny. The T1 plants were challenged with SCMV inoculum in greenhouse. 12 resistant plants confirmed to contain both cp gene and crylacm gene by PCR assay was selected for propagating T2 progeny. The percentage of double-resistant plants from one line was higher than 50% in challenging incubation of both SCMV and corn borer in field and the double resistant plants from the line were confirmed to contain both cp gene and crylacm gene by PCR assay.A marker-free antisense SCMV cp gene expression vector, named pACP, was constructed. Maize immature embryos were co-cultured with Agrobacterium carrying two vectors, one marker-free pACP and one vector harboring bar gene as the selective marker. Resistant calli were recovered by selection on medium containing Biolaphos and regenerated plantlets from resistant calli were obtained. The percentages of resistant plants from 2 T1 lines were higher than 70%. Further PCR assay confirmed that two resistant plants from one line contained acp gene but not bar gene.