Dev't of Marker-free and RSV-resistant GM Rice using a Twin T-DNA System

One of the convenient techniques in developing selectable marker-free transgenic plants is a twin T-DNA system. Thus, Yayuan Jiang from Shandong Agricultural University and other scientists developed a twin T-DNA system wherein the standard transformation plasmid (pCAMBIA 1300) was modified into a binary vector with two separate T-DNAs. One of the T-DNAs contained the hygromycin phosphotransferase (hpf) marker gene. Using the binary vector, two vectors were constructed expressing inverted-repeat structures targeting the rice stripe virus (RSV) coat protein (CP) gene and the special-disease protein (SP) gene.

Through Agrobacterium-mediated transformation, GM rice lines were generated. Seven independent clones were obtained which harboured both the hpt marker gene and the target genes (RSV CP or SP) in the primary transformants of pDTRSVCP and pDTRSVSP, respectively. The segregation frequencies of the target gene and the marker gene in the T₁ plants were 8.72% for pDTRSVCP and 12.33% for pDTRSVSP. Two of the pDTRSVCP lines and three pDTRSVSP lines harbouring the homozygous target gene, but not the hpt gene, were found to be strongly resistant to RSV.

The researchers conducted a molecular analysis of the resistant GM plants and confirmed the integration and expression of the desired genes. The resistant GM plants displayed lower levels of the transgene transcripts and specific small interfering RNAs, which may imply that gene silencing caused the viral resistance.

Read the abstract at http://link.springer.com/article/10.1007/s12038-013-9349-0.