Breeding Of Elite Transgenic Rice Lines Pyramided With Multigenes Conferring Resistance To Disease, Insect And Herbicide

Rice (Oryza sativa L.) is one of the most serious suffering cereal crops by pests, diseases and weeds. The ideal rice varieties performed not only high yield and good quality but also multiple resistance. Therefore, development of transgenic rice conferring multiple resistance will provide an important insurance for protecting against yield loss, and lead to a dramatic reduction in chemical pesticide useage. In this study, we developed stable elite transgenic rice lines pyramided with multigenes for resistance to insect pests, disease and herbicide through both transgenic and conventional breeding technology. Four target genes, including the Ap1 gene provides resistance against rice diseases, the Bt gene gives active against the Lepidopteran insects, the GNA gene conferring resistance to Homopteran insects, and the herbicide-tolerance Bar gene, were pyramided into the japonica rice variety Guanglinxiangjing with good agronomic traits and grain quality. Several trials were carried out to evaluate the resistance and field performance of the selected multi-gene transgenic rice lines. The main results were as follows:1. Selection of elite transgenic rice lines with multiple target genes. The transgenic rice line 3015-1-1 harboring GNA and Bar genes, was crossed with another transgenic line D10 or D19 containing Bt and Ap1 genes. After several generations self-crossing and molecular marker-assisted selection, three new types of selectable-marker free multi-gene transgenic rice lines were obtained, including two lines containing double transgenes (Ap1+Bt or GNA+Bar), two lines with triple transgenes (Ap1+GNA+Bar or Bt+GNA+Bar), one line with quadrivalent transgenes (Ap1+Bt+GNA+Bar). The multiple transgenes were confirmed to be homozygous and stable inherit by PCR and Southern blotting analyses.2. The evaluation of resistance of the multi-gene transgenic rice. Bioassay tests were carried out on multi-gene transgenic rice lines both in field and laboratory, and the results revealed that the selected multi-gene transgenic rice lines conferred multiple resistance as prospective. The multi-gene transgenic rice lines containing Ap1 gene showed resistance to rice bacterial blight strain KS-1-20 and sheath blight strain RH-9, while it was at infection and mid-infection level, respectively, for the wild type control. The neonate larval mortality rates of rice leaf folder (Cnaphalocrocis medinalis) reached 100% in an insect bioassay in vitro after fed with the multi-gene transgenic rice harboring Bt gene, which also revealed high-level resistance to striped stem borer (Chilo suppressalis), yellow stem borer(Tryporyza incertulas) and rice leaf folder in the paddy field bioassay. The multi-gene transgenic rice containing GNA gene showed significantly resistance to the brown planthopper (Nilaparvata lugens). The wild type rice was not resistant to all of the above insects. Furthermore, the multi-gene transgenic rice containing Bar gene showed significantly resistance to herbicide.3. The main agronomic characters and grain quality traits of the multi-gene transgenic rice lines. The results from small-scale field release trials showed that there were no significant differences on most of agronomic characters and quality traits between the transgenic rice lines and their wild type, except several agronomic characters were a little changed compared with wild type, for example, a little higher in plant height, more in plant productive tillers, and increased grain weight.From the integrative analyses of resistance and agronomic characters, the multi- resistant transgenic rice lines we obtained will be promisingly and widely applied in future breeding programs.