Development Of Dwarf And Male Sterile Rice By RNA Interference

Rice is not only one of the most important food crops in the world, feeding nearly 50% of the world?s population, but also a model crop for plant molecular genetics and breeding. In traditional rice breeding, rice anthers or pollens must be emasculated in preparation of crossing because for its self-pollinating nature. Emasculation of rice anthers or pollens is a time-consuming work, making recurrent selection very difficult in rice breeding. Recent advances in molecular biology have raised possibility of development of new plant traits through introduction of specific genes from one species into another. The rice semi-dwarfing phenotype conferred by the green revolution gene sd-1 and male sterility trait related with hybrid rice breeding have been constantly exploited to improve the rice productivity. In order to regulate plant height and male sterility of rice synergistically, we have adopted RNAi strategy to suppress expressions of endogenous genes Os GA20ox2(Green Revolution gene sd-1) and RTS(RICE TAPETUM-SPECIFIC GENE) or DTD(DELAYED TAPETUM DEGENERATION) simultaneously to develop a kind of dwarf and male sterile(DMS) rice that can be used a genetic tool for recurrent selection of multiple traits or genes in rice breeding. The main results are as follows:1. We have constructed two hairpin RNAi vectors p TCK-RIIR and p TCK-DIID. For p TCK-RIIR, the DNA fragments of Os GA20ox2 and RTS were joined in the hairpin structure; for p TCK-DIID, the DNA fragments of Os GA20ox2 and DTD were joined for the hairpin structure.2. The two RNAi vectors were used to transform callus generated from Z0201, a japonica rice variety, by Agrobacterium-mediated transformation. From the regenerated T0 plants in controlled condition, 100 PCR-positive plants were selected by PCR analysis with hygromycin phosphotransferase gene(Hyg) specific primers, among which 70 plants were generated from transformation with vector p TCK-RIIR and 60 plants from transformation with p TCK-DIID.3. By phenotypic identification of the T0 transgenic rice plants, 37 p TCK-RIIR-induced(from transformation with vector p TCK-RIIR) and 46 p TCK-DIID-induced DMS rice plants were obtained; their fertility was almost completely sterile and plant height was in a range of 40-60% compared with the wild-type(non-transgenic Z0201) plants. Unfortunately, the p TCK-RIIR-induced DMS rice plants could not be examined in next generation due to the failure of normal seed production. Nonetheless, the p TCK-DIID-induced DMS rice plants produced seeds normally in cross pollination or open pollination. The progenies of the p TCK-DIID-induced DMS rice were clearly distinguishable from the wild-type plants in T0, T1 and T2 populations, and the DMS phenotype was inherited following the Mendelian ratio(1:1) in T1 and T2 generations.4. RT-PCR and q RT-PCR analysis showed that expression of the endogenous target genes, Os GA20ox2 and DTD, was significantly reduced in the DMS rice plants, indicating that the RNAi vector p TCK-DIID could synergistically silence expression of the two endogenous target genes in DMS rice plants from generation to generation. Except for the traits of plant height and fertility, the other yield relevant traits of the p TCK-DIID-induced DMS rice plants were not significantly different from those of the wild-type.5. Additionally, we have crossed simultaneously some panicles of the DMS rice plants with other pollen-donor parents for testing the ability of recurrent crossing in T0, T1 and T2 generations. The obtained results exhibited that the recurrent DMS rice also produced 50% of DMS rice plants and 50% of tall and male fertile(TMF) plants, and as expected, no T-DNA was detected in the TMF plants. Since it is very easy to identify the DMS rice plants based on the shorter plant height and crossing can be easily done without emasculation.6. In conclution, DMS rice provides a favorable tool for rice recurrent selection, or a genetic tool for highly efficient pyramiding of multiple genes in rice breeding. Since the DMS rice progeny populations always release 50% of TMF plants with no T-DNA, elite lines from the TMF plants can be commercialized without biosafety concerns of GM crops.