Engineering higher yield and herbicide resistance in rice by Agrobacterium-mediated multiple gene transformation

The Vitreoscilla hemoglobin gene (VHb), trans-zeatin secretion gene (tzs), and the modified 5-enolpyruvyishikimate-3-phosphate synthase gene (EPSPS), as linked expression cassettes, were simultaneously introduced into immature embryos of the rice (Oryza sativa L.) cultivars Xiushui-11, Qiufeng, Youfeng, and Hanfeng by Agrobacterium tumefaciens. A total of 1153 transgenic lines composed of 4222 plants were obtained through selection for hygromycin (hyg) B resistance. Genomic polymerase chain reaction (PCR), southern and northern blotting analyses, and other relative tests showed that all transgenes had been integrated into the rice genome and expressed effectively. Approximately 90.2% of the transgenic lines harbored all the transgenes. Expression analysis revealed that all transgenes coexpressed stably in transgenic plants, and the frequency of coexpression was about 85%. Statistically significant increases were observed in plant height, particle length, total grains per panicle, and filled grains per panicle in transgenic plant lines compared with the control. Our study demonstrates a possible way to introduce different transgenes as linked expression cassettes within a single vector into the plant genome. Moreover, this transgenic approach has great potential in developing new rice cultivars with increased productivity and enhanced tolerance to the herbicide glyphosate.