Marker-free Transgenic Fragrant Rice Achieved Via Artificial MicroRNA Through Co-transfomation

Rice is the most important food crops in the world, and is the staple food for nearly half the population on the earth. With the improvement of people’s living standard, the demand for high quality rice is growing exponentially. Fragrance is an important eating quality property of rice. The price of fragrant rice with an unique flavor character is higher than unfragrant rice in the market. Therefore, fragrant rice breeding is one of the important contents of the modern rice breedingIn this study, we used WMD3(Web MicroRNA Designer3) http://wmd3.weigelworld.org to design amiRNA for betaine aldehyde dehydrogenase gene BADH2controlling rice fragrance. amiRNA driven by Ubi constitutive promoter and GluC endosperm specific promoter respectively was integrated into the co-transformed expression vector pSB130, then transformed into rice varieties of Nipponbare and Minghui86. After the smell identification and the determination of fragrance substance2AP, fragrance was detected in the Ubi::BADH2amiRNA transgenic plants but not in the GluC::BADH2amiRNA transgenic plants. In the Ubi::BADH2amiRNA transgenic fragrant plants, the expression of BADH2was specifically interfered by amiRNA. The expression of BADH2was significantly decreased to almost20-40%of the level of the expression in untransformed plants. The difference in the expression of a close homolog gene BADH1did not achieve significantly level. In the Ubi::BADH2amiRNA transgenic plants, fragrance substance2AP was detected definitely, however, the content was significantly lower than that of fragrant rice cultivars. while2AP was not detected in untransformed plants.Through continuous detection on targeted fragment amiRNA and selective marker gene hygromycin in the Ubi::BADH2amiRNA transgenic TO generation and its offspring T1, T2generation, the segregation of double T-DNA regions was observed in T1generation. amiRNA was detected in some transgenic frangrant lines without selective marker gene. The segregation ratio of amiRNA in offspring of part of the T1lines was fit to1:3statistically. Therefore, amiRNA in these lines was a single-copy insertion.The results of proline content determination showed that proline content in leaf of Ubi::BADH2amiRNA regeneration fragrance plants was slightly higher than that of untransformed plants in transgenic plants and the expression of PRODH and P5CS in transgenic plants were down-regulated and up-regulated respectively compared to untransformed plants...