Construction and introduction of OsAER1::LeAlaAT cassette to improve the nitrogen use efficiency in rice cv. Mekongga

Abstract

Nitrogen use efficiency (NUE) in rice can be improved by developing a variety that absorbs and use nitrogen (N) more efficiently. We have successfully isolated the Lycopersicon esculentum alanine aminotransferase (LeAlaAT) gene from tomato, which is responsible for improved efficiency of N use in plants, and Oryza sativa alkenal reductase (OsAER1) promoter from rice cv. Awan Kuning. These gene and promoter can be used to develop NUE rice through the genetic engineering approach. The purposes of this study were to construct an OsAER1::LeAlaAT expression plasmid cassette, to introduce the constructed plasmid cassette into genome of rice cv. Mekongga, and to test the transgenic rice lines at several levels of N concentration. The cassette was constructed by replacing the β-glucuronidase (GUS) gene derived from the pCAMBIA1300_OsAER1::GUS cassette with LeAlaAT gene. The recombinant plasmid was then transformed into Agrobacterium tumefaciens vector strain LBA4404 and introduced into rice cv. Mekongga. The plant transformation technique was carried out at with immature embryos as explants. Molecular analysis was performed using PCR to evaluate the presence of targeted gene construct introgression in the rice lines and the efficacy test of NUE was carried out hydroponically to determine the performance of lines at several levels of N concentrations. The results showed that the OsAER1 promoter and LeAlaAT gene were successfully constructed in pCAMBIA1300 vectors and introduced to Agrobacterium vector. Rice transformation cv. Mekongga resulted in 50 transgenic rice lines (T0). Hydroponic rapid screening of NUE in 50 T1 transgenic rice lines using Yoshida solution with ¼ strength of N, resulted in five selected rice lines based on superior biomass dry weight compared to control, namely M40, M41, M45, M50, and M54 lines. The T1 generation of rice lines was tested hydroponically to evaluate the response and plant performance at several N concentrations treatment. Compared to the control, the transgenic lines showed the best performance at ¼ strength of N concentration. Further NUE testing is required to determine the performance of selected rice lines in greenhouses and confined field trials.