Developing a long-term and powerful in vitro culture and Agrobacterium-mediated transformation system for Miscanthus sinensis (Poaceae)

Abstract

Miscanthus sinensis (Poaceae) is one of the perennial non-food biomass crops, whose germplasms have extensive use in Miscanthus breeding owing to its strong ecological adaptability and high level of genetic variation. However, the lack of a simple and powerful transformation system for this Miscanthus species remains a challenge for improving the trait performances and understanding their genetic mechanisms. In the present study, an extremely high percentage of embryogenic calli was obtained for different genotypes of M. sinensis using their mature seeds as the explants. Furthermore, the embryogenic calli were found to maintain a long-term high regeneration capacity (more than 2 years) when they were subcultured timely. The callus propagation, shoot regeneration, and root production of M. sinensis exhibited different tolerance ability to the selective agent including hygromycin, G418, and basta, and as a result, a novel selective culture strategy for identifying resistant transformants was developed to improve the efficiency of selection and transformation, in which the concentration of selective agent was decreased with different developmental stage. Optimizing the co-cultivation method and duration significantly increased the transformation frequency as well. Using the optimized protocol, a transformation frequency of 62.22 % or a positive transgenic plant frequency of 65.44 % was obtained for M. sinensis via Agrobacterium tumefaciens strain GV3101 harboring binary vector pCAMBIA2301. However, GUS enzyme activity in the transgenic lines of M. sinensis failed to be accurately detected by histochemical staining assay as well as by quantitative fluorescent assay, suggesting that GUS expression was disturbed in the transgenic plants and as a consequence, its results needed to be carefully interpreted. This study provided an efficient tool for cultivar improvement and genetic analysis of M. sinensis or other Miscanthus species.