Preliminary Establishment of an Efficient Regeneration and Genetic Transformation System for Hemerocallis middendorffii Trautv. & C. A. Mey.

Hemerocallis middendorffii is widely used in the landscaping of Northern China for its exceptional ornamental and ecological attributes. It is also the focus of a substantial body of germplasm development and stress tolerance research. However, the absence of an efficient regeneration and genetic transformation system has been a critical barrier to conducting gene function studies on this species. In this research, the aerial parts of seed-derived H. middendorffii plantlets were used as explants, and the callus induction, proliferation, subculture, differentiation, and rooting conditions in the in vitro regeneration process were optimized. A callus induction rate of 95.6% was achieved, with a regeneration rate of 84.4%. Based on this procedure, a simple and effective Agrobacterium-mediated genetic transformation system was preliminarily developed using a hygromycin-based selection system. The system comprised an Agrobacterium tumefaciens culture solution optical density at 600 nm (OD600) of 0.6, an acetosyringone concentration of 100 μmol·L−1 in both the A. tumefaciens infection solution and the co-cultivation medium, a sterilization culture with Timentin at 300 mg·L−1, and a selection culture with hygromycin at 9 mg·L−1. Transgenic H. middendorffii T0 rooted plants were produced within a 5-month period, with a transformation rate of 11.9% and positive rate of 32.8%. The regeneration and genetic transformation system established in this study should help advance functional gene research and genetic improvement in H. middendorffii. However, the genetic transformation was only validated in the T0 plants. To confirm stable integration and long-term transgene stability, future research on the phenotypic and molecular characterization of T1 progeny, including segregation analysis and Southern blot verification, will be conducted.