Development of an Efficient Agrobacterium-Mediated Transformation Protocol for the Tetraploid Medicinal and Vegetable Species Solanum villosum Mill.

Abstract

Genetic transformation is a cornerstone approach for functional genomics and molecular breeding in plants. Solanum villosum Mill. is a tetraploid species of the genus Solanum that possesses both nutritional and medicinal properties. However, its genetic improvement has been constrained by the lack of efficient transformation systems. In this study, a robust protocol was established for shoot regeneration and Agrobacterium-mediated transformation of S. villosum using leaf explants and A. tumefaciens strain EHA105/pBI35S::GFP, which harboring the Neomycin phosphotransferase II (NPT II) selectable marker and the green fluorescence protein (GFP) reporter gene. The results showed that 100% leaf explants produced adventitious shoots on semi-solid full-strength MS medium containing 1.0 mg L−1 thidiazuron (TDZ,) 0.1 mg L−1 α-naphthaleneacetic acid (NAA), 0.7% agar and 10 mg L−1 AgNO3, with an average of 7.8 shoots per explant after four weeks. Furthermore, transformation frequency was 81%, and over 90% of kanamycin-resistant plantlets showed a normal phenotype and stable GFP fluorescence. Moreover, transgene integration was verified by molecular analyses and fluorescence imaging of flowers, fruits, and T1 seedlings. Taken together, these results demonstrate that the tissue culture and transformation system established for S. villosum provides a valuable platform for large-scale propagation, gene function studies, and biotechnological improvement of this species.