Efficient Embryogenic Callus Induction and Agrobacterium-Mediated Transformation of the Elite Foxtail Millet (Setaria italica L.) Variety Jingu51

Foxtail millet (Setaria italica L.) is an important food crop worldwide and serves as a model for studying C4 photosynthesis and drought resistance. However, its application in functional genomics and molecular breeding has been severely constrained by the absence of a reliable genetic transformation system. In this study, 66 varieties were systematically evaluated for embryogenic callus induction capacity, with rates ranging from 0 to 70.22%. Among these, five varieties exhibited high embryogenic potential (>50%). Jingu51, an elite cultivated variety in northern China, was selected as the transformation recipient due to its rapid embryogenic response and high induction frequency. Optimization of culture conditions revealed that Murashige & Skoog (MS) medium supplemented with 30 g·L−1 sucrose, 3 g·L−1 phytagel, 1 mg·L−1 CuSO4, and 1 mg·L−1 2,4-dichlorophenoxyacetic acid combined with 0.5 mg·L−1 6-benzylamino purine was optimal for embryogenic callus induction. Key parameters of Agrobacterium-mediated transformation, including 100 μM acetosyringone concentration, callus pretreatment (heat shock, followed by ice bath), bacterial density (OD600 = 0.3), and cocultivation temperature (19 °C), were optimized. Under these conditions, a robust genetic transformation system was successfully established, yielding an average efficiency of 9.25% by PCR across multiple expression vectors; half transgenic event exhibited single copy. Notably, the genetic transformation system of Jingu51, an elite variety of foxtail millet, provided a robust platform for functional genomics research and accelerated molecular breeding efforts in this important crop.