An Agrobacterium-Mediated Transformation for Zingeria biebersteiniana

Zingeria biebersteiniana, a grass species with the lowest known chromosome number among angiosperms (2n = 2x = 4), offers a distinctive platform for cytogenetic and grass research. Despite its unique karyotype and potential for molecular and educational applications, no transformation system has previously been reported for this species. Here, we establish a reproducible Agrobacterium tumefaciens-mediated transformation protocol for Z. biebersteiniana, optimized through comparative evaluation of three tissue culture media. A modified Khromov medium with Plant Preservative Mixture supported robust callus induction and plant regeneration, enabling the successful introduction of a GFP–mouse talin1 fusion construct driven by the rice Actin-1 promoter. Transgenic lines were validated via PCR amplification of the hygromycin resistance gene, and GFP signals were observed in transformed individuals. However, the expression pattern was less specific than previously reported in rice, potentially due to species-specific differences in mouse Talin1 protein localization. Although actin filament visualization in mature pollen remained unspecific, the protocol provides a foundational tool for future molecular and functional genomics and genetics studies. This work represents the first documented genetic transformation of Z. biebersteiniana, expanding its utility as a model system in plant biology and genomics.