A Rapid and Visual Soybean Hairy Root Transformation Protocol Using the RUBY Reporter

Agrobacterium rhizogenes-mediated hairy root transformation provides a rapid platform for gene function analysis prior to stable whole-plant transformation. However, most existing hairy root transformation methods rely on tissue culture and require chemical or fluorescence-based selection, which increases experimental complexity. Here, we describe a tissue culture-free soybean hairy root transformation protocol incorporating the RUBY visual reporter system. While this work does not introduce a new transformation concept, it presents a streamlined implementation of established soybean hairy root methodologies that emphasizes procedural simplicity, reduced handling, and faster access to functional root material. Transgenic roots expressing RUBY can be directly identified by red pigmentation with the naked eye. In RUBY-positive roots, candidate genes driven by the CaMV 35S promoter showed higher expression levels than those in empty-vector controls, indicating that the system supports effective gene expression. Using this procedure, clearly identifiable transgenic hairy roots can be obtained within 20 days. Overall, this protocol simplifies induction and screening while reducing operational complexity and equipment requirements. Key features • Induces soybean hairy roots without tissue culture, simplifying the transformation workflow. • Incorporates RUBY reporter for direct naked-eye identification of transgenic roots. • Generates identifiable transgenic hairy roots within 20 days, enabling rapid functional analysis. • Applicable to gene and protein function studies, metabolite profiling, and root-microbe interaction assays.