A simplified and rapid hairy root transformation system for functional promoter and symbiosis studies in Fenugreek (Trigonella foenum-graecum)

Trigonella foenum-graecum (fenugreek) is a leguminous plant valued for its nutritional and medicinal properties. Despite its agricultural importance, molecular studies in fenugreek have been limited due to the lack of efficient genetic transformation tools. In this study, we developed a simplified Agrobacterium rhizogenes -mediated hairy root transformation protocol using strains K599 and A4. The K599 strain, carrying CaMV 35S:eGFP or DR5::GUS constructs, demonstrated superior transformation efficiency (92%) compared to A4 (58%), along with significantly higher root number and length. Fluorescence microscopy confirmed eGFP expression in transformed roots, and molecular validation through polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) confirmed stable integration and expression of the transgene. DR5::GUS reporter activity showed strong auxin-responsive expression in primary and lateral root tips, indicating the system's suitability for promoter functional analysis. Furthermore, transgenic hairy roots retained full symbiotic competence, as evidenced by successful colonization with Rhizophagus irregularis and nodule formation following inoculation with Sinorhizobium meliloti , comparable to wild-type roots. This simple, rapid, tissue culture-free transformation system supports robust gene expression and functional assays without compromising root physiology, offering a valuable platform for functional genomics, promoter studies, and symbiosis research in fenugreek and other non-model legumes. • Rapid hairy root transformation system developed for fenugreek. • Agrobacterium rhizogenes K599 showed high transformation efficiency. • eGFP and DR5::GUS reporters validated promoter activity in hairy roots. • Transgenic roots retained AM and rhizobial symbiotic competence. • Platform enables functional genomics in non-model legume fenugreek.