Functional analysis of ARR, MYC, and FLS genes in alfalfa under salt stress using Agrobacterium rhizogenes-mediated...

To develop a rapid platform for gene function analysis in alfalfa, two Agrobacterium rhizogenes strains, K599 and Arqua1, were compared for their ability to induce transgenic roots. Composite plants (with transgenic roots only) were generated using a root‒tip coating method, and K599, which showed higher induction and transformation efficiency, was selected for subsequent experiments. Guided by our previously published transcriptome atlas, composite plants overexpressing three flavonoid-associated genes, cytokinin-response regulators (ARRs), MYC transcription factors (MYCs), and flavonol synthase (FLS) were produced and subjected to short-term salt stress (150 mmol/L NaCl for 1 h). Under these conditions, FLS-overexpressing plants exhibited increased stress sensitivity, whereas ARR- and MYC-overexpressing plants showed improved early stress responses compared with those of empty-vector control plants. Consistently, the total flavonoid content was elevated in ARR and MYC lines but reduced in FLS lines. These results demonstrate the distinct roles of ARR, MYC, and FLS genes in modulating early salt stress responses and flavonoid accumulation in alfalfa, and highlight the utility of the composite plant system for rapid functional analysis of candidate genes.