Physiological and Transcriptomic Evaluation of Drought Tolerance in Alfalfa (Medicago sativa L.) and Identification of Resilient Germplasm

Drought stress is a major constraint on alfalfa (Medicago sativa L.) production. Screening for drought tolerance at the seedling stage can accelerate the identification of resilient germplasm. In this study, six alfalfa cultivars were selected and subjected to drought stress at the seedling stage. Morphological traits (stem diameter, plant height, biomass, and root–shoot ratio) and oxidative/antioxidant indicators (malondialdehyde (MDA), superoxide (O₂•⁻), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR), and ascorbate peroxidase (APX) activity) were quantified in leaves and roots. Drought stress significantly reduced plant height (by up to 42.4% in ZL2) and biomass (by up to 30% in some cultivars), but increased the root–shoot ratio (by 50–166%). MDA and O₂•⁻ levels increased by 10–174% in leaves and 8–65% in roots across cultivars. Antioxidant enzyme activities rose markedly: SOD by 23–125% in leaves and 2–100% in roots; POD by 47–240% (leaves) and 38–166% (roots); CAT by 9–129% (leaves) and 30–227% (roots); GR by 35–107% (leaves) and 23–172% (roots); APX by 8–175% (leaves) and 3–89% (roots), indicating a coordinated leaf–root antioxidant defense. Transcriptome analysis of the tolerant cultivar ZM3 revealed 853 differentially expressed genes, which were enriched in pathways such as the non-homologous end-joining DNA repair pathway. Multivariate assessment of seedling-stage performance identified ZM3 and ZL2 as the most drought-tolerant cultivars. Collectively, these findings provide germplasm leads and empirical evidence for coordinated leaf–root antioxidant strategies in alfalfa, informing the selection and improvement of drought-tolerant cultivars.