Comprehensive Assessment of Alfalfa Aluminum Stress Resistance Using Growth and Physiological Trait Analysis

Alfalfa (<i>Medicago sativa</i> L.) is an important perennial leguminous forage; however, its high sensitivity to aluminum (Al) stress severely restricts its cultivation in regions with acidic soil. Therefore, this study conducted an integrated assessment of Al stress tolerance by performing systematic evaluations of 11 growth and physiological parameters across 30 alfalfa cultivars under Al stress, and calculated the Al tolerance coefficients based on these parameters. The results revealed that Al stress markedly inhibited root growth and biomass accumulation in alfalfa, thereby triggering increased malondialdehyde (MDA) content in roots across most cultivars, the scope of increase is 0.19–183.07%. Moreover, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased by 7.50–121.44%, 2.50–135.89%, and 3.84–70.01%, respectively. Based on the comprehensive evaluation value (<i>D</i>) obtained via principal component analysis and membership function, the 30 alfalfa cultivars were categorized into four distinct groups: 4 highly Al-tolerant cultivars, 11 moderately high-Al-tolerant cultivars, 9 moderately low-Al-tolerant cultivars, and 6 low-Al-tolerant cultivars. Stepwise linear regression analysis identified root elongation rate, root-to-shoot ratio, root volume, SOD, MDA, CAT, root dry weight, POD, and root length as pivotal indicators for predicting and evaluating Al stress tolerance in alfalfa cultivars. The qRT-PCR analysis showed dynamic changes in ABC transporter gene expression in alfalfa roots over time under aluminum stress. Therefore, this study comprehensively evaluated Al tolerance by systematically investigating the morphophysiological effects of Al stress across 30 alfalfa cultivars using principal component analysis (PCA), membership function, and hierarchical clustering analysis. It provides a practical solution for expanding alfalfa planting in acid soil and improving feed production in acidic environments.