Cold Tolerance in Alfalfa: Physiological Mechanisms, Regulatory Networks, and Molecular Breeding Prospects

Alfalfa (Medicago sativa L.) is a perennial legume forage whose winter survival determines stand persistence, spring green-up, and long-term yield in cold regions. Low-temperature stress causes membrane phase transitions, cellular dehydration, impaired photosynthetic electron transport, and excessive reactive oxygen species (ROS) accumulation. Tolerance depends on coordinated antioxidant protection, osmotic adjustment, membrane stabilization, photosynthetic maintenance, and carbon-nitrogen allocation to the root crown. At the molecular level, this process is mediated by calcium ion signaling, phytohormone pathways, and the cold-responsive ICE-CBF/DREB-COR regulatory cascade. Reported molecular resources include calmodulin-like protein modules (MsCML10 and MsCML50), CBF/DREB factors, autophagy and circadian regulators, sugar-metabolism genes, and genes from yellow-flowered alfalfa. This review integrates low-temperature injury, physiological responses, molecular regulatory networks, evaluation methods, genetic resources, and breeding progress. It emphasizes that evidence strength differs among cultivated-alfalfa functional studies, heterologous validation, and omics-based candidate discovery. Major gaps include inconsistent phenotyping, insufficient root-crown and overwintering-bud characterization, limited validation under natural overwintering, and weak translation of candidate genes into breeding populations. Future research should combine standardized multi-environment phenotyping with multi-omics, functional validation in alfalfa, marker development, genomic selection, and multi-trait improvement of cold tolerance, yield, forage quality, and stress adaptation.