Dynamic Analysis of Physiological and Biochemical Substances in Two Types of Root Type Alfalfa during Overwintering Period

Background: Cold resistance in alfalfa (Medicago sativa L.) is significantly influenced by root system type. The root system plays a crucial role in water absorption and soil stress response. This study investigates the physiological and biochemical responses of creeping-rooted and taprooted alfalfa to cold stress during early winter and mid-winter periods. Methods: Samples were collected on November 3,2023 and January 7,2024, from the experimental plot. Roots at a depth of 20 cm were cleaned with distilled water and stored in cryopreservation tubes at ultra-low temperatures. Free proline, soluble sugars, soluble proteins, malondialdehyde (MDA), superoxide dismutase (SOD) activity and catalase (CAT) activity were measured using standard biochemical methods. Result: Results indicated that with decreasing temperatures, the contents of soluble sugars, soluble proteins, free proline, MDA and CAT activity increased, whereas SOD activity decreased. In the early overwintering stage, creeping-rooted alfalfa exhibited higher soluble sugar content and SOD activity compared to taprooted alfalfa. During the overwintering period, creeping-rooted alfalfa maintained higher levels of soluble sugars, soluble proteins, MDA and CAT and SOD activities. Principal component analysis identified CAT, MDA, soluble sugars and soluble proteins as key indicators for evaluating cold resistance in alfalfa.