Aging deterioration of seeds is inevitable during storage, which is mainly manifested by mitochondrial dysfunction and reactive oxygen species (ROS) accumulation. Mitochondria, as the center of cellular energy metabolism, and its ascorbic acid-glutathione (AsA-GSH) cycle are the key defense to scavenging ROS. This experiment was designed to investigate the antioxidant mechanism of the mitochondrial AsA-GSH cycle and the rules of endogenous hormones changed in the radicle during imbibition of aged alfalfa (Medicago sativa L.) seeds, and to lay a foundation for revealing the relationship between the metabolism of the radicle cells and the imbibed process in the aged alfalfa seeds.

In this experiment, seed samples were treated with controlled deterioration and obtained at a medium level of alfalfa seed vigor, and unaged seeds were used as the control. After the seed germination characteristics were tested, the changes in mitochondrial antioxidant enzyme activity and antioxidant content of the radicle during imbibition (6, 12, 24 and 36 h), and the changes in hormone content of the radicle during imbibition (12, 24 and 36 h).

Controlled deterioration treatment inhibited the seed germination and seedling growth, which mainly presented the decrease in seed germination potential, germination percentage and root length of seedlings. The activities of catalase (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR), as well as the contents of ascorbic acid (AsA) and glutathione (GSH) were decreased in the radicle mitochondria of aged seeds within 36 h imbibition; and the activities of peroxidase (POD) and glutathione reductase (GR) increased in the radicle mitochondria of aged seeds after 24 h of imbibition. With the progress of imbibition, the difference of AsA/oxidized ascorbic acid (DHA) and GSH/oxidized glutathione (GSSG) ratios between aged and unaged seeds was gradually reduced. The contents of abscisic acid (ABA), gibberellin (GA₃), growth hormone (IAA) and oleuropein lactone (BR) in the radicles presented the similar tendency during the imbibition.

During the process of imbibition, ROS accumulated in the mitochondria of the radicle, resulting in oxidative damage and delayed seed germination. After 24 h imbibition of aged seeds, POD and GR activities in radicle mitochondria increased to maintain a certain antioxidant capacity and germination ability. It could be suggested that the enhancement of POD and GR activities would play an important role in the maintenance of seed vigor level during the imbibition of aged alfalfa seeds, which provided a basis for an in-depth exploration of the mechanism of the role of the AsA-GSH cycle in antioxidant responses, and provided a reference for optimizing the conservation strategy of forage germplasm bank and prolonging seed life.