Cold-Induced Biochemical and Molecular Changes in Alfalfa Populations Selectively Improved for Freezing Tolerance

Synthetic populations obtained after successive cycles of recurrent selection for superior tolerance to freezing were used to probe the molecular bases of superior adaptation to cold in alfalfa (Medicago sativa L.). The initial genetic backgrounds and populations derived from the cultivars Apica and Evolution were assessed for their freezing tolerance and cold-induced molecular changes using plants acclimated to natural hardening conditions in an unheated greenhouse. Significant increase in freezing tolerance in response to recurrent selection was confirmed by the determination of the lethal temperature for 50% of the plants (LT(50)). Improvement of freezing tolerance was associated with differences in cold-induced molecular changes. Starch reserves in crowns progressively declined during fall hardening with a more extensive mobilization observed in advanced cycles of selection. Concentrations of cryoprotective sugars and of the amino acids proline, asparagine, and arginine increased during fall hardening and were significantly higher in recurrently selected populations than in the initial backgrounds. Transcript levels of two cold-regulated (COR) genes, cas15 and GaS, were strongly up-regulated during fall hardening and significantly increased in response to selection mostly in the Apica background. Our results provide evidence that recurrent selection for superior freezing tolerance in alfalfa induces marked changes in traits associated with the cold acclimation process.