Applying Freezing Test to Quantify Cold Acclimation in Medicago truncatula Gaertn.

Understanding cold acclimation is important for concurrently improving autumn yield and winter survival in alfalfa (Medicago sativa L.).  Medicago truncatula Gaertn., an annual relative of alfalfa, can be used for cold acclimation studies if the ability and conditions required for its cold acclimation are determined. The major objectives of this study were to develop a laboratory screening procedure to quantify cold acclimation and determine freezing tolerance of M. truncatula. Two genotypes, Jemalong-6 and W6 5018 were grown in non acclimation (NA) and three cold acclimation (CA1, CA2, CA3) regimes. Cold acclimation was quantified by measuring freezing tolerance, as estimated by ion leakage (IL) from leaf tissues. Percent injury and LT₅₀ (the freeze temperature at which 50% tissues were injured) were derived from freeze injury data. Cold-acclimated plants had reduced stem length (SL), number of leaves (LVS) and shoot dry weight (SDW) compared to control. Root-to-shoot ratio (RS) and root dry weight (RDW) were higher in cold-acclimated than in control plants. Acclimation regimes resulted in differential percent injury in tissues. While 80% injury was induced by -7°C in NA plants, -20°C only induced 52% injury in CA3 plants. In NA plants, 80% injury was recorded at -7°C while in CA3, only 52% at -20°C. Average LT₅₀ temperatures were -3, -8, -7.8 and -12.5 °C in NA, CA1, CA2 and CA3 regimes, respectively. Percent injury was lower in ‘Jemalong-6’ than in ‘W6 5018’ for all cold acclimation regimes. Our results demonstrated the capability of M. truncatula to cold acclimate under the controlled cold acclimation regimes and the possible use of IL as a rapid laboratory method to quantify cold acclimation.