Leakage of Intracellular Substances as an Indicator of Freezing Injury in Alfalfa

Abstract

Alfalfa (Medicago sativa L.) freezing tolerance is commonly determined by measuring ion leakage (by electrical conductivity) after freezing. This study was conducted to evaluate other measurements of freeze‐induced leakage of intracellular substances as indicators of freezing tolerance in alfalfa cultivars varying in fall dormancy and winterhardiness. Alfalfa crowns and roots were harvested in late fall of the seeding year and subjected to a −8 °C freezing treatment. Freezing injury was determined by measuring electrical conductivity, ultraviolet (UV) light absorbance, and oxidized nicotinamide‐adenine dinucleotide (NAD+)‐malate dehydrogenase (MDH) activity of leakage from roots after freezing. The optimal wavelength for determining freeze‐induced leakage of intracellular substances and for delineating differences in leakage among cultivars was 265 nm (A265). Percent electrical conductivity was linearly related to A265 (r2 = 0.92). Quadratic relationships were found between MDH activity and percent electrical conductivity (R2 = 0.88) and A265 (R2 = 0.90). Differences among cultivars were significant and cultivar rankings were similar for all leakage measurements. These data demonstrate that A265 and MDH activitiy of plant leakage after freezing are useful indicators of freezing injury, and that cellular rupture results from the −8 °C freezing treatment commonly used in alfalfa freezing tolerance tests. The MDH assay is more timeconsuming and variable than A265 or electrical a conductivity and is probably most useful when absolute measures of cellular rupture are important. Measuring A265 was quick and provided a good alternative to measuring electrical conductivity.