Leakage of Intracellular Substances from Alfalfa Roots at Various Subfreezing Temperatures

Alfalfa ( Medicago sativa L.) freezing tolerance is often determined by measuring leakage of electrolytes from root tissue subjected to only one subfreezing temperature, ‐8 °C. This study was conducted to evaluate the effects of a wide range of subfreezing temperatures on the leakage of intracellular substances from very cold‐hardy (Heinrichs and Vernal) and less cold‐hardy (5432 and Nitro) alfalfa cultivars. Roots of all cultivars were harvested in late antumn of the seeding year and subjected to seven subfreezing temperatures, from ‐4 to ‐16 °C. Injury was determined by measuring the leakage of electrolytes, substances absorbing light at 265 nm (A 265 ), and malate dehydrogenase (MDH) activity from roots after thawing. All measurements of leakage of intracellular substances increased with decreasing subfreezing temperatures, except that those of the less coldhardy caltivars tended to plateau from ‐6 to ‐10 °C. All three leakage measurements followed a similar general trend across subfreezing temperatures, indicating that leakage of electrolytes and A~5 generally reflected substantial membrane damage (leakage of MDH). However, the leakage of electrolytes from roots of very coldhardy cultivars was much more apparent than the leakage of MDH activity at relatively warmer (e.g., ‐4 to ‐6 °C) subfreezing temperatures. This was not true for cultivars with low freezing tolerance, indicating that they suffered substantial membrane damage at all subfreezing temperatures used in this study. The ‐8 °C freezing treatment did not maximize differences among the cuitivars used. Differences in cultivars with high and lower levels of cold‐hardiness were most apparent at temperatures above ‐8 °C and below ‐8 °C, respectively.