Physiological Evaluation and Selection of Cold-Tolerant Individuals in Zoysia japonica ‘Zenith’ Based on Electrolyte Leakage, Cell Death, and ROS Accumulation

This study evaluated cold tolerance and selected superior individuals from 100 Zoysia japonica ‘Zenith’ plants. Following pre-cold treatment at 4 °C for 24 h, candidates were screened using electrolyte leakage (EL) with a 10% selection intensity. Selected candidates and cold-sensitive controls were exposed to 0, −5, and −10 °C for 24 h and assessed using visual injury, EL, Evans blue staining, and 3,3′-diaminobenzidine (DAB) staining. Visual injury increased with decreasing temperature but did not clearly distinguish cold tolerance. Individual 11 exhibited the lowest lethal temperature causing 50% injury (LT50) based on both EL (−10.4 °C) and Evans blue staining (−11.0 °C). The two LT50 estimates were strongly correlated (r = 0.996) and concordant (Lin’s concordance correlation coefficient = 0.909). DAB-based recovery analysis showed effective hydrogen peroxide (H2O2) clearance in individuals 5, 11, 21, 23, and 40, with individual 23 exhibiting the lowest reactive oxygen species area under the curve (121.5). A modified Generalized Value Index integrating visual injury, mean LT50, oxidative stress, and recovery slope identified individuals 5, 11, 18, 23, and 30 as superior cold-tolerant selections. Therefore, combining membrane-damage and oxidative-recovery indicators can provide a robust framework for selecting cold-tolerant Z. japonica ‘Zenith’ for field validation and breeding.