Enhancing Cold Tolerance Evaluation in Camellia sinensis and Camellia japonica Through Multimethod Analysis and Predictive Modeling

Abstract

Cold stress is a critical factor restricting the cultivation of subtropical evergreen species such as Camellia sinensis and C. japonica in temperate climates. This study aimed to develop an integrated framework for evaluating cold tolerance by combining visual assessment, electrolyte leakage (EL), Evans blue staining, and nonlinear regression modeling. All experiments were conducted with n = 3 samples per treatment, and statistical analyses were performed at a significance level of alpha = 0.05. Under freezing treatment at -6 degrees C, C. japonica exhibited faster and more severe damage, including leaf curling and vein darkening, compared to C. sinensis. Electrolyte leakage and cell death increased rapidly in C. japonica, and a sharp rise in cell death occurred in both species when EL exceeded 55%. Logistic regression of EL data estimated LT50 values of -10.96 degrees C for C. sinensis and -9.38 degrees C for C. japonica, while EL Temp50 values were -9.59 degrees C and -8.97 degrees C, respectively, indicating higher membrane stability in C. sinensis. The difference in LT50 between the two species was statistically significant (p < 0.05). Biochemical and heatmap analyses from 25 degrees C to -12 degrees C showed that C. sinensis maintained higher chlorophyll, antioxidant activity, and sugar levels, reflecting stronger cold tolerance. In contrast, C. japonica accumulated more proline and MDA, indicating higher stress sensitivity and membrane damage. This study presents a reproducible, quantitative framework for evaluating cold tolerance in Camellia species, offering valuable insights for breeding and expanding cultivation under climate change.