Effects of High-Temperature Stress on Physiological Characteristics and Propagation Potential of Seed-Derived Pholidota chinensis Plantlets

Pholidota chinensis is an orchid with both medicinal and ornamental value. Due to overharvesting and limited natural reproduction, its wild populations have declined sharply. Although aseptic sowing enables large-scale propagation, climate-driven extreme heat poses a threat to seedling production. However, the physiological responses and propagation potential of P. chinensis plantlets under heat stress remain poorly understood. This study examined six-month-old in vitro-grown plantlets under four temperatures (34 °C, 36 °C, 38 °C, 40 °C) for up to 96 h. The common 0 h measurements were used as a pre-treatment baseline, and no parallel time-matched 25 °C control was included. Photosynthetic pigment characteristics were assessed using UV-Vis spectroscopy, while PSII photochemical efficiency was evaluated using chlorophyll fluorescence (Fv/Fm). Pigment estimates showed non-monotonic variation among the four chamber-temperature conditions and exposure durations. At 34 °C, total chlorophyll concentration decreased from the common 0 h pre-treatment baseline of approximately 6.9 mg L−1 to approximately 5.5 mg L−1 at 96 h, corresponding to approximately 80% of the initial value. Under the 36–40 °C conditions, total chlorophyll concentrations exhibited substantial temporal fluctuations rather than a consistent decline with increasing temperature or exposure duration. In contrast, Fv/Fm showed a clearer numerical decline across the four chamber–temperature conditions at the 96 h endpoint. Selected spectral absorbance variables showed significant positive covariation across treatments (r = 0.708–0.983, all raw p < 0.001). At the 96 h endpoint, mean Fv/Fm showed a progressive decrease across the four chamber–temperature conditions, with values of 0.50, 0.45, 0.41, and 0.25 under the 34 °C, 36 °C, 38 °C, and 40 °C conditions, respectively. Because each temperature was imposed in a single incubator, these between-condition differences should be interpreted descriptively and cannot be separated from potential chamber-specific effects. At 36 °C, Fv/Fm showed a modest increase from 0.40 at 72 h to 0.45 at 96 h, whereas Fv/Fm remained more persistently suppressed at 38 °C. Contrasting response patterns were observed under the 36 °C and 38 °C chamber-temperature conditions; however, whether this pattern represents a reproducible temperature-dependent transition requires confirmation using independently replicated incubators and finer temperature intervals. Across the 16 chamber-temperature × stress-duration combinations, mean Fv/Fm showed a positive descriptive association with mean proliferation coefficient (Pearson’s r = 0.798, 95% CI = 0.500–0.927, n = 16). Because the analysis was based on treatment-combination means grouped within four single-incubator temperature conditions, the association does not establish Fv/Fm as an independently validated predictor of regenerative performance. These findings indicate that absorbance-based pigment estimates and PSII photochemical efficiency exhibited distinct response patterns under the tested conditions. The association between Fv/Fm and regenerative performance suggests that chlorophyll fluorescence may have potential value as a physiological indicator, although its predictive capacity requires further validation.