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Strawberry (Fragaria x ananassa) is cultivated worldwide for its high nutritional value, but high temperature remains a major challenge in its production. Calcium priming (CP) and heat acclimation (HA) have been reported to improve plant thermotolerance; however, their effects on strawberry plants are not well understood. Hence, this study aimed to investigate the impact of CP and HA on the thermotolerance of strawberry plants. Fragaria x ananassa ‘Taoyuan No.1’ microplants were primed with calcium chloride (20 or 40 mM for 24 h), either alone or combined with HA (30 to 42℃ for 5 h), before exposure to a lethal temperature (LT) challenge (48℃ for 4 h). Morphological, physiological, and antioxidant enzyme responses, along with the durability of induced thermotolerance, were evaluated. Results indicated that HA was essential for the strawberry microplants to survive under LT stress; however, 40 mM CP combined with HA further enhanced their thermotolerance. The CP/HA-treated microplants exhibited superior morphology (characterized by lower leaf damage rates and higher adventitious root counts), improved physiological parameters (including higher total chlorophyll content, lower H2O2 levels, and an increased AsA/DHA ratio), and elevated antioxidant enzyme activities (CAT, POD, GR) compared to the HA-only treated microplants following the LT challenge. Additionally, microplants recovered from the initial LT challenge were able to survive a subsequent LT re-challenge after 56 days, indicating that the induced thermotolerance can be classified as long-term acquired thermotolerance (LAT).

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