Winter chilling regulates seedling development in Mediterranean terrestrial orchids cultivated in vitro

Effective ex situ propagation is critical for the conservation and restoration of threatened terrestrial orchids. However, large-scale propagation remains constrained by developmental bottlenecks including the transition from protocorms to plantlets with storage organs. Although cold exposure is often recommended to improve propagation success, the thermal conditions governing survival and post-dormancy performance remain poorly understood. We experimentally exposed protocorms of the Mediterranean terrestrial orchids Anacamptis coriophora, A. laxiflora, Himantoglossum hircinum, and H. robertianum to eight thermal treatments for 8 weeks, including controlled laboratory conditions and outdoor sites along an altitudinal gradient. Survival, shoot growth, and tuber development were quantified before and after summer dormancy, and their relationship with temperature descriptors was analysed using a model selection approach. Plant performance was consistently better explained by cumulative chilling exposure than by mean temperature alone, with different developmental processes responding to distinct thermal thresholds. Exposure to moderately low temperatures (10°C-12°C) determined survival and reserve accumulation before dormancy, while chilling below 6°C-7°C doubled post-dormancy shoot length in responsive species, revealing carry-over effects across growing seasons. Species differed markedly in their thermal responses; increased chilling reduced post-dormancy survival by ∼50% in A. coriophora but increased it two-fold in A. laxiflora, whereas H. robertianum developed leaves only following cold exposure. Winter chilling therefore appears to be a key regulator of seedling development in Mediterranean terrestrial orchids. The identified thermal thresholds provide a basis for improving ex situ propagation and restoration protocols, while highlighting species-specific vulnerabilities and responses to future climate change.