Cold treatment benefits Mediterranean orchid seedlings cultivated in vitro

Gwenaëlle Deconninck , Argyrios Gerakis Gerakis, Victoria Chatzopoulou, Alexandra Thoma, Eirini Katsalirou

1. Effective ex situ propagation is increasingly critical for the conservation and restoration of terrestrial orchids threatened by habitat loss, over-collection and climate change. However, large-scale propagation remains constrained by developmental bottlenecks during the transition from protocorms to established plantlets with storage organs. Cold exposure is often recommended to improve propagation success in temperate orchids, yet the thermal conditions required to maximize survival and post-dormancy performance remain poorly understood.
2. We experimentally exposed protocorms of four Mediterranean terrestrial orchids (Anacamptis coriophora, A. laxilora, Himantoglossum robertianum, H. hircinum) to eight thermal environments for eight weeks, including controlled laboratory conditions and outdoor treatments distributed along a steep altitudinal gradient. We quantified survival, shoot growth, and tuber development before and after summer dormancy, and evaluated the relative importance of multiple temperature descriptors using an information-theoretic model selection approach.
3. 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 (10–12 °C) controlled survival and reserve accumulation before dormancy, while chilling below 6–7 °C promoted post-dormancy shoot growth, indicating delayed carry-over effects on subsequent development. These responses generated a positive feedback between winter chilling and growth during the following season. Thermal requirements differed markedly among species, suggesting species-specific climatic adaptations that may influence propagation success and restoration outcomes under future warming conditions.
4. Synthesis and applications. Our results demonstrate that winter chilling is a key determinant of propagation success in Mediterranean terrestrial orchids and identify temperature thresholds that can be directly integrated into ex situ cultivation and restoration protocols. Species-specific chilling requirements further suggest that climate warming may alter the suitability of current propagation practices and reintroduction strategies. Optimizing cold treatments could substantially improve the efficiency of orchid conservation programmes and support climate-adapted restoration planning.