Cold treatment benefits Mediterranean orchid seedlings cultivated in vitro

This is a Preprint and has not been peer reviewed. This is version 1 of this Preprint. You must log in to post a comment. There are no comments or no comments have been made public for this article. This is a Preprint and has not been peer reviewed. This is version 1 of this Preprint. Add a Comment You must log in to post a comment. Comments There are no comments or no comments have been made public for this article. 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. https://doi.org/10.32942/X2W97F Biology, Ecology and Evolutionary Biology, Life Sciences, Other Ecology and Evolutionary Biology, Plant Biology, Plant Sciences Anacamptis, asymbiotic propagation, climate change, dormancy, geophytes, Himantoglossum, orchidaceae, protocorm development Published: 2026-05-11 16:26 Last Updated: 2026-05-11 16:26 CC BY Attribution 4.0 International Conflict of interest statement: None Data and Code Availability Statement: The data supporting the results and the code used are freely available on Zenodo.org (https://doi.org/10.5281/zenodo.20120715). The DOI represents all versions and will always resolve to the latest one. Language: English