Cold treatment benefits Mediterranean orchid seedlings cultivated in vitro

#### Publication title: Cold treatment benefits Mediterranean orchid seedlings cultivated in vitro #### Publication authors: Anonymous #### Code author: Anonymous #### Last Update: May 2026 This repository houses the codes (Orchid_analysis.R and Temperature_analysis.R) and datasets that were used for the article ‘Cold treatment benefits Mediterranean orchid seedlings cultivated in vitro. ABSTRACT 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. R session info: - R version 2026.01.1+403 - Platform: x86_64-apple-darwin17.0 (64-bit) - Running under: macOS 26.3.1 Metadata for the data files are supplied below: #code folder Contains .R files with code for analysis of temperature (Temperature_analysis.R) and orchid growth (Orchid_analysis.R). #data_orchids folder Contains .csv files with raw data for growth of four orchid species exposed to different temperature treatments: Anacamptis coriophora (A_cor.csv), A. laxiflora (A_lax.csv), Himantoglossum robertianum (H_rob.csv) and H. Hircinum (H_hir.csv). Columns of the files: - Genera: Orchid genus name - Species: Orchid species name - Location: site of the treatment - MGRS: Military Grid Reference System coordinate of the site - Altitude: Elevation of the site above sea level - Treatment: Experimental treatment applied (# 1 to 8) - Vessel: Identifier of the culture vessel - Protocorm: Identifier of protocorm - Height_1: Shoot length in mm measured at time point 1 (week 8) - Brown_1: Presence of browning at time point 1 (week 8) - Height_2: Shoot length in mm measured at time point 2 (week 12) - Brown_2: Presence of browning at time point 2 (week 12) - Height_3: Shoot length in mm measured at time point 3 (week 16) - Brown_3: Presence of browning at time point 3 (week 16) - Height_4: Shoot length in mm measured at time point 4 - Brown_4: Presence of browning at time point 4 - Tuber_height: Height in mm of the developed tuber - Tuber_width: Width in mm of the developed tuber - Tuber_mass_per_vessel: Total tuber mass in g measured per vessel - No_tubers_per_vessels: Number of tubers produced per vessel at the end of the experiment - Shoot_h_postd: Shoot length in mm measured after dormancy - Tuber_mass_postd: Tuber mass in g measured after dormancy #data_temperature folder Contains .csv files for each treatment location with two columns: - Time: date and time of the temperature measurement - Temp: temperature measured in °C #output_orchids folder Contains the figures (FIGX.jpg and FIGX.png) of the article and the selection of the top five models fitting the data (Top5_models_per_trait.csv). #output_temperature folder Contains the supplementary figure (FIGS1.jpg and FIGS1.png) of the article and the table of the cumulative hours below a temperature threshold for all locations (cumhours.csv). For any inquiries or bugs, please contact corresponding author Anonymous (added later).