Orchid species are increasingly threatened, necessitating both ex situ and in situ conservation efforts. Asymbiotic seed germination can enable the propagation of orchids, but optimal media formulations are often species-specific. Ophrys reinholdii subsp. straussii and Comperia comperiana are typical tuberous and terrestrial orchid species grown in the Euro-Mediterranean basin and the Iran plateau. This research aimed to investigate the suitability of five basic culture media — Malmgren, Orchimax, P723, ¼ Murashige and Skoog (MS), and ¼ Gamborg (B5) — for in vitro asymbiotic seed germination and optimal plantlet growth of O. reinholdii subsp. straussii and C. comperiana. Based on the results, the highest average germination percentages for O. reinholdii subsp. straussii were obtained using ¼ B5 (87.31%) and Malmgren (83.83%) respectively, and the lowest germination percentages were obtained with ¼ MS and Orchimax at 65.86% and 60.5%, respectively. For C. comperiana, the highest germination percentage (14.04%) was observed using P723, and the lowest (4.5%) was with ¼ B5 probably due to special nutritional requirement or as physiological or embryo dormancies. Seedlings were successfully obtained only for O. reinholdii subsp. straussii, tubers forming in vitro on both P723 and ¼ B5 media. The highest seedling height (120.03 mm) was achieved on the ¼B5 medium, whereas the lowest seedling height (59.64 ± 2.39 mm) was observed using Malmgren basal medium. This research indicates that there is no single culture medium for the optimal propagation of all terrestrial orchids. Different terrestrial orchid species exhibit varied responses to different culture media, likely due to genetic factors and the structure and compounds present in their seeds.
