Terrestrial orchids belonging to the Orchis genus are difficult to propagate and are under great pressure in their natural habitats. Studies regarding the influence of photoperiod and temperature regimes on Orchis militaris germination and morphological changes during immature seed development in vitro are scarce. Our aim was to identify photoperiod, temperature, and different nutrient media requirements for optimization of O. militaris seed germination and vigorous seedling production. Post-germination morphological changes were recorded with O. militaris seeds collected from 32-day-old fruits, where the percentage of O. militaris seeds without embryo was 38.4%. The highest rate of O. militaris seed germination (82.6%) was obtained on Malmgren modified terrestrial orchid medium (mM), enriched by 5% coconut water, 5% birch sap, and 0.1% AC. Nine percent of seedlings were able to reach the advanced seedling stage (stage 6) after 12 months of maintenance on this medium. In all 3 modified media (Harvais, Knudson С and Malmgren), regeneration was via the production of protocorms and seedlings without callus formation. It was proved that more abundantly vigorous protocorms were formed on the modified Harvais 2 under continuous darkness, while the subculture in Knudson C medium with AC addition could be necessary to stimulate their further development. The regeneration success of the species for in vitro conditions was increased by following its natural seasonal cycle. This study demonstrated an efficient micropropagation system for O. militaris using immature seeds and thus widely opened the perspectives for its conservation in nature. The favorable conditions of seed germination periods for in vitro culture, identified as the definite shift of temperatures and photoperiod regimes intrinsic to the species in nature, could improve seedling survival of this medicinally important orchid.

中文翻译：

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西伯利亚濒危兰花兰花的非共生种子萌发和离体幼苗发育

兰花属的陆生兰花难以繁殖，并且在其自然栖息地中承受着巨大的压力。关于光周期和温度制度对兰花萌发和体外未成熟种子发育过程中形态变化的影响的研究很少。我们的目标是确定光周期、温度和不同营养介质的需求，以优化 O. militaris 种子发芽和旺盛的幼苗生产。从32天龄果实中采集的蛹虫草种子记录萌发后的形态变化，其中无胚蛹虫草种子的百分比为38.4%。在含有 5% 椰子水、5% 桦树汁和 0.1% AC 的 Malmgren 改良陆生兰花培养基 (mM) 上获得了最高的 O. militaris 种子发芽率 (82.6%)。在该培养基上维持 12 个月后，9% 的幼苗能够达到高级幼苗阶段（第 6 阶段）。在所有 3 种改良培养基（Harvais、Knudson С 和 Malmgren）中，再生是通过产生原球茎和幼苗而没有愈伤组织形成。证明在连续黑暗下改良的 Harvais 2 上形成了更丰富的活力原球茎，而添加 AC 的 Knudson C 培养基中的继代培养可能是刺激它们进一步发育的必要条件。通过遵循其自然季节性循环，该物种在体外条件下的再生成功率得到提高。这项研究证明了使用未成熟种子的 O. militaris 的有效微繁殖系统，从而广泛地为其在自然界中的保护开辟了前景。