Micropropagation of Dactylorhiza umbrosa (Kar. & Kir.) Nevski through asymbiotic seed germination and somatic embryogenesis: A promising tool for conservation of rare terrestrial orchids

We aimed to explore in vitro seed germination optimizing and somatic embryogenesis induction as well as to identify phytochemical constituents of endangered terrestrial orchid, Dactylorhiza umbrosa (Kar. & Kir.) Nevski. The highest germination rate (93.04±0.90%) was achieved with the combination of pineapple juice (PJ) and casein hydrolysate (CH). Moreover, the individual application of organic nitrogen compounds (Vamin, peptone, and casein hydrolysate) led to the higher germination efficiencies, with Pep being the most effective in shortening the germination time (the number of days from seed sown to embryo enlargement and seed testa rupture) (2.9 times faster compared to PJ+Pep+CH). Young protocorm explants treated with 1 mg L   −   1 2,4-D and 2 mg L   −   1 Kin showed the highest embryogenesis rate (39.99±5.72%), while this explant treated with 0.5 mg L   −   1 2,4-D and 2 mg L   −   1 Kin produced the highest embryo number (5.37±0.21). Morpholine, 4-(4-methyl-1-cyclohexen-1-yl) was the predominant phytochemical in tuber methanolic extract (60.63±3.23%), while 1,2,3-propanetriol was found at the highest percentage in leaf methanolic extract (47.48±3.23%). The extracts derived from the tubers had more content of total phenolics (TP) and total flavonoids (TF), as well as a greater antioxidant activity (TAA) compared to the extract obtained from the leaves. The recorded glucomannan content (31.31±0.60%) was lower than that of starch (32.65±1.22%), however both carbohydrates were responsible for the recorded viscosities of Salep samples. Due to high viscosity of products from Salep powder obtained from D. umbrosa, its tubers are used as superior crude materials in Iranian herbal markets. Taking into account the endangered status of D. umbrosa populations in the Euro-Mediterranean and the Middle East basins caused by climate change and human activities, our method may be exploited for large-scale propagation of in vitro raised plants and reintroduce them into their natural habitats.