Optimized Organic–Inorganic Nitrogen Balance Improves Asymbiotic In Vitro Seed Germination and Early Seedling Development of the Endangered Terrestrial Orchid Anacamptis coriophora

Anacamptis coriophora is a threatened tuberous orchid of the Irano-Turanian calcareous grasslands, subject to unsustainable harvesting for salep, increasing pressure from habitat loss and climate change. Asymbiotic in vitro culture is the principal method for its ex situ propagation; however, nitrogen nutrition, which is a decisive determinant of early morphogenesis, remains poorly optimized for Iranian terrestrial orchids, whose dust-like, endosperm-free seeds require external nutritional support for germination. Using a modified Malmgren basal medium, we investigated how nitrogen form and concentration affected asymbiotic seed germination and early seedling development in A. coriophora. Organic nitrogen (Aminoven at 25%, 50%, and 75% of full strength) was supplied in combination with inorganic nitrogen (KNO3 and (NH4)2SO4 in four combinations (balanced-low, nitrate-dominant, ammonium-dominant and high-inorganic). Germination declined markedly from 66% under balanced-low treatment to 13% under high inorganic nitrogen treatment, whereas organic nitrogen consistently enhanced germination across all inorganic backgrounds. Increasing Aminoven significantly promoted shoot elongation and biomass accumulation under low and moderate inorganic nitrogen levels but had little effect under high inorganic nitrogen concentrations. Root growth was enhanced by organic nitrogen across all inorganic treatments, whereas plantlet diameter increased under ammonium-dominant conditions, a signature of stress-induced radial swelling rather than enhanced vigor. Under the optimal medium (75% organic nitrogen, with low inorganic nitrogen), germination rate, shoot length, root length and fresh biomass reached 72%, 12.2 mm, 6.4 mm, and 44 mg plantlet−1, respectively. These findings provide a practical and scalable propagation protocol for this pharmaceutically and ecologically valuable Iranian orchid, thereby supporting conservation strategies aimed at mitigating salep overharvesting and environmental degradation.