Himantoglossum affine is a threatened terrestrial orchid. We aimed to optimize asymbiotic seed germination and direct embryogenesis and to analyze the phytochemical profile and physicobiochemical analysis of leaf and tuber. The individual use of organic nitrogen compounds resulted in higher germination efficiencies, while the shortest times to germination were observed using coconut water plus casein hydrolysate. Plantlets grown on media supplemented with pineapple juice and peptone had the highest plantlet length and weight. For embryogenesis, the highest regeneration rate (44%) and embryo number/explant (10.12 ± 2.08) were observed in young protocorm-like body (PLB) explants with 0.5 mg/L naphthalene acetic acid (NAA) and 1 mg/L thidiazuron (TDZ). During the acclimatization process, the scattered vascular tubes converted to fully developed vascular tissues, ensuring maximum sap flux. Gas chromatography–mass spectrometry analysis identified 1,2,3- propanetriol, monoacetate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and 2-butenedioic acid, 2-methyl-, (E)- as the most prevalent compounds. We reported higher contents of total phenolics and flavonoids and antioxidant activity compared to other terrestrial orchids. The glucomannan content (36.96%) was also higher than starch content (31.31%), comparable to those reported in other tuberous orchids. Based on the fragmentation of H. affine populations in the Middle East and Euro-Mediterranean countries due to over-harvesting, climate change, and/or human impact, our procedure offers a tool for the re-introduction of in vitro-raised plants to threatened areas.
