In vitro germination and seedling development of two European orchid species, Himantoglossum jankae Somlyay, Kreutz & Óvári and Spiranthes spiralis (L.) Chevall

New protocols for successful asymbiotic seed germination of two European orchid species, Himantoglossum jankae Somlyay, Kreutz & Óvári and Spiranthes spiralis (L.) Chevall., were established in this study. The influence of two basal media, organic supplements, illumination, and cytokinins on germination, protocorm formation, and seedling development was examined. A strong species-specific dependence on illumination conditions and nutrient medium composition was observed. Improved germination under continual darkness indicated that H. jankae had negatively photoblastic seeds, while S. spiralis seeds could germinate under both light conditions examined in this study, but slightly faster development was achieved under the 16-h photoperiod protocol. The H. jankae seeds cultured on Knudson C. medium failed to germinate in any medium combination, irrespective of the organic supplement use. The highest germination rate of H. jankae was achieved on Malmgren medium supplemented with coconut water, peptone, and L-glutamine, indicating that germination of this species was amino aciddependent. Although S. spiralis seeds germinated on all medium combinations, Knudson basal medium (without supplements) yielded better results when compared with the Malmgren medium. The best S. spiralis seedling development was achieved on the medium supplemented with 6-(γ,γ-dimethylallylamino) purine, while the cytokinin 6-benzyladenine promoted H. jankae seedling development. With the implementation of these protocols, well-developed seedlings were acclimatized to greenhouse conditions after 7 mo in culture.