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

  • Jovana Dulić
  • Mirjana Ljubojević
  • Vladislav Ognjanov
  • Goran Barać
  • Tamara DulićEmail author


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 acid–dependent. Although S. spiralis seeds germinated on all medium combinations, Knudson C 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.


Asymbiotic seed germination Illumination Nutrient requirements Orchid conservation Protocorm formation 


Funding information

This research was supported by the project entitled “Improvement of agricultural production in Vojvodina (Serbia) in accordance with EU Regulations.”

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© The Society for In Vitro Biology 2019

Authors and Affiliations

  • Jovana Dulić
    • 1
  • Mirjana Ljubojević
    • 1
  • Vladislav Ognjanov
    • 1
  • Goran Barać
    • 1
  • Tamara Dulić
    • 2
    • 3
    Email author
  1. 1.Faculty of Agriculture, Department of Fruit Growing, Viticulture, Horticulture and Landscape ArchitectureUniversity of Novi SadNovi SadSerbia
  2. 2.Faculty of Science and EngineeringAbo Akademi UniversityTurkuFinland
  3. 3.Faculty of Sciences, Department of Biology and EcologyUniversity of Novi SadNovi SadSerbia

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