Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 3, pp 419–432 | Cite as

In vitro propagation of Epipactis flava Seidenf., an endangered rheophytic orchid: a first study on factors affecting asymbiotic seed germination, seedling development and greenhouse acclimatization

  • Boworn Kunakhonnuruk
  • Phithak Inthima
  • Anupan KongbangkerdEmail author
Original Article


Epipactis flava Seidenf., is an endangered Thai rheophytic orchid that has decreased rapidly throughout its natural habitat and urgently requires conservation using in vitro techniques. Effect of pollination types (self- and cross-pollination) and seed from different capsule ages (2, 4, 6 and 8 weeks after pollination) on asymbiotic seed germination efficiency was performed on semi-solid VW medium supplemented with 150 mL/L coconut water and 50 g/L potato extract. Highest germination rate was 79.2% with definite rate of protocorm developmental stage 5 at 24.0% using seeds from 6 week old cross-pollination capsules. Anatomical investigation revealed young seed has been formed at 6 weeks old capsules. Identical seeds were then cultured on semi-solid and liquid systems of five different media including modified-VW, MS, BM, MM and KC for 2 months. Results showed that semi-solid and liquid VW medium promoted the highest seed germination rates at 70.2% and 70.4%, respectively, with the highest definite rate of protocorm developmental stage 5 at 54% found on semi-solid BM medium. In vitro seedlings were transferred to culture on both semi-solid and liquid VW and MS media for 2 months. Higher growth parameters, as indicated by shoot number and fresh weight, were obtained on liquid MS medium. Clumps of plantlets containing 5–8 shoots were acclimatized and transplanted into different potting media for 3 months. Survival percentages in all tested substrates were recorded at nearly 100% with no significant differences, while 63–68% of the living plantlets produced new shoots.


Epipactis flava Rheophytic orchid Asymbiotic germination Acclimatization 



The authors highly appreciate assistance provided by the Plant Tissue Culture Research Unit, Department of Biology, Faculty of Science, Naresuan University, Thailand and all colleagues for use of the facilities. The first author offers thanks to the Science Achievement Scholarship of Thailand (SAST) for provision of the supporting budget.

Author contribution

BK conducted the experiments and prepared draft manuscript. PI take response for research suggestion and editing the manuscript. AK designed experiments, supporting facilities and finalized the manuscript. All authors have read and approved the manuscript.


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Boworn Kunakhonnuruk
    • 1
  • Phithak Inthima
    • 1
  • Anupan Kongbangkerd
    • 1
    Email author
  1. 1.Plant Tissue Culture Research Unit, Department of Biology, Faculty of ScienceNaresuan UniversityPhitsanulokThailand

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