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

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 150mL/L coconut water and 50g/L potato extract. Highest germination rate was 79.2% with definite rate of protocorm developmental stage 5at 24.0% using seeds from 6 week old cross-pollination capsules. Anatomical investigation revealed young seed has been formed at 6weeks 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 2months. 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 5at 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 3months. 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.