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Cryopreservation the seeds of a Taiwanese terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification

Wei Hsin Hu1, Yue Han Yang2, Song Iuan Liaw3 and Chen Chang2*

Author Affiliations

1 Department of Biology, National Museum of Natural Science, Taichung 404, Taiwan

2 Department of Horticulture, National Chung Hsing University, Taichung 402, Taiwan

3 Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan

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Botanical Studies 2013, 54:33  doi:10.1186/1999-3110-54-33

Published: 12 September 2013



The cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant. This investigation was to establish a method for the vitrification and cryopreservation of seeds of B. formosana that may be suitable for the long term storage of Taiwan native orchid germplasm for conservation purposes.


The germination rate and morphological stability of seeds from spontaneous-dehiscent capsules of Bletilla formosana (Hayata) Schltr. were evaluated after cryopreservation by vitrification. The germination rates of cryopreserved seeds varied according to immersion time and the vitrification method used. Seeds that were dehydrated by immersion in loading solution (LS; 2.0 M glycerol, 0.4 M sucrose) for 10 min to 30 min then transferred to plant vitrification solution 2 (PVS2) for 30 min prior to freezing in liquid nitrogen (LN) showed significantly higher germination rates than seeds immersed in PVS2 only. The optimal immersion times were 10 min for LS and 30 min for PVS2, resulting in an in vitro germination rate of 91%. Germination was not observed for cryopreserved seeds that were dehydrated by immersion in LS only. Seed viabilities and germination rates did not vary significantly for cryostorage times from 10 minutes to 1 year.


This study improve, an efficient protocol was established that maintained seed viability and enhanced the germination rates of seeds, compared with previously described cryopreservation methods, and the germinated seeds showed normal morphology of both vegetative and reproductive organs.

Germination; Loading; PVS2; Viability; Vitrification