Abstract
BackgroundThe 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.ResultsThe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-33) contains supplementary material, which is available to authorized users.
Highlights
The cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant
After the loading solution (LS) was removed, the seeds were dehydrated on ice for 30 min with 1.0 ml plant vitrification solution 2 (PVS2, Sakai et al, 1990) containing 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide in 1/2 MS basal medium supplemented with 0.4 M sucrose
Seeds treated with loading solution for 0, 10, 20, or 30 minutes before cryopreservation had in vitro germination rates of 86%, 91%, 93%, and 94%, respectively, after 4 weeks of culture (Figure 1)
Summary
The cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant. There are many techniques available for the conservation of plant genetic resources of endangered species. These include in-situ and ex-situ conservation practices, micropropagation, seed germination, regeneration from explants, and cryopreservation (Nitzsche, 1983; Rick, 1984; Stanilova et al, 1994; Chang et al, 2000). Compared with traditional methods of storage, cryopreservation is cost-effective, Though many reports of the cryopreservation of orchids exist in the literature, studies of the effects of cryopreservation on the seeds of wild orchids are scant (Pritchard, 1984; Popova et al, 2003; Hirano et al, 2005a, 2005b, 2009). It eliminates the need for controlled slow freezing, and permits cells and meristems to be cryopreserved by direct transfer into liquid nitrogen (LN) (Thammasiri and Soamkul, 2007; Tsai et al, 2009)
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