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.

A protocol for plant regeneration via direct induction of protocorm-like bodies (PLBs) from leaf segments of Tolumnia Snow Fairy was developed as a basis for mass production. Ten-month-old, in vitro–grown donor plantlets were obtained by inducing shoots from buds on the flower stalk. Leaf segments harvested from plantlets of different heights and from expanding leaves at different positions were compared, as were two BA concentrations with 0.5 mg·L−1 NAA. The greatest rate of PLB induction (16.7%) was observed when leaf segments taken from 1- to 2-cm height plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 2 mg·L−1 BA and 0.5 mg·L−1 NAA after 16 weeks of culture. When using leaf explants, only inner, expanding leaves cultured on MS basal medium supplemented with 4 mg·L−1 BA and 0.5 mg·L−1 NAA resulted in PLB induction, at an average rate of 25.5 PLBs per explant. After 16 weeks of culture, histological and scanning electron microscopy (SEM) observations revealed that PLBs originated from epidermal cells of leaf explants. PLBs of 1 to ≤2 mm in diameter continued to proliferate after 4 weeks of culture. These secondary PLBs could be produced from either whole PLBs or the upper side of PLBs. Finally, PLBs were regenerated into plantlets. After ≈14 months of culture, fully developed plants exhibiting well-developed roots and shoots were acclimatized. These plants grew well, with 1-year survival rates of nearly 73%, for plants originating as explants taken from 1- to 2-cm tall plants, and of 79%, for plants originating as explants taken from inner leaves. Some mature plants flowered 1 year after transplantation. This study presents a simple system that can provide a large number of PLBs for mass propagation in a short time that can be converted into plants and also used for the new cultivars of Tolumnia orchids.

Additional index words. plant growth regulator, light quality, shoot organogenesis, Begonia Abstract. Begonia montaniformis 3 Begonia ningmingensis var. bella hybrids have high ornamental potential. Hence, the aim of this study was to determine the optimal conditions for the micropropagation of a Begonia montaniformis 3 Begonia ningmingensis var. bella F 1 progeny by using various concentrations of plant growth regulators (PGRs) and varying light spectra in half-strength Murashige and Skoog (1/2 MS) medium. The results showed that the explant regeneration was optimal when the lamina was incubated in a medium supplemented with 2.0 mM N 6 -benzylaminopurine and 0.8 mM a-naphthaleneacetic acid (NAA). Under such conditions, 98% of the explants regenerated adventitious shoots after 8 weeks, and 41 buds were produced per explant on average. The mean shoot length was 9.6 mm, and on average, 4.5 shoots per explant were more than 2 mm long. Subsequently, the induced adventitious shoots were transferred into rooting medium consisting of 1/2 MS and various NAA concentrations. After 4 weeks, the shoots subcultured in this medium showed ' '93% root induction and an average of 3.5 adventitious roots per explant. Furthermore, the applied light spectrum significantly influenced shoot regeneration, and optimal results were achieved under an equal distribution of blue, red, and infrared light. The histological sections of shoots regenerated from direct organogenesis were observed through scanning electron microscopy (SEM). Afterward, the rooting adventitious shoots were subcultured in PGR-free medium for 8 weeks. The seedlings were successfully acclimated 4 weeks after being transferred to soil and bloomed after 11 months in a greenhouse. Thus, the PGR composition in micropropagation efficiently shortened the time to blooming from 25 to 16 months.