RESUMEN
BACKGROUND: Cypripedium subtropicum is a unique, endangered lady's slipper orchid with evergreen leaves on non-dormant shoots that is native to southwestern China. This study documents the major developmental events in C. subtropicum seed development from fertilization to seed maturity, determines the optimum period for seed collection, and examines the cytokinin requirements for asymbiotic germination and protocorm survival. RESULTS: Structural studies revealed that embryo development proceeded after successful fertilization at 60 days after pollination (DAP). At 105 DAP, a globular embryo with the shrinking inner seed coat was observed, and seeds collected at this time point exhibited optimal germination. After 120 DAP, most seeds had a mature embryo within the capsule, and within the cells of the embryo proper, numerous proteins/lipid bodies were present as the main storage products. In addition, the inner seed coat had compressed into a thin layer that tightly enclosed the embryo, while the outer seed coat had progressively elongated, resulting in a hair-like appearance of the mature seed. Histochemical staining using Nile red and toluidine blue O (TBO) indicated that the lignified inner and outer seed coats may lead to coat-imposed dormancy. Seeds collected at this stage germinated poorly. Analyses of cytokinin preferences and optimal concentrations for germination and protocorm survival showed that both 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-benzylaminopurine (BA) enhanced germination compared with the control, although higher concentrations of BA (4 and 8 µM) suppressed germination. The protocorm survival rate improved with increasing 2iP concentration. CONCLUSIONS: This study provides a reproducible procedure for culturing immature seeds of C. subtropicum based on a defined time schedule of seed development. In addition, the cytokinin 2iP was shown to improve germination and protocorm survival. This study provides a scientific basis for seedling establishment through asymbiotic seed culture for further reintroduction efforts.
RESUMEN
A molecular analysis was performed on 56 taxa in the orchid genus Cypripedium using nrDNA ITS and five chloroplast regions (trnH-psbA, atpI-atpH, trnS-trnfM, trnL-F spacer, and the trnL intron). The genus Cypripedium was confirmed as monophyletic. Our data provided strong support for monophyletic grouping of eight infrageneric sections (Subtropica, Obtusipetala, Trigonopedia, Sinopedilum, Bifolia, Flabelinervia, Arietinum, and Cypripedium) defined in earlier taxonomic treatments, and paraphyletic grouping of two sections (Irapeana and Retinervi). Within the genus Cypripedium, the first divergent lineage consisted of two Mesomaerican species, and subsequently the Cypripedium debile lineage from eastern Asia was split. Our study did not support the notion that two Asian species (Cypripedium subtropicum and Cypripedium singchii) were closely related to either Mesoamerican Cypripedium irapeanum or North American Cypripedium californicum, as indicated by previous interpretations based on morphological evidences. In addition, one pair of vicariant species, Cypripedium plectrochilum (eastern Asia) and Cypripedium arietinum (North America), unique to section Arietinum, was confirmed. Furthermore, within the monophyletic section Cypripedium two previously recognized subsections, Cypripedium and Macrantha, were shown to be paraphyletic. Our results suggested that this section split into two groups based on distribution (North America vs. Eurasia) instead of such previously used, morphological traits as flower color, and the shape of the lips (labellum) and lateral petals.