Comparative seed germination and seedling development of the ghost orchid, Dendrophylax lindenii (Orchidaceae), and molecular identification of its mycorrhizal fungus from South Florida.

BACKGROUND AND AIMS: The endangered leafless ghost orchid, Dendrophylax lindenii, one of the most renowned orchids in the world, is difficult to grow under artificial conditions. Published information on asymbiotic and symbiotic (co-culture with a mycobiont) seed germination, seedling anatomy and developmental morphology of this leafless orchid is completely lacking. This information is critical for the development of efficient procedures for ghost orchid production for successful reintroduction. METHODS: Ghost orchid seedling early development stages were morphologically and anatomically defined to compare germination, embryo and protocorm maturation and seedling development during asymbiotic and symbiotic culture with one of two mycorrhizal strains (Dlin-379 and Dlin-394) isolated from ghost orchid roots in situ KEY RESULTS: Seeds symbiotically germinated at higher rates when cultured with fungal strain Dlin-394 than with strain Dlin-379 or asymbiotically on P723 medium during a 10-week culture period. Fungal pelotons were observed in protocorm cells co-cultured with strain Dlin-394 but not Dlin-379. Some 2-year-old seedlings produced multinode inflorescences in vitro Production of keikis from inflorescence nodes indicated the capacity for clonal production in the ghost orchid. CONCLUSIONS: Ghost orchid embryo and seedling development were characterized into seven stages. Fungal strain Dlin-394 was confirmed as a possible ghost orchid germination mycobiont, which significantly promoted seed germination and seedling development. Internal transcribed spacer sequencing data confirmed that Dlin-394 belongs within the genus Ceratobasidium These results offer the opportunity to examine the benefits of using a mycobiont to enhance in vitro germination and possibly ex vitro acclimatization and sustainability following outplanting.

Asymbiotic germination response to photoperiod and nutritional media in six populations of Calopogon tuberosus var. tuberosus (Orchidaceae): evidence for ecotypic differentiation.

BACKGROUND AND AIMS: Ecotypic differentiation has been explored in numerous plant species, but has been largely ignored in the Orchidaceae. Applying a specific germination protocol for widespread seed sources may be unreliable due to inherent physiological or genetic differences in localized populations. It is crucial to determine whether ecotypic differentiation exists for restoration and conservation programmes. Calopogon tuberosus var. tuberosus, a widespread terrestrial orchid of eastern North America, is a model species to explore ecotypic differences in germination requirements, as this species occupies diverse habitats spanning a wide geographical range. METHODS: Mature seeds were collected from south Florida, north central Florida, three locations in South Carolina, and the upper Michigan peninsula. Effects of three photoperiods (8/16, 12/12, 16/8 h L/D) were examined on asymbiotic in vitro seed germination and seedling development of C. tuberosus. Germination and early development was monitored for 8 weeks, while advanced development was monitored for an additional 8 weeks. In an additional experiment, asymbiotic seed germination and development was monitored for 8 weeks on six culture media (BM-1 terrestrial orchid medium, Knudson C, Malmgrem, half-strength MS, P723, and Vacin and Went). A tetrazolium test for embryo viability was performed. KEY RESULTS: Short days promoted the highest germination among Florida populations, but few differences among photoperiods in other seed sources existed. Different media had little effect on the germination of Michigan and Florida populations, but germination of South Carolina seeds was higher on media with higher calcium and magnesium. Tetrazolium testing confirmed that South Carolina seeds exhibited low viability while viability was higher in Florida seeds. Seed germination and corm formation was rapid in Michigan seeds across all treatments. Michigan seedlings allocated more biomass to corms compared with other seed sources. CONCLUSIONS: Rapid germination and corm formation may be a survival mechanism in response to a compressed growing season in northern populations. Ecotypic differentiation may be occurring based on seed germination and corm formation data.

Compatible host/mycorrhizal fungus combinations for micropropagated sea oats. I. Field sampling and greenhouse evaluations.

Micropropagation technology promises to improve the supply of sea oats for restoring Florida's eroded beaches, but concerns about genetic diversity need to be addressed. These dune plants are colonized by a wide array of arbuscular mycorrhizal (AM) fungi, yet little is know of the diversity of these fungal communities. Our goal was to test the level of functional diversity that exists among communities of AM fungi that are present in divergent Florida dunes. Community pot cultures were established from samples collected from ten transects in two Gulf coast and two Atlantic coast locations in Florida, and these were used to conduct two greenhouse studies. The objective of the first study was to evaluate within-location variance in the mycorrhizal function of different AM fungal communities associated with endemic sea oats. The objective of the second study was to evaluate among-location responses of plant and fungal ecotypes using selected combinations obtained from the first experiment. Within locations, the AM fungal community had significant impacts on shoot mass and shoot-P contents, confirming a range of symbiotic effectiveness exists within the beach-dune system. Among locations, there was a tendency for greater root colonization between host clones and fungal communities from the same location, indicating a degree of specificity between host ecotypes and their symbiotic fungi. Relative to plant growth response, one fungal community was superior across plant genotypes from all locations, while one plant genotype tended to have the best response across all fungal communities. These data suggest that while it is possible to select effective AM fungal-host combinations for outplanting, origin of host and AM fungi have little predictive value in screening these combinations.

Periplasmic cuticular calcium oxalate crystal deposition in Dracaena sanderiana.

• The chemical nature and biological basis for crystal deposition in epidermal subcuticular areas are reported here for the angiosperm Dracaena sanderiana. • Position, development and identification of crystals in adult leaves of D. sanderiana was carried out using X-ray diffraction, crystal morphology and scanning and transmission electron microscopy techniques. • Numerous small (< 1-6 µm) calcium oxalate monohydrate crystals were found between the primary epidermal cell wall and the cuticle. Their formation was highly specific and predictable with respect to location and relative timing of development during leaf ontogeny. The crystals were perisplasmic as, at formation, the nascent epidermal cell wall was external to the crystals. Cuticular crystallization of calcium oxalate monohydrate in D. sanderiana occurred in crystal chambers situated between the plasma membrane and the primary cell wall. Crystal deposition did not occur in developing guard cells. • The spatial pattern of calcium oxalate monohydrate within the epidermal cells, orientation of the crystallographic axes and the existence of crystal chambers outside the plasma membrane suggest biologically controlled crystal deposition in D. sanderiana.