Asymbiotic Media Research Articles

Symbiotic Seed Germination and Seedling Development of Epidendrum geminiflorum Knuth from Ecuador

A greater understanding of the relationship between native orchids and their mycorrhizal symbionts is needed to ensure more effective orchid conservation strategies. A protocol for symbiotic seed germination and seedling development was developed for E. geminiflorum. Mature seeds were collected from a naturally occurring orchid population in Ecuador. Putative mycorrhizal fungi isolated from other native orchid species were used to screen their ability to facilitate germination and seedling development in vitro in either a 0/24 h or 12/12 h light/dark photoperiod at 20 °C. The mycorrhizal fungus Tulasnella calospora (UAMH 9824) isolated from Spiranthes brevilabris in Florida, USA, was also included in this study. Sterilization treatments using 0.3%, 0.5% sodium hypochlorite/ethanol or 2% calcium hypochlorite were tested for their effectiveness as sterilant and their subsequent effects on seed germination percentage. Effective surface seed sterilization was achieved with either 0.5% NaClO/ethanol or 2% calcium hypochlorite. However, significantly higher percentages of germinated embryos developed into protocorms when NaOCl solutions were used compared to the other treatments. Seed germination occurred in both photoperiods tested; however, delayed germination was observed under complete darkness. Seeds of E. geminiflorum germinated without fungal inoculation; however, co-culture with Tulasnella strains improved germination significantly. Seedling development was only observed when seeds were cultured in asymbiotic medium or co-cultured with T. caloscopa (UAMH 9824). Significantly longer seedlings were obtained when T. calospora was present in the culture compared with seedlings cultured in asymbiotic medium. The establishment of mycorrhizal associations was confirmed by the presence of pelotons in the roots of E. geminiflorum seedlings.

Seed Micromorphology, In Vitro Germination, and Early-Stage Seedling Morphological Traits of Cattleya purpurata (Lindl. & Paxton) Van den Berg

In the context of a symbiotic plant-fungus interaction study concerning Cattleya purpurata, we focused on some aspects of seed morphology and biology, and the early stages of seedling development. Seed morphology was characterized using light and scanning electron microscopy. In vitro seed germination capability was evaluated, comparing symbiotic and asymbiotic methods. The morphology of the seeds was overall comparable to that of other congeneric species, showing classical adaptations related to the aerodynamic properties and to the wettability of seeds, but calcium oxalate druses were identified inside the suspensor cells. Asymbiotic seed germination was successful in all tested media (17.1–46.5%) but was higher on 1/2 Murashige & Skoog. During symbiotic interaction with the fungal strain MUT4178 (Tulasnella calospora), germination rate was significantly lower than that obtained with the best three asymbiotic media, suggesting a low fungal compatibility. Seedling morphology was in line with other taxa from the same genus, showing typical characteristics of epiphytic species. Our observations, in particular, highlighted the presence of stomata with C-shaped guard cells in the leaves, rarely found in Cattleyas (where usually they are reniform), and confirm the presence of tilosomes in the roots. Idioblasts containing raphides were observed in both roots and leaves.

Cryopreservation without vitrification suitable for large scale cryopreservation of orchid seeds

BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Symbiotic seed germination of an endangered epiphytic slipper orchid, Paphiopedilum villosum (Lindl.) Stein. from Thailand

Paphiopedilum villosum (Lindl.) Stein is a native epiphytic slipper orchid in Thailand. This species is now being threatened and endangered. Propagation of this species is essential for conservation and reintroduction purposes. In this study, the propagation of P. villosum was achieved through the in vitro asymbiotic and symbiotic seed germination. Seeds of P. villosum sown on asymbiotic media, Murashige and Skoog (MS), Vacin and Went (VW) and Thomale GD (TH), did not germinate within 16weeks. Seven different fungal strains were isolated from roots of this orchid species. The germination rate index (GRI) and the development rate index (DRI) of P. villosum seeds in treatments inoculated with fungal isolates PVCP01, PVCP05, and PVCP06 was significantly higher than uninoculated control treatments. Fungal isolate PVCP01 significantly increased the GRI and DRI of every stage of protocorm development, whereas fungal isolates PVCP05 and PVCP06 were only able to promote seed germination and protocorm development to stage 2. As for the wild orchid species, P. villosum, a compatible fungus is therefore required for promoting seed germination and protocorm development. Based on analysis of morphological characters and sequences of the nuclear ribosomal transcribed spacer (ITS), fungal isolates PVCP01, PVCP 05, and PVCP06 were identified as Tulasnella sp., Ceratobasidium sp., and Flavodon sp., respectively. The information obtained from this study will be used to propagate other threatened Thai orchids for conservation and reintroduction programs.

Symbiotic propagation of seedlings of Cyrtopodium glutiniferum Raddi (Orchidaceae)

In nature, orchid seeds obtain the nutrients necessary for germination by degrading intracellular fungal structures formed after colonization of the embryo by mycorrhizal fungi. Protocols for asymbiotic germination of orchid seeds typically use media with high concentrations of soluble carbohydrate and minerals. However, when reintroduced into the field, seedlings obtained via asymbiotic germination have lower survival rates than do seedlings obtained via symbiotic germination. Tree fern fiber, the ideal substrate for orchid seedling acclimatization, is increasingly scarce. Here, we evaluated seed germination and protocorm development of Cyrtopodium glutiniferum Raddi cultivated in asymbiotic media (Knudson C and Murashige & Skoog) and in oatmeal agar (OA) medium inoculated with the mycorrhizal fungus Epulorhiza sp., using non-inoculated OA medium as a control. We also evaluated the performance of tree fern fiber, pine bark, eucalyptus bark, corncob and sawdust as substrates for the acclimatization of symbiotically propagated plants. We determined germination percentages, protocorm development and growth indices at 35 and 70 days of cultivation. Relative growth rates and the effects of substrates on mycorrhizal formation were calculated after 165 days of cultivation. Germination efficiency and growth indices were best when inoculated OA medium was used. Corncob and pine bark showed the highest percentages of colonized system roots. The OA medium inoculated with Epulorhiza sp. shows potential for C. glutiniferum seedling production. Corncob and pine bark are promising substitutes for tree fern fiber as substrates for the acclimatization of orchid seedlings.

Protocorms of an epiphytic orchid (Epidendrum amphistomum A. Richard) recovered in situ, and subsequent identification of associated mycorrhizal fungi using molecular markers

Epiphytic orchids have received considerable study, yet little has been published on their germination requirements in situ involving mycorrhizal fungi. Such research has been hampered by the small, dust-like size of seeds and leafless seedlings (protocorms) which are difficult to pinpoint on natural substrates, especially those on arboreal substrates (tree limbs). We report a novel seed sowing and retrieval method, modified from one applied to terrestrial orchids, used in the acquisition of epiphytic orchid protocorms from the Florida Panther National Wildlife Refuge. Seeds from two epiphytic orchid species (Epidendrum amphistomum A. Richard, E. nocturnum Jacquin) were placed in separate nylon mesh packets secured within 35 mm plastic slide mounts, and affixed to tree bark using gutter mesh and a staple gun. To confirm that the embryos were viable, some seeds were also sown on asymbiotic media in the laboratory which subsequently germinated after 52 days incubation. Of 60 packets distributed among 18 tree limb sites, one packet - harboring seeds of E. amphistomum affixed to pop ash (Fraxinus caroliniana Mill.) on a moss substrate - harbored protocorms after 267 days. Using molecular markers, a fungus assignable to the Ceratobasidiaceae, appears to be the mycorrhizal associate of these protocorms suggesting that this fungus may be associated with the germination process in situ.

Seed propagation and re-introduction of the U.S. federally endangered Hawaiian endemic, Platanthera holochila (Hbd.) Krzl. (Orchidaceae)

Platanthera holochila (Hbd.) Krzl. [syn = Peristylus holochila (Hbd) N. Hallé] is the rarest of three orchids endemic to the Hawaiian Archipelago. As of 2011, 33 individual plants of this U.S. Federally endangered species remained on three islands with only one specimen known to occur on Kauai. This paper presents a summary of experiments aimed at cultivating this species from seed leading to the reintroduction of seedlings. We describe: 1) the mycorrhizal fungi acquired from P. holochila protocorms on Molokai, 2) the role of light vs. dark pretreatment on symbiotic seed germination using a mycorrhizal fungus from Florida, and 3) asymbiotic germination on three media (Murashige and Skoog, Knudson C, P723). Protocorms recovered in situ using seed packets yielded seven strains of a mycorrhizal fungus assignable to the anamorphic genus Epulorhiza Moore, but none of these strains prompted seed germination in vitro. Using the mycorrhizal fungus from Florida, no significant differences were detected between light pre-treatment vs. dark incubation on seed germination or development, but statistical differences were evident among two agar types tested. Seeds sown on acidified (pH 5.0) asymbiotic medium P723 (PhytoTechnology Labs) developed to the leaf-bearing stage 351 days after sowing and incubation in darkness at 16-19 C. Seedlings illuminated 451 days after sowing were eventually established on soil in a greenhouse (ex vitro). A total of 85 seedlings were promptly transported to Hawaii in March 2011. A minimum of 3.1 years is required for the propagation of P. holochila from seed using acidified asymbiotic medium P723.

Aspects of the reproductive biology of Brassavola cebolleta Rchb.f. (Orchidaceae)

This survey assessed some aspects of the reproductive biology of Brassavola cebolleta Rchb. f. (Orchidaceae) in the Municipality of Dourados, Mato Grosso do Sul State, Brazil. Floral biology, breeding systems, floral visitors and seed germination were analyzed. Differential success and fruit production rate were calculated. Pollination system indicated the pollen vector dependence and absence of pre-zygotic barriers related to self-incompatibility. Pollination occurred at night, being the potential pollen vector a Lepidoptera-Notodontidae of the genus Hemiceras. Differential success of male was 19%, female 9%, and the rate of effective fructification was 6.3%. Fruits produced after pollinator visits were larger than those generated by manual cross pollination, and despite the lower number of potentially viable seeds produced by the first, they presented a higher germination rate in asymbiotic media. This study warns to the vulnerability of the studied species, since the habitat fragmentation associated with pollinator scarcity and seed predation may significantly decrease new recruitment into populations.

Asymbiotic seed germination and in vitro seedling development of Epidendrum ibaguense Kunth. (Orchidaceae)

Effects of four asymbiotic media; Murashige and Skoog (MS), Phytamax (Sigma, USA; PM), Mitra et al. (M) and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development in Epidendrum ibaguense Kunth. Besides, effects of peptone, activated charcoal and plant growth regulators (6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)) were also studied. Maximum seed germination was achieved on M and PM (~90%) fortified with 2 gl-1 peptone. Medium supplemented with 1.0 mgl-1 BAP improved seed germination in contrast to basal media whereas 2,4-D either singly or in combination with BAP remarkably reduced the germination rate. Activated charcoal in the medium enhanced germination of seed, induced significantly large size protocorms (1.63±18) and early rooting in protocorms. Within two weeks of culture, spherules emerged out due to cracking of the seed coat. The spherules developed into protocorms with a leaf primordium at apical portion after 3 to 4 weeks and gradually produced complete seedlings. Strong and stout root system was induced in in vitro seedlings on transferring in half strength PM and M media fortified with 0.5-1.0 mgl-1 indole acetic acid (IAA). Well-rooted seedlings were transferred to green house with 90% survival.

Axenic Seed Germination and in Vitro Seedling Development of Restrepia brachypus (Orchidaceae)

Montane rain forests in Central and South America are threatened by human activities and climate change. Consequently, epiphytic plant genera such as Restrepia are also endangered, making their ex-situ conservation vital. For success, this conservation strategy requires affordable, efficient, and reliable protocols for axenic seed germination as well as protocorm and seedling development prior to establishment ex vitro. In our study, effects of four asymbiotic media (Murashige and Skooge, Phytotech P668, Vacin and Went, and Western) on seed germination and early protocorm development of Restrepia brachypus were compared. In addition, their effects with and without banana pulp were examined on in vitro seedling development. Western medium produced the highest mean germination rate (53%), the second highest mean protocorm diameter (325 µm) and, with banana, the largest mean seedling length (3.6 mm). These data provide a simple protocol using commercially available media that is suitable for ex-situ conservation of Restrepia. These media may also be of use for the micropropagation and conservation of other related orchid genera.

Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae)—preliminary evidence for the symbiotic culture advantage

Eulophia alta (Linnaeus) Fawcett & Rendle seeds collected from the Florida Panther National Wildlife Refuge (Collier County, FL; FPNWR) were used in a screen of five asymbiotic orchid seed germination media to determine their effectiveness in promoting germination and protocorm development. In addition, 10 fungal isolates collected from the roots of E. alta at sites in the FPNWR, Highlands County (FL), and Goethe State Forest (Levy County, FL; GSF), and a fungal isolate from the roots of Spiranthes brevilabris collected from GSF were screened for their effectiveness at promoting in vitro symbiotic germination of E. alta seeds. After 18 weeks asymbiotic culture, seeds sown on PhytoTechnology Orchid Seed Sowing Medium germinated to a higher percentage (87.9%) and had a higher percentage of protocorms with developing protomeristems (32.7%) than seeds cultured on Knudson C, Malmgren Modified Terrestrial Orchid Medium, ½-strength Murashige & Skoog, or Vacin & Went Modified Orchid Medium. Significantly more leaf-bearing protocorms were observed on PhytoTechnology Orchid Seed Sowing Medium (0.8%) and Vacin & Went Modified Orchid Medium (1.3%) than other media tested. Of the fungi tested, one fungal isolate (Ealt-396) promoted germination to 69.0%, two isolates promoted germination to less than 0.75% and did not support further protocorm development, and eight isolates did not support germination. Seeds co-cultured in darkness with Ealt-396 grew more rapidly than asymbiotic seedlings following germination. In addition, co-cultured (=symbiotic) seedlings continued to develop more rapidly than asymbiotic seedlings upon transfer to 16/8 h light/dark photoperiod. Symbiotic seed culture of E. alta may be a more desirable method of propagation since protocorms develop more rapidly than seeds sown on asymbiotic media. Symbiotic seedlings may be more appropriate for reintroduction to natural areas than asymbiotic seedlings since symbiotic seedlings could serve to inoculate soils with a germination promoting mycobiont.

Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

Continuing loss of native orchid habitat has lead to an increased emphasis on orchid conservation. Major obstacles in the production of native orchid seedlings for use in conservation have been: (1) development of efficient and reliable seed germination protocols and (2) an understanding of early seedling growth and development. Effects of six asymbiotic media (Modified Lucke, Murashige & Skoog, Lindemann, Vacin & Went, Malmgren Modified, Knudson C), four exogenous cytokinins (BA, Zea, Kin, 2-iP), and three photoperiods (0/24, 16/8, 24/0 h L/D) were examined on seed germination and early protocorm development of Habenaria macroceratitis, a rare native Florida terrestrial orchid. Finally, the effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on in vitro seedling development were examined. Percent seed germination was highest on both LM and KC after seven weeks culture (LM = 89.1%, KC = 89.2%); however, protocorm development was enhanced on MM after both seven and 16 weeks. Both zeatin and kinetin at 1 µM enhanced seed germination (Zea = 58.1%, Kin = 47.2%). Final percent seed germination (91.7%) and protocorm development (Stage 4) was increased in the absence of light (0/24 h L/D). In␣vitro seedlings cultured under 8/16 h L/D conditions produced the highest number of tubers per seedling (1.06) with the greatest tuber (42.7 µg) and shoot (fwt = 69.5 µg) biomass and tuber diameter (3.1 mm).