The Cytokinin Preference for in vitro Germination and Protocorm Growth of Cypripedium candidum

Cytokinin preference and optimum levels were investigated forin vitro germination and protocorm growth of Cypripedium candidumMuhl. ex Willd. (Lady's Slipper Orchid) using seed collected8 weeks after pollination. Benzyl adenine (BA) and 6-(     

Endophytic fungi from Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand

Eight endophytic fungi were isolated from roots of the threatened terrestrial orchid, Pecteilis susannae (L.) Rafin. Phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that seven isolates belonged to the genus Epulorhiza and one to Fusarium. All fungal isolates were cultured with orchid seeds collected from three field sites near Doi Suthep-Pui National Park, Chiang Mai, Thailand. Seed germination and protocorm development were evaluated up to 70 days after sowing. Percent symbiotic seed germination was highest (86.2%) when seeds were cultured with Epulorhiza (CMU-Aug 013). The protocorm development was the most advanced up to stage 2, continued embryo enlargement, or rupture of the testa, and the highest percentage was 17.8% when seeds were cultured with Epulorhiza (CMU-Aug 007). Without fungi, seed germination and protocorm development were 62.1% and 11.1%, respectively. The dependency of P. susannae on fungal symbionts for early seedling development is yet to be determined. Optimizing seed germination and seedling fitness will assist the conservation of this threatened orchid in Thailand.     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.     

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).     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

Developmental processes of achlorophyllous orchid, <Emphasis Type="Italic">Epipogium roseum</Emphasis>: from seed germination to flowering under symbiotic cultivation with mycorrhizal fungus

We have achieved the symbiotic cultivation of an apparently achlorophyllous orchid, Epipogium roseum Lindl., with a mycorrhizal fungus isolated from an underground organ of this orchid. Although the seed germination rate was extremely low, subsequent growth from protocorm to flowering was induced in a medium containing volcanic soils and sawdust. Stolons elongated from each protocorm, and rhizomes were formed at certain intervals on the stolons. Some of the rhizomes developed into a coralloid form, and tubers were formed from the coralloid rhizomes. The coralloid rhizomes degenerated concurrently with maturation of the tubers. Six months after seed sowing, around 80 tubers were produced from a single protocorm. An inflorescence appeared from each of the large tubers, and the process to flowering was observed in one of these. Consequently, the developmental processes from seed to flowering in E. roseum was clearly revealed in this study.     

Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand

Symbiotic seed germination of Pecteilis susannae (L.) Rafin was investigated using 11 fungal isolates recovered from roots of four Thai terrestrial orchids (P. susannae, Eulophia spectabilis, Paphiopedilum bellatulum and Spathoglottis affinis). Seed germination and protocorm development were evaluated up to 133 days after sowing. Protocorm development was most advanced, up to stage 5 (elongation of the first leaf), when seeds were cultured with 4 Epulorhiza isolates obtained from roots of P. susannae (CMU-Aug 028, 4.3%, CMU-Aug 007, 4.2%, and CMU-Aug 013, 2.2%) and E. spectabilis (CMU-STE 014, 3.9%). Moreover, stage 4 protocorm development (emergence of the first leaf) occurred with fungal isolates CMU-STE 011, 5.7%, (Epulorhiza sp.) and CMU-AU 212, 4.3%, (Tulasnella sp.) obtained from roots of E. spectabilis and S. affinis respectively. When seed was incubated without fungi (control), development was limited to stage 3 of protocorm development (appearance of promeristem). This is the first report of protocorm stage 5 development in P. susannae using compatible fungal symbionts. Optimization of seed germination and seedling fitness will assist the conservation and propagation of this orchid species and other terrestrial orchids in Thailand.     

Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.     

兰科杓兰属（Cypripedium）植物种子非共生萌发研究进展

兰科杓兰属(Cypripedium)植物主要分布于东亚、北美等温带地区和亚热带山地。杓兰不仅具有极高的观赏价值,而且其经济价值和科研价值也越来越受到人们的重视。近年来对杓兰属植物人工繁殖的相关研究不断深入,主要集中于种子的非共生萌发等方面。本文对濒危兰科杓兰属植物进行了简要介绍,并就其种子非共生萌发研究从种子成熟度、预处理和有机添加物的作用、培养基的配置等方面进行综述,为目标杓兰种类的非共生萌发试验方案的制定奠定基础,将有助于温带/高山兰科植物保育研究的发展。     

Proteome changes in Oncidium sphacelatum (Orchidaceae) at different trophic stages of symbiotic germination

Mutualistic symbioses between plants and fungi are a widespread phenomenon in nature. Particularly in orchids, association with symbiotic fungi is required for seed germination and seedling development. During the initial stages of symbiotic germination, before the onset of photosynthesis, orchid protocorms are fully mycoheterotrophic. The molecular mechanisms involved in orchid symbiotic germination and development are largely unknown, but it is likely that changes in plant energy metabolism and defense-related responses play a central role in these processes. We have used 2D-LC-MS/MS coupled to isobaric tagging for relative and absolute quantification to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. We identified and quantified 88 proteins, including proteins putatively involved in energy metabolism, cell rescue and defense, molecular signaling, and secondary metabolism. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense-related responses, and phytoalexins and carotenoid biosynthesis. Our results suggest profound metabolic changes in orchid protocorms during the switch from the fully mycoheterotrophic to the photosynthetic stage. Part of these changes may be also related to the obligatory nature of the interaction with the endomycorrhizal fungus.     

Improved assessment of viability and germination of Cattleya (Orchidaceae) seeds following storage

Effective seed storage methods for orchid species provide a unique opportunity to preserve large amounts of germplasm. Early evaluation of the physiological status of seeds is useful for selecting seed lots for conservation. To score seed quality, seeds from nine Cattleya species (Cattleya granulosa, Cattleya hegeriana, Cattleya intermedia, Cattleya mossiae, Cattleya purpurata, Cattleya sanguiloba, Cattleya tenuis, Cattleya tigrina and Cattleya walkeriana) were germinated on Knudson C medium and viability was measured using a tetrazolium (TZ) test following pre-conditioning in a 10% sucrose solution, both before and after storage at −18°C for 3 mo. Tetrazolium test results were quantified using digital imaging of germinating seeds on a flatbed scanner. Seed viability (with or without pre-conditioning in sucrose), germination, Germination Velocity Index (GVI) and the correlation between viability measurements and germination were also evaluated. Both the tetrazolium test and the germination results showed that there was no loss of viability after storage, although in five seed lots, the GVI decreased. Pre-conditioning improved the correlation between viability exhibited in the tetrazolium test and seed germination. Image analysis proved to be a powerful tool for analysing these small seeds.     

Mycorrhizal compatibility and symbiotic reproduction of Gavilea australis,an endangered terrestrial orchid from south Patagonia

Gavilea australis is a terrestrial orchid endemic from insular south Argentina and Chile. Meeting aspects of mycorrhizal fungi identity and compatibility in this orchid species is essential for propagation and conservation purposes. These knowledge represent also a first approach to elucidate the mycorrhizal specificity of this species. In order to evaluate both the mycorrhizal compatibility and the symbiotic seed germination of G. australis, we isolated and identified its root endophytic fungal strains as well as those from two sympatric species: Gavilea lutea and Codonorchis lessonii. In addition, we tested two other strains isolated from allopatric terrestrial orchid species from central Argentina. All fungal strains formed coilings and pelotons inside protocorms and promoted, at varying degrees, seed germination, and protocorm development until seedlings had two to three leaves. These results suggest a low mycorrhizal specificity of G. australis and contribute to a better knowledge of the biology of this orchid as well as of other sympatric Patagonian orchid species, all of them currently under serious risk of extinction.     

Structural Changes During the in vitro Germination of Vanilla planifolia (Orchidaceae)

A study is reported of histogenesis and organogenesis duringthe processes leading up to seedling formation in cultures ofVanilla planifolia. Prior to germination, all cells of the embryoincreased in size rupturing the seed coat and initiating theprotocorm stage. The cells of the protocorm were heavily ladenwith starch grains. Although all of the cells of the matureembryo were heavily laden with protein bodies, these were confinedto the terminal cell descendents on emergence of the embryofrom the seed coat, and they disappeared during differentiationof the meristem, indicating that some reserves were mobilizedand utilized during germination. The terminal locus of embryonal axis did not differentiate intoa cotyledon and epicotyl as in other angiosperm embryos butformed a thick meristematic layer. Bipolar differentiation withinthe meristem produced the shoot, and after a few leaves hadbeen formed, the first root differentiated endogenously fromthe base of the meristem. Subsequent roots, however, appearedto originate more superficially. The chain of events appearsto be quite unique to Vanilla amongst the angiosperms. Vanilla planifolia, protocorm, starch grains, protein bodies     

A rapid double-staining technique to improve seed viability testing in terrestrial orchids

Abstract

The need to optimize seed banking efforts has stimulated research for rapid methods to estimate quality in seed-lots. For terrestrial orchids, viability testing using tetrazolium (TTC) staining requires chemical scarification, as seeds have an impermeable testa. Different seed-coat permeability may affect TTC staining, thus affecting the results. The aim of this study was to perform a permeability test to assess the effectiveness of the used scarification method and its usefulness to correct TTC viability results. Mature seeds of Anacamptis laxiflora were subjected to eight scarification treatments with sodium hypochlorite solutions with different concentration and duration. Viability tests were performed using the basic TTC methodology, followed by a permeability test performed by means of trypan blue dye. The different scarification methods resulted in estimated TTC viability ranging from 0% and 94% for the same seed lot of A. laxiflora seeds. Our results proved that the used scarification protocol significantly affects both seed coat permeability and subsequent TTC staining (two-way ANOVA, p?< 0.0001). We describe a new rapid protocol that can be used to test terrestrial orchid seed viability. This double-staining method, providing rapid information on seed coat permeability, can be useful to avoid under-estimation of TTC results.     

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.Key words: Asymbiotic germination, corm development, Calopogon tuberosus, ecotypic differentiation, native orchid, orchid seed germination, seedling development     

铁皮石斛种子的室内共生萌发

由于人为的采挖和原生境的破坏,使得铁皮石斛野生资源已濒临灭绝。因此,保护铁皮石斛野生资源及其生境,加快其野生资源的繁殖显得非常重要。以铁皮石斛种子(TTC染色显示种子生活力为77.65%)为材料,与分离自2种野生兰科植物根部的4株共生真菌 (C20来自铁皮石斛,L12,L24b 和 L28来自美花石斛)在燕麦培养基上进行共生萌发。经过18周的共生培养,4株真菌均不同程度地促进了铁皮石斛种子的萌发,其中菌株L24b (Epulorhiza)和L28 (Epulorhiza)显著提高了种子的萌发率,分别比对照高出26.51%和12.20%,但未形成幼苗,只是处于原生分生组织阶段(阶段3);菌株C20(Epulorhiza)和L12 (Alternaria) 虽没有显著提高种子的萌发率,但对原球茎的发育和幼苗的生长有明显的促进作用;而对照的种子仍然处于膨大转绿期,即萌发阶段(阶段2)。同时发现,TTC染色显示的铁皮石斛种子生活力要高于种子共生萌发的萌发率(除了菌株L24b)。研究结果表明:种子生活力染色检测的活力值只代表种子所具有的潜在的萌发能力,而不能代表实际的萌发率。在异地条件下,铁皮石斛与共生真菌间没有严格的专一性,可以与瘤菌根菌属、链格孢属真菌形成共生关系。菌株C20和L12能促进萌发后的种子进一步分化成幼苗。这两个菌株为铁皮石斛的人工优质栽培和野外种群的建立提供了可能。     

Surprisingly high orchid diversity in travertine and forest areas in the Huanglong valley, China, and implications for conservation

The presence of such a large number of terrestrial orchid species in a small area (ca. 1 km²) of the Huanglong valley in southwestern China is uncommon for this country. Studying the relationship between the distribution patterns of these orchid species and their microenvironments may help us understand this uncommon phenomenon. We established 662 1 m × 1 m plots, measured the cover of each species and found that there were 33 orchid species distributed mainly in two different habitats, i.e. travertine areas and forest. In the travertine areas, 30 orchid species were found; the six most common ones being Cypripedium bardolphianum, Cypripedium flavum, Cypripedium tibeticum, Galearis diantha, Ponerorchis chusua and Phaius delavayi. However in the forested habitat, we found 21 orchid species; the most common ones being Tipularia szechuanica and Goodyera repens. Travertine areas had a higher number of orchid species as well as higher numbers of orchid species per plot as compared to forest. Light availability seems critical to the performance and distribution of orchid species. Stream flow through the travertine area during the orchids growing season appears to be an important factor in shaping and maintaining stable microenvironments favorable to the growth and reproduction of orchids. The results presented in this study suggest that some orchid species in the travertine area might be threatened if the travertine stream flows were to change or be disrupted.     

In vitro symbiotic germination of myco-heterotrophic Gastrodia elata by Mycena species

Symbiotic germination was analyzed in the myco-heterotrophic orchid Gastrodia elata. Seeds were placed on water agar medium containing a leaf-disc of Quercus accutissima that had been previously inoculated with one of six different Mycena species. Among the six fungi, KFRI1212 (HQ662845) and KFRI2121 (HQ662846) germinated 60.1 and 47.0 % seeds, respectively, while others germinated less than 3.5 %. Although KFRI1212 induced a significantly higher germination rate than KFRI2121, initiation of protocorm development was induced much faster by KFRI2121 than by KFRI1212. Molecular identification and phylogenetic analysis showed that the two fungi belonged to one clade that includes Mycena chlorophos, M. amicta and M. tenerrima, suggesting that seed germination of G. elata depends on a narrow taxonomic range of fungi. In conclusion, this study not only showed fungal preference of G. elata for seed germination but also confirmed molecular identities of mycorrhizal fungi for the first time, which will allow us to better understand the process of symbiotic events at the germination stage of G. elata.     

同色兜兰的非共生萌发与快速繁殖研究

以授粉后不同发育时期的同色兜兰种子为材料,观察其形态特征和萌发过程,并探讨建立同色兜兰高效快繁体系的最佳条件。结果表明,种子发育中后期即授粉后210~240d为较适宜的采收期,授粉后210d的种子萌发率最高(达77.79%);1/4 MS和1/2MS为同色兜兰适宜的基本培养基,添加100mL/L椰乳或1g/L蛋白胨对种子萌发及原球茎生长和分化有明显的促进作用;添加1g/L活性炭对原球茎褐化有一定的抑制作用,但添加剂量不宜过大;添加香蕉汁和苹果汁对同色兜兰种子萌发和原球茎生长分化有抑制作用;暗处理对同色兜兰种子萌发无影响;分化后的原球茎在壮苗和生根培养基上培养120d即可得到4~5片叶、高3~5cm的同色兜兰健壮试管苗。