Wild Orchid Species Research Articles

How Effective Is Environmental Protection for Ensuring the Vitality of Wild Orchid Species? A Case Study of a Protected Area in Italy.

Orchids are among the plants most threatened by anthropic impact and environmental changes. Therefore, all known orchid species are protected in several countries by regional, national and international legislation. Several studies have cast doubts on the effectiveness of legislation to ensure the protection of wild orchids. We assessed the vitality of four orchid populations in a coastal area in Northern Italy, by monitoring the vegetative and reproductive traits of the orchid populations growing both in the protected sites comprising the Natura 2000 network, and in non-protected sites. We also monitored the level of environmental threat to orchid vitality. The early-flowering deceptive species (Ophrys sphegodes and Anacamptis morio) exhibited high vegetative vitality and experienced similar levels of environmental threat in the protected and non-protected areas. However, their reproductive success was strongly jeopardized, probably by a failed pollination that impeded the fruit set. The late-flowering, partially or totally rewarding species (Anacamptis pyramidalis and Anacamptis coriophora) were more strongly impacted by spring mowing and ungulate herbivory and alien species. Only for A. coriophora were the herbivory and alien species invasions lower at the protected vs. non-protected sites, which ensured a higher population vitality at the protected sites. We conclude that the environmental protection in our study area is ineffective for preserving orchids without targeted actions against specific environmental threats.

Induction of Protocorm-like Bodies (PLBs) and Plant Regeneration in Cymbidium chloranthum (Orchidaceae)

Cymbidium chloranthum Lindl. is an indigenous orchid native to Sabah and several tropical countries in Asia. The significant horticultural value of orchids has led to many wild orchid species being threatened with extinction due to overcollection and habitat destruction. In this study, an efficient protocol for the in vitro regeneration and mass propagation of this orchid was developed. Protocorms of C. chloranthum were cultured on Murashige and Skoog (MS) basal medium containing plant growth regulators (PGRs) such as meta-Topolin (mT) and 6-benzylaminopurine (BAP) at concentrations of 0.5 or 1.0 mg/L, and coconut water (CW) at 5 or 10% (v/v). The cultures were incubated at 25±2℃ under 12h of photoperiod. The MS basal medium devoid of any PGRs served as a control. After 90 days of culture, the combination of 0.5 mg/L mT and 0.5 mg/L BAP significantly promoted 2.97±0.53 shoots and 3.83±1.07 roots, respectively. The same treatment also promoted protocorms multiplication with an average production of 2.24±0.55 PLBs. During the acclimatization phase, 66% of the regenerated plants survived. The fully acclimatized plants were reintroduced into their natural habitat in Tenom Orchid Centre. The current approach offers a sustainable way to meet commercial demand while conserving the remarkable species in its natural habitat.

UTILIZING GIS AND REMOTE SENSING FOR MODELING THE SPATIAL DISTRIBUTION OF WILD ORCHID SPECIES IN PHU FAEK FOREST PARK

Thailand's wild orchids, cherished for their beauty, face threats from habitat loss and over-collection. Urgent conservation is needed, with efforts including protected areas and sustainable practices. This study aims to model the distribution of wild orchids through the utilization of remote sensing and GIS techniques. The methodology involves comprehensive surveys of wild orchid species within Phu Faek forest park. Spatial regression analysis explores intricate relationships between wild orchid density and environmental factors such as NDWI, forest type, elevation, basin density, and aspect. The results of field surveys identified 28 orchid species with diverse distribution patterns, including dominant species like Aerides falcata Lindl. & Paxton, Aerides falcata Lindl., and Cleisostoma fuerstenbergianum F.Kranzl. The spatial regression model revealed distribution patterns, with higher density in central and north regions. The NDWI indicator, which reflects moisture content, provided additional insights into the distribution of orchids

\(\textit{In vitro}\) regeneration of \(\textit{Renanthera imschootiana}\) Rolfe from protocorm-like body

Renanthera imschootiana Rolfe is an extremely rare and endangered tropical epiphytic orchid. Studies on in vitro culture of R. imschootiana were conducted in order to conserve and increase the genetic pool of this wild orchid species. In this study, the effect of plant growth regulators (BA and NAA), ripe bananas in combination with potato, humic acid, and silver nanoparticles (AgNPs) were investigated to find a suitable condition for in vitro plant regeneration from R. imschootiana protocorm-like body (PLB). The results showed that after 45 days of culture, MS medium supplemented with 100 g ripe banana/liter and 100 g potato/liter was suitable for regenerating shoots from the protocorm-like body (PLB) (17.56 shoots/PLBs and 100.00% of shoot-regenerating PLBs); MS medium supplemented with 2.0 mg/L BA, 0.3 mg/L NAA and 4 ppm AgNPs was the most suitable for the growth of shoots after 60 days of culture (10.00 leaves/shoot and shoot length of 4.22 cm). The in vitro shoots were transferred to half-strength MS supplemented with humic acid and AgNPs to investigate the root formation of R. imschootiana. After 60 days of culture, the best rooting was obtained at 2.0 mg/L humic acid and 6 ppm AgNPs (8.33 roots/shoot; root length of 4.00 cm and 100.00% root formation). The findings suggest that the in vitro micropropagation from R. imschootiana protocorm-like body provides a useful alternative tool for the conservation of this endangered species.

The effect of the use of salep powder obtained from different wild orchid species in Turkey on the rheological, thermal, and sensory properties of ice cream

The aim of this study is to investigate the effect of salep species grown in different regions of Turkey on the rheological and microstructural properties of ice cream mix and the thermal and sensory properties of ice cream. For this purpose, ten different types of salep grown in different regions (Mersin, Yozgat, Muğla, Kahramanmaraş, Adana, Van, Muş) in the natural microflora of Turkey were used in a formulation of an ice cream mix. The flow behavior, frequency sweep, and 3-ITT rheological properties of ice cream mixes were studied. All samples showed shear thinning, viscoelastic solid-like, and recoverable character. The K and n values ​​for the ice cream mixes were determined as 0.03-35.08 Pasn and 0.33-0.80, respectively, and significantly differed according to salep varieties (p < 0.05). The zeta potential values of ice cream mix samples ranged from -25.87 mV to -33.95 mV and were significantly affected by salep varieties (p < 0.05). The use of different salep varieties significantly affected the thermal properties of ice cream such as freezing point temperature (Tf), temperature range (ΔT), and enthalpy of fusion (ΔHf). In conclusion, the results of this study showed that the use of different salep varieties can significantly affect the rheological, thermal, and sensory properties of ice cream, and the selection of salep varieties may be vital for the desired quality of ice cream.

Molecular identification of seed‐feeding flies dissected from herbarium specimens clarifies the 100‐year history of parasitism by Japanagromyza tokunagai in Japan

AbstractInsect pests, particularly seed‐feeding flies, can cause serious damage to the fruits of many wild orchid species. Evidence of fly damage has been observed in 55 orchid species, belonging to 25 genera, distributed throughout Japan. Although some authors suggested that the observed increase in fly damage to orchids in recent years is related to the introduction of exotic species, the timing and route of spread remain unclear. We examined herbarium specimens of seven orchid species, collected between 1923 and 2016, to collect coarctate pupae and empty puparia, with minimal damage to the specimens. Genomic DNA was then extracted from the insect tissue and species were identified using mitochondrial cytochrome oxidase subunit 1 barcode regions. Long amplicons (660 base pairs [bp]) were obtained from 45.2% of samples and short amplicons (162 bp) were obtained from 90.3% of samples. In total, 51 of 62 samples had nucleotide sequences homologous to Japanagromyza tokunagai, with specimens distributed across collection years and geographic locations. Therefore, the severe damage to Japanese orchids observed in recent years is not related to the recent introduction of an exotic species; it may be related to a rapid increase in a native species. This work highlights the value of herbaria as important resources for elucidating the history and distribution of parasitic insects.

Orchids of Mount Ungaran (Indonesia) compiled from a decade of data collections between 2010 and 2021

Abstract. Kurniawan FH, Nazar L, Anjarwati R, Sasono HD, Rahayuningsih M. 2021. Orchids of Mount Ungaran (Indonesia) compiled from a decade of data collections between 2010 and 2021. Nusantara Bioscience 13: 245-259. Orchid is well known as a plant group widely used as ornamental plants because of their uniqueness. But, high exploitation, deforestation, and land-use changes have threatened orchid species and triggered extinction. It also happened in Mount Ungaran, Central Java, Indonesia, despite designation as an essential ecosystem area. This research was conducted to add an updated list of wild orchid species that can serve as biodiversity baseline data for conservation management in Mount Ungaran. The study was carried out through exploration activities from 2010 to 2021. The study area covered eleven sites namely Gentong Hills, Kalisidi, Pasigitan, Banyuwindu, Gajahmungkur, Watuondo, Mawar Camp, Indrakila, Gondang and Puncak which were all included in the buffer zone of Mount Ungaran. A total of 115 species from 54 genera of orchids were successfully identified, including 78 species of epiphyte and 37 species of terrestrial orchids. Among the species recorded, at least 27 species were known to be endemic to Indonesia, and 8 species were endemic to the island of Java, which four species were listed in the IUCN Red List. In addition, all the species found were listed in Appendix II of the CITES, except one species, Paphiopedilum javanicum which was listed in Appendix I. This study recorded the new distribution records of several species of orchids that were previously only found in West Java or East Java, i.e., Ania penangiana, Pholidota convallariae, and Crepidium junghuhnii. This study has also documented a species from genus Crepidium, which morphological characters have not been described in any identification guides. The Gentong area has potential as a priority for in-situ orchid protection area by the highest number of species were reached 54% of all orchids found on Mount Ungaran.

Evaluation of salep obtained from different wild orchid species of Turkey and their use in Maras type ice cream

The study was aimed to investigate the effects of different salep samples on Maras type ice cream quality characteristics. For this purpose, 10 different wild orchid species, a significant number of which have not been studied before, were collected from different regions of Turkey. pH, moisture, total protein, ash, glucomannan, starch contents, and rheological properties of salep obtained from their tubers were evaluated. Then salep (group 1) and salep-stabilizer-emulsifier (group 2) were used in Maras type ice cream production. Especially glucomannan contents of salep affected important quality parameters (phase separation, hardness, rheological, melting, and sensory properties) of the product. The data obtained from group 2 and multidimensional scaling analysis showed that those parameters were improved with the use of stabilizer-emulsifier. The results of the study provide significant information on the conservation of plant diversity in terms of wild orchids, as well as ensuring the sustainability of Maras type ice cream. Novelty impact statement Salep obtained from Serapias lingua, Orchis mascula, Dactylorhiza majalis, Ophrys sphegodes, and Habenaria repens wild orchid species is more appropriate in Maras type ice cream production. Ophrys apifera and Himantoglossum hircinum species should not be collected from the nature considering their suitability for Maras type ice cream. Replacing some of the salep amount used in the production of Maras type ice cream with a stabilizer-emulsifier mixture will not only protect the sustainability of the product but also protect the plant diversity in terms of wild orchids.

Micropropagation of Paphiopedilum x Dalatense

Paphiopedilum x dalatense is a beautiful orchid species with large flowers in variable colors and leaves covered with stripes and beautiful unseen mosaic spots. Recently, many people exploit this species, causing it becomes very rare. In this study, we studied the effects of various organic matter: potato, banana and tryptone, yeast powder, peptone on the growth and development of P. dalatense shoots as well as the effects of NAA and humic acid on in vitro rooting of this orchid were investigated. The research results showed that MS medium supplemented with 100 g/L banana in combination with 100 g/L potato (5,4 shoots/sample, 18,8 mm/shoot, 4,5 leaves/shoot, and shoots survival rate of 100%) or MS medium supplemented with 1 g/L peptone (4,19 shoots/sample, 15 mm/shoot, 4 leaves/bud, and 92% of shoots survival rate) were the best response for the shoot formation and development. In addition, the half strength MS culture medium supplemented with 1 mg/L NAA (5,2 leaves/sample, 4,6 roots/buds, 3,56 cm/root, and 100% rate for rooting) was the suitable medium for the in vitro rooting of P. dalatense. Being cultured on half strength MS medium supplemented with 2 mg/L humic acid, the rooting rate reached 100% with the greatest root number and the longest root (5 roots/shoots, 5,5 cm/root). The obtained results on the in vitro propagation on this orchid helps contribute to the conservation and increases the genotic pool of this precious wild orchid species, as well as the rapid multiplication of healthy plantlets serving the commercialization of precious orchid species.

The complete chloroplast genome of Cymbidium dayanum (Orchidaceae)

Cymbidium dayanum, a wild orchid species in the Orchid family (Orchidaceae), is considered highly valuable because of its long flowering period and beautiful plant shape. We sequenced the complete chloroplast genome of C. dayanum using the Illumina Hiseq platform (Illumina, San Diego, CA). The size of the C. dayanum chloroplast genome is 155,408 bp, with an average GC content of 36.76%. This chloroplast genome has containing a large single-copy (LSC) region of 84,189 bp, a small single-copy (SSC) region of 17,991 bp, and two inverted (IRa and IRb) repeat regions of two 26,614 bp. A total of 118 unique genes were annotated, including 76 protein-coding genes, 38 tRNA genes, and 4 rRNA genes. A maximum-likelihood phylogenetic tree indicated that C. dayanum is closely related to C. tracyanum in the genus Cymbidium based on 9 whole chloroplast genome sequences.

. In vitro propagation of the new orchid Dendrobium trankimianum T. Yukawa

Dendrobium trankimianum T. Yukawa is a beautiful, endemic orchid of Vietnam, a new species with a first - published description in 2004. It is very rare and expected to be added to the IUCN Red List status - CR. In vitro studies of orchid D. trankimianum T. Yukawa were conducted in order to conserve and increase the genetic pool of this precious wild orchid species. The results showed that full-strength MS medium supplemented with 2.0 mg/L BA and 0.5 mg/L NAA (10.24 PLBs/explant; 90.11% explants formed PLBs) or full-strength MS medium supplemented with 1.5 mg/L TDZ and 0.5 mg/L NAA (14.11 PLBs/explant; 92.06% explants formed PLBs) were the most suitable for protocorm formation. For subculture, suitable growth of shoots were obtained on full-strength MS medium supplemented 1.5 mg/L BA (22.35 shoots/explant; shoots length of 1.96 cm) and full-strength MS medium supplemented with 60 g ripe banana per liter (25.11 shoots/explant; shoots length of 2.12 cm). The shoots in vitro were transferred to half-strength MS supplemented with different concentrations of IAA, IBA and NAA to investigate root formation. The best rooting occurred at 0,5 mg/L NAA (7.91 roots/shoot; root length of 4.01 cm; 98.51% root formation). The plantlets with uniform growth were planted on different substrate: Eco clean soil, Coconut fiber, Fern fiber, 50% Rice husk in combination with 50% Eco clean soil for research the most suitable substrate. After 60 days of transplantion and acclimatization, the result showed that Fern fiber was suitable substrate for plantlet growth in a nursery garden (8.0 roots/ explant; root length of 5.5 cm; survival rate of 93.29%).

Cytotoxic effect of selected wild orchids on two different human cancer cell lines

Majority of the orchid species are used in the traditional medicines for the treatment of several diseases. They are the sources of polysaccharides, phenanthrenes, bibenzyl derivatives, revesteral, stilbenoids and polyphenol compounds. This study explored the cytotoxic activity of seven wild orchid species and identification of medicinally active compounds. The extracts of orchid species were screened for cytotoxic effect on the human cervical cancer cells (HeLa) and human glioblastoma cells (U251) using an MTT assay. The medicinally active compounds of high cytotoxic extracts were identified by GC-MS resulting in many stilbenoids and phenolic derivatives. The extract of Dendrobium transparens (DTs) and Vanda cristata (VCw) showed high cytotoxic effect towards the HeLa and U251 cell lines (IC50 of DTs: 382.14 μg/ml and 75.84 μg/ml respectively and IC50 of VCw: 317.23 μg/ml and 163.66 μg/ml respectively). This study concludes that they could be used as cancer therapeutics.

Assessing conservation efforts against threats to wild orchids in China

Orchids are of special interest to plant conservationists because of their high species diversity as well as their obligate dependencies on one or more biotic interactions, which often pose additional challenges in their conservation. In this study, we analyze threats to all known Chinese orchid species and evaluate whether current in situ and ex situ conservation efforts match these threats. Of the 1582 orchids included in this study, 653 (43.5%) were categorized as threatened (i.e., critically endangered, endangered, or vulnerable), 191 (12.0%) were traded using wild-collected plants, 531 (33.6%) were Chinese endemics, 826 (52.2%) were found in one or more protected areas, 802 (50.7%) were in ex-situ collections, 136 (8.4%) had seeds in storage; and only 43 (2.7%) were subject to conservation translocation actions within China. Proportionally, more threatened species were protected in situ and ex situ than non-threatened species. However, the current version of the Chinese Redlist assessment assigned over harvesting as a threat factor to 15 Chinese orchids only (2.3%), a gross underestimation of this threat considering both the Chinese and international orchid markets. We propose establishing small nature reserves to protect wild orchid species that do not currently occur in any protected area, creating ex situ collection networks to strengthen conservation capacity (including institutions that are concerned with utilization only), and establishing a dynamic list of wild orchids in trade, along with trade volumes, to better-gauge conservation threats posed by wild harvest.

MICROPROPAGATION OF RHYNCHOSTYLIS GIGANTEA ORCHID BY SOMATIC EMBRYOGENIC CULTURES

Rhynchostylis gigantea is a wild orchid species commonly grown. Flowering at the traditional Tet holiday, the flower season always blooms in December of the lunar calendar. The flower has a faint aroma, so it is called Nghinh spring and is a kind of orchid with a national soul and national essence. Rhynchostylis gigantea is found in many areas in Vietnam as it is distributed in the South Central Highlands, especially the areas bordering Laos and Cambodia at low altitudes &lt;600m. Rhynchostylis gigantea is a type of heat-resistant orchid, with suitable temperatures for orchid being from 26-30oC. Rhynchostylis gigantea species have many different colors such as red, white, shrimp brick color, blue and pink. The single-member tree does not assume pseudobulbs, grows vertically, has many pneumatophores and grows straight from the stem. Rhynchostylis gigantea grows slowly and regenerates with seeds. Due to high genetic diversity, it is difficult to maintain the parent’s characteristics. Applying techniques of somatic embryo culture in conservation and development of the Rhynchostylis gigantea tree to meet the increasing demand for seedlings is urgent.

Micropropagation of dendrobium heterocarpum lindl.

Dendrobium heterocarpum Lindl. is an endangered species which is currently used as ornamental pot plant for its beautiful flowers. An increase in collection for trade or any other purposes may lead to a dramatic decrease in the population of this species, thus becoming rare or endangered species in the near future. In this study, effects of plant growth regulators (BA, NAA, IAA, IBA, TDZ) and natural supplements (carrot, potato, and banana extracts) on protocorm like bodies (PLBs) formation; growth and development of shoot; and root regeneration of D. heterocarpum Lindl. as well as type of substrates on acclimatization and growth of seedlings were investigated. The results showed that PLBs formation was optimal on MS medium supplemented with 2.0 mg/L BA and 1.0 mg/L NAA (7.11 PLBs/explant; PLBs formation percentage of 68.9%) or MS medium supplemented with 1 mg/L TDZ and 0.5 mg/L NAA (7.29 PLBs/explant; PLBs formation percentage of 75.53%). For subculture, MS medium supplemented 1.5 mg/L BA and 60 g/L banana extract (22.40 shoots/explant; shoot length of 2 cm) was the most suitable for shoot regeneration and growth. Additionally, root formation was the most suitable on ½ MS medium supplemented with 1 mg/L NAA (4.4 roots/shoot; root length of 3.12 cm; root formation of 95.56%). Finally, the sufficiently rooted plantlets were transferred to greenhouse for hardening. After 60 days, coconut fiber substrate was the most suitable for seedling growth and development (with survival rate of 97.78%, root number of 5 and shoot length of 3.4 cm). The results of propagation in vitro Dendrobium heterocarpum Lindl. contribute to conservation and sustainable development as well as towards the rapid multiplication of seedlings for commercial commercialization of this wild orchid species.

Germination and Protocorm Formation of Ophrys Sphegodes Mill. – In Vitro Protocol for a Rare Orchid Species

Summary Ophrys sphegodes Mill. is a wild orchid species which is threatened and protected due to its pollination biology, small seed and habitat destruction. The aim of this study was to establish asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media Knudson C (KC) and Malmgren (MM), supplemented with organic additives (peptone (PE), L-glutamin (A)e, folic acid, casein hydrolysate (CA)) added separately and control media KC--C and MM--C were used in the present research. All the nutrition media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to 5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. The seeds were examined under two illumination conditions, 0/24 light/dark (L/D) and 16/8 L/D. The presented results indicate a huge influence of illumination and nutrition media on the seed germination and protocorm formation. The seed germination was overall significantly more successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). Protocorm, rhizoids and shoot formation were achieved only on the seeds cultured on MM medium, while the KC medium caused only swelling of the embryo. Organic additives had positive influence on the germination rate. According to the obtained results, the best germination rate and seedling development were achieved on MM-PE media, cultured in dark. The presented procedure accelerates the germination period and can provide a large number of plants in a relatively short period of time so it can be used for conservation programs and mass production protocol.

Diversity and conservation strategy of orchids under anthropogenic influence in Taman Wisata Alam Curug Setawing, Yogyakarta

Kurniawan FY, Setiaji A, Putri F, Suyoko A, Semiarti E. 2018. Keanekaragaman. Pros Sem Nas Masy Biodiv Indon 4: 173-177. Yogyakarta has unique ecoregions with a high diversity of orchids. Forest in Taman Wisata Alam Curug Setawing (TWACS),Yogyakarta is located in a community protected forest area and keeps a high diversity of orchids. Relative to other plant families,orchids are subject to high levels of threat, such as over-collection, land clearing and climate change. In this study, we describe thediversity of orchids in newly opened tourist areas and their influencing factors and conservation strategies under anthropogenic areas.Ten imaginary plots of 20 m x 20 m each were established randomly by selecting points along the two trail in location. All collectedorchids are identified by matching the collection sample with orchid flora books. 15 species of wild orchids can be found in TWACS.Based on subfamily distribution, 73.33% of species including Epidendroideae, 20% Orchidoideae, and 6.66% Vanilloideae. Based ongrowth form, 46.66% of species belong to epiphytes and 53.33% terestrial orchids. Dendrobium crumenatum has the highest of IVIcompared to other species. Zeuxine sp. found only one individual because it is likely to enter the dormancy period. The study revealsthat to improve the population size, trees (mainly Albizia chinensis, Syzygium aromaticum, bamboo, Artocarpus heterophyllus, andAlbizia saman) should be planted in areas where the orchid species is recently missing through regular inventory.

Perspectives of Children Orphaned by HIV/AIDS on Ecology and Gathering of Wild Orchids in Tanzania

Orchid gathering in the Southern Highlands of Tanzania became an income generating strategy for HIV/AIDS-affected households, particularly for children orphaned by HIV/AIDS. Being orphaned might have implications for children's local ecological knowledge leading to indiscriminate harvesting of both preferred and non-preferred orchids. Using qualitative and quantitative mixed methods, we assessed the knowledge of wild orchid species among gatherers in four categories: children orphaned by HIV/AIDS, children not affected by HIV/AIDS, HIV/AIDS-affected adults, and non-HIV/AIDS-affected adults. Data collection took place from February 2006 until February 2007. More than 70% of the children orphaned by HIV/AIDS relied on tuber taste and texture to distinguish preferred and non-preferred species. HIV/AIDS-affected adults were competent at using orchid morphology to distinguish preferred from non-preferred orchid species. Gatherers stated preferred orchid species were primarily found in restricted areas, and non-preferred species were widespread. Cramer's V analysis revealed an association between the HIV/AIDS status of a gatherer and the gatherer's views on the conservation status of preferred and non-preferred species. Gatherers perceived non-preferred species as having higher natural regeneration potential than preferred species. Cramer's V analysis also showed a difference in gathering knowledge acquisition between HIV/AIDS-affected double orphans who emigrated from urban settings, and those residing in their natal rural village under guardian care. Both types of orphans relied primarily on other children for gathering knowledge, but those with guardians also gleaned knowledge from guardians. Across categories, children also gained knowledge from middlemen, who buy and resell the preferred orchids.

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.

Seasonality and mycorrhizal colonization in three species of epiphytic orchids in southeast Mexico

Orchids establish symbiosis with Rhizoctonia mycorrhizal fungi, forming the characteristic pelotons within the cells of the root cortex. Under natural conditions, terrestrial and epiphytic orchids have different levels of dependence upon the fungal symbiont, although various authors have mentioned that once orchid plants reach maturity the interaction becomes weaker and intermittent. Recent evidence shows that in some epiphytic orchid species mycorrhization is constant and systematic. In three species of wild orchids from southeast Mexico, we show that mycorrhization is systematically present in roots of different ages, in the wet and dry seasons. We demonstrate that the volume of the root that is colonized depends upon the quantity of rainfall and the diameter of the root, and that rainfall also determines the presence of fresh, undigested pelotons. In very thin roots, mycorrhizal colonization occupies a considerable proportion of the cortex, whereas in thicker roots the proportion of the volume of the root cortex colonized is lower.