Orchid Seeds Research Articles

Cryopreservation of Orchids - A Review.

The orchids are one of the beautiful creations of nature which stand apart from any other assemblage of flowering plants. They are highly evolutionary and ecologically significant group of plants that have effectively occupied almost every habitat on the earth. Indiscriminate collections and extermination of their natural habitats have threatened many species of orchids with extinction, resulting in a severe reduction of their genetic resources in nature according to recent patents. It is necessary to adopt sound scientific protocols for the preservation of orchid species. This cost-effective technique provides large storage time for the conservation of germplasm. Presently, efforts have been made to explore various cryopreservation techniques utilized so far and factors affecting the longevity of the propagules (in vivo and in vitro) while cryopreserving them. The sample to be cryopreserved is freeze-preserved in two ways, a) stepwise at two different subzero temperatures and b) in the rapid method, the samples are placed directly in the liquid nitrogen. The orchid seeds and pollen are the most suitable propagules for cryopreservation of orchids due to their minute size and less space requirement. Among the tissues (such as seeds, pollen, protocorms etc.) seeds are the most reliable. The present article reviews the cryopreservation techniques and factors effecting the cryopreservation, for in vitro conservation of orchid gene pool.

YUNHAN LEI, The Linsly School, Wheeling, WV, 26003. Pre-germination inoculation with 11 species of endomycorrhizal fungi slightly improves early growth of lettuce (Lactuca sativa) seeds but not that of radish (Raphanus raphanistrum) or beans (Phaseolus vulgaris).

Mycorrhizal associations of fungi with established and mature plant roots are well-known to benefit the plant. Mycorrhizal fungi, by penetrating into plant’s root cells and increasing their surface area, improve a plant’s absorption of water and mineral nutrients. In return, the fungi absorb carbohydrates from the plant. Less well known are the effects mycorrhizal fungi have on germination and early root growth. Tiny orchid seeds contain low quantities of nutrients and need early contact with mycorrhizal fungi for germination success. This study asks whether inoculation of lettuce, radish and bean seeds improves seed germination or seedling growth, and whether any effect is seed size dependent. Ten groups of seeds of lettuce, radish and beans were treated with a powder consisting of 11 different species of endomycorrizal fungi while ten seeds for each species served as controls. Seeds were placed in plastic petri dishes lined with paper toweling and provided equal amounts of distilled water. All six groups had 100% germination rates, except for 90% germination in the mycorrhizal fungi radish group. In comparing mycorrhizal fungi treatment groups to the control, average root length in lettuce increased by 12% (p=0.15, t-test), but only by 8% (p=0.71, t-test) in radish and 5% (p=0.91, t-test) in beans. Only the effect on lettuce seeds approached statistical significance. A larger trial with more seeds growing longer might yield significant results. Since mycorrhizal fungi treated seeds absorbed water faster and left drier paper toweling, seed coat proteins may be triggered to absorb water faster, aiding germination.

First record of orchid subfossil seeds – The abundant occurrence of Epipactis palustris (L.) Crantz and Dactylorhiza spp. seeds in early Holocene sediments from Central Europe

Numerous subfossil seeds of Epipactis palustris (L.) Crantz, and Dactylorhiza spp. (D. incarnata/maculata complex) found in early Holocene sediments in northern Poland are the first record of orchid seeds in geological formations of any age and location. The macro- and micromorphological characters of the seeds were examined to determine their taxonomic affinity. The seeds were found among numerous macroremains of aquatic and terrestrial organisms, which are distinct in effective buoyancy, enabling air and water dispersal. The orchid populations developed in a floodplain meadow where summer inundation was probably among the main agents facilitating seed dispersal and deposition in lake sediments. We discuss potential environmental factors enabling the formation and preservation of the subfossil assemblages and suggest some technical issues among the reasons for the general poverty of orchid fossil data. Our data provide the first direct evidence for the early post-glacial establishment of populations of E. palustris and species of the D. incarnata/maculata complex in the northern part of Central Europe.

New Insights into the Symbiotic Relationship between Orchids and Fungi

Mycorrhizas play an important role in plant growth and development. In mycorrhizal symbioses, fungi supply soil mineral nutrients, such as nitrogen and phosphorus, to their host plants in exchange for carbon resources. Plants gain as much as 80% of mineral nutrient requirements from mycorrhizal fungi, which form associations with the roots of over 90% of all plant species. Orchid seeds lack endosperms and contain very limited storage reserves. Therefore, the symbiosis with mycorrhizal fungi that form endomycorrhizas is essential for orchid seed germination and protocorm development under natural conditions. The rapid advancement of next-generation sequencing contributes to identifying the orchid and fungal genes involved in the orchid mycorrhizal symbiosis and unraveling the molecular mechanisms regulating the symbiosis. We aim to update and summarize the current understanding of the mechanisms on orchid-fungus symbiosis, and the main focus will be on the nutrient exchange between orchids and their fungal partners.

Orchid Mycorrhizal Interactions on the Pacific Side of the Andes from Chile. A Review

In order to confront the constant decline in global biological diversity, amelioration strategies are needed for threatened species to design reintroduction policies, particularly in plants with critical reproduction steps, such as orchids. Orchids are part of a highly diverse plant family, with several species under imminent extinction risk. This is the case of Chilean Orchidaceae, which has shown a constant decay in their populations due to an increase in the alteration processes of their natural distribution habitats. Successful orchid reintroductions require a full understanding of orchid mycorrhizal fungi and their dynamic according to different developmental stages and environmental conditions because orchid seeds need mycorrhizal fungi to obtain nutritional compounds at early developmental stages. This article performed a critical literature review of the ecological studies conducted on Chilean orchids and their relationships with mycorrhizal fungi in order to focus on the best scientific approach to achieve successful restoration programs involving orchid seeds and compatible mycorrhizal fungi.

Seed storage and micropropagation of pencil orchid (Papilionanthe hookeriana) in Bogor Botanic Gardens

Abstract. Handini E. 2019. Seed storage and micropropagation of pencil orchid (Papilionanthe hookeriana) in Bogor Botanic Gardens. Pros Sem Nas Masy Biodiv Indon 5: 7-12. Papilionanthe hookeriana as “Queen of Orchid” is found in Bengkulu and Bangka Belitung as potential ornamental plants. This orchid has terete leaves and lives naturally in aquatic or semi-aquatic habitats. Seed storage at-20?C and orchid seed viability testing is intended to extend the shelf life of seeds and maintain the availability of orchid seeds. Propagation of these orchids is carried out in vitro using seeds. Seeds from broken fruit after being stored in a desiccator for five to seven days, some are placed in the freezer at-20 ?C. Orchid seeds are sterilized using 10% and 5% Clorox, then rinsed with sterile water three times. This viability test was carried out in four media, namely KC, VWS, HS, and KCA. Regeneration of Pencil Orchid PLBs using T1 and VWS media in subculture I, while subculture II used VT5, KC and ½ P. Seed viability tests at a shelf life of 26 months showed the percentage of sprouts was significantly different between HS 18, 03% and KCA 9,49%. While the percentage of sprouts in VWS 14,4% was not significantly different from KC 11,02%. Decrease in the percentage of sprouts occurs in the seed viability test the shelf life of 31 months. The results of PLBs regeneration showed that the plantlets were able to grow with VW, KC and MS base media until they were ready for acclimatization. Until now, acclimatized orchids are still alive at the LIPI Botanical Garden (a.k.a Bogor Botanic Gardens).

Storage and in vitro germination of orchids (Orchidaceae) seeds from Atlantic Forest - Brazil.

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.

THE INTRAPOPULATION HETEROSPERMIA OF SOME ORCHID SPECIES

The aim of the work was a comparative morphological study of the peculiarities of the structure of seeds of certain Orchidaceae species from various subfamilies growing on the territory of the mainland Ukraine and Crimea. Methods. In order to carry out the morphological analysis , the seeds were examined on a scanning electron microscope JSM-6060 LA (JEOL, Japan). The size of the structures on the obtained microphotographs was determined using the UTHSCSA Image Tool 3.0 program. The morphometric parameters of the seeds were determined in accordance with the average size of viable seeds for a particular species of orchids. The seed index , the embryo index , and the relative size of the embryo were analyzed. The results of the comparative study of features of the structure of seeds of orchids ( Orchidaceae family) of domestic flora: ( Cephalanthera longifolia (L.) Fritsch., Epipactis palustris (L.) Crantz, Dactylorhiza incarnata (L.) Soo. s.l., Anacamptis coriophora (L.) R.M. Bateman) are presented. The significant heterogeneity of seeds of the studied species is revealed. The morphological analysis established that at the time of dissemination the major part of the studied seeds of orchids were nonviable. Species of orchids with the greatest part of viable seeds with large embryos ( D. incarnata and C. longifolia ) are considered as perspective reintroducents. Conclusion . The results of the research can contribute to the scientific and practical developments aimed at creation of reserve genetic collections in botanical gardens and in reserved areas and also at restitution of a number of natural populations of rare and endangered species.

Functional Insights into the Roles of Hormones in the Dendrobium officinale-Tulasnella sp. Germinated Seed Symbiotic Association.

Dendrobium is one of the largest genera in the Orchidaceae, and D. officinale is used in traditional medicine, particularly in China. D. officinale seeds are minute and contain limited energy reserves, and colonization by a compatible fungus is essential for germination under natural conditions. When the orchid mycorrhizal fungi (OMF) initiates symbiotic interactions with germination-driven orchid seeds, phytohormones from the orchid or the fungus play key roles, but the details of the possible biochemical pathways are still poorly understood. In the present study, we established a symbiotic system between D. officinale and Tulasnella sp. for seed germination. RNA-Seq was used to construct libraries of symbiotic-germinated seeds (DoTc), asymbiotic-germinated seeds (Do), and free-living OMF (Tc) to investigate the expression profiles of biosynthesis and metabolism pathway genes for three classes of endogenous hormones: JA (jasmonic acid), ABA (abscisic acid) and SLs (strigolactones), in D. officinale seeds and OMF under symbiotic and asymbiotic conditions. Low concentrations of endogenous JA, ABA, or SLs were detected in the D. officinale-Tulasnella symbiont compared with the asymbiotic tissues. Gene annotation results suggest that the expression of DEGs (differentially expressed genes) related to JA and ABA biosynthesis from D. officinale were down-regulated, while most of the key DEGs related to SL biosynthesis from D. officinale were up-regulated in the symbiotic germinated seeds compared with the asymbiotic germinated seeds. Moreover, in the OMF, we found a significantly up-regulated differential expression of the JA and ABA biosynthesis-related genes in the symbiotic interaction, with the opposite expression trends to those found in Dendrobium. This indicates that Dendrobium seed symbiotic germination may be stimulated by the apparent involvement of the OMF in the production of hormones, and relatively low concentrations of endogenous JA, ABA, or SLs might be maintained to promote the growth of the D. officinale-Tulasnella symbiotic protocorm-like body. These results will increase our understanding of the possible roles played by endogenous hormones in the regulation of the orchid-fungus symbiosis.

Caracterización morfométrica de semillas de cuatro especies de orquídeas terrestres nativas de Argentina

Las semillas de orquídeas aunque producidas en abundancia son muy pequeñas y extremadamente livianas. El objetivo del presente trabajo fue caracterizar morfométricamente las semillas de cuatro especies de orquídeas terrestres, Cyclopogon elatus (Sw.) Schltr; Bipinnula pennicillata (Rchb. f.) Sisternas & Salazar; Chloraea membranacea Lindl. y Chloraea philippii Rchb. f.. Las mediciones de la semilla y el embrión se realizaron sobre las microfotografías digitales. Para el cálculo del volumen de las semillas y del embrión se utilizaron fórmulas geométricas. Las semillas presentaron, en general, forma ahusada a levemente ahusada, tanto alargadas como truncadas, con colores del pardo oscuro a amarillo y sus volúmenes variaron entre 0,01 mm3 (C. philippii) y 0,001 mm3 (C. elatus). La estructura de la testa es reticulada para todas las especies, excepto para C. elatus siendo ésta estriada. C. philippii es la que presentó el mayor porcentaje de aire (85,72%), mientras que C. membranacea el menor (42,61%).

Complex fine-scale spatial genetic structure in Epidendrum rhopalostele: an epiphytic orchid.

Orchid seeds are presumably dispersed by wind due to their very small size and thus can potentially travel long distances. However, the few related studies indicate that seeds fall close to their mother plants. Because seed dispersal and colonization patterns can have relevant consequences for long-term species persistence, we assessed the fine-scale genetic structure of the epiphytic orchid Epidendrum rhopalostele to provide insight into these patterns. All individuals in the studied population were georeferenced and genotyped with AFLP-markers. Genetic structure was evaluated at two levels (forest and tree) using three approaches: principal coordinates analysis, model-based clustering, and spatial autocorrelation analysis. Results showed two genetic groups, composed of individuals from almost every tree with orchids. Spatial autocorrelation analysis at the forest level found no significant genetic structure when all individuals were considered, but a pattern of genetic patches was revealed when the analysis was performed separately for each group. Genetic patches had an estimated diameter of 4 m and were composed of individuals from more than one tree. A weak genetic structure was detected at the tree level at distances less than 1.5 m. These results suggest that many seeds fall close to the mother plant and become established in the same host tree. Additionally, a sequential colonization process seems to be the predominant mode of expansion, whereby progeny from orchids in one tree colonize neighboring trees. Thus, the existence of two distinct genetic groups and the presence of genetic patches should be considered when seed sampling for ex situ conservation.

Increased Expression of 9-Cis-Epoxycarotenoid Dioxygenase, PtNCED1, Associated With Inhibited Seed Germination in a Terrestrial Orchid, Phaius tankervilliae.

The phytohormone abscisic acid (ABA) is involved in regulating seed dormancy and germination. A crucial step of ABA biosynthesis in higher plants is the oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED). Seed development in orchids is unusual because the embryos are minute in size, without obvious histodifferentiation, and lack endosperm. To understand the regulation of ABA biosynthesis in orchid seeds, we isolated and characterized a full-length cDNA encoding an NCED homolog, PtNCED1, from developing seeds of an ornamental orchid, Phaius tankervilliae. Germination percentage was high at 90 days after pollination (DAP), when a globular embryo proper with a degenerating suspensor was evident. After 90 DAP, seed maturation was accompanied by a decrease in water content and a concomitant increase in ABA content and PtNCED1 mRNA level along with a marked decrease in germination percentage. Mature seeds pretreated with NaOCl solution lowered ABA content and improved seed germination. Moreover, after seed germination, developing protocorms could respond to dehydration stress. Dehydration of protocorms stimulated an increase in PtNCED1 level along with ABA content. Our results provide evidence of the involvement of PtNCED1 in regulating endogenous ABA content in developing seeds and protocorms. The accumulation of endogenous ABA content in orchid seeds may have a critical role in seed dormancy and the protocorm response to water stress after seed germination.

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.

Cryopreservation of orchid seeds through rapid and step freezing methods

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

Orchid seed removal by ants in Neotropical ant-gardens.

Most plants that inhabit ant-gardens (AGs) are cultivated by the ants. Some orchids occur in AGs; however, it is not known whether their seeds are dispersed by AG ants because most orchid seeds are tiny and dispersed by wind. We performed insitu seed removal experiments, in which we simultaneously provided Azteca gnava ants with seeds of three AG orchid species and three other AG epiphyte species (Bromeliaceae, Cactaceae and Gesneriaceae), as well as the non-AG orchid Catasetum integerrimum. The seeds most removed were those of the bromeliad Aechmea tillandsioides and the gesneriad Codonanthe uleana, while seeds of AG orchids Coryanthes picturata, Epidendrum flexuosum and Epidendrum pachyrachis were less removed. The non-AG orchid was not removed. Removal values were positively correlated with the frequency of the AG epiphytes in the AGs, and seeds of AG orchids were larger than those of non-AG orchids, which should favour myrmecochory. Our data show that Azt. gnava ants discriminate and preferentially remove seeds of the AG epiphytes. We report for the first time the removal of AG orchid seeds by AG ants in Neotropical AGs.

Cryopreservation of orchid seeds through rapid and step freezing methods.

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

Development of C-lignin with G/S-lignin and lipids in orchid seed coats – an unexpected diversity exposed by ATR-FT-IR spectroscopy

AbstractMembers of the orchid family occupy many germination niches, in terrestrial, epiphytic and epilithic environments. How orchid seeds attach to their substrate and survive after dispersal is largely unknown. C-lignin is a recently discovered specialized lignin, found in seed coats of some plants, including orchid species, but its functional and biological significance is obscure. We studied seed coat ontogenesis in three species (Neuwiedia veratrifolia,Cypripedium formosanumandPhalaenopsis aphrodite) that represent basal and advanced branches in orchid phylogeny and divergent life forms. From each species, controlled pollination yielded several stages of seed development, from which seed coats (testa) were isolated and analysed by ATR-FT-IR spectroscopy. The use of the ATR set-up ensured that the chemical information originated only from the integral outer seed surface layers. The FT-IR bands of C-lignin are presented here for the first time, and distinguished from bands of G/S-lignin. In the seed coats, C-lignin developed after G/S-lignin inN. veratrifoliaandC. formosanum, while only G/S-lignin developed inP. aphrodite. We discuss C-lignin properties and possible function in relation to seed coat properties. The species differed with respect to sequence and amounts of deposition, not only of lignins but also lipids, resulting in differences in mature seed coat compositions. Thus we revealed an unexpected and marked diversity among orchids with respect to seed surface chemistry, with possible implications for seed and germination ecology.

Metagenomic Analyses of the Viruses Detected in Mycorrhizal Fungi and Their Host Orchid.

In nature, mycorrhizal association with soilborne fungi is indispensable for orchid families. Fungal structures from compatible endo-mycorrhizal fungi in orchid cells are digested in cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until shoots develop (leaves) and become photoautotrophic. Seeds of all orchid species surely geminate with the help of their own fungal partners, and this specific partnership has been acquired for a long evolutional history between orchids and fungi.We have studied the interactions between orchids and mycorrhizal fungi and recently conducted transcriptome analyses (RNAseq) by a next-generation sequencing (NGS) approach. It is possible that orchid RNA isolated form naturally grown plants is contaminated with RNAs derived from mycorrhizal fungi in the orchid cells. To avoid such contamination, we here prepared aseptically germinated orchid plants (i.e., fungus-free plants) together with a pure-cultured fungal isolate and field-growing orchid samples. In the cDNA library prepared from orchid and fungal tissues, we found that partitivirus-like sequences were common in an orchid and its mycorrhizal fungus. These partitivirus-like sequences were closely related by a phylogenetic analysis, suggesting that transmission of an ancestor virus between the two organisms occurred through the specific relation of the orchid and its associated fungus.

Optimal disinfection times for seeds of Mediterranean orchids propagated on nutrient media

A time-consuming yet mandatory step during in vitro sexual propagation of orchids is the treatment of seeds with a disinfecting solution that also serves to scarify the seeds. If the seeds are not properly disinfected, microorganisms grow within the culture vessel, thus reducing the efficacy of the process and burdening the operation with unnecessary materials and labour. On the other hand, a long period of disinfection may damage the seed. The literature is inconclusive with respect to the proper combination of solution strength and duration of the treatment, especially with respect to Mediterranean orchids. The objective of this research is to determine optimal disinfection/scarification times for two species with thin and thick seed coats, respectively. Seeds of Anacamptis laxiflora (Lam.) and Himantoglossum robertianum (Loisel.) were treated in 1% NaClO solution for 5, 15, 25, 35 and 45 minutes and sown in modified organic Malmgren medium. Logistic regression models were fitted to the results. Due to the small number of observations per treatment, regression models of infection rates on treatment duration had no more predictive ability than the mean infection rate. On the other hand, regression models of germination rates on treatment duration proved statistically significant or nearly so. Treatment of only a few minutes in 1% NaClO seems to be optimal for seeds with relatively permeable seed coats such as those of A. laxiflora (Lam.). Conversely, treatment of 45 minutes in 1% NaClO may be inadequate for seeds with relatively impermeable seed coats such as those of H. robertianum (Loisel.).

Cyrtopodium paludicolum germination with two Tulasnella isolates

ABSTRACT Symbiosis between orchid seeds and mycorrhizal fungi has been reported to be a determining factor in the success of germination and protocorm development in vitro. The aim of this study was to isolate and identify by molecular analysis the mycorrhizal fungus associated with Cyrtopodium paludicolum, and to evaluate its efficiency in facilitating seed germination and development. Germination experiments were carried out using a fungus isolated from C. paludicolum (CH01) and Epidendrum secundum (M65), which has been successfully used a number of times in symbiotic germination. The experiments were conducted in a completely randomized design with treatments of CH01, M65 as well as under asymbiotic conditions. The mycobiont CH01 was successfully isolated from Cyrtopodium paludicolum and identified as Tulasnella sp. Treatments with both fungi reached a higher germination percentage than under asymbiotic conditions, indicating no specificity in the relationship between Cyrtopodium paludicolum and the fungi. The results presented have the potential to advance research into the propagation and conservation of C. paludicolum, a native of the Cerrado biome.