Synthetic Tetraploid of Oncidium crispum Lodd. (Orchidaceae).

In ornamental plants, artificial polyploidization has enabled the creation of new cultivars. Due to their high commercial value in the international flower market and their ornamental characteristics, such as the shape, size, color, and durability of their flower, orchids have received great attention in studies of artificial polyploidization. Here we described the protocol used for polyploid induction in Oncidium crispum, an epiphyte species native of southeastern Brazil, of great ornamental interest and widely sold in flower shops. The species stands out for having inflorescence with large flowers, brown in color with yellow spots. In addition, O. crispum has great potential for use in genetic improvement programs since the species is widely used in interspecific crosses. Closed capsules containing mature O. crispum seeds were subjected to running sterilized water for 10 min and then to a 1.5% sodium hypochlorite solution for 10 min. Small portions of seeds were introduced into 50 mL of water-soluble fertilizer with macro- and micronutrients (B>M) plus 0.7% agar. Explants originating from seeds previously in vitro germinated were submitted to 0.05% and 0.1% of colchicine for 4 days and 8 days. Flow cytometry and chromosome counts confirmed that the protocol successfully produced synthetic polyploid plants.

Evaluation of in vitro cytotoxicity and in vivo potential toxicity of the extract from in vitro cultivated Anoectochilus roxburghii Lindl.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding.

The process through induction, proliferation and regeneration of protocorm-like bodies (PLBs) is one of the most advantageous methods for mass propagation of orchids which applied to the world floricultural market. In addition, this method has been used as a tool to identify genes of interest associated with the production of PLBs, and also in breeding techniques that use biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular studies developed have used model plants as species of Phalaenopsis, and interestingly, despite similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for conservation and collection purposes, the flower market is supported by hybrid cultivars, usually polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous. However, studies of other factors and new technologies affecting PLB production such as the use of temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools for advancing the technique with increasing PLB production efficiency. In addition, recent studies on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the molecular characterization of plantlets obtained from this technique currently allow the technique to be evaluated in a more comprehensive way regarding its real applications and main limitations aiming at mass propagation, such as somaclonal variation.

Morphological Developments of Orchis purpurea Huds. Salep Orchids

Abstract This article aims to monitor the development of Orchis purpurea Huds., salep orchids, of different sizes over a period of two years, and to investigate the relationship between the parameters studied. In the first step, the measurements taken at the time of planting and harvesting of tubers divided into eight different groups according to their size were subjected to variance and Duncan's test. In the second step, the relationship between the parameters was investigated by ignoring seedling groups. The relationship between the two variables was determined by correlation analysis. The significance of the relationships between planting and harvest data sets, and variable contributions were determined by canonical correlation analysis. Finally, leaf area prediction modeling was performed by applying multiple regression analysis. In variance analysis all parameters were significant. The canonical correlation between the first pair of canonical variables was 0.988 (p<0.01). The data obtained from the tubers made the greatest contribution to the explanatory power of the canonical variables. The leaf area model was formulized as LA (mm2) = -1237.0204 + 57.7912 × LW + 16.6211 × LL where LA is leaf area, LW is leaf width, LL is leaf length and a, b, and c are coefficients.

Development of Growth Models Based on Years for Salep Orchid Planted in Natural Environment

Abstract The orchids that produced tuber are known as salep orchids and have been collected from nature for centuries as a medicinal and aromatic plant. These orchids are endangered species because of their limited vegetative and generative production possibilities. Salep orchids, seed germination has been achieved with in-vitro studies, but no source has been found regarding the development of germinated seeds over the years. In this study, Dactylorhiza euxina (Nevski) Czerep. was used as research material. Seeds that were determined to germinate in nature were observed to develop in their own environment for five years and some morphological characteristics (plant height, number of leaves, leaf length, leaf width, total weight, tuber width, and tuber weight) were determined at the end of each vegetation year. Plants that start to develop with seed germination produce one leaf and a tuber of 0.115 g in the first year. When the tubers obtained from the first year were planted again, the plants was obtained 2-3 leaves and they were formed 0.465 grams of tubers. In the third year plants bloom for the first time. The period until flowering in the plant was called maturity period and Dactylorhiza euxina (Nevski) Czerep. was found to have a 3-year maturity period. Plants produced tuber 0.850 g in the third year, 1.585 g in the fourth year and 2.522 g in the fifth year. According to the mathematical modeling, the relationship between year (Y) and fresh tuber weight (FTW) was found Y = 1.22 + (1.61 × FTW) and there was a significant relationship among year, fresh tuber weight, and total biomass (TBM). This equation Y = 1.05 + (0.76 × FTW) + (0.14 × TBM) shows us the mathematical relations of year, fresh tuber weight, and total biomass. The mathematical relations among to fresh tuber weight, total biomass, and plant height (PH) was produced Y = 1.74 - (0.11 × FTW) + (0.57 × TBM) - (0.09 × PH). R2 values were found 0.95-0.99. All R2 values and standard errors were found to be significant at the p < 0.01 level.

[Effect of cultivation substrate on growth and active component contents of Anoectochilus roburghii from three different origins].

Three different origins of Anoectochilus roburghii were used as experimental materials to study the effect of three different substrate( peat soil-river sand-peanut shell) radio on survival rate,plant height,stem diameter,plant fresh weight,root number,the longest root length,root diameter,and the contents of polysaccharide,flavonoids,and polyphenol. The results showed that when the substrate ratio was 4 ∶2 ∶2,the survival rate of A. roburghii from different origins was the highest,and the plant height,stem diameter,plant fresh weight,the longest root length and root diameter were also the largest. The cultivation substrate had no significant effect on the polysaccharide content of A. roxburghii and A. formosanus. When the substrate ratio was 4 ∶ 2 ∶ 2,the polysaccharide content of A.chapaensis was significantly lower than that of the other two combinations. When the substrate ratio was 4 ∶2 ∶1,the flavonoid content of A. formosanus was higher than that of the other two combinations. When the substrate ratio was 4 ∶2 ∶2,A. formosanus and A. chapaensis had higher polyphenol content.

Antioxidants and improved regrowth procedure facilitated cryoconservation of Paphiopedilum insigne Wall. Ex. Lindl. - An Endangered Slipper Orchid.

Orchids and their sustainability are very important issues that need global conservation efforts. Paphiopedilum insigne (an endangered orchid), is one of the most excessively exploited species of orchids and is mentioned in the IUCN Red List and Appendix I of CITES. The prospect for conservation and commercialization of this species would be strengthened with the development of improved cryopreservation techniques. This study reports on successful cryopreservation of protocorms of P.insigne after cryopreservation using vitrification (Vit) and encapsulation-vitrification (E-Vit) techniques. The study compared the addition of four antioxidants to the pretreatment and recovery stages, three growth media, and agitated vs. semisolid culture medium for initial recovery. Recovery after cryopreservation for the control was 27% for Vit and 37% for E-Vit. In both cases agitated culture produced improved recovery by about 10%, but with significantly better recovery with E-Vit. The best recovery (51.2 ±â€¯0.9%) was recorded for 0.5 M sucrose precultured encapsulated protocorms treated for 45 min with PVS2 and recovered in ½ MS (L/S) liquid medium for 10 days under agitation, followed by transfer to semi-solid medium. This recovery was further enhanced (62.7 ±â€¯0.5%) with the incorporation of 30 µM glutathione in both liquid preculture and the liquid and semisolid regrowth medium. This new protocol improved the E-Vit cryopreservation recovery from the initial 37%-63%, providing a suitable technique for storage of this threatened orchid.

Induction and Characterization of Tetraploids from Seeds of Bletilla striata (Thunb.) Reichb.f.

Bletilla striata (Thunb.), an ornamental and medicinal plant, is on the list of endangered plants in China. Its pseudobulb is abundant in polysaccharide and has been used for centuries as a herbal remedy. However, a recent rise in demand has placed it at risk of extinction, and therefore, research on its propagation and genetic improvement is essential. Since polyploids tend to possess advantageous qualities, we incubated B. striata seeds with colchicine with the aim of creating tetraploid plantlets. Aseptic seeds treated with 0.1% colchicine for 7 days showed the highest tetraploid induction rate of 40.67 ± 0.89%. Compared with the wild-type, the tetraploids could be identified by their morphological characteristics including larger stomata at a lower density, larger leaf blades, and a thicker petiole. Contents of polysaccharide and phenolic compounds were also determined in the tetraploid pseudobulbs, revealing significantly higher values than in the wild-type. In vitro colchicine treatment can therefore be used to successfully produce B. striata tetraploids with superior pseudobulbs.

Agrobacterium-mediated transformation of the wild orchid Cattieya maxima Lindi / Transformación mediada por Agrobacterium de la orquídea silvestre Cattleya maxima Lindl / Transformação da orquídea silvestre Cattleya maxima Lindl mediada por Agrobacterium

Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.

Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.

Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.

Expression profiles of five FT-like genes and functional analysis of PhFT-1 in a Phalaenopsis hybrid

Background: Phalaenopsis is an important ornamental flowering plant that belongs to the Orchidaceae family and is cultivated worldwide. Phalaenopsis has a long juvenile phase; therefore, it is important to understand the genetic elements regulating the transition from vegetative phase to reproductive phase. In this study, FLOWERING LOCUS T (FT) homologs in Phalaenopsis were cloned, and their effects on flowering were analyzed. Results: A total of five FT-like genes were identified in Phalaenopsis. Phylogenetic and expression analyses of these five FT-like genes indicated that some of these genes might participate in the regulation of flowering. A novel FT-like gene, PhFT-1, distantly related to previously reported FT genes in Arabidopsis and other dicot crops, was also found to be a positive regulator of flowering as heterologous expression of PhFT-1 in Arabidopsis causes an early flowering phenotype. Conclusions: Five FT homologous genes from Phalaenopsis orchid were identified, and PhFT-1 positively regulates flowering.

Effects of naphthalene acetic acid (NAA) on the plant growth and sugars effects on the cut flowers Mokara chark kuan orchid / Efeitos do ácido naftaleno acético (naa) sobre o crescimento das plantas e de açúcares sobre flores cortadas da orquídea Mokara chark kuan

A study was carried out to investigate the effects of Naphthalene Acetic Acid (NAA) and sugars on the plants and flowers quality of Mokara Chark Kuan orchid respectively. Parameters were monitored between November 2014 to March 2015, with the application of three concentrations of NAA at 25, 50 and 100 mg/L in the field. The application of 25 mg/L NAA significantly increases the plant height, number of roots and total soluble solid content of Mokara Chark Kuan orchid plants. But, there was no significant effect on the leaves number, chlorophyll content and number of flowers. In case of storage experiment, the cut Mokara Chark Kuan flowers were treated with 3 % and 6 % glucose and sucrose. Physiological parameters: fresh weight, water uptake, petal thickness, discoloration and vase life were evaluated. It can be concluded that spraying with 25 mg/L NAA enhanced the plant growth and development of Mokara Chark Kuan orchid. The study also showed that 6 % sucrose was the best treatment for maintaining the post-harvest quality as well as vase life of cut Mokara Chark Kuan orchid flowers.

Um estudo foi realizado para investigar os efeitos do ácido naftaleno ácido acético (NAA) no crescimento de e açúcares no plantas e na qualidade de flores da orquídea Mokara Chark Kuan . Os parâmetros foram monitorados entre novembro de 2014 e março 2015, com a aplicação de três concentrações de ANA em 25, 50 e 100 mg / L sob condições de campo. A aplicação de 25 mg / L de ANA aumentou significativamente a altura da planta, o número de raízes e o teor de sólidos solúveis totais de plantas de orquídeas Mokara Chark Kuan. Todavia, não houve nenhum efeito significativo sobre o número de folhas, o teor de clorofila e o número de flores. No caso do experimento de armazenamento, flores cortadas de Mokara Chark Kuan foram tratadas com 3% e 6% de glicose e de sacarose. Os parâmetros fisiológicos: peso fresco, absorção de água, espessura pétala, descoloração e vida de vaso foram avaliados. Pode concluir-se que a pulverização com 25 mg / L de NAA aumentou o crescimento e o desenvolvimento das plantas de orquídea Mokara Chark Kuan. O estudo também mostrou que 6% de sacarose foi o melhor tratamento para a manutenção da qualidade pós-colheita, bem como vida de vaso em flores de corte da orquídea Mokara Chark Kuan .

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris.

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein-protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division.

Effects of exogenous proline and a natural ventilation system on the in vitro growth of orchids / Efeitos da prolina exógena e sistema de ventilação natural no crescimento in vitro de orquídeas

Micropropagation is an alternative to produce orchid plants in large scale. However, this process presents losses during acclimatization. Exogenous proline use in vitro plant tissue culture can reduce the stress of the plant acclimatization phase. We aimed to verify the growth of orchids in different micropropagation systems with the addition of proline in the culture medium. Cattleya walkeriana plants were obtained from the germination of seeds in culture medium. Seeds were germinated in MS medium, added 20 g. L-1 of sucrose, solidified with 6 g. L-1 of agar and pH adjusted for 5,8. The cultures were incubated in a growth room with temperature of 24 ± 2 0C, under photoperiod of 16 h. After 5 months, 1-cm long seedlings were placed in a culture vessel according to the treatments, which were composed of two micropropagation systems (conventional and natural ventilation) and three proline concentrations (0, 1, and 2 g·L-1). The experiment was carried out in an entirely randomized design consisting of a 2 × 3 factorial, for a total of 6 treatments, each with 5 replicates. The natural ventilation system with the use of proline (1 g·L-1) promoted higher dry mass accumulation and better control of water loss by plants.

A micropropagação é uma alternativa para a produção de plantas de orquídeas em larga escala. Entretanto, este processo apresenta perdas durante a fase de aclimatização. O emprego de prolina exógena na cultura de tecidos vegetais é uma alternativa para reduzir o estresse das plantas na fase de aclimatização. O objetivo da presente pesquisa foi verificar o crescimento de orquídeas em diferentes sistemas de micropropagação com prolina adicionada no meio de cultura. Plantas de Cattleya walkeriana foram obtidas a partir da germinação de sementes em meio de cultura. Sementes foram germinadas em meio MS, adicionado de 20 g. L-1 de sacarose, solidificado com 6 g. L-1 de ágar e pH ajustado para 5,8. Após 5 meses, plântulas com 1 cm de comprimento foram inoculadas nos frascos de cultivo de acordo com os tratamentos, os quais foram compostos por dois sistemas de micropropagação (convencional e ventilação natural) em combinação com prolina (0, 1 e 2 g L-1). O experimento foi conduzido em esquema inteiramente casualizado, constando de um fatorial 2x3, totalizando 6 tratamentos com 5 repetições. O sistema de ventilação natural com o uso de prolina (1 g L-1) promoveu o maior acúmulo de massa seca e melhor controle da perda de água das plantas.

A Low Glutathione Redox State Couples with a Decreased Ascorbate Redox Ratio to Accelerate Flowering in Oncidium Orchid.

Glutathione (GSH) plays multiple roles in plants, including stress defense and regulation of growth/development. Previous studies have demonstrated that the ascorbate (AsA) redox state is involved in flowering initiation in Oncidium orchid. In this study, we discovered that a significantly decreased GSH content and GSH redox ratio are correlated with a decline in the AsA redox state during flowering initiation and high ambient temperature-induced flowering. At the same time, the expression level and enzymatic activity of GSH redox-regulated genes, glutathione reductase (GR1), and the GSH biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and glutathione synthase (GSH2), are down-regulated. Elevating dehydroascorbate (DHA) content in Oncidium by artificial addition of DHA resulted in a decreased AsA and GSH redox ratio, and enhanced dehydroascorbate reductase (DHAR) activity. This demonstrated that the lower GSH redox state could be influenced by the lower AsA redox ratio. Moreover, exogenous application of buthionine sulfoximine (BSO), to inhibit GSH biosynthesis, and glutathione disulfide (GSSG), to decrease the GSH redox ratio, also caused early flowering. However, spraying plants with GSH increased the GSH redox ratio and delayed flowering. Furthermore, transgenic Arabidopsis overexpressing Oncidium GSH1, GSH2 and GR1 displayed a high GSH redox ratio as well as delayed flowering under high ambient temperature treatment, while pad2, cad2 and gr1 mutants exhibited early flowering and a low GSH redox ratio. In conclusion, our results provide evidence that the decreased GSH redox state is linked to the decline in the AsA redox ratio and mediated by down-regulated expression of GSH metabolism-related genes to affect flowering time in Oncidium orchid.

Glacial Refugia and Future Habitat Coverage of Selected Dactylorhiza Representatives (Orchidaceae).

The intensively discussed taxonomic complexity of the Dactylorhiza genus is probably correlated with its migration history during glaciations and interglacial periods. Previous studies on past processes affecting the current distribution of Dactylorhiza species as well as the history of the polyploid complex formation were based only on molecular data. In the present study the ecological niche modeling (ENM) technique was applied in order to describe the distribution of potential refugia for the selected Dactylorhiza representatives during the Last Glacial Maximum. Additionally, future changes in their potential habitat coverage were measured with regard to three various climatic change scenarios. The maximum entropy method was used to create models of suitable niche distribution. A database of Dactylorhiza localities was prepared on the grounds of information collected from literature and data gathered during field works. Our research indicated that the habitats of majority of the studied taxa will decrease by 2080, except for D. incarnata var. incarnata, for which suitable habitats will increase almost two-fold in the global scale. Moreover, the potential habitats of some taxa are located outside their currently known geographical ranges, e.g. the Aleutian Islands, the western slopes of the Rocky Mountains, Newfoundland, southern Greenland and Iceland. ENM analysis did not confirm that the Balkans, central Europe or central Russia served as the most important refugia for individual representatives of the Dactylorhiza incarnata/maculata complex. Our study rather indicated that the Black Sea coast, southern Apennines and Corsica were the main areas characterized by habitats suitable for most of the taxa.

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Córdoba (Argentina) / Hongos micorrízicos aislados de orquídeas terrestres nativas de regiones pristinas en Córdoba (Argentina)

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Córdoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25°C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITS1 and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the association of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

La Familia Orchidaceae se encuentra estrechamente relacionada con hongos micorrízicos que pertenecen al complejo Rhizoctonia, e incluyen los géneros Ceratorhiza, Moniliopsis, Epulorhiza y Rhizoctonia. Esta asociación es esencial para el desarrollo de nuevas plantas ya que favorecen el proceso de germinación de las semillas. Por lo tanto, el conocimiento de la naturaleza de esta interacción es importante para que los resultados de los programas de conservación de orquídeas sean efectivos. La fragmentación del bosque Chaqueño Serrano en el centro de Argentina, ha alcanzado un punto crítico en los últimos años, afectando el funcionamiento del ecosistema. El objetivo de este trabajo fue: a) aislar y caracterizar hongos micorrízicos presentes en orquídeas terrestres de la provincia de Córdoba (Argentina) y b) conocer el hábitat natural y las asociaciones fúngicas que se establecen en esta región prístina. A partir de las raíces de orquídeas terrestres, se obtuvieron 23 aislamientos de hongos micorrízicos que pertenecen al complejo Rhizoctonia. Estos aislamientos fueron caracterizados con base en caracteres morfológicos y moleculares. Las características morfológicas (color y textura de las colonias, cinética de crecimiento, diámetro y largo de la hifa y presencia de esclerocios) fueron observados en PDA y MEA a 25ºC. El número de núcleos por célula se observó en cultivos crecidos en AA (agar-agua) y teñidos con una solución de Safranine-O. La región ITS se amplificó usando los primers ITS1 e ITS4. Con base en las características morfológicas de la colonia, los aislamientos fueron agrupados en binucleados (MSGib) y multinucleados (MSGim). De acuerdo al cladograma obtenido con las secuencias de ADN, los aislamientos Q23 y Q29 están relacionados a especies de Ceratobasidium, aisladas de raíces de orquídeas. Los aislamientos Q24 y Q4 se asocian con el grupo de anastomosis de Rhizoctonia AG-C. Finalmente, se observó una alta variabilidad en el grado de especificidad existente en la simbiosis que se establece entre las raíces de estas orquídeas terrestres y los aislamientos obtenidos a partir de ellas. Este es el primer reporte de la asociación entre el grupo de anastomosis AG-C y orquídeas terrestres. Dado que estos aislamientos se obtuvieron de orquídeas terrestres nativas, podrían ser incorporados como nuevos patrones para micorrizas de orquídeas terrestres en Argentina. Este trabajo contribuye al conocimiento de la relación simbiótica que se establece entre orquídeas y hongos micorrízicos, así como también al desarrollo de estrategias de conservación de orquídeas terrestres nativas del bosque Chaco Serrano.

A molecular framework for understanding the phylogeny of Spiranthes (Orchidaceae), a cosmopolitan genus with a North American center of diversity.

UNLABELLED: • PREMISE OF THE STUDY: Spiranthes is a genus of small terrestrial orchids that are most diverse in North America, yet unusually cosmopolitan, including many of conservation concern. Taxonomy based on morphology alone is problematic, but molecular evidence could help resolve evolutionary relationships within the genus. The phylogeny of Spiranthes was reconstructed to evaluate these patterns, particularly among North American and Old World lineages, determine the systematic value of chromosome numbers and phenology, consider aspects of historical biogeography, and provide evidence for the taxonomic status of vulnerable species.• METHODS: DNA sequences were produced from 219 samples representing 30 Spiranthes taxa plus one outgroup. Both parsimony and Bayesian inference analyses were applied to individual and combined data matrices generated for nuclear (nrITS) and plastid (trnL, trnS-G, matK) regions.• KEY RESULTS: Two major clades were recovered. One contains primarily summer-blooming species from western North America with haploid chromosome number of n = 22. The other clade (largely n = 15) contains midwestern and eastern North American species varying in phenology, although most autumn-flowering taxa within it are monophyletic. Whereas an Old World subclade is embedded within the tree, derived from New World ancestors, no genetic differentiation was found between trans-Atlantic specimens of S. romanzoffiana. Most species for which multiple individuals were sampled reflect monophyly in the combined gene tree (including S. lucida positioned on an unusually long branch), but some demonstrate dubious taxonomic status.• CONCLUSIONS: This is the most complete phylogenetic reconstruction of Spiranthes published to date and is likely to influence future taxonomic decisions, with important implications for conservation of several threatened orchids. Our discoveries about species distributed outside North America offer important evidence for repeated long-distance dispersal, often coupled with subsequent speciation-an uncommon phenomenon in Orchidaceae.

Evaluación de diferentes medios de cultivo in vitro en el desarrollo de híbridos de Phalaenopsis (Orchidaceae) / Evaluation of different in vitro culture media in the development of Phalaenopsis hybrid (Orchidaceae)

Los híbridos de Phalaenopsis tienen una gran importancia económica a nivel mundial, como flor cortada y planta ornamental, debido a sus flores vistosas y a la capacidad de adaptación a diferentes condiciones ambientales. Las técnicas de cultivo in vitro resultan indispensables para mejorar la eficacia germinativa, el crecimiento y desarrollo de orquídeas con fines comerciales e investigativos. En esta investigación se determinó el medio de cultivo más apropiado para la germinación in vitro de un híbrido de Phalaenopsis. Inicialmente se evaluó la viabilidad de las semillas utilizando la prueba de tetrazolio (TZ). Las semillas se desinfectaron y se cultivaron aplicando el método de la jeringuilla. El porcentaje de viabilidad en promedio fue de 92,2 % (P≤ 0,05: Tukey HSD), con un porcentaje de germinación entre todos los medios de 95,1 % (P≤ 0,05: Tukey HSD). El medio de cultivo más eficiente para la germinación de híbridos de Phalaenopsis a las 18 semanas de cultivo fue el Murashige & Skoog (MS) suplementado con agua de coco, y jugo de piña con diferencias estadísticamente significativas (P≤ 0,05: Tukey HSD), con respecto a los demás medios de cultivo, contribuyendo de esta manera al uso de componentes orgánicos con el fin de mejorar la germinación y desarrollo de Phalaenopsis.

The Phalaenopsis hybrids have a significant economic importance throughout the world, as ornamental flower or plant. It is because of its attractive flowers and its adaptation capacity into different environments. The different culture media in vitro are vital to improve the efficacy of germination, growing and development of the Orchids for commercial and research purposes. In this research, the most appropriated medium for in vitro propagation of Phalaenopsis hybrid was determined. At first, the seeds viability was evaluated by using tetrazolium test (TZ). The seeds were disinfected and cultivated by means of the syringe method. The viability percentage average was 92.2 % (P≤ 0.05: Tukey HSD), with a percentage of germination of 95.1 % (P≤ 0.05: Tukey HSD) in all the environments. The most efficient culture Medium for Phalaenopsis hybrid phenological development, at 16 weeks, was Murashige & Skoog (MS). Coconut water and pineapple juice were used as supplement showing statistically significant differences (P≤ 0,05: Tukey HSD), in comparison with the other culture media, contributing this way to the usage of organic components, which will be employed to improve the germination and development of the Phalaenopsis.

Ploidy-specific symbiotic interactions: divergence of mycorrhizal fungi between cytotypes of the Gymnadenia conopsea group (Orchidaceae).

Polyploidy is widely recognized as a major mechanism of sympatric speciation in plants, yet little is known about its effects on interactions with other organisms. Mycorrhizal fungi are among the most common plant symbionts and play an important role in plant nutrient supply. It remains to be understood whether mycorrhizal associations of ploidy-variable plants can be ploidy-specific. We examined mycorrhizal associations in three cytotypes (2x, 3x, 4x) of the Gymnadenia conopsea group (Orchidaceae), involving G. conopsea s.s. and G. densiflora, at different spatial scales and during different ontogenetic stages. We analysed: adults from mixed- and single-ploidy populations at a regional scale; closely spaced adults within a mixed-ploidy site; and mycorrhizal seedlings. All Gymnadenia cytotypes associated mainly with saprotrophic Tulasnellaceae (Basidiomycota). Nonetheless, both adults and seedlings of diploids and their autotetraploid derivatives significantly differed in the identity of their mycorrhizal symbionts. Interploidy segregation of mycorrhizal symbionts was most pronounced within a site with closely spaced adults. This study provides the first evidence that polyploidization of a plant species can be associated with a shift in mycorrhizal symbionts. This divergence may contribute to niche partitioning and facilitate establishment and co-existence of different cytotypes.

Flower-specific KNOX phenotype in the orchid Dactylorhiza fuchsii.

The KNOTTED1-like homeobox (KNOX) genes are best known for maintaining a pluripotent stem-cell population in the shoot apical meristem that underlies indeterminate vegetative growth, allowing plants to adapt their development to suit the prevailing environmental conditions. More recently, the function of the KNOX gene family has been expanded to include additional roles in lateral organ development such as complex leaf morphogenesis, which has come to dominate the KNOX literature. Despite several reports implicating KNOX genes in the development of carpels and floral elaborations such as petal spurs, few authors have investigated the role of KNOX genes in flower development. Evidence is presented here of a flower-specific KNOX function in the development of the elaborate flowers of the orchid Dactylorhiza fuchsii, which have a three-lobed labellum petal with a prominent spur. Using degenerate PCR, four Class I KNOX genes (DfKN1-4) have been isolated, one from each of the four major Class I KNOX subclades and by reverse transcription PCR (RT-PCR), it is demonstrated that DfKNOX transcripts are detectable in developing floral organs such as the spur-bearing labellum and inferior ovary. Although constitutive expression of the DfKN2 transcript in tobacco produces a wide range of floral abnormalities, including serrated petal margins, extra petal tissue, and fused organs, none of the vegetative phenotypes typical of constitutive KNOX expression were produced. These data are highly suggestive of a role for KNOX expression in floral development that may be especially important in taxa with elaborate flowers.