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.

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 .

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.

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.

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog’s (MS) medium containing 2.5 mg L-1 6-benzylaminopurine (BAP), 1.0 mg L-1 kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L-1 each and 50 mg L-1 ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and ~40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L-1 CH, 2.5 mg L-1 BAP and 1.0 mg L-1 Kin with 30 PLBs and 6 shoots per callus mass (~5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L-1 indole-3-butyric acid, 200 mg L-1 activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

Mass propagation of Cymbidium giganteum Wall. ex Lindl. using in vitro seedlings.

In vitro seedlings were used as explants for protocorm like bodies (PLBs) production which in turn were used for regeneration purpose. PLBs were induced from the base of seedlings (1.0-1.5 cm in size) in MS + BAP (8.88 µM). After 90 days of inoculation, PLBs production rate started declining and most of the PLBs turned into plantlets. Preculture of seedlings in 1.0 µM thidiazuron (TDZ) for 7 days and transfer to BAP supplemented medium resulted in production of 16 PLBs per seedling within 90 days of culture. Increase of TDZ concentration to 2.5 µM and preculture time 15 days, resulted in induction of highest number of PLBs (19 PLBs per seedling) in the basal medium. The results emphasized the importance of thidiazuron (TDZ) concentration and preculture time for PLBs proliferation from the base of seedlings. The PLBs thus produced were used for regeneration studies. Irrespective of single, segmented or clumps of PLBs, the regeneration response was 100% in 2,4-D (4.52 µM) and KN (4.64 µM) but when KN was replaced by BAP (8.88 µM), response was observed only in clumps of PLBs, whereas in single and segmented ones it was 99 and 97%, respectively. Regenerants developed stout root system in half strength M medium supplemented with 2.84 µM of IAA and transferred to greenhouse with 90% survival. The present study holds tremendous potential as the mother plant is not destroyed and PLBs are produced as a continuous system.

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.

Cryopreservation of Cyrtopodium hatschbachii Pabst (Orchidaceae) immature seeds by encapsulation-dehydration

The aim of the present study was to investigate the efficiency of the encapsulation-dehydration technique for cryopreservation of Cyrtopodium hastchbachii Pabst seeds. Immature seeds of this species were cryopreserved by an encapsulation-dehydration technique. Seeds of five immature pods, 120 days after pollination, were encapsulated in 3% calcium alginate matrix and pretreated in liquid medium supplemented with 0.08 M sucrose (24 h), 0.15 M sucrose (24 h), 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h) in shaker at 60 rpm. Alginate beads were dehydrated 5 h in silicagel and immersed in liquid nitrogen for 12 h. Cryopreserved beads were thawed at 30°C for 1 min, rehydrated using the same liquid mediums (0.75 M sucrose (24 h), 0.5 M sucrose (24 h), 0.25 M sucrose (48 h) and 0.15 M sucrose (24 h)) and cultivated in half strength Murashige & Skoog medium (1962) with the addition of 2 g/L activated charcoal. Sixty four percent of seeds survived and developed into acclimatized plants after being cryopreserved. In this work, the encapsulation-dehydration technique was employed for first time in Cyrtopodium hatschbachii.

Acclimatization of phalaenopsis and cattleya obtained by micropropagation / . Aclimatización de phalaenopsis y cattleya obtenidas por micropropagación

The quality of micropropagated plants relies on the acclimatization stage. This research intends to develop an efficient protocol to obtain the acclimatization of Phalaenopsis and Cattleya. Plants of Phalaenopsis obtained from protocorms were selected. They came from flowering stalks grown at modified Murashige and Skoog (MS) (1962) medium and classified by growth ranks and put into moss, mesquite wood shaving and perlite (1:1:1), into a humidity chamber. The protocorms were multiplied at MS from Cattleya sown in Knudson C (1946) medium; regenerated plants of 1-2 cm were selected, and implanted in humidity chamber on: moss, coal and perlite (1:1:1) MCP; mesquite wood shavings, coal and perlite (1:1:1) ACP; moss and perlite (1:1) MP; mesquite wood shaving and perlite (1:1) AP. The following results were obtained: Phalanopsis: a) Survival: 44% in R0 and 100% in RI and RII of the. b) Number of leaves: RI gave on average 1 more leaf than the range 0; c) Roots number and length: RI and RII gave on average 2 more roots than R0, and there were no significant differences in length. d) Height: RII presented greater growth than RI and Ro. Cattleya: a) The survival in MCP was 0%, MP 16 %, ACP 32% and AP 80%. b) The height in MP was significantly superior to the ones in ACP and AP. Plants from both genera need to achieve a 2 to 4 cm growth rank in vitro to endure the greenhouse conditions. MAP was the best substrate in Phalaenopsis and moss-perlite in Cattleya.

La calidad final de las plantas producidas por micropropagación depende de la etapa de aclimatización. Se intenta desarrollar un protocolo eficiente para la aclimatización de Phalaenopsis y Cattleya. Se seleccionaron plantas de Phalaenopsis, obtenidas de protocormos provenientes de estacas florales cultivadas en Murashige y Skoog modificado (MS) (1962), por rangos de crecimiento e implantadas en musgo, viruta de algarrobo y perlita (1:1:1), en cámara húmeda. De siembras de Cattleya en medio de Knudson C (1951) se multiplicaron protocormos en MS; se seleccionaron plantas regeneradas de 1-2 cm, e implantadas en cámara húmeda en los sustratos: musgo, carbón y perlita (1:1:1) MCP; viruta de algarrobo, carbón y perlita (1:1:1) ACP; musgo y perlita (1:1) MP; viruta de algarrobo y perlita (1:1) AP. Se obtuvieron los siguientes resultados: en Phalaenopsis: a) Supervivencia: para R0 de 44% y RI y RII del 100%; b) número de hojas: RI generó en promedio 1 hoja más que el rango 0; c) número y longitud de raíces: RI y RII generaron en promedio dos raíces más que R0, no detectándose diferencias significativas en longitud; d) altura: RII presentó mayor crecimiento que RI y R0 En Cattleya: a) La supervivencia en MCP fue 0%, MP 16%, ACP 32% y AP 80%; b) La altura en MP resultó significativamente superior que en ACP y AP. Ambos géneros necesitan alcanzar un crecimiento de 2 a 4 cm in vitro para tolerar las condiciones de invernáculo. El mejor sustrato fue MAP en Phalaenopsis, y la mezcla musgo-perlita en Cattleya.

Considerações sobre a Família Orquidacea: taxonomia, antropismo, valor econômico e tecnologia: [revisão] / Considerations on the Family Orchidaceae: taxonomy, anthropism, economic value and technology: [revision] / Consideraciones acerca de la Familia Orchidaceae: taxonomía, antropismo, valor económico y tecnología: [revisión]

O presente trabalho tem como finalidade divulgar o estado da arte das Orquidacea e discorrer sobre a sua sistemática evolutiva, antropismo e valor econômico. O trabalho teve como base uma cuidadosa revisão bibliográfica, bem como a análise da coleção do Orquidário do Estado de São Paulo e exsicatas do herbário “Maria Eneyda P. K. Fidalgo”, do Instituto de Botânica do Estado de São Paulo, provenientes de todo o Brasil.

The present work aims to present the state-of-the-art of Orchidaceae and discuss its evolutive systematic, anthropism and economical value. The work took as its bases a careful bibliographical survey as well as the analysis of the collection of the Orchidarium of Sao Paulo State and exsiccates of the herbarium “Maria Eneyda P. K. Nobleman”, of the Institute of Botany of Sao Paulo State, comingfrom all parts of Brazil.

Este trabajo pretende presentar el actual nivel de conocimiento de las Orchidaceae y hablar de su sistemática evolutiva, antropismo y valor económico. El trabajo tomó como sus bases una revisión bibliográfica cuidadosa así como el análisis de la colección del Orchidarium del estado de São Paulo y exsicatos del herbario “Maria Eneyda P. K. Nobleman”, del Instituto de Botánica del Estado de São Paulo, que viene de todas las partes de Brasil.

Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook.

In vitro mass production of C. suaveolens (Lindl.) Hook, an endangered orchid with its snowy white flowers having horticultural potential was accomplished through immature seed culture, and subsequent plant regeneration. The developmental stage of the immature seeds and nutrient media significantly influenced the germination frequency. Seeds at 13 months after pollination cultured on 3% sucrose containing Murashige and Skoog (MS) medium with 9 microM alpha-naphthaleneacetic acid (NAA), and 15% coconut water exhibited 93% germination after 40 days of culture. Upon subculture, the germinated shoots on MS medium with 9 microM BA, 6 microM NAA, 3% casein hydrolysate and 0.1% activated charcoal (AC) yielded >12 shoots per shoot or bud. Addition of AC favoured the enlargement of pseudobulbs and better rooting. The plantlets transferred to community potting mix after in vitro hardening (8-10 wk) displayed 85% survival.

Hardening of the national flower of Colombia, the threatened Cattleya trianae (Orchidaceae), from in vitro culture with previous invigoration phase

Cattleya trianae is an endemic species from the tropical rainforest in the Colombian Andes. Its survival is currently threatened due to habitat loss and commercial overexploitation. This study evaluates ten substrates, some organic (pine bark, coconut fiber and wood shavings), some inert icopor (polystyrene foam), vegetable coal and their combinations, and the effects these have on morphometric and phenotypic traits in the hardening phase of 250 plants of C. trianae cultivated in vitro. Recorded data include percent survival, length of longest leaf, biomass (wet weight) and number of roots and leaves at the beginning and at the end of the experiment. After the hardening phase, the plants were taken to a greenhouse and later to the natural environment. Coconut fiber alone or mixed in equal parts with pine bark and coal was the most efficient substrate when percent survival (80±SE=0.3742), biomass, and leaf length were evaluated. Hardened plants displayed qualitative characteristics such as vigor, hardness and waxy texture, strength of green coloration in the leaves, and velamen formation. Under greenhouse conditions, plants grew better with filtered light, relative humidity bordering on 80 %, permanent aeration, misting with water, and an average temperature of 25±2 °C. Invigorated plants were firmly anchored on their host trees. Rev. Biol. Trop. 55 (2): 681-691. Epub 2007 June, 29.

Cattleya trianae es una especie endémica de los bosques tropicales de los Andes colombianos. Actualmente se encuentra amenazada por la disminución de su hábitat natural y la sobreexplotación con fines comerciales. En este estudio se evaluó el efecto de diez tratamientos con sustratos biológicos (corteza de pino, fibra de coco y viruta) e inertes (esferitas de "icopor" y carbón vegetal) en diferentes combinaciones, sobre aspectos morfométricos y fenotípicos en la etapa de endurecimiento de 250 vitroplantas de C. trianae. Se registró porcentaje de supervivencia, longitud de la hoja, biomasa en peso fresco, número de raíces y hojas al inicio y al final del experimento. Al finalizar la fase de endurecimiento, las vitroplantas fueron llevadas a invernadero y posteriormente a ambiente natural. La fibra de coco sola ó mezclada en partes iguales con pino y carbón vegetal, fue el sustrato más eficiente cuando se evaluó el porcentaje de supervivencia (80 % ±SE=0.3742), biomasa en peso fresco y longitud de hoja. Las plantas endurecidas mostraron características cualitativas como vigorosidad, textura coriácea y cerosa, verdor intenso en sus hojas y velamen. En condiciones de invernadero las plantas se desarrollan mejor con luz filtrada, humedad relativa alrededor del 80 %, aireación continua, nebulización y temperatura promedio de 25±2 °C. Las plantas vigorizadas mostraron buen anclaje y adaptación en árboles.

Influência da caseína hidrolisada no cultivo in vitro deOncidium baueri (Orchidaceae) / Influence of hydrolized casein on the Oncidium baueri(Orchidaceae) in vitro cultivation

O nitrogênio possui grande importância para a maioria das espécies cultivadas in vitro. Está envolvidonos processos fisiológicos e bioquímicos associados ao controle de crescimento, diferenciação emorfogênese, sendo a caseína uma das principais fontes de suplementação do nitrogênio. O objetivodeste trabalho foi avaliar a influência de diferentes concentrações de caseína hidrolisada no crescimentovegetativo e enraizamento in vitro de Oncidium baueri (Orchidaceae). Os tratamentos testados foram:T1- 0,0 g.L-1; T2- 0,5 g.L-1 de caseína hidrolisada; T3- 1,0 g.L-1 de caseína hidrolisada; T4- 2,0 g.L-1 decaseína hidrolisada e T5- 4,0 g.L-1 de caseína hidrolisada. O meio de cultura utilizado foi o Murashige eSkoog (1962), modificado com metade da concentração de macronutrientes. Após seis meses do iníciodo experimento foram avaliados: massa fresca total; altura da parte aérea; comprimento da maior raiz; númerode raízes e número de brotações laterais. Não se observou efeito benéfico no desenvolvimento vegetativo eradicular de Oncidium baueri em meio de cultura suplementado com caseína hidrolisada. A porcentagem desobrevivência foi 100%, 95% e 94%, respectivamente para T1, T2 e T3, e 80% para T4 e T5.

The nitrogen is essentially important for most in vitro cultivated species. It is regarded to the physiologicaland biochemical processes involved in growth control, differentiation and morphogenesis, and casein isone of the main nitrogen supplementation sources. The objective of this work was to evaluate theinfluence of different concentrations of hydrolized casein on the invitro vegetative growth and rootingof Oncidium baueri (Orchidaceae). The treatments tested were: T1- 0.0 g.L-1; T2- 0.5 g.L-1; T3- 1.0 g. L-1;T4 ­2.0 g.L-1; T5- 4.0 g.L-1 of hydrolized casein. The culture medium used was the Murashige and Skoogmodified with half macronutrients concentration. Total fresh mass, aerial part height, biggest root length,roots number and lateral sprouts number were evaluated after six months from the beginning of theexperiment. It was not detected any beneficial effects of casein on vegetative and root development ofOncidium baueri. The percentage of surviving plants was 100%, 95 % and 94%, respectively for T1, T2and T3, and 80% of surviving plants for T4 and T5

In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture.

Rapid in vitro propagation of the terrestrial orchid, M. khasiana through immature seed culture was achieved. Immature seeds of 8-9 week after pollination (WAP) cultured on MS medium (2% sucrose) supplemented with 500 mgl(-1) casein-hydrolysate and 1 microM N6-benzyladenine (BA) exhibited germination of 75% seeds after 107 days of culture and subsequently supported the development of PLBs. Subsequent culture on MS medium enriched with 6 microM of indole-3-acetic acid (IAA), 18 microM each of BA and kinetin induced multiple shoots and plantlets. Transfer of PLBs to MS medium with 0.1% activated charcoal (AC) facilitated rapid proliferation of PLBs, while AC at 0.2% favored shoot bud induction and rhizome enlargement. The plantlets, developed on medium with IAA, BA and kinetin, after hardening in vitro for 8-10 weeks were planted in community pots and transferred to poly-house. The plantlets showed 65% survival under field conditions.

Evaluación de medios alternativos al Phytamax TM para la micropropagación de Schomburkia undalata (orquídea) / Evaluation of means alternative to Phytamax TM for the microspread of schomburkia undalata (orchid)

El PhytamaxTM es un medio de cultivo muy utilizado para la micropropagación de orquídeas, el mismo es producido por Corporación Sigma-Aldrich. El presentetrabajo utilizo cuatro medios de cultivo para la multiplicación in vitro de orquídeas, donde se plantean dos medios alternativos al PhytamaxTM los mismos que tienen una composición similar a este.

Propagación in vitro de Phalaenopsis (Orchidaceae) a partir de protocormos, mediante el sistema de inmersión temporal RITA / In vitro propagation of Phalaenopsis (Orchidaceae) from protocorms, using the temporary inmersion system RITA

Protocormos in vitro de Phalaenopsis de tres meses de edad se transfirieron a contenedores RITA® con el fin depropagarlos masivamente. Los factores evaluados fueron la concentración de sacarosa en el medio y la frecuencia de inmersión. Se dispusieron cinco pares de contenedores RITA® con medio de cultivo líquido a concentraciones de sacarosa de 0, 15, 30, 45 y 60 g/L. El medio utilizado fue el MS a la mitad de la concentración de las sales, suplementado con vitaminas y tidiazuron (5 mg/L). El experimento se realizó en dos etapas, cada una con duración de dos meses. La primera etapa con una frecuencia de inmersión de cuatro horas y la segunda con una frecuencia de inmersión de ocho horas, ambas con un tiempo de inmersión de un minuto. Los resultados mostraron que la mejor respuesta proliferativa, con 8,2 protocormos adventicios por protocormo por mes, se obtuvo en el medio con 15 g/L de sacarosa y un tiempo de inmersión de un minutocada cuatro horas.

In order to massively propagate Phalaenopsis orchids, three months old in vitro protocorms were transferred to RITA® vessels. The evaluation factors were the sucrose concentration in the culture medium and the frequency immersion. There was a set of five pairs of RITA® vessels with liquid culture medium containing sucrose at 0, 15, 30, 45, 60 g/L. A half-strength MS medium plus vitamins, supplemented with vitamins and thidiazuron (5 mg/L) was used. The experiment had two stages, each lasting two months. Each stage had a one minute immersion. The first stage had a four hour immersion frequency and the second one had an eight hour immersion frequency. Results showed that the best proliferating response was reached in a 15 g/L of sucrose medium with one minute of immersion time every four hours, resulting in 8,2 adventitious protocorms per protocorm per month.

Regeneration of plantlets from in vitro raised leaf explants of Cleisostoma racimeferum Lindl.

Protocorm like bodies (PLBs), callus and shoot buds developed in culture from in vitro raised foliar explants of Cleisostoma racimeferum. Among the different basal media, better result was obtained on MS medium containing sucrose (3%) and BA (2 microM) with approximately 80% frequency after 40 days of culture. Young leaves (15 week old) produced better PLBs. Whole leaf placed vertically upside-up orientation can regenerate PLBs and shoot buds (80%). PLBs and shoot buds formed on entire surface of the leaves. Cultures on BA and NAA (2 and 2 microM respectively in combination) stimulated callus mediated regeneration (68%). The rooted plantlets regenerated within 8-10 week from PLBs and shoot buds on MS medium containing IAA and kinetin (2 microM each in combination). BA containing medium triggered multiple shoot bud formation, while NAA alone or in combination with other growth regulators was inhibitory. Incorporation of activated charcoal (0.01%) in the medium stimulated formation of repetitive PLBs and multiple shoot buds. Rooted plants were ready for harvest after 20-22 week of initiation of culture. About 65% of the potted plants survived after 3 months in the poly house.