Orchid Protocorms Research Articles

RESPON PERTUMBUHAN PROTOKORM ANGGREK SENDU (Grammatophyllum stapeliiflorum) DENGAN PENAMBAHAN BEBERAPA JENIS PUPUK DAUN SECARA IN VITRO

Indonesia has 26,000 species of orchids that are very potential and profitable. Grammatophllum stapeliiflorum is an orchid species that is increasingly difficult to find in its natural habitat. The addition of foliar fertilizer containing macro and micro nutrients can increase plant growth. The purpose of this study was to determine the effect of foliar fertilizer type, concentration and the combination of the two factors on the growth of the orchid protocorm G. stapeliiflorum. This study used a factorial completely randomized design with two research factors, namely the type of foliar fertilizer with 2 treatment levels, B1 = Gandasil-D and B2 = Growmore. The second factor, fertilizer concentration with 4 treatment levels, namely A0 = control (without foliar fertilizer), A1 = 1 g/L, A2 = 2 g/L, and A3 = 3 g/L. This study consisted of 8 treatment combinations which were repeated 4 times, resulting in 32 experimental units. The parameters observed were the percentage of live explants, the percentage of browning, and the percentage of protocorms growth stages. Data were analyzed by Analysis of Variance (ANOVA) and continued with Duncan's Multiple Range Test (DMRT) at a 5% level. These results indicated that the application of foliar fertilizers with Gandasil-D and Growmore affects the percentage of live explants and protocorm growth phases and also increase the percentage browning until of 32,5%. Gandasil-D is better for protocorm growth from globular to torpedo. The control is better for forming shoot of 22,5% compared to the use of foliar fertilizer.

Plant and fungal gene expression coupled with stable isotope labeling provide novel information on sulfur uptake and metabolism in orchid mycorrhizal protocorms.

Orchid mycorrhiza (OM) represents an unusual symbiosis between plants and fungi because in all orchid species carbon is provided to the host plant by the mycorrhizal fungus at least during the early stages of orchid development, named a protocorm. In addition to carbon, orchid mycorrhizal fungi provide the host plant with essential nutrients such as phosphorus and nitrogen. In mycorrhizal protocorms, nutrients transfer occurs in plant cells colonized by the intracellular fungal coils, or pelotons. Whereas the transfer of these vital nutrients to the orchid protocorm in the OM symbiosis has been already investigated, there is currently no information on the transfer of sulfur (S). Here, we used ultra-high spatial resolution secondary ion mass spectrometry (SIMS) as well as targeted gene expression studies and laser microdissection to decipher S metabolism and transfer in the model system formed by the Mediterranean orchid Serapias vomeracea and the mycorrhizal fungus Tulasnella calospora. We revealed that the fungal partner is actively involved in S supply to the host plant, and expression of plant and fungal genes involved in S uptake and metabolism, both in the symbiotic and asymbiotic partners, suggest that S transfer most likely occurs as reduced organic forms. Thus, this study provides originalinformation about the regulation of S metabolism in OM protocorms, adding a piece of the puzzle on the nutritional framework in OM symbiosis.

Precision measurement of physical properties of orchid synthetic seeds produced under various encapsulation conditions using Image J platform

Synthetic seed technology is a popular method in plant biotechnology and agricultural science because of its wide application in plant multiplication, short-term conservation, and germplasm exchange. The orchid is a precious ornamental plant that was studied in this project for encapsulation due to its medicinal and commercial value. The main objectives of this study were determining the physical properties of orchid synthetic seeds by image J platform and comparing experimentally two magnetic and air-bubble stirring methods for the production of artificial seed. Physical properties of encapsulated Phalaenopsis ' ‘Beijing’ orchid protocorms produced under three Na-alginate concentrations (3 %, 4 %, and 5 %), three CaCl2-2H2O concentrations (100, 125, and 150 mM), and two stirring methods (magnetic and air bubbling) were investigated. Image J processing platform was applied to determine the physical properties (length, width, sphericity index (SI), and concentric index (CI)) of orchid artificial seeds extracted from digital images. Analysis of variance (ANOVA) demonstrated that interaction terms (Na-alginate * stirring method) and (Na-alginate * CaCl2-2H2O) significantly affected all the dependent variables including weight, volume, SI, and CI (P < 0.05). Response Surface Method (RSM) was utilized for the prediction and optimization of the response variables. The optimum conditions were obtained with the air bubble stirring method having the desirability of 0.879, a weight of 0.15 g, volume of 0.14 cm3, sphericity index of 0.87, and concentric index of 0.13. This is while, the optimum conditions of the encapsulation process using the magnetic method with the desirability of 0.786 were recorded with weight, volume, SI, and CI values of 0.137 g, 0.169 cm3, 0.72, and 0.13, respectively. A comparison of the results showed that the air bubble method demonstrates better encapsulation performance in terms of sphericity index and concentricity index, while capsules produced using the magnetic method had higher weight and volume. Results of this study show the potential of the image J processing as a precision measurement tool which has been successfully applied here to determine the physical properties of orchid synthetic seeds.

Integrative Study Supports the Role of Trehalose in Carbon Transfer From Fungi to Mycotrophic Orchid.

Orchids rely on mycorrhizal symbiosis, especially in the stage of mycoheterotrophic protocorms, which depend on carbon and energy supply from fungi. The transfer of carbon from fungi to orchids is well-documented, but the identity of compounds ensuring this transfer remains elusive. Some evidence has been obtained for the role of amino acids, but there is also vague and neglected evidence for the role of soluble carbohydrates, probably trehalose, which is an abundant fungal carbohydrate. We therefore focused on the possible role of trehalose in carbon and energy transfer. We investigated the common marsh orchid (Dactylorhiza majalis) and its symbiotic fungus Ceratobasidium sp. using a combination of cultivation approaches, high-performance liquid chromatography, application of a specific inhibitor of the enzyme trehalase, and histochemical localization of trehalase activity. We found that axenically grown orchid protocorms possess an efficient, trehalase-dependent, metabolic pathway for utilizing exogenous trehalose, which can be as good a source of carbon and energy as their major endogenous soluble carbohydrates. This is in contrast to non-orchid plants that cannot utilize trehalose to such an extent. In symbiotically grown protocorms and roots of adult orchids, trehalase activity was tightly colocalized with mycorrhizal structures indicating its pronounced role in the mycorrhizal interface. Inhibition of trehalase activity arrested the growth of both symbiotically grown protocorms and trehalose-supported axenic protocorms. Since trehalose constitutes only an inconsiderable part of the endogenous saccharide spectrum of orchids, degradation of fungal trehalose likely takes place in orchid mycorrhiza. Our results strongly support the neglected view of the fungal trehalose, or the glucose produced by its cleavage as compounds transported from fungi to orchids to ensure carbon and energy flow. Therefore, we suggest that not only amino acids, but also soluble carbohydrates are transported. We may propose that the soluble carbohydrates would be a better source of energy for plant metabolism than amino acids, which is partially supported by our finding of the essential role of trehalase.

A Transcriptomic Approach Provides Insights on the Mycorrhizal Symbiosis of the Mediterranean Orchid Limodorum abortivum in Nature.

The study of orchid mycorrhizal interactions is particularly complex because of the peculiar life cycle of these plants and their diverse trophic strategies. Here, transcriptomics has been applied to investigate gene expression in the mycorrhizal roots of Limodorum abortivum, a terrestrial mixotrophic orchid that associates with ectomycorrhizal fungi in the genus Russula. Our results provide new insights into the mechanisms underlying plant–fungus interactions in adult orchids in nature and in particular into the plant responses to the mycorrhizal symbiont(s) in the roots of mixotrophic orchids. Our results indicate that amino acids may represent the main nitrogen source in mycorrhizal roots of L. abortivum, as already suggested for orchid protocorms and other orchid species. The upregulation, in mycorrhizal L. abortivum roots, of some symbiotic molecular marker genes identified in mycorrhizal roots from other orchids as well as in arbuscular mycorrhiza, may mirror a common core of plant genes involved in endomycorrhizal symbioses. Further efforts will be required to understand whether the specificities of orchid mycorrhiza depend on fine-tuned regulation of these common components, or whether specific additional genes are involved.

Enhancement Propagation of Protocorms in Orchid (Cymbidium tracyanum L. Castle) by Cold Atmospheric Pressure Air Plasma Jet

Propagation of orchid (Cymbidium tracyanum L. Castle) treated by using an air cold atmospheric pressure plasma jet at room temperature was investigated. The peak-to-peak voltage discharge and flow rate of the air atmospheric pressure plasma jet were set at 10.5 kV and 10 L/min, respectively. The constituent particles of the air cold atmospheric pressure plasma jet were revealed by optical emission spectroscopy. At the end of the jet nozzle, the constituent particles were observed, such as hydroxyl radical ˙OH, N2 first negative system (N2 1 −), N2s positive system (N2 2 +), and atomic oxygen. The average size, average number of buds, fresh and dry weights simultaneously increased by cold air plasma treatment compared with the control ones. The highest positive effects of treated protocorms on average size, average number of buds, fresh and dry weight were significantly improved by 238.2, 475.9, 85.6, and 152.5%, respectively. The transmission electron microscope showed the cytoplasmic cell wall in orchid protocorm disturbed by air cold plasma. Thus, the effects of constituent particles of air plasma might attack in the cell wall of protocorm leading to cell loosening, which improved elongation and released bud dormancy. These results indicated that the propagation of orchid protocorms depended on the effects of air cold plasma treatment with different plasma time durations.

Expression regulation of MALATE SYNTHASE involved in glyoxylate cycle during protocorm development in Phalaenopsis aphrodite (Orchidaceae)

Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.

In Vitro Response of Protocorm Grammatophyllum stapeliiflorum (Teijsm. &amp; Binn.) J.J.Sm. Orchid in Growth on Several Media Composition

Family Orchidaceae has about 800 genera which are already difficult to find and almost extinct, including the orchid genus Grammatophyllum. A species of rare orchids, Grammatophyllum stapeliiflorum, is hard to find in original habitat. The addition of Growmore, BAP and 15% coconut water is expected to increase the growth and development of the G. stapeliiflorum orchid protocorms. This study use a Completely Randomized Design, consisting of 6 treatments (control, 1 and 3 mg/l BAP, 1 and 3 g/l Growmore, and 150 ml/l coconut water) in MS media with 5 replicates with observation for 4 weeks after planting. The results showed did not differ markedly in the number of protocorms, number of shoots and number of browning protocorms, but differ markedly in the parameter protocorm color based on DMRT test. Treatment of 3 mg/l BAP gives the best results on the number of shoots 2,60 protocorms and number of browning protocormss at the least amount 0,80 protocorms. The number of protocorms most widely on the treatment of 1 g/l Growmore 14,40 protocorms. Protocorms color is best found in 3 g/l Growmore with green color. This research managed to multiply the number of protocorms and induces protocorms shoots from Grammatophyllum stapeliiflorum orchid.

An overview of the life of an orchid protocorm – a developmental perspective

An overview of the life of an orchid protocorm – a developmental perspective

Pengaruh Konsentrasi Zat Pengatur Tumbuh Naphthalene Acetic Acid dan Benzil Amino Purin terhadap Pertumbuhan Protokorm Anggrek Dendrobium spectabile pada Kultur In Vitro

The aim of the article is to study the interaction effect between NAA and BAP concentration on protocorm growth and to know the proper concentration for growth of Dendrobium spectabile orchid protocorm. This research method using an experimental method which is conducted in the Laboratory of Agriculture Faculty of Winaya Mukti University, Tanjungsari Subdistrict, Sumedang District. The experiment was conducted from June 2017 until September 2017. The experiment used was a Completely Randomized Design (CRD) with the factorial pattern, consisting of two factors and repeated twice. the first factor was the effect of NAA concentration which consisted of five levels, namely without NAA, 0.5 mg kg-1, 1.0 mg kg-1, 1.5 mg kg-1, and 2.0 mg kg-1. The second factor is the BAP concentration which consists of five levels, namel without BAP, 1.0 mg kg-1, 2.0 mg kg-1, 3.0 mg kg-1, and 4.0 mg kg-1. Eksplant is protocorm from orchid D. spectabile which is grown on MS medium (Murashig and Skoog) half recipe as base medium accompanied by each treatment for 12 weeks. The experimental results show that there is an interaction between the effect of NAA and BAP concentration on the number of leaves only. Without NAA or 1.5 mg kg-1 NAA concentration with BAP 2.0 mg kg-1 gives more leaves. Independent of NAA or BAP concentrations did not affect the number of buds, number of roots, root length, fresh and dry weight of plantlets, and also growth ability of plantlets. BAP concentration only affect plant height, and the highest plantlet height is found without add of BAP in medium culture

IRADIASI AKUT DENGAN SINAR GAMMA PADA PROTOKORM ANGGREK Dendrobium macrophyllum A. Richard DAN Dendrobium undulatum M. A. Clem & D. L. Jones

Increasing genetic variation in plants can be done by irradiation technique. The purpose of this study was to improve genetic variation in Dendrobium macrophyllum and D. undulatum orchids by gamma irradiation to obtain superior seeds. Accute Irradiation treatment was perfomed using orchid protocorms. Irradiation doses given: 0, 10, 20, and 25 Gy. The observed parameters were the number of explants, shoots, dead shoots, and leaves at five months after treatment (MAT) and number of roots in seven MAT. Calculation of Lethal Dosage (LD) 20 and 50 was carried out on three MAT. The results of Curve Expert analysis showed that different levels of radiosensitivity in D. macrophyllum were more sensitive than D. undulatum . D. macrophyllum showed LD 20 at an irradiation dose of 13,98 Gy, LD 50 at an irradiation dose 48,42 Gy, while D. undulatum experienced LD 20 at an irradiation dose of 195,67 Gy, LD 50 at an irradiation dose of 490,67 Gy. Acute irradiation effects were seen in protocorm D. macrophyllum at seven MAT in the presence of inhibition at 10 Gy irradiation doses, which was a decrease in the number of shoot of 2,66, decrease in leaf number 18,84 and decrease in root number 5,17 compared to control

PERKECAMBAHAN DAN PERTUMBUHAN PROTOKORM ANGGREK PHALAENOPSIS PADA BEBERAPA KOMBINASI KOMPOSISI MEDIA DAN AIR KELAPA

The composition of artificial media used is one of the factors that determine the speed of germination and growth of orchid seedling in the bottle. The study aims to: 1) obtain a good combination of media composition and coconut water for the germination and growth of Phalaenopsis amabilis orchids protocorm; and 2) to know whether the leaf fertilizer can replace the use of MS base media in germinating the seeds and helping in the growth of Phalaenopsis orchids protocorm. The study was conducted from September to November 2017, at the Tissue Culture Laboratory of Politeknik Negeri Lampung. There are 9 treatment combinations tested: P1 = MS without addition of coconut water, P2 = MS plus coconut water 75 ml. l-1, P3 = MS plus coconut water 150 ml. l-1, P4 = Kristalon Hijau without coconut water, P5 = Kristalon Hijau plus 75 ml coconut water. l-1, P6 = Kristalon Hijau plus 150 ml coconut water. l-1, P7 = Growmore without coconut water, P8 = Growmore plus 75 ml coconut water. l-1, and P9 = Growmore plus 150 ml of coconut water. l-1. Observations were made on the time needed for the seeds to germinate and the growth speed of protocorm until they were ready to be subcultured. The results of this research showed: 1) The best media composition for the germination and growth of Phalaenopsis orchid protocorm is basic media MS with 75 ml coconut water addition. l-1 followed by MS media with the addition of 150 ml of coconut water. l-1; 2) Kristalon Hijau Fertilizers with the addition of coconut water 75 ml. l-1 as well 150 ml. l-1 can be used for germination and growing of Phalaenopsis orchid protocorm to become seedling, but with longer time needed than MS medium.

In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development.

Pyroloids, forest sub-shrubs of the Ericaceae family, are an important model for their mixotrophic nutrition, which mixes carbon from photosynthesis and from their mycorrhizal fungi. They have medical uses but are difficult to cultivate ex situ; in particular, their dust seeds contain undifferentiated, few-celled embryos, whose germination is normally fully supported by fungal partners. Their germination and early ontogenesis thus remain elusive. An optimized in vitro cultivation system of five representatives from the subfamily Pyroloideae was developed to study the strength of seed dormancy and the effect of different media and conditions (including light, gibberellins and soluble saccharides) on germination. The obtained plants were analysed for morphological, anatomical and histochemical development. Thanks to this novel cultivation method, which breaks dormancy and achieved up to 100 % germination, leafy shoots were obtained in vitro for representatives of all pyroloid genera (Moneses, Orthilia, Pyrola and Chimaphila). In all cases, the first post-germination stage is an undifferentiated structure, from which a root meristem later emerges, well before formation of an adventive shoot. This cultivation method can be used for further research or for ex situ conservation of pyroloid species. After strong seed dormancy is broken, the tiny globular embryo of pyroloids germinates into an intermediary zone, which is functionally convergent with the protocorm of other plants with dust seeds such as orchids. Like the orchid protocorm, this intermediary zone produces a single meristem: however, unlike orchids, which produce a shoot meristem, pyroloids first generate a root meristem.

Evidence for Brassinosteroid-Mediated PAT During Germination of Spathoglottis plicata (Orchidaceae).

Polar auxin transport (PAT) is facilitated by polar localization of PIN-FORMED (PIN) efflux carriers, which direct auxin flow and regulate developmental events. Brassinosteroids (BRs) and auxin work synergistically to promote growth, and in root geotropisms this cross-talk involves BR-directed polarization of PIN through the mobilization of F-actin. However, the role of BR in PAT during shoot growth, hair formation, and embryogenesis has not been well studied. Orchid seed are mature at a point in development that is analogous to the globular-stage of embryogenesis in typical angiosperms. Thus, this system provided a unique opportunity to study the effects of BR on PAT during embryogenesis-like events, including meristem/first leaf formation and protocorm/stem development, which is followed by protocorm hair formation. In this work, the degree to which BRs rescued embryo-like protocorms from the impact of PAT-disrupting agents, such as PAT inhibitors or high auxin levels, was determined based on growth responses. This study first established that auxin and BRs work together synergistically to promote seedling elongation in Spathoglottis. Repressed seedling growth caused by the PAT-disrupting agents was alleviated with eBL, suggesting that BRs enhance PAT in embryogenesis-like stages of young protocorms. However, similar responses were not evident in seed embryos. Results from this study also suggested that BRs may enhance orchid protocorm elongation by regulating auxin transport through an F-actin-mediated mechanism. With regard to protocorm hairs, increased eBL levels inhibited formation, whereas reduced BR biosynthesis altered hair patterning, and prevented outgrowth of auxin-stimulated hairs. Moreover, PAT inhibitors and repression of BR biosynthesis caused hair bud formation without hair outgrowth, suggesting a role for BR in PAT during protocorm hair development.

Micropropagation of Dendrobium phalaenopsis Orchid Through Overexpression of Embryo Gene AtRKD4

To increase the efficiency of crop production from Dendrobium phalaenopsis orchids, mass propagation has been performed by inducing somatic embryogenesis through Agrobacterium-mediated transformation of the Arabidopsis embryo gene AtRKD4 into orchid protocorm (developing orchid embryo). The three-week-old protocorms of D. phalaenopsis were genetically transformed with T-DNA carrying 35S :: GAL4 :: AtRKD4 :: GR through A. tumefaciens strain EHA 105. The cultures were maintained in VW medium with 10 mg L-1 Hygromycin. Due to the existence of glucocorticoid response element (GR) in the T-DNA construct, the transformed protocorms were transferred into VW medium with the addition of 15 μM Dexamethasone in 6 weeks after transformation to activate the transgene. A total of 12% protocorms has been confirmed for Hyg + by using PCR. The expression of embryo gene AtRKD4 was confirmed by cDNA analysis using AtRKD4 specific primers and Actin primers as a positive control experiment. The expression level of AtRKD4 in 2.5-month-old D. phalaenopsis transformant shoots was 7 times higher than non-transformant plants, and increased to 86 times higher in 8-months, that much higher than that of non-transformant. These results provide an improved method for genetic transformation of D. Phalaenopsis and will (eventually) increase production efficiency in the future.

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.

Utilization of exogenous saccharides by protocorms of two terrestrial orchids

Orchid protocorms are completely mycoheterotrophic structures. Although saccharides are proposed as the main energy and carbon (C) sources provided by fungi, there is only limited knowledge on their effects. For the first time, utilization of a wide range of saccharides by in vitro axenic protocorms of two terrestrial orchids from two subfamilies, Ophrys iricolor subsp. lojaconoi and Oeceoclades, was tested. Protocorm size and, in the first of these also rhizoid length and soluble saccharide contents, were analysed. The endogenous saccharide spectra reflected the supplied saccharides and their metabolism. In both species, sucrose supported protocorm growth best. Surprisingly, fructose inhibited O. iricolor subsp. lojaconoi protocorm growth while O. decaryana ones grew well on it. Interestingly, mannitol abundant in mycorrhizal fungi was not utilized while sorbitol not found in fungi was usable. Galactose was toxic at pre-germination stage. Protocorm rhizoid length correlated with protocorm size but revealed several signalling effects of some saccharides. In conclusion, the orchid’s ability to utilize various saccharides reflects more likely species life strategy rather than phylogenetic relations or saccharide abundance in mycorrhizal fungi.

Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien”

Phalaenopsis “Sogo Vivien “is a mini orchid hybrid with beautiful flowers and numerous inflorescences.Mass propagation of this orchid is needed to meet the market demand. Objective of this research was toinduce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNAcontaining 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated byAgrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid inductionsystem, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developedinto protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocormsdeveloped from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specificprimers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantletswere positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryoswere also emerged from leaf explants of transformant on hormone-free NP medium and became normalplantlets. It is probably due to the high activity of AtRKD4 genes in orchids

Aplicación de visión por computadora en la evaluación del desarrollo in vitro de protocormos de Oncidium sphacelatum Lindl., (Orchidaceae)

&lt;p&gt;Se presenta una aplicación de visión por computadora diseñada para monitorear el desarrollo de protocormos de orquídea en condiciones &lt;em&gt;in vitro&lt;/em&gt;, con el objetivo de evaluar el proceso de germinación de manera eficiente, rápida y objetiva. A partir de fotografías digitales, se evaluó el desarrollo de protocormos de &lt;em&gt;Oncidium sphacelatum&lt;/em&gt; Lindl. (Orchidaceae) en términos de tamaño (medido a través de su área en pixeles), el cambio de etapa fenológica dentro del proceso de germinación (medido a través del factor de redondez) y el estado de salud (por tonalidad de color). La programación desarrollada para que el ordenador sea capaz de este tipo de análisis, se compone de una etapa de segmentación para discernir los pixeles de interés (protocormos) del fondo, una de entrenamiento-aprendizaje para establecer criterios de segregación y una última de clasificación. La programación se realizó mediante la plataforma Matlab R2012a. Las características obtenidas por el sistema permiten proporcionar de manera automática, digerida y objetiva, información útil para la evaluación del proceso de germinación de las orquídeas en condiciones in vitro, con un significativo ahorro en termino de horas/trabajo y desgaste físico del operador humano, sin perturbar la calidad de la evaluación.&lt;/p&gt;

Proteomic and morphometric study of the in vitro interaction between Oncidium sphacelatum Lindl. (Orchidaceae) and Thanatephorus sp. RG26 (Ceratobasidiaceae).

Orchidaceae establish symbiotic relationships with fungi in the Rhizoctonia group, resulting in interactions beneficial to both organisms or in cell destruction in one of them (pathogenicity). Previous studies have focused mostly on terrestrial species with a few, preliminary studies, on epiphytes. To further our understanding of the molecular mechanisms involved in these symbioses, we evaluated the interaction between Oncidium sphacelatum Lindl. and the mycorrhizal fungus Thanatephorus sp. strain RG26 (isolated from a different orchid species) in vitro using morphometric and proteomic analyses. Evidence from the morphometric and microscopic analysis showed that the fungus promoted linear growth and differentiation of orchid protocorms during 98days interaction. On day 63, protocorm development was evident, so we analyzed the physiological response of both organisms at that moment. Proteome results suggest that orchid development stimulated by the fungus apparently involves cell cycle proteins, purine recycling, ribosome biogenesis, energy metabolism, and secretion that were up-regulated in the orchid; whereas in the fungus, a high expression of proteins implicated in stress response, protein-protein interaction, and saccharides and protein biosynthesis were found in the symbiotic interaction. This is the first work reporting proteins differentially expressed in the epiphytic orchid-fungus interaction and will contribute to the search for molecular markers that will facilitate the study of this symbiosis in both wild orchids and those in danger of extinction.