Desarrollo de una nueva metodología de transformación genética no tradicional, como estrategia potencial para incluir resistencia a infecciones fúngicas en vainilla (Vainilla Planifolia).

Proyecto de Investigación (Código: 5402-2160-2701) Instituto Tecnológico de Costa Rica. Vicerrectoría de Investigación y Extensión (VIE). Escuela de Ciencias y Letras, 2012Este estudio consistió en la evaluación de las condiciones experimentales para laransformación genética de Vanilla planifolia por medio de un sistema de biobalística de baja presión. Los meristemos radicales de aproximadamente 0.5 cm, provenientes de microestacas desarrolladas en un medio líquido MS básico en agitación, se colocaron en un medio osmótico 16 hr antes y 24 hr después del bombardeo, posteriormente se subcultivaron en medio MS suplementado con 1 y 3 mg/L de benciladenina. Se incluyeron controles de bombardeo sin plásmido y controles de explantes, con y sin estrés osmótico. 48 hr después del bombardeo se realizó la prueba de expresión transitoria con el X-Gluc y se observó una tinción azulada en la epidermis de las terminales radicales bombardeadas con el plásmido así como también en los controles. A las 2 semanas también se presentó tinción en las regiones externa e interna de las estructuras iniciales que conducen a la formación de los PLBs en todos los tratamientos evaluados y los controles. La presencia de la tinción azul correspondiente a la degradación del sustrato X-Gluc en los controles, evidencia la expresión de enzimas endógenas en los tejidos de la planta que podrían interferir con la interpretación de los resultados de expresión transitoria. Este sería el primer reporte de actividad -glucoronidasa endógena en materiales in vitro de vainilla. De acuerdo a la evidencia obtenida, el estrés inducido por el choque osmótico en las terminales radicales de vainilla, puede provocar contaminación por un aumento en el crecimiento de potenciales microorganismos endógenos y por ende, la pérdida de material vegetal. Aunque es factible utilizar meristemos radicales de vainilla para la transformación genética de esta especie, se propone que una estructura más apropiada para este fin podría ser el estadio inicial observado cuando se forma el PLB a partir de los meristemos radicales. _______________________________________________________________________ Abstract: This study consisted in the evaluation of the experimental conditions for genetic transformation of Vanilla planifolia by a biolistic system under low pressure. Root tips of 0.5 cm of length, from seedlings developed in basic MS liquid medium under agitation, were placed in an osmotic medium, 16 hr before and 24 hr after bombarding, subsequently they were placed in MS culture medium supplemented with 1 and 3 mg/L benzyladenine. The assay included bombarding controls without plasmid and controls of explants with and without osmotic stress. After 48 hr of bombarding, root tips were exposed to X-Gluc and a blue staining was observed in the epidermis of the root tips, as well as in the controls. After 2 weeks, the blue staining was also present in the external and internal areas of the initial structures that become PLBs, in all the treatments evaluated and controls. The presence of the staining corresponding to X-Gluc substrate degradation in the controls evidences the expression of endogenous enzymes in plant tissue, which could interfere with interpretation of transient expression results. This would be the first report of endogenous -glucuronidase activity in in vitro vanilla materials. According to the obtained evidence, it is also important to take into consideration that stress induced by osmotic shock in the terminal tips of vanilla plants, could cause contamination by an increase in the growth of potential endogenous microorganisms and, consequently the loss of plant material. Although it is possible to utilize vanilla root meristems for genetic transformation of this species, it is postulated that a more appropriated structure for transformation could be the initial stage on the process of PLB formation from root meristems

Caracterización de Quassia amara para su manejo sostenible en ecosistemas naturales y agroforestales en Costa Rica

Proyecto de Investigación (Código: 5402 – 2151 – 6201) Instituto Tecnológico de Costa Rica. Vicerrectoría de Investigación y Extensión (VIE). Escuela de Ingeniería en Agronomía, 2008El Hombre grande (Quassia amara) es una planta medicinal y tiene actividad biocida, la cual la hace ser de gran importancia en la producción de cultivos orgánicos. Sin embargo, existe una serie de limitaciones, como son la falta de una semilla apropiada para el establecimiento de las plantaciones. El objetivo principal de este proyecto fue caracterizar los genotipos de Quassia amara y su clonación. El estudio realizó una serie de evaluaciones para determinar la metodología para la micropropagación de esta planta, así como estudios preliminares de extracción de ADN para la caracterización molecular del hombre grande. Los resultados de estas investigaciones permitieron desarrollar una metodología para la extracción del ADN, pero no la caracterización molecular de los genotipos. Además, determinaron, que el explante recomendado para la micropropagación es el uso de nudos, la desinfección de estos explanes no se encuentra definida, el medio de cultivo para el establecimiento de nudos que dio el mejor resultado fue el medio de Gamborg o B5, suplementado con 1.5 mg/l de ANA

DETECCIÓN DEL VIRUS DEL MOSAICO DEL Cymbidium (CymMV) Y DEL VIRUS DE LA MANCHA ANILLADA DEL Odontoglossum (ORSV) EN ORQUÍDEAS CULTIVADAS EN COSTA RICA

El virus del mosaico del Cymbidium (CymMV) y el virus de la mancha anillada de Odontoglossum (ORSV) son los agentes virales más importantes en orquídeas debido a su alta incidencia y a los daños económicos que causan. Ambos virus fueron detectados simultáneamente en orquídeas provenientes de dos viveros costarricenses por medio de la técnica TD/RT-PCR (Touchdown/Retrotranscription-Polimerase Chain Reaction). Los productos de PCR fueron analizados por electroforesis en geles de agarosa y por medio de un sistema de electroforesis automatizada por microchip (MultiNa-Shimadzu). El 52% de las 21 plantas analizadas resultaron positivas para la presencia de al menos uno de los dos virus. El CymMV presentó la mayor infección con 33%, en comparación con el 14% de plantas infectadas con el ORSV. Solamente una planta evidenció infección mixta por ambos virus. Se encontraron además dos plantas asintomáticas infectadas con el CymMV. Se detectó también la infección por CymMV en la orquídea Lycaste aromatica, una especie nativa de Costa Rica. La utilización de la técnica TD/RT-PCR en combinación con la electroforesis automatizada por microchip permitió detectar simultáneamente el CymMV y el ORSV con una mayor sensibilidad, rapidez y con menor costo en comparación con otras técnicas como el ELISA

PROPAGACIÓN MASIVA Y FORMACIÓN DE CALLOS PROTOCÓRMICOS DE VAINILLA A PARTIR DE ÁPICES RADICALES

El objetivo principal de esta investigación fue el desarrollo de una metodología de propagación masiva para Vanilla planifolia, así como la inducción de estructuras vegetales que por sus características permitan el desarrollo e implementación de técnicas biotecnológicas modernas para el mejoramiento genético no tradicional de esta especie tropical de gran valor económico y cultural. Como resultado directo de esta investigación se desarrolló una metodología de micropropagación  eficiente y rápida de vainilla a partir de ápices radicales. Por otra parte, se lograron establecer las condiciones y protocolos para la formación de una estructura indiferenciada y totipotente a la cual hemos dado el nombre de callo protocómico, debido a que tiende a formar en última instancia brotes y estructuras similares a los PLBs (protocorm-like badies) característicos en diferentes especies de orquídeas. Los callos protocórmicos se formaron a su vez a partir de una estructura indiferenciada y transitoria generada a partir de los ápices radicales cultivados en ausencia de luz, en un medio líquido MS suplementado con 30 g/L de sacarosa, 1 mg/L de BAP y 1 g/L de caseína hidrolizada. Esta estructura a la cual se le dio el nombre de precallo, presentó una gran diversidad morfológica en los diferentes tratamientos experimentales utilizados, razón por la cual se estableció una clasificación para su identificación. El mayor porcentaje de callos protocórmicos se formó en el medio de cultivo base MS sólido suplementado con 0.5 mg/L de 2,4-D en oscuridad, con un 72%.Abstract: The main research objective was the development of an efficient and rapid mass propagation methodology for Vanilla planifolia Jacks. ex Andrews (Orchidaceae), as well as to generate plant structures based on their characteristics of totipotency, undifferentiation and regeneration capacities, which would allow the development and implementation of modern biotechnological techniques for non-traditional genetic improvement of this tropical species, which has an enormous cultural and economic value. As a result of this research, the conditions and protocols for the formation of an undifferentiated and totipotent structure were established, which was named protocormic callus, because it tends to form shoots and similar structures in appearence to those PLBs (protocorm-like bodies) characteristic of different species of orchids, under regeneration conditions. The protocormic callus were formed from a transitory and undifferentiated structure generated by root tips cultivated without the presence of light, in a liquid MS medium supplemented with 30 g/L of sucrose, 1 mg/L BAP and 1 g/L of hydrolyzed casein. This structure was named pre-callus and presented a great morphological diversity when different experimental treatments were used, for this reason a morphotypic classification was established. Most of the protocormic callus formed in a solid MS basal medium with 0.5 mg/L of 2,4-D in the dark, with 72% (p<0.05). Two vanilla mass micropropagation methodologies, through indirect organogenesis (by pre-callus and protocormic callus induction), were established for the rapid and big scale reproduction of this species from a unique explant, which produced a high number of plants and decreased the time for their obtention. High propagation from pre-callus allows continuous autopropagation sustained in an exponential way through several consecutive cycles of multiplying, due to the ability that these structures present to form a big amount of shoots, which also develop roots, which form pre-callus again, in the same  culture cycle, in liquid medium.

Registro de galhas em plantas da Área de Conservação Guanacaste, Costa Rica, como conceito integrado de um banco de dados biológico

Galling insects are specialist herbivorous that have the ability of manipulating plant tissue to form complex biological structures called galls. Even though different organisms have the ability to induce galls in plants, insect galls have the highest degree of structural complexity. The main goal of this study was to obtain a preliminary systematic record of plant gall morphotypes from the Guanacaste Conservation Area in Costa Rica and integrate the information into a biological database. Plant gall morphotypes were recorded, characterized and deposited into a specialized herbarium established as a reference for the inventory. Moreover, organisms associated with gall morphotypes were included in the inventory when it was possible to obtain and identify them. Galls were collected in the rainy season over a period of three years. In total, we recorded forty-four families, seventy genera, and eighty-seven host plant species. One hundred thirty-one morphotypes of plant galls were identified in the Guanacaste Conservation Area. The family with the highest number of gall morphotypes was Fabaceae (8.4%). Leaves were the organ with the largest number of galls (71%), followed by stems (17.6%), and apical buds (6.9%). The predominant gall shape was globular (25.2%), followed by discoid (18.3%). Fifty-nine percent of the galls had a glabrous texture, which was most common on leaves, with 77%. One hundred twenty of our field records (91.6%) of plant galls were new morphotypes not only for Costa Rica but also the world. As a consequence of this research and considering the prospect of future increases in new gall records (and associated organisms), we proposed having the biological entities resulting from the inventory placed in a cecidiarium. This repository represents a standardized and comprehensive way to manage the data and biological materials associated with the plant galls. We also suggest a nomenclature for standardizing gall morphotype registries and identifications. This work is the first and most detailed inventory of plant galls carried out thus far in the Guanacaste Conservation Area.Instituto Tecnológico de Costa Rica/[5402- 2160- 3101]/TEC/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

Microbiome and plant cell transformation trigger insect gall induction in cassava

Several specialised insects can manipulate normal plant development to induce a highly organised structure known as a gall, which represents one of the most complex interactions between insects and plants. Thus far, the mechanism for insect-induced plant galls has remained elusive. To study the induction mechanism of insect galls, we selected the gall induced by Iatrophobia brasiliensis (Diptera: Cecidomyiidae) in cassava (Euphorbiaceae: Manihot esculenta Crantz) as our model. PCR-based molecular markers and deep metagenomic sequencing data were employed to analyse the gall microbiome and to test the hypothesis that gall cells are genetically transformed by insect vectored bacteria. A shotgun sequencing discrimination approach was implemented to selectively discriminate between foreign DNA and the reference host plant genome. Several known candidate insertion sequences were identified, the most significant being DNA sequences found in bacterial genes related to the transcription regulatory factor CadR, cadmium-transporting ATPase encoded by the cadA gene, nitrate transport permease protein (nrtB gene), and arsenical pump ATPase (arsA gene). In addition, a DNA fragment associated with ubiquitin-like gene E2 was identified as a potential accessory genetic element involved in gall induction mechanism. Furthermore, our results suggest that the increased quality and rapid development of gall tissue are mostly driven by microbiome enrichment and the acquisition of critical endophytes. An initial gall-like structure was experimentally obtained in M. esculenta cultured tissues through inoculation assays using a Rhodococcus bacterial strain that originated from the inducing insect, which we related to the gall induction process. We provide evidence that the modification of the endophytic microbiome and the genetic transformation of plant cells in M. esculenta are two essential requirements for insect-induced gall formation. Based on these findings and having observed the same potential DNA marker in galls from other plant species (ubiquitin-like gene E2), we speculate that bacterially mediated genetic transformation of plant cells may represent a more widespread gall induction mechanism found in nature

Desarrollo de una nueva metodología de transformación genética no tradicional, como estrategia potencial para incluir resistencia a infecciones fúngicas en vainilla (Vainilla Planifolia).

Proyecto de Investigación (Código: 5402-2160-2701) Instituto Tecnológico de Costa Rica. Vicerrectoría de Investigación y Extensión (VIE). Escuela de Ciencias y Letras, 2012Este estudio consistió en la evaluación de las condiciones experimentales para laransformación genética de Vanilla planifolia por medio de un sistema de biobalística de baja presión. Los meristemos radicales de aproximadamente 0.5 cm, provenientes de microestacas desarrolladas en un medio líquido MS básico en agitación, se colocaron en un medio osmótico 16 hr antes y 24 hr después del bombardeo, posteriormente se subcultivaron en medio MS suplementado con 1 y 3 mg/L de benciladenina. Se incluyeron controles de bombardeo sin plásmido y controles de explantes, con y sin estrés osmótico. 48 hr después del bombardeo se realizó la prueba de expresión transitoria con el X-Gluc y se observó una tinción azulada en la epidermis de las terminales radicales bombardeadas con el plásmido así como también en los controles. A las 2 semanas también se presentó tinción en las regiones externa e interna de las estructuras iniciales que conducen a la formación de los PLBs en todos los tratamientos evaluados y los controles. La presencia de la tinción azul correspondiente a la degradación del sustrato X-Gluc en los controles, evidencia la expresión de enzimas endógenas en los tejidos de la planta que podrían interferir con la interpretación de los resultados de expresión transitoria. Este sería el primer reporte de actividad -glucoronidasa endógena en materiales in vitro de vainilla. De acuerdo a la evidencia obtenida, el estrés inducido por el choque osmótico en las terminales radicales de vainilla, puede provocar contaminación por un aumento en el crecimiento de potenciales microorganismos endógenos y por ende, la pérdida de material vegetal. Aunque es factible utilizar meristemos radicales de vainilla para la transformación genética de esta especie, se propone que una estructura más apropiada para este fin podría ser el estadio inicial observado cuando se forma el PLB a partir de los meristemos radicales. _______________________________________________________________________ Abstract: This study consisted in the evaluation of the experimental conditions for genetic transformation of Vanilla planifolia by a biolistic system under low pressure. Root tips of 0.5 cm of length, from seedlings developed in basic MS liquid medium under agitation, were placed in an osmotic medium, 16 hr before and 24 hr after bombarding, subsequently they were placed in MS culture medium supplemented with 1 and 3 mg/L benzyladenine. The assay included bombarding controls without plasmid and controls of explants with and without osmotic stress. After 48 hr of bombarding, root tips were exposed to X-Gluc and a blue staining was observed in the epidermis of the root tips, as well as in the controls. After 2 weeks, the blue staining was also present in the external and internal areas of the initial structures that become PLBs, in all the treatments evaluated and controls. The presence of the staining corresponding to X-Gluc substrate degradation in the controls evidences the expression of endogenous enzymes in plant tissue, which could interfere with interpretation of transient expression results. This would be the first report of endogenous -glucuronidase activity in in vitro vanilla materials. According to the obtained evidence, it is also important to take into consideration that stress induced by osmotic shock in the terminal tips of vanilla plants, could cause contamination by an increase in the growth of potential endogenous microorganisms and, consequently the loss of plant material. Although it is possible to utilize vanilla root meristems for genetic transformation of this species, it is postulated that a more appropriated structure for transformation could be the initial stage on the process of PLB formation from root meristems

Cytotoxic effect induced by retinoic acid loaded into galactosyl-sphingosine containing liposomes on human hepatoma cell lines

Two retinoids, ATRA and 13cisRA, were incorporated into liposomes of different composition and charge and added to two hepatoma cell lines with different degree of transformation to measure cytotoxicity by MTT assay. Retinoid-free cationic liposomes were more toxic than the other kinds (anionic and made only of PC) but were also the best delivery system for retinoic acid to induce specific cytotoxic effects on these tumor hepatoma cell lines. Galactosyl-sphingosine containing cationic liposomes increased the cytotoxic effect induced by ATRA on Hep3B cells when compared to glucosyl-sphingosine cationic liposomes, but did not improve the effect induced by free retinoid or ATRA loaded into liposomes without glycolipids. This suggests that in this cell line, ATRA is being incorporated by a mechanism mediated by the asialoglycoprotein receptor, but at the same time, non-specific sugar-independent capture is also taking place as well as free diffusion of ATRA directly through the membrane. Galactose-specific effect was not observed in HepG2 cells treated with ATRA or both cell lines treated with 13cisRA. In fact, treatment of HepG2 cells with retinoids entrapped into liposomes likely induces proliferation instead of cytotoxicity, a result that interferes with the measurement of cell death by MTT. Compared to the specific effect of ATRA entrapped into cationic liposomes, vesicles made only by PC, did not mediate a specific mechanism, since differences between ATRA in galactosyl- and glucosyl-shpingosine PC-liposomes were not statistically significant. The specific mechanism was not present in the myoblastic cell line C2C12, where ATRA incorporated into galactosyl- and glucosyl-sphingosine containing cationic and PC-liposomes, was able to induce cytotoxicity at the same extent. Micelles containing ATRA and galactosyl-sphingosine had a significantly more toxic effect than the retinoid administered together with glucosyl-sphingosine, in Hep3B cells. Also, micelles containing ATRA were more toxic than glycolipid-containing liposomes with ATRA, for both kinds of sphingosines. The same effect was not observed in C2C12 cells, where glycolipid-containing liposomes worked better than micelles, and a sugar-specific mechanism was not seen. This suggests that, even though galactose-containing cationic liposomes could be a promising approach, a galactose-specific emulsion system could be the best strategy to specifically deliver retinoic acid to liver tumor cells, since it shows tissue specificity (perhaps induced by ASGPR-mediated internalization) and a stronger cytotoxic effect than the retinoid incorporated into liposomes.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 mM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteasesUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Citrus huanglongbing associated with ‘Candidatus liberibacter asiaticus’ present in the northern region of Costa Rica but has not been detected in other citrus-growing areas

Since 2011, citrus Huanglongbing (HLB, ex-greening) disease has been detected in Los Chiles, Costa Rica, close to the Nicaraguan border, by the State Phytosanitary Service (Servicio Fitosanitario del Estado; SFE) and by TicoFrut’s Agricultural Diagnostic Laboratory (Arredondo-Bernal et al. 1999; SFE 2011). However, there has not been a formal scientific report confirming its presence by two independent methods, nor has there been an update on the spread of the disease to other parts of the country. Sweet orange (Citrus sinensis) trees in Los Chiles showing zinc deficiency, vein yellowing, blotchy mottle, and corky veins resembling HLB disease were sampled. Genomic DNA was extracted from leaf midrib and petiole, and Asian citrus psyllid (Diphorina citri) samples using DNeasy Plant, and Blood and Tissue extraction kits (Qiagen, Hilden, Germany). Quantitative polymerase chain reaction (qPCR; TaqMan probes) assays (Li et al. 2006) detected the presence of ‘Candidatus Liberibacter asiaticus’ in 30 symptomatic tree samples. The intergenic region between rplA and rplJ of the β-operon (Hocquellet et al. 1999) was amplified from eight plant and two psyllid samples containing five insects each, and was directly sequenced (Macrogen, Korea) to corroborate the presence of ‘Ca. L. asiaticus’. All sequences obtained from plants and D. citri shared 100% identity with each other, and a BLAST search showed a 99% identity to different sequences of ‘Ca. L. asiaticus’ available in GenBank, e.g., some from India (KC477384). Although D. citri is present in the country (Villalobos et al. 2005), the disease has not been detected in other growing areas. Three hundred seventeen trees and 32 psyllid samples were tested as described above for the presence of ‘Ca. L. asiaticus’ by qPCR in July through September 2013, and April through May 2014. Sampled trees were grown in the San Jose, Alajuela, and Puntarenas provinces, and showed chlorosis-like zinc deficiency. None of the samples tested positive for ‘Ca. L. asiaticus’. Ct values were above 36 or undetected, while internal control (COX) values ranged from 17 to 23. Considering that Costa Rica is a small country, the containment or the failure to detect ‘Ca. L. asiaticus’ in other regions was unexpected. This phenomenon can be due to the SFE’s and the private sector’s quarantine and containment plans (Arredondo-Bernal et al. 2013); however, there may be other factors that are containing the disease that have not been studied. © 2015 The American Phytopathological Society