Competition may explain the fine-scale spatial patterns and genetic structure of two co-occurring plant congeners

1. The spatial distribution of individual plants within a population and the population’s genetic structure are determined by several factors, like dispersal, reproduction mode or biotic interactions. The role of interspecific interactions in shaping the spatial genetic structure of plant populations remains largely unknown. 2. Species with a common evolutionary history are known to interact more closely with each other than unrelated species due to the greater number of traits they share. We hypothesize that plant interactions may shape the fine genetic structure of closely related congeners. 3. We used spatial statistics (georeferenced design) and molecular techniques (ISSR markers) to understand how two closely related congeners, Thymus vulgaris (widespread species) and T. loscosii (narrow endemic) interact at the local scale. Specific cover, number of individuals of both study species and several community attributes were measured in a 10 × 10 m plot. 4. Both species showed similar levels of genetic variation, but differed in their spatial genetic structure. Thymus vulgaris showed spatial aggregation but no spatial genetic structure, while T. loscosii showed spatial genetic structure (positive genetic autocorrelation) at short distances. The spatial pattern of T. vulgaris’ cover showed significant dissociation with that of T. loscosii. The same was true between the spatial patterns of the cover of T. vulgaris and the abundance of T. loscosii and between the abundance of each species. Most importantly, we found a correlation between the genetic structure of T. loscosii and the abundance of T. vulgaris: T. loscosii plants were genetically more similar when they were surrounded by a similar number of T. vulgaris plants. 5. Synthesis. Our results reveal spatially complex genetic structures of both congeners at small spatial scales. The negative association among the spatial patterns of the two species and the genetic structure found for T. loscosii in relation to the abundance of T. vulgaris indicate that competition between the two species may account for the presence of adapted ecotypes of T. loscosii to the abundance of a competing congeneric species. This suggests that the presence and abundance of close congeners can influence the genetic spatial structure of plant species at fine scales.

Effectiveness of AFLPs and retrotransposon-based markers for the identification of portuguese grapevine cultivars and clones

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.

Localization and Tracking in Energy-Constrained Networks with Dynamic Connectivity

Systems used for target localization, such as goods, individuals, or animals, commonly rely on operational means to meet the final application demands. However, what would happen if some means were powered up randomly by harvesting systems? And what if those devices not randomly powered had their duty cycles restricted? Under what conditions would such an operation be tolerable in localization services? What if the references provided by nodes in a tracking problem were distorted? Moreover, there is an underlying topic common to the previous questions regarding the transfer of conceptual models to reality in field tests: what challenges are faced upon deploying a localization network that integrates energy harvesting modules? The application scenario of the system studied is a traditional herding environment of semi domesticated reindeer (Rangifer tarandus tarandus) in northern Scandinavia. In these conditions, information on approximate locations of reindeer is as important as environmental preservation. Herders also need cost-effective devices capable of operating unattended in, sometimes, extreme weather conditions. The analyses developed are worthy not only for the specific application environment presented, but also because they may serve as an approach to performance of navigation systems in absence of reasonably accurate references like the ones of the Global Positioning System (GPS). A number of energy-harvesting solutions, like thermal and radio-frequency harvesting, do not commonly provide power beyond one milliwatt. When they do, battery buffers may be needed (as it happens with solar energy) which may raise costs and make systems more dependent on environmental temperatures. In general, given our problem, a harvesting system is needed that be capable of providing energy bursts of, at least, some milliwatts. Many works on localization problems assume that devices have certain capabilities to determine unknown locations based on range-based techniques or fingerprinting which cannot be assumed in the approach considered herein. The system presented is akin to range-free techniques, but goes to the extent of considering very low node densities: most range-free techniques are, therefore, not applicable. Animal localization, in particular, uses to be supported by accurate devices such as GPS collars which deplete batteries in, maximum, a few days. Such short-life solutions are not particularly desirable in the framework considered. In tracking, the challenge may times addressed aims at attaining high precision levels from complex reliable hardware and thorough processing techniques. One of the challenges in this Thesis is the use of equipment with just part of its facilities in permanent operation, which may yield high input noise levels in the form of distorted reference points. The solution presented integrates a kinetic harvesting module in some nodes which are expected to be a majority in the network. These modules are capable of providing power bursts of some milliwatts which suffice to meet node energy demands. The usage of harvesting modules in the aforementioned conditions makes the system less dependent on environmental temperatures as no batteries are used in nodes with harvesters--it may be also an advantage in economic terms. There is a second kind of nodes. They are battery powered (without kinetic energy harvesters), and are, therefore, dependent on temperature and battery replacements. In addition, their operation is constrained by duty cycles in order to extend node lifetime and, consequently, their autonomy. There is, in turn, a third type of nodes (hotspots) which can be static or mobile. They are also battery-powered, and are used to retrieve information from the network so that it is presented to users. The system operational chain starts at the kinetic-powered nodes broadcasting their own identifier. If an identifier is received at a battery-powered node, the latter stores it for its records. Later, as the recording node meets a hotspot, its full record of detections is transferred to the hotspot. Every detection registry comprises, at least, a node identifier and the position read from its GPS module by the battery-operated node previously to detection. The characteristics of the system presented make the aforementioned operation own certain particularities which are also studied. First, identifier transmissions are random as they depend on movements at kinetic modules--reindeer movements in our application. Not every movement suffices since it must overcome a certain energy threshold. Second, identifier transmissions may not be heard unless there is a battery-powered node in the surroundings. Third, battery-powered nodes do not poll continuously their GPS module, hence localization errors rise even more. Let's recall at this point that such behavior is tight to the aforementioned power saving policies to extend node lifetime. Last, some time is elapsed between the instant an identifier random transmission is detected and the moment the user is aware of such a detection: it takes some time to find a hotspot. Tracking is posed as a problem of a single kinetically-powered target and a population of battery-operated nodes with higher densities than before in localization. Since the latter provide their approximate positions as reference locations, the study is again focused on assessing the impact of such distorted references on performance. Unlike in localization, distance-estimation capabilities based on signal parameters are assumed in this problem. Three variants of the Kalman filter family are applied in this context: the regular Kalman filter, the alpha-beta filter, and the unscented Kalman filter. The study enclosed hereafter comprises both field tests and simulations. Field tests were used mainly to assess the challenges related to power supply and operation in extreme conditions as well as to model nodes and some aspects of their operation in the application scenario. These models are the basics of the simulations developed later. The overall system performance is analyzed according to three metrics: number of detections per kinetic node, accuracy, and latency. The links between these metrics and the operational conditions are also discussed and characterized statistically. Subsequently, such statistical characterization is used to forecast performance figures given specific operational parameters. In tracking, also studied via simulations, nonlinear relationships are found between accuracy and duty cycles and cluster sizes of battery-operated nodes. The solution presented may be more complex in terms of network structure than existing solutions based on GPS collars. However, its main gain lies on taking advantage of users' error tolerance to reduce costs and become more environmentally friendly by diminishing the potential amount of batteries that can be lost. Whether it is applicable or not depends ultimately on the conditions and requirements imposed by users' needs and operational environments, which is, as it has been explained, one of the topics of this Thesis.

Localización personal en entornos interiores con tecnología RFID

Este trabajo presenta un sistema de posicionamiento local (LPS) para personas en entornos interiores basado en la combinación de tecnología RFID activa y una metodología bayesiana de estimación de la posición a partir de la fuerza de las señales de RF recibidas. La complejidad inherente a la propagación de las ondas de RF en entornos interiores causa grandes fluctuaciones en el nivel de la fuerza de la señal, por lo que las técnicas bayesianas, de naturaleza estadística, tienen ventajas significativas frente a métodos de posicionamiento más comunes, como multilateración, minimización cuadrática o localización por fingerprinting. En la validación experimental del sistema RFID-LPS se consigue un error de posicionamiento medio de 2.10 m (mediana de 1.84 m y 3.89 m en el 90% de los casos), en un área abarcada de 475 m2 con 29 tags RFID, y con velocidades de desplazamiento de hasta 0.5 m/s, prestaciones iguales o superiores a otros sistemas del estado del arte. Aunque existen precedentes en Robótica móvil, la combinación de métodos bayesianos y tecnología RFID activa usada en este trabajo es original en el marco de los sistemas de localización de personas, cuyos desplazamientos son generalmente más impredecibles que los de los robots. Otros aspectos novedosos investigados son la posibilidad de alcanzar una estimación conjunta de posición y orientación de un usuario con dos métodos distintos (uso de antenas directivas y aprovechamiento de la atenuación de la señal de RF por el cuerpo humano), la escalabilidad del sistema RFID-LPS, y la estimación de la posición por técnicas bayesianas en sistemas simples que pueden detectar los marcadores RFID, pero no medir su fuerza de señal.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Contribución a la localización en interiores basada en comunicaciones inalámbricas

Determinar con buena precisión la posición en la que se encuentra un terminal móvil, cuando éste se halla inmerso en un entorno de interior (centros comerciales, edificios de oficinas, aeropuertos, estaciones, túneles, etc), es el pilar básico sobre el que se sustentan un gran número de aplicaciones y servicios. Muchos de esos servicios se encuentran ya disponibles en entornos de exterior, aunque los entornos de interior se prestan a otros servicios específicos para ellos. Ese número, sin embargo, podría ser significativamente mayor de lo que actualmente es, si no fuera necesaria una costosa infraestructura para llevar a cabo el posicionamiento con la precisión adecuada a cada uno de los hipotéticos servicios. O, igualmente, si la citada infraestructura pudiera tener otros usos distintos, además del relacionado con el posicionamiento. La usabilidad de la misma infraestructura para otros fines distintos ofrecería la oportunidad de que la misma estuviera ya presente en las diferentes localizaciones, porque ha sido previamente desplegada para esos otros usos; o bien facilitaría su despliegue, porque el coste de esa operación ofreciera un mayor retorno de usabilidad para quien lo realiza. Las tecnologías inalámbricas de comunicaciones basadas en radiofrecuencia, ya en uso para las comunicaciones de voz y datos (móviles, WLAN, etc), cumplen el requisito anteriormente indicado y, por tanto, facilitarían el crecimiento de las aplicaciones y servicios basados en el posicionamiento, en el caso de poderse emplear para ello. Sin embargo, determinar la posición con el nivel de precisión adecuado mediante el uso de estas tecnologías, es un importante reto hoy en día. El presente trabajo pretende aportar avances significativos en este campo. A lo largo del mismo se llevará a cabo, en primer lugar, un estudio de los principales algoritmos y técnicas auxiliares de posicionamiento aplicables en entornos de interior. La revisión se centrará en aquellos que sean aptos tanto para tecnologías móviles de última generación como para entornos WLAN. Con ello, se pretende poner de relieve las ventajas e inconvenientes de cada uno de estos algoritmos, teniendo como motivación final su aplicabilidad tanto al mundo de las redes móviles 3G y 4G (en especial a las femtoceldas y small-cells LTE) como al indicado entorno WLAN; y teniendo siempre presente que el objetivo último es que vayan a ser usados en interiores. La principal conclusión de esa revisión es que las técnicas de triangulación, comúnmente empleadas para realizar la localización en entornos de exterior, se muestran inútiles en los entornos de interior, debido a efectos adversos propios de este tipo de entornos como la pérdida de visión directa o los caminos múltiples en el recorrido de la señal. Los métodos de huella radioeléctrica, más conocidos bajo el término inglés “fingerprinting”, que se basan en la comparación de los valores de potencia de señal que se están recibiendo en el momento de llevar a cabo el posicionamiento por un terminal móvil, frente a los valores registrados en un mapa radio de potencias, elaborado durante una fase inicial de calibración, aparecen como los mejores de entre los posibles para los escenarios de interior. Sin embargo, estos sistemas se ven también afectados por otros problemas, como por ejemplo los importantes trabajos a realizar para ponerlos en marcha, y la variabilidad del canal. Frente a ellos, en el presente trabajo se presentan dos contribuciones originales para mejorar los sistemas basados en los métodos fingerprinting. La primera de esas contribuciones describe un método para determinar, de manera sencilla, las características básicas del sistema a nivel del número de muestras necesarias para crear el mapa radio de la huella radioeléctrica de referencia, junto al número mínimo de emisores de radiofrecuencia que habrá que desplegar; todo ello, a partir de unos requerimientos iniciales relacionados con el error y la precisión buscados en el posicionamiento a realizar, a los que uniremos los datos correspondientes a las dimensiones y realidad física del entorno. De esa forma, se establecen unas pautas iniciales a la hora de dimensionar el sistema, y se combaten los efectos negativos que, sobre el coste o el rendimiento del sistema en su conjunto, son debidos a un despliegue ineficiente de los emisores de radiofrecuencia y de los puntos de captura de su huella. La segunda contribución incrementa la precisión resultante del sistema en tiempo real, gracias a una técnica de recalibración automática del mapa radio de potencias. Esta técnica tiene en cuenta las medidas reportadas continuamente por unos pocos puntos de referencia estáticos, estratégicamente distribuidos en el entorno, para recalcular y actualizar las potencias registradas en el mapa radio. Un beneficio adicional a nivel operativo de la citada técnica, es la prolongación del tiempo de usabilidad fiable del sistema, bajando la frecuencia en la que se requiere volver a capturar el mapa radio de potencias completo. Las mejoras anteriormente citadas serán de aplicación directa en la mejora de los mecanismos de posicionamiento en interiores basados en la infraestructura inalámbrica de comunicaciones de voz y datos. A partir de ahí, esa mejora será extensible y de aplicabilidad sobre los servicios de localización (conocimiento personal del lugar donde uno mismo se encuentra), monitorización (conocimiento por terceros del citado lugar) y seguimiento (monitorización prolongada en el tiempo), ya que todos ellas toman como base un correcto posicionamiento para un adecuado desempeño. ABSTRACT To find the position where a mobile is located with good accuracy, when it is immersed in an indoor environment (shopping centers, office buildings, airports, stations, tunnels, etc.), is the cornerstone on which a large number of applications and services are supported. Many of these services are already available in outdoor environments, although the indoor environments are suitable for other services that are specific for it. That number, however, could be significantly higher than now, if an expensive infrastructure were not required to perform the positioning service with adequate precision, for each one of the hypothetical services. Or, equally, whether that infrastructure may have other different uses beyond the ones associated with positioning. The usability of the same infrastructure for purposes other than positioning could give the opportunity of having it already available in the different locations, because it was previously deployed for these other uses; or facilitate its deployment, because the cost of that operation would offer a higher return on usability for the deployer. Wireless technologies based on radio communications, already in use for voice and data communications (mobile, WLAN, etc), meet the requirement of additional usability and, therefore, could facilitate the growth of applications and services based on positioning, in the case of being able to use it. However, determining the position with the appropriate degree of accuracy using these technologies is a major challenge today. This paper provides significant advances in this field. Along this work, a study about the main algorithms and auxiliar techniques related with indoor positioning will be initially carried out. The review will be focused in those that are suitable to be used with both last generation mobile technologies and WLAN environments. By doing this, it is tried to highlight the advantages and disadvantages of each one of these algorithms, having as final motivation their applicability both in the world of 3G and 4G mobile networks (especially in femtocells and small-cells of LTE) and in the WLAN world; and having always in mind that the final aim is to use it in indoor environments. The main conclusion of that review is that triangulation techniques, commonly used for localization in outdoor environments, are useless in indoor environments due to adverse effects of such environments as loss of sight or multipaths. Triangulation techniques used for external locations are useless due to adverse effects like the lack of line of sight or multipath. Fingerprinting methods, based on the comparison of Received Signal Strength values measured by the mobile phone with a radio map of RSSI Recorded during the calibration phase, arise as the best methods for indoor scenarios. However, these systems are also affected by other problems, for example the important load of tasks to be done to have the system ready to work, and the variability of the channel. In front of them, in this paper we present two original contributions to improve the fingerprinting methods based systems. The first one of these contributions describes a method for find, in a simple way, the basic characteristics of the system at the level of the number of samples needed to create the radio map inside the referenced fingerprint, and also by the minimum number of radio frequency emitters that are needed to be deployed; and both of them coming from some initial requirements for the system related to the error and accuracy in positioning wanted to have, which it will be joined the data corresponding to the dimensions and physical reality of the environment. Thus, some initial guidelines when dimensioning the system will be in place, and the negative effects into the cost or into the performance of the whole system, due to an inefficient deployment of the radio frequency emitters and of the radio map capture points, will be minimized. The second contribution increases the resulting accuracy of the system when working in real time, thanks to a technique of automatic recalibration of the power measurements stored in the radio map. This technique takes into account the continuous measures reported by a few static reference points, strategically distributed in the environment, to recalculate and update the measurements stored into the map radio. An additional benefit at operational level of such technique, is the extension of the reliable time of the system, decreasing the periodicity required to recapture the radio map within full measurements. The above mentioned improvements are directly applicable to improve indoor positioning mechanisms based on voice and data wireless communications infrastructure. From there, that improvement will be also extensible and applicable to location services (personal knowledge of the location where oneself is), monitoring (knowledge by other people of your location) and monitoring (prolonged monitoring over time) as all of them are based in a correct positioning for proper performance.

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

Cinchona officinalis (Rubiaceae), especie endémica del Valle de Loja, ubicado en la región sur del Ecuador, es un recurso forestal de importancia medicinal y ecológica, además la especie ha sido catalogada como planta nacional y es un ícono de la región sur por su aporte a la farmacopea mundial. Esta especie, entre los siglos XVII-XIX sufrió una gran presión en sus poblaciones debido a la extracción masiva de la corteza para la cura del paludismo. Aunque la actividad extractiva generó grandes ingresos a la Corona Española y a la región Sur del Ecuador, ésta fue poco o nada sustentable ecológicamente, provocando la desaparición de la especie en muchos sitios de la provincia, pues, en su momento, no se consideraron alternativas de recuperación de las poblaciones naturales. Actualmente la extracción y consumo de la corteza en la zona de origen es baja o nula, sin embargo esta zona enfrenta nuevas amenazas. La deforestación a causa de proyectos de desarrollo en infraestructuras, la práctica de actividades agrícolas y de ganadería, y los efectos del cambio climático han ocasionado, en estos últimos años, la fragmentación de los ecosistemas. La mayoría de los bosques del sur del Ecuador se han convertido en parches aislados (los bosques en los que se distribuye C. officinalis no son la excepción) siendo esta la principal causa para que la especie se encuentre en estado de amenaza. Los individuos de la especie tienen una alta capacidad de rebrote y producen semillas durante todo el año; sin embargo la capacidad germinativa y la tasa de sobrevivencia son bajas, además de estas dificultades la especie requiere de la asociación con otras especies vegetales para su desarrollo, lo cual ha limitado su distribución en pequeños parches aislados. Con esta problemática, la recuperación natural de las poblaciones es una necesidad evidente. Varios trabajos y esfuerzos previos se han realizado a nivel local: i. Identificación de la distribución actual y potencial; ii. Determinación de la fenología y fructificación iii. Programas de educación ambiental, iv. Análisis moleculares para determinar la diversidad genética. v. Ensayos de propagación vegetativa; y otras acciones de tipo cultural. No obstante, el estado de conservación y manejo de las poblaciones naturales no ha mejorado significativamente, siendo necesaria la aplicación de estrategias integradas de conservación in situ y ex situ, que permitan la recuperación y permanencia de las poblaciones naturales a largo plazo. El presente trabajo tiene como fin dar alternativas para el cultivo de tejidos in vitro de Cinchona officinalis centrados en la propagación masiva a partir de semillas, análisis de la fidelidad genética y alternativas de conservación de tejidos. Los objetivos específicos que se plantean son: i. Analizar el proceso de germinación y proliferación in vitro. ii. Evaluar la estabilidad genética en explantes cultivados in vitro, mediante marcadores ISSR. iii. Establecer protocolos de conservación in vitro mediante limitación del crecimiento y criopreservación de segmentos nodales y yemas. Los resultados más significativos de esta investigación fueron: i. El desarrollo de protocolos eficientes para mejorar los porcentajes de germinación y la proliferación de brotes en explantos cultivados in vitro. Para evaluar el efecto de los fenoles sobre la germinación, se determinó el contenido total de fenoles y el porcentaje de germinación en semillas de C. officinalis comparados con una especie de control, C. pubescens. Para inducir a proliferación, se utilizaron segmentos nodales de plántulas germinadas in vitro en medio Gamborg (1968) suplementado con diferentes combinaciones de reguladores de crecimiento (auxinas y citoquininas). Los resultados obtenidos sugieren que el contenido de compuestos fenólicos es alto en las semillas de C. officinalis en comparación con las semillas de C. pubescens. Estos fenoles pueden eliminarse con peróxido de hidrógeno o con lavados de agua para estimular la germinación. La formación de nuevos brotes y callos en la mayoría de las combinaciones de reguladores de crecimiento se observó en un período de 45 días. El mayor porcentaje de proliferación de brotes, formación de callos y presencia de brotes adventicios se obtuvo en medio Gamborg (B5) suplementado con 5.0 mg/l 6-bencil-aminopurina y 3.0 mg/l de ácido indol-3-butírico. ii. La evaluación de la fidelidad genética de los explantes obtenidos con distintas combinaciones de reguladores de crecimiento vegetal y diversos subcultivos. Se realizó el seguimiento a los explantes obtenidos de la fase anterior, determinando el índice de multiplicación y analizando la fidelidad genética de los tejidos obtenidos por las dos vías regenerativas: brotación directa y regeneración de brotes a partir de callos. Este análisis se realizó por amplificación mediante PCR de las secuencias ubicadas entre microsatélites-ISSR (Inter simple sequence repeat). El medio Gamborg (B5) con 3.0 mg/l de AIB y 5.0 mg/l de BAP usado como medio de inducción en la primera etapa de cultivo generó el mayor índice de proliferación (11.5). Un total de 13 marcadores ISSR fueron analizados, 6 de éstos fueron polimórficos. El mayor porcentaje de variación somaclonal fue inducido en presencia de 1.0 mg/l 2,4-D combinado con 0.2 mg/l Kin con un 1.8% en el segundo sub-cultivo de regeneración, la cual incrementó a 3.6% en el tercer sub-cultivo. Todas las combinaciones con presencia de 2,4-D produjeron la formación de callos y presentaron variación genética. Por su parte la fidelidad genética se mantuvo en los sistemas de propagación directa a través de la formación de brotes a partir de meristemos preformados. iii. El establecimiento de protocolos de conservación in vitro y crioconservación de segmentos nodales y yemas. Para la conservación limitando el crecimiento, se cultivaron segmentos nodales en los medios MS y B5 en tres concentraciones de sus componentes (25, 50 y 100%); y en medio B5 más agentes osmóticos como el manitol, sorbitol y sacarosa en diferentes concentraciones (2, 4 y 8%); los cultivos se mantuvieron por 12 meses sin subcultivos. Para el establecimiento de protocolos para la crioconservación (paralización del metabolismo) se usaron yemas axilares y apicales a las cuales se les aplicaron los métodos de encapsulación-deshidratación y vitrificación. La efectividad de los protocolos usados se determinó en función de la sobrevivencia, reducción del crecimiento y regeneración. Los resultados obtenidos en este apartado reflejan que un crecimiento limitado puede mantener tejidos durante 12 meses de almacenamiento, usando medio B5 más manitol entre 2 y 8%. En los protocolos de crioconservación, se obtuvo el mayor porcentaje de recuperación tras la congelación en NL en el tratamiento control seguido por el método crioprotector de encapsulación-deshidratación. Este trabajo brinda alternativas para la propagación de C. officinalis bajo condiciones in vitro, partiendo de material vegetal con alta diversidad genética. El material propagado puede ser fuente de germoplasma para la recuperación y reforzamiento de las poblaciones naturales así como una alternativa de producción para las comunidades locales debido a la demanda actual de corteza de la zona de origen para la elaboración de agua tónica. ABSTRACT Cinchona officinalis (Rubiaceae) is endemic to the Loja Valley, located in the southern area of Ecuador. The importance of this plant as medical and ecological resource is so great that it has been designated as the national flower and is an icon of the southern region for its contribution to the world pharmacopoeia. Between XVII-XIX centuries its population suffered great reduction due to massive harvesting of the bark to cure malaria. Although extraction activity generated large revenues to the Spanish Crown and the southern region of Ecuador, this was not ecologically sustainable, causing the disappearance of the species in many areas of the province, because during that time alternatives to prevent extinction and recover natural populations were not taken in account. Currently the extraction and consumption of bark in the area of origin is almost absent, but this species faces new threats. Deforestation due to infrastructure development, the practice of farming and ranching, and the effects of climate change had led to the fragmentation of ecosystems during the recent years. Most of the forests of southern Ecuador have become isolated patches, including those where C. officinalis is diffused. The lack of suitable habitat is today the main threat for the species. The species has a high capacity for regeneration and produces seeds throughout the year, but the germination rate is low and the growth is slow. In addition, the species requires the association with other plant species to develop. All these factors had limited its distribution to small isolated patches. The natural recovery of populations is essential to face this problem. Several studies and previous efforts had been made at local level: i. Identification of current and potential distribution; ii. Phenology determination. iii. Environmental education programs, iv. Molecular analisis to determine the genetic diversity. v. Testing of vegetative propagation; and other actions of cultural nature. Despite these efforts, the state of conservation and management of natural populations has not improved significantly. Implementation of integrated in situ and ex situ conservation strategies for the recovery and permanence of long-term natural populations is still needed. This work aims to provide alternatives for in vitro culture of tissue of Cinchona officinalis focused on mass propagation from seeds, genetic fidelity analysis and tissue conservation alternatives. The specific aims are: i. Analyze the process of germination and proliferation in vitro. ii. To evaluate the genetic stability of the explants cultured in vitro by ISSR markers. iii. Establish protocols for in vitro conservation by limiting growth and cryopreservation of nodal segments and buds. The most significant results of this research were: i. The development of efficient protocols to improve germination rates and proliferation of buds in explants cultured in vitro. To study the effect of phenols on germination, the total phenolic content and percentage germination was measured in C. officinalis and in a control species, C. pubescens, for comparison. The content of phenolic compounds in C. officinalis seeds is higher than in C. pubescens. These phenols can be removed with hydrogen peroxide or water washes to stimulate germination. To analyze the regeneration, we used nodal explants from seedlings germinated in vitro on Gamborg medium (1968) supplemented with different combinations of growth regulators (auxins and cytokinins) to induce proliferation. The formation of new shoots and calluses was observed within a period of 45 days in most combinations of growth regulators. The highest percentage of shoot proliferation, callus formation and adventitious buds were obtained in B5 medium supplemented with 5.0 mg/l 6-benzyl-aminopurine and 3.0 mg/l indole-3-butyric acid. ii. Evaluating genetic fidelity explants obtained with various combinations of plant growth regulators and different subcultures. The genetic fidelity was analyzed in tissues obtained by the two regenerative pathways: direct sprouting and shoot regeneration from callus. This analysis was performed by PCR amplification of the sequences located between microsatellite-ISSR (Inter Simple Sequence Repeat). Among a total of 13 ISSR markers analyzed, 6 were polymorphic. The highest percentage of somaclonal variation was induced in the presence of 1.0 mg/l 2,4-D combined with 0.2 mg/l Kin with 1.8% in the second round of regeneration, and increased to 3.6% in the third round. The presence of 2,4-D induced genetic variation in all the combinations of growth regulators. Meanwhile genetic fidelity remained systems propagation through direct shoot formation from meristems preformed. iii. Establishing conservation protocols in vitro and cryoconservation of nodal segments and buds. For medium-term conservation (limited growth) nodal segments were cultured in MS and B5 media at three concentrations (25, 50 and 100%); we tested B5 medium with different concentrations of osmotic agents such as mannitol, sorbitol and sucrose (2, 4 and 8%); cultures were maintained for 12 months with regular subculturing. To establish protocols for cryoconservation (cessation of metabolism) different methods of encapsulation-dehydration and vitrification were applied to axillary and apical buds. The effectiveness of the used protocols is determined based on the survival, growth and regeneration success. The results show that these tissues can be maintained in storage for 12 months, using B5 medium plus mannitol between 2 and 8%. The cryoconservation protocol with highest percentage of recovery was obtained by contral treatment, followed by freezing in NL with encapsulation-dehydration method. This work provides alternatives for the propagation in vitro of C. officinalis, starting from plant material with high genetic diversity. The obtained material represents a source of germplasm to support the recovery and strengthening of natural populations as well as a creation of alternative sources for local communities due to the current demand of bark for the preparation of tonic water.