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.

Infrastructure Finance in Developing Countries by State and Multilateral Development Banks

Infrastructure concession is an alternative widely used by governments to increase investment. In the case of the road sector, the main characteristics of the concessions are: long-term projects, high investments in the early years of the contract and high risks. A viability analysis must be carried out for each concession and consider the characteristics of the project. When the infrastructure is located in a developing country, political and market growth uncertainties should be add in the concession project analysis, as well as economic instability, because they present greater risks. This paper is an analysis of state bank participation in road infrastructure finance in developing countries. For this purpose, we studied road infrastructure financing and its associated risks, and also the features of developing countries. Furthermore, we considered the issue of state banks and multilateral development banks that perform an important role by offering better credit lines than the private banks, in terms of cost, interest and grace period. Based on this study, we analyzed the Brazilian Development Bank - BNDES – and their credit supply to road infrastructure concessions. The results show that BNDES is the main financing agent for long-term investment in the sector, offering loans with low interest rates in Brazilian currency. From this research we argue that a single state bank should not alone support the increasing demand for finance in Brazil. Therefore, we conclude that there is a need to expand the supply of credit in Brazil, by strengthening private banks in the long-term lending market.

Development of the infrastructure for a multi-agent traffic management system simulation

This document contains detailed description of the design and the implementation of a multi-agent application controlling traffic lights in a city together with a system for simulating traffic and testing. The goal of this thesis is to design and build a simplified intelligent and distributed solution to the problem with the traffic in the big cities following different good practices in order to allow future refining of the model of the real world. The problem of the traffic in the big cities is still a problem that cannot be solved. Not only is the increasing number of cars a reason for the traffic jams, but also the way the traffic is organized. Usually, the intersections with traffic lights are replaced by roundabouts or interchanges to increase the number of cars that can cross the intersection in certain time. But still there are places where the infrastructure cannot be changed and the traffic light semaphores are the only way to control the car flows. In real life, the traffic lights have a predefined plan for change or they receive information from a centralized system when and how they have to change. But what if the traffic lights can cooperate and decide on their own when and how to change? Using this problem, the purpose of the thesis is to explore different agent-based software engineering approaches to design and build a non-conventional distributed system. From the software engineering point of view, the goal of the thesis is to apply the knowledge and use the skills, acquired during the various courses of the master program in Software Engineering, while solving a practical and complex problem such as the traffic in the cities.

The role of private sector in development: The relation between public-private investment in infrastructure and agricultural exports in developing countries

Increasing foreign private investment in developing countries explains why the Public-Private Investment (PPI) is becoming a key tool to reach the development goal. This article analyzes the relation between PPI in infrastructure and agricultural exports in developing countries. We use the panel data approach (52 countries and 17 years). Results show that PPI in infrastructure has a positive impact on agricultural exports of developing countries. The impact is greater in developing countries with higher income rates. This suggests that the lower income countries require the intervention of public sector without which private investment cannot help to economic development.

End-User Development Success Factors and their Application to Composite Web Development Environments

The Future Internet is expected to be composed of a mesh of interoperable Web services accessed from all over the Web. This approach has not yet caught on since global user-service interaction is still an open issue. Successful composite applications rely on heavyweight service orchestration technologies that raise the bar far above end-user skills. The weakness lies in the abstraction of the underlying service front-end architecture rather than the infrastructure technologies themselves. In our opinion, the best approach is to offer end-to-end composition from user interface to service invocation, as well as an understandable abstraction of both building blocks and a visual composition technique. In this paper we formalize our vision with regard to the next-generation front-end Web technology that will enable integrated access to services, contents and things in the Future Internet. We present a novel reference architecture designed to empower non-technical end users to create and share their own self-service composite applications. A tool implementing this architecture has been developed as part of the European FP7 FAST Project and EzWeb Project, allowing us to validate the rationale behind our approach.

Floating car data augmentation based on infrastructure sensors and neural networks

The development of new-generation intelligent vehicle technologies will lead to a better level of road safety and CO2 emission reductions. However, the weak point of all these systems is their need for comprehensive and reliable data. For traffic data acquisition, two sources are currently available: 1) infrastructure sensors and 2) floating vehicles. The former consists of a set of fixed point detectors installed in the roads, and the latter consists of the use of mobile probe vehicles as mobile sensors. However, both systems still have some deficiencies. The infrastructure sensors retrieve information fromstatic points of the road, which are spaced, in some cases, kilometers apart. This means that the picture of the actual traffic situation is not a real one. This deficiency is corrected by floating cars, which retrieve dynamic information on the traffic situation. Unfortunately, the number of floating data vehicles currently available is too small and insufficient to give a complete picture of the road traffic. In this paper, we present a floating car data (FCD) augmentation system that combines information fromfloating data vehicles and infrastructure sensors, and that, by using neural networks, is capable of incrementing the amount of FCD with virtual information. This system has been implemented and tested on actual roads, and the results show little difference between the data supplied by the floating vehicles and the virtual vehicles.

Quantity of CO2 Emissions in the Life Cycle of a Highway Infrastructure

There is strong evidence to indicate that carbon dioxide and other greenhouse gases are accumulating at unprecedented concentrations in out atmosphere contributing to global climate change. Evidence is equally strong that human activities, mainly the burning of fossil fuels, are driving force in this process (IPCC 2007). While different industries contribute varying amounts to total anthropogenic greenhouse gases, it is incumbent upon each to understand its contribution and search for sensible ways to reduce overall greenhouse gas production. The aim of this paper is the development of a methodology to determine the amount of CO2 emissions of a highway, allowing providing solutions that can improve the energy footprint and reduce its emissions

Renewable energy infrastructure. A challenge for Venezuelan industrial construction

The relevance of renewable energy has grown significantly in our global society. Important efforts are oriented to sustain it. Renewable energy depends on different technical, financial environmental and social complex processes. From the point of view of industrial construction sector this research evaluates some of the current trends in energy generation and use in Venezuela as well as environmental consequences and risks that derive from these. Additionally, authors highlight the importance of infrastructure as key issue to sustain renewable energy generation and use. The study present references of some energy renewable projects in process in Venezuela and the main problems that constrain their performance. Conclusions evidence the complex nature of industrial construction and suggest the need to improve industrial construction competitivenes as a strategy oriented to enhance renewable energy offer in the country. Additionally it is proposed to all stakeholders to work toghether to correct the conditions that currently limit industrial construction development. This is part of ongoing research.

The technofusion project for fusion technology development: opportunities for the inertial community

Technofusion will be the new Spanish singular scientific-technical installation for fusion research. The research activities will be focused on seven areas of materials research considered the most relevant ones for further technological developments of fusion energy. In principle, most of the infrastructure in Technofusion will be useful for both, magnetic (MC) and inertial (IC) confinement fusion communities and most of the research and developments carried out for one of the fusion concepts will be valid and transferable to the other. However, some aspects related to first wall materials strongly differ in MC and IC approaches. This is due to the very different typical ion energy and deposited powers in both cases.

Development of a methodology and tool to evaluate the impact of ICT measures on road transport emissions

The paper presents the main elements of a project entitled ICT-Emissions that aims at developing a novel methodology to evaluate the impact of ICT-related measures on mobility, vehicle energy consumption and CO2 emissions of vehicle fleets at the local scale, in order to promote the wider application of the most appropriate ICT measures. The proposed methodology combines traffic and emission modelling at micro and macro scales. These will be linked with interfaces and submodules which will be specifically designed and developed. A number of sources are available to the consortium to obtain the necessary input data. Also, experimental campaigns are offered to fill in gaps of information in traffic and emission patterns. The application of the methodology will be demonstrated using commercially available software. However, the methodology is developed in such a way as to enable its implementation by a variety of emission and traffic models. Particular emphasis is given to (a) the correct estimation of driver behaviour, as a result of traffic-related ICT measures, (b) the coverage of a large number of current vehicle technologies, including ICT systems, and (c) near future technologies such as hybrid, plug-in hybrids, and electric vehicles. The innovative combination of traffic, driver, and emission models produces a versatile toolbox that can simulate the impact on energy and CO2 of infrastructure measures (traffic management, dynamic traffic signs, etc.), driver assistance systems and ecosolutions (speed/cruise control, start/stop systems, etc.) or a combination of measures (cooperative systems).The methodology is validated by application in the Turin area and its capacity is further demonstrated by application in real world conditions in Madrid and Rome.

Enhancing K-12 science education through a multi-device web tool to facilitate content integration and e-infrastructure access

An effective K-12 science education is essential to succeed in future phases of the curriculum and the e-Infrastructures for education provide new opportunities to enhance it. This paper presents ViSH Viewer, an innovative web tool to consume educational content which aims to facilitate e-Science infrastructures access through a next generation learning object called "Virtual Excursion". Virtual Excursions provide a new way to explore science in class by taking advantage of e-Infrastructure resources and their integration with other educational contents, resulting in the creation of a reusable, interoperable and granular learning object. In order to better understand how this tool can allow teachers and students a joyful exploration of e-Science, we also present three Virtual Excursion examples. Details about the design, development and the tool itself are explained in this paper as well as the concept, structure and metadata of the new learning object.

An online e-Learning authoring tool to create interactive multi-device learning objects using e-Infrastructure resources

Education can take advantage of e-Infrastructures to provide teachers with new opportunities to increase students' motivation and engagement while they learn. Nevertheless, teachers need to find, integrate and customize the resources provided by e-Infrastructures in an easy way. This paper presents ViSH Editor, an innovative web-based e-Learning authoring tool that aims to allow teachers to create new learning objects using e-Infrastructure resources. These new learning objects are called Virtual Excursions and are created as reusable, granular and interoperable learning objects. This way they can be reused to build new ones and they can be integrated in websites or Learning Management Systems. Details about the design, development and the tool itself are explained in this paper as well as the concept, structure and metadata of the new learning objects. Lastly, some real examples of how to enrich learning using Virtual Excursions are exposed.

EU support mechanisms to promote public private partnerships for financing Trans-European transport infrastructure

Since the mid 80ies the Trans ‐European Transport Network (TEN‐T) policy has been setting the framework for the development of infrastructure for the smooth functioning of the internal market within the European Union (EU). Public Private Partnerships (PPPs) have always been regarded by the EU as a key instrument to promote the TEN‐T.

Identifying Potential Risks and Benefits of Using Cloud in Distributed Software Development.

Cloud-based infrastructure has been increasingly adopted by the industry in distributed software development (DSD) environments. Its proponents claim that its several benefits include reduced cost, increased speed and greater productivity in software development. Empirical evaluations, however, are in the nascent stage of examining both the benefits and the risks of cloud-based infrastructure. The objective of this paper is to identify potential benefits and risks of using cloud in a DSD project conducted by teams based in Helsinki and Madrid. A cross-case qualitative analysis is performed based on focus groups conducted at the Helsinki and Madrid sites. Participants observations are used to supplement the analysis. The results of the analysis indicated that the main benefits of using cloud are rapid development, continuous integration, cost savings, code sharing, and faster ramp-up. The key risks determined by the project are dependencies, unavailability of access to the cloud, code commitment and integration, technical debt, and additional support costs. The results revealed that if such environments are not planned and set up carefully, the benefits of using cloud in DSD projects might be overshadowed by the risks associated with it.

Beyond the Regional Development Paradigm: The Limits of Transport-Based Policies in Spain

This paper reviews the transport and economic development trends for the last 20 years in Spain at a detailed (province or NUTS3) level. As Spain has sustained a significant transport investment effort in this period, with the support of EU funding, this review offers an excellent perspective to put some further light on how the transport-and-regional-development paradigm has shaped decision-making in the transport sector. The paper reviews changes in gross domestic product (GDP), population and motorway endowment for the 47 provinces in mainland Spain. Regional development trends seem to be closely associated to particular local conditions, not clearly associated to transport (motorway) infrastructure endowment. This is consistent with the fact that transport infrastructure has not generally been a critical bottleneck for trade and economic activity during this period. The paper concludes that, in general terms, transport infrastructure investment does not seem to be clearly associated to the otherwise substantial differences in regional development among Spanish mainland provinces during this period.