Somatic embryogenesis as a regeneration method for clonal propagation of Mediterranean forest species

La mejora y conservación de recursos genéticos en especies forestales lleva siglos de retraso con respecto a las especies agrícolas. Los recursos forestales se han considerado tradicionalmente como recursos �mineros�, en los que primaba la mera extracción dejando exclusivamente a la regeneración natural la labor de sostenibilidad en los montes y dehesas o montados. Hoy en día, el necesario desarrollo del medio rural obliga a la explotación racional de los recursos como medio de garantizar su sostenibilidad. Por ello se está empezando a extender el criterio de que las especies forestales se pueden y deben �cultivar� en determinados espacios. Las características biológicas de las especies forestales las hacen, a menudo, recalcitrantes a las técnicas de mejora y conservación de recursos genéticos tradicionalmente aplicadas a especies agrícolas. En particular, la propagación vegetativa se ha utilizado ampliamente en muchos cultivos leñosos como una herramienta muy poderosa para capturar todo el potencial genético de combinaciones genéticas valiosas. En especies forestales, en particular en las mediterráneas, esta posibilidad raramente se ha podido aplicar debido a la baja capacidad morfogénica de estas especies y la fuerte influencia de la maduración o cambio de fase. En los últimos años la biotecnología forestal ha tenido un desarrollo espectacular. En particular las técnicas de regeneración clonal de plantas basadas en técnicas de cultivo in vitro, fundamentalmente vía embriogénesis somática, se están ya aplicando por muchas empresas privadas e instituciones públicas a nivel semi-operativo con diversas especies, para la conservación de material selecto y el establecimiento de ensayos clonales. Nuestros grupos de trabajo están desarrollando protocolos de regeneración por embriogénesis somática en distintas especies forestales. En esta comunicación se presenta el estado actual de los conocimientos en dos especies típicamente mediterráneas, el alcornoque (Quercus suber L.) y el pino piñonero (Pinus pinea L.), destacando los principales cuellos de botella para su aplicación a gran escala.

Growth data from a field trial of Quercus suber plants regenerated from selected trees and their half-sib progenies by somatic embryogenesis

The development of reliable clonal propagation technologies is a requisite for performing Multi-Varietal Forestry (MVF). Somatic embryogenesis is considered the tissue culture based method more suitable for operational breeding of forest trees. Vegetative propagation is very difficult when tissues are taken from mature donors, making clonal propagation of selected trees almost impossible. We have been able to induce somatic embryogenesis in leaves taken from mature oak trees, including cork oak (Quercus suber). This important species of the Mediterranean ecosystem produces cork regularly, conferring to this species a significant economic value. In a previous paper we reported the establishment of a field trial to compare the growth of plants of somatic origin vs zygotic origin, and somatic plants from mature trees vs somatic plants from juvenile seedlings. For that purpose somatic seedlings were regenerated from five selected cork oak trees and from young plants of their half-sib progenies by somatic embryogenesis. They were planted in the field together with acorn-derived plants of the same families. After the first growth period, seedlings of zygotic origin doubled the height of somatic seedlings, showing somatic plants of adult and juvenile origin similar growth. Here we provide data on height and diameter increases after two additional growth periods. In the second one, growth parameters of zygotic seedlings were also significantly higher than those of somatic ones, but there were not significant differences in height increase between seedlings and somatic plants of mature origin. In the third growth period, height and diameter increases of somatic seedlings cloned from the selected trees did not differ from those of zygotic seedlings, which were still higher than data from plants obtained from somatic embryos from the sexual progeny. Therefore, somatic seedlings from mature origin seem not to be influenced by a possible ageing effect, and plants from somatic embryos tend to minimize the initial advantage of plants from acorns

Plant regeneration from an endangered valuable cork oak tree by somatic embryogenesis.

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

Use of calculus of variations to determine the shape of hovering rotors of minimum power and its application to micro air vehicles

In this paper, calculus of variations and combined blade element and momentum theory (BEMT) are used to demonstrate that, in hover, when neither root nor tip losses are considered; the rotor, which minimizes the total power (MPR), generates an induced velocity that varies linearly along the blade span. The angle of attack of every blade element is constant and equal to its optimum value. The traditional ideal twist (ITR) and optimum (OR) rotors are revisited in the context of this variational framework. Two more optimum rotors are obtained considering root and tip losses, the ORL, and the MPRL. A comparison between these five rotors is presented and discussed. The MPR and MPRL present a remarkable saving of power for low values of both thrust coefficient and maximum aerodynamic efficiency. The result obtained can be exploited to improve the aerodynamic behaviour of rotary wing micro air vehicles (MAV). A comparison with experimental results obtained from the literature is presented.

Dynamic response of hydro power plants to load variations for providing secondary regulation reserves considering elastic water column effects

In this paper, the dynamic response of a hydro power plant for providing secondary regulation reserve is studied in detail. S pecial emphasis is given to the elastic water column effects both in the penstock and the tailrace tunnel. For this purpose, a nonline ar model based on the analogy between mass and momentum conservation equations of a water conduit and those of wave propagation in transmission lines is used. The influence of the plant configuration and design parameters on the fulfilment of the Spanish Electrical System Operator requirem ents is analysed.

Early common markers of microspore and somatic embryogenesis in Quercus suber.

Early common markers of microspore and somatic embryogenesis in Quercus suber.

Ratio-based temperature-sensing technique hardened against nanometer process variations

This letter presents a temperature-sensing technique on the basis of the temperature dependency of MOSFET leakage currents. To mitigate the effects of process variation, the ratio of two different leakage current measurements is calculated. Simulations show that this ratio is robust to process spread. The resulting sensor is quite small-0.0016 mm2 including an analog-to-digital conversion-and very energy efficient, consuming less than 640 pJ/conversion. After a two-point calibration, the accuracy in a range of 40°C-110°C is less than 1.5°C , which makes the technique suitable for thermal management applications.

Modelling experiments of the variations of the calving front position of Hansbreen, Svalbard

Hansbreen is a tidewater glacier in Svalbard, with grounded tongue, about 16 km in length and ca. 2.5 km in width at its tongue. The calving front position has shown, over the recent decades, a general retreating trend, often rather smooth but with some occasional abrupt changes. We apply a full-Stokes model of glacier dynamics, incorporating a crevasse-depth calving model, with the aim of reproducing the glacier front positions observed since 1936 and analyzing the sensitivity of the model to environmental parameters.

Conductivity variations of multi-walled carbon nanotubes oriented in liquid crystal cells

The control of carbon nanotubes conductivity is generating interest in several fields since it may be relevant for a number of applications. The self-organizing properties of liquid crystals may be used to impose alignment on dispersed carbon nanotubes,thus control-ling their conductivity and its anisotropy. This leads to a number of possible applications in photonic and electronic devices such as electrically controlled carbon nanotube switch- es and crossboards. In this work, cells of liquid crystals doped with multi-walled nanotubes have been prepared in different configurations. Their conductivity variations upon switching have been investigated. It turns out that conductivity evolution depends on the initial configuration (either homogeneous, homeotropic or in-plane switching), the cell thickness and the switching record. The control of these manufacturing paramenters allows the modulation of the electrical behavior of carbon nanotubes.

Correction of the Colour Variations under Uncontrolled Lighting Conditions

Esta tesis trata sobre métodos de corrección que compensan la variación de las condiciones de iluminación en aplicaciones de imagen y video a color. Estas variaciones hacen que a menudo fallen aquellos algoritmos de visión artificial que utilizan características de color para describir los objetos. Se formulan tres preguntas de investigación que definen el marco de trabajo de esta tesis. La primera cuestión aborda las similitudes que se dan entre las imágenes de superficies adyacentes en relación a su comportamiento fotométrico. En base al análisis del modelo de formación de imágenes en situaciones dinámicas, esta tesis propone un modelo capaz de predecir las variaciones de color de la región de una determinada imagen a partir de las variaciones de las regiones colindantes. Dicho modelo se denomina Quotient Relational Model of Regions. Este modelo es válido cuando: las fuentes de luz iluminan todas las superficies incluídas en él; estas superficies están próximas entre sí y tienen orientaciones similares; y cuando son en su mayoría lambertianas. Bajo ciertas circunstancias, la respuesta fotométrica de una región se puede relacionar con el resto mediante una combinación lineal. No se ha podido encontrar en la literatura científica ningún trabajo previo que proponga este tipo de modelo relacional. La segunda cuestión va un paso más allá y se pregunta si estas similitudes se pueden utilizar para corregir variaciones fotométricas desconocidas en una región también desconocida, a partir de regiones conocidas adyacentes. Para ello, se propone un método llamado Linear Correction Mapping capaz de dar una respuesta afirmativa a esta cuestión bajo las circunstancias caracterizadas previamente. Para calcular los parámetros del modelo se requiere una etapa de entrenamiento previo. El método, que inicialmente funciona para una sola cámara, se amplía para funcionar en arquitecturas con varias cámaras sin solape entre sus campos visuales. Para ello, tan solo se necesitan varias muestras de imágenes del mismo objeto capturadas por todas las cámaras. Además, este método tiene en cuenta tanto las variaciones de iluminación, como los cambios en los parámetros de exposición de las cámaras. Todos los métodos de corrección de imagen fallan cuando la imagen del objeto que tiene que ser corregido está sobreexpuesta o cuando su relación señal a ruido es muy baja. Así, la tercera cuestión se refiere a si se puede establecer un proceso de control de la adquisición que permita obtener una exposición óptima cuando las condiciones de iluminación no están controladas. De este modo, se propone un método denominado Camera Exposure Control capaz de mantener una exposición adecuada siempre y cuando las variaciones de iluminación puedan recogerse dentro del margen dinámico de la cámara. Los métodos propuestos se evaluaron individualmente. La metodología llevada a cabo en los experimentos consistió en, primero, seleccionar algunos escenarios que cubrieran situaciones representativas donde los métodos fueran válidos teóricamente. El Linear Correction Mapping fue validado en tres aplicaciones de re-identificación de objetos (vehículos, caras y personas) que utilizaban como caracterísiticas la distribución de color de éstos. Por otra parte, el Camera Exposure Control se probó en un parking al aire libre. Además de esto, se definieron varios indicadores que permitieron comparar objetivamente los resultados de los métodos propuestos con otros métodos relevantes de corrección y auto exposición referidos en el estado del arte. Los resultados de la evaluación demostraron que los métodos propuestos mejoran los métodos comparados en la mayoría de las situaciones. Basándose en los resultados obtenidos, se puede decir que las respuestas a las preguntas de investigación planteadas son afirmativas, aunque en circunstancias limitadas. Esto quiere decir que, las hipótesis planteadas respecto a la predicción, la corrección basada en ésta y la auto exposición, son factibles en aquellas situaciones identificadas a lo largo de la tesis pero que, sin embargo, no se puede garantizar que se cumplan de manera general. Por otra parte, se señalan como trabajo de investigación futuro algunas cuestiones nuevas y retos científicos que aparecen a partir del trabajo presentado en esta tesis. ABSTRACT This thesis discusses the correction methods used to compensate the variation of lighting conditions in colour image and video applications. These variations are such that Computer Vision algorithms that use colour features to describe objects mostly fail. Three research questions are formulated that define the framework of the thesis. The first question addresses the similarities of the photometric behaviour between images of dissimilar adjacent surfaces. Based on the analysis of the image formation model in dynamic situations, this thesis proposes a model that predicts the colour variations of the region of an image from the variations of the surrounded regions. This proposed model is called the Quotient Relational Model of Regions. This model is valid when the light sources illuminate all of the surfaces included in the model; these surfaces are placed close each other, have similar orientations, and are primarily Lambertian. Under certain circumstances, a linear combination is established between the photometric responses of the regions. Previous work that proposed such a relational model was not found in the scientific literature. The second question examines whether those similarities could be used to correct the unknown photometric variations in an unknown region from the known adjacent regions. A method is proposed, called Linear Correction Mapping, which is capable of providing an affirmative answer under the circumstances previously characterised. A training stage is required to determine the parameters of the model. The method for single camera scenarios is extended to cover non-overlapping multi-camera architectures. To this extent, only several image samples of the same object acquired by all of the cameras are required. Furthermore, both the light variations and the changes in the camera exposure settings are covered by correction mapping. Every image correction method is unsuccessful when the image of the object to be corrected is overexposed or the signal-to-noise ratio is very low. Thus, the third question refers to the control of the acquisition process to obtain an optimal exposure in uncontrolled light conditions. A Camera Exposure Control method is proposed that is capable of holding a suitable exposure provided that the light variations can be collected within the dynamic range of the camera. Each one of the proposed methods was evaluated individually. The methodology of the experiments consisted of first selecting some scenarios that cover the representative situations for which the methods are theoretically valid. Linear Correction Mapping was validated using three object re-identification applications (vehicles, faces and persons) based on the object colour distributions. Camera Exposure Control was proved in an outdoor parking scenario. In addition, several performance indicators were defined to objectively compare the results with other relevant state of the art correction and auto-exposure methods. The results of the evaluation demonstrated that the proposed methods outperform the compared ones in the most situations. Based on the obtained results, the answers to the above-described research questions are affirmative in limited circumstances, that is, the hypothesis of the forecasting, the correction based on it, and the auto exposure are feasible in the situations identified in the thesis, although they cannot be guaranteed in general. Furthermore, the presented work raises new questions and scientific challenges, which are highlighted as future research work.

Spatial and temporal variations of trace element distribution in soils and street dust of an industrial town in NW Spain: 15 years of study

Extensive spatial and temporal surveys, over 15 years, have been conducted in soil in urban parks and street dusts in one of the most polluted cities in western Europe, Avilés (NW Spain). The first survey was carried out in 1996, and since then monitoring has been undertaken every five years. Whilst the sampling site is a relatively small town, industrial activities (mainly the steel industry and Zn and Al metallurgy) and other less significant urban sources, such as traffic, strongly affect the load of heavy metals in the urban aerosol. Elemental tracers have been used to characterise the influence of these sources on the composition of soil and dust. Although PM10 has decreased over these years as a result of environmental measures undertaken in the city, some of the “industrial” elements still remain in concentrations of concern for example, up to 4.6% and 0.5% of Zn in dust and soil, respectively. Spatial trends in metals such as Zn and Cd clearly reflect sources from the processing industries. The concentrations of these elements across Europe have reduced over time, however the most recent results from Avilés revealed an upward trend in concentration for Zn, Cd, Hg and As. A risk assessment of the soil highlighted As as an element of concern since its cancer risk in adults was more than double the value above which regulatory agencies deem it to be unacceptable. If children were considered to be the receptors, then the risk nearly doubles from this element.

Study of PM10 and PM2.5 levels in three European cities: Analysis of intra and inter urban variations

In the present paper, 1-year PM10 and PM 2.5 data from roadside and urban background monitoring stations in Athens (Greece), Madrid (Spain) and London (UK) are analysed in relation to other air pollutants (NO,NO2,NOx,CO,O3 and SO2)and several meteorological parameters (wind velocity, temperature, relative humidity, precipitation, solar radiation and atmospheric pressure), in order to investigate the sources and factors affecting particulate pollution in large European cities. Principal component and regression analyses are therefore used to quantify the contribution of both combustion and non-combustion sources to the PM10 and PM 2.5 levels observed. The analysis reveals that the EU legislated PM 10 and PM2.5 limit values are frequently breached, forming a potential public health hazard in the areas studied. The seasonal variability patterns of particulates varies among cities and sites, with Athens and Madrid presenting higher PM10 concentrations during the warm period and suggesting the larger relative contribution of secondary and natural particles during hot and dry days. It is estimated that the contribution of non-combustion sources varies substantially among cities, sites and seasons and ranges between 38-67% and 40-62% in London, 26-50% and 20-62% in Athens, and 31-58% and 33-68% in Madrid, for both PM10 and PM 2.5. Higher contributions from non-combustion sources are found at urban background sites in all three cities, whereas in the traffic sites the seasonal differences are smaller. In addition, the non-combustion fraction of both particle metrics is higher during the warm season at all sites. On the whole, the analysis provides evidence of the substantial impact of non-combustion sources on local air quality in all three cities. While vehicular exhaust emissions carry a large part of the risk posed on human health by particle exposure, it is most likely that mitigation measures designed for their reduction will have a major effect only at traffic sites and additional measures will be necessary for the control of background levels. However, efforts in mitigation strategies should always focus on optimal health effects.

Development of somatic embryogenesis for cloning and conservation of mature holm oak trees (Quercus ilex L.)

La encina (Quercus ilex L.) es una de las especies forestales mediterráneas más importantes. Constituye gran parte del estrato arbóreo de dehesas o montados, produce bellota como alimento del ganado y establece simbiosis con hongos micorrizógenos de gran valor económico. La encina está considerada como una especie recalcitrante en términos de conservación de semillas y capacidad morfogénica, lo que dificulta los programas de conservación de recursos genéticos y la mejora de la especie. La propagación vegetativa es una potente herramienta de los programas de mejora, por lo que es preciso desarrollar protocolos de regeneración somática en encina. La embriogénesis somática está considerada como la modalidad más adecuada de regeneración basada en técnicas de cultivo de tejidos vegetales utilizada en biotecnología forestal. Este trabajo se centra en el estudio de determinados aspectos de la embriogénesis somática para la regeneración clonal de encinas adultas. La memoria de esta tesis se ha dividido en capítulos que se corresponden con diferentes aspectos del sistema embriogénico. La embriogénesis somática se indujo en tegumentos maternos de óvulos en desarrollo procedentes de bellotas inmaduras de encinas adultas. A pesar de las bajas frecuencias de inducción, las líneas embriogénicas generadas se amplificaron mediante embriogénesis secundaria observándose cierta pérdida de la capacidad de diferenciación con el tiempo. Tanto el genotipo como la formulación del medio de cultivo influyeron en la respuesta embriogénica, concluyendo que la formulación de macronutrientes de Schenk y Hildebrant del medio sin reguladores de crecimiento fue la combinación más efectiva en la inducción. Los resultados sugirieron la existencia de una ventana en el desarrollo del óvulo más sensible a la inducción. El genotipo in[luyó en la capacidad proliferativa de los cultivos y en la conversión de los embriones somáticos, que se incrementó suplementando el medio con ácido indol-3-butírico y 6-benciladenina. El cultivo en medio líquido de líneas embriogénicas en condiciones de inmersión transitoria incrementó el crecimiento, dependiendo del genotipo, con respecto al cultivo en medio semisólido. Sin embargo, no mejoró la capacidad de diferenciar embriones cotiledonares aislados. Se estableció un protocolo de inicio y mantenimiento de cultivos en suspensión para varias líneas embriogénicas mediante inoculación en alta densidad de agregados embrionarios procedentes del medio semisólido. Para evitar la pérdida de vigor y la capacidad morfogénica debida al cultivo prolongado se desarrolló un protocolo de crioconservación de líneas embriogénicas mediante vitrificación. Al determinar la influencia de los agentes crioprotectores antes y después de su inmersión en nitrógeno líquido se concluyó que las respuestas de capacidad de crecimiento y de diferenciación del material embriogénico son independientes, además de estar bajo influencia del genotipo y el tipo de material crioconservado. La combinación de sacarosa y PVS2 previa a la inmersión en nitrógeno líquido proporcionó la mayor tasa de recuperación. Cuando las líneas fueron crioconservadas 30 días la capacidad de diferenciación se perdió en todas ellas. El análisis de SSR detectó variación somaclonal en el material crioconservado a corto plazo. SSR y RAPD mostraron importantes diferencias genéticas entre los árboles donantes y el material embriogénico que dependieron del genotipo. El grado de detección dependió del marcador empleado. Ambos marcadores revelaron baja inestabilidad intraclonal. Los RAPD revelaron variación genética intra-individuo en las encinas donantes. Se discuten la variación genética pre-existente en encina, su aparición durante las primeras fases de la inducción de embriogénesis, y la presencia de tejidos provenientes de la fertilización en el explanto materno. Esto hace preciso definir la identidad genética del material donante y acometer ensayos de detección precoz de variación somaclonal. ABSTRACT Holm oak (Quercus ilex L.) is one of the most important Mediterranean forest species. It conforms the tree layer of dehesas or montados, it produces acorns to feed the livestock and it establishes symbiosis with profitable mycorrhizal fungi. Holm oak is considered as recalcitrant species in terms of seed conservation and morphogenic capacities, which complicates the development of genetic conservation and improvement programs. Vegetative propagation is one of the mightiest tools for breeding programs therefore; developing protocols for clonal regeneration of holm oak is essential. Somatic embryogenesis is considered the best tissue culture-based way of plant regeneration in forest biotechnology. The present study is focused on the study of certain aspects of somatic embryogenesis for clonal regeneration of mature holm oak. This thesis manuscript is divided into several chapters that match with different aspects of the embryogenic system. Somatic embryogenesis induction was achieved on maternal teguments of developing ovules from immature acorns of adult holm oak trees. Despite the low induction frequencies, the generated embryogenic lines were amplified by secondary embryogenesis. A decline in the differentiation capacity over time was also observed. It was concluded that both genotype and culture media formulation influenced the embryogenic response, being the Schenk and Hildebrandt´s macronutrients formulation from culture medium and the lack of plant growth regulators the most effective combination for the induction of the embryogenic response. It has been suggested the existence of a developmental window in which ovules are prone to induction. Genotype influenced the proliferation capacity and the plant conversion of somatic embryos, which was also favoured by the presence of indol-3-butyric acid and 6-bencyladenine. The use of temporary immersion systems as proliferation in liquid culture of the embryogenic lines increased the growth depending on genotype, when compared to semisolid cultures. However, it did not improve the differentiation of single cotyledonary embryos. A protocol for the initiation and maintenance of embryogenic suspension cultures was established for several embryogenic lines with highly dense inoculi of embryogenic clusters from proliferating semisolid cultures. In order to avoid the loss of vigour and morphogenic ability of embryogenic lines due to prolonged cultures, a cryopreservation protocol for embryogenic lines of holm oak has been developed. During the determination of the influence of cryoprotective agents on the growth and differentiation capacities before and after liquid nitrogen immersion, it was concluded that both responses were independent from each other and also under the influence of genotype and the type of cryopreserved material. The combination of sucrose and PVS2 prior liquid nitrogen immersion provided higher recovery rates. When the same embryogenic lines were cryopreserved for 30 days, none was able to differentiate. The SSRs analysis of the short-term cryopreserved material detected somaclonal variation. Both SSR and RAPD markers showed high sensitivity to detect genetic differences between the donor trees and the generated embryogenic material. Nevertheless, the degree of instability detection depended on the marker. The SSR analysis indicated a relationship between genotype, the studied loci and the located polymorphisms. Also, both markers revealed low intraclonal genetic variation. The RAPD detected genetic variation within the donor trees. The presence of pre-existent genetic variation within mature trees, in addition to its occurrence during the early stages of the embryogenic induction, and the presence of tissues of fertilisation origin within the maternal explants are all discussed. Nonetheless, the determination of the genetic identity of donor material is required, in addition to early detection methods of somaclonal variation.

Development of somatic embryogenesis for cloning and conservation of mature holm oak trees (Quercus ilex L.)

La encina (Quercus ilex L.) es una de las especies forestales mediterráneas más importantes. Constituye gran parte del estrato arbóreo de dehesas o montados, produce bellota como alimento del ganado y establece simbiosis con hongos micorrizógenos de gran valor económico. La encina está considerada como una especie recalcitrante en términos de conservación de semillas y capacidad morfogénica, lo que dificulta los programas de conservación de recursos genéticos y la mejora de la especie. La propagación vegetativa es una potente herramienta de los programas de mejora, por lo que es preciso desarrollar protocolos de regeneración somática en encina. La embriogénesis somática está considerada como la modalidad más adecuada de regeneración basada en técnicas de cultivo de tejidos vegetales utilizada en biotecnología forestal. Este trabajo se centra en el estudio de determinados aspectos de la embriogénesis somática para la regeneración clonal de encinas adultas. La memoria de esta tesis se ha dividido en capítulos que se corresponden con diferentes aspectos del sistema embriogénico. La embriogénesis somática se indujo en tegumentos maternos de óvulos en desarrollo procedentes de bellotas inmaduras de encinas adultas. A pesar de las bajas frecuencias de inducción, las líneas embriogénicas generadas se amplificaron mediante embriogénesis secundaria observándose cierta pérdida de la capacidad de diferenciación con el tiempo. Tanto el genotipo como la formulación del medio de cultivo influyeron en la respuesta embriogénica, concluyendo que la formulación de macronutrientes de Schenk y Hildebrant del medio sin reguladores de crecimiento fue la combinación más efectiva en la inducción. Los resultados sugirieron la existencia de una ventana en el desarrollo del óvulo más sensible a la inducción. El genotipo in[luyó en la capacidad proliferativa de los cultivos y en la conversión de los embriones somáticos, que se incrementó suplementando el medio con ácido indol-3-butírico y 6-benciladenina. El cultivo en medio líquido de líneas embriogénicas en condiciones de inmersión transitoria incrementó el crecimiento, dependiendo del genotipo, con respecto al cultivo en medio semisólido. Sin embargo, no mejoró la capacidad de diferenciar embriones cotiledonares aislados. Se estableció un protocolo de inicio y mantenimiento de cultivos en suspensión para varias líneas embriogénicas mediante inoculación en alta densidad de agregados embrionarios procedentes del medio semisólido. Para evitar la pérdida de vigor y la capacidad morfogénica debida al cultivo prolongado se desarrolló un protocolo de crioconservación de líneas embriogénicas mediante vitrificación. Al determinar la influencia de los agentes crioprotectores antes y después de su inmersión en nitrógeno líquido se concluyó que las respuestas de capacidad de crecimiento y de diferenciación del material embriogénico son independientes, además de estar bajo influencia del genotipo y el tipo de material crioconservado. La combinación de sacarosa y PVS2 previa a la inmersión en nitrógeno líquido proporcionó la mayor tasa de recuperación. Cuando las líneas fueron crioconservadas 30 días la capacidad de diferenciación se perdió en todas ellas. El análisis de SSR detectó variación somaclonal en el material crioconservado a corto plazo. SSR y RAPD mostraron importantes diferencias genéticas entre los árboles donantes y el material embriogénico que dependieron del genotipo. El grado de detección dependió del marcador empleado. Ambos marcadores revelaron baja inestabilidad intraclonal. Los RAPD revelaron variación genética intra-individuo en las encinas donantes. Se discuten la variación genética pre-existente en encina, su aparición durante las primeras fases de la inducción de embriogénesis, y la presencia de tejidos provenientes de la fertilización en el explanto materno. Esto hace preciso definir la identidad genética del material donante y acometer ensayos de detección precoz de variación somaclonal. ABSTRACT Holm oak (Quercus ilex L.) is one of the most important Mediterranean forest species. It conforms the tree layer of dehesas or montados, it produces acorns to feed the livestock and it establishes symbiosis with profitable mycorrhizal fungi. Holm oak is considered as recalcitrant species in terms of seed conservation and morphogenic capacities, which complicates the development of genetic conservation and improvement programs. Vegetative propagation is one of the mightiest tools for breeding programs therefore; developing protocols for clonal regeneration of holm oak is essential. Somatic embryogenesis is considered the best tissue culture-based way of plant regeneration in forest biotechnology. The present study is focused on the study of certain aspects of somatic embryogenesis for clonal regeneration of mature holm oak. This thesis manuscript is divided into several chapters that match with different aspects of the embryogenic system. Somatic embryogenesis induction was achieved on maternal teguments of developing ovules from immature acorns of adult holm oak trees. Despite the low induction frequencies, the generated embryogenic lines were amplified by secondary embryogenesis. A decline in the differentiation capacity over time was also observed. It was concluded that both genotype and culture media formulation influenced the embryogenic response, being the Schenk and Hildebrandt´s macronutrients formulation from culture medium and the lack of plant growth regulators the most effective combination for the induction of the embryogenic response. It has been suggested the existence of a developmental window in which ovules are prone to induction. Genotype influenced the proliferation capacity and the plant conversion of somatic embryos, which was also favoured by the presence of indol-3-butyric acid and 6-bencyladenine. The use of temporary immersion systems as proliferation in liquid culture of the embryogenic lines increased the growth depending on genotype, when compared to semisolid cultures. However, it did not improve the differentiation of single cotyledonary embryos. A protocol for the initiation and maintenance of embryogenic suspension cultures was established for several embryogenic lines with highly dense inoculi of embryogenic clusters from proliferating semisolid cultures. In order to avoid the loss of vigour and morphogenic ability of embryogenic lines due to prolonged cultures, a cryopreservation protocol for embryogenic lines of holm oak has been developed. During the determination of the influence of cryoprotective agents on the growth and differentiation capacities before and after liquid nitrogen immersion, it was concluded that both responses were independent from each other and also under the influence of genotype and the type of cryopreserved material. The combination of sucrose and PVS2 prior liquid nitrogen immersion provided higher recovery rates. When the same embryogenic lines were cryopreserved for 30 days, none was able to differentiate. The SSRs analysis of the short-term cryopreserved material detected somaclonal variation. Both SSR and RAPD markers showed high sensitivity to detect genetic differences between the donor trees and the generated embryogenic material. Nevertheless, the degree of instability detection depended on the marker. The SSR analysis indicated a relationship between genotype, the studied loci and the located polymorphisms. Also, both markers revealed low intraclonal genetic variation. The RAPD detected genetic variation within the donor trees. The presence of pre-existent genetic variation within mature trees, in addition to its occurrence during the early stages of the embryogenic induction, and the presence of tissues of fertilisation origin within the maternal explants are all discussed. Nonetheless, the determination of the genetic identity of donor material is required, in addition to early detection methods of somaclonal variation.