Determinación de la higroscopicidad y comportamiento termodinámico de la madera juvenil y madura a través de sus isotermas de sorción

La respuesta higroscópica de la madera varía a lo largo de la dirección radial del árbol. El aumento de corta de ejemplares jóvenes y el uso de troncos de pequeños diámetros en la industria de los productos forestales, hacen preciso estudiar el comportamiento higroscópico tanto de la madera juvenil como de la madura. Su determinación proporciona información para comprender los mecanismos de sorción. Asimismo, la obtención de las propiedades termodinámicas de ambos tipos de madera facilita la modelización de procesos industriales como el secado o el encolado. En el presente trabajo, se ha comparado el comportamiento higroscópico y las propiedades termodinámicas de la madera juvenil y madura de Abies pinsapo Boissier, Abies alba Mill., Pinus canariensis C. Sm. ex DC., Pinus nigra Arnold, Pinus uncinata Mill. ex Mirb. y Pinus pinea L. Para este propósito se han utilizado las isotermas de sorción obtenidas mediante el método tradicional de sales saturadas descrito por COST Action E8 a 15, 35 y 50ºC en Abies pinsapo, Abies alba, Pinus nigra, Pinus uncinata y Pinus pinea, y a 35 y 50ºC en Pinus canariensis. Igualmente, se ha empleado el equipo dynamic vapor sorption (DVS) en la obtención de las isotermas de Pinus pinea a 35 y 50ºC. El ajuste de las curvas se ha realizado mediante el modelo Guggenheim, Anderson y de Boer-Dent (GAB), cumpliendo todas las muestras los criterios de aceptación establecidos. En el estudio de las isotermas se ha calculado el coeficiente y área de histéresis entre el proceso de adsorción y desorción para cada una de las muestras. Con el fin de comprender el comportamiento higroscópico experimentado por cada madera se ha determinado la composición química, espectros de infrarrojos (FTIR) y difractogramas de rayos X de cada una. Los parámetros termodinámicos - calor isostérico neto y total heat of wetting - se han obtenido a partir de las isotermas de sorción mediante el método de integración de la ecuación de Clausius-Clapeyron. Finalmente, se han comparado los datos obtenidos con el método tradicional de sales saturadas y con dynamic vapor sorption con el propósito de conocer la existencia de similitudes entre ambas metodologías. Los resultados mostraron que los puntos de equilibrio son, en la mayor parte de los casos, superiores en la madera madura frente a la juvenil, y por tanto las isotermas de la madera madura se encuentran siempre por encima de las de la juvenil, debido principalmente a la composición química. Respecto a las propiedades termodinámicas, se ha determinado que la energía involucrada en los procesos de sorción es superior en la madera madura que en la madera juvenil, siendo mayor en el proceso de desorción frente al de adsorción. En la comparación de las metodologías de sales saturadas y dynamic vapor sorption no se han detectado casi diferencias significativas en el proceso de adsorción, mientras que sí se han obtenido en el de desorción. ABSTRACT The hygroscopic response of wood varies throughout the radial direction of the tree. The longer cut of young trees and the use of small-diameter trunks in the forest product industry make it necessary to study the hygroscopic behaviour of both juvenile and mature wood. Determining this behaviour in both types of wood provides information for understanding the sorption mechanisms. Similarly, obtaining the thermodynamic properties of juvenile and mature wood facilitates modelling of industrial processes such as drying and bonding. In this study a comparison was made of the hygroscopic behaviour and thermodynamic properties of juvenile and mature wood of Abies pinsapo Boissier, Abies alba Mill., Pinus canariensis C. Sm. ex DC., Pinus nigra Arnold, Pinus uncinata Mill. ex Mirb. and Pinus pinea L. This was done by obtaining the sorption isotherms using the traditional saturated salt method described by COST Action E8 at 15, 35 and 50ºC in Abies pinsapo, Abies alba, Pinus nigra, Pinus uncinata and Pinus pinea, and at 35 and 50ºC in Pinus canariensis. In addition, dynamic vapour sorption (DVS) was used to obtain the isotherms of Pinus pinea at 35 and 50ºC. The curves were fitted using the Guggenheim, Anderson and de Boer- Dent (GAB) model and all samples met the established acceptance criteria. In the study of the isotherms, the hysteresis coefficient and area of the hysteresis loop between adsorption and desorption were calculated for each sample. To understand the hygroscopic behaviour of juvenile and mature wood, the chemical composition, infrared spectra (FTIR) and X-ray diffractograms of each type of wood were determined. The thermodynamic parameters - net isosteric heat and total heat of wetting - were obtained from the sorption isotherms by applying the integration method of the Clausius-Clapeyron equation. The data obtained using the traditional saturated salt method and with dynamic vapour sorption were compared to determine the similarities between the two methods. The results showed that the equilibrium points are greater in the mature wood than in the juvenile wood in most cases, and therefore the mature wood isotherms are always above the juvenile wood isotherms, mainly because of the chemical composition. As regards the thermodynamic properties, it was determined that the energy involved in the sorption processes is greater in the mature wood than in the juvenile wood, and is greater in the desorption process than in the adsorption process. On comparing the saturated salt and dynamic vapour sorption methods, almost no significant differences were detected in the adsorption process, but significant differences were obtained in the desorption process.

Análisis de envejecimiento en paneles sándwich de madera/ Ageing tests study on wood-based sandwich panels

Los paneles sándwich de madera son un producto de creciente aplicación en la edificación de nuestro país. Este ascendente uso del material debe estar acompañado de las garantías necesarias avaladas por un estudio previo de sus prestaciones. Como es preceptivo y entre otros, se evalúa su durabilidad frente a las condiciones climatológicas, clave en los productos derivados de la madera, acorde a la normativa actual definida con tal fin, la Guía ETAG 016. Sin embargo, debido a la clase de uso del material, se ha detectado que dicha normativa tal y como está concebida no es capaz de valorar su envejecimiento adecuadamente. En este trabajo se proponen ensayos alternativos al establecido tras exhaustivos aná- lisis que recrean las condiciones reales de uso y más acordes a los productos de madera. Se concluye que la incorporación de una lámina impermeable, pero permeable al vapor de agua hacia el exterior, como las utilizadas en el montaje, aportan el mejor procedimiento de ensayo. Composite lightweight wood panels are being increasingly used in construction in Spain. Their growing use should be accompanied by necessary guarantees based on studies of their properties. As it is prescriptive and in addition to others tests, in the present work is examinated the durability of these panels when exposed to the climatic conditions, a characteristic of great importance for wood products, according to Guide ETAG 016, the current standard defining the ageing tests to be used. However, due to the use class of this material, there are indications that the testing outlined in this Guide is inappropriate for assessing the ageing of wood-based sandwich panels. Alternative tests are here proposed that recreate rather better the real conditions under which these products are used. Covering the samples in a waterproof sheeting permeable to the outward movement of water vapour, which is in fact used in the installation, provided the best procedure for testing these panels.

Palaeobiogeographical perspectives on pinus pinea, a controversial and enigmatic mediterranean pine

The origins of some species of economic importance occurring over the Mediterranean Basin have been a traditional matter of debate that has important implications for land management. The case of Pinus pinea L. (Stone pine) is probably one of the most controversial, due to its documented long-term interaction with humans and its presence as a symbolic tree in certain areas of the Mediterranean (e.g., southwestern Iberia and Tuscany). Among the rest of the Mediterranean pines, several features make this pine unique (it has a characteristic crown shape, an edible kernel, cones that require three years to mature, and a very depauperate genetic diversity across its range). In addition, its palaeoecological information is rather limited, as the taxonomic precision attained by pollen analysts is insufficient for this tree and macroremains (such as kernels or anatomically well preserved wood) are needed to unequivocally detect the species in the fossil record. Recent findings of macrofossils of Pinus pinea in inland Iberia (Duero Basin) extend the late- Holocene range of the species, but the palaeobiogeographical information and the exhaustive genetic data available still suggest a very limited natural area (but still not sufficiently well defined) and a long and intense history of linkage to humans.

Spectral Biomimetic Technique for Wood Classification

Palatal clicks are most interesting for human echolocation. Moreover, these sounds are suitable for other acoustic applications due to their regular mathematical properties and reproducibility. Simple and nondestructive techniques, bioinspired by synthetized pulses whose form reproduces the best features of palatal clicks, can be developed. The use of synthetic palatal pulses also allows detailed studies of the real possibilities of acoustic human echolocation without the problems associated with subjective individual differences. These techniques are being applied to the study of wood. As an example, a comparison of the performance of both natural and synthetic human echolocation to identify three different species of wood is presented. The results show that human echolocation has a vast potential.

Seasonal evolution of soil respiration in a mixed forestry plantation of walnuts (Juglans × intermedia Carr.) and alders (Alnus cordata (Loisel.) Duby) in Domaine de Restinclières (France)

The ecological intensification of crops is proposed as a solution to the growing demand of agricultural and forest resources, in opposition to intensive monocultures. The introduction of mixed cultures as mixtures between nitrogen fixing species and non nitrogen fixing species intended to increase crop yield as a result of an improvement of the available nitrogen and phosphorus in soil. Relationship between crops have received little attention despite the wide range of advantages that confers species diversity to these systems, such as increased productivity, resilience to disruption and ecological sustainability. Forests and forestry plantations can develop an important role in storing carbon in their tissues, especially in wood which become into durable product. A simplifying parameter to analyze the amount allocated carbon by plantation is the TBCA (total belowground carbon allocation), whereby, for short periods and mature plantations, is admitted as the subtraction between soil carbon efflux and litterfall. Soil respiration depends on a wide range of factors, such as soil temperature and soil water content, soil fertility, presence and type of vegetation, among others. The studied orchard is a mixed forestry plantation of hybrid walnuts(Juglans × intermedia Carr.) for wood and alders (Alnus cordata (Loisel.) Duby.), a nitrogen fixing specie through the actinomycete Frankia alni ((Woronin, 1866) Von Tubeuf 1895). The study area is sited at Restinclières, a green area near Montpellier (South of France). In the present work, soil respiration varied greatly throughout the year, mainly influenced by soil temperature. Soil water content did not significantly influence the response of soil respiration as it was constant during the measurement period and under no water stress conditions. Distance between nearest walnut and measurement was also a highly influential factor in soil respiration. Generally there was a decreasing trend in soil respiration when the distance to the nearest tree increased. It was also analyzed the response of soil respiration according to alder presence and fertilizer management (50 kg N·ha-1·año-1 from 1999 to 2010). None of these treatments significantly influenced soil respiration, although previous studies noticed an inhibition in rates of soil respiration under fertilized conditions and high rates of available nitrogen. However, treatments without fertilization and without alder presence obtained higher respiration rates in those cases with significant differences. The lack of significant differences between treatments may be due to the high coefficient of variation experienced by soil respiration measurements. Finally an asynchronous fluctuation was observed between soil respiration and litterfall during senescence period. This is possibly due to the slowdown in the emission of exudates by roots during senescence period, which are largely related to microbial activity.

Holocene distribution of woody taxa at the westernmost limit of the Circumboreal/Mediterranean boundary: evidence from wood remains

Información paleoecológica relativa a la distribución de táxones arbóreos durante el holoceno, basado en restos fósiles leñosos.

Antigenic Proteins Involved in Occupational Rhinitis 1 and Asthma Caused by Obeche Wood (Triplochiton Scleroxylon)

Background Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. Objective To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma Materials and methods An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. Results Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA−), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. Conclusions Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

La silvicultura como primera operación de transformación de la madera Forestry as the first operation of wood transformation

Se analiza la influencia de las principales actuaciones selvicolas en la calidad de la madera. Turno, Espaciamiento, Poda, Abonado, Riego y Tratamiento contra plagas.

Spectral Biomimetic Technique for Wood Classification Inspired by Human Echolocation

Los Clics palatales son sonidos interesantes para la ecolocalización humana pero tambien pueden utilizarse para determinar propiedades de la madera como para poder identificar tres especies diferentes de madera. Los resultados muestran que la ecolocalización humana tiene un enorme potencial.

Mechanical properties of wood from Pinus sylvestris L. treated with Light Organic Solvent Preservative and with waterbone Copper Azole

El objetivo del estudio es determinar el efecto de tratamiento de la madera de Pinus sylvestris con sustancias protectoras en las propiedades mecánicas. Para ello se utilizan 40 muestras de madera libre de defectos de Pinus sylvestris L. tratándose con protectores orgánicos (Vacsol Azure WR 2601) 50 con protectores hidrosolubles (Tanalith E 3492) y 40 muestras de control sin tratamiento. Se evaluó la resistencia mecánica a la flexión estática, módulo de elasticidad y la fuerza de compresión paralela a la fibra fueron comparados con madera no tratada. El análisis de regresión entre la penetración y la fuerza de compresión paralela se realizó con las muestras tratadas con conservante a base de agua. Resultados principales: Los resultados indican que la madera tratada (con cualquiera de los productos) presenta un aumento estadísticamente significativo de la resistencia mecánica en todas las tres características mecánicas. Los resultados obtenidos difieren de estudios anteriores llevada a cabo por otros autores. No hubo correlación entre la resistencia a la compresión en paralelo y el grado de impregnación de la madera con base de agua de cobre azoles. La explicación más probable para estos resultados se refiere a cambios en la presión durante el tratamiento. El uso de muestras de control no tratadas en lugar de las muestras tratadas sólo con agua es más probable para producir resultados significativos en los estudios de resistencia mecánica . La investigación pone de relieve que la madera tratada presenta un aumento estadísticamente significativo en el Modulo de Elasticidad, módulo de rotura a la flexión estática y resistencia a la compresión paralela. No hubo correlación entre la resistencia a la compresión en paralelo y el grado de impregnación con conservante hidrosoluble.

Variation throughout the tree stem in the physical-mechanical properties of the wood of Abies alba Mill. from the Spanish Pyrenees

This study analyses the variation of main physical-mechanical properties of wood along the longitudinal and radial directions of the tree for Abies alba Mill. growing in the Spanish Pyrenees. Small clear specimens were used to study the properties of volumetric shrinkage (VS), density (?), hardness (H), bending strength (MOR), modulus of elasticity (MOE), maximum compressive strength parallel to the grain (MCS) and impact strength (K). Several models of properties variation in the longitudinal and radial directions were analyzed. Main trends of variation of properties throughout the tree stem were identified although none of them could be fitted to predictive statistical models. Along the longitudinal direction, the properties studied followed a downward trend from the base to the crown, which was not significant in all cases, indicating that no differences in quality existed. Throughout the radial direction the trend is upward for the first 40-50 growth rings, after which it slopes downwards, more gently at first until rings 70-75 and then more steeply. This behaviour is related to variation in wood structure from the pith to the bark, depending on whether the wood is juvenile, sapwood or heartwood, and to wood maturity and microfibril angle. Authors encourage carrying further studies on other populations of A. alba in the Spanish Pyrenees to check if the trends found in this study apply to other provenances.

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.

NDT to identify biological damage in wood

Nondestructive techniques are widely used to assess existing timber structures. The models proposed for these methods are usually performed in the laboratory using small clear wood specimens. But in real situations many anomalies, defects and biological damage are found in wood. In these cases the existing models only indicate that the values are outside normality without providing any other information. To solve this problem, a study of non-destructive probing methods for wood was performed, testing the behaviour of four different techniques (penetration resistance, pullout resistance, drill resistance and chip drill extraction) on wood samples with different biological damage, simulating an in-situ test. The wood samples were obtained from existing Spanish timber structures with biotic damage caused by borer insects, termites, brown rot and white rot. The study concludes that all of the methods offer more or less detailed information about the degree of deterioration of wood, but that the first two methods (penetration and pullout resistance) cannot distinguish between pathologies. On the other hand, drill resistance and chip drill extraction make it possible to differentiate pathologies and even to identify species or damage location. Finally, the techniques used were compared to characterize their advantages and disadvantages.