Nutrient cycling aspects in stands of Pinus-elliottii engelm var elliottii under differents resining intensities .2. nutrients contents in litter

Annual and monthly quantities of nutrients wich returned to the soil through litter fall in Pinus elliottii Engelm. var. elliottii stand were estimated durint a two years period, in Experimental Station of Mogi-Guacu, SP. From the trees of the stand was extracted oleoresin according two differents techniques and control. The annual average of nutrients that returned to the soil was 72.2 Kg/ha, in following order: N > Ca > K > Mg > P. No relationship appeared between nutrient concentrations plus quantities when they were compared with oleoresin yields treatments and control. The differences in annual quantities were possibly due to climatic factors. The soil profile showed short concentrations in nutrient contents. Thus, the forest may be suffering by nutrient deficiences.

Phenological aspects of flowering and fruiting at the Ecological Station of Paulo de Faria-SP-Brazil

The present work was carried out at 'Estacao Ecologica de Paulo de Faria', north of the state of Sao Paulo, southeast of Brazil (19°55'-19°58'S and 49°31'-49°32'W). The flowering and fruiting phenology of trees, shrubs, and lianas species were observed from April 1993 to December 1995. The results were compaired with those surveyed at other forests from Sao Paulo state: Santa Genebra (Campinas). Serra do Japi (Jundiai) and lianas of Mata Atlantica. The majority of species of lianas flowered from January to April and trees and shrubs between September to December. The most intense frutification period occurred during May to August. Similarly to Santa Genebra, the flowering season at 'Estacao Ecologica de Paulo de Faria' started at the beginning of the wet season while frutification during the dryest period of the year. At Serra do Japi, the flowering and frutification occurred from the end of dry and beginning of the rainy season. Considering the liana at Mata Atlantica presented two peaks of flowering, while at 'Estacao Ecologica de Paulo de Faria' occurs only one. Regarding the fruiting phenology is similar for both area, most species producing fruits during the first semestre.

Planting coconuts in Indian villages: ethnoecological aspects and evaluation of a "sustainable development" project

Apesar de cientistas e tomadores de decisão abraçarem o conceito geral de "desenvolvimento sustentável", há pouco acordo sobre como se atingir esta meta em situações específicas. Assim, projetos de desenvolvimento sustentável são amplamente criticados por etno-cientistas quanto à forma como são inter-culturalmente formulados. Aqui reportamos um caso controverso de plantio de coco-da-Bahia em aldeias Kayapó do sul do Pará, e fazemos a nossa autocrítica. Nós partimos da premissa de que o reflorestamento e o estado geral de saúde/nutrição caminham lado a lado. Portanto, o desenvolvimento da cultura de cocos por si só deve contribuir para a conservação da floresta no longo prazo e, simultaneamente, contribuir para o bom estado nutricional do povo Kayapó que protege a floresta da ameaça de práticas não-sustentáveis. Nós buscamos descobrir como que o comportamento dos Kayapó afeta o desenvolvimento da cultura de cocos quando amparada com suporte externo. Nós apresentamos resultados de duas viagens de campo para a terra Kayapó, onde detectamos fatores sócio-ecológicos relevantes para o sucesso de nosso projeto de apoio à cultura de coqueiros nas aldeias indígenas. Primeiro, em novembro de 2007, nós visitamos as aldeias Kikretum, Moikarakô e Aukre (dentre 10 aldeias que receberam mudas de coqueiros de nosso programa de apoio) para entregar um segundo carregamento de mudas de coqueiro (o primeiro carregamento aconteceu em abril de 2006). E descrevemos quantitativamente um aspecto do comportamento dos dispersores de sementes de coco (os Kayapó). Especificamente, como as palmeiras pré-existentes nas aldeias são distribuídas dentre as famílias dos índios e como este carregamento sobreviveu a fatores etno-ecológicos. Segundo, em julho de 2008 nós visitamos as aldeias Kokraimoro e Pykararankre e estimamos a posição dos coqueiros pré-existentes e dos novos em relação a outras árvores cultivadas, fazendo uso de censos partindo do centro das aldeias para seus limites exteriores. Nas três aldeias indígenas visitadas em 2007, virtualmente todos os coqueiros pré-existentes pertenciam a poucas famílias e a distribuição de frutos era, na maior parte dos casos, altamente concentrada dentre os membros destas famílias. Entretanto, assumindo que todos os coqueiros jovens que sobreviveram ao primeiro ano chegarão à maturidade (do primeiro carregamento em abril de 2006), eles representam um aumento considerável no numero projetado de coqueiros adultos nas três aldeias visitadas (48, 195 e 101% em Kikretum, Moikarakô e Aukre, respectivamente). E uma redução substancial na desigualdade de acesso aos cocos. Na expedição de 2008, encontramos que os índios geralmente plantam coqueiros bem próximos das suas casas onde a competição com outras árvores cultivadas podem limitar o desenvolvimento das palmeiras.

Agronomic performance of peach trees grown in subtropical region

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evolving decision trees with beam search-based initialization and lexicographic multi-objective evaluation

Decision tree induction algorithms represent one of the most popular techniques for dealing with classification problems. However, traditional decision-tree induction algorithms implement a greedy approach for node splitting that is inherently susceptible to local optima convergence. Evolutionary algorithms can avoid the problems associated with a greedy search and have been successfully employed to the induction of decision trees. Previously, we proposed a lexicographic multi-objective genetic algorithm for decision-tree induction, named LEGAL-Tree. In this work, we propose extending this approach substantially, particularly w.r.t. two important evolutionary aspects: the initialization of the population and the fitness function. We carry out a comprehensive set of experiments to validate our extended algorithm. The experimental results suggest that it is able to outperform both traditional algorithms for decision-tree induction and another evolutionary algorithm in a variety of application domains.

Salt stress responses in pear and quince: physiological and molecular aspects

The productivity of agricultural crops is seriously limited by salinity. This problem is rapidly increasing, particularly in irrigated lands. Like almost all the fruit tree species, Pyrus communis is generally considered a salt sensitive species, but only little information is available on its behavior under saline conditions. Previous studies, carried out in the Department of Fruit Tree and Woody Plant Science (University of Bologna), focused their attention on pear and quince salt stress responses to understand which rootstock would be the most suitable for pear in order to tolerate a salt stress condition. It has been reported that pear and quince have different ability in the uptake, translocation and accumulation of chloride (Cl-) and sodium (Na+) ions, when plants were irrigated for one season with saline water (5 dS/m). The aim of the present work was to deepen these aspects and investigate salt stress responses in pear and quince. Two different experiments have been performed: a “short-term” trial in a growth chamber and a “long-term” experiment in the open field. In the short-term experiment, three different genotypes usually adopted as pear rootstocks (MC, BA29 and Farold®40) and the pear variety Abbé Fétel own rooted have been compared under salt stress conditions. The trial was performed in a hydroponic culture system, applying a 90 mM NaCl stress to half of the plants, after five weeks of normal growth in Hoagland’s solution. During the three-weeks of salt stress treatment, physiological, mineral and molecular analyses were performed in order to monitor, for each genotype, the development of the salt stress responses in comparison with the corresponding “unstressed” plants. Farold®40 and Abbé Fétel own rooted showed the onset of leaf necrosis, due to salt toxicity, one week before quinces. Moreover, quinces displayed a significant delay in premature senescence of old leaves, while pears emerged for their ability to regenerate new leaves from apparently dead foliage with the salt stress still running. Physiological measurements, such as shoots length, chlorophyll (Chl) content, and photosynthesis, have been carried out and revealed that pears exhibited a significant reduction in water content and a wilting aspect, while for quinces a decrease in Chl content and a growth slowdown were observed. At the end of the trial, all plants were collected and organs separated for dry weight estimation and mineral analyses (Cu, Fe, Mn, Zn Mg, Ca, K, Na and Cl). Mineral contents have been affected by salinity; same macro/micro nutrients were altered in some organs or relocated within the plant. This plant response could have partially contributed to face the salt stress. Leaves and roots have been harvested for molecular analyses at four different times during stress conditions. Molecular analyses consisted of the gene expression study of three main ion transporters, well known in Arabidopsis thaliana as salt-tolerance determinants in the “SOS” pathway: NHX1 (tonoplast Na+/H+ antiporter), SOS1 (plasmalemma Na+/H+ antiporter) and HKT1 (K+ high-affinity and Na+ low-affinity transporter). These studies showed that two quince rootstocks adopted different responsive mechanisms to NaCl stress. BA29 increased its Na+ sequestration activity into leaf vacuoles, while MC enhanced temporarily the same ability, but in roots. Farold®40, instead, exhibited increases in SOS1 and HKT1 expression mainly at leaf level in the attempt to retrieve Na+ from xylem, while Abbé Fétel differently altered the expression of these genes in roots. Finally, each genotype showed a peculiar response to salt stress that was the sum of its ability in Na+ exclusion, osmotic tolerance and tissue tolerance. In the long-term experiment, potted trees of the pear variety Abbé Fétel grafted on different rootstocks (MC, BA29 and Farold®40), or own rooted and also rootstocks only were subjected to a salt stress through saline water irrigation with an electrical conductivity of 5 dS/m for two years. The purposes of this study were to evaluate salinity effects on physiological (shoot length, number of buds, photosynthesis, etc.) and yield parameters of cultivar Abbé Fétel in the different combinations and to determine the salt amount that pear is able to tolerate over the years. With this work, we confirmed the previous hypothesis that pear, despite being classified as a salt-sensitive fruit tree, can be cultivated for two years under saline water irrigation, without showing any salt toxicity symptoms or severe drawbacks on plant development and production. Among different combinations, Abbé Fétel grafted on MC resulted interesting for its peculiar behaviors under salt stress conditions. In the near future, further investigations on physiological and molecular aspects will be necessary to enrich and broaden the knowledge of salt stress responses in pear.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Eros and Transgression in an Age of Immanence: Georges Bataille’s (Religious) Critique of Kinsey

This paper explores the religious implications of eroticism in Western culture since the Sexual Revolution, a period at once applauded for its open and immanent view of sexuality and denounced for its shamelessness and promiscuity. After discussing the work and effects of Alfred C. Kinsey, the father of the Sexual Revolution, I focus on a critical appraisal of Kinsey written by French theorist Georges Bataille (“Kinsey, the Underworld and Work,” in L’Erotisme, 1957). Bataille situates contemporary Western sexuality within a larger historical movement towards the “desacralization” of all aspects of human life: sex, under the scientific gaze of the Kinsey team, became simply another “object” to be analyzed and classified, and “good” sex defined solely in terms of frequency and explosiveness of orgasm. For many, including Hugh Hefner, this approach to sex occasioned a refreshing awakening from the long dark night of Victorian sexual repression. However, as Bataille’s protégé Foucault has shown, the scientific approach to sexuality often masks a desire to control and delimit sexual behaviour, not “liberate” it. Moreover, Bataille makes the point that the desacralization of sexuality denudes sex of a vital component—eroticism—which is necessary for real pleasure and ecstasy. Beyond the “moral” critiques one often hears leveled against Kinsey and his work, Bataille provides a “religious” critique, one that stands, perhaps surprisingly, on the “near side” of sexuality.

Collingwood on religious atonement

R. G. Collingwood’s philosophical analysis of religious atonement as a dialectical process of mortal repentance and divine forgiveness is explained and criticized. Collingwood’s Christian concept of atonement, in which Christ TeX the Atonement (and also TeX the Incarnation), is subject in turn to another kind of dialectic, in which some of Collingwood’s leading ideas are first surveyed, and then tested against objections in a philosophical evaluation of their virtues and defects, strengths and weaknesses. Collingwood’s efforts to synthesize objective and subjective aspects of atonement, and his proposal to solve the soteriological problem as to why God becomes flesh, as a dogma of some Christian belief systems, is finally exposed in adversarial exposition as inadequately supported by one of his main arguments, designated here as Collingwood’s Dilemma. The dilemma is that sin is either forgiven or unforgiven by God. If God forgives sin, then God’s justice is lax, whereas if God does not forgive sin, then, also contrary to divine nature, God lacks perfect loving compassion. The dilemma is supposed to drive philosophy toward a concept of atonement in which the sacrifice of Christ is required in order to absolve God of the lax judgment objection. God forgives sin only when the price of sin is paid, in this case, by the suffering and crucifixion of God’s avatar. The dilemma can be resolved in another way than Collingwood considers, undermining his motivation for synthesizing objective and subjective facets of the concept of atonement for the sake of avoiding inconsistency. Collingwood is philosophically important because he asks all the right questions about religious atonement, and points toward reasonable answers, even if he does not always deliver original philosophically satisfactory solutions.

‘Idolatry’ and Religious Diversity: Thinking about the Other in Early Modern Europe

Idolatry is a key concept in the history of Western thinking about religion, as an all-encompassing category in which all religions more or less alien to the Christian tradition could be subsumed. From Late Antiquity to the Modern period, we can follow how the notion was put to work within Christian discourse to think about the religious “other. ” In fact, the word is almost ubiquitous in pre-modern debates on religion and the origins of religion. Theories on the nature and causes of “idolatry” framed much of the issue of “Religion” vs. the “religions,” and largely provided the conceptual space, in early modern Europe, in which religious anthropology would emerge. The present paper will investigate some aspects of the early modern discourse on idolatry, and its place in early modern discussions on the “diversity” of religions.

The Psychological Outcome of Religious Coping with Stressful Life Events in a Swiss Sample of Church Attendees

Background: Recent research suggested thatreligious coping, based on dispositional religiousness and spirituality (R/S), is an important modulating factor in the process of dealing with adversity. In contrast to the United States, the effect of R/S on psychological adjustment to stress is a widely unexplored area in Europe. Methods: We examined a Swiss sample of 328 church attendees in the aftermath of stressful life events to explore associations of positive or negative religious coping with the psychological outcome. Applying a cross-sectional design, we used Huber’s Centrality Scale to specify religiousness and Pargament’s measure of religious coping (RCOPE) for the assessment of positive and negative religious coping. Depressive symptoms and anxiety as outcome variables were examined by the Brief Symptom Inventory. The Stress-Related Growth Scale and the Marburg questionnaire for the assessment of well-being were used to assess positive outcome aspects. We conducted Mann-Whitney tests for group comparisons and cumulative logit analysis for the assessmentof associations of religious coping with our outcome variables. Results: Both forms of religious coping were positively associated with stress-related growth (p < 0.01). However, negative religious coping additionally reduced well-being (p = 0.05, β = 0.52, 95% CI = 0.27–0.99) and increased anxiety (p = 0.02, β = 1.94, 95% CI = 1.10–3.39) and depressive symptoms (p = 0.01, β = 2.27, 95% CI = 1.27–4.06). Conclusions: The effects of religious coping on the psychological adjustment to stressful life events seem relevant. These findings should be confirmed in prospective studies.

Renewal of Religious Leadership According to an Ancient Model. Bishop Joseph Hubert Reinkens and Bishop Martin of Tours

Im Jahr 1866 verfasste der damals in Breslau lehrende römisch-katholische Kirchenhistoriker Joseph Hubert Reinkens eine der ersten historisch-kritischen Studien in deutscher Sprache über Martin von Tours, in der er sich u.a. mit Martins bischöflichem Leitungsdienst befasste. Nach dem Ersten Vatikanischen Konzil (1870) wurde Reinkens 1873 der erste Bischof für die Alt-Katholiken im Deutschen Reich. Der Beitrag beschreibt den Einfluss, den Reinkens' Martin-Rezeption auf sein theologisches und praktisches Verständnis des Bischofsamts hatte.

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.