Einfluss der Habitatfragmentierung durch Überflutung auf Laufkäferpopulationen (Coleoptera; Carabidae)

Innerhalb dieser Dissertation wurde zwischen den Jahren 2002 und 2005 mit Hilfe von Barberfallen die Laufkäferfauna der Auwälder am nördlichen Oberrhein zwischen Mainz und Bingen erfasst. Dabei dienten verschiedene Rheininseln und ufernahe Festlandgebiete als Probeflächen. Fünf der typischen Bewohner dieser Flächen (Agonum afrum, Nebria brevicollis, Oxypselaphus obscurus, Platynus assimilis, Pterostichus anthracinus) dienten weiterhin als Modellarten für die Untersuchung der genetischen Diversität zwischen den einzelnen Populationen mittels RAPD-Analysen. Alles in allem konnten im Untersuchungsgebiet über 20.000 Individuen aus 101 Carabidenarten gefangen werden. Die häufigsten Vertreter waren Platynus assimilis, Pterostichus melanarius und Agonum afrum. Hohe Diversitäts- und Dominanzindices auf allen Flächen sprechen für die Dynamik des Lebensraumes und somit die Intaktheit der untersuchten Auwälder. Einen weiteren Hinweis auf die ständig wechselnden Lebensbedingungen durch immer wiederkehrende Überflutungen zeigt das Auftreten verschiedener ökologischer Gruppen. Überall dominierten deutlich die Arten, die mit gewissen Störungen des Habitates auskommen oder durch ihr hohes Ausbreitungspotential davor fliehen können. Das sind die Imaginalüberwinterer, makropteren, hygrophilen und kleinen Spezies. Auch das Geschlechterverhältnis weist auf deutliche Anzeichen für regelmäßige Beeinträchtigungen der Flächen hin. Knapp die Hälfte der beobachteten Arten im Untersuchungsgebiet steht auf einer der Roten Listen von Deutschland, Rheinland-Pfalz oder Hessen. Somit besteht für das gesamte Gebiet ein hoher Schutzbedarf. Das Hauptaugenmerk dieser Arbeit lag bei den Einflüssen der Hochwasserstände auf die Artenzusammensetzungen und die genetischen Diversitäten der Laufkäferpopulationen. Deshalb untersuchte man auch die Wirkung derjenigen Faktoren, die ihrerseits unmittelbar von den Extremwasserständen beeinflusst werden. Hier sind vor allem der Auentyp (Weichholz/Hartholz) und die Lage der Flächen auf Insel oder Festland zu nennen, die die deutlichsten Unterschiede in Artendiversität und Genetik der einzelnen Populationen zeigten. Aber auch weitere Faktoren, wie Wasserstandsdynamik, Auwaldbreite, Entfernung vom Fluss und Lage zum Damm weisen Zusammenhänge mit bestimmten ökologischen Gruppen auf. Lediglich die Habitatgröße scheint keinen Einfluss auf die Diversitäten zu nehmen. Abschließend konnten auch für das Jahr 2003, in dem extrem heiße und trockene Bedingungen herrschten, negative Effekte auf die Laufkäfergemeinschaften gezeigt werden.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Simultaneous, bilateral anterior ischemic optic neuropathy (AION) in polycythemia vera: a case report

BACKGROUND: Polycythemia vera (PV) is a hemopoetic disorder. Apparently, although thrombosis accounts for the majority of morbidity, AION has not been associated with PV so far. PATIENT AND FINDINGS: A 63 y-old woman with PV was hospitalized because of acute liver failure. She also experienced bilateral painless loss of vision. Bilateral, pale optic disc swelling with flame-like hemorrhages, more pronounced in the right eye, constricted visual fields, and relative afferent papillary defect (RAPD) on the right side were present. Computer tomography scan revealed no signs of intraorbital pathology, elevated intracranial pressure or hemorrhages. CLINICAL COURSE: We interpreted the findings as AION associated with the hyperviscosity syndrome. Liver transplantation had to be carried out in the next days. Three weeks later, vision improved slightly, but RAPD persisted, and disc pallor developed in both eyes. The patient died two months later. DISCUSSION: Central retinal artery and vein occlusions have been described as complications of Essential thrombocythemia, but not of PV. We observed a rare case of bilateral neuropathy suggestive of AION. This condition has so far not been associated with PV.

Demonstration of Rhodanese Activity in Polyacrylamide Gels.

Rhodanese activity from crude extracts of Thiobacillus sp. strain IV-85 was demonstrated in polyacrylamide gels after incubation in the reaction mixture by staining with dichloroindophenol in the presence of methylphenazonium methosulfate. The sensitivity of the staining system was found to be 8 x 10 moles of sulfite.

La homogeneidad varietal en viveros de olivo estudiada con marcadores moleculares

La difusión de variedades tradicionales de olivo (Olea europea L.) y la obtención de nuevas variantes fenotípicas han generado confusión en la correcta identificación y denominación de algunas de las aprox. 2 000 variedades conocidas a nivel mundial. En la Argentina, en los últimos años se ha triplicado la superficie implantada con olivo principalmente a partir de plantas de viveros locales e importadas. Con el fin de evaluar la homogeneidad genotípica del material comercializado en Mendoza, se probaron 7 marcadores RAPD altamente reproducibles en muestras de 5 viveros, correspondientes a 5 variedades de olivo. Los marcadores RAPD fueron previamente desarrollados para caracterizar las variedades del INTA Junín. Arbequina y Arauco fueron los materiales más homogéneos. En ambas variedades, todos los individuos compartieron el 100 % de los marcadores utilizados. Los lotes de muestras de Empeltre, Farga y Aloreña no fueron genotípicamente homogéneos, observándose 2-3 patrones diferentes por lote. Todas las variedades ensayadas -en alguna de sus muestras- tuvieron diferencias con su respectiva variedad del INTA Junín. Arbequina y Arauco también compartieron el 100 % de marcadores entre sí, no pudiéndose separar ambos grupos.

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.

Estudio de la estabilidad genética asociada a los procesos de crioconservación de ápices de menta

La crioconservación se ha descrito como una técnica de conservación ex situ a largo plazo que ha sido aplicada con éxito a numerosas especies, y resulta especialmente importante en aquellas con propagación vegetativa, infértiles o amenazadas, en las que sistemas de conservación ex situ más sencillos, como los bancos de semillas, no son posibles. También presenta ventajas frente a la conservación in vitro, ya que logra disminuir o eliminar problemas como la excesiva manipulación del material, evitando los subcultivos periódicos y disminuyendo así el riesgo de contaminaciones y de aparición de variación somaclonal. Sin embargo, someter al material vegetal a los procedimientos que implica la crioconservación provoca distintos estreses. Entre ellos, el estrés oxidativo puede potencialmente producir daños en membranas, proteínas, carbohidratos y en el ADN. En este trabajo se han evaluado diversos sistemas de crioconservación en ápices de Mentha × piperita L., híbrido estéril entre Mentha aquatica L. y Mentha spicata L. Se han utilizado ápices de dos genotipos (‘MEN 186’y ‘MEN 198’) en los cuales se compararon dos técnicas de crioconservación, encapsulación-deshidratación y vitrificación-droplet. El análisis de la supervivencia y capacidad de regeneración del material sometido a los tratamientos de crioconservación, junto con el análisis de la estabilidad genética de dicho material mediante marcadores moleculares (RAPD y AFLP) han permitido comparar los distintos protocolos y tratamientos establecidos. El estudio sobre el tipo de protocolo empleado reveló una mayor variabilidad genética en la técnica de encapsulación-deshidratación, especialmente en el genotipo ‘MEN 186’, ya que ‘MEN 198’ resultó ser más estable en todos los análisis. La inestabilidad encontrada en esta técnica no fue exclusiva de aquellos explantos crioconservados, sino que los pasos previos a la inmersión en nitrógeno líquido (NL) también provocaron variaciones en el ADN. Según el tipo de muestra analizada se encontraron diferencias en la estabilidad: muestras provenientes de callos presentaron una mayor inestabilidad que aquellas de hojas (brotes). Se utilizaron tres medios para la recuperación de los ápices tras la crioconservación con el uso de diferentes combinaciones de reguladores de crecimiento: “Reed” (0,5 mgL-1 6-bencilaminopurina, BAP), “Senula” (0,5 mgL-1 6-dimetilalilamino-purina, 2-iP + 0,1 mgL-1 ácido α-naftalen-acético, ANA) y “Nudos” (0,5 mgL-1 BAP + 0,1 mgL-1ANA). El medio “Reed” produjo un aumento en la supervivencia y recuperación de los ápices en ambos genotipos y técnicas, y disminuyó la formación de callo. Sin embargo, no tuvo un efecto significativo en la estabilidad genética. El medio “Senula” provocó una mayor estabilidad genética en el genotipo más inestable, ‘MEN 186’. Para reducir el daño oxidativo producido durante la encapsulación-deshidratación, e incrementar la recuperación de los ápices manteniendo su estabilidad genética, se comparó el efecto de añadir sustancias antioxidantes en el precultivo de los ápices (ácido ascórbico, vitamina E y glutatión). No se obtuvo la respuesta esperada y estos tratamientos no presentaron efectos significativos tanto en la estabilidad como en la recuperación. Para entender mejor qué sucede durante todo el proceso de encapsulación-deshidratación, se evaluó cada paso del protocolo por separado y su efecto en la estabilidad y la recuperación. Además, se determinó el estado de oxidación en cada etapa mediante la cuantificación de malondialdehído y la detección de la formación de radicales libres (mediante el ensayo del ácido tiobarbitúrico, y sondas fluorescentes específicas, respectivamente). Se determinó que a partir de los primeros pasos se genera estrés oxidativo, el cual aumenta a medida que se avanza por el protocolo hasta la inmersión en nitrógeno líquido. Esto se ve reflejado en la disminución progresiva tanto de la recuperación como de la estabilidad genética. Con el uso de antioxidantes en el precultivo (ácido ascórbico y vitamina E) no se obtuvo un efecto positivo en el mantenimiento de la estabilidad genética, y tan sólo con el uso de vitamina E se observó una recuperación mayor en uno de los pasos estudiados (después de la desecación). Sin embargo, cuando se utilizó ácido ascórbico durante el precultivo o la deshidratación osmótica se consiguió disminuir de forma significativa la formación de MDA y la acumulación del radical superóxido (O2•-) en la mayoría los pasos analizados, aunque esta reducción no parece tener un efecto directo en la estabilidad genética del material recuperado. ABSTRACT Cryopreservation has been described as an effective technique for the long term of ex situ conservation that has been successfully applied to numerous species, and is of especial relevance for those with vegetative propagation, infertile or endangered, in which simpler systems of ex situ conservation, such as seed banking, are not feasible. It also has advantages over in vitro conservation, as it reduces or eliminates excessive material handling, avoids periodic subcultures and thus limits the risk of contamination and the appearance of somaclonal variation. However, plant material is subjected to different treatments involved in the cryopreservation procedures, which impose several stresses. Among them, oxidative stress can potentially cause damage to membranes, proteins, carbohydrates and DNA. In this work, two cryopreservation techniques have been evaluated in Mentha × piperita L. shoot tips, sterile hybrid between Mentha aquatica L. and Mentha spicata L. Two genotypes ('MEN 186' and 'MEN 198') were used to compare two techniques: encapsulation-dehydration and droplet-vitrification. The analysis of survival and recovery capacity of the material after the cryopreservation treatments, and the analysis of the genetic stability by molecular markers (RAPD and AFLP) have enabled the comparison between protocols and treatments. The study of the two cryopreservation procedures revealed a higher genetic variability in the encapsulation-dehydration technique, especially in genotype 'MEN 186', as 'MEN 198' was more stable in all analyses. The instability generated in this technique was not exclusive of cryopreserved explants, pretreatments prior to immersion in NL also caused DNA variations. The type of sampled plant material revealed also differences in the stability: callus samples showed greater instability than shoots. Three different culture media were used for the recovery of shoot tips after cryopreservation, using different combinations of growth regulators: "Reed" (0.5 mgL-1 6-benzylaminopurine, BAP), "Senula" (0.5 mgL-1 6-dimetilalilamino-purine, 2-iP + 0.1 mgL-1 α-naphthalene acetic acid, ANA) and "Nodes" (0.5 mgL-1 BAP + 0.1 mgL-1 ANA). "Reed" medium increased survival and recovery of shoot tips in both genotypes and techniques and decreased callus formation. However, it didn`t have a significant effect on genetic stability. "Senula" medium caused a higher genetic stability in the most unstable genotype, 'MEN 186'. To reduce oxidative damage during encapsulation-dehydration, and increase shoot tip recovery and maintain genetic stability, the effect of added antioxidants (ascorbic acid, vitamin E and glutathione) in the shoot tip preculture medium was studied. These treatments had no significant effect on both stability and recovery. To better understand the events during the encapsulation-dehydration process, the effect of each step of the protocol on stability and recovery was evaluated separately. Moreover, the oxidation level was determined by quantifying malondialdehyde (MDA) formation and detecting free radical accumulation (using the thiobarbituric acid assay, and specific fluorescent probes, respectively). The oxidative stress was detected from the first steps and increased throughout the protocol until the immersion in liquid nitrogen. This was also reflected in the gradual decline of recovery and genetic stability. The use of antioxidants (ascorbic acid and vitamin E) in the shoot tip preculture medium had no effect in maintaining genetic stability; only vitamin E increased recovery in one of the steps studied (after desiccation). However, when ascorbic acid was used during the preculture or during the osmotic dehydration, a significantly decrease was observed in MDA formation and superoxide radical accumulation in most of the steps analyzed, although this reduction did not seem to have a direct effect on the genetic stability of recovered material.

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May–July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as “tags” for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.

Identification of Acremonium isolates from grapevines and evaluation of their antagonism towards Plasmopara viticola

Some endophytic fungal genera in Vitis vinifera, including Acremonium, have been reported as antagonists of Plasmopara viticola. Endophytic Acremonium isolates from an asymptomatic grapevine cultivar Inzolia from Italy were identified by morphological features and multigene phylogenies of ITS, 18S and 28S genes, and their intra-specific genomic diversity was analyzed by RAPD analysis. Culture filtrates (CFs) obtained from Acremonium isolates were tested in vitro for their inhibitory activity against the P. viticola sporangia germination. Among 94 isolates, 68 belonged to the Acremonium persicinum and 26 to the Acremonium sclerotigenum. RAPD analysis grouped the A. persicinum isolates into 15 clusters and defined 31 different strains. The A. sclerotigenum isolates, instead, were clustered into 22 groups and represented 25 strains. All A. persicinum CFs inhibited sporangia germination of P. viticola, while not all those of A. sclerotigenum had inhibitory effect. A different degree of inhibition was observed between strains of the same species, while some strains of different species showed identical inhibitory effect. No correlation was found between RAPD groups and inhibitory activity in both Acremonium species.

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Crystallization and preliminary X-ray analysis of sulfite dehydrogenase from Stargeya novella

Crystals of purified heterodimeric sulfite dehydrogenase from Starkeya novella have been grown using vapour diffusion. X-ray diffraction data have been collected from crystals of the native protein at lambda=1.0 Angstrom and close to the iron absorption edge at lambda=1.737 Angstrom. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a=97.5, b=92.5, c=55.9 Angstrom. Native data have been recorded to 1.8 Angstrom resolution and Fe-edge data to 2.5 Angstrom.

A comparison of molecular markers for genetic analysis of macadamia

Various marker systems exist for genetic analysis of horticultural species. Isozymes were first applied to the woody perennial nut crop, macadamia, in the early 1990s. The advent of DNA markers saw the development, for macadamia, of STMS (sequence-tagged microsatellite site), RAPD (randomly amplified polymorphic DNA), and RAF (randomly amplified DNA fingerprinting). The RAF technique typically generates dominant markers, but within the dominant marker profiles, certain primers also amplify multi-allelic co-dominant markers that are suspected to be microsatellites. In this paper, we confirm this for one such marker, and describe how RAF primers can be chosen that amplify one or more putative microsatellites. This approach of genotyping anonymous microsatellite markers via RAF is designated RAMiFi (randomly amplified microsatellite fingerprinting). Several marker systems were compared for the type, amount, and cost-efficiency of the information generated, using data from published studies on macadamia. The markers were also compared for the way they clustered a common set of accessions. The RAMiFi approach was identified as the most efficient and economical. The availability of such a versatile tool offers many advantages for the genetic characterisation of horticultural species.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.