Comparing two different superovulation protocols on ovarian activity and fecal glucocorticoid levels in the brown brocket deer (Mazama gouazoubira)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze)

Brazilian pine (Araucaria angustifolia (Bert) O. Ktze) is the only native conifer species with economic importance in Brazil. Recently, due to intensive exploitation Brazilian pine was included in the official list of endangered Brazilian plants, under the "vulnerable" category. Biotechnology tools like somatic embryogenesis (SE) are potentially useful for mass clonal propagation and ex situ conservation strategies of commercial and endangered plant species. In spite of that, numerous obstacles still hamper the full application of SE technology for a wider range of species, including Brazilian pine. To enhance somatic embryogenesis in Brazilian pine and to gain a better understanding of the molecular events associated with somatic embryo development, we analyzed the steady-state transcript levels of genes known to regulate somatic embryogenesis using semiquantitative reverse transcription polymerase chain reaction (sqRT-PCR). These genes included Argonaute (AaAGO), Cup-shaped cotyledon1 (AaCUC), wushel-related WOX (AaWOX), a S-locus lectin protein kinase (AaLecK), Scarecrow- like (AaSCR), Vicilin 7S (AaVIC), Leafy Cotyledon 1 (AaLEC), and a Reversible glycosylated polypeptide (AaRGP). Expression patterns of these selected genes were investigated in embryogenic cultures undergoing different stages of embryogenesis, and all the way to maturation. Up-regulation of AaAGO, AaCUC, AaWOX, AaLecK, and AaVIC was observed during transition of somatic embryos from stage I to stage II. During the maintenance phase of somatic embryogenesis, expression of AaAGO and AaSCR, but not AaRPG and AaLEC genes was influenced by presence/ absence of plant growth regulators, both auxins and cytokinins. The results presented here provide new insights on the molecular mechanisms responsible for somatic embryo formation, and how selected genes may be used as molecular markers for Brazilian pine embryogenesis.

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

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

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.

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

Cinchona officinalis (Rubiaceae), especie endémica del Valle de Loja, ubicado en la región sur del Ecuador, es un recurso forestal de importancia medicinal y ecológica, además la especie ha sido catalogada como planta nacional y es un ícono de la región sur por su aporte a la farmacopea mundial. Esta especie, entre los siglos XVII-XIX sufrió una gran presión en sus poblaciones debido a la extracción masiva de la corteza para la cura del paludismo. Aunque la actividad extractiva generó grandes ingresos a la Corona Española y a la región Sur del Ecuador, ésta fue poco o nada sustentable ecológicamente, provocando la desaparición de la especie en muchos sitios de la provincia, pues, en su momento, no se consideraron alternativas de recuperación de las poblaciones naturales. Actualmente la extracción y consumo de la corteza en la zona de origen es baja o nula, sin embargo esta zona enfrenta nuevas amenazas. La deforestación a causa de proyectos de desarrollo en infraestructuras, la práctica de actividades agrícolas y de ganadería, y los efectos del cambio climático han ocasionado, en estos últimos años, la fragmentación de los ecosistemas. La mayoría de los bosques del sur del Ecuador se han convertido en parches aislados (los bosques en los que se distribuye C. officinalis no son la excepción) siendo esta la principal causa para que la especie se encuentre en estado de amenaza. Los individuos de la especie tienen una alta capacidad de rebrote y producen semillas durante todo el año; sin embargo la capacidad germinativa y la tasa de sobrevivencia son bajas, además de estas dificultades la especie requiere de la asociación con otras especies vegetales para su desarrollo, lo cual ha limitado su distribución en pequeños parches aislados. Con esta problemática, la recuperación natural de las poblaciones es una necesidad evidente. Varios trabajos y esfuerzos previos se han realizado a nivel local: i. Identificación de la distribución actual y potencial; ii. Determinación de la fenología y fructificación iii. Programas de educación ambiental, iv. Análisis moleculares para determinar la diversidad genética. v. Ensayos de propagación vegetativa; y otras acciones de tipo cultural. No obstante, el estado de conservación y manejo de las poblaciones naturales no ha mejorado significativamente, siendo necesaria la aplicación de estrategias integradas de conservación in situ y ex situ, que permitan la recuperación y permanencia de las poblaciones naturales a largo plazo. El presente trabajo tiene como fin dar alternativas para el cultivo de tejidos in vitro de Cinchona officinalis centrados en la propagación masiva a partir de semillas, análisis de la fidelidad genética y alternativas de conservación de tejidos. Los objetivos específicos que se plantean son: i. Analizar el proceso de germinación y proliferación in vitro. ii. Evaluar la estabilidad genética en explantes cultivados in vitro, mediante marcadores ISSR. iii. Establecer protocolos de conservación in vitro mediante limitación del crecimiento y criopreservación de segmentos nodales y yemas. Los resultados más significativos de esta investigación fueron: i. El desarrollo de protocolos eficientes para mejorar los porcentajes de germinación y la proliferación de brotes en explantos cultivados in vitro. Para evaluar el efecto de los fenoles sobre la germinación, se determinó el contenido total de fenoles y el porcentaje de germinación en semillas de C. officinalis comparados con una especie de control, C. pubescens. Para inducir a proliferación, se utilizaron segmentos nodales de plántulas germinadas in vitro en medio Gamborg (1968) suplementado con diferentes combinaciones de reguladores de crecimiento (auxinas y citoquininas). Los resultados obtenidos sugieren que el contenido de compuestos fenólicos es alto en las semillas de C. officinalis en comparación con las semillas de C. pubescens. Estos fenoles pueden eliminarse con peróxido de hidrógeno o con lavados de agua para estimular la germinación. La formación de nuevos brotes y callos en la mayoría de las combinaciones de reguladores de crecimiento se observó en un período de 45 días. El mayor porcentaje de proliferación de brotes, formación de callos y presencia de brotes adventicios se obtuvo en medio Gamborg (B5) suplementado con 5.0 mg/l 6-bencil-aminopurina y 3.0 mg/l de ácido indol-3-butírico. ii. La evaluación de la fidelidad genética de los explantes obtenidos con distintas combinaciones de reguladores de crecimiento vegetal y diversos subcultivos. Se realizó el seguimiento a los explantes obtenidos de la fase anterior, determinando el índice de multiplicación y analizando la fidelidad genética de los tejidos obtenidos por las dos vías regenerativas: brotación directa y regeneración de brotes a partir de callos. Este análisis se realizó por amplificación mediante PCR de las secuencias ubicadas entre microsatélites-ISSR (Inter simple sequence repeat). El medio Gamborg (B5) con 3.0 mg/l de AIB y 5.0 mg/l de BAP usado como medio de inducción en la primera etapa de cultivo generó el mayor índice de proliferación (11.5). Un total de 13 marcadores ISSR fueron analizados, 6 de éstos fueron polimórficos. El mayor porcentaje de variación somaclonal fue inducido en presencia de 1.0 mg/l 2,4-D combinado con 0.2 mg/l Kin con un 1.8% en el segundo sub-cultivo de regeneración, la cual incrementó a 3.6% en el tercer sub-cultivo. Todas las combinaciones con presencia de 2,4-D produjeron la formación de callos y presentaron variación genética. Por su parte la fidelidad genética se mantuvo en los sistemas de propagación directa a través de la formación de brotes a partir de meristemos preformados. iii. El establecimiento de protocolos de conservación in vitro y crioconservación de segmentos nodales y yemas. Para la conservación limitando el crecimiento, se cultivaron segmentos nodales en los medios MS y B5 en tres concentraciones de sus componentes (25, 50 y 100%); y en medio B5 más agentes osmóticos como el manitol, sorbitol y sacarosa en diferentes concentraciones (2, 4 y 8%); los cultivos se mantuvieron por 12 meses sin subcultivos. Para el establecimiento de protocolos para la crioconservación (paralización del metabolismo) se usaron yemas axilares y apicales a las cuales se les aplicaron los métodos de encapsulación-deshidratación y vitrificación. La efectividad de los protocolos usados se determinó en función de la sobrevivencia, reducción del crecimiento y regeneración. Los resultados obtenidos en este apartado reflejan que un crecimiento limitado puede mantener tejidos durante 12 meses de almacenamiento, usando medio B5 más manitol entre 2 y 8%. En los protocolos de crioconservación, se obtuvo el mayor porcentaje de recuperación tras la congelación en NL en el tratamiento control seguido por el método crioprotector de encapsulación-deshidratación. Este trabajo brinda alternativas para la propagación de C. officinalis bajo condiciones in vitro, partiendo de material vegetal con alta diversidad genética. El material propagado puede ser fuente de germoplasma para la recuperación y reforzamiento de las poblaciones naturales así como una alternativa de producción para las comunidades locales debido a la demanda actual de corteza de la zona de origen para la elaboración de agua tónica. ABSTRACT Cinchona officinalis (Rubiaceae) is endemic to the Loja Valley, located in the southern area of Ecuador. The importance of this plant as medical and ecological resource is so great that it has been designated as the national flower and is an icon of the southern region for its contribution to the world pharmacopoeia. Between XVII-XIX centuries its population suffered great reduction due to massive harvesting of the bark to cure malaria. Although extraction activity generated large revenues to the Spanish Crown and the southern region of Ecuador, this was not ecologically sustainable, causing the disappearance of the species in many areas of the province, because during that time alternatives to prevent extinction and recover natural populations were not taken in account. Currently the extraction and consumption of bark in the area of origin is almost absent, but this species faces new threats. Deforestation due to infrastructure development, the practice of farming and ranching, and the effects of climate change had led to the fragmentation of ecosystems during the recent years. Most of the forests of southern Ecuador have become isolated patches, including those where C. officinalis is diffused. The lack of suitable habitat is today the main threat for the species. The species has a high capacity for regeneration and produces seeds throughout the year, but the germination rate is low and the growth is slow. In addition, the species requires the association with other plant species to develop. All these factors had limited its distribution to small isolated patches. The natural recovery of populations is essential to face this problem. Several studies and previous efforts had been made at local level: i. Identification of current and potential distribution; ii. Phenology determination. iii. Environmental education programs, iv. Molecular analisis to determine the genetic diversity. v. Testing of vegetative propagation; and other actions of cultural nature. Despite these efforts, the state of conservation and management of natural populations has not improved significantly. Implementation of integrated in situ and ex situ conservation strategies for the recovery and permanence of long-term natural populations is still needed. This work aims to provide alternatives for in vitro culture of tissue of Cinchona officinalis focused on mass propagation from seeds, genetic fidelity analysis and tissue conservation alternatives. The specific aims are: i. Analyze the process of germination and proliferation in vitro. ii. To evaluate the genetic stability of the explants cultured in vitro by ISSR markers. iii. Establish protocols for in vitro conservation by limiting growth and cryopreservation of nodal segments and buds. The most significant results of this research were: i. The development of efficient protocols to improve germination rates and proliferation of buds in explants cultured in vitro. To study the effect of phenols on germination, the total phenolic content and percentage germination was measured in C. officinalis and in a control species, C. pubescens, for comparison. The content of phenolic compounds in C. officinalis seeds is higher than in C. pubescens. These phenols can be removed with hydrogen peroxide or water washes to stimulate germination. To analyze the regeneration, we used nodal explants from seedlings germinated in vitro on Gamborg medium (1968) supplemented with different combinations of growth regulators (auxins and cytokinins) to induce proliferation. The formation of new shoots and calluses was observed within a period of 45 days in most combinations of growth regulators. The highest percentage of shoot proliferation, callus formation and adventitious buds were obtained in B5 medium supplemented with 5.0 mg/l 6-benzyl-aminopurine and 3.0 mg/l indole-3-butyric acid. ii. Evaluating genetic fidelity explants obtained with various combinations of plant growth regulators and different subcultures. The genetic fidelity was analyzed in tissues obtained by the two regenerative pathways: direct sprouting and shoot regeneration from callus. This analysis was performed by PCR amplification of the sequences located between microsatellite-ISSR (Inter Simple Sequence Repeat). Among a total of 13 ISSR markers analyzed, 6 were polymorphic. The highest percentage of somaclonal variation was induced in the presence of 1.0 mg/l 2,4-D combined with 0.2 mg/l Kin with 1.8% in the second round of regeneration, and increased to 3.6% in the third round. The presence of 2,4-D induced genetic variation in all the combinations of growth regulators. Meanwhile genetic fidelity remained systems propagation through direct shoot formation from meristems preformed. iii. Establishing conservation protocols in vitro and cryoconservation of nodal segments and buds. For medium-term conservation (limited growth) nodal segments were cultured in MS and B5 media at three concentrations (25, 50 and 100%); we tested B5 medium with different concentrations of osmotic agents such as mannitol, sorbitol and sucrose (2, 4 and 8%); cultures were maintained for 12 months with regular subculturing. To establish protocols for cryoconservation (cessation of metabolism) different methods of encapsulation-dehydration and vitrification were applied to axillary and apical buds. The effectiveness of the used protocols is determined based on the survival, growth and regeneration success. The results show that these tissues can be maintained in storage for 12 months, using B5 medium plus mannitol between 2 and 8%. The cryoconservation protocol with highest percentage of recovery was obtained by contral treatment, followed by freezing in NL with encapsulation-dehydration method. This work provides alternatives for the propagation in vitro of C. officinalis, starting from plant material with high genetic diversity. The obtained material represents a source of germplasm to support the recovery and strengthening of natural populations as well as a creation of alternative sources for local communities due to the current demand of bark for the preparation of tonic water.

Tetrazolium test for seed viability and germinability of Melocactus ernestii Vaupel subsp. ernestii and Melocactus zehntneri (Briton & Rose) Luetzelb. (Cactaceae)

Despite its ornamental value, some Melocactus species (Cactaceae) are threatened by several factors and only sexual propagation is possible. Thus, artificial seed banks are an appropriate method for their ex situ conservation. Suitable methods to germinate and to monitor viability are necessary for the seeds of these species. This work explored the use of tetrazolium test for monitoring seed viability of two Melocactus species. There was a correlation between the percentage of stained embryos, considering different cover area or tone stain, and the germination percentage. Unviable embryos did not stain. The embryos curved shape can explain the partial stain on the most of cases.

Domestication for Conservation of an Endangered Species: The Case of the Wollemi Pine

A small population of tall slender conifers was discovered in 1994 in a deep rainforest canyon of the Wollemi National Park, New SouthWales, Australia. The living trees closely resembled fossils that were more than 65 million years old, and this ‘living fossil’ was recognised as a third extant genus in the Araucariaceae (Araucaria, Agathis and now Wollemia). The species was named the Wollemi pine (W. nobilis). Extensive searches uncovered very few populations, with the total number of adult trees being less than 100. Ex situ collections were quickly established in Sydney as part of the Wollemi Pine Recovery Plan. The majority of the ex situ population was later transferred to our custom-built facility in Queensland for commercial multiplication. Domestication has relied very heavily on the species’ amenability to vegetative propagation because seed collection from the natural populations is dangerous, expensive, and undesirable for conservation reasons. Early propagation success was poor, with only about 25% of cuttings producing roots. However, small increases in propagation success have a very large impact on a domestication program because plant production can be modelled on an exponential curve where each rooted cutting develops into a mother plant that, in turn, provides more rooted cuttings. An extensive research program elevated rooting percentages to greater than 80% and also provided in vitro methods for plant multiplication. These successes have enabled international release of the Wollemi pine as a new and attractive species for ornamental horticulture.

The effectiveness of botanic garden collections in supporting plant conservation: a European case study

Europe has the greatest concentration of botanic gardens in the world, they cultivate extensive collections of plants that include samples of European threatened plant species. This study looks at the effectiveness of these collections in supporting species conservation. A three part study is presented: (1) the results of a survey and assessment of threatened plants in botanic gardens, as defined by the Bern Convention; (2) case studies illustrating current issues in the ex situ management of European threatened plant species; and (3) presentation of policy recommendations on further improving botanic garden contributions to European plant conservation. The survey indicated that of 119 European botanic gardens in 29 European countries, 105 are cultivating 308 of the 573 threatened plant species listed by the Bern Convention. The survey identified 25 botanic gardens in 14 countries undertaking 51 conservation projects focused on 27 Bern listed species. In particular this survey has established that the majority of taxa are held in a small number of collections, dominated by non-wild origin accessions, and are not adequately documented. The majority of specimens in botanic gardens are cultivated out of the range country and not contributing to a specific conservation project. We review the genetic representation and documentation of origin in collections. Existing plant collections contain representatives of populations, now lost in the wild and maintain samples of at least nine European plant taxa identified as 'Extinct in the Wild'. However, inadequate standards of record keeping has compromised the conservation value of many collections. We highlight the dangers of hybridisation and disease in ex situ collections. The results suggest that botanic garden collections are skewed towards horticulturally robust and ornamental species and do not fully reflect priorities as defined by the Bern Convention. Recognising the limitations of traditional botanic garden collections we propose that botanic gardens more effectively utilise their two core competencies, namely scientific horticulture and public display and interpretation. The unique horticultural skills resident in European botanic gardens could be more effectively utilised through the application of horticulture to the management of wild populations.

Caracterização in situ e diversidade genética de algodoeiros mocós (Gossypium hissutum raça marie galante) da Região Nordeste do Brasil

Brazil is one of the major centers of diversity for polyploid cotton plants; these plants belong to the genus Gossypium, which has three known species: G. hirsutum, G. barbadense and G. mustelinum. The Northeast is the only region where the three species occur, the last group being endemic. Northeast s cotton plants can be important sources of variability for genetic breeding. It is believed that great part of local diversity is being lost, due to economic, political, cultural and agricultural problems. In an attempt to mitigate this loss and delineate conservation strategies it is necessary to know how the species are found where they occur. The objective was to characterize and determine how plants are maintained in situ in the states of Maranhão, Piauí, Ceará, Rio Grande do Norte and Paraíba at the beginning of the XXI century. The in situ characterization of G. hirsutum and G. barbadense was conducted through structured interviews with the cotton plants owners and through the analysis of the environment. The data were collected during expeditions undertaken between the years 2004 to 2005. Twenty-two plants were collected in the state of Paraíba, forty-four in the state of Rio Grande do Norte, one hundred and forty-six in the state of Ceará, forty in the state of Maranhão and ninety-one plants in the state of Piauí. All plants collected in the states of Paraíba and Rio Grande do Norte belonged to moco type. Moco cotton plants also predominated in the other states, representing 92%, 62% and 78% of plants collected in Ceará, Piauí and Maranhão, respectively. The other cotton plants collected belong to the species G. barbadense. The cotton plants were found in situ as dooryard plants, roads side, feral populations, cultivation or local varieties. Great part were dooryard plants (45.2%), being major in Piauí and Maranhão. Cultivation predominated in Ceará; in Rio Grande do Norte feral populations were the most frequent and, in Paraíba, local varieties. The maintenance of moco plants is related, mainly, to the phytotherapic domestic use (20.9%) and to confection of lamp wicks (29.7%). Few inhabitants in Paraíba, Rio Grande do Norte, Piauí and none in Maranhão used harvest the plants, storage the seeds or gin; however, in Ceará, 40.5% of owners affirmed that they harvested and commercialized the fiber. It was found that the maintenance of species is dependent of the fragile cultural habits of local inhabitants, therefore the maintenance in situ is not a suitable way to conservation of genetic resources. The efforts must be directed to the continuity of collections, maintenance and characterization ex situ

Caracterização genética e in situ de Gossypium barbadense na região norte do Brasil

Brazil has been considered one of the diversity centers of Gossypium barbadense species. It is believed that a relatively big erosion genetic process occurs with the species, due to economic, cultural and agricultural problems. A local diagnostic about species situation is the first step for reducing the diversity loss and establishing conservation strategies in situ. This research aimed the identification of the presence of Gossypium populations, characterization, determination of the main risks and collection of the accesses to store in germoplam banks, in Para and Amapa States. Expeditions were conducted in November 2004. An interview was carried out with the plant proprietor for characterizing in situ of G. barbadense species and of the environment where the plants were inserted. On hundred seventy nine plants in 22 municipal districts were collected in Para State and 117 plants in nine municipal districts in Amapa State. The majority of plants belong to G. barbadense species (98% in Amapa and 94% in Para). Plants occur in back yards, beside roads and spontaneously. That ones from back yards were more abundant (97% in Amapa and 95% in Para) and maintained as medicinal plants as the principal reason. Plants in natural environments in both states evaluated were not found, therefore, the creation of reserves and the application of others conventional methods of maintenance in situ are not applicable. The plant proprietors do not use to store or process seeds. Seed storage was reported as a practice by only 1% of the plant proprietors from Para and 11% from Amapa. The most plants collected were from two to three years of age (58% in Amapa and 93% in Para). As conclusions G. barbadense is the species most spread in the two studied states and are found in back yards. In Amapa State the botanical variety barbadense or Quebradinho is predominant, whereas in Para State the predominant variety is brasiliense or Rim-de-boi. Adequate conservation of thestudied species must be carried out in germoplasm collections maintained ex situ

Polinização de Cacauí (Theobroma speciosum Willd. ex Spreng. - Sterculiaceae): implicações para conservação

O cacauí (Theobroma speciosum) é uma fruteira típica da Região Norte, cujas flores sapromiofílicas atraem dípteros. Os recursos que os insetos utilizam são muito variados e entre eles estão as flores, as quais podem ser utilizadas como alimento e local para reprodução. Este trabalho visou fazer uma comparação do sistema de polinização de T. speciosum em duas áreas de preservação distintas, contribuindo para a avaliação das estratégias de conservação da espécie. O trabalho foi realizado nos anos de 2000 a 2002, no Centro de Pesquisa do Trópico Úmido da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA/CPATU) e na Estação Científica Ferreira Penna (ECFPn/MPEG). Foram realizados estudos sobre estrutura populacional e fenologia de T. speciosum nas duas áreas de preservação, observação comportamental dos insetos no campo, coletas de insetos visitantes das flores de cacauí, análise de carreamento de pólen pelos insetos e verificação dos insetos emergentes no laboratório. As árvores de cacauí, em ambos os locais, florescem em períodos mais secos e frutificam em períodos mais úmidos. As populações de cacauí nas duas áreas diferem pela distribuição espacial e etária dos seus indivíduos. A falta de jovens e subadultos na área de preservação ex situ, mostra falha no "tum over' populacional, demonstrando fragilidade no sistema estabelecido. As inflorescências de T. speciosum são utilizadas por uma grande diversidade de invertebrados. Nove ordens de insetos, e pelo menos duas ordens de outros invertebrados, visitaram as flores de cacau'. As ordens com maior quantidade de indivíduos foram Hymenoptera, Thysanoptera, Diptera e Coleoptera. Os visitantes das flores de cacauí foram classificados como: oportunistas, predadores e polinizadores. Drosofilideos foram indicados como polinizadores de T speciosum, com a possibilidade da participação de alguns forídeos. As guildas dos drosofilídeos foram distintas nas duas áreas de preservação e mais diversa na área de preservação in situ. Quatro espécies foram comuns as duas áreas. A perda de diversidade e alteração na composição na guilda de polinizadores parece não ter afetado a população de T. speciosum na área de preservação ex situ. No caso em estudo o sistema de polinização apesar de profundamente alterado, ainda apresenta sinais de viabilidade.

Estudo hierárquico do sistema de reprodução entre e dentro de frutos, fluxo de pólen e estrutura genética espacial em um fragmento e em árvores isoladas na pastagem de hymenaea stigonocarpa mart. ex hayne na região de cerrado

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Mapping Genetic Diversity of Cherimoya (Annona cherimola Mill.): Application of Spatial Analysis for Conservation and Use of Plant Genetic Resources.

There is a growing call for inventories that evaluate geographic patterns in diversity of plant genetic resources maintained on farm and in species' natural populations in order to enhance their use and conservation. Such evaluations are relevant for useful tropical and subtropical tree species, as many of these species are still undomesticated, or in incipient stages of domestication and local populations can offer yet-unknown traits of high value to further domestication. For many outcrossing species, such as most trees, inbreeding depression can be an issue, and genetic diversity is important to sustain local production. Diversity is also crucial for species to adapt to environmental changes. This paper explores the possibilities of incorporating molecular marker data into Geographic Information Systems (GIS) to allow visualization and better understanding of spatial patterns of genetic diversity as a key input to optimize conservation and use of plant genetic resources, based on a case study of cherimoya (Annona cherimola Mill.), a Neotropical fruit tree species. We present spatial analyses to (1) improve the understanding of spatial distribution of genetic diversity of cherimoya natural stands and cultivated trees in Ecuador, Bolivia and Peru based on microsatellite molecular markers (SSRs); and (2) formulate optimal conservation strategies by revealing priority areas for in situ conservation, and identifying existing diversity gaps in ex situ collections. We found high levels of allelic richness, locally common alleles and expected heterozygosity in cherimoya's putative centre of origin, southern Ecuador and northern Peru, whereas levels of diversity in southern Peru and especially in Bolivia were significantly lower. The application of GIS on a large microsatellite dataset allows a more detailed prioritization of areas for in situ conservation and targeted collection across the Andean distribution range of cherimoya than previous studies could do, i.e. at province and department level in Ecuador and Peru, respectively.

Cryopreservation of somatic embryos for avocado germplasm conservation

Presently avocado germplasm is conserved ex situ in the form of field repositories across the globe including Australia. The maintenance of germplasm in the field is costly, labour and land intensive, exposed to natural disasters and always at the risk of abiotic and biotic stresses. The aim of this study was to overcome these problems using cryopreservation to store avocado (Persea americana Mill.) somatic embryos (SE). Two vitrification-based methods of cryopreservation were optimised (cryovial and droplet-vitrification) using four avocado cultivars (‘A10′, ‘Reed’, ‘Velvick’ and ‘Duke-7′). SE of the four cultivars were stored for short-term (one hour) in liquid nitrogen using the cryovial-vitrification method and showed a viability of 91%, 73%, 86% and 80% respectively. While when using the droplet vitrification method viabilities of 100%, 85% and 93% were recorded for ‘A10′, ‘Reed’ and ‘Velvick’. For long-term storage, SE of cultivars ‘A10′, ‘Reed’ and ‘Velvick’ were successfully recovered with viability of 65–100% after 3 months of LN storage. For cultivar ‘Reed’ and ‘Velvick’ SE were recovered after 12 months of LN storage with viability of 67% and 59%, respectively. The outcome of this work contributes towards the establishment of a cryopreservation protocol that is applicable across multiple avocado cultivars.