Establishment of cryopreserved gene banks of European chestnut and cork oak

Cryopreservation of selected genotypes of European chestnut and cork oak was carried out in two laboratories in a project involving conservation of field collections. Plant material was selected on the basis of disease resistance (chestnut), growth habit, phytosanitary performance and cork quality (cork oak). The cryopreservation technique comprised of vitrification of shoot apices isolated from in vitro stock shoot cultures (chestnut) and somatic embryos (cork oak). Forty-three out of 46 chestnut genotypes assayed survived the freezing process, but only 63% recovered their capacity to produce new shoots. After completion of multiplication and rooting steps, the surviving shoots produced plants that were morphologically identical to those derived from non-supercooled material. All 51 cork oak genotypes withstood freezing and were able to produce new somatic embryos through a process of secondary embryogenesis. Multiplication and germination of the recovered embryos enabled production of plants that were morphologically identical to those derived from non-supercooled material. In light of the results obtained, long-term cryopreservation of these species is feasible, thereby ensuring conservation of valuable genotypes during field evaluation.     

Simultaneous determination of four biogenic and three volatile amines in anchovy by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

A new method has been developed and validated for the simultaneous determination of four biogenic (putrescine, cadaverine, histamine, and tyramine) and three volatile amines (trimethylamine, triethylamine, and tripropylamine) in anchovy. Separation and determination of the selected compounds were carried out by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), using an electrospray ionization source (ESI) in positive ion mode. Chromatographic separation was carried out using an aqueous solution of formic acid (0.1%) and methanol as mobile phase in gradient mode. The method was validated, and mean recoveries were evaluated at three concentration levels (75, 150, and 250 μg/kg), ranging from 70 to 110% at the three levels assayed. Intra- and interday precision, expressed as relative standard deviation (RSD), were lower than 15% and 20%, respectively. Limits of quantitation (LOQs) were 25 μg/kg for all cases, except for that of TMA, which was set at 60 μg/kg. The developed procedure was applied to determine the target compounds in anchovy samples stored during 7 days at 4 °C, observing the increasing in the concentration of these compounds at longer storage time.     

Oxygen Consumption and Development of Volatile Sulfur Compounds during Bottle Aging of Two Shiraz Wines. Influence of Pre- and Postbottling Controlled Oxygen Exposure

The evolution of different volatile sulfur compounds (VSCs) during bottle maturation of two Shiraz wines submitted to controlled oxygen exposure prior to bottling (through micro-oxygenation, MOX) and postbottling (through the closure) was investigated. H(2)S, methyl mercaptan (MeSH), and dimethyl sulfide (DMS) were found to increase during aging. Lower postbottling oxygen exposure, as obtained by different degrees of oxygen ingress through the closure, resulted in increased H(2)S and methyl mercaptan. In one wine MOX increased the concentration of H(2)S and methyl mercaptan during maturation. Dimethyl disulfide and DMS were not affected by any form of oxygen exposure. Overall, postbottling oxygen had a stronger influence than MOX on the evolution of VSCs. Data suggest that dimethyl disulfide was not a precursor to methyl mercaptan during bottle maturation. For the two wines studied, a consumption of oxygen of 5 mg/L over 12 months was the most effective oxygen exposure regimen to decrease accumulation of MeSH and H(2)S during bottle aging.     

An Exploratory Study of Particulate PAHs in Low-Polluted Urban and Rural Areas of Southwest Spain: Concentrations, Source Assignment, Seasonal Variation and Correlations with Other Air Pollutants

A set of 72?PM₁₀ samples from low-polluted urban and rural locations belonging to the regional air monitoring network of Extremadura (Spain) were collected in a 1?year sampling period. Sample pre-treatment and analytical determination by gas chromatography?Cion trap mass spectrometry were optimised and validated for the analysis of the priority 16 US Environmental Protection Agency polycyclic aromatic hydrocarbons (PAHs). The influence of meteorological conditions (temperature, relative humidity and solar radiation) and other atmospheric pollutants (O₃, NO₂, SO₂, PM₁₀) has been covered in detail and Pearson correlation test were used for this purpose. Spatial distribution of particulate PAHs was evaluated and the comparison with other European sites was also established. Possible emission sources were identified and assigned by using molecular diagnostic criteria.     

Ecotoxicological Assessment of Contaminated River Sites as a Proxy for the Water Framework Directive: an Acid Mine Drainage Case Study

Metal contamination of freshwater bodies resulting from mining activities or deactivated mines is a common problem worldwide such as in Portugal. Bra?al (galena ore) and Palhal (pyrrhotite, chalcopyrite, galena, sphalerite, and pyrite ore), located in a riverside position, are both examples of deactivated mining areas lacking implemented recovery plans since their shutdown in the early mid-1900s. In both mining areas, effluents still flow into two rivers. The purpose of this work was to evaluate the potential hazard posed by the mining effluents to freshwater communities. Therefore, short- and long-term ecotoxicological tests were performed on elutriates from river sediments collected at each site using standard test organisms that cover different functional levels (Vibrio fischeri, Pseudokirchneriella subcapitata, Lemna minor, and Daphnia sp.). The results show that elutriates from the sediments of Palhal were very toxic to all tested species, while in contrast, elutriates from Bra?al showed generally no toxicity for the tested species. Our study highlights the usefulness of using an ecotoxicological approach to help in the prioritization/scoring of the most critical areas impacted by deactivated mines. This ecotoxicological test battery can provide important information about the ecological status of each concerning site before investing in the application of time-consuming and costly methods defined by the Water Framework Directive or can stand as a meaningful complementary analysis.     

Evaluation of preflooding effects on iron extractability and phytoavailability in highly calcareous soil in containers

Previous pot cropping and laboratory incubation experiments were consistent with field observations showing that temporary flooding before cropping can increase the availability of soil Fe to plants. To study the effect of temporary flooding on changes in soil Fe phytoavailability we used 24 highly calcareous, Fe chlorosis–inducing soils to carry out a pot experiment where peanut and chickpea were successively grown after flooding for 30 d. At the end of the cropping experiment, the preflooded soil samples exhibited higher concentrations of acid oxalate‐, citrate/ascorbate‐ and diethylenetriaminepentacetic acid (DTPA)–extractable Fe (Fe_ox, Fe_ca, and Fe_DTPA, respectively) than the control (nonflooded) samples. Also, Fe_ox and Fe_ca exhibited no change by effect of reflooding of the cropped soils or three wetting–drying cycles in freeze‐dried slurries of soils previously incubated anaerobically for several weeks. Leaf chlorophyll concentration (LCC) in both peanut and chickpea was greatly increased by preflooding. The best predictor for LCC was Fe_ox, followed by Fe_ca and Fe_DTPA. The LCC–soil Fe relationships found suggest that the Fe species extracted by oxalate and citrate/ascorbate from preflooded soils were more phytoavailable than those extracted from control soils. However, the increased phytoavailability of extractable Fe forms was seemingly limited to the first crop (peanut). Flooding dramatically increased Fe_DTPA; however, high Fe_DTPA levels did not result in high LCC values, particularly in the second crop. Therefore, this test is a poor predictor of the severity of Fe chlorosis in preflooded soils.     

Anatomy,Morphometry and Radiography in the thoracic limb bones of the Patagonian Huemul Deer (Hippocamelus bisulcus)

The aim of this study was to provide morphometric, anatomic and radiographic data of the thoracic limb bones of the Patagonian Huemul (Hippocamelus bisulcus) including a functional interpretation of this, as a reference for clinical use, biomedical research and teaching purposes. Currently, the Patagonian huemul deer is in danger of becoming extinct due to multiple causes. Research carried out for its conservation has focused mainly on its ecology and pathology, leaving gaps in biological knowledge, which is basic and important for its comprehension. This study was conducted to reveal the gross osteology and radiology features of the thoracic limb bones of the Patagonian huemul deer. The osteological findings suggest the presence of powerful flexor muscles in the scapulohumeral and elbow joints, useful to cushion the jumps. Also, the principal nutrient foramen of Patagonian huemul differs in position with respect to domestic ungulates, which may be important to consider during surgical procedures. Finally, the radiographic data can provide new information about the tissue loading conditions in Patagonian huemul, so that this new knowledge can be of great importance for a better understanding of mechanically induced or adaptive changes in bone produced by habitat or other ecological phenomena.     

Effects of irrigation water quality on loquat plant nutrition: Sensitivity of loquat plant to salinity

An investigation was conducted to study the possible use of municipal wastewater for watering loquat plants, and to determine the effects of this water on nutrient status of two‐year‐old loquat plants (Eriobotriae japonica L.) during two complete vegetative cycles. The plants, grafted on franco and Anger stocks, were planted on major soils of the area of La Marina Babea. Plants (grafted on franco stock), irrigated with this wastewater, suffered from foliar necrosis, and all of them died at the end of the experiment, whereas only 12.5% of the plants watered with municipal drinking water died at the same period of time. The Anger stock proved to be much more resistant to salinity than the franco stock. Therefore, medium quality waters (wastewater and saline water) can be used for the irrigation of loquat trees grafted on Anger stock, without causing damages on plant nutrition.     

Use of CLSA and SPME-headspace techniques followed by GC-MS analysis to extract and identify the floral odorants

Flowers of Ziziphus spina christi are known to be attractive for parasitoids and predators. In Y-tube olfactometer experiments, the dried flowers attracted significantly (p < 0.001) the female parasitoids Aphelinus abdominalis. The flower volatile compounds were analyzed to understand which compounds could be specifically responsible for this attractiveness. The volatile compounds of Ziziphus flowers were extracted by closed-loop-stripping-analysis (CLSA) and also by solid phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) analysis. The main chemical classes of the volatile compounds are aldehydes, monoterpene-alcohols, ketones and hydrocarbons. Flower extract and some specific compounds will be further tested for their responsiveness to predators and parasitoids in behavioural and electrophysiological experiments.