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Identification and functional characterization of cation-chloride cotransporters in plants
No full textChloride (Cl-) is an essential nutrient and one of the most abundant inorganic anions in plant tissues. We have cloned an Arabidopsis thaliana cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Deduced plant CCC proteins are highly conserved, and phylogenetic analyses revealed their relationships to the sub-family of animal K+:Cl- cotransporters. In Xenopus laevis oocytes, the A. thaliana CCC protein (At CCC) catalysed the co-ordinated symport of K+, Na+ and Cl-, and this transport activity was inhibited by the 'loop' diuretic bumetanide, a specific inhibitor of vertebrate Na+:K+:Cl- cotransporters, indicating that At CCC encodes for a bona fide Na+:K+:Cl- cotransporter. Analysis of At CCC promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules and anthers. Plants homozygous for two independent T-DNA insertions in the CCC gene exhibited shorter organs such as inflorescence stems, roots, leaves and siliques. The elongation zone of the inflorescence stem of ccc plants often necrosed during bolt emergence, while seed production was strongly impaired. In addition, ccc plants exhibited defective Cl- homeostasis under high salinity, as they accumulated higher and lower Cl- amounts in shoots and roots, respectively, than the treated wild type, suggesting At CCC involvement in long-distance Cl- transport. Compelling evidence is provided on the occurrence of cation-chloride cotransporters in the plant kingdom and their significant role in major plant developmental processes and Cl- homeostasis
Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus
No full textA nucleic acid sequence-based amplification method Coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens. (c) 2006 Elsevier B.V. All rights reserved
Efficiency of in vivo recovery of rabbit ovules by laparoscopy
No full textLa obtención de óvulos es una etapa imprescindible tanto para los programas de
conservación ex situ de recursos genéticos (Holt y Pickard et al., 1999; FAO), como
para su aplicación en diferentes técnicas reproductivas como ICSI, FIV o transferencia
nuclear. En conejo, es habitual que las recuperaciones de ovocitos tengan lugar postmortem. Para la obtención de ovocitos inmaduros se suelen utilizar las técnicas de
cortes foliculares o “slicing” (Lorenzo et al., 1997) o de punción y aspiración de los
mismos (Viudes de Castro et al., 1999; Mocé et al. 2002). En cambio, si se desea
recuperar óvulos maduros, la técnica utilizada con más frecuencia es la recuperación
post-mortem mediante lavado oviductal realizado a las 14 horas tras la inducción de la
ovulación (Viudes de Castro et al. 2005; Zhao et al., 2006). La recuperación in vivo
llevada a cabo por laparotomía (Liu et al. 2004) es bastante inusual.
La obtención de embriones se ha llevado a cabo en numerosas ocasiones in vivo
por laparotomías (Forcada y López, 2000) y laparoscopias repetidas (Besenfelder et
al., 1998; Lavara et al., 2005; Mehaisen et al., 2006) para optimizar el número de
embriones recuperados por hembra. Debido a la ventaja que supone recuperar in vivo,
especialmente cuando se utilizan poblaciones con escaso número de individuos o
animales de gran valor, en este trabajo planteamos la recuperación de óvulos maduros
por laparoscopia repetida en una misma hembra, haciendo uso de una técnica similar
a la utilizada en la recuperación in vivo de embriones por laparoscopia
Safety assessment of transgenic plums and grapevines expressing viral coat protein genes: New insights into real environmental impact of perennial plants engineered for virus resistance
No full textThe potential impact of transgenic plums and grapevines expressing viral coat protein (CP) gene constructs on the diversity and dynamics of virus populations was assessed under open and confined conditions in the frame of a research program sponsored by the European Commission. Across all field trials conducted in different locations (France, Romania, and Spain) and environments (continental and Mediterranean), transgenic plums expressing the CP gene of Plum pox virus (PPV) and transgenic grapevines expressing the CP gene of Grapevine Janleaf virus (GFLV) had no detectable effect on the emergence of recombinant PPV and GFLV species over eight-ten and three years, respectively. Also, no statistically significant difference was found in the number and type of aphids, including viruliferous individuals, and other arthropods that visited transgenic and nontransgenic plum trees. In addition, Apple chlorotic leaf spot virus, Prune dwarf virus, and Prunus necrotic ringspot virus did not influence the stability of the engineered resistance to PPV in co-infected transgenic plums over three dormancy periods. Further, under confined conditions, no recombinant virus was found to detectable level in transgenic grapevines expressing the CP gene of Grapevine virus A (GVA) or Grapevine virus B (GVB) that were challenged with the homologous or heterologous virus, despite high accumulation of transgene transcripts. Also, translocation of transgene-derived products, i.e. protein, mRNAs and siRNAs, did not occur to detectable level from transgenic grapevine rootstocks expressing the GFLV CP gene to nontransgenic scions. Altogether, our transgenic plums and grapevines expressing viral genes had a neutral impact on virus populations and non-target organisms over extended time. These findings provide new insights into the environmental impact of transgenic perennial crops engineered for virus resistance. It is expected that they will assist national and international regulatory authorities in making scientifically based decisions for the release of virus-resistant transgenic crops
El programa de mejora genética del albaricoquero en el IVIA: primeras obtenciones
Get PDFLa producción de albaricoques en la Comunidad Valenciana (CV) se ha reducido a la tercera parte en los últimos 10 anos, debido principalmente a la expansión del virus de la Sharka. Se describen en este trabajo las causas de la importancia económica de esta enfermedad, su detección y difusión en España, los intentos de erradicación en la CV y las razones que explican por qué han fallado dichos intentos. Se presenta el programa de obtención de variedades resistentes del IVIA como la única forma de convivir con la enfermedad. Se describen los objetivos del programa de cruzamientos, los genitores utilizados, las etapas del proceso de selección de las descendencias, el método de determinación de la resistencia y las primeras obtenciones, algunas de las cuales van a ser presentadas a Registro para que puedan ser comercializadas en breve tiempo
SaPI operon I is required fpr SaPI packaging and is controlled by LexA
No full textTransfer of Staphylococcus aureus pathogenicity islands (SaPls) is directly controlled by the cellular repressor LexA. We have found that transcription of the SaPIbov1 operon I is repressed by LexA and is therefore SOS-induced. Two copies of the LexA binding site consensus (Cheo box) are present in the 5' region of this operon, at the same location in all of 15 different SaPIs analysed. Both of these boxes bind LexA protein. Furthermore, replacement of the chromosomal lexA with a non-cleavable mutant LexA (G94E) greatly diminished expression of SaPlbovl operon I and differentially reduced the production of SaPI transducing particles in comparison with the production of plaque-forming particles. In concordance with this finding, deletion of operon I blocked the formation of SaP1 transducing particles but had no effect on replication of the island. Operon I contains a gene encoding a homologue of the phage terminase small subunit plus two other genes that direct the assembly of the small sized SaPlbovl capsids. Interestingly, mutations affecting the latter two genes were not defective in SaPI transfer, but rather encapsidated the island in full-sized phage heads, which would have to contain a multimeric SaPI genome
Evaluation of plant-based water status indicators in mature apple trees under field conditions
No full textThe performance of different indicators of plant water status as a tool for irrigation management was evaluated in mature field grown 'Golden Delicious' apple trees during the late summer of 1998. Control (C) and stress (S) treatments were studied. In the C treatment trees were irrigated daily at 100% ETc whereas in the S treatment water was withheld during 31 days (DOY's 236-266). Predawn water potential (Psi(pd)) and midday stem water potential (Psi(stem)) were measured several times a week during the experimental period. Three daily measurements of stomatal conductance (g(s)) and stem water potential were made during five consecutive days in mid-September. Trunk diameter changes (TDC) were recorded by LVDT sensors, and from these measurements, maximum daily shrinkage (MDS), daily growth (DG), and cumulative growth (CG) were calculated. Midday Psi(stem) showed the best ratio between the response to moderate water stress and tree variability ("signal/noise" ratio) among the indicators studied here, followed closely by Psi(pd). On the other hand, the poorest water status indicator was g(s). Due to the low trunk growth rate of the trees, and its high variability, DG and CG were not adequate indicators. MDS showed a lower sensitivity to water stress and a higher variability (CV = 0.19) than midday Psi(stem) (CV = 0.08) and Psi(pd) (CV = 0.10). However, MDS correlated well with ET0 and with midday Psi(stem) (R 2= 0.79) thus, making this parameter an interesting and promising tool for irrigation management in apple orchards. More research needs to be done in order to define reference values for MDS and plant water potential indicators, in relation to evaporative conditions and in different phenological periods, and to quantify the relationship between water status indicators values and apple tree yield and fruit quality
Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism
No full textUnlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts
Effects of X-ray irradiation and sodium carbonate treatments on postharvest Penicillium decay and quality attributes of clementine mandarins
No full textThe integration of sodium carbonate (SC; dips at 20 °C for 150 s in aqueous 3% SC solutions) treatments and X-ray irradiation (at doses of 510 and 875 Gy) was evaluated on artificially inoculated ‘Clemenules’ clementine mandarins for the control of postharvest green and blue molds, caused by Penicillium digitatum and Penicillium italicum, respectively. Although significant, the reduction of both disease incidence (number of infected fruit) and severity (lesion diameter) on fruit either incubated at 20 °C for 7 days or cold-stored at 5 °C for 21 days was not sufficient for satisfactory disease control under hypothetical commercial conditions. Therefore, the combined treatments could not be a substitute for conventional chemical fungicides. However, pathogen sporulation was greatly inhibited on infected clementines, thus X-irradiation could be of value for management of Penicillium resistant strains and to reduce inoculum levels in citrus packinghouses. X-ray irradiation at 195, 395, 510, and 875 Gy did not influence either decay incidence or the area under the disease progress curve (AUDPC) of lesions of green and blue molds on mandarins inoculated with the pathogens 2, 3, or 6 days after irradiation and incubated for 7 days at 20 °C. Therefore, X-ray treatment did not induce disease resistance in the rind of irradiated fruit. Although X-irradiation at doses up to 875 Gy followed by either 14 days at 20 °C or 60 days at 5 °C caused very slight rind pitting, minor decreases in fruit firmness, and modest increases in juice acetaldehyde and ethanol contents, these changes had no practical impact on fruit quality. Rind color, titratable acidity, soluble solids concentration, maturity index and juice yield were not influenced by irradiation. ‘Clemenules’ can be considered as a clementine cultivar highly tolerant to X-irradiation
Efecto del 1-MCP en cuatro variedades de ciruela
Get PDFEl efecto de la aplicación de 1-metilciclopropeno (1-MCP), como tratamiento para alargar la conservación de frutas y hortalizas esta siendo objeto de estudio desde hace unos anos. En el Centro de Postcosecha del Instituto Valenciano de Investigaciones Agrarias (IVIA) se ha estado evaluando la respuesta al tratamiento con 1-MCP en diferentes productos, como fruta de hueso (ciruelas y albaricoques) y caqui, investigaciones que han estado enmarcadas en el proyecto financiado por la empresa AgrofeshTM para obtener el registro del producto en España. El 1-MCP es un ciclopropeno sintético con fórmula C4H6 que actúa uniéndose a los receptores de etileno, bloqueando así la acción de éste, siendo la afinidad del 1-MCP por los receptores de aproximadamente 10 veces mayor que la del etileno (Sisler y Serek, 1997). El 1-MCP tiene un modo de acción no tóxico, no deja residuos y es activo a muy bajas concentraciones. Para su uso comercial, el 1-MCP aparece formando complejo con ciclodextrina. El método de aplicación se basa en la generación del 1-MCP como gas cuando se mezcla el producto comercial con agua