Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

DRIS norms for 'Valencia' sweet orange on three rootstocks

Diagnosis and Recommendation Integrated System (DRIS) applies nutrient ratios instead of the isolated concentration values of each nutrient in interpretation of tissue analysis. The objectives of this research were to establish adequate DRIS norms for 'Valencia' sweet orange irrigated commercial groves budded on three rootstocks and correlate indexes of nutrition balance with yield. Experiments were conducted in São Paulo State, Brazil. Rootstocks Rangpur lime, Caipira sweet orange, and Poncirus trifoliata, with more than six years old and yield above 40 ton ha-1 were utilized. Data referred to yield, tree spacing, rootstock and foliar concentrations of N, P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B in non fruiting terminals for each grove were processed for the years 1994 through 1998. DRIS indexes were calculated by Nick criterion for choosing the ratio order of the nutrients and Jones calculation method of the ratio functions. Indexes of nutritional balance calculated from DRIS norms presented high correlation with yield for the three scion/rootstock combinations. DRIS norms defined in this research are valid, since leaf sampling is done on non fruiting terminals and the grove is irrigated.

Sweet orange trees grafted on selected rootstocks fertilized with nitrogen, phosphorus and potassium

The majority of citrus trees in Brazil are grafted on 'Rangpur lime' (Citrus limonia Osb.) rootstock. Despite its good horticultural performance, search for disease tolerant rootstock varieties to improve yield and longevity of citrus groves has increased. The objective of this work was to evaluate yield efficiency of sweet oranges on different rootstocks fertilized with N, P, and potassium. Tree growth was affected by rootstock varieties; trees on 'Swingle' citrumelo [Poncirus trifoliata (L.) Raf. × C. paradisi Macf.] presented the smallest canopy (13.3 m³ in the fifth year after tree planting) compared to those on 'Rangpur lime' and 'Cleopatra' mandarin [C. reshni (Hayata) hort. ex Tanaka] grown on the same grove. Although it was observed an overall positive relationship between canopy volume and fruit yield (R² = 0.95**), yield efficiency (kg m-3) was affected by rootstocks, which demonstrated 'Rangpur lime' superiority in relation to Cleopatra. Growth of citrus trees younger than 5-yr-old might be improved by K fertilization rates greater than currently recommended in Brazil, in soils with low K and subjected to nutrient leaching losses.

Genetic linkage maps of 'Pêra' sweet orange and 'Cravo' mandarin with RAPD markers

The objective of this work was to construct linkage maps of 'Pêra' sweet orange [Citrus sinensis (L.) Osbeck] and 'Cravo' mandarin (Citrus reticulata Blanco) using RAPD markers and the pseudo-testcross strategy. The parents were chosen according to the resistance/susceptibility to citrus variegate chlorosis (CVC). The segregation of 176 markers was analyzed in 94 progeny of F1 hybrids, which were obtained from controlled crossings. The linkage map of 'Pêra' sweet orange had 117 markers defined by 12 linkage groups, which spanned 612.1 cM. Only six markers could not be linked to the linkage group and 48.7% of the markers showed segregation distortion. The linkage map of 'Cravo' mandarin had 51 markers defined by 12 linkage groups, which spanned 353.3 cM. Only two markers did not link to the groups and 15.7% showed segregation distortion. The construction of linkage maps is relevant to future mapping studies of the inheritance of CVC, citrus canker and leprosis.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Characterization of potato genotypes using molecular markers

The objective of this work was to characterize 27 potato genotypes, using molecular markers. Polyacrylamide gel electrophoresis, RAPD techniques and isozymes of esterase, phosphoglucomutase and soluble proteins were analyzed in tubers, and isocitrate dehydrogenase, aspartate transaminase, phosphoglucomutase and peroxidase, in leaves. Eighteen primers were tested and four were chosen, kits OPX (01, 04 and 09) and OPY (07), to analyze RAPD markers in leaf extracts. Similarity and cluster analysis were conducted using Jaccard coefficient and the unweighted pair-group method using arithmetic average. Despite the differences detected in the analysis of proteins and isozymes in the tubers, as well as of isozymes in the leaves, the characterization of all genotypes through gel electrophoresis was not possible, while RAPD markers were efficient to characterize all the 27 genotypes.

Pollination efficiency of the stingless bee Melipona subnitida on greenhouse sweet pepper

The present study was carried out in Northeastern Region of Brazil, in order to investigate the use of stingless bee Melipona subnitida Ducke in the pollination of greenhouse sweet pepper (Capsicum annuum L.). Treatments of hand cross-pollination, hand self-pollination, pollination by bees and restricted pollination were performed. Results showed that despite sweet pepper flowers are considered autogamous, this crop benefits from pollination by M. subnitida, producing fruits significantly heavier and wider, containing a greater number of seeds and of better quality (lower percentage of malformed fruits) than self-pollinated sweet pepper. Thus, M. subnitida can be considered an efficient pollinator of greenhouse sweet pepper.

Respiratory viruses in HIV-infected patients with suspected respiratory opportunistic infection.

OBJECTIVE: To assess the incidence and epidemiological pattern of respiratory viruses in HIV-infected patients and to evaluate their potential clinical impact. DESIGN AND METHODS: A prospective population-based cohort study was conducted at three Swiss university hospitals. Study participants were HIV-infected patients who underwent a bronchoalveolar lavage to rule out an opportunistic event. All bronchoalveolar lavage specimens were screened using a set of real-time reverse transcriptase-polymerase chain reaction assays targeting 17 different respiratory viruses. RESULTS: Between November 2003 and November 2006, 59 bronchoalveolar episodes from 55 HIV-infected patients were analysed. Eleven of 59 episodes (18.6%) were positive for at least one respiratory virus. Coronavirus OC43 was identified in three cases (27.3%) followed by influenza A in two (18.2%). Parainfluenza virus (PIV) 2, PIV 3, PIV 4, bocavirus, human rhinovirus A and human metapneumovirus were each identified in one case (9%). In the majority of these cases (63.6%) no other concomitant microorganism was isolated. CONCLUSIONS: Clinical investigation of respiratory viral infections in HIV-infected patients should not be restricted to prototype viruses and also need to target all the different family of viruses as it seems likely that these viruses contribute to pulmonary complications and morbidity in this population.

Airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses.

We conducted a 12-year retrospective study to determine the effects that the community respiratory-virus species and the localization of respiratory-tract virus infection have on severe airflow decline, a serious and fatal complication occurring after hematopoietic cell transplantation (HCT). Of 132 HCT recipients with respiratory-tract virus infection during the initial 100 days after HCT, 50 (38%) developed airflow decline < or =1 year after HCT. Lower-respiratory-tract infection with parainfluenza (odds ratio [OR], 17.9 [95% confidence interval {CI}, 2.0-160]; P=.01) and respiratory syncytial virus (OR, 3.6 [95% CI, 1.0-13]; P=.05) independently increased the risk of development of airflow decline < or =1 year after HCT. The airflow decline was immediately detectable after infection and was strongest for lower-respiratory-tract infection with parainfluenza virus; it stabilized during the months after the respiratory-tract virus infection, but, at < or =1 year after HCT, the initial lung function was not restored. Thus, community respiratory virus-associated airflow decline seems to be specific to viral species and infection localization.

Respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults.

BACKGROUND: The epidemiology of respiratory viruses and their potential clinical impact when recovered in lower respiratory specimens has not been established in the hospital setting. A study was performed to investigate the association between positive viral detection and respiratory infection in an at-risk population. METHODS: 299 adult patients who underwent bronchoalveolar lavage (BAL) procedures were enrolled in a hospital-based prospective cohort study. Descriptive epidemiology is presented of 17 different respiratory viruses detected by reverse transcription-polymerase chain reaction assays in BAL fluid specimens. Multivariate analysis was conducted to identify the clinical characteristics independently associated with the presence of virus. RESULTS: Of 522 BAL fluid specimens analysed, 81% were collected in adult transplant recipients or other immunocompromised patients. Overall, PCR assays identified viral nucleic acid in 91 BAL fluid samples (17.4%). Similar rates of virus-positive BAL fluid were found in the different subpopulations studied (p = 0.113). Coronaviruses were the most frequent (32.3%), followed by rhinovirus (22.6%), parainfluenza (19.5%), influenza (9.7%), respiratory synctial virus (8.6%), human metapneumovirus (4.2%) and bocavirus (3.1%). Multivariate analysis using mixed models showed that respiratory viral infections were associated with a lack of antibiotic treatment response (OR 2.2, 95% CI 1.2 to 4.1) and the absence of radiological infiltrate (OR 0.3, 95% CI 0.2 to 0.8). In lung transplant recipients in whom a respiratory infection was suspected, the respiratory viral detection rate was 24.4% compared with 13.8% overall in other patients (p = 0.02). CONCLUSIONS: In this cohort of hospitalised adults, respiratory viruses detected in BAL fluid specimens were associated with respiratory symptoms, absence of radiological infiltrates and a poor response to antibiotic therapy.

Anti-apoptotic strategies of lymphotropic viruses.

Induction of apoptosis of virus-infected cells is an important host cell defence mechanism. However, some viruses have incorporated genes that encode anti-apoptotic proteins or modulate the expression of cellular regulators of apoptosis. Here, Edgar Meinl and colleagues discuss recent evidence that viral interference with host cell apoptosis leads to enhanced viral replication, and to evasion of cytotoxic T-cell effects.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Potato plant growth and macronutrient uptake as affected by soil tillage and irrigation systems

The objective of this study was to evaluate potato plant growth and macronutrient uptake, as affected by soil tillage methods, in sprinkle and drip irrigated experiments. Eight treatments were set: T1, no tillage, except for furrowing before planting; T2, one subsoiling (SS); T3, twice rotary hoeing (RH); T4, one disc plowing (DP) + twice disc harrow leveling (DL); T5, 1DP + 2DL + 1RH; T6, 1DP + 2DL + 2RH; T7, 1SS + T6; T8, one moldboard plowing (MP) + 2DL. Treatments were arranged in a randomized block design with four replications. In both irrigation systems, plants presented higher emergence velocity index (EVI), when the soil was not tillaged, and the EVI was inversely related to the maximum tuber dry mass production. In both experiments, a functional direct relationship was found between the leaf area index and maximum tuber dry mass yield. The growth of plant organs (tuber, leaf, stem and root) and the macronutrient (N, P, K, Ca and Mg) contents in potato plant responded positively to a deeper soil revolving caused by plowing, especially with moldboard plow.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.