Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model "diploid embryogenic callus protoplast + diploid mesophyll-derived protoplast". Protoplasts were isolated from embryogenic calli of 'Pera' and 'Westin' sweet orange cultivars (Citrus sinensis) and from young leaves of 'Fremont', Nules', and 'Thomas' mandarins (C. reticulata), and 'Nova' tangelo [C. reticulata x (C. paradisi x C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when 'Pera' sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of 'Pera' sweet orange + 'Fremont' mandarin, 3 'Pera' sweet orange + 'Nules' mandarin, and 2 'Pera' sweet orange + 'Nova' tangelo, and all the diploid regenerated plants showed the 'Pera' sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96 h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.

Variations in peak expiratory flow measurements associated to air pollution and allergic sensitization in children in Sao Paulo, Brazil

Background In the last 20 years, there has been an increase in the incidence of allergic respiratory diseases worldwide and exposure to air pollution has been discussed as one of the factors associated with this increase. The objective of this study was to investigate the effects of air pollution on peak expiratory flow (PEF) and FEV1 in children with and without allergic sensitization. Methods Ninety-six children were followed from April to July, 2004 with spirometry measurements. They were tested for allergic sensitization (IgE, skin prick test, eosinophilia) and asked about allergic symptoms. Air pollution, temperature, and relative humidity data were available. Results Decrements in PEF were observed with previous 24-hr average exposure to air pollution, as well as with 310-day average exposure and were associated mainly with PM10, NO2, and O3 in all three categories of allergic sensitization. Even though allergic sensitized children tended to present larger decrements in the PEF measurements they were not statistically different from the non-allergic sensitized. Decrements in FEV1 were observed mainly with previous 24-hr average exposure and 3-day moving average. Conclusions Decrements in PEF associated with air pollution were observed in children independent from their allergic sensitization status. Their daily exposure to air pollution can be responsible for a chronic inflammatory process that might impair their lung growth and later their lung function in adulthood. Am. J. Ind. Med. 55:10871098, 2012. (c) 2012 Wiley Periodicals, Inc.

Size distributions and temporal variations of biological aerosol particles in the Amazon rainforest characterized by microscopy and real-time UV-APS fluorescence techniques during AMAZE-08

As a part of the AMAZE-08 campaign during the wet season in the rainforest of central Amazonia, an ultraviolet aerodynamic particle sizer (UV-APS) was operated for continuous measurements of fluorescent biological aerosol particles (FBAP). In the coarse particle size range (> 1 mu m) the campaign median and quartiles of FBAP number and mass concentration were 7.3x10(4) m(-3) (4.0-13.2x10(4) m(-3)) and 0.72 mu g m(-3) (0.42-1.19 mu g m(-3)), respectively, accounting for 24% (11-41%) of total particle number and 47% (25-65%) of total particle mass. During the five-week campaign in February-March 2008 the concentration of coarse-mode Saharan dust particles was highly variable. In contrast, FBAP concentrations remained fairly constant over the course of weeks and had a consistent daily pattern, peaking several hours before sunrise, suggesting observed FBAP was dominated by nocturnal spore emission. This conclusion was supported by the consistent FBAP number size distribution peaking at 2.3 mu m, also attributed to fungal spores and mixed biological particles by scanning electron microscopy (SEM), light microscopy and biochemical staining. A second primary biological aerosol particle (PBAP) mode between 0.5 and 1.0 mu m was also observed by SEM, but exhibited little fluorescence and no true fungal staining. This mode may have consisted of single bacterial cells, brochosomes, various fragments of biological material, and small Chromalveolata (Chromista) spores. Particles liquid-coated with mixed organic-inorganic material constituted a large fraction of observations, and these coatings contained salts likely from primary biological origin. We provide key support for the suggestion that real-time laser-induce fluorescence (LIF) techniques using 355 nm excitation provide size-resolved concentrations of FBAP as a lower limit for the atmospheric abundance of biological particles in a pristine environment. We also show some limitations of using the instrument for ambient monitoring of weakly fluorescent particles < 2 mu m. Our measurements confirm that primary biological particles, fungal spores in particular, are an important fraction of supermicron aerosol in the Amazon and that may contribute significantly to hydrological cycling, especially when coated by mixed inorganic material.

Glutathione improves early somatic embryogenesis in Araucaria angustifolia (Bert) O. Kuntze by alteration in nitric oxide emission

In this work, it was observed a straight relationship between the manipulation of the reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio, nitric oxide emission and quality and number of early somatic embryos in Araucaria angustifolia, a Brazilian endangered native conifer. In low concentrations GSH (0.01 and 0.1 mM) is a potential NO scavenger in the culture medium. Furthermore, it can increase the number of early SE formed in cell suspension culture media in a few days. However, the maintenance in this low redox state lead to a loss of early somatic embryos polarization. In gelled culture medium, high levels of GSH (5 mM) allows the development of globular embryos presenting a high NO emission on embryo apex, stressing its importance in the differentiation and cell division. Taken together these results indicate that the modification of the embryogenic cultures redox state might be an effective strategy to develop more efficient embryogenic systems in A. angustifolia. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Immune transcript variations among Aedes aegypti populations with distinct susceptibility to dengue virus serotype 2

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RTPCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain. (C) 2012 Elsevier B.V. All rights reserved.

Assessment of variations of the mandibular canal through cone beam computed tomography

The neurovascular bundle may be vulnerable during surgical procedures involving the mandible, especially when anatomical variations are present. Increased demand of implant surgeries, wider availability of three-dimensional exams, and lack of clear definitions in the literature indicate that features of anatomical variations should be revisited. The objective of the study was to evaluate features of anatomical variations related to mandibular canal (MC), such as bifid canals, anterior loop of mental nerve, and corticalization of MC. Additionally, bone trabeculation at the submandibular gland fossa region (SGF) was assessed and related to visibility of MC. Cone beam computed tomography exams from 100 patients (200 hemimandibles) were analyzed and the following parameters were registered: diameter and corticalization of MC; trabeculation in SGF region; presence of bifid MC, position of bifurcations, diameter, and direction of bifid canals; and measurement of anterior loops by two methods. Corticalization of the MC was observed in 59% of hemimandibles. In 23%, MC could be identified despite absence of corticalization. Diameter of MC was between 2.1 and 4 mm for nearly three quarters of the sample. In 80% of the sample trabeculation at the SGF was either decreased or not visible, and such cases showed correlation with absence of MC corticalization. Bifid MC affected 19% of the patients, mostly associated with additional mental foramina. Clinically significant anterior loop (> 2 mm of anterior extension) was observed in 22-28%, depending on the method. Our findings, together with previously reported limitations of conventional exams, draw attention to the unpredictability related to anatomical variations in neurovascularization, showing the contribution of individual assessment through different views of three-dimensional imaging prior to surgical procedures in the mandible.

Comparative study of reserve lipid accumulation during somatic and zygotic Acca sellowiana (O. Berg.) Burret embryogenesis

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

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.

Matrix metalloproteinase-9 genetic variations affect MMP-9 levels in obese children

Objective: Matrix metalloproteinase-9 (MMP-9) is involved in the atherosclerotic process and functional polymorphisms in the MMP-9 gene affect MMP-9 expression/activity, and are associated with cardiovascular diseases. However, no study has tested the hypothesis that functional MMP-9 polymorphisms could affect MMP-9 levels in obese children. We investigated whether three MMP-9 gene polymorphisms (C-1562T (rs3918242), 90(CA)((14-24)) (rs2234681) and Q279R (rs17576)), or haplotypes, affect MMP-9 levels in obese children. Methods: We studied 175 healthy control children and 127 obese children. Plasma MMP-9, tissue inhibitor of MMPs (TIMP)-1 and adiponectin concentrations were measured using enzyme-linked immunosorbent assay. Results: We found similar MMP-9 genotypes, allelic and haplotypes distributions in the two study groups (P > 0.05). However, we found lower plasma MMP-9 concentrations in obese subjects carrying the CC or the QQ genotypes for the C-1562T and the Q279R polymorphisms, respectively, in obese children compared with children with the other genotypes, or with non-obese children with the same genotypes (all P < 0.05). Moreover, we found lower MMP-9 levels and lower MMP-9/TIMP-1 ratios (which reflect net MMP-9 activity) in obese children carrying the H2 haplotype (which combines the C, H and Q alleles for the three polymorphisms, respectively) when compared with obese children carrying the other haplotypes, or with non-obese children carrying the same haplotype (P < 0.05). Conclusions: Our findings show that MMP-9 genotypes and haplotypes affect MMP-9 levels in obese children and adolescents, and suggest that genetic factors may modify relevant pathogenetic mechanisms involved in the development of cardiovascular complications associated with obesity in childhood. International Journal of Obesity (2012) 36, 69-75; doi:10.1038/ijo.2011.169; published online 16 August 2011

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. Key message Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Spatial-temporal variations in phytoplankton size and colored detrital matter absorption at global and regional scales, as derived from twelve years of SeaWiFS data (1998-2009)

A procedure has been proposed by Ciotti and Bricaud (2006) to retrieve spectral absorption coefficients of phytoplankton and colored detrital matter (CDM) from satellite radiance measurements. This was also the first procedure to estimate a size factor for phytoplankton, based on the shape of the retrieved algal absorption spectrum, and the spectral slope of CDM absorption. Applying this method to the global ocean color data set acquired by SeaWiFS over twelve years (1998-2009), allowed for a comparison of the spatial variations of chlorophyll concentration ([Chl]), algal size factor (S-f), CDM absorption coefficient (a(cdm)) at 443 nm, and spectral slope of CDM absorption (S-cdm). As expected, correlations between the derived parameters were characterized by a large scatter at the global scale. We compared temporal variability of the spatially averaged parameters over the twelve-year period for three oceanic areas of biogeochemical importance: the Eastern Equatorial Pacific, the North Atlantic and the Mediterranean Sea. In all areas, both S-f and a(cdm)(443) showed large seasonal and interannual variations, generally correlated to those of algal biomass. The CDM maxima appeared in some occasions to last longer than those of [Chl]. The spectral slope of CDM absorption showed very large seasonal cycles consistent with photobleaching, challenging the assumption of a constant slope commonly used in bio-optical models. In the Equatorial Pacific, the seasonal cycles of [Chl], S-f, a(cdm)(443) and S-cdm, as well as the relationships between these parameters, were strongly affected by the 1997-98 El Ni o/La Ni a event.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): a model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies. (c) 2012 Elsevier Inc. All rights reserved.