Evaluation of diagnostic molecular markers for DUS phenotypic assessment in the cereal crop, barley (Hordeum vulgare ssp. vulgare L.)

The deployment of genetic markers is of interest in crop assessment and breeding programmes, due to the potential savings in cost and time afforded. As part of the internationally recognised framework for the awarding of Plant Breeders’ Rights (PBR), new barley variety submissions are evaluated using a suite of morphological traits to ensure they are distinct, uniform and stable (DUS) in comparison to all previous submissions. Increasing knowledge of the genetic control of many of these traits provides the opportunity to assess the potential of deploying diagnostic/perfect genetic markers in place of phenotypic assessment. Here, we identify a suite of 25 genetic markers assaying for 14 DUS traits, and implement them using a single genotyping platform (KASPar). Using a panel of 169 UK barley varieties, we show that phenotypic state at three of these traits can be perfectly predicted by genotype. Predictive values for an additional nine traits ranged from 81 to 99 %. Finally, by comparison of varietal discrimination based on phenotype and genotype resulted in correlation of 0.72, indicating that deployment of molecular markers for varietal discrimination could be feasible in the near future. Due to the flexibility of the genotyping platform used, the genetic markers described here can be used in any number or combination, in-house or by outsourcing, allowing flexible deployment by users. These markers are likely to find application where tracking of specific alleles is required in breeding programmes, or for potential use within national assessment programmes for the awarding of PBRs.

Identification and expression analyses of Cytosolic Glutamine Synthetase genes in barley (Hordeum vulgare L.)

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, particularly during seed development. Three cytosolic GS isoforms (HvGS1) were identified in barley (Hordeum vulgare L. cv Golden Promise). Quantitation of gene expression, localization and response to N supply revealed that each gene plays a non-redundant role in different tissues and during development. Localization of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that it is important in N transport and remobilization. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localized to the leaf mesophyll cells, in the cortex and pericycle of roots, and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in leaves with an increasing supply of N, suggesting its role in the primary assimilation of N. HvGS1_3 was specifically and predominantly localized in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH+4, suggesting that it has a primary role in grain N assimilation and also in the protection against ammonium toxicity in roots. The expression of HvGS1 genes is directly correlated with protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

Cultura de anteras e embriogênese de genótipos selecionados de cevada (Hordeum vulgare L.ssp.vulgare)

A eficiência da técnica de cultura de anteras, em escala comercial, ainda pode ser considerada baixa quando medida em número de plantas duplo-haplóides férteis obtidas para cada antera estabelecida in vitro. Dessa forma, o presente trabalho é pioneiro no estudo detalhado da embriogênese in vitro do micrósporo e do grão de pólen de cevada (Hordeum vulgare L. ssp. vulgare). Com o objetivo de contribuir para o aperfeiçoamento da técnica de cultura de anteras foi analisada a embriogênese, com especial ênfase na etapa da indução, através de análises citológicas e histológicas de anteras cultivadas in vitro. Foram analisadas uma cultivar brasileira de cevada, em comparação com linhagens de duas outras cultivares brasileiras, que foram selecionadas, por seleção divergente para maior ou para menor resposta na indução da rota embriogênica e, respectivamente, para menor ou para maior capacidade de regenerar plântulas verdes. Somente foram estabelecidas em cultivo in vitro as anteras que apresentaram micrósporos e pólens jovens, das linhagens selecionadas da cultivar A-05 (S3A22 e S3A23), e da cultivar BR-2(S3B63 e, apenas na cultura de anteras, S3B61), bem como da cultivar MN-599 (nãoselecionada). Para as análises histológicas, foram fixadas, a cada dois dias, duas anteras, correspondentes a cada fileira da mesma espiga, após o início do cultivo in vitro. As anteras em cultivo e respectivas estruturas multicelulares foram fixadas em FAA 50%, desidratadas em série etílica e incluídas em hidroxietilmetacrilato. Os blocos de resina polimerizada foram secionados longitudinalmente com 3 mm de espessura. Para as análises citológicas foram fixadas, de cada espiga recém-coletada, três espiguetas sendo uma da base, outra do meio e outra do ápice. Após o pré-tratamento à baixa temperatura (5 °C), porém antes do cultivo in vitro, foram fixadas três anteras (amostras utilizadas como controles). A cada três dias, durante o cultivo, três anteras foram fixadas (até 18 dias). As anteras em cultivo e estruturas multicelulares foram fixadas em Farmer e FAA 50%, transferidas após 24 horas para etanol 70%. Na cultura in vitro das anteras houve diferenças entre uma das linhagens da cultivar A-05 em relação a cultivar MN- 599, na produção inicial de estruturas embriogênicas, diferença que desapareceu na produção total. Entretanto, houve diferenças na formação dos xiii embriões: a cv.MN-599 formou embriões bem diferenciados ao passo que a linhagem S3A22 produziu um número aparentemente menor, sendo que os embriões não eram bem diferenciados. A linhagem S3B63 não apresentou embriões até o final da análise histológica. Considerando que a amostra dessa linhagem, mantida em cultura, formou plantas verdes, pode-se propor que a formação de embriões deve ocorrer posteriormente ao desenvolvimento da cv.MN-599. Cabe destacar que houve diferenças significativas entre as cultivares A-05 e BR-2 quanto à regeneração de plântulas verdes. Esses resultados indicam ter havido maior eficiência da seleção em relação à etapa da regeneração. Com relação às categorias classificatórias dos micrósporos e grãos de pólen, constatou-se que desde o início da análise histológica (2o dia de cultivo in vitro) até o final (34o dia), foram observados micrósporos, o mesmo tendo sido observado na análise citológica. Os grãos de pólen multinucleados ocorreram praticamente em todo o período de cultivo in vitro, em ambas análises; não ocorrendo nos controles da citologia (antes do cultivo); os multinucleados foram observados a partir do 3o dia, enquanto que os multicelulares a partir do 4o dia de cultivo. As estruturas multicelulares foram observadas a partir do 8o dia. A quantidade e o tamanho das estruturas multicelulares foram variáveis ao longo da análise histológica, sendo que do 14o ao 20o dia foram encontradas as de maiores dimensões, resultantes da proliferação celular por mitoses sucessivas. A partir do 22o dia (cultivar MN- 599), a ocorrência de estruturas multicelulares no interior dos lóculos da antera diminuiu, predominando o processo de proliferação externo às anteras. Para as linhagens, a partir do 18o dia foram observadas estruturas multicelulares liberadas das anteras. A análise das estruturas multicelulares permitiu classificá-las em quatro categorias: 1. SFD: Sem forma definida; 2. MAC: meristema apical caulinar; 3. MAR: meristema apical radical embrionário adventício; e 4. Embriões. As estruturas amorfas apareceram em maior número, quando comparadas com as outras categorias. Em síntese: as linhagens selecionadas e a cultivar diferiram não apenas no tempo necessário para a formação dos embriões, mas também no desenvolvimento dos mesmos, que foi mais diferenciado na cultivar MN-599, porém sendo observados mais cedo na linhagem S3A22 e S3A23, do que na cultivar MN-599.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Sazonalidade dos ductos secretores e óleo essencial de Foeniculum vulgare var. vulgare Mill. (Apiaceae)

Este trabalho teve como objetivo analisar os ductos secretores e o óleo essencial das folhas de Foeniculum vulgare em diferentes épocas do ano. Para esta finalidade, foram realizados estudos de caracterização anatômica, bem como anatomia comparada dos ductos secretores e testes histoquímicos das folhas. O óleo essencial foi obtido de folhas e frutos, por hidrodestilação em aparelho de Clevenger e analisados quantitativamente e qualitativamente por cromatografia em fase gasosa acoplada ao espectrômetro de massa, realizando-se análises seguidas de três réplicas para folhas coletadas durante o inverno e primavera, e frutos no verão. Os resultados encontrados para os ductos secretores de óleo corresponderam à redução do teor de óleo essencial nas folhas coletadas no final da primavera. O componente majoritário do óleo essencial de folhas e frutos foi o trans-anetol, durante todas as estações do ano. Portanto, evidenciou-se que os ductos secretores e teor de óleo essencial estão relacionados, bem como os constituintes químicos também estão sujeitos a sazonalidade, conforme o estágio fenológico da planta.

Antioxidant potential of oregano (Oreganum vulgare L.), basil (Ocimum basilicum L.) and thyme (Thymus vulgaris L.): application of oleoresins in vegetable oil

Por razões econômicas e de proteção à saúde, pesquisas têm sido dirigidas para ampliar a estabilidade dos óleos vegetais. Existe uma tendência para a adição de antioxidantes naturais, em particular, um crescente interesse em ervas e especiarias. Desta forma, os objetivos deste trabalho foram avaliar o potencial antioxidante das oleorresinas de orégano, manjericão e tomilho e seu comportamento ao serem aplicadas ao óleo de soja em diferentes concentrações. Numa primeira etapa foi determinada a atividade antioxidante pelo sistema β-caroteno/ácido linoleico e a quantificação de compostos fenólicos totais. Posteriormente, foram adicionadas ao óleo de soja diferentes concentrações das oleorresinas (500 a 3000 mg.kg-1) e analisado seu potencial antioxidante por meio da estabilidade oxidativa utilizando o Rancimat. A concentração de 3000 mg.kg-1das oleorresinas de orégano e tomilho foi a que apresentou melhor estabilidade oxidativa ao óleo de soja, o que as tornam alternativa natural na conservação de óleos vegetais.

Efeitos de potenciais de água no solo, em diferentes estádios fenológicos da cultura da cevada (Hordeum vulgare L.)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Supercritical fluid extraction from fennel (Foeniculum vulgare): global yield, composition and kinetic data

Supercritical fluid extraction (SFE) from solids has proven to be technically feasible for almost any system; nonetheless, its economical viability has been proven for a restricted number of systems. A common practice is to compare the cost of manufacturing of vegetable extracts by a variety of techniques without deeply considering the huge differences in composition and functional properties among the various types of extracts obtained; under this circumstance, the cost of manufacturing do not favor SFE. Additionally, the influence of external parameters such as the agronomic conditions and the SFE system geometry are not considered. In the present work, these factors were studied for the system fennel seeds + CO2. The effects of the harvesting season and the degree of maturation on the global yields for the system fennel seeds + CO2 were analyzed at 300 bar and 40 degrees C. The effects of the pressure on the global yields were determined for the temperatures of 30 and 40 degrees C. Kinetics experiments were done for various ratios of bed height to bed diameter. Fennel extracts were also obtained by hydrodistillation and low-pressure solvent extraction. The chemical composition of the fennel extracts were determined by gas chromatography. The SFE maximum global yield (12.5%, dry basis) was obtained with dry harvested fennel seeds. Anethole and fenchone were the major constituents of the extract; the following fat acids palmitic (C16H32O2), palmitoleic stearic (C18H36O2), oleic (C18H34O2), linoleic (C18H32O2) and linolenic (C18H30O2) were also detected in the extracts. A relation between amounts of feed and solvent, bed height and diameter, and solvent flow rate was proposed. The models of Sovova, Goto et al. and Tan and Lion were capable of describing the mass transfer kinetics. (c) 2005 Elsevier B.V. All rights reserved.

Oxalate determination in urine using an immobilized enzyme on Sorghum vulgare seeds in a flow injection conductimetric system

A flow-injection (FI) method was developed for the determination of oxalate in urine. It was based on the use of oxalate oxidase (E.C. 1.2.3.4) immobilized on ground seeds of the BR-303 Sorghum vulgare variety. A reactor was filled with this activated material, and the samples (200 μL) containing oxalate were passed through it, carried by a deionized water flow. The carbon dioxide produced by the enzyme reaction permeated through a microporous PTFE membrane, and was received in a water acceptor stream, promoting conductivity changes proportional to the oxalate concentration in the sample. The results obtained showed a useful linear range from 0.05 to 0.50 mmol dm-3. The proposed method, when compared with the Sigma enzymatic procedure, showed good correlation (Y = 0.006(±0.016) + 0.98(±0.019)X; r = 0.9995, Y = conductivity in μS, and X = concentration in mmol dm-3), selectivity, and sensitivity. The new immobilization approach promotes greater stability, allowing oxalate determination for 6 months. About 13 determinations can be performed per hour. The precision of the proposed method is about ± 3.2 % (r.s.d).

Eficácia do extrato aquoso do Triticum vulgare no tratamento de radiodermatites geradas pelo uso da radioterapia em gatos

The use of ionizing radiation in the treatment of oncological diseases in veterinary medicine has grown in recent years. In general, these animals are very well tolerated by the treatment with radioactive elements; however, the harmful effects that may occur are not always able to be controlled, particularly those relating to skin burns. Then, the purpose of this study was to evaluate the effects of aqueous extract of Triticum vulgare in treat of 10 cats radiodermatitis with squamous cell carcinoma (SCC), which were treated with radiotherapy as single modality treatment. The drug was used topically and daily until complete healing of wounds. Based on the findings, it may concluded beneficial effects of the drug, because it allowed the formation of granulation tissue and epithelization of wounds with epidermal repair not exuberant, good-quality cosmetics and, considering the deleterious effects that can result from cancer treatments.

Protective effect of oregano (Origanum vulgar L.) and thyme (Thymus vulgaris L.) oleoresins in stored soybean oil

Purpose: The objective of this study was to evaluate the antioxidant effect of oregano and thyme extracts isolatedly and combinedly applied in soybean oil. Design/methodology/approach: Soybean oil containing 3,000 mg/kg of oregano and thyme oleoresins and the mixture of both, as well as soybean oil containing TBHQ (50 mg/kg) and soybean oil free of antioxidants, were subjected to accelerated oven test (60°C/10 days). Samples were collected every two days and analyzed as to their peroxide and conjugated diene values. Findings: The mixture of oleoresins and consequent increase of concentration were as effective as the antioxidant TBHQ. Practical implications: These studies may prove to be beneficial to the exploitation of natural antioxidant sources for the preservation and/or extension of raw and processed food shelf life. Therefore, they could also be applied in the area of pharmaceuticals for the protection of human life. Originality/value: This study offers information on the use of natural antioxidants as an alternative to the use of synthetic antioxidants, which might be considered toxic. © Emerald Group Publishing Limited.

Caracterização bioquímica e centesimal das espécies Astrocaryum vulgare Mart. (tucumã) e Endopleura uchi (Huber) Cuatrec. (uxi) nativas da região Amazônica

Pós-graduação em Biotecnologia - IQ