QUALIDADE FISIOLÓGICA DE SEMENTES DE Tabernaemontana fuchsiaefolia A. DC EM FUNÇÃO DO TESTE DE ENVELHECIMENTO ACELERADO1

Tabernaemontana fuchsiaefolia A. DC é uma espécie da família Apocynaceae, nativa do Brasil, com característica invasora, possuindo potencial para uso em recuperação de áreas degradadas. O trabalho teve o objetivo de avaliar a influência dos métodos de envelhecimento acelerado sobre a qualidade fisiológica e germinação de sementes, utilizando-se o método tradicional e em solução de NaCl, empregando-se a metodologia de gerbox, formando minicâmaras de envelhecimento, em três temperaturas (41, 43 e 45°C) e cinco tempos de envelhecimento (0, 24, 48, 72 e 96 horas). As sementes, após o envelhecimento, foram colocadas para germinar em rolos de papel germitest, em quatro repetições de 25 sementes, em câmaras BOD a temperatura de 30°C, com fotoperíodo de 8 horas. O efeito da temperatura foi comparado pelo teste de Tukey em nível de 5% de probabilidade e o efeito do tempo de envelhecimento, pela análise de regressão. Após 35 dias, foram avaliados percentual, índice de velocidade, tempo médio e frequência da germinação, bem como comprimento de raiz e parte aérea, massa seca de plântulas e percentual de plântulas normais. Observou-se que o método alternativo de envelhecimento com solução de NaCl teve pouca influência na qualidade das sementes, não sendo indicado para testes de vigor para a espécie. O método tradicional, na temperatura de 45°C em período de envelhecimento de 96 horas, promoveu queda na germinação e pode ser utilizado para testar o vigor de sementes de leiteira.

Atividade bioinseticida e mecanismo de ação de vicilinas de sementes Erythrina velutina sobre moscas-das frutas Ceratitis capitata

The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0,13% and 0,14% (w/w), respectively for the larvae. EvV deleterious effects were related to the binding to chitin structures presented in peritrophic membrane and gut epithelial cells, associated with its low digestibility in C. capitata digestive tract. The results described herein are the first demonstration of the larvicidal effects of plant protein on C. capitata larvae. EvV may be part of the pest management programs, in the toxic bait composition, or an alternative in plant improvement program

SUBSTRATOS E TEMPERATURAS PARA TESTE DE GERMINAÇÃO EM SEMENTES DE MYRTACEAE

As espécies brasileiras de Myrtaceae compreendem diversas plantas arbóreas e arbustivas que podem ser utilizadas na produção de frutos para consumo in natura ou para industrialização. Informações sobre avaliação da qualidade de sementes dessas espécies não estão bem definidas, principalmente as relacionadas à adequação do teste de germinação. Objetivou-se testar substratos e temperaturas para realização do teste de germinação em sementes de Acca sellowiana (O. Berg) Burret. (goiaba-serrana), Campomanesia xanthocarpa O. Berg (guabiroba), Eugenia involucrata DC. (cereja-do-mato) e Eugenia pyriformis Camb. (uvaia). Sementes de diferentes procedências foram submetidas ao teste de germinação em substratos areia e rolo de papel tipo germitest, umedecidos com água destilada, e nas temperaturas 15 °C, 25 °C, 30 °C, 35 °C e 20-30 °C, sob luz constante, em germinadores tipo BOD. O delineamento experimental utilizado foi o inteiramente casualizado com quatro repetições de 25 sementes/tratamento/lote/espécie, e as médias dos tratamentos foram comparadas pelo teste de Tukey (P < 0,01). O teste de germinação pode ser conduzido na temperatura de 25 ºC e em substrato rolo de papel para Eugenia involucrata e Eugenia pyriformis. Ambos os substratos podem ser utilizados para o teste de germinação de Acca sellowiana, a 25 ºC. A temperatura de 25 ºC e alternância de 20-30 ºC são indicadas para Campomanesia xanthocarpa, assim como, os substratos areia e rolo de papel.

Purificação, caracterização e análise da atividade bioinseticida, de um inibidor de tripsina em sementes de catanduva (Piptadenia moniliformis)

One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic

Efeito inseticida de vicilinas isoladas de sementes de erythrina velutina em condições de semi-campo para moscas das frutas (ceratitis capitata)

Chitin-binding vicilins from legume seeds (Erythrina velutina. Canavalia ensiformes and Phaseolus vulgares) were isolated by ammonium sulfate followed by affinity chromatography on a chitin column. Effect of these vicilins on female adults of Ceratitis capitata was examined by bioassay and in a semi-field assay model. Mechanism of action of the vicilins was determined by in vivo digestibility and chitin affinity. Among the tested vicilins, E. velutina when added to diet caused strong effect on mortality at 10% dose. This insecticidal property was tested in a semi-field assay which showed the same effect observed in laboratory conditions, where doses of 10% and 15% were lethal to female adults of C. capitata. These deleterious effects were not only associated to the binding to chitin structures present in peritrophic membrane, but principally to its low digestibility in the C. capitata digestive tract. This fact was confirmed because chiting binding proteins as WGA and the other tested vicilins were not toxic to female adults of C. capitata due susceptibility of these proteins to digestive enzymes of the insects. By other side EvV was more resistant to digestive enzymes, causing deleterious effects on female adults of C. capitata. These results showed that EvV may be part of the pest management programs or an alternative in plant improvement program in the population control of this fruticulture pest

Avaliação das atividades anti-inflamatória, anticoagulante e antiproliferativa do inibidor de quimotripsina das sementes de erythrina velutina (EvCI)

Studies indicate that several components were isolated from medicinal plants, which have antibacterial, antifungal, antitumor and anti-inflammatory properties. Sepsis is characterized by a systemic inflammation which leads to the production of inflammatory mediators exacerbated by excessive activation of inflammatory cells and disseminated intravascular coagulation (DIC), in which the human neutrophil elastase plays an important role in its pathogenesis. Several epidemiological studies suggest that components of plants, especially legumes, can play a beneficial role in reducing the incidence of different cancers. A chymotrypsin inhibitor of Kunitz (Varela, 2010) was purified from seeds of Erythrina velutina (Mulungu) by fractionation with ammonium sulfate, affinity chromatography on Trypsin-Sepharose, Chymotrypsin-Sepharose and ion exchange chromatography on Resource Q 1 ml (GE Healthcare) in system FPLC / AKTA. The inhibitor, called EvCI, had a molecular mass of 17 kDa determined by SDS-PAGE. The purified protein was able to inhibit human neutrophil elastase (HNE), with an IC50 of 3.12 nM. The EvCI was able to inhibit both pathways of HNE release stimulated by PAF and fMLP (75.6% and 65% respectively). The inhibitor also inhibited leukocyte migration in septic mice about 87% and prolonged the time of coagulation and inhibition factor Xa. EvCI showed neither hemolytic activity nor cytotoxicity. EvCI showed a selective antiproliferative effect to HepG2 cell lines with IC50 of 0.5 micrograms per milliliter. These results suggest EvCI as a molecule antagonist of PAF / fMLP and a potential use in fighting inflammation related disorders, disseminated intravascular coagulation (DIC) and cancer

Um novo inibidor de tripsina multifuncional de sementes de Erythrina velutina: caracterizacao e propriedades farmacologicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

O banco de sementes do solo: uma ferramenta para a avaliação do estado de conservação dos charcos temporários mediterrânicos

Os Charcos temporários mediterrânicos (3170*) são habitats prioritários devido à enorme biodiversidade que albergam e ao grau de ameaça sob o qual que se encontram. A singularidade deste habitat resulta da coexistência de diferentes comunidades de plantas no mesmo espaço mas em períodos de tempo diferentes, o que é viabilizado pela existência de bancos de sementes abundantes e diversificados. A avaliação da composição específica e abundância do banco de sementes do solo permitiu determinar a distribuição espacial das sementes e o potencial de recuperação dos charcos. Com esta avaliação concluiu-se que existe uma maior riqueza específica nas cinturas externa e média dos charcos do que na cintura interna. Concluiu-se também que o conhecimento do banco de sementes dos charcos pode contribuir para a definição de estratégias de gestão/recuperação dos charcos, nomeadamente daqueles que possuem grau de conservação intermédio, funcionando como um valioso instrumento de gestão; The soil seed bank - a tool for assessing the conservation status of Mediterranean temporary ponds Abstract: Mediterranean temporary ponds (3170*) are priority habitats due to the huge biodiversity they shelter and because they are menaced. The uniqueness of this habitat results from the coexistence of different plant communities in the same space at different time periods, what is allowed by the existence of abundant and diversified soil seed banks. The assessment of the specific composition and abundance of soil seed bank, made possible to ascertain the spatial distribution of seeds within ponds and their recovery potential. This assessment showed that there is more specific richness in intermediate and outer belts of ponds than in the inner belt. It also showed how valuable can this information be regarding the design of management or recovery strategies for ponds, particularly for those with intermediate degree of conservation.

O Menir do Patalou - Nisa - entre Contextos e cronologias, in Terra e Água escolher sementes invocar a Deusa, estudos em homenagem a Victor S. Gonçalves

Publicam-se neste capítulo a data de radiocarbono obtida para o Menhir do Patalou e discutem-se as outras datas disponíveis para os menhires portugueses com especial destaque para a data do menhir da Meada.

Vicilinas de sementes de leguminosas selvagens:purificação, caracterização, efeito de deletério e mecanismo de ação para bruquídeos e para fungos fitopatogênicos

Globulins fractions of legume seeds of Crotalaria pallida, Erytrina veluntina and Enterolobium contortisiliquum were isolated and submitted to assays against serine, cysteine and aspartic proteinases, as also amylase present in midgut of C. maculatus and Z. subfasciatus. Hemagglutination assays indicated presence of a lectin in E. veluntina globulin fractions. This lectin had affinity to human erythrocytes type A, B and O. Vicilins were purified by chromatography on Sephacryl S-300 followed of a chromatography on Sephacryl S-200, which was calibrated using protein markers. Vicilins from C. pallida (CpV) and E. veluntina (EvV) seeds had a molecular mass of 124.6 kDa and E. contortisiliquum a molecular mass of 151kDa. Eletrophoresis in presence of SDS showed that CpV was constituted by four subunities with apparent molecular mass of 66, 63, 57 and 45 kDa, EvV with three subunities with apparent molecular mass of 45kDa and EcV four subunities, two with 37.1 kDa and two with 25.8 kDa. Non denaturantig eletrophoresis displayed single bands with high homogeneity, where CpV had lower acidic behavior. All vicilins are glycoproteins with carbohydrate contents at 1 to1.5%. Bioassays were done to detect deleterious effects of vicilins against C. maculatus and Z. subfasciatus larvae. CpV, EvV and EcV exhibited a WD50 of 0.28, 0.19 and 1.03%; LD50 0.2, 0.26, and 1.11% respectively to C. maculatus. The dose responses of CpV, EvV and EcV to Z. subfasciatus were: WD50 of 0.12, 0.14, 0.65% and LD50 of 0.09, 0.1, and 0.43% respectively. The mechanism of action of these proteins to bruchids should be based on their properties of bind to chitin present in mid gut of larvae associated with the low digestibility of vicilin. In assays against phytopatogenous fungus, only EcV was capable of inhibit F. solani growth at concentrations of 10 and 20 µg and its action mechanism should be also based in the affinity of EcV to chitin present in the fungi wall

Vicilina de sementes da leguminosa selvagem Anadenanthera macrocarpa: purificação, caracterização, efeito deletério e mecanismo de ação para Callosobruchus maculatus

Grains and legume seeds are foods that form the basis of the diets of many cultures around the world, winch contritbute to the daily nutrient requirements of humans. Vicilins (7S globulin) are storage proteins found in legume seeds, and may have an additional function constitutive defense of the embryo against pests and pathogens. In this work the vicilin from Anadenanthera macrocarpa - AmV (red-angico), was purified and partially characterized, its effect on development and larval survival and adult emergence of Callosobruchus maculatus was evaluated by determination of LD50, WD50 and ED50 in system bioassay. Purification of vicilin was initiated by the chitin affinity chromatography and then gel filtration (Superdex 75 Tricorn 10x300 mm) FPLC system followed by reverse phase chromatography (C8 phenomenex) on HPLC system. Bioassays WD50 and LD50 for larvae were 0.32% and 0.33% (w:w) respectively, since the ED50 for adults was 0.096%. The probable mechanism of action was evaluated by testing digestibility of AmV in vitro, and observed for the involvement of two fragments vicilins immunoreactive against polyclonal Anti-vicilin from Erythrina velutina (Anti-EvV) about of 22 and 13 kDa chitin binding. The AmV in its native form has been recognized by the anti-EvV, indicating that there is a conserved region in the vicilin and is probably corresponding to the chitin binding domains. These results point to a new vicilin chitin binding that can subsequently be used as a possible biopesticide protein source, in order to control insect pest C. maculatus and confirm literature findings that demonstrate vicilin in the presence of different kinds of ligands to conserved regions chitin not yet characterized

Dispersão de sementes por aves na galeria ripícola e matriz envolvente

As galerias ripícolas são habitats com uma grande disponibilidade de recursos alimentares e abrigo para a fauna, estando entre os habitats mais importantes e vulneráveis do mundo. São uma fonte de produção de frutos, o que atrai muitos animais, como as aves frugívoras, que são os principais dispersores de sementes na bacia do Mediterrâneo. O objetivo principal deste estudo foi estudar os padrões de dispersão de sementes por aves na interface galeria ripícola-matriz florestal envolvente (Montado). Foram marcadas com fluorescência plantas-mãe produtoras de frutos, e utilizadas armadilhas para sementes e transectos, para a recolha dos dejetos de aves onde, posteriormente, se detetaram as sementes dispersadas. Constatou-se que a dispersão de sementes e a abundância de plantas-mãe foi superior a distâncias mais próximas da ribeira, diminuindo à medida que esta distância aumenta. A espécie mais dispersada foi Smilax aspera e as espécies com sementes de menor dimensão, foram dispersadas a uma maior distância da planta-mãe, provavelmente devido a este tipo de sementes permanecerem mais tempo no trato intestinal das aves. Os resultados deste estudo apresentam algumas implicações para a colonização da matriz adjacente por plantas produtoras de frutos; ABSTRACT: Riparian galleries are habitats with a large availability food resources and shelter for animals, being among the most important and vulnerable habitats in the world. These zones are a great source of fruits attracting many animals, such as frugivorous birds, which are the main seed dispersers in the Mediterranean basin. The objective of this thesis is the study of seed dispersal patterns by birds, in the riparian gallery and in the surrounding forest matrix (Montado). Fruits of several plant species were used to collect bird droppings and detect fluorescent seeds in those droppings. Fruit abundance and seed dispersal were higher close to the stream, and decreased rapidly with an increase in distance from the stream. From all species, the most dispersed was Smilax aspera and species with smaller seed were found at a significantly larger distance from parents plants than species with large seeds. This can be explained by the fact that smaller seed stay longer in the intestinal tract of the birds. The results of this study present some implications for the colonization of freshly fruit plants from the riparian gallery into the adjacent matrix.

Germinação e armazenamento de sementes de COCA (Erythroxylum ligustrinum DC. - Erythroxylaceae)

O objetivo deste trabalho foi avaliar o efeito da temperatura sobre a germinação e o armazenamento de sementes de Erythroxylum ligustrinum DC. As sementes foram colocadas germinar caixas de plásticas sobre duas folhas de papel, umedecidas com Nistatina 0, 2%, mantidas em germinadores nas temperaturas constantes de 5, 10, 15, 20, 25, 30, 35, 40 e 45ºC e com fotoperíodo de 12 horas. Para o armazenamento, as sementes foram colocadas em sacos de papel, mantidos à temperatura ambiente e em geladeira. em seguida, mensalmente, foi avaliada a germinação das sementes. A temperatura mínima, a faixa de temperatura ideal e a temperatura máxima para germinação de sementes de coca estão entre 10 e 15ºC, 25 e 30ºC e 40 e 45ºC, respectivamente. O armazenamento de sementes coca por longo período é inviável em embalagens de papel na geladeira (10ºC ± 2) e em temperatura ambiente (27ºC ± 5).

Estudo dos parâmetros dosimétricos de sementes de Iodo-125 desenvolvidas pelo IPEN-CNEN/SP utilizadas em braquiterapia por simulação computacional pelo método de Monte Carlo

Dissertação (Mestrado em Tecnologia Nuclear)

Desenvolvimento e construção de sistema automatizados para controle de qualidade na produção de sementes de iodo-125

Tese (Doutorado em Tecnologia Nuclear)