Transformação genética em cevada por bombardeamento de partículas

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.

The field resistance of potato cultivars to foliar late blight and its relationship with foliage maturity type and skin type in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização da resistência do algodoeiro a Ramularia areola e variabilidade molecular do patógeno

This research was conducted with the aim to study the genetic and pathogenic structure of Ramularia areola isolates collected in Brazil and to characterize the resistance response in cotton plants to ramularia spot. The genetic variability of 28 isolates of R. areola was studied using RAPD markers. The pathogenicity evaluation was realized by the inoculation of 6 isolates on cotton varieties Guazuncho-2 (Gossypium hirsutum) and VH8-4602 (Gossypium barbadense). The inheritance of disease resistance was studied using an artificially inoculated population of F2 individuals derived from the intercross of Guazuncho-2 (susceptible variety) end VH8-4602 (resistant variety), and also the parents and F1 individuals. Molecular polymorphism between the G. hisutum varieties DeltaOpal (suscetible) and CNPA CO-11612 (resistant) was estimated by 118 SSR and 24 AFLP markers. The parental genotypes Guazuncho-2 and VH8-4602 were selected for mapping, and then Recombinant Inbred Lines (RIL´s) derived from this crossing were evaluated with SSR 12 markers. The analysis of population structure of R. areola revealed that the three subpopulations were genetically simillar (Gst=0.18), and the isolates from Goiás and Minas Gerais were more similar to each other (0,92). This probability can be related to the relatively high gene flow among the three subpopulations (Nm=2.20). The isolates R. areola 9.1, from Minas Gerais State and 8.1 and 8.3 from Goiás State were the most aggressive ones to the susceptible variety Guazuncho-2. The variety VH8-4602 presented high level of resistance to ramularia spot. No differential interaction was observed between the pathogens and the analyzed varieties, and the resistance was classified as horizontal. The quantification of disease by number of necrotic lesions and number of spores in individual plants of F1 and F2 generations from the crossing between the varieties Guazuncho-2 and VH8-4602 presented continuous distribution, suggesting polygenic resistance. The resistance is probabilly recessive, since necrotic lesions and sporulation were observed on F1 plants. The molecular polymorphism between DeltaOpal e CNPA CO-11612 lineages was low (6%), then would be difficult to accomplish molecular mapping of disease resistance using this intercross. With the genotyping of the RIL s it was verified that 25% of the markers segregated in the proportions proposed by Mendel s Law and 75% of the studied markers presented segregation distortion in favor to the parental G. hirsutum. Both the low genetic variability of the pathogen and the number of resistance genes suggest that durable genetic resitance may be achieved

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

A herança da resistência ao oídio na cultivar de ervilha MK-10 e alguns aspectos histológicos da infecção foram estudados. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas. Nas avaliações histológicas observou-se a porcentagem de conídios germinados, porcentagem de conídios que formaram apressório, porcentagem de conídios que estabeleceram colônia e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração, característica da presença do par de alelos recessivos er1er1.

Diallel analysis of maize resistance to Phaeosphaeria maydis

Em programas de melhoramento visando resistência genética a doenças, a estimativa de parâmetros genéticos que governam a resistência permite direcionar a introdução de resistência em germoplasmas. O objetivo deste trabalho foi estimar os efeitos heteróticos, a capacidade geral (CGC) e específica (CEC) de combinação, utilizando-se de dois métodos de avaliação da resistência, à Phaeosphaeria maydis através da análise dialélica de 36 híbridos F1 e de suas nove linhagens genitoras, em experimentos conduzidos em três ambientes. Foi utilizado um delineamento experimental em blocos casualizados com três repetições e a parcela experimental foi representada por uma fileira de 5 m. As diferenças entre as estimativas da capacidade de combinação, em diferentes ambientes e para os dois métodos de avaliação, apresentaram efeitos significativos (P < 0.01) para ambientes (E), CGC e CGC x E. O efeito de CEC e a interação CEC x E não foi significativa para os dois métodos de avaliação. Os efeitos de CGC foram mais importantes que CEC nesse conjunto de linhagens, sugerindo que efeitos genéticos aditivos são mais importantes como fonte de variação para resistência a esta doença. Efeitos heteróticos para resistência foram estimados, sendo possível identificar combinações híbridas específicas entre linhagens com alto potencial para o controle genético deste patógeno. Resultados para os dois métodos de avaliação foram praticamente idênticos, embora o método PI seja de maior praticidade de uso.

Genetics of resistance to soybean cyst nematode, Heterodera glycines ichione (Race 3), in a brazilian soybean population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A novel technique to produce polygenic resistance to anthracnose in Stylosanthes capitata

An improved cultivar, based on 17 genotypes of S. capitata and six of S.macrocephala, was developed at the wEmbrapa Beef Cattle Research Center, Campo Grande, Brazil. The aim was to create durable, race non-specific resistance to anthracnose controlled by polygenic factors. A mass hybridisation technique was employed to produce a high degree of genetic diversity and sizeable quantities of seed of hybrid-derived progenies of Brazilian and Venezuelan genotypes of S. capitata. Outcrossing resulted in a significant improvement in the forage production of progeny of Venezuelan accessions. The multicross was evaluated in multilocational trials, each representing a large tract of country in the Cerrados ecosystem along a north-south transect from lat.6degrees S to 20degrees S. The genetic shift that occurred in S. capitata was a key element in the formation of the new cultivar. It is a complex mixture of two species, and a recombination of much desired forage traits of Brazilian x Venezuelan genotypes, high forage and seed yields coupled with anthracnose resistance. The new cultivar with its diverse genetic make-up has a wide application in the acid-soil savannas of tropical America. It was released by Embrapa for the Cerrados in 2000.

Identification of quantitative trait loci affecting resistance to gastrointestinal parasites in a double backcross population of Red Maasai and Dorper sheep

A genome-wide scan for quantitative trait loci (QTL) affecting gastrointestinal nematode resistance in sheep was completed using a double backcross population derived from Red Maasai and Dorper ewes bred to F1 rams. This design provided an opportunity to map potentially unique genetic variation associated with a parasite-tolerant breed like Red Maasai, a breed developed to survive East African grazing conditions. Parasite indicator phenotypes (blood packed cell volume PCV and faecal egg count FEC) were collected on a weekly basis from 1064 lambs during a single 3-month post-weaning grazing challenge on infected pastures. The averages of last measurements for FEC (AVFEC) and PCV (AVPCV), along with decline in PCV from challenge start to end (PCVD), were used to select lambs (N = 371) for genotyping that represented the tails (10% threshold) of the phenotypic distributions. Marker genotypes for 172 microsatellite loci covering 25 of 26 autosomes (1560.7 cm) were scored and corrected by Genoprob prior to qxpak analysis that included BoxCox transformed AVFEC and arcsine transformed PCV statistics. Significant QTL for AVFEC and AVPCV were detected on four chromosomes, and this included a novel AVFEC QTL on chromosome 6 that would have remained undetected without BoxCox transformation methods. The most significant P-values for AVFEC, AVPCV and PCVD overlapped the same marker interval on chromosome 22, suggesting the potential for a single causative mutation, which remains unknown. In all cases, the favourable QTL allele was always contributed from Red Maasai, providing support for the idea that future marker-assisted selection for genetic improvement of production in East Africa will rely on markers in linkage disequilibrium with these QTL.

An alternative field method for screening soybean genotypes for resistance to Heterodera glycines

The soybean cyst nematode (Heterodera glycines) has become an increasingly severe problem in soybean production areas in Brazil. The development and use of resistant cultivars is the most efficient method of minimizing losses due to this pathogen. Our objective was to test the efficiency of an alternative method for screening soybean genotypes for resistance to H. glycines in field plots. The alternative method was compared to the standard method of sowing the test genotypes in fields found to be infested during the previous crop season. In the alternative method, the test genotypes are sown in the furrow following the uprooting of 45-day-old infected plants. The alternative method resulted in twice the cyst population and fewer escapes, and more consistent results than the standard method. The major advantage of the alternative method is that it permits screening in a more homogeneous distribution of H. glycines in the soil.

Physical exercise modulates peripheral levels of brain-derived neurotrophic factor (BDNF): A systematic review of experimental studies in the elderly

The objective of this study was to conduct a systematic review of studies that analyzed the effect of physical exercise on the peripheral levels of BDNF in elderly individuals. Method: We conducted a search in PsycINFO, Biological Abstracts, Pubmed, Web of Science, and Science Direct from 1990 to 2011, using the following keywords: physical exercise , physical activity , physical therapy , training , BDNF , neuroplasticity , neurotrophins , neuroplasticity proteins , aged , older , elderly The articles were considered for inclusion in the review if they were studies with elderly, assessed peripheral (serum and/or plasma) BDNF and evaluated an acute exercise or chronic exercise (training). Results: Five randomized controlled trial and one randomized non-controlled trial studies were analyzed. Five out of six studies reported a significantly higher BDNF response to aerobic acute exercise and to aerobic or strength training program in healthy elderly and elderly with different pathologies. Conclusion: It was not possible to establish a recommendation protocol for the type and intensity of physical exercise required to produce an increase in levels BDNF. However, physical exercise, particularly, moderate-intensity exercises seem to be more effective to promote increase the peripheral levels of BDNF in the elderly. © 2012 Elsevier B.V.

Elucidating capacitance and resistance terms in confined electroactive molecular layers

Electrochemical analyses on confined electroactive molecular layers, herein exemplified with electroactive self-assembled monolayers, sample current contributions that are significantly influenced by additional nonfaradaic and uncompensated resistance effects that, though unresolved, can strongly distort redox analysis. Prior work has shown that impedance-derived capacitance spectroscopy approaches can cleanly resolve all contributions generated at such films, including those which are related to the layer dipolar/electrostatic relaxation characteristics. We show herein that, in isolating the faradaic and nonfaradaic contributions present within an improved equivalent circuit description of such interfaces, it is possible to accurately simulate subsequently observed cyclic voltammograms (that is, generated current versus potential patterns map accurately onto frequency domain measurements). Not only does this enable a frequency-resolved quantification of all components present, and in so doing, a full validation of the equivalent circuit model utilized, but also facilitates the generation of background subtracted cyclic voltammograms remarkably free from all but faradaic contributions. © 2012 American Chemical Society.

Physiology and endocrinology symposium: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Plant-derived essential oils affecting settlement and oviposition of Bemisia tabaci (Genn.) biotype B on tomato

The silverleaf whitefly Bemisia tabaci (Genn.) biotype B (Hemiptera: Aleyrodidae) is an economically important pest of tomatoes Solanum lycopersicum (L.), causing irregular ripening on fruits and transmitting several plant pathogenic geminiviruses. The management of this pest is commonly based on repetitive spraying with synthetic pesticides, causing serious environmental damages and increase of resistance by insect population. In the present study, essential oils from the leaves of Artemisia camphorata Vill., Ageratum conyzoides L., Foeniculum vulgare Mill., Lippia alba (Mill.) N. E. Br., Plectranthus neochilus Schltr., and Tagetes erecta L. were investigated for their possible repellent and oviposition-deterrent effects against B. tabaci biotype B on tomato. In a multi-choice assay, P. neochilus essential oil was the most active repellent and oviposition deterrent. Essential oils of A. conyzoides and T. erecta significantly deterred the female B. tabaci biotype B from laying eggs on treated tomato leaflets compared with the control. (E)-Caryophyllene (30.67 %) and the monoterpenes α-pinene (15.02 %) and α-thujene (11.70 %) were identified as the major constituents of the essential oil of P. neochilus. Our findings demonstrated the potential of essential oil of P. neochilus and other oils in the reduction of settlement and oviposition of B. tabaci biotype B on tomato. © 2012 Springer-Verlag Berlin Heidelberg.