Zebrafish (Danio rerio) Gynogenetic Production by Heat Shock: Comparison Between Mitotic and Meiotic Treatment.

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm2) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm2 was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Avaliação imunológica dos camundongos geneticamente selecionados para diferente capacidade de produção de anticorpo

INTRODUCTION: The potentiality of the inflammatory response or the specific immune response of the individual are complex characteristics that vary continuously and have a normal distribution in a heterogeneous population, since they are under polygenic control. Aiming at the study of the genetic regulation of specific immunity, our laboratory developed strains of heterogeneous mice genetically selected for high – HIII and low – LIII ability to produce antibodies to certain antigens through the process of genetic selection bidirectional. For some genetic and immunological studies, isogenic mice must be used. Therefore, we produced isogenic trunks of these parental lines in the vivarium of the Immunogenetics laboratory of the Butantan Institute, which after the selection process were subjected to inbreeding. OBJECTIVE: Evaluate the ability to produce antibodies as well as cells – B and T lymphocytes (CD4 and CD8) after stimulating the immune system with diphtheria anatoxin, comparing isogenic trunks with parental lineages. METHODOLOGY: Isogenic mice from Stem A (High) and Stem E (Low) and heterogeneous (HIII and LIII) were immunized with diphtheria anatoxin. The primary and secondary humoral immune response was evaluated in serum by quantifying the production of anti-diphtheria IgG antibodies using the ELISA technique. For the characterization of the T and B lymphocyte cell response profile, the spleen and lymph nodes were collected and the cells were identified by surface markers (CD4, CD8, B220) and evaluated by flow cytometry. RESULTS: The results show significant increases in the concentration of antibodies produced by the groups when comparing normal serum with primary and secondary responses. The production of antibodies in the secondary response was higher in animals from the HIII and Stem A strains compared to the other groups. In the cellular response, a significant increase in T lymphocytes (CD4+and CD8+) and B lymphocytes was seen in the lymph node in the experimental HIII group compared to the other groups, however in the spleen a significantly increased T cell response was detected in the immunized animals in the LIII group. Regarding the isogenic trunks, the animals from Stem A showed an increase in CD4+ T lymphocytes when compared to the experimental Stem E in splenic cells. CONCLUSIONS: Animals selected for high antibody production capacity showed greater responses both in IgG secretion in the secondary response and in the higher frequency of T and B cells in the lymph nodes. It is important to in-depth study of the modulation mechanism of the immune response in these strains to understand the multi-specific genetic effects against certain antigens.

INTRODUÇÃO: A potencialidade da resposta inflamatória ou da resposta imune específica do indivíduo são características complexas que variam de forma contínua e têm uma distribuição normal numa população heterogênea, pois estão sob controle poligênico. Visando o estudo da regulação genética da imunidade específica nosso laboratório desenvolveu linhagens de camundongos heterogênicos geneticamente selecionadas para alta (“High” - HIII) e baixa (“Low” - LIII) capacidade de produção de anticorpos a certos antígenos através do processo de seleção genética bidirecional. Para alguns estudos genéticos e imunológicos devem ser utilizados camundongos isogênicos, assim, produzimos no Biotério do laboratório de Imunogenética do Instituto Butantan troncos isogênicos destas linhagens parentais, que após o processo seletivo foram submetidas a cruzamentos consanguíneos. OBJETIVO: Avaliar a capacidade de produção de anticorpos assim como as células – linfócitos B e T (CD4+ e CD8+) após o estímulo do sistema imune com anatoxina diftérica comparando os troncos isogênicos com as linhagens parentais. METODOLOGIA: Camundongos isogênicos (High – Tronco A e Low – Tronco E) e heterogênicos (HIII e LIII) foram imunizados com anatoxina diftérica. A resposta imune humoral primária e secundária foram avaliadas no soro pela quantificação da produção de anticorpos IgG antidiftéricos através da técnica de ELISA. Para a caracterização do perfil de resposta celular de linfócitos T e B, foram coletados o baço e linfonodos e as células foram identificadas por marcadores de superfície (CD4, CD8, B220) e avaliadas por citometria de fluxo. RESULTADOS: Os resultados mostram aumentos significativos na concentração de anticorpos produzidos pelos grupos quando comparadas o soro normal com as respostas primária e secundária. A produção de anticorpos na resposta secundária foi maior nos animais das linhagens HIII e Tronco A em relação aos outros grupos. Na resposta celular foi visualizado no linfonodo um aumento significante de linfócitos T (CD4+e CD8+) e linfócitos B no grupo HIII experimental em relação aos outros grupos, entretanto no baço a resposta significativamente aumentada das células T foi detectada nos animais imunizados do grupo LIII. Em relação aos troncos isogênicos, os animais do Tronco A apresentaram um aumento de linfócitos T CD4+ quando comparados ao Tronco E experimental nas células esplênicas. CONCLUSÕES: Os animais selecionados para alta capacidade de produção de anticorpos apresentaram respostas maiores tanto na secreção de IgG na resposta secundária como na maior frequência de células T e B nos linfonodos. Sendo importante o estudo aprofundado do mecanismo de modulação da resposta imune nessas linhagens para entendimento de efeitos genéticos multiespecíficos frente a determinados antígenos.

Fitness costs associated with chlorantraniliprole resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) strains with different genetic backgrounds.

BACKGROUND: Spodoptera frugiperda (J.E. Smith) is a difficult pest to manage mainly because of its resistance to insecticides and Bt proteins. We evaluated fitness costs of S. frugiperda resistant strains to diamide insecticides with different genetic backgrounds aiming to highlight the importance of using isogenic strains. We established a near-isogenic strain of S. frugiperda resistant to diamides (Iso-RR), using a chlorantraniliprole resistant strain (RR) selected from a field-collected population and a susceptible reference strain (SS). Fitness costs were assayed using strains with close-related genetic backgrounds (Iso-RR and SS) and strains with distant-related genetic backgrounds (RR and SS). RESULTS: No fitness cost associated with chlorantraniliprole resistance in S. frugiperda was observed using the Iso-RR strain, based on life history traits. The only parameter that differs between Iso-RR and SS strains was the mean length of a generation (T), whereas the Iso-RR strain presented T = 35.8 and SS strain showed T = 34.6. On the other hand, a significant fitness cost was detected using the RR strain. All population growth parameters differ between RR and SS strains. Based on the intrinsic rate of population increase (rm ) parameter, the relative fitness estimated was 1.02 for the Iso-RR strain and 0.64 for the RR strain. CONCLUSION: The genetic background of the resistant strains alters fitness cost outcomes. The RR strain showed fitness costs associated with resistance, but the Iso-RR did not. Our work supports the decision-making process of resistance management programs and adds to the growing body of research that enlightens the importance of strain genetics in fitness cost experiments.

Photosensitizers and exposure times to light showed tissue compatibility in isogenic mice / Fotossensibilizadores e tempo de exposição a luz em relação a compatibilidade de tecidos em ratos isogênicos

Objective: The aim of this study was to evaluate the subcutaneous tissue response after different protocols to photodynamic therapy (PDT). In Phase 1, were tested the diode laser (used for 1min) associated to the photosensitizer phenothiazine chloride solution (PCS) in different concentrations. In Phase 2 ­ the diode laser and LED were tested associated to two different photosensitizers, PCS and Curcumin, in different exposure times of light application. Material and Methods: After 7, 21 and 63-days the animals were euthanized and the subcutaneous tissue processed to histological analysis. Qualitative and semi-quantitative descriptions of the inflammatory process and immunohistochemical technique were performed. The obtained data were analyzed by Kruskal-Wallis and Dunn's post-test (α= 0.5). Results: On Phase 1, the tissue response was very similar among the groups. For the inflammatory infiltrate, PCS with concentration of 10mg/mL exhibited the most intense reaction (p > 0.05). On Phase 2, at 7-days period, the analyzed parameters presented small magnitude and after 21 and 63-days, all the parameters demonstrated tissue compatibility. Conclusion: Both photosensitizers presented proper tissue compatibility regardless the different concentrations used on Phase 1 and different durations of light exposure on Phase 2 (AU)

Objetivo: Este estudo avaliou a resposta do tecido subcutâneo após terapia fotodinâmica, utilizando na Fase 1 - laser diodo por 1min e solução fotossensibilizadora de cloreto de fenotiazina (CF) em diferentes concentrações e Fase 2 - laser diodo e LED e dois fotossensibilizadores, CF e Curcumina, em diferentes tempos de exposição da aplicação de luz. Material e Métodos: Após 7, 21 e 63 dias, foram realizadas descrições qualitativas e semiquantitativas do processo inflamatório e técnica de imunoistoquímica. Os dados foram analisados pelo pós-teste de Kruskal-Wallis e Dunn (α = 0,5). Resultados: Na Fase 1, a resposta do tecido foi muito semelhante. O infiltrado inflamatório, na concentração de 10 mg / mL, exibiu reação mais intensa (p > 0,05). Na Fase 2, aos 7 dias, os parâmetros analisados apresentaram pequena magnitude. Aos 21 e 63 dias, todos os parâmetros demonstraram compatibilidade com o tecido. Conclusão: Ambos os fotossensibilizadores apresentaram compatibilidade de tecido adequada, independentemente das diferentes concentrações utilizadas na Fase 1 e diferentes durações de exposição à luz na Fase 2 (AU)

Tissue response after subcutaneous implantation of different glass ionomer-based cements

Abstract The aim of this study was to evaluate the subcutaneous connective tissue response of isogenic mice after implantation of different glass ionomer-based cements (EQUIA® Forte Fil, EQUIA® Fil and Ketac™ Universal Aplicap™). Eighty-seven isogenic BALB/c mice were allocated in 12 groups, 9 were considered as experimental groups (Ketac, E. Fil and E. Forte at 7, 21 and 63 days) and 3 controls (empty polyethylene tubes at 7, 21 and 63 days). After the experimental periods, the subcutaneous connective tissue surrounding the implanted material was removed and subjected to histotechnical processing and staining with hematoxylin and eosin. A histopathological description of the tissue reaction surrounding each material and a semi-quantitative analysis of collagen fiber formation and inflammatory infiltrate were performed. Additionally, the thickness of the granulomatous tissue in contact with each material was measured. Data were analyzed statistically (α=0.05) by the Kruskal-Wallis test, followed by Dunn post-test. Initially, the collagen fiber formation was not different among all the tested materials (p>0.05) but was different at 21 days with the control group presenting the most advanced stage of collagen fiber formation. At 63 days, EQUIA® Forte Fil group showed the most advanced stage of collagen fiber formation, compared to EQUIA® Fil group (p<0.05). The inflammatory infiltrate was not different among the tested materials in any experimental period (p>0.05). The thickness of the granulomatous tissue was greater in the E. Forte group, compared to control in all periods. All glass ionomer-based cements showed tissue compatibility, according to the evaluated parameters.

Resumo O objetivo deste estudo foi avaliar a resposta subcutânea do tecido conjuntivo de camundongos isogênicos após o implante de diferentes cimentos à base de ionômero de vidro (EQUIA® Forte Fil, EQUIA® Fil e Ketac ™ Universal Aplicap ™). Oitenta e sete camundongos isogênicos BALB/c foram alocados em 12 grupos, 9 como grupos experimentais (Ketac, E. Fil e E. Forte aos 7, 21 e 63 dias) e 3 controles (tubos de polietileno vazios aos 7, 21 e 63 dias). Após os períodos experimentais, o tecido conjuntivo subcutâneo ao redor do material implantado foi removido e submetido ao processamento histotécnico e coloração com hematoxilina e eosina. Uma descrição histopatológica da reação tecidual envolvendo cada material e uma análise semi-quantitativa da fibrose e infiltrado inflamatório foram realizadas. Além disso, foi realizada a mensuração da espessura do tecido granulomatoso em contato com cada material. Os dados foram analisados estatisticamente (α=0,05) pelo teste de Kruskal-Wallis, seguido do pós-teste de Dunn. Inicialmente, a fibrose não foi diferente entre todos os materiais testados (p>0,05), mas foi diferente aos 21 dias, com o grupo controle apresentando o estágio mais avançado de fibrose. Aos 63 dias, o grupo EQUIA® Forte Fil apresentou o estágio mais avançado de fibrose, comparado ao grupo EQUIA® Fil (p<0,05). O infiltrado inflamatório não foi diferente entre os materiais testados em nenhum período experimental (p>0,05). A espessura do tecido granulomatoso foi maior no grupo E. Forte, comparado ao controle em todos os períodos. Todos os cimentos à base de ionômero de vidro apresentaram compatibilidade tecidual, de acordo com os parâmetros avaliados.

Effect of genetic background on the stability of sunflower fatty acid composition in different high oleic mutations.

BACKGROUND: The effect of genetic background on the stability of fatty acid composition in sunflower near isogenic lines (NILs) carrying high-oleic Pervenets (P) or high-oleic NM1 mutations was studied. The materials were field-tested in different locations and at different sowing dates to evaluate a wide range of environmental conditions. Relationships were established between the fatty acids and the minimum night temperature (MNT) and the response was characterized. RESULTS: A genetic background effect for the fatty acid composition was found in both groups of NILs. The NM1-NILs showed an oleic level higher than 910 g kg-1 and they were more stable across environments with a zero or low dependence on the genetic background; on the other hand, high oleic materials bearing the P mutation showed lower levels of oleic acid, with a higher variation in fatty acid composition and a highly significant dependence on the genetic background. CONCLUSION: The NM1 mutation is the best option to develop ultra-high oleic sunflower oil that is stable across environments and genetic backgrounds, making its agronomical production more efficient and predictable. © 2018 Society of Chemical Industry.

Genetic impact of Rht dwarfing genes on grain micronutrients concentration in wheat.

Wheat is a major staple food crop providing about 20% of dietary energy and proteins, and food products made of whole grain wheat are a major source of micronutrients like Zinc (Zn), Iron (Fe), Manganese (Mn), Magnesium (Mg), Vitamin B and E. Wheat provides about 40% intake of essential micronutrients by humans in the developing countries relying on wheat based diets. Varieties with genetically enhanced levels of grain micronutrient concentrations can provide a cost-effective and sustainable option to resource poor wheat consumers. To determine the effects of commonly deployed dwarfing genes on wheat grain Zn, Fe, Mn and Mg concentrations, nine bread wheat (Triticum aestivum) and six durum wheat (T. turgidum) isoline pairs differing for Rht1 (= Rht-B1b) and one bread wheat pair for Rht2 (= Rht-D1b) dwarfing genes were evaluated for three crop seasons at N.E. Borlaug Research Station, Cd. Obregon, Sonora, Mexico. Presence of dwarfing genes have significantly reduced grain Zn concentration by 3.9 ppm (range 1.9-10.0 ppm), and Fe by 3.2 ppm (range 1.0-14.4 ppm). On the average, about 94 ppm Mg and 6 ppm Mn reductions occurred in semidwarf varieties compared to tall varieties. The thousand kernel weight (TKW) of semidwarf isolines was 2.6 g (range 0.7-5.6 g) lower than the tall counterparts whereas the plant height decreased by 25 cm (range 16-37 cm). Reductions for all traits in semidwarfs were genotype dependent and the magnitude of height reductions did not correlate with reductions in micronutrient concentrations in wheat grain. We conclude that increased grain yield potential of semidwarf wheat varieties is associated with reduced grain micronutrient concentrations; however, the magnitude of reductions in micronutrients varied depending on genetic background and their associated pleiotropic effect on yield components.

Controle reprodutivo em biotérios de criação de animais de laboratório com ênfase em roedores / Reproductive control in Rodents animals facilities

A pesquisa experimental utiliza em sua grande maioria ratos e camundongos. Os animais de laboratório sãocaracterizados pela sua genética (isogênicos ou heterogênicos) definida através de práticas reprodutivas sistemáticas(acasalamentos irmão x irmão ou randômicos). O conhecimento da biologia e genética destes é fundamental paraque se estabeleçam controles reprodutivos capazes de evitar desperdícios espaciais, financeiros e sobretudo odescarte excessivo de excedentes. O uso de animais isogênicos elimina a variabilidade genética em experimentos,uma vez que possuem ao menos 98,2% de homozigose em seus alelos e formam uma linhagem onde ascaracterísticas genéticas e fenotípicas são únicas e exclusivas. O uso de linhagens permite a reprodutibilidade deexperimentos ao longo do tempo e em diferentes laboratórios. Ao contrário, animais heterogênicos, chamados deestoque ou colônias que objetivam mimetizar a variabilidade genética de uma população, são mantidos emacasalamentos randômicos, não possuem uma genética definida e suas características fenotípicas podem variar entrelaboratórios. Tanto um quanto o outro exigem esquemas reprodutivos rígidos para garantir a qualidade efidedignidade dos experimentos.(AU)

The great majority of animals used in research are rats and mice. Laboratory animals are characterized by theirgenetic patterns (isogenic or heterogenic) defined through systematic breeding practices (brother vs. brother orrandom mating). It is fundamental to know their biology and genetics to establish reproductive controls in order toavoid spatial and financial waste and, more important, to avoid the excessive discarding of surpluses. The use ofisogenic animals eliminates genetic variability in experiments, since they have at least 98.2% homozygoze in theiralleles and form a lineage where the genetic and phenotypic characteristics are unique and exclusive. The use oflineages allows the reproducibility of experiments in different laboratories and along the time. On the other hand,heterogenic animals, called stock or colonies, that aim to mimic the genetic variability of a population, are kept inrandom mating, they do not have a defined genetic profile and their phenotypic characteristics can vary amonglaboratories. Both ways of breeding (isogenic or heterogenic) demand rigid reproductive schemes to ensure thequality and reliability of the experiments.(AU)

Controle reprodutivo em biotérios de criação de animais de laboratório com ênfase em roedores / Reproductive control in Rodents animals facilities

A pesquisa experimental utiliza em sua grande maioria ratos e camundongos. Os animais de laboratório sãocaracterizados pela sua genética (isogênicos ou heterogênicos) definida através de práticas reprodutivas sistemáticas(acasalamentos irmão x irmão ou randômicos). O conhecimento da biologia e genética destes é fundamental paraque se estabeleçam controles reprodutivos capazes de evitar desperdícios espaciais, financeiros e sobretudo odescarte excessivo de excedentes. O uso de animais isogênicos elimina a variabilidade genética em experimentos,uma vez que possuem ao menos 98,2% de homozigose em seus alelos e formam uma linhagem onde ascaracterísticas genéticas e fenotípicas são únicas e exclusivas. O uso de linhagens permite a reprodutibilidade deexperimentos ao longo do tempo e em diferentes laboratórios. Ao contrário, animais heterogênicos, chamados deestoque ou colônias que objetivam mimetizar a variabilidade genética de uma população, são mantidos emacasalamentos randômicos, não possuem uma genética definida e suas características fenotípicas podem variar entrelaboratórios. Tanto um quanto o outro exigem esquemas reprodutivos rígidos para garantir a qualidade efidedignidade dos experimentos.

The great majority of animals used in research are rats and mice. Laboratory animals are characterized by theirgenetic patterns (isogenic or heterogenic) defined through systematic breeding practices (brother vs. brother orrandom mating). It is fundamental to know their biology and genetics to establish reproductive controls in order toavoid spatial and financial waste and, more important, to avoid the excessive discarding of surpluses. The use ofisogenic animals eliminates genetic variability in experiments, since they have at least 98.2% homozygoze in theiralleles and form a lineage where the genetic and phenotypic characteristics are unique and exclusive. The use oflineages allows the reproducibility of experiments in different laboratories and along the time. On the other hand,heterogenic animals, called stock or colonies, that aim to mimic the genetic variability of a population, are kept inrandom mating, they do not have a defined genetic profile and their phenotypic characteristics can vary amonglaboratories. Both ways of breeding (isogenic or heterogenic) demand rigid reproductive schemes to ensure thequality and reliability of the experiments.

Fitness costs and stability of Cry1Fa resistance in Brazilian populations of Spodoptera frugiperda.

BACKGROUND: The presence of fitness costs of resistance to Bacillus thuringiensis (Bt) insecticidal proteins in insect populations may delay or even reverse the local selection of insect resistance to Bt transgenic crops, and deserves rigorous investigation. Here we assessed the fitness costs associated with Cry1Fa resistance in two strains of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), derived from field collections in different Brazilian regions and further selected in the laboratory for high levels of resistance to Cry1Fa using leaves of TC1507 corn. RESULTS: Fitness components were compared using paired resistant and susceptible strains with similar genetic backgrounds and F1 generations from reciprocal crosses, all of them reared on non-transgenic corn leaves. No apparent life history costs in the larval stage were observed in the Bt-resistant strains. Moreover, the resistance remained stable for seven generations in the absence of selection, with no decrease in the proportion of resistant individuals. Larval respiration rates were also similar between resistant and susceptible homozygotes, and heterozygotes displayed respiration rates and demographic performance equal or superior to those of susceptible homozygotes. CONCLUSION: In combination, these results indicate the lack of strong fitness costs associated with resistance to Cry1Fa in the fall armyworm strains studied. These findings suggest that Cry1Fa resistance in S. frugiperda populations is unlikely to be counterselected in Cry1Fa-free environments. © 2016 Society of Chemical Industry.

Allelic differences in a vacuolar invertase affect Arabidopsis growth at early plant development.

Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level.

A mutation that eliminates bundle sheath extensions reduces leaf hydraulic conductance, stomatal conductance and assimilation rates in tomato (Solanum lycopersicum).

Bundle sheath extensions (BSEs) are key features of leaf structure whose distribution differs among species and ecosystems. The genetic control of BSE development is unknown, so BSE physiological function has not yet been studied through mutant analysis. We screened a population of ethyl methanesulfonate (EMS)-induced mutants in the genetic background of the tomato (Solanum lycopersicum) model Micro-Tom and found a mutant lacking BSEs. The leaf phenotype of the mutant strongly resembled the tomato mutant obscuravenosa (obv). We confirmed that obv lacks BSEs and that it is not allelic to our induced mutant, which we named obv-2. Leaves lacking BSEs had lower leaf hydraulic conductance and operated with lower stomatal conductance and correspondingly lower assimilation rates than wild-type leaves. This lower level of function occurred despite similarities in vein density, midvein vessel diameter and number, stomatal density, and leaf area between wild-type and mutant leaves, the implication being that the lack of BSEs hindered water dispersal within mutant leaves. Our results comparing near-isogenic lines within a single species confirm the hypothesised role of BSEs in leaf hydraulic function. They further pave the way for a genetic model-based analysis of a common leaf structure with deep ecological consequences.

Nutritional characteristics of bmr mutant and normal sorghum genotypes used for cutting and grazing / Características nutricionais de genótipos de sorgo mutantes BMR e normais utilizados para corte e pastejo

This study evaluated the nutritional characteristics of BMR mutant and normal sorghum genotypes, eleven BMR mutants and nine normal. Seeds were sown in a randomized block design with three blocks, in which each genotype was a treatment. The nutritional characteristics were analyzed at 42 days of regrowth after the first cut, determined for DM, EE, ash, CP, NDIN, NIDA, NDF, ADF, NFC, CT, HCEL, CEL and NGLs. Regarding DM, EE, ash, NDIN, NIDA, NDF, FDA and HCEL, there were no differences between genotypes. As for CP, there were differences, with mean levels ranging from 9.77% for BR001AXTX2785bmr to 16.45% for BR001AxTX2784. Considering CT and NFC, there were differences in the mean levels that ranged from 75.21 to 83.28% for BR007AxTX2785bmr and BR001AXTX2785bmr, and from 17.46 to 25.51% for CMSXS156AxTX2785bmr and IS10428xTX2784, respectively. In relation to CEL and LGN significant difference were detected, the mean levels varied between 22.30 and 27.94% for the IS10428xTX2784 and TX635AxTX2785bmr, and from 3.08 to 7.31% for BR007AxTX2785bmr and BR001AxTX2784, respectively. The genotypes IS10428xTX2784 and BR007AxTX2784 are the most suitable for use in ruminant feed.

Objetivou-se avaliar as características nutricionais de genótipos de sorgo mutantes BMR e normais, sendo onze mutantes BMR e nove normais. A semeadura foi realizada em blocos casualizados, com três blocos onde cada genótipo foi um tratamento. As características nutricionais foram analisadas aos 42 dias de rebrotação após o primeiro corte, sendo determinados os teores de MS, EE, cinzas, PB, NIDN, NIDA, FDN, FDA, CNF, CT, HCEL, CEL e LGN. Em relação à MS, ao EE, às cinzas, ao NIDN, ao NIDA, à FDN, à FDA e à HCEL, não houve diferença entre os genótipos. Quanto à PB, houve diferença, cujos teores médios oscilaram de 9,77% para o BR001AXTX2785bmr a 16,45% para o BR001AxTX2784. Em relação aos CT e CNF, houve diferença, os teores médios variaram de 75,21% para o BR007AxTX2785bmr a 83,28% para o BR001AXTX2785bmr e de 17,46 a 25,51% para o CMSXS156AxTX2785bmr e IS10428xTX2784, respectivamente. Quanto à CEL e LGN houve diferença, os teores médios variaram de 22,30% para o IS10428xTX2784 a 27,94% para o TX635AxTX2785bmr e de 3,08% para o BR007AxTX2785bmr a 7,31% para o BR001AxTX2784, respectivamente. Os genótipos IS10428xTX2784 e o BR007AxTX2784 são os mais indicados para se utilizar na alimentação de ruminantes.

Nutritional characteristics of bmr mutant and normal sorghum genotypes used for cutting and grazing / Características nutricionais de genótipos de sorgo mutantes BMR e normais utilizados para corte e pastejo

This study evaluated the nutritional characteristics of BMR mutant and normal sorghum genotypes, eleven BMR mutants and nine normal. Seeds were sown in a randomized block design with three blocks, in which each genotype was a treatment. The nutritional characteristics were analyzed at 42 days of regrowth after the first cut, determined for DM, EE, ash, CP, NDIN, NIDA, NDF, ADF, NFC, CT, HCEL, CEL and NGLs. Regarding DM, EE, ash, NDIN, NIDA, NDF, FDA and HCEL, there were no differences between genotypes. As for CP, there were differences, with mean levels ranging from 9.77% for BR001AXTX2785bmr to 16.45% for BR001AxTX2784. Considering CT and NFC, there were differences in the mean levels that ranged from 75.21 to 83.28% for BR007AxTX2785bmr and BR001AXTX2785bmr, and from 17.46 to 25.51% for CMSXS156AxTX2785bmr and IS10428xTX2784, respectively. In relation to CEL and LGN significant difference were detected, the mean levels varied between 22.30 and 27.94% for the IS10428xTX2784 and TX635AxTX2785bmr, and from 3.08 to 7.31% for BR007AxTX2785bmr and BR001AxTX2784, respectively. The genotypes IS10428xTX2784 and BR007AxTX2784 are the most suitable for use in ruminant feed.(AU)

Objetivou-se avaliar as características nutricionais de genótipos de sorgo mutantes BMR e normais, sendo onze mutantes BMR e nove normais. A semeadura foi realizada em blocos casualizados, com três blocos onde cada genótipo foi um tratamento. As características nutricionais foram analisadas aos 42 dias de rebrotação após o primeiro corte, sendo determinados os teores de MS, EE, cinzas, PB, NIDN, NIDA, FDN, FDA, CNF, CT, HCEL, CEL e LGN. Em relação à MS, ao EE, às cinzas, ao NIDN, ao NIDA, à FDN, à FDA e à HCEL, não houve diferença entre os genótipos. Quanto à PB, houve diferença, cujos teores médios oscilaram de 9,77% para o BR001AXTX2785bmr a 16,45% para o BR001AxTX2784. Em relação aos CT e CNF, houve diferença, os teores médios variaram de 75,21% para o BR007AxTX2785bmr a 83,28% para o BR001AXTX2785bmr e de 17,46 a 25,51% para o CMSXS156AxTX2785bmr e IS10428xTX2784, respectivamente. Quanto à CEL e LGN houve diferença, os teores médios variaram de 22,30% para o IS10428xTX2784 a 27,94% para o TX635AxTX2785bmr e de 3,08% para o BR007AxTX2785bmr a 7,31% para o BR001AxTX2784, respectivamente. Os genótipos IS10428xTX2784 e o BR007AxTX2784 são os mais indicados para se utilizar na alimentação de ruminantes.(AU)

Asymmetric effects of loss and gain of a floral trait on pollinator preference.

Shifts in pollination syndromes involve coordinated changes in multiple floral traits. This raises the question of how plants can cope with rapid changes in pollinator availability by the slow process of accumulation of mutations in multiple genes. Here we study the transition from bee to hawkmoth pollination in the genus Petunia. Interspecific crosses followed by single locus introgressions were used to recreate putative intermediate evolutionary stages in the evolution of moth pollination. The effect of the loss/gain of petal color was asymmetric: it had no influence on the established pollinator but enhanced visitation by the new pollinator. Therefore, shifts in pollination syndromes may proceed through intermediate stages of reduced specialization and consequently enhanced reproductive assurance. The loss of petal color in moth-pollinated Petunia involves null mutations in a single regulatory gene, An2. Such simple genetic changes may be sufficiently rapid and frequent to ensure survival during pollinator failure.

Chromosomal locations of the maize (Zea mays L.) HtP and rt genes that confer resistance to Exserohilum turcicum

We used 125 microsatellite markers to genotype the maize (Zea mays L.) near isogenic lines (NIL) L30HtPHtPRtRt and L30htphtpRtRt and the L40htphtprtrt line which contrast regarding the presence of the recently described dominant HtP and the recessive rt genes that confer resistance to Exserohilum turcicum. Five microsatellite markers revealed polymorphisms between the NIL and were considered candidate linked markers for the HtP resistance gene. Linkage was confirmed by bulked segregant sample (BSS) analysis of 32 susceptible and 34 resistant plants from a BC1F1 population derived from the cross (L30HtPHtPRtRt x L40htphtprtrt) x L40htphtprtrt. The bnlg198 and dupssr25 markers, both located on maize chromosome 2L (bin 2.08), were polymorphic between bulks. Linkage distances were estimated based on co-segregation data of the 32 susceptible plants and indicated distances of 28.7 centimorgans (cM) between HtP and bnlg198 and 23.5 cM between HtP and dupssr25. The same set of susceptible plants was also genotyped with markers polymorphic between L30HtPHtPRtRt and L40htphtprtrt in order to find markers linked to the rt gene. Marker bnlg197, from chromosome 3L (bin 3.06), was found linked to rt at a distance of 9.7 cM. This is the first report on the chromosomal locations of these newly described genes.

Comportamiento de una cepa salvaje de Saccharomyces cerevisiae killer y su isogénica sensible respecto de diferentes fuentes de nitrógeno en cultivos mixtos / Behaviour of a wild Saccharomyces cerevisiae killer yeast and its isogenic sensitive one with respect to different nitrogen sources in mixed cultures

La composición química del vino constituye el fundamento de la posterior respuesta sensorial del producto y está determinada por varios factores, como las relaciones levadura-levadura. Se denomina fenómeno killer a la secreción por parte de ciertas cepas de levadura de una proteína tóxica que mata a células denominadas sensibles. El conocimiento del comportamiento en condición aeróbica de cultivos mixtos killer-sensible es útil para relacionarlo con la primera fase de la fermentación enológica, ya que en ella puede definirse la prevalencia o no de la cepa killer. Además, el empleo de mutantes con el plásmido curado permite comparaciones más precisas. El objetivo fue analizar el mecanismo de competencia por sustrato en levaduras killer de Saccharomyces cerevisiae y su mutante sensible con el plásmido curado, empleando distintas fuentes de nitrógeno. Si las muestras se incuban a temperatura de inactivación de la toxina, se evita la infraestimación de células sensibles. Los resultados del co-cultivo de las cepas en proporciones iguales muestran el rol desempeñado por la fuente de nitrógeno en la actividad killer. Cuando el inóculo es 10%K-90%S, el modelo de exclusión competitiva planteado para levaduras killer deja paso a otras variables de competencia.

Wine chemical composition is the outcome of complex chemosensory interactions that are difficult to predict because of the influences of many variables, like as yeast-yeast interactions. Killer phenomenon implicates the secretion of a toxic protein by some yeasts, that kill other yeasts called sensitive. The knowledge of the behaviour of killer-sensitive mixed cultures in aerobic conditions is useful to be related with the first stages of oenological fermentation. In these stages it can be defined the killer prevalence in the medium. Also, the use of cured plasmid mutants allows better comparisons. The objective was to analyse the mechanism of substrate competition in Saccharomyces cerevisiae killer strains and its sensitive cured plasmid mutant, using different nitrogen sources. When samples were incubated at the toxin inactivation temperature, the infraestimation of sensitive cells is avoided. Results obtained in co-cultures (50%K-50%S) show the role of the nitrogen source in killer activity. Results obtained with 10%K-90%S inoculum, show that there are another competence variables than the competitive exclusion model for killer yeasts.

Regeneração de plantas de trigo (Triticum aestivum L.) de linhas isogênicas do genótipo Maringá com genes de porte baixo (Rht1 e Rht2)

Different genotypes show different responses for in vitro plant regeneration. In previous work with lhe Maringá genotype a result of 100% of in vitro plant regeneration was obtained. The purpose of this study was to investigate the capacity of plant regeneration of Maringá genotype and two of it's isogenic lines with one and two shorts heigth genes (Rht1 and Rht2). Imature embryos from: (a) Maringá, (b) the isogenic lines and (c) the crosses between these genotypes were placed on MS medium (modified by MILACH et al., 1991) with saccharose, agar and decreasing doses of 2,4D (2mg/l; 0.5mg/l; 0.1mg/l and 0.0mg/l) according to the stage of culture. The possibility of suppressing one of the stages of culture, (which uses 0.1mg/1 of 2.4D) was also evaluated. The statistic analisys demonstrated that the different genotypes gave different responses, therefore this indicates that the Rht genes had influence on plant regeneration rates. The suppression of the stage of culture which uses 0.1mg/l of 2,4D did not have any influence on regeneration, showing that this stage can be eliminated. Different genotypes had the same responses on the different culture medium sequences. The intercection between genotype x medium was not significam.

Existem variações entre genótipos de trigo quanto à regeneração de plantas in vitro. Em trabalho precursor com o genótipo Maringá obteve-se 100% de regeneração de plantas. Este trabalho visou investigar a capacidade de regeneração de plantas do Maringá e duas linhas isogênicas com um e dois genes de estatura reduzida (Rht1 e Rht2). Foram utilizados embriões imaturos de Maringá, das linhas isogênicas e das F2's dos cruzamentos entre estes genótipos, que foram colocados em meio de cultura MS modificado (MILACH et al., 1991) com sacarose, ágar e doses decrescentes de 2,4D conforme a etapa de cultivo (2mg/l; 0,5mg/l; 0,1mg/l e sem 2,4D). Também foi avaliada a possibilidade de supressão de um subcultivo, quando os calos não passariam pelo meio com 0,1mg/l de 2,4D. As análises estatísticas demonstraram que os genótipos diferiram quanto à regeneração de plantas, evidenciando a influência do gene Rht, que reduziu o número de plantas regeneradas por calo. A retirada do meio com 0,1mg/l de 2,4D não alterou a regeneração, podendo-se eliminar esta etapa de cultivo. Os genótipos responderam da mesma forma nas diferentes sequências de subcultivo, não havendo interação entre estes dois caracteres.

Regeneração de plantas de trigo (Triticum aestivum L.) de linhas isogênicas do genótipo Maringá com genes de porte baixo (Rht1 e Rht2)

Different genotypes show different responses for in vitro plant regeneration. In previous work with lhe Maringá genotype a result of 100% of in vitro plant regeneration was obtained. The purpose of this study was to investigate the capacity of plant regeneration of Maringá genotype and two of it's isogenic lines with one and two shorts heigth genes (Rht1 and Rht2). Imature embryos from: (a) Maringá, (b) the isogenic lines and (c) the crosses between these genotypes were placed on MS medium (modified by MILACH et al., 1991) with saccharose, agar and decreasing doses of 2,4D (2mg/l; 0.5mg/l; 0.1mg/l and 0.0mg/l) according to the stage of culture. The possibility of suppressing one of the stages of culture, (which uses 0.1mg/1 of 2.4D) was also evaluated. The statistic analisys demonstrated that the different genotypes gave different responses, therefore this indicates that the Rht genes had influence on plant regeneration rates. The suppression of the stage of culture which uses 0.1mg/l of 2,4D did not have any influence on regeneration, showing that this stage can be eliminated. Different genotypes had the same responses on the different culture medium sequences. The intercection between genotype x medium was not significam.

Existem variações entre genótipos de trigo quanto à regeneração de plantas in vitro. Em trabalho precursor com o genótipo Maringá obteve-se 100% de regeneração de plantas. Este trabalho visou investigar a capacidade de regeneração de plantas do Maringá e duas linhas isogênicas com um e dois genes de estatura reduzida (Rht1 e Rht2). Foram utilizados embriões imaturos de Maringá, das linhas isogênicas e das F2's dos cruzamentos entre estes genótipos, que foram colocados em meio de cultura MS modificado (MILACH et al., 1991) com sacarose, ágar e doses decrescentes de 2,4D conforme a etapa de cultivo (2mg/l; 0,5mg/l; 0,1mg/l e sem 2,4D). Também foi avaliada a possibilidade de supressão de um subcultivo, quando os calos não passariam pelo meio com 0,1mg/l de 2,4D. As análises estatísticas demonstraram que os genótipos diferiram quanto à regeneração de plantas, evidenciando a influência do gene Rht, que reduziu o número de plantas regeneradas por calo. A retirada do meio com 0,1mg/l de 2,4D não alterou a regeneração, podendo-se eliminar esta etapa de cultivo. Os genótipos responderam da mesma forma nas diferentes sequências de subcultivo, não havendo interação entre estes dois caracteres.

Regeneração de plantas de trigo (Triticum aestivum L. ) de linhas isogênicas do genótipo Maringá com genes de porte baixo (Rht1 e Rht2) / Plant regeneration of isogenic lines of the Maringá genotype of wheat (Triticum aestivum L. ) with short height genes (Rh1 and Rht2)

Existem variações entre genótipos de trigo quanto à regeneração de plantas in vitro. Em trabalho precursor com o genótipo Maringá obteve-se 100 por cento de regeneração de plantas. Este trabalho visou investigar a capacidade de regeneração de plantas do Maringá e duas linhas isogênicas com um e dois genes de estatura reduzida (Rht1 e Rht2). Foram utilizados embriões imaturos de Maringá, das linhas isogênicas e das F2's dos cruzamentos entre estes genótipos, que foram colocados em meio de cultura MS modificado (MILACH et al., 1991) com sacarose, ágar e doses decrescentes de 2,4D conforme a etapa de cultivo (2mg/l; 0,5mg/l; 0,1mg/l e sem 2,4D). Também foi avaliada a possibilidade de supressão de um subcultivo, quando os calos não passariam pelo meio com 0,1mg/l de 2,4D. As análises estatísticas demonstraram que os genótipos diferiram quanto à regeneração de plantas, evidenciando a influência do gene Rht, que reduziu o número de plantas regeneradas por calo. A retirada do meio com 0,1mg/l de 2,4D não alterou a regeneração, podendo-se eliminar esta etapa de cultivo. Os genótipos responderam da mesma forma nas diferentes sequências de subcultivo, não havendo interação entre estes dois caracteres.

Different genotypes show different responses for in vitro plant regeneration. In previous work with lhe Maringá genotype a result of 100 percent of in vitro plant regeneration was obtained. The purpose of this study was to investigate the capacity of plant regeneration of Maringá genotype and two of it's isogenic lines with one and two shorts heigth genes (Rht1 and Rht2). Imature embryos from: (a) Maringá, (b) the isogenic lines and (c) the crosses between these genotypes were placed on MS medium (modified by MILACH et al., 1991) with saccharose, agar and decreasing doses of 2,4D (2mg/l; 0.5mg/l; 0.1mg/l and 0.0mg/l) according to the stage of culture. The possibility of suppressing one of the stages of culture, (which uses 0.1mg/1 of 2.4D) was also evaluated. The statistic analisys demonstrated that the different genotypes gave different responses, therefore this indicates that the Rht genes had influence on plant regeneration rates. The suppression of the stage of culture which uses 0.1mg/l of 2,4D did not have any influence on regeneration, showing that this stage can be eliminated. Different genotypes had the same responses on the different culture medium sequences. The intercection between genotype x medium was not significam.