Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Herança genética e marcadores moleculares associados à resistência de Euphorbia heterophylla L. aos herbicidas inibidores da ALS e PROTOX

This study aimed to assess the genetic inheritance, determine the better DNA isolation protocol for this species and to identify molecular markers associated with the Wild Poinsettia (Euphorbia heterophylla L.) resistance ALS- and PROTOX- inhibiting herbicides and. The genetic inheritance of resistance was determined from crosses between E. heterophylla biotypes susceptible (S) and resistant (R), backcrosses and F2 generation. The complete dominance of resistance was confirmed with dose response curves. Ten adjusted methods for DNA isolation described in the literature were tested. The specific primers for ALS and PROTOX genes were designed from the consensus DNA sequence of these genes, obtained by aligning the gene sequences of the species Manihot esculenta and Ricinus communis L. Additionally, it was assessed the transferability of twenty SSR (simple sequence repeat) markers designed for Manihot esculenta, because among the species of Euphorbiaceae with more developed SSRs markers, because it is the closest relative phylogenetic species of E. heterophylla. Regarding genetic inheritance, the frequencies observed in the F1, F2, RCs and RCr did not differ significantly from the expected frequencies for a trait controlled by two dominant genes for multiple resistance and a single dominant gene for simple resistance to ALS- and PROTOX-inhibiting herbicides. The similar levels of resistance to dosage up to 2000 g i.a. ha-1 of fomesafen and dosage up to 800 g i.a. ha-1 of imazethapyr observed in F1 (heterozygous) and homozygous R biotype confirm the complete dominance of resistance to PROTOX- and ALS-inhibiting herbicides, respectively. The 0.2%BME protocol allowed the isolation of 7,083 ng μL-1 DNA, significantly (P=0.05) higher than other methods. Co-isolation of phenolic compounds was observed in FENOL and 3%BME+TB methods, but the addition of polyvinylpyrrolidone (PVP40) in the protocol extraction buffer 3%BME+TA solved this problem. The primers designed for ALS and PROTOX genes amplified but not showed no visible polymorphism in agarose gel between the S and R biotypes of E. heterophylla. Regarding the SSR transferability, ten markers were transferred to E. heterophylla, however, these six primers showed polymorphism among S and R biotypes.

Mandioca.

2016

Diversidade da fauna edáfica e epigeica de invertebrados em consórcio de mandioca com adubos verdes.

2016

Diversidade genética entre acessos de mandioca avaliados a partir de caracteres morfo-agronômicos.

A avaliação de acessos locais representa uma etapa importante do programa de melhoramento da mandioca, com vistas à identificação e recomendação de materiais superiores. Serve também para quantificar a diversidade genética entre progenitores visando futuros cruzamentos, sendo a análise multivariada um instrumento bastante utilizado pelos melhoristas para estimar a diversidade genética. O objetivo deste trabalho foi identificar acessos de mandioca produtivos e divergentes que possam ser utilizados em programa de melhoramento envolvendo cruzamentos. Doze caracteres morfo-agronômicos foram utilizados para a obtenção do calculo da divergência genética por meio da analise de agrupamento em que a medida de dissimilaridade utilizada foi a distancia euclidiana media padronizada e o método de agrupamento de otimização de Tocher, envolvendo 45 acessos de mandioca, coletados no Estado do Amapá e avaliados em dois anos. Constatou-se que os acessos foram mais contrastantes tendo como referencia os caracteres altura da planta e ramificação com 94,67% da variação. Contudo, a produção de raiz, apesar de ser muito importante, apresentou baixa contribuição para a divergência. As distancias euclidianas medias padronizadas classificaram os acessos em oito grupos distintos, confirmando a presença de divergência genética entre os acessos e perspectivas promissoras na obtenção de ganho por meio da seleção de progênies provenientes de cruzamentos envolvendo o acesso Feifim 2, que apresentou a segunda maior produtividade de raiz e maior divergência genética entre os acessos analisados.

Ocorrência de bactérias diazotroficas e fungos micorrizicos arbusculares na cultura da mandioca.

Este trabalho teve como objetivo avaliar a ocorrencia, isolar e identificar fungos micorrízicos carbunculares associados a cultura da mandioca (Manihot esculenta). Amostras de solo rizosférico e de varias partes da planta (raízes, tubérculos, manivas e folhas) de locais nos Estados do Rio de Janeiro, São Paulo e Paraná, foram inoculadas nos meios LGI-P, NFb-malato e NFb-GOC, avaliando-se o numero mais provável de células e a atividade de redução de acetileno. Bactérias diazotróficas foram isoladas de todas as partes da planta, com exceção das folhas, sendo identificadas como Klebsiella sp., Azospirillum lipoferum e uma bactéria denominada "E", provavelmente pertencente ao gênero Burkholderia. A bactéria E acumulou de 7,63 mg a 14,84 mg de N/g de C em meio semi-solido, isento de N, e conseguiu manter a capacidade de fixação biológica de N, mesmo apos uma dezena de repicagens consecutivas. A colonização micorrízica variou de 31% a 69%, e a densidade de esporos de 10 a 384 esporos/100 mL de solo, predominando as espécies Entrophospora colombiana e Acaulospora scrobiculata no Rio de Janeiro, A. scrobiculata e Scutellospora heterogama no Paraná e em Piracicaba (São Paulo) e A. appendicula e S. pellucida em Campinas (São Paulo). This study was performed to evaluate the occurrence and to isolate and identify diazotrophic bacteria and arbuscular mycorrhizal fungi associated with the cassava (Manihot esculenta) crop. Samples from rhizosperical soil, roots, tubers, stems and leaves from several localities of the States of Rio de Janeiro, Sao Paulo and Parana, in Brazil, were inoculated in three media specific for diazotrophic associative bacteria, LG1-P, NFb-malate and NFb-GOC, evaluating the most probable number of cells and the acetylene-reducing activity. Diazotrophic bacteria were detected in all plant parts except for the leaves, and were identified as Klebsiella sp., Azospirillum lipoferum and a bacterium called "E", probably belonging to the Burkholderia genus. Bacterium E was able to accumulate, in the N-free semi-solid media, from 7.63 to 14.84 mg of N/g of C and to maintain N fixation capacity after ten consecutive transferences. Mycorrhizal root colonization varied from 31% to 69% and spore density from 10 to 384 spores/100 mL of soil, predominanting the species Entrophospora colombiana and Acaulospora scrobiculata in Rio de Janeiro, A. scrobiculata and Scutellospora heterogama in Parana and in Piracicaba (Sao Paulo), and A. appendicula and S. pellucida in Campinas (Sao Paulo).

Somatic embryogenesis, organogenesis and plant regeneration in taro (Colocasia esculenta var. esculenta)

Callus was initiated in three different ‘‘esculenta’’ taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4- dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and *72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.

Improving taro (Colocasia esculenta var. esculenta) production using biotechnological approaches

Taro (Colocasia esculenta L. Schott) is an important crop worldwide but is of particular significance in many Pacific Island countries where it forms part of the staple diet and serves as an export commodity. Escalating pest and disease problems are jeopardizing taro production with serious implications to food security and trade. Biotechnological approaches to addressing pest and disease problems, such as somatic embryogenesis and transgenesis, are potentially viable options. However, despite biotechnological advancements in higher profile agronomic crops, such progress in relation to Colocasia esculenta var. esculenta has been slow. This paper reviews taro biology, highlights the cultural and economic significance of taro in Pacific Island countries and discusses the progress made towards the molecular breeding of this important crop to date.

Initiation of embryogenic cell suspensions of taro (Colocasia esculenta var. esculenta) and plant regeneration

Embryogenic callus was initiated by culturing in vitro taro corm slices on agar-solidified half-strength MS medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by transfer to 1.0 mg/L thidiazuron (TDZ). Callus was subsequently proliferated on solid medium containing 1.0 mg/L TDZ, 0.5 mg/L 2,4- D and 800 mg/L glutamine before transfer to liquid medium containing the same components but with reduced glutamine (100 mg/L). After 3 months in liquid culture on an orbital shaker, cytoplasmically dense cell aggregates began to form. Somatic embryogenesis was induced by plating suspension cells onto solid media containing reduced levels of hormones (0.1 mg/L TDZ, 0.05 mg/L 2,4-D), high concentrations of sucrose (40–50 g/L) and biotin (1.0 mg/L). Embryo maturation and germination was then induced on media containing 0.05 mg/L benzyladenine (BA) and 0.1 mg/L indole-3-acetic acid (IAA). Histological studies of the developing embryos revealed the presence of typical shoot and root poles suggesting that these structures were true somatic embryos. The rate of somatic embryos formation was 500–3,000 per mL settledcell volume while approximately 60% of the embryos regenerated into plants.

Assessment and rationalization of genetic diversity of Papua New Guinea taro (Colocasia esculenta) using SSR DNA fingerprinting

Taro (Colocasia esculenta) accessions were collected from 15 provinces of Papua New Guinea (PNG). The collection, totalling 859 accessions was collated for characterization and a core collection of 81 accessions (10%) was established on the basis of characterization data generated on 30 agro-morphological descriptors, and DNA fingerprinting using seven SSR primers. The selection of accessions was based on cluster analysis of the morphological data enabling initial selection of 20% accessions. The 20% sample was then reduced and rationalized to 10% based on molecular data generated by SSR primers. This represents the first national core collection of any species established in PNG based on molecular markers. The core has been integrated with core from other Pacific Island countries, contributing to a Pacific regional core collection, which is conserved in vitro in the South Pacific Regional Germplasm Centre at Fiji. The core collection is a valuable resource for food security of the South Pacific region and is currently being utilized by the breeding programmes of small Pacific Island countries to broaden the genetic base of the crop.

Competition of sorghum cultivars and densities with Japanese millet (Echinochloa esculenta).

Field studies were conducted at two locations in southern Queensland, Australia during the 2003-2004 and 2004-2005 growing seasons to determine the differential competitiveness of sorghum (Sorghum bicolor L. Moench) cultivars and crop densities against weeds and the sorghum yield loss due to weeds. Weed competition was investigated by growing sorghum in the presence or absence of a model grass weed, Japanese millet (Echinochloa esculenta). The correlation analyses showed that the early growth traits (height, shoot biomass, and daily growth rate of the shoot biomass) of sorghum adversely affected the height, biomass, and seed production of millet, as measured at maturity. "MR Goldrush" and "Bonus MR" were the most competitive cultivars, resulting in reduced weed biomass, weed density, and weed seed production. The density of sorghum also had a significant effect on the crop's ability to compete with millet. When compared to the density of 4.5 plants per m2, sorghum that was planted at 7.5 plants per m2 suppressed the density, biomass, and seed production of millet by 22%, 27% and 38%, respectively. Millet caused a significant yield loss in comparison with the weed-free plots. The combined weed-suppressive effects of the competitive cultivars, such as MR Goldrush, and high crop densities minimized the yield losses from the weeds. These results indicate that sorghum competition against grass weeds can be improved by choosing competitive cultivars and by using a high crop density of > 7.5 plants per m2. These non-chemical options should be included in an integrated weed management program for better weed management, particularly where the control options are limited by the evolution of herbicide resistance.

Efecto de dos condiciones de humedad del suelo y tiempo de cosecha sobre el rendimiento de malanga (Colocasia esculenta L. Schott) para exportacion, Boaco - Nicaragua 2011

La malanga ( Colocasia esculenta. (L.) Schott) , familia Aráceae, es el tubérculo de mayor exportación en Nicaragua, pero es escasa la información sobre su agrotecnia. Se evaluó el efecto que tienen el tiempo de cosecha y dos condiciones de humedad del suelo sobre el rendimiento de Malanga Lila para exportación. Se establecieron dos ensayos. En el ensayo I se evaluó el efecto de cosechar las plantas a los 7 meses (7M) y 9 meses (9M) en suelo seco. Se establecieron 2 lotes de plantas de 0.7 ha cada uno, el primero en noviembre 2009 y el otro en enero 2010, ambos lotes cosechados en agosto 2010. En el ensayo II se evaluó el efecto de siembra en secano (7MS) y fangoso (7MF) sobre el rendimiento. En ambos ensayos se establecieron cuatro submuestras de 10 plantas (observaciones) por lote. En el Ensayo I se evaluaron variables morfológicas a 127, 150, 180 y 215 días después de la siembra (dds) en relación a plantas 9MS y a los 96, 120, 150 y 185 dds a plantas 7M. El ensayo II se evaluó de igual forma que las plastas 7M del ensayo I. A los datos de las variables morfológicas se realizó la prueba de T-Student y las de rendimiento un ANDEVA. Se determinó la incidencia del Virus del Mosaico del Dasheen (DsMV) mediante la prueba ELISA, se evaluó la concentración de materia seca de los cormos de plantas madres. Las plantas 9M y 7M registraron valores de las variables morfológicas y rendimiento similares (45.15 y 42.47 t ha-1 respectivamente), el momento ó ptimo de cosecha es 9 meses. Las plantas 7MF crecieron más lentamente que las platas 7MS, las que no lograron el máximo desarrollo foliar y presentaron mayor incidencia de enfermedades fungosas y bacterianas en cormos, raíces y hojas. Las plantas de ambos ensayos mostraron síntomas del virus. Las plantas 7MF presentaron cormos alargados y menos anchos que las plantas 7MS.

Influencia de la forma del corte apical y de la posición de la estaca, en la producción de raiz en yuca (Manihot utilissima polh)

Con el objeto de determinar si la posición y el corte en el extremo apical, influían en el rendimiento de la raíz de yuca, se hizo un ensayo en el cual se utilizaron tres posiciones diferentes: a) Horizontal, b) Inolinada y c) Vertical; con tres cortes diferentes en el extremo apical enterrado: 1) Redondo, 2) Chaflan y 3) Punta de lápiz. Se uso el diseño experimental de bloques completos al azar con arreglo de parcelas sub-divididas, empleándose seis repeticiones. La cosecha se hizo a los siente meses de la siembra, obteniéndose la mayor producción con la interacción posición inclinada corte chaflan, la cual mostró diferencia significativa para uno por ciento de error. La posición de siembra influyo significativamente sobre el rendimiento de raíz de yuca; observándose que la posición inclinada fue la que facilito mas el cultivo. Se hizo dos labores de limpia en todo el periodo de ensayo. El porcentaje de enraizamiento de plantas, altura de plantas numero de raíces, tamaño de raíces y no mostraron diferencias estadísticas significativas. Este ensayo se realizo en los campos de la Escuela Nacional de Agricultura y Ganadería (ENAG), durante el periodo de julio de 1968 a febrero de 1969. La variedad empleada fue la Cubana, cortándose para este estacas de cuarenta a cuarenta y cinco centímetros de longitud de todo el tallo, con excepción de la parte muy cerca de la raíz y la muy tierna cerca del extremo a pical.

Estudio de la densidad de población en el cultivo de yuca (Manihot utilissima (Pohl)

El presente ensayo se realizo en el Centro de Diversificación Agrícola "Campos Azules", Masatepe, durante los meses de julio de 1970 a mayo de 1971. Se estudiaron las combinaciones posibles de distancias de 0.50, 1.00 metro entre matas de una sola planta. Se utilizo la variedad Cubana que se emplea tanto en la alimentación humana como en la industria. El diseño experimental usado fue de bloques al azar con arreglo de parcelas dividida con cuatro repeticiones. Las plantas alcanzaron mayor altura cuando las densidades de población fueron mayores. Se observo que a mayores distancias se obtienen mayor numero de raíces por planta y de mayor longitud. Con la combinación de 0.50 x 0.25 metros se obtuvo el mayor rendimiento de raíz consistente en 25 toneladas por hectárea. El estudio de costos de producción, demostró que con distancias de 1.00 metros entre surcos por 0.75 metro entre matas de una sola planta se obtiene el mayor beneficio económico; con el tratamiento 1.50 x 1.00 metro se logra la menor inversión y un beneficio/costo tan alto como el obtenido con el tratamiento 1.00 x 0.75 metros.

Ensayo comparativo de cuatro herbicidas en el cultivo de la yuca (Manihot utilissima Polh)

Con el objetivo de estudiar el comportamiento de varios herbicidas sobre el control de malezas en el cultivo de la yuca, se llevo a efecto un ensayo en los suelos de la Escuela Nacional de Agricultura y Ganadería en Nicaragua. Al mismo tiempo se estudio la factibilidad económica del empleo de estos herbicidas. La evaluación se hizo en base a peso de las raíces de yuca al momento de la cosecha. También se uso una escala visual y el numero de especies de malezas por parcela. Todos los herbicidas se aplicaron de presiembra en tres dosis. Se presentaron diferencias significativas entre los herbicidas, pero no entre las dosis de estos, ni en la interacción de los herbicidas y las dosis. Gesagard en dosis de 1.0 kilogramos por hectárea de material activo produjo una ganancia neta de C209,20 por hectárea. El Karmer en dosis de 0,53 kilogramos por hectárea de material activo produjo una ganancia de C123,30 por hectárea, mientras que Treflan y Gesaprim resultaron antieconomicos produciendo perdidas de C24,15 y C177,20 respectivamente. Gesagard y Gesaprim en sus tres dosis controlaron totalmente las malezas de hoja ancha. Gesapard permitió el desarrollo normal del cultivo, mientras que Gesaprim produjo efectos fitotoxicos sobre el cultivo. Karmex y Treflan no controlaron malezas de hoja ancha como Euphorbia spp. (golondrina), Argemon mexicana (cerdo santo), Phylantus niruri (huevos de rana), Portulaca oleracea (verdolega), Euphorbia scabrella, Amaranthus spinosos (bledo) y Physalis ixocarpa (popa), ni malezas de hojas angostas como Cyperus rotundus (coyolillo), Cenchrus equinatus (mozote) y Cynodon dactilon Pers. (pasto bermuda). En Treflan se noto que no ejerció ningún buen control los primeros treinta días y luego se comporto como un tratamiento regular.