Efficiency of biological control for fall armyworm resistant to the protein Cry1F / Eficiência do controle biológico da lagarta-do-cartucho resistente à proteína Cry1F

Abstract Understanding the ecological and toxicological relationship between genetically modified cultivars (GM) and biological control agents is of great importance for discussions related to the compatability of GM cultivars and integrated management strategies for pest resistance. The present study evaluated the search behavior and predatory capacity of Orius insidiosus (Say) (Hemiptera: Anthocoridae) and Doru luteipes (Scudder) (Dermaptera: Forficulidae) on eggs and caterpillars of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistant or not to the protein Cry1F expressed in Bt corn. To determine the search time, a stopwatch was run until the capture of the first prey, predation capacity was evaluated by counting the prey remaining after 24 hours of infestation. The injuries of S. frugiperda in genetically modified and conventional corn in the presence and absence of predators was also evaluated. The predators were not able to distinguish between resistant and susceptible prey (eggs or caterpillars), given the predatory behaviour observed. There was no difference in searching time or predatory capacity between the predators for eggs and caterpillars of either resistant or susceptible S. frugiperda. In the presence of predators, the injury scores for resistant S. frugiperda on the Bt corn plants were lower. It was concluded that O. insidiosus and D. luteipes did not notice the presence of the protein Cry1F in the prey S. frugiperda, which may facilitate the combined use of GM corn and biological control in integrated management programs and for management of pest resistance.

Resumo O entendimento de relações ecológicas e toxicológicas envolvendo culturas geneticamente modificadas (GM) e agentes de controle biológico é de grande importância para discussões relativas à compatibilidade de culturas GM com estratégias de manejo integrado e manejo de resistência de pragas. Este trabalho avaliou o comportamento de busca e a capacidade predatória de Orius insidiosus (Say) (Hemiptera: Anthocoridae) e Doru luteipes (Scudder) (Dermaptera: Forficulidae) sobre ovos e lagartas de Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) resistente ou não à proteína Cry1F expressa em milho Bt. Para determinar o tempo de busca foi utilizado um cronômetro que foi disparado até a captura da primeira presa; a capacidade de predação foi avaliada através da contagem das presas remanescentes 24 h após infestação. Também foram avaliadas as injúrias de S. frugiperda em milho transgênico e milho convencional na presença ou ausência dos predadores. Os predadores não foram capazes de distinguir entre presas (ovos ou lagartas) resistentes e suscetíveis, considerando os comportamentos predatórios avaliados. Não houve diferença no tempo de busca e capacidade predatória sobre ovos e lagartas de S. frugiperda resistente ou suscetível entre os predadores. Na presença dos predadores, as notas de injúria de S. frugiperda resistente nas plantas de milho Bt foram menores. Conclui-se que O. insidiosus e D. luteipes não percebem a presença da proteína Cry1F na presa S. frugiperda, o que pode contribuir para o uso integrado de milho GM e controle biológico em programas de manejo integrado e manejo de resistência de pragas.

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Identificación de marcadores moleculares (SNP) y genes candidatos asociados a la tolerancia genética al complejo mancha de asfalto en variedades nativas de maíz (Zea mays L) / Identification of molecular markers (SNP) and candidate genes associated to genetic tolerance to tar spot complex disease in maize (Zeamays L) landraces

El complejo mancha de asfalto (CMA) en maíz (ZeamaysL.), causado por los hongos Phyllachora maydis Maubl. Y Monographella maydis Müller & Samuels, es una enfermedad de importancia económica en Guatemala, que ha causado pérdida en el rendimiento entre 30 a 50%, inclusive del 100% si las condiciones son favorables. El objetivo de esta investigación fue identificar marcadores de un solo nucleótido o SNP (Single Nucleotide Polymorphism, por sus siglas en inglés) y genes candidatos asociados a la tolerancia genética al CMA. Para ello se analizaron 463 poblaciones nativas y 329,692 SNP, y se compararon dos modelos genómicos, single markery BayesB, para la identificación de regiones asociadas a la tolerancia genética al CMA. Se identificaron 40 marcadores SNP asociados significativamente a la tolerancia genética al CMA con ambos modelos. La proporción de variación fenotípica total explicada (PVE) por los 40 SNPs fue de 56%, atribuida a efectos genéticos aditivos. Múltiples genes de resistencia a enfermedades fueron identificados en las regiones señaladas por los marcadores SNP. Sus funciones principales son receptores y transductores de señal, factores de transcripción que regulan positivamente la expresión de genes de tolerancia y genes de la familia kinasa, por lo que potencialmente están involucrados en el mecanismo de defensa al CMA.

The tar spot complex (TSC) diseasein maize (ZeamaysL.), caused by the fungi Phyllachora maydis Maubl. And Monographella maydis Müller & Samuels, is an economic important disease in Guatemala, producing yield losses between 30 to 50%, inclusive of 100% if the conditionsare favorable. The objective of this researchwasto identify single nucleoti depolymorphism markers (SNP) and candidate genes associated withgenetictoleranceto TSC. Asetof 463 native populations and 329,692 SNP were analyzed with two genomic models, single marker and BayesB, for the identification of regions associated with genetic tolerance to TSC. Forty SNP markers were significantly associated with the genetic tolerance to TSC with both models. The proportion of total phenotypic variation explained (PVE) by the 40 SNPs was 56%, attributed to additive genetice ffects. Multiple candidate genes for disease resistance were identified in the región sindicated by the SNP markers. Their main functionsare signal transducersand receptors, transcription factors that positively regulatethe expression of tolerance genes and family kinase genes, there fore, they are potentially involved in the defense mechanism to TSC.

Effect of Salinity Stress on Various Growth and Physiological Attributes of Two Contrasting Maize Genotypes

Abstract The response of two local maize (Zea mays L.) genotypes designated as Sahwal-2002 (salt-tolerant) and Sadaf (salt-sensitive) to salt stress was investigated under controlled growth conditions. The role of phenylalanine and seed priming under salt stress in maize with different morphological parameters were studied. The genotype Sadaf, being salt-tolerant, experienced more oxidative damage than the Sahiwall-2002 genotype under salt stress. The salinity affected both growth and physiological attributes of the maize species whereas the phenylalanine successfully increased the salinity tolerance in maize species at the seedling stage.

Influência da qualidade micotoxicológica e nutricional de híbridos de milho no custo da ração de frangos de corte / Influence of mycotoxicological and nutritional quality of maize hybrids on broiler chickens feed cost

Objetivou-se avaliar as variáveis micotoxicológicas e nutricionais de híbridos de milho com diferentes características que influenciam no custo da ração para frangos de corte. Foram avaliados 26 híbridos de milho geneticamente modificados nas safrinhas de 2016 e 2017, com diferentes germoplasmas, textura de endosperma e duração do ciclo. Nos híbridos, foram avaliados grãos avariados, fumonisinas (B1+B2) (FUM), aflatoxinas (B1+B2+G1+G2) (AFLA), zearalenona (ZEA), deoxinivalenol (DON), umidade, proteína bruta (PB), energia metabolizável aparente corrigida para balanço de nitrogênio (EMAn), aminoácidos digestíveis para aves (lisina, metionina, cistina e treonina) e o respectivo custo da ração inicial para frangos de corte, que foi calculada pelo custo mínimo. A prevalência de FUM, AFLA, ZEA e DON foi de 90, 17, 33 e 0%, com médias de 3067, 1, 38 e 0µg/kg nos dois anos, respectivamente. A média de EMAn e PB foi de 3264kcal/kg e 8,02%, respectivamente, e diferiu (P<0,05) nos dois anos. O custo da ração foi influenciado significativamente (P<0,05) por FUM, PB, EMAn nos dois anos. Híbridos com tecnologia Viptera apresentam menor concentração por FUM e menor custo da ração. Híbridos de ciclo precoce têm menor concentração de FUM, maiores percentuais de PB e de aminoácidos digestíveis e menor custo da ração.(AU)

The objective of this study was to evaluate the mycotoxicological and nutritional variables of maize hybrids with different characteristics that influence the broiler chicken's feed costs. In 2016 and 2017 winter crops, 26 genetically modified hybrids of maize with different germplasm, endosperm texture and cycle duration were evaluated. The analyzed variables were damaged grains, fumonisins (B 1 +B 2 ) (FUM), aflatoxins (B 1 +B 2 +G 1 +G 2 ) (AFLA), zearalenone (ZEA), deoxynivalenol (DON), moisture, crude protein (CP), apparent metabolizable energy corrected for nitrogen balance (AMEn), digestible amino acids for poultry (lysine, methionine, cystine and threonine) and the respective cost of the initial feed for broiler chickens calculated at the minimum cost. The prevalence of FUM, AFLA, ZEA and DON was 90, 17, 33 and 0%, with means of 3067, 1, 38 and 0µg/kg in the two years, respectively. The mean of AMEn and CP was 3264kcal/kg and 8.02%, respectively, and differed (P< 0.05) in the two years. The feed cost was significantly influenced (P<0.05) by FUM, PB, AMEn in two years. Hybrids with Viptera technology show lower concentration per FUM and lower feed cost. Early cycle hybrids have lower concentrations of FUM, higher percentages of CP and digestible amino acids, and lower feed costs.(AU)

High-throughput micro-phenotyping measurements applied to assess stalk lodging in maize ( Zea mays L. )

BACKGROUND: The biomechanical properties of maize stalks largely determine their lodging resistance, which affects crop yield per unit area. However, the quantitative and qualitative relationship between micro-phenotypes and the biomechanics of maize stalks is still under examined. In particular, the roles of the number, geometry, and distribution of vascular bundles of stalks in maize lodging resistance remain unclear. Research on these biomechanical properties will benefit from high-resolution micro-phenotypic image acquisition capabilities, which have been improved by modern X-ray imaging devices such as micro-CT and the development of micro-phenotyping analysis software. Hence, high-throughput image analysis and accurate quantification of anatomical phenotypes of stalks are necessary. RESULTS: We have updated VesselParser version 1.0 to version 2.0 and have improved its performance, accuracy, and computation strategies. Anatomical characteristics of the second and third stalk internodes of the cultivars 'Jingke968' and 'Jingdan38' were analyzed using VesselParser 2.0. The relationships between lodging resistance and anatomical phenotypes of stalks between the two different maize varieties were investigated. The total area of vascular bundles in the peripheral layer, auxiliary axis diameter, and total area of vascular bundles were revealed to have the highest correlation with mechanical properties, and anatomical phenotypes of maize stalk were better predictors of mechanical properties than macro features observed optically from direct measurement, such as diameter and perimeter. CONCLUSIONS: This study demonstrates the utility of VesselParser 2.0 in assessing stalk mechanical properties. The combination of anatomical phenotypes and mechanical behavior research provides unique insights into the problem of stalk lodging, showing that micro phenotypes of vascular bundles are good predictors of maize stalk mechanical properties that may be important indices for the evaluation and identification of the biomechanical properties to improve lodging resistance of future maize varieties.

Caracterização in silico dos genes envolvidos no metabolismo do nitrogênio em milho (Zea mays) / In silico characterization of genes involved in nitrogen metabolism in maize (Zea mays) / Caracterización in silico de genes implicados en el metabolismo de nitrógeno en maíz (Zea mays)

O N é o nutriente mineral requerido pelas plantas em maior quantidade e frequentemente limita o crescimento e produtividade. A partir de uma análise in silico foram identificados 26 genes envolvidos na assimilação do nitrogênio: quatro genes que codificam a enzima nitrato redutase (ZmNR), oito nitrato redutase de transporte (ZmNRT), uma nitrito redutase (ZmNRi), uma nitrito de transporte (ZmNRiT), seis glutamina sintetase (ZmGS), quatro glutamato sintase (ZmGOGAT) e duas glutamato desidrogenase (ZmGDH). A árvore filogenética foi construída onde foi possível observar a formação de cinco grupos distintos de acordo com as funções. A análise da estrutura dos genes mostrou que o número de íntrons variou de 0 a 32. A análise dos domínios conservados mostrou que a maioria dos genes identificados possuem o domínios específicos a função que desempenham na rota de assimilação do N em milho. Além disso, esses genes apresentaram padrões de expressão diferenciais em tecidos e órgãos. Os dados gerados neste trabalho forneceram subsídios para selecionar genes-candidatos para futuras análises funcionais a serem utilizados nos programas de melhoramento de milho.

Nitrogen is a mineral highly requested by plants and often limits both growth and productivity. From an in-silico analysis, a total of 26 genes were identified as being involved in nitrogen assimilation: four genes encoding nitrate reductase enzyme (ZmNR), eight encoding nitrate reductase transporters (ZmNRT), one encoding nitrite reductase (ZmNRi), one encoding nitrite transporter (ZmNRiT), six encoding glutamine synthesis (ZmGOGAT) and two encoding glutamate dehydrogenase (ZmGDH). A phylogenetic tree was generated where the formation of five distinct clusters could be observed according to gene function. Structural genes analysis showed that introns varied from 0 to 32. The analysis of conserved domains showed that most of the identified genes play a domain-specific function in the N assimilation pathway in maize. Moreover, these genes present a differential expression pattern in tissues and organs. Data from this study will provide subsidies to select candidate genes for further functional analyses in maize breeding programs.

N es el nutriente mineral requerido por las plantas en mayor cantidad y a menudo limita el crecimiento y la productividad. Desde un análisis in silico se ha identificado 26 genes implicados en la asimilación de nitrógeno: cuatro genes que codifican la enzima nitrato reductasa (ZmNR), ocho nitrato reductasa de transporte (ZmNRT), un nitrito reductasa (ZmNRi), un nitrito de transporte (ZmNRiT), seis glutamina sintetasa (ZmGS), cuatro sintasa de glutamato (ZmGOGAT) y dos glutamato deshidrogenasa (ZmGDH). El árbol filogenético se ha construido donde ha sido posible observar la formación de cinco grupos distintos de acuerdo con sus funciones. El análisis de la estructura de los genes ha mostrado que el número de intrones ha variado de 0 a 32. El análisis de los dominios conservados mostró que la mayoría de los genes identificados poseen dominios específicos a la función que desempeñan en la ruta de asimilación de N en maíz. Además, esos genes mostraron patrones de expresión diferenciales en tejidos y órganos. Los datos generados en este estudio proporcionan subvenciones para la selección de genes candidatos para posterior análisis funcional, para ser utilizados en programas de mejoramiento de maíz.

Estudo proteômico de variedades de milho ( Zea mays L. ) obtidas por melhoramento clássico e por recombinação genética / Proteomic study of maize ( Zea mays L. ) varieties obtained by classical breeding and genetic recombination.

O melhoramento genético clássico de sementes milho (Zea mays L.) permitiu desenvolver inúmeras variedades, incluindo o milho com qualidade proteica melhorada (Quality Protein Maize, QPM), que visava aumentar os teores proteicos e as propriedades nutricionais. Por outro lado, novas variedades comerciais foram obtidas por vegetais geneticamente modificados (GM), com foco em parâmetros agronômicos. Em ambos os casos, a segurança dessas variedades para uso como alimento é uma das principais preocupações dos desenvolvedores e dos órgãos de regulamentação. A Equivalência Substancial é a base do sistema de avaliação da segurança de culturas geneticamente modificadas, no entanto alterações na expressão de proteínas não são devidamente analisadas e esclarecidas. As abordagens proteômicas complementam as técnicas de avaliação de biossegurança para alimentos GM, bem como permitem investigar possíveis efeitos indesejáveis derivados do melhoramento clássico. Os objetivos do presente estudo foram caracterizar e comparar os perfis proteicos de variedades de milhos convencionais melhorados (QPM) e geneticamente modificados (GMs), contra suas respectivas linhas convencionais utilizando técnicas proteômicas como eletroforese bidimensional (2-DE) e bottom up shotgun (gel-free). Num primeiro estudo, foram utilizadas três amostras de milho, sendo duas variedades convencionais com QPM (QP1 e QP2) e uma variedade convencional normal (CN). No segundo estudo, foram analisadas duas cultivares de milho GM (GM1 e GM2) e seus respectivos convencionais genitores (CG1 e CG2). As composições químicas de todas as amostras também foram avaliadas quanto a Equivalência Substancial. O extrato bruto proteico foi submetido à análise de eletroforese unidimensional (1-DE), bidimensional (2-DE) e bottom up shotgun (gel-free). As imagens dos mapas proteicos foram analisadas pelo software Image Master 2D Platinum 7.0 (GE). Os spots diferencialmente expressos e selecionados foram sequenciados por MS. Pela composição química das principais frações das amostras de milho foi possível identificar a equivalência substancial entre as amostras convencionais e GMs, bem como QPMs e sua convencional dentro das faixas de variabilidade esperadas da espécie. Nos géis 1-DE foram observadas bandas proteicas com perfis similares entre os grupos de amostras avaliadas para ambos estudos. Nas imagens dos géis 2-DE não houveram alterações extremas entre as amostras de milhos GMs e seus respectivos convencionais genitores (CGs), mas apenas diferenças na intensidade dos spots proteicos. As variedades QPMs e CN apresentaram diferenças devido à distribuição dos spots. Os mapas proteicos das amostras CG1 x GM1 e CG2 x GM2 apresentaram maior semelhança com porcentagens de matchings superiores a 70 %, enquanto as porcentagens de matchings entre variedades diferentes (QPMs e CN) foram menores. No total foram identificadas 219 proteínas das amostras CGs x GMs e QPMs x CN, classificadas quanto aos seus processos biológicos e função molecular. Em conclusão, foram encontradas diferenças entre os cultivares GMs e CGs, indicando uma variação normal entre variedades de milho, que não comprometem a segurança alimentar das amostras estudadas. Quanto às amostras com QPM e CN as diferenças encontradas são devido à sua distância nas linhagens ou germoplasma

The classic genetic breeding of corn seeds (Zea mays) has enabled the development of many varieties, including corn with improved protein quality (Quality Protein Maize, QPM), which aimed to increase protein levels and nutritional properties. On the other hand, new commercial varieties have been obtained out of genetically modified (GM) vegetables, with a focus in agronomic parameters. In both cases, the safety of these varieties for food use is one of the main concerns for the developers and for the regulatory agencies. Substantial Equivalence is the basis of the safety evaluation system for genetically modified crops, however, alterations in the protein expressions are not been properly analyzed and clarified. The protein approaches complement the techniques of biosafety evaluation for GM foods, as well as allow for possible undesirable effects derived from classic improvement to be investigated. The goals of the current studies were to characterize and compare the protein profiles of the different varieties of conventionally improved (QPM) and genetically modified (GM) corn, against their respective conventional lines using proteomic techniques, such as, two-dimensional electrophoresis (2-DE), bottom up shotgun (gel-free) and masses spectrometry (MS). In a first instance of the study, three samples of corn were used, two of conventional varieties with QPM (QP1 and QP2) and one conventional normal variety (CN). In a second instance of the study, two cultures of GM corn (GM1 and GM2) were analyzed and their respective conventional genitors (CG1 and CG2). The chemical compositions of all the samples were also evaluated for their Substantial Equivalence. The protein raw extract was submitted to analysis of one-dimensional (1-DE), two-dimensional (2-DE) electrophoresis, and bottom up shotgun (gel-free). The protein image maps were analyzed by the Image Master 2D Platinum 7.0 (GE) software. The spots which were expressed and selected differentially were sequenced by MS. By the chemical composition of the main fractions of the samples of corn, it was possible to identify the substantial equivalence between the conventional samples and GMs, likewise with OPMs and their conventional in the ranges of variability which were expected for the species. On the 1-DE gel, it was observed protein bands with similar profiles amongst the groups of evaluated samples for both studies. In the images of the 2-DE gel, there were no alterations between the GM corn and their respective conventional genitors (CGs), but only differences in intensity of the protein spots. The OPM and CN varieties presented differences due to the distribution of the spots. The protein maps of samples CG1 vs. GM1 and CG2 vs. GM2 presented greater similarities with the percentages of matchings superior to 70%, while the percentage of matchings among different varieties (QPMs and CN) were smaller. In total, there were 219 proteins identified in the samples CGs vs. GMs and QPMs vs. CN, classified by the biologic processes and molecular function. In conclusion, there were found differences between the cultures of GMs and CGs, indicating a normal variation among the corn varieties, which do not affect the food security of the studied samples. As per the samples with QPM and CN, the differences found were due to the line distances or germplasm

Population structure and genetic diversity of Brazilian popcorn germplasm inferred by microsatellite markers

Background The genetic diversity and structure of 31 popcorn accessions of the germplasm bank of the State University of Maringá were assessed using 30 microsatellite primers. Results 127 alleles were identified from 30 evaluated loci. The number of alleles per locus ranged from two to eight. The overall mean of the polymorphic loci averaged 79.89%. The primers UMC1549 and UMC1072 detected polymorphism in all accessions analyzed. The mean observed heterozygosity ranged from 0.07 to 0.30 and the highest proportion of heterozygous plants was observed in accession BOZM 260 (Ho = 0.30). The analysis of molecular variance revealed that 60% of the total genetic variation was found within accessions and 40% was found between accessions. The Bayesian clustering approach grouped the 31 accessions into two genetically differentiated clusters. The dendrogram revealed that accessions TATU 2 and ARZM 05 083 are genetically less similar than the others. Conclusions The analysis allowed to identify microsatellite loci with high levels of heterozygosity (UMC1549 and UMC1072). These loci can be indicated as promising for detecting polymorphisms in popcorn accessions and in the monitoring of genetic improvement programs. Moreover, allowed to identify heterozygous accessions (BOZM 260), this accession showed allelic variation at all analyzed microsatellite loci and can be recommended for crosses with plants that have desirable agronomic characteristics, with a view to the broadening of the genetic base of popcorn accessions and developing new cultivars.

Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)

Background Head smut of maize, which is caused by Sporisorium reilianum f. sp. zeae (Kühn), is a serious disease in maize. In order to reveal the molecular mechanism of the resistance to head smut in maize, a microarray containing ~ 14,850 probes was used to monitor the gene expression profiles between a disease resistant near isogenic line (NIL) and a highly susceptible inbred line after S. reilianum was injected with an artificial inoculation method. Results Levels of expression for 3,532 genes accounting for 23.8% of the total probes changed after inoculation. Gene Ontology analysis revealed that the differentially expressed genes participated in physiological and biochemical pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that plant-pathogen interaction, natural killer cell mediated cytotoxicity and benzoxazinoid biosynthesis pathways play important roles in resistance to head smut. Three head smut resistance-related candidate genes, CLAVATA1, bassinosteroid insensitive 1-associated receptor kinase 1 and LOC100217307 with leucine-rich repeat (LRR) conserved domains were identified, each of which is in maize mapping bin 2.09, a region previously shown to include a major QTL for head smut resistance. Furthermore, LOC100217307 was validated by quantitative real-time (qRT)-PCR inferring that this gene may be involved in the resistance to head smut of maize. Conclusions This study provided valuable information for cloning, functional analysis and marker assisted breeding of head smut resistance genes.

Differential antioxidative response of tolerant and sensitive maize (Zea mays L.) genotypes to drought stress at reproductive stage.

The role of oxidative stress management was evaluated in two maize (Zea mays L.) genotypes — Parkash (drought-resistant) and Paras (drought-sensitive), subjected to drought stress during reproductive stage. Alterations in their antioxidant pools — glutathione (GSH) and ascorbic acid (AsA) combined with activities of enzymes glutathione reductase (GR), ascorbate peroxidase (APX), peroxidase (POX) and catalase (CAT) involved in defense against oxidative stress and stress parameters, namely chlorophyll (Chl), hydrogen peroxide (H2O2) and malondialdehyde (MDA) were investigated in flag leaves from silk emergence till maturity. The drought caused transient increase in GR, APX, POX and CAT activities in drought-tolerant genotype (Parkash) which decreased at later stages with the extended period of drought stress. However, in Paras, drought stress caused decrease in activities of GR and CAT from initial period of stress till the end of experiment, except for POX which showed slight increase in activity. A significant increase in GSH content was observed in Parkash till 35 days after silking (DAS), whereas in Paras, GSH content remained lower than irrigated till maturity. Parkash which had higher AsA and Chl contents, also showed lower H2O2 and MDA levels than Paras under drought stress conditions. However, at the later stages, decline in antioxidant enzyme activities in Parkash due to severe drought stress led to enhanced membrane damage, as revealed by the accumulation of MDA. Our data indicated that significant activation of antioxidant system in Parkash might be responsible for its drought-tolerant behavior under drought stress and helped it to cope with the stress up to a definite period. Thus, the results indicate that antioxidant status and lipid peroxidation in flag leaves can be used as indices of drought tolerance in maize plants and also as potential biochemical targets for the crop improvement programmes to develop drought-tolerant cultivars.

Iron bioavailability of different maize genotypes developed in a breeding program: in vitro and in vivo studies

The objective of this study was to evaluate iron bioavailability of maize genotypes, and analyze the correlation between in vitro and in vivo methods. Dialysable iron was analyzed in 13 genotypes from which 5 were selected for the biological assay. Mean iron content of the genotypes (n=13) was 17.93±2.93 mg kg-1. Phytate varied from 0.77% to 1.03%; phytate: iron molar ratio from 30.64 to 55.41; and soluble iron from 13.17 to 39.63%. The highest value for dialysable iron was 19.14%. In the biological assay, the control group, that received ferrous sulphate, did not present significant difference between the genotypes for Hb gain, Hb gain per gram of iron consumed and HRE. Hb gain did not present a significant correlation with in vitro assay. However, there were positive correlations varying from 0.653 to 0.809. The maize genotypes evaluated presented a good bioavailability since the genotypes showed the same result in hemoglobin gain than control group.

Biodisponibilidade de ferro de diferentes genótipos de milho desenvolvidos em programa de melhoramento genético: estudos in vitro e in vivo. O objetivo deste estudo foi avaliar a biodisponibilidade do ferro de genótipos de milho e analisar a correlação entre métodos in vitro e in vivo. Ferro dialisável foi analisado em13 genótipos, a partir do qual 5 foram selecionados para o ensaio biológico. A média de teor de ferro dos genótipos (n= 13) foi 17,93 ± 2,93 mg kg-1. O teor de fitato variou de 0,77% a 1,03%; razão molar fitato:ferro de 30,64 a 55,41; e ferro solúvel de 13,17 a 39,63%.O valor mais alto para o ferro dialisável foi 19,14%. No ensaio biológico, o grupo controle, que recebeu sulfato ferrso, não apresentou diferença significativa entre os genótipos no ganho Hb, ganho de Hb por grama de ferro consumido e HRE. Ganho de Hb não apresentou correlação significativa com o ensaio in vitro. No entanto, houve correlações positivas variando de 0,653 a 0,809. Os genótipos de milho avaliados apresentaram uma boa biodisponibilidade uma vez que os genótipos apresentaram o mesmo resultado quanto ao ganho de hemoglobina em relação ao grupo controle.

Somatic embryogenesis and plant regeneration capacity in Argentinean maize (Zea mays L. ) inbred lines

Somatic embryogenesis, which is still the method of choice for tissue culture, regeneration and transformation of maize, is largely considered highly genotype-dependent. The Hi II, a highly embryogenic genotype, has been extensively used in transformation protocols. However, this is not an inbred line; instead, it has a proportion of the undesirable A-188 background, and the progeny segregates for phenotypic characteristics and shows poor agronomic performance. In an effort to identify genotypes that combine a high somatic embryogenic response and good agronomic performance, we evaluated 48 advanced inbred lines developed at INTA. Callus development and somatic embryogenesis capacity were measured in 200 immature embryos per line. Embryogenic capacity [EC (mature somatic embryos/callus evaluated) x 100], Regeneration Capacity (RC) and Fertile Plant Recovery in greenhouse (FPR, fertile plants/regenerated plants) were recorded. A total of 17 lines reached an EC > 50 percent, and 14 out of those 17 lines regenerated seedlings. The FPR ranged between 50 and 100 percent. Also, we selected three promising lines with high agronomic performance, as alternatives to Hi II, in order to be included in a maize transformation scheme via somatic embryogenesis. In addition, we report the usefulness of Single Sequences Repeat (SSRs) in the determination of genetic diversity among 14 divergent lines for somatic embryogenesis response. The seven lines displaying good in vitro behaviour can be crossed to obtain hybrids combining desirable alleles for somatic embryogenesis response and different genetic backgrounds.

Detecting adventitious transgenic events in a maize center of diversity

Background: The genetic diversity of maize in Peru includes several landraces (within race clusters) and modern open pollinated and hybrid cultivars that are grown by farmers across various regions, thereby making this country a secondary center of diversity for this crop. A main topic of controversy in recent years refers to the unintended presence of transgenic events in locally grown cultivars at main centers of crop diversity. Peru does not yet have biosafety regulations to control or permit the growing of genetically modified crops. Hence, the aim of this research was to undertake a survey in the valley of Barranca, where there were recent claims of authorized transgenic maize grown in farmers fields as well as in samples taken from feed storage and grain or seed trade centers. Results: A total of 162 maize samples (134 from fields, 15 from local markets, eight from the collecting centers of poultry companies, from the local trading center and four samples from seed markets) were included for a qualitative detection by the polymerase chain reaction (PCR) of Cauliflower Mosaic Virus (CaMV) 35S promoter (P35S) and nopaline synthase terminator (Tnos) sequences, as well as for six transgenic events, namely BT11, NK603, T25, 176, TC1507 and MON810. The 134 maize samples from farmers fields were negative for Cry1Ab delta-endotoxin insecticidal protein and enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) using lateral flow strips. The PCR analysis did not detect any of the six transgenic events in samples from farmers fields, local markets, seed trading shops and the local collecting center. There were four transgenic events (T25, NK603, MON810 and TC1507) in grain samples from the barns of poultry companies. Conclusions: This research could not detect, at the 95 percent probability level, transgenes in farmers' fields in the valley of Barranca. The four transgenic events in grain samples from barns of poultry companies...

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.

Monitoring the threat of unintentional transgene flow into maize gene banks and breeding materials

The use of transgenic crops is steadily increasing around the world, led by soybean (based on total area) and maize (in terms of total number of countries). Transgenic maize is grown in at least 17 countries across four continents: Africa, America, Asia and Europe. The comprehensive global spread of transgenic maize has significant implications for organizations involved in germplasm conservation and genetic enhancement; particularly as some countries require a GMO-free declaration when receiving shipments of maize germplasm. This article describes the protocol used by the International Maize and Wheat Improvement Center (CIMMYT) for monitoring unintentional transgene flow in maize genebank and breeding plots. The protocol is based on polymerase chain reaction (PCR) markers for detecting specific recombinant DNA sequences in bulked samples collected from sentinel plots. To date, no unintentional transgene flow has been detected in CIMMYT fields of maize genebank accessions or breeding materials.

Genetic diversity of breeding popcorn lines determined by SSR markers / Diversidad genética de líneas de mejoramiento del Zea mays determinada por marcadores SSR

Information about genetic dissimilarity is very important to corroborate genealogical relationships and to predict the most heterozygotic hybrid combinations. Eight popcorn S6 lines of diverse germplasm types were evaluated using simple sequence repeats (SSR) markers. Of a total of 51 evaluated polymorphic primers, 15 were used for polymerase chain reaction (PCR) amplification. The genetic distance was estimated by Rogers’ modified distance. The different popcorn breeding programs in Brazil are possibly using highly similar base-populations. The genetic similarity of lines P1-3 and P8-1 was lowest, while P3-3 and P8-2 were genetically more similar. The cophenetic correlation showed that the Unweighted Pair-Group Method Using Arithmetic Averages (UPGMA) was reliable to discriminate the genotypes in five groups. The clusters were consistent with the estimates of genetic identity. There was a moderate coincidence degree between the groups and genealogy of lines. Higher levels of heterozygosity are expected from crosses between the group containing lines P3-3 and P7-3 with that of P1-3 and P7-4. Crosses between lines P1-3 and P8-1 are also promising.

QTL identification of grain protein concentration and its genetic correlation with starch concentration and grain weight using two populations in maize (Zea mays L.).

Protein is one of the three main storage chemical components in maize grains, and is negatively correlated with starch concentration (SC). Our objective was to analyse the influence of genetic backgrounds on QTL detection for protein concentration (PC) and to reveal the molecular genetic associations between PC and both SC and grain weight (GWP). Two hundred and eighty-four (Pop1) and 265 (Pop2) F(2:3) families were developed from two crosses between one high-oil maize inbred GY220 and two normal maize inbreds 8984 and 8622 respectively, and were genotyped with 185 and 173 pairs of SSR markers. PC, SC and GWP were evaluated under two environments. Composite interval mapping (CIM) and multiple interval mapping (MIM) methods were used to detect single-trait QTL for PC, and multiple-trait QTL for PC with both SC and GWP. No common QTL were shared between the two populations for their four and one PC QTL. Common QTL with opposite signs of effects for PC and SCGWP were detected on three marker intervals at bins 6.07-6.08, 8.03 and 8.03-8.04. Multiple-traits QTL mapping showed that tightly-linked QTL, pleiotropic QTL and QTL having effects with opposite directions for PC and SCGWP were all observed in Pop1, while all QTL reflected opposite effects in Pop2.

Development of a Brazilian maize core collection

The aim of this study was to evaluate methods for developing a Brazilian maize core collection. For an initial survey of the active collection, passport information, as well as characterization and evaluation of accessions, were taken into consideration, these then being divided according to geographic region and kernel-type. Multiple sampling methods were evaluated. The strategy of constant sampling generated extensive alterations in extract accession frequency. The multivariate strategy with dispersion graphs and principal components associated with the Tocher method was considered efficient for identifying the most divergent genotypes. The multivariate strategy generated greater alterations in the variance of traits. The average number of traits revealed few modifications with the various sampling strategies used. Therefore, the active collection could be considered as possessing a satisfactory amount of information for most of its accessions. Moreover, the multivariate strategy generated modifications in the variance of the traits, independent of sampling intensity.

Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.