Resposta androgênica de genótipos brasileiros de trigo a diferentes pré-tratamentos das espigas e a um agente gelificante

The objective of this work was to analyze the androgenic response of Brazilian wheat genotypes to different pretreatments of the spikes, prior to the culture of isolated microspores, and to the effect of a gelling agent in the induction culture medium. Five genotypes were evaluated for embryo formation, green plantregeneration, and spontaneous chromosome duplication. Wheat spikes were subjected to two pretreatments:cold, at 4°C for 21 days; and 2-hydroxynicotinic acid, at 32°C for two days. Culture media were evaluated with or without Ficoll as a gelling agent. Cold produced more embryos and green plants than the chemical pretreatment in four out of five genotypes. Only two genotypes treated with 2-hydroxynicotinic acid were able to produce plants, and one of them produced a single albino plant. Medium containing Ficoll produced moreembryos than liquid medium and promoted a higher number of plants. Spontaneous chromosome duplication varies between genotypes and pretreatments, and shows high variability.O objetivo deste trabalho foi analisar a resposta androgênica de genótipos brasileiros de trigo a diferentes pré-tratamentos das espigas, antes da cultura de micrósporos isolados, e ao efeito de um agente gelificante no meio de cultura de indução. Cinco genótipos foram avaliados quanto à formação de embriões, regeneração de plantas verdes e à duplicação espontânea dos cromossomos. Espigas de trigo foram submetidas a dois pré-tratamentos: frio, a 4°C por 21 dias; e ácido 2-hidroxinicotínico, a 32°C por dois dias. Os meios decultura foram avaliados com ou sem Ficoll como agente gelificante. O frio produziu mais embriões e plantas verdes do que o pré-tratamento químico, em quatro dos cinco genótipos testados. Apenas dois genótipos tratados com ácido 2-hidroxinicotínico foram capazes de produzir plantas, e um deles produziu uma única planta albina. O meio com Ficoll produziu mais embriões do que o meio líquido e gerou maior número de plantas. A duplicação espontânea dos cromossomos varia entre os genótipos e os pré-tratamentos e apresentaalta variabilidade

Identification and partial characterization of genes differentially expressed in the tomato-Meloidogyne incognita interaction

Meloidogyne incognita é um nematóide endoparasita sedentário que induz a formação de galhas nas raízes das plantas suscetíveis. Em tomateiros com o gene Mi-1, as células próximas ao estilete dos juvenis de segundo estágio exibem reação de hipersensibilidade (HR) a partir de oito a doze horas após a inoculação. Esse trabalho objetivou identificar e caracterizar parcialmente genes envolvidos nas vias de transdução de sinais e nas respostas de defesa do tomateiro a Meloidogyne incógnita mediadas pelo gene Mi-1 utilizando a técnica de hibridização subtrativa seguida de amplificação por PCR (suppressive subtractive hybridization, SSH). A partir dos cultivares quase isogênicas de tomateiros Moneymaker (suscetível) e Motelle (resistente) inoculados com M. incognita foram construídas duas bibliotecas subtrativas enriquecidas para genes diferencialmente expressos na interação incompatível (MoI 24h e MoI 72h) e uma biblioteca para a interação compatível (MMI 24h). Foram seqüenciados 81 clones da biblioteca MoI 72h, 26 da MMI 24h e 620 da MoI 24h. As análises subseqüentes se concentraram nesta última porque as outras duas apresentaram elevada redundância ou muitos ESTs (Expressed Sequence Tags) com similaridade a genes não caracterizados. Na análise de expressão por macroarranjo, 126 (41%) entre os 307 clones não redundantes de cDNAs apresentaram expressão diferencial. A análise de Northern blot não apresentou sensibilidade suficiente para evidenciar diferenças nos níveis de expressão. Vinte e um genes foram submetidos à análise de expressão por PCR quantitativa e, entre estes, oito apresentaram indução duas horas após a inoculação. A classificação dos ESTs em categorias funcionais em potencial mostrou que 31% deles têm relação com resposta de resistência, resposta a patógenos, proteção à célula, sinalização celular ou a estresse abiótico. Os ESTs classificados como não caracterizados corresponderam a 26%. Entre os ESTs com similaridade a genes induzidos nessa interação relatados previamente, estão inibidores de proteases, quitinase, aquaporina, fenilalanina amônia liase, Ki1 e peroxidase. Entre os ESTs relacionados com defesa relatados em outros patossistemas estão os similares a genes que codificam para MAPK4, defensinas, proteínas de transferência de lipídios, PR10 e proteína 14-3-3. Os genes semelhantes a MtN19, D13F MYB ST1 e cinase dependente de cálcio parecem apresentar mais de uma cópia no genoma e os genes semelhantes a Myb1, gene relacionado à resposta de resistência, gene envolvido com a resistência sistêmica adquirida, Ki1 e a defensina parecem estar presentes em cópia única no genoma. Os transcritos dos genes correspondentes aos clones SSH08D08 (MtN19), SSH09A10 (peroxidase 10), SSH10H09 (semelhante a Ki1) e SSH09D04 (defensina) possuem cerca de 2,5 kb, 1,2 kb, 1,8 kb e 0,5 kb, respectivamente. A presença de muitos genes relacionados com defesa e a ausência de genes do patógeno na biblioteca demonstra que a subtração foi eficiente para enriquecer para genes diferencialmente expressos. A grande quantidade de genes identificados e parcialmente caracterizados fornece subsídios para o estudo funcional visando caracterizar as vias de respostas de defesa mediadas pelo gene Mi-1.In the compatible interaction between tomato and root-knot nematode Meloidogyne incognita, galls are formed on the roots. The tomato gene Mi-1 confers resistance to M. incognita accompanied by a hypersensitive response. To elucidate the mechanisms underlying these interactions, three cDNA libraries enriched for transcripts differentially expressed in susceptible (MMI 24h) and in resistant (MoI 24h and MoI 72h) plants, respectively, were generated by suppressive subtractive hybridization (SSH). Twenty-six ESTs were generated from MMI 24h, 81 from MoI 72h and 620 from MoI 24h library. The library MoI 24 was further characterized because the first two were highly redundant or had many ESTs similar to unknown genes. The cDNA macroarray analysis of 307 nonredundant clones revealed that 126 (41%) corresponded to differentially expressed transcripts. Northern blot analysis did not show enough sensitivity to detect differential expression. Quantitative PCR was performed to investigate the expression patterns of 21 selected genes. Transcript accumulation occurred for eight out of the 21 genes two hours after M. incognita challenge in resistant plants. Classification of the ESTs into several putative functional categories showed that 31% of these ESTs represented genes involved in resistance response, pathogen and stress response, cell protection or signaling. Twenty-six percent of ESTs corresponded to genes with unknown functions. Among ESTs similar to genes known to be involved in plant- nematode interactions were trypsin-alpha amylase inhibitor, chitinase, aquaporin, phenylalanine ammonia-lyase, Ki1 and peroxidase. Among ESTs similar to genes reported in other pathosystems were MAPK4, defensins, lipid transfer proteins, PR10 and 14-3-3 proteins. The genes similar to MtN19, to D13F MYB ST1 and to calcium-dependent protein kinase 3 apparently are present in more than one copy in the tomato genome and the genes similar to Myb1, to disease resistance response protein, to systemic acquired resistance- related protein, to Ki1 and to plant defensin apparently have only one copy. The transcripts corresponding to SSH08D08 (MtN19), SSH09A10 (peroxidase 10), SSH10H09 (similar to Ki1) and SSH09D04 (plant defensin) clones have approximately 2,5 kb, 1,2 kb, 1,8 kb and 0,5 kb, respectively. The presence of various defense related genes and the absence of pathogen genes in the libraries give evidence of successfully subtraction for genes differentially expressed. The identification and partial characterization of these cDNAs will assist functional analysis aimed to elucidate the pathways activated by the Mi-1 gene.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Molecular identification based on coat protein sequences of the Barley yellow dwarf virus from Brazil

Yellow dwarf disease, one of the most important diseases of cereal crops worldwide, is caused by virus species belonging to the Luteoviridae family. Forty-two virus isolates obtained from oat (Avena sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), corn (Zea mays L.), and ryegrass (Lolium multiflorum Lam.) collected between 2007 and 2008 from winter cereal crop regions in southern Brazil were screened by polymerase chain reaction (PCR) with primers designed on ORF 3 (coat protein - CP) for the presence of Barley yellow dwarf virus and Cereal yellow dwarf virus (B/CYDV). PCR products of expected size (~357 bp) for subgroup II and (~831 bp) for subgroup I were obtained for three and 39 samples, respectively. These products were cloned and sequenced. The subgroup II 3' partial CP amino acid deduced sequences were identified as BYDV-RMV (92 - 93 % of identity with "Illinois" Z14123 isolate). The complete CP amino acid deduced sequences of subgroup I isolates were confirmed as BYDV-PAV (94 - 99 % of identity) and established a very homogeneous group (identity higher than 99 %). These results support the prevalence of BYDV-PAV in southern Brazil as previously diagnosed by Enzyme-Linked Immunosorbent Assay (ELISA) and suggest that this population is very homogeneous. To our knowledge, this is the first report of BYDV-RMV in Brazil and the first genetic diversity study on B/CYDV in South America

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

AbstractLow transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciensovergrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

Abstract Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22°C and 25°C, and a 400 mM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future

Adaptation of in vitro regeneration protocol for Brazilian wheat genotypes

ABSTRACT: The availability of an efficient protocol for in vitro regeneration is imperative for genetic transformation. Using genotypes adapted to the target region as a transgenic platform accelerates the development of cultivars. Therefore, this study aimed to adapt an in vitro regeneration protocol for Brazilian wheat genotypes. For this purpose, the in vitro regeneration capacity of immature embryos from six Brazilian wheat genotypes using two protocols of regeneration of somatic embryos was analysed. Furthermore, combinations of 2,4-D and picloram in the callus induction medium were tested in order to improve regeneration efficiency. Genotypes with higher regeneration efficiency were BR18-Terena and PF020037, yielding 0.42 and 1.13 shoots per explant using the Hu and the Wu protocol, respectively. Adding 1mgL-1 2,4-D in the callus induction medium was the most favourable, producing 3.73 and 3.07 shoots per explant for PF020037 and BR18-Terena, respectively. In conclusion, a protocol for regeneration for two Brazilian wheat genotypes recommended and developed to be cultivated at the Cerrado region has been adapted

Characterization of entomopathogenic nematodes and symbiotic bacteria active against Spodoptera frugiperda (Lepidoptera: Noctuidae) and contribution of bacterial urease to the insecticidal effect

AbstractEntomopathogenic nematodes carrying symbiotic bacteria represent one of the best non-chemical strategies for insect control. Infective juveniles of Heterorhabditidae and Steinernematidae nematodes actively seek the host in the soil, penetrating through insect’s openings to reach the hemocoel where symbiotic bacteria in the genera Photorhabdus or Xenorhabdus, respectively, are released. The bacteria replicate and produce virulence factors that rapidly kill the insect host, providing nutrients for the nematodes development and reproduction within the insect cadaver. More studies are necessary to better understand the factors implicated in the nematode-bacteria association, particularly focusing the bacterial symbionts, the final effectors of the insect death. Our group has shown that ureases are lethal to some groups of insects and may contribute to the entomopathogenic properties of the symbiotic bacteria.The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is one of the major insect pests in corn (Zea mays) crops in Brazil, with infestations resulting in reduction up to 39% yield and losses amounting US$ 500 million annually. Native strains of entomopathogenic nematodes active against S. frugiperda represent a promising alternative to the intensive use of chemical insecticides to control fall armyworm population in corn plantations.In this study we screened soil nematodes collected in the south region of Brazil for pathogenicity against S. frugiperda. Symbiotic bacteria associated with these nematodes were isolated and characterized. We also evaluated urease production by the symbiotic bacteria in vitro and along the course of infection in S. frugiperda and demonstrated that urease production correlated positively to their entomopathogenicity