Leaf anatomy of Crambe abyssinica Hochst. during in vitro shoot induction

This study aimed to characterize and evaluate possible modifications in the leaf anatomy of crambe during the process of shoot induction based on micropropagation protocol. The anatomic characteristics of the leaves, and also the morphological characteristics of crambe plantlets, were evaluated during the shoot induction phase of the micropropagation. The shoots were induced by the cytokinins, 6-benzylaminopurine (BAP), kinetin (KIN) and thidiazuron (TDZ), at distinct concentrations on Murashige and Skoog (MS) medium during 120 days of culture. Comparing the leaf anatomy, it was observed that, at day 30, only the adaxial epidermis and the palisade parenchyma presented significant differences in relation to the tested concentrations, independently of the type of cytokinin employed. At 120 days, the anatomic analysis of the mesophyll demonstrated no marked difference among the cytokinins at 5 μM. Therefore, the various sources and concentrations of cytokinins applied in this work did not promote marked changes in the sense of altering the organization and/or thickness compared to the control.Key words: Oilseeds, micropropagation, mesophyll, histology, plant morphology

Genetic diversity of bromeliaceae species from the atlantic forest

The Bromeliaceae family includes a range of species used for many purposes, including ornamental use and use as food, medicine, feed, and fiber. The state of Espírito Santo, Brazil is a center of diversity for this family in the Atlantic Forest. We evaluated the genetic diversity of five populations of the Bromeliaceae family, including specimens of the genera Aechmea, Billbergia (subfamily Bromelioideae), and Pitcairnia (subfamily Pitcairnioidea), all found in the Atlantic Forest and distributed in the state of Espírito Santo. The number of alleles per locus in populations ranged from two to six and the fixation index (F), estimated for some simple sequence repeats in bromeliad populations, was less than zero in all populations. All markers in the Pitcairnia flammea population were in Hardy-Weinberg equilibrium (P < 0.05). Moreover, significant deviations from Hardy-Weinberg equilibrium were observed at some loci in populations of the five bromeliad species. In most cases, this can be attributed to the presence of inbreeding or the Wahlund effect. The genetic diversity indices of five species showed greater allelic richness in P. flammea (3.55). Therefore, we provide useful information for the characterization of genetic diversity in natural populations of Aechmea ramosa, Aechmea nudicaulis, Billbergia horrid, Billbergia euphemia, and P. flammea in Atlantic Forest remnants in the south of Espírito Santo state. © 2017 The Authors

Quantification of the diversity among common bean accessions using Ward-MLM strategy

O objetivo deste trabalho foi avaliar a divergência de acessos de feijoeiro-comum por suas características agronômicas, morfológicas e moleculares, com base no procedimento Ward-MLM. Uma coleção de 57 acessos do banco de germoplasma da Universidade Federal do Espírito Santo foi utilizada neste estudo, dos quais: 31 acessos locais, pertencentes à comunidade Fortaleza, no Município de Muqui, ES; 20 acessos fornecidos pela Embrapa Trigo; e 6 cultivares comerciais. Foram avaliados cinco caracteres agronômicos (ciclo da planta, número de sementes por vagem, número de vagens por planta, peso de 100 grãos e produtividade de grãos), cinco caracteres morfológicos (hábito de crescimento, porte da planta, formato da semente, cor da semente e grupo comercial) e 16 iniciadores microssatélites. Detectou-se ampla variabilidade genética pelos dados morfológicos, agronômicos e moleculares nos 57 acessos de feijão. O procedimento Ward-MLM mostrou que cinco foi o número ideal de grupos, de acordo com os critérios do pseudo F e pseudo t2 . Os acessos de origem andina tiveram sementes mais pesadas do que os outros e ficaram em um mesmo grupo. O procedimento Ward-MLM é uma técnica útil para detectar divergência genética e agrupar genótipos pelo uso simultâneo de descritores morfológicos, agronômicos e moleculares.The present work aimed at evaluating the divergence among common bean accessions by their agronomic, morphological and molecular traits, based on the Ward-MLM procedure. A collection of 57 accessions from the gene bank of Universidade Federal do Espírito Santo was used in this study, from which: 31 were landraces belonging to the community Fortaleza, in the municipality of Muqui, ES, Brazil; 20 accessions were provided by Embrapa Trigo; and 6 were commercial cultivars. Five agronomic traits (plant cycle, number of seeds per pod, number of pods per plant, weight of 100 seeds, and grain yield), five morphological traits (growth habit, plant size, seed shape, seed color, and commercial group) and 16 microsatellite primers were evaluated. High genetic variability was detected considering morphological, agronomic and molecular traits in the 57 common bean accessions studied. The Ward-MLM procedure showed that the ideal number of groups was five, according to the pseudo F and pseudo t2 criteria. The accessions from Andean origin had heavier seeds than others and formed a cluster. The Ward-MLM statistical procedure is a useful technique to detect genetic divergence and to cluster genotypes by simultaneously using morphological, agronomic and molecular data

Construction of co-suppression cassettes for the oleoil desaturase gene and genetic transformation of somatic soybean embryos

A introdução de características agronomicamente importantes em soja pode ser realizada por transformação genética de plantas como um método alternativo ao melhoramento tradicional. Alterações nas proporções relativas dos ácidos graxos na fração óleo podem ser alcançadas por meio do silenciamento gênico de enzimas dessaturases, responsáveis pela síntese de ácidos graxos polinsaturados. Os objetivos deste trabalho foram: isolar um fragmento do gene da oleoil dessaturase de soja, construir cassete de expressão para a co-supressão deste gene e estabelecer metodologias para a extração e quantificação de ácidos graxos de embriões somáticos. A análise por RT-PCR mostrou que o gene Fad2-1 é expresso em todos os estádios de desenvolvimento da semente. A expressão desse gene aumentou a partir dos estádios iniciais de desenvolvimento e diminuiu em sementes maduras. Um fragmento de cDNA de cerca de 1 kb foi clonado nos vetores binários pCAMBIA 3301 e 1304, cujos T- DNAs apresentam genes para resistência a herbicida e antibiótico, respectivamente. A clonagem foi confirmada por meio de PCR, restrição enzimática e seqüenciamento. A construção clonada no vetor pCAMBIA 3301 foi utilizada para a transformação de soja via Agrobacterium. Embriões somáticos foram originados a partir de cotilédones imaturos cultivados na presença de 40 mg/L de 2,4-D. Agregados de embriões globulares foram transformados com suspensão bacteriana mediante sonicação e adição de acetoseringona e mantidos em meio seletivo com 3 mg/L e 5 mg/L do herbicida Finale → . A maioria dos embriões germinou normalmente em meio sem regulador de crescimento, embora alguns não tenham sido capazes de regenerar plantas. Apenas cinco plantas foram aclimatadas em casa de vegetação, mas não eram transgênicas. As análises moleculares com plântulas e/ou embriões somáticos cotiledonares permitiram a detecção de vários eventos de transformação. O conteúdo de ácidos graxos dos diferentes estádios de desenvolvimento embrionário foi determinado por cromatografia gasosa. Foram observadas mudanças na composição de ácidos graxos no estádio globular, com aumento dos conteúdos de ácido oléico (18:1) e linoléico (18:2) e diminuição de ácido linolênico (18:3). A partir do estádio torpedo, a composição de ácidos graxos não variou significativamente até o final do desenvolvimento, apresentando resultados similares aos de sementes maduras. A análise por RT-PCR mostrou um aumento no acúmulo de transcritos do gene Fad2-1 do estádio globular para torpedo. A expressão desse gene permaneceu relativamente constante até o estádio cotiledonar maduro, com redução da expressão após o processo de dessecação. Os dados quanto aos perfis de ácidos graxos de 138 embriões cotiledonares transformados foram analisados por meio de estatística descritiva para verificar a freqüência dos diferentes eventos de transformação. Oitenta e quatro embriões apresentaram conteúdo de 18:1 semelhante ao controle não transformado (13-21%), 18 apresentaram níveis inferiores (9-13%) e 36 apresentaram níveis aumentados (22-61%), provavelmente como possível conseqüência de silenciamento gênico. Em relação ao conteúdo de 18:2, 88 embriões apresentaram conteúdo semelhante ao controle não transformado (49-59%), 17 apresentaram níveis superiores (59-64%) e 33 apresentaram níveis inferiores de 18:2 (6-49%). Em 101 embriões o conteúdo de 18:3 foi semelhante ao controle não transformado (8-15%), 30 mostraram níveis superiores (15-27%) e 7 mostraram níveis inferiores (4-8%). Os resultados mostraram a existência de modificação na composição de ácidos graxos nos embriões cotiledonares analisados, mas não se pode afirmar que essas alterações sejam causadas por fenômenos de silenciamento gênico. Estes resultados podem ter sofrido a influência de variações somaclonais ou da auxina 2,4-D.The introduction of agronomically important traits into soybean may be accomplished by genetic transformation of the plants as an alternative method to traditional breeding. Alterations in the relative proportions of the fatty acids in the oil fraction may be reached through gene silencing of desaturase enzymes that are responsible for the synthesis of the polyunsaturated fatty acids. The objectives of this work were: to isolate a fragment of the soybean oleoil desaturase gene; to construct expression cassettes for co-suppression of this gene; and to establish methodologies for the extraction and quantification of fatty acids in soybean somatic embryos. The analysis based on RT-PCR showed that the Fad2-1 gene is expressed in all developmental stages of the seed. The expression of the gene increases after the first stages of development and decreases in the dry seeds. One cDNA fragment around 1 kb was cloned in the binary vectors pCAMBIA 3301 and 1304, in which the T-DNAs harbor genes for resistance to herbicide and antibiotic, respectively. Cloning was confirmed through PCR, enzymatic restriction and sequencing. The constructs cloned in pCAMBIA 3301 were used for the soybean transformation via Agrobacterium. Somatic embryos were obtained from immature cotyledons cultivated in the presence of 2,4-D (40 mg/L). The globular embryo clusters were transformed with bacterial suspension using sonication and addition of acetosyringone and kept in selective medium with 3 mg/L and 5 mg/L of the Finale ® herbicide. Most embryos germinated in medium without growth regulator, however, some of them were not able to regenerate plants. Only five plants were acclimated in the greenhouse, but they were not transgenic. The molecular analyses of seedlings and/or cotyledone somatic embryos allowed the detection of several transformation events. Fatty acid content at different stages of the embryonic development was determined by gas chromatography. Changes in the fatty acid xcompositions at the globular stage were observed. There was an increase on oleic (18:1) and linoleic (18:2) acids contents and a decrease on linolenic acid (18:3) content. From the torpedo stage, the composition of the fatty acids did not vary significantly until the end of the development. The values obtained were similar to those of the dry seeds. The RT-PCR analysis showed an increase on the accumulation of transcripts for the Fad2-1 gene from globular to the torpedo stage. The expression of this gene remained relatively constant until the ripe cotyledonal stage, but there was a reduction on expression after the desiccation process. The data relative to the fatty acid profiles of 138 cotyledonal transformed embryos were analyzed by descriptive statistics in order to verify the frequency of the different transformation events. Eighty-four embryos showed a content of 18:1 similar to the non-transformed control (13-21%), 18 presented lower levels (9- 13%) and 36 presented increased levels (22-61%), probably a possible consequence of gene silencing. In relation to 18:2, 88 embryos presented a content similar to that of the non-transformed control (49-59%), 17 presented higher levels (59-64%) and 33 presented lower levels of 18:2 (6-49%). In 101 embryos, the content of 18:3 was similar to the non-transformed control (8-15%), 30 showed higher levels (15-27%) and 7 showed lower levels (4-8%). The results show that the cotyledonal embryos analyzed had a modified fatty acid composition, but one cannot state that these alterations were caused by the gene silencing phenomenon. These results might have been affected by the influence of either somaclonal variations or the auxin 2,4-D .Conselho Nacional de Desenvolvimento Científico e Tecnológic

Genome size and base composition of Bromeliaceae species assessed by flow cytometry

Flow cytometry (FCM) has been used to estimate the nuclear DNA content of Bromeliaceae species, which constitutes relevant information for studies of taxonomy, evolution, genetic diversity, and reproductive biology in bromeliads. Nevertheless, C values have only been estimated for 58 out of the 3,140 existing Bromeliaceae species. Aiming to contribute to the genome database of Bromeliaceae, the current study was carried out to measure the nuclear DNA content and base composition of Bromelioideae and Tillandsioideae species occurring in the Atlantic Rainforest. The most adequate FCM procedure provided histograms exhibiting G0/G1 peaks with coefficients of variation below 5%, so that these histograms were used to measure the mean 2C and AT% values for all collected Bromelioideae and Tillandsioideae species. These values were statistically compared, and dendrograms were plotted. A second comparison was performed among all mean 2C values reported for Pitcairnioideae, Tillandsioideae, and Bromelioideae species. In accordance with previous statistical comparisons, two groups were formed: cluster 1, composed by Tillandsia loliacea, Tillandsia usneoides, and Tillandsia cyanea, and cluster 2, gathering other 69 species. Based on these results, we concluded that FCM was a rapid, accurate, and reliable technique to assess genome size and base composition. Furthermore, the FCM data reported here will contribute to the Monocot and Bromeliaceae database, which still displays several ongoing gaps, especially for endemic species

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group

Induction of in vitro shoots of Billbergia euphemiae E. Morren (Bromeliaceae) from leaf explants

Bromeliads are an important group for the maintenance of the Atlantic Forest, with many threatened species due to exacerbated extraction and destruction of their natural habitats. Considering the need of developing protocols for the conservation of these species, the aim of this work was to evaluate the effect of different growth regulators in the in vitro induction of shoots of Billbergia euphemiae. Leaf explants were excised from seedlings derived from in vitro germination and grown on MS medium supplemented with NAA (0, 1 or 2 μM) and BA (0, 2, 4 or 6 μM) combinations. The evaluation of the number of shoots per explant, shoot length, number of leaves per shoot and longest leaf length average was carried out after 30 and 60 days of culture. The best in vitro responses were observed in the presence of 1 μM NAA after 60 days of culture, which induced the best production of shoots per explant (16.39), as well as the highest rates of shoot length (1.08 cm), number of leaves per shoot (5.00) and the longest leaf length (0.56 cm). This work determined the best conditions for shoot production from leaf explants of B. euphemiae, being the first report on micropropagation of this species.As bromélias constituem um importante grupo para a manutenção da Floresta Atlântica, com várias espécies ameaçadas de extinção pelo extrativismo exacerbado e a destruição dos habitats naturais. Considerando a necessidade do desenvolvimento de protocolos para a conservação destas espécies, este trabalho teve como objetivo avaliar o efeito de diferentes reguladores de crescimento na indução de brotos de Billbergia euphemiae. Explantes foliares foram excisados de plântulas derivadas da germinação in vitro e inoculados em meio MS suplementado com combinações de ANA (0, 1 ou 2 μM) e BA (0, 2, 4 ou 6 μM). O número de brotos por explantes, comprimento dos brotos, número médio de folhas por broto e comprimento médio da maior folha foram avaliados após 30 e 60 dias de cultura. As melhores respostas foram observadas na presença de ANA a 1 μM, após 60 dias de cultura, que induziu a maior produção de brotos por explante (16,39), assim como as maiores taxas de comprimento dos brotos (1,08 cm), número médio de folhas por broto (5,00) e comprimento da maior folha (0,56 cm). Este trabalho determinou as melhores condições para produção de brotos de B. euphemiae a partir de explantes foliares, representando o primeiro relato de micropropagação desta espécie

Diversidade genética de acessos de feijão comum por caracteres agronômicos Genetic diversity of common bean accessions by agronomic traits

Este experimento teve como objetivo avaliar a divergência genética entre 57 acessos de feijão comum, sendo 31 genótipos locais do sul do Espírito Santo, 20 da EMBRAPA e seis cultivares comerciais. Foram realizados três experimentos no município de Alegre, ES nos anos agrícolas de 2008/2009 e de 2009/2010. O delineamento utilizado foi o de blocos casualizados com três repetições. Os dados foram submetidos à análise de variância pelo teste F. Foi utilizada análise multivariada para avaliar a divergência genética entre os acessos utilizando o método de agrupamento UPGMA e variáveis canônicas com base na distância generalizada de Mahalanobis. As variáveis que mais contribuíram para a separação dos acessos foram: peso de cem sementes (P100) com 24,01%, período vegetativo (PV) com 20,39%, período reprodutivo (PR) com 17,16% e comprimento da semente (CS) com 14,87%. Os acessos menos divergentes foram o F15 e o F18 (9,18) e os mais divergentes foram F10 e F08 (1308,62). Verificou-se baixa dissimilaridade genética entre as cultivares comerciais e também entre os acessos provenientes da EMBRAPA e entre ambas. Os acessos locais demonstraram uma diversidade genética significativa. Tanto a análise de agrupamento quanto a de variáveis canônicas foram capazes de separar os acessos de acordo com os centros de origem.This research aimed to evaluate the genetic divergence among 57 accessions of common bean genotypes being 31 genotypes from southern Espírito Santo, Brazil, 20 cultivars from EMBRAPA and 6 commercial cultivars. Three experiments were conducted in the agricultural years 2008/2009 and 2009/2010, two in the experimental area and a CCA-UFES at IFES, both sites in the municipality of Alegre, ES, Brazil. The desing was a randomized block with tree replications. Data were subjected to analysis of variance test by F. By multivariate analysis assessed the genetic diversity among accessions using the UPGMA clustering method and canonical variables based on the Mahalanobis distance. The variables that contributed most to the separation of the accessions were one hundred seed weight (P100) with 24.01%, growing season (PV) with 20.39%, reproductive period (PR) with 17.16% and seed length (CS) with 14.87%. The accessions were less divergent the F15 and F18 (9.18) and the most divergent F10 and F08 (1308.62). Multivariate analysis showed a low genetic similarity between cultivars and also between accessions from EMBRAPA and between them. Site accesses demonstrated significant genetic diversity. Both the cluster analysis and the canonical variables were able to separate the access according to the centers of origin