Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)

Detalhes bibliográficos
Autor(a) principal: Araújo, Fernanda Santos
Data de Publicação: 2008
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/4664
Resumo: Carica papaya is one of the most economically important species of the Caricaceae family, with many industrial applications. Despite its importance, there is little information covering the cytogenetic and cytometric characterization of this species. Hence, this study is justified by the need to increase the knowledge related to papaya karyotype, nuclear DNA content and base composition. The specific objectives of this study were to: characterize papaya chromosomes, using image analysis resources; apply the acridine orange staining to mitotic chromosomes of C. papaya to identify nucleolar organizers regions (NORs); estimate the nuclear DNA content and determine the base composition of male, female and hermaphrodite plants of C. papaya, by the use of flow cytometry and verify if the cytometric data can differentiate the three sex types with regard to their DNA content and base pair frequencies. Root tips from hermaphrodite plants were pre-treated with amiprophosmethyl (APM) 3 µM for 15h, at 4ºC and fixed. Slides were prepared by cellular dissociation and air drying, and submitted to Giemsa and acridine orange staining. The association of cellular dissociation and air drying techniques to Giemsa conventional staining resulted in metaphasic and prometaphasic cells with individual and well spread chromosomes, without overlaps, with well defined primary and secondary constrictions. These characteristics enabled the homologues pairing and morphological classification of the chromosomes, with karyograms assembly of a hermaphrodite plant, which had 18 chromosomes, being 7 pairs metacentrics (1, 2, 3, 4, 5, 7 and 8) and 2 submetacentrics (6 and 9). The acridine orange staining allowed the identification of yellowish green bands in two chromosomes (1 and 2). The strong bands corresponded to NOR flanking regions, as sustained by the localization of chromosome 1 associated to the nucleolus. Considering the obtained results by the cytogenetic techniques, there were no morphological differences that could evidence a possible heteromorphic pair of chromosomes in the hermaphrodite plant of C. papaya. For the application of flow cytometry, leaves from the three sexual forms of papaya were submitted to the chopping procedure in an isolation buffer, followed by a staining buffer. The obtained suspension was passed through a filter and analyzed in a flow cytometer, to determine the total nuclear DNA content and base composition. The cytometric analyses evidenced histograms with peaks of G0/G1 nuclei with coefficients of variation (CVs) lower than 5%. The analysis of male, female and hermaphrodite plants nuclei, stained with propidium iodide (PI), allowed the calculation of the mean 2C DNA values, which were 0.67, 0.65 and 0.65 pg, respectively, and statistically equal, by the F test, for the three sex forms. The analysis of nuclei stained with 4 ,6-diamidino-2-phenylindole (DAPI) and chromomicin A3 (CMA3) enabled the calculation of AT/GC base pair frequencies of the three sex types, also statically equal, by the F test. Therefore, there was no significant difference between the three sex forms of papaya in relation to their total nuclear DNA content and base pair composition. In relation to the obtained mean values of nuclear DNA content and considering the morphological similarity of papaya chromosomes, each one of the 18 chromosomes would have, on average, 0.036 to 0.037 pg of DNA. Hence, in order to identify a probable pair of heteromorphic chromosomes, a tiny portion of the chromosome would have to be distinguished, corresponding to an amount of DNA under the limits of resolution of the cytogenetic and cytometric techniques employed in this study.
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spelling Araújo, Fernanda Santoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4759994T2Otoni, Wagner Camposhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6Pompolo, Silvia das Graçashttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780564A2Carvalho, Carlos Roberto dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0Tavares, Mara Garciahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798782P4Caixeta, Eveline Teixeirahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728636Z72015-03-26T13:42:01Z2008-05-262015-03-26T13:42:01Z2008-02-26ARAÚJO, Fernanda Santos. Cytogenetic and cytometric studies in papaya (Carica papaya L.). 2008. 58 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008.http://locus.ufv.br/handle/123456789/4664Carica papaya is one of the most economically important species of the Caricaceae family, with many industrial applications. Despite its importance, there is little information covering the cytogenetic and cytometric characterization of this species. Hence, this study is justified by the need to increase the knowledge related to papaya karyotype, nuclear DNA content and base composition. The specific objectives of this study were to: characterize papaya chromosomes, using image analysis resources; apply the acridine orange staining to mitotic chromosomes of C. papaya to identify nucleolar organizers regions (NORs); estimate the nuclear DNA content and determine the base composition of male, female and hermaphrodite plants of C. papaya, by the use of flow cytometry and verify if the cytometric data can differentiate the three sex types with regard to their DNA content and base pair frequencies. Root tips from hermaphrodite plants were pre-treated with amiprophosmethyl (APM) 3 µM for 15h, at 4ºC and fixed. Slides were prepared by cellular dissociation and air drying, and submitted to Giemsa and acridine orange staining. The association of cellular dissociation and air drying techniques to Giemsa conventional staining resulted in metaphasic and prometaphasic cells with individual and well spread chromosomes, without overlaps, with well defined primary and secondary constrictions. These characteristics enabled the homologues pairing and morphological classification of the chromosomes, with karyograms assembly of a hermaphrodite plant, which had 18 chromosomes, being 7 pairs metacentrics (1, 2, 3, 4, 5, 7 and 8) and 2 submetacentrics (6 and 9). The acridine orange staining allowed the identification of yellowish green bands in two chromosomes (1 and 2). The strong bands corresponded to NOR flanking regions, as sustained by the localization of chromosome 1 associated to the nucleolus. Considering the obtained results by the cytogenetic techniques, there were no morphological differences that could evidence a possible heteromorphic pair of chromosomes in the hermaphrodite plant of C. papaya. For the application of flow cytometry, leaves from the three sexual forms of papaya were submitted to the chopping procedure in an isolation buffer, followed by a staining buffer. The obtained suspension was passed through a filter and analyzed in a flow cytometer, to determine the total nuclear DNA content and base composition. The cytometric analyses evidenced histograms with peaks of G0/G1 nuclei with coefficients of variation (CVs) lower than 5%. The analysis of male, female and hermaphrodite plants nuclei, stained with propidium iodide (PI), allowed the calculation of the mean 2C DNA values, which were 0.67, 0.65 and 0.65 pg, respectively, and statistically equal, by the F test, for the three sex forms. The analysis of nuclei stained with 4 ,6-diamidino-2-phenylindole (DAPI) and chromomicin A3 (CMA3) enabled the calculation of AT/GC base pair frequencies of the three sex types, also statically equal, by the F test. Therefore, there was no significant difference between the three sex forms of papaya in relation to their total nuclear DNA content and base pair composition. In relation to the obtained mean values of nuclear DNA content and considering the morphological similarity of papaya chromosomes, each one of the 18 chromosomes would have, on average, 0.036 to 0.037 pg of DNA. Hence, in order to identify a probable pair of heteromorphic chromosomes, a tiny portion of the chromosome would have to be distinguished, corresponding to an amount of DNA under the limits of resolution of the cytogenetic and cytometric techniques employed in this study.O mamoeiro, Carica papaya, é a espécie da família Caricaceae considerada mais importante economicamente, possuindo diversas aplicações industriais. Apesar de sua importância, são poucos os dados envolvendo a caracterização citogenética e citométrica dessa espécie. Portanto, o presente estudo visa ampliar o conhecimento com relação ao cariótipo, ao tamanho do genoma e à relação de bases do mamoeiro. Os objetivos específicos foram: caracterizar morfologicamente os cromossomos de C. papaya, utilizando recursos de análise de imagem; aplicar a coloração de laranja de acridina aos cromossomos mitóticos de C. papaya, para a identificação de regiões organizadoras do nucléolo (RONs); estimar o conteúdo de DNA nuclear e determinar a composição de bases de plantas masculinas, femininas e hermafroditas de C. papaya, por meio da técnica de citometria de fluxo e verificar se os dados citométricos possibilitam diferenciar os três tipos sexuais quanto ao tamanho genômico e à relação de bases AT/GC. Para isso, raízes de plantas hermafroditas foram pré-tratadas com amiprofos-metil (APM), 3 μM, por 15h, a 4ºC e fixadas. Lâminas foram preparadas por dissociação celular e secagem ao ar e submetidas à coloração com Giemsa e com laranja de acridina. A associação das técnicas de dissociação celular e secagem ao ar, com a coloração convencional com Giemsa resultou na obtenção de células metafásicas e prometafásicas com cromossomos individualizados, bem espalhados na lâmina e sem sobreposições, com constrições primárias e secundárias bem definidas. Essas características possibilitaram o pareamento dos homólogos e a classificação morfológica dos cromossomos, com a montagem de cariogramas de uma planta hermafrodita de C. papaya, que apresentou número cromossômico de 2n = 18, sendo sete pares metacêntricos (1, 2, 3, 4, 5, 7 e 8) e dois submetacêntricos (6 e 9). A coloração com laranja de acridina possibilitou a identificação de bandas verde-amareladas em dois cromossomos (1 e 2). As bandas que emitiram maior intensidade de fluorescência corresponderam às regiões flanqueadoras das RONs, como salientado pela localização do cromossomo 1 associado ao nucléolo. Considerando os resultados obtidos por meio das técnicas citogenéticas, não houve diferenças morfológicas que evidenciassem um possível par de cromossomos heteromórficos na planta hermafrodita. Para a aplicação da citometria de fluxo, folhas de plantas dos três tipos sexuais de mamão foram submetidas ao procedimento de cortes seqüenciais em tampão de extração, seguidos do tampão de coloração. A suspensão obtida foi filtrada e analisada em citômetro de fluxo, para a determinação do conteúdo de DNA nuclear total e da composição de bases AT e GC. As análises citométricas realizadas possibilitaram a geração de histogramas com picos de núcleos G0/G1 com coeficientes de variação (CVs) menores do que 5%. A análise dos núcleos de plantas masculinas, femininas e hermafroditas de C. papaya, corados com iodeto de propídio (IP), permitiu calcular os valores médios 2C de DNA, que foram 0,67, 0,65 e 0,65 pg, respectivamente, sendo estatisticamente iguais, pelo teste F, para os três sexos. A análise dos núcleos corados com 4 ,6- diamidino-2-fenilindol (DAPI) e cromomicina A3 (CMA3) possibilitou o cálculo das freqüências de pares de bases AT e GC para os três tipos sexuais, também estatisticamente iguais, de acordo com o teste F. Portanto, não houve diferença significativa quanto ao tamanho genômico ou quanto à relação de pares de bases entre os três sexos do mamoeiro. Com relação aos valores médios de conteúdos absolutos de DNA nuclear observados, e considerando a semelhança morfológica dos cromossomos de C. papaya, cada um dos 18 cromossomos teria, em média, 0,036 a 0,037 pg de DNA. Conseqüentemente, para a identificação de um provável par de cromossomos heteromórficos, ter-se-ia de se distinguir uma porção muito pequena do cromossomo, com uma quantidade de DNA que estaria abaixo do limite de resolução das técnicas citogenéticas e citométricas utilizadas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaMestrado em Genética e MelhoramentoUFVBRGenética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; MeCarica papayaCromossomosCitogenéticaCarica papayaChromosomesCytogeneticsCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETALEstudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)Cytogenetic and cytometric studies in papaya (Carica papaya L.)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf492128https://locus.ufv.br//bitstream/123456789/4664/1/texto%20completo.pdf4591b57e36d0c8c37588137394b8de46MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain94793https://locus.ufv.br//bitstream/123456789/4664/2/texto%20completo.pdf.txtb52028618f24487ee5b9bfd4d4eadde2MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3738https://locus.ufv.br//bitstream/123456789/4664/3/texto%20completo.pdf.jpg055262a632224226ca1c432a56ce5d97MD53123456789/46642016-04-10 23:12:22.987oai:locus.ufv.br:123456789/4664Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:12:22LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
dc.title.alternative.eng.fl_str_mv Cytogenetic and cytometric studies in papaya (Carica papaya L.)
title Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
spellingShingle Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
Araújo, Fernanda Santos
Carica papaya
Cromossomos
Citogenética
Carica papaya
Chromosomes
Cytogenetics
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
title_short Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
title_full Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
title_fullStr Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
title_full_unstemmed Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
title_sort Estudos citogenéticos e citométricos em mamoeiro (Carica papaya L.)
author Araújo, Fernanda Santos
author_facet Araújo, Fernanda Santos
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4759994T2
dc.contributor.author.fl_str_mv Araújo, Fernanda Santos
dc.contributor.advisor-co1.fl_str_mv Otoni, Wagner Campos
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6
dc.contributor.advisor-co2.fl_str_mv Pompolo, Silvia das Graças
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780564A2
dc.contributor.advisor1.fl_str_mv Carvalho, Carlos Roberto de
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0
dc.contributor.referee1.fl_str_mv Tavares, Mara Garcia
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798782P4
dc.contributor.referee2.fl_str_mv Caixeta, Eveline Teixeira
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728636Z7
contributor_str_mv Otoni, Wagner Campos
Pompolo, Silvia das Graças
Carvalho, Carlos Roberto de
Tavares, Mara Garcia
Caixeta, Eveline Teixeira
dc.subject.por.fl_str_mv Carica papaya
Cromossomos
Citogenética
topic Carica papaya
Cromossomos
Citogenética
Carica papaya
Chromosomes
Cytogenetics
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
dc.subject.eng.fl_str_mv Carica papaya
Chromosomes
Cytogenetics
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
description Carica papaya is one of the most economically important species of the Caricaceae family, with many industrial applications. Despite its importance, there is little information covering the cytogenetic and cytometric characterization of this species. Hence, this study is justified by the need to increase the knowledge related to papaya karyotype, nuclear DNA content and base composition. The specific objectives of this study were to: characterize papaya chromosomes, using image analysis resources; apply the acridine orange staining to mitotic chromosomes of C. papaya to identify nucleolar organizers regions (NORs); estimate the nuclear DNA content and determine the base composition of male, female and hermaphrodite plants of C. papaya, by the use of flow cytometry and verify if the cytometric data can differentiate the three sex types with regard to their DNA content and base pair frequencies. Root tips from hermaphrodite plants were pre-treated with amiprophosmethyl (APM) 3 µM for 15h, at 4ºC and fixed. Slides were prepared by cellular dissociation and air drying, and submitted to Giemsa and acridine orange staining. The association of cellular dissociation and air drying techniques to Giemsa conventional staining resulted in metaphasic and prometaphasic cells with individual and well spread chromosomes, without overlaps, with well defined primary and secondary constrictions. These characteristics enabled the homologues pairing and morphological classification of the chromosomes, with karyograms assembly of a hermaphrodite plant, which had 18 chromosomes, being 7 pairs metacentrics (1, 2, 3, 4, 5, 7 and 8) and 2 submetacentrics (6 and 9). The acridine orange staining allowed the identification of yellowish green bands in two chromosomes (1 and 2). The strong bands corresponded to NOR flanking regions, as sustained by the localization of chromosome 1 associated to the nucleolus. Considering the obtained results by the cytogenetic techniques, there were no morphological differences that could evidence a possible heteromorphic pair of chromosomes in the hermaphrodite plant of C. papaya. For the application of flow cytometry, leaves from the three sexual forms of papaya were submitted to the chopping procedure in an isolation buffer, followed by a staining buffer. The obtained suspension was passed through a filter and analyzed in a flow cytometer, to determine the total nuclear DNA content and base composition. The cytometric analyses evidenced histograms with peaks of G0/G1 nuclei with coefficients of variation (CVs) lower than 5%. The analysis of male, female and hermaphrodite plants nuclei, stained with propidium iodide (PI), allowed the calculation of the mean 2C DNA values, which were 0.67, 0.65 and 0.65 pg, respectively, and statistically equal, by the F test, for the three sex forms. The analysis of nuclei stained with 4 ,6-diamidino-2-phenylindole (DAPI) and chromomicin A3 (CMA3) enabled the calculation of AT/GC base pair frequencies of the three sex types, also statically equal, by the F test. Therefore, there was no significant difference between the three sex forms of papaya in relation to their total nuclear DNA content and base pair composition. In relation to the obtained mean values of nuclear DNA content and considering the morphological similarity of papaya chromosomes, each one of the 18 chromosomes would have, on average, 0.036 to 0.037 pg of DNA. Hence, in order to identify a probable pair of heteromorphic chromosomes, a tiny portion of the chromosome would have to be distinguished, corresponding to an amount of DNA under the limits of resolution of the cytogenetic and cytometric techniques employed in this study.
publishDate 2008
dc.date.available.fl_str_mv 2008-05-26
2015-03-26T13:42:01Z
dc.date.issued.fl_str_mv 2008-02-26
dc.date.accessioned.fl_str_mv 2015-03-26T13:42:01Z
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dc.identifier.citation.fl_str_mv ARAÚJO, Fernanda Santos. Cytogenetic and cytometric studies in papaya (Carica papaya L.). 2008. 58 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/4664
identifier_str_mv ARAÚJO, Fernanda Santos. Cytogenetic and cytometric studies in papaya (Carica papaya L.). 2008. 58 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2008.
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