Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo
Ano de defesa: | 2012 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Viçosa
|
Programa de Pós-Graduação: |
Mestrado em Genética e Melhoramento
|
Departamento: |
Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
|
País: |
BR
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://locus.ufv.br/handle/123456789/4770 |
Resumo: | Tissue culture techniques are often associated with the occurrence of somaclonal variation, characterized by a genetic/epigenetic alteration found in cells, embryos, organs or in vitro grown plants, due to stress conditions imposed by the system. The propagation process involving the calogenesis step has a higher susceptibility to variation, because of many cell divisions. Calli are disorganized cell masses, whose cells divide at different rates depending on where they are located in relation to the culture medium, therefore, they are not considered homogeneous materials. Flow cytometry is a tool that has been used to evaluate the competence of somatic embryogenesis in callus, both to identify the different types as possible and to detect early changes in DNA content of their cells. However, there are no reports in the literature of the flow cytometry use to determine the existence of heterogeneity in relation to the DNA content in callus. In this perspective, the present work adapted this methodology for friable Passiflora cincinnata calli in order to verify the existence of vertically regionalized heterogeneity in relation to DNA ploidy level, and relate it to factors inductors of somaclonal variation, such as cultivation time and subculture numbers. Initially, P. cincinnata cotyledonary leaves were inoculated onto semi-solid medium containing 2, 4-D and BA, for friable embryogenic callus induction. The calli were multiplied and subcultured at different times, thus generating three callus types: new, intermediate and old. Twenty calli of each type were subjected to sagittal cuts resulting two layers (I, II) in new calli, and three layers (I, II and III) in intermediate and old calli, totalizing 160 layers, which were analyzed by flow cytometry. Six different DNA ploidy levels and multiploidies were detected in the layers. In new calli, 25% were heterogeneous, i.e. at least one callus layer showed different DNA ploidy level from the others, differently from intermediate and old calli, which 75% and 63% were heterogeneous, respectively. The homogeneous new calli were predominantly diploid. The highest DNA ploidy levels were observed in the layers in contact with the culture medium on intermediate calli, and 4C and 4C/8C cells were more evident in older calli. These observations suggest that the appearance of new DNA ploidy levels between the layers and between the different callus types is related to the exposure time to the medium containing relatively high concentration of 2,4-D. It was also possible to verify that the layer in contact with the culture medium is more proliferative when compared the percentages of the G2 peaks in homogeneous diploid calli. The heterogeneity in DNA ploidy level between the layers was observed, and it implies that parts of a callus cannot infer about a whole callus. This work suggests that time influences the appearance of different DNA ploidy levels in calli induced in vitro. |
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Silva, Thaís Cristina Ribeiro dahttp://lattes.cnpq.br/9114202993101367Motoike, Sérgio Yoshimitsuhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728221T8Koehler, Andréa Diashttp://lattes.cnpq.br/2563890461457295Carvalho, Carlos Roberto dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0Clarindo, Wellington Ronildohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H92015-03-26T13:42:26Z2013-03-212015-03-26T13:42:26Z2012-07-17SILVA, Thaís Cristina Ribeiro da. Vertical heterogeneity of DNA ploidy in Passiflora cincinnata calluses analyzed by flow cytometry. 2012. 50 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2012.http://locus.ufv.br/handle/123456789/4770Tissue culture techniques are often associated with the occurrence of somaclonal variation, characterized by a genetic/epigenetic alteration found in cells, embryos, organs or in vitro grown plants, due to stress conditions imposed by the system. The propagation process involving the calogenesis step has a higher susceptibility to variation, because of many cell divisions. Calli are disorganized cell masses, whose cells divide at different rates depending on where they are located in relation to the culture medium, therefore, they are not considered homogeneous materials. Flow cytometry is a tool that has been used to evaluate the competence of somatic embryogenesis in callus, both to identify the different types as possible and to detect early changes in DNA content of their cells. However, there are no reports in the literature of the flow cytometry use to determine the existence of heterogeneity in relation to the DNA content in callus. In this perspective, the present work adapted this methodology for friable Passiflora cincinnata calli in order to verify the existence of vertically regionalized heterogeneity in relation to DNA ploidy level, and relate it to factors inductors of somaclonal variation, such as cultivation time and subculture numbers. Initially, P. cincinnata cotyledonary leaves were inoculated onto semi-solid medium containing 2, 4-D and BA, for friable embryogenic callus induction. The calli were multiplied and subcultured at different times, thus generating three callus types: new, intermediate and old. Twenty calli of each type were subjected to sagittal cuts resulting two layers (I, II) in new calli, and three layers (I, II and III) in intermediate and old calli, totalizing 160 layers, which were analyzed by flow cytometry. Six different DNA ploidy levels and multiploidies were detected in the layers. In new calli, 25% were heterogeneous, i.e. at least one callus layer showed different DNA ploidy level from the others, differently from intermediate and old calli, which 75% and 63% were heterogeneous, respectively. The homogeneous new calli were predominantly diploid. The highest DNA ploidy levels were observed in the layers in contact with the culture medium on intermediate calli, and 4C and 4C/8C cells were more evident in older calli. These observations suggest that the appearance of new DNA ploidy levels between the layers and between the different callus types is related to the exposure time to the medium containing relatively high concentration of 2,4-D. It was also possible to verify that the layer in contact with the culture medium is more proliferative when compared the percentages of the G2 peaks in homogeneous diploid calli. The heterogeneity in DNA ploidy level between the layers was observed, and it implies that parts of a callus cannot infer about a whole callus. This work suggests that time influences the appearance of different DNA ploidy levels in calli induced in vitro.Técnicas de cultura de tecidos estão frequentemente associadas com a ocorrência de variação somaclonal, caracterizada pela alteração genética e/ou epigenética encontrada em células, embriões, órgãos ou plantas cultivadas in vitro, em decorrência das condições de estresse impostas pelo sistema. O processo de propagação que envolve a etapa de calogênese apresenta maior susceptibilidade à ocorrência de variação, em função das inúmeras divisões celulares. Calos são massas celulares não organizadas, cujas células se dividem a diferentes velocidades dependendo de onde se localizam em relação ao meio de cultura; portanto, não são considerados materiais homogêneos. A citometria de fluxo é uma ferramenta que tem sido utilizada para avaliar a competência da embriogênese somática em calos, tanto para identificar seus diferentes tipos quanto para detectar precocemente possíveis alterações no conteúdo de DNA de suas células. No entanto, não há relatos na literatura da utilização da citometria de fluxo para verificar a existência da heterogeneidade quanto ao conteúdo de DNA em calos. Nesta perspectiva, o presente trabalho adaptou essa metodologia para calos friáveis de Passiflora cincinnata, a fim de verificar a existência de heterogeneidade verticalmente regionalizada, em relação à ploidia de DNA, e relacioná-la com fatores de indução de variação somaclonal, como tempo de cultivo e número de subcultivos. Inicialmente, folhas cotiledonares de P. cincinnata foram inoculadas em meio semissólido contendo 2,4-D e BA, para indução de calos embriogênicos friáveis. Os calos foram multiplicados e subcultivados em diferentes tempos, gerando, assim, três tipos de calos, denominados como novos, intermediários e velhos. Vinte calos de cada tipo foram submetidos a cortes sagitais que resultaram duas camadas (I e II) em calos novos, e três camadas (I, II e III) em calos intermediários e velhos, totalizando 160 amostras que foram analisadas por citometria de fluxo. Seis diferentes ploidias de DNA e multiploidias foram detectadas nas camadas. Em calos novos, 25% deles foram heterogêneos, ou seja, pelo menos uma camada apresentou nível de ploidia de DNA diferente das demais, diferentemente dos calos intermediários e velhos, os quais 75% e 63% foram heterogêneos, respectivamente. Os calos novos homogêneos foram predominantemente diploides. As ploidias de DNA mais altas foram observadas nas camadas em contato com o meio de cultura em calos intermediários, e células 4C e 4C/8C foram mais evidentes em calos velhos. Essas observações sugerem que o surgimento de novos níveis de ploidia entre as camadas e entre os calos de diferentes tipos está relacionado com o tempo de exposição ao meio contendo concentração relativamente alta de 2,4-D. Foi possível, também, verificar que a camada em contato com o meio é mais proliferativa quando se comparou os percentuais dos picos G2 de calos diploides homogêneos. A heterogeneidade quanto ao nível de ploidia de DNA entre as camadas foi constatada e isto implica que partes de um calo não podem inferir sobre um calo inteiro. Esse trabalho sugere que o tempo influenciou o surgimento de diferentes ploidias de DNA em calos induzidos in vitro.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaMestrado em Genética e MelhoramentoUFVBRGenética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; MeCitometria de fluxoNível de ploidiaEmbriogênese somáticaCalosPassifllora cincinnataFlow cytometryPloidy levelSomatic embryogenesisCallusesPassifllora cincinnataCNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETALHeterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxoVertical heterogeneity of DNA ploidy in Passiflora cincinnata calluses analyzed by flow cytometryinfo: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/pdf881362https://locus.ufv.br//bitstream/123456789/4770/1/texto%20completo.pdf0e498263c87a06e3c091110afa80ddfeMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain94757https://locus.ufv.br//bitstream/123456789/4770/2/texto%20completo.pdf.txta4575776f378b4a01684092b883a9485MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3521https://locus.ufv.br//bitstream/123456789/4770/3/texto%20completo.pdf.jpgd2e7cfa739ced95852b23afeb0977a61MD53123456789/47702016-04-10 23:03:57.061oai:locus.ufv.br:123456789/4770Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:03:57LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
dc.title.alternative.eng.fl_str_mv |
Vertical heterogeneity of DNA ploidy in Passiflora cincinnata calluses analyzed by flow cytometry |
title |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
spellingShingle |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo Silva, Thaís Cristina Ribeiro da Citometria de fluxo Nível de ploidia Embriogênese somática Calos Passifllora cincinnata Flow cytometry Ploidy level Somatic embryogenesis Calluses Passifllora cincinnata CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
title_short |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
title_full |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
title_fullStr |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
title_full_unstemmed |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
title_sort |
Heterogeneidade vertical da ploidia de DNA em calos de Passiflora cincinnata analisada por citometria de fluxo |
author |
Silva, Thaís Cristina Ribeiro da |
author_facet |
Silva, Thaís Cristina Ribeiro da |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/9114202993101367 |
dc.contributor.author.fl_str_mv |
Silva, Thaís Cristina Ribeiro da |
dc.contributor.advisor-co1.fl_str_mv |
Motoike, Sérgio Yoshimitsu |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728221T8 |
dc.contributor.advisor-co2.fl_str_mv |
Koehler, Andréa Dias |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://lattes.cnpq.br/2563890461457295 |
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 |
Clarindo, Wellington Ronildo |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H9 |
contributor_str_mv |
Motoike, Sérgio Yoshimitsu Koehler, Andréa Dias Carvalho, Carlos Roberto de Clarindo, Wellington Ronildo |
dc.subject.por.fl_str_mv |
Citometria de fluxo Nível de ploidia Embriogênese somática Calos Passifllora cincinnata |
topic |
Citometria de fluxo Nível de ploidia Embriogênese somática Calos Passifllora cincinnata Flow cytometry Ploidy level Somatic embryogenesis Calluses Passifllora cincinnata CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
dc.subject.eng.fl_str_mv |
Flow cytometry Ploidy level Somatic embryogenesis Calluses Passifllora cincinnata |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL |
description |
Tissue culture techniques are often associated with the occurrence of somaclonal variation, characterized by a genetic/epigenetic alteration found in cells, embryos, organs or in vitro grown plants, due to stress conditions imposed by the system. The propagation process involving the calogenesis step has a higher susceptibility to variation, because of many cell divisions. Calli are disorganized cell masses, whose cells divide at different rates depending on where they are located in relation to the culture medium, therefore, they are not considered homogeneous materials. Flow cytometry is a tool that has been used to evaluate the competence of somatic embryogenesis in callus, both to identify the different types as possible and to detect early changes in DNA content of their cells. However, there are no reports in the literature of the flow cytometry use to determine the existence of heterogeneity in relation to the DNA content in callus. In this perspective, the present work adapted this methodology for friable Passiflora cincinnata calli in order to verify the existence of vertically regionalized heterogeneity in relation to DNA ploidy level, and relate it to factors inductors of somaclonal variation, such as cultivation time and subculture numbers. Initially, P. cincinnata cotyledonary leaves were inoculated onto semi-solid medium containing 2, 4-D and BA, for friable embryogenic callus induction. The calli were multiplied and subcultured at different times, thus generating three callus types: new, intermediate and old. Twenty calli of each type were subjected to sagittal cuts resulting two layers (I, II) in new calli, and three layers (I, II and III) in intermediate and old calli, totalizing 160 layers, which were analyzed by flow cytometry. Six different DNA ploidy levels and multiploidies were detected in the layers. In new calli, 25% were heterogeneous, i.e. at least one callus layer showed different DNA ploidy level from the others, differently from intermediate and old calli, which 75% and 63% were heterogeneous, respectively. The homogeneous new calli were predominantly diploid. The highest DNA ploidy levels were observed in the layers in contact with the culture medium on intermediate calli, and 4C and 4C/8C cells were more evident in older calli. These observations suggest that the appearance of new DNA ploidy levels between the layers and between the different callus types is related to the exposure time to the medium containing relatively high concentration of 2,4-D. It was also possible to verify that the layer in contact with the culture medium is more proliferative when compared the percentages of the G2 peaks in homogeneous diploid calli. The heterogeneity in DNA ploidy level between the layers was observed, and it implies that parts of a callus cannot infer about a whole callus. This work suggests that time influences the appearance of different DNA ploidy levels in calli induced in vitro. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-07-17 |
dc.date.available.fl_str_mv |
2013-03-21 2015-03-26T13:42:26Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:42:26Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
SILVA, Thaís Cristina Ribeiro da. Vertical heterogeneity of DNA ploidy in Passiflora cincinnata calluses analyzed by flow cytometry. 2012. 50 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2012. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/4770 |
identifier_str_mv |
SILVA, Thaís Cristina Ribeiro da. Vertical heterogeneity of DNA ploidy in Passiflora cincinnata calluses analyzed by flow cytometry. 2012. 50 f. Dissertação (Mestrado em Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me) - Universidade Federal de Viçosa, Viçosa, 2012. |
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http://locus.ufv.br/handle/123456789/4770 |
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Universidade Federal de Viçosa |
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Mestrado em Genética e Melhoramento |
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UFV |
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BR |
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Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me |
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Universidade Federal de Viçosa |
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