Multiploidia em calos provenientes de anteras de tomate
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/123456789/4771 |
Resumo: | The anther culture has been applied in tomato as an attempt to induce callogenesis followed by regeneration of haploid and doubled haploid plants. By this technique, homozygous lines can be obtained, which has been considered relevant to breeding programs and molecular techniques. The androgenetic response can be influenced by several factors, especially the development stage of microsporogenesis. In this perspective, this study adapted a protocol by associating of flow cytometry and cytogenetic techniques in order to relate the development stages of microsporogenesis in tomato anthers to their size, aiming to select anthers profiles to be inoculated into culture medium. After a flow cytometry protocol was adapted for calli obtained in vitro, in order to monitor the DNA ploidy level. The nuclear suspensions analyzed by flow cytometry were obtained from digestion of anther cells and further homogenization using a mini mixer. The histograms showed low CV s, being considered suitable for cytometric analysis. Thus, anthers small (0.5 0.7 mm), showing cells in interphase/prophase I, exhibited a histogram profile similar to that observed in diploid leaf, and anthers large (1.6 1.9 mm), containing tetrad and microspores, showed an haploid peak and a G2 peak enlarged due to the presence of tetrads. From these analyzes, anthers of flower buds 1 5 mm, with size corresponding to those analyzed by flow cytometry and cytogenetics (0.5 1.9 mm), were inoculated in the callogenesis induction medium, to identify the phases more responsive to the condition in vitro. The basal MS medium supplemented with 2 mg L-1 AIA and 1 mg L-1 2 ip, induced 21.7% of callogenesis, and the highest percentage of calli was obtained from anthers of flower buds 2.0 3.9 mm, corresponding to the phases prophase I to anaphase II. The calli were analyzed by flow cytometry to identify the DNA ploidy. The chopping performed on calli afforded the nuclei isolation, and histograms with CV s considered suitable were obtained. The cytometric analysis revealed that the all calli were multiploids, each of which presented one of five classes of DNA ploidy levels, namely: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. A total of 44.4% of calli analyzed showed five levels of DNA ploidy. A statistical test of interaction showed that the size of flower buds had no effect on polyploidization occurred in the calli. These data evidence the somaclonal variation occurrence, which, probably, results from the interaction of genotype with growth regulators added to the induction medium. |
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Julião, Sirlei Aparecidahttp://lattes.cnpq.br/3011849999892572Koehler, Andréa Diashttp://lattes.cnpq.br/2563890461457295Cruz, Cosme Damiãohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788274A6Carvalho, Carlos Roberto dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4780878T0Motoike, Sérgio Yoshimitsuhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728221T8Clarindo, Wellington Ronildohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H92015-03-26T13:42:26Z2013-03-262015-03-26T13:42:26Z2012-07-16JULIÃO, Sirlei Aparecida. Multiploidy in calli from tomato anther. 2012. 63 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/123456789/4771The anther culture has been applied in tomato as an attempt to induce callogenesis followed by regeneration of haploid and doubled haploid plants. By this technique, homozygous lines can be obtained, which has been considered relevant to breeding programs and molecular techniques. The androgenetic response can be influenced by several factors, especially the development stage of microsporogenesis. In this perspective, this study adapted a protocol by associating of flow cytometry and cytogenetic techniques in order to relate the development stages of microsporogenesis in tomato anthers to their size, aiming to select anthers profiles to be inoculated into culture medium. After a flow cytometry protocol was adapted for calli obtained in vitro, in order to monitor the DNA ploidy level. The nuclear suspensions analyzed by flow cytometry were obtained from digestion of anther cells and further homogenization using a mini mixer. The histograms showed low CV s, being considered suitable for cytometric analysis. Thus, anthers small (0.5 0.7 mm), showing cells in interphase/prophase I, exhibited a histogram profile similar to that observed in diploid leaf, and anthers large (1.6 1.9 mm), containing tetrad and microspores, showed an haploid peak and a G2 peak enlarged due to the presence of tetrads. From these analyzes, anthers of flower buds 1 5 mm, with size corresponding to those analyzed by flow cytometry and cytogenetics (0.5 1.9 mm), were inoculated in the callogenesis induction medium, to identify the phases more responsive to the condition in vitro. The basal MS medium supplemented with 2 mg L-1 AIA and 1 mg L-1 2 ip, induced 21.7% of callogenesis, and the highest percentage of calli was obtained from anthers of flower buds 2.0 3.9 mm, corresponding to the phases prophase I to anaphase II. The calli were analyzed by flow cytometry to identify the DNA ploidy. The chopping performed on calli afforded the nuclei isolation, and histograms with CV s considered suitable were obtained. The cytometric analysis revealed that the all calli were multiploids, each of which presented one of five classes of DNA ploidy levels, namely: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. A total of 44.4% of calli analyzed showed five levels of DNA ploidy. A statistical test of interaction showed that the size of flower buds had no effect on polyploidization occurred in the calli. These data evidence the somaclonal variation occurrence, which, probably, results from the interaction of genotype with growth regulators added to the induction medium.A cultura de anteras tem sido aplicada em tomate como tentativa à indução de calogênese seguida da regeneração de plantas haplóides e duplo-haplóides. Por meio desta técnica, linhas homozigóticas podem ser obtidas, o que tem sido considerado relevante para programas de melhoramento e técnicas moleculares. A resposta androgenética pode ser influenciada por diversos fatores, destacando-se, dentre eles, o estádio de desenvolvimento da microsporogênese. Nesta perspectiva, este estudo adaptou um protocolo associando técnicas de citometria de fluxo e citogenética para relacionar as fases de desenvolvimento da microsporogênese em anteras de tomate com o tamanho das mesmas. O objetivo desta associação foi selecionar perfis de anteras a serem inoculadas em meio de cultura. Posteriormente, adaptou-se um protocolo de citometria de fluxo para os calos obtidos in vitro, a fim de monitorar o nível de ploidia de DNA. As suspensões nucleares analisadas pela citometria de fluxo foram obtidas com digestão das anteras e posterior homogeneização das células usando um mini mixer. Os histogramas obtidos mostraram baixos CV s, sendo considerados adequados para a análise citométrica. Dessa forma, anteras pequenas (0,5 0,7 mm), na fase de intérfase/prófase I, apresentaram um perfil de histograma semelhante ao observado em folha diplóide, e anteras grandes (1,6 1,9 mm) na fase de tétrades e micrósporos apresentaram um pico haplóide e um aumento em G2 em função da presença de tétrades. A partir destas análises, as anteras de botões florais de 1 5 mm, com tamanho correspondente aos daquelas analisadas por citometria de fluxo e citogenética (0,5 1,9 mm) foram inoculadas em meio de indução de calogênese, visando identificar as fases mais responsivas a esta condição in vitro. O meio MS basal, suplementado com 2 mg L-1 de AIA e 1 mg L-1 de 2 ip, induziu 21,7% de calogênese, e o maior percentual de calos foi obtido a partir de anteras de botões florais com 2,0 3,9 mm, correspondendo às fases prófase I a anáfase II. Os calos foram analisados pela citometria de fluxo a fim de identificar a ploidia de DNA. O chopping realizado nos calos proporcionou o isolamento dos núcleos, e histogramas com CV s considerados adequados foram obtidos. A análise citométrica evidenciou que todos os calos eram multiplóides, sendo que cada um apresentou uma das cinco classes de níveis de ploidia de DNA, a saber: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. Um total de 44,4% dos calos analisados apresentaram cinco níveis de ploidia de DNA. Um teste estatístico de interação mostrou que o tamanho dos botões florais não influenciou na poliploidização ocorrida nos calos. Estes dados evidenciam a ocorrência de variação somaclonal que, provavelmente, é resultado da interação do genótipo com os reguladores de crescimento acrescidos ao meio de indução.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; MeCultura de anterasMeiócitosCalosPloidia de DNACitometria de fluxoTomateAnther cultureMeiocytesCalos, DNA PloidyFlow cytometryTomatoCNPQ::CIENCIAS BIOLOGICAS::GENETICAMultiploidia em calos provenientes de anteras de tomateMultiploidy in calli from tomato antherinfo: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/pdf1076965https://locus.ufv.br//bitstream/123456789/4771/1/texto%20completo.pdf39f371186cedd4587ea0ad27b341a26bMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain102475https://locus.ufv.br//bitstream/123456789/4771/2/texto%20completo.pdf.txt94e036f20effdaac3652aa0122e93e4dMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3628https://locus.ufv.br//bitstream/123456789/4771/3/texto%20completo.pdf.jpgdfd608a7234bbe20ea5784ed3fb5a113MD53123456789/47712016-04-10 23:04:07.092oai:locus.ufv.br:123456789/4771Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-11T02:04:07LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
Multiploidia em calos provenientes de anteras de tomate |
dc.title.alternative.eng.fl_str_mv |
Multiploidy in calli from tomato anther |
title |
Multiploidia em calos provenientes de anteras de tomate |
spellingShingle |
Multiploidia em calos provenientes de anteras de tomate Julião, Sirlei Aparecida Cultura de anteras Meiócitos Calos Ploidia de DNA Citometria de fluxo Tomate Anther culture Meiocytes Calos, DNA Ploidy Flow cytometry Tomato CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
title_short |
Multiploidia em calos provenientes de anteras de tomate |
title_full |
Multiploidia em calos provenientes de anteras de tomate |
title_fullStr |
Multiploidia em calos provenientes de anteras de tomate |
title_full_unstemmed |
Multiploidia em calos provenientes de anteras de tomate |
title_sort |
Multiploidia em calos provenientes de anteras de tomate |
author |
Julião, Sirlei Aparecida |
author_facet |
Julião, Sirlei Aparecida |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/3011849999892572 |
dc.contributor.author.fl_str_mv |
Julião, Sirlei Aparecida |
dc.contributor.advisor-co1.fl_str_mv |
Koehler, Andréa Dias |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/2563890461457295 |
dc.contributor.advisor-co2.fl_str_mv |
Cruz, Cosme Damião |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788274A6 |
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 |
Motoike, Sérgio Yoshimitsu |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4728221T8 |
dc.contributor.referee2.fl_str_mv |
Clarindo, Wellington Ronildo |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4702819H9 |
contributor_str_mv |
Koehler, Andréa Dias Cruz, Cosme Damião Carvalho, Carlos Roberto de Motoike, Sérgio Yoshimitsu Clarindo, Wellington Ronildo |
dc.subject.por.fl_str_mv |
Cultura de anteras Meiócitos Calos Ploidia de DNA Citometria de fluxo Tomate |
topic |
Cultura de anteras Meiócitos Calos Ploidia de DNA Citometria de fluxo Tomate Anther culture Meiocytes Calos, DNA Ploidy Flow cytometry Tomato CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
dc.subject.eng.fl_str_mv |
Anther culture Meiocytes Calos, DNA Ploidy Flow cytometry Tomato |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::GENETICA |
description |
The anther culture has been applied in tomato as an attempt to induce callogenesis followed by regeneration of haploid and doubled haploid plants. By this technique, homozygous lines can be obtained, which has been considered relevant to breeding programs and molecular techniques. The androgenetic response can be influenced by several factors, especially the development stage of microsporogenesis. In this perspective, this study adapted a protocol by associating of flow cytometry and cytogenetic techniques in order to relate the development stages of microsporogenesis in tomato anthers to their size, aiming to select anthers profiles to be inoculated into culture medium. After a flow cytometry protocol was adapted for calli obtained in vitro, in order to monitor the DNA ploidy level. The nuclear suspensions analyzed by flow cytometry were obtained from digestion of anther cells and further homogenization using a mini mixer. The histograms showed low CV s, being considered suitable for cytometric analysis. Thus, anthers small (0.5 0.7 mm), showing cells in interphase/prophase I, exhibited a histogram profile similar to that observed in diploid leaf, and anthers large (1.6 1.9 mm), containing tetrad and microspores, showed an haploid peak and a G2 peak enlarged due to the presence of tetrads. From these analyzes, anthers of flower buds 1 5 mm, with size corresponding to those analyzed by flow cytometry and cytogenetics (0.5 1.9 mm), were inoculated in the callogenesis induction medium, to identify the phases more responsive to the condition in vitro. The basal MS medium supplemented with 2 mg L-1 AIA and 1 mg L-1 2 ip, induced 21.7% of callogenesis, and the highest percentage of calli was obtained from anthers of flower buds 2.0 3.9 mm, corresponding to the phases prophase I to anaphase II. The calli were analyzed by flow cytometry to identify the DNA ploidy. The chopping performed on calli afforded the nuclei isolation, and histograms with CV s considered suitable were obtained. The cytometric analysis revealed that the all calli were multiploids, each of which presented one of five classes of DNA ploidy levels, namely: 2C-4C-8C-16C; 2C-4C-8C-16C-32C; 4C-8C; 4C-8C-16C; 8C-16C-32C. A total of 44.4% of calli analyzed showed five levels of DNA ploidy. A statistical test of interaction showed that the size of flower buds had no effect on polyploidization occurred in the calli. These data evidence the somaclonal variation occurrence, which, probably, results from the interaction of genotype with growth regulators added to the induction medium. |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-07-16 |
dc.date.available.fl_str_mv |
2013-03-26 2015-03-26T13:42:26Z |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:42:26Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
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masterThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
JULIÃO, Sirlei Aparecida. Multiploidy in calli from tomato anther. 2012. 63 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/123456789/4771 |
identifier_str_mv |
JULIÃO, Sirlei Aparecida. Multiploidy in calli from tomato anther. 2012. 63 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/123456789/4771 |
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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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