Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora

Autores
Souza, M. M.; Urdampilleta, Juan Domingo; Forni Martins, E. R.
Año de publicación
2010
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.
Fil: Souza, M. M.. Universidade Estadual de Santa Cruz. Departamento de Ciências Biológicas; Brasil
Fil: Urdampilleta, Juan Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinar de Biología Vegetal (p). Grupo Vinculado Centro de Relevamiento y Evaluacion de Recursos Agricolas y Naturales; Argentina
Fil: Forni Martins, E. R.. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Biologia Vegetal; Brasil
Materia
PASSION FLOWER
CYTOGENETIC
PECTINEX
FISH
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/14980
Acceder
id CONICETDig_43ef876ede15148f2d81a8a8351f433c
oai_identifier_str oai:ri.conicet.gov.ar:11336/14980
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Improvements in cytological preparations for fluorescent in situ hybridization in PassifloraSouza, M. M.Urdampilleta, Juan DomingoForni Martins, E. R.PASSION FLOWERCYTOGENETICPECTINEXFISHhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.Fil: Souza, M. M.. Universidade Estadual de Santa Cruz. Departamento de Ciências Biológicas; BrasilFil: Urdampilleta, Juan Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinar de Biología Vegetal (p). Grupo Vinculado Centro de Relevamiento y Evaluacion de Recursos Agricolas y Naturales; ArgentinaFil: Forni Martins, E. R.. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Biologia Vegetal; BrasilFundacao de Pesquisas Científicas de Riberao Preto2010-11info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/14980Souza, M. M.; Urdampilleta, Juan Domingo; Forni Martins, E. R.; Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora; Fundacao de Pesquisas Científicas de Riberao Preto; Genetics And Molecular Research; 9; 4; 11-2010; 2148-21551676-5680enginfo:eu-repo/semantics/altIdentifier/url/http://www.funpecrp.com.br/gmr/year2010/vol9-4/pdf/gmr951.pdfinfo:eu-repo/semantics/altIdentifier/doi/10.4238/vol9-4gmr951info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-29T12:11:09Zoai:ri.conicet.gov.ar:11336/14980instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-29 12:11:10.104CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
title Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
spellingShingle Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
Souza, M. M.
PASSION FLOWER
CYTOGENETIC
PECTINEX
FISH
title_short Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
title_full Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
title_fullStr Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
title_full_unstemmed Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
title_sort Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
dc.creator.none.fl_str_mv Souza, M. M.
Urdampilleta, Juan Domingo
Forni Martins, E. R.
author Souza, M. M.
author_facet Souza, M. M.
Urdampilleta, Juan Domingo
Forni Martins, E. R.
author_role author
author2 Urdampilleta, Juan Domingo
Forni Martins, E. R.
author2_role author
author
dc.subject.none.fl_str_mv PASSION FLOWER
CYTOGENETIC
PECTINEX
FISH
topic PASSION FLOWER
CYTOGENETIC
PECTINEX
FISH
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.
Fil: Souza, M. M.. Universidade Estadual de Santa Cruz. Departamento de Ciências Biológicas; Brasil
Fil: Urdampilleta, Juan Domingo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinar de Biología Vegetal (p). Grupo Vinculado Centro de Relevamiento y Evaluacion de Recursos Agricolas y Naturales; Argentina
Fil: Forni Martins, E. R.. Universidade Estadual de Campinas. Instituto de Biologia. Departamento de Biologia Vegetal; Brasil
description Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F₁ progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples.
publishDate 2010
dc.date.none.fl_str_mv 2010-11
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/14980
Souza, M. M.; Urdampilleta, Juan Domingo; Forni Martins, E. R.; Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora; Fundacao de Pesquisas Científicas de Riberao Preto; Genetics And Molecular Research; 9; 4; 11-2010; 2148-2155
1676-5680
url http://hdl.handle.net/11336/14980
identifier_str_mv Souza, M. M.; Urdampilleta, Juan Domingo; Forni Martins, E. R.; Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora; Fundacao de Pesquisas Científicas de Riberao Preto; Genetics And Molecular Research; 9; 4; 11-2010; 2148-2155
1676-5680
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://www.funpecrp.com.br/gmr/year2010/vol9-4/pdf/gmr951.pdf
info:eu-repo/semantics/altIdentifier/doi/10.4238/vol9-4gmr951
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Fundacao de Pesquisas Científicas de Riberao Preto
publisher.none.fl_str_mv Fundacao de Pesquisas Científicas de Riberao Preto
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.10058

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