Artículos de revistas
Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora
Registro en:
Genetics And Molecular Research. Funpec-editora, v. 9, n. 4, n. 2148, n. 2155, 2010.
1676-5680
WOS:000284324400016
10.4238/vol9-4gmr951
Autor
Souza, MM
Urdampilleta, JD
Forni-Martins, ER
Institución
Resumen
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 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 F(1) 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 (R) 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. 9 4 2148 2155 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Universidade Estadual de Santa Cruz (UESC) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Programa de Pos-Graduacao em Producao Vegetal (PPGPV/UESC) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)