RESUMO
Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (-0.5 to -0.7 MPa), severe (-1.1 to -1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.
Assuntos
Perfilação da Expressão Gênica/normas , Genes de Plantas , Poaceae/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Estresse Fisiológico , Secas , Folhas de Planta/genética , Folhas de Planta/fisiologia , Poaceae/genética , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Fatores de TempoRESUMO
Cotyledons were used as explants in order to test the effect of different vitamin mixtures and BAP concentrations on yellow passion fruit (Passiflora edulis f. flavicarpa Deg.) in vitro direct organogenesis. They basal medium was constituted of MS salts, supplemented with 1 and 2mg. img SRC="http:/img/fbpe/cr/v32n2/a09img01.gif"> -1 of BAP (6-benzilaminopurina). Vitamin mixtures MS and B5 were tested. Every assay was done with 60 explants. The data were analyzed by Tukey's test. The medium supplemented with B5 vitaminic complex induced an increase of the capacity of cotyledons to regenerate in the presence of both BAP concentrations, as well for the number of explants with buds as for the number of buds per explant. The analysis of ploidy level of regenerated plants showed a number of chromosomes apparently normal and typical for this specie (2n=18).
Cotilédones obtidos a partir de sementes germinadas in vitro foram usados como explantes com o objetivo de avaliar o efeito de dois complexos vitamínicos e de duas concentrações de 6-benzilaminopurina (BAP) na organogênese direta do maracujá amarelo (Passiflora edulis f. flavicarpa Deg.). Empregou-se o meio Murashige & Skoog (1962) suplementado com as vitaminas MS ou B5 (vitaminas de GAMBORG et al., 1968) e 1 ou 2 mg. img SRC="http:/img/fbpe/cr/v32n2/a09img01.gif"> -1 de BAP. Foram testados quatro tratamentos, sendo cada um constituído de 60 explantes. O experimento foi inteiramente casualizado, com dez repetições. Os dados, analisados pelo teste de Tukey, demonstraram que os meios de cultura suplementados com o complexo vitamínico B5 induziram gemas em mais de 70% dos explantes, obtendo-se até 8 brotos por explante, com um enraizamento superior a 90%. A análise de ploidia das plantas regeneradas indicou um número aparentemente normal de cromossomos típicos dessa espécie (2n=18)
RESUMO
Cotyledons were used as explants in order to test the effect of different vitamin mixtures and BAP concentrations on yellow passion fruit (Passiflora edulis f. flavicarpa Deg.) in vitro direct organogenesis. They basal medium was constituted of MS salts, supplemented with 1 and 2mg. img SRC="http:/img/fbpe/cr/v32n2/a09img01.gif"> -1 of BAP (6-benzilaminopurina). Vitamin mixtures MS and B5 were tested. Every assay was done with 60 explants. The data were analyzed by Tukey's test. The medium supplemented with B5 vitaminic complex induced an increase of the capacity of cotyledons to regenerate in the presence of both BAP concentrations, as well for the number of explants with buds as for the number of buds per explant. The analysis of ploidy level of regenerated plants showed a number of chromosomes apparently normal and typical for this specie (2n=18).
Cotilédones obtidos a partir de sementes germinadas in vitro foram usados como explantes com o objetivo de avaliar o efeito de dois complexos vitamínicos e de duas concentrações de 6-benzilaminopurina (BAP) na organogênese direta do maracujá amarelo (Passiflora edulis f. flavicarpa Deg.). Empregou-se o meio Murashige & Skoog (1962) suplementado com as vitaminas MS ou B5 (vitaminas de GAMBORG et al., 1968) e 1 ou 2 mg. img SRC="http:/img/fbpe/cr/v32n2/a09img01.gif"> -1 de BAP. Foram testados quatro tratamentos, sendo cada um constituído de 60 explantes. O experimento foi inteiramente casualizado, com dez repetições. Os dados, analisados pelo teste de Tukey, demonstraram que os meios de cultura suplementados com o complexo vitamínico B5 induziram gemas em mais de 70% dos explantes, obtendo-se até 8 brotos por explante, com um enraizamento superior a 90%. A análise de ploidia das plantas regeneradas indicou um número aparentemente normal de cromossomos típicos dessa espécie (2n=18)