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      Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine ( Vitis  vinifera L., cv. Mencía)

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          Abstract

          The low induction rates of somatic embryogenesis are one of the main limitations in its routine application in the grapevine ( Vitis vinifera L.). The use of an induction medium containing histone deacetylase inhibitors (trichostatin A and, mainly, sodium butyrate) resulted in an improvement of the embryogenic responses in grapevine (cv. Mencía) cotyledonary and recently germinated somatic embryos. The relative expression of several grapevine genes related to embryogenic competence or encoding histone deacetylase enzymes was studied in cotyledonary somatic embryos that were cultured in the presence of 0.5 mM sodium butyrate. The results showed a significant overexpression of the BBM and VvSERK2 genes after 24 h of culture, whereas the VvWOX2 gene was underexpressed less in treated versus untreated explants. The results suggest that the inhibitor may trigger a molecular response related to an increase in embryogenic competence and changes in the expression of associated genes. The treatment with sodium butyrate also produced significant variations in the expression of several histone deacetylase enzyme-encoding genes. These results may enhance the possibility of obtaining somatic embryos, reducing the seasonal constraints associated with the use of floral explants in grapevines.

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          The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.

          Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader's ability to evaluate critically the quality of the results presented or to repeat the experiments. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.
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            Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR.

            Real-time reverse transcription followed by polymerase chain reaction (RT-PCR) is the most suitable method for the detection and quantification of mRNA. It offers high sensitivity, good reproducibility and a wide quantification range. Today, relative expression is increasingly used, where the expression of a target gene is standardised by a non-regulated reference gene. Several mathematical algorithms have been developed to compute an expression ratio, based on real-time PCR efficiency and the crossing point deviation of an unknown sample versus a control. But all published equations and available models for the calculation of relative expression ratio allow only for the determination of a single transcription difference between one control and one sample. Therefore a new software tool was established, named REST (relative expression software tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group, for reference and up to four target genes. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio results of the four investigated transcripts are tested for significance by a randomisation test. Herein, development and application of REST is explained and the usefulness of relative expression in real-time PCR using REST is discussed. The latest software version of REST and examples for the correct use can be downloaded at http://www.wzw.tum.de/gene-quantification/.
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              Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data

              Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as ‘fold-difference’ results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.
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                Author and article information

                Contributors
                Role: Academic Editor
                Role: Academic Editor
                Journal
                Plants (Basel)
                Plants (Basel)
                plants
                Plants
                MDPI
                2223-7747
                08 June 2021
                June 2021
                : 10
                : 6
                : 1164
                Affiliations
                [1 ]Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain; oscarmtroncoso@ 123456gmail.com (Ó.M.); arjonesgonzalezveronica@ 123456gmail.com (V.A.)
                [2 ]Departamento de Biología Funcional, Universidad de Santiago de Compostela, Campus Sur, 15872 Santiago de Compostela, Spain; mvictoria.gonzalez@ 123456usc.es
                Author notes
                [* ]Correspondence: mrey@ 123456uvigo.es ; Tel.: +34-986812596
                Author information
                https://orcid.org/0000-0001-8170-0022
                https://orcid.org/0000-0002-1445-5538
                Article
                plants-10-01164
                10.3390/plants10061164
                8228518
                34201224
                15822ed1-62d7-40c9-be5d-72c230b7dd0d
                © 2021 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 15 May 2021
                : 04 June 2021
                Categories
                Article

                somatic embryogenesis,vitis vinifera,gene expression,sodium butyrate,trichostatin a,baby boom,serk2,histone deacetylase-encoding genes

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