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5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels

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Abstract

DNA methylation is an epigenetic regulatory mechanism of gene expression which can be associated with developmental phases and in vitro morphogenetic competence in plants. The present work evaluated the effects of 5-azacytidine (AzaC) and 2,4-dichlorophenoxyacetic acid (2,4-D) on Acca sellowiana somatic embryogenesis (SE) and global DNA methylation levels by high-performance liquid chromatography mass spectrometry (HPLC/MS/MS). 2,4-D-free treatments revealed no somatic embryo formation in both accessions tested. Treatments supplemented with 2,4-D pulse plus AzaC in the culture medium resulted in increased embryo formation. In AzaC-free treatment, HPLC/MS/MS analysis showed a gradual increase in methylation levels in cultures of both accessions tested during SE induction. Treatment with AzaC and 2,4-D-free resulted in a marked decrease in methylation for both accessions, ranging from 37.6 to 20.8 %. In treatment with 2,4-D and AzaC combined, the 85 accession showed increasing global methylation levels. Otherwise, the 101X458 accession, in the same treatment, showed a decrease between 10 and 20 days, followed by an increase after 30 days (39.5, 36.2 and 41.6 %). These results indicate that 2,4-D pulse combined with AzaC improves SE induction. However, the conversion phase showed that although positively influencing SE induction, AzaC had a dysregulatory effect on the stage of autotrophic plant formation, resulting in significantly lower conversion rates. The results suggest that DNA methylation dramatically influences SE in Acca sellowiana, and global DNA methylation dynamics are related to morphogenetic response.

Key message 5-Azacytidine combined with 2,4-D increases the number of Acca sellowiana somatic embryos. Global DNA methylation is directly affected by these compounds.

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Acknowledgments

The authors thank Prof. Faruk José Nome Aguilera for helping in the mass spectrometry analysis. This work was supported by CNPq, FAPESC and CAPES.

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Authors and Affiliations

  1. Graduate Program in Plant Genetic Resources, Plant Developmental Physiology and Genetics Laboratory, Federal University of Santa Catarina, Rod. Admar Gonzaga, Km 3, Florianópolis, SC, 88034-001, Brazil

    Hugo P. F. Fraga, Leila N. Vieira, Clarissa A. Caprestano, Rosete Pescador & Miguel P. Guerra

  2. Biosomatica Institute, Holambra, SP, 13825-000, Brazil

    Douglas A. Steinmacher

  3. Department of Chemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil

    Gustavo A. Micke & Daniel A. Spudeit

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  1. Hugo P. F. Fraga
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  2. Leila N. Vieira
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Correspondence to Miguel P. Guerra.

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Communicated by L. Jouanin.

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Fraga, H.P.F., Vieira, L.N., Caprestano, C.A. et al. 5-Azacytidine combined with 2,4-D improves somatic embryogenesis of Acca sellowiana (O. Berg) Burret by means of changes in global DNA methylation levels. Plant Cell Rep 31, 2165–2176 (2012). https://doi.org/10.1007/s00299-012-1327-8

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