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Plant Cell Reports

, Volume 36, Issue 8, pp 1311–1322 | Cite as

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level

  • Dimitrij Tyč
  • Eva Nocarová
  • Lenka Sikorová
  • Lukáš FischerEmail author
Original Article

Abstract

Key message

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level.

Abstract

Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Keywords

5-Azacytidine De novo regeneration Methylation Reactivation TGS Transgene silencing 

Abbreviations

AzaC

5-Azacytidine

GFP

Green fluorescent protein

NPTII

Neomycin phosphotransferase

PTGS and TGS

(Post)transcriptional gene silencing

Notes

Acknowledgements

We thank Filip Vomáčka for English correction. This work was supported by the project LO1417 of the Ministry of Education, Youth and Sports of the Czech Republic.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

299_2017_2155_MOESM1_ESM.docx (17 kb)
Online Resource 1 List of primers used for methylation and qRT PCR analysis. (DOCX 16 kb)
299_2017_2155_MOESM2_ESM.tif (1.7 mb)
Online Resource 2 Reactivation of GFP in potato calli after the AzaC treatment. Leaf segments (including petioles) from in vitro grown potato lines R17 and R28 with the silenced expression of GFP and nptII genes were cultured for 3 days on a callus-inducing medium and then for 4 days on the same medium with or without 10 μM AzaC. Thereafter the explants were transferred on a shoot-inducing medium with 50 mg/l kanamycin. (a, b) Reactivation of GFP expression in individual dedifferentiated cells of R17 line after 3 days of AzaC treatment; (c) Detail of R17 leaf petiole 10 days after AzaC treatment (arrow indicates a callus with GFP fluorescence) and (d) control R17 petiole without AzaC treatment; (e–h) gradual proliferation of selected reactivated cell/callus of R28 line (e) just after AzaC treatment, (f) 6 days, (g) 14 days and (h) 72 days after AzaC treatment. (a-d) fluorescence captured with the FITC filter set: bright green fluorescence of GFP, red fluorescence of chlorophyll and yellowish fluorescence of damaged and dead cells; (e-h) fluorescence with the EGFP filter set. Scale bars: 100 μm in (a, e, f), 200 μm in (b), 400 μm in (g, h), 500 μm in (c, d). (TIFF 1707 kb)
299_2017_2155_MOESM3_ESM.tif (1.6 mb)
Online Resource 3 GFP fluorescence in shoots of selected potato lines. Shoots of in vitro grown plants of (a, e) untransformed Désirée; (b, f) spontaneously silenced line R28; (c, g) line R17A with reactivated GFP and nptII; (d, h) line R28A with reactivated nptII only. Images were captured with (a-d) EGFP filter set and in (e-h) bright field. Scale bar: 3 mm. (TIFF 1680 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Plant Experimental Biology, Faculty of ScienceCharles UniversityPrague 2Czech Republic

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