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

Abstract

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. 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.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

“…DNA-methylation analysis was performed using bisulfite conversion with the EpiTect Bisulfite Kit (QIAGEN) as described previously [74]. Primers for amplification of P35S region (Additional file 8: Table S1) were designed to anneal on the converted DNA, irrespective of its original methylation state.…”

Annual growth cycle observation, hybridization and forcing culture for improving the ornamental application of Paeonia lactiflora Pall. in the low-latitude regions

“…5-azacytidine is a cytosine analogue that can intercalate into DNA[50,51], leading to the inactivation of methylase enzymes, and is known as a “demethylation/demethylating agent” or a “DNA-hypomethylating molecule”[49,50,52–54]. Therefore, 5-azacytidine could partially replace the effect of cold, potentially promoting the progress of DNA demethylation, accelerating the dormancy release and finally inducing earlier sprouting and/or flowering[47,53,55].…”

Epigenetics for Crop Improvement in Times of Global Change

“…Another group of applications focuses on the fact that the activity of the transgene in genetically transformed tissues is silenced by DNA methylation of the promoter contained in the transgenic construct. Treatment of such silenced transformants with 5-AzaC then caused reactivation of silenced transgenes [ 137 , 138 ]. Due to the commonly discussed role of DNA methylation in the field of priming or in transgenerational memory, treatment with 5-AzaC or Zebularine is also used for deeper understanding of DNA methylation in stress-memory-related phenomena.…”