CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time.

Piotr T Bednarek,Renata Orłowska

doi:10.3390/ijms21124401

Abstract

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations (‘Cu*Ag’) acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of ‘Cu*Ag’, and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis

Highlights

Studies on in vitro tissue culture-induced variation (TCIV) revealed that plant regeneration requires cell reprogramming and implicate a broad range of genetic and epigenetic modifications [1]
The in vitro anther tissue cultures of barley were performed under nine distinct conditions varying in the Cu2+, Ag+ ion concentrations, and the time of in vitro culture (Table 1) allowed the regeneration of 35 plants from a single donor plant
The metAFLP quantitative characteristics related to demethylation (DMV) of the CG, CHG (H = C, A, and T) sequence contexts, demethylation de novo methylation (DNMV) and sequence variation (SV) were evaluated (Table 1)

Summary

Introduction

Studies on in vitro tissue culture-induced variation (TCIV) revealed that plant regeneration requires cell reprogramming and implicate a broad range of genetic and epigenetic modifications [1]. In this context, DNA methylation (de novo methylation and demethylation) plays a key role for in vitro plant regeneration [2]. De novo methylation is itself under a well-recognized epigenetic control [7] It is the outcome of a common substrate S-adenosyl-L-methionine (SAM) reactivity [8,9] that coordinately with the SAM-dependent DNA methyltransferase transfers methyl group on 5 position of cytosine [10]

Methods

Results

Discussion

Conclusion