DNA methylation changes and TE activity induced in tissue cultures of barley (Hordeum vulgare L.)

Abstract

BackgroundIn vitro plant regeneration via androgenesis or somatic embryogenesis is capable of inducing (epi)mutations that may affect sexual progenies. While epimutations are associated with DNA methylation, mutations could be due to the movement of transposons. The common notion is that both processes are linked. It is being assumed that demethylation activates transposable elements (TEs). Analysis of methylation changes and their relation with TEs activation in tissue cultures requires uniquely derived donor plants (Ds), their regenerants (Rs) and respective progeny (Ps) that would allow discrimination of processes not related to changes introduced via in vitro cultures. Moreover, a set of methods (RP-HPLC, SSAP, and MSTD) is needed to study whether different TEs families are being activated during in vitro tissue culture plant regeneration and whether their activity could be linked to DNA methylation changes or alternative explanations should be considered.ResultsThe in vitro tissue culture plant regeneration in barley was responsible for the induction of DNA methylation in regenerants and conservation of the methylation level in the progeny as shown by the RP-HPLC approach. No difference between andro- and embryo-derived Rs and Ps was observed. The SSAP and MSTD approach revealed that Ds and Rs were more polymorphic than Ps. Moreover, Rs individuals exhibited more polymorphisms with the MSTD than SSAP approach. The differences between Ds, Rs and Ps were also evaluated via ANOVA and AMOVA.ConclusionsStressful conditions during plant regeneration via in vitro tissue cultures affect regenerants and their sexual progeny leading to an increase in global DNA methylation of Rs and Ps compared to Ds in barley. The increased methylation level noted among regenerants remains unchanged in the Ps as indicated via RP-HPLC data. Marker-based experiments suggest that TEs are activated via in vitro tissue cultures and that, independently of the increased methylation, their activity in Rs is greater than in Ps. Thus, the increased methylation level may not correspond to the stabilization of TEs movement at least at the level of regenerants. The presence of TEs variation among Ds that were genetically and epigenetically uniform may suggest that at least some mobile elements may be active, and they may mask variation related to tissue cultures. Thus, tissue cultures may activate some TEs whereas the others remain intact, or their level of movement is changed. Finally, we suggest that sexual reproduction may be responsible for the stabilization of TEs.

Precise evaluation of tissue culture-induced variation during optimisation of in vitro regeneration regime in barley

“…One of the hypotheses of tissue culture-induced variation considers that sequence changes may be due to DNA demethylation and activation of mobile elements (Azman et al 2014;Orłowska et al 2016;Xu et al 2004). Our data indicate that DNA methylation/demethylation in barley affects symmetric sites and depends on the trial.…”

“…It is assumed that variation induced in regenerants may be partly inherited by generative progeny (Han et al 2018). Thus, regenerants subjected to generative cycle(s) should produce more uniform plants than regenerants (Machczyńska et al 2014b(Machczyńska et al , 2015Orłowska et al 2016Orłowska et al , 2020. Our recent studies demonstrated that, depending on the species and the number of generative cycles, the genome stabilisation level may vary (Machczyńska et al 2014a).…”

“…Regenerated barley plants displayed increased DNA methylation levels compared with donor plants (source of explants), whereas regenerated triticale plants displayed reduced DNA methylation within analysed restriction sites. Methylation changes were linked to the activation of transposons that might be responsible for genetic variation (Gimenez et al 2016;Orłowska et al 2016). Plants regenerated in vitro from somatic tissues also display numerous (epi)mutations (Bednarek and Orłowska 2019) as indicated in triticale (Machczyńska et al 2014b), although those changes did not appear to affect plant regenerants at the morphological level (Machczyńska et al 2014b(Machczyńska et al , 2015.…”

“…The alterations in DNA methylation patterns were species-specific. In barley, the DNA methylation level increased in regenerant plants and remained unchanged during a generative cycle (Orłowska et al 2016). Reduced DNA methylation was observed in triticale regenerants, and it continued decreasing for the next few generative cycles before slightly increasing (Machczyńska et al 2014a).…”

“…Reduced DNA methylation was observed in triticale regenerants, and it continued decreasing for the next few generative cycles before slightly increasing (Machczyńska et al 2014a). Thus, tissue culture-induced variation at the molecular level can be transmitted to progeny (Rosato et al 2016), and several generative cycles may or may not stabilise it (Machczyńska et al 2014a;Orłowska et al 2016). Further studies are needed to determine whether regenerant variation can be controlled.…”