Abstract
Tissue culture is an essential tool for the regeneration of uniform plant material. However, tissue culture conditions can be a source of abiotic stress for plants, leading to changes in the DNA sequence and methylation patterns. Despite the growing evidence on biochemical processes affected by abiotic stresses, how these altered biochemical processes affect DNA sequence and methylation patterns remains largely unknown. In this study, the methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach was used to investigate de novo methylation, demethylation, and sequence variation in barley regenerants derived by anther culture. Additionally, we used Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy to identify the spectral features of regenerants, which were then analyzed by mediation analysis. The infrared spectrum ranges (710–690 and 1010–940 cm−1) identified as significant in the mediation analysis were most likely related to β-glucans, cellulose, and S-adenosyl-L-methionine (SAM). Additionally, the identified compounds participated as predictors in moderated mediation analysis, explaining the role of demethylation of CHG sites (CHG_DMV) in in vitro tissue culture-induced sequence variation, depending on the duration of tissue culture. The data demonstrate that ATR-FTIR spectroscopy is a useful tool for studying the biochemical compounds that may affect DNA methylation patterns and sequence variation, if combined with quantitative characteristics determined using metAFLP molecular markers and mediation analysis. The role of β-glucans, cellulose, and SAM in DNA methylation, and in cell wall, mitochondria, and signaling, are discussed to highlight the putative cellular mechanisms involved in sequence variation.
Highlights
It is commonly believed that sequence variation during in vitro tissue culture is either caused by the activation of transposons [1], resulting in DNA demethylation [2] during cell reprogramming [3], or by the presence of modified cytosines [4], which undergo further modifications [5] but escape DNA repair mechanisms [6]
The 710–690 cm−1 band region is putatively associated with cellulose, whereas another band at approximately 665 cm−1 was detected in the spectra of the examined material [47,48]
Mediation analysis did not show a significant effect of the 665 cm−1 band, possibly because of the presence of cellulose in dissimilar forms and/or at different crystallinity levels, generating separate peaks
Summary
It is commonly believed that sequence variation during in vitro tissue culture is either caused by the activation of transposons [1], resulting in DNA demethylation [2] during cell reprogramming [3], or by the presence of modified cytosines [4], which undergo further modifications [5] but escape DNA repair mechanisms [6]. DNA demethylation occurs in two sequence contexts: symmetric and asymmetric [7]. Symmetric DNA demethylation affects CG and CHG sequences (where H = A, T, and C) predominantly in gene-rich euchromatic regions, whereas asymmetric DNA demethylation affects CHH sequences mostly in heterochromatic regions. The maintenance of both types of DNA methylation patterns is regulated by distinct mechanisms [7]. Two mechanisms of DNA demethylation have been identified: passive and active. Passive demethylation has been reported in the male gametophyte and endosperm with reduced RNA-directed DNA methylation (RdDM) factors [9]
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