Time of In Vitro Anther Culture May Moderate Action of Copper and Silver Ions that Affect the Relationship between DNA Methylation Change and the Yield of Barley Green Regenerants

Plant anther culture allows for the regeneration of uniform and homozygous double haploids. However, off-type regenerants may appear as a result of so-called tissue culture-induced variation (TCIV). In addition, the presence of Cu(2+)and Ag(+)ions in the culture medium might influence the number of green plants. The regenerants were obtained via anther cultures of barley under varying Cu(2+)and Ag(+)ion concentrations in the induction medium during distinct time conditions. DArTseqMet markers were evaluated based on regenerants and donor plants and delivering data on DNA demethylation (DM) and de novo methylation (DNM) and changes in methylation (Delta). The number of green regenerated plants per 100 anthers (GPs) was evaluated. The Cu(2+)and Ag(+)ion concentrations moderated relationships between Delta and the number of green plants conditional on time of tissue cultures. Depending on the ions, moderated moderation is valid within the different time of anther culture. When the highest concentration of copper is analyzed, plant regeneration is possible under short 'Time' (21 days) of anther culture wherein Delta is negative or under elongated Time when Delta is positive. Under 21 days of culture, the highest concentration of silver ions and when Delta is negative, some regenerants could be evaluated. However, under high Ag(+)concentration when Time of culture is long and Delta positive, the highest number of green plants could be obtained.