Abstract
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 Cu2+ 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 Cu2+ 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 Cu2+ 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.
Highlights
Plant regeneration via anther cultures requires precise tuning of the in vitro tissue culture conditions, including the concentration of ingredients that may influence cellular processes promoting plant regeneration
We have shown that under varying conditions of Cu2+, Ag+ and time of in vitro tissue cultures differences in DNA methylation change may appear [35]
In vitro anther tissue cultures performed under nine distinct conditions varying in the Cu2+, Ag+ ion concentrations and time (Time) allowed the regeneration of 35 plants
Summary
Plant regeneration via anther cultures requires precise tuning of the in vitro tissue culture conditions, including the concentration of ingredients (i.e., the balanced concentration of ions) that may influence cellular processes promoting plant regeneration. Such ingredients may encourage the induction of cell reprogramming [1,2,3,4] and potentially change the balance of biochemical pathways. The addition of Cu2+ ions may affect mitochondrial Complex IV [5] belonging to the electron transport chain and is involved in the copper-delivery pathway and creates functional ethylene receptors [6]. Copper ions are responsible for generation of hydroxyl radicals, which may attack the DNA bases in a site-specific manner [10,11]
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