Modulation of genetic and androgenic response by copper and silver ions in wheat regenerants (Triticum aestivum L.)

Abstract The embryogenesis of wheat microspores is a process strongly influenced by genotype and in vitro culture conditions, which limits its efficiency in the production of doubled haploids and biotechnological applications. It is known that copper and silver ions used in plant tissue culture affect the efficiency of plant regeneration; however, their combined effect on the efficiency of regeneration and the genetic and epigenetic stability of wheat is poorly understood. The study evaluated the effect of Cu²⁺ and Ag⁺ concentrations and anther incubation time on the green plant regeneration efficiency (GPRE) and tissue culture-induced variation in wheat ( Triticum aestivum L.) anther cultures. Regeneration occurred in nine medium variants with different concentrations of copper sulfate, silver nitrate, and anther incubation time. GPRE and (epi)genetic variation were estimated using metAFLP, determining sequence variation (SV), DNA demethylation (DMV), and de novo methylation (DNMV) in the contexts of CG, CHG, and CHH sequences. GPRE differed significantly among culture treatments, ranging from 8.92 to 52.94 green regenerants per 100 plated anthers. Differences were also detected in metAFLP-derived sequence variation, DNA demethylation, and de novo methylation. Exploratory ANCOVA models indicated associations between culture conditions, metAFLP-derived variation components, and GPRE. Because the molecular dataset comprised 44 regenerants from one genotype and the fitted models included interaction terms, these results should be interpreted as hypothesis-generating rather than confirmatory. Among the fitted models, CHG-associated sequence variation showed the strongest model fit, suggesting that this component may be an informative marker of the androgenic response under the tested conditions, but not evidence of a causal role in regeneration. Significant two- and three-way interactions between Cu²⁺, Ag⁺, anther incubation time, and metAFLP variables demonstrated that GPRE was highly context-dependent.