5-azacytidine improves microspore embryogenesis initiation by decreasing global DNA methylation while it impairs embryo progression in rapeseed and barley

Microspores are reprogrammed by stress treatments in vitro, changing their developmental pathway to embryogenesis; the process, microspore embryogenesis, represents an important tool in plant breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification which changes during plant cell differentiation and proliferation, regulating gene expression. Recently, we have shown modifications in global DNA methylation that accompany the microspore reprogramming to embryogenesis. 5-Azacytidine (AzaC), known analog of 5-cytosine, cannot be methylated and leads to DNA hypomethylation in eukaryotic cells, constituting a useful demethylating agent to study DNA epigenetic dynamics, with potential application to improve the efficiency of microspore embryogenesis. This work analyzes for the first time effects of short and long AzaC treatments on microspore embryogenesis induction and progression in two species, the dicot Brassica napus (rapeseed) and the monocot Hordeum vulgare (barley). Quantitative analyses of pro-embryos formed after induction and embryo production, quantification of global DNA methylation levels, 5mdC immunofluorescence at confocal microscope were performed in 2.5 µM AzaC-treated and untreated cultures. Short AzaC treatments produced significant increases of embryogenesis induction rates, by decreasing global DNA methylation levels and modifying 5mdC distribution patterns. In contrast, longer AzaC treatments dramatically diminished embryo production. Similar effects were found in both species indicating that DNA demethylation favors microspore reprogramming, totipotency acquisition and embryogenesis initiation, while subsequent embryo differentiation requires de novo DNA methylation and is impaired by AzaC. Results give new insights into the role of epigenetic mark dynamics by epigenetic inhibitors, like AzaC, in stress-induced microspore embryogenesis, improving its efficiency in biotechnology and agronomic breeding programs.