Análise da metilação no DNA e acúmulo de auxina durante a indução da embriogênese somática em Eucalyptus grandis

Abstract

Somatic embryogenesis (SE) is a process of great importance for clonal micropropagation in vitro, presenting benefits when applied to tree species,especially for the Eucalyptus species, since its regeneration by somatic embryos is recalcitrant. It has been proposed that during SE, de-differentiation of target cells and acquisition of embryogenic competence can be modulated by DNA methylation and by phytohormones, such as picloram.The objectives of this study was (1) to analyze the effect of the 5-Azacytidine (5-AzaC) DNA methylation inhibitor on the induction of calogenesis in Eucalyptus grandis by testing different concentrations of the demethylating agent, and (2) to identify early process markers of SE, through the accumulation of auxin and DNA methylation in the cells, testing different concentrations of auxin picloram. For the experiments, hypocotyls were used after 20 days of germination. The results showed that the concentration of 0.5 μM of the 5- AzaC demethylating agent was more efficient in promoting hypomethylation of the overall DNA of the calli without causing cellular toxicity to the explants, promoting a reduction of 76.5% in methylation levels after 60 days of exposure to 5-AzaC. The concentration of 40 μM picloram was more efficient in the induction of embryogenic callus, with 19.80% of friable callus formation at 60 days, and it was possible to visualize, at 180 days, the first groupings of cell divisions, giving rise to the proembryos.The results revealed that auxin accumulation and DNA hypermethylation were induced during the early stages of SE, differently found in embryogenic cells of the callus, whereas they were not present in non-embryogenic cells after the onset of SE induction.It assumes that these factors represent processes that can be interconnected and involved in the regulation of cellular reprogramming.