Using pumpkin as a model to study stress-induced somatic embryogenesis

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium but also on hormone-free medium containing 1 mM ammonium (NH4+) as a sole source of nitrogen. NH4Cl as the sole source of nitrogen permitted the establishment of highly uniform cultures in which preglobular and globular embryos comprised more than 70% of tissue mass. Proembryogenic cell clusters consisted of the small spherical cells with dense cytoplasm and callose-rich cell walls. The domination of preglobular and globular embryos correlated with high level of global DNA methylation. Globular embryos proceeded to maturity only after the re-supply of nitrogen in form of nitrate or L-glutamine and development was followed with a significant increase of global DNA methylation. The activities of antioxidative enzymes superoxide dismutase (SOD) and soluble peroxidase (POD) were high in tissues growing into NH4+ medium, and decreased after re-supply of nitrogen what indicated oxidative stress in the tissue assimilating only NH4+. In addition, NH4+-induced embryogenic tissue was characterized by elevated activity of esterase, the early marker of somatic embryogenesis, what was in accordance with highly proembryogenic state of the culture. Together, results show that the induction and development of embryogenic competence in pumpkin tissue is associated with the oxidative stress induced by nitrogen limitation.