Genotoxic effects due to in vitro culture and H2O2 treatments in Petunia x hybrida cells monitored through DNA diffusion assay, FPG-SCGE and gene expression profile analyses

In the present work, Petunia × hybrida leaf discs maintained on regeneration medium for 8 days were used to assess the effects of genotoxic stress induced by in vitro culture. The investigation was carried out by comparing the response of intact leaves excised from Petunia × hybrida plantlets grown in vitro and the regenerating leaf discs. In situ detection by histochemical staining and alkaline-Single Cell Gel Electrophoresis (SCGE) analysis demonstrated that both reactive oxygen species accumulation and DNA damage were enhanced in explants cultured in vitro. Significant up-regulation of the PhOGG1 (8-oxoguanine DNA glycosylase/lyase), PhAPX (ascorbate peroxidase) and PhMT2 (metallothionein) genes involved in DNA repair and antioxidant defence was observed in the explants cultured in vitro, compared to intact leaves. The Petunia × hybrida leaf discs were exposed to increasing (0, 100, 150, 200 and 400 mM) doses of the model genotoxic agent hydrogen peroxide (H₂O₂) and then analysed. The DNA diffusion assay highlighted the dose- and time-dependent fluctuations of programmed cell death/necrosis events in response to H₂O₂. Leaf discs treated with increasing H₂O₂ concentration and untreated controls were analysed by FPG-SCGE to assess the level of oxidative DNA damage at different time points following treatments. The PhOGG1, PhAPX and PhMT2 genes were significantly up-regulated in response to H₂O₂, reaching the highest transcript levels with the 150 mM dose. Based on the reported data, these genes might be used as molecular indicators of the genotoxic stress response in Petunia × hybrida cells.