Developmental processes during in vitro culture are influenced by many environmental factors, which are required to start regeneration programs encoded in the genome. Isolation of explants and their in vitro culture might cause oxidative imbalance in cells. The aim of the experiment was to determine the effects of osmotic potential of medium on the in vitro regeneration focusing on the oxidative stress. Regenerating callus of Triticum aestivum and Vicia faba ssp. minor were obtained from immature embryos and non-regenerating from mature embryos. Callus after four week induction was placed for three weeks on the media with 3% sucrose (ok. -0.42 MPA), 6% sucrose (ok. -0.71 MPA), and 3% sucrose together with 7% mannitol (ok. -1.33 MPA). After that, the amount of endogenous H₂O₂ and activity of SOD, CAT and POD were measured during the subsequent days of callus culture. 6% sucrose inhibited regeneration of wheat callus, and addition of 7% mannitol stimulated shoot regeneration in comparison with 3% sucrose, whereas did not change regeneration potential of field bean callus. Regenerating callus of wheat accumulated less endogenous H₂O₂ than nonregenerating one and showed a higher activity of SOD, CAT and POD. Regenerating callus of field bean showed a higher amount of endogenous H₂O₂ and antioxidative enzymes. The results of the experiments indicated that the oxidative stress caused by changes of osmotic potential of medium and type of carbohydrates play an important role in regulation of regeneration processes. The activities of antioxidative enzymes and endogenous level of H₂O₂ can be used in prognosis for the ability of tissues to differentiate and regenerate.
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