To gain further insight into the role played by sporophytic anther tissues in the early stages of the androgenic process, we have compared the cytology and ultrastructure of barley embryogenic pollen grains obtained by anther culture with those obtained by isolated-microspore culture. The microspores behaved similarly in both culture systems but ultrastructural studies detected a significant difference: the presence of electron-dense deposits on the intine of embryogenic pollen grains generated by isolated-microspore culture compared to their absence in grains generated by anther culture. To discover the nature of these deposits, we applied proteinase K and EDTA treatments to ultrathin sections. We also subjected the deposits to X-ray microanalysis and found that they contained iron. Anthers and isolated microspores were cultured in media containing different concentrations of iron so as to evaluate the presence of these deposits on the intine. Deposits were not found in anther cultures at any iron concentration used or in microspore cultures when concentrations were lower than 40 mg/L. The Fe deposits on the intine appear to derive from an excess of Fe in the isolated-microspore culture medium which, if allowed to pass through the cell wall, could well be toxic to the embryogenic development of the microspores. (C) 2004 Elsevier Inc. All rights reserved.