Cellular Approach to the Study of Androgenesis in Maize Anthers: Immunocytochemical Evidence of the Involvement of the Ubiquitin Degradative Pathway in Androgenesis Induction

Maize anthers in the late-uninucleate stage, corresponding to a DH 109 x ZK 191/a F 1 hybrid line, were induced to produce embryoids by cold pre-treatment and incubation onto a modified YP medium. LM observation of sections of the anthers showed the existence of bicellular microspores two days after starting the culture. Several types of multicellular microspores (MCM) and multinucleate microspores (MNM) appeared from the fifth day after culture onwards, together with many degenerated, non-induced microspores. This abundance of forms reveals the presence of different patterns of division and development. Ultrastructural observation of these cellular forms showed particular features, such as the presence of numerous nuclear pores in early stages of induction and the formation of a thick independent cell wall underneath the microspore intine. A certain degree of polarity in these structures was also frequently observed. Proembryoids appeared after 10–13 days of culture, whereas embryo-like structures were observed several days later (16 days of culture). Immunocytochemical studies were carried out on semithin sections of the anthers using a commercially available polyclonal antibody to ubiquitin. Slight labeling was consistently found in most cell types, excluding degenerated and non-induced microspores. The intensity of the labeling was found to be substantially greater in putative androgenic MCM. A model for the involvement of the ubiquitin-mediated degradation pathway in pollen cell cycle control and androgenesis induction is proposed.