Cell wall reformation by pollen tube protoplasts of olive (Olea europaea L.): structural comparison with the pollen tube wall

. Mature pollen grains of olive (Olea europaea L.) were germinated in vitro in Brewbaker and Kwack medium, and emerging pollen tubes were then enzymatically digested in the presence of high osmoticum. This treatment resulted in simultaneous degradation of pollen tube walls and fragmentation of their cytoplasm, giving rise to numerous protoplasts of different sizes and different numbers of nuclei. After the protoplasts had been purified, they were cultured in Murashige and Skoog medium supplemented with auxin and cytokinin. The initial steps of cell wall reformation were studied after 12 h and 24 h of culture with a series of cytochemical techniques including periodic acid-Schiff reagent and phosphotungstic acid, as well as with electron microscopy and immunocytochemical techniques using monoclonal antibodies directed against pectins and #-(1̅)-glucan (callose). Among the components of new wall in the protoplasts, callose proved to be the earliest and most abundantly secreted polysaccharide, whereas the deposition of pectins recognized by the antibody JIM7 started several hours later. Pectins that bind JIM5 antibody were not detected in this early stage of development. Cell wall components deposited by protoplasts were compared with those present in growing pollen tubes. Callose secreted by protoplasts formed a relatively thicker layer than that found in the tubes, and pectins recognized by JIM7 were highly abundant, mostly within the cytoplasm and in the apical zone of the tubes.