ULTRASTRUCTURE OF CELL-WALL REGENERATION BY ISOLATED PROTOPLASTS OF PALMARIA-PALMATA (RHODOPHYTA)

Viable protoplasts were isolated from a spontaneous mutant clone of Palmaria palmata (L.) O. Kuntze by enzymatic degradation of the cell walls. Different enzyme mixtures and osmotica were tested for protoplast isolation and viability. Fifteen to 20% of the protoplasts completed cell wall regeneration and survived for longer than one month. Only 1-2% of these surviving cells underwent cell division twice. Changes in cellular organization and cell wall formation were studied by transmission and scanning electron microscopy (SEM) and by cytochemical methods. In the initial stages of cell wall regeneration, endoplasmic reticulum (ER) and Golgi bodies became abundant. Electron-dense bodies began to appear in the cytoplasm of a few cells during enzymatic digestion but before protoplast release. After several hours in culture, similar bodies were observed in all regenerating protoplasts. These bodies were associated with the ER and their contents were later discharged outside the cells. These results suggest that the ER probably produced enzymes that synthesized precursors of cell walls within the electron-dense bodies. The precursors were then transported to the plasma membrane and discharged. Many small electron-dense granules appeared near the plasma membrane prior to cell wall fibril formation. These granules contained neither polysaccharides nor lipids. They might be proteins that were involved somehow in fibril production. After 6 h in culture, cell wall fibrils began to appear unevenly on the cell surfaces, giving a patchy appearance under SEM. A thin layer of wall covered the entire cell surface after 24 h in culture, but the surface was still rough. By 48 h a thick cell wall had formed and the cell surface now appeared smooth with only small irregularities.