CELL STRUCTURE AND FUNCTION 2, 249-255 (1977) C, by Japan Society for Cell Biology Preparation and Culture of Spirogyra and Zygnema Protoplasts Tateshige Ohiwa Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan ABSTRACT. Protoplasts were enzymatically released from Spirogyra and Zygnema. The released protoplasts cultivated in liquid media regenerated a wall, grew in filamentous fashion and usually divided with an incomplete septum. When the media osmolarity was gradually reduced, the regenerated cells grew further. These regenerated cells appeared to be the same as the original cells. Although enzymatic procedures have been established for producing protoplasts from various higher plants and yeasts, experiments with eukaryotic algae are limited. Protoplasts have not been obtained from Spirogyra and Zygnema. Diverse enzymatic methods were developed for various algal species (1, 2, 5, 16). Protoplasts isolated from various plants regenerate a wall and revert in some cases to complete plants (6, 13, 14, 15). The reversion process has thus been described and is of interest in the study of cell wall synthesis and cell shape development. An attempt was made in the present study to release protoplasts from Spirogyra and Zygnema by an enzymatic method and to revert them to normal filamentous form. MATERIALS AND METHODS Algae. Spirogyra sp. (cell diameter, 34-39 µm; number of chloroplast bands, 1) collected in the Osaka area and Zygnema extenue Jao supplied by the Culture Collection of Algae at Indiana University, Bloomington, Indiana, U.S.A. were used. The osmolarity of the Spirogyra cells, measured by the plasmolysis method, was 0.38 M sucrose equivalent. Cells of Zygnema extenue (19-24,ƒÊm in cell diameter) had an osmolarity of 0.27 M. Axenic culture. A procedure combining charcoal application with dragging through a gelatin gel was used. The algal filament was first transferred to a medium containing charcoal. The filament with its surface covered with charcoal was then dragged through gelatin (5-7 %) gel, so that the charcoal was detached from the algal surface. After both procedures were repeated two to three times, the filament was cut into pieces of about 2 mm and cultured on an agar plate prepared with the medium described below. The absence of contaminants was confirmed with media containing yeast extract and sugars (glucose, sucrose). Stock cultures were grown on an agar (0.7 %) plate containing a Volvox medium (Darden, 1966) of which glycylglycine was replaced with Tricine (Dotite) (500 mg/1) and to which thiamine (10 Mg/1) was added. This medium was also used for experiments and is referred to as the algal medium. About five days prior to releasing protoplasts, the algal filament was transferred to the liquid medium by filling the agar plate. These cultures were kept under 16-h light (2,000-3,500 lux)-8-h dark regime at 20•Ž. Preparation of protoplasts Driselase (Kyowa Hakko Kogyo, Tokyo), dissolved in the algal 249 250 T. Ohiwa medium with 5 mM maleate buffer (pH 5.2), was desalted by passing through a Biogel P-6 column. The algal medium containing 20 % driselase, 450 mM sorbitol, 2 mM CaC12 and 5 mM maleate buffer (pH 5.2) was used for producing protoplasts from Spirogyra. The enzyme solution used for producing Zygnema protoplasts contained 10 % driselase, 250 mM NaCl and 5 mM CaCl2 (pH 5.2). The enzyme solution was sterilized by filtration through a Mil lipore filter HA. Pieces of Spirogyra and Zygnema filaments were transferred to the respective enzyme solution in the latter part (6-8 h) of the dark period. For Spirogyra protoplasts, incubation lasted sequentially for 30 min at 20•Ž, 12-14 h at 4-6•Ž and 2 h at 20•Ž. For Zygnema protoplasts, incubation lasted for 30 min at 20•Ž then for 12-14 h at 4-6•Ž. The released protoplasts were suspended in the algal medium containing 300 mM glycerol and 50 mM CaC12, and washed by centrifuging for 10 min at about 2•~g for Spirogyra protoplasts and at about 6•~g for Zygneina protoplasts. The same procedures were repeated three times. Culture of protoplasts. Spirogyra and Zygnema protoplasts were cultured in the algal medium supplemented with chemicals: Spirogyra protoplasts-algal medium, 350 mM sorbitol, 20 mM CaCl2 and 5 mM KCl; and Zygnema protoplasts-algal medium, 250 mM sorbitol, 20 mM CaC12 and 5 mM KCl. The media were slightly hypertonic to the respective original cells. Petri dishes were sealed with parafilm and kept under continuous light (ca. 1,000 lux) at 20•Ž. RESULTS Protoplasts of Spirogyra and Zygnema were completely released from actively growing cells by driselase, while cellulase Onozuka R-10 (Kinki Yakult, Nishinomiya) and snail digestive juice (Helicase; Industrie Biologique Francaise, Gennevilliers) were ineffective. Freshly released protoplasts (Figs. 1 and 7) were osmotically sensitive and did not fluoresce when stained with Calcofluor (American Cyanamid, N.J., 12). Regeneration of Spirogyra protoplasts. Protoplasts of this alga regenerated a wall in osmotically stabilized liquid medium. Most protoplasts showed weak fluorescence with Calcofluor after one day of culture and became oval after two days. Subseque ntly, most protoplasts (60-80%) began to grow in filamentous fashion. Aside from regular elongation, some budded out when the cytoplasm retreated from the newly formed wall. The diameter of the freshly released protoplasts was about 45-60 ƒÊm in hypertonic culture medium, whereas that of the original cells was 34-39 ,ƒÊm. As protoplasts elongated, their diameter tended to decrease (Fig. 6). The direction of protoplast elongation did not always coincide with the axis of the chloroplast helix. Thus, the regular shape of the helix was distorted in the elongated protoplast (Fig. 2). Karyokinesis occurred after 7-8 days of culture. About 50% of elongated proto plasts were divided at 15 days of culture. Although two sister nuclei separated from each other regularly, the septum formation was usually incomplete. Remnants of the septum were scattered circumferentially or absent (Fig. 3). The second division oc curred after 10 days of culture. Septum formation was usually still incomplete, though not as defective as the septum of the first division. After 20 days of culture, many regenerated protoplasts gradually became brownish or shrank and eventually deteriorated. A few, however, continued to survive, elongat ing and repeating divisions. Some of them underwent karyokinesis two to three times during 30 days usually forming incomplete septa. The diameter of the filament grow ing in the hypertonic medium was locally variable over the length of the same filament. The shape and arrangement of the chloroplast were also altered (Fig. 4). Spirogyra and Zygnema Protoplasts 251 Fig. 1. Freshly released Spirogyra protoplasts. Fig. 2. Elongating Spirogyra protoplast with a single, distorted ribbon of chloroplast after 6 days of culture. Magnification for Figs. 2-5 is indicated by the scale bar in Fig. 5. Fig. 3. Regenerating Spirogyra protoplast after 7 days of culture. Nuclear division occurred with incomplete formation of septum. Arrows indicate remnants of the septum. Fig. 4. Regenerating Spirogyra protoplast after 20 days of culture. Arrows indicate incomplete septa. The chloroplasts have irregular shapes and arrangements. Fig. 5. Cells of Spirogyra regenerated from protoplasts after the hypertonic medium was re placed with algal medium. Note the fatty cell with two nuclei (n). 252 T. Ohiwa Fig. 6. Diameter perpendicular to the long axis (-•ü-), long axis length (-•œ-) and volume (---•ü---) in the course of regenerating Spirogyra protoplasts. Each point is the mean of 25 specimens. Volume was calculated from diameter and length under the assumption that elongating protoplasts assume a cylindrical shape with hemispherical ends. The medium osmolarity was reduced after 5-10 days to allow regenerating pro toplasts to grow and develop. Since the regenerated wall was vulnerable to osmotic shock, the original medium with 350 mM sorbitol was replaced stepwise with media of decreasing sorbitol concentrations (300, 250, 200, 100 and finally 0 mM) over an interval of 2-3 days. The growth of regenerated cells was enhanced by this procedure. The algal filament which developed (Fig. 5) consisted mostly of cells with a single nu cleus, and also of cells with two nuclei and remnants of the septum. The diameter of the algal filament remained constant except for fatty cells with two nuclei. The shape and arrangement of the chioroplast in regenerated cells were the same as those in the original cells, although some variants were found in cells with two nuclei. Regeneration of Zygnema protoplasts. Regeneration of the wall was detected in protoplasts of this alga within one day of culture in liquid medium. Most protoplasts assumed an oval shape after 2 days of culture, then began to elongate. Again, the diameter of the regenerating protoplasts tended to decrease as they elongated (Fig. 11). Two chloroplasts were not always located along the long axis of the regenerating protoplasts, but often obliquely to the long axis (Fig. 8). Nuclear division occurred after 4-5 days. More than 80 % of cultured protoplasts divided within 10 days. Two daughter nuclei separated from each other and two chloroplasts also divided, but the formation of the septum was usually incomplete (Fig. 9). The regenerating protoplasts grew to 2 weeks of culture in filamentous fashion, repeated nuclear division two to three times and formed a complete or incomplete septum. Most regenerating cells failed to survive further, although some cells in the filaments continued to grow and divide even after 20 days of culture. Since regenerating Zygnema protoplasts were vulnerable osmotically, just as Spirogyra protoplasts, the sorbitol concentration of the medium was reduced after about 5 days of culture to 200 mM, and then stepwise to 150, 100 and 0 mM over an Spirogyra and Zygnema Protoplasts 253 Fig.