Planta
Planta 139, 257-260 (1978)
9 by Springer-Verlag 1978
Studies on ChloreUa Protoplasts Demonstration of the Protoplastic Nature and the Regeneration of the Cell Wall H.G. Aach, Sabine Bartsch, a n d V. F e y e n BotanischesInstit ut der Rheinisch-WestffilischenTechnischen Hochschule, Hainbuchenstr. 24, D-5100Aachen, Federal Republic of Germany
Abstract.
Protoplasts of Chlorella saccharophila (Krfiger) N a d s o n were o b t a i n e d by cellulase digestion of the microfibrillar i n n e r c o m p o u n t of the cell wall after the resistant o u t e r m o s t layer had been scratched with sea sand. The absence of the cell wall was d e m o n strated i m m u n o l o g i c a l l y , electron microscopically a n d by staining, thus c o n f i r m i n g the protoplastic nature of the treated cells. After transfer to a n enzymefree m e d i u m r e g e n e r a t i o n of a thin cell wall was observed. The r e g e n e r a t i o n of the cell wall o b v i o u s l y followed the same steps as does the cell wall developm e n t of the autospores. At least 50% of the protoplasts were able to form colonies when plated o n a suitable agar m e d i u m . Key words: Cell wall staining - Cell wall r e g e n e r a t i o n - ChlorelIa - Protoplasts.
Introduction Some Chlorella cells a n d other algae are protected against a n e n z y m a t i c attack from w i t h o u t by a resistant t r i l a m i n a r Iayer c o n t a i n i n g s p o r o p o l l e n i n outside of the microfibrillar, cellulase digestable, layer of the cell wall ( A t k i n s o n et al., 1972). Therefore cells of C. Jusca resisted efforts aimed at p r o d u c i n g n a k e d protoplasts by e n z y m a t i c d e g r a d a t i o n of the cell wall. Even in experiments with C. saccharophila, which has a fairly distinct, instead of the t r i l a m i n a r , outer zone it took nearly four days before osmotically labile, spherical cells a p p e a r e d within a n e n z y m e - e n r i c h e d culture m e d i u m ( B r a u n a n d Aach, 1975). The observ a t i o n that isolated cell walls of C. saccharophila were m u c h more rapidly degraded by cellulase t h a n cell walls of native cells i n d u c e d us to try scratching Chlorella cells by shaking them with sea sand before a d d i n g cellulase to the osmotically s t a n d a r d i z e d cul-
ture m e d i u m (Feyen, 1977). The state of the o b t a i n e d cells was investigated a n d the r e g e n e r a t i o n of the cell wall was followed.
Materials and Methods Chlorella saccharophila (Kr/iger) Nadson 211-9a was obtained from the algae collection in G6ttingen. The cells were cultured in Ruppel's medium (1962) enriched with 4 g glucose, 1 g Difco yeast extract, and 1 g Difco nutrient broth per liter. The cells were grown in rotating test tubes containing 10 ml culture medium under continuous illumination without aeration of the medium. The cultures were bacteria-free. Before enzyme treatment the cells were washed once with 2 mi of 1 M sorbitol/mannitol (1 : 1) solution, the packed cells were shaken with sea sand for 30 s, and incubated in the enzyme solution overnight. The enzyme solution was composed of 2 parts of culture medium and 3 parts of 1 M sorbitol/mannitol solution. Onozuka SS cellulase was added for a final concentration of 4% w/v. The pH was adjusted to 6.5. The mixture was sterilized by passing it through a Sartorius membrane filter with 0.2 gm pore diameter. Up to this point, we had been unable to synchronize the division rhythms of Chlorella saccharophila cells. Therefore autospores were isolated to obtain a cell population of a similar state of development. Cultured cells were transferred on the top of a discontinuous gIucose gradient of three layers with 2 ml each of 50%, 40%, and 30% glucose concentration respectively. After 1 rain of centrifugation, the autospores could be collected from the 40% glucose layer. To demonstrate cell walls, the cells were incubated with 0.1 mg Calcofluor white per ml medium, then washed twice in 1 M sorbitol/mannitol solution. Fluorescence originated by UV light (365 rim) was observed in a Zeiss fluorescence microscope through a 435-nm light filter (see Nagata and Takebe, 1970).
Experiments and Results Enzymatic Degradation o f Isolated Cell Walls A q u a n t i t y of 8 • 109 cells was centrifuged from liquid cultures. After a d d i t i o n of sea s a n d the pellet was shaken vigorously on a v i b r a t o r y micro shaker. To
H.G. Aach et al.: Studies on Chlorella Protop[asts
258
Table 1. The influence of sea sand treatment and cell constitution on the yield of protoplasts State of culture
Original cell concentration (cells/ml) ~
Sea sand treatment (s)
Stationary
2 • 10 a
Stationary
2 • 10 a
30 and 60
Exponentially growing
107
30
20-40
24-h-old cells from growing cultures
107
30
60 80
8tg~
24-h-old cells from growing cultures
107
60
20-30
n
0
Percentage of protoplasts b
0 0 0.1
f: C
Ul
a The final concentration of enzyme-treated cells was always 107 cells/ml b The protoplasts were counted 12 h after incubation in the enzyme solution
~f
5
10
15 min
Fig. 1. Degradation of isolated Chlorella cell walls after sea sand
treatment of 2 (A), 4 ([]), or 12 (o) min. The cell wall fragments were incubated in 2% Onozuka ceilulase at 25~ C and the suspension diluted with the enzyme solution to As0o 0.5. The declining of the extinction was examined in a Zeiss spectrophotometer. (Feyen, 1977)
obtain cell wall fragments of different sizes, the period of sea sand treatment was varied from 2 to 12 min. The cell fragments were washed from the sand. The pellets then collected by centrifugation at 1800 g for 5 min were washed by repeated suspension and recentrifugation. Some chlorophyll still visible in the preparation was extracted nearly completely by ethanol. The ethanol-destained fragments were incubated in 2% cellulase and the clearing of the suspension due to the successive degradation of wall material was examined in a spectrophotometer at a wavelength of 500 nm. The speed of degradation increased with the degree of fineness due to prolonged sea sand treatment (Fig. 1). Cell walls originating from cells that were shaken with sea sand for 12 rain lost 40% of their light absorption within 60 min; 24 h later 99% of the isolated cell wall fragments were no longer detectable.
Protoplast Preparation Microscopic examination of Chlorella cells transferred to cellulase solution just after shaking with sea sand revealed the development, within a few mi-
nutes, of some cells with one or more protoplasmatic buds. I f some water was added cautiously the protoplasts sometimes slipped out of their cell wall through the fissure. Measurements of the diameter of the now spherical protoplasts resulted in a fourfold increase in volume in comparison with the elliptical cells. For preparative work, however, the yield was inadequate. Table 1 shows the results of experiments to determine conditions for higher protoplast yields. When 24-h-old cells had been shaken with sea sand for 30 s, 60-80% of the cells w e r e t r a n s f o r m e d to protoplasts. To obtain these cells, isolated autospores (see Materials and Methods) had been cultivated for one day under continuous illumination.
Cell Surface Properties of the Protoplasts When protoplasts were incubated together with the smaller elliptical cells in ChloreIla antiserum, diluted 1:10, both cell forms agglutinated with each other almost exclusively. If sometimes protoplasts were found bound to elliptical cells these aggregates were not stable and could be disrupted easily by moving the cover slide slightly. The small number of elliptical cells involved in the formation of hybrid agglutinates, mostly showed plasmic buds. Calcofluor-stained cell walls and cell wall fragments showed intense light blue fluorescence when irradiated with UV light of a wavelength of 365 nm (Nagata and Takebe, 1970). Thus all elliptical cells
H.G. Aach et ah : Studies on Chlorella Protoplasts f l u o r e s c e d i n t e n s i v e l y . T h e cell walls o f s o m e s p h e r i c a l .cells, a p p a r e n t l y c o n t a i n i n g a u t o s p o r e s , w e r e also stained, b u t t h e s p h e r i c a l p r o t o p l a s t s s h o w e d n o f l u o r e s c e n c e at all (Fig. 2). E l e c t r o n - m i c r o s c o p i c e x a m i n a t i o n s s h o w e d all spherical protoplasts absolutely naked without remn a n t s o f t h e cell w a l l (Fig. 3). U n t r e a t e d cells r e v e a l e d a t h i c k celt wall w i t h a d a r k o u t e r z o n e t h a t o b v i o u s l y h i n d e r s the e n z y m a t i c d e g r a d a t i o n o f t h e cell wall from without.
259
Cell Wall Regeneration I f t h e p r o t o p l a s t s h a d b e e n k e p t 48 h in a n e n z y m e free m e d i u m u n d e r c o n t i n u o u s i l l u m i n a t i o n at 23 ~ C, t h e s u r f a c e s o f t h e cells s t a b i l i z e d a p p r e c i a b l y . T h u s t h e y c a n n o l o n g e r be c o n s i d e r e d as t r u e p r o t o p l a s t s . W h e n w a t e r was a d d e d s u c h cells e x p a n d e d e i g h t f o l d or m o r e in v o l u m e b e f o r e t h e y f i n a l l y burst. T h e s u r f a c e s o f t h e s e cells, h o w e v e r , w e r e s t a i n a b l e only after bursting. Autospores that had frequently
Fig. 2. Calcofluor-white fluorescence. A mixture of protoplasts and normal cells were stained with Calcofluor and examined under the fluorescence microscope (see Materials and Methods). To discriminate cells with and without cell walls, the object was illuminated with UV and visible light simultaneously. The nonfluorescing spherical cells are thought to be protoplasts while the elliptical ones as well as the spherical cell in the center are not. When water was added only the nonfluorescing spherical cells could be induced to burst, x 600
Fig. 3a and b. Chlorella saccharophila. Cross section through the outer part of a cell x 200,000. a Native cell surrounded by a double membrane and a thick cell wall. Note the dark outer zone on the wall, which perhaps hinders enzymatic degradation, h Protoplast bordered by the plasma membrane only. PM Plasma cell membrane, CW cell wall. Preparation according to Sprey and Laetsch (1975). Examination in a JEM-200A electron microscope of the Common Laboratory for Electron Microscopy of the RWTH Aachen
260
developed within these protoplasts regenerating their cell walls were stainable as soon as they came in contact with the dye. The remaining spherical cells could be stained with Calcofluor only after being kept in an enzyme-free medium for 100 h, but the color of the fluorescence of the regenerated cell walls was still somewhat different from that of the native cells. To test the viability of the protoplasts, the cells were embedded in 2 ml isotonic soft agar and plated on both normal ChlorelIa agar and agar containing 0.6 M sorbitol/mannitol. The difference in the number of colonies on the two media after 12 days was considered to represent the number of viable protoplasts. It always exceeded 50% of the protoplasts counted previously.
Discussion
The elliptical cells of Chlorella saccharophila were transformed to larger spherical cells that were osmotically labile after treatment with sea sand and cellulase. The mere appearance of spherical cells, however, does not provide sufficient information as to whether cell walls are present or not (Fig. 2). Furthermore the lability against lowered osmotic pressure is proof only of a certain weakness of the cell surface. We consider the following features to be characteristic for true protoplasts : (1) perfect spherical shape of the cells; (2) the absence of any Calcofluor fluorescence; (3) sudden bursting when osmotic pressure is lowered without leaving a visible envelope or budding before bursting; (4) no aggregation with normal cells when incubated with anti-ChIorella serum; (5) absence of cell wall material as shown by electron microscopy. The protoplastic nature of our sea sand and cellulase treated cells was confirmed, based on these considerations. Points 1-3, however, may be sufficient when quick screening is needed. The ability of our protoplasts to regenerate a cell wall as well as to proliferate has been demonstrated. Two phases of cell wall regeneration could be discer-
H.G. Aach et al. : Studies on Chlorella Protoplasts
ned: (1) stabilization of the cell surface that cannot be stained with Calcofluor from without; (2) formation of a rigid Calcofluor-positive cell wall. These observations are in accord with some descriptions of cell wall development of autospores. As Wanka (1968) and Atkinson et al. (1972) showed for C. fusca, the outer component (Calcofluor-negative in our case) forms first and subsequently the fibrillar inner component is synthesized (Calcofluor-positiv in C. saccharophila). Obviously the formation of autospores is independent of phase (2) of cell wall regeneration, but cells without a rigid wall containing autospores may have been transformed to protoplasts after the beginning of autospore formation. Since the writing of this article, the publication of M. Berliner (1977) has become available. She observed protoplasts of Chlorella vulgaris after cellulase treatment of native cells in chamber slides. We thank Dr. Sprey from Kernforschungsanstalt Jtilich for advice and generous help in preparing the electron micrographs.
References Atkinson, Jr. A.W., Gunning, B.E.S., John, P.C.L. : Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry and incorporation of 14C-acetate, studied in synchronous cultures, Planta, 107, 1-32 (1972) Berliner, M.D.: Protoplast induction in Chlorella vulgaris. Plant Sci. Lett. 9, 201-204 (1977) Braun, Elke, Aach, H.G. : Enzymatic degradation of the cell wall of Chlorella. Planta 126, 181-185 (1975) Feyen, V. : Versuche zur somatischen Hybridisation von Chlorella saccharophila, Thesis, Rheim Westf. Technische Hochschule, Aachen 1977, W.-Germany Nagata, T., Takebe, J. : Cell walI regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92, 301-308 (1970) Ruppel, H.G.: Untersuchungen tiber die Zusammensetzung yon Chlorella. Flora 152, 113-138 (1962) Sprey, B., Laetsch, W.M.: Chloroplast envelopes of Spinacia oleracea L. Z. Pflanzenphysiol. 75, 38-52 (1975) Wanka, F. : Ultrastructural changes during normal and colchicineinhibited cell division of Chlorella. Protoplasma 66, 105 130 (1968) Received 20 October; accepted 20 December 1977
Planta 139, 257-260 (1978)
9 by Springer-Verlag 1978
Studies on ChloreUa Protoplasts Demonstration of the Protoplastic Nature and the Regeneration of the Cell Wall H.G. Aach, Sabine Bartsch, a n d V. F e y e n BotanischesInstit ut der Rheinisch-WestffilischenTechnischen Hochschule, Hainbuchenstr. 24, D-5100Aachen, Federal Republic of Germany
Abstract.
Protoplasts of Chlorella saccharophila (Krfiger) N a d s o n were o b t a i n e d by cellulase digestion of the microfibrillar i n n e r c o m p o u n t of the cell wall after the resistant o u t e r m o s t layer had been scratched with sea sand. The absence of the cell wall was d e m o n strated i m m u n o l o g i c a l l y , electron microscopically a n d by staining, thus c o n f i r m i n g the protoplastic nature of the treated cells. After transfer to a n enzymefree m e d i u m r e g e n e r a t i o n of a thin cell wall was observed. The r e g e n e r a t i o n of the cell wall o b v i o u s l y followed the same steps as does the cell wall developm e n t of the autospores. At least 50% of the protoplasts were able to form colonies when plated o n a suitable agar m e d i u m . Key words: Cell wall staining - Cell wall r e g e n e r a t i o n - ChlorelIa - Protoplasts.
Introduction Some Chlorella cells a n d other algae are protected against a n e n z y m a t i c attack from w i t h o u t by a resistant t r i l a m i n a r Iayer c o n t a i n i n g s p o r o p o l l e n i n outside of the microfibrillar, cellulase digestable, layer of the cell wall ( A t k i n s o n et al., 1972). Therefore cells of C. Jusca resisted efforts aimed at p r o d u c i n g n a k e d protoplasts by e n z y m a t i c d e g r a d a t i o n of the cell wall. Even in experiments with C. saccharophila, which has a fairly distinct, instead of the t r i l a m i n a r , outer zone it took nearly four days before osmotically labile, spherical cells a p p e a r e d within a n e n z y m e - e n r i c h e d culture m e d i u m ( B r a u n a n d Aach, 1975). The observ a t i o n that isolated cell walls of C. saccharophila were m u c h more rapidly degraded by cellulase t h a n cell walls of native cells i n d u c e d us to try scratching Chlorella cells by shaking them with sea sand before a d d i n g cellulase to the osmotically s t a n d a r d i z e d cul-
ture m e d i u m (Feyen, 1977). The state of the o b t a i n e d cells was investigated a n d the r e g e n e r a t i o n of the cell wall was followed.
Materials and Methods Chlorella saccharophila (Kr/iger) Nadson 211-9a was obtained from the algae collection in G6ttingen. The cells were cultured in Ruppel's medium (1962) enriched with 4 g glucose, 1 g Difco yeast extract, and 1 g Difco nutrient broth per liter. The cells were grown in rotating test tubes containing 10 ml culture medium under continuous illumination without aeration of the medium. The cultures were bacteria-free. Before enzyme treatment the cells were washed once with 2 mi of 1 M sorbitol/mannitol (1 : 1) solution, the packed cells were shaken with sea sand for 30 s, and incubated in the enzyme solution overnight. The enzyme solution was composed of 2 parts of culture medium and 3 parts of 1 M sorbitol/mannitol solution. Onozuka SS cellulase was added for a final concentration of 4% w/v. The pH was adjusted to 6.5. The mixture was sterilized by passing it through a Sartorius membrane filter with 0.2 gm pore diameter. Up to this point, we had been unable to synchronize the division rhythms of Chlorella saccharophila cells. Therefore autospores were isolated to obtain a cell population of a similar state of development. Cultured cells were transferred on the top of a discontinuous gIucose gradient of three layers with 2 ml each of 50%, 40%, and 30% glucose concentration respectively. After 1 rain of centrifugation, the autospores could be collected from the 40% glucose layer. To demonstrate cell walls, the cells were incubated with 0.1 mg Calcofluor white per ml medium, then washed twice in 1 M sorbitol/mannitol solution. Fluorescence originated by UV light (365 rim) was observed in a Zeiss fluorescence microscope through a 435-nm light filter (see Nagata and Takebe, 1970).
Experiments and Results Enzymatic Degradation o f Isolated Cell Walls A q u a n t i t y of 8 • 109 cells was centrifuged from liquid cultures. After a d d i t i o n of sea s a n d the pellet was shaken vigorously on a v i b r a t o r y micro shaker. To
H.G. Aach et al.: Studies on Chlorella Protop[asts
258
Table 1. The influence of sea sand treatment and cell constitution on the yield of protoplasts State of culture
Original cell concentration (cells/ml) ~
Sea sand treatment (s)
Stationary
2 • 10 a
Stationary
2 • 10 a
30 and 60
Exponentially growing
107
30
20-40
24-h-old cells from growing cultures
107
30
60 80
8tg~
24-h-old cells from growing cultures
107
60
20-30
n
0
Percentage of protoplasts b
0 0 0.1
f: C
Ul
a The final concentration of enzyme-treated cells was always 107 cells/ml b The protoplasts were counted 12 h after incubation in the enzyme solution
~f
5
10
15 min
Fig. 1. Degradation of isolated Chlorella cell walls after sea sand
treatment of 2 (A), 4 ([]), or 12 (o) min. The cell wall fragments were incubated in 2% Onozuka ceilulase at 25~ C and the suspension diluted with the enzyme solution to As0o 0.5. The declining of the extinction was examined in a Zeiss spectrophotometer. (Feyen, 1977)
obtain cell wall fragments of different sizes, the period of sea sand treatment was varied from 2 to 12 min. The cell fragments were washed from the sand. The pellets then collected by centrifugation at 1800 g for 5 min were washed by repeated suspension and recentrifugation. Some chlorophyll still visible in the preparation was extracted nearly completely by ethanol. The ethanol-destained fragments were incubated in 2% cellulase and the clearing of the suspension due to the successive degradation of wall material was examined in a spectrophotometer at a wavelength of 500 nm. The speed of degradation increased with the degree of fineness due to prolonged sea sand treatment (Fig. 1). Cell walls originating from cells that were shaken with sea sand for 12 rain lost 40% of their light absorption within 60 min; 24 h later 99% of the isolated cell wall fragments were no longer detectable.
Protoplast Preparation Microscopic examination of Chlorella cells transferred to cellulase solution just after shaking with sea sand revealed the development, within a few mi-
nutes, of some cells with one or more protoplasmatic buds. I f some water was added cautiously the protoplasts sometimes slipped out of their cell wall through the fissure. Measurements of the diameter of the now spherical protoplasts resulted in a fourfold increase in volume in comparison with the elliptical cells. For preparative work, however, the yield was inadequate. Table 1 shows the results of experiments to determine conditions for higher protoplast yields. When 24-h-old cells had been shaken with sea sand for 30 s, 60-80% of the cells w e r e t r a n s f o r m e d to protoplasts. To obtain these cells, isolated autospores (see Materials and Methods) had been cultivated for one day under continuous illumination.
Cell Surface Properties of the Protoplasts When protoplasts were incubated together with the smaller elliptical cells in ChloreIla antiserum, diluted 1:10, both cell forms agglutinated with each other almost exclusively. If sometimes protoplasts were found bound to elliptical cells these aggregates were not stable and could be disrupted easily by moving the cover slide slightly. The small number of elliptical cells involved in the formation of hybrid agglutinates, mostly showed plasmic buds. Calcofluor-stained cell walls and cell wall fragments showed intense light blue fluorescence when irradiated with UV light of a wavelength of 365 nm (Nagata and Takebe, 1970). Thus all elliptical cells
H.G. Aach et ah : Studies on Chlorella Protoplasts f l u o r e s c e d i n t e n s i v e l y . T h e cell walls o f s o m e s p h e r i c a l .cells, a p p a r e n t l y c o n t a i n i n g a u t o s p o r e s , w e r e also stained, b u t t h e s p h e r i c a l p r o t o p l a s t s s h o w e d n o f l u o r e s c e n c e at all (Fig. 2). E l e c t r o n - m i c r o s c o p i c e x a m i n a t i o n s s h o w e d all spherical protoplasts absolutely naked without remn a n t s o f t h e cell w a l l (Fig. 3). U n t r e a t e d cells r e v e a l e d a t h i c k celt wall w i t h a d a r k o u t e r z o n e t h a t o b v i o u s l y h i n d e r s the e n z y m a t i c d e g r a d a t i o n o f t h e cell wall from without.
259
Cell Wall Regeneration I f t h e p r o t o p l a s t s h a d b e e n k e p t 48 h in a n e n z y m e free m e d i u m u n d e r c o n t i n u o u s i l l u m i n a t i o n at 23 ~ C, t h e s u r f a c e s o f t h e cells s t a b i l i z e d a p p r e c i a b l y . T h u s t h e y c a n n o l o n g e r be c o n s i d e r e d as t r u e p r o t o p l a s t s . W h e n w a t e r was a d d e d s u c h cells e x p a n d e d e i g h t f o l d or m o r e in v o l u m e b e f o r e t h e y f i n a l l y burst. T h e s u r f a c e s o f t h e s e cells, h o w e v e r , w e r e s t a i n a b l e only after bursting. Autospores that had frequently
Fig. 2. Calcofluor-white fluorescence. A mixture of protoplasts and normal cells were stained with Calcofluor and examined under the fluorescence microscope (see Materials and Methods). To discriminate cells with and without cell walls, the object was illuminated with UV and visible light simultaneously. The nonfluorescing spherical cells are thought to be protoplasts while the elliptical ones as well as the spherical cell in the center are not. When water was added only the nonfluorescing spherical cells could be induced to burst, x 600
Fig. 3a and b. Chlorella saccharophila. Cross section through the outer part of a cell x 200,000. a Native cell surrounded by a double membrane and a thick cell wall. Note the dark outer zone on the wall, which perhaps hinders enzymatic degradation, h Protoplast bordered by the plasma membrane only. PM Plasma cell membrane, CW cell wall. Preparation according to Sprey and Laetsch (1975). Examination in a JEM-200A electron microscope of the Common Laboratory for Electron Microscopy of the RWTH Aachen
260
developed within these protoplasts regenerating their cell walls were stainable as soon as they came in contact with the dye. The remaining spherical cells could be stained with Calcofluor only after being kept in an enzyme-free medium for 100 h, but the color of the fluorescence of the regenerated cell walls was still somewhat different from that of the native cells. To test the viability of the protoplasts, the cells were embedded in 2 ml isotonic soft agar and plated on both normal ChlorelIa agar and agar containing 0.6 M sorbitol/mannitol. The difference in the number of colonies on the two media after 12 days was considered to represent the number of viable protoplasts. It always exceeded 50% of the protoplasts counted previously.
Discussion
The elliptical cells of Chlorella saccharophila were transformed to larger spherical cells that were osmotically labile after treatment with sea sand and cellulase. The mere appearance of spherical cells, however, does not provide sufficient information as to whether cell walls are present or not (Fig. 2). Furthermore the lability against lowered osmotic pressure is proof only of a certain weakness of the cell surface. We consider the following features to be characteristic for true protoplasts : (1) perfect spherical shape of the cells; (2) the absence of any Calcofluor fluorescence; (3) sudden bursting when osmotic pressure is lowered without leaving a visible envelope or budding before bursting; (4) no aggregation with normal cells when incubated with anti-ChIorella serum; (5) absence of cell wall material as shown by electron microscopy. The protoplastic nature of our sea sand and cellulase treated cells was confirmed, based on these considerations. Points 1-3, however, may be sufficient when quick screening is needed. The ability of our protoplasts to regenerate a cell wall as well as to proliferate has been demonstrated. Two phases of cell wall regeneration could be discer-
H.G. Aach et al. : Studies on Chlorella Protoplasts
ned: (1) stabilization of the cell surface that cannot be stained with Calcofluor from without; (2) formation of a rigid Calcofluor-positive cell wall. These observations are in accord with some descriptions of cell wall development of autospores. As Wanka (1968) and Atkinson et al. (1972) showed for C. fusca, the outer component (Calcofluor-negative in our case) forms first and subsequently the fibrillar inner component is synthesized (Calcofluor-positiv in C. saccharophila). Obviously the formation of autospores is independent of phase (2) of cell wall regeneration, but cells without a rigid wall containing autospores may have been transformed to protoplasts after the beginning of autospore formation. Since the writing of this article, the publication of M. Berliner (1977) has become available. She observed protoplasts of Chlorella vulgaris after cellulase treatment of native cells in chamber slides. We thank Dr. Sprey from Kernforschungsanstalt Jtilich for advice and generous help in preparing the electron micrographs.
References Atkinson, Jr. A.W., Gunning, B.E.S., John, P.C.L. : Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry and incorporation of 14C-acetate, studied in synchronous cultures, Planta, 107, 1-32 (1972) Berliner, M.D.: Protoplast induction in Chlorella vulgaris. Plant Sci. Lett. 9, 201-204 (1977) Braun, Elke, Aach, H.G. : Enzymatic degradation of the cell wall of Chlorella. Planta 126, 181-185 (1975) Feyen, V. : Versuche zur somatischen Hybridisation von Chlorella saccharophila, Thesis, Rheim Westf. Technische Hochschule, Aachen 1977, W.-Germany Nagata, T., Takebe, J. : Cell walI regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92, 301-308 (1970) Ruppel, H.G.: Untersuchungen tiber die Zusammensetzung yon Chlorella. Flora 152, 113-138 (1962) Sprey, B., Laetsch, W.M.: Chloroplast envelopes of Spinacia oleracea L. Z. Pflanzenphysiol. 75, 38-52 (1975) Wanka, F. : Ultrastructural changes during normal and colchicineinhibited cell division of Chlorella. Protoplasma 66, 105 130 (1968) Received 20 October; accepted 20 December 1977
