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Protoplast induction in Chlorella vulgaris
Plant Science Letters, 9 (1977) 201--204
201
© Elsevier/North-Holland Scientific Publishers, Ltd.
P R O T O P L A S T INDUCTION IN C H L O R E L L A V U L G A R I S MARTHA D. BERLINER Department of Biology, Simmons College, 300 The Fenway, Boston, Mass. 02115 (U.S.A.) (Received November 26th, 1976) (Accepted January 21st, 1977)
SUMMARY
Cells of the unicellular green alga Chlorella vulgaris formed osmotically fragile protoplasts when incubated for 4--6 h in a defined medium plus 0.4 M mannitol plus 0.5% Cellulysin. The protoplasts were extruded through breaks in the cell wall and n o t by dissolution of the wall. Protoplasts kept in the inducing medium remained bright green for several days and tended to adhere into chains, rings or clusters. Budding of protoplasts was also observed. INTRODUCTION Reports of rapid protoplast formation in green algae are limited to desmids [1,2], whereas in one species of Chlorella the process took several days [3]. Chlorella is one of the most studied algal genera, particularly as potential sources of singie-cell protein [4] b u t protoplasts have been difficult to induce because most species possess a cell wall constituent, sporopoUenin, which is unusually resistant to enzymatic degradation. Previous work with rapid protoplast induction in yeasts and desmids by using high osmotic pressure [1] with or without added enzymes, led to studies with C. vulgaris which is larger and grows faster than C. saccharophila [3]. METHODS C. vulgaris (Carolina Biological) was grown in flasks of Chlorella Broth (Difco) on a reciprocal shaker with a 15 h light/9 h dark cycle for 7--10 days at 21°C. Cells were collected and incubated for 18 h in the dark in Chlorella broth plus 0.4 M mannitol. Cellulysin (Calbiochem) was then added for a final enzyme concentration of 0.5% w/v. The chamber slides (Lab-Tek) were incubated at room temperature in the light for the duration of the experiments. Preparation of media, glassware, observation and photographic procedures
202
l
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V
v v
M,
i Oi O
Figs. 1--4. Protoplast formation in Chlorella vulgaris. A, autospores; B, budding; C, cluster; N, normal non-protoplasting vegetative ceil; P, protoplast; R, ring of adhering protoplasts; W, empty cell wall after protoplast release.
203
have been reported [1,2]. Viability was determined by Trypan Blue exclusion [5]. RESULTS
Within 4 h in the induction medium, most of the vegetative cells were in the process of releasing a single protoplast (Fig. 1) and in 6 h more than 95% of the cells were protoplasts. They were bright green and very dense so that no internal structures could be discerned. They were at least twice the diameter of vegetative cells (Figs. 1 and 3). Autospores did n o t form protoplasts. The e m p t y cell walls remained intact and were n o t dissolved. They tende~t to sink to the b o t t o m whereas the protoplasts floated to the top of the induction medium. At least 80% of the protoplasts were viable. When protoplasts were placed in either Chlorella broth or in distilled water, blebs or vesicles quickly formed on the surface {Figs. 2 and 3) and the contents then poured o u t of one or more breaks in the membrane {Fig. 3). After 24 h in the induction medium, and for several days thereafter, the protoplasts remained green and osmotically fragile b u t became less dense (Fig. 4). They tended to adhere to each other in clusters, rings or chains and some appeared to be budding (Fig. 4). The latter was difficult to ascertain because the protoplasts burst shortly after a cover slip is applied. DISCUSSION
The results reported here differ significantly from those of Braun and Aach [3] in several ways. The protoplasts were actually observed to emerge from a split in the cell wall whereas they concluded that the walls of C. saccharophila were dissolved; indeed the e m p t y cell walls of C. vulgaris remained intact in the induction medium. The process t o o k 4--6 h whereas they reported a minimum of 4 days to achieve 80% protoplast formation. They also did n o t determine protoplast viability nor did they report whether the protoplasts were green.
Fig. 1. Four hours of incubation in induction medium. Arrow indicates hole in cell wall through which one protoplast emerged from a pair of dividing cells. This hole is probably the site of a previous division scar. Fig. 2. Protoplast in Chlorella broth without osmotic stabilizer. Outer membrane forms blebs which will rupture. Fig. 3. Protoplast in distilled water. Contents have started to exude through ruptured cell membrane. Fig. 4. Protoplasts after 24 h in induction medium. Size bar = 25 ~m.
204
It appears that Cellulysin [2,3] which is an undefined mixture of Trichoderma enzymes, can attack some weak point in algal cell walls and is aided by the high osmotic pressure of the mannitol. In desmids the weak point is the isthmus and in Chlorella it is probably the vegetative division scar as i n fission yeasts [6]. This awaits confirmation by electron microscopy. Adhesion or fusion of protoplasts in the inducing medium occurred w i t h o u t the addition of polyethylene glycol or Ca+ ions [7,8]. W~ether true plasmogamy and wall regeneration will occur in these protoplasts is now being determined. ACKNOWLEDGEMENTS
This research was supported by grants from the Simmons College Fund for Research, by the Cottrell Undergraduate Science Fund of the Research Corporation and by a grant from General Foods. REFERENCES 1 M.D. Berlinerand K.A. Wenc, Microbios Lett., 2 (1976)39. 2 M.D. Berliner and K.A. Wenc, Protoplasma, 89 (1976) 389. 3 E. Braun and H.G. Aach, Planta, 126 (1975) 181. 4 M.D. Berlinerand K.A. Wenc, Appl. Environ. Microbiol., 32 (1976) 436. 5 P.J. Larkin, Planta~ 128 (1976) 213. 6 M.D. Berliner,Mycologia, 63 (1971) 819. 7 J. Anne and J.F. Pederby, Arch. Microbiol., 105 (1975) 201. 8 J.B. Power and E.C. Cocking, Sci. Prog. (London), 59 (1971) 181.
201
© Elsevier/North-Holland Scientific Publishers, Ltd.
P R O T O P L A S T INDUCTION IN C H L O R E L L A V U L G A R I S MARTHA D. BERLINER Department of Biology, Simmons College, 300 The Fenway, Boston, Mass. 02115 (U.S.A.) (Received November 26th, 1976) (Accepted January 21st, 1977)
SUMMARY
Cells of the unicellular green alga Chlorella vulgaris formed osmotically fragile protoplasts when incubated for 4--6 h in a defined medium plus 0.4 M mannitol plus 0.5% Cellulysin. The protoplasts were extruded through breaks in the cell wall and n o t by dissolution of the wall. Protoplasts kept in the inducing medium remained bright green for several days and tended to adhere into chains, rings or clusters. Budding of protoplasts was also observed. INTRODUCTION Reports of rapid protoplast formation in green algae are limited to desmids [1,2], whereas in one species of Chlorella the process took several days [3]. Chlorella is one of the most studied algal genera, particularly as potential sources of singie-cell protein [4] b u t protoplasts have been difficult to induce because most species possess a cell wall constituent, sporopoUenin, which is unusually resistant to enzymatic degradation. Previous work with rapid protoplast induction in yeasts and desmids by using high osmotic pressure [1] with or without added enzymes, led to studies with C. vulgaris which is larger and grows faster than C. saccharophila [3]. METHODS C. vulgaris (Carolina Biological) was grown in flasks of Chlorella Broth (Difco) on a reciprocal shaker with a 15 h light/9 h dark cycle for 7--10 days at 21°C. Cells were collected and incubated for 18 h in the dark in Chlorella broth plus 0.4 M mannitol. Cellulysin (Calbiochem) was then added for a final enzyme concentration of 0.5% w/v. The chamber slides (Lab-Tek) were incubated at room temperature in the light for the duration of the experiments. Preparation of media, glassware, observation and photographic procedures
202
l
|•
V
v v
M,
i Oi O
Figs. 1--4. Protoplast formation in Chlorella vulgaris. A, autospores; B, budding; C, cluster; N, normal non-protoplasting vegetative ceil; P, protoplast; R, ring of adhering protoplasts; W, empty cell wall after protoplast release.
203
have been reported [1,2]. Viability was determined by Trypan Blue exclusion [5]. RESULTS
Within 4 h in the induction medium, most of the vegetative cells were in the process of releasing a single protoplast (Fig. 1) and in 6 h more than 95% of the cells were protoplasts. They were bright green and very dense so that no internal structures could be discerned. They were at least twice the diameter of vegetative cells (Figs. 1 and 3). Autospores did n o t form protoplasts. The e m p t y cell walls remained intact and were n o t dissolved. They tende~t to sink to the b o t t o m whereas the protoplasts floated to the top of the induction medium. At least 80% of the protoplasts were viable. When protoplasts were placed in either Chlorella broth or in distilled water, blebs or vesicles quickly formed on the surface {Figs. 2 and 3) and the contents then poured o u t of one or more breaks in the membrane {Fig. 3). After 24 h in the induction medium, and for several days thereafter, the protoplasts remained green and osmotically fragile b u t became less dense (Fig. 4). They tended to adhere to each other in clusters, rings or chains and some appeared to be budding (Fig. 4). The latter was difficult to ascertain because the protoplasts burst shortly after a cover slip is applied. DISCUSSION
The results reported here differ significantly from those of Braun and Aach [3] in several ways. The protoplasts were actually observed to emerge from a split in the cell wall whereas they concluded that the walls of C. saccharophila were dissolved; indeed the e m p t y cell walls of C. vulgaris remained intact in the induction medium. The process t o o k 4--6 h whereas they reported a minimum of 4 days to achieve 80% protoplast formation. They also did n o t determine protoplast viability nor did they report whether the protoplasts were green.
Fig. 1. Four hours of incubation in induction medium. Arrow indicates hole in cell wall through which one protoplast emerged from a pair of dividing cells. This hole is probably the site of a previous division scar. Fig. 2. Protoplast in Chlorella broth without osmotic stabilizer. Outer membrane forms blebs which will rupture. Fig. 3. Protoplast in distilled water. Contents have started to exude through ruptured cell membrane. Fig. 4. Protoplasts after 24 h in induction medium. Size bar = 25 ~m.
204
It appears that Cellulysin [2,3] which is an undefined mixture of Trichoderma enzymes, can attack some weak point in algal cell walls and is aided by the high osmotic pressure of the mannitol. In desmids the weak point is the isthmus and in Chlorella it is probably the vegetative division scar as i n fission yeasts [6]. This awaits confirmation by electron microscopy. Adhesion or fusion of protoplasts in the inducing medium occurred w i t h o u t the addition of polyethylene glycol or Ca+ ions [7,8]. W~ether true plasmogamy and wall regeneration will occur in these protoplasts is now being determined. ACKNOWLEDGEMENTS
This research was supported by grants from the Simmons College Fund for Research, by the Cottrell Undergraduate Science Fund of the Research Corporation and by a grant from General Foods. REFERENCES 1 M.D. Berlinerand K.A. Wenc, Microbios Lett., 2 (1976)39. 2 M.D. Berliner and K.A. Wenc, Protoplasma, 89 (1976) 389. 3 E. Braun and H.G. Aach, Planta, 126 (1975) 181. 4 M.D. Berlinerand K.A. Wenc, Appl. Environ. Microbiol., 32 (1976) 436. 5 P.J. Larkin, Planta~ 128 (1976) 213. 6 M.D. Berliner,Mycologia, 63 (1971) 819. 7 J. Anne and J.F. Pederby, Arch. Microbiol., 105 (1975) 201. 8 J.B. Power and E.C. Cocking, Sci. Prog. (London), 59 (1971) 181.