Planta (Berl.) 103, 348 355 (1972) 9 by Springcr-Verlag 1972
Cell Division and Plant Development from Protoplasts of Carrot Cell Suspension Cultures* H . J . G r a m b o w **, K . N . K a o , R . A . Miller, a n d 0 . L. G a m b o r g National Research Council, Prairie Regional Laboratory, Saskatoon, Saskatchewan Received October 22, December 9, 1971
Summary. Cell regeneration and sustained division have been observed in protoplasts from carrot cell suspension cultures. Carrot plants were produced from the protoplasts by embryogcnesis. Introduction
P r o t o p l a s t s can be p r o d u c e d from c u l t u r e d cells b y e n z y m e t r e a t m e n t (Schenk a n d H i l d e b r a n d t , 1969; E r i k s s o n a n d Jonasson, 1969; K e l l e r et al., 1970; C h u p e a u a n d Morel, 1970). The p r o t o p l a s t s m a y be c u l t u r e d a n d s u s t a i n e d division of r e g e n e r a t e d cells has been o b s e r v e d in p r o t o p l a s t s of s o y b e a n a n d Haplopappus gracilis ( K a o et al., 1970, 1971) a n d of t o b a c c o m e s o p h y l l (Takebe etal., 1971; N a g a t a a n d T a k e b e , 1971). I n a r e c e n t r e p o r t T a k e b e et al. (1971) h a v e described t h e p r o c e d u r e for r e g e n e r a t i n g t o b a c c o p l a n t s from p r o t o p l a s t s of t o b a c c o leaf cells. P r o t o p l a s t s h a v e been p r e p a r e d from c a r r o t cells b y R e i n e r t a n d I-Iellm a n n (1971). The p r e s e n t r e p o r t outlines t h e conditions for cell r e g e n e r a t i o n of p r o t o p l a s t s from c a r r o t cell cultures a n d t h e p r o d u c t i o n of p l a n t s b y embryogenesis. Material and Methods
Cell Cultures The cell cultures of carrot (I)aucu8 carota cv. Royal Chantenay) were obtained from seedlings grown under sterile conditions. Root and petiole sections were placed on agar containing B5 medium with 0.1 mg per liter of 2,4-dichloro-phenoxyacetic acid (2,4-D) (Gamborg et al., 1968). The callus, which formed in about three weeks, was transferred to 25 ml of liquid B5 medium with the same concentration of 2,4-D. New cultures were started at intervals of about 6 months, and used after 2-3 months of subculturing at the rate of twice a week. After this period the cultures consisted of mixtures of cells, cell aggregates, and embryoids. The cultures used for protoplast production were grown in 250 ml De Long flasks each containing a * NRCC No. 12268. ** Visiting Scientist.
Plant Development from Carrot Protoplasts
349
total volume of 40 ml of cells and medium. The cells were transferred to new medium 2 days before protoplast production and grown in continuous light of 2000 lux at 27 ~ C.
Protoplast Production Carrot protoplasts were produced by techniques similar to those of Kao et al. (1971). Cell clumps in the size range between 44 ~m and 61 ~zm obtained by filtering the cell suspension through stainless steel mesh proved to be suitable for protoplast production. The cell fraction collected on the 44 Ezm filter was washed with B5 medium lacking sucrose and 2,4-D, but enriched with 0.56 1~ mannito] or sorbitol to yield a final calculated molarity of 0.60, and suspended in the same medium. About 2 ml of this cell suspension containing about 100 mg cells (fresh weight) were mixed with the same volume of washing medium containing 1% w/v desalted cellulase "Onozuka P 5 000" (All J a p a n Biochemical Co. Ltd.) (Kao et al., 1971), and 0.5% desalted hemicellulase ("Rhozyme H P 150", Rhom and Haas Co. of Canada). The same concentration of desalted pectinase (Sigma) could be used in place of the hemicellnlase. The conditions for protoplast formation and culture are summarized in Table 1. The first protoplasts appeared within 2 hours. After 10 to 12 hours the protoplast production usually was complete except for some remaining elongated " a g e d " cells which never formed protoplasts and some cells still sticking together in clumps. These clumps could be removed by filtering the protoplasts suspension through a 44 ~m steel mesh. The protoplasts were washed free of enzyme on an 8 tzm Millipore filter using 80 ml of medium. The liquid on the filter was never allowed to drain completely. Table 1. Conditions for production and culture of carrot protoplasts Production
Washing
Culture
Nutrients
B5
B5
B5
Sugar
--
--
0.05 M glucose
Growth regulator
--
--
0.1 rag/1 2,4-D
Osmotic agent
0.56 M mannitol or sorbitol
0.56 M mannitol or sorbitol
0.57 M mannitol or sorbitol
Total molarity (calculated)
0.60
0.60
0.60
pH
5.5
5.5
5.5
Temperature
Ambient a
Ambient
Ambient
Enzymes
0.5 % cellulase 0.25% hemicellulase
About 25 ~ C.
Protoplast Culture After washing, the protplasts were collected and mixed 1 : I (v/v) in B 5 medium containing 0.1 M glucose instead of sucrose, 0.2 mg/I 2,4-D and 0.46 M mannitol or sorbitol. The final molarity of the protoplast culture medium was calculated to be 0.60. The protoplasts were normally cultured in 50-100 ~l drops in 30 • 1.5 cm
Fig. 4. Embryoid cell group from protoplasts. Two weeks old
Fig. 3. Cells regenerated fl'om a protoplast. One week old
Fig. 2. Protoplasts after wall form&tion. Two days old
Fig. 1. Protoplasts from carrot cell cultures
9
~z
P
O
P l a n t Development from Carrot Protoplasts
351
plastic dishes (Fig. 1). However, to facilitate observation of individual protoplasts over an extended period under the microscope, drops of 5 ILl containing about 150-200 protoplasts frequently were used. The dishes were sealed with Parafilm and kept a t 23 ~ C in light at 800 1000 Lux. Addition of fresh medium was started at a stage which is shown in Fig. 3 and repeated every three to four days. For this purpose B 5 medium containing 2% sucrose and lacking any growth regulator was added very carefully in amounts of 5-10% by volume (Kao et al., 1971).
Production o/ Protoplast, /rein Carrot Lea/ The leaves of 3 4 cm sterile plantlets which were grown from protoplasts of carrot suspension cultures were shredded with a scalpel and incubated with the enzyme mixture for 8 hours. The washing procedure and culturing conditions were the same as for the production of protoplasts from suspension culture.
Results After 1 day in culture, more than 80% of the protoplasts (Fig. 2) became oval shaped. This deviation from a spherical shape is indicative of wall formation (Kao et al., 1970) which was verified by labelling with Calcoiluor white (American Cyanamid Co., Bound Brook, New Jersey). The first cell divisions usually started after two days of protoplast culture. About 10-20% of the regenerated cells underwent one or two divisions within 6 days (Fig. 3). Many dividing protoplasts formed two symmetrical daughter cells which eventually went through a second division, but then deteriorated slowly. After 8-10 days 5-30% of the dividing protoplasts had formed clusters (Figs. 4, 5) which produced embryoids when fed with fresh medium. Four-weeks old embryoids (Fig. 6) were washed on an 88 ~m steel mesh filter to remove the rest of the growth regulator and some of the cell debris, and plated on B 5 medium (0.7% agar). After three more weeks 3-6 mm plantlets were transferred onto 2,4-D-free B5 agar slants in test tubes. Finally after having reached a size of 3-5 cm the plants were transferred to vermiculite containing a I-Ioagland nutrient medium and placed in the greenhouse. A number of carrot plants which were obtained in vitro by this procedure reached a size of 20 cm four months after starting the protoplast culture. Their appearance was normal as shown in Fig. 7. Best results were obtained by starting the protoplast culture with either eellobiose or glucose as the carbohydrate source and gradually replacing it with sucrose by adding fresh medium to the protoplast culture. When sucrose at 0.05 M was used throughout the culture period the frequency of divisions was lowered to 20-50 % of that obtained when starting with glucose. Using sucrose also delayed the initiation of division by i-2 days. Healthy protoplasts were produced when working in a range of molarity between 0.55 and 0.75 (calculated). Lower molarity
352
H . J . Grambow, K. N. Kao, R. A. Miller, and O. L. Gamborg:
0
.~
-
Fig. 8. Fusion of carrot protoplasts during protoplas~ formation. - - Fig. 9. Polynucleate carrot protoplast. - - Fig. 10. Protoplasts from carrot leaves. Arrow indicates dividing cell
o
g
C~
O
5"
g
354
H . J . Grambow, K. N. Kao, 1~. A. Miller, and O. L. Gamborg:
caused deterioration to most of the protoplasts during incubation with the enzymes. After washing the protoplasts it was possible, but not necessary, to lower the molarity slowly b y about 10%.
Culture o/Carrot Lea/Protoplasts The protoplasts from the leaves could be cultured for several weeks. There were indications t h a t wall formation was taking place, and division was observed in a few protoplasts (Fig. 10). To date it has not been possible to achieve cell regeneration and sustained division of the leaf protoplasts. Discussion
Sustained division was observed in the regenerated cells from carrot protoplasts. The embryos were formed from protoplasts, although the present technique did not permit continuous observation of individual protoplasts. After cell wall regeneration, the development of the proembryoids appeared to take place as indicated in Figs. 4-6. The new cell increased in density, and divisions occurred within two weeks to produce a structure composed of tightly packed cells containing a large number of cell inclusions. These structures developed into embryoids within one month. I n addition to the direct production of embryoid structures, the protoplasts also gave rise to compact white and yellowish cell clumps, but only when they were transferred to agar 10-20 days after production. These cell clumps looked different from the original carrot cells on agar or in suspension, indicating t h a t modifications or selection of cells had occurred during the procedures described above. From these cells on agar it has also been possible to obtain carrot plantlets. Reinert and Hellmann (1971) have reported the occurrence of polynuclear protoplasts in preparations of carrot protoplasts and they suggested t h a t this condition resulted from mitosis. We observed protoplast fusions in m a n y experiments (Figs. 8, 9). These occurred during preparation, indicating t h a t multinucleate protoplasts were formed initially by fusion and not b y nuclear division. Similar observations have been made with protoplasts from soybean cells (Miller et al., 1971). This study showes t h a t division and formation of plants can be achieved in regenerated cells from carrot protoplasts using a defined medium. These results are encouraging and add substantial support to the feasibility of producing plants by protoplast and cell hybridization techniques. A grant of Deutsche Forschungsgemeinschaft to the senior author is gratefully acknowledged.
Plant Development from Carrot Protoplasts
355
References Chupeau, Y., Morel, G. : 0btention de protoplastes de plantes sup~rieures a partir de tissues cultiv4s in vitro. C. 1%. Acad. Sci. (Paris.), Set. D 270, 2659 2962 (1970). Eriksson, T., Jonasson, K.: Nuclear division in isolated protoplasts from ceils of higher plants grown in vitro. Planta (Berl.) 89, 85-89 (1969). Gamborg, O.L., Miller, l~.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). Kao, K.N., Gamborg, O.L., Keller, W.A., Miller, R.A.: Cell division in cells regenerated from protoplasts of soybean and Haplopappus gracilis. Nature (Lond.) 282, 124 (1971). Kao, K.N., Keller, W.A., Miller, I~.A.: Cell division in newly formed cells from nude protoplasts of soybean. Exp. Cell Res. 62, 338-340 (1970). Keller, W.A., Harvey, B., Gamborg, O.L., Miller, R.A., Eveleigh, D.E.: Plant protoplasts for use in somatic cell hybridization. Nature (Lond.) 226, 280-282 (1970). Miller, I~.A., Gamborg, O.L., Keller, W.A., Kao, K.~. : Fusion and division of nuclei in multinucleated soybean protoplasts. Canad. J. Genet. Cytol. 13, 347-353 (1971). Nagata, T., Takebe, J. : Plating of isolated tobacco mesophyll-protoplasts on agar medium. Planta (Berl.) 99, 12-20 (1971). Reinert, J., I-Iellmann, S. : Mechanism of the formation of polynuclear protoplasts from cells of higher plants. Naturwissenschaftcn 58, 419 (1971). Schenk, R.U., Hildebrandt, A.C. : Production of protoplasts from plant cells in liquid culture using purified commercial cellulases. Crop. Sci. 9, 629-631 (1969). Takebe, J., Labib, G., Melchers, G.: Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwisseschaften 58, 318-320 (1971). Dr. H.J. Grambow Institut fiir Pflanzenphysiologie Ruhr-Universit~t D-4630 Bochum Germany
Cell Division and Plant Development from Protoplasts of Carrot Cell Suspension Cultures* H . J . G r a m b o w **, K . N . K a o , R . A . Miller, a n d 0 . L. G a m b o r g National Research Council, Prairie Regional Laboratory, Saskatoon, Saskatchewan Received October 22, December 9, 1971
Summary. Cell regeneration and sustained division have been observed in protoplasts from carrot cell suspension cultures. Carrot plants were produced from the protoplasts by embryogcnesis. Introduction
P r o t o p l a s t s can be p r o d u c e d from c u l t u r e d cells b y e n z y m e t r e a t m e n t (Schenk a n d H i l d e b r a n d t , 1969; E r i k s s o n a n d Jonasson, 1969; K e l l e r et al., 1970; C h u p e a u a n d Morel, 1970). The p r o t o p l a s t s m a y be c u l t u r e d a n d s u s t a i n e d division of r e g e n e r a t e d cells has been o b s e r v e d in p r o t o p l a s t s of s o y b e a n a n d Haplopappus gracilis ( K a o et al., 1970, 1971) a n d of t o b a c c o m e s o p h y l l (Takebe etal., 1971; N a g a t a a n d T a k e b e , 1971). I n a r e c e n t r e p o r t T a k e b e et al. (1971) h a v e described t h e p r o c e d u r e for r e g e n e r a t i n g t o b a c c o p l a n t s from p r o t o p l a s t s of t o b a c c o leaf cells. P r o t o p l a s t s h a v e been p r e p a r e d from c a r r o t cells b y R e i n e r t a n d I-Iellm a n n (1971). The p r e s e n t r e p o r t outlines t h e conditions for cell r e g e n e r a t i o n of p r o t o p l a s t s from c a r r o t cell cultures a n d t h e p r o d u c t i o n of p l a n t s b y embryogenesis. Material and Methods
Cell Cultures The cell cultures of carrot (I)aucu8 carota cv. Royal Chantenay) were obtained from seedlings grown under sterile conditions. Root and petiole sections were placed on agar containing B5 medium with 0.1 mg per liter of 2,4-dichloro-phenoxyacetic acid (2,4-D) (Gamborg et al., 1968). The callus, which formed in about three weeks, was transferred to 25 ml of liquid B5 medium with the same concentration of 2,4-D. New cultures were started at intervals of about 6 months, and used after 2-3 months of subculturing at the rate of twice a week. After this period the cultures consisted of mixtures of cells, cell aggregates, and embryoids. The cultures used for protoplast production were grown in 250 ml De Long flasks each containing a * NRCC No. 12268. ** Visiting Scientist.
Plant Development from Carrot Protoplasts
349
total volume of 40 ml of cells and medium. The cells were transferred to new medium 2 days before protoplast production and grown in continuous light of 2000 lux at 27 ~ C.
Protoplast Production Carrot protoplasts were produced by techniques similar to those of Kao et al. (1971). Cell clumps in the size range between 44 ~m and 61 ~zm obtained by filtering the cell suspension through stainless steel mesh proved to be suitable for protoplast production. The cell fraction collected on the 44 Ezm filter was washed with B5 medium lacking sucrose and 2,4-D, but enriched with 0.56 1~ mannito] or sorbitol to yield a final calculated molarity of 0.60, and suspended in the same medium. About 2 ml of this cell suspension containing about 100 mg cells (fresh weight) were mixed with the same volume of washing medium containing 1% w/v desalted cellulase "Onozuka P 5 000" (All J a p a n Biochemical Co. Ltd.) (Kao et al., 1971), and 0.5% desalted hemicellulase ("Rhozyme H P 150", Rhom and Haas Co. of Canada). The same concentration of desalted pectinase (Sigma) could be used in place of the hemicellnlase. The conditions for protoplast formation and culture are summarized in Table 1. The first protoplasts appeared within 2 hours. After 10 to 12 hours the protoplast production usually was complete except for some remaining elongated " a g e d " cells which never formed protoplasts and some cells still sticking together in clumps. These clumps could be removed by filtering the protoplasts suspension through a 44 ~m steel mesh. The protoplasts were washed free of enzyme on an 8 tzm Millipore filter using 80 ml of medium. The liquid on the filter was never allowed to drain completely. Table 1. Conditions for production and culture of carrot protoplasts Production
Washing
Culture
Nutrients
B5
B5
B5
Sugar
--
--
0.05 M glucose
Growth regulator
--
--
0.1 rag/1 2,4-D
Osmotic agent
0.56 M mannitol or sorbitol
0.56 M mannitol or sorbitol
0.57 M mannitol or sorbitol
Total molarity (calculated)
0.60
0.60
0.60
pH
5.5
5.5
5.5
Temperature
Ambient a
Ambient
Ambient
Enzymes
0.5 % cellulase 0.25% hemicellulase
About 25 ~ C.
Protoplast Culture After washing, the protplasts were collected and mixed 1 : I (v/v) in B 5 medium containing 0.1 M glucose instead of sucrose, 0.2 mg/I 2,4-D and 0.46 M mannitol or sorbitol. The final molarity of the protoplast culture medium was calculated to be 0.60. The protoplasts were normally cultured in 50-100 ~l drops in 30 • 1.5 cm
Fig. 4. Embryoid cell group from protoplasts. Two weeks old
Fig. 3. Cells regenerated fl'om a protoplast. One week old
Fig. 2. Protoplasts after wall form&tion. Two days old
Fig. 1. Protoplasts from carrot cell cultures
9
~z
P
O
P l a n t Development from Carrot Protoplasts
351
plastic dishes (Fig. 1). However, to facilitate observation of individual protoplasts over an extended period under the microscope, drops of 5 ILl containing about 150-200 protoplasts frequently were used. The dishes were sealed with Parafilm and kept a t 23 ~ C in light at 800 1000 Lux. Addition of fresh medium was started at a stage which is shown in Fig. 3 and repeated every three to four days. For this purpose B 5 medium containing 2% sucrose and lacking any growth regulator was added very carefully in amounts of 5-10% by volume (Kao et al., 1971).
Production o/ Protoplast, /rein Carrot Lea/ The leaves of 3 4 cm sterile plantlets which were grown from protoplasts of carrot suspension cultures were shredded with a scalpel and incubated with the enzyme mixture for 8 hours. The washing procedure and culturing conditions were the same as for the production of protoplasts from suspension culture.
Results After 1 day in culture, more than 80% of the protoplasts (Fig. 2) became oval shaped. This deviation from a spherical shape is indicative of wall formation (Kao et al., 1970) which was verified by labelling with Calcoiluor white (American Cyanamid Co., Bound Brook, New Jersey). The first cell divisions usually started after two days of protoplast culture. About 10-20% of the regenerated cells underwent one or two divisions within 6 days (Fig. 3). Many dividing protoplasts formed two symmetrical daughter cells which eventually went through a second division, but then deteriorated slowly. After 8-10 days 5-30% of the dividing protoplasts had formed clusters (Figs. 4, 5) which produced embryoids when fed with fresh medium. Four-weeks old embryoids (Fig. 6) were washed on an 88 ~m steel mesh filter to remove the rest of the growth regulator and some of the cell debris, and plated on B 5 medium (0.7% agar). After three more weeks 3-6 mm plantlets were transferred onto 2,4-D-free B5 agar slants in test tubes. Finally after having reached a size of 3-5 cm the plants were transferred to vermiculite containing a I-Ioagland nutrient medium and placed in the greenhouse. A number of carrot plants which were obtained in vitro by this procedure reached a size of 20 cm four months after starting the protoplast culture. Their appearance was normal as shown in Fig. 7. Best results were obtained by starting the protoplast culture with either eellobiose or glucose as the carbohydrate source and gradually replacing it with sucrose by adding fresh medium to the protoplast culture. When sucrose at 0.05 M was used throughout the culture period the frequency of divisions was lowered to 20-50 % of that obtained when starting with glucose. Using sucrose also delayed the initiation of division by i-2 days. Healthy protoplasts were produced when working in a range of molarity between 0.55 and 0.75 (calculated). Lower molarity
352
H . J . Grambow, K. N. Kao, R. A. Miller, and O. L. Gamborg:
0
.~
-
Fig. 8. Fusion of carrot protoplasts during protoplas~ formation. - - Fig. 9. Polynucleate carrot protoplast. - - Fig. 10. Protoplasts from carrot leaves. Arrow indicates dividing cell
o
g
C~
O
5"
g
354
H . J . Grambow, K. N. Kao, 1~. A. Miller, and O. L. Gamborg:
caused deterioration to most of the protoplasts during incubation with the enzymes. After washing the protoplasts it was possible, but not necessary, to lower the molarity slowly b y about 10%.
Culture o/Carrot Lea/Protoplasts The protoplasts from the leaves could be cultured for several weeks. There were indications t h a t wall formation was taking place, and division was observed in a few protoplasts (Fig. 10). To date it has not been possible to achieve cell regeneration and sustained division of the leaf protoplasts. Discussion
Sustained division was observed in the regenerated cells from carrot protoplasts. The embryos were formed from protoplasts, although the present technique did not permit continuous observation of individual protoplasts. After cell wall regeneration, the development of the proembryoids appeared to take place as indicated in Figs. 4-6. The new cell increased in density, and divisions occurred within two weeks to produce a structure composed of tightly packed cells containing a large number of cell inclusions. These structures developed into embryoids within one month. I n addition to the direct production of embryoid structures, the protoplasts also gave rise to compact white and yellowish cell clumps, but only when they were transferred to agar 10-20 days after production. These cell clumps looked different from the original carrot cells on agar or in suspension, indicating t h a t modifications or selection of cells had occurred during the procedures described above. From these cells on agar it has also been possible to obtain carrot plantlets. Reinert and Hellmann (1971) have reported the occurrence of polynuclear protoplasts in preparations of carrot protoplasts and they suggested t h a t this condition resulted from mitosis. We observed protoplast fusions in m a n y experiments (Figs. 8, 9). These occurred during preparation, indicating t h a t multinucleate protoplasts were formed initially by fusion and not b y nuclear division. Similar observations have been made with protoplasts from soybean cells (Miller et al., 1971). This study showes t h a t division and formation of plants can be achieved in regenerated cells from carrot protoplasts using a defined medium. These results are encouraging and add substantial support to the feasibility of producing plants by protoplast and cell hybridization techniques. A grant of Deutsche Forschungsgemeinschaft to the senior author is gratefully acknowledged.
Plant Development from Carrot Protoplasts
355
References Chupeau, Y., Morel, G. : 0btention de protoplastes de plantes sup~rieures a partir de tissues cultiv4s in vitro. C. 1%. Acad. Sci. (Paris.), Set. D 270, 2659 2962 (1970). Eriksson, T., Jonasson, K.: Nuclear division in isolated protoplasts from ceils of higher plants grown in vitro. Planta (Berl.) 89, 85-89 (1969). Gamborg, O.L., Miller, l~.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968). Kao, K.N., Gamborg, O.L., Keller, W.A., Miller, R.A.: Cell division in cells regenerated from protoplasts of soybean and Haplopappus gracilis. Nature (Lond.) 282, 124 (1971). Kao, K.N., Keller, W.A., Miller, I~.A.: Cell division in newly formed cells from nude protoplasts of soybean. Exp. Cell Res. 62, 338-340 (1970). Keller, W.A., Harvey, B., Gamborg, O.L., Miller, R.A., Eveleigh, D.E.: Plant protoplasts for use in somatic cell hybridization. Nature (Lond.) 226, 280-282 (1970). Miller, I~.A., Gamborg, O.L., Keller, W.A., Kao, K.~. : Fusion and division of nuclei in multinucleated soybean protoplasts. Canad. J. Genet. Cytol. 13, 347-353 (1971). Nagata, T., Takebe, J. : Plating of isolated tobacco mesophyll-protoplasts on agar medium. Planta (Berl.) 99, 12-20 (1971). Reinert, J., I-Iellmann, S. : Mechanism of the formation of polynuclear protoplasts from cells of higher plants. Naturwissenschaftcn 58, 419 (1971). Schenk, R.U., Hildebrandt, A.C. : Production of protoplasts from plant cells in liquid culture using purified commercial cellulases. Crop. Sci. 9, 629-631 (1969). Takebe, J., Labib, G., Melchers, G.: Regeneration of whole plants from isolated mesophyll protoplasts of tobacco. Naturwisseschaften 58, 318-320 (1971). Dr. H.J. Grambow Institut fiir Pflanzenphysiologie Ruhr-Universit~t D-4630 Bochum Germany
