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Partial synchrony in soybean cell suspension cultures induced by ethylene
Printed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form resent-d ISSN 00144827
Experimental
PARTIAL
SYNCHRONY CULTURES
Cell Research
IN
105 (1977) 263-268
SOYBEAN
INDUCED
CELL
SUSPENSION
BY ETHYLENE1
F. CONSTABEL, W. G. W. KURZ, K. B. CHATSON and J. W. KIRKPATRICK Prairie
Regional Laboratory, National Saskatoon, Saskatchewan
Research S7NOW9,
Council Canada
of Canada,
SUMMARY Partial synchrony of cell division in continuous cultures of soybean cell suspensions was obtained bv flushine the cultures with ethvlene at intervals of 36 h. The most pronounced synchrony resulted from flushing the suspensions with 3 % ethylene for 3 h, followed immediately by 3 % CO2 for 3 h and 30 h aeration prior to the next ethylene treatment. Soybean cells responded to this regime of gassing also with a significant enhancement of growth.
Availability of synchronized populations of plant cells cultured in vitro would greatly facilitate investigations of molecular and physiological events in the cell cycle and of the control mechanisms which govern mitosis [Ill. In addition, synchronized populations of cells would permit studies on cell competence for the uptake and integration of foreign genetic information and on the compatibility of nuclei in heterokaryons k31. Progress in methods to induce and maintain synchrony of division is hampered by three factors: the cells (1) generally have long generation times; (2) tend to grow in clumps; and (3) respond with temporary growth retardation to drastic changes in their medium (such as may be required for the addition or removal of growth regulators). Earlier experiments on the synchronization of soybean cell suspension cultures by flushes of gaseous nitrogen, ’ NRCC No. 15770.
creating temporary anaerobic conditions, yielded encouraging results [4]. The generation time was found to be relatively short, the tendency to form callus was low, and changes of the medium were unnecessary. The cultures were characterized by 30 h cycles MI (mitotic index) (max)= 12-15 %, MI (min)= l-2 %. A more pronounced synchrony was expected from employing ethylene as the synchrony inducing factor. Ethylene appears to be a regular metabolite of plant cells in culture [5]. The production of the compound is not associated with a particular growth phase, although the rates of production appeared to increase at the beginning of the stationary growth phase. When added to the medium, 20 mM ethylene reduced the growth of rose and Ruta cultures by 20 and 30%, respectively [6]. In pea seedlings, a sharp decline in the frequency of cell division occurred within 2 h of ethylene treatment [7] and a reduced rate of DNA synthesis was found to be associated with the Exp
Cell
RPS 105 (1977)
264
Constabel
0
40
et al.
80
120
160
200
240
Figs I-3. Abscissa: time (hours); ordinate: (a) MI (%); (b) mg dry weight/l00 ml cell suspension. Fig. 1. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. 0,3 % ethylene; A, 5 % ethylene; 0. control.
inhibition of cell division. On the other hand, tobacco cell suspension cultures responded to an application of 25 ppm ethylene, 4,7 and 10 days after inoculation, with a three-fold increase of (fresh and dry) weight over controls [8]. Treatment of soybean cultures with flushes of ethylene may not only arrest the cells in a particular phase of their cycle and, thus, induce synchrony but may also help to further elucidate the effect of ethylene on cultured cells.
MATERIAL
AND
METHODS
Eight 2-day-old subcultures of soybean (Glycine mm (L.) Merr.) cells grown in liquid l-B5 medium [9] as 100 ml batches at 28°C in continuous light on a gyratory shaker (150 rpm) were combined to form an inoculum (ca 20 g cell mass), and introduced into the 2 1 growth vessel of the fermentor with enough fresh medium to reach a total volume of 1.8 I. Exp
Cd
Res
/OS (1977)
The fermentor system used was the same as described earlier [IO, 141. After the growth vessels had been filled, the suspensions were agitated and aerated with flushes of filtered, compressed sterile air at 5-10 psi for 0.1 set at intervals of 3-4 sec. The fermentors were operated as chemostats and kept at 28°C in continuous light of about 800 lux. The flow rate of fresh medium into, and of cell suspension out of, the fermentors was adjusted to maintain a population density equaling a cell dry weight of -100 mg/lOO ml culture. When experiments required a supplement of ethylene and COz, these gases were injected into the air supply lines ahead of the sterile filter by way of syringe needles. For dosing the different gases into the fermentor, two normally closed solenoid valves B2D199 175 (Skinner Electric Valve, New Britain, Conn.) and a timer MC12, gear rack Al8 (Industrial Timer Corp., Parsippany, N.J.) were used; one solenoid each for ethylene and COz. The cams on the timer were adjusted through independently shifting cams to supply gas to the fermentor for the required time. The percentage of cells in mitosis, mitotic index (MI), was determined by counting mitotic nuclei (late prophase to telophase) in two samples, 1000 cells each. The data represent average figures. Samples were fixed in acetic acid/ethanol, 1 : 3, and stained with carbol-fuchsin in 45% acetic acid and 0.2 M sorbitol[4].
RESULTS The inoculum, 40-44 h subcultures of the soybean cell strain, was characterized by a MI=5% (3-6%). Dilution of the inoculum with fresh medium (1 : 3) at the beginning of the culture generally caused a drop in mitotic activity for 8-10 h. Thereafter the frequency of cell division increased, peaked 40-60 h after inoculation and gradually fell back to the initial MI of 3-5 % during the rest of the 10 day culture period (fig. 1). In a number of experiments the mitotic activity, after initial decline, returned to a constant frequency of 3-6 % (figs 2 and 3). Treatment of the cells with flushes of ethylene (3 h duration, at intervals of 36 h) beginning 8 h after inoculation induced partial synchrony of cell division with a 36 h cycle. Synchrony was most pronounced with flushes of 3 and 5% ethylene, MI (max)=8-12 %, MI (min)=O.l-2 %. The synchrony extended over the entire culture period of 10 days (fig. 1). Treatment with
Partial
synchrony
g:, --I.---* ‘“\.-, ---01, 80.#J \.,I.-. --.. I
,O0
40
60
120
160
200
240
Fig. 2. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. Cl, 1% ethylene; 0, control.
1% ethylene resulted in only 34 cycles of mitotic activity (fig. 2). The amplitudes of these cycles decreased steadily, and about 150 h after inoculation the cycles turned into perturbations with mitotic indices within the range of the control culture. When the cells were subjected to flushes of 10% ethylene oscillations of the mitotic index retained a duration of 36 h, the amplitudes, however, were considerably smaller than under the regime of 3 % ethylene (fig. 3). The cell population reacted with a regular perturbation rather than with synchrony of cell division. When the cultures were treated with flushes of l-5% ethylene, growth of the cells seemed to be initially enhanced (figs l-2). Ten percent ethylene caused growth inhibition towards the end of the culture period (fig. 3). Partial synchrony of cell division was greatly improved by subjecting the cultures to a regime of 3 % ethylene for 3 h, followed immediately by 3 h of 3 % CO, and 30 h of
in soybean cell suspension
cultures
265
aeration prior to the next ethylene treatment. This sequence of gases made the amplitude of cell cycles extend from MI (min)=0.5-2% to MI (max)=15-20%. As the duration of the cycles remained constant at about 36 h, the oscillations in the frequency of division became more prominent than with 3 or 5% ethylene (fig. 4). The cycles of cell division were of a similar magnitude when the cultures were treated with a combination of 3 % ethylene and 3 % CO, for 3 h, followed by 33 h of aeration (fig. 4). When, however, the sequence of gases was reversed to 3 h of 3 % CO*, followed by 3 h of 3 % ethylene and 30 h of aeration, the resulting synchrony was reduced to the magnitude observed with 3 % ethylene alone. Growth of the cells increased significantly under the regime of ethylene followed by CO, (fig. 4) and a combination of ethylene and CO, (not shown). When ethylene was preceded by COz, growth of
60 /~‘+*<“.=
.
40
\
.-* 010>“2/.
0
o-0 1
0
--40
60
I 120
/ 160
-T&F-
240
Fig. 3. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. 0, 10% ethylene; 0, control. Exp Cd
Rrs
IO5 (1977)
266
Constabel
et al.
The pH of the cultures increased slightly from an initial pH 4.8 to 5.6 after 10 days. In the preceding experiments, soybean cells initially were flushed with ethylene 8 h after inoculation, i.e. at a period of decreasing mitotic activity due to the dilution of the inoculum. As a result, there was a temporary loss of almost all mitotic activity followed by a pronounced increase and peak of cell division 10-15 h later. The subsequent treatments, again, fell into periods of decreasing activity, following a mitotic wave. By treating with ethylene when mitotic activity is increasing, one can learn whether it prevents cells from entering mitosis and, if so, cells require a long or short period to reestablish the mitotic process. In this experiment (fig. 6a), the culture had been flushed twice for 3 h with 3 % ethylene at 8 h and 44 h after inoculation and had passed through one cycle of division. Sev-
160
0
40
60
120
160
200
240
time (hours); ordinate: (a, b) MI (%); (c) mg dry weight/l00 ml cell suspension. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene and CO, flushes (arrow) applied at 36 h intervals. (a) 0, 3 % ethylene 3 h followed by 3 % CO2 3 h; 0, control; (b) 0,3 % ethylene and3%COr3h;0... 0,3%ethylene3h;(c)0,3% ethylene 3 h followed by 3% CO, 3 h; O...O, 3% ethylene 3 h; 0, control.
Fig. 4. Abscissa:
the cultures increased only slightly as compared with the controls. Three percent COs, flushed through the cell cultures for 3 h at intervals of 36 h, did not produce synchronous cycles, but resulted only in one moderate wave of mitotic activity followed by a slow increase in the frequency of cell division towards the end of the culture period (fig. 5). There was a net increase in cell mass in cultures treated with flushes of 3 % COz. Exp Cd/
Res 105 (1977)
I 1 O 0
1 40
1 60
I 120
I 160
I 200
I 240 ’
5. Abscissa: time (hours); ordinnre: (a) MI (%); (b) mg dry weight/l00 ml cell suspension. Variation in the frequency of cell division, growth and pH of suspension cultures of soybean with time under a regime of CO, flushes (arrow) applied at 36 h intervals. 0.3% CO, 3 h; A, pH.
Fig.
Partial
I
ETHY,LENE
synchrony
in soybean cell suspension
cultures
267
AIR
Fig.
6. (a, b) Abscissa: time (hours); ordinate: mitotic index (%). Variation in the frequency of cell division of a partially synchronized suspension culture of soybean with
time under a regime of ethylene flushes applied 70 h (125 h) after inoculation and during the 2nd (4th) cell cycle. 0, 3 % ethylene 3 h.
enty hours after inoculation (instead of 80 h as usual) a 3 h treatment with 3 % ethylene was applied. The cells responded with continued increasing mitotic activity for only 1 h. Thereafter, the frequency of division dropped sharply for 3 h until a burst of cell division occurred for about 2 h. A second peak in the frequency of mitosis, although lower in magnitude, appeared 10 h after release of the culture from ethylene. In repeating this experiment ethylene was applied 125 h after inoculation and during the beginning of the fourth mitotic cycle (fig. 6b). The response of the cells was essentially the same. This time, however, the second peak of mitotic activity, 8 h after release of the culture from ethylene, was more pronounced.
CO, for 3 h each. Although only partial synchrony was achieved, maximal mitotic indices were higher than those obtained earlier by flushing the cultures with nitrogen [4]. Possibly the synchrony could be improved further by variation of the doses of ethylene and COe. Synchrony was maintained over a lo-day period and probably could have been extended. The system, therefore, appears suitable to provide partially synchronized cell cultures in fair quantities at any time. The degree of synchrony obtained may permit interpretation of conventional biochemical analyses of cells in terms of their mitotic cycles. Flushing soybean cells with 3 % or 5 % ethylene for 3 h at intervals of 36 h slightly enhanced the growth of the culture. This observation would support results of Freytag et al. [8] obtained with tobacco cells. The ethylene action resulting in division synchrony was deduced from experiments in which ethylene flushes were applied to cultures approaching a peak in the frequency of division. The response of the cultures to ethylene was a decline of the mitotic activity for 3 h followed by two peaks,
DISCUSSION Cell division in a continuous culture of soybean cells was synchronized by flushing the culture with 3 % or 5 % ethylene for 3 h at 36 h intervals. The synchrony was even more pronounced when the cells were treated with a sequence of 3 % ethylene and 3 %
Exp
Cdl
Rrs
105 (1977)
268
Constabel et al.
2-3 and 8-10 h after release of the cultures from ethylene. These results may be interpreted as the consequence of an arrest of cells in two zones of the interphase. Comparison with data on the duration of the cell cycle and its phases in suspension cultures of carrot [ 1 l] and soybean cells [ 121 suggests that under a regime of ethylene flushes the cells were arrested and accumulated in G2 and S. As in all other experiments (figs l-4) ethylene was applied during a period of decreasing mitotic activity and a minimum quantity of cells in G2, the induction of synchrony in the cultures may have been facilitated by an arrest and accumulation of cells in S rather than G2. Flushing soybean cells with CO, enhanced growth of the cultures. A similar observation was reported for sycamore cultures [13]. CO, did not contribute to synchrony in a direct way, but possibly by improving the culture conditions in general and increasing the percentage of cells participating in cell cycles or lowering the number of cells sequestering from cell cycles and proceeding to maturity.
Exp Cell
Res 105 (1977)
REFERENCES
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Yeoman, M M, Tissue culture and plant science (ed H E Street) p. 7. Academic Press, London & New York (1974). Gamborg, 0 L, Adv exp med bio162 (1975) 45. Constabel, F, In vitro (1976). In press. Constabel, F, Kurz, W Cl W, Chatson, B & Gamberg, 0 L, Exp cell res 85 (1974) 105. Abeles, F B, Ethylene in ulant biology. Academic Press, New York& London (1973). -. LaRue, T A G & Gamborg, 0 L, Plant physio148 (1971) 394. Burg, S P, Apelbaum, A, Eisinger, W & Kang, B G, Hort science 6 (1971) 359. Freytag, A H, Lira, E P & Widholm, J M, Plant physiol56 (1975) 58 (Abstract). Gambotg, 0 L, Miller, R A & Ojima, K, Exp cell res 50 (1968) 151. Kurz, W G W, Tissue culture: Methods and applications (ed P F Kruse, Jr & M K Patterson) p.359. Academic Press, New York (1973). Bayliss, M W, Exp cell res 92 (1975) 31. Chu. Y & Lark. K G. Planta (1976). In press. Gathercole, R W E, Conf. IAPTC,‘ Leicester (1974) (Abstract). Kurz, W G W, Plant tissue culture methods (ed 0 L Gamborg & L R Wetter) p. 74. National Research Council of Canada, Ottawa (1975).
Received September 9, 1976 Accepted November 3, 1976
Experimental
PARTIAL
SYNCHRONY CULTURES
Cell Research
IN
105 (1977) 263-268
SOYBEAN
INDUCED
CELL
SUSPENSION
BY ETHYLENE1
F. CONSTABEL, W. G. W. KURZ, K. B. CHATSON and J. W. KIRKPATRICK Prairie
Regional Laboratory, National Saskatoon, Saskatchewan
Research S7NOW9,
Council Canada
of Canada,
SUMMARY Partial synchrony of cell division in continuous cultures of soybean cell suspensions was obtained bv flushine the cultures with ethvlene at intervals of 36 h. The most pronounced synchrony resulted from flushing the suspensions with 3 % ethylene for 3 h, followed immediately by 3 % CO2 for 3 h and 30 h aeration prior to the next ethylene treatment. Soybean cells responded to this regime of gassing also with a significant enhancement of growth.
Availability of synchronized populations of plant cells cultured in vitro would greatly facilitate investigations of molecular and physiological events in the cell cycle and of the control mechanisms which govern mitosis [Ill. In addition, synchronized populations of cells would permit studies on cell competence for the uptake and integration of foreign genetic information and on the compatibility of nuclei in heterokaryons k31. Progress in methods to induce and maintain synchrony of division is hampered by three factors: the cells (1) generally have long generation times; (2) tend to grow in clumps; and (3) respond with temporary growth retardation to drastic changes in their medium (such as may be required for the addition or removal of growth regulators). Earlier experiments on the synchronization of soybean cell suspension cultures by flushes of gaseous nitrogen, ’ NRCC No. 15770.
creating temporary anaerobic conditions, yielded encouraging results [4]. The generation time was found to be relatively short, the tendency to form callus was low, and changes of the medium were unnecessary. The cultures were characterized by 30 h cycles MI (mitotic index) (max)= 12-15 %, MI (min)= l-2 %. A more pronounced synchrony was expected from employing ethylene as the synchrony inducing factor. Ethylene appears to be a regular metabolite of plant cells in culture [5]. The production of the compound is not associated with a particular growth phase, although the rates of production appeared to increase at the beginning of the stationary growth phase. When added to the medium, 20 mM ethylene reduced the growth of rose and Ruta cultures by 20 and 30%, respectively [6]. In pea seedlings, a sharp decline in the frequency of cell division occurred within 2 h of ethylene treatment [7] and a reduced rate of DNA synthesis was found to be associated with the Exp
Cell
RPS 105 (1977)
264
Constabel
0
40
et al.
80
120
160
200
240
Figs I-3. Abscissa: time (hours); ordinate: (a) MI (%); (b) mg dry weight/l00 ml cell suspension. Fig. 1. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. 0,3 % ethylene; A, 5 % ethylene; 0. control.
inhibition of cell division. On the other hand, tobacco cell suspension cultures responded to an application of 25 ppm ethylene, 4,7 and 10 days after inoculation, with a three-fold increase of (fresh and dry) weight over controls [8]. Treatment of soybean cultures with flushes of ethylene may not only arrest the cells in a particular phase of their cycle and, thus, induce synchrony but may also help to further elucidate the effect of ethylene on cultured cells.
MATERIAL
AND
METHODS
Eight 2-day-old subcultures of soybean (Glycine mm (L.) Merr.) cells grown in liquid l-B5 medium [9] as 100 ml batches at 28°C in continuous light on a gyratory shaker (150 rpm) were combined to form an inoculum (ca 20 g cell mass), and introduced into the 2 1 growth vessel of the fermentor with enough fresh medium to reach a total volume of 1.8 I. Exp
Cd
Res
/OS (1977)
The fermentor system used was the same as described earlier [IO, 141. After the growth vessels had been filled, the suspensions were agitated and aerated with flushes of filtered, compressed sterile air at 5-10 psi for 0.1 set at intervals of 3-4 sec. The fermentors were operated as chemostats and kept at 28°C in continuous light of about 800 lux. The flow rate of fresh medium into, and of cell suspension out of, the fermentors was adjusted to maintain a population density equaling a cell dry weight of -100 mg/lOO ml culture. When experiments required a supplement of ethylene and COz, these gases were injected into the air supply lines ahead of the sterile filter by way of syringe needles. For dosing the different gases into the fermentor, two normally closed solenoid valves B2D199 175 (Skinner Electric Valve, New Britain, Conn.) and a timer MC12, gear rack Al8 (Industrial Timer Corp., Parsippany, N.J.) were used; one solenoid each for ethylene and COz. The cams on the timer were adjusted through independently shifting cams to supply gas to the fermentor for the required time. The percentage of cells in mitosis, mitotic index (MI), was determined by counting mitotic nuclei (late prophase to telophase) in two samples, 1000 cells each. The data represent average figures. Samples were fixed in acetic acid/ethanol, 1 : 3, and stained with carbol-fuchsin in 45% acetic acid and 0.2 M sorbitol[4].
RESULTS The inoculum, 40-44 h subcultures of the soybean cell strain, was characterized by a MI=5% (3-6%). Dilution of the inoculum with fresh medium (1 : 3) at the beginning of the culture generally caused a drop in mitotic activity for 8-10 h. Thereafter the frequency of cell division increased, peaked 40-60 h after inoculation and gradually fell back to the initial MI of 3-5 % during the rest of the 10 day culture period (fig. 1). In a number of experiments the mitotic activity, after initial decline, returned to a constant frequency of 3-6 % (figs 2 and 3). Treatment of the cells with flushes of ethylene (3 h duration, at intervals of 36 h) beginning 8 h after inoculation induced partial synchrony of cell division with a 36 h cycle. Synchrony was most pronounced with flushes of 3 and 5% ethylene, MI (max)=8-12 %, MI (min)=O.l-2 %. The synchrony extended over the entire culture period of 10 days (fig. 1). Treatment with
Partial
synchrony
g:, --I.---* ‘“\.-, ---01, 80.#J \.,I.-. --.. I
,O0
40
60
120
160
200
240
Fig. 2. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. Cl, 1% ethylene; 0, control.
1% ethylene resulted in only 34 cycles of mitotic activity (fig. 2). The amplitudes of these cycles decreased steadily, and about 150 h after inoculation the cycles turned into perturbations with mitotic indices within the range of the control culture. When the cells were subjected to flushes of 10% ethylene oscillations of the mitotic index retained a duration of 36 h, the amplitudes, however, were considerably smaller than under the regime of 3 % ethylene (fig. 3). The cell population reacted with a regular perturbation rather than with synchrony of cell division. When the cultures were treated with flushes of l-5% ethylene, growth of the cells seemed to be initially enhanced (figs l-2). Ten percent ethylene caused growth inhibition towards the end of the culture period (fig. 3). Partial synchrony of cell division was greatly improved by subjecting the cultures to a regime of 3 % ethylene for 3 h, followed immediately by 3 h of 3 % CO, and 30 h of
in soybean cell suspension
cultures
265
aeration prior to the next ethylene treatment. This sequence of gases made the amplitude of cell cycles extend from MI (min)=0.5-2% to MI (max)=15-20%. As the duration of the cycles remained constant at about 36 h, the oscillations in the frequency of division became more prominent than with 3 or 5% ethylene (fig. 4). The cycles of cell division were of a similar magnitude when the cultures were treated with a combination of 3 % ethylene and 3 % CO, for 3 h, followed by 33 h of aeration (fig. 4). When, however, the sequence of gases was reversed to 3 h of 3 % CO*, followed by 3 h of 3 % ethylene and 30 h of aeration, the resulting synchrony was reduced to the magnitude observed with 3 % ethylene alone. Growth of the cells increased significantly under the regime of ethylene followed by CO, (fig. 4) and a combination of ethylene and CO, (not shown). When ethylene was preceded by COz, growth of
60 /~‘+*<“.=
.
40
\
.-* 010>“2/.
0
o-0 1
0
--40
60
I 120
/ 160
-T&F-
240
Fig. 3. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene flushes (arrow) applied for 3 h at 36 h intervals. 0, 10% ethylene; 0, control. Exp Cd
Rrs
IO5 (1977)
266
Constabel
et al.
The pH of the cultures increased slightly from an initial pH 4.8 to 5.6 after 10 days. In the preceding experiments, soybean cells initially were flushed with ethylene 8 h after inoculation, i.e. at a period of decreasing mitotic activity due to the dilution of the inoculum. As a result, there was a temporary loss of almost all mitotic activity followed by a pronounced increase and peak of cell division 10-15 h later. The subsequent treatments, again, fell into periods of decreasing activity, following a mitotic wave. By treating with ethylene when mitotic activity is increasing, one can learn whether it prevents cells from entering mitosis and, if so, cells require a long or short period to reestablish the mitotic process. In this experiment (fig. 6a), the culture had been flushed twice for 3 h with 3 % ethylene at 8 h and 44 h after inoculation and had passed through one cycle of division. Sev-
160
0
40
60
120
160
200
240
time (hours); ordinate: (a, b) MI (%); (c) mg dry weight/l00 ml cell suspension. Variation in the frequency of cell division and growth of suspension cultures of soybean with time under a regime of ethylene and CO, flushes (arrow) applied at 36 h intervals. (a) 0, 3 % ethylene 3 h followed by 3 % CO2 3 h; 0, control; (b) 0,3 % ethylene and3%COr3h;0... 0,3%ethylene3h;(c)0,3% ethylene 3 h followed by 3% CO, 3 h; O...O, 3% ethylene 3 h; 0, control.
Fig. 4. Abscissa:
the cultures increased only slightly as compared with the controls. Three percent COs, flushed through the cell cultures for 3 h at intervals of 36 h, did not produce synchronous cycles, but resulted only in one moderate wave of mitotic activity followed by a slow increase in the frequency of cell division towards the end of the culture period (fig. 5). There was a net increase in cell mass in cultures treated with flushes of 3 % COz. Exp Cd/
Res 105 (1977)
I 1 O 0
1 40
1 60
I 120
I 160
I 200
I 240 ’
5. Abscissa: time (hours); ordinnre: (a) MI (%); (b) mg dry weight/l00 ml cell suspension. Variation in the frequency of cell division, growth and pH of suspension cultures of soybean with time under a regime of CO, flushes (arrow) applied at 36 h intervals. 0.3% CO, 3 h; A, pH.
Fig.
Partial
I
ETHY,LENE
synchrony
in soybean cell suspension
cultures
267
AIR
Fig.
6. (a, b) Abscissa: time (hours); ordinate: mitotic index (%). Variation in the frequency of cell division of a partially synchronized suspension culture of soybean with
time under a regime of ethylene flushes applied 70 h (125 h) after inoculation and during the 2nd (4th) cell cycle. 0, 3 % ethylene 3 h.
enty hours after inoculation (instead of 80 h as usual) a 3 h treatment with 3 % ethylene was applied. The cells responded with continued increasing mitotic activity for only 1 h. Thereafter, the frequency of division dropped sharply for 3 h until a burst of cell division occurred for about 2 h. A second peak in the frequency of mitosis, although lower in magnitude, appeared 10 h after release of the culture from ethylene. In repeating this experiment ethylene was applied 125 h after inoculation and during the beginning of the fourth mitotic cycle (fig. 6b). The response of the cells was essentially the same. This time, however, the second peak of mitotic activity, 8 h after release of the culture from ethylene, was more pronounced.
CO, for 3 h each. Although only partial synchrony was achieved, maximal mitotic indices were higher than those obtained earlier by flushing the cultures with nitrogen [4]. Possibly the synchrony could be improved further by variation of the doses of ethylene and COe. Synchrony was maintained over a lo-day period and probably could have been extended. The system, therefore, appears suitable to provide partially synchronized cell cultures in fair quantities at any time. The degree of synchrony obtained may permit interpretation of conventional biochemical analyses of cells in terms of their mitotic cycles. Flushing soybean cells with 3 % or 5 % ethylene for 3 h at intervals of 36 h slightly enhanced the growth of the culture. This observation would support results of Freytag et al. [8] obtained with tobacco cells. The ethylene action resulting in division synchrony was deduced from experiments in which ethylene flushes were applied to cultures approaching a peak in the frequency of division. The response of the cultures to ethylene was a decline of the mitotic activity for 3 h followed by two peaks,
DISCUSSION Cell division in a continuous culture of soybean cells was synchronized by flushing the culture with 3 % or 5 % ethylene for 3 h at 36 h intervals. The synchrony was even more pronounced when the cells were treated with a sequence of 3 % ethylene and 3 %
Exp
Cdl
Rrs
105 (1977)
268
Constabel et al.
2-3 and 8-10 h after release of the cultures from ethylene. These results may be interpreted as the consequence of an arrest of cells in two zones of the interphase. Comparison with data on the duration of the cell cycle and its phases in suspension cultures of carrot [ 1 l] and soybean cells [ 121 suggests that under a regime of ethylene flushes the cells were arrested and accumulated in G2 and S. As in all other experiments (figs l-4) ethylene was applied during a period of decreasing mitotic activity and a minimum quantity of cells in G2, the induction of synchrony in the cultures may have been facilitated by an arrest and accumulation of cells in S rather than G2. Flushing soybean cells with CO, enhanced growth of the cultures. A similar observation was reported for sycamore cultures [13]. CO, did not contribute to synchrony in a direct way, but possibly by improving the culture conditions in general and increasing the percentage of cells participating in cell cycles or lowering the number of cells sequestering from cell cycles and proceeding to maturity.
Exp Cell
Res 105 (1977)
REFERENCES
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Yeoman, M M, Tissue culture and plant science (ed H E Street) p. 7. Academic Press, London & New York (1974). Gamborg, 0 L, Adv exp med bio162 (1975) 45. Constabel, F, In vitro (1976). In press. Constabel, F, Kurz, W Cl W, Chatson, B & Gamberg, 0 L, Exp cell res 85 (1974) 105. Abeles, F B, Ethylene in ulant biology. Academic Press, New York& London (1973). -. LaRue, T A G & Gamborg, 0 L, Plant physio148 (1971) 394. Burg, S P, Apelbaum, A, Eisinger, W & Kang, B G, Hort science 6 (1971) 359. Freytag, A H, Lira, E P & Widholm, J M, Plant physiol56 (1975) 58 (Abstract). Gambotg, 0 L, Miller, R A & Ojima, K, Exp cell res 50 (1968) 151. Kurz, W G W, Tissue culture: Methods and applications (ed P F Kruse, Jr & M K Patterson) p.359. Academic Press, New York (1973). Bayliss, M W, Exp cell res 92 (1975) 31. Chu. Y & Lark. K G. Planta (1976). In press. Gathercole, R W E, Conf. IAPTC,‘ Leicester (1974) (Abstract). Kurz, W G W, Plant tissue culture methods (ed 0 L Gamborg & L R Wetter) p. 74. National Research Council of Canada, Ottawa (1975).
Received September 9, 1976 Accepted November 3, 1976