Printed in Sweden Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
Experimental Cell Research92 (1975) 31-38
THE DURATION
OF THE
CELL
IN VIVO
CYCLE
AND
OF DAUCUS
CAROTA
L.
IN VITRO
M. W. BAYLISS Botanical Laboratories,
University of Leicester, Leicester LEl 7RH, UK
SUMMARY The duration of the cell cycle in Daucus carota seedling root-tips was measured by scoring the fraction of labelled metanhases after nulse labelling with PH-TdR). This gave a total cycle duration of 7.5 h almost equally divided between th< component stages, G-l, S, G2 and M. The duration of the cell cycle in established diploid and tetraploid suspension cultures grown in the presence of 2,4-D was measured by continuous labelling with 8H-TdR. This gave mean cell cycle times almost identical with the mean cell number doubling times (snecific growth rates) obtained from cell number growth curves. Specific growth rates were then‘used as estimates of the mean cell cvcle duration for dioloid and tetranloid lines grown in the nresence and absence of 2.4-D. As the continuous labeiling experimenis showed that 93 % of cells could incorporate 8H-TdR, cell cycle phase durations were calculated from measurements of the proportions of cells in G 1, S, G2 and M. These proportions were determined by a combination of autoradiography, microdensitometry and mitotic index determination on culture samples incubated for short periods with sH-TdR. In the presence of 2,4-D, both diploid and tetraploid cultures had cell cycle times of 45-58 h. In the absence of 2,4-D the diploid culture actively produced embryo plantlets with a mean cell cycle time of 33 h, whereas the tetraploid culture showed a cell cycle time of 82 h and remained undifferentiated. The extension and variation of the cell cycle in culture relative to the root-tip meristem was due almost entirely to changes in the duration of G 1. However, except in the root-tip and the differentiating diploid culture, Gl and G2 occupied constant proportions of the cell cycle. S phase duration under all cultural conditions was similar to that found in the root-tips but mitotic duration was variably increased. Despite this variation in mitotic duration, mitotic phase proportions in culture were mostly similar to those of the root-tip.
The duration and control of the somatic cell cycle has been studied in a large number of organisms [ll]. Though some data has been derived from studies of whole plants [17], current knowledge is based mainly upon work with cultures of micro-organisms and mammalian cells. The sophisticated techniques [16] now available for growing plant cells in culture under precisely defined nutrient conditions, and the capacity of many such cultures to undergo differentiation in vitro, suggest that these cultures will now prove particularly valuable for cell cycle studies. In the present investigation, 3-751807
the basic parameters of the cell cycle have been defined for root-tip meristems of Daucus carota and compared with the cell cycles of differentiating and undifferentiated cell suspension cultures of this species. MATERIALS
AND METHODS
Root-tip meristems Seed of a partially inbred, diploid (2n = 18), nucleus stock of Daucus carob L. (kindly donated by Elsoms’ Ltd, Spalding, Lines.) was germinated in the dark at 25°C for 72 h. Seedlings showing uniform development of roots l-2 cm long were then immersed in a solution containing 0.5 &i/ml 8H-TdR (methyl labelled, spec. act. 5 Ci/mM, Radiochemical Centre, Exptl
Cell Res 92 (197.5)
32
M. W. Bayliss
Amersham) for 15 min, followed by a 15 min chase period in 2.5 x lo-” M unlabelled TdR. The seedlings were then washed with distilled water and returned to the dark 25°C incubator. Roots from six seedlings were collected then and subsequently at 90 min intervals for 18 h and fixed in acetic alcohol (1: 3). Autoradioaravhs of Feulaen-orcein sauash vrevarations of th;: terminal 0.5 -mm of the roots were prevared with Kodak AR10 strivvina film and exvosed at 3°C for 11 days. The percenta& of labelled metaphases was scored in 25@300 metaphases from 4-6 roots at each sample time. All slides were coded and randomised before scoring. The percentage labelled interphases was scored in 2 000 nuclei from each of five roots fixed immediately after the chase with cold TdR. The mitotic index was scored for 1 000 nuclei in each of 10 squashes selected at random from all sample times. Proportions of the different mitotic stages were scored in 200 dividing nuclei from four of the mitotic index slides. Analysis of the cell cycle was by the method of Quastler & Sherman [13]. The total cvcle duration was taken from the 30 % intercepts of the ascending portions of the first and second peaks. The durations of G2 + a M and S +labelling time were taken at the 50 % intercepts of the first peak and the ordinate.
Suspensioncultures Two culture lines were used, both originally derived from seedling root segments of the seed stock described above. One line was 95 % diploid and had been in culture for twenty l&day passages. The second line was wholly tetraploid and had been in culture for forty-seven Is-day passages (fig. 4 illustrates the relative nuclear DNA distributions of these lines). Both lines were normally maintained in 250 ml Erlenmeyer flasks on liquid Murashige & Skoog medium 1121containing 0.1 mg/l 2,4-dichlorovhenoxvacetic acid (24-D) 1151. The cells of each hne g&n in the absence of 2,4-D were in their second passage in this medium during the experiment. Cell counts were performed using a chromic acid maceration technique [9] and specific growth rates (mean cell number doubling times) were determined by fitting linear regressions to logarithmic transformations of the cell count data. For the continuous labelling experiment, the cells were grown in batch fermentors [18] containing one litre of medium in a 2 1 vessel. After the cultures had been growing exponentially for 116 h, 2 ml of sterile *H-TdR solution was added to each vessel to aive a label concentration of 0.125 $i/ml in the medium. Similar quantities of 3H-TdR were added 12,24 and 36 h later to give a final concentration of approx. 0.5 @/ml. Samples of the cultures were taken 20 min after the first label addition, just before the subsequent label additions and 12 and 36 h after the final label addition. The samples were fixed by addition of an equal volume of 98 % v/v formic acid. For the experiments conducted in shake-flasks, cells were inoculated into 150 ml of medium in 500 ml Erlenmeyer flasks. Twenty ml samples were withdrawn aseptically 118 and 182 h after inoculation and added to a solution of 3H-TdR to give a final concentration of 0.5 &i/ml. These samples were incubated under normal cultural Exptl
Cell Res 92 (1975)
conditions for 20 min and then fixed in 50 % aqueous formic acid. Fixed cells were Feulgen stained after hydrolysis for 12 min in 1 N HCl at 60°C. Slides for mitotic and labelling index determination were counter stained with propionic orcein. Autoradiographs were prepared and exposed as described for the root meristems. Nuclear DNA measurements were made with a Vickers M85 scanning microdensitometer at 565 nm. DNA distributions were compiled from measurements of 300 nuclei for each samvle. Mitotic and labelling indices were measured in-a total of 5 000 nuclei and mitotic stage proportions in a total of 400 dividing nuclei for each sample. In all cases the total number of nuclei scored was made up from replicate scores performed on several different slides.
RESULTS The results of the pulse labelling experiment performed on the seedling root-tips is shown in fig. 1. The labelled metaphase plot gave an overall cycle time of 7.5 h which was sub-divided as shown in table 2. For comparison between the cell cycles in the roottips and in the various suspension cultures, the percentages of the cell cycle occupied by the various phases were calculated from their durations (table 1). Growth data obtained for the diploid and tetraploid cultures grown in batch fermentors is shown in fig. 2a. Linear regressions fitted to this data gave mean cell number doubling times of 51.2 and 44.9 h for the diploid and tetraploid lines respectively, though these values were not significantly different. Successive additions of 3H-TdR to these cultures from 116 to 152 h after inoculation caused a progressive rise in the labelling index for the 48 h following first addition of label (fig. 2b). No further change in labelling index occurred after this and the final values were 92.4 and 92.9% for the diploid and tetraploid lines respectively (means of 48 and 72 h values). The time at which the labelling index first reaches 100 % is an estimate of the mitotic cycle time (T) minus S phase duration [5]. Linear regressions were thus fitted to the
data up to and including the 48 h value (fig. 2b). The regression lines for the diploid and tetraploid cultures, which were not significantly different in slopes or 100 % intercepts, gave values for T-S of 48.2 h and 43.3 h respectively. These figures are remarkably similar to the mean doubling times calculated from the cell count data and as more than 90 % of the cells were capable of incorporating SH-TdR with 48 h, suggest that the cell number doubling time is a good estimate of the mean cell cycle time in these cultures. The curvature of the labelling index plots probably indicates variation in cycle time between different cells within the cultures. As a measure of this, the 95 % confidence limits of the 100 % intercepts of the regression lines are 34.9 to 61.6 h for the diploid culture and 20.1 to 66.6 h for the tetraploid culture. To determine the proportions of cells in the different stages of mitosis and of the cell cycle, measurements of the labelling and mitotic indices, mitotic stage proportions and the frequency distribution of nuclear DNA contents in unlabelled interphase nuclei were made on samples fixed 20 min after the first label addition. As the continuous labelling experiments indicated 9293 % of cells were actively proliferating, the proportions of cells in the different cell cycle phases provide a reasonable estimate of the relative durations of these phases when the total cycle time is known. Using the cell number doubling time as an estimate of mean cell cycle time, these proportions were thus converted to durations using corrections for the non-random distribution of cells around the cell cycle during exponential growth [5]. These values are given in tables 1 and 2. When these culture lines were grown in the absence of 2,4-D, the diploid line differentiated into embryo plantlets whereas the
60
60
40
20
0
2
4
6
8
10
12
I4
16
Fig. I. Abscissa: time from end of pulse label(hours); ordinate: % metaphasesshowinglabel.
Variation in the frequencyof labelledmetaphases in Duucus root-tius with time after u&e labelling with *H-TdR. 0; means;0, stand&d errors talc culatedfrom thereplicateslidesscoredat eachsample time.Threeparameters wereestimatedfrom thisplot: A, G2 -I-8 M; B, S + labelbg time; C, total cellcycle time.
tetraploid line did not. A second experiment was therefore conducted to analyse the cell cycles in cultures with and without 24-D. To accommodate the increased number of treatments, this experiment was performed on cells grown in Erlenmeyer flasks on a gyrorotatary shaker. In the presence of 2,4-D (0.1 mg/l) there was no significant difference between the regression slopes for diploid and tetraploid lines (fig. 3), the mean cell number doubling times being 50.9 and 57.8 h respectively. This diploid value was virtually identical with that obtained in the batch fermentor, though the doubling time of the tetraploid was significantly longer. In the absence of 2,4-D, the diploid line, which was actively differentiating into embryos from the late globular to early heart-shaped stages, had a significantly shorter mean doubling time of 32.9 h. The tetraploid line which, though slightly more aggregated in the absence of 2,4-D did not differentiate, had a significantly longer mean doubling time of 81.6 h. In a separate long-term experiment, it was possible to maintain the tetraploid culture indefinitely Exptl Cell Res 92 (1975)
34
M. W. Bay&
Table 1. Percentages of cells in the various stages of the cell cycle and mitosis in root-tips and suspension cultures of Daucus % of mitosis occupied by % of cell cycle occupied by Material
Gl
s
G2
M
prophase
metaphase
ana -t telophase
Root tip
17.3
36.0
38.7
8.0
47.9
21.5
30.6
Diploid culture Batch fermentor + 2,4-D Shake flask f 2,4-D -2,4-D
83.4
5.4
8.9
3.2
50.0
16.7
33.2
84.6 70.2
6.0 7.5
1;:;
2.3 2.4
49.5 49.8
15.7 18.7
34.8 31.5
Tetraploid culture Batch fermentor +2,4-D Shake flask + 2,4-D -2,4-D
84.5
3.6
7.7
4.2
48.7
16.7
34.6
85.6 84.5
2.3 3.3
10.0 10.8
2.1 1.4
48.5 61.5
14.7 13.2
36.8 25.3
in the absence of 2,4-D, suggesting its lowered growth rate in this medium was not due to carry-over of 2,4-D with the inoculum. The percentages of cells in G 1, S, G2 and M and the mitotic stages did not differ
significantly between the two times (118 and 182 h) at which samples of each culture were labelled and fixed (fig. 3). The results wera thus pooled and the mean values are shown in table 1. Examples of the DNA distributions
Table 2. Durations of the cell cycle and mitosis and their constituent stagesin root-tips and suspension cultures of Daucus
Material Root tips Dipioid culture Batch fermentor +2,4-D Shake flask + 2,4-D -2,4-D Tetraploid culture Batch fermentor + 2,4-D Shake flask -2,4-D +2,4-D
Duration (hours)
of cell cycle and stages
Total
Gl
Duration (how
s
G2
M
of mitotic stages
prophase
metaphase
ana + telophase
7.5
1.3
2.7
2.9
0.6
0.3
0.1
0.2
51.2
39.6
3.0
6.2
2.4
1.2
0.4
0.8
50.9 32.8
2:
4::
5.0 8.5
1.7 1.1
0.8 0.6
0”::
0.6 0.3
44.9
35.4
2.1
4.7
2.7
1.3
0.4
1.0
57.8 81.6
46.5 64.5
:::
1.7
0.8 1.0
i::.
X5
Exptl Cell Res 92 (1975)
1::;
Cell cycle of Daucus 35
for the different lines and treatments are shown in fig. 4. The durations of Gl, S, G2 and M and the mitotic stages were 6.0 a
1
Fig. 3. Abscissa: time after inoculation (hours); ordinate: log,, cell number/ml culture. Semi-logarithmic plot of cell number against time for diploid (A) and tetraploid (B) cultures in the absence ( l ) and presence ( q ) of 0.1 mg/l2,4-D. The vertical dotted lines show the two time points at which samples were labelled and fixed. The following are the parameters of the fitted regression lines (b, slope constant; r, correlation coefficient): Diploid2,4-D absent, b, 0.00916~0.002; r, 0.983; diploid2,4-D present, b, 0.0059f0.0003, r, 0.982; tetraploid -2,4-D absent, b, 0.00369 +0.0002; r, 0.974; tetraploid - 2,4-D present; b, 0.0052 ~0.0003, r, 0.978.
OT 0
20
40
60
80
Fig. 2. (a) Abscissa: time after inoculation (hours); ordinate: log,, cell number/ml culture. Semi-logarithmic plot of cell number against time for 0, diploid and q , tetraploid suspension cultures. The vertical bars represent times of label addition. A mark sample times for labelling index determination. The following are the parameters of the fitted regression lines (b, slope constant; r, correlation coefficient): Diploid (-); b, 0.00588+0.0004; r, 0.980. Tetraploid (- -); b, 0.00670f0.0003; r, 0.990. (b) Abscissa: time after first labelad dition (hours); ordinate: % nuclei labelled. Increase in labelling index during continuous exposure of l , diploid, and q , tetraploid suspension cultures to SH-TdR. The following are the parameters of the regressions fitted to the points up to and including the 48 h values: Diploid (-a-); b, 1.80+0.17; r, 0.980, 100% intercept=48.2 h. Tetraplotd (--); 6, 1.99kO.31; r, 0.940, 100 % intercept =43.3 h.
calculated as before and the resulting time values are given in table 2. The results obtained both in the batch fermentors and in the shake-flasks showed that cultured Daucus cells have mean cell cycles greatly extended beyond the root-tip value due largely to an increase in Gl duration (table 2). However, both Gl and G2 occupied approximately constant proportions of the cell cycle in all undifferentiated suspensions with doubling times of greater than 45 h (table 1). In the embryogenie diploid culture, with a significantly shorter doubling time, the proportions of Gl and G2 nuclei changed significantly towards the values found in the root tips. Changes in labelling index generally correlated with cell cycle duration in culture to give an approximately constant S phase duration, equal to that of the root-tips for both diploid and tetraploid lines. In the shake flask experiment, mitotic index varied to give a constant mitotic duration in the fiptl
Cell Res 92 (197.5)
36
M. W. Bayliss
o-1
I
I
t
1
absence of 2,4-D. This culture showed a significant increase in the percentage of prophase nuclei accompanied by reductions in the percentages of the other mitotic stages (table 1). DISCUSSION
soD
0 1
A
Alli OS
to
20
40
‘ a0
Fig. 4. Abscissa: DNA content/nucleus(g x lo-IS); ordinate: no. of nuclei.
Examplesof frequency distributionsof nuclear DNA contentsin exponentialculturesof Duucus. A, diploid- 2,4-Dabsent;B, diploid- 2,4-D present; C, tetraploid-2,4-D absent;D, tetraploid- 2,4-D present.
undifferentiated suspensions and a duration significantly reduced towards the roottip value in the differentiating diploid suspension. In the batch fermentors, mitotic indices were significantly higher than those in shake flasks with similar growth rates, implying longer mitotic durations in the batch fermentors. Despite the variations in mitotic duration, the proportions of the different mitotic stages were constant in all material except the tetraploid suspension growing in the Exptl Cell Res 92 (197.5)
Analysis of the cell cycle in root meristems by pulse labelling with 3H-TdR has been used to give values for at least 30 plant species [4]. The precise mathematical analysis of results from pulse labelling experiments is complex [lo] and no single method is yet applicable to all data. The simple graphical analysis used here can, however, yield results compatible with those from more complex approaches (cf [2 and 51) albeit without estimate of error limits. Cell cycle information from most systems so far studied [6, 121 points to Gl as being the most variable phase of the cell cycle. Extensive data from different areas within root-tips of Zea [2] showed that those cells with the minimum cycle time had a short or non-existent G 1, whereas other areas of the root-tip contained cells with Gl durations of up to 135 h. Root-tips of most species probably exhibit a similar heterogeneity of cycle times and the interpretation of cycle durations obtained from squash preparations, where identification of different zones in the root-tip is impossible, must be based on the overall Gl durations produced. It seems reasonable to assume, as those cells with the shortest cycle will be the first to reappear in the second peak of labelled mitoses, that the data presented for Daucus root-tips, giving a total cycle time of 7.5 h and a Gl of 1.7 h, approaches quite closely the minimum cell cycle duration for this species at 25°C. These values are shorter than those previously reported for herbaceous plant species [4, 71 and probably reflect the
Cell cycle of Daucus
37
The 45-50 h doubling time obtained for extremely low DNA content (2C= 1.21 x the Daucus cultures grown in the presence lo-l2 g [S]) of Daucus. In suspension cultures of Daucus, the of 2,4-D seems to be a stable minimum evidence that cell number doubling time value (cf [15]) representing the limit of adapprovided a good estimate of mean cell tion of the cells to their cultural conditions. cycle time confirms similar findings [8] in Only when embryogenesis was induced in Acer suspension cultures where pulse labelling the absence of 2,4-D was the cell cycle experiments were also shown to agree with shortened, presumably in response to the continuous labelling and cell count data. greater degree of tissue organisation. An Though the continuous labelling experi- increased labelling index in embryogenic ments on Daucus did not give a final labelling cultures, probably due to a shortened cell index of lOO%, this does not necessarily cycle, has been previously reported for imply the unlabelled cells were outside the Daucus [19]. cell cycle. As no measurements were made The extension of the cell cycle in the of the life of 3H-TdR in the culture medium, tetraploid culture in the absence of 2,4-D, cells entering S phase a short time after the coupled with lack of embryogenesis, suggests last label addition, or cells with longer cell that though this line can be maintained cycles may not have had label available for indefinitely under these conditions, it is incorporation. The variation of cell cycle only partially autotrophic for auxin and is duration about the mean, indicated by the limited in growth rate by a limiting rate of continuous labelling data, does not seem endogenous auxin production. of sufficient magnitude to suggest the preThe constancy of S phase duration and the sence of some cells in culture with cycle limited extension of mitosis suggests that times as short as those of the root-tip. How- these processes are less affected by cultural ever, it remains a possibility that differences conditions than the other phases of the cell in mean cycle time between different experi- cycle. The constancy of mitotic stage promental conditions could be caused by changes portions in all but the most extended cell in the proportions of cells with different cycle suggests that once initiated, the procycle times. gramme of mitosis is relatively insensitive This contrast between cell cycles in vivo to the cellular and cultural environment. and in vitro observed for Daucus is similar The low frequency of abnormal mitoses to that shown in Haplopuppus [l, 7, IS], observed in Daucus cultures [3] may thus where extension of the cell cycle in suspen- reflect division in cells which are also unusual sion culture resulted mainly from elongation in other respects, for example in being highly of Gl, although S and G2 were also slightly vacuolated. extended. Mitosis had a similar duration to I gratefully acknowledge the help and advice of that in the root-tip. Variation in Gl was Professor H. E. Street and the technical assistance also the cause of the variably extended cell of Mrs Helen Morris. This work was supported by a grant from the Nuffield Foundation. cycle in Acer suspension cultures [8] and it seems probable from the generally low REFERENCES growth rates observed in plant cell cultures 1. Ames, I & Mitra, J, The nucleus 9 (1966) 61. that present cultural methods do not allow 2. Barlow, P W & MacDonald, P D M, Proc roy sot B 183 (1973) 385. plant cells to grow at their maximum attain3. Bayliss, M W, Nature 246 (1973) 529. able rates. 4. Bennett, M D, Proc roy sot B181 (1972) 109. Exptl Cell Res 92 (1975)
38 M. W. Bayliss 5. Cleaver, J E. Thymidine metabolism and cell cycle kinetics.‘Norih-Holland, Amsterdam (1967). 6. Eriksson. T. Phvsiol nlant 20 (1967) 348. 7. Evans, 6 &I, kees,‘H, Sneli, C ‘L & Sun, S, Chromosomes today 3 (1970) 24. 8. Gould, A R, Bayliss, M W & Street, H E, J exptl bot 25 (1974) 468. 9. Henshaw, G G, Jha, K K, Mehta, A R, Shakeshaft, D J & Street, H E, J exptl bot 17 (1966) 362. 10. Mendelsohn, M L & Takahashi, M, The cell cycle and cancer (ed R Baserga). Dekker, New York (1971). 11. Mitchison, J M, The biology of the cell cycle. Cambridge University Press (1971). 12. Murashige, T & Skoog, F, Physiol plant 15 (1962) 473.
Exptl Cell Res 92 (1975)
13. Ouastler. H & Sherman. , F G.,_Exntl cell res 17 (i959) 4io. 14. Sparvoli, E, Gay, H & Kaufman, B P, Caryologia 19 (1959) 420. 15. Smith. S M & Street. H E. Ann bot 38 (1974) 223. 16. Street; H E, Plant iissue’and cell cult&e. glackwell, Oxford (1973). 17. Van? Hof, J & Kovacs, C J, Advances in experimental medicine and biology 18 (1972) 15. 18. Wilson, S B, King, P J & Street, H E, J exptl bot 21 (1971) 177. 19. Wochok, Z S, Biologia plantarum (Praha) 15 (1973) 107. Received September 19, 1974
Experimental Cell Research92 (1975) 31-38
THE DURATION
OF THE
CELL
IN VIVO
CYCLE
AND
OF DAUCUS
CAROTA
L.
IN VITRO
M. W. BAYLISS Botanical Laboratories,
University of Leicester, Leicester LEl 7RH, UK
SUMMARY The duration of the cell cycle in Daucus carota seedling root-tips was measured by scoring the fraction of labelled metanhases after nulse labelling with PH-TdR). This gave a total cycle duration of 7.5 h almost equally divided between th< component stages, G-l, S, G2 and M. The duration of the cell cycle in established diploid and tetraploid suspension cultures grown in the presence of 2,4-D was measured by continuous labelling with 8H-TdR. This gave mean cell cycle times almost identical with the mean cell number doubling times (snecific growth rates) obtained from cell number growth curves. Specific growth rates were then‘used as estimates of the mean cell cvcle duration for dioloid and tetranloid lines grown in the nresence and absence of 2.4-D. As the continuous labeiling experimenis showed that 93 % of cells could incorporate 8H-TdR, cell cycle phase durations were calculated from measurements of the proportions of cells in G 1, S, G2 and M. These proportions were determined by a combination of autoradiography, microdensitometry and mitotic index determination on culture samples incubated for short periods with sH-TdR. In the presence of 2,4-D, both diploid and tetraploid cultures had cell cycle times of 45-58 h. In the absence of 2,4-D the diploid culture actively produced embryo plantlets with a mean cell cycle time of 33 h, whereas the tetraploid culture showed a cell cycle time of 82 h and remained undifferentiated. The extension and variation of the cell cycle in culture relative to the root-tip meristem was due almost entirely to changes in the duration of G 1. However, except in the root-tip and the differentiating diploid culture, Gl and G2 occupied constant proportions of the cell cycle. S phase duration under all cultural conditions was similar to that found in the root-tips but mitotic duration was variably increased. Despite this variation in mitotic duration, mitotic phase proportions in culture were mostly similar to those of the root-tip.
The duration and control of the somatic cell cycle has been studied in a large number of organisms [ll]. Though some data has been derived from studies of whole plants [17], current knowledge is based mainly upon work with cultures of micro-organisms and mammalian cells. The sophisticated techniques [16] now available for growing plant cells in culture under precisely defined nutrient conditions, and the capacity of many such cultures to undergo differentiation in vitro, suggest that these cultures will now prove particularly valuable for cell cycle studies. In the present investigation, 3-751807
the basic parameters of the cell cycle have been defined for root-tip meristems of Daucus carota and compared with the cell cycles of differentiating and undifferentiated cell suspension cultures of this species. MATERIALS
AND METHODS
Root-tip meristems Seed of a partially inbred, diploid (2n = 18), nucleus stock of Daucus carob L. (kindly donated by Elsoms’ Ltd, Spalding, Lines.) was germinated in the dark at 25°C for 72 h. Seedlings showing uniform development of roots l-2 cm long were then immersed in a solution containing 0.5 &i/ml 8H-TdR (methyl labelled, spec. act. 5 Ci/mM, Radiochemical Centre, Exptl
Cell Res 92 (197.5)
32
M. W. Bayliss
Amersham) for 15 min, followed by a 15 min chase period in 2.5 x lo-” M unlabelled TdR. The seedlings were then washed with distilled water and returned to the dark 25°C incubator. Roots from six seedlings were collected then and subsequently at 90 min intervals for 18 h and fixed in acetic alcohol (1: 3). Autoradioaravhs of Feulaen-orcein sauash vrevarations of th;: terminal 0.5 -mm of the roots were prevared with Kodak AR10 strivvina film and exvosed at 3°C for 11 days. The percenta& of labelled metaphases was scored in 25@300 metaphases from 4-6 roots at each sample time. All slides were coded and randomised before scoring. The percentage labelled interphases was scored in 2 000 nuclei from each of five roots fixed immediately after the chase with cold TdR. The mitotic index was scored for 1 000 nuclei in each of 10 squashes selected at random from all sample times. Proportions of the different mitotic stages were scored in 200 dividing nuclei from four of the mitotic index slides. Analysis of the cell cycle was by the method of Quastler & Sherman [13]. The total cvcle duration was taken from the 30 % intercepts of the ascending portions of the first and second peaks. The durations of G2 + a M and S +labelling time were taken at the 50 % intercepts of the first peak and the ordinate.
Suspensioncultures Two culture lines were used, both originally derived from seedling root segments of the seed stock described above. One line was 95 % diploid and had been in culture for twenty l&day passages. The second line was wholly tetraploid and had been in culture for forty-seven Is-day passages (fig. 4 illustrates the relative nuclear DNA distributions of these lines). Both lines were normally maintained in 250 ml Erlenmeyer flasks on liquid Murashige & Skoog medium 1121containing 0.1 mg/l 2,4-dichlorovhenoxvacetic acid (24-D) 1151. The cells of each hne g&n in the absence of 2,4-D were in their second passage in this medium during the experiment. Cell counts were performed using a chromic acid maceration technique [9] and specific growth rates (mean cell number doubling times) were determined by fitting linear regressions to logarithmic transformations of the cell count data. For the continuous labelling experiment, the cells were grown in batch fermentors [18] containing one litre of medium in a 2 1 vessel. After the cultures had been growing exponentially for 116 h, 2 ml of sterile *H-TdR solution was added to each vessel to aive a label concentration of 0.125 $i/ml in the medium. Similar quantities of 3H-TdR were added 12,24 and 36 h later to give a final concentration of approx. 0.5 @/ml. Samples of the cultures were taken 20 min after the first label addition, just before the subsequent label additions and 12 and 36 h after the final label addition. The samples were fixed by addition of an equal volume of 98 % v/v formic acid. For the experiments conducted in shake-flasks, cells were inoculated into 150 ml of medium in 500 ml Erlenmeyer flasks. Twenty ml samples were withdrawn aseptically 118 and 182 h after inoculation and added to a solution of 3H-TdR to give a final concentration of 0.5 &i/ml. These samples were incubated under normal cultural Exptl
Cell Res 92 (1975)
conditions for 20 min and then fixed in 50 % aqueous formic acid. Fixed cells were Feulgen stained after hydrolysis for 12 min in 1 N HCl at 60°C. Slides for mitotic and labelling index determination were counter stained with propionic orcein. Autoradiographs were prepared and exposed as described for the root meristems. Nuclear DNA measurements were made with a Vickers M85 scanning microdensitometer at 565 nm. DNA distributions were compiled from measurements of 300 nuclei for each samvle. Mitotic and labelling indices were measured in-a total of 5 000 nuclei and mitotic stage proportions in a total of 400 dividing nuclei for each sample. In all cases the total number of nuclei scored was made up from replicate scores performed on several different slides.
RESULTS The results of the pulse labelling experiment performed on the seedling root-tips is shown in fig. 1. The labelled metaphase plot gave an overall cycle time of 7.5 h which was sub-divided as shown in table 2. For comparison between the cell cycles in the roottips and in the various suspension cultures, the percentages of the cell cycle occupied by the various phases were calculated from their durations (table 1). Growth data obtained for the diploid and tetraploid cultures grown in batch fermentors is shown in fig. 2a. Linear regressions fitted to this data gave mean cell number doubling times of 51.2 and 44.9 h for the diploid and tetraploid lines respectively, though these values were not significantly different. Successive additions of 3H-TdR to these cultures from 116 to 152 h after inoculation caused a progressive rise in the labelling index for the 48 h following first addition of label (fig. 2b). No further change in labelling index occurred after this and the final values were 92.4 and 92.9% for the diploid and tetraploid lines respectively (means of 48 and 72 h values). The time at which the labelling index first reaches 100 % is an estimate of the mitotic cycle time (T) minus S phase duration [5]. Linear regressions were thus fitted to the
data up to and including the 48 h value (fig. 2b). The regression lines for the diploid and tetraploid cultures, which were not significantly different in slopes or 100 % intercepts, gave values for T-S of 48.2 h and 43.3 h respectively. These figures are remarkably similar to the mean doubling times calculated from the cell count data and as more than 90 % of the cells were capable of incorporating SH-TdR with 48 h, suggest that the cell number doubling time is a good estimate of the mean cell cycle time in these cultures. The curvature of the labelling index plots probably indicates variation in cycle time between different cells within the cultures. As a measure of this, the 95 % confidence limits of the 100 % intercepts of the regression lines are 34.9 to 61.6 h for the diploid culture and 20.1 to 66.6 h for the tetraploid culture. To determine the proportions of cells in the different stages of mitosis and of the cell cycle, measurements of the labelling and mitotic indices, mitotic stage proportions and the frequency distribution of nuclear DNA contents in unlabelled interphase nuclei were made on samples fixed 20 min after the first label addition. As the continuous labelling experiments indicated 9293 % of cells were actively proliferating, the proportions of cells in the different cell cycle phases provide a reasonable estimate of the relative durations of these phases when the total cycle time is known. Using the cell number doubling time as an estimate of mean cell cycle time, these proportions were thus converted to durations using corrections for the non-random distribution of cells around the cell cycle during exponential growth [5]. These values are given in tables 1 and 2. When these culture lines were grown in the absence of 2,4-D, the diploid line differentiated into embryo plantlets whereas the
60
60
40
20
0
2
4
6
8
10
12
I4
16
Fig. I. Abscissa: time from end of pulse label(hours); ordinate: % metaphasesshowinglabel.
Variation in the frequencyof labelledmetaphases in Duucus root-tius with time after u&e labelling with *H-TdR. 0; means;0, stand&d errors talc culatedfrom thereplicateslidesscoredat eachsample time.Threeparameters wereestimatedfrom thisplot: A, G2 -I-8 M; B, S + labelbg time; C, total cellcycle time.
tetraploid line did not. A second experiment was therefore conducted to analyse the cell cycles in cultures with and without 24-D. To accommodate the increased number of treatments, this experiment was performed on cells grown in Erlenmeyer flasks on a gyrorotatary shaker. In the presence of 2,4-D (0.1 mg/l) there was no significant difference between the regression slopes for diploid and tetraploid lines (fig. 3), the mean cell number doubling times being 50.9 and 57.8 h respectively. This diploid value was virtually identical with that obtained in the batch fermentor, though the doubling time of the tetraploid was significantly longer. In the absence of 2,4-D, the diploid line, which was actively differentiating into embryos from the late globular to early heart-shaped stages, had a significantly shorter mean doubling time of 32.9 h. The tetraploid line which, though slightly more aggregated in the absence of 2,4-D did not differentiate, had a significantly longer mean doubling time of 81.6 h. In a separate long-term experiment, it was possible to maintain the tetraploid culture indefinitely Exptl Cell Res 92 (1975)
34
M. W. Bay&
Table 1. Percentages of cells in the various stages of the cell cycle and mitosis in root-tips and suspension cultures of Daucus % of mitosis occupied by % of cell cycle occupied by Material
Gl
s
G2
M
prophase
metaphase
ana -t telophase
Root tip
17.3
36.0
38.7
8.0
47.9
21.5
30.6
Diploid culture Batch fermentor + 2,4-D Shake flask f 2,4-D -2,4-D
83.4
5.4
8.9
3.2
50.0
16.7
33.2
84.6 70.2
6.0 7.5
1;:;
2.3 2.4
49.5 49.8
15.7 18.7
34.8 31.5
Tetraploid culture Batch fermentor +2,4-D Shake flask + 2,4-D -2,4-D
84.5
3.6
7.7
4.2
48.7
16.7
34.6
85.6 84.5
2.3 3.3
10.0 10.8
2.1 1.4
48.5 61.5
14.7 13.2
36.8 25.3
in the absence of 2,4-D, suggesting its lowered growth rate in this medium was not due to carry-over of 2,4-D with the inoculum. The percentages of cells in G 1, S, G2 and M and the mitotic stages did not differ
significantly between the two times (118 and 182 h) at which samples of each culture were labelled and fixed (fig. 3). The results wera thus pooled and the mean values are shown in table 1. Examples of the DNA distributions
Table 2. Durations of the cell cycle and mitosis and their constituent stagesin root-tips and suspension cultures of Daucus
Material Root tips Dipioid culture Batch fermentor +2,4-D Shake flask + 2,4-D -2,4-D Tetraploid culture Batch fermentor + 2,4-D Shake flask -2,4-D +2,4-D
Duration (hours)
of cell cycle and stages
Total
Gl
Duration (how
s
G2
M
of mitotic stages
prophase
metaphase
ana + telophase
7.5
1.3
2.7
2.9
0.6
0.3
0.1
0.2
51.2
39.6
3.0
6.2
2.4
1.2
0.4
0.8
50.9 32.8
2:
4::
5.0 8.5
1.7 1.1
0.8 0.6
0”::
0.6 0.3
44.9
35.4
2.1
4.7
2.7
1.3
0.4
1.0
57.8 81.6
46.5 64.5
:::
1.7
0.8 1.0
i::.
X5
Exptl Cell Res 92 (1975)
1::;
Cell cycle of Daucus 35
for the different lines and treatments are shown in fig. 4. The durations of Gl, S, G2 and M and the mitotic stages were 6.0 a
1
Fig. 3. Abscissa: time after inoculation (hours); ordinate: log,, cell number/ml culture. Semi-logarithmic plot of cell number against time for diploid (A) and tetraploid (B) cultures in the absence ( l ) and presence ( q ) of 0.1 mg/l2,4-D. The vertical dotted lines show the two time points at which samples were labelled and fixed. The following are the parameters of the fitted regression lines (b, slope constant; r, correlation coefficient): Diploid2,4-D absent, b, 0.00916~0.002; r, 0.983; diploid2,4-D present, b, 0.0059f0.0003, r, 0.982; tetraploid -2,4-D absent, b, 0.00369 +0.0002; r, 0.974; tetraploid - 2,4-D present; b, 0.0052 ~0.0003, r, 0.978.
OT 0
20
40
60
80
Fig. 2. (a) Abscissa: time after inoculation (hours); ordinate: log,, cell number/ml culture. Semi-logarithmic plot of cell number against time for 0, diploid and q , tetraploid suspension cultures. The vertical bars represent times of label addition. A mark sample times for labelling index determination. The following are the parameters of the fitted regression lines (b, slope constant; r, correlation coefficient): Diploid (-); b, 0.00588+0.0004; r, 0.980. Tetraploid (- -); b, 0.00670f0.0003; r, 0.990. (b) Abscissa: time after first labelad dition (hours); ordinate: % nuclei labelled. Increase in labelling index during continuous exposure of l , diploid, and q , tetraploid suspension cultures to SH-TdR. The following are the parameters of the regressions fitted to the points up to and including the 48 h values: Diploid (-a-); b, 1.80+0.17; r, 0.980, 100% intercept=48.2 h. Tetraplotd (--); 6, 1.99kO.31; r, 0.940, 100 % intercept =43.3 h.
calculated as before and the resulting time values are given in table 2. The results obtained both in the batch fermentors and in the shake-flasks showed that cultured Daucus cells have mean cell cycles greatly extended beyond the root-tip value due largely to an increase in Gl duration (table 2). However, both Gl and G2 occupied approximately constant proportions of the cell cycle in all undifferentiated suspensions with doubling times of greater than 45 h (table 1). In the embryogenie diploid culture, with a significantly shorter doubling time, the proportions of Gl and G2 nuclei changed significantly towards the values found in the root tips. Changes in labelling index generally correlated with cell cycle duration in culture to give an approximately constant S phase duration, equal to that of the root-tips for both diploid and tetraploid lines. In the shake flask experiment, mitotic index varied to give a constant mitotic duration in the fiptl
Cell Res 92 (197.5)
36
M. W. Bayliss
o-1
I
I
t
1
absence of 2,4-D. This culture showed a significant increase in the percentage of prophase nuclei accompanied by reductions in the percentages of the other mitotic stages (table 1). DISCUSSION
soD
0 1
A
Alli OS
to
20
40
‘ a0
Fig. 4. Abscissa: DNA content/nucleus(g x lo-IS); ordinate: no. of nuclei.
Examplesof frequency distributionsof nuclear DNA contentsin exponentialculturesof Duucus. A, diploid- 2,4-Dabsent;B, diploid- 2,4-D present; C, tetraploid-2,4-D absent;D, tetraploid- 2,4-D present.
undifferentiated suspensions and a duration significantly reduced towards the roottip value in the differentiating diploid suspension. In the batch fermentors, mitotic indices were significantly higher than those in shake flasks with similar growth rates, implying longer mitotic durations in the batch fermentors. Despite the variations in mitotic duration, the proportions of the different mitotic stages were constant in all material except the tetraploid suspension growing in the Exptl Cell Res 92 (197.5)
Analysis of the cell cycle in root meristems by pulse labelling with 3H-TdR has been used to give values for at least 30 plant species [4]. The precise mathematical analysis of results from pulse labelling experiments is complex [lo] and no single method is yet applicable to all data. The simple graphical analysis used here can, however, yield results compatible with those from more complex approaches (cf [2 and 51) albeit without estimate of error limits. Cell cycle information from most systems so far studied [6, 121 points to Gl as being the most variable phase of the cell cycle. Extensive data from different areas within root-tips of Zea [2] showed that those cells with the minimum cycle time had a short or non-existent G 1, whereas other areas of the root-tip contained cells with Gl durations of up to 135 h. Root-tips of most species probably exhibit a similar heterogeneity of cycle times and the interpretation of cycle durations obtained from squash preparations, where identification of different zones in the root-tip is impossible, must be based on the overall Gl durations produced. It seems reasonable to assume, as those cells with the shortest cycle will be the first to reappear in the second peak of labelled mitoses, that the data presented for Daucus root-tips, giving a total cycle time of 7.5 h and a Gl of 1.7 h, approaches quite closely the minimum cell cycle duration for this species at 25°C. These values are shorter than those previously reported for herbaceous plant species [4, 71 and probably reflect the
Cell cycle of Daucus
37
The 45-50 h doubling time obtained for extremely low DNA content (2C= 1.21 x the Daucus cultures grown in the presence lo-l2 g [S]) of Daucus. In suspension cultures of Daucus, the of 2,4-D seems to be a stable minimum evidence that cell number doubling time value (cf [15]) representing the limit of adapprovided a good estimate of mean cell tion of the cells to their cultural conditions. cycle time confirms similar findings [8] in Only when embryogenesis was induced in Acer suspension cultures where pulse labelling the absence of 2,4-D was the cell cycle experiments were also shown to agree with shortened, presumably in response to the continuous labelling and cell count data. greater degree of tissue organisation. An Though the continuous labelling experi- increased labelling index in embryogenic ments on Daucus did not give a final labelling cultures, probably due to a shortened cell index of lOO%, this does not necessarily cycle, has been previously reported for imply the unlabelled cells were outside the Daucus [19]. cell cycle. As no measurements were made The extension of the cell cycle in the of the life of 3H-TdR in the culture medium, tetraploid culture in the absence of 2,4-D, cells entering S phase a short time after the coupled with lack of embryogenesis, suggests last label addition, or cells with longer cell that though this line can be maintained cycles may not have had label available for indefinitely under these conditions, it is incorporation. The variation of cell cycle only partially autotrophic for auxin and is duration about the mean, indicated by the limited in growth rate by a limiting rate of continuous labelling data, does not seem endogenous auxin production. of sufficient magnitude to suggest the preThe constancy of S phase duration and the sence of some cells in culture with cycle limited extension of mitosis suggests that times as short as those of the root-tip. How- these processes are less affected by cultural ever, it remains a possibility that differences conditions than the other phases of the cell in mean cycle time between different experi- cycle. The constancy of mitotic stage promental conditions could be caused by changes portions in all but the most extended cell in the proportions of cells with different cycle suggests that once initiated, the procycle times. gramme of mitosis is relatively insensitive This contrast between cell cycles in vivo to the cellular and cultural environment. and in vitro observed for Daucus is similar The low frequency of abnormal mitoses to that shown in Haplopuppus [l, 7, IS], observed in Daucus cultures [3] may thus where extension of the cell cycle in suspen- reflect division in cells which are also unusual sion culture resulted mainly from elongation in other respects, for example in being highly of Gl, although S and G2 were also slightly vacuolated. extended. Mitosis had a similar duration to I gratefully acknowledge the help and advice of that in the root-tip. Variation in Gl was Professor H. E. Street and the technical assistance also the cause of the variably extended cell of Mrs Helen Morris. This work was supported by a grant from the Nuffield Foundation. cycle in Acer suspension cultures [8] and it seems probable from the generally low REFERENCES growth rates observed in plant cell cultures 1. Ames, I & Mitra, J, The nucleus 9 (1966) 61. that present cultural methods do not allow 2. Barlow, P W & MacDonald, P D M, Proc roy sot B 183 (1973) 385. plant cells to grow at their maximum attain3. Bayliss, M W, Nature 246 (1973) 529. able rates. 4. Bennett, M D, Proc roy sot B181 (1972) 109. Exptl Cell Res 92 (1975)
38 M. W. Bayliss 5. Cleaver, J E. Thymidine metabolism and cell cycle kinetics.‘Norih-Holland, Amsterdam (1967). 6. Eriksson. T. Phvsiol nlant 20 (1967) 348. 7. Evans, 6 &I, kees,‘H, Sneli, C ‘L & Sun, S, Chromosomes today 3 (1970) 24. 8. Gould, A R, Bayliss, M W & Street, H E, J exptl bot 25 (1974) 468. 9. Henshaw, G G, Jha, K K, Mehta, A R, Shakeshaft, D J & Street, H E, J exptl bot 17 (1966) 362. 10. Mendelsohn, M L & Takahashi, M, The cell cycle and cancer (ed R Baserga). Dekker, New York (1971). 11. Mitchison, J M, The biology of the cell cycle. Cambridge University Press (1971). 12. Murashige, T & Skoog, F, Physiol plant 15 (1962) 473.
Exptl Cell Res 92 (1975)
13. Ouastler. H & Sherman. , F G.,_Exntl cell res 17 (i959) 4io. 14. Sparvoli, E, Gay, H & Kaufman, B P, Caryologia 19 (1959) 420. 15. Smith. S M & Street. H E. Ann bot 38 (1974) 223. 16. Street; H E, Plant iissue’and cell cult&e. glackwell, Oxford (1973). 17. Van? Hof, J & Kovacs, C J, Advances in experimental medicine and biology 18 (1972) 15. 18. Wilson, S B, King, P J & Street, H E, J exptl bot 21 (1971) 177. 19. Wochok, Z S, Biologia plantarum (Praha) 15 (1973) 107. Received September 19, 1974
