Growth inhibition, mitotic cycle time and cell number in chronically irradiated root meristems of Pisum

Radiation Botany, 1963, Vol. 3, pp. 239 to 247. Pergamon Press Ltd. Printed in Great Britain.

GROWTH INHIBITION, M I T O T I C CYCLE TIME AND CELL NUMBER IN CHRONICALLY IRRADIATED R O O T MERISTEMS OF PISUM* J. V A N ' T H O F a n d A. H . S P A R R O W Biology Department, Broolthaven National Laboratory, U p t o n , N.Y.

(Received 2 May 1963) Abstract--Seedlings of Pisum sativum were chronically irradiated with gamma rays from a Co 6° source at daily exposure rates sufficient to produce growth inhibition. Exposure rates of 500 and 1000 r/day reduced root growth by the fourth day. In order to determine the factor(s) that might be responsible for the reduced growth rate, the following measurements were made : (1) the number of ceils comprising the root meristem was determined daily for seven days, (2) the percentage of aberrant cells at anaphase was scored after three days exposure, and (3) the minimum mitotic cycle time for meristematic cells was established, also after three days of exposure. Cell counts showed an approximate reduction of 5, 25, and 45 per cent compared to control on the third day of exposure at rates of 250, 500 and 1000 r per day. The minimum mitotic cycle time was measured by producing a tetraploid cell population with colchicine and noting the span of time between the production of this population and its initial appearance in the subsequent mitosis. These measurements indicated no change in the minimum cycle time of the cells in the irradiated merlstems as compared to the unirradiated controls. Analysis of cells at anaphase showed an increase in the number of cells with broken chromosomes with increased exposure rate. Therefore, the reduction in growth observed in irradiated seedlings was attributed to the loss of reproductive integrity not due to a general slowing down of the cells' division rate but rather to genetic loss and/or unbalance and mitotic arrest of meristematic ceils. R,~smm&--Des plantules de Pisum sativum ont ~t~ irradi~es d'une mani~re chronique par des rayons gamma d'une source de Co 6° ~ des d~bits de dose journaliers suffisants pour produire une inhibition de croissance. Des d~bits de dose de 500 et 1000 r/jour ont r~duit la croissanee radiculaire au quatri~me jour. Afin de d&erminer le ou les facteur(s) qui pourraient fitre responsables de la croissance rfiduite, les mesures suivantes ont ~tfi faites: (1) le hombre de cellules du mfirist~me radiculaire a ~tfi d&ermin~ pour sept jours; (2) le pourcentage de cellules aberrantes en anaphase a dt~ relevd apr~s le troisi~me jour d'exposition; (3) le temps minimum du cycle mitotique pour les cellules m~rist~matiques a ~t~ &abli 6galement apr~s le troisi6me jour d'exposition. Des comptages de cellules ont montrd une rdduction approximative de 5, 25 et 45 pour cent eomparativement au tdmoin le troisi6me jour de l'exposition apr~s des d6bits de dose de 250, 500 et 1000 r/jour. Le temps minimum du cycle mitotique a 6t6 mesur6 en produisant une population cellulaire t&raploide avec de la colchicine et en notant le ddcalage de temps entre la production de cette population et sa premiere apparition la mitose ult6rieure. Les mesures n'ont pas indiqu6 de changement darts le temps minimum du cycle mitotique des mdrist~mes irradids comparativement aux tdmoins non irradi6s. L'analyse des cellules ~ l'anaphase a montrd un accroissement avec la dose de cellules poss~dant des chromosomes cass6s. D~s lors, la rdduction de croissance dans les plantules irradi6es a dt6 attribude ~ la perte de l'int6grit6 de reproduction. Cette int6grit6 n'est pas due ~ la rdduction gdn6rale de la vitesse de division cellulaire mais plut6t ~t une perte g6n&ique et ou/par ddbalancement g6n&ique et arr& des cellules m6rist6matiques. *Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. 239

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GROWTH INHIBITION IN R O O T MERISTEMS OF PISUM Zus~mme~assmag--Keimlinge von Fisum safivum erhielten eine chronische GammaBestrahlung aus einer Co*°-QueUe. Die t/igliehen Bestrahlungsraten waren ausreichend, um das Wachstum zu hemmen. Bestrahlungsraten yon 500 und I000 r/Tag hemmten das Wurzelwachstum vom 4. Tag an. Um die Faktoren zu bestimmen, die die Hemmung verursachen, wurden die folgenden Messungen durchgeftihrt: (1) Die Anzahl yon ZeUen, aus denen das Wurzelmeristem besteht, wurde t/iglich w~ihrend 7 aufeinanderfolgender Tage ausgez/ihlt. (2) Der Prozentsatz aberranter Anaphasen wurde nach 3 t/igiger Bestrahlung festgesteUt. (3) Die Mindestzeit ftir einen Mitosezyklus bei meristematischen Zellen wurde bestimmt, ebenfalls nach 3-t/igiger Bestrahlung. Zellz/ihlungen (zu I) ergaben eine Reduktion von ca. 5, 25 und 45 Prozent gegeniiher den Kontrollen am 3. Tag der Bestrahlung mit 250, 500 und 1000 r pro Tag. Um die Mindestzeit ffir einen Mitosezyklus (3) zu bestimmen, wurde mittels Colchicin, eine tetraploide Zellpopulation erzeugt und die Zeitspanne bestimmt, die zwischen der Erzeugung dieser Population und ihrem ersten Auftreten in tier folgenden Mitose verstreieht. Die Messungen ergaben keine Unterschiede gegeniiber den KontroUen. Die Analyse yon Zellen in Anaphase (2) ergab einen Anstieg der ZeUen mit Chromosomenbrtichen bei ansteigender Bestrahlungsrate. Die Wachstumshemmung, die bei bestrahlten Keimlingen zu beobachten ist, wird deshalb auf eine St6rung der Teilungsf/ihigkeit zurfickgefiihrt, wobei diese St6rung sich nicht aus einer allgemeinen Verlangsamung der Zellteilungsrate herleitet, sondern vielmehr aus Verlusten von genetischem Material und/oder Mitosest6rungen und Mitosestillstand in meristematischen Zellen.

GROWTH inhibition resulting from acute or chronic exposure is a well-known radiobiological effect. The causes of growth inhibition produced by ionizing radiation have been attributed to chromosome deletion,el0) and changes in a variety of biochemical and physiological systems.(5) While it seems reasonable that growth inhibition could result from mitotic inhibition, such a relationship is difficult to demonstrate experimentally. For example, a decrease in the number of cells produced per unit time could be the result of either an increase in the duration of the average cell cycle or a reduction in the number of cells proliferating. The object of these experiments was to attempt to relate radiationinduced growth inhibition with three cellular parameters, namely, the number of cells per meristem, the minimum mitotic cycle time, and the frequency of cells with chromosome aberrations.

nutrient solution to about 3/8 in. anu thus minimized shielding by the solution. The plates and seedlings were placed in a controlled environment chamber, in the dark, at about 20°C, with continual aeration and were exposed to a Co 6° source for 20 hr/day. T h e doors of the chamber were opened randomly and intermittently to expose the seedlings to light thus eliminating the possibility of photo-induced synchrony of cell division. Control seedlings were grown under similar conditions in a growth chamber which received no radiation.

Mitotic cycle measurements

T h e full details of the technique used are published elsewhere~O1,1~) In general it involves the production of a tetraploid population of ceils in diploid roots. T h e tetraploid or tagged cells are produced by a 30 min treatment with colehieine at time zero. T h e affected cells carry out karyokinesis but no cytokinesis and then MATERIAL A N D M E T H O D S proceed into interphase. T h e time between the Pea seedlings (Pisum sativum, var. Alaska) production of these 4n ceils and their appearance were germinated at approximately 21°C for in the following division is a measurement of three days in vermiculite moistened with dis- the m i n i m u m mitotic cycle time. Any changes tilled water. Seedlings with a primary root of produced by various treatments can be detected 2-3 cm in length were placed in plastic bags by comparing the characteristics of the control containing Hoagland's nutrient solution. The 4n population with that of the treated cells. bags were made rigid by inserting a plexiglass T h e experiments described below were generplate which reduced the depth of the bag and ally performed in the same manner with respect

j. VAN'T HOF and A. H. SPARROW to germination and conditions in the growth chamber. Specifically, however, each experiment was slightly different. I n the first experiment the seedlings were removed from the growth chamber to the laboratory for cycle time determination after three days of chronic exposure. A colchicine concentration of 5.02 X 10 -4 M was used to produce a 4n cell population. In the second experiment, cycle time determination was performed in the growth chamber with continuous irradiation. Consequently these seedlings were exposed to more radiation than those of the first experiment. T h e third experiment was performed like the first and second with the exception that a lower concentration of colchicine was used (4"39 X 10 -~ M) and the distribution of the tetraploid populations was measured. The data from these experiments are expressed as the polyploid index, which is the number of dividing tetraploid cells per thousand cells. Each index is the average of data from three slides, each slide contained one meristem (the terminal 2 ro_m), and a thousand cells were counted per slide.

Cell counts These were performed on material grown in the same growth chamber under the conditions mentioned above. G a m m a irradiation was chronic and three samples were removed per day for counting. To obtain cell counts, the root meristem was defined as that portion which showed a vivid Feulgen stain. T h e meristem defined in this manner includes differentiated cells such as those of the root cap as well as proliferating cells. T h e demarcation between deeply stained and lightly stained portions was determined by placing the root on frosted glass which was illuminated from beneath. T h e deeply stained portion was removed and placed in the well of a concave slide and macerated. T h e separated cells were suspended in clear Schiff's reagent and cell counts made with a haemocytometer.

Growth measurements Seedlings were cultured under the same conditions mentioned above. Root length measure-

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ments were made daily on ten seedlings per dose.

Chromosome aberration T o obtain an estimate of the amount of genet?c damage produced by exposure to radiation, dividing ceils were scored at anaphase for chromosome aberrations. I t was understood that this analysis would not produce data about the type or number of breaks per cell. The data obtained, however, would produce information concerning the percentage of cells damaged by irradiation. Analyses were made on Feulgenstained cells from meristems (the terminal 2 m m ) that had been treated with 5"02 × 10 -4 M colchicine 10 hr before fixation. To determine the percentage of damaged cells, fifty anaphases were scored from each of three meristems per dose rate. R E S U L T S A N D DISCUSSION

Figure 1 shows the progressive decrease in size of the Feulgen-stained portion of the root. There was little difference in the size of the stained pprtion after one day of irradiation (Fig. l d). After three, four, and seven days a gradual decrease in meristem size of irradiated roots was discerned. Following three days of irradiation the size of the stained portion decreased with the dose rate or cumulative exposure. A control at day 7 was eliminated because it had well:developed lateral roots and thus represented a somewhat different system of growth. Cell counts were made to obtain a more precise measurement of the meristem size. Counts made on control meristems showed a gradual decrease in number with time, an observation which substantiated other experiments performed with Pisum.a~ The change in the number of ceils in the Feulgen-stained portion of irradiated roots expressed as per cent of control is represented in Fig. 2. Any significant decrease in cell number in a meristem would be expected to be reflected.in gross measurements such as length. Fig. 3 shows the increase in root length with time in control and irradiated seedlings. T h e data were expressed as a ratio of the length at day 0 to that of day 1, 2, 3, and 4. D a t a expressed in this

242

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was determined on the third day of irradiation at dose rates of 250, 500, and 1000 r/day. T h e intercepts of the vertical line in Fig. 2 show the relative cell number per meristem per dose rate at the time cell cycle measurements were performed. Seedlings were treated with colchicine, thereby tagging or marking all cells that passed through metaphase while colchicine was effective. At the subsequent metaphase these cells were tetraploid and their appearance in the irradiated seedlings occurred at the same time as in the unirradiated control. At 10 and 12 hr after colchicine tagging no significant difference between the means of control and irradiated samples was detected by the "Student" t test at the 0.05 level. I f an appreciable elongation of the cycle had occurred due to irradiation, it should have been revealed at these times. I t is possible that the change from diploi~ to tetraploid m a y have had an advantageous effect on injured cells and thus prevented an increase in m i n i m u m cycle time. This possibility is highly improbable, however, for the following reasons: 3.5

I I I I FIO. 2. Reduction in the number of c.ells in the Feulgen-stained portion of chronically irradiated pea / , root tip meristems expressed as per cent of control. / CONTROL The vertical line indicates the time at which mitotic 3.G cycle time was measured. manner eliminated any differences that m a y / .Y have existed when the experiment was initiated. The ratio for the control at day 3 was 3.39-J-0.16 O while those of 500 and 1000 r/day were 2"644Y i '~ 2.5 " 0"24 and 2.56-4-0.17 respectively. The expression of decreased length, therefore, lagged one or two days behind that of visible decrease in meristem size (Fig. 1) as well as cellular reduction (Fig. 2). '-' 2.0 The apparently unaltered increase in length during the first and second days of irradiation was probably due to cells that had composed the elongation area of the root at the time the 1.5 experiment was initiated. Once these cells enlarged they were not replaced, thus resulting in an observable inhibition of growth. This apparent lack of effect on cell elongation substantiates the concept that cell enlargement is I.O~ I l I i I 2 3 4 relatively insensitive to doses of radiation that DAYS OF CHRONIC y l R R A D I A T I O N (n} produce observable effects on proliferating Fxo. 3. Change in the length of roots of pea seedlings cells.(4, s) expressed as a ratio of the length after n days of T h e m i n i m u m duration of the mitotic cycle chronic irradiation to the length at day zero.

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Fro. I. The size of the Feulgen-stained portion of pea roots after being exposed to chronic irradiation for various lengths of time. Reading from left to right : ld, one day of exposure, control, 250 r/day, 500 r/day, 1000 r/day; Bd, three days of exposure, 250 r/day, 500 r/day, 1000 r/day; 4d, four days of exposure, control, 250 r/day, 500 r/day, 1000 r/day; 7d, seven days of exposure, 250 r/day, 500 r/day, 1000 r/day.

J. VAN'T HOF and A. I-I. SPAILR.OW (l) colchicine tagging took place at metaphase after the cells had been exposed to irradiation for approximately six complete cycles, a length of time sufficient for the establishment of altered cycle rates, (2) it was unlikely that a tetraploid condition would accelerate the cell cycling rate but rather decrease the rate, and (3) since the diploid cells w e r e randomly tagged with colchicine the marked population would include cell having damaged chromosomes and a permanent impairment possessed by the diploid cells at the time of colchicine treatment would be replicated in the tetraploid cell T h e data in Fig. 4 are from an experiment conducted in the laboratory without chronic irradiation. I t seemed possible that the unaltered cycle time might have resulted from a compensatory flush of divisions as observed with acute irradiation.(4) To ascertain whether or not such events were taking place, another experiment was performed in the growth chamber with chronic irradiation. Again, as in the previous experiment, cycle time measurements were commenced on day 3. The results substantiated those of the previous experiment in that the duration of the nuclear cycle was approximately

243

the same in irradiated as in the unirradiated control meristems (Fig. 5). T h e "Student" t test at the 0.05 level showed no significant difference between the means of the control and irradiated meristems at 10 hr. There was a significant difference between the means of control and 1000 r/day at 12 hr but it was not detected at 14 hr. I f the cycle were elongated by 1000 r/day, a significant difference would have been noted at 10, 12 and 14 hr. T h e same argument applies to the results from seedlings exposed at 250 r/day. In this case the means were the same at 10 and 12 hr but were different at 14 hr. T h e drop in polyploid index at 15 hr in meristems exposed to 250 r and 1000 r/day was probably due to the fact that most of the tetraploid cells had divided and these decreased indices represented the tailing portion of the tagged cells. Such a decrease was not only inevitable, but desirable in instances where some notion of the population distribution was the object of experimentation as in Fig. 6. Perhaps it is appropriate at this time to inject a word of I

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Fio. 6, The appearance and distribution of two tetraploid populations in the first division after colchicine tagging. caution about comparing the polyploid indices from various experiments. Such comparisons should not be made because the cytological effectiveness of colchicine appears to vary from batch to batch and from container to container. Therefore, the only bona fide comparisons are those made within an experiment. Figure 2 indicated a decrease in meristematic cells in roots of exposed seedlings. Therefore, it seemed reasonable that the size of the tagged population likewise should be decreased. T o test this hypothesis an experiment was performed similar to the previous two experiments with the exception that less colchicine was used (see Material and Methods) and samples were taken over a greater span of time. The experiment consisted of unirradiated controls and seedlings exposed to 1000 r/day for three days. The results (Fig. 6) showed no difference in cycle time but a large difference in the number of tagged cells. No evidence supporting an alteration in minimum cycle time due to irradiation was produced. There was also no evidence suggesting that cells which normally have a cycle time longer than that of the average cell were

preferentially affected by the gamma rays. However, if these slower cycling cells were preferentially affected, it would be difficult to evaluate what effect this would have on the reduction of cells per meristem and on growth inhibition. Perhaps the best estimate would be obtained by means of an index such as t h e ratio of the number of proliferating cells to the average cycle time of these cells. A given set of indices for cells having unequal cycle times plus an estimate of the number of cells cycling at a given rate would permit an approximation of the relative contribution made by each group of cells to the total cell population per unit time. Table 1 illustrates this point. Table l a indicates the number of cells contributed to a subsequent proliferating population by four subpopulations equal in cell number but unequal in cycle time. After one cycle the proliferating population is completely altered in constituency. Subpopulations B, C, and D have contributed fewer cells due to their longer cycle time relative to subpopulation A. Table la shows also that after the subpopulation having the shortest cycle time completed a single cycle, a steady-state condition was established. In this instance, the steady-state is defined as that process wherein the products of a single division, on the average, result in one daughter cell undergoing differentiation and the other daughter cell retaining reproductivity. Table lb shows the net increase in cell number produced by each subpopulation. The sum of all the cells produced results in growth. This increase in total number of cells is shown graphically in Fig. 7. Table l c shows the percentage of cells contributed by each subpopulation to the total number of cells f o r m e d after each minimum cycle. The percentage of cells produced per subpopulation per cycle is a constant and is a function of the ratio of the subpopulation's cycle time to the minimum cycle time (Fig. 8). Consideration of this theoretical population indicates that the general meristematic cell population is composed mostly of cells having the shortest cycle time and that growth is primarily the result of cells produced by this population. The information in Table 1 also reveals that an increase in cycle time of cells which normally pass through the cycle at less than average rates

j . VAN'T HOF and A. H. SPARROW

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Table 1a. A theoretical heterogeneouspopulation of proliferating cells and the relative contribution of cells with different cycle times

Subpopulation

Number of new proliferating cells in subpopulations after completion of each cycle (Cr)*

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Number of cycles (C/-) A B

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*Meristems represent a cell proliferating system that increases linearly. Therefore, the product of a single division, on the average, results in one daughter cell undergoing differentiation and the other remaining reproductive. would produce a rather minor reduction in the cell n u m b e r of the total population. T h e conclusions drawn from this model assume that the number of ceils in each subpopulation is initially equal. I t is probable that the n u m b e r of ceils per subpopulation would be greater for those with longer cycle times. This would tend to equate the net cell production of a subpopulation having relatively t~w cells with a short cycle time and a subpopulation having m a n y ceils with a long cycle time. T h e curves in Fig. 6 indicate that disproportion of cell number probably does not exist. I f the faster cycling ceils were fewer in n u m b e r the initial p a r t of the control curve would be less steep and the peak would be observed at 16 or 18 hr. This peak would represent the large n u m b e r of relatively

slow cycling cells. Thus, an adequate explanation of growth inhibition should apply to all cells irrespective of cycle time. An explanation that seems acceptable is that of genetic loss due to chromosome damage. Such damage has previously been proposed as a factor involved in growth inhibition.(2, 8. s-z0) Assuming that chromosome damage reflects genetic damage, meristems exposed to 250, 500 and 1000 r/day for three days were analyzed for aberrant anaphases. The results showed an increase in the percentage of damaged cells with increased dose rate (Fig. 9). T h e high percentage of abnormal anaphase cells in the control was probably due to the colchicixte tagging which all seedling received. In similar nonirradiated seedlings that were not treated

GROWTH INHIBITION IN ROOT MERISTEMS OF PISUM

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Fzo. 8. Percentage of cells produced by various subpopulations of proliferating cells having a different mitotic cycle time. with colchicine the percentage of abnormal anaphase cells approached zero. The question remains how the reduction in cell number was produced. At least two explanations should be considered. The first is cell death due to chromosome damage. I f chromosomes are fragmented during interphase in such a manner that unequal distribution of the genetic material takes place at the subsequent mitosis, there is a high probability that either one or both of the daughter cells would be genetically deficient and death or loss of reproductive integrity would result. The relationship between the decrease in dry weight and the increase in chromosome damage with dose supports this

concept.(a) An addition to these data is the interesting observation on Luzula, a plant whose cells contain chromosomes with diffuse centromeres. The diffuse centromeres reduce the amount of genetic material normally lost or unequally distributed following irradiation and therefore this plant is m u c h more radioresistant than would be expected when other nuclear parameters are considered.(S, z°) A second cause of a reduction in cell number could be the production of permanent interphase (mitotic arrest) in a portion of proliferating ceils. This effect could be considered as either the m a x i m u m in cycle time elongation or as the result of genetic damage or finally as the consequence of radiation-induced changes in other systems.(5) I f a cell was damaged so that it could function in almost every way but could not divide, it would contribute nothing to continual growth or population size. Although it is unlikely that these two factors would be equally responsible for the reduction of cells in the meristem, both

J. V A N ' T H O F and A. H. S P A R R O W possibilities r e q u i r e investigation for it w o u l d be very interesting to k n o w the relative c o n t r i b u t i o n of each factor to g r o w t h i n h i b i t i o n a n d tissue atrophy. S i m i l a r e x p e r i m e n t s to those m a d e w i t h Pisum have been r e p o r t e d for intestinal c r y p t cells o f mice~ 7) a n d rats.(6) A t a dose r a t e o f 12 r / d a y for 200 days, the cycle t i m e in m i c e was s h o r t e n e d b y 3 h r a n d n u m b e r o f c r y p t cells slightly r e d u c e d . A t 415 r a t s / d a y , u p to five days, similar cells in the r a t were u n a l t e r e d in cycle time, b u t the n u m b e r o f cells was reduced. T h e cell decrease in the intestinal crypts o f the r a t was n o t as extensive as t h a t observed in Pisum. H o w e v e r , this m a y be d u e to the t e m p o r a r y n a t u r e o f the p r i m a r y meristem. T h e m e r i s t e m of Pisum is n o t a steady-state system w h e n considered over a p e r i o d o f several days for there is an obvious decrease in cells.O) Therefore, the f o r m a t i o n of a r a d i o r e s i s t a n t p o p u l a t i o n such as t h a t in the r a t intestinal crypts was i m p r o b a b l e because the p r i m a r y m e r i s t e m n o r m a l l y diminishes in size a n d t e r m i n a t e s following the full p r o d u c t i o n o f l a t e r a l roots. I n conclusion, the e x p e r i m e n t a l results indic a t e d t h a t g r o w t h i n h i b i t i o n was d u e to a r e d u c t i o n in the n u m b e r o f m e r i s t e m a t i c cells. T h e decrease in the n u m b e r o f m e r i s t e m a t i c cells was n o t a t t r i b u t e d to a l e n g t h e n e d m i t o t i c cycle b u t r a t h e r to m i t o t i c arrest. M i t o t i c arrest was considered to be the result o f genetic loss or u n b a l a n c e a c c o m p a n y i n g c e r t a i n kinds of c h r o m o s o m e a b e r r a t i o n s .

Acknowledgements--The authors wish to thank Miss ALEXANDRAJAHN for her aid in the performance of the experiments, to Dr. K~.ITH THOMPSON for his advice concerning the statistics, and to Dr. GRACE M. DONNELLY, Mr. LLOYD A. SCH~UP.ER and Miss VIROINIA POND for their aid in preparing the manuscript. REFERENCES 1. BROWN R. (1951) The effects of temperature on the division of different stages of cell division in the root-tip. 07. Exptl. Botany 2, 96-110.

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