Thymidine and The Measurement of Rates of Mitosis in Meristems

New Phytol (1974) 73, 919-925.
THYMIDINE AND THE MEASUREMENT OF RATES

OF MITOSIS IN MERISTEMS
BY C . DE LA TORRE AND F . A. L. CLOWES
Botany School, University of Oxford
{Received 25 January 1974)
SUMMARY
A series of pulses of [^Hjthymidine was used to measure cell flow through the mitotic cycle in
four regions of the root meristem of Zea. For comparison, colchicine was used to measure cell
flow after the supply of pulses of non-radioactive thymidine. Thymidine itself does not, at
normal labelling concentrations, disrupt the pattern of cell proliferation within the meristem
although it does so in other circumstances. [^HJthymidine, in contrast, does, at labelling concen-
trations, change the behaviour of the meristem, lengthening cell cycles in the regions normally
with rapid cell division and accelerating cell cycles in the quiescent centre. The significance
of this disruption is discussed in relation to the use of thymidine in investigating the cell kinetics
of meristems.
INTRODUCTION
Tritium-labelled thymidine is widely used to measure the rate of mitosis in meristems.

Its behaviour is well known (e.g. Cleaver, 1967) and it has several advantages over other
precursors of DNA. Although not on the direct pathway of normal DNA synthesis, it is
incorporated, via its monophosphate, into little except nuclear DNA in the normal
DNA-synthetic period {S) of most proliferating or endoreduplicating cells. Experience
shows that we can neglect endoreduplication, metabolieally labile DNA and gene
amplification as sources of errors of interpretation so long as we consider only cells
within meristems. Apical cells of ferns are possible exceptions for these appear to behave
differently from any cells in the root meristems of higher plants (Avanzi and D'Amato,
1970). The disadvantages are that not all cells make thymidine available to their nuclei,
not all have a thymidine kinase, though one may be induced by supplying the substrate,
and that the energy of decay is given largely to those nuclei that incorporate the com-
pound and so may cause differential damage in cell populations.
However, ever since Quastler and Sherman (1959) used [^H]thymidine to pulse-label
dividing cells this has been the favourite method of obtaining data about the time para-
meters of the mitotic cycle (for a critical evaluation see Mendelsohn and Takahashi
(1971)). In many systems it has been shown that the supply of [^HJthymidine to an
organism over a short period constitutes a pulse since removing the supply ends the
availability of the precursor. This is true of root tips placed in the labelled solution for
30 min or less and then returned to an unlabelled solution. The fraction of all cells that
is labelled remains constant over at least 24 h in Zea (Clowes, 1971) showing that labelled
thymidine is not made available to the meristem even if there is a leak of radioactivity
from non-meristematic regions after the end of the pulse. The pulse-labelled mitosis
curve enables us to estimate the duration of all the phases of the cell cycle and we may
919
920 C. DE LA TORRE AND F . A. L. CLOWES
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also score the same meristem to find the fraction of cells in cycle (growth fraction). Such
confidence in the technique exists that elaborate statistical methods have been invented
to match data with theoretical curves (e.g. Macdonald, 1970; Gilbert, 1972). Such
treatments are needed because there are no better ways of obtaining some of the time
parameters of the cycle: this also means that fitting data to a theoretical average cycle is
a somewhat subjective exercise. Cells in a population behave in such a complex manner
that any estimates that we make of their time parameters are necessarily simplifications
and no calculations elucidate the true position.
There' have been reports that thymidine itself upsets the cell kinetics of populations
and some that it has no important effects (Barr, 1963; MacLeod, 1971). A more serious
possibility is that radioactivity upsets cell kinetics. Clowes (1961) has shown that high
doses of [^Hjthymidine (20 //Ci/ml) can cause most of the meristematic cells of a root
tip to stop dividing. After such treatments the root survives and renews growth by the
establishment of a new meristem derived from the meristem's quiescent centre. The
cells of the quiescent centre survive the treatment because their rates of cycling are low
and only a few cells incorporate the thymidine whereas cells elsewhere in the meristem
suffer from the ^-radiation in their nuclei. The nature of the stimulation of the quiescent
centre is not fully understood, but the relief from pressure exerted by the expansion of
the surrounding cells and enhanced nutrition due to reduced demand elsewhere possibly
combine with a direct stimulation by the radiation during feeding to force quiescent cells
into cycle as after X-irradiation (Clowes, 1970, 1972)-
Twenty /iCi/ml is a concentration not commonly reached in labelling experiments,
but pulse-labelling often uses i /iCi/ml and there is a possibility that the pattern of cell
proliferation in a tissue is upset by the technique. To find out, we have used a triple
pulse method for measuring the cell fiow in different regions of a root meristem and we
have used metaphase accumulation to test the effect of non-radioactive thymidine.
METHODS
Primary root tips from Zea mays (cv. Golden Bantam) seedlings grown at 23° C in
darkness were used. Labelling was carried out by immersing the roots in an aerated
solution of [6-^H]thymidine (0.5 /iCi/ml and 23.3 Ci/mM) for 20 min, washing, growing
in aerated water for 3 h, relabelling for 20 min and repeating the growth period and
labelling pulse. Mitosis is not synchronized by such treatments in Zea. Roots were
sampled at the end of each of the three pulses and autoradiographs made from 5 /im
longitudinal sections of the apices on stripping film. Four regions of median sections
were scored for labelled and unlabelled interphases and mitoses and the fraction of cells
labelled after the three pulses was used to estimate the rates of mitosis in each region
between the first and second and between the second and third pulses.
The first pulse labels all cells in ^S; the second labels those cells that have entered S in
the 3.33 h since the start of the first pulse as well as continuing to label cells still moving
through iS (3 h was chosen to give an inter-pulse period shorter than S). The difference
in the fraction of cells labelled after the first two pulses enables us to calculate the cell
flow through the mitotic cycle and hence the rate of division. The duration of the
mitotic cycle (T) so obtained is the average for all cells including non-cycling cells; it is
therefore the cell-doubling time and comparable with the value obtained by metaphase
accumulation which was the method used to measure T after subjecting roots to un-
labelled thymidine. To obtain a cycle duration equivalent to that derived from the pulse-
Mitosis in meristems 921
labelled mitosis curve we have to multiply the cell-doubling time by the fraction of cells
with a fast cycle, for only these contribute to the curve. The number of cells with fast
cycles may be estimated by measuring the areas under plots of the fractions of cells that
are in mitosis and in labelled mitosis at intervals over a cell cycle after a pulse (Clowes,
1971). The times spent in DNA synthesis and mitosis may be derived from T and the
initial labelling and mitotic indices assuming either exponential or steady-state kinetics.
For the effect of non-radioactive thymidine on the cell cycle, colchicine was used to
accumulate metaphases over 3 h after growth in thymidine. Accumulation between i and
3 h was used to calculate T directly from cell flow. Used thus, colchicine does not alter
entry into prophase and there is no escape from metaphase in Zea. In one set of experi-
ments the roots were grown for 20 min in thymidine at a concentration of 5.2 /ig/litre
and then transferred to a 0.05% colchicine solution and sampled after o, i and 3 h. In
another, after the first period in thymidine the roots were transferred to water for 3 h
and then replaced in thymidine for a second 20 min before colchicine treatment. The
concentration of thymidine used (2.15 x 10"^ M) is similar to that used for pulse-labelling
with [^Hjthymidine. Continuous treatments with unlabelled thymidine over 3 and 40 h
at 3.3iox~^ M were also used for metaphase accumulation to match those used by
MacLeod (1971) in his investigation of thymidine kinase in roots of Victa faba.
In all the experiments the regions of the meristem scored were the cap initials (CI),
which are the normally most rapidly dividing cells in the root lying at the proximal
boundary of the cap, the quiescent centre (QC) of the meristem at the pole of the stele
and cortex, the stele just above (STi) and at 200 fiin above (ST2) the quiescent centre.
Each sample was of twenty roots.
RESULTS
The percentages of cells labelled with [^Hjthymidine (labelling index) and in mitosis
(mitotic index) after each of the three pulses and the percentages of cells in metaphase
after i and 3 h in colchicine after one and two pulses of non-radioactive thymidine at the
labelling concentration are given in Table i. Table 2 sets out the durations of the
mitotic cycles calculated as cell-doubling times from these observations and also from
the pulse-labelled mitosis method and after the treatments with thymidine at higher
than labelling concentrations.
In the multiple pulse experiment the labelling index increases after each pulse more
in the cap initials and less in the quiescent centre than in the stele. In calculating T we
have assumed that the cells proliferate exponentially. This is probably the best assump-
tion when dealing with small regions of the meristem and measuring over small fractions
of the cell cycle as here. Hence for example, if LI^ is the labelling index after the first
pulse and LI2 the index after the second,
3.33 X IOO xln2
for the first interpulse interval and a similar equation is used for metaphases by a direct
cell flow method. The method of Evans, Neary and Tonkinson (1957) uses a more sophis-
ticated approach which involves measuring the proportion of cells between metaphase
and telophase before treatment with colchicine. The values for T derived from the same
metaphase accumulations but calculated by the method of Evans et al. for roots after one
922 C. DE LA T O R R E AND F . A. L. CLOWES
Table i . Data {±standard errors) obtained by pulse-labelling with
thymidine and by accumulating metaphases after thymidine treatments in roots
of7jQ2i mays at 2.1° C. The four regions of the meristem scored are the cap initials
{CI), the quiescent centre {QC), the stele just above the quiescent centre (STi)
and the stele 200 fim above the quiescent centre {STz)
CI QC STI ST2
(a) •'H-thymidine-labelled roots
Labelling index, first pulse 22.1 ±2.1 IO.I ±1.6 20.3 ±1.4
Labelling index, second pulse 40.6 ±3.0 ii.4±i-3 34.6 ±2.0 32.3 + 1.9
' Labelling index third pulse 49.9 ±2.6 16.I±1.2 4I.2±2.I
Mitotic index first pulse 7-4±i-S 2.8±o.8 6.7±o.6 6.9 ±0.7
Mitotic index second pulse 3.6±o.7 i.8±o.7 4.6 ±0.8 5-3±o.9
Mitotic index third pulse i.4±o.4 1.3 ±0.4 3-7 ±0.4 3-5 ±0.4
(b) Colchicine-treated roots
( i ) 2 . i 5 X i o ~ * M thymidine 20 min
Metaphase index at i h 3.1 ±0.6 0.8 ±0.4 i.3±o.8 i.8±o.5
Metaphase index at 3 h II.6±2.O i.4±o.7 6.7± 1.0 7.8±o.Q
(2)2.i5Xio~^M thymidine 20 min
water 3 h, thymidine 20 min
Metaphase index at i h i.o±o.4 i.o±o.4 0.6 ±0.3 i.6±o.5
Metaphase index at 3 h 7.8 ±0.9 i.8±o.5 7-7±i.3
pulse of unlabelled thymidine at 2.15 x 10 ^ M are similar (17.3, 231.0, 27.9 and 24.8 h
compared with 16.3, 231.0, 25.7 and 23.1 by the direct method for CI, QC, STi and
ST2 respectively). The values for T given for the pulse-labelled mitosis method are not
those normally calculated, which refer to fast cycling cells, but those calculated from the
width of the first peak of labelled mitoses and the initial labelling index and they are
cell-doubling times (Clowes, 1971).
The results obtained after a single 20-min pulse of thymidine, radioactive or not,
show T to follow the same pattern within the meristem as in untreated roots. The cap
initials divide faster than any other cells and a quiescent centre is maintained. The actual
rates of mitosis are within the range found at 23° C in untreated roots by metaphase
accumulation or from pulse-labelled mitosis curves. These rates vary slightly with the
Table 2. Durations of complete mitotic cycles {cell-doubling times in

hours) calculated by cell flow methods after multiple pulses of[^H]thymi-
dine and after colchicine treatment and by the pulse-labelled mitosis
method in the four regions of the root meristem given in Table i
CI QC STI ST2
(a) [^H]thymidine-labelled
Between first and second pulse 12.5 i77-S 16,1 15.8
Between second and third pulse 24.8 49.1 35.0 26.2
Pulse-labelled mitosis method (calculated as
cell-doubling time) 15.i 182.i 23.9 17.6
(b) Colchicine-treated
After 20 min in 2.15 X10""^M thymidine 16.3 231.0 25.7 23.1
After two 20-min pulses of2.i5Xio~^M
thymidine separated by 3 h 20 •3 173 •3 29 •4 2 2 •7
24 h after 3 h i n 3 . 3 X i o ~ ^ M thymidine 2 1 •3 77 . 0 17 •5 1 2 •7
After 4 o h i n 3 . 3 X i o ~ ^ M thymidine 27 . 2 63 . 0 2 2 .0 26 •7
Controls in water 16 •5 185 •7 21 •4 2 2 •4
age of the seed and with other conditions of growth: the important observations are on
the relationships between the regions of the meristem.
Cell flow, however, changes considerably after the second pulse of [^HJthymidine in a
manner that is characteristic of these meristems when injured. The rate of mitosis drops
in the regions that are normally meristematic and rises in the quiescent centre.
DISCUSSION
De la Torre, Gonzalez-Fernandez and Gim^nez-Martin (1971) point out the advantages

of using cell flow rather than the pulse-labelled mitosis method when using radioactive
precursors of DNA to measure rates of mitosis. The cell flow method is quicker and
therefore less damaging to the cells as well as being less laborious. Their method of
calculating a rate of mitosis differs from the one presented here because they used whole
meristems in which steady state kinetics may more appropriately be assumed to apply.
The pulse-labelled mitosis curve has the advantage of providing a direct estimate of S
and of G2 and the labelled material can also yield a value for the growth fraction. The
duration of the cell cycle determined from the pulse-labelled mitosis curve applies only
to those cells with relatively fast cycles whereas cell flow methods provide cell-doubling
times because they count non-cycling cells as well as cycling cells. In the quiescent centres
of roots the growth fraction may be quite small (0.4-0.5 in Zea at 23° C) and there is
therefore a large difference between the cycle times for the relatively fast cells and for
the population as a whole. The cycle times for Zea grown in the same conditions as here,
but calculated from the pulse-labelled mitosis curve are 10, 40, 14 and 14 h for fast cells
displayed by the curves for CI, QC, STi and ST2 respectively (Clowes, 1971).
The double pulse technique used by Wimber and Quastler (1963) is essentially a cell
flow method, but it uses two isotopes with different ^-particle energies. It is possible to
distinguish the labelled cells from the scatter of silver grains in the double autoradio-
graphic film. The basic calculation here is that of S and it is necessary to score labelled
mitoses to obtain a value for T. The method therefore has some of the advantages and
disadvantages of both the pulse-labelled mitosis curve and of the simple cell flow method
that we have used here. With plant cells it is also possible to use caffeine to measure cell
flow as Gim6nez-Martm et al. (1971) have done.
The results obtained here by measuring the durations of the cycles after pulses of
non-radioactive thymidine at labelling concentrations suggest that the change in the
organization of the meristem after radioactive pulses is due mainly to the tritium rather
than the thymidine. The effect of a second 20-min pulse of unlabelled thymidine is small
though possibly indicating slight toxicity. The effect of a second 20-min pulse of labelled
thymidine is to lengthen cycle durations in the normally meristematic regions by factors
of 2.0 in CI, 2.2 in STi and 1.7 in ST2 and to shorten the cycle in the quiescent centre
by a factor of 3.6. We have no means of determining exactly how these changes are
brought about, but probably changes in the growth fraction as well as changes in the
cycles of dividing cells occur.
We cannot use the growth fractions previously calculated for roots of Zea growing at
23° C to compare the rates of mitosis in cycling cells only since the change in the average
duration of the mitotic cycle after the second pulse of [^H]thymidine could itself be
brought about by a change in the fraction of cells cycling. However, if we use the
fractions of cells with fast cycles calculated by Clowes (1971) and the cell-doubling times
after the first pulse, we obtain cycle durations of 8.3, 30.2, 9.5 and 12.6 h for the four
924 C. DE LA TORRE AND F . A. L. CLOWES
regions Cl, QC, STi, ST2 respectively. These are equivalent to the cycles measured on
pulse-labelled mitosis curves, but are shorter possibly because of the shorter radiation
effect.
The changes in cell flow after the second pulse of [^H]thymidine show how damaging
the j5-radiation is to the meristem even when the thymidine is supplied as a pulse of 20
min. The changes are of the magnitude observed within i h of the meristem receiving a
large dose of X-rays (Clowes, 1972). But the mechanism causing the change must be
different, for X-rays hit all cells and, apart from statistical considerations, the only
differential effects within the meristem are due to the cycle phase and the nature of the
cells. In ^-irradiation these differences are enhanced by the quiescent centre having a
smaller proportion of its cells subject to the radiation during the periods between pulses.
A point worth noting is the fall in the mitotic index throughout the meristem during
the course of the pulse-label experiment. In the quiescent centre this is accompanied
by a rise in the rate of mitosis from 0.006 to 0.020 per h and is due to a shortening of
mitosis greater than the shortening of the rest of the cycle from 7.2 to 1.3 h.
Several workers have commented on the effect that [^HJthymidine has on the cell
cycles it is employed to measure. Moser (1967) found that it alters the rate of DNA
synthesis and modifies the chronology of the cell cycle in mouse ascites mast cells.
Wimber and Quastler (1963) found the 'normal' cycle disturbed if the labelling solution
amounted to 4 /iCi/ml for 30 min in whole root tips of Tradescantia. MacLeod (1971)
showed that thymidine kinase could be induced by supplying roots of Vicia faba with
thymidine at 3.3 x io~^ M. This also stimulates the incorporation of thymidine into
DNA, but has no effect on root elongation. Barr (1963) found that thymidine prolongs
metaphase at 2 x io~^ M, but has no effect on root growth in Hordeum vulgare at io~^ M.
He ascribes reports of a stimulation of mitosis by thymidine as due to the illegitimate use
of the mitotic index to indicate rates of mitosis. Our own results on Zea show that pulses
of unlabelled thymidine at normal labelling concentrations (2.15 x 10"^ M) do not upset
the organization of the meristem, but at the higher concentrations used by MacLeod on
Vicia we find in Zea a slight slowing of cell division in the cap initials and an appreciable
acceleration in the quiescent centre. This is a much more sensitive test of disturbance
in mitotic cycles than has been used by other workers and the average rate of mitoses
over the whole meristem could well be unchanged.
Since most meristems in both animals and plants consist of complexly organized
populations of dividing cells, we must consider the kind of effect demonstrated here
whenever we use [•'Hjthymidine to measure cell cycle parameters.
ACKNOWLEDGMENT
We are grateful to the Royal Society for a grant enabling Dr de la Torre to work in
Oxford on leave from the Instituto de Biologia Celular at Madrid.
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AvANZi, S. & D'AMATO, F . (1970). Cytochemical and autoradiographic analyses on root primordia and root
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BARR, H . J. (1963). An eflFect of exogenous thymidine on the mitotic cycle. X cell. comp. PhysioL, 61, 119.
CLEAVER, J. E. (1967). Thymidine Metabolism and Cell Kinetics. Amsterdam, North-Holland.
CLOWES, F . A. L. (1961) EfTeets of )9-radiation on meristems. Expl. Cell Res., 25, 529.
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New Phytol (1974) 73, 919-925.

THYMIDINE AND THE MEASUREMENT OF RATES

BY C . DE LA TORRE AND F . A. L. CLOWES

Botany School, University of Oxford

{Received 25 January 1974)

Tritium-labelled thymidine is widely used to measure the rate of mitosis in meristems.

Table 2. Durations of complete mitotic cycles {cell-doubling times in

De la Torre, Gonzalez-Fernandez and Gim^nez-Martin (1971) point out the advantages

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