International Journal of Radiation Biology and Related

Studies in Physics, Chemistry and Medicine

ISSN: 0020-7616 (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/irab19

The Effect of Ionizing Radiation on the Survival of

Free Plant Cells Cultivated in Suspension Cultures

P.A.Th.J. Werry & K.M. Stoffelsen

To cite this article: P.A.Th.J. Werry & K.M. Stoffelsen (1979) The Effect of Ionizing Radiation
on the Survival of Free Plant Cells Cultivated in Suspension Cultures, International Journal of
Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 35:3, 293-298, DOI:
10.1080/09553007914550351

To link to this article: https://doi.org/10.1080/09553007914550351

Published online: 03 Jul 2009.

Submit your article to this journal

Article views: 184

View related articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=irab20
INT . J . RADIAT . BIOL ., 1979, VOL . 35, NO . 3, 293-298

The effect of ionizing radiation on the survival of

free plant cells cultivated in suspension cultures
P. A . Th . J . WERRY and K . M . STOFFELSEN
Radiation Biophysics Group, Association EURATOM-ITAL, P .O . Box 48,
6700 AA Wageningen, The Netherlands .

(Received 5 yuly 1978 ; accepted 23 October 1978)

1. Introduction
For mammalian organisms numerous reports have been published dealing with
the cellular response to radiation with respect to cell survival, mutation induction,
induction of neoplasms, repair of lesions, etc . Mammalian cells cultured in vitro are
excellent experimental material since they occur as unicellular units, and suitable
techniques have been developed to study quantitatively the fate of irradiated cells .
There are some problems, however, which cannot be studied by using
mammalian cells : for example, the effect of ploidy level on the radiation sensitivity of
cells (haploid, diploid, polyploid), the significance of radiation-induced lesions for
differentiation, or genetical characterization by means of classical methods (crossing
after regeneration of cells into complete organisms) . Free plant cells can offer the
possibility of studying these aspects since they can be cultivated at different ploidy
levels and differentiation and regeneration to complete and fertile plants is possible
for many species (Reinert and Bajaj 1977) . An approach similar to that for
mammalian cells, however, was not possible since plant cells cultivated in vitro occur
mostly as multicellular aggregates . Furthermore, when free cells were selected from
suspensions and plated on solid growth medium in low, easily scorable numbers the
plating efficiency was rather poor (Bergmann 1960, Nagata and Takebe 1971,
Logemann and Bergmann 1974, Engvil 1974, Vardi, Spiegel-Roy and Galun 1975) .
Galun and Raveh (1975) used naked protoplasts immediately after isolation . The
properties of free plant cells can indeed be studied in this way, but also here the
plating efficiency is rather low .
Recently a new plating technique has been developed, which yields a high plating
efficiency (> 80 per cent), independent of the concentration of plated cells and even
for low numbers of plated cells (Werry and Stoffelsen 1978) . This note describes the
survival of free cells of the annual composite Haplopappus gracilis after irradiation
with X-rays and fission neutrons .

2. Materials and methods

The cell suspension of H . gracilis (Nutt) Gray which was used throughout this
investigation was initiated and maintained as described previously (Werry and
Stoffelsen 1978) . Regular cytological observations revealed that consistently 80 per
cent of the cells were diploid, whereas the remaining 20 per cent were mostly
tetraploid ; only a few were haploid or aneuploid .
Free cells were obtained by filtering a suspension culture through a series of
nylon sieves having successive mesh widths of 297, 210 and 88 ym . The resulting
suspension contains about 2 . 5 x 10 3 viable units per millilitre . Of these units,
approximately 80 per cent are unicellular and 20 per cent bicellular ; a negligible
proportion are multicellular .


294 Correspondence

Free cells suspended in their own growth medium were irradiated with X-rays
-1 )
(l60kVp, filtration 0 . 25 mm Cu and 1 . Omm Al, dose rate 3 . 1 Gymin and fission
neutrons (mean energy 1 . 7 MeV, gamma contamination less than 5 per cent of the
-1
neutron dose, dose rate 1 . 0 Gymin ) ( BARN reactor ; Chadwick and Oosterheert
1969).
Immediately after irradiation the replating technique was employed (for a critical
evaluation see Werry and Stoffelsen (1978)) : an aliquot of the irradiated cells was
rapidly mixed with 2 . 3 volumes of liquid (40 °C) B-5 agar medium and poured on top
of feeder agar (preplating step) . After 2 weeks' incubation at 28 ° C in darkness, the
soft agar containing small colonies was carefully mixed with liquid (40 °C) soft agar so
as to give a maximum density of 0 . 5 x 10 3 small colonies per millilitre . Then 1 ml of
this mixture was poured on top of solid feeder agar in Petri dishes (replating step) .
The Petri dishes were sealed with parafilm and incubated at 28°C in darkness .
After 2 weeks those cells that had developed into a colony large enough to be
observed by eye-after about 12 cell divisions-were defined as survivors and
counted . The plating efficiency (PE) is defined as

(number of visible colonies)

PE= x 100 per cent .
(number of preplated units)

Survival S is defined as the PE at a given dose relative to the PE of the unirradiated

control .
Corrections for the occurrence of bi- or tricellular aggregates in the filtered
suspensions were made by applying the functions (Howland 1977)

Sobs =1- L
where Sobs is the observed fractional colony survival and L the observed fractional
colony inactivation,
L=pf1 +p 2f2+p 3f3
where fl , f2 and f3 are the fractions of uni-, bi- and tricellular aggregates in the
irradiated suspension . (these proportions are determined microscopically in each
experiment) and p is the fraction of inactivated free cells (it is assumed that p has the
same value for free cells and cells in aggregates) and
S=1-p
where S is the free cell survival .

3. Results
The suspension cultures of H. gracilis used in this study exhibit a logarithmic
growth phase for 7 days after inoculation, after which the culture grows into a
stationary phase . It has been demonstrated for suspension cultures of Acer
pseudoplatanus that cells in a stationary phase of growth occur mainly in the G 1 phase
of the cell cycle (Rembur 1977) . Therefore we have studied whether or not free cells
taken from stationary growing cultures ('stationary cells') and cells from exponen-
tially growing cultures ('exponential cells') show different sensitivities to radiation .
In addition, the effect of X-rays and fission neutrons on the survival of both types of
 Correspondence 295

1 .00
0 .70
0 .50

0 .30

0.10

0.05

0.01

0,005

0 001

Figure 1 . Free cell survival of H . gracilis after X-irradiation (3 . 12 Gy/min ; °, exponential cells ; 0,
stationary cells) and fission-neutron irradiation (1 Gy/min ; /, exponential cells ; j, stationary cells) .

cells has been studied . The dose-effect relationships resulting from these experi-
ments, which are shown in figure 1, give rise to the following observations .
Fission neutrons are more effective than X-rays in killing cells from both
stationary and exponentially growing cultures . The dose-effect relationship for
neutron irradiation seems to be purely exponential .
Stationary cells are more sensitive to X-rays and fission neutrons than
exponential cells .
Up to doses of about 4 Gy of X-rays, the plating efficiency of irradiated cells is
higher than that of the unirradiated control cells . This `stimulation' never
exceeds 20 per cent .
It should be borne in mind that the absolute plating efficiency in the control is always
about 80 per cent and the `stimulated' absolute plating efficiency can never be higher
than 100 per cent in a properly executed experiment . Extensive and repeated
counting of aggregates before and after the replating step showed that the increased
PE can not be attributed to fragmentation of aggregates in the replating step . A more
detailed study of this stimulation phenomenon is presented in figure 2, from which it
can be seen that the optimum dose for this stimulation is about 2 . 5 Gy of X-rays .
At survival rates higher than 1 per cent, morphology and size distribution of the
colonies are no different from those of the unirradiated control . At survival rates
lower than 1 per cent, the morphology of many colonies was altered and the average
growth rate was reduced . No search for particular microscopic radiation effects has
yet been undertaken .


296 Correspondence

x- exp . cells

0 1 .25 2 .50 3.75 5 .00

dose (Gy)

Figure 2. Free cell survival of H. gracilis cells after X-irradiation (x , exponential cells ; E], stationary
cells) .

4. Discussion
The observed increase in plating efficiency at X-ray doses of about 2 . 5 Gy is an
unexpected and unique phenomenon in cellular radiation biology . The following
arguments lead to a plausible explanation of the stimulation phenomenon .
(1) It has been shown previously (Werry and Stoffelsen 1978) that the volume of
the inoculum at the replating step is the main factor influencing the plating
efficiency . From this observation it can be inferred that, at a given volume of
inoculum, and therefore a given depth of the soft agar layer, the size of the
plated aggregates determines the plating efficiency . In the procedure
employed in this research the diameter of the aggregates, resulting from
unirradiated control cells, is such that approximately 20 per cent of the
aggregates are covered with a layer of soft agar and therefore cannot develop
into visible colonies because of inadequate gas exchange .
(2) Preliminary experiments in our laboratory have shown that extracts of
Haplopappus cells grown in suspension for 3-weeks after irradiation with
X-rays to doses of 2 . 5 and 5 Gy stimulated growth when applied to growing
suspension cultures . This stimulation was expressed as increased fresh
weight ; however, no increase in dry weight was found, indicating that the
size of the cells was increased but not the number of cells . Growth
stimulation in callus cultures of Haworthia mirabilis effected by radiation-
induced cytokinins has recently been demonstrated by Pander, Sabharwal
and Kemp (1978) .
(3) It is conceivable from these arguments that at X-ray doses of 2 . 5 Gy the
diameter of cells, and consequently the size of the aggregates at the replating
step, is increased . This would result in a higher plating efficiency which,
when the radiation damage is not severe, could exceed that of the controls .
From this reasoning it can be concluded that the survival curve for X-rays shown
in figure 1 is the result of two different radiation effects-cell elongation and cell
inactivation . Stimulation of plating efficiency is observed only when the cell
inactivation effect of radiation is very low .


Correspondence 297

The stimulation of plating efficiency resembles, at least phenomenologically, the

radiation-stimulation effect in crop plants (for an extensive review see Simon and
Bhattachariya (1977)) . Irradiation of seeds, mostly at doses between 2 and 20 Gy,
often results in significantly higher yields, whatever criterion is used for yield . It is
conceivable that radiation-induced increase of cell size in a critical phase of growth of
the crop plants could be the basic mechanism underlying the radiation-stimulation
effect .
It is clear from figure 1 that stationary cells of H . gracilis are more sensitive to
ionizing radiation than exponential cells . Rembur (1977) showed that, in the
stationary phase of growth, cells of A . pseudoplatanus occur mainly in the G 1 phase of
the cell cycle . The different sensitivity to radiation of stationary cells as compared to
exponential cells could be related to their supposed occurrence in the G 1 phase . On
the other hand, the transition from exponential to stationary phase of growth is
coincident with nearly complete exhaustion of the energy source in the growth
medium (saccharose) . Therefore, deprivation of nutrients may also in itself explain
the higher sensitivity of stationary cells.
Eriksson (1967) found a substantially lower inactivation effect of X-rays for H .
gracilis than is found in this study . Since the conditions of irradiation employed by
Eriksson are quite similar to those used in this study, the discrepancy in the results
must be due to the aggregational quality of the irradiated cell suspensions . No
qualification is given by Eriksson other than `less than 1'0%of the cells are tetraploid
or aneuploid under the culture conditions used' . Eriksson applied no correction to
convert survival to cellular survival . Dose-effect relationships for survival after
irradiation have been reported for haploid and diploid protoplasts of Datura inoxia
(Schieder 1976), citrus protoplasts (Vardi et al . 1975) and tobacco protoplasts
(Galun and Raveh 1975) . In addition to the difficulties mentioned in the introduc-
tion (i .e . low plating efficiency and very high concentration of plated cells),
inadequate specification of irradiation procedures (Schieder 1976) or essentially
different irradiation conditions (Vardi et al . 1975, Galun and Raveh, 1975) also
prevent a correct comparison of results .
The conditions for the neutron irradiations employed in this study differ in
energy spectrum and dose rate from those that are mostly used in mammalian
systems (Broerse and Zoetelief 1978, Takeshita and Sawada 1974, Richold and Holt
1973, Antoku 1975) . Nevertheless, it seems justified to state that, with respect to
colony-forming capacity and shape of the survival curve, Haplopappus cells respond
to neutron irradiation in a similar way to mammalian cells . At survival lower than 0. 5,
the r .b .e . of neutrons as employed in this study is 3-5, as this value is in accordance
with r .b .e . factors found for mammalian cells (Davids, Barendsen and Broerse 1971,
Berry 1971) . At higher survival an r .b .e . value cannot be calculated because the
stimulation effect interferes with the cell inactivation effect of X-rays .
In summary it seems justified to state that plant cells respond to ionizing
radiation in a way quite similar to that of mammalian cells with respect to single-cell
survival and r .b .e . of fission neutrons . However, at low doses of X-rays the plating
efficiency of plant cells (under the conditions employed) is increased, most probably
as a consequence of radiation-induced endogenous cytokinine synthesis .


298 Correspondence

ACKNOWLEDGMENTS
Stimulating discussions with our colleagues from the radiation biophysics group,
Drs . K . J . Puite, H. P . Leenhouts, K . H . Chadwick and M . M . J. D . Litwiniszyn,
and the technical assistance of Mr . C . Blom, are gratefully acknowledged .

REFERENCES
ANTOKU, S ., 1975, Int . Y. Radiat . Biol., 27, 287 .
BERGMANN, L ., 1960, Y . gen . Physiol ., 43, 841 .
BERRY, R . J ., 1971, Eur ., . Cancer, 7, 145 .
BROERSE, J . J ., and ZOETELIEF, J ., 1978, Int . Y. Radiat . Biol ., 33, 383 .
CHADWICK, K . H ., and OOSTERHEERT, W. F ., 1969, Atompraxis, 3, 178 .
DAVIDS, J . A . G ., BARENDSEN, G . W ., and BROERSE, J . J ., 1971, Int . Conf . peaceful Uses atom . Energy, 4, 3 .
ENGVILA, K . C ., 1974, Physiologia Pl ., 32, 290 .
ERIKSSON, T ., 1967, Physiologia Pl ., 20, 507 .
GALUN, E ., and RAVEH, D ., 1975, Radiat . Bot ., 15, 79 .
HoWLAND, G ., 1977, Plant Cell, Tissue and Organ Culture, edited by J . Reinert and Y .P .S . Bajaj (Berlin :
Springer-Verlag), p . 731 .
LOGEMANN, H ., and BERGMANN, L ., 1974, Planta, 121, 283 .
NAGATA, T ., and TAKEBE, I ., 1971, Planta, 99, 12 .
PANDEY, K . N ., SABHARWAL, P . S ., and KEMP, T. R ., 1978, Nature, Lond ., 271, 449 .
REINERT, J ., and BAJAJ, Y . P . S . (editors), 1977, Plant Cell, Tissue and Organ Culture (Berlin : Springer-
Verlag) .
REMBUR, J ., 1977, Z. PflPhysiol., 81, 102 .
RICHOLD, M ., and HOLT, P . D ., 1973, Biological Effects of Neutron Irradiation (Vienna : IAEA), p . 237 .
SCHIEDER, 0 ., 1976, Molec . gen . Genet ., 149, 251 .
SIMON, J ., and BHATTACHARIYA, S ., 1977, The Present Status and Future Prospect of Radiation Stimulation
in Crop Plants (Budapest : Phylaxia) .
TAKESHITA, K ., and SAWADA, S ., 1973, Biological Effects of Neutron Irradiation (Vienna : IAEA), p .245 .
VARDI, A ., SPIEGEL-Roy, P ., and GALLN, E ., 1975, Pl . Sci. Lett., 4, 231 .
WERRY, P . A . TH . J ., and STOFFELSEN, K . M ., 1978, Theor . appl . Genet ., 51, 161 .