H E R E 55-16

GENETIC CHANGES INDUCED BY SEMIACUTE Y -IRRADIATION OF POLLEN

MOTHER CELLS IN LARIX LEPTOLEPIS
(SIEB ET ZUCC.) GORD
By COSTA ERIKSSON, INGER EKBERG, LARS EHRENBERG,
and BRANIL4 BEVILACQU.4
DEPARTMENT OF FOREST GENETICS, ROYAL COLLEGE OF FORESTRY, STOCKHOLM

50

AND INSTITUTE O F RADIOBIOLOGY, UNIVERSITY O F STOCKHOLM, SWEDEN

(Received April 4th, 1966)

I. INTRODUCTION

N several plant species induction of mutation has proven to be a valuable means of increasing variation for plant breeding work. During
the past two decades mutation breeding of a few annual or biennial selffertilizing species has led to decided progress. This is especially true for
barley (cf. GUSTAFSSON,1963) and peas (GELIN, 1954). Relatively rapid
progress with such species is partly dependent on the availability of test
systems which permit the determination of optimum conditions for efficient exploitation of mutagenesis in general. Thus, the chlorophyll
mutation technique in barley (GUSTAFSSON,1940) and the corresponding method for peas (BLIXT et al., 1958) constitute good examples of
useful test systems. It should be added, however, that even in crossfertilizing species such as white mustard (cf. ANDERSSONand OLSSON,
1954) mutagenically created variation has proven to be useful in plant
breeding.
In conifers and other forest trees, hybridization, selection, and allied
methods of plant breeding will certainly continue to be used for a considerable time. It may be stated, however, that the role mutation could
play in these materials is practically unknown. It is worth mentioning
that several important clones of fruit trees are mutants (cf. GRANHALL,
1954).
A successful mutation 1)reeding prograin for forest trees will pro-

Pernianeiit address: Department o f Forest Genetics and Dendrology, Forestry

Faculty, University of Zagreb, Yugoslavia.

214

G. ERIKSSON, I. EKBERG, L. EHRENBERG, AND B. BEVILACQUA

TABLE 1. Studies concerning the effectso f irradiation in growing forest

trees
Species

Studies of

Chamaecyparis obtusa and

pisifera, Cryptomeria japonica Larix leptolepis
Pinus densiflora, Thunbergii
Alnus Betula
Paulownia tomentosa
Populus Quercus acutissima
Pinus elliottii
,, taeda
Pinus elliottii
,) palustris
,, taeda
Pinus monophylla
Pinus rigida
Quercus sp.

Various radioinduced effects

after chronic and acute1
y-irradiation

OHBA

Depression of CO, evolution

and photosynthesis
Growth and survival of
seedlings

HADLEY
and WOODWELL,
1965
DAVIS,1962

Morphology and growth

Reproductive capacity,
flower abnormalities,
growth in descendents
Growth after several years
of chronic irradiation3
Growth; changes in insect
defoliation
Survival after chronic irradia
tion

BRANDENBURC

Anatomical examination of
male strobili and cytological
evaluation of aberrations
induced during microsporogenesis
Radial increments of different parts of the stems
Survival, height, growt,
productivity, and cytology
Tolerances of young plants
to acute and chronic
radiation

hfERCEN

Mortality, growth inhibition,

seed setting

SPARROW
el el. 1965

Pinus rigida
,, strobus
Pinus rigida
Quercus alba
Pinus rigida

Taxus media hatfieldii

Pinus rigida

Pinus rigida
Pinus rigida
Pinus rigida P. strobus
Picea glauca, Larix leptolepis, Abies balsamea
Pseudotsuga taxifolia,
Taxus media, Juniperus
uirgineana, Thuja occidentalis, Sambucus canadensis
Quercus rubra, Fraxinus
americana, Acer saccharum
Acer rubrum, Betula lutea
Pinus rigida

Iteferences
19642

et a!., 196;
MERCENand STAIRS,196:

SPARROW
and WOODWELL
1962
WOODWELL,
1962
SPARROW
et al., 1961

and JOHANSES,

1963

WOODWELL
and RIILLsn,
1963
MERCEN and TIIIELGES
1966
SPARROW,
1965

215

IRRADIATION OF LARIX

TABLE 1. (cont.)
Species

Pinus silueslris, Populus

robusta, serotina and variet
No. 297
Pinus siluestris, Populus
robusla and tremula
Pinus siluestris, Picea abies
Populus robusta, serotina
and clone 297
Pinus strobus

Studies of

References

Growth and survival of

graftings

GUSTAFSSON
and SIMAK,
1958

Survival of graftings

WETTSTEIN
e f , al., 1959

Growth and survival of

graftings

GUSTAFSSON,
1962

Sensitivity in dormant and

growing seedlings
Pinus taeda
Reproductive capacity,
lethality
Pinus taeda
Lethality
Pinus rigida, Quercus alba
Shoot growth and tree
Q. coccinea, Q. ilicifolia
mortality
Quercus coccinea, Q. ilicifoli, Seed production
Quercus alba
Morphological and histolo,, uelutina
gical damage
Taxus media
Bud formation and its
connection to histological
changes

SPARROW
el al., 1963
PEDIGO,
1963
PLATT,
1963
WOODWELL
and SPARROW,
1963
STAIRS1964
MERICLEel al., 1962
MIKSCHE el al., 1962

Acute was used in a wide sence including irradiations lasting for some months.
This author also refers t o some works published in Japanese.
* A brief summary of tolerance data for several species was given by SPARROW
and
GUNCKEL(1956).

bably haveto concentrate on induction of bud mutations, sports (cf.

GUSTAFSSON, 1962). It is of fundamental importance to study the further
development of the chimeric tissue obtained in its competition with unmutated tissue ( c f . BAUER,1957; NYBOM,1961) irrespective of whether
the mutation will be propagated sexually or vegetatively.
In maize and barley it has been possible to use the occurrence of pollen grains of a deviating phenotype (so called waxy pollen grains) as a
sensitive measure of induced mutation (ERIKSSON,
1962, 1963, 1965 b;
EHRENBERG
and ERIKSSON,
1964; ERIKSSON
and TAVRIN,1965). This
technique has also permitted the determination of the distribution of
the mutated sector following irradiation or chemical treatment of the
1965 a ) .
(multicellular) embryo of barley kernels (ERIKSSON,
The use of a n inherited pollen character for the estimation of mutation rates and for the elaboration of suitable conditions for mutation

216

G. ERIKSSON, I. EKBERG, L. EHRENBERG, AND B. BEVILACQUA

induction would seem to be especially advantageous in forest trees,

since a time consuming study of the following generation (after vegetative or sexual reproduction) could be avoided. The purpose of the
present investigation is primarily to clarify the possibilities of using the
"waxy system", or its biochemical counterpart, for the measurement of
mutation rates and for the estimation of the distribution of the mutated
sectors in forest trees.
In a preliminary test, pollen grains from h e r , Alnus, Betula, Corylus,
Larix, Picea, Pinus, Salix, and Ulmus were examined with respect to
their staining affinity to iodine solution. Only the pollen grains of Larix,
which are relatively large, gave a strong positive starch reaction. However, by improving the screening procedure it seems possible to use
some of the other species also for similar tests. The composition of
coniferal starch in branches ( c f .BLUKET, 1963) is similar to that of the
angiosperms studied. The occurrence of waxy mutants in the latter was
therefore thought at least to have a counterpart in Larix.
I n order to obtain more complete information about the radiation
effects induced, the rate of chromosomal aberrations induced in the
pollen mother cells (PMC) were determined for the same material. The
aberration rates were estimated at the second meiotic anaphase. Percentages of sterile and giant pollen grains were also recorded.
So far some 20 investigations of radiation effects in growing forest
trees have been carried out. A compilation of studies performed in this
field is made in Table 1 (References concerning seed irradiation are not
included.). The studies reported have been mostly concerned with
radioecological problems. Besides the references listed it may be added
that GUSTAFSSON (personal communication) obtained two mutants in
Populus after repeated y-ray exposure. One of the mutants contained
a higher amount of anthocyanin than the original strain. This mutant
also showed a reduced growth ability. Another mutant deviated with
respect to branching and contained a low amount of anthocyanin.
Chronic y-irradiation of Larix leptolepis and L . sibirica revealed that
they were extremely sensitive to the induction of certain physiological
changes (SIMAR, personal communication) which may be related to
those described for Taxus obtained after such low daily doses as 3.75 R
(MIKSCHEet al., 1962). I n contrast to this high sensitivity of Larix and
Taxus it has been shown that grafts of Alnus were uneffected by
several times higher doses (8000 Tad, semiacute treatment) than those
used for Larix and Taxus (EKLUNDH-EHRENBERG,personal communication).

IRRADIATION OF LARIX

21i

Material and methods

Material from a clone (Skarnas, L 1003) of Lariz leptolepis (SIEB.
et ZUCC.) GORD. grafted in 1958 was used for the present study. The
plants were placed in large pots in the autumn of 1964 so that they
could be easily transported to the y-field at Bogesund, Sweden. During
the semi-acute irradiation, which lasted for 24 hours, the plants were
placed at the distances of 3, 5, 7, and 10 meters from the y-source ('*'Cs,
about 890 Ci). Doses received by the plants varied from 500-45 rad. At
irradiation the control plants were transported to the y-field and placed
about 150 meters from the y-source in a shielded position. The dose
received by the control plants amounted to 0 . 2 4 . 4 mrad.
The appearance of normal buds containing the pollen mother cells
has already been described by BARNER
and CHRISTIANSEN(1960). To be
able to determine the stage of meiosis at irradiation male strobili were
fixed in acetic alcohol (1:3). The irradiation was carried out on February 13th, 1965. At that time the PMC had reached the first part of
the diplotene stage. The material was fixed on various days after irradiation in order to include the anaphase I1 stage of meiosis among the
fixed PMC. This stage was frequent about one month after treatment.
During irradiation the temperature was about kOo C, and after irradiation it was below 0' C until March 9th (except for February 21st
when $4' C was noted at 1 P M ) . Frost was observed during the period
following ending on April loth, however, only rarely during daytime.
From this date until May lst, when fixing of the pollen was performed,
no temperatures below 0' C were noted.
Entire male strobili containing mature pollen grains were fixed in
70 % alcohol. The preparations for screening of waxy pollen grains
were made with the aid of a microhomogenizer. The size of a pollen
grain population of a slide was estimated by the sum of 16 counted
sections over the surface of the slide, multiplied by a constant. This
technique has been worked out by NELSON (personal communication).
The percentage of pollen sterility was estimated in a lactophenol fuchsin solution. The classification procedure is described below ( c f . p. 7).
The pollen mother cells were dissected and squashed in 2 % acetoorcein. The frequency of bridges, fragments, micronuclei, lagging chromosomes and any other abnormalities were determined in each examined anaphase I1 division. The size of 200 pollen grains from each
treated material was determined.

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G . ERIKSSON, I. EKBERG, L. EHRENBERG, AND B. BEVILACQUA

11. RESULTS AND DISCUSSION

1. Waxy mutants

The analysis of the larch pollen grains showed that it is possible to

induce a change which phenotypically resembles the waxy phenotype
of maize and barley pollen grains. Therefore, it is possible to analyse
the pollen grains of branches originating from irradiated buds in order
to determine the further development of a n induced waxy mutation.
The number of mutants as well as the number of pollen grains analysed are shown in Table 2. I n Fig. l the relationship between mutation
rate and dose is demonstrated.
The spontaneous rate of mutants is somewhat lower than that reported for barley (EHRENBERG
and ERIKSSON,
1964) and several times lower
1963, 1965b; ERIKSSON
and
than that obtained in maize (ERIKSSON
TAVRIN, 1965).
Fig. 1 suggests that the mutation rate reached a plateau after as low
a dose as 90 rad. Due to the high sterility induced by 500 rad, no
screening for waxy mutants was carried out after this treatment.
Although the mutation rates given in Table 2 and Fig. 1 are expressed
as the fraction of waxy type among fertile pollen grains it seems probable that induced sterility masks several mutants induced by a dose
higher than 90 rad. A comparison with the data obtained after irradiation (24 hours treatment) of growing maize plants shows that the mutation rates in larch and maize pollen are of about the same order of
magnitude in spite of great physiological differences between the materials (ERIKSSON, unpublished).
Meiosis in P M C of Larix leptolepis starts in the autumn. The PMC
remains in the diplotene stage for a relatively long time (EKBERG
and
ERIKSSON,
unpublished). By appropriate cultivation techniques it ought
TABLE 2. Number of rvaxy resembling pollen grains and the mutation
rritcs after 24 hours y-irrccdiatiorz of pollen mother cells of 1,arix
leptolepis during the diplotene stage.
Dose

Control
45 rad
90 rad
180 rad

1113000
358 000
264 000
129000

2
7
24
12

0.02
0.20
0.91
0.93

219

IRRADIATION OF LARIX

1.5-

1.0-

T
L%

Fig. 1. The relationship between the induced rate of pollen grains resembling the
waxy phenotype and y-ray dose. 95 O/o confidence intervals are given.

to be possible to induce the development of later meiotic stages in the

PMC. Therefore, Larix and similar materials should be suitable for
studies of variations in radiosensitivity during different stages of meiosis.
2. Chromosomal aberrations

In the cytological examination of the irradiated materials several

types of chromosomal aberrations such as bridges, fragments, micronuclei, ring chromosomes, and lagging chromosomes were detected.
'Often only 11 of the 12 bivalents were oriented in the equatorial plane
during metaphase I in irradiated PMC. The appearance of such a cell
is seen in Fig. 2 A. Cells containing one or more chromosomal bridges
during anaphase I and I1 are illustrated in Fig. 2 B-D. After 500 rad
of irradiation a high degree of fragmentation was observed. Besides
fragmentation, the formation of micronuclei was also frequent (cf. Fig.
2 D-E). Some of the bridges occurring during the first division seemed
to remain until the second division, as seen from Fig. 2 F.

220

G.

ERIKSSON, I. EKBERG, L. EHRENBERG, AND B. BEVILACQUA

TABLE 3. T h e frequency of various types of chromosomal aberrations

appearing at the anaphase I I stage of meiosis after 24 hours irradiation
of pollen mother cells of Larix leptolepis during the diplotene stage as
well as the percentage of sterility among the mature pollen grains.

Dose

0
45
90
180
500

1 1
Normal
divisions

151
102
111
35

Abberrant divisions

X aberra-

others

total

tions per
rad

2
1
5
12
47

3
10
26
26
112

1.9
8.9
19
43
96

0.16
0.19
0.23
0.19

bridges

1
2
13
8
13

fragments

8
18
17
101

iterility in
mature
pollen
grains %

16
20
63
72
85

All treatments resulted in a marked increase of the rate of chromosomal aberrations as shown in Table 3 and Fig. 3. Only a few abnormal
divisions were detected in the control material. I n Table 3 two common
aberration types are listed separately; the others are listed as a group.
Cells containing several types of aberrations were found, especially
following 500 rad of irradiation. In this material the percentage of cells
containing fragments was as high as 86 %. The frequency of divisions
containing two or more fragments was estimated to be 58 %.
That climatic conditions might be responsible for the chromosomal
aberrations (cf. CHRISTIANSEN,1960) is excluded because only 3 out of
150 divisions in the control material showed abnormalities. Therefore,
it is concluded that irradiation was responsible for the increase in aberrant divisions revealed after the various y-ray treatments. On the other
hand it is most probable that the level of radiation induced aberrations
was influenced by climatic conditions, especially temperature, during
and after treatment ( c f . SAXand ENZMANN,
1939; SPARROW
et d.,
1961).
1
comprise
studies
of
effects
of
Most references listed in Table
chronic irradiation on different variables. As far as we are aware,
analyses of induced chromosomal aberrations after acute treatment
have not, as yet been carried out. In herbaceous plants some studies of
induced chromosomal aberrations after irradiation of the diplotene
stage have been reported. Several references will be found in Table 1
in the paper by ERIKSSON
and TAVRIN(1965, p. 157). However, in the
present investigation the irradiation lasted for 24 hours whereas only
short treatments were used by the investigators listed in the above

222

G . ERIKSSON, I. E K B E R G , L. E H R E N B E R G , A N D B. BEVILACQUA

3. Pollen sterility

Estimation of pollen sterility was carried out in a lactophenol fuchsin

solution. The appearance of a fertile pollen grain is demonstrated i n
Fig. 4 A. The criteria for a fertile pollen grain are that the cytoplasm
of the pollen should show a smooth surface and that it should contain
three nuclei in addition to the two small nuclei of the degenerative
(1941) there should
prothallial cells (Fig. 4 A). According to SCHNARF
be three nuclei in the pollen grains of Larix, uiz., the pollen tube, the
body, and the stalk nuclei. Some of the pollen grains which were classified as sterile contained neither cytoplasm nor nucleus. Sometimes
the surface of the cytoplasm of sterile pollen grains was rough and it
contained only starch granules (Fig. 4 C ) . Pollen grains completely
lacking starch granules were also classified as sterile (Fig. 4 B) . Borderline cases might occur in the estimation of the sterility as the change
from one pollen type to another is more or less continuous. This arbitrary classification makes the sterility analysis somewhat unreliable.
The induced pollen sterility seemed to rise rapidly between the doses
-c fir - - A m
A t hiahor r l n c p c R flnttrnine of the dose response curve

223

IRRADIATION OF LARIX
25CONTROL

254 5 RAD

90 RAD

8
a

'

25500 RAD

35

40

45

so

55

60

65

70

75

80

SIZE OF POLLEN GRAINS IN RELATIVE UNITS ( s i = i o o ~

Fig. 5. The distribution of the sizes of the pollen grains after the different treatments.

what smaller average for two trees of Larix decidua was obtained by
CHRISTIANSEN(1960). The pollen grains measured after treatment with
180 rad showed a n average size of 91 p. The other materials contained
averages between this value and the control value of 86 [i. Thus, all
treatments gave rather similar results with respect to the average size
of the pollen grains. However, the size distributions were quite different
for the various doses as seen from Fig. 5. The doses in the range of
90-500 rad caused a broadening of the distribution both to lower and
higher diameters. It is difficult to fix a borderline between normal and
giant pollen grains as there is a more or less continuous distribution.
If, however, all pollen grains with diameters 30 % larger than the
average diameter (corresponding to a doubling of the volume, cf.
CHRISTIANSEN, 1960), are so classed, the following percentages of giant
pollen grains will be obtained:
control
45 rad
90
180 ,,
500
9,

99

0.5
0
3.5
3
7

224

G. ERIKSSON. I. EKBERG. L. EHRENBERG. AND B. BEVILACOUA

It is of particular interest that most giant pollen grains showed a

fertile appearance which suggests that they might be usable in crossing
experiments where the use of unreduced diploid pollen grains is of
value (cf. NILSSON-EHLE,1938).
Acknowledgement. - The plant material was kindly supplied by Dr. hl. SIMAK,
Stockholm. Miss ELLENKILIAN and hlr. KJELL LANNERHOLM
have given skilful tcclivisited Sweden as stipendiate of
nical assistance. One of us (BRANKA
BEVILACQUA)
the International Atomic Energy Agency, Vienna (contract No. YUG/G41), which is
acknowledged. The investigation was aided by grants from the Swedish Forest Hescarch Foundation and the National Institute of Health (project G M 08877-04). The
radiation source was financed by grants from the Agricultural and Natural Sciences
Research Councils as well as the Knut and slice Wallenberg foundation.

SUMMARY
The usefulness of waxy mutants as a n indicator system in mutation
research on forest trees is pointed out. Pollen grains resembling the
waxy pollen grains in maize and barley were induced after 24 hours
y-irradiation (Cs) of the pollen mother cells in a clone of Larix leptolepis. The dose range used was 45-500 rad. The dose response curves
for induction of waxy pollen grains and of pollen sterility were found
to be sigmoidal, whereas the percentage of aberrant divisions seemed
to increase more linearly with the doses. The doses in the range of 90500 rad gave appreciably higher rates of giant (possibly diploid) pollen
grains than was found in the control material.

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Inarkct
variety selected in X-ray treated material. - Acta Agric. Scand. 4 : 574-577.
BARNER,1. and CHRISTIANSEN,
H. 1960. The formation of pollen, the pollination
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pollination in Larix. - Silvae Genet. 9: 1-11,
BAUER,
H. 1057. The induction of vegetative mutations in Rihes nigrum. - Heriditas
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RLIXT,S., EIIRENBERG,
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225

EHRENBERG,
L. and ERIKSSON,
G. 1964. Mutation in the waxy character in barley
pollen grains following aoSr-incorporation a t low activities. - Mutat. Res. 2:
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- 1966. The dose dependence of mutation rates in the rad range, in the light of
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G. 1962. Radiation induced reversions of a waxy allele in barley. - Radiat.
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- 1965 a. The size of the mutated sector in barley spikes estimated by means of
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pollen mother cells i n maize. - Hereditas 54: 1 5 6 1 6 9 .
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I. 1954. Spontaneous and induced bud mutations in fruit trees. - Ibid.
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A. The mutation system of the chlorophyll apparatus. - Lunds Univ.
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HADLEY,
E. B. and WOODWELL,
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MERGEN,F. and THIELGES,
B. A. 1966. Effects of chronic exposures to Coeoradiation
on Pinus riyida seedlings. - Ibid. 6 : 203-210.
MERICLE,L. W., MERICLE,R. P. and SPARROW,
A. H. 1962. Cumulative radiation
damage in oak trees. - Ibid. 2: 265-271.
MIKSCHE,J. P., SPARROW,
A. H. and ROGERS,A. F. 1962. The effect of chronic
gamma irradiation on the apical meristem and bud formation of Taxus media.
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NILSSON-EHLE,
H. 1938. Darstellung tetraploider Apfel und ihre Bedeutung fur die
praktische Apfelzuchtung Schwedens. - Hereditas 24: 195-209.
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OHBA,K. 1964. Studies on radiosensitivity and induction of somatic mutations in
forest trees. - Gamma Field Symposia No. 3 Mutat. Quant. Traits 1964.
15 - Hereditas 55

226

G. ERIKSSON. I. EKBERG. L. EHRENBERG. AND B. BEVILACOUA

PEDIGO,R. A. 1963. Effects of ionizing radiation on Pinus taetlu L. - Proc. Nat.

Symp. Radioecol. lst, Fort Collins, Colo. Sept. 10-15, 1961, p. 295-299.
PLATT,R. B. 1963. Ecological effects of ionizing radiation on organisms, communities
and ecosystems. - Ibid., p. 243-255.
SAX,K . and ENZMANN,
E. V. 1939. Effect of temperature on X-ray-induced chroniosome aberrations. - Proc. Nat. Acad. Sci. U S . 25: 397-405.
SCHNARF,
K. 1941. Vergleichende Cytologie des Geschlechtsapparates der Kormophyten. - Berlin.
SPARROW,
A. H. 1965. Comparisons of the tolerances of higher plant species to acute
and chronic exposures of ionizing radiation. - Jap. J. Genet. Suppl. 40: 12-37.
SPARROW,
A. H., CUANY,R. L., hfIKSCHE, J. P. and SCHAIRER,
L. A. 1961. Some
factors affecting the responses of plants to acute and chronic radiation exposures. - Radiat. Bot. 1 : 10-34.
SPARROW,
A. H. and GUNCKEL,J. E. 1956. The effects on plants of chronic exposure
to gamma radiation from radiocobalt. - Proc. Intern. Conf. Peaceful Uses At.
Energy, Geneva 1955 12: 52-51).
SPARROW,
A. H., SHAIRER,
L. A,, SPARROW,
R. C. and CAMPBELL,
W. F. 1963. The
radiosensitivity of gymnosperms. I. The effect of dormancy on the response of
Pinus strobus seedlings to acute gamma irradiation. - Ibid. 3 : 169-173.
SPARROW,
A. H. SCHAIRER,
L. A. and WOODWELL,
G. M. 1965. Tolerance of Pinus
rigida trees to a ten-year exposure to chronic gamma irradiation from cobalt-60.
- Ibid. 5: 7-22.
SPARROW,
A. H. and WOODWELL,
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