Genetic Changes Induced

H E R E 55-16

GENETIC CHANGES INDUCED BY SEMI- ACUTE 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 OF RADIOBIOLOGY, UNIVERSITY OF STOCKHOLM, SWEDEN

(Received April 4th, 1966)

I. INTRODUCTION

N several plant species induction of mutation has proven to be a val- I uable means of increasing variation for plant breeding work. During the past two decades mutation breeding of a few annual or biennial self- fertilizing 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 ef- ficient 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 cross- fertilizing species such as white mustard (cf. ANDERSSON and 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 con- siderable 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 effects o f irradiation in growing forest trees

Species

Chamaecyparis obtusa and pisifera, Cryptomeria ja- ponica 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.

Pinus rigida

Pinus rigida Quercus alba Pinus rigida

,, strobus

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 occiden- talis, Sambucus canadensis Quercus rubra, Fraxinus americana, Acer saccharum Acer rubrum, Betula lutea Pinus rigida

Studies of

Various radioinduced effects after chronic and acute1 y-irradiation

Depression of CO, evolution and photosynthesis Growth and survival of seedlings

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

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

Mortality, growth inhibition, seed setting

Iteferences

OHBA 19642

HADLEY and WOODWELL, 1965 DAVIS, 1962

BRANDENBURC et a!., 196; MERCEN and STAIRS, 196:

SPARROW and WOODWELL 1962 WOODWELL, 1962

SPARROW et al., 1961

hfERCEN and JOHANSES, 1963

WOODWELL and RIILLsn,

1963 MERCEN and TIIIELGES 1966 SPARROW, 1965

SPARROW el el. 1965

IRRADIATION OF LARIX 215

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

Pinus taeda

Pinus taeda Pinus rigida, Quercus alba Q. coccinea, Q. ilicifolia Quercus coccinea, Q. il icifoli , Quercus alba

Taxus media ,, uelutina

TABLE 1. (cont.)

Studies of

Growth and survival of graftings

Survival of graftings

Growth and survival of graftings

Sensitivity in dormant and growing seedlings Reproductive capacity, lethality Lethality Shoot growth and tree mortality Seed production Morphological and histological damage Bud formation and its connection to histological changes

References

GUSTAFSSON and SIMAK, 1958

WETTSTEIN e f , al., 1959

GUSTAFSSON, 1962

SPARROW el al., 1963

PEDIGO, 1963

PLATT, 1963 WOODWELL and SPARROW, 1963 STAIRS 1964 MERICLE el 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 un- mutated tissue (cf . 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 (multicellular) embryo of barley kernels (ERIKSSON, 1965 a) .

The use of an inherited pollen character for the estimation of mutation rates and for the elaboration of suitable conditions for mutation


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. How- ever, 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.

In 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. Per- centages 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 (MIKSCHE et al., 1962). In 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 Feb- ruary 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 PM) . 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 % aceto- orcein. 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.

218 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 an induced waxy mutation.

The number of mutants as well as the number of pollen grains ana- lysed are shown in Table 2. In 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 than that obtained in maize (ERIKSSON 1963, 1965b; ERIKSSON and 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 PMC 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

1113000 358 000 264 000 129000

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.

2 0.02 7 0.20

24 0.91 12 0.93

Dose

Control 45 rad 90 rad

180 rad


1.5-

1.0-

-

I i

L%

T

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.


8 18 17

101

TABLE 3. The 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.

2 1 5

12 47

Normal ~

Dose’ 1 divisions 1 bridges

45 90

180 500

0 I 151 I 1 102 2 111 13

35 8 5 13

Abberrant divisions

others fragments

total

3 10 26 26

112

%

1.9 8.9

19 43 96

X aberrations per

rad

0.16 0.19 0.23 0.19

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. In 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 (cf . SAX and ENZMANN, 1939; SPARROW et d., 1961).

Most references listed in Table 1 comprise studies of effects of 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. EKBERG, L. E H R E N B E R G , AND 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 in 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 prothallial cells (Fig. 4 A). According to SCHNARF (1941) there should 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) . Border- line cases might occur in the estimation of the sterility as the change from one pollen type to another is more or less continuous. This arbi- trary 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 lncpc R flnttrnine of the dose response curve


25-

CONTROL

25- 4 5 RAD

90 RAD 8 a

3 '

25- 500 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 an 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 0.5 45 rad 0 90 9 , 3.5

180 ,, 3 500 9 9 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 ELLEN KILIAN and hlr. KJELL LANNERHOLM have given skilful tccli- nical assistance. One of us (BRANKA BEVILACQUA) visited Sweden as stipendiate of the International Atomic Energy Agency, Vienna (contract No. YUG/G41), which is acknowledged. The investigation was aided by grants from the Swedish Forest Hesc- arch 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 an 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 90- 500 rad gave appreciably higher rates of giant (possibly diploid) pollen grains than was found in the control material.

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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

mechanism, and the determination of the most favourable time for controlled pollination in Larix. - Silvae Genet. 9: 1-11,

BAUER, H. 1057. The induction of vegetative mutations in Rihes nigrum. - Heriditas

RLIXT, S., EIIRENBERG, L. and GELIN, 0. 1958. Quantitative studies of induced mutn- tions in peas. I. Methodological investigations. - Agri Hort. Genet. 16: 238-250.

BLUBET, N. A. 1963. Starch in gymnosperms. - Dokladi TSHA 83: 367-372. BRANDENBURG, hf. K., MILLS, H. L., RICKARD, W. H. and SHIELDS, L. hl. 1962. Effects

of acute gamma radiation on growth and morphology in Pinus monophylln TORR and FREM. (Pinyon pine). - Radiat. Bot. 2: 252-263.

CHRISTIANSEN, H. 1960. On the effect of low temperature on meiosis and pollen fertility in Larir decidua MILL. - Silvae Genet. 9 : 72-78.

DAVIS, T. S. 1962. Effect of cobalt-GO gamma radiation on pine seed and one-year- old seedlings. - Forest Sci. 8: 4 1 1 4 1 2 .

43: 323-337.


EHRENBERG, L. and ERIKSSON, G. 1964. Mutation in the waxy character in barley pollen grains following aoSr-incorporation at low activities. - Mutat. Res. 2:

- 1966. The dose dependence of mutation rates in the rad range, in the light of

ERIKSSON, G. 1962. Radiation induced reversions of a waxy allele in barley. - Radiat.

- 1963. Induction of waxy mutants in maize by acute and chronic gamma irradiation. - Hereditas 50: 161-178.

- 1965 a. The size of the mutated sector in barley spikes estimated by means of waxy mutants. - Ibid. 53: 307-326.

- 1965 b. Variations in radiosensitivity during meiosis of pollen mother cells in barley and maize. - Proc. Symp. Mutat. Process, Praha 1965. In press.

ERIKSSON, G. and TAVRIN, E. 1965. Variations in radiosensitivity during meiosis of pollen mother cells in maize. - Hereditas 54: 1 5 6 1 6 9 .

GELIN, 0. E. V. 1954. X-ray mutants in peas and vetches. - Acta Agric. Scand. 4: 55&568.

GRANHALL, I. 1954. Spontaneous and induced bud mutations in fruit trees. - Ibid. 4: 5 9 4 4 0 0 .

GUSTAFSSON, A. The mutation system of the chlorophyll apparatus. - Lunds Univ. Arsskr. N.F. 36: 1 4 0 .

- 1962. Polyploidy and mutagenesis in forest-tree breeding. - Proc. 5th World Forestry Congr., p. 793-805.

- 1963. Productive mutations induced in barley by ionizing radiations and chemical mutagens. - Hereditas 50: 211-263.

GUSTAFSSON, A. and SIMAK, M. 1958. Effect of X- and y-rays on conifer seed. - Medd. Stat. Skogsforskningsinst. 48: 5: 1-20.

HADLEY, E. B. and WOODWELL, G. M. 1965. Effects of ionizing radiation on rates of CO, exchange of pine seedlings. - Radiat. Res. 24: 650-4356.

MERGEN, F. and JOHANSEN, T. S. 1963. Effect of ionizing radiation on microsporo. genesis in Pinus rigida MILL. - Radiat. Bot. 3 : 321-331.

MERGEN, F. and STAIRS, G. R. 1962. Low level chronic gamma irradiation of a pitch pine-oak forest-its physiological and genetical effects on sexual reproduction. - Ibid. 2:205-216.

MERGEN, F. and THIELGES, B. A. 1966. Effects of chronic exposures to Coeo radiation 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. - Ibid. 2 : 125-129.

NILSSON-EHLE, H. 1938. Darstellung tetraploider Apfel und ihre Bedeutung fur die praktische Apfelzuchtung Schwedens. - Hereditas 24: 195-209.

NYBOM, N. 1961. The use of induced mutations for the improvement of vegetatively propagated plants. - Mutat. Plant Breed. NAS-NRC 892: 252-294.

OHBA, K. 1964. Studies on radiosensitivity and induction of somatic mutations in forest trees. - Gamma Field Symposia No. 3 Mutat. Quant. Traits 1964.

139-145.

experiments with higher plants. - Acta Radiol. Suppl. 254: 7 3 - 8 1 .

Bot. 2: 35-39.

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 chronio- some aberrations. - Proc. Nat. Acad. Sci. U S . 25: 397-405.

SCHNARF, K. 1941. Vergleichende Cytologie des Geschlechtsapparates der Kormo- phyten. - 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, G. hl. 1962. Prediction of the sensitivity of plants to chronic gamma irradiation. - Ibid. 2: 9-26.

STAIRS, G. R. 1964. Effects of chronic gamma radiation on seed production in an oak forest. - Advancing Frontiers Plant Sci. 9: 207-216.

SWANSON, C. P. 1957. Cytology and cytogenetics. - Prentice-Hall. SYRACH LARSEN, C. and WESTERGAARD, hi. 1938. Contributions to the cytogenetics of

forest trees. I. A triploid hybrid between Larir decidua MILLER and Larir occi- dentalis NUTT. - J. Genet. 36: 523-530.

WETTSTEIN, D. VON, GUSTAFSSON, a. and EHRENBERG, I,. 1959. hlutationsforschung und Ziichtung. - Arbeitsgemeinsch. f. Forsch. Land. Nordrhein - Westf. Ed. L. BRANDT. Koln und Opladen. 73: 7-48, 59-60.

WOODWELL, G. hI. 1962. Effects of ionizing radiation on terrestrial ecosystems. Ex- periments show how ionizing radiation may alter normally stable patterns of ecosystem behavior. - Science 138: 572-577.

WOODWELL, G. hl. and MILLER, L. N. 1963. Chronic gamma radiation affects the distribution of radial increment in Pinus rigidu stems. - Ibid. 139: 222-223.

WOODWELL, G . hi. and SPARROW, A. H. 1963. Predicted and observed effects of chronic gamma radiation on a near-climax forest ecosystem. - Radiat. Bot. 3: 23 1-23 7 .

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Genetic Changes Induced