A.H. Sparrow

Some factors affecting the responses of plants to acute and chronic radiation exposures

Abstract The radiobiological responses of a large number of species of higher plants have been studied after acute or chronic irradiation of growing plants mostly with γ radiation from Co60. Some acute treatments were made with X-rays. The radiation responses studied were of four main types: chromosome breakage, somatic mutation, growth inhibition and lethality. Somatic mutations in petals of several species of plants heterozygous for flower colour show responses most readily explicable on the basis that they result from deletion of the dominant locus, i.e., in most respects they behave as chromosome breakage events and not as point mutations. The yield of somatic mutations is reduced by dose fractionation of acute exposures or, within limits, by reduced dose rates with chronic exposures. The frequency per r of somatic mutations and of chromosome deletions produced under chronic irradiation is higher during periods of slow growth than during periods of fast growth. Definite evidence of long-term cumulative dosage effects was described for Pinus and Taxus. Over a long period (several years) at dose rates as low as 5 r/day, cumulative effects are expressed as severe morphological deformity, severe growth inhibition, or even death. A correlation was shown between the acute and daily chronic doses necessary to produce severe growth inhibition in young plants of several different species. To produce equivalent effects the acute dose required averages about thirteen times the daily chronic dose. In diploid species a clear relationship was shown between the average nuclear volume of apical meristem cells and tolerance to chronic gamma radiation. The larger the nuclear volume the greater the sensitivity of the nucleus and ultimately of the whole plant. The frequency of somatic mutation per r and nuclear size were shown to be directly related. Preliminary data also indicate a similar relationship between nuclear volume and frequency of chromosome aberration and for three species between average amount of DNA per nucleus and radiosensitivity (the more DNA the greater the sensitivity). Two polyploid series (Chrysanthemum and Sedum) showed increasing resistance as the degree of polyploidy increased. Preliminary data suggest that increasing chromosome number without known polyploidy and without a changing nuclear volume has a protective effect. A theoretical model was presented describing a possible explanation for the protective effect. The protection offered by doubling the chromosome number without polyploidy may be greater than that afforded by polyploidy at a doubling level. Some theoretical aspects relating to the interrelationship of certain major factors determining radiosensitivity are presented. Analysis of the relationship between sensitivity of different species and nuclear volume indicates that a constant (or nearly constant) number of ionizations per nucleus is required to produce growth inhibition or death. This relationship supports the concept that a cluster effect such as chromosome breakage (deletion) may be the critical event or events. Differential sensitivity results in part from the different doses required to produce a unit number of breaks in nuclei of different average volumes. The protective effect associated with increasing chromosome number or polyploidy is assumed to result from the reduced seriousness of an average break or deletion as the chromosome number increases. Clarification of the manner in which radiosensitivity is determined in different species should make predictions of radiosensitivities possible in the very near future. This should hold for animal species as well as for plants. Extrapolation of certain kinds of radiobiological data from one species to another can now be made on a reasonably sound basis.

Mutations induced in Tradescantia by small doses of X-rays and neutrons - Analysis of dose-response curves.

Dose-response curves for pink somatic mutations in Tradescantia stamen hairs were analyzed after neutron and x-ray irradiation with doses ranging from a fraction of a rad to the region of saturation. The dose-effect relation for neutrons indicates a linear dependence from 0.01 to 8 rads; between 0.25 and 5 rads a linear dependence is indicated for x-rays also. As a consequence the relative biological effectiveness reaches a constant value (about 50) at low doses. The observations are in good agreement with the predictions of the theory of dual radiation action and support its interpretation of the effects of radiation on higher organisms. The doubling dose of x-rays was found to be nearly I rad.

Tradescantia Stamen Hairs: A Radiobiological Test System Applicable to Chemical Mutagenesis

Several species in the family Commelinaceae, of which Tradescantia is a member, have features particularly well suited for certain radiobiological studies. Effects produced by ionizing radiations which are easily studied include (1) chromosome aberrations in microspores, root tips, ovaries, and stamen hairs; (2) somatic mutations in petals and stamen hairs in clones heterozygous for flower color; (3) pollen abortion; (4) loss of reproductive integrity in stamen hairs; and (5) whole plant or seedling death. The stamen hairs of Tradescantia clone 02 have also proved to be sensitive to radiation-induced mutations at doses in the millirad region.(1)

Comparison of somatic mutation rates induced in tradescantia by chemical and physical mutagens

Summary When genes for flower color and other phenotypic characteristics are present in a Heterozygous condition in Tradescantia plants, they can be used as genetic markers for the study of induced mutation. The procedure used is to expose cuttings containing young flower buds to chemical or physical mutagens. 1 to 3 weeks later the resultant mutant pink or colorless cells can be seen in mature flowers, and after relatively simple scoring procedures somatic mutation rates can be calculated. Dose-response curves for ethyl methanesulfonate (EMS) and 1,2-dibromoethane (DBE) were obtained and compared with existing X-ray dose-response curves. Two clones, 02 and 4430 were exposed to known levels (from 5–250 ppm) of EMS in gaseous form for periods of from 1–19 h. After exposure to X-rays pink mutation rates showed similar sensitivities for both clones but the rate for colorless was much higher for clone 4430 and the slope much steeper. After treatment with EMS both colorless and pink rates were appreciably higher for clone 4430 than for clone 02. Preliminary data show similar differences in clonal sensitivity following exposures to DBE. The large differences in mutation rates between loci and between clones after chemical and physical mutagen treatments are not understood at present. Possibly differences in mutation rates between the two clones after EMS treatment may be associated with a larger amount of heterochromatin or a greater number of SAT-chromosomes in clone 4430. The genetic basis for the phenotypic changes (pink and colorless) is not definitely known but may be associated with chromosome breakage, gene mutation, chromosome non-disjunction, or somatic crossing over.

Relationship between Nuclear Volumes, Chromosome Numbers, and Relative Radiosensitivities.

An inverse relationship between a volume estimated to be associated with interphase chromosomes and acute lethal exposure to x-or gamma radiation has been found in 16 plant species. The apparent differences in radiosensitivities found would seem spurious, since the estimated average energy absorbed in the nucleus per chromosome (3.6 x 106 ev) approaches a constant (variation less than fourfold) in spite of wide ranges of lethal exposures (0.6 to 75 kr), of nuclear volumes (43 to 1758 �3), and of somatic chromosome numbers (6 to 136). The regression line obtained can be used to predict the radiosensitivities of other plant species if their nuclear volumes and chromosome numbers are known.

The Use of Radiocobalt as a Source of Gamma Rays and Some Effects of Chronic Irradiation on Growing Plants

Radiocobalt has been used as a source of gamma rays in a study of the effect of chronic irradiation on growing plants. The installation and method of operation of sources of approximately 16 and 145 curies is described. The Co60 was placed in the center of an experimental plot in such a fashion that it could be operated safely by remote control. By placing plants in concentric circles (or arcs) around the source at various distances it was possible to study the effects of a wide range of radiation intensities. Actual dosages used varied from less than 0.1 r per day up to dosages in excess of 2,000 r per day. The tolerance to ionizing radiation of different species of plants studied varied widely. Preliminary tolerances are given for nineteen different species of plants. Tradescantia paludosa showed severe effects at 30 r per day, whereas Gladiolus showed relatively little effect after 2,000 r per day for 42 days. Mutation rates of four endosperm characters of corn revealed the following: (1) There was no increase in mutation rate over the control when the radiation was less than 5 r/day. (2) Above 5 r/day there was a gradual increase up to 31 r/day with a more marked increase from 32 to 57 r/day. From 57 r/day to 127 r/day the increase in mutation rate was even more marked. The increase in mutation rate at the higher intensities of radiation was greater than expected if the increase is linearly proportional to dose. (3) The logarithm of the mutation rate seemed to show a linear relationship with dose received. (4) The gene with the greatest spontaneous mutation frequency (R) showed a smaller percentage increase in induced changes than the other three genes Su, Sh and Pr. Under chronic gamma radiation the percentage of abnormal pollen grains in corn increased with an increase in radiation. There was no marked increase over the control below 2.6 r/day and the maximum effect noted was 57 per cent. abnormal grains at 226 r/day. Chromosome fragments were scored in somatic and meiotic (first) anaphase of Tradescantia after 47 days exposure. Fragmentation in meiosis was more than tenfold greater than that observed in somatic cells. Micronuclei derived from chromosome breaks were scored in microspores of chronically exposed Tradescantia. A statistically significant increase in number of micronuclei was found at a dose rate as low as 0.41 r per day. The numbers of micronuclei did not increase significantly after 32 and 64 days as compared to that found after 16 days. Other effects observed included killing, various degrees of growth inhibition, inhibition of flowering, pollen abortion, reduced fertility (or complete sterility in some cases) abnormal growth pattern of both floral and vegetative parts as well as a strong tendency to proliferative growth in some cases. The effect of irradiation on production of crown gall in tomatoes is also briefly reported.

Prediction of the sensitivity of plants to chronic gamma irradiation

The principal site of damage to plants by ionizing radiation is the cell nucleus. The effect on plant growth, measured as size or weight, used as the criterion of sensiviti to chronic gamma irradiation, shows the variation in sensitivity between different species of higher plants to approach 500-fold. This variation is correlated with chromosomal and nuclear characteristics: that is, plants which have low chromosome numbers and large nuclear volumes are most sensitive while polyploids and plants with high chromosome numbers and small nuclear volumes are highly resistant. The rate of nuclear division is also important. A slow rate increases the exposure time of each interphase nucleus to chronic irradiation and thereby increases the nuclear damage. Any environmental factor which influences the rate of growth will by influencing the rate of cell division, affect sensitivity. Environmental factors influencing the degree of response caused by ionizing radiation fall into four general categories: (1) those modifying dosage, dose rate or dose fractionation; (2) those involving type of ionizing radiation; (3) those influencing growth rate or rate of cell division; and (4) those affecting recovery from radiation damage. Consideration of these nuclear and environmental factors permits the prediction of radiosensitivity of plant species not previously irradiated. At the plant population level, chronic irradiation can probably be expected to have its most severe effects on sexual reproduction because during and after meiosis: (1) nuclear volume is high; (2) chromosome number is reduced after meiosis; (3) the rate of nuclear division may be low, some species requiring 2 years between meiosis and full maturation of seeds; (4) meiotic pairing and reduction tend to enhance the damage wrought by aberrations which may survive in diploid somatic cells. The high sensitivity of sexual reproduction is probably further enhanced by seed dormancy, during which damage accumulates. The relative dosage levels necessary to produce specified responses in growth rate, reproductive capacity or in degree of mortality vary greatly within a species. Both vegetative growth and the integrity of the sexual reproductive process of pines appear to be highly susceptible to damage from ionizing radiation and the genus Pinus includes some of the most radiosensitive plants known. Chronic exposures averaging about 8 months per year for 9 years produced detectable effects on growth of P. rigida at average dose rates as low as 2 r/day. The lethal acute dose for P. strobus seedlings of 600 r also indicates a high radiosensitivity. The principal reasons for these high sensitivities are the large nuclear volume of pines and, for chronic exposures, the long period between production of the meiocytes and the maturation of seed. It is predicted that many other gymnosperms will have a radiosensitivity approaching that of pines. A comparison of the estimated maximum exposure of vegetation from fallout occurring in New York City in 1958 with the minimum chronic radiation levels known to influence growth and reproduction of Pinus rigida indicates that these levels of fallout are approximately 1/800th of the 2 r/day necessary to produce visible morphological effects within several years. A forest stand representative of the Long Island oak-pine forest is currently being irradiated with chronic gamma radiation from about 9500 c of cesium-137 in an effort to provide quantitative estimates of radiation effects at the population and community levels. Present information makes possible the prediction, on the basis of nuclear characteristics, that the first years exposure will cause selective killing. All trees within 35 m of the source (85 r/day) will die leaving a shrub community dominated by Gaylussacia baccata, probably one of the most resistant of the species present. The pitch pine (Pinus rigida), with its large nuclear volume, is the most sensitive plant present and will probably be killed by the second year at distances up to 75 m (15 r/day). This radiation facility is available to research workers interested in the general problem of radiation effects on organisms.

Chromosomes and cellular radiosensitivity. I. The relationship of D0 to chromosome volume and complexity in seventy-nine different organisms.

Cellular radiosensitivity (D0) was correlated with the chromosome volume of seventy-nine organisms ranging from viruses to higher plants and animals. A plot of D0 versus interphase chromosome volum...

RADIOSENSITIVITY STUDIES WITH WOODY PLANTS. I. ACUTE GAMMA IRRADIATION SURVIVAL DATA FOR 28 SPECIES AND PREDICTIONS FOR 190 SPECIES.

Abstract Survival curves were determined for 12 species of woody angiosperms and 16 gymnosperms given 16-hr γ-irradiations during the early period of spring growth. The plants were scored approximately one year after irradiation and annually thereafter for up to 6 years. Since second-year scoring has been found to be a reasonable approximation of ultimate survival, second-year survival data have been used throughout. ld 10 s, ld 50 s and ld 90 s were determined from survival curves fitted by probit approximation; ld 100 s were actual values (first exposure which killed all plants). ld 50 s in the gymnosperm species ranged from 460 to 1203 R (average 826 ± 54 R) and in the angiosperms from 477 to 17,500 R (with 8 of the 12 species between about 3000 and 8000 R). The relationship previously found between interphase chromosome volume (ICV) and radiosensitivity for other end points was confirmed for the above end points (the larger the ICV, the greater the sensitivity). The regressions obtained by plotting ld 10 s, ld 50 s, ld 90 s, and ld 100 s against ICVs had similar slopes (average −0·73). Predicted radiosensitivities of species not irradiated can be obtained from these regressions if the ICV is known. Within a species, nuclei of actively growing woody plants average 1·65 times larger than those of dormant plants, and nuclei of terminal buds, 1·25 times those of lateral buds. These differences are reflected in the expected differences in radiosensitivities. ICVs were determined for 190 additional unirradiated species of gymnosperms and woody angiosperms, mainly for the purpose of predicting ld 50 s and other end points. A wide range of ICVs was found, with gymnosperms tending to have large ICVs, and most angiosperms relatively small ICVs with very little overlap between the two groups. The rank order of ICVs and predicted ld 50 s is given for 45 plant families (mostly based on a small number of species per family). About 50 per cent of the 120 gymnosperms studied have predicted ld 50 s of 1 kR or less. Only five species have ld 50 s above 2 kR and none of these is of major economic value. The predicted ld 50 s for 95 angiosperm species tend to be much higher, with over 80 per cent between 2 and 8 kR, only 11 species at 2 kR or less and only 4 above 8 kR. Since exposure times much shorter than the 16-hr periods used here (but common in mammalian radiation studies) would lower the plant ld 50 s appreciably, it appears that the radiosensitivity of a high percentage of acutely irradiated gymnosperm species would be in the same general range as that of mammals. These predictions can be used as a rough estimate of the exposures required to produce a specified amount of radiation-induced injury in the planning of future experiments or in rough predictions of the consequences of the γ-component of fallout radiation. The usefulness of such predictions and some of the factors limiting their accuracy are discussed.

Comparative effects of ionizing radiation and two gaseous chemical mutagens on somatic mutation induction in one mutable and two non-mutable clones of tradescantia

Abstract The X-ray dose responses of mutable clone 0106 of Tradescantia (mutable for blue to pink), and its parent clone 02 have been determined for pink and colorless mutations in stamen hair cells, and are compared to the previously determined X-ray response for pink mutations of a third unrelated clone, clone 4430 (hybrid of T. subacaulis and T. hirsutiflora). X-ray response curves are compared to the response curves of the same three clones after exposure to the gaseous phase of the alkylating agent ethyl methanesulfonate (EMS) and the fumigant and gasoline additive 1,2-dibromoethane (DBE). X-irradiation induces a pink mutation rate in mutable clone 0106 that is significantly higher than that of the nearly identical pink mutation rates in clones 02 and 4430. However, the colorless mutation rates of clones 02 and 0106 are not significantly different from one another. In clones 02 and 0106, pink mutations occur more frequently than colorless mutations at lower doses, but colorless dose—response curves saturate at higher doses than do those for pink mutations. Exposure—response curves for EMS and DBE have characteristics similar to those of X-ray response curves: exponential rise followed by an area of saturation. However, it was found that the relative sensitivities of the three clones to the gaseous mutagens and to ionizing radiation do not parallel one another. Where clones 02 and 4430 are equally sensitive to X-rays, at equal mutagen concentration clone 4430 is 6–7 times more sensitive to EMS and 7–9 times more sensitive to DBE than is clone 02. Mutable clone 0106 shows intermediate sensitivities to both EMS and DBE.