differ markedly in their quantitative effects.34' 5 The sensitivity ratio, about 3

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1 INDUCED POLLEN LETHALS FROM SEEDS OF DA TURA STRAMONIUM EXPOSED TO RADIATION FROM A NUCLEAR DETONATION* BY J. L. SPENCER AND A. F. BLAKESLEE UNIVERSITY OF MASSACHUSETTS AND SMITH COLLEGE GENETICS EXPERIMENT STATION Communicated April 9, 1954 Biological effects of ionizing radiations produced by an explosion of a nuclear device may be complicated by the heterogeneity and extreme intensity of the emissions. Changes in biological systems may be induced that are not attributable to blast, heat, or single-type radiation effects. Many data and discussions have resulted from laboratory studies of radiations, but until recently scant information was available on the effects induced by the unparalleled intensity of radiation from an explosion of a nuclear device. This study presents some of the results of the combined biological tests on the effectiveness of a nuclear detonation, reported in part by Conger' and Kirby-Smith and Swanson.2 The present investigations were particularly concerned with the biological efficiency of fast neutrons from a nuclear device, as compared to other types of radiation, especially X-radiation. Neutrons and X-rays produce the same kinds, or qualitative types, of rearrangements for chromatid as well as for chromosome effects, but the two radiations differ markedly in their quantitative effects.34' 5 The sensitivity ratio, about 3 or 4 for fast neutrons as compared to X-rays, is different for different kinds of aberrations. Fast neutrons have been observed to be about three times as efficient as X-rays in inducing single gene mutations in Drosophila.6 Thermal neutrons have been reported to have a cytogenetic effect of about eleven times that of X-rays per unit dose.' The difference in effectiveness between thermal neutrons and X- rays is believed to be due largely to the internal origin of the protons and alpha rays which result from capture reactions. However, the discrepancy that apparently exists between the two types of neutrons is thought to be a manifestation of the relative ease with which thermal neutrons are captured. Pollen lethals in Datura, as employed in studies of aged seeds8 and thermalneutron effects,9 furnish a method for determining with considerable accuracy the biological efficiency of fast neutrons as well as the dose estimates in roentgen equivalents. Additional advantages afforded by rates of pollen abortion in Datura include (1) the rapidity and ease by which large numbers of plants can be handled; (2) an indication of the type of aberration present, viz., chromosomal or gene, with an accuracy of over 8 per cent; and (3) a convenient method for detecting and delimiting the aberrant sectors. The source of neutrons furnished by a nuclear detonation made it possible to determine the relative effect of fast neutrons on mutation rates in comparison with plants from gamma-irradiated and X-irradiated control seeds. Experimental Procedures and Results.-Seeds of Datura stramonium, Line 1, dried to a constant moisture content of 4.9 per cent and sealed in aluminum vials, were exposed to fast-neutron radiation from a nuclear detonation. These seeds were shielded from blast, heat, and much of the gamma radiation, as noted by Conger,' by being inclosed in a temperature-controlled chamber capped with a lead hemis- 441

2 442 GENETICS: SPENCER AND BLAKESLEE PROC. N. A. S. phere 7 inches in thickness. Seeds of the same Line 1-in fact, from the same capsules-and possessing the same moisture content were treated with gamma rays from Co6w and with X-rays at Brookhaven National Laboratory. No seed was used from flowers that yielded over 1 per cent bad pollen. Germination was carried out in the greenhouse, and the plants were subsequently transferred to the field, where they matured and were studied for types of pollen abortion and for chromosomal aberrations.'4 Pollen was examined from a single flower from each of the two main forks of the plant tested and the condition of the pollen recorded. In general, three types of grains were recognized: (1) the normal grain; (2) the chromosomal type, or small, shriveled, and empty grain; and (3) the gene type, or subnormal to very small size grain, often showing degenerative changes in the character of its contents. If a flower showed abnormal pollen, additional adjacent flowers were examined in order to delimit the sector exhibiting the pollen abnormality. The Atomic Energy Commission furnished all dose estimates used in this report. The roentgen equivalent physical (rep) is based on 93 ergs/gm., which is the energy absorption of 1 roentgen in water. Problems concerning the dosimetry for the detonation have been noted by Sheppard and Darden" and by Kirby-Smith and Swanson.2 The gamma ray contamination is probably between 25 and 35 per cent. The authors are indebted to Dr. Seymour Shapiro, of the Brookhaven National Laboratory, for exposing the seed to gamma and X-rays. TABLE 1 GERMINATION OF Datura SEED Per Cent Per Cent Exposure Seed Exposed Germinating Maturing Hemisphere rep (Estimate) C 7 6 D 4 6 * G I L o , r , r ,r Control 3 tested * A few seeds germinated but did not survive. The lethality effects of the detonation on Datura seeds from hemispheres C and D (Table 1) were very pronounced; none of the plants matured, although a few of the seeds germinated. Normally, germination percentages for Datura are above 8, and 75 per cent or more of the plants usually mature. For these tests, 84 per cent of the control seeds (3) germinated, and all the seedlings matured. Fifty per cent or more of the exposed seeds from hemispheres G, I, L, and germinated, and 3, 55, 74, and 75 per cent of these plants matured. Although the same percentage of seeds exposed to gamma radiation of 1, r and seeds exposed to 246 rep from the detonation germinated, fewer plants from the latter treatment ma-

3 VOL. 4, 1954 GENETICS: SPENCER AND BLAKESLEE 443 tured. The seeds given a dose of 1, r X-radiation showed somewhat lower germination (71 per cent) and maturation (65 per cent). A summary of the data on plants tested and possessing pollen lethals is given in Table 2. Approximately the equivalent percentages of plants bearing pollen abnormalities were yielded (1) by exposure to 1, r of gamma rays, (2) by the same dosage of X-rays, and (3) by a neutron dose estimate of 76 rep. Extrapolation indicates that a dose estimate of about 8 rep would more closely approximate the figures yielded by gamma and X-ray exposure of 1, r. Thoroughly positive conclusions concerning relative biological efficiencies may not be possible to arrive at, in view of the small numbers employed in these gamma ray and X-ray tests. However, the data at hand, from the tests noted here and from previous work, demonstrate that fast neutrons are between 12 and 15 times more efficient than gamma radiation or X-radiation when mutation rates based on pollen lethals in Datura are compared. TABLE 2 INDUCED POLLEN LETHALS IN Datura Plants with Exposure Plants Tested* Pollen Lethals Hemisphere Sulfur flux rep C i 7 Lethal... D 1.4 X 11 4 Lethal... G 9.43 X (93%) I 8.37 X (86%) L (59%) 2.5 X (21%) t 1.9 X 11' ( 9%) 1, r (64%) 2, r (76%) 1, r (67%) Control 218 ( %) * Two flowers per plant examined. t Outside hemisphere, i.e., not placed within lead-capped chamber. The data accumulated on the types of pollen lethals induced by the three kinds of exposures, i.e., nuclear detonation, gamma rays, and X-rays, suggest that the dose estimate of 76 rep of fast neutrons from the nuclear device was roughly equivalent to 1, r of either gamma rays or X-rays (Table 3). These tabulations were not limited to two flower samples employed for comparisons in the previous table (Table 2), and, as a result, the additional gamma and X-ray data afford further reliability for evidence of relative biological efficiencies. If the flowers found to contain both types of pollen lethals (Table 3) are apportioned between the single types noted, then the dose estimate of 76 rep induced about 4 per cent of the flowers to show a chromosomal type of pollen lethal and 1 per cent to possess a gene type of pollen lethal. On the same basis, gamma radiation of 1, r induced about 28 per cent chromosomal type of aberration and 18 per cent gene type. The X-irradiated material treated by 1, r appears to be more comparable to the 76 rep of fast neutrons than the gamma-exposed plants, since X-rays of this dosage apparently induced about 35 per cent of the flowers to

4 444 GENETICS: SPENCER AND BLAKESLEE PROC. N. A. S. bear chromosomal types of pollen abortion and 6 per cent to have gene types. The highest dose estimate of fast neutrons from the nuclear device from which mature Datura plants were obtained, 2 rep, yielded approximately 65 per cent of the flowers with chromosomal-type pollen abortion and approximately 2 per cent with gene-type lethals. Exposure Hemisphere G I L 1, r 2, r 1, r Control * Outside hemisphere. TABLE 3 TYPES OF INDUCED POLLEN LETHALS IN Datura POLLEN LETHAL TYPES FOUND IN --INDIVIDUAL FLOWERS (PER CENT)- Flowers Chromosomal Tested Chromosomal Gene and Gene rep Exposure Hemisphere rep G 2 99 I L t , r 44 2, r 15 1, r 16 Control * Based on sample of two flowers per plant. t Outside hemisphere. TABLE 4 INDUCED POLLEN LETHAL SECTORS IN Datura Plants Tested I PLANTS and Possessing Chromosomal/ Gene/Normal Pollen Lethals8 Normal Type 9 ( 9%) 16 ( 9%) 44 (22%) 16 (25%) 9 (39%) 11(25%) 9 (56%) WITH SECTORS BETWEEN* ^ Chromosomal/ Type Gene Type 1( 1%) 6( 3%) 6 ( 3%) 5( 8%) 4 (17%) 1 (23%) 2( 3%) 31(32%) 83 (44%) 25 (13%) 2( 3%) 2( 9%) 3 ( 7%) 4(3%) 1( 5%) The relative biological efficiency of detonation fast neutrons as indicated by data on chromosomal and gene aberrations based on types of pollen lethals in Datura flowers (Table 3) appears to be somewhat higher than the efficiency index arrived at by comparing two-flower samples. However, the ample number of flowers tested of the gamma and X-ray material gives a degree of reliability to these data not accorded to that summarized in Table 2. The results recapitulated in Table 3 suggest an index of about 15.

5 VOL. 4, 1954 GENETICS: SPENCER AND BLAKESLEE 4-1 The analysis of data concerning pollen lethal sectors in Datura furnishes another method of estimating and comparing relative mutation rates. The induced changes generally occur in limited sectors of the plant; however, the entire plant may be involved. These sectors develop from aberrant initials in the second layer The X-ray data are too limited to be of comparative of the plumule in the embryo. value (Table 4); however, results obtained by analysis of two-flower samples from plants that arose from gamma-ray-treated seed indicate that the equivalency for detonation fast neutrons does not exceed 8 rep by much, if at all, and there is a strong suggestion that the figure may be lower. A number of plants tested were found to bear the same type of mutation in both flowers. In other words, no sectoring was noted in these plants, and therefore they were not included in Table 4. Furthermore, the data show a direct correlation between the induction of pollen lethals involving the entire plant and severity of exposure (Table 3). Plants possessing a single type of pollen aberration usually bear the chromosomal kind. The data indicate that a higher percentage of plants possessing a single over-all pollen-abortion type resulted from exposure to 76 rep of fast neutrons than resulted from either a higher or a lower dose estimate. In fact, 122 plants of a total number of 197 with pollen lethals induced by exposure to a dose estimate of 76 rep of fast neutrons show a single type of aberration. Pollenabortion types involving the entire plant are thought to arise when only a single, lightly hit cell of the second layer survives, the adjacent cells of this layer having been killed. Discussion and Conclusions.-The marked difference between the relative biological efficiency of fast neutrons from a nuclear device and either gamma radiation or X-radiation is demonstrated by analysis of mutation rates as indicated by pollen lethals arising from exposed Datura seed. Fast neutrons apparently induced the same qualitative types of pollen lethals as the gamma rays and X-rays, but the sensitivity ratio, as determined by quantitative differences, is probably 13 or 14. The dose estimate of 76 rep of fast neutrons is comparable with 1, r of either gamma or X-radiation. Giles, in his early study of the comparative effects of fast neutrons and X-rays,3 noted that neutrons were more efficient than X-rays in breaking Tradescantia chromosomes. Previous to this report, Marshak'2 and Marshak and Mallock5 Various sensitivity ratios be- had shown the superior efficiency of fast neutrons. tween 2 and 6 were obtained from these data, and the greater sensitivity of the neutrons was attributed to the greater number of ionizations induced by protons than by the electrons of X-rays. Thoday and Lea4 reported different sensitivity ratios for different types of chromosomal aberrations, and they obtain ratios as high as 1 for interchanges. The present work indicates the same relative sensitivity ratio for fast neutrons to X-rays whether chromosomal types of pollen lethals or gene types are compared. Either the same kinds of aberrations, in the same ratios, are caused by the different exposures, or the distinction between the two basic types of pollen lethals is considerably less reliable than is manifested by previous work.8' 9 1 A recent study of injurious actions of ionizing radiations on seeds reports several differences in the actions of fast neutrons as compared to X-rays.13 Seeds were shown to be 2-3 times more sensitive to fast neutrons than to X-rays, and this sensitivity to neutrons was less

6 446 GENETICS: SPENCER ANDfBLAKESLEE PROC. N. A. S. dependent on the physiological state of the seeds. Thermal neutrons have been shown to be about 11 or 12 times more efficient in inducing cytogenetic change than are X-rays.7 The results reported here, indicating a biological efficiency for fast neutrons from a nuclear device of about 13 or 14, suggest a restudy of the effects of thermal neutrons in relation to the cytogenetic effects of fast neutrons and X-rays, since the former superiority or greater effectivity of thermal neutrons was thought to result from the relative ease by which they are captured. Also, the reliability and accuracy which allow pollen lethals to be employed as criteria in distinguishing chromosomal aberrations from genic changes need to be substantiated and established. In summary, the relative effectiveness of fast neutrons from a nuclear detonation appears to be about 13 or 14 times that of either gamma rays or X-rays when mutation rates based on pollen lethals in Datura are compared. Further data concerning relative sensitivities are given and discussed in the following companion report. 14 * A portion of the Atomic Energy Commission "Report on Genetic Effects of Fast Neutrons from Nuclear Detonations." Contribution from the Department of Botany, Smith College, New Ser., No. 59. This work has been aided by a grant from the Atomic Energy Commission. The authors wish to thank Dr. Harold Plough, of Amherst College, formerly assistant chief of the Biology Branch, Division of Biology and Medicine, Atomic Energy Commission, for his guidance and help in this undertaking. 'A. D. Conger, Science, 119, 36-42, J. S. Kirby-Smith and C. P. Swanson, Science, 119, 42-44, N. H. Giles, Jr., Genetics, 28, , J. M. Thoday and D. E. Lea, J. Genetics, 43, , A. Marshak and W. S. Mallock, Genetics, 27, , P. T. Ives, R. P. Levine, and H. T. Yost, these PROCEEDINGS, 4, , A. D. Conger and N. H. Giles, Jr., Genetics, 35, , J. L. Cartledge and A. F. Blakeslee, these PROCEEDINGS, 2, 13-11, J. L. Spencer, W. R. Singleton, and A. F. Blakeslee, these PROCEEDINGS, 39, , H. T. Yost, Jr., W. R. Singleton, and A. F. Blakeslee, these PROCEEDINGS, 39, , C. W. Sheppard and E. B. Darden, Jr., Science, 119, 44-45, A. Marshak, Proc. Soc. Exper. Biol. Med., 41, , N. Nybom, X. Gustafsson, and L. Ehrenberg, Bot. Notiser, 4, , H. T. Yost, Jr., J. Cummings, and A. F. Blakeslee, these PROCEEDINGS, 4, , 1954.