224 Cytologia 17

A Study of X-ray-induced Chromosomal Aberrations in Barley1

Richard S. Caldecott2 and Luther Smith3

Received June 19, 1952

Introduction The mechanism of, and the steps involved in, the production of radiation-induced chromatinic aberrations is of fundamental importance to those interested in studies on radiation effects. For that reason, many reports and thoughts on chromosomal aberrations, sometimes re ferred to as mutations, have appeared in the literature. Over a period of years, the writers and their associates have studied many thousands of cells which were derived from irradiated seeds. These observations have provided an opportunity to develop certain theories with regard to the manner of origin and behavior of chromo somal breaks, which in some respects differ from, or expand, current concepts on these points. It is hoped that the present report will be a contribution to a better understanding of the behavior of irradiated chromosomes. Further, it is hoped that the considerable number of photomicrographs, illustrating various kinds of chromosomal aberrations, will be of value to cytogeneticists in research and teaching.

Review of literature Many investigators have discussed the mode of origin and the types and frequencies of chromosomal aberrations induced by irradiating somatic and meiotic nuclei. Thus, our present knowledge is a result of the contributions of a number of workers -particularly Carlson (1938 a, b, 1940, 1941 a, b, 1942), Mather and Stone (1933), Riley (1936), Sax (1938, 1940, 1941 a, b) and Stone (1933). It has been established that the types of induced aberrations depend on whether the chromosomes are effectively monopartite or bipartite at the time of irradiation. If the chromosomes are "effectively" monopartite when irradiated, the aberrations are of the chromosome type; if bipartite, they are primarily of the chromatid type. (See Lea 1947 pp. 194-195 for a diagrammatic presentation of the types of chromosome and chromatid aberrations). Chromatinic bridges are one of the most commonly observed aber

-1 Scientific Paper No. 1129, Washington Agricultural Experiment Stations, Pullman. Project No. 1058.2 Research Fellow, American Cancer Society and Research Associate in Agronomy during the investigation, now Assistant Professor in Agronomy, University of Nebraska, Lincoln. 3 Professor in the Department of Agronomy , State College of Washington, Pullman. 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 225 rations at somatic anaphase following X-radiation. According to Carlson (1938 b), who studied irradiated cells of the grasshopper neuroblast , th ese bridges may result from: 1) The union of two centric chromo some fragments; 2) the union of two centric chromatid fragments; 3) or the fusion of sister chromatids at their broken ends . Mather and Stone (1933) showed a pair of similar chromatid bridges in the crocus . They presumably were formed by the chromatids involved in a dicentric translocation. Sax (1941 a) found that nuclei irradiated in the interphase or resting stage in Allium root tips often had a pair of dicentric chro matid bridges at anaphase. Such bridges were considered to result from the union of two centric chromosome fragments , followed by the separation of the sister strands. The study of the mitotic behavior of acentric chromatinic frag ments is important for a better understanding of chromosome mecha nics. In the grasshopper, Carlson (1938 a) showed that such fragments, induced by x-raying neuroblast cells, separated into chromatids at the same time as their centric counterparts, and gave rise to V's, rings, and pairs of rods. As anaphase progressed, these fragments could move and contact the spindle, and ultimately undergo poleward move ment characteristic of chromosomes and centric fragments. Catcheside et al. (1946) noted that acentric fragments, induced by x-raying Trades cantia microspores, may or may not form sister unions. Koller (1943) indicated that acentric fragments, induced by irradiating pollen grain nuclei of Tradescantia, frequently formed micronuclei. Mather and Stone (1933), working with x-rayed root tips from the crocus and tulip, and Sax (1941 a) working with root tips from x-rayed dormant seeds and bulbs of Allium, were of the opinion that acentric fragments usually lie free in the cytoplasm and are not included in the daughter nuclei. A number of cytogeneticists have expressed the opinion that the ultimate fate of cells undergoing division, i.e. whether or not they or their progeny survive, is primarily dependent on the nature and extent of induced chromosomal changes and gene mutations rather than physio logical changes (Sax, 1942, Koller, 1943, and Lea 1947). For this reason it is of importance to know the fate of acentric fragments and whether or not, when included in the nucleus, they maintain their genetic potential. Carlson (1938, 1941 a) suggested that acentric fragments may persist through the resting stage, and that delayed attachments could occur between the fragments and broken ends of bridges, to produce a normally-behaving translocated chromosome. A rather unique interpretation of radiation-induced breaks and reunions has been suggested by Matsuura (1950) and Matsuura and Haga (1950). These workers irradiated pollen mother cells of Trillium kam tschaticum in the resting stage. They reportgd finding both chromo

- 15* 226 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

some and chromatid breaks. But, more significantly, it was concluded that a broken end might unite with an unbroken end, as well as with another broken end. They were of the opinion that broken ends do not often reunite. They also felt that broken ends could persist and form paired chromatids which "bridged together at their broken ends" in the first mitosis in the pollen grains.

Material and methods

Dormant seeds of Himalaya barley were used in the present study.

The seeds came from a line derived from a single plant, and hence were as genetically uniform as possible. Their moisture content was not determined, but for most tests they were kept in a humidity controlled desiccator for a least six weeks before treatment. The radiation was unfiltered and was obtained from a Beryllium window

A. E. G. 50 Machlett x-ray tube operated at 34 KVP and 27 ma. Under these conditions the half value for Aluminum is 0.060mm. It was calculated that approximately 75% of the radiation would effectively pass through 0.5 cm. of tissue with a specific gravity of one. For most tests the seeds were irradiated at room temperature with the embryo uppermost. In different experiments the dosage varied from 0 to 32,000r.

For observations on root tips, seeds were germinated (after ir radiation) in petri dishes at about 18•Ž. In most cases root tips were removed 24 to 40 hours later. It was usually necessary to take root tips somewhat later from seeds given the heavier dosages. On the basis of exploratory observations, it was believed that observations were made on the first cycle of cell divisions following irradiation.

For observations on pollen mother cells, the irradiated seeds were planted directly in the field, except in a few cases when seeds receiv ing the highest dosages were planted in plant bands and the few sur viving seedlings later transplanted to the field. Spikes at the meiotic stage were taken at random from each test. For both root tip and pollen mother cell analyses, the specimens were fixed in Carnoy's 6:3:1

(ethyl alcohol: chloroform: glacial acetic acid) solution, and smears were made with acetocarmine.

In observations on root tips a conscious effort was made to select cells at a stage such that delayed separation or stickiness was no longer present, yet before true bridges were broken. Identifying two bridges is usually no problem at the late anaphase stage. However, if the two centromeres of a dicentric chromosome are close together , a bridge may break or become very thin at relatively early anaphase . By examing all cells observed under the oil immersion objective, at about a

1,000 diameter magnification, such thin bridges could usually be detected. 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 227

Experimental observations

Chromatinic bridges in root-tip cells

The frequency of chromatinic bringes at mid to late anaphase in cells of root tips grown from irradiated seeds was one of the principal criteria used to measure the effects of ionizing radiations. These bridges are one of the most common, readily detectable aberrations resulting from such radiations. They commonly occur in pairs and, as a matter of fact, in an analysis of 78 cells aimed directly at determining the frequency of single bridges, not one of the 78 had a single bridge. In Table 1 data are presented on the frequencies of paired somatic bridges at various dosages, ranging from 0 to 32,000r. The table also includes data on the frequencies of interchanges observed at meiosis in spikes grown from seeds receiving the same treatments. The paired bridges may lie: 1) Separate and more or less parallel; 2) apparently touching, in which case they frequently lie across each other; or 3) occasionally interlocked. The latter two types usually are not easily distinguishable. In one study involving 792 cells in which two bridges were present, 483 pairs crossed or interlocked, and 209 were parallel, or not crossed. Figs. 1 to 7 illustrate various bridge configurations and probable dicentric chromosomes which form bridges at anaphase.

Table 1. A comparison of the frequencies of interchanges in root-tip cells and pollen mother cells of plants grown from x-rayed dormant seeds of barley

1 An interchange at mitosis is considered to be represented by two (a pair of) bridges. 2 Calculated on a ring-of-four basis with all ring configurations considered to be the result of reciprocal translocations. Thus, a ring-of-six was calculated to be equi valent to two rings-of-four. 228 RICHARDS. CALDECOTTand LUTHERSMITH Cytologia 17

Acentric fragments in root-tip cells Acentric fragments also were used-as-a, measure-of radiation effects.

Figs. 1-8. 1-7, Interchanges and bridges in somatic mitoses. 1, a (rare) single chro matinic bridge at anaphase of somatic mitosis. 2, 3, dicentric chromosomes at prophase and metaphase respectively. 4 to 7, separation of dicentric chromosomes at anaphase (4, 6. 7, are typical cells but the interpretation of 5 is not clear). 7, 8, several re latively long pairs of acentric fragments at anaphase. 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 229

On the average, there were about three times as many such fragments as there were chromatinic bridges. However, this ratio varied some what with dosage, and will be dealt with in another report. Fragments also usually occurred in pairs, ranging in size from minute dots to rods which appeared to be as long as a whole chromosome, although , rods as long as or longer than a chromosome arm were relatively uncommon. In addition to the types of acentric fragments described above, ring fragments occasionally were observed. In many cases it was not possible to determine whether or not these ring fragments had centro meres, but in a considerable number of cases, centromeres were present, as evidenced by apparent stress(es) on the fragment at anaphase. Ring fragments were too infrequent to be important in the data on frequen cies. Figs. 7-14 were selected to illustrate some of the more typical acentric and ring fragments which were observed.

Chromosomal aberrations which persist to meiosis Reciprocal interchanges were by far the most common type of aberration observed at meiosis in spikes of plants grown from x-rayed dormant seeds. Data on the frequency of interchange configurations are presented in Tables 1 and 2. The most frequent type of inter change configuration at meiosis was a ring-of-four plus five bivalents. The next most frequent was two rings-of-four and three bivalents. A ring-of-six and four bivalents was less common than two rings-of-four. More complicated configurations, such as a ring-of-eight were rare and obtained only with higher dosages of radiation. Photomicrographs of pollen mother cells with various interchange configurations are shown in Figs. 15 to 21.

Table 2. Interchange types observed in analyses of pollen mother cells from X-rayed dormant seeds of barley (Dosages ranged from 0 to 32,000r)

What appeared to be iso-chromosomes were the next most common aberation observed at microsporogenesis. In all, 31 separate spikes were found which contained "iso-chromosomes". Structurally, they seemed to be of two types. In the first and most common type, the "iso -chromosomes" occurred in pairs. The usual configuration was six bivalents and two "iso-chromosomes" but in some spikes additional 230 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17 aberrations were also present. Typical pollen mother cells with this type of "iso-chromosome" are depicted in Figs. 22-30.

Figs. 9-14. Fragments in somatic mitoses. 9, long, paired and small acentric frag ments at anaphase, and chromatid bridges. This figure illustrates how close observation is sometimes necessary to determine that more than one bridge is present. 10, acentric dot fragments, a ring fragment and crossed bridges at anaphase. 11 to 13, ring frag ments at prophase, metaphase and anaphase respectively. 14, interlocked centric ring fragments at anaphase. 1952 A Study of X-ray-induced Chromosomal Aberrations in Ba rley 231

Figs. 15-21. Interchanges at microsporogenesis. 15, 16, a ring and chain-of-four and five bivalents, respectively. 17, chain-of-six and four bivalents. 18, two rings-of-four and three bivalents. 19, 20, ring and chain-of-eight and three bivalents, respectively. 21, ring-of-four, chain-of-six and two bivalents. 232 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

In the second type, the "iso-chromosomes" occurred singly. Only three spikes were found with this type of alteration as compared with the 28 spikes which had two (four in one case) "iso-chromosomes". In

Figs. 22-27. Pseudo-iso-chromosomesat microsporogenesis, 22, two pseudo-iso-chromo somesand six closedbivalents. 23, two pseudo-iso-chromosomesof different sizes, and six bivalents, one of which is open. 24, 25, a "y-shaped" associationinvolving two pseudo-iso-chromosomes and two additional chromosomes, plus five bivalents, (from the same spike as 23). 26- two pseudo-iso-chromosomes, a ring-of-four, and four bivalents. 27, four pseudo-iso-chromosomes and five bivalents, one of which is open. 1952 A Study of X-ray-induced Chromosomal Aberrations in Barle y 233

the first spike with one "iso-chromosome" the usual configuration con sisted of five closed bivalents, a ring-of-four , and the single "iso-chro mosome", a total of 15 chromosomes . In the second spike, typical con figurations showed five closed pairs , a ring-of-three and the "iso-chro mosome", a total of 14 chromosomes . Microsporocytes in the third

Figs. 28-33. 28-30, Pseudo-iso-chromosomes at microsporogenesis. 28, 29, two associated pseudo-iso-chromosomes (arrow), two free iso-chromosomes, and five bivalents. 30, four lagging pseudo-iso-chromosomes, and five normal chromosomes nearing each pole at first anaphase. 31-34, single iso-chromosomes at microsporagenesis, 31, 32, an iso chromosome, ring-of-four, and five bivalents. 33, eight chromosomes approaching one pole, seven approaching the other (from the same spike as 31 and 32). 234 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

Figs. 34-37. Single iso-chromosomes at microsporogenesis. 34, an iso-chromosome, ring-of-three, and five bivalents. 35, 36, an iso-chromosome, ring-of-three, ring-of-four, and three bivalents. 37. an iso-chromosome, ring-of-four, a univalent and four biva lents (from the same spike as 35 and 36). Fig. 33 at second anaphase, others of first metaphase. All figs. •~1000 approx.

spike contained either four closed bivalents, a ring-of-four, one univalent

and an "iso-chromosome"; or they contained three closed bivalents,

closed rings-of-three and-four, and one "iso-chromosome", a total of 14

chromosomes. Photomicrographs showing these various configurations

are presented in Figs. 31 to 37.

A consideration of the types of aberrations which were observed

at mitosis and meiosis, their probable origins, etc. will be taken up in the discussion.

Dicussion

A preponderance of the aberrations observed in cells of root tips grown from x-rayed dormant seeds of barley originate from chromo some rather than chromatid breaks. That is, the chromosomes respond to x-rays as though they are monopartite, and the evidence indicates 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 235 that reunions occur before the chromosomes become "effectively" bipartite. Evidence in this connection is the fact that most bridges and fragments observed at anaphase occurred in pairs . Pairs of bridges could occassionally be seen to have unlike arms , emphasizing the likeli hood that they were produced by translocations between non -homologous chromosomes which later divided to form the paired bridges . Based on their appearance at prophase and metaphase , acentric f ragments seem to undergo longitudinal splitting at the same time as do centric fragments and chromosomes . These fragments also commonly are paired at anaphase, indicating a lack of opposing poleward attrac tion and movement characteristic of centric chromatin material . Acentric fragments may lie anywhere in the cytoplasm , although they were found more often in the region of the equatorial plate. However, such fragments sometimes were observed near the poles and hence, by chance, presumably were included in the daughter nuclei in these cells. Circular dot fragments (Fig. 10) which ranged in size from bare microscopic visibility to the diameter of a normal chromosome, were observed frequently. It is difficult to perceive the manner of origin of the smaller dots, but in many cases they were paired, indicating that they had the power of reduplication, and were undoubtedly chromatinic in nature. Presumably they were tiny pieces of chromatin which did not possess the ability to accumulate matrix material in sufficient quan tity to attain a diameter equal to that of a normal chromatid. Acentric fragments which were longer than a normal chromosome arm occasionally were observed. They are not easily accounted for. They could originate from the inactivation of a centromere, but there is no positive evidence for this theory. Another possibility is that they arise from the fusion of two or more acentric chromosome fragments. However, if such fusions occur, they do not do so frequently or else long fragments would be observed more often than they are. It seems certain that a fusion between two acentric fragments is much less likely to occur than a fusion between a centric and an acentric, or between two centric fragments. Altered chromosomes resulting from fusions involving one or two centric fragments occur commonly, which indicates that the centromere plays an important role in the reunion of fragments. A union between a centric and an acentric fragment results in an interchange which may be detected at meiosis, whereas a union between two centric chromosome, fragments results in paired bridges detectable at somatic anaphase. From Table 1 it appears that paired bridges at somatic anaphase and interchanges at meiosis occur with about equal frequencies at the same dosage. Since this is so it seems evident that 236 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

unions between a centric and an acentric fragment and between two centric fragments are about equally likely to occur. Considerable evidence has been accumulated in the present study which indicates that "iso-chromosomes" may originate from two different kinds of chromosomal aberrations. The first and most common type apparently involved translocations between opposite arms of homologous chromosomes. It seems appropriate to call these cases pseudo-iso-chro mosomes. The pollen mother cells of spikes in which they were found characteristically had six bivalents and two pseudo-iso-chromosomes. In most cases the pseudo-iso-chromosomes showed no tendency to associate with any of the other chromosomes or with each other. This would indicate that the breaks which formed the pseudo-iso-chromosomes occurred close to the centromeres. However, in two spikes other con figurations were observed in some microsporocytes. In the first spike, six bivalents and two separate pseudo-iso-chromosomes were usually seen. However, in a few pollen mother cells, the pseudo-iso-chromo somes were associated with one of the bivalents forming a V-shaped configuration (Figs. 24 and 25). This indicates that there had been an exchange of a small segment between one of the homologs which formed the pseudo-iso-chromosomes and another chromosome. The second instance of associations involving the pseudo-iso-chromo some occurred in a spike which usually had five bivalents and two pairs of pseudo-iso-chromosomes. In a few cells two of the pseudo-iso-chro mosomes were associated and appeared as a centrally-constricted biva lent (Figs. 28-29). The association between these two pseudo-iso-chro mosomes probably resulted from pairing between interstitial segments lying near the centromeres. This interstitial segment was evidently too small to result in an association between the members of the pseudo iso-chromosome in most pollen mother cells-at least the association did not persist to first meiotic metaphase. A characteristic of all the "iso-chromosomes" observed was that they tended to lag at anaphase of the first and second divisions (Figs. 30 and 33). For the reasons mentioned, the aberrations present in the 28 spikes discussed above seem to be more properly classified as "pseudo-iso-chro mosomes" rather than as true iso-chromosomes. The second type of aberration observed, which involved pairing between the two arms of one chromosome, apparently was a typical iso-chromosome, i.e. a chromosome formed by the deletion of one arm, producing a telocentric fragment, which reduplicated. This would result in a chromosome with two identical arms a true iso-chromosome. This type of alteration was observed in three separate spikes. In each of these three spikes, at least one interchange was also present (Figs. 31 - 37). In two of the spikes, an association of three chromosomes was 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 237 present in some of the microsporocytes. These two cases will be dis cussed below in dealing with delayed fusions between broken ends of chromosomes. In one spike in which a single iso-chromosome was present there were 15 chromosomes instead of the usual 14 (Fig . 33). Since species with higher numbers of chromosomes (and presumably numbers of genes) frequently are more advanced taxonomically than species with lower chromosome numbers, it seems possible that iso-chromosomes may be one way in which chromosome number is increased . That is, iso-chro mosomes may have evolutionary significance. Unions between sister strands occurred infrequently , if at all. E vidence for this is found in the fact that paired bridges were the rule, and single bridges which could result from the fusion of sister chromatids at a common breakage point, never were positively identi fied. This was true even at low dosages where the coincidence of chromosome breaks which could result in an interchange that would produce a pair of bridges at somatic anaphase would be low in com parison with the frequency of single breaks which could produce a single bridge. Further proof that broken sister strands do not usually fuse was indicated by the fact that acentric fragments ordinarily occurred in pairs, and rarely in V's. This observation is at variance with some reports on the behavior of chromosomes broken during pro phase (see Lea, 1947, for pertinent literature). It seems to be more or less accepted that reunions between X-ray induced chromatinic fragments occur shortly after breakage. The pre sent observations may be taken to support this hypothesis, even though the chromosomes were in dormant seeds when irradiated. Possibly the best evidence for this conclusion is the fact that bridges in the root tip cells were almost invariably paired. That is, evidently the broken chromosomes had fused before the prophase split "effectively" occurred. However, there is some evidence that delayed unions may some times occur. Occasional single bridges were observed, and in some of these cases (Fig. 1) there was no indication that another bridge had been present but had broken. These single bridges suggest that a chromatid translocation had occurred after the X-rayed chromosomes became "effectively" bipartite, although they were presumably all monopartite when irradiated. With reference to the bridge in Fig. 1, there is little doubt that it resulted from chromatid translocation since one arm has a satellite whereas the other lacks it. Some additional evidence was obtained that delayed unions occurred. It is believed that the rings-of-three in spikes possessing a single iso chromosome (Figs. 34-36). can best be explained on the basis of such 238 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17 delayed attachments.4 In order for three chromosomes to form a closed ring-of-three in some cells, and a closed bivalent and univalent in others, it is presumably necessary for each of the three chromosomes to have a segment homologous with a segment in each of the other two. That is, the three chromosomes are apparently quadruplicate for at least a few loci. In the dormant seed, at the time of x-radiation, the chromo somes apparently are "effectively" monopartite, and there could be only two homologous segments of any chromosome present. Only after the chromosomes split would four homologous segments be present in a cell. This split presumably occurs during germination of the seeds - several hours after irradiation. Delayed unions between broken chromosomes have been reported in x-rayed Drosophila sperm (Muller, 1940), and in x-rayed pollen mother cells (Matsuura and Haga 1950). Rings-of-six seem to be induced at a considerably lower frequency than two-rings-of-four, although the data in Table 2 are not extensive enough for adequate proof. This would suggest that a ring-of-six is usually the result of four breaks rather than three, because four breaks would be less common than three. Further, it would suggest that interchanges are usually reciprocal, and that they probably ordinarily result from breaks which are close together. Muller (1940) was also of the opinion that in Drosophila, chromosome ends derived from breaks which are near together are much more apt to unite with one another than with ends, simultaneously present, which are "distal". The data in Table 1 suggest that the frequency of paired bridges in somatic cells of root tips, and the frequency of interchanges in pollen mother cells are equal. This would be a very interesting and useful relationship if adequately established. However, since bridges in somatic cells presumably result in the death of the cell; whereas cells with interchanges which persist to meiosis are viable, there should be a selective advantage for cells with monocentric chromosomes rather than dicentric chromosome interchanges. This would mean that as the dosage increases an increasingly higher proportion of the surviving cells would have monocentric interchange chromosomes. More data are needed, particularly at the higher dosages, to determine whether or not this is the case. Also data are needed comparing the frequencies of aberra tions in the first division of shoots in germinating seeds with the 4 Rings-of-three were previously considered to be due to non-homologus pairing (Caldecott and Smith 1952). However, it should be pointed out that pairing between non-homologouschromosomes at meiotic metaphase in barley, is extremely rare. In fact in the many thousands of spikes which the writers -have-examined, they have not observed a single positive case of such an association. One readily detectable evidence of non-homologouspairing would be an occasional pollen mother cell with a ring-of-four in a spike which characteristically did not have an interchange present. 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 239

corresponding frequencies of aberrations in root-tip cells to determine whether or not a given dosage of x-radiation produces comparable aberration frequencies in each organ. It would be of considerable ontogenetic interest to know how many cells in the dormant embryo of barley are involved in the formation of the spike on a single culm. It is felt that some evidence was ob tained indicating that all the sporophyte tissue on a spike usually results from a single cell. This conclusion was based on two lines of evidence. In the first place, it was found that in the large percentage of cases, all the pollen mother cells observed in a given spike had the same chromosomal constitution. That is, if one floret had a particular chromosomal aberration , such as a R4, R6, 2R4, iso-chromosome, etc., anthers from other florets in the same spike usually had the same aberration. This was true even when the florets examined were from both upper and lower spikelets on the spike. For rings-of-four data were collated only from dosages up to and including 8,000r. With these dosages, it was considered that the likelihood of the coincidental induction of a ring-of-four (the most common aberration at meiosis) in two or more adjacent cells in the embryo, whose descendents would be jointly involved in the formation of a spike, would be remote. With more complicated meiotic inter changes and "iso-chromosomes", etc., data were obtained from all dosages. Sixty spikes had a ring-of-four in both upper and lower florets. Only seven spikes lacked the aberration in both positions. Fourteen spikes with a ring-of-six, 23 with two rings-of-four, and 25 with "iso chromosomes" all had the aberration in both positions. Therefore, it seems reasonable to conclude that the microsporocytes, and probably the megasporocytes, in a barley spike usually are the descendants of a single cell in the dormant embryo. This conclusion was further borne out by mutation studies on head progenies of spikes not used for cytological observations. It was found that, in segregating head progenies, 15.1% of the seedlings were mutants. This frequency is slightly lower (about 5%) than reported by Moh and Smith (1951) for the percentage of mutant seedlings in the progenies of heterozygous X2, and later generation, plants. Moh and Smith's data were obtained from a study of atomic bomb and x-ray induced mutants. This relatively close correspondence between the percentages of mutant progeny in X2 and later generatians would not be expected if different florets in a spike were frequenctly descended from more than one cell in the embryo.

Cytologia 17, 1952 16 240 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

Summary Observations are presented on chromosomal aberrations in root-tip mitoses and microsporogenesis in plants grown from x-rayed dormant seeds of barley. The study was made as part of a considerable number of experiments over a period of five years. In all tests x-radiation was applied at 34 KVP and 27 ma. Dosages ranged from 0 to 32,000r. It was concluded that chromosomes in the resting embryo ordinarily responded to x-radiation as though they were monopartite, because in duced breaks were almost always chromosomal breaks rather than chromatid breaks. This conclusion was based on: 1) the high frequency of paired, apparently identical dicentric chromosomal bridges and acentric fragments at anaphase in somatic cells of root tips; 2) the presence of unlike ends on some of these dicentric chromosomes; 3) the relatively rare occurrence of single bridges in these somatic anaphases. The rarity of single bridges at anaphase in somatic cells, in contrast with the frequency of paired bridges (even at low dosages), also was interpreted as indicating that the fusion of sister chromatids at a com mon breakage point, or following splitting of a chromosome during seed germination, did not often occur. Unlike ends on a dicentric chromo some also would not be expected if broken ends of sister strands fused. Usually two acentric chromatinic fragments do not join. Evidence for this is the fact that acentric fragments usually occurred in pairs (ranging in size from minute dots to rods as long as a normal chromo some) rather than as V's or rings as would be the case if their broken ends united. Thus, it seems that the centromere plays an important part in the union of broken ends of chromosome fragments. The observations suggest that acentric fragments are able to under go one division, but do not persist beyond the subsequent resting stage. This opinion is based on the fact that these fragments usually occurred in pairs at somatic metaphase and anaphase, as though a piece of chromatin had reduplicated. If these fragments went into the telophase stage, returned to the condensed state and redivided again, quadruplet fragments should have been seen occasionally, but were not. However, it is true that most of the experimental observations were believed to have been made on the first cell division following radiation. At anaphase the acentric fragments were distributed freely in the cytoplasm, although they seemed to occur most frequently in the region of the equatorial plate. There was no evidence of any attraction be tween the fragments and the pole. Evidently a centric fragment attached itself as often to an acentric as to another centric fragment. This conclusion is based on the fact that there was a reasonably close parallelism between the frequencies 1952 A Study of X-ray-induced Chromosomal Aberrations in Barley 241

of pairs of dicentric bridges in root tip cells (resulting from a union between two centric fragments) and interchanges in pollen mother cells (resulting from a union between a centric and an acentric fragment). If this relationship is proved to be true , it should be possible to predict fairly accurately the frequency of spikes with interchanges in their microsporocytes from the frequency of bridges in cells of root tips taken from seeds given the same x-ray treatment. Evidence is presented (see Discussion) which indicates that ordinarily , broken ends of chromosomes unite shortly after the breakage takes place. That is, most of the results were consistent with the theory that exchanges between irradiated chromosomes are usually the result of simultaneous breakage and fusion. However, a few observations seem to be accounted for most logically if it is assumed that delayed attachments between fragments occasionally do occur. Observations at meiosis in 3,509 spikes revealed that the most common aberration was a ring-of-four (in 371 spikes), resulting from a simple reciprocal translocation. The frequencies of two rings-of-four, "pseudo-iso-chromosomes" , rings-of-six and, true "iso-chromosomes" fol lowed in that (descending) order (in 32, 29, 18, and 3 spikes, respectively). The 28 spikes with "pseudo-iso-chromosomes" had aberrations which were considered to be the result of interchanges between homologous chromosomes. Such a high proportion of interchanges between homolo gous chromosomes would not be expected if breakage and exchange between the 14 somatic chromosomes were determined purely by chance. Evidence is presented which strongly suggests that all of the microsporocytes in a barley spike usually arise from a single cell in the dormant embryo. Acknowledgment. -Support of this study by the United States Public Health Service is gratefully acknowledged. The senior author is especially indebted to the American Cancer Society under whose auspices he was a Research Fellow during a major part of the study. Sincere thanks are tendered to Helga Gunthardt, Lucille P. Caldecott and Florence Burroughs for invaluable assistance in the cytological observations.

Literature cited Caldecott, R. S. and Smith, L. 1952. The influence of heat treatments on the injury and cytogenetic effects of x-rays in barley. Genetics 37: 136-157. Carlson, J. G. 1938 a. Mitotic behavior of induced chromosomal fragments lacking spindle attachements in the neuroblasts of the grasshopper. Proc. Nat. Acad. Sci. 24: 500-507.- 1938b. Some effects of x-radiation on the neuroblast chromosomes of the gras shopper Chortophaga viridifasciata. Genetics 23: 596-609.- 1940. Immediate effects of 250r of x-rays on the different stages of mitosis in neuroblasts of grasshopper, Chortophaga viridifasciata, J. Morph. 66: 11-23. 16* 242 RICHARD S. CALDECOTT and LUTHER SMITH Cytologia 17

Carlson, J. G. 1941 a. An analysis of x-ray induced single breaks in neuroblast chromosomes of the grasshopper (Chortophaga viridifasciata). Proc. Nat. Acad. Sci. 27: 42-47.- 1941 b. Effects of x-radiation on grasshopper chromosomes. Cold Spring Harbor Symposia on Quant. Bio. 9: 104-111.- 1942. Immediate effects of 31r of x-rays on the different stages of mitosis in neuroblasts of Chortophaga. J. Morph. 71: 449-462.- 1950. Effects of radiation on mitosis. J. Cell. and Comp. Physiol. 35: 89-101. Catcheside, D. G., Lea, D. E. and Thoday, J. M. 1946. The production of chromosome structural changes in Tradescantia microspores in relation to dosage, intensity, and temperature. J. Genet. 47: 137-149. Koller, P. C. 1943. Effects of radiation on pollen grain development, differentiation, and germination. Proc. Roy. Soc. Edinb. B 61: 398-429. Lea, D. E. 1947. Actions of radiation on living cells. McMillan Co., New York. Mather, K. and Stone, L. H. A. 1933. The effect of x-radiation upon somatic chromo somes, J. Genet. 28: 1-24. Matsuura, H. 1950. Chromosome studies on Trillium kamtschaticum Pall. and its allies XIX. Chromatid breakage and reunion at chiasmata. Cytologia 16: 48-57. Matsuura, H. and Haga, T. 1950. Chromosome studies on Trillium kamtschaticum Pall. and its allies. IX. Chromosome aberrations induced by x-ray treatment. Cytologia 16: 37-47. Moh, C. C. and Smith, L. 1951. An analysis of seedling mutants (spontaneous, atomic bomb-radiation, and X-ray-induced) in barley and durum wheat. Genetics 36: 629-640. Muller, H. J. 1940. An analysis of the process of structural change in chromosomes of Drosophila. J. Genet. 40: 1-66. Riley, H. P. 1936. The effect of x-rays on the chromosomes of Tradescantia gigantea. Cytologia 7: 131-142. Sax, K. 1938. Chromosome aberrations induced by x-rays. Genetics 23: 494-516. - 1940. An analysis of x-ray induced chromosomal aberrations in Tradescantia. Genetics 25: 41-68.- 1941 a. The behavior of x-ray induced chromosomal aberrations in Allium root tip cells. Genetics 26: 418-425.- 1941 b. Types and frequencies of chromosomal aberrations induced by x-rays. Cold Spring Harbor Symp. Quant. Biol. 9: 93-101.- 1942. The mechanism of x-ray effects on cells. J. Gen. Physiol. 25: 533-537. Stone, L. H. A. 1933. The effect of x-radiation on the meiotic and mitotic divisions of certain plants. Ann. Bot. 47: 815-826.