“HE PRODUCTION OF HOMOZYGOUS DEFICIENT TISSUES WITH MUTANT CHARACTERISTICS BY MEANS OF THE ABERRANT MITOTIC BEHAVIOR OF RING-SHAPED CHROMOSOMES* BARBARA McCLINTOCK Cornell University, Ithaca, New York and University of Missouri, Columbia, Missouri Received February 25, 1938 : TABLE OF CONTENTS PAGE [ Introduction... 0.0.0.0. ett tte 315 ll The mitotic behavior of ring-shaped chromosomes. . . 318 ILL The nature of the Ba f-bm I variegation... ......--.-..--5 200 - 333 IV Types of functional gametes produced by the two original plants... 336 \ Production of plants mosaic for homozygous deficiencies... .....- 346 \'L Simulation of the 6 / phenotype through loss of the Bm J locus. ..... 357 VIL The production and appearance of plants homozygous for Def2 R2... 360 VILL The phenotypic effect of altered ring-chromosomes........----++--- 365 IN Discussion.......0.0...006. 0000 0b eee eee eee 368 Summary. oc... ee eee 373 Literature cited... 00 eee ete rere tess 375 I. INTRODUCTION ‘T IS the purpose of this paper to describe the method by which viable tissues, homozygous deficient for a known region of a chromosome, may be produced in maize. The chromosomal region involved includes the locus of the gene Bm J in chromosome V (allele of bm If, brown mid- rib, producing a brown color in the lignified cell walls). The lignified cell walls of the homozygous deficient tissue exhibit the features characteristic of the known recessive gene bm 1 although the locus of this gene is absent. The method of obtaining the homozygous deficient tissue is related to the unique behavior of ring-shaped chromosomes during somatic mitosis. This behavior has been briefly mentioned in previous publications (Mc- CLINTOCK 1932; RHOADES and MCCLINTOCK 1935). Ring-shaped chromo- somes do not always maintain themselves unaltered through successive nuclear cycles in the maize plant. They may (1) increase in size through duplication and reduplication of segments of the original ring, (2) decrease in size by deletions of segments from the ring, (3) be totally lost from the nuclei or (4) be present in increased numbers in the different nuclei. What- ever the method by which a change in size occurs, only ring chromosomes are produced from ring chromosomes. In maize it has been found that deficiencies in certain regions of the * The cost of the half-tone illustrations in this paper is met by the Galton and Mendel Memorial Fund. 1 Contribution from the Department of Botany in codperation with the Department of Field Crops, Genetics Research Project, Missouri Agricultural Experiment Station, Journal Series No. 570. Generics 23: 315 July 1938 316 BARBARA McCLINTOCK chromosomes may be transmitted successfully through the egg but not through the pollen (BURNHAM 1932; STADLER 1935). Pollen possessing a deficient chromosome plus a ring-shaped fragment chromosome should be functional if the ring-shaped fragment completely compensates for the deficiency. By utilizing a deficiency transmissible through the eggs and rendered non-lethal in the pollen by the inclusion of a ring fragment covering the deficiency, a zygote with two deficient chromosomes plus a ring chromosome can be produced. This zygote is heterozygous for thé Ficure 1.—Two sides of a stalk of a variegated plant. The leaves at the two nodes have been removed. The dark bands are mJ, the light bands, Bu J. deficiency. This heterozygosity in the resulting individual would be main- tained as long as an unaltered ring chromosome was present. Should the ring chromosome be lost in subsequent nuclear divisions, or should it change in size through loss of a segment within it, the tissues arising after such loss or alteration would be homozygous deficient for the entire de- ficiency in the first case or for regions within the limits of the deficiency in the second case. Two cases of deficient rod chromosomes with complementary ring chromosomes were available for this study. The two cases arose in the RING CHROMOSOMES IN MAIZE 317 progeny of X-rayed pollen containing a normal haploid complement with the dominant gene Bm JI. This pollen, when placed upon silks of bm 1 plants with a normal chromosome complement, gave rise, among a progeny of 466, to two individuals which were variegated for Bm I and bm I (figure 1). Aberrant behavior of a ring chromosome produced by the X-ray treatment and carrying the gene Bm I was suspected to be the cause of the variegation. I Ir G 5 4 Sou 4 3 2O1 aC) I —»13 2 {Ai I 1 oo a b FIcurE 2.-a. Diagram of a normal chromosome V. The slightly bulging section represents the spindle fiber attachment region. The sets of arrows, I and II, point to the positions of breaks which gave rise to the two deficient red chromosomes and their compensating ring chromosomes illustrated in I and II of b. The deficient rod and compensating ring chromosomes of I are referred to in the text as Def1 and R 1 respectively, those of II as Def2 and R2 respectively Examination of synaptic configurations in sporocytes revealed not only the presence of a small ring-shaped chromosome in each plant but also a deficiency in one chromosome V. In each case, the size of the ring-shaped chromosome and the extent of the deficiency in the rod-chromosome were comparable. The deficiency in both cases involved a section of the short arm immediately adjacent to the spindle fiber attachment region. Since the ring fragment in both cases possessed a small but definite spindle fiber attachment region, these regions being clearly visible in meiotic prophases, 318 BARBARA McCLINTOCK it was assumed that in each case the deficient rod and its compensating ring chromosome arose as the result of two breaks in the normal chromo- some V, one break passing through the spindle fiber attachment region, the other breaking the chromosome at a distance from the spindle fiber attachment region equal to approximately 1/20 (Case I) and 1/7 (Case IT) of the total length of chromosome V (figure 2). Fusions two by two of the broken ends resulted in a deficient rod and a compensating ring chromosome each with a section of the original spindle fiber attachment region. Since both the deficient rod and the ring chromosome possessed a section of the spindle fiber attachment region, both could be maintained through nuclear cycles. Proof that the ring chromosome represented the region for which the rod chromosome was deficient was furnished by the synaptic configura- tions produced by homologous associations of the three chromosomes: the normal chromosome V contributed by the female parent, the deficient rod chromosome V and the small ring chromosome contributed by the male parent (figures 25 and 26 and photographs of the same, 17 and 18, Plate IT). Cytological examination of different portions of the tassel disclosed the loss of the ring chromosome in several branches. Similarly, within a single anther, groups of cells were found lacking the ring chromosome. It was suspected, therefore, that the ring chromosome carried the locus of Bm 1, its loss during somatic mitoses being responsible for the presence of the bm I (brown) streaks in these plants. Conclusive proof for this was derived from the progeny of these two plants when crossed to normal bm I plants. The progeny included variegated (Bm 1 and bm 1) and bm I plants. Of the variegated plants, microsporocytes of 148 individuals were examined for the presence of the ring chromosome. The ring frag- ment was found in 146 of these individuals although in many plants several branches of the tassel lacked the ring fragment. In two plants no ring chromosome was found in the several branches of the tassel which were collected. Of the totally bm 1 plants, 47 were examined. In no case was a ring chromosome found. In a bm J tiller of a variegated plant, a considerably reduced ring chromosome was found. It is probable in this case that the Bm J locus had been deleted from the ring chromo- some through somatic alterations to be described in the next section. In- dividual collections were made on the two-sides of plants which were approximately half bm J and half variegated. In these cases, the presence of the ring chromosome could be established only on the variegated side. Ii. THE MITOTIC BEHAVIOR OF RING-SHAPED CHROMOSOMES The interpretation of the variegation and of the production of homozy- gous deficient tissues has been based on a knowledge of the behavior of RING CHROMOSOMES IN MAIZE . 3190 ring-shaped chromosomes in somatic nuclear cycles. A description of what has been observed regarding the appearance and behavior of the ring chromosomes in meristematic regions is therefore necessary before the individual cases can be considered. Although the primary cause of irregu- Jarities in the nuclear cycles is undoubtedly the same for large and small ring-shaped chromosomes, the subsequent behavior and the genetical con- sequences vary in these two extremes. The behavior of large ring-shaped chromosomes will be considered first; this will be followed by an account of the small ring-shaped chromosomes; finally, correlations and conclusions will be drawn regarding ring-shaped chromosomes in general. Mitotic behavior of large ring-shaped chromosomes Since the two ring-shaped chromosomes of cases I and II, figure 2, are both small, a large ring-shaped chromosome originally representing most of chromosome II has been examined (McCLINTOCK 1932). The observa- tions were made on longitudinal sections of actively growing root tips. Observations at meiotic prophase in this plant had clearly indicated that changes in size and hence chromatin content of the ring chromosome were occurring in the premeiotic nuclei. Groups of related cells usually had similar ring chromosomes but the differences in unrelated cells were very great. In a few cells the altered ring chromosome was larger than the normal chromosome II. In some cells it had been reduced to only a few chromomeres. All gradations between these two extremes were found in different sporocytes of this same plant. The smallest ring chromosome has obviously undergone a great loss of chromatin. The original ring chromo- some possessed a single knob. Evidence for duplication of segments other than the obvious increase in size of the ring chromosome was clearly registered in some cells by the increase in the number of knobs. Rings with two, three and four knobs were found. It was suspected that the alteration in chromatin content of the ring was related to the division cycle of the chromosome. Observations of mitoses in root tip meristems suggested the manner in which the altera- tions occur without, however, revealing the primary cause. If one assumes that during the splitting process or after the split has occurred, a cross- over took place between the two sister chromatids, a double-sized, con- tinuous ring with two spindle fiber attachment regions would be produced. A second crossover between the two sister chromatids could result in an interlocking of the sister ring chromosomes provided the second crossover did not counteract the first. The presence of double-sized rings with two spindle fiber attachment regions at late anaphase and early telophase was clearly evident in a number of cells (figures 5, 7, 15, 16; photographs 4, 5, 8, Plate I). Unfortunately the presence of interlocking rings could not 320 BARBARA McCLINTOCK be determined directly since the chromosomes of maize in somatic cells are relatively small. Many anaphase figures were su ggestive but none could be definitely distinguished from double-sized rings with a twist at the mid-region. From the point of view of the origin of such configurations it would be important to know the relative percentage of each type. From actual counts it is certain that the double-sized rings are present in at least one-third of the aberrant figures. The actual number of late anaphase and early telophase figures with chromatin bridges produced by double- sized or interlocked rings amounted to approximately 8 percent of a total of 1145 figures recorded in roots whose ring chromosome had not materially reduced in size in most of the cells (D, table 1). : TABLE I The frequency of normal and aberrant somatic anaphase and early telophase configurations in plants with different ring chromosomes. RING CHROMOSOME NORMAL ABERRANT % ABERRANT A Ri . 605 I 0.16 B R2 1195 14 1.1 Cc R2 plus enlarged R2 1169 76 6.1 D Large ring chromosome IT 1053 g2 8.1 Since the fate of the double-sized or interlocked rings is not the same in all late anaphase and telophase figures, a number of types of behavior from anaphase to late telophase have been diagrammed in figure 3. Repre- sentative drawings from different cells are given in figures 5 to 24 and photographs of Plate I. In the diagrams, the behavior of double-sized rings has been emphasized since this type could be clearly recognized in many cells. They are either clearly open or show a twist at the mid-region. Some of the interlocked rings should produce figures resembling those shown in the diagram and would not be easily distinguished from them. In most of the mitotic cycles the ring chromosome splits along a single plane, separation of the two halves proceeding normally at anaphase, figure 4. In the late anaphase figures the double-sized rings produce 4 double bridge the chromatin of which is pulled taut (figure 5, photograph 1, Plate I). It is suspected that breakage of the chromatin bridges some- times occurs during this period (photograph 10, Plate I). Since such figures were not included in the counts mentioned above, the 8 percent of anaphase and telophase figures with bridges represent the minimum number of cells in which double-sized or interlocked rings occurred. Some of the telophase figures suggest an early breakage of one or both strands of the double bridge (figures 8 and 9 and photographs 5 and 6, Plate I. RING CHROMOSOMES IN MAIZE ‘ 321 3 é (Sl) (le) Cid E 8 $s a “(S| Q O 4 h i FIGURE 3.—Diagrams illustrating the behavior in somatic mitosis of double-sized ring chromo- somes with two spindle fiber attachment regions produced from the two split halves of a single ting chromosome. a. Successive stages from mid-anaphase to mid-telophase of a medianly placed double-sized ting chromosome. The cell plate determines the positions at which breaks will occur in the two chromatin bridges. b. Similar to a except that a twist is present in the bridge strands of the double-sized ring chromosome. c. Appearance in early and mid-telophase of a double-sized ring which was non-medianly placed in the spindle figure. The components entering each daughter nucleus vary in chromosome length and constitution. d. Similar to c except that the upper portion of the double-sized ring chromosome is not in- cluded in the reorganizing telophase nucleus. Such behavior results in the loss of a component of the ring chromosome from one of the daughter nuclei. e. Appearance at mid-telophase of a double-sized ring chromosome with one broken bridge strand. f. Appearance at mid-telophase of a double-sized ring chromosome with both strands broken. g. Appearance at very early telophase suggesting an early breakage of bridge strands of a double-sized ring chromosome (or two interlocked sister ring chromatids). h. Comparable situation as illustrated in d except that the strands of the bridges are twisted at the cell-plate region. i. Fine bridge of chromatin between two resting nuclei suggesting that a breaking of the Strands had not occurred at telophase. - 322 BARBARA McCLINTOCK In many cases, breakage of the strands composing the bridge does not occur at anaphase; compare photographs 9 and 10, Plate I. The moving apart of the spindle fiber attachment regions in the double-sized rings is retarded by the tension of the chromatin bridges. The subsequent behavior is conditioned by the position in the spindle figure of this retarded ring or of two retarded interlocked rings. As the telophase sets in there is an ‘inp 89g < Q eed Ficure 4.—Normal separation of a ring chromosome in somatic anaphase. Ficure 5.—A double-sized ring chromosome in early telophase. Ficure 6.—A double-sized ring chromosome at a slightly later stage than that shown in figure 5s. FicurE 7.—Double-sized ring chromosome at mid-telophase. The bridge strands close to the cell-plate have become very thin. The shape of the chromosome within the nuclei has become discernible. FIcuRE 8.—Mid-telophase. Early breakage at the cell-plate region of two bridge strands of the double-sized ring. See comparable figure, photograph 6, Plate I. FicuRE g.—Mid-telophase. Early breakage at the cell-plate region of one bridge strand of a double-sized ring. See photograph 5, Plate I. FiGuRE 10.—Late telophase. Breakage of bridge strands of a double-sized ring chromosome at the cell-plate region and withdrawal of the chromatin into the nucleus at the lower part of the figure. immediate release of tension on the chromatin bridges produced through the swelling of the forming nuclei (photograph 2, Plate I). As the nuclei continue to swell and approach the cell-plate, the chromatin of the ring within the nuclei is relaxed, allowing the form of the ring chromosome t0 be clearly defined (figures 8, 9, 11, 16; photographs 3, 4, 5 and 8, Plate I). At this stage the tension on the chromatin threads from the nuclear mem- RING CHROMOSOMES IN MAIZE 323 Ficure 11.—Double-sized ring at mid-telophase. See photograph 3, Plate I. Fricure 12.—Similar stage to that shown in figure 10 resulting from a previous double-sized ring with a twist at the mid-region or from two sister ring chromatids which were interlocked. FIGURE 13.—Sister nuclei at late telophase. The positions of the ring chromosomes suggest a previous bridge formation which has broken. Ficure 14.—Resting stage. Sister nuclei with a fine connecting chromatin bridge. Frcure 15.—Non-median position of a double-sized ring chromsome at late anaphase. See photograph 7, Plate I. In the photograph there is a twist in the ring chromosome. FIGURE 16.—Mid-telophase. The result of a non-median placement of a double-sized ring chromosome at anaphase. See photograph of the same, 8, Plate I. FIGURE 17,—Mid-telophase. The result of a non-median placement of a double-sized ring chromosome with a twist, or of two interlocked sister ring chromatids. brane to the cell-plate again increases. The threads become thin and taut as if being pulled into the nuclei (figures 7, 16, 17; photograph 8, Plate I). In a few cases, these fine chromatin threads are seen in relatively late telo- phase nuclei (figure 14). Since they usually do not persist into late stages, breakage must usually occur during the earlier telophase period. There were many sister telophase nuclei observed in which the ring chromosome in each nucleus was close to the region of the nuclear membrane lying nearest the cell-plate (figures 12 and 13). Such figures probably represent the last stage in the progress of the previously double-sized or interlocked rings. It should be emphasized that fusions of broken ends must occur after such breakage, since only ring chromosomes have been found to arise from ring chromosomes although rod chromosomes might be ex- pected. It sometimes happens that the passage of one spindle fiber attachment 324 BARBARA McCLINTOCK region of a double-sized ring proceeds toward its pole in advance of the opposing spindle fiber region. Consequently, the double-sized ring is not medially placed in the spindle figure. The cell-plate then intercepts the chromatin bridges in a non-median position (figure 15, photograph 7, Plate I). As a result, the components of the double-sized ring entering sister telophase nuclei will be unequal in size and chromatin constitution. One segment of the double-sized ring is sometimes not included in the telophase nucleus on its side of the cell-plate (figures 16 and 17, photo- graph 8, Plate I). If the chromosome is not split at anaphase, fusions of broken ends could give rise in the next division to normally disjoining sister ring chromo- somes, or if twists are present in the chromonema before fusion, to a con- tinuous double-sized ring or interlocked sister ring chromosomes when EXPLANATION OF PLATE I All magnifications are approximately X 1100. Plate I.—Individual cells from longitudinal sections of the growing points of roots. Photo- graphs 1 to ro are of the large chromosome II ring. Photographs 11 to 14 show an enlarged R2 chromosome. Photographs 15 and 16 are of the normal R2 chromosome. Photograph 1. Late anaphase. Bridge produced by separation of the split halves of a ring- shaped chromosome which is in the form of a double-sized continuous ring. There is a twist of the strands at the mid-region. Photograph 2. Early telophase. Beginning of relaxation of tension on the strands of the double bridge. Photograph 3. Mid-telophase. Complete relaxation of tension on strands of bridge. Photograph 4. Mid-telophase. Double-sized ring chromosome. Photograph s. Mid-telophase. A double-sized ring chromosome. The strand to the right ap- pears to be broken. Photograph 6. Mid-telophase. A double-sized ring chromsome. Both strands appear to be broken at the cell-plate region. Photograph 7. Late anaphase. Non-median placement in the spindle figure of a double-sized ring chromosome. : Photograph 8. Mid-telophase. The result of a non-median placement in the spindle figure of a double-sized ring chromosome. The strands adjacent to the cell-plate have become attenuated. The lower segment of the ring chromosome was excluded from the forming nucleus. Photograph 9. Very early telophase. Chromatin bridge produced by a double-sized ring chro- mosome with twisted strands, or possibly two interlocked sister ring chromatids. Photograph 10. Very early telophase. Figures such as this suggest an early breakage of the strands of a double-sized ring chromosome or of interlocked sister ring chromatids. Photograph 11. Typical late anaphase position of a small ring-shaped chromosome which will be excluded from the reforming telophase nuclei. Photograph 12. Early telophase. Excluded ring chromosome which was previously non- medianly placed in the spindle figure. The cell-plate has passed below it. Photograph 13. Late anaphase. Stage in the process of exclusion of two closely associated ring chromosomes. Photograph 14. Similar to photograph 13. Photograph 13. Typical late anaphase position of a small ring-shaped chromosome which will be excluded from the telophase nuclei. Photograph 16. Mid-telophase. The result of a previously excluded ring-shaped chromosome. The cell-plate has passed below the ring. 325 N MAIZE RING CHROMOSOMES I PuaTE J BARBARA McCLINTOCK 326 PLATE II RING CHROMOSOMES IN MAIZE : 327 simple assumptions are made regarding the method of splitting or re- duplication of a chromonema along a single plane. If the chromosomes are split at anaphase, two by two fusions of the two adjacent broken ends of sister chromatids could result immediately in a continuous double-sized ring. If fusions two by two took place between the non-adjacent broken ends, continuous double-sized rings or interlocked sister ring chromatids could result. If the single (no anaphase split) threads were very much twisted or the double (split present at anaphase) threads coiled about one another, complex configurations would appear in the next anaphase. Only rarely was a figure found suggesting any complexity. If such behavior were the secondary cause of double-sized or interlocked ring chromatids (it cannot be the primary cause, see discussion), adjacent cells in the longitudinal rows could be expected to show chromatin bridges in an appreciable percent of the cases. They were present in a number of longi- tudinally adjacent cells. However, a very large number of such figures would be necessary to allow a satisfactory statistical study to be made. Although a large number of anaphase and telophase figures with aberrant configurations have been observed, the numbers of these in adjacent cells were insufficient for such a study. The mitotic behavior of small ring-shaped chromosomes The mitotic behavior of small ring-shaped chromosomes differs from that of large ring-shaped chromosomes in (1) the reduced frequency with EXPLANATION OF PxLaTE IT PLate II.—All photographs are of pachytene configurations in microsporocytes. X 1100. Photograph 17. Synaptic association of a normal chromosome V, a Def 2 chromosome V and an R2 chromosome. See text figure 25. Photograph 18. Similar to photograph 17. See text figure 26. Photograph rg. For description, see text figure 27. Photograph 20. Synaptic association of a normal chromosome V and a Def 2 chromosome V. See text figure 28. , Photograph 21. From a sporocyte of a plant with a normal chromosome V, a Def2 chromo- some V and two R2 chromosomes. The two rod-chromosomes have associated with one another (note buckle at spindle fiber attachment region). The two ring chromosomes have associated with one another. Photograph 22. Synaptic association of two R 2 chromosomes. Photograph 23. Late pachytene. The arrow points to the tiny R1 chromosome. Photograph 24. Collapsed R1 (upper) and R2 (lower) chromosomes associated at their spindle fiber attachment regions. Photograph 25. Collapsed R1 (left) and R2 (right) chromosomes associated at their spindle fiber attachment regions. Photograph 26. R1 chromosome (arrow), Its spindle fiber attachment region is stuck to that of bivalent chromosome VIII. Photograph 27. Collapsed R 1 chromosome (arrow) whose spindle fiber attachment region is associated with that of a normal chromosome V bivalent. Photograph 28. Synaptic association of a normal chromosome V and a Def1 chromosome V. See text figure 3o. 328 BARBARA McCLINTOCK which double-sized or interlocked rings arise; (2) the more frequent loss of the ring chromosomes from the nuclei; (3) the considerably less frequent occurrence of changes in size of the ring chromosomes and (4) the occa- sional increase in the number of rings in a nucleus. The two small rings in cases J and II, figure 1, have been used to study the behavior of small ring-shaped chromosomes in mitosis. In the sub- sequent discussions these two ring chromosomes will be referred to as R1 and R2 respectively. Cytological examination of the sporocytes in different branches of the tassel in plants with either of these rings had indicated that loss of the ring chromosome was occurring far more fre- quently than changes in size of the ring. This is in direct contrast to the behavior of large ring chromosomes, where changes in size are more fre- quent than loss. To obtain evidence on the method of loss, examinations of the meristematic regions of the roots of such plants were made. The tiny R 1 ring chromosome is clearly visible in the prophase nuclei of these cells. However, the description will confine itself to the behavior of the larger of these two rings, R 2, and one of its enlarged derivatives, since anaphases showing aberrant configurations of the R 1 chromosome are found only very rarely. The aberrant anaphase and telophase configurations are characterized by the median or nearly median position of the double-sized (or inter- locked) ring chromosome in the spindle figure (photograph 15 for the normal R 2 and photograph 11 for the enlarged R 2, Plate 1). However, they occasionally lie some distance from this position (figure 22 and photo- graph 12, Plate I). The ring chromosome in these configurations, as with the large ring chromosomes, frequently appears to be double-sized. In roots in which most of the nuclei contained the normal R 2 chromosome, 14 of the 1209 anaphase and telophase figures counted, or 1.1 percent showed these aberrant configurations (B, table 1). They were observed many times in roots where counts were not made. The fate of the delayed double-sized ring depends upon its position in the spindle figure as the cell-plate appears. If it is in the middle, the cell plate passes through it, dividing it into relatively equal or decidedly un- equal segments (figures 19 and 20). If it is not medially placed, the cell- plate passes to one side and the double-sized ring remains in the cytoplasm of one of the daughter cells (figures 21 and 22, and photographs of same, 12 and 16, Plate I). If it lies rather far away from the mid-region, it may be included in one of the nuclei. If this occurs and if normal splitting of this double-sized ring with two spindle fiber attachment regions follows in the next division, two double-sized rings, each with two spindle fiber regions should then be found lying close together in the spindle figure when the two spindle fiber regions on the same chromatid pass to opposite RING CHROMOSOMES IN MAIZE. 329 poles. Several configurations have been observed in which two rings were lying very close together (figures 23 and 24, and photographs of the same, 13 and 14, Plate I). The exact contours of the individual rings could not be accurately followed and therefore have not been shown in the drawings. Since the contours of the two ring chromosomes could not be accurately ue © oO & aS —— () % Co iy - Ficure 18.—Simultaneous loss at late anaphase of two enlarged R2 chromosomes. FicuRE 19.—Late telophase appearance after loss of an R2 chromosome. The double-sized ring chromosome has been intercepted by the cell-plate. Ficure 20.—Similar to figure 19 but a later stage. FycurE 21.—Telophase. The excluded R2 chromosome has not been intercepted by the cell-plate. See photograph 16, Plate I. FicurE 22.—Telophase. The excluded enlarged R2 chromosome was not medianly placed in the spindle figure. See photograph of the same, 12, Plate I. Ficure 23.—Late anaphase. Two enlarged R2 chromosomes lying close together at the cell- plate region. For description, see text. See photograph of the same, 13, Plate [. Ficure 24.—Similar to figure 23. See photograph of the same, 14, Plate I. followed, such figures could represent two interlocked ring chromosomes from a similar condition to that described above, if instead of a double- sized ring, two interlocked sistér ring chromosomes had been included in the preceding telophase nucleus. Since anaphases and telophases with the ring chromosome lying in the middle of the spindle figure are the most frequent types of aberrant con- figurations, and since these rings are subsequently excluded from the 330 BARBARA McCLINTOCK telophase nuclei, the frequently observed absence of the ring chromosome from branches of the tassel or from groups of cells in an anther can be ex- plained. In connection with the problem of the mechanism of movement of chromosomes in the spindle it would be of interest to explain why these small double-sized rings do not appear to be under greater tension as their spindle fiber regions pass toward opposite poles. It is possible that move- ment toward opposite poles is initiated at the spindle fiber region of the chromosome at early anaphase but that continued movement is made possible by other forces exerted on the chromosomes when they have reached a region in the spindle which is some distance away from the equatorial plate. These double-sized ring chromosomes may be too small to reach this region by the pull exerted at the spindle fiber region. The behavior of intermediate sized rings lends support to this assumption since these sometimes remain in the equatorial plate either with or without evidence of tension. From cytological examination of sporocytes it was known that changes in size of the small ring chromosomes do occur though with relatively low frequency. The R 2 chromosome has been observed to decrease to several chromomeres and also to increase to seven or eight times its original size. Photograph 15, Plate I, represents a normal R 2 chromosome, photographs 11 and 12, an enlarged R2 chromosome. It has also been seen that an increase in the number of these rings in a nucleus, usually with alterations in size, sometimes occurs. As many as six ring chromosomes in the nuclei of a sector of a plant which very probably possessed but one ring in the zygote have been observed in a single instance. If a double-sized ring is included in one of the daughter nuclei, as described above, a start in the direction of increase in number of rings has been made. The chance of loss of this ring chromosome in subsequent divisions is great. However, as already shown, the ring chromosome may be directly broken in two by opposed poleward forces at anaphase, or the poles of the spindle may lie so close together that the spindle fiber regions of the ring are readily included in the two nuclei, after which the resulting chromatin bridges are cut through by the cell-plate and drawn into the nuclei. When two double-sized sister ring chromosomes each with two spindle fiber regions are present in an anaphase figure, and when each of these is subsequently broken and the broken ends drawn into the telophase nuclei, the initial event in the production of a sector of tissue with two altered ring chromosomes has occurred. It is apparent, on this basis, why increase in number of these small ring chromosomes is relatively rare: one infrequent event must be followed by another. RING CHROMOSOMES IN MAIZE 331 Conclusions regarding ring chromosome behavior The foregoing account has indicated the probable method by which rings are altered in size and genic constitution or are lost from the nuclei altogether. From both cytological and genetical observations it has been concluded that the rate at which this occurs is dependent upon the length of the chromonema composing the ring: the longer the chromonema, the more frequent the occurrence of aberrant mitoses involving the ring. Counts of the aberrant ring chromosome configurations in late anaphase and very early telophase are given in table 1. The counts are from roots in which the ring chromosome was present in most of the cells. In A, the R1 chromosome of case I, figure 1, is represented. One aberrant configura- tion of the ring chromosome was observed in these roots which were counted. Judging from the relatively small amount of bm 1 tissue shown by plants with this R 1 chromosome, loss of the ring chromosome must be relatively infrequent. In B, the small R2 chromosome of case II is repre- sented. The observed aberrant configurations of the ring chromosome in these roots amounted to 1.1 percent of all the figures recorded. As stated above, these aberrant configurations result mainly in loss of the ring chromosome from both nuclei. Variegation, expressed by the bm I tissue in these plants, is considerably greater than in the plants from which the counts of table 1, A, were obtained. Table 1, C, represents the counts from a plant which possessed two ring chromosomes, 4 normal R 2 and an R2 enlarged approximately three times, In the roots from which the counts were made, both rings were present in many of the nuclei. Aberrant configurations involving the enlarged R2 chromosome were more frequent than those involving the normal R2 chromosome. The size of the enlarged R2 chromosome lies at the border line between those rings whose aberrant configurations lead mainly to exclusion from the telophase nuclei (small rings) and those whose aberrant configurations lead mainly to changes in size of the ring chromosomes (large ring chromosomes). For the enlarged R 2, both types of configurations were frequently encountered. In table 1, D, from a plant with a ring chromosome approximately twice the size of the enlarged R 2 chromosome, the aberrant configurations amounted to 8 percent of the total number recorded. In this case, as described above, the figure is possibly too low. The telophase figures here were characterized mainly by changes in size of the ring chromosome rather than loss from the nuclei. If a ring chromosome in a given ‘ndividual carried a dominant gene and the two normal rod chromosomes carried a recessive gene, the expression of variegation produced by losses or changes in constitution of the ring chromosome would depend upon (1) the size of the ring chromosome and 332 BARBARA McCLINTOCK (2) the position of the gene with respect to the spindle fiber attachment region. In relatively small ring chromosomes, which are mainly lost from the nuclei, the expression of variegation is directly dependent upon the actual size of the ring chromosome: the larger the ring chromosome the greater the amount of variegation exhibited. This is strikingly illustrated by the two ring chromosomes, R 1 and R 2. The variegation produced by R1 is very much less than that produced by R 2. To check this conclusion without prejudice, cultures were obtained in which either R1 or R2 or both R1 and R 2 chromosomes were expected to be present in individual plants TABLE 2 Comparisons of the predicted and observed ring chromosome constitutions in cultures segregating plants with one, two and three ring chromosomes, PREDICTION: PRED.CTION: PREDICTION: PREDICTION: 1iR1 1R2 2 rings 3 rings Correct Deviation Correct Deviation Correct Deviation Correct Deviation 5 (x ring)* 1 1(Ri+R2) 1§ 3(R 1) 24 1(3 rings)t 3 ° * Three showed one R.1; one showed one R2 in an estimated two R2 plant; one showed one R2 in an estimated R1 plus R2 plant. Complete agreement in all cases could not be expected in two- and three-ring plants from sporocyte examinations since loss of one of the ring chromosomes in the developmental stages of the tassel is expected in some cases. This particularly applies to the R2 chromosome. } The estimate for this plant was two Ri. Some of the two R1 plants have practically no bm 1 tissue. A two R1 plant could be difficult to distinguish from a three R 2 plant. of the culture. In some of these cultures plants with three ring chromo- somes were expected. From the expression of the variegation exhibited by each plant a prediction was made as to the ring chromosome constitu- tion of the plant. Cytological observations were subsequently made to determine the correctness of these predictions. Table 2 shows the correla- tion of these observations with the predictions. Cultures of plants with the R1 chromosome can readily be separated from cultures whose individ- uals possess the R 2 chromosome through observations of the variegation alone (see following section for more complete discussion). With relatively large ring chromosomes, which are characterized mainly by changes in size of the ring chromosomes, the expression of variegation would depend upon the nearness of the locus of the gene to the spindle fiber region. The farther away the locus, the greater is the amount of varie- gation that should be expressed. ; The method of alteration of the ring chromosomes as suggested by the somatic anaphase and telophase configurations should produce rod-shaped chromosomes. Although thousands of microsporocytes have been exam- ined in many of which an alteration of the ring chromosome has been ob- RING CHROMOSOMES IN MAIZE , 333 served, no rod-fragments have been recognized. It can not be stated that they do not occasionally occur, but certainly their frequency must be exceedingly low. If the method by which ring chromosomes change in size has been correctly interpreted from the study of somatic anaphase and telophase figures, one is forced to conclude that the broken ends of the chromosomes unite, thus reéstablishing a ring. It might be stated here that when two ring chromosomes are present in the nuclei of a plant, it is rare that both rings show aberrant configurations in the same cell (figure 18). Each ring chromosome apparently acts inde- pendently with regard to the formation of double-sized or interlocked rings. That the behavior of ring chromosomes in maize is a consequence of their form and not of their genic constitution can be definitely stated, since a number of different ring chromosomes, each involving segments of chromosomes not strictly comparable, have been found so far. These in- clude segments from chromosomes I, V, VI, VIII and IX. Most of them were detected by the variegation which they produced but three were isolated independently of any visible effect. Ill. THE NATURE OF THE Bm 1—-bm I VARIEGATION The gene bm 1 when homo- or hemizygous produces a brown color of the cell walls. The color appears in the walls as soon as lignification sets in. It is not present before this period. The depth of color, on external exam- ination of bm 1 plants, is greatest in those tissues which are composed largely of thickened cell walls, such as the midrib of the leaf, the veins in the leaf sheath and the stalk tissue. The brown color is not easily detected in the leaf tissues other than the midrib since the cell walls are thin and the color is masked by the chlorophyll. As the plant matures in the field, the brown color has been noted to fade considerably in exposed regions of the plant but remains deep in those regions which are well protected from light. It was suspected that direct sunlight was causing a change in the structure of the brown pigment which resulted in loss of color. To determine if this was correct, black paper was placed about exposed parts of several bm 1 plants when the brown color was intense. The bands of black paper remained about these parts for a period of three weeks. When the paper was removed, the tissues protected from light had retained their original deep brown; the brown color in the tissues above and below the protected region had faded con- siderably. In plants possessing two normal chromosomes V with bm J (or one normal chromosome V with bm I and one of the deficient chromosomes V) and a ring chromosome with Bm 1, streaks of bm 1 tissues are pro- 334 BARBARA McCLINTOCK duced and can be seen by external examination of the plants (figure 1). Over 7000 variegated plants have been examined in the progeny of the two original variegated plants. Cytological observations have indicated that loss of the ring chromosome carrying Bm J is the primary cause for the appearance of the bm I tissues.-Losses can occur anywhere in the ontogeny of the plant. The patterns of the bm I tissues should give some indication of where and when these losses occurred. Although wide or narrow bands on the stalk (figure 1) indicate an early or late loss of the ring chromosome, respectively, cross sections of the stem, where most of the cell walls are heavily lignified, give even a better indication of the time of loss. If loss occurred early in ontogeny, the whole plant would be bm 1. If the first loss occurred in one of the cells which is to give rise to the part of the plant above the ground, a wide sector of bm JI would be produced. Still later losses would produce sectors of various widths in the stem. Very late losses would produce streaks or patches composed of a few cells only. All of these types of variegation patterns have been ob- served. When a stalk with a relatively wide external band of dm 1 tissue is cross-sectioned and examined with low magnification, the brown-walled tissue is seen to be composed of a V-shaped sector with the tip of the V pointing toward the center of the stalk. Many narrow surface streaks are produced by similar sectors but the V is smaller and the tip considerably removed from the center of the stalk. Very narrow streaks may be com- posed of only a few cells. Such streaks are visible on external examination of the stalk only if they lie at or close to the surface. Patches of bm 1 cells not close to the surface cannot be seen by external examination. Dilution of color in the brown (0m 1) cell walls on the side of the wall adjacent to the white (Bm 1) cell walls was a striking feature of the variegation in all plants. That it is a dilution produced by the adjacent Bm 1 cells and not a spreading of the brown color from the bm J cell walls is suggested by the considerable reduction in intensity of color in the brown walls of the very small patches composed of only a few cells, and by the dilution of color of a row of dm I epidermal cells on the side adjacent to inner Bm 1 cells. The variegation in plants possessing an R2 chromosome is expressed by a few totally 5m 1 plants where the ring chromosome has been lost before the cells which are to produce the stem meristem have been differ- entiated, to plants which are composed of many bm I streaks of different widths. Cross sections of the stems of the average variegated plant show wide V-shaped sectors, smaller V-shaped sectors and many irregular patches of bm 1 composed of few to many cells. The variegation patterns in plants with the R1 chromosome were similar RING CHROMOSOMES IN MAIZE , 335 to those produced by plants with the R2 chromosome but the total amount of bm 1 tissue was very much less. There were fewer sectors of all types in these plants, making cultures of the two types of variegated plants readily distinguishable. This is expected from the cytological examinations since the smaller ring chromosome is lost less frequently in somatic divi- sions than the larger ring chromosome. The extent of variegation is a direct expression of the rate of loss of the ring chromosome. Plants with two ring chromosomes show considerably less variegation than plants with one ring chromosome. Loss of one ring chromosome followed later by loss of the second ring chromosome or simultaneous losses of both ring chromosomes must occur before the bm 1 tissue could be produced. The patterns of the bm 1 tissues in cross-sections of the stem clearly show this relationship. These fall into three main types of sectors: (1) solid V-shaped sectors, (2) spotted V-shaped sectors, and (3) small patches of bm I tissue. | The solid V-shaped sectors are interpreted as relatively early losses of one ring followed slightly later by loss of the second ring chromosome or by occasional simultaneous losses of both rings. The spotied V-shaped sectors reveal more closely the relationship between loss of one ring fol- lowed considerably later by losses of the second ring. They are detected as a Cluster of bm J patches in an isolated region of a stem which other- wise shows very few bm 1 patches. When each of the brown patches in such a cluster is traced with a camera lucida and lines drawn joining the outer boundaries of the outermost patches, the lines converge in the direc- tion of the center of the stem. They clearly define a V-shaped sector. Such spotted V-shaped sectors would be expected if loss of one ring carry- ing Bm 1 is followed later in development by losses in different cells of the the second ring chromosome carrying Bm 1. The small patches of brown walled tissue, usually composed of only a few cells, can be interpreted as relatively late, successive, or occasionally simultaneous, losses of the two rings. There are three types of plants with two ring chromosomes: (1) those with two R1, (2) those with one R1 and one R2 chromosome, and (3) those with two R2 chromosomes. Since somatic loss of the R2 chromosome is considerably more frequent than the R1 chromosome, the amount of bm 1 tissue produced in each of these plants is progressively greater. Plants with two R2 chromosomes have considerable amounts of bm J tissue; those with one R1 plus one R2, very much less, and those with two R1 chromosomes exceedingly ‘little bm J tissue. In this latter type of plant it is often necessary to examine cross-sections of the stem to determine if any bm 1 tissue is present. Such tissue, when not close to the surface, cannot be detected from field examinations of the plants. 336 ‘ BARBARA McCLINTOCK Plants with three ring chromosomes of the constitution two R2 plus one R1 chromosome or one R2 plus two Ri chromosomes, have been obtained. These plants frequently show no external evidence of bm 1 tissues. In all cases, however, careful examinations of the stalks have revealed small patches of bm J cells. The bm 1 cells could arise only after loss (mainly successive) of all three ring chromosomes from the nuclei. For the sake of comparison, the stalks of a number of plants with a normal chromosome constitution carrying Bm I in one chromosome V and bm JI in its homologue were examined. In no case was there any evidence of bm I tissue. In conclusion it can be emphasized that the genetic expression of varie- gation is in full agreement with expectation on the basis of the cytological observations given in the previous section. In these plants with small ring chromosomes whose aberrant mitotic configurations are followed mainly by loss of the ring chromosome from the nucleus, the extent of variegation is a direct indication of the length of the chromonema composing the ring chromosome, the larger the ring chromosome the higher the rate of loss and thus, the greater the amount of exhibited variegation. Loss of the ring chromosome can occur at any stage in the development of the plant, early loss giving rise to a totally bm J plant, later loss to wide sectors of bm 1 and very late losses to small patches of bm I cells. The patterns exhibited by two and three ring chromosome plants are those expected from the cytological observations where it has been shown that simultane- ous loss of the several ring chromosomes from a nucleus is rare. The cause of the aberrant mitotic configuration arises independently in each ring chromosome. Knowledge gained from a study of variegation in these plants has been utilized in the analysis of tissues of plants mosaic for homozygous de- ficiencies (section V). IV. TYPES OF FUNCTIONAL GAMETES PRODUCED BY THE TWO ORIGINAL VARIEGATED PLANTS Each of the two original variegated plants possessed one normal chromo- some V with bm I, one deficient chromosome V and a ring-shaped frag- ment chromosome corresponding in size to the deficiency in the rod chromosome (figure 2). In case II (Def 2, R2) prophase meiotic associa- tions had indicated the homology of the ring chromosome with the region in the rod chromosome which had been deleted (figures 25 and 26; photo- graphs of the same, 17 and 18, Plate II). Most frequently, the ring chromo- some did not associate with its homologous section in the normal rod RING CHROMOSOMES IN MAIZE 337 chromosome but remained separate and collapsed (for meiotic prophase behavior of ring-shaped chromosomes, see MCCLINTOCK 1933). In all cells the deficient rod chromosome V and the normal chromosome V were asso- ciated. The normal V had to buckle to compensate for the deletion in the deficient V. Figures 27 and 28, and photographs of the same, 19 and 20, Tlate II, illustrate this association. In the plant from which figure 27 FicurE 25.—Pachytene association of a normal chromosome V, a Def2 chromosome V and its compensating R2 chromosome. The ring chromosome has been drawn with a finer line. The spindle fiber attachment region has been drawn as a slight bulge. The dark bodies toward the ‘end to the right are the knobs. See photograph of the same, 17, Plate IT. was drawn, two ring chromosomes were present. They are separate and collapsed. Another figure from the same plant, photograph 21, Plate II, shows the not infrequent association of the two R2 chromosomes to form a true ring-shaped configuration and also the association of the normal and Def 2 chromosomes. FicureE 26.—Similar situation to that shown in figure 25. See photograph of the same, 18, Plate II. In case I (Def1, R1), no figures were observed showing the association of the ring chromosome with its homologous section in the normal chromo- some. It is probable that it occurred in a small percentage of the cases but 338 BARBARA McCLINTOCK would be difficult to detect except in the most favorable figures because of the smallness of the deficiency and the ring chromosome. Figures 29 and 30 illustrate the pachytene association of the Def1 chromosome with FicurE 27.—Pachytene association in a microsporocyte of a plant with one normal chromo- some V, one Def2 chromosome V and two R2 chromosomes. Note the buckle in the normal chromosome V at the spindle fiber attachment region and the two unassociated, collapsed ring chromosomes. The ring chromosomes have a similar chromatin constitution to that of the buckle. See photograph of the same, 19, Plate IT. a normal chromosome V. The small ring chromosome (R 1) lies free and is collapsed. In figure 30 and photograph of the same, 28, Plate II, non- homologous associations about the deficient region have resulted in a y FicurE 28.—Pachytene association of a normal chromsome V and a Def2 chromosome V. See photograph of the same, 20, Plate IT. separation of the spindle fiber attachment regions of the two chromosomes (for expected non-homologous associations, see McCurntock 1933). The small buckle below the lower spindle fiber region or the distance between RING CHROMOSOMES IN MAIZE 339 the two spindle fiber regions represents the extent of the deficiency. Photographs 23, 26 and 27, Plate I, illustrate the appearance of the R1 ring at meiotic prophase. In photograph 23, very early diplotene, the VY Ficure 29.—Pachytene association in a microsporocyte of a plant with one normal chromo- some V, one Def 1 chromosome V and an R 1 chromosome. The ring chromosome is collapsed and is not associated with its homologous region (buckle) in the normal chromosome V. ring shape of the chromosome is clear. In photograph 26 the spindle fiber region of the Ri chromosome is stuck to that of chromosome VIII. In photograph 27, the spindle fiber region of the collapsed R1 chromosome is adjacent to that of a normal chromosome V bivalent. Photographs 24 > FicuRE 30.—Pachytene association of a normal chromosome V and a Def1 chromosome V. Through non-homologous associations, the buckle which compensates for the deficiency, has shifted into the long arm of the normal chromosome V. Note the displacement of the spindle fiber attachment regions. See photograph of same, 28, Plate IT. and 25, Plate II, illustrate the relative sizes of R1 and R2 when both are present in the same sporocyte. In both photographs the collapsed ring chromosomes are associated by their spindle fiber attachment regions. In photograph 24 the R1 chromosome is above, the R2, below. In photo- graph 25 the R1 chromosome is to the left, the R2 to the right. In plants heterozygous for either deficiency and its compensating ring 340 BARBARA McCLINTOCK chromosome, the deficient rod chromosome and its normal homologue proceed quite normally during the meiotic mitoses, two spores of a quartet receiving the deficient chromosome, two the normal chromosome. The behavior of the ring chromosome, on the other hand is irregular. In the case of R1 chromosome the split halves separate and pass to opposite poles at anaphase I along with the disjoining bivalents (except where double- sized or interlocked ring chromosomes are formed). The split halves of the R2 chromosome likewise separate at I, either at the same time that the bivalents disjoin or slightly later. They are nearly always included in the first division telophase nuclei. In the second meiotic mitosis, the behavior of the ring chromosome is variable. They do not divide again but either pass to one of the poles along with the other chromosomes of the complement or remain in the spindle figure and are excluded from the telophase nuclei. The behavior of the rings in the two sister cells is not always the same. As a result of meiosis, four types of spores are to be expected. They carry the following chromosomes: 1. Normal chromosome V. 2. Deficient chromosome V. 3. Normal chromosome V plus the ring fragment. 4. Deficient chromosome V plus the ring fragment. The percentage of each type in an anther would depend upon (1) the proportion of the sporocytes which lacked a ring chromosome, giving only types 1 and 2 above, and (2) the percentage of cases in which the ring chromosome, when present, was included in the second meiotic telophase nucleus. Examination of the pollen has given some indication of the percentage of each of these four types which are present in an anther. Pollen from a bm sector of a plant known to have a normal chromosome V (carrying bm 1), a deficient chromosome V (Def1), and an R1 chromosome, showed three types of grains: (1) large well filled grains, (2) small partially filled grains and (3) small totally empty grains. The proportions of each type are shown in table 3, A. In these dm/ sectors it is assumed on good evi- dence (see sections I and II) that the ring chromosome has been lost. Equal proportions of type 1 and type 2 grains should be present. If the small partially filled grains represent those with the Def 1 chromosome, the large filled grains, those with the normal chromosome V, they should be present in equal proportions. A total of 5535 normal pollen grains to 5547 small partially filled grains clearly indicates this association. The 347 small empty grains represent 3.3 percent of the total. In all samples of RING CHROMOSOMES IN MAIZE . 341 pollen from normal maize plants there is a small percentage of these empty grains. They probably represent the products of abnormalities in meiosis which are not infrequently observed in normal plants. Anthers from Bm 1 regions of the plant, in which the ring chromosome is present, give a higher proportion of normal well-filled grains (table 3, B). The difference is interpreted as due to the presence of the ring chromosome in some of the grains which have a deficient rod chromosome (type 4, above). Since the ring chromosome, if unaltered, covers the deficiency, a normal appearing pollen grain is expected. On this interpretation, the number of each type in a particular anther can be estimated. Pollen types 1 and 2 should be present in equal numbers. Likewise, types 3 and 4 - should be present in equal numbers. Type 2 grains can be directly re- corded. An equal number of the normal appearing grains should belong to type 1. When this number is subtracted from the total number of normal appearing grains, the remainder can be equally distributed to types 3 and 4. The estimates of each type of grain from the Bm 1 anthers in table 3 B, are: type 1, 1994; type 2, 1994; type 3, 743; type 4, 743, OF 36.5 percent each of types 1 and 2 and 13.5 percent each of types 3 and 4. In one plant, heterozygous for Defi R1, four types of pollen grains were present, 619 large well-filled grains, 170 small but well-filled grains, 426 small partially-filled grains (type 2) and 49 small empty grains. If it is assumed that the small well-filled grains represent type 4 with an altered ring chromosome which does not completely cover the deficiency, pollen types 2 and 4 can be directly recorded. If, on the other hand, these grains are included in the normal appearing class, and calculations made as above, the proportion of types are: type 1, 426; type 2, 426; type 3, 184; type 4, 184. It is obvious that there is a close agreement in the cal- culated number of 184 for type 4 grains and the 170 grains which have been assumed to represent this type. In plants heterozygous for Def2 and R2, the type 2 pollen grains are large but almost completely empty. These grains cannot be distinguished from the few empty grains produced by other causes than the presence of the deficiency in chromosome V. However, if these latter grains are assumed to represent two percent of all the grains, an approximate esti- mate can be made of the number of grains with each of the four chromo- somal constitutions. The counts from the bm anthers are given in table 3 C, and similar counts from the Bm anthers, with estimates of propor- tions of types, in table 3 D. , The functional capacity of each of the four types of gametes can best be illustrated by reference to the types and numbers of individuals result- ing from the crosses given in table 4. Section A in the table represents the 342 BARBARA McCLINTOCK TABLE 3 : A. Pollen counts from bm1 anthers of plants with: the constitution Def 1/bm1/R1. PLANT LARGE FILLED SMALL PARTIALLY EMPTY % EMPTY GRAINS FILLED GRAINS GRAINS GRAINS 5984-2 1181 1148 49 2.0 598A-2 832 O17 38 2.1 597A-2 775 751 66 4.1 597A-2 610 611 21 1.6 598A-16 692 678 42 2.9 598A-3 523 526 70 6.2 597B-6 922 g16 61 3.2 Totals 5535 5547 347 3-3 B. Pollen counts from Bm1 anthers of a plant with the constitution Def1/om1/R 1. LARGE SMALL ESTIMATES OF FOUR TYPES PARTIALLY EMPTY % EMPTY OF GRAINS IN PERCENT PLANT FILLED FILLED GRAINS GRAINS GRAINS GRAINS I 2 3 4 598A-2 1431 802 47 2.0 36 36 14 14 598A-2 1094 561 26 1.5 34 34 16 16 598A-2 956 631 28 1.7 40 4° 10 10 Totals 3481 1994 101 1.8 C. Pollen counts from bm 1 anthers of a plant with the constitution Def2/bm1/R2. PLANT NORMAL GRAINS EMPTY GRAINS 953B-6 681 733 9538-6 943 918 953B-6 864 g62 953B-6 761 . 779 953B-6 1036 1079 Totals 4285 4471 D. Pollen counts from Bm 1 anthers of plants with the constitution Def 2 /obm1/R2. ESTIMATE OF FOUR TYPES OF GRAINS IN PERCENT PLANT NORMAL GRAINS EMPTY GRAINS I 2 3 4 1009-10 906 864 48 48 2 2 1009-8 055 623 38 38 12 12 1009-8 1281 847 38 38 12 12 1009-8 1109 822 41 41 9 9 Totals 4251 3156 RING CHROMOSOMES IN MAIZE 343 progeny from crosses of the two original variegated plants. Section B rep- -esents CIOSSES of plants from A of this table, which were heterozygous cor the deficiency and compensating ring, with normal bm 1 plants. It can be seen that all four gametes produced by plants heterozygous for Deil R1 can be transmitted through the eggs. Through the pollen, cametes of type 1 and 3 are readily transmitted but gamete type 2 is not transmitted and gamete type 4 only rarely in competition with gametes I and 3. TABLE 4 CONSTITUTION OF PLANTS RESULTING FROM CROSSES TYPE OF CROSS bmi PLANTS VARIEGATED PLANTS (Female parent to left) bm1/bml Def/bmi . bm1/bm1/R Def/bmi/R Defi/om1/R1Xbm1 218 9 16 12 AL bm 1