The Pennsylvania State College The Graduate School Department of Botany Studies on X-ray Treatment of Phlox by John Stuver Bangson A Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at The Pennsylvania State College . August, 1940 Approved: Professor of Botany STUDIES ON X - R A Y TREATMENT OF PHLOX. Contents. I. Introduction 1 II. Materials and Method of Procedure 1 1. The Material 1 2. The Experiments 2 3. The X-ray Machine • 3 4-. Unit of Measurement for Dosages 3 5. Experiment I - Material, Technique 4 6. Experiment II - Material, Technique 6 7. Experiment III - Material, 7 8. Experiment IV - Material, Technique 8 9. Experiment V - Materials, Technique 10 Technique III. R e s u l t s . A. Physiological Results a. Buds - reaction, seed-setting 12 b. Seeds 1. S pe e d of germination 15 2. Percentages of germination 15 c. Pollen - reaction, seed-setting 20 d. Seedlings - abnormal appearance 23 e. Lethal effects 24 B. Genetical Results 1. Foliage Color Studies a. Yellow - origin, heredity, discussion 27 b. Mottled - origin, 29 heredity, discussion 2. Flower Pigmentation a. White - origin, discussion 33 b. Violet Flash - origin, heredity, d i s cussion 35 c. Spinel Pink - origin, heredity, d i s cussion 37 3. Stature a. Giant - origin, discussion 39 b. Dwarf - origin, heredity, discussion 42 4. Aborted Flowers - origin, 5. Lethals - origin, discussion IV. Summary and Conclusions V. Literature discussion 45 46 47 50 STUDIES ON X-RAY TREATMENT OP PHLOX. This investigation was undertaken during the summer of 1931. The purpose was twofold. It was considered desirable to as certain if X-radiation might prove as effective in changing the constitution of genes and chromosomes of Phlox Drummondii as it was shown to be when used with Nicotians spp., Pordeum s a ti vu m, Gossypturn hirsutum, Zea ma;/s, and other plant material, and Drosophila melanogaster among animals. There was also a desire for a supply of variations which would be available for the prosecution of further studies involving Phlox D r u m m o n d i l . The Material - The material for the investigation consist ed of two families of Phlox Drummondil which had been self pollinated for several previous generations. Each was uni form in X'Ggard to stature, and flower and foliage colors, and on the basis of progeny tests of untreated lines, both must have been homozygous when the experiments were initiat ed.. One family (Fig.lb) had light bluish-violet**'" petals with a star-eye. The eye consisted of a flash in the center of the petal near the throat, and two bars, one on each side of the flash, and slightly nearer to the base of the throat. The pigment of the star-eye was more intense than the other parts of the petal. *"'" kidgway's COLOR STANDARDS AP'D NOMENCLATURE was used in determining all colors mentioned in this paper. The foliage of this famil3r was slightly lighter than spin ach green. In the greenhouse the plants displayed a tend ency to produce a main st e m with slight branching, while in the field several main branches developed, each of which had many secondary branches. The greenhouse cultures of this family were about 33 cm. in height. The flower color of the other family (Pig.la) was tyrian pinlc, and there was a star-eye that was slightly darker than spinel red. The foliage v/as grass green; house height was 25-30 cm. the green vhe habit of growth was the same as the other family. The Experiments - Five experiments were undertaken. In the first experiment the treated material was immature buds; dry seeds were used in the second experiment, while soaked seeds were treated in experiment three. Ivlature and imma ture pollen was irradiated in experiment four, last experiment pollen and buds were exposed. and for the The X-ray Machine - The machine employed for irradiating this material was an upright type (Fig.2) with the tube housing surmounting a shelf which surrounded it entirely, hear the base of the housing there were fifteen windows, three-fourths of an inch square, closing covers. and with automatically It was through these windows that the material extended while being treated. Except for the last experiment, the X-ray tube had a molybdenum target; in that experiment a tube with a copper target was em ployed. This machine was the property of the X-ray Labor tory, The Pennsylvania State College. Unit of measurement - The mllliainpere-minute*v“ ' was the unit of measurement that was used to calculate dosages. It is computed, as follo ws : Hilliampere-minute (MAM) 0.251 t I E d 0.251 x t x I x E 2 -------------------d2 - correction. - time of raying in minutes. - current through the tube inrailliamperes. - voltage across the tube inkilivolts. - distance from the target to the specimen in cm. * Permission for its use was generously granted by Doctor W.P.Davey, Research Professor of Physics and Chemistry. Suggested by Doctor W.P.Davey. Experiment I - As was previously stated, the treated mater ial in this experiment consisted of immature "buds. greenhouse phloxes grow strong terminal buds. generally very little branching, quent. In the There is and tillers are infre In preparing the first portion of the material that was treated in this experiment, the terminal bud was pinch ed out when the seedlings were about four inches tall. This gave two equally strong lateral branches. diating was done, the buds of one branch were treated, the buds of the other branch were controls. of treating, When the irra During the process the control buds were well protected from X- rays, not only by the walls of the tube housing, but also by very thick portions of lead which were placed between the outside walls of the machine and the control buds. The clusters of buds to be treated were very carefully ad justed so that they would be at the proper distance (10 cm) from the target of the X-ray tube. very difficult to attain. Sometimes this was It is very obvious that the dis tance between the treated material and the target, ences greatly the dosage of the treatment. influ Cotton and small corks were also used to protect the delicate stems of the plants from injury through contact with the window covers and the walls of the machine. After treatment, the plants were placed in the greenhouse. Before any of the buds opened, glassine bags of an appro priate size were fastened over the clusters. Later, when the buds opened, the flowers were self-pollinated. The phlox flowers which were used in all of these experi ments were salver-shape cl.. There are five stamens which are adnate on t h e tube of the is accomplished easily. flower. Self-pollination The tube is severed longitudi nally on opposite sides down to the level of the stigma; the pollen is then pushed down upon it v/ith a sterile needle. All needles were sterilized in the flame of an alcohol lamp. Experiment II - Dry Seeds. This experiment dealt with dry seeds. The seeds of each plant were divided into two groups of about equal numbers. Each portion was confined in small squares of double two cheesecloth. The seeds of one of the/packages from the same plant were treated; the other seeds were used as con trols. All control seeds were left in the greenhouse. The seeds to be treated were placed inside the windows of the X-ray machine at the proper distance from the target, diated. and irra A check was made to ascertain if the cheesecloth absorbed any energy; the results were negative. After the containers were removed from the X-ray machine, they were placed outside the treatment room with those yet to be treated, where they received no additional X-rays. After the treatments were completed, to the greenhouse, and treated and control seeds were sown in soil from the same mixing. were transplanted, the same mixing. the seeds were taken they, also, When the seedlings were put into soil from Experiment III - Soaked Seeds. The seeds which were selected for this experiment were soak ed in tap water for forty-two hours. The time of soaking was arbitrarily determined as the time after which soaking was started when the cells of the embryo were is a very active condition. It was felt that the genes and chromo somes of active cells would be more susceptible to altera tions through the action of X-rays than dry seeds. The seeds of each plant were soaked separately in prepara tion dishes. At the end of the soaking period, the seeds were divided into two almost equal portions, one portion of the seeds of each plant was designated for treatment, other portion v;as used as controls. the As a precaution to prevent evaporation during the process of irradiation, the seeds were kept moist in wet paper, and the paper with its seeds was contained in the same type of cheesecloth sacks as were used for the treated seeds in Experiment II. Tests indicated that the paper did not absorb any appreciable quantity of energy. Aside from soaking, the seeds in this experiment were hand led in the same manner as the dry seeds in the previous ex periment. Treated and untreated seeds were germinated separately under the same conditions of moisture and tem perature, and the seedlings and mature plants were grown in close proximity in the greenhouse, or in the field. Experiment IV - Pollen In this experiment pollen in various degrees of maturity was used as experimental material. It will be recalled that in Phlox Drurnmondii the stamens are attached to the corolla tube, so that when the corolla is removed, stamens are removed with it. In a mature, the or almost mature flower, the removal of the corolla does not interfere with the pistil. Just previous to the opening of the flower, the anthers were examined to determine whether or not they had dis charged their pollen. If the anthers had not opened, they were selected for treatment. The matter of knowing the approximate time when the anthers will open is not difficult, for there is a definite correlation between the opening of the flower and the discharge of the pollen by the anthers. The pollen of about one-half of the anthers of a plant was used for self-pollinating their own flowers, and these flowers served as controls. The corollas of the anthers which wei’e selected for irra diation were split on opposite sides, and then they were placed in double cheesecloth sacks for treatment. The same technique was followed as was used in the other ex periments • Seventy-three plants were used in this experiment. dosages varied from 64 1/IA.M to 1051 MAM. The The material from any one plant was given the same dosage. Treated pollen was placed u pon the flowers of the plant from which it was taken, hut no attempt was made to pollinate flowers with their own pollen. Experiment V - Pollen and Buds. The material dealt with in this experiment consisted of buds of varying degrees of maturity, instances the pollen was mature, and pollen. In most or nearly mature. The same technique was employed in this experiment as was used in the previous experiments where the same materials were involved. The target of the tube was copper. Plants arising from dry and soaked treated seeds, seeds resulting from treated buds and flowers, from from seeds resulting from treated pollen and untreated ovules, and from all of the control material, were self-pollinated, when viable, for from four to seven generations. The plants were grown in the greenhouse and in the gar dens of the Department of Botany, The Pennsylvania State College, and in the greenhouse and garden of the Depart ment of Biology of Berea College. When feasible, treated and control plants were grown in adjacent rows in the field, and in the garden they were in close proximity. Each plant was critically examined for any divergence from the original types, and records were made. Differ ences which were observed in all of the progenies from treated and from untreated germ cells, were painstakingly examined, and their flowers were self-pollinated to as certain whether or not the variations were heritable. PHYSIOLOGICAL RESULTS. Buds - In many instances, particularly after the heavier dosages were administered, large numbers of buds were not able to survive the treatment. off. Many were able to survive, In a few days they fell and they developed seeds, but not always in abundance. Following (Tables I & II) are summaries of the seedsetting percentages of treated and untreated buds. These computations were based upon three seeds to the cap sule, as is normal for Phlox Drummondi i. The number of flowers was multiplied by three to get the number of poten tial seeds. The number of seeds which were produced was divided by the number of potential 3eeds to determine the seed percentages. Table I - Summary of Seed-Setting Percentages of Treated and Untreated Buds, Target of Tube Molybdenum UNTREATED Dosage No.FIs. No.Sds. MAM PercM o.Fls. No.Sds. Perc1 26 32 75 78 20 55 91 53 28 71 85 19 52 91 62 352 684 85 246 685 93 125 948 1730 61 787 2145 91 187 526 651 41 468 1288 91 252 1108 1097 53 1155 3014 87 312 647 383 20 600 1638 91 378 1052 347 11 832 2171 87 437 250 120 16 235 627 89 509 920 303 11 701 1893 90 567 337 1112 11 292 697 91 630 1105 245 7 867 2367 91 700 131 43 11 158 431 91 765 514 93 6 396 1081 91 908 234 4-2 6 175 436 33 1025 380 57 5 293 836 96 1071 96 11 4 118 382 91 14 Table II - A Summary of Seed-Setting Percentages of Treated and Untreated Suds, Target of Tube Copper. TREATED Dosage No.FIs. MAM UNTREATED No.Sds. Perc't No.FIs. No.Sds. P e r c ’t 61 159 420 88 146 423 97 123 162 334- 69 128 366 95 185 185 381 69 135 377 93 246 222 349 52 138 444 94 311 178 200 38 155 396 85 372 172 125 24 131 359 91 I Germination - a. Speed of germination - An immediate effect of X-rays upon dry and soaked seeds was a delay in germina tion. Generally from ten days to two weeks are required for the germination of phlox seeds, and the untreated seeds of these experiments displayed that habit. But the treated seeds were delayed a week or more in coming through the soil. There seemed to be a correlation between the intensity of the treatment and the extent of the delay in germination (Pigs. 3 & 4). Those seeds which were given relatively liglit dosages displayed very little delay. Goodspeed (1929) reported delayed germination for seeds of Nicotiana tabacum which had been X-rayed. Our dosages were in excess to those administered by Goodspeed. b. Percentages of germination - The germina tion percentages are interesting. tables III o: IV. They are illustrated in Irradiation did not interfere greatly with the percentages of germination of dry seeds, but the higher dosages were disastrous to a large portion of the treated soaked seeds. Eighty-seven percent of all/dry seeds ger minated, while 93/;’ of their controls germinated. But only 33,o of all treated soaked seeds germinated, as compared with 92>o of their controls. It is obvious that X-rays are more deleterious to active seeds than to inactive seeds. The following tables (III & IV) show the germination per centages of dry and soaked treated seeds, and their controls. 16 Table III- A Summary of Germination Percentages of Treated and Untreated Dry Seefe. TREATED )osage MAM Uj,!TREA TED No.Sds. No.Sds. Germ P e r c ’t Germ No.Sds. No.Sds. Germ. Perc’ Genr 626 78 66 85 66 54 82 745 80 72 90 68 52 77 868 80 56 70 44 4-4 100 969 214 196 91 148 141 95 1070 110 150 88 114 112 98 1175 140 112 80 94 36 92 1276 63 57 90 52 36 70 1577 40 24 60 28 20 71 1476 72 48 67 38 38 100 1574 56 52 93 42 42 100 1684 68 56 83 48 48 100 1807 61 60 98 60 60 100 1842 96 90 94 83 77 93 1908 50 50 100 40 40 100 1988 96 90 94 82 78 93 2008 51 42 80 41 39 95 2108 66 56 85 46 46 100 2127 100 94 94 68 60 88 2239 98 92 94 68 60 88 2209 51 45 88 50 48 96 Table III - concluded. TREATED UNTREATED Dosage MAM No.Sds. No.Sds. Germ. 2309 55 52 94 44 42 95 2409 55 52 94 60 60 100 2 51 0 57 51 89 45 45 100 2 71 1 80 56 70 66 58 88 Perc't. Germ. No.Sds. No.Sds. Germ. P e r c ’t Germ 1-8 Table IV - A Summary of Germination Percentages of Soaked Seeds, Treated and Untreated. TREATED No.Sds. Germ. UNTREATED P e r c' t. Germ osage MAM No.Sds. Tr'd. 195 21 16 76 18 16 89 261 31 29 94 27 25 93 293 25 20 80 21 18 86 380 42 36 86 35 34 97 389 26 22 85 18 16 89 490 32 26 81 27 24 89 524 35 30 86 31 29 94 588 32 22 69 27 22 81 652 33 30 91 27 25 93 686 33 20 61 25 25 100 762 29 22 76 25 22 88 785 26 15 58 23 22 96 819 25 15 60 22 20 91 981 33 22 67 32 26 88 1050 40 21 53 20 19 95 1080 22 11 50 18 15 03 1175 192 97 51 162 152 92 No.Sds. No.Germ. P e r c ’' Germ m Table IV - concluded TREATED Dosage MAM UNTREATED No.Sds. No.Sds. P e r c 1' T r 1d. Germ. Germ rlo.Sds. No.Germ. P e r c ’t. Germ. 1275 130 60 46 113 109 96 1306 28 12 43 25 23 92 1380 89 33 37 82 73 89 1441 20 7 33 28 25 89 1475 150 27 18 131 123 94 1575 114 19 17 87 84 97 1585 25 1 4 21 20 95 1703 23 8 35 20 19 95 Assembling the data of the experiment, it will be ob served that the soaked seeds which were treated have a germination percentage of 49$, while 92$ of the control seeds germinated. Whe n these data are compared with those gotten from treated and untreated dry seeds, we have, Treated Untreated Dry Seeds 87$ 9 0/6 Soaked 49$ 92/o Seeds Collins and maxwell X-rays* (1936) treated dry maize seeds v/ith The seeds were always irradiated with the embryo side facing the source of the X-rays. They found that very high dosages did not affect adversely the germination of seeds. Goodspeed (1929a, 1929b) reported that soaked seeds of Hicotlana tabacum were more sensitive to X-rays than were dry seeds. Iienshaw and Francis (1933) found the same to be true for seeds of Triticum vulgare. This was also the observation of Lambert (1933), and Stadler (1928) from work v/ith barley. Pollen - X-rays had a deleterious effect upon pollen, the following tables (V and VI) indicate. as 21 Table V - Summary of Seed Production Percentages from Treated and Untreated Pollen, j; I V Target of Tube Molybdenum. TREATED Dosage itlAi/i i UNTREATED No.FIs. LTo.Sds. Perc't. No.Pis. No.Sds. t Perc't. 64 104 109 35 81 217 89 127 107 123 43 83 211 80 195 129 26 6 58 114 29 8 87 260 165 308 62 133 355 89 325 130 217 56 10 5 278 88 388 117 192 55 107 256 80 450 130 213 55 110 283 86 528 18 8 284 50 1 59 408 86 600 139 186 4.5 135 347 86 668 13 6 174 43 107 ’ 307 96 732 136 157 38 112 29 1 86 795 122 138 38 10 1 22 1 73 859 91 102 37 73 198 90 923 67 62 31 50 1 31 87 987 14 12 29 9 21 73 1051 23 21 30 20 53 88 . m 22 Table VI - A Summary of Seed Production Percentages of Treated and Untreated Pollen; Copper Target. TREATED UNTREATED Dosage No.FIs. N o . S d s . P e r c ’t . No.FIs. No.Sds. Perc iiiA ivi 63 137 333 81 104 284 91 125 121 273 75 83 225 90 191 121 255 76 93 262 94 252 152 341 75 124 34-2 92 316 135 233 58 128 368 96 378 165 192 39 125 381 94 441 162 149 31 136 369 90 505 186 46 8 65 180 92 569 79 10 4 74 213 96 The Seedlings - It will he observed from figure 3 that seedlings from irradiated seeds, which were able to sur vive the treatment, appeared stunted and weak. This ef fect persisted for several weeks, but gradually the plants assumed a normal appearance. At maturity there was no evidence of this earlier retardation, and the seeds which these plants produced were as viable as the seeds of the control plants. Stunted progeny were observed from dry and soaked seeds alike. 24 Another effect of X-ray treatment of dry and soaked seeds was the appearance of abnormalities that were observed in some of the first few leaves. Sometimes they were twist ed; some of the leaves were bifurcated; they were very small. in other instances Some of these forms are illustrat ed in figures 5, 6 and 7. These abnormal leaves never as sumed a normal appearance, and they never reached the oroportions of normal leaves. Further leaves which these plants produced were normal. Goodspeed (1029) observed distorted leaves in his progenies from X-rayed seed3 of ilicotiana tabacum, and Horlacher and Killough (1931) made the same observation in the progenies of Gossypium hirsutum from treated dry seeds. Lethal Effects - In the second and third generations after treatment numerous instances were encountered where appar ently normal seeds failed to germinate. A peculiar attri bute of some of these cultures was that frequently the entire culture was not viable, but among the sibs from treated ancestry, the germination was high* Details of several of these lethals follow: a. Twenty-three buds of culture 396 were given a dosage of 625 i.iAi'.i; 11 seeds developed, able to germinate, hone of these seeds was although they appeared normal, but 53 seeds from 13 control buds of the same plant germinated end developed into normal plants. b. Twelve buds of culture 126 were treated with a dosage of 437 MAM. Twenty-two seeds resulted. these was able to germinate. None of Ten untreated buds of the same plant developed 26 seeds; all of these were viable. c. Albino - This form arose from bud raying. of 430 MAM was administered to 12 buds; A dosage one seed resulted. The plant from this seed developed chlorophyll, and it presented the appearance of a normal plant. pollination, Prom self- 33 plants resxilted in the next generation. All of them were albino, ho albino plants were observed among the 271 controls. As far as it is known, albino was never encountered pre viously in Phlox D r u m a o n d l i , although this character ap pears abundantly in nature. It is found in Antirrhinum ma.jus, Delphinium spp., horde urn sativum, bicotiana spp., Gossypium liirsutum, Trltioum spp., and Zea m a y s , to m en tion a limited number (katsuura, 1933). Pew references in the literature have been observed which indicate that albinism has been induced in plants through X-rays, although Stadler (1931) reports that in maize "white seedling is the most common mutant type." GENETXCAL RESULTS Variations which were ohserved over a period of from 4-7 generations, and lethal and other forms which were lost, but which were genetical, arrange themselves in the foll owing grouping. 1. Foliage Color Studies. a. Yellow (cosse green) - from grass green. b. Mottled - from grass green. 2. Flower Pigmentation. a. White - fr om the violet type. b. Flash - from the violet type. c. Spinel pink - from the violet type. 3. Stature. a. Giant - from the violet type. b. Dwarf - two appearances, one from the violet type, one from the pink type of flower. 4. Aborted flowers. 5. Lethal - two appearances. As will be demonstrated subsequently, all of these varia tions are gene differences, except, perhaps, the giant and the white-flowered type. We shall now consider in more detail the characteristics, origin and inheritance of these diverse forms. 1. Foliage Color Studies. a. Yellow - This form (Fig.8) arose in the second generation after treating 56 dry seeds of culture Til with a dosage of 1574 MAY. lings. It was observed in 3 seed The foliage of the ancestry of these seeds was slightly darker than grass green. The mutation was uni formly colored from the first observation, was retained throughout its existence. control seeds. and this color There were 52 In this second generation there were 975 individuals from treated parentage. consisted of 854 plants, The control group and no yellow plants appeared among them. This mutation, as far as it is known, never appeared pr e viously in Phlox D r u m m o n d ! i > although what appears to be analogous is known in many other plant species, as Antir rhinum m a j u s , Crepis canillaris, ilicotlana tabacum, Pisum spp., Triticum spp., Zea m a y 3 , and others (Yatsuura, 1933 DeKann, 1933). This variation has bred true through self-pollination for six generations, and when it was combined reciprocally with unrelated green plants, segregation occurred in the B’g in true simple mendelian fashion. led in the following table (VII). The data are assernb Table VII - Summary of Second Generation Progenies of Re ciprocal Grosses Between Yellow and Green. 142.3 (yellow) x 321.1 (green) 33 green 9 yellow 115.8 (yellow) x 321.2 (green) 41 green 11 yellow 172.3 (green) x 118.4 (yellow) 27 green 7 yellow 132.3 (green) x 142.9 (yellow) 50 green 12 yellow Total 151 green 39 yellow Calculated 142.5 green 47.5 yellow f8.5 green -8.5 yellow Deviation The deviation divided by the probable error of the ratio*' and indicates (D/P.B.) is 2.1,/a good fit of theory to fact. Stadler (1930, 1931b) reports that various yellows were induced by X-rays in maize, and also in barley. them behave as simple recessives. mutations are recognizable All of In barley 90/ of all in the seedling stage, and nearly all of them are chlorophyll deficiencies, of which about 15/ are yellow. in the same manner. In maize yellow mutations behave Horlacher and K i H o u g h (1931, 1932, 1933) induced virescent yellow in cotton through X-rays. It proved to be monofactoral in its heredity. An inter esting fact is that a reverse mutation was produced by X-radiation P.E. - through treating a ye H o y / virescent plant. .6745 / P x Q, x n - .6745/.75 x .25 x n 29 The reverse mutation was "larger, thriftier and more vigor ous than the virescent yellow plants from the same bolls." Horlacher and Killough (1932) described a yellow mutation which was devoid of chlorophyll, and lethal. It segregated as a simple recessive. MacArthur (1954) was able to induce yellow-white variations in the tomato by the use of X-rays. He did not kn o w the heredity of this mutation, but he sug gested that it may be either a lethal, or a dominant. b. Mottled - A second foliage variation, mottled, non-mottied arose from the/grass green stock. This mottling may be described as irregularly angular areas of grass green and yellow of almost equal dimensions. In instances it seems that the yellow areas are more closely associated with the veins of the leaves. It is shown in figure 0. Mottled arose from dry seeds of culture Til which were given a dosage of 1684 MAM. It was first observed in one plant. There were 48 treated and 46 untreated seeds. In the second generation after treatment there were 752 plants from treat ed ancestry, and 651 plants in the control group. in this generation that mottled appeared. It was mottled has bred true for six generations through self-pollination. Follow ing are the Fg progenies of reciprocal crosses between mot tled and unrelated grass green plants. Table VIII - Summary of Second Generation Progenies of Reciprocal Crosses Between Mottled and Green. 210.3 (mottled) x 318.2 (green) 18 green 4 mottled 210.6 (mottled) x 318.6 (green) 43 green 12 mottled 219.4 (mottled) 39 gr e en 12 mottled (mottled) 43 green 11 mottled 174.8 (green) X 326.8 (mottled) 35 green 9 mottled x 318.5 (green) 173.9 (green) X 326.7 318.1 (green) X 210.2 (mottled) 21 green 4 mottled Totals 199 green 52 mottled Calculated 188. 25 green 62. 75 mottled Deviation *10. 75 green -10. 75 mottled The difference divided by the probable error of the ratio is 2.3 which indicates that fact and theory fit. Yellow and mottled were crossed reciprocally. generation was green, are not alleles. crosses are shown. The first indicating that jellow and mottled In table IX the ? 2 progenies of these Table XX - Summary of Second Generation Progenies of Recip rocal Crosses Between Mottled and Yellow. Yellow x Mottled F2 115.2 x 210.1 71 green 25 mottled 31 yellow 116.5 x 210.2 42 green 17 mottled 20 yellow 142.7 x 325.4 55 green 21 mottled 24 yellow 142.8 x 325.3 30 green 6 mottled 12 yellow Totals 198 green 69 mottled 87 yellow Calculated 199.125 " 66.375 " 83. 5 " f1.125 " -2.625 " + 1. 5 " 210.1 x 142.5 63 green 18 mottled 20 yellow 326.5 x 115.1 52 green 14 mottled 19 yellow 209.6 x 115.3 27 green 6 mottled 8 yellow Totals 142 green 38 mottled 47 yellow Calculated 127.3675 42.5625 56. 75 Deviation 4-14.3125 -4.5625 -9. 75 Deviation tattled x Yellow nation of reciproc al totals 340 green 107 mottled 134 yellow Calculated 326.8125 108.9375 145. 25 Deviation f13.1875 -1.9375 -11. 25 Analyzing the s e data by the Chi^ Liethod, we h a v e : (0-C) Obs. Calc. (0-C) (0-C)2 c Green 340 326.8125 f13.1375 173.91 .532 Mottled 107 108.9375 -1.9375 3.75 .0o4 Yellow 134 145.25 126.56 .871 Totals 581 581 -11.25 v2 Prom Fisher's tableh“^ J ^ chi square of 1.437 is A 1.437 that the value of P. for 3 rf£ wh i c h indicates that the theo- retie 9:3:4 interpretation verj satisfactorily fits the ex perimental facts. Four segregates would be expected, green, mottled, yellow, end mottled-yellow, in the F q , but it was impossible to distinguish between the last two classes, so all of the mottled-yellow individuals were classified as yellow. It will be observed that there are too few of the mottled, yellow and mottled-yellow classes. It may be that a larger proportion of these perished in the seedling stage than of the green individuals. were healthy, Generally, mottled and yellow plants in the field particularly. Statistical Methods for Research W o r k e r s . they reached almost the proportions of normal green plants. Mosaics, particularly eye mosaics, are found frequently in Drosophila melanogaster as a result of X-rays which are ap plied to parental cells. Patterson (1930a, 1930b), and Timofeeff-Ressovsky (1929) induced eye mosaics v/hich were somatic. induced mosaic areas in the Goodspeed (1929) leaves of Nicotiana tabacum through irradiation, and Stadler (1930) suggested that In barley chlorophyll deficiencies v/hich he obtained as a result of X-rays, least 15/o of mosaics. consisted of at lie also reported mosaic endosperms; he explained their origin as being due to a deletion. Ii'orlacher (1932), and Horlacher and Killough (1932) report ed that splotched and angular variegations were found in the leaves of Gossypium h i r s u t u m . These variations were undoubtedly due to "abnormal cytoplasmic behavior induced by X-rays." Angular variations of another kind seemed to be due to nuclear changes. 2. Flower Pigmentation. a. White - It was previously stated that the flower color of one of the families that was used in this investipigmented gation had light bluish-violet petals, and a more intensely/ star-eye. In the second generation after treating 22 soak ed seeds of culture T49 with a dosage of 1080 MAM, there ap peared a giant plant (Figs. 9 & 10) which had white flowers. The giant aspects of this plant will be discussed later; here we are interested in flower pigmentation only. the 22 treated seeds, 15 germinated. consisted of 1R seeds; 15 Of The control group of them germinated. In the second generation after treatment there were 727 plants from treated seeds, and 698 plants from untreated control seeds. Kelly (1920) considered that flower color in the petal of Phlox Drummondii is due to at least 3 factors. "The three factors are conceived to be, first, one that leads to chromogen formation; secondly, production of an enzyme; one --- , that leads to the and lastly, one that leads to the production of an activator of the enzyme, ---." Violet flowers, zyme factor, then, would have a chromo,_.en factor, and an enzyme-activating factor. an en Vv'hite flowers may result from a number of circxmistances in which any one of the above three factors m a y be absent, the other two be ing present; of the third; to a lack of any two of them, and the presence and to an absence of all three factors. The white-flowered giant was self-pollinated for a large next generation, but its fertility was limited. three seeds developed, and they were plump, and somewhat larger than the average seed. The germination percentage was very poor, as only one seedling resulted. ling was very weak, Thirty- This seed and in a few weeks it was lost, so further analysis of this character was interrupted. Pollen from the giant white-flowered plant was used on flowers of distantly related control plants which were light bluish-violet. In the generation that followed these crosses, the flowers displayed a more intense p ig mentation, a shade near blue-violet. self-pollinated, These plants were and a large crop of seeds developed. Eut through disaster, only three plants were produced, so the situation could not be analyzed further. Young (1940) reported that through X-rays which were ad ministered by the late Doctor J. J. Taubenhaus to Liarglobe tomato seeds, some white-flowered plants arose. This character in the tomato bred true for two generations, and it behaved as a simple recessive. b. Violet Plash - As was indicated previously, star- eye of Phlox Drummondli consists of two bars at the throat of each petal, and a flash which occupies the center of the petal slightly distal to the bars. In the second genera tion after treating buds of culture 127a with a dosage of 1041 DAM, there appeared a plant in a culture of 14 whose flowers had color only in the flash part of the petal. The bars of the eye, and other portions of the petal were white. There was no variation in the intensity of the pigment of the flash from that of the parent, and the sibs. rio variations were observed among the control material of 537 plants. Reciprocal crosses were made between the variant and. violetcolored plants of remote relationship. The distribution of pigment in the Fg is shown in table X. Table X - Summary of Fg Progenies of Reciprocal Grosses Between the Mutation Violet Flash and Light Bluish-Violet. 342 (flash) x 110 (violet) 35 violet 15 violet flash 346 (flash) x 115 (violet) 23 violet 11 violet flash 118 (violet) x 351 (flash) 41 violet 15 violet flash 121 (violet) x 247 (flash) 53 violet 13 violet flash Observed 152 violet 59 violet flash Calculated 158.25 violet 52.75 violet fl. -G.25 violet *6.25 violet fl. Deviation The deviation divided by the probable error of the ratio I; 1.4-7, which means that the results approach very closely to theoretical expectation. Kelly (1920, 1934) showed that the star-eye of Phlox Drum- mondii is not transmitted as a unit. There are genes for pigmentation of the blade, of the bars, of which are inherited independently. and of flash, all In addition to the pigment genes, there is a gene for an enzyme, and an ac tivating gene. In some instances there is a gene for bluing. It would seem that the X-rays eliminated the genes for pigment in the blade and in the bars, or at least inactivated them. The genes for the enzyme, and for the enzyme-activating factor were active, b e cause there was pigment in the flash. c. Spinel Pink - A group of 6 spinel pink plants appeared in the second generation after irradiating 51 dry seeds of culture T51 of the violet-flowered stock with a dosage of 1275 MAM. Twenty-two of these seeds germinated, while 41 of the 45 control seeds were vi able. In this second generation there were 767 progeny from treated seeds, and 645 plants in the control group. This character has bred true for five generations, and it was confined to the treated plants. Pollen from three of these mutant plants was used to pollinate control flowers of the violet stock; the flowers of the other 3 plants received pollen from the same control stock. All of the offspring of these re ciprocal crosses were violet-colored. Following is a table (XII) which shows the distribution of pigment in the second generation. Table XII - Summary of Fg Progenies of Reciprocal Grosses Between Spinel Pink and Light Bluish-Violet. 29.3 (sp.pink) x 122 (violet) 61 violet 15 spinel pink 29.4 (sp.pink) x 125 (violet) 53 violet 14 spinel pink 29.5 (sp.pink) x 124 (violet) 43 violet 12 spinel pink 133 (violet) x 29.1 (sp.pink) 29 violet 8 spinel pink 131 (violet) x 29.2 (sp.pink) 31 violet 8 spinel pink 142 (violet) x 29.6 (sp.pink) 27 violet 10 spinel pink Observed 244 violet 67 spinel pink Calculated 233. 25 violet 77. 75 sp.pink Deviation +10. 75 vblet -10. 75 sp.pink The probable error of the ratio divided into the devia tion is 2.09. The assumption that the ratio is due to one pair of genes reasonable fits the data. When Kelly (1920) crossed a white-flowered Phlox Drummond!i with a dark-eyed, white-flowered type, in the F]_ the flower color was close to rhodamine purple. segregated. In the Pg, nine types "Sight of the types possessed color of some kind in the flowers and these obviously fell into pairs, one of each pair being, 'bluer1 than its mate." Types Ila and lib formed one. Phlox purple' in type Ilaj blade color was about Among the pairs, "The color was about in its 'redder' mate, 'amaranth pink.'" 'light lib, the The flower of type H a possesses the following genes, a gene, chromogen factor, an enzyme factor, (P), a (E), the factor A, an activator of E, and a "hluing" factor, (B). Type lib, the 'redder' mate, possesses all of the dominant genes for flower color, except the gene for "bluing," (B ) . This spinel pink flower seems to be analogous to the "red der" type lib above. It would seem that the "bluing" fac tor was eliminated through X-ray action, vated. or, it was inacti The transmission of spinel pink conforms to the findings of Kelly (1920), "bluing" factor, (B), for he demonstrated that the in Phlox Drummondii behaves as a dominant. 3. Stature. a. viously. 9). Giant - The origin of this plant was given pre It was observed early in the seedling stage (Fig. The difference in stature between it and its sibs, and control plants, was quite evident. As it increased in age, the difference became increasingly conspicuous, as figures 9 and 10 indicate. height of 57.7 cm. At maturity, (22.7 inches); the diameter of the stem at the surface of the soil was 7.2 mm. sturdy plant. it reached a It was a very Wo other plant was ever observed among the thousands of individuals which vmre examined in the progress of this investigation that could compare with it in size. Kelly (1915) states that "there are varieties that are from 12-20 Inches tall", but individuals of the latter propor tions, or even approaching them, were not Included in this study, except this giant mutant. The giant, its sibs, and the control plants, were all greenhouse cultures. All of the plants received as near identical environmental con ditions as it was possible to give them. Following is a table which indicates the dimensions of all of the experimental progeny, control plants. same parent. and a representative group of Cultures 316 and 372 are offspring of the Table XXI - Dimensions of Giant, Sibs, and Representative Controls. Number Relation Height Diameter 316.4 Giant 57.7 cm. 7.2 mm. 316.1 Sib 31.3 3.3. 316.2 Sib 32.5 3.5 316.3 Sib 33.6 3.6 316.5 Sib 33.2 3.5 372.1 Control 29.6 3.3 372.2 Control 33.8 3.6 372.3 Control 32.9 3.7 372.4 Control 34.1 3.6 372.5 Control 30.7 3.5 372.6 Control 34.3 3.5 372.8 Control 32.9 3.4 372.9 Control 34.5 3.5 Giant 57.7 cm. 7.2 mm. Sibs 32.65 3.475 mm. Controls 32.35 cm. Averages cm. 3.4 mm. The -unusual proportions of giant are not clear. The offspring, previously mentioned, which were obtained when pollen of the giant plant was used to fertilize flowers of normal-statured plants, were not abnormally large, for 31 plants in the field averaged 31 cm. in height. b. Dwarf - In two instances dwarf plants were p r o duced (Figs. 11 & 12). Both were very short, and their offspring, whether they were grown in the greenhouse, or in the field, were likewise very small. They were diffi cult to handle on account of their short stature. In some instances the plants were very weak; other plants were more sturdy. Screen door wire covers were construct- edjto use over them, not only foi' protection from the weather, but also to guard against cross-pollination. One of these mutations arose in the second generation following the irradiation of soaked seeds of T'46 with a dosage of 735 Mini. Fifteen of the 2G treated seeds ger minated, an d produced mature plants. control seeds were viable. Twenty-two of 23 In this second generation there were 477 plants from treated seeds, and 607 control plants. The other mutation came from culture 024.5. given a dosage of 380 IIAm. Buds were Thirteen seeds were set f r o m this treated material, while control branches of the same plant produced 66 seeds. irradiation, In the second generation after the generation in v/hich this mutation was ob served, there were 515 plants from treated buds; the con trol plants numbered 407. Progeny from both of these forms were crossed reciprocally with unrelated control plants which were normal fox'* height. All of the F-j_ plants were normal in size. ing table (XIII) acters in the F’2 * In the follow is shown the distribution of these char 44 Table XXII - Summary of Fo Progenies of Reciprocal Crosses Between Tall and Dwarf. < 171.3 (31.4 cm. ) X 426.2 (7.5 c m . ) 25 tall 6 dwarf 171.5 (29.5 cm. ) X 426.5 (7.6 c m . ) 36 tall 9 dwarf 171.7 (33.7 cm. ) X 426.6 (7.4 cm. ) 43 tall 11 dwarf 133.5 (34.5 c m . ) X 84.2 (6.2 c m . ) 37 tall 12 dwarf 133.7 (30.4 c m . ) X 84.3 (6.5 c m . ) 45 tall 13 dwarf 85.2 (9.2 cm. ) X 155.5 (55.2 c m . ) 38 tall 11 dv/arf 97.9 (9.0 cm. ) y 155.5 (35.2 c m . ) 9 tall 2 dwarf 97.11 (8.3 cm. ) X 155.7 (31.3 c m . ) 10 tall 1 dwarf 97.5 (7.7 cm. ) 171.2 (31.3. cm. ) 11 tall 3 dwarf 97.12 (9.0 cm. ) X 171.2 (31.1 c m . ) 15 tall 3 dv/arf Observed 267 tall 71 dwarf Calcula ted 253. 5 tall 34. 5 dw. Deviation +13. 5 tall -13. 5 dw. The X probable error of the r a t i o is 5.37; the deviation di vided by the probable error is 2.51, which indicates that the simple mendelian interpretation is correct for these dwarf mutations. 45 The relatively smaller number of dv/arf plants as compared with the number of normal plants, was undoubtedly due to the fact that in general the dwarf plants were weaker, and it was difficult to get them started. Dwarf forms have been induced in various plants through rays. reports a dwarf recessive in Zea Stadler (1951) X- mays, and Horlacher and Killough (1951) record two types of dwarfs in Gossypium h i r s u t u m . ultra-dwarf, dwarf. One of them was called and the other was designated as intermediate Both of them can be recognized early in the seed ling stage, normal, intermediate dwarf and ultra-dwarf form a progressive series. 4. Aborted Flowers - Aborted flowers appeared twice in the treated progeny of the violet-flowered family. One appearance followed the treatment of 22 soaked seeds of culture T49 which were given a dosage of 1080 LAli. Eleven of the seeds germinated; the second generation there were 15 control plants. In after treatment this plant arose, among 727 plants from treated seeds; there were G98 con trol plants in this generation. The other plant whose flowers aborted arose in the second generation after irradiating soaked seeds of culture T44 with a dosage of 588 LLAi.i. Twenty-two of 39 treated seeds germinated; in the control group there were 27 seeds, and 22 of them germinated.. In this second generation there were 559 plants from treated seeds, and 637 plants from The first of these was shorter than its 10 sibs and 15 control plants. Both the leaves and the flowers were very small (Fig.13). The other individual (Fig.14) had very small flowers and leaves, and the stem was etiolated. The anthers and ovules of both plants were small and shriveled, and contained no viable pollen and ovules. Through X-radiation, sterility is produced frequently in Drosophila m e l a n o g a s t e r , and in other animals. tations in Zea mays (Stabler, 1931a., 1931b) show reduced fertility, and frequently complete sterility. likewise true of barley. I.iany mu This is In both of these sterility may be due to gene or to chromosomal alterations. 9-oodspeed (1929a, 1929b) reports that in hi cot fans. tabacum "various degrees of gametic and zygotic sterility are involved." 4. Lethals - Two instances of physiological sterility have been presented.; 2 genetic forms will now be discussed. a. Fifty-six seeds of culture T15 were given a treatment of 868 UAL. minated, not good. Seventy percent of the seeds ger but the seed-setting ability of the plants was In the next generation the seeds of 7 plants in a population of 43 plants, failed to germinate. There was good germination among the control seeds, and no seed losses were observed. b. Of the 33 soaked seeds of culture T48 which were treated (981 BIA.Z.I), only 22 germinated. Seventeen plants developed, and all of them set a normal quantity of seeds. But in the following generation, not germinate, although the control plants gave almost per fect germination, freely. the seeds of 4 plants did and those which were not lethal germinated Lethals are common among progeny of Drosophila melanogaster w hi c h have been treated.hy X-rays during some stage of the life cycle. Summary and Comclusions: 1. Buds, dry and soaked seeds, and pollen served as material for these experiments. 2. Dosages. A. Unit of measurement - the milliampere-mimute was the unit of measurement that was employed to calculate dosages. B. Materials. a. Buds - the minimum dosage was 61 MAM; the maximum, 1168; the interval 2|- minutes. b. D r y seeds - minimum dosage, 626 MAM; maximum, 2711; interval 5 minutes. c. Soaked seeds - minimum dosage, 195 MAwI; maxi mum, 1705; int erval, 5 minutes. d. Pollen - minimum dosage, 64 MAM; maximum, 1051 LIAM; interval, 2-b minutes. e. Pollen - minimum dosage, 65 MAM; maximum, 568 IvIAi.i; interval, 2i minutes. f • Buds - minim um dosage, 61 IvlAM; maximum, 568 MAH; interval, 2-g minutes. 3. Results. A. Physiological Results. a. Many of the treated buds dropped off within a few days after irradiation. But many of the buds were able to withstand the deleterious effects of treatment, and they set normal seeds in normal quantities. b. There was a delay of a week or more in the germination of all treated seeds, dry and soaked. c. The germination percentages of treated seeds were lower than that of the control seeds; the higher dosages were injurious to the soaked seeds. d. The seedlings of most of the treated seeds were stunted, but those seedlings which survived develop ed into normal plants. They set a normal quantity of seeds which gave as high germination percentages as the control seeds. e. When higher dosages were administered, much of the pollen was inactivated, tilize many ovules, but treated pollen did fer and normal seed was set. f. In some instances the X-rays displayed a "di rect death-dealing effect upon the seeds." g. In the Fo albino plants v/ere among the progeny of a normal plant. B. Genetical Results. a. Yellow foliage from green, a simple recessive. b. Mottled foliage from green, a simple recess ive. c. v/hite flower from violet-colored flower, poss ibly due to chromosomal aberration. d. Flash from violet-colored flower, a simple re cessive . e. Spinel pink from violet-colored flower, a simple recessive. f. Giant from normal stature, probably due to chromosomal aberration. g. Dwarf from normal stature, recessive; appear ed twice. h. Aborted flowers from normal flowers, i. Lethal, sterile. two instances. Acknowledgements - l.iy sincere gratitude is due Doctor J.P. Kelly for suggesting the problem, .md for guiding me through all of its perplexities; to the Department of Botany, The Pennsylvania State College, facilities, and for a scholarship; the use of the X-ray equipment, for the use of its to Doctor W.P.Davey for and for technical advice; to Doctor Harry R.Kiehl for technical assistance; to Jr. A.F.Hildebrandt for his advice in growing the plants; and to my wife, Sllen S.Eangson, of seeds. for harvesting the many crops 50 LITERATURE Collins, G.N. and L.R.Maxwell, maize seedlings with X-rays. DeHann, II., 1933. 1936. Delayed killing of Science 83:375-376. Inheritance of chlorophylldeficiencies. Bibliographia Genetica 10: 357-416. Goodspeed, T . H . , 1929a. Cytological and other features of variant plants produced from X-rayed sex cells of Ni cotians tabacum. Bot.Gaz. >87:563-582. 1929b. The effects of X-rays and radium on species of Nicot ia n a. 1930. J.Tiered. 20: 243-259. Inheritance in Nicotiana tabacum IX. Mutations following treatments with X-rays and radium. Univ.Gal.Bub.Bot. Goodspeed, T.H. 11: 285-293. and F.II.Tiber, 1939. cyto-genetics. Bot.Rev. Radiation and plant 5: 1-43. Henshaw, P.S. and D.S.Francis, 1933. Growth rate and radio-sensitivity in Triticum vul,gare . Comp.Physiol. Horlacher, 4: 111-122. ,;.R., 1932. Production of invitations in Ameri can upland cotton by radiations. tics 2: 37-90. Jour.Cell. 5; Sixth Int.Gong.Gene 51 Horlacher, W.R. and D.T.Killough, variation in cotton. 1931. Radiation-induced Jour.I-Iered. 22: 253-262. 1932. Chlorophyll defi ciencies induced in cotton (Gossypium h i r s u t u m ) by ra diations. Trans.Tex.Acad.Sci. 15: 33-30. 1933. Progressive m u t a tions induced in Gossypium hirsutum by radiations. Am.Hat. 67: 532-538. Johnsom, E.L., 1931. Effect of X-radiation upon growth and reproduction of tomato. Kelly, J.P., 1915. Plant Physiol. 6: 685-694. Cultivated varieties of Phlox Emmmondii Jour. N.Y.Bot.Garden 16: 179-191. 1920. The genetical study of flower form and flower color in Phlox D rummondli. 1934. Genetics 5: 189-248. The "eye11 of Phlox. Jour. He red. 25: 183-186. Lambert, J . , 1933. Recherches sur les factairs be la radio- sensibilite tissulaire en dehors des phenomenas morphologiques. Les proprietes biologiques des tissus ietents. Archiv.Biol. 44: 621-739. MacArthur, John W., 1934. Jour.Hered. 25: 75-78. X-ray mutations in the tomato. m Matsuura, Hajimem 1933. genetics. A Bibliographical monograph of Asppor, Iloore, C.N. and C.P.Haskins, 1935. X-ray induced modifica tion of flower color in the petunia. Jour.Hered. 26: 249-355. 'duller, H.J., 1930. Radiation and genetics. Am.Nat. 64: 1934. Radiation genetics. Quart.Rev.Biol. 220-251. Oliver, C.P., 9: 381-408. Patten, Ruth E.P. on seeds. and S.B.Widoger, 1929. Effect of X-rays Nature 123: 606. Ridgway, R . , 1912. Color standards and color nomenclature. Washington, D.G. Stadler, L.J., 1928. Genetic effects of Z-rays in 'maize. Proc.Nat.Acad.Sci. 14: 69-75. 19285. Nutations in barley induced by X-rays and radium. Science 68: 186-187. 1928c. The rate of induced imitation in re lation to dormancy, temperature and dosage. Anat.Rec. 41: 97. 1930. plants. Some genetic effects of X-rays in Jour.Hered. 21: 3-19. Stadler, L.J., 1931a. The experimental modification of heredity in crop plants. gularities. Sci.Agr. I. Induced chromosomal irre 11: 557-572. II. Induced mutation. Sci. Agr. 11: 645-661. 1932. On the genetic nature of induced mutations in plants. Proc.Sixth Int.Gong.Genetics 1: 274-294. Timifeeff- Ressovsky, N.V/., 1929. The effect of X-rays in producing somatic genovariations of a definite locus in different directions in Drosophila melanogaster. Am.Nat. 63: 118-124. Young, P . A . , 1940. White-flowered character from X-rays treatment of tomato seed. Jour.Hered. 31: 78-79. Figure 1. - The plant on the left (a) is a representa tive of family 2; the flowers are tyrian pink, and th star-eye is more intensely pigmented. right (b) represents family 1. The plant on t The flowers are light bluish-violet with a more deeply colored star-eye. it r Figure 2. - The machine which, was used in administer ing the X-rays to the experimental material. Figure 3. - These seedlings illustrate the effect of X-rays upon germination. The seedlings on the right (b) are from dry seeds whic h were given a dosage of 2209 FIAX. Those on the left (a) are controls of the same age. The picture was taken 29 days after the seeds were sown. mVmik feNSw Bill i ^ l l l lr%>M X* --;•$!-t w A'^4 V .^ <*f ? >*,»V Figure 5. - Showing the effect of X-rays upon growth end development of some of the first leaves. The leaves on the right (b) are from seedlings whose seeds were given a dosage of 626 MAX. The leaves on the left (a) are from control plants. All of these leaves were taken at the same level. '■" ■ • „ m Figure 6. - iere are some of the _abnormal shapes assumed b;; a few of the first leaves of seedlings from treated dry seeds. The T25A plants are from seeds which were given a dosage of 626 MAM. The T165.1 plant came from a dry seed..- which was given a dosage of 1684 I :21.9 . 13 I Figure 7. - Deformed first leaves of plants from t reated dry seeds. shape. Future leaves were normal in size and in The T21 series received a dosage of 2209 -.i/i.a ) that given the T22 plant was 2309 lift.:.:. Figure 3. - The plant in the upper left (a) is mottled. It arose in the second generation after 'treating dry seeds with a dosage of 1634 nAII. (b) is yellow. In the foreground It was first observed in the second generation after irradiating dry seeds with a dosage of 1574 normal plant. The plant in the upper right (c) is a Figure 9. - The giant plant (b) arose from soaked seeds which had been irradiated with a dosage of 1080 MALI. The plant on the left (a) ’is a sib of the giant plant; control plant. the plant on the right (c) is a ^7! V ,• ' 4 •• - Xj. ' A *«» 2 ’'■,i ;sS5?*SS** «&• —’*f? ‘’igure 10. - The giant (b) is in the center, the left, sib (a) on and control (c) plant on the right. This picture was taken 16 days after the picture which is figure 9. Figure 12. - The plants at the left (a) are dwarf mutations; sib. the plant at the right (b) is a normal The dwarf plants came from treated buds which were given a dosage of 380 I.IAM. igure 14. - Aborted flowers. small, The leaves are very and the stem is etiolated. This plant arose from a soaked seed which had been given a dosage of 588 SIAM. See 'figure 15 for a control plant.