PLEASE HANDLE WITH CARE University of Connecticut Libraries Digitized by the Internet Archive in 2011 with funding from LYRASIS members and Sloan Foundation http://www.archive.org/details/geneticsmorpholoOOmang Bulletin 279 June, 1926 / Qlimtterttntt Agrirultural lExprrtmrttt Station Nrm Haunt, (Smtnrrtiritt The Genetics and Morphology of Some Endosperm Characters in Maize P. C. MANGELSDORF The Bulletins of this Station are mailed free to citizens of Connecticut who apply for them, and to other applicants as far as the editions permit. CONNECTICUT AGRICULTURAL EXPERIMENT STATION OFFICERS AND STAFF as of June, 1926 BOARD OF CONTROL His Excellency, John H. Trumbull, ex-officio, President. Charles R. Treat, Vice President Orange George A. Hopson, Secretary Mount Carmel Wm. L. Slate, Jr., Treasurer New Haven Joseph W. Alsop Avon Elijah Rogers Southington Edward C. Schneider Middletown Francis F. Lincoln Cheshire STAFF. E. H. Jenkins, Ph.D., Director Emeritus. Administration. Wm. L. Slate, Jr., B.Sc, Director and Treasurer. Miss L. M. Brautlecht, Bookkeeper and Librarian. Miss J. V. Bekger. Stenographer and Bookkeeper. Miss Mary E. Bradley, Secretary. G. E. Graham, In charge of Buildings and Grounds. Chemistry: E. M. Bailey, Ph.D., Chemist in Charge. Analytical C. E. Shepard ~| Laboratory. Owen L. Nolan I Assistani Chemists. Harry J. Fisher, A.B. f W. T. Mathis J Frank C. Sheldon, Laboratory Assistant. V. L. Churchill, Sampling Agent. Miss Mabel Bacon, Stenographer. Biochemical T. B. Osborne, Ph.D., Chemist in Charge. Laboratory. H. B. Vickery, Ph.D., Biochemist. Miss Helen C. Cannon, B.S., Dietitian. Botany. G. P. Clinton, Sc.D., Botanist in Charge. E. M. Stoddard, B.S., Pomologist. Miss Florence A. McCormick, Ph.D., Pathologist. Willis R. Hunt, Ph.D., Assistant in Botany. A. D. McDonnell, General Assistant. Mrs. W. W. Kelsey, Secretary. Entomology. W. E. Britton, Ph.D., Entomologist in Charge; State Entomologist. B. H. Walden, B.Agr. » M. P. Zappe, B.S. [ Assistant Entomologists. Philip Garman, Ph.D. ) Roger B. Friend, B.Sc, Graduate Assistant. John T. Ashworth, Deputy in Charge of Gipsy Moth Work. R. C. Botsford, Deputy in Charge of Mosquito Elimination. Miss Grace A. Foote, B.A., Secretary. Forestry. Walter O. Filley, Forester in Charge. H. W. Hicock, M.F., Assistant Forester. J. E. Riley, Jr., M.F., In charge of Blister Rust Control. Miss Pauline A. Merchant, Stenographer. Plant Breeding. Donald F. Jones, S.D., Geneticist in Charge. P. C. Mangelsd.orf, S.D., Assistant Geneticist. H. R. Murray, B.S., Graduate Assistant. Soil Research. M. F. Morgan, M.S., Investigator. George D. Scarseth, B.S., Assistant. Tobacco Sub-station Paul J. Anderson, Ph.D., Pathologist in Charge. at Windsor. N. T. Nelson, Ph.D., Plant Physiologist. THE TUTTLE, MOREHOUSE & TAYLOR COMPANY TABLE OF CONTENTS PAGE Introduction 5*3 Definitions S x 4 Acknowledgments 5*4 PART I Defective Seeds 516 Widespread Distribution in Germplasm 517 Origin by Mutation 518 Source of Material 521 Simple Mendelian Recessives 523 Crossing Experiments 523 Method of Crossing 523 Results of Crosses 525 The F 2 Generation 526 Summary of Crosses 529 The Morphology of Defective Seeds 531 The Cytologial Mechanism of Endosperm Formation 531 Development After Fertilization 532 Development of Defective Seeds 533 Regular Development in Early Stages 534 The Pericarp 534 The Nucellus 534 The Endosperm 535 Starch Grain Formation 536 The Aleurone Layer 536 The Embryo 537 General Aspects 538 Physiology of Defective Seeds 539 Rate of Growth, Final Weight and Germination of Normal Seeds 540 Rate of Growth, Final Weight and Germination of Defective Seeds 541 The Influence of Lethal Factors in Heterozygous Condition .... 545 Effect upon the Gametophyte 546 Linkage Relations 548 Linkage Between Su and de Factors 550 Linkage of Defectives with Each Other 554 Linkage of Defectives with Growth Factors 558 A Plant Character for Defective Seeds 559 Discussion 562 Summary 562 PART II Non-Hereditary Defective Seeds 564 Parthenocarpic Defectives 564 Some Conditions Which Influence the Frequency of Partheno- carpic Defectives in Maize 566 Influence of Age of Silks 568 Influence of the Age of Pollen 569 Arrested Development 572 Irregularities in the Fertilization Mechanism . .'. 573 Summary 578 PART III Genetic Factors Which Influence the Texture of the Endo- sperm 579 Brittle Endosperm 579 Sugary x Brittle 580 Shrunken x Brittle 582 Brittle Endosperm from Two Varieties 583 Shrunken Endosperm from Two Sources 584 Waxy Endosperm in China and America 584 Constant Variation in the Storage Material of the Endosperm . . 585 The Relative Development of Endosperm Characters 586 Discussion 587 Summary 589 PART IV Premature Germination of Maize Seeds and Genetic Factors Which Govern Dormancy 590 Complementary Factors 590 Description Summarized 594 Phenotypical and Genetic Differences 594 Duplicate Factors ; 596 Ratios of 8 : 1 and 41 : 1 598 Summary of Breeding" Behavior 509 Linkage Relations 599 gei x Su 600 ge» x su 601 ge 3 and get, x su 601 Apparent Linkage with Endosperm Color Factors 602 Premature Digestion and Pigment Formation 603 The Relation of Premature Germination to Chlorophyll Develop- ment 604 Physiology of Premature Germination 605 The Effects of Premature Germination on the Growth of the Seed 605 Discussion 607 Summary 607 Conclusion 608 Literature Cited 610 Explanation of Plates 614 The Genetics and Morphology of Some Endosperm Characters in Maize : P. C. Mangelsdorf Introduction When Nawaschin in 1898 discovered the phenomenon of double fertilization in Lilium it was generally believed by botanists that such a peculiar mechanism was confined to this species and per- haps a few closely related ones. Later investigations have shown that it is widely distributed and that the endosperm of angio- sperms, with perhaps a few exceptions, is the product of a sexual fusion, quite apart from that which gives rise to the embryo, and differing from the latter in that one male nucleus combines with two or more female nuclei, while the embryo results from a fusion in which both parents contribute equally. Thus the endosperm of angiosperms is unique in several respects. It resembles its near relative, the embryo, in its sexual origin, but differs from the latter in structure, in capacity for continued development, and in ability to reproduce. This unique sporophyte, if indeed, it may be called a sporophyte, achieves its highest development in the cereals, in which it con- stitutes the part which makes these plants of such great economic importance, and in which it gives evidence of its sexual origin by the expression of the hereditary factors which it receives from its two parents. Mendelian characters which have their expression in the endo- sperm have been found in wheat, rye, barley, rice and maize. They are apparently most numerous in maize and a number of characters which affect the color of the endosperm or aleurone layer and the texture of the endosperm tissue of this species have long been familiar, and have played an important part in the researches leading up to the re-discovery of Mendel's Law _ * Parts I-IV of a thesis submitted to the Faculty of the Bussey Institu- tion of Harvard University in partial fulfillment of the requirements for the degree of Doctor of Science, June, 1925. Part V, on "Genetic Factors Which Affect the Development of the Gametophyte and Their Relation to Some Endosperm Characters," has been combined with researches by Dr. D. F. Jones on the same subject and will appear in Genetics, Vol. XI, under the title "The Expression of Mendelian Factors in the Gametophyte of Maize." 514 CONNECTICUT EXPERIMENT STATION BULLETIN 279 in 1900 and in the accumulation of a vast amount of genetic evidence since its re-discovery. The endosperm characters of maize are of unusual value to the geneticist because, like all endosperm characters, they are visible sooner than those which affect other parts of the plant, and because they are readily studied in large numbers without the necessity of devoting a great amount of land or labor to the purpose. The average ear of maize bears from several hundred to a thousand seeds and one pollination on a single plant produces a large popu- lation which is readily classified because the environment has been remarkably alike for all its members. In recent years the widespread application of a new method of corn improvement which involves the extensive inbreeding of this crop by artificial self-pollination, has brought to light many new characters which influence the development of the endosperm. Because of their possible phylogenetic significance, and because they represent new material which may prove of value in charting the germplasm of this important species, these characters merit a thorough study. The following pages are devoted to the pre- liminary investigations of a number of these new characters, their breeding behavior, morphology, effect upon development, and their relation to each other and to other characters. DEFINITIONS Two terms are used so frequently throughout the pages which follow that they deserve to be defined and limited. The endosperm generation is the period beginning with fertil- ization and ending with the disappearance of the endosperm through absorption or digestion. In the cereals, the endosperm persists until the germination of the seed but in some plants it is almost completely lost in the early stages of development. This period has also been termed the xenia generation by some writers. In crosses the F 2 endosperm generation is borne on F x plants. An endosperm character is any character which has its expres- sion in the endosperm generation. The term does not imply that the character in question affects only the endosperm, in fact some of the endosperm characters produce their major effect upon the embryo. ACKNOWLEDGMENTS Grateful acknowledgment is made to Dr. E. M. East, under whose supervision this investigation was made and these pages written, for his helpful advice and kindly criticism. A word of appreciation is due the Connecticut Agricultural Experiment Station for an arrangement enabling the writer to complete these studies while a member of its staff, and particularly to Dr. D. F. ENDOSPERM CHARACTERS IN MAIZE 5*5 Jones for his suggestion of the problem, his generous provision of material including a number of preliminary crosses, and his con- stant encouragement throughout. Acknowledgment is also due Dr. Florence McCormick for advice in regard to the preparation of material for histological studies, to Helen Parker Mangelsdorf, for assistance in the statistical work, and to numerous investi- gators who have contributed material. 5*6 CONNECTICUT EXPERIMENT STATION BULLETIN 279 PART I Defective Seeds Defective seeds are lethal or semi-lethal characters which affect the development of the endosperm and embryo between the time •of fertilization and maturity. These characters were first reported by Jones (1920) who described them as "aborted seeds with either entirely empty pericarps or badly shrivelled seeds, completely lethal in some cases and partially so in others," He found these char- acters to be inherited as simple Mendelian recessives. Previous to 1920 aborted seeds had frequently been noted on open-pollinated ears of maize but had generally been regarded as due to imperfect pollination or other external factors. Self-pollinated ears in which approximately one fourth of the seeds were aborted, had also undoubtedly appeared in the cultures of many investigators before 1920 but these lethal characters were not noted or were not regarded as heritable. In an early edition of Bailey's "Plant Breeding" appears a photograph of two ears grown shortly after the re-discovery of Mendel's Law, illustrating the alternative inheritance of the starchy and sugary conditions of the endosperm. One of these ears is clearly segregating for defective seeds in addition to the other two characters. The segregation is so well defined that the normal and aborted seeds on three of the rows of grain can be counted from the photograph. Seventy-five normal and 23 defec- tive seeds are noted. The investigator who pollinated these ears to prove or disprove to his own satisfaction the newly re-dis- covered Law of Mendel, had more evidence of its correctness than he probably realized. Richey (1923) found several defective seeds on an ear of maize, believed to be many centuries old, unearthed from an Indian graveyard in Peru. He concludes from this discovery that defec- tive seeds are characters of considerable antiquity. Although his conclusion is probably correct, it is scarcely justified from this evidence alone, since it is equally possible that the few aborted seed on this ancient ear are of the non-hereditary types described in Part II. In the past few years many experiment stations in this country, Canada, South America and Europe have undertaken projects for the improvement of corn by the method outlined by East and Jones (1919) and by Jones (1920) and known as "Selection in Self-fertilized Lines." Thousands of self-pollinations in many varieties have been made every year, and this extensive inbreeding of a naturally cross-fertilized species has brought to light many recessive variations previously covered up by the remarkable heterozygosity which exists in the average variety of maize. ENDOSPERM CHARACTERS IN MAIZE 5 ! 7 Among these variations have been a large number of defective seed types. The writer (Mangelsdorf, 1923) has noted defective seeds in self-pollinated ears of more than 30 representative American varieties as well as several from Spain, Italy, China and Peru. Since 1920, defective seeds have been reported under various names by numerous investigators. Lindstrom (1920, 1923) has described "abortive," "flint defectives," and "sweet defectives." Demerec (1923) has reported a condition which he calls "germ- less," Eyster (1922) a peculiar defect to which he gives the term Fig. 51. — Self-pollinated ears of three New England varieties which are segregating for defective seeds. The third, sixth and seventh ears from the left represent the original ears of stocks dei, dez and de% respectively. "scarred" and Wentz (1924) a type known as "miniature germ." Garber and Wade (1924) report a semi-lethal type of defective seed in their cultures. All of these characters may be considered as variations of the "defective" condition since all of them represent a seed develop- ment considerably below normal and most of them are lethal or semi-lethal in a homozygous condition. WIDESPREAD DISTRIBUTION IN GERM PLASM Some conception of the frequency with which defective seeds-, occur may be gained from the following figures taken from self- pollinations made in typical American varieties. 5 18 CONNECTICUT EXPERIMENT STATION BULLETIN 279 Defective seeds were first noted in a lot of 86 self-pollinated ears of four New England varieties. Thirteen of these ears, or 15.0 per cent, were segregating. In 1922, 575 self-pollinated ears of six regional strains of Sanf ord White Flint were examined for these variations. Nine- teen of these ears, or 3.3 per cent, were found to be segregating. Hutchison (1922), in making a systematic search for variations of all sorts, self-pollinated 2,110 ears representing 468 different lots of seed which had been obtained from seed companies and experiment stations. These lots contained most of the varieties commonly grown in the Northern states and included sweet, pop, dent, and flint types. Sixty-seven of these ears, or 3.2 per cent, were found to be segregating for defective seeds. The percentage of segregating ears, following the first self- pollination, in the lot of 575 ears of Sanford White, agrees so closely with the percentage found in Hutchison's 2,110 ears, that these figures, 3.3 and 3.2, probably represent the average condi- tion of most varieties of maize. In other words, about one plant in every 30 in the average variety is heterozygous . for a lethal factor which causes defective seeds. In some varieties this pro- portion is probably higher, depending to some extent on the amount of natural self-pollination which has occurred in past generations. ORIGIN BY MUTATION The relatively high frequency of these lethal characters in most varieties suggests that maize, like Drosophila, is constantly under- going factor changes at various points in the germplasm and that a large proportion of these changes may be lethal in their effect. Muller and Altenburg (1919), in a study to determine the fre- quency of mutation in the X chromosome of Drosophila, find that characters which are lethal or semi-lethal are the most frequent to occur. They estimate that the X chromosome in Drosophila produces a new lethal, on the average, about once in every 100 generations. Recent evidence from homozygous inbred strains of maize indicates that the frequency of lethal mutations in this species may be as high or perhaps considerably higher. In 1 92 1 an inbred strain of maize which had been self -polli- nated for thirteen generations and which was apparently homozy- gous for its genetic factors, as demonstrated by a test made by Jones (1924), began to segregate for defective seeds. The sud- den appearance of this new character was clearly due to a germ- inal change since nothing of this kind had previously been noted, although a careful search for new variations in all inbred strains had been constantly maintained. Nor could this new character have been the result of a segregation following accidental cross- ing since outcrossing with unrelated stocks is immediately apparent ENDOSPERM CHARACTERS IN MAIZE 519 by the increased vigor and productiveness of the hybrid plants. With the exception of the segregation for defective seeds, the mutant stock differed in no detail from the original inbred strain. This new character originating by mutation in a homozygous stock is a typical defective seed, is completely lethal in its effect Fig. 52.— Ears of a strain of Learning which mutated to defective seeds after thirteen generations of inbreeding. The ear at the right is segregating for the mutant character. and is inherited as a simple Mendelian recessive. Ears of this inbred strain which are segregating for defective seeds are shown in Fig ; 52. A sister strain separated from this one after seven generations of inbreeding also showed defective seeds in the thirteenth genera- tion when grown by H. A. Wallace at Des Moines, Iowa. This, 520 CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 same strain, though of slightly different pedigree, produced defec- tive seeds on two ears in 1924 after 17 generations of inbreeding. The parental ears of both segregating progenies were normal in 1923 so two separate mutations must have occurred. In other words, four separate mutations have appeared in the germplasm of this strain in the past four years and each time a lethal char- acter, defective seeds, resulted. The pedigree, showing the genera- tions in which the mutations were first noted, is given below : T-I-I-I-I-K^ 1 ) 5 ^- ^ 4-4-2-1 < (l) >(2) seg. de. 1 -6- 1 -3-4-4-4-2 <^ 3 K 1 ) se &»- ae - ^5-5-2-i-i-(i) seg. de. The two defectives which were noted in 1924 are similar in appearance but both are quite different from the one found in 1921. Though no crosses have yet been made between these four separate mutations, it is certain that they are of at least two distinct types phenotypically and probably genetic differences will be found as well. In the past four years only a few ears have been self-pollinated each season from this strain. The fact that four separate muta- tions have been noted in this rather small sample indicates that germinal changes are now occurring rather frequently. Previous to 1921, however, no mutations in this stock had ever been noted, though an active search for variations was constantly maintained and in some seasons a large number of ears were self-pollinated. The only other mutations ever found in inbred strains have been red cobs in a white cob strain, dwarf plants in another strain and a chlorophyll deficiency in a third. From the limited experience with these long-inbred and rela- tively homozygous strains of maize it is evident that germinal changes do occur and, perhaps, very frequently. As in Droso- phila, a high proportion of these changes probably result in lethal factors. The dominant lethals, if they occur at all, are imme- diately lost, because individuals which carry them do not live to reproduce. The recessive factors, unless they have a marked deleterious effect in the heterozygous condition, may be carried along for generations. It is not at all surprising, therefore, to find these lethal factors in almost every variety of maize. In- breeding brings them to light and demonstrates that about one plant in every thirty is heterozygous for one or more of them. How many genetically distinct lethal seed factors there are in maize can not be estimated at the present time, but some indica- tion of the enormous number which probably exists may be gained from the following pages. ENDOSPERM CHARACTERS IN MAIZE . 52 1 SOURCE OF MATERIAL As already mentioned, defective seeds were noted by the writer in more than 30 varieties of maize. It soon became evident, that in order to make a thorough investigation it would be impossible to study the breeding behavior of more than half this number. Accordingly only those stocks in which defective seeds had appeared at least two successive generations, and in which the segregating ears gave clear-cut 3:1 ratios, were continued. Fourteen stocks met these requirements and were retained. It is almost certain that, in confining the investigation to those strains in which simple 3 :i ratios were obtained, lethals which are due to duplicate or triplicate factors were eliminated. It was con- sidered best, however, to study first the inheritance of the simple recessives without the complications brought in by duplicate or triplicate factors, especially since defectives which segregate in 15:1 or 63:1 ratios would be difficult to distinguish from the various types of non-hereditary defectives which occur in small numbers on almost every ear and which are discussed in some detail in Part II. The source of the fourteen types of defective seeds used in this investigation is given below. The de numbers under which they are discussed were assigned after all the types had been arranged in a series on the basis of their "defectiveness." Those with high numbers such as de 13 and de 14 are the most defective in appear- ance, while those with low numbers, such as de 1} de 2 , and de 3 most nearly approach the normal condition and are only semi-lethal in effect, being sometimes obtained in a homozygous condition. de x This type appeared following the first self-pollination of Gold Nugget, an eight rowed, large-eared, yellow, flint variety. Twenty self-pollinated ears were obtained of which three were segregating for defective seeds. Only one strain has been kept heterozygous for defective seeds. de 2 This defective was obtained from a self-pollinated ear of an eight-rowed yellow flint type grown by Dr. E. G. Anderson, then at Cornell University. This is probably the same stock in which Lindstrom found his "flint defective" and, if so, was obtained originally from Dr. W. E. Castle of Harvard University. de z Defective seeds appeared on a self-pollinated ear of Century Dent, a many-rowed, medium early, yellow, New England dent variety. Five ears out of 18 which were self -pollinated, segre- 52 2 CONNECTICUT EXPERIMENT STATION BULLETIN 279 gated for defective seed, but only this and one other, de & , have been continued. de± This type was first noted in the third generation of inbreeding in two ears of a strain of Beardsley's Learning, a many-rowed, fairly late, yellow dent variety. No defectives had been noted in this strain in the first two generations and their appearance in the third may have been due to mutation. de 5 This stock originated from a segregating self-pollinated ear of Reid's Yellow Dent received from Dr. J. R. Holbert of Bloomington, Illinois. de Q A self-pollinated ear of Luce's Favorite, a large eared, New England dent variety received from Dr. R. A. Emerson, segre- gated for defective seeds of this type. de 7 A mutation in an inbred strain of Chester's Learning which had been self-pollinated for thirteen successive generations and was apparently homozygous, gave rise to this defective. de 8 This stock originated from another ear of the same lot of self-pollinated ears of Century Dent as de z . de a This type was found on a self -pollinated ear of Cornell No. 12, a selection of Funk's 90 Day, obtained from Dr. R. A. Emerson. de 10 This defective appeared in a stock of "fine striped" which had been obtained some years previously from Cornell University. de xx The source of this stock was a self-pollinated ear of Clarage Dent, a typical Western yellow dent variety received from Professor M. T. Meyers, Ohio University. de 12 Seeds of this type appeared in a self-pollinated ear of Burbank's "Rainbow," a novelty purchased from Peter Henderson Co., New York. ENDOSPERM CHARACTERS IN MAIZE 523 de 13 This type was isolated from a cross made by Mr. H. A. Wallace of Des Moines, Iowa. The seed parent was a hybrid combination of four inbred strains ; the pollen parent a plant of "Illinois Two Ear" which Mr. Wallace believed to be homozygous for defective seeds. de 14: This defective appeared in the second generation of inbreeding of a strain of Beardsley's Learning, the same variety which gave rise to de±. Defective seeds were not noted in this strain in the first generation of inbreeding. SIMPLE MENDELIAN RECESSIVES Except for the fact that there is often a slight deficiency of recessives, whereas an excess might be expected because of the regular occurrence of non-hereditary defectives on almost every ear, all of these fourteen types appear to be inherited as simple Mendelian recessives. CROSSING EXPERIMENTS The first defectives studied, de t , de 2 , de z , and de 8) showed slight phenotypical differences, the first three being "partial" defectives ; the last a "complete" defective. Crosses of these four strains made by Dr. D. F. Jones, the later generations of which were classified by the writer, indicated that these four defectives were genetically distinct. The next step was to determine the number of factors involved in the remaining ten stocks. METHOD OF CROSSING Throughout the investigation the general method of crossing two stocks has been as follows : A number of tassels, five or more, of the strain to be used as pollen parent were bagged. When pollinations were made the pollen from all of the bagged plants was collected, combined and mixed. Theoretically two- thirds of the plants in any segregating stock are heterozygous for the lethal factor and one-third are homozygous for the dominant allelomorph. Half of the pollen grains of the heterozygous plants should carry the lethal factor while the remaining half, as well as all of the pollen from the homozygous plants, should lack the lethal factor. Assuming an equal production of pollen by the heterozygous and homozygous plants, a composite collection of pollen made in this way should contain, on the average, one-third of the pollen grains carrying the lethal factor and two-thirds carrying its dominant allelomorph. 524 CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 The seed parent of the cross should, like the pollen parent, pro- duce heterozygous and homozygous plants in the proportion of 2 :i. When the composite mixture of pollen is applied to homozy- gous plants, only normal seeds should be produced. When applied to the heterozygous plants, of which half the ovules carry the lethal factor, one-sixth of the seeds should be defective if the two stocks which are crossed are alike in their lethal factors, but all of the seeds should be normal if the lethal factors of the two parental stocks are unlike. Since on the average two-thirds of the plants of a segregating stock are heterozygous for the lethal factor, the odds against obtaining no heterozygous plants when pollinations are made by this method are as follows : ^0. Ears P jllinated Odds I 2:1 2 8:i 3 26:1 4 8o:i 5 242:1 In order to be reasonably certain of including at least one heterozygous plant in every cross and to allow for failure to secure seed, it was customary to pollinate five ears. The pollen was always collected from five or more plants and it is practically certain that some pollen from heterozygous plants was always included in the mixtures. This method of making the crosses between stocks, rather than between individual plants, has the advantage of being very rapid, a large number of pollinations being made from a single collec- tion of pollen. In several cases in which crosses were made between strains which regularly bear two ears, one of the ears was self-fertilized to determine the composition of the plant, the other was crossed. At harvest, only the crosses between known heterozygotes were retained. This method requires so much additional time and labor that its possible advantages are offset by the fact that only a limited number of crosses can be made in a season. The same objections were found to the method used by Demerec (1923) who, in making crosses between white seedling stocks, pollinated the ears with a mixture of own and foreign pollen and separated the selfed and crossed seeds by the effects of xenia. Only stocks which differ in their endosperm color or texture can be crossed by this method. That the method of crossing at random without determining the composition of the plants used, gives the results which are theo- retically expected, is shown by the following experiment, which incidentally shows that these lethal factors retain their srenetic ENDOSPERM CHARACTERS IN MAIZE 525 identity as do any other Mendelian characters. A stock in which defective seeds had appeared for three successive generations was crossed with another stock originally from the same source, but which had been crossed with an entirely unrelated strain and the defective seeds recovered in the second generation. The pedigree of these two stocks is shown below : >i-i = Stock A 105-9-7 \Cross-9-1 = Stock B Six ears of Stock A were pollinated by a mixture of pollen collected from six plants of B. Four of the six ears proved to be segregating for defective seeds. A count of the normal and defective seeds on these four segregating ears is shown in Table I. Table i. Ratios Resulting when Plants Heterozygous for a Lethal Factor are Pollinated with a Composite Mixture of Pollen from Homozygous and Heterozygous Plants. ar No. Normal Defective 448 449 450 451 184 Il6 ISI 164 22 32 32 43 Total 615 129 Ex. 5 :i Deviation 620 5 124 The agreement of the actual results with the theoretical expec- tation is surprisingly good. Exactly two-thirds of the ears proved to be segregating and exactly one-sixth of the seeds on these ears were defective. RESULTS OF CROSSES It was believed that the most rapid progress in determining the total number of lethals involved in the fourteen stocks, would be made by crossing the first four, which were apparently all differ- ent, with the remaining ten, on the assumption that some of the untested stocks would prove to be carrying the same lethal factors as the first four and these could then be eliminated from further investigation. Thirty-nine crosses were made in 1922 with the astonishing results that the F t seeds were normal in every case. This indi- cated that not one of the ten stocks carried the same genetic factors for defective seeds as the four original strains by which they had been crossed, 526 CONNECTICUT EXPERIMENT STATION BULLETIN 279 The next step was to cross the stocks in all combinations among themselves. This program involved a total of 91 crosses of which 45 had already been made. Since the remainder could not all be made in a single season, it was decided to cross first only the stocks in which the defective seeds showed some' resem- blance. In appearance the fourteen types range from complete defectives in which the caryopsis consists of little more than the flattened, transparent, pericarp to the partial defectives in which the recessive seeds are about half the size of normal seeds. Between these two extremes are all gradations and within each type there is a certain amount of variation. Several representa- tive types of defectives are shown in Fig. 53. The fourteen types were arranged in a series on the basis of the average appearance of the recessive seeds. The plan was to cross each type with the two or three others nearest to it in the series. Although it was realized that resemblance in appearance did not necessarily mean genetic relationship, it seemed only reasonable to suppose that types resembling each other phenotypically were more likely to be alike genetically than those which were wholly different in appearance. Twenty-seven crosses between types close together in the series were made in 1923 and again every cross produced only normal seeds. Four additional crosses were made in 1924 and eight more in 1925; these, with the six preliminary crosses of 1920 and 1921, bring the total number to 84. Two of the crosses were recipro- cals, however, so that the actual number of distinct combinations is only 82. This leaves 9 of the possible 91 crosses which are not yet made. Of all these crosses, only one, a combination of de 5 and de llf gave defective seeds in F a . This shows that de 5 and de lt are genetically identical, and that the results of any crosses made with one of these stocks applies as well to the other. Taking this fact into consideration only seven combinations remain to be made. THE F 2 GENERATION In order to be certain that segregating plants had always been included in making the crosses, and to obtain additional evidence that the two types entering the cross were genetically distinct in each case, F 2 progenies of a large proportion of the crosses have been grown. Since the heterozygous crossed ears could not be distinguished from the homozygous ones, all of the ears of a cross were com- bined by counting off an equal number of seeds from each. As ENDOSPERM CHARACTERS IN MAIZE 5 2 7 one-third of the pollen grains of a composite collection of pollen are expected to carry the lethal factor of the pollen parent, and Fig. 53. — Six types of defective seeds showing the gradation from complete to partial defectives. Normal seeds from same ears are shown at left. one-third of the ovules on a composite collection of ears the lethal factor of the seed parent, then a mixture of seed obtained from 528 CONNECTICUT EXPERIMENT STATION BULLETIN 279 such crosses would be expected to give on the average the fol- lowing F 2 progenies : 4 Ears not segregating. 2 Ears segregating for defective of pollen parent. 2 Ears segregating for defective of seed parent. 1 Ear segregating for both defectives. The di-hybrid ears are expected only once in nine times. How- ever, if the defectives contributed by the two parents show slight phenotypical difference, then the reappearance of two distinct types in F 2 may be regarded as fairly conclusive evidence that heterozygous plants of both parents were included in making the cross and that the parental types are therefore genetically unlike. In order to have better than an even chance of obtaining di- hybrid ears, it was customary to self -pollinate fifteen to twenty plants of each cross. Though this method of making crosses in a wholesale manner and self -pollinating F 2 progenies on the same prodigious scale, may appear to entail unnecessary labor, in reality, it proved to be the most economical procedure. It is true that by growing only the crosses between plants known to be heterozygous, di-hybrid ears would be expected once out of every four trials instead of once in every nine. Self-pollinating vigorous F x plants on a large scale can be done very rapidly, however, and it has been found easier to make the crosses at random and pollinate twice as many F 2 progenies, than to make individual crosses between numbered plants and self-pollinate fewer F 2 progenies. A total of 1089 F 2 progenies of crosses made by this method have been self -pollinated. The ratio of non-segregating to mono- hybrid and di-hybrid ears is given in Table 2. Table 2. Non-segregating, Mono-hybrid, and Di-hybrid Progenies Obtained in F 2 from Crosses Made at Random. Found Ears not segregating 428 Ears segregating one type 552 Ears segregating both types 109 The number of dy-hybrid ears obtained agrees very closely with the theoretical expectation. There is a significant excess of the mono-hybrid ears, however. These are expected only as fre- quently as the non-segregating ears, actually they have appeared in considerable excess. A greater production of pollen by the heterozygous plants in some of the stocks, or the occurrence of heterozygous plants more frequently than two out of three, might account for this. Expected Deviation 484 484 -56 68 121 — 12 ENDOSPERM CHARACTERS IN MAIZE 5 2 9 SUMMARY OF CROSSES The diagram in Fig. 54 gives a picture of the situation with respect to these fourteen stocks.* Squares with vertical cross hatching represent crosses in which the F x seeds were normal. Those with horizontal cross hatching represent crosses in which dej dej de4 des de6 *07 de 8 deg , Seeds Normal Two Types in Fg Dl-hybrid Ears in P 2 Fig. 54.— Diagram showing the crosses which have been made among the fourteen defective seed stocks. The types were arranged in the order of their "defectiveness." the F x seeds were normal and two distinct types of defectives appeared in F 2 though no di-hybrid ears were obtained. Squares with diagonal cross hatching represent crosses in which the F ± * For reasons of economy the detailed data showing the segregation in the individual ears of the fourteen parental stocks and their crosses are not included. Any marked deviations from expectation are noted, however, and are discussed in this and other papers. 53° CONNECTICUT EXPERIMENT STATION BULLETIN 279 seeds were normal and one or more di-hybrid ears were obtained in F 2 , while the single case in which defective seeds appeared in F x is shown by a solid square. As has already been pointed out, the production of only normal seeds in F t is fairly good evidence that the two types entering the cross are genetically distinct providing that three or more ears have been crossed. The reappearance of two distinct types in F 2 is still better evidence, while the occurrence of one or more di-hybrid ears may be safely regarded as conclusive proof. The squares are numbered diagonally from top to bottom and from left to right. Thus 1-14 represent self-pollinations: 15-27 the crosses between types immediately adjacent in the series ; 28-39 crosses between types one degree apart in the series and 40-50 crosses between types two steps apart. In other words the crosses 15-50 represent the most important combinations. It will be noted that all of these 36 crosses have been made. In 27 of these combinations di-hybrid ears have been obtained in F 2 . In five crosses, Nos. 18, t,7> 4°> 43> an d 46, two distinct types of defectives reappeared in F 2 , while three of the combinations, Nos. 21, 33 and 41, have not been tested further than F x . Since de 5 and de lt have proven to be identical, the crosses 24 and 38 represent the same combinations as 65 and 89. This leaves only 7 distinct combinations which have not yet been made. There is, however, some additional evidence in several of the untested combinations which indicates that the defectives involved are not alike. Crosses 88 and 94, representing combinations of de± by de 12 and de 13 respectively, will certainly give di-hybrid segregation in F 2 because de 4 is a "germless" defective while de 12 and de rj both produce embryos. Crosses 75 and 90, representing com- binations of de 6 by de 12 and de 14 respectively, should also give de- hybrid segregation because de e is linked with sugary, as recorded later, while de 12 and de 14: are not. The same is true - of the cross 100 between de x and de 12 . The former is linked with sugary, the latter not. This leaves only two combinations, 59 and 69, about which there is any doubt. The evidence, then, is almost conclusive in indicating that thirteen distinct genetic factors for defective seeds are involved in the four- teen stocks tested. There is, of course, the possiblity that one of the two doubtful combinations will reveal an additional case of two stocks which are genetically identical, even though these com- binations are between defectives which differ decidedly in appear- ance. It must be remembered that two characters may be genetically identical in one main factor and yet differ pheno- typically because of minor modifying factors. This is shown by the cross between de 5 and de 1± which proved these two defective's to be identical although they were far apart in the arbitrary series which had been arranged on the basis of external appearance. ENDOSPERM CHARACTERS IN MAIZE 53 1 These results give some indication of the enormous number of ■distinct defective seed types which probably occur in maize. A sample of fourteen types was taken at random from the grab bag which constitutes the germplasm of maize, and thirteen of these proved to be genetically distinct. The total number of distinct lethal seed factors in the germplasm of this species can only be conjectured, but it probably equals or exceeds the number of dis- tinct varieties of maize which are now grown. THE MORPHOLOGY OF DEFECTIVE SEEDS In order to determine the irregularities in development which cause one-fourth of the seeds on a segregating ear to be defective while the remainder are normal, and to find, if possible, constant differences which distinguish some of the types from others, a histological examination of all fourteen types, in various stages of development, has been made. The material was killed and fixed in Carnoy's solution, a mix- ture of three parts of absolute alcohol to one of glacial acetic acid. Two other fixing agents, Benda's solution and a concen- trated solution of picric acid, were also tried. The former gave excellent results, but was discontinued because of the high cost of osmic acid, its most important constituent. The picric acid solution proved to be very unsatisfactory because of the difficulty of removing all traces of the discoloration from such large sec- tions. All material was imbedded in paraffin, cut in sections of ten microns and stained in Delafield's haematoxylin. Some of the sections were also stained in a dilute solution of iodine and potassium iodide to bring out possible differences in the starch grains. THE CYTOLOGICAL MECHANISM OF ENDOSPERM FORMATION The cytological details of the mechanism leading up to the formation of the endosperm in maize are fairly well established. When Nawaschin, in 1898, made the discovery that the endosperm of Lilium is the product of a sexual fusion entirely apart from that which gives rise to the embryo, three investigators, DeVries (1899), Correns (1899), and Webber (1900), simultaneously and independently reached the conclusion that this mechanism was probably responsible for the phenomenon of xenia in maize, although it was not until 1901 that Guignard furnished the cyto- logical evidence of double fertilization in this species. More recently Weatherwax (1919) and Miller (1919) have independently repeated Guignard's researches and both have given detailed descriptions and illustrations of the entire process lead- ing up to fertilization. In most respects the accounts of these two writers agree very well, though Miller believed that all four 53 2 CONNECTICUT EXPERIMENT STATION BULLETIN 2JO, megaspores functioned while Weather wax observed the disinte- gration of three megaspores, with only one persisting. In an earlier paper, however, Weatherwax (1917) also had made the observation that all four megaspores functioned and his dis- covery that only one persisted was made only after attention was called to the disagreement between his earlier cytological observa- tion and certain well established facts regarding the genetic behavior of the endosperm. When the pollen tube enters the micropyle, two identical sperm are emptied into the embryo sac. One of these fuses with the egg and an embryo is produced ; the other fuses with one of the polar nuclei which lie close together in the embryo sac. Almost immediately the fusing nuclei are joined by the second polar nucleus, this process constituting the ''triple fusion" characteristic of many angiosperms. It is of importance to note, in connection with the possible explanation of some of the forms of non-hered- itary defectives described later, that both Weatherwax and Miller, in repeated observations, never found the two polar nuclei fusing before fertilization of one of them had occurred. The endosperm of angiosperms is unique in that it is the product of a fusion in which the two parents do not contribute equally. Two maternal nuclei, with their assortment of chromosomes bear- ing the hereditary factors, combine with one male nucleus. The female parent, therefore, contributes two sets of chromosomes and a double dose of the assortment of hereditary factors while the pollen parent contributes only one set of chromosomes and a single dose of factors. This peculiar situation enabled Hayes and East (191 5) to demonstrate the fallacy of the "presence and absence" conception of dominant and recessive factors. These writers found that in crosses between flint and flour varieties the inheritance was always apparently maternal, a double dose of the maternal condition being always dominant to a single dose of the alternative condition. In other words, two "absences" were dominant to a single "presence." DEVELOPMENT AFTER FERTILIZATION The general features of the development of the endosperm and embryo in the cereals are fairly well established. Details of development which distinguish maize from other grasses are gradually being added as special phases are investigated. True (1893) and Poindexter (1903) studied the general development of the caryopsis. Reed (1904) has investigated the secreting cells of the scutellum of maize. Sargent and Robertson (1905) have made a very thorough study of the anatomy of the scutellum. The aleurone layer has been the subject of cytological studies especially by Liidtke (1890), Haberlandt (1890) and Groom ENDOSPERM CHARACTERS IN MAIZE 533 (1893). The successive stages in the development of the embryo have been described and figured by Weather wax (1920). The general development of the caryopsis in maize is briefly as follows : The endosperm fusion nucleus begins division almost at once and the rapidly growing endosperm soon' fills the embryo sac. The embryo nucleus does not divide immediately after fusion and the first division usually does not occur until after the nuclei of the endosperm number 20 or more. (Miller, 1919.) The nucellus soon begins to disintegrate and is partly absorbed, the remainder being compressed into a thin integument between the pericarp and endosperm. By the time that the early milk stage is reached, the endosperm occupies the entire space within the pericarp and exerts consider- able pressure. (See Plate XXI, Fig. 1.) ,The embryo on the other hand is still rather rudimentary. Fom this point on, the embryo develops more rapidly than the endosperm, the latter undergoing only slight additional increase in size while the former grows rapidly, pushing further and further into the endosperm tissue. Small starch grains are found in the outer cells of the endo- sperm in the early milk stage, which in the writer's material occurred at about 15-20 days after pollination. By the time that the late milk stage is reached at about 25 days to four weeks after pollination, the cells in the upper part of the endosperm are com- pletely packed with starch grains, although those in the lower part are still relatively clear. In material fixed after this stage, the contents of the cells drop out in sectioning and in most cases no histological studies of further changes have been made. The aleurone layer is present in most specimens in the early milk stage, though no color can be detected in this layer in un- sectioned material at this period. DEVELOPMENT OF DEFECTIVE SEEDS In preliminary experiments of 1922, seeds were fixed at inter- vals of 1, 2, 4, 7, 10, 20 and 30 days after pollination. The •defective seeds could not be distinguished from the normal seeds in the early stages and it was necessary to examine sections from a large number of seeds in order to be certain that defectives were included. In 1923 no material was collected until the normals and defectives on segregating ears could be distinguished from each other. This point was usually reached in the blister or early milk stage, or at about fifteen to twenty days after pollination in most of the types. Some of the partial defectives, however, could not be distinguished from normal seeds on the same ears until later. Defectives and normal seeds were always removed in pairs for comparison and the seeds were usually taken from the middle of 534 CONNECTICUT EXPERIMENT STATION BULLETIN 279' the ear to avoid differences due to unequal development at butts and tips of the ears. Before dropping the seeds into the fixing agent, as much tissue as possible on either side of the embryo was removed to permit a more rapid penetration of the solution. REGULAR DEVELOPMENT IN EARLY STAGES The inheritance of all of the defective seed types as simple Mendelian recessives pointed to a regular functioning of the fertil- ization mechanism and the fusion of male and female gametes. The specimens collected in the early stages bore out this assump- tion. All of the types of which the early stages after pollination were studied showed the normal beginning of endosperm and embryo formation and no marked differences between normal and defective seeds were noted. In specimens fixed after the blister or early milk stage, how- ever, the differences between normal and defective seeds were very striking. The various types of defectives differed from each other, however, only in general development and, with the excep- tion of one type, no specific morphological differences, which always distinguish one type from another, have been found. A general account of the development of these various types of aborted seeds follows. THE PERICARP No matter how defective the endosperm and embryo may be, the pericarp usually attains a normal or almost normal develop- ment. This affords a striking illustration of the complete inde- pendence of these two tissues which, though borne on the same plant, represent distinct sporophyte generations. The pericarp is maternal in its origin and with the exception of the stimulus from pollination which sets off its development, it does not appear to be influenced by the hereditary composition of the new sporophyte which it encloses. In normal seeds, the pericarp is constantly distended by the pres- sure of the growing endosperm. In the defectives, there is always a space between these tissues. In early stages this space is filled, partly with nucellar tissue and partly with a clear watery solution. In later stages the walls of the ovule are pushed together by the pressure of the normal seeds on either side and the space dis- appears as shown in Plate XXI, which shows three successive stages in the development of the de 14 type of defective seeds. THE NUCELLUS In normal seeds the nucellus rapidly disintegrates following fertilization. Part of it is probably absorbed while the remainder is compressed into a thin integument between the endosperm and ENDOSPERM CHARACTERS IN MAIZE 535 pericarp and soon loses its identity as a separate tissue. (Plate XXL) The nucellus, like the pericarp, is of maternal origin and is not influenced by the sporophyte with which it is in constant contact, except that in the absence of a vigorous and rapidly grow- ing endosperm it is permitted to persist as a distinct tissue for longer periods than it does in normal seeds. THE ENDOSPERM The endosperm of the defective seeds differs from that of normal seeds in degree rather than in kind. In no case does it attain the size of the normal endosperm, but in details of develop- ment no marked differences are noted. With regard to the development of the endosperm, the defectives of these fourteen stocks form a continuous series ranging from the de lz and de xi Fig. 55. — Three successive stages of development of defective seeds of dew. No aleurone layer is found at any stage. (Figures 55-58 represent a magnification of approximately 6.5 diameters.) types in which only a small mass fo tissue is present, to the de x and de 5 types, in which the endosperm is fully half size. With regard to the rate at which the endosperm increases in size from week to week the same gradation is found. In the de 14: stock, for example, the defective seeds at the early stage have only a small mass of endosperm tissue. At the late milk stage no increase in size is noted, and at the early dough stage the develop- ment of the endosperm still remains practically the same, as shown in Plate XXI. In partial defectives, such as those of the de 5 stock, for example, the endosperm gradually increases in size from week to week though its rate of growth is considerably retarded as compared to the endosperm of normal seeds and its 536 CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 final size is considerably less than that of a normal endosperm. (Plate XXII.) The order of the fourteen stocks of defective seeds on the basis of their endosperm development as shown by these histological studies is not exactly the same as the arrangement made on the basis of external appearance alone. In general, however, the two series agree very well. STARCH GRAIN FORMATION The same general differences noted in size of endosperm are also found with regard to the formation of the starch grains. In the defectives at the lower end of the series no starch grains were found at any stage examined. Types further up in the series produce small starch grains in the periphery of the endosperm in later stages, while in the partial defectives, starch grains are found Fig. 56. — Defective seeds of den at early milk stage ; dei, late milk ; den, late milk. in every stage examined, and in later stages the cells of the endo- sperm are packed with starch grains which are apparently normal in size and structure. No characteristic differences in the structure of starch grains were noted with the exception of the defective seeds of de±, In the recessive seeds of this strain the starch grains have the appear- ance of undergoing hydrolosis many of them being completely broken up. THE ALEURONE LAYER The defectives at the foot of the series produce no aleurone layer at any stage examined and this might be expected since an aleurone layer is usually formed only in later stages of normal development. Beginning with de lt , however, an aleurone layer ENDOSPERM CHARACTERS IN MAIZE 537 is found in the later stages examined. In some cases this layer extends only part way around the endosperm, in others it com- pletely surrounds this structure except, of course, at the base where no aleurone layer is found even in normal seeds. Draw- ings in which the aleurone layer has been enlarged out of propor- tion to the other structures are shown in figures 55-58. In defectives higher in the series than de 10 , an aleurone layer is found in almost all stages examined. THE EMBRYO With the exception of de± all of the types were found to con- tain embryos. This type is similar to the "germless" seeds reported by Demerec (1923) which at maturity contain no embryo. Fig. 57. — Defective seeds ,of den de- 3 and des at approxi- mately the same stage, showing difference in degree of develop- ment of embryo, endosperm, and aleurone layer. No paraffin sections of this defective were secured because the recessive seeds are distinguishable from the normals only shortly before maturity, at which time the seeds are too corneous to be sectioned by the paraffin method. Free-hand dissection in early stages of seeds from ears later proven to be segregating for this character, showed that an embryo was present in all the seeds. Apparently the embryo is digested and absorbed later. The cavity which remains on the germinal side of the seed is partly filled with a hard brittle mass of substance with no definite structure. As already mentioned, the endosperm of this type also shows evidence of digestion. 538 CONNECTICUT EXPERIMENT STATION BULLETIN 279 In general, there is a marked correlation between the develop- ment of the embryo and that of the endosperm. The complete defectives in which the endosperm remains practically stationary have very rudimentary, undifferentiated embryos which show no further development after the early milk stage. (Plate XXL) Defectives higher in the series show some increase in size from week to week, but no clear differentiation of various parts of the embryo is apparent though a "growing point" is indicated by a greater concentration of nuclei in certain regions. Beginning with de s , a scutellum, coleptile and several rudimentary leaves are distinguished, while in some of the partial defectives the develop- ment of the embryo is fairly normal. The embrvos of the defective seeds often show considerable dis- Fig. 58. — Three successive stages of den. No aleurone layer is found in the early stage but a partial or com- plete layer is formed later. Note the distortion of the embryo in the final stage. tortion in shape. In later stages this may be attributed to the pressure exerted by the normal seeds on either side as shown in Plate XXIII, but in some cases a distortion is noted even when the defective seeds have not been under pressure. The embryo tends to be short and "blocky" as shown in Plate XXII, which may be compared to Plate XXIV which shows the successive stages in the development of the normal embryo. GENERAL ASPECTS Aside from the interesting demonstration that the pericarp, once its development is stimulated by pollination, proceeds quite inde- pendently of the tissues which it encloses, the most outstanding ENDOSPERM CHARACTERS IN MAIZE 539 feature of the morphology of the defective seeds is the marked correlation between the development of the endosperm and embryo. An external examination of some of the defective seeds had led to the assumption that these, in many cases, contained no tissue whatever. It was surprising, therefore, to find distinct, though rudimentary, endosperm and embryo at some stage in every type examined. It was even more surprising to find that the delete- rious influence of the lethal factors, which they carried, affected both of these structures to almost the same degree. It might be supposed that some factors would affect specifically the embryo, permitting the endosperm to continue in a more or less normal fashion. Others might be expected to inhibit particularly the endosperm. This has not been found to be the case. Even in the de i type, which lacks an embryo at maturity, the development of both structures proceeds normally in the early stages and the influence which later destroys the embryo also has a very marked effect upon the endosperm. Although there is still some difference of opinion among morph- ologists as to the real nature of the endosperm in angiosperms, from the standpoint of the geneticist it has always been considered a sporophyte, differing from its near relative the embryo, in struc- ture, in capacity for continued development, and in ability to repro- duce. In the expression of the hereditary factors which they receive, the endosperm and embryo are fundamentally alike and eventually the morphologists, too, may come to regard the endo- sperm of angiosperms as a modified sporophyte. Table 3. Average Weight of Seeds from Ears Harvested at Successive Stages of Maturity. Days after r — Av. wt. in mg- "V Relative Pollination 1 2 3 Av. Development 14 5 5 5 5 1-5 21 25 27 37 30 9.1 28 82 86 71 80 24.2 35 in 97 126 III 33-6 41 159 166 189 171 51.8 51 234 222 226 227 68.8 75 374 319 306 330 1 00.0 PHYSIOLOGY OF DEFECTIVE SEEDS Histological examinations of the normal and defective seeds were confined largely to a rather brief period beginning with the blister stage and ending when the seeds had reached the dough stage. In order to determine the differences between normal and defective seeds in later stages of development, a comparison of their rate of growth, final dry weight, percentage of germination 54° CONNECTICUT EXPERIMENT STATION BULLETIN 2/0, and effect upon the sporophyte and gametophyte in the haploid condition was made. BATE OF GROWTH, FINAL WEIGHT AND GERMINATION OF NORMAL SEEDS The rate of growth of normal seeds of maize was determined as follows : A large number of plants of an F 1 hybrid of homozy- gous inbred strains were grown under uniform soil conditions. These plants, all being genetically alike, came into silk and were pollinated at about the same time. At two weeks after pollina- tion, and at intervals thereafter until maturity, three ears were taken at random from these plants. The ears were reduced to an air dry condition after which the kernels were removed, counted, weighed "en masse" and the average weight at each stage determined by simple division. It is quite possible that in ears harvested at various stages of maturity in this way there is a transfer of materials from the cob to the kernels during the drying out process. Such an exchange, if it occurs at all, would probably be proportionate for the various stages and is not regarded as a serious source of error. Table 4. Percentage Germination of Corn Seeds Harvested at Successive Stages of Maturity. Days After Pollination Relative Development Percent Germination 14 1-5 O 21 9.1 28 28 24.2 56 35 33-6 72 41 51.8 92 51 68.8 96 75 1 00.0 98 The average weights in milligrams and the relative weights, as compared to the final weight at maturity, of these seeds harvested at various stages of development, are given in Table 3. Figures 1 and 2 of Plate XXV show respectively a representative ear at each stage and 50 seeds from each ear. The 50 seeds representing each stage of development were planted in sand in the greenhouse with the results shown by the photograph in Plate XXV, Figure 3. Some germination occurred in every lot except the first which was harvested at fourteen days after pollination. The percentage of germination of each lot and the relative weight of the seeds from which it was grown are given in Table 4. The normal seeds of maize, like those of barley (Harlan and Pope, 1922), are apparently capable of some germination at all stages of development but the very earliest. ENDOSPERM CHARACTERS IN MAIZE 541 RATE OF GROWTH, FINAL WEIGHT, AND GERMINATION OF DEFECTIVE SEEDS The rate of growth of defective seeds as compared to normal seeds from the same ears was determined for one type, de 10 . Ears from ah F x hybrid of this stock crossed with an unrelated inbred strain were harvested when the segregation on the ears first became apparent and at intervals thereafter until maturity. When all the ears had been reduced to dryness, the kernels were shelled off, the normal and defective seeds separated, counted and weighed, and the average weight of each class determined. The growth curves of the normal and defective seeds from ears segre- gating for de 10 are shown in Figure 59. Although the rate of 300 200 100 » De de 20 30 40 DAYS AFTER POLLINATION 50 60 Fig. 59. — Growth curves of normal and defective seeds from the same segregating ears of the dew stock. increase in dry weight is very low for the defective seeds, the two curves appear to be of the same general type. In determining the relative development at maturity of the four- teen types, dry weight was used as a criterion. Obviously, the relative development cannot be determined with any high degree of accuracy because it is influenced to some extent by the environ- ment and considerably by the hereditary constitution of the stocks, certain of the types showing more development in crosses than in inbred strains. In order to make the determinations as nearly comparable as possible, the figures on relative development have, with one exception, been taken from crosses which were all iden- tical with respect to one of the parents, and all grown the same season. In this way the defectives are compared under condi- tions in which hereditary and environmental differences are reduced to a minimum. 54 2 CONNECTICUT EXPERIMENT STATION BULLETIN 279 The relative development of defective seeds at maturity of each of the fourteen stocks is shown in Table 5. Table 5. Relative Development in Defective Seeds of Fourteen Stocks as Compared to Normal Seeds on Same Ears. • Stock No. Ears Relative Devel dei 6 504 de s 3 58.8 de 3 3 29-9 dei 3 37-3 des 2 18.0 des 3 34-0 der 3 18.8 des 3 15.0 dea 3 5-9 deio 2 13.7 den 3 7-3 dei2 3 40 den 2 4-5 den 3 2.4 The types show a range in development from 2.4 per cent in the de 14: type to 58.8 in the de 2 type. It is noted that the order of these types based on their relative development is not the same as that arranged on the basis of external appearance alone. This is partly due to the fact that the original arrangement was based on inbred strains while the relative development has been deter- mined from hybrids. Even when all are crossed with the same unrelated stock, some of the types, particularly 2, 7, and 10, show a greatly increased development after crossing. In some crosses the de 2 type reappears in the second generation so altered in appearance that it is scarcely recognized. The recessive seeds are almost equal to normal seeds in size and weight, and differ from the latter only in a paler color and a mottled appearance. Whether this increase in development which follows crossing is due to the greater vigor of the plants on which they are borne, or to the action of modifying factors contributed by one or both parents, is not known. That some of the defectives are influ- enced by modifying factors, is almost certain. The de 5 and de lt types, for example, are genetically alike, yet" differed in appearance, not only in the original stocks, but in hybrids in which both were crossed to the same unrelated stocks. Judging from the dry weight alone, the fourteen types of defectives correspond to various stages in the development of the normal seed. This is shown diagramatically in Figure 73 (Part III) in which the relative development of each type is represented by a point on the normal growth curve of maize seeds. At first glance, the defective seeds are comparable to normal seeds which have had their development arrested at an early stage, ENDOSPERM CHARACTERS IN MAIZE 543 as has occurred in the ears harvested at successive stages of development, shown in Plate XXV. When these various defec- tive seeds are tested for germination, however, it is found that they are by no means equal to normal seeds of the same relative development. This is shown by the figures in Table 6, in which the germination of each type of defective is compared to the theoretical germination of immature normal seeds of the same relative development. The theoretical germination of the normal seeds was determined by interpolation of the figures in Table 4. It is noted that the defective seeds in every case show a much lower germination than would be expected from normal seeds of Fig. 60. — Seedlings from normal and defective seeds of dez. Only rarely do the seedlings from the recessive seeds survive more than a few weeks. similar development. In fact, the types below de 7 showed no germination whatever in this test with the exception of one seed from a total of 131 seeds of the de lx type. Defective seeds of de s have also shown slight germination in some cases, but in the ears used in this particular test no germination occurred. Not only are the defective seeds less able to germinate . than normal seeds of the same development, but those which do succeed in sprouting produce very weak seedlings which are lacking in vigor and soon die. Seedlings from normal and defective seeds of the de 2 stocks are shown in Figure 60. By planting large numbers of seeds in the greenhouse and transplanting the most 544 CONNECTICUT EXPERIMENT STATION BULLETIN 279 vigorous of the seedlings to the field, it has been possible to obtain homozygous plants of stocks i, 2, 3 and 6. The behavior of the plants grown from homozygous defectives is in striking contrast to those which result when immature normal seed is grown. In appearance the two lots of seed are almost identical, both being badly shrivelled and aborted. Both types give a low germination and the seedlings are, in both cases, very weak and spindling. The plants which are genetically normal, however, soon recover from the handicap of the poor food supply which the aborted seed affords, and by flowering time are fully normal in stature, and yield practically as much grain as plants which grow from well filled, mature seed. The homozygous defectives, on the other hand, remain very weak and spindling throughout the season, produce only a small amount of pollen, and rarely any ears. At least one pure defective ear has been obtained, however, from each of the four stocks mentioned above, during the past five seasons. Figure 61 shows a normal and pure defective ear from the de 3 stock. Table 6. The Percentage of Germination in Defective Seeds of Fourteen Stocks Compared to the Theoretical Germination of Normal Seeds of the Same Relative Development. Stock Defectives Theoretical Normal d& 45-6 91.0 de* 44.9 94-0 de& 54-0 66.5 de* 3-5 75-5 de& 1 1.2 47-5 dee 11.8 72.5 dd 19.6 49.0 de& 42.0 det 17.0 deio 39-0 den 0.8 22.0 dei2 10.0 de-a 12.5 den 3-5 The fact that the germination of the defective seeds is con- siderably lower than that of immature normal seeds of the same relative development, that the seedlings of all types are extremely weak, and that even the most vigorous ones which are able to survive make a very feeble growth throughout the season, indi- cates that these lethal and semi-lethal factors do more than merely arrest the seeds at a certain stage; apparently these factors in a homozygous condition have a deleterious influence on the sporo- phyte at any stage in which they are given an opportunity for expression. In most of the types the deleterious influence is so ENDOSPERM CHARACTERS IN MAIZE 545 marked that the career' of the new sporophyte is brought to an end in the seed stage and the lethal factors have no opportunity for further damage past this period. Fig. 6i. — Left; ear segregating de 3 . Center; self-pollinated ear from a plant homozygous for de 3 . Right ; open-pollinated ear from homozygous plant. THE INFLUENCE OF LETHAL FACTORS IN HETEROZYGOUS CONDITION Since the effect of the lethals is so marked when they are in homozygous condition, it might be questioned whether they have 546 CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 not a similar unfavorable influence, though in a smaller degree, in the heterozygous condition. The only evidence bearing on this question is that secured from a comparison of the height of segregating and non-segregating plants among the 1089 self -pollinated F 1 plants which were grown in 1923 and 1924. In 1923 the height to base of tassel on 398 F 1 plants was measured. The average heights of the plants which later proved to be free of these lethal factors was 77.9 inches as compared to 77.2 for those which segregated for a single type of defective and 73.7 for those which segregated for two types. In 1924, 659 F x plants were measured. The non-segregating plants averaged 73.9 inches in height as compared to 71.9 for the other two groups. Though the difference in height is not significant either year, the fact that the two groups of segregating plants are somewhat lower both years than the non-segregating plants may be of some significance. Table 7 gives the frequency distribution of the segregating and non-segregating plants for these two years. Table 7. Frequency Distribution in Height of Fi Plants from Crosses of Defective Seed Stocks. , Mid-Class Values in Inches s Type of Plant 42 47 52 57 62 67 72 77 82 87 92 97 102 Total Mean Segregating 3:1 3 7 9 22 51 72 86 117 98 52 12 2 .. 531 . 73.85 ± .20 Segregating 9:7 .. .. 1 6 9 15 23 20 17 9 100 73.30 ± .5; Total segregating plants 3 7 10 28 60 87 109 137 115 61 12 2 .. 631 73.80 ±.3f Not segregating .. 5 8 21 29 45 78 89 69 54 15 2 1 416 74.90 ± .3? Difference in favor of non-segregating plants i.io± 4<. Although no data on the yield of segregating and non-segre- gating plants can be secured because the ears are artificially polli- nated and full ears are seldom obtained, it has often been noted that the segregating ears have a tendency to be smaller than those which are uniformly normal. These results and observations cannot be regarded as more than mere indications that the lethals do have an effect in the heterozy- gous condition ; they certainly do not afford conclusive evidence in favor of such an assumption. The effect, if any, of the lethals when in combination with their dominant allelomorphs is so slight that it could be accurately detected only by a delicate test. Such a test is now being made with the inbred strains which have mutated to defective tseeds, and which are presumably homozygous for all factors with the exception of a single pair involving the defective factor and its dominant allelomorph. EFFECT UPON THE GAMETOPHYTE The gametophyte generation is ordinarily assumed to be inde- pendent of the influences of the genetic factors distributed on the chromosomes which it carries. Recent evidence (Jones, 1924^1 ENDOSPERM CHARACTERS IN MAIZE 547 (Mangelsdorf and Jones, 1926) indicates, however, that there are exceptions to the rule and that in some cases, the gametophytes from the same plant are not all alike in their ability to reach the micropyle and accomplish fertilization. This brings up the ques- tion of the effect of the lethal factors on the gametophyte genera- tion. Since these factors have such a marked deleterious influence on the sporophyte at all stages, might they not have some degree of expression in the gametophyte generation as weil ? It has already been noted that there is a deficiency of recessive seeds on segregating ears of a number of these types and such a condition might be explained by slower rate of growth of the pollen tubes carrying the lethal factors. In order to answer this question, a large number of segregating ears of the de 3 stock which regularly shows a deficiency of the recessives were divided arbitrarily into top and bottom halves and the normal and defective seeds in each half were counted separately. If there were a constant difference in the rate of growth between pollen tubes carrying the lethal factors and those carrying the dominant allelomorphs, then the greater difference which the pollen tubes were forced to travel in reaching the ovules at the base of the ears would act against those carrying the lethal factors and cause a greater deficiency of the defective seeds in the lower halves of the ears. The results of making counts in the upper and lower half of fifteen ears segregating for de 3 are shown in Table 8. Table 8. Normal and Defective Seeds on Top and Bottom Halves of Ears Segregating dez. 1 Top Half , , — Bottom Half -, ,— -Percent D efective- Ear No. Normal Defective Normal Defective Top Bottom IO I65 60 204 50 26.7 197 29 I46 48 158 48 24.7 23-3 30 154 44 145 39 22.2 21.2 33 89 24 78 36 21.2 31-6 45 IOX) 57 207 54 23.I 20.7 46 210 63 200 65 23.1 24-5 5i 156 44 178 36 22.0 16.8 55 155 45 185 55 22.5 22.9 56 138 40 172 60 22.5 25.9 58 145 56 171 50 27.9 22.6 120 119 32 144 48 21.2 25.0 135 119 32 79 3i 21.2 28.2 139 145 47 96 3i 24.5 24.4 148 125 4i 145 42 24.7 22.5 158 115 46 112 41 28.6 26.8 Total 2171 679 2274 686 23.8 23.2 Ex 3:1 2137-5 712.5 2220 740 Deviations 33-5 46 P. E. 15.6 15-9 Dev./P. E. 2.2 2.9 54§ CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 Although the percentage of recessives in the bottom halves of these ears is lower than that in the top halves, the difference is not significant. When the ears are examined individually it is noted that the percentage of recessives is lower in the top half almost as frequently as in the bottom half. From these results it may be concluded that the lethal factor? have very little, if any, effect upon the rate of pollen tube growth. Even very slight differences in the ability of the pollen tubes to reach the micropyle would cause marked distortions in the ratios and should result in different proportions of defective seeds in the upper and lower halves of the inflorescences. LINKAGE RELATIONS Although no special study of the linkage relations of these char- acters has yet been made, it was to be expected that some indica- tions of linkage would be encountered as by-products of the other investigations. This proved to be the case. The first linkage to appear was that between de 2 and a factor for albino seedlings. This linkage has been briefly mentioned in a previous paper. (Mangelsdorf, 1922.) Table 9. Seedlings from Normal and Defective Seeds Showing Linkage Between de-i and w. r -Norrr al \ < _ Defect ve N r No. Green White Green White 2 126 15 30 20 4 158 19 23 19 5 79 19 O 19 7 130 20 I 39 8 119 I 28 32 Total 612 74 82 129 As already noted, the de 2 character is peculiar in that it is greatly modified in certain crosses. In a cross between the de 1 and de. 2 stocks the recessive seeds of the latter reappeared, so altered in appearance, and so well developed, that it was considered feasible to plant a row of them in the field in order to secure a stock homozygous for this factor. A week later when the seedlings had emerged, this row was easily the most conspicuovis one in the field. With one exception all of the seedlings were albinos, and this solid row of pure white seedlings in striking contrast to the normal green plants on either side furnished a most striking demonstration of linkage. The remaining ears of this cross were tested in the greenhouse and the results are given in Table 9. Figure 62 shows the seed- lings grown from the normal and defective seeds from one of these ears. ENDOSPERM CHARACTERS IN MAIZE 549 The amount of crossing over between de 2 aire, w is 18 per cent as determined from the normal seeds and 21.5 per cent as deter- mined from the recessive seeds. These values are believed to be somewhat high because of pos- sible inaccuracies in the classification of normal and defective seeds on these ears. A large number of F 3 progenies were there- fore grown. In these the development of the defective seed was reduced, and the segregation so well defined that, it is believed, Fig. 62. — Seedlings from normal and defective seeds of dei show- ing linkage between defective seeds and a factor for white seedlings. a fairly accurate separation was made. The results of planting the normal and defective seeds from 17 F 3 ears are given in Table 10. The amount of crossing over as determined from the normal seeds is 11.7; from the defective seeds, 10.5. As nearly as can be determined from the records the de 2 stock is the same one in which the w 2 factor for white seedlings was found. (Lindstrom, 1924.) The w 2 factor is known to belong to the second linkage group in maize and it is probable, there- fore, that the de 2 factor is also a member of this group although further tests are necessary to substantiate such an assumption. Some additional evidence for it exists in the fact that Lindstrom (1923) has also found a case of close linkage between w 2 and a defective seed which answers the description of the de type. 55° CONNECTICUT EXPERIMENT STATION BULLETIN 279 LINKAGE BETWEEN SU AND de FACTORS The relation between the factor for sugary endosperm, a repre- sentative of the third linkage group, and the fourteen factors for defective seeds, may be determined from the crosses between de. and the thirteen remaining types. The de 7 stock, originally starchy, had been changed over to sugary before any crosses were made. Table 10. Seedlings from Normal and Defective Seeds of F 3 Progenies Showing Linkage Between de» and w. -Normal . , Defective- Green White Green White 84 3 5 28 89 5 16 22 79 17 o 12 83 9 2 32 91 1 27 32 54 7 1 16 58 2 1 2 42 o o 6 8302 63 3 1 25 21 232 84 8 1 26 81 12 12 33 51 004 74 9 1 11 68 4 o 15 59 2 2 15 1089 87 72 283 Since the defective seeds, with the exception of two or three types, cannot be accurately classified with regard to their endo- sperm texture, their linkage relations with sugary must be determined from the normal seeds alone. Linkage of sugary endosperm with any of the lethal factors would be indicated by a distortion of the normal 3 starchy : 1 sugary ratio. An excess of sugary seeds is expected when the sugary and defective seed factors enter the cross from opposite parents and the recessive factor of one is linked with the dominant allelomorph of the other. With complete linkage between Sn and de, 2>ZV3 P er cent sugary seeds in the normal class are expected. Thus the range in cross- ing over from 50 per cent to o, corresponds to a range in per- centage of sugary seeds of 25 to ZZ T A- This is shown diagram- matically in Figure 63. F 2 progenies of crosses between sugary endosperm and all of the defective seed types have been grown. The results of sepa- rating and counting the starchy and sugary seeds in the normal class, on ears from all of these crosses, are shown in Table 11. ENDOSPERM CHARACTERS IN MAIZE 551 PERCENTAGE OF CROSSING-OVER Fig. 63. — Diagram showing the theoretical distortion of the starchy : sugary ratio among the normal seeds when a defective seed factor is linked with the normal allelomorph of sugary. With complete linkage ZZVz per cent sugary seeds are expected in the normal class. 55 2 CONNECTICUT EXPERIMENT STATION BULLETIN 279 Table ii. Segregation of Starchy and Sugary among the Normal Seeds of Ears also Segregating for Defectives. Cross Ear No. Starchy Sugary % Sugary deixsu 947 71 36 95i 213 89 953 219 81 956 98 32 Total 601 238 28.4 dei x su 1024 217 91 1034 131 42 1035 173 52 Total 521 185 26.2 de 3 x su 1013 255 1016 352 1017 223 Total 830 287 25.7 dd x su 1045 185 75 1047 150 37 1049 244 87 Total 579 199 25.6 de 5 x su 1056 334 97 1058 330 115 1068 421 128 Total 1085 340 23.0 den xsu 1 107 66 37 1 108 138 55 Total 204 92 3 1. 1 de-txsu 1072 187 58 1073 291 114 1084 252 90 Total 730 262 26.4 deixsu 2432 287 in 2433 252 75 Total 539 186 25.7 dCn X SU IO96 200 62 1098 177 50 " 1 1 02 242 72 Total 619 184 22.9 ENDOSPERM CHARACTERS IN MAIZE 553 Table II (cont'd). Segregation of Starchy and Sugary among the Normal Seeds of Ears also Segregating for Defectives. Cross Ear No. Starchy Sugary % Sugary dew x su 996 278 88 24.O den x su 969 970 971 295 137 22$ 104 43 54 Total 660 201 23-3 dei2 x su 937 938 940 306 298 170 95 105 45 Total 774 245 24.0 devi x su 1075 1079 222 135 70 5i Total 357 121 25.3 den x su « 981 988 992 162 345 231 55 in 83 Total 738 249 25.2 With the exception of the cross involving de 7 , the lethal factors and the factor for sugary endosperm have been contributed by opposite parents and linkage would be indicated by an excess of sugary seeds. In the cross of de,, the de and su factors were introduced by the same parent and linkage would be indicated by a deficiency of sugary seeds. . The only crosses in which there is a noticeable distortion ot the starchy-sugary ratio are those involving the de x and de 6 factors. In the former 28.4 per cent of the normal seeds are sugary. This is an excess of sugary seeds of 3.4 times the prob- able error and indicates linkage with crossing over of 38.5 per cent. Fortunately the defective seeds of this type cap also be readily classified as to their endosperm texture and this is done in Table 12. _ ■ . Among the defective seeds there is a marked deficiency ot sugary individuals, only 20.6 per cent being found as compared to 28.4 per cent in the normal class. The amount of crossing over as determined from the defective seeds is 39 per cent. This agrees very closely with the percentage as determined from the normal seeds. The evidence is fairly good, therefore, that the de 1 and su factors are linked. 554 CONNECTICUT EXPERIMENT STATION BULLETIN 279 Table 12. Segregation of Starchy and Sugary among the Defective Seeds from Ears of a Cross dei x su r Ear No. Starchy 947 42 95i 75 953 85 Sugary % Sugary 10 27 18 9 19.2 26.5 17-5 16.7 64 78 20.9 25.0 956 45 Total 247 Ex. 3:1 233 Deviation 14 — 14 4.4 Probable Error: 5.15 In the cross of de Q x su, 31.1 per cent of the normal seeds were sugary. This is an excess of 3.6 times the probable error and indicates linkage with crossing over of 26 per cent. Unfortu- nately the defectives on these ears could not be classified with regard to their endosperm texture and it is not so certain that linkage between de 6 and su exists, although it is strongly indicated. An excess of sugary seeds of 3.6 times the probable error would be expected as a chance deviation only once in about 65 trials. The cross between the de x and de 6 factors shows independent inheritance of these two characters as is shown in Table 13. This would be expected even though both are linked with the su factor providing that the loci of the two lethals were on opposite sides of the su locus. Table 13. Segregation in F 2 of a Cross between dd and de& Showing Independent Inheritance. Ear No. Normal Defective 166 168 175 178 124 117 l6l 195 103 83 142 131 Total Ex. 9:7 Deviation 597 594 3 459 462 —3 LINKAGE OF DEFECTIVES WITH EACH OTHER In most varieties of maize there are ten pairs of chromosomes. (Kuwada 191 5, Kiesselbach and Petersen 1925.) Therefore, in crossing thirteen different factors in all combinations some cases of linkage are almost certain to occur. The difficulty lies in their detection. With independent inheritance two defectives when crossed give a 9 7 ratio in F 2 . With complete linkage these two defectives should give a 1 :i ratio. Thus the entire rangfe of cross- ENDOSPERM CHARACTERS IN MAIZE 555 50 49 a 48 P An fl 47 8 o % 46 I 45 44 43.75 y»1 10 20 30 40 50 PERCENTAGE OP CROSSING OVER Fig. 64. — Diagram showing how linkage between two defective seed factors would distort the normal 9:7 di-hybrid ratio. If each defective is completely linked with the dominant allelomorph of the other, a 1 :i ratio is expected. ing over from 50 per cent to o corresponds to a range of 43.75 to 50 in the percentage of defectives. This is shown diagrammatic- 556 CONNECTICUT EXPERIMENT STATION BULLETIN 279 ally in Figure 64. Since deviations of two and three per cent are expected to occur fairly frequently by chance alone, the difficulty of detecting any but the closest linkages is at once apparent. However, the fact that defectives often show a deficiency and very 100 2 80 a m & o p^ o s u 8 « 44.4 PERCENTAGE OF CROSSING OVER Fig. 65. — Diagram showing how linkage between two lethal factors brought in from opposite parents causes an increase in the proportion of heterozygotes. rarely an excess, makes it necessary to regard any di-hybrid ears which produce more than 43.75 per cent defectives as possible cases of linkage. Further evidence must then be obtained by growing additional di-hybrid ears or by examining F 3 progenies. If the high percentage of defectives in F 2 is due to linkage, then ENDOSPERM CHARACTERS IN MAIZE 55 7 the F 3 should produce an excess of heterozygous plants as well as an excess of recessive seeds on a majority of the di-hybrid ears. In other words, when two defectives, whose factors occupy loci on homologous chromosomes, are brought together, a condi- tion of balanced lethals is automatically set up. With complete linkage these two lethals, when once brought together, can never be separated and only di-hybrid ears all of which give I :i ratios will be produced thereafter. The diagram in Figure 65 shows how, with such a condition of balanced lethals, the proportion of double heterozygotes in F 3 is expected to increase with the intensity of the linkage. Balanced lethals were first suggested by Renner (1916) as a possible explanation of some of the peculiar results from the breeding experiments with Oenothera. De Vries (1916) adopted the explanation to account for the production of twin hybrids in crosses of this species, but he failed to appreciate the full signifi- cance of the effects of lethals in a balanced condition. It remained for Muller (1918) in his classic contribution on the inheritance of the beaded wing character in Drosophila to show the bearing of balanced lethals on constant hybridity, the sporadic appearance of certain "mutants" due to crossing-over, and the production of twin hybrids. Of the 59 crosses shown in Figure 54, in which di-hybrid ears have been obtained, those which have given marked excesses of defective seeds are shown in Table 14. Table 14. Crosses of Defective Seeds which Produced an Excess of Recessives in F 2 . Cross Normal Defective % Defective dew x dez 418 376 474 dea x de 5 270 235 46.5 deio x de« 64 59 48.0 den x den 275 251 47-7 F 3 progenies of only one of these crosses, de 10 x de z , have been grown. Twelve self-pollinated F 3 progenies were obtained of which five were segregating for both types of defective seeds. Although the di-hybrid ears are not in excess, as would be expected under balanced lethal conditions, the sample is too small to permit any final conclusion on this point. When the normal and defec- tive seeds on these five di-hybrid ears are counted, it is found that there is again an excess of the recessive seeds as is shown in Table 15. When these five progenies are combined with the two F 2 prog- enies already shown, making a total of 1060 normal to 916 defec- tives, the deviation is 52 ± 13. The average percentage of defec- tive seeds is found to be 46.3, which indicates linkage with cross- ing over of 38.5 per cent. 55^ CONNECTICUT EXPERIMENT STATION BULLETIN 279 Table 15. Normal and Defective Seeds from F 3 Progenies of a Cross of deio x dez Indicating Linkage between These Factors. Ear No. Normal Defective % Defective 858 112 99 46.9 859 130 100 43-5 862 132 112 45-9 867 125 105 45-7 868 143 124 46.4 Total 642 540 45-7 Expected 665 517 43-7 Deviation —23 P. E. = 11.5 It should be mentioned, that with a condition of balanced lethals, occasional progenies are expected in F 3 in which there is a deficiency of recessives instead of an excess. This condition would be brought about through crossing over so that the two lethal factors, originally on homologous chromosomes, are now borne on the same chromosome. Thus unless the linkage between the two lethals were fairly close, so that the excesses and deficien- cies within each progeny were sufficient to permit a separation, the two types of F 3 progenies would tend to balance each other and linkage would be almost impossible to detect. LINKAGE OF DEFECTIVES WITH GROWTH FACTORS The method of improving corn by selection in self-fertilized lines aims at the removal of all recessive abnormalities such as white seedlings and defective seeds. There seems to be a general belief that these factors have a deleterious effect, even in the heterozygous condition. Lindstrom (1920) suggests that these recessive abnormalities, if they do have an unfavorable effect in the heterozygous condi- tion, are permitted to persist in the germplasm only when they are linked with particularly good growth factors, and that in remov- ing them by inbreeding, some of the best germplasm is lost. Jones and Mangelsdorf (1925) have shown, however, that inbred strains from which all recessive abnormalities have been eliminated, yield fully as well as sister strains which still carry one or more of these abnormal characters. Apparently nothing of value was lost through their elimination ; neither was there any marked improve- ment when their supposedly unfavorable influence was removed. Still assuming that these factors have an influence in the heterozy- gous state, a probable explanation of these conflicting results is that the defective seeds and other lethal abnormalities are per- mitted to persist and accumulate, not because they are linked with especially good factors for development, as Lindstrom has sug- gested, but because their presence tends to keep short sections of the chromosomes which they occupy in a continued state of hetero- ENDOSPERM CHARACTERS IN MAIZE 559 zygosity. The increased vigor which results from such enforced heterozygosity of the accompanying growth factors enables the recessive abnormalities to survive in the germplasm even though they have an unfavorable influence in themselves. Furthermore, when two such lethal factors which occupy homologous chromosomes are brought together, a condition of balanced lethals is set up which may so increase the vigor of the stock by keeping whole chromosomes or large sections of chromo- somes in a continued state of heterozygosity, that the lethals are actually given an advantage and are able to survive even though they are linked with especially poor growth factors instead of particularly good ones. Shull (1923) has pointed out that varieties of Oenothera which carry lethal factors are, in general, more vigorous than those which lack these characters. The mechanism of crossing over in Oenothera appears to be different from that in most species as is shown by both cytological and genetic studies; Shull (1923), Cleland (1925). All of the characters so far studied in this species fall into a single linkage group and the amount of cross- ing over between the members of the group is relatively low. It is possible, therefore, that lethal factors in Oenothera keep all of the chromosomes, with their hereditary factors for growth and development, in a continued state of enforced heterozygosity. If such is the case; then the increased vigor brought about in Oenothera by the presence of lethal factors is probably more marked than would occur in other species where there are as many linkage groups as chromosomes. A PLANT CHARACTER FOR DEFECTIVE SEEDS In addition to the thirteen endosperm characters which cause one-fourth of the seeds on segregating ears to be defective, a plant character which causes defectiveness in all the seeds on one-fourth of the plants has been found. This character appeared in the de 13 stock which was received from Mr. H. A. Wallace of Des Moines, Iowa. Mr. Wallace had found among the plants of the variety "Illinois Two Stalk" several which produced only aborted seeds, and which appeared to be homozygous for defective seeds. Pollen from one of these plants applied to a hybrid of inbred strains known to be free of hereditary defectives produced only normal seeds. The ¥ 1 plants grown from these normal seeds were selfed and produced some ears which were segregating for defective seeds of the type which has already been described as de 13 . Not all of the ears were segregating, however, as should have been the case, had one of the parents been homozygous for the defective factor. Nor did the recessive seeds on the segregating F 1 ears resemble the 560 CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 aborted seeds of the pollen parent. The extracted recessives on the Fj ears were completely aborted, appeared to have no endo- sperm tissue and showed no germination whatever. It was diffi- cult to understand how this type could have been obtained in a homozygous condition, or why, if the pollen parent was homp- zygous for defective seeds, only part of the F 1 ears were segregating for the character. This confusing situation was cleared up, however, when a large number of F 2 ears, which had been grown for another purpose, were harvested. A total of 201 F 2 ears were examined and of these 51, or almost exactly one-fourth, bore only aborted seeds and were identical in appearance to the ears of the grandparental pollen parent. The other 150 ears were normal in appearance although some were segregating for defective seeds and others were not. Apparently the plant of "Illinois Two Stalk" which served as the pollen parent for this cross, in addition to being heterozygous for a recessive endosperm character de 13 , was homozygous for a recessive plant character, to which the symbol de p \ may be given. On this hypothesis the genetic composition of the parental stocks and the F 1 seeds is as follows : Pollen parent De 13 de lz de v \ de P i Seed parent Z>Five single factors, any one of which in a homozygous ge± recessive condition causes premature germination. a e ~\ A pair of independent duplicate factors which cause pre- g e \ mature germination when both are present in the reces- sive condition a e \ A pair of linked duplicate factors causing premature ger- % e * f urination when both are present. Crossing over is about 6 °-> 34 per cent. A third factor in this set, ge 10 , is also indicated. LINKAGE RELATIONS A detailed study of the linkage relations of these factors with other well known'characters has not yet been undertaken. Data are available, however, to show the relation of the ge lt ge 2 , ge s , and ge 5 genes with the factor for sugary endosperm. In all these cases the germinating seeds cannot be accurately classified with regard to endosperm texture and linkage must be detected by the distortion in the starchy : sugary ratio among the dormant seeds. In the repulsion phase the percentage of sugary seeds in 600 CONNECTICUT EXPERIMENT STATION BULLETIN 279 the dormant class should vary between 25 and 33^3, depending- on the intensity of the linkage. This is shown diagrammatically in Figure 63, Part I. In the coupling phase, linkage would be expected to cause a deficiency of sugary seeds in the dormant class, the percentage ranging from 25 to o. ge x x Su The linkage relations of ge x with Su are determined from the cross between the ge 1 and ge 2 stocks. The former is a yellow flint variety, the latter a yellow sugary. Eight ears which were segregating for ge 1 were obtained from this cross and the propor- tion of sugary seeds in the dormant class on these ears is shown in Table 27. gregation in Dormant Seeds from Ears Segregating gei. Table 27. Starchy Sugary Seg Seg Ear No. Starchy 710 145 715 159 721 179 722 146 723 162 725 110 726 164 727 153 Total I2l8 Ex. 3:1 1269 Dev. 51 P. E. 12.0 Sugary Percent Sugary 60 29-3 6l 27.7 73 29.O 69 32.1 44 2I.4 43 28.1 71 30.2 53 25-7 474 28.0 423 25.0 It is noted that there is an excess of sugary seeds, amounting to 4.25 times the probable error. Such a deviation would be expected by chance alone only once in about 250 trials. The excess, though it occurs in all but one of the eight ears, would not be regarded as significant if seeds from a smaller number of ears were counted. When all the ears are combined, however, the accumulation of small deviations is one direction results in a total deviation which can scarcely be attributed to chance. The excess of sugary seeds in this class can be explained by assuming linkage between the genes ge 1 and Su with approximately 40 per cent crossing over. It is realized, of course, that linkage values determined by the distortion in a single class necessarily have a large probable error and the value given must not be regarded as more than an approximation. That the excess of sugary seeds in the dormant class is due to some relation with the ge x factor and not to errors in classifica- tion or to genetic factors affecting the rate of pollen tube growth, is further indicated by a count of the sugary seeds on eight ears ENDOSPERM CHARACTERS IN MAIZE 6oi from the same cross which are not segregating' for ge x . On these ears the percentage of sugary seeds is very close to expectation and deviations are minus almost as frequently as plus, as is indi- cated in Tahle 28. Table 28. Starchy :Sugary Segregation in Ears Not Segregating gei from Same Cross as Ears in Table 27. Ear No. Starchy Sugary Percent Sugary 728 192 48 20.0 729 221 80 26.6 730 211 65 23.6 731 204 69 25-3 733 163 57 25-9 734 185 82 30.7 735 227 68 23.0 736 175 79 3i. 1 Total 1578 548 25.8 Ex. 3:1 1594 532 25.0 Dev. 16 P. E. 13.5 ge 2 X su The relation of ge 2 and su is shown by ears from this same cross which are segregating for ge 2 and sugary but not for ge x . The counts from seven such ears appear in Table 29. Linkage in this case would be indicated by a deficiency of sugary seeds since the two factors entered the cross in the coupling phase. Actually there is a slight but not significant excess of sugary seeds in the dormant class and it appears safe to conclude that ge 2 and su are independent. Table 29. Starchy : Sugary Segregation in Dormant Seeds of Segregating gei from Same Cross as Ears in Tables 27 and 28. Ears Ear No. Starchy 705 706 707 708 121 172 I46 158 716 134 717 155 718 152 Total Ex. 3:1 Dev. 26 IO38 IO64 P. E. 1 1.0 Sugary Percent Sugary 44 26.7 59 25-5 67 3i.5 52 24.8 4i 23-4 54 25.8 64 29.6 38i 26.8 355 25.0 ge 3 and ge 5 x su Five of the ears represented in Table 24 are segregating for su, ge 3 and ge 5 . The two types of germinating seeds cannot be distinguished but if either one is linked with sugary a distortion 602 CONNECTICUT EXPERIMENT STATION BULLETIN 279 of the starchy : sugary ratio in the dormant seeds would be expected. A count of the two types of seed in the dormant class on these five ears appears in Table 30. The agreement with expectation on the basis of independent inheritance is very good and it seems certain that both factors are independent of sugary. Table 30. Starchy : Sugary Segregation in Dormant Seeds of Ears Segregating ge 3 and ges. Ear No. Starchy Sugary Percent Sugary 2039 99 23 18.9 2041 121 44 26.7 2043 96 35 26-7 2044 154 58 27.4 2046 99 29 22.7 2047 124 47 27.5 Total 693 236 25.4 Ex. 3:1 697 232 25.0 Dev. 4 P. E. 8.9 APPARENT LINKAGE WITH ENDOSPERM COLOR FACTORS As has already been mentioned, there is a strong association between germinating seeds and absence of color in the endosperm. In the case of ge r , ge z and ge 5 the recessive seeds are, with few exceptions, completely white, while in the ge 2 strain the germinat- ing seeds are a pale yellow. Table 31. Apparent Linkage between gei and White Endosperm. , Yellow s , White N Ear No. Ge ge Ge ge 710 204. I I 66 715 219 I 56 721 252 I 67 722 213 I 2 74 723 205 I 62 726 234 I 61 754 205 I 74 Total 1532 3 7 460 Occasionally, however, germinating seeds with yellow endo- sperm are found as well as white seeds which have not sprouted. The frequency of these exceptions in stocks 1 and 3 is shown in Tables 31 and 32. At first glance the situation represents a clear cut case of close linkage with less than 1 per cent of crossing over. Eyster (1924) has assumed this to be the situation in his stock and has calculated the amount of crossing over as 1.26 per cent. The writer (1923) had previously suggested physiological correlation as an explanation of these results and the evidence ENDOSPERM CHARACTERS IN MAIZE 603 indicates that this is probably the correct interpretation in the stocks reported here. There are at least three series of facts which are not compatible with a linkage hypothesis : 1. All of the stocks in which the association between pre- mature germination and color of endosperm appears have originated from varieties which were homozygous for yellow endosperm. White seeds might be expected to arise occasionally by mutation, but the appearance of four genetically distinct factors for white seeds, each one closely linked with a factor for germi- nating seeds, cannot reasonably be assumed. 2. When pollen from plants which are segregating for germi- nating seeds is applied to silks of a white variety, only yellow seeds are produced. If the segregating plants were heterozy- gous for endosperm color, as they appear to be, such crosses should produce I :i ratios, providing that the white endosperm of the germinating seeds has the same genetic basis as the white endosperm of common white varieties. 3. The apparent cross overs, white seeds which fail to sprout, should breed true if it is assumed that they are homozygous for a recessive endosperm color factor. Only a small number of these seeds have been available but all those which were grown pro- duced only plants segregating for white seeds which germinated, with few exceptions. Table 32. Apparent Linkage between ge 3 and White Endosperm. , Yellow -, , White , Ear No. Ge ge Ge ge 758 70 I 22 760 60 17 761 IOI 27 Total 231 1 o 66 If the association between germinating seeds and endosperm color is not due to linkage, to what may it be attributed ? A histo- logical study of the germinating seeds of the ge 1 stock has given evidence which seems to have some bearing on this question. PREMATURE DIGESTION AND PIGMENT FORMATION Some of the white seeds were removed from the ear at an early stage, killed and fixed, imbedded in paraffin, sectioned, and stained. It was found that even at this early stage the processes of germination had already begun. The cells in the epithelial layer of the scutellum had elongated and the invaginations of this layer, so characteristic of mature seeds of maize, were already apparent. Sargant and Robertson (1905) have made a thorough 604 CONNECTICUT EXPERIMENT STATION BULLETIN 279 study of the scutellum of maize and are of the opinion that these invaginations are glandular in nature and that their function is the secretion of diastase. There is some appearance of digestion in the cells of the endosperm of germinating seeds even at the early milk stage, and in material gathered from the same ears a week later, the digestion is quite marked. It is possible that the normal production of color in the cells of the endosperm cannot proceed while digestion is occurring in these cells. The yellow color in the endosperm is found in the matrix which surround the starch grains, and if this matrix is being digested as rapidly, or more rapidly, than new material is being supplied by the plant, it is hardly to be expected that pig- ment formation would proceed in the normal fashion. The apparent cross overs, the yellow seeds which germinate and the white seeds which remain dormant, may be merely variations of this condition. Some of the germinating seeds probably remain yellow because the digestion does not begin soon enough or is not rapid enough to inhibit the formation of endosperm color. This would appear to be the case in the stock where the duplicate factors ge 6 and ge- are involved. In this stock the pale yellow is usually confined to an area adjacent to the embryo and the dorsal side of the seed retains the full yellow color. The other class of apparent cross overs, the white dormant seeds, are more difficult to explain. The fact that all of these seeds which have ever been grown have given ears segregating for germinating seeds might suggest that this character occa- sionally manifests itself in the heterozygous condition. THE RELATION OE PREMATURE GERMINATION TO CHLOROPHYLL DEVELOPMENT Evidently there is also a physiological relation of some sort between premature germination and chlorophyll development. Types which begin germination at a very early stage, such as ge~ and ge 5 always produce pure white sprouts. The ge 1 type, in which germination begins somewhat later, ordinarily produces white plumules but occasionally these show a tinge of green. The remaining types in which germination begins only after the kernels are well developed, produce only normal green sprouts. Apparently the premature germination, if it begins early enough, completely inhibits the formation of chorophyll just as it pre- vents the laying down of yellow pigment in the endosperm. This association, too, is characterized by occasional exceptions. Germinating seed of ge 1} ge 3 and ge 5 are sometimes found which produce sprouts of normal green color, but dormant seeds which Sfive albino seedlings when germinated have never been observed. ENDOSPERM CHARACTERS IN MAIZE 605 PHYSIOLOGY OF PREMATURE GERMINATION Oppenheimer (1922) has found that in seeds of tomato, gourd, cucumber and Nicotiana rustica, germination can be suppressed by surrounding the seeds with crushed tissue of the receptacles of the mother plants or by growing them on filter paper saturated with an extract from these tissues. The degree of suppression is approximately proportional to the amount of tissue present or the concentration of the extract. This suppression can be overcome by heating the tissue or extract to ioo° C. Apparently the mother plants of these species nor- mally supply the growing seed with inhibiting substances which prevent germination while the seeds are still attached to the plant. Maze (1910) is of the opinion, and presents some evidence in favor of his view, that dormancy in seeds, buds, bulbs, and tubers is due, in some cases, to the action of volatile esters which prevent growth until they are eliminated. Oppenheimer did not include seeds of maize in his experiments but a test made by the writer, in an effort to determine at what stage of development germination in the normal seed still attached to the plant could be induced, may have some bearing on the problem. An ear in the early milk stage was stripped down and wrapped with cotton. Around this were wrapped several layers of cloth. The ends of the cloth were submerged in a vessel of water and served as a wick, keeping the cotton surrounding the ear constantly saturated. The grains swelled considerably, indi- cating that water was being absorbed, but no germination occurred. A number of seeds which had been removed from this ear and placed in an ordinary germinator at approximately the same temperature and with the same moisture supply, began to sprout after about ten days. This is much longer than the time required by immature, dry seeds to germinate under the same con- ditions and indicates that inhibiting substances were first elimi- nated before germination could begin. THE EFFECTS OF PREMATURE GERMINATION ON THE GROWTH OF THE SEED An attempt was made to determine whether the germinating seeds receive the normal amount of nourishment from the plant while germination is going on or whether these seeds cease their development after germination begins. Ears which were segre- gating for germinating seeds (ge t ) were harvested at three weeks after pollination, and at intervals of one week thereafter, until maturity. These ears were dried on a rack until thoroughly dry, at which time the kernels were shelled off, the dormant and ger- minating seeds separated, counted and weighed, and the average weight of each class determined by simple division. The results 6o6 CONNECTICUT EXPERIMENT STATION BULLETIN 279 are shown in two curves in Fig. 76. It will be seen that already in the early milk stage there was a noticeable difference in the relative development of the two types as represented by their dry weights. This difference increased in the second week under observation and thereafter the germinating seeds no longer in- creased in weight and actually fell off somewhat during the last 400 ^ 300 a SZ5 C5 H I 200 100 i L-— -~~~~~ """ """"" lA ^r , 1 iB WEEKS AFTER POLLINATION Fig. 76. — Growth curves of dormant and germinating (gci) seeds from the same segregating ears. The sprouting seeds do not receive enough nourishment from the plant to replace the material lost in germination. three weeks. The normal seeds on the same ears showed an increase in dry matter during every week of the test. It is evident from these two growth curves that the amount of nourishment supplied to the aberrant seeds by the plant is not sufficient to replace that consumed in germination. In fact, it is quite likely that the germinating seeds become partially or wholly "physi- ologically isolated" from the plant during the later periods. At maturity the germinated seeds weighed only 51 per cent as much as the dormant seeds from the same ears. ENDOSPERM CHARACTERS IN MAIZE 607 DISCUSSION For a period of several weeks, while the seed is in the milk or dough stage, natural conditions for germination are almost at an optimum. The temperature is fairly high and the moisture supply is abundant. The embryos are sufficiently developed to produce plants capable of surviving and the endosperm contains enough food material to nourish the seedling until it begins to manufacture food for itself. That this is true is shown by the behavior of immature seeds harvested at these early stages when many of them are capable not only of germinating but of produc- ing almost a normal yield of grain. (See Part I.) Why is it, then, that the partially developed seed ordinarily never germinates while still attached to the plant? Apparently the mother plant, though it provides conditions suitable for ger- mination, at the same time supplies inhibiting substances which prevent germination from beginning. The physiological processes involved in maintaining a period of dormancy, which permits the embryo to attain a maximum devel- opment and the endosperm to accumulate a mass of food material, are probably very complicated. It is not at all surprising, there- fore, to find a number of distinct genetic factors operating during this period. Every stage in the ontogeny of the sporophyte is evidently controlled by many genetic factors and the maintenance of a normal period of dormancy which prevents premature ger- mination with its disastrous effects, and permits the sporophyte to pass safely through unfavorable seasons, is no exception. Nine Mendelian factors which govern this stage have already been identified. Many others will undoubtedly be found as maize is investigated more extensively. Summary — Part IV 1. Nine Mendelian factors involved in the maintenance of a normal period of dormancy in maize seeds have been identified. 2. Five of these are complementary factors. When any one of these is lacking the seed germinates prematurely. Plants heterozygous for one, two or three factors give 3:1, 9:7, and 27 :37 ratios respectively. 3. A pair of independent duplicate factors results in ratios of 15 dormant: 1 germinating when plants are heterozygous for both. 4. A pair of linked duplicate factors gives 8 :i ratios when plants are heterozygous for both. Crossing over is about 34 per cent. 5. The ge x factor appears to be linked with the gene for sugary endosperm. Crossing over is about 40 per cent. ge 2 , ge z and ge 5 are found to be independent of sugary. 608 CONNECTICUT EXPERIMENT STATION BULLETIN 279 6. An apparent case of close linkage between endosperm color and several types of germinating seeds is probably due to the physiological effects of premature germination upon the accumula- tion of pigment in the cells of the endosperm. 7. A similar association between germinating seeds and white seedlings may also be due to physiological complications. Seeds which germinate at early stage produce only white plumules ; those which germinate later have normal green sprouts. 8. Premature germination is apparently caused by the lack or loss of inhibiting substances normally supplied by the plant to the growing seeds. 9. It is suggested that many genetic factors are involved in the maintenance of a normal period of dormancy in maize seeds. Conclusion The mature, dormant seed of maize with its well developed embryo and the cells of its endosperm packed with starch grains, represents a real organic achievement. Each ovule has its separate style ; each style, in order for a seed to develop, must receive a pollen grain capable of germinat- ing and producing a tube sufficiently vigorous to reach the micro- pyle. Failure of the growing tube to attain its goal results in the production of "parthenocarpic" defectives without endosperm or embryo. After the pollen tube has entered the micropyle, a very precise mechanism of fertilization begins to function. Failure of this intricate mechanism in any detail may cause the formation of "germless" seeds, lacking an embryo, "miniature" seeds in which the endosperm is greatly reduced in size or perhaps aborted seeds of several other types. The fertilization mechanism having functioned properly, the growing seed begins to receive the influence of various genetic factors. Thirteen distinct factors have been found which arrest the development of the seed and cause it to be defective and in- capable of normal growth and germination. Five additional factors may affect the nature of the stored food material to such a degree that the seed is handicapped and cannot attain a maximum development. In addition to the 18 genetic factors so far found which retard development to a greater or lesser degree, nine other factors have appeared which stimulate certain functions prematurely, with equally disastrous consequences. The seed, in order to reach maturity and pass safely through unfavorable seasons must remain dormant while still attached to the plant, even though it is capable of germination at this stage and the conditions favor- ing germination are almost optimum. Five complementary factors ENDOSPERM CHARACTERS IN MAIZE 609 and two pairs of duplicate factors are involved in the mainten- ance of dormancy during development. The loss of any one or pair of these causes the seed to germinate prematurely with fatal results. A fully mature, normally developed, dormant, white, starchy seed, then, represents the cumulative action of 27 Mendelian factors of which we know the mode of inheritance. How many additional factors are involved would be difficult to estimate, but since all these permanent departures from the normal condition of the germplasm have been found in a limited amount of material, it is certain that many more hereditary factors of a similar nature will appear. This gives some clue as to the infinitely large num- ber of genes always working to produce a normal seed. The majority of these can not be known because they do not vary. All hereditary units here studied concern only the seed which comprises a brief period betweeen fertilization and the resting stage of the embryo. What, therefore, must be involved in the ontogeny of the entire plant? The young seedling, the growing plant, the chlorophyll processes, reproductive machinery and even the gametophyte generation are all controlled by genetic factors the tabulation of which has onlv been started. 6lO CONNECTICUT EXPERIMENT STATION BULLETIN 2/9 LITERATURE CITED. Bailey, L. H., 1906. Plant Breeding. Fourth Edition. The Macmillan Co., New York. Cleland, R., 1925. Meiosis in pollen mother cells of Oenothera franciscana sulfurea. Bot. Gaz. jj: 149-170. Collins, G. N., 1020. Waxy maize from upper Burma. Science, n. s. 52:48-51. Correns, C, 1899. Untersuchungen iiber die Xenien bei Zea mays. Bericht. Deutsch. Bot. Gesell. 17 -.^io-^ij. Demerec, M., 1923. Heritable characters of maize XV — Germless seeds. Jour. Heredity 14:297-300. Demerec, M., 1923. Inheritance of white seedlings in maize. Genetics 8 :56i-593- DeVries, H., 1899, Sur la fecondation hybride de Talbumen. Compt. Rend. 129:973-975. DeVries, H., 1916. Gute, harte, und leere Samen von Oenothera. Zeitschr. indukt. Abstamm. u. Vererb. 16:239-292. East, E. M., 1913. Xenia and the endosperm of angiosperms. Bot. Gaz. 56:217-224. East, E. M., and Hayes, H. K., 191 1. Inheritance in maize. Connecticut Agric. Exp. Sta. Bull. 167:1-142. East, E. M., and Jones, D. F., 1919. Inbreeding and outbreeding. J. B. Lippincott Co., Philadelphia, Pa. Emerson, R. A., 1921. Genetic evidence of aberrant chromosome behaviour in maize endosperm. Amer. Jour. Bot. 8:411-424. Ewert, R., 1909. Neuere Untersuchungen iiber Parthenokarpie bei Obst- baumen und einigem anderen fruchttragenden Gewachsen. Landw. Jahr. 38: 767-839- Ewert, R., 1910. Parthenokarpie bei der Stachelbeere. Landw. Jahr. 39 :463-470. Eyster, William H., 1922. Scarred endosperm and size inheritance in kernels of maize. Missouri Agric. Exp. Sta. Bull. 52. Eyster, William H, 1924. A primitive sporophyte in maize. Amer. Jour. Bot. 11 :7-i4. Eyster, William H., 1924. A second factor for primitive sporophyte in maize. Amer. Nat. 68 : 436-439. Garber, R. J., and Wade, B. L., 1924. Another instance of defective endo- sperm in maize. Jour. Heredity 15:69-71. Groom, P., 1893. The aleurone layer of the seeds of grasses. Annals of Bot. 7 :387-392. Guignard, L., 1901. La double fecondation dans le mais. Jour. Botanique. IS : 37-50. Guignard, L., 1901. La double fecondation dans le Naias major. Jour. Botanique 15 : 205-213. Haberlandt, G, 1890. Die Kleberschlicht des Gras-Endosperms. Bericht. Deutsch. Bot. Gesell. 8 : 40-48. Harlan, Harry V. and Pope, M. N., 1922. The germination of barley seeds harvested at different stages of growth. Jour. Heredity 13 : 7-~74- Harlan, Harry V. and Pope, M. N., 1925. Some cases of apparent single fertilization in barley. Amer. Jour. Bot. 12:50-53. Hayes, H. K. and East, E. M., 1915. Further experiments on inheritance in maize. Connecticut Agric. Exp. Sta. Bull. 188. Hutchison, C. B., 1921. Heritable characters of maize VII. Shrunken endosperm, jour. Heredity 12:76-82. Hutchison, C. B., 1922. Heritable variations in maize. Jour. Agrom 14 : 73-78. Jones, D. F., 1920. Heritable characters of maize IV. A lethal factor- Defective seeds. Jour. Heredity 11:161-167. ENDOSPERM CHARACTERS IN MAIZE 6l I Jones, D. F., 1920. Selection in self-fertilized lines as the basis for corn improvement. Jour. Agron. 12:77-100. Jones, D. F., 1924. The attainment of homozygosity in inbred strains of maize. Genetics 9:405-418. Jones, D. F. and Mangelsdorf, P. C, 1925. The improvement of natur- ally cross-pollinated plants by selection in self-fertilized lines. I. The production of inbred strains of corn. Connecticut Agric. Exp. Sta. Bull. 266. Kempton, J. H.. 192 1. Waxy endosperm in Coix and Sorghum. Jour. Heredity 12 : 396-400. Kiesselbach, T. A. and Peterson, N. F., 1925. The chromosome number of maize. Genetics 10:80-85. Kuwada, Yoshinari, 191 5. Uber die Chromosomenzahl von Zea mays L. Bot. Mag. (Tokyo) 29:83-89. Lindstrom, E. W., 1920. Chlorophyll factors in maize. Jour. Heredity 11 : 269-277. Lindstrom, E. W., 1923. Heritable characters of maize : XIII. Endosperm defects-sweet defective and flint defective. Jour. Heredity 14: 126- 135- Lindstrom, E. W., 1924. Complementary genes for chlorophyll develop- ment in maize and their linkage relations. Genetics 9:305-326. Longley, Albert E., 1924. Chromosomes in maize and maize relatives. Jour. Agric. Res. 28 : 673-682. Liidtke, F., 1890. Beitrage zur Kenntniss der Aleuronkorner. Jahrb. f. Wiss. Bot. 21 : 62-123. Mangelsdorf, P. C, 1922. Heritable characters of maize XII — Mealy endosperm. Jour. Heredity 13 : 359-365. Mangelsdorf, P. C, 1923. The inheritance of defective seeds in maize. Jour. Heredity 14: 1 19-125. Mangelsdorf, P. C. and Jones, D. F., 1926. The expression of Mendelian factors in the gametophyte of maize. Genetics 11 (in press). Maze, P., 1910. Maturation provoquee des graines.. Action antigermina- tive de l'alde'hyde ethylique. Compt. Rend. Acad. Sci. (Paris) 151: 1383-1386. Miller, Edwin C, 1919. Development of the pistillate spikelet and ferti- lization in Zea mays L. Jour. Agric. Res. 18:255-266. Muller, H. J., 1917. An Oenothera-like case in Drosophila. Proc. Nat. Acad. Sci. 3 : 619-626. Muller, H. J., 1918. Genetic variability, twin hybrids and constant hybrids, in a case of balanced lethal factors. Genetics 3 : 422-499. Muller, H. J. and Altenburg, E.. 1919. The rate of change of hereditary factors in Drosophila. Proc. Soc. Exp. Biol. Med. 17:10-14. Nawaschin, S., 1898. Resultate einer Revision der Befruchtung svor- gange bei Lilium Martagon und Fritillaria tenella. Bull. Acad. Imp. Sci. St. Petersbourg 9 : 377-382. Noll, F., 1902. Uber Fruchtbildung ohne vorausgegangene Bestaubung (Parthenocarpie) bei der Gurke. Sitzungsber. d. Gesell. f. Natur. Bonn. Oppenheimer, H., 1922. Keimungshemmende Substanzen in der Frucht von Solarium Lycopersicum und in anderem Pflanzen. Sitzungber. Akad. Wiss. Wien 131 : 59-65. Oppenheimer, H., 1922. Das Unterbleibung der Keimung in den Behal- tern der Mutterpflanze. Sitzungber. Akad. Wiss. Wien 131 : 279-312. Poindexter, C. C, 1903. The development of the spikelet and grain of corn. Ohio Nat. 4:83-89. Reed, H. S.. 1904. A study of the enzyme secreting cells in the seedling of Zea Mais and Phoenix dactylifera Annals Bot. 18 : 267-287. Renner, O., 1916. Befruchtung und Embryobildung bei Oenothera Lamarckiana und einigen verwandten Arten. Flora, N. F. 7: 115-150. 6l2 CONNECTICUT EXPERIMENT STATION BULLETIN 279 Richey, F. D., 1923. Defective seeds in maize — An ancient character. Jour. Heredity 14 : 359-360. Sargant, Ethel and Robertson, Agnes, 1905. The anatomy of the scutel- lum in Zea Mais. Annals of Bot. 19: 1 15-124. Shull, George H., 1923. Further evidence of linkage with crossing-over in Oenothera. Genetics 8 : 154-167. Shull, George H., 1923. Linkage with lethal factors in the solution of the Oenothera problem. Rept. 2d Internat. Congr. Eugenics 1 : 86-99. True, Rodney H., 1893. On the development of the caryopsis. Bot. Gaz. 18 : 212-226. Weatherwax, Paul, 1917. The development of the spikelets of Zea mays. Bull. Torrey Bot. Club 43:483-496. Weatherwax, Paul, 1919. Gametogenesis and fecundation in Zea mays as the basis of Xenia and heredity in the endosperm. Bull. Torrey Bot. Club 46 : 73-90. Weatherwax, Paul, 1920. Position of scutellum and homology of coleop- tile in maize. Bot. Gaz. 69: 179-182. Weatherwax, Paul, 1922. A rare carbohydrate in waxy maize. Genetics 7:568-572. Webber, H. J., 1900. Xenia, or the immediate effect of pollen in Maize. U. S. Dept. Agric. Div. Veg. Phys. and Path. Bull. 22. Wellington, Richard, 1913. Studies of natural and artificial partheno- genesis in the genus Nicotiana. Amer. Nat. 47 : 279-306. Wentz, John B., 1924. Heritable Characters of Maize XVIII — Miniature germ. Jour. Heredity 15 : 269-272. Winkler, Hans, 1908. Parthenogenesis und Apogamie in Pflanzenreiche. Progr. rei bot. 2 : 293-454. 6 14 CONNECTICUT EXPERIMENT STATION BULLETIN 2~9 EXPLANATION OF PLATES. Plate XXI. Longitudinal sections of normal seed and three successive stages of development of defective seeds of den stock, x n. 1. Normal seed at early milk stage. 2. Defective seed, early milk.* 3. Defective seed, late milk. 4. Defective seed, dough. Plate XXII. Three successive stages in development of defective seeds of the des stock, x 11. 1. Defective seed, early milk. 2. Defective seed, late milk. 3. Defective seed, dough. Plate XXIII. Three successive stages in development of defective seeds of the des stock, x 11. 1. Defective seed, early milk. 2. Defective seed, late milk. 3. Defective seed, early dough. Plate XXIV. Successive stages in the development of the normal embryo x 15. Blister stage. Early milk. Milk. Late milk. Dough. Plate XXV. 1. Ears of a uniform first generation hybrid harvested at successive stages of maturity. From left to right the ears represent stages of 14, 21, 28, 35, 41, 51 and 75 days after pollination. 2. Fifty seeds from each of the ears. In appearance and dry weight these normal seeds harvested at successive stages of development resemble various types of hereditary defectives. 3. The results of planting the fifty seeds shown in 2. In ability to germinate the immature normal seeds are superior to hereditary defectives of the same relative development. Plate XXVI. Four morphologically distinct types of seeds which may occur on any ear of maize, x 11. 1. Normal seed with well developed endosperm and embryo. 2. Hereditary defective with aborted endosperm and embryo. 3. Germless seed containing endosperm but no embryo. This type is probably due to single fertilization. 4. Parthenocarpic defective with neither endosperm nor embryo. This type is caused by pollination which fails to accomplish fertilization. * The relative stage of development, in every case, is that of normal seeds from the same, ear, and not of the defectives themselves. PLATE XXI. \ CO OJ PLATE XXII. PLATE XXIII. CO /T*~ '.\ - a PLATE XXIV, o f PLATE XXV x .fr- kk fllfe 1 PLATE XXVI. University of Connecticut Libraries 3tnb3UZ»yb4Zb3