Digitized by the Internet Archive in 2011 with funding from LYRASIS members and Sloan Foundation http://www.archive.org/details/effectsofinbreedOOjone CONNECTICUT •42 AGRICULTURAL EXPERIMENT STATION NEW HAVEN, CONN. BULLETIN 207 SEPTEMBER, 1918 THE EFFECTS OF INBREEDING AND CROSSBREEDING UPON DEVELOPMENT BY D. F. JONES The Bulletins of this Station are mailed free to citizens of Connecti- cut who applyTor them, and to others as far as the editions permit. CONNECTICUT AGRICULTURAL EXPERIMENT STATION OFFICERS AND STAFF BOARD OF CONTROL. His Excellency, Marcus H. Holcomb, ex-officio, President. James H. Webb, Vice President Hamden George A. Hopson, Secretary Wallingford E. H. Jenkins, Director and Treasurer New Haven Joseph W. Alsop Avon Wilson H. Lee Orange Elijah Rogers Southington William H. Hall South Willington Administration. Chemistry, Analytical Laboratory, Protein Research. Botany. Entomology. Forestry. Plant Breeding. Vegetable Growing. E. H. Jenkins, Ph.D., Director and Treasurer. Miss V. E. Cole, Librarian and Stenographer. Miss L. M. Brautlecht, Bookkeeper and Stenographer. William Veitch, In charge of Buildings and Grounds. *John Phillips Stree*t, M.S. E. Monroe Bailey, Ph.D., Chemist in charge. *C. B. Morison, B.S., C. E. Shepard, ] M. d'Esopo, Ph.B. I Assistants. H. D. Edmond, B.S. j Miss A. H. Moss, Clerk. V. L. Churchill, Sampling Agent. T. B. Osborne, Ph.D., D.Sc, Chemist in Charge. Miss E. L. Ferry, M.S., Assistant. G. P. Clinton, Sc.D., Botanist. E. M. Stoddard, B.S., Assistant Botanist. Florence A. McCormick, Ph.D., Scientific Assistant. G. E. Graham, General Assistant. W. E. Britton, Ph.D., Entomologist; State Entomologist. B. H. Walden, B.Agr., First Assistant. *I. W. Davis, B.Sc, M. P. Zappe, B.S., Assistants. Miss Martha de Bdssy, B.A., Stenographer. Walter O. Filley, Forester- also State Forester and State Forest Fire Warden. A. E. Moss, M.F., Assistant State and Station Forester. Miss E. L. Avery, Stenographer. Donald F. Jones, S.D., Plant Breeder. C. D. Hubbell, Assista7it. W. C. Pelton, B.S. * Absent on leave. In service of the United States. CONTENTS Page Introduction 5 Definitions .* 8 Early investigations with plants 9 The observations of Darwin upon plants 12 Recent investigations with plants 14 Investigations with animals 18 Universality of heterosis 21 A theoretical consideration of inbreeding 22 The results of inbreeding the naturally cross-pollinated maize plant 27 The approach to complete homozygosity • . . . . 44 The effect of heterozygosis on vegetative luxuriance 47 The value of inbreeding in plant and animal improvement 59 The effect of heterozygosis upon endosperm development and selective fertilization 61 The effect of heterozygosis upon longevity, hardiness and viability. 69 The effect of heterozygosis upon the time of flowering and maturing. 76 The relation of the effects of heterozygosis and of the environment. 78 Summary of the effects of inbreeding and crossbreeding 81 A Mendelian interpretation of heterosis 82 The part that heterosis has played in the establishment of sex 93 Literature cited 96 The Effects of Inbreeding and Crossbreed- ing Upon Development* INTRODUCTION. Among the higher seed plants certain groups are characterized by almost universal and continuous self-fertilization. On the other hand certain other groups are completely, or to a large extent, cross-fertilized in every generation. Between these two extremes every gradation in the degree of self- and cross-fertiliza- tion can be illustrated. The structure and function of the floral organs have become more or less clearly adapted to the customary mode of sexual reproduction characteristic of each species. In the thallophytes, bryophytes and pteridophytes much the same situation exists whereby the gametes which enter into a sexual fusion may arise either from the same or from different organisms. In the lower animals the same variation in the mode of sexual reproduction exists as in plants. Among the higher animals, however, hermaphroditism is replaced entirely by bisexuality; and sexual reproduction, except when parthenogenesis takes place, results only from the union of gametes originating in different organisms. This array of facts has naturally led to searching inquiries as to the purpose of sexual reproduction as compared to other methods of propagation as well as to the effects of. artificial in- breeding in bisexual animals and in naturally cross-fertilized plants. Bound up with this latter problem is that which is con- cerned with the effects of cross-fertilization in all types of animals and plants of different degrees of relationship. The development of the Mendelian theory of heredity, carrying with it the conception of definable, hereditary units which are sufficiently stable in their transmission from generation to genera- tion to be recognized and their somatic expression to be described, * Submitted to the Faculty of the Bussey Institution of Harvard University in partial fulfillment of the requirements for the degree of Doctor of Science, December, 1917. 6 CONNECTICUT EXPERIMENT STATION BULLETIN 207. lias made possible an attack upon these problems which has opened a way towards their solution. From the knowledge of alternate inheritance it is possible to ascribe, very definitely and surely, certain of the results of in- breeding to the segregation and isolation of hereditary factors which results were formerly thought to be due solely to inbreeding as a cause in itself. Certain pathological, abnormal or otherwise undesirable conditions occurring more frequently in animals and plants produced by matings between nearly related individuals were formerly attributed to inbreeding as the cause, and it was thought that inbreeding must always show such undesirable results. It is now known that many of these pathological and abnormal conditions resulting from inbreeding do not owe their origin to that process, but are due solely to the segregation, into a pure state of the hereditary factors causing the anomalies which factors were present in the organisms previous to their being inbred. Inbreeding, then, has nothing to do with the origin of the undesirable characters under consideration but merely brings them into visible expression, and whether or not they appear depends upon their presence originally in the stock before inbreeding takes place. There still remains a conviction, however, that all the manifestations attending inbreeding and the converse effects of cross breeding cannot be accounted for solely on the basis of the operation of definable, hereditary factors, but that there is a stimulating effect resulting from crossing, which is lost by inbreeding, and that this stimulation differs somewhat from the expression of hereditary factors which can be transferred and fixed in different organisms. This stimulation is supposed to be of a physiological nature appearing when dis- similar germ-plasms are united, and disappearing as the germinal heterogeneity disappears in subsequent recombinations. Since this physiological stimulation has always been purely hypothetical, having never been definitely proven, and since it has been used to account for certain facts heretofore inexplicable in any other way, the existence of such a stimulation may fairly be questioned, in so far as the facts can be logically accounted for in other ways. Recent advances in the knowledge of the methods of inheritance have made it possible to meet certain objections previously held against the view that the effects of inbreeding and crossbreeding can be attributed solely to the INTRODUCTION. / operation of hereditary factors without assuming an additional hypothetical stimulation. Some of the previous work bearing upon the effects of inbreed- ing and crossbreeding is reviewed here and with this are given original data obtained from the naturally cross-fertilized corn plant, Zea mays L. The facts at hand co-ordinate with the exist- ing knowledge of heredity in such a way that it seems to the writer unnecessary any longer to make the fundamental dis- tinction between the effects of inbreeding and crossbreeding and of heredity in development. , , No attempt is made to canvas the extensive literature on hybridization (a bibliography of which alone would fill a volume) in order to list all the cases in which crossing does or does not result in increased development and inbreeding in a reduction. It does not take one long in reading over the many published results of crossing in animals and plants to become convinced that an increase in development following a cross is a frequent occurrence. It is hoped that sufficient references are given to show something as to the universality and nature of the phenom- enon and a review of the more important contributions is made in order to sketch briefly the development of the ideas concerning the cause of the stimulation and the part it has played in evo- lution and in breeding practice. The experiments on inbreeding, which have resulted in the material from which the data given here have been gathered, were started by Professor E. M. East at the Connecticut Agri- cultural Experiment Station and carried on by him and subse- quently by Professor H. K. Hayes and later by the writer. From time to time reports on these experiments have been made and conclusions drawn from the facts as observed. These include various publications under the titles " Inbreeding in Corn," " The Distinction between Development and Heredity in In- breeding " published by Professor East in the Report of the Connecticut Experiment' Station and in the American Naturalist and " Heterozygosis in Evolution and in Plant Breeding " by Professors East and Hayes in a Bureau of Plant Industry bulletin. Under the title of " Dominance of Linked Factors as a Means of Accounting for Heterosis " the writer had proposed a different view as to the cause of hybrid vigor. This was published in Genetics and its application is discussed here in more detail. 8 CONNECTICUT EXPEEIMENT STATION BULLETIN 207. Further publications are planned which will discuss more ade- quately much of the data which are scantily treated here. The significance which these investigations may have for the practical improvement of plants and animals has only been briefly alluded to here. This phase of the subject has been reserved for another time when the methods which have suggested them- selves as the result of these investigations have been more thor- oughly tested. Finally this collection of facts and theories should be viewed as a report of progress rather than a well rounded presentation of the subject of inbreeding and crossbreeding. The writer is especially indebted to his predecessors whose work has made these experiments possible. Grateful acknowl- edgement is due Dr. E. M. East for his careful supervision of the work and for his kindly advice and helpful criticism as to the presentation of the results obtained. The writer alone, however, must assume the responsibility for the opinions expressed. Much credit is due Mr. C. D. Hubbell, Dr. Charles Drechsler and Mr. G. A, Adsit for their careful assistance in the collection and • preparation of the data. Definitions. The knowledge of a stimulating effect resulting from a cross between different animals and between different plants which gives progeny which may excel their parents in general vigor, size or other visible characteristics has naturally led to the use of terms to describe this effect. This stimulation is variously spoken of as "vigor due to crossing" or "hybrid vigor." Since hybrid vigor occurs only in crosses of which the parents are dis- similar in hereditary constitution more exact and comprehensive terms were needed. The zygote resulting from a union of unlike gametes is spoken of as a heterozygote (following the usage of Bateson), hence the term heterozygosis (used by Spillman, '09) refers to that germinal heterogeneity which results from the union of unlike gametes, and the stimulation to development which accom- panies such a condition is spoken of as a "stimulus of heterozy- gosis," or "heterozygotic stimulation," meaning the stimulating effects of hybridity or the stimulation due to differences in uniting gametes. The converse fact of a reduction in vigor accompanying a return to a homozygous condition is therefore said to be due to, EARLY INVESTIGATIONS WITH PLANTS. 9 or result from, homozygosis. Shull ('14) has proposed the term "heterosis" to designate this increase in development which may result from a heterozygous condition; hence, heterosis, as used here, will be considered synonymous with "hybrid vigor" or "stimulus accompanying heterozygosis," in whatever form this may be manifested or whatever cause or causes it may be due to. Shull proposed this term, as he says, "... .to avoid the implication that all the genotypic differences which stimulate cell-division, growth, and other physiological activities of an organism, are Mendelian in their inheritance and also to gain in brevity of ex- pression. ..." Hence the term heterosis is not meant as a mere contraction of heterozygosis and is not synonymous with it. The adjective "heterotic" has also been proposed and such an ex- pression as "heterotic stimulation" is synonymous with heterosis. Early Investigations with Plants. Certain evidence' remains from the carvings of the ancient Egyptians to show that they had some conception of a sexuality in plants. However, it was not until the last of the 17th century, when Camerarius first demonstrated such condition, that interest in the production of artificial hybrids began. Tt is significant that the first artificial hybrids to be systematically studied, those of Kolreuter (1776), furnished some of the best examples of heterosis. Kolreuter made many interspecific crosses in Nicotiana, Dianthus, Verbascum, Mirabilis, Datura and others, many of which astonished their producer by their greater size, increased number of flowers and general vegetative vigor, as compared to" the parental species entering into the cross. Concerning one of the tobacco crosses he says: (pp. 57-58) "Hybrids obtained from the cross of Nicotiana maj. 9 and glut, d71 produced a far greater number of flowers and grew to an uncommonly greater height and a much greater circumference than the pure species under the same conditions ; the height of the plants which were kept in the hot bed or were set out in the field after they had ob- tained full growth, amounted to eight feet and 1 to 10 inches; the whole circumference of the branches to 24 feet; the largest diameter of the stalks from 2 inches to 2 inches and 3 lines; and the largest leaves were 2 feet, 2 inches and 9 lines long and 1 foot and 4 inches wide. Never has anyone seen more magnificent tobacco plants than these were." 10 CONNECTICUT EXPERIMENT STATION BULLETIN 207. Thomas Andrew Knight (1799) was one among several at that time who experimented with hybrids with the view of producing more desirable varieties of vegetables, flowers and fruits. Knight observed many instances of high vigor resulting from crossing; among these we note the following remarks about a cross between two varieties of peas. (P. 200) "By introducing the farina of the largest and most luxuriant kinds into the blossoms of the most diminutive and by reversing this process, I found that the powers of the male and female in their effects on the offspring, are exactly equal. The vigor of the growth, the size of the seeds produced, and the season of maturity, were the same, though the one was a very early, and the other a very late variety. I had, in this experiment, a striking instance of the stimulative effects of crossing the breeds; for the smallest variety, whose height rarely exceeded two feet, was increased to six feet; whilst the height of the large and luxuriant kind was very little diminished." It is evident that, in these crosses, Knight was dealing with dwarf and standard peas and the dominance of standardness is expected. A sufficient number of cases, however, were observed in which the crosses were more vigorous than an average of the parents to convince him that "nature intended that a sexual intercourse should take place between neighboring plants of the same species." It was this principle which Darwin elaborated 50 years later. Sageret ('26) reports vigorous hybrids in Nicotiana and also between different types of the Cucurbitaceae. Among other 'things he notes that in human crosses between one individual which shows a hereditary pathological condition and a normal individual, that the disease disappeared in the first generation but reappeared in the second and following generations. Wiegmann ('28) gives instances of hybrids in the Cruciferae which showed distinct evidences of heterosis. Probably the most extensive series of experiments on hybridiza- tion were those of Gartner ('49) and of Focke ('81). According to Lindley ('52) Gartner made 10,000 crosses between 700 different species and produced 250 different hybrids. Many of these hy- brids showed distinct evidences of heterosis, and this phenomenon was manifested in many different ways. Gartner speaks especially of their general vegetative luxuriance, increase in root develop- ment, in height, in number of flowers and their hardiness and early EAELY INVESTIGATIONS WITH PLANTS. 11 and prolonged blooming. Focke made equally extensive observa- tions and catalogues his own experiments with many of those made previously. His valuable book shows clearly that the phe- nomenon of heterosis is widespread and may be expected in the gymnosperms and pteridophytes as well as in the angiosperms. Both the works of Gartner and of Focke have been so thoroughly reviewed in recent times (East and Hayes '12) in connection with the problem in hand that it would be a needless repetition to say more about their results here. Special points in their observations, as they supplement the experiments recorded here, will be referred to later. While the work of Gartner and Focke must always rank high as contributions to our knowledge of genetics one cannot refrain from remarking that they both missed by their extensive studies of many species the point which Mendel discovered by his inten- sive and careful study in one species. Naudin ('65) next to Mendel will always be remembered, no doubt, as the first to conceive of a method in the uniformity of the first generation and the variability of the second. His con- ception of the segregation of parental qualities as a whole leads up naturally to Mendel's law whereby the characters of the parents segregate as units and when finally appreciated the chaotic observations of Gartner, Focke and their contemporaries began to be understood as orderly facts. In Naudin's classical experiments there are many excellent examples of heterosis. Out of 36 interspecific crosses which he made in Papaier, Mirabilis, Primula, Datura, Nicotiana, Petunia, Digitalis, Linaria, Luffa, Coccinea and Cucumis, 24 show positive evidence of het- erosis. Among the most notable crosses in this respect was that of Datura Stramonium with D. Tatula in which both reciprocal hybrids were twice as tall as either parent. Concerning the Datura crosses Naudin says : "A shape very much taller than the two parental types, and the pre- mature falling off of the flowers in the first dichotomies, which leads to tardy fructification are the principal characteristics of this hybrid of which all the plants in the collection present the greatest uniformity. We shall see that these different characteristics appear in all the hybrids of this section of the genus Datura." Mendel ('65) also records instances of heterosis in his pea hybrids as is shown in the following passage : 12 CONNECTICUT EXPERIMENT STATION BULLETIN 207. "The longer of the two parental stems is usually exceeded by the hybrid, a fact which is possibly only attributable to the greater luxuriance which appears in all parts of plants when stems of very different lengths are crossed. Thus, for instance, in repeated experiments, stems of 1 foot and 6 feet in length yileded without exception hybrids which varied in length between 6 feet and 7K feet." The Observations of Darwin upon Plants. Of all the contributors to our knowledge of the effects of in- breeding and crossbreeding no one has collected as many facts as Darwin ('75, '77). Although undoubtedly much confusion and misunderstanding have resulted from Darwin's conclusions on this problem, one cannot but admire his painstaking efforts to accumulate facts from the behavior of many species of plants through many generations of crossing and selfing before advancing his conclusions. No one was more frank to acknowledge the discrepancies between the facts as he found them and the con- clusions he drew from them. Those parts of his results which were not clear to Darwin are clearer to us through our knowledge of Mendelism of which he was not permitted to know. Since his method of experimentation, and the results obtained are familiar to all interested in the problem at hand no extensive review of his work is necessary. Only a brief summary of the results obtained and the conclusions which he drew from them will be given here, reserving a more detailed review of special parts for a later part of this paper. Among animal breeders in Darwin's time it was a common belief that whatever evil effects resulted from more or less close inbreeding were due to the accumulation of abnormal, diseased, or morbid tendencies in the offspring of parents which possessed such tendencies. Darwin refused to ascribe any large part of the effects of inbreeding to this cause because he knew so many cases were weakened and reduced types of both plants and animals which gave vigorous progeny when crossed among themselves. Instead of an accumulation of the undesirable traits of both parents the very reverse seemed to be true. Had Darwin known of the way by which recessive characters may exist for many generations without making their appearance, doubtless his views on this point would have differed materially. Darwin clearly thought that the evil effects of inbreeding kept on accumulating until eventually a plant or animal propagated THE OBSERVATIONS OF DARWIN UPON PLANTS. 13 in that manner was doomed to extinction. His Own results came far short of proving such an assumption. The two wild plants with which inbreeding was practiced the longest — Ipomea and Mimulus — showed very little further loss of vigor after the first generation. What these experiments did show, most clearly, was that there was segregation of the inbred stock into diverse types which differed in minor, visible, heriditary characters and which also differed in their ability to grow. In both species plants appeared which were superior to other plants derived from the same source and some were even equal or superior in vigor to the original cross-pollinated stock. They differed from this race, however, most noticeably in the uniformity of all visible characteristics. After several generations of inbreeding Darwin found that it made no difference in the resulting vigor, whether the plants in an inbred lot were selfed or were crossed among themselves. This he correctly attributed to the fact that the members of such an inbred strain had become germinally alike. From his views on the effect of the environment on organisms, it is easy to see why he attributed this approach to similarity in inherited qualities to the fact that the plants were grown for several generations under the same- conditions. This view he thought was supported by the fact that crosses of his selfed lines with the intercrossed lines (also inbred, but to a less degree) did not give as great increase in vigor as the crosses of either lines with a fresh stock from distant regions. The crosses between two inbred lines did give a noticeable increase in vigor, in many cases, equaling the original variety. This is illustrated in the Dianthus crosses in which the selfed line was crossed with the intercrossed line and with a fresh stock. The ratio of both crosses to the selfed plants in height, number of capsules and weight of seed produced is as follows: Selfed Selfed X X Inter-crossed Fresh stock Height, compared to selfed 100:95 100:81 No. Capsules, compared to selfed 100:67 100:39 Weight of seed, compared to selfed 100:73 100:33 Like Darwin we now attribute the greater increase of vigor in a cross with distinct stocks to a greater germinal diversity although we may differ in our ideas as to the way in which that 14 CONNECTICUT EXPERIMENT STATION BULLETIN 207. diversity was brought about. Whatever may be the explanation of that, credit is due Darwin for being the first to see that it was not the mere act of crossing which induced vigor but the union of different germinal complexes. This he states clearly in the following sentences (Cross and Self Fert., p. 270) : " These several cases taken together show us in the clearest manner that it is not the mere crossing of any two individuals which is beneficial to the offspring. The benefit thus derived depends on the plants which are united differing in some manner, and there can hardly be a doubt that it is in the constitution or nature of the sexual elements. Anyhow, it is certain that the differences are not of an external' nature, for two plants which resemble each other as closely as the individuals of the same species ever do, profit in the plainest manner when intercrossed, if their progenitors have been exposed during the several generations to different conditions." Recent Investigations with Plants. Although Darwin was the first to attack the problem from the standpoint of determining the effects of inbreeding, it is doubtful if he clearly recognized that the same phenomenon was concerned in both inbreeding and crossbreeding. It remained for Shull ('08, '09, '10, '11 and '14), East ('08, '09) and East and Hayes ('12) to bring out clearly the fundamental similarity of both processes and to put the matter in such a light that a far clearer under- standing of the nature of the effects of inbreeding has resulted. Their conclusions in regard to the causes of the effects of inbreeding and crossing were for the most part entirely new and dependent for their support upon the Mendelian principle of the segregation and recombination of inherited qualities as inde- pendent units and upon Johannsen's genotype conception of heredity. Stated briefly their main tenets, based upon their own careful experiments and a survey of previous results bearing upon the problem, are as follows: 1. Inbreeding automatically sorts out into homozygous, pure breeding lines, the diverse and variating complex of hereditary characters found in a naturally cross-pollinated species. 2. Although complete homozygosity is difficult to attain in practice, after several generations of selfing, members of the resulting inbred lines are uniform among themselves but the respective lines may differ greatly among each other in visible RECENT INVESTIGATIONS WITH PLANTS. 15 hereditary characters. The strains may also differ in their power of development, some being larger, stronger and more productive than others at normal maturity. Some individuals are often isolated which are so lacking in necessary characters that they perish because of inability to reproduce themselves. 3. Those inbred strains which are able to survive finally be- come constant; no further reduction in vigor or change in visible characters is to be expected by continued inbreeding. These constant types are thus quite comparable to naturally self- fertilized species and may exist indefinitely. 4. When these pure breeding types are crossed there is com- monly an immediate and striking increase in general size and vigor to be expected in the resulting first hybrid generation. To account for this increase in development, following a cross, a physiological stimulation was postulated which accompanied heterozygosity of hereditary factors and disappeared as the organisms approached homozygosity. As an illustration the union of factor "A" with it allelomorph "a" was considered to evolve developmental 'energy which was lacking when either "A" or "a" were united with themselves. This stimulus to devel- opment was considered to be due to the union of unlike factors alone and to have an effect quite different from whatever part each factor had by itself in the development of the organism. Stated in their own words the main conclusions of East and Hayes ('12) are as follows (p. 8): " 1. Mendel's law — "that is, the segregation of character factors in the germ cells of hybrids and their chance recombinations in sexual fusions — is a general law. 2. Stimulus to development is greater when certain, or possibly all, characters are in the heterozygous condition than when they are in a homozygous condition. 3. This stimulus to development is cumulative up to a limiting point and varies directly with the number of heterozygous factors in the organism although it is recognized that some of the factors may have a more powerful action than others." It was clearly apparent to recent investigators that many of the unfavorable characters which appear on inbreeding a naturally cross-pollinated species are recessive characters which are segre- gated out of the original complex. In a naturally crossed species, these are hidden from sight on account of being continually 16 CONNECTICUT EXPERIMENT STATION BULLETIN 207. crossed with dominant characters. That dominance of factors could in any way be an essential factor in the vigor and excellence of hybrids, an idea first proposed by Keeble and Pellew ('10) and also by Bruce ('10), has not been accepted by most writers on this subject. They considered dominance to be totally in- adequate to account for the widespread and almost universal occurrence of heterosis in plants and animals and the fact that nearly all naturally cross-fertilized domesticated species are reduced by inbreeding. Collins ('10) has shown clearly that many crosses between varieties of Indian corn already widely crossed among themselves and grown in the same regions may not give any increase in productiveness, but when these same varieties are crossed with varieties from distinct geographical regions great increases in productiveness are obtained. Further evidence as to the occurrence of heterosis is seen in the many publications which have appeared from time to time urging the commercial utilization of this hybrid vigor as a method of increasing production in many plants. Among these are Beal (76- 82), McCleur ('92), Morrow and Gardner ('93-94), Swingle and Webber ('97), Hayes and East ('11), Hartley ('12), Wellington ('12), Hayes ('13), Hayes and Jones ('16). In view of the innumerable cases in which an increase in devel- opment, in some character, results from crossing and the converse fact of reduction following subsequent inbreeding, of which the preceding paragraphs refer to only a small fraction, it is surpris- ing to note such radically diverse opinions as are held by Burck {'08) and championed by Stout ('16). Stout attributes the following statements to Burck: (p. 418) "That (1) plants that are regularly self-fertilized show no benefits from crossing and that (2) nowhere in wild species is there evidence of an injurious effect from self-fertilization, and that there is abundant evidence of continued vigor and high fertility resulting from long con- tinued self-fertilization." If by the first statement is meant that crossing between members of the same variety or between individuals of a uniform species does not give an increase in development such a result would be expected because of the germinal similarity brought about by long continued selfing and elimination by selection, either natural EECENT INVESTIGATIONS WITH PLANTS. 17 or artificial, of all but one type. On the other hand, there is abundant evidence to show that crossing between different vari- eties or between different wild species of self-pollinated plants often results in striking increases in size and vigor. It is only necessary to refer to the work of Kolreuter, Knight, Gartner, Naudin and Mendel where many crosses between different species or between distinct types of Nicotiana, Pisum, and Lathyrus — plants which are naturally self-fertilized — give unmistakable evidence of heterosis. Turning to the effects of inbreeding, almost no long-continued experiments have been carried out with strictly wild cross-polli- nated species of plants. Collins ('18) in a brief note states that teosinte, a semi-wild relative of maize, is not affected by in- breeding to the extent that maize is. That there is "abundant evidence of continued vigor and high fertility resulting from long continued self-fertilization" no one longer doubts. There is, however, hardly enough evidence from plants, so far on record, to justify the sweeping statement, which the quotation implies, that cross-fertilized wild species are never reduced by inbreeding. What evidence there is indicates that naturally crossed wild species are not reduced by inbreeding to anything like the extent that domesticated races are. More will be said about this differ- ence between wild and domesticated races later. There is some evidence, however, to show that strictly wild species are affected by inbreeding. Darwin compared the progeny of artificially self- fertilized plants with the progeny of artificially intercrossed plants of many wild species. Many of these species were such as were for one cause or another almost completely cross-fertilized in their natural state at all times. Although the difference may be slightly exaggerated there can be no question but that the difference in the first generation which Darwin obtained between the selfed plants and the intercrossed plants represents in many cases the effect which inbreeding has upon these plants. As examples of widely crossed wild species in which a reduction in the first generation of inbreeding was obtained by Darwin, one can, therefore, cite: Digitalis purpurea, Linaria vulgaris, Saro- thamnus scoparius and Reseda lutea. Moreover, no matter how much domestication may change plants from the wild, one cannot cast aside, as of no consequence, the results obtained from cultivated plants. 18 connecticut experiment station bulletin 207. Investigations with Animals. According to Darwin, the mule, that classic example of hybrid vigor, was known in the time of Moses, when its hardihood and general good qualities doubtless endeared this animal to the Jews no less than to the Southern cotton planters of to-day. A similar cross of the ass with the wild zebra according to Riley ('10) gives a first generation hybrid animal of considerable merit. In the early history of the establishment and fixation of breeds of livestock we note in Darwin's "Animals and Plants under Domestication" that certain crosses between different breeds often resulted in progeny excelling individuals of either parent breed; just as to-day it is not an uncommon practice for livestock raisers to cross certain well-established breeds to produce crossed animals to feed for market. In looking over the reports of experiments designed to test the effects of crossing in both wild and domesticated animals there is little disagreement as to the results usually obtained. All are practically in accord that crossing diverse breeds or races of animals, if not too distantly related, may frequently result in vigorous, large and fertile offspring, excelling either parent in one or more respects. For example, Castle et al ('06) find that crossing diverse stocks of Drosophila results in an increase in fertility and that matings between different inbred lines give progeny with increased fertility up to or beyond that of the more fertile parental race. In Meriones Bonhote ('15) states that fertility and size are increased by crossing. Castle ('16) has crossed domesticated races of guinea-pigs with the wild species from Peru with the result that there is a noticeable increase in body weight over either pure parent. Gerschler ('14) crossed different genera of fishes and obtained large increases in size in the first hybrid generation. Xiphophorus strigatus, of which the males were 43.0" cm. long and the females 52.0 cm., when crossed with Platypoecilius viaculatus, of which the males were 26.0 and the females 31.0 cm. in length, gave hybrid males 54.0 cm. and females 57.5 cm. He speaks of their "gigantic size." Fischer ('13) in his study of the Rehoboth hybrids, a race in South Africa resulting from a mixture of Hottentots and Boers, states that their average height is somewhat greater than either the Hottentots or the Hollanders and South Germans of whom INVESTIGATIONS WITH ANIMALS. 19 statistics are available. All the members of this new race are not first generation crosses by any means, but they are not many generations removed and crossing with the pure Hottentots, the shorter parental race, is frequent. When, however, the literature on the effects of inbreeding in animals is examined one finds the greatest diversity of facts and opinions. We find the extreme views of Kraemer ('13) who states that "continued inbreeding always must result in weakened con- stitution, through its own influence" together with the equally extreme and biased opinion of Huth ('75) that in mankind there is no injurious effect resulting from consanguineous marriages which cannot be accounted for on other grounds. Crampe ('83), Ritzema-Bos ('94), Guaita ('98), Fabre-Domengue ('98) and Weismann ('04) by inbreeding mammals and birds found that the process was accompanied by decreased fertility, attended more or less commonly by lack of vigor, diminution in size, and pathological malformations. Castle, Carpenter et al ('06) inbreeding extensively the fruit fly, Drosophila, maintained fer- tility by selection, so that at the end of 59 generations of brother and sister matings in one line the fecundity was no less at the end of the experiment than it was at the start. There was some indi- cation of reduction in size of inbred flies when compared to nor- mally crossed stock flies reared under the same conditions. Fur- thermore, fertility was increased by crosses between certain inbred, lines and between the inbred lines and stock flies. From this fact and from the fact that their experiments show that the number of flies in a brood fluctuates greatly, due to temperature and food conditions, it is not positive that inbreeding was wholly without injurious effects. It is evident that their experiments do show clearly: 1. That inbreeding results in strains of unequal fertility. 2. That the occurrence of absolute sterility was pronounced in the first part of the experiment with the "A" line but almost entirely disappeared in the later part of the experiment. The figures as I have calculated them from their table I, p. 736, are as follows: Percent of matings totally sterile Generations 6 to 24 17 . 80 25 to 42 18.47 43 to 59 3.37 20 CONNECTICUT EXPERIMENT STATION BULLETIN 207. This result is to be expected on the view that inbreeding isolates homozygous individuals and these whenever sterile are, of course, eliminated. Moenkhaus ('11) and Hyde ('14) by similar inbreeding experi- ments with Drosophila have also found that sterility is increased in the first stages of inbreeding but tends to be eliminated after this process is long continued. Hyde found definite evidence that inbreeding caused reduction in size, vigor, rate of growth, longevity and fecundity and that there was a return to the normal condition on crossing. As in the other experiments Hyde found that selec- tion was an effective agent in controlling sterility. Both Whitney (12a) and A. F. Shull (12a) have shown that inbreeding and crossbreeding have considerable effect upon the rotifer, Hydatina Senta, in the size, of family, number of eggs laid per day, rate of growth and in the difficulty of rearing the animals. King ('16) has obtained results with albino rats which are quite in agreement with those of Castle. By growing about one thousand rats in each inbred generation, and selecting the best individuals for mating, animals have been carried through 22 generations of brother and sister matings without loss of size, fertility, longevity, resistance to disease and with constitutional vigor unimpaired. This writer states: "The results so far obtained with these rats indicate that close inbreed- ing does not necessarily lead to a loss of size or constitutional vigor or of fertility, if the animals so mated came from sound stock in the beginning and sufficient care is taken to breed only from the best individuals." Here, as in Drosophila, inbreeding isolates diverse types of different degrees of excellence. In this case individuals are ob- tained which surpass the original stock before inbreeding. Thus we have "Goliaths" among inbred rats as Darwin found "Heroes" in morning-glories. Castle ('16) has found that in inbred rats "races of fair vigor and fecundity can be maintained under these conditions, but that when two of these inbred races are crossed with each other, even though they have their origin in a small common stock many generations earlier, an imme- diate and striking increase of fecundity occurs." The evidence from relationship marriages in human stocks is even more conflicting and conclusions still more difficult to draw. Huth ('75) has certainly done a service in showing that consan- UNIVERSALITY OF HETEROSIS. 21 guineous marriages seldom result in the disastrous effects usually attributed to them. He has shown that incest was not a rare custom and that races which have undergone such practices are many of them far from weak. Certainly, races have practiced close intermarriage for many generations with no marked deterio- ration. The Persians, Spartans, the ruling classes among the Egyptians and Polynesians are cited by Huth in support of this assertion. The data from human matings, however, are of little value since the close unions are seldom continued many genera- tions in succession, and the results from isolated communities mean little, since often the original stock is exceedingly diverse so as to make the resulting races extremely heterogeneous in hereditary constituents. This is particularly true of the Rehoboths and the Pitcairn Islanders which are cited as instances of close inter- marrying without loss of racial vigor. Looking over the experiments upon animals it seems as unwise to expect that inbreeding may not have some deleterious effects, which, in some cases at least, cannot be overcome by the most rigid selection, as it is to hold that inbreeding must always result injuriously. It is to be expected that all breeds of domestic animals and wild species will not be equally affected by inbreeding. Domesticated animals in many cases are more widely crossed and diversified than wild species, and those characters affected by inbreeding are more accentuated. Certain wild species, which, by their mode of life, are forced to endure long periods of isolation, and consequently more or less close inbreeding, would be expected to show less change under artificial inbreeding. Finally, as I shall attempt to show that there is no longer a question as to whether or not inbreeding, in itself, is injurious, the effect which inbreeding will have on any organism depends solely on the hereditary constitution of that organism at the time the inbreed- ing process is commenced. Universality of Heterosis. From the literature on the subject of crossbreeding it is to be observed, therefore, that the occurrence of an incentive to in- creased development accompanying germinal heterogeneity is widespread, as it has been noted in plants in the angiosperms, gymnosperms and pteridophytes, and according to Britton ('98) 22 CONNECTICUT EXPERIMENT STATION BULLETIN 207. there is even some slight evidence that heterosis occurs in the sporophyte of the bryophytes. In animals the mammals, birds, fishes, insects and rotifers show the phenomenon of heterosis although in some of the unicellular animals, as we shall see later, the evidence is not so clear. I shall now take up, in some detail, experiments on inbreeding and crossbreeding in cultivated plants, principally in maize. A Theoretical Consideration of Inbreeding. Up to the present time it has been maintained that the effects of inbreeding were of two kinds, an isolation of homozygous biotypes together with a loss of a physiological stimulation which was considered to be roughly proportional to the number of heter- ozygous allelomorphs present in the organism at any time. The reduction of the number of heterozygous allelomorphs in an inbred population is automatic and varies with the closeness of inbreeding. Pearl ('15) on the basis of the number of ancestors which make up the pedigree of any individual has worked out a coefficient of inbreeding which is an indication of the degree to which that individual has been inbred. The fewer the number of ancestors the greater the degree of inbreeding which may vary from no inbreeding, in which no one ancestor appears more than once in the pedigree of an individual, to the closest kind of inbreeding in which no more than one ancestor is concerned in any one generation in the production of an individual (self-fertilization). The latter degree is only approached by hermaphroditic plants and animals, which are capable of self-fertilization and in function- ally bisexual animals and plants by brother and sister matings. This statement of inbreeding must, of course, leave out of con- sideration any germinal change which might take place by means other than hybridization and as Castle ('16) has pointed out is modified by the differences in heterozygosity of the ancestors making up the pedigree. The automatic reduction in the number of heterozygous allelo- morphic pairs in an inbred population, by self-fertilization, follows the well known Mendelian formula by which any hetero- zygous pair forms in the next generation 50 percent homozygotes and 50 percent heterozygotes in respect to that pair. Since the homozygotes must always remain homozygous and the hetero- zygotes are halved each time and one half added to the homo- A THEORETICAL CONSIDERATION OF INBREEDING. 23 zygotes the reduction in the number of heterozygous elements proceeds as a variable approaching a limit by one half the differ- ence in each generation. The curve illustrating this condition is shown as No. 1 in Fig. I. Various formulae dealing with 100$ Percent of Heterozygous Individuals in Each Selfed Generation when the Number of Allelomorphs Concerned Are: 1,5,10,15. 10 Segregating Generations Figure I. The percent of heterozygous individuals and the percent of heterozygous allelomorphic pairs in the whole population in each generation of self-fertilization. inbreeding have been discussed by East and Hayes ('12), Jennings ('12, '16), Pearl ('15) and Bruce ('17). It should be remembered that this reduction applies only to the whole population in which every member is inbred and all 24 CONNECTICUT EXPERIMENT STATION BULLETIN 207. the progeny grown in every generation. In practice, in an inbreeding experiment, only one individual in self-fertilization or two individuals in brother and sister matings are used to produce the next generation. Thus the rate at which complete homo- zygosis is approached depends on the heterozygosity of the individuals chosen. Theoretically in any inbred generation the progenitors of the next generation may either be completely homozygous or completely heterozygous or any degrees in between depending upon chance. The only condition which must follow in self-fertilization is that no individual can ever be more hetero- zygous than its parent but may be the same or less. Thus it is seen that inbreeding, as it is practiced, may theoretically never cause any reduction in heterozygosity, or it may bring about complete homozygosity in the first inbred generation. In other words the rate at which homozygosity is approached may vary greatly in different lines. However, as the number of heter- ozygous factors at the commencement of inbreeding increases the more nearly will the reduction to homozygosity follow the curve shown because the chance of choosing a completely homozygous or completely heterozygous individual in the first generations will become less. In Table 1 is shown the theoretical classification of the progeny of a self-fertilized organism which was heterozygous with respect to 15 independent mendelizing units. . It will be seen that the bulk of the individuals lie between classes 6 and 11 where none of the members are heterozygous for more than 10 factors nor less than 5. In other words any individual selected for the progenitor of the next generation would probably come from the middle classes and therefore it would be heterozygous for about half the factors that its parent was. The chance that this individual would not come from the mid-classes between 6 and 11 would be about 1 out of 10. The chance that it would be completely homo- zygous or completely heterozygous would be 1 out of 32,768. If 20 instead of 15 factors were concerned the chances would be 1 out of 1,048,576. This condition by which the progenitor of each generation tends to be half as heterozygous as its parent holds true for any number of factors and in every generation. Also in Table 1 it can be seen that the progeny as a whole has an equal number of heterozygous factor pairs as homozygous factor pairs in respect to those A THEORETICAL CONSIDERATION OF INBREEDING. 25 H W « o w Q o W t> H i-l & o H > M fc W *H h In |x| w « h o y 15 M o £ o "izi u H t— 1 << H P5 M ffl g -i < in" H H « fn U H )H P H W W H i-i w 1-1 n «i H a O 1 m ;,t >> ra § J5 o X o .." m g >> ft OiOOiOOiOOifOiOOOOiCOiO C '-> j3 CJ t-h>— icDriD>— iCO'^00-*CO'-HCDcD^Hi— i g^ ^ o3 NM-#OOO^OOOt)(MN -d 0 § o ' c 'fl a 6 1 HIOICOWHIOCWOH m -c> ca .a i-H CO - 43 a a £ hD 'cj ioO"ooiooLcomoioo>ooLoo ^^ >> a HrtOOrHCO^OO'^COrHtOCO'-lrH 1 1 NCO^OOO^OOO^MIN «£ fl -3 HlOlOOl^HlPOCIOH Th-!N ,- a 1— 1 CO ■* lO "HH CO r-H !N +3 m o o W "" +3 -^ rj fcH Jp a O M >> 2 t3 O B OHNM^'OONKffiC'HNMTtHO IQ PI o I-H +3 QJ o -a 03 -t= W °l - £ is £ a ■So03 M 6 ■** g >> 3 -m £ lOT^CON'HOaoONOiO'^COlMr-iO LO pj « s ^ O T— H r^ T—l i-H ^M t— 1 1— 1 ■ ■ H^OltDHHtDO'H/H & ' o s -S HIMNH ii a c ft o 13

1— 1 ° '> S C a Eh ra 1—1 -1 '-HtMCOTfitOcOt^GOasO'-HtMCO^iCcD 26 CONNECTICUT EXPERIMENT STATION BULLETIN 207. characters in which the parent was heterozygous. So it is that in practice the reduction in growth accompanying inbreeding (on the assumption that heterosis is correlated with heterozygosity) is greatest at first, rapidly becomes less and finally ceases for all practical purposes. If there were no deviating factors the curve of reduction should, in the majority of cases, approximate curve 1 in Fig. I. However, it has never been assumed that the amount of heterosis was perfectly correlated with the number of heterozygous factors. Moreover, since the heterozygous individuals are more vigorous than the homozygous, selection, either unconscious or purposeful, would favor the more heterozygous so that the tendency might be that the actual approach to homozygosity would not proceed at as fast a rate as the theoretical curve would indicate. Self-fertilization is the quickest and surest means of obtaining complete homozygosis for the reason that whenever any pair of allelomorphs becomes homozygous it must always remain so long as self-fertilization takes place, whereas in brother and sister mating a homozygote may be mated to a heterozygote. Thus we see from Jennings' ('16) tables that 6 generations of self- fertilization are more effective than 17 generations of brother and sister matings in bringing about homozygosis. The reduction in heterozygous allelomorphs in a population as a whole follows curve 1 in Fig I irrespective of the number of factors concerned, provided, as stated before, that a random sample of all the different classes of individuals are selfed and used as progenitors for the next generation and that there is equal productiveness and equal viability. If the heterozygotes are more productive, as in many cases they are, the reduction to complete homozygosity will be delayed. The number of completely homozygous individuals in any gener- ation, inbred by self-fertilization, differs according to the number of heterozygous factors concerned at the time that the inbreeding process is commenced. The curves showing the reduction in the number of individuals heterozygous in any factors, where, 1, 5, 10 and 15 factors are concerned at the start are given in Fig. I calculated from the formula given by East and Hayes ('12). The curve for the reduction in heterozygous individuals, where one factor only is concerned, is identical with the curve showing the reduction in heterozygous factors in an inbred population RESULTS OF INBREEDING. 27 where any number of factors are concerned. In any case almost complete homozygosity is reached in about the tenth generation on the average, although theoretically it may be reached in the first generation, or may never be reached when a single individual is used in each generation to perpetuate the line. Assuming, then, that the loss of the stimulation, accompanying heterozygosity, is correlated with the reduction in the number of heterozygous allelomorphs we should expect to find the decrease of heterosis greatest in the first generations, rapidly becoming less until no further loss is noticeable in any number of subsequent generations of inbreeding, and that, on the average, the loss will become negligible at about the eighth generation and from then on no further marked change will take place. Some cases are to be expected in which stability is reached before this generation and some cases in which it is not reached until later or may even theoretically never be reached. With these assumptions in mind let us see what are the actual results of long continued inbreeding in maize. The Results of Inbreeding the Naturally Cross-pollinated Maize Plant. The behavior of maize during six generations of inbreeding by self-fertilization has already been reported by East and Hayes ('12). The same inbred strains have been continued and in some cases the results up to the eleventh generation are given here. In the previous publication it was stated that a loss of vege- 'tative vigor has followed every case of inbreeding in maize. Some plants had been obtained which were unable to reproduce themselves. Those strains which were maintained became uni- form but differed considerably from each other. It was con- sidered at the end of the period of inbreeding that some strains were appreciably better than others in their ability to yield. Six additional years of inbreeding with this material has confirmed, in the main, these conclusions. A further appreciable reduction in productiveness, however, has taken place in all lines together with certain changes in various parts of the plants. The original experiment began with four individual plants obtained from seed of a commercial variety of Learning dent corn grown in Illinois. This variety was given the number 1 28 CONNECTICUT EXPERIMENT STATION BULLETIN 207. and the four plants which were self-pollinated and selected for continuation of the inbreeding experiment were numbered 1-6, 1-7, 1-9 and 1-12. These four strains were continued each year by self-pollination. In the second inbred generation two self- pollinated plants in the 1-7 line were saved for seed and from them two inbred lines were split off which therefore came originally from one line inbred two generations. These are numbered 1-7-1-1 etc, and 1-7-1-2 etc. In a similar way these, and the other inbred lines, were further split up in subsequent generations. After the experiment was started with the dent corn inbreeding was commenced with other material. Two inbred strains of floury corn, Nos. 10-3 and 10-4, originally from the same variety, have been maintained and also two strains of flint, Nos. 5 and 29, and two strains of popcorn Nos. 64 and 65. Chief attention has been paid to the inbred strains of Learning corn (the longest inbred) and most of the data presented here have resulted from this material. Many other varieties besides these have been inbred for many generations in connection with other investi- gations and while they are not specifically mentioned the observa- tions as a whole include these. In Tables 2 and 3 the yield and height of some of these inbred strains are given. In 1916 seed of the original Learning variety was obtained which had been grown in the meantime in the same locality whence it was originally secured and was grown for comparison with the inbred strains. This variety in Illinois in 1905 yielded at the rate of 88 bushels per acre, and in Connecticut in 1916 at the rate of 74.7 bushels. While there is no proof that any change has not taken place in the original variety there is no reason to suppose that it has changed to any great extent. Grown under the same conditions in 1916 the four inbred Learning strains yielded from one-third to one-half as much as the original non-inbred variety. With regard to rate of reduction in yield or the constancy of the varieties during the later generations it is difficult to draw conclusions from these figures owing to the fluctuation in yield from year to year due to seasonal conditions and to the difficulty of accurate testing in field plot work, which is recognized by all who have made such tests. As was stated in the first report the yields for 1909 were too low and in 1911 much too low on account of poor seasons. No yields were taken on any of the strains in RESULTS OF INBREEDING. 29 Table 2. The effect of INBREEDING on the yield and height of maize. Year grown No. of genera- tions selfed Four inbred strains derived from a variety of Learning dent corn. 1-6-1-3-eto. 1-7-1-1-eto. 1-7-1-2-etc. 1-9-1-2 -etc. Yield bu. per acre Height inches Yield bu. per acre Height inches Yield bu. per acre Height inches Yield bu. per acre Height inches 1916 1905 1906 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 0 0 1 2 3 4 5 6 7 8 9 10 11 74.7 88.0 59.1 95.2 57.9 80.0 27:7 117.3 86.7 74.7 88.0 60.9 190759 . 3 190846 . 0 63.2 25.4 111.3 81.1 74.7 88.0 60.9 190759 . 3 190859 . 7 68.1 41.3 117.3 90.5 74.7 88.0 42.3 51.7 35.4 47.7 26.0 191338.9 191445 _ 4 191521.6 19l630.6 191731.8 117 3 76 5 41.8 78.8 25.5 32.8 46.2 96.0 97.7 103.7 39.4 47.2 24.8 32.7 42.3 85 0 83.5 58.5 88.0 78 82 7 84.9 78.6 19.2 37.6 86.9 83.8 4 Table 3. The effect of inbreeding on the yield of maize. Two inbred strains of One inbred strain of floury corn flint corn Year No. of genera- 10-3-7-ete. 10-4-S-etc. 5-8-6-etc. grown tion's Yield Yield Yield " selfed bu. per bu. per bu. per acre acre acre 1908 0 70.5 70.5 75.7 1909 1 56.0 43.0 47.5 1910 2 67.0 48.7 36.1 1911 3 39.1 29.3 11.5 1912 4 1913 5 32.2 49.5 30.4 1914 6 52.6 38.1 1915 7 1916 8 13.9 16.6 18.3 1917 9 26.6 24.0 30 CONNECTICUT EXPERIMENT STATION BULLETIN 207. 1912. The yields in 1914 are too high and in 1915 too low for the- same reasons. Also in 1915 the yields are unreliable because only a few plants were available to calculate yields from as most of them were used for hand pollination. During the last three years of the test samples of corn have been dried to a uniform moisture basis and the yields calculated to bushels of shelled corn per acre with 12 per cent, moisture. This has probably had a tendency to reduce the yields somewhat as these inbred strains are very late in maturing and consequently contain large amounts of water. With these points in mind an examination of the table shows that from the beginning of the experiment to the ninth generation there has been a tremendous drop in productiveness, so that in that generation the strains are approximately only one-third as productive as the variety before inbreeding. From the ninth to the eleventh generation there has been at least no reduction in productiveness, and practically no change in visible plant or ear characters. In the previous publication it was stated (U. S. Dept. of Agric, B. P. I. Bull. 243, pp. 23-24) that " strain No. 6, is a remarkably good variety of corn even after five generations of inbreeding. It yielded eighty bushels per acre- in 1910. The yield was low in 1911, but since all yields were low that year it can hardly be doubted that this strain will continue to produce good normal yields of grain The poorest strain, No. 12, is partially sterile, never fills out at the tip of the ear and can hardly exist alone. In 1911 it yielded scarcely any corn but will no doubt continue its exist- ence as a partly sterile variety." These statements will have to be modified somewhat. Although No. 6 is, in the eleventh generation, still the most vigorous inbred strain, as a producer of grain, however, it can hardly be considered to give " good normal yields." The plants, nevertheless, are perfectly healthy and functionally normal in every way except for an extreme reduction in the amount of pollen which they produce. The strain No. 12 was lost. Since the difficulty of carrying along any inbred strain is very great owing to failure to pollinate at the right time, attacks of fungus on the ear enclosed in a paper bag, and poor germination in the cold, wet weather common in New England at corn planting time, the loss of this strain might be easily accounted for without supposing that it RESULTS OF INBREEDING. 31 -simply ran out. It may be that this strain could have been perpetuated if sufficient effort had been put forth to do so. In view of the further reduction in the other strains, however, the maintaining of. this strain would have been extremely difficult. Complete records on the height of plant are wanting for many of the generations, and, unfortunately, in the first part of the inbreeding period. What figures are available certainly show that very little change in height has taken place in all four strains during the last seven generations. Strain No. 6 has increased in height, if anything. Height is less affected by environmental factors than is yield and in that respect is a more reliable indicator. However, great changes in the structure, size and productiveness may take place without height of plant being greatly altered. , From the figures given in Table 2 there is some evidence that these strains have reached about the limit of reduction in pro- ductiveness and that there has been very little change in the last three years. This, however, is not proven. The continuation of inbreeding is necessary for conclusive evidence on this point. As the crosses between individual plants within these inbred strains have given very little increase over the selfed strains, as will be shown later, and from the fact that almost no visible change has taken place in these four strains during the past three years that I have had them under observation, it seems apparent to me that the reduction in vegetative vigor and productiveness is very nearly at an end. Jn Tables 4, 5, 6 and 7 are given the frequency distributions of height, length of ear, number of nodes and the number of rows of grain on the cob of the original, non-inbred Learning variety and several inbred strains derived "from this variety after nine or ten generations of selfing. All the plants from which the data were taken were grown on the same field in the same year. Four different plots of the variety were grown in different parts of the field and the data on these plots are given separately and totaled in the tables. It can be seen from these that.no great variations in range, mean, standard deviation or coefficient of variability were caused by environmental factors. The pedigree numbers show the relationship of the several inbred strains to each other. From these tables it can be seen that both height of plant and length of ear have been reduced, but in different degrees in different lines. In some strains reduction in height amounts to 40 inches and in length of ear to 3.5 inches. The reduction in CO t*< lO CC- OS lO >C lO lO C^l o t- co cm. CM CM --h CO ■* t- CO CD CO CO CO lO CN CM 00 H tH CO 41 41 -H -H -H « CO O CD tM CN 41 4) O 03 41 41 CO «* Is- o oo oo oo oo cc -# -* co >o o o m t-- Tf T^ lO G i1 CO CO CD CO CO CO CO f-i CO CN CM rf CO 41 41 41 41 41 41 41 41 41 41 41 41 41 -H 41 41 41 o o m o co in lO W lO lO lO ITS m US 1 05 -* co CN OS O t- oo ■* to h SfflO t» N ># t- 41 41 41 41 41 O CM CO CO CO 41 41 41 41 »NON 41 41 41 41 U3 CM IN t- 41 41 CO CM 41 41 O CO -# es co a Is- 00 CM O 00 t- 00 OS 00 CM i-l Is- O CM 00 CO 00 U5 "5 "O CO Q o a « ~H H OM * CM lO CO CO CO CO CO Is- 00 i-l CD i-l CD O CO as oo cs co cm 1-h o oo oo Is- N if m N M io -*-*•* r~ CM rH o Is- CM lO rH rH rH CM CD t> m oo oo Tf lO OS CO Ol 00 CD co oo rH co OS CM CD CM 00 rH f O 00 rH • rH rH rH CM O o » J j CO ~4 rH rH rH ri ?i Tl rH CO *JH IH w co to rH t- lO ■* rH ■<*< w Tt< ■* no m CM CN ** -# rH rH CO CO CM cn o «5 -* ■* -r f Is- Is- t» i> CN CN l> t^ ■* -r -Is -* ■* •# ** Tj< OS OS to to TH -" H. •* rH rH rH rH CN CN »* -* CO CO CO co ■v 7 ^ ■7 CN (N CN CN °? to to to t". t- ti Is- t~. t- os OS RESULTS OF INBREEDING. 33 CO tJH IO 00 CD CO 00 CM iO iO CO ^j 1-1 (M t>o Oo0 g NiOOOJCD HCOCBO OS CO 10 CO I>CM CS IO < i— i i— I i— 1 H i-H 1—1 1— 1 1— 1 1— 1 1—1 1—1 En > -H -H -H -H -M -H -H -H -H -H-H-H-H ■u-n ■ti-H CO d O CD ■* OS b- i> co t> co 00 O 10 CD co-* l> CM Q OCOINH O HOHN oOnn co O l> CO fa CS CM !> iO OS lr^ co co co iO O CM OS coco COOS P5 HNrtrHH ^-HMrH i-h CM CM ^H M I-H ■^ 00 i— i 00 00 iO NiCHN ■*cDON OS (-- CO tHH O^h OO O OOi-iO OOOO 00 00 Q 0 ■U-H-H-H-H ^^4i^ 4^414] -H-tt ■H-H 02 MhhOCO lOHN CM t- T« i-H CO OS IO ^H »0 CO CD CO CM t(h OOOlOrt CO 00 OS 00 l-H 00 00 LO fa ,-i^^h ^^_, ,-H T-Hi-H ,-H 5 g (NiQHOtO OS b- CO O co 00 00 cs CM OS 00 10 fa i— i i— I i— I i— i O O O^H >"H 000 c i-H C 0 0 o «i -H-H-H-H-H -H 4H 4H -H ■ti-H-H + ^ + ^ O CM CO CS »0 HNOO CO CM O CM i-H CO i-H OS fa t>t> t-t> b- CO CO CD CD Tt* "^ TH t+ iO IC co 10 % 1> i— 1 i— I OS 00 CI 00 CD CD CM CD r~- CO COCN LO OS a fa iC iO CD iO CM CM lO IO Ttl lO CO to iO CO •^ -si" IO IO n (M i—l ... l—l £ o 3 OH ^H -CM < £ fa ^ o i— i co co co o ^ 1— 1 P5 K i— i co 1— 1 1— 1 fa fa o t> o a 00 CO i— 1 OS GO i-H i-H OO i— 1 i— 1 i— 1 i— 1 CO 1—1 1 — 1 w « a M fa u O (M CS THiO OS OS 00 »0 ^hh r^ CS LO cj NHHHC5 CM — 1 CM fa O CO CM05I> tJHCM CO CS 1> CD ,_, ,_ ■* a iO tH fa A i—i co 1-1 CM CM •<* O o 1 NOi-iHin 00 I> 1— t id 00 cm 00 -^ i-H LT 00 os o P-H 1— 1 1-1 CM CM i-h 1- ^H^ EH [ • CM CM i-h »C U3 CM 1> CO t^o l-H O fa CO CM 1— 1 1— 1 i-H i-l !M O ^H CV P T}H 1— 1 IO CO CO T-ll> It ) -HH ,- ( T)H CO © fa r* thh to lO (M C<) 4h t 1— 1 1- H CO CO W CM CM CM CM lOirsiOir ) l-H 1- H XO IO fe • £ S "0 tHH Tf 1^ *HH t> t^ t^I> (M Cn 1 I>-1>. id to -9 — = , 05 p-2.5^ •»H — — — 1 CJC'T1 S 05 1 1 1 1 TJH ^HH ^* tHH 1 1 1 1 t4^ ^ Tf " c 1 1 1 1 1 1 n < 1 1 1 1 CO CO CO CO 1 1 1 1 1 1 os os H 34 CONNECTICUT EXPERIMENT STATION BULLETIN 207. H H « 0 w PH pq fc * c fc «! H ft n s DQ o w M o ft c 'A c ft ft i> o a « w ft G a 3 ft t-> H fc < « ft H OS H ft C fc o H t= pq Ph H ft p * CO CO iO COCO CO 1> COTJHCOcOTflCOLOGOb- I> i-H t^ Oi CI GO CO CO O CO OrJH CO i— i i— i i— i i— i O CO id GO CO oooo NOt-O anon O GO OO b- CD OO ■ti-H-H-H-H CO GO Tt* GO tH -H -H -H -H j>iot*co -H-H-H41 GO CO CO i> 4141 i-hO OS i—l ■^coTt< co-* NHNN T-H i-H CO CO co co (MCO 01 HHirlHTf i— I i— I lO CO CO i— I 13 1-2 O o COCMiHH l l l l -hh ^« ic>iO CO CO r«-* i i i i (DHNO i i I CO COCO (N I I i I T^H tHH ^H rH/t I I I I TjH -HH T^ -HH I I I I ^* "HH tH ^1 I I iO lO iO >0 t-h tH J>1>1>.1> CO CO I I I I II "*cH "o HIO CO CO CO i— I CO lO CO O CO CO t-h lO >0 CO TfH t-h COCO CO TH 1— I T-H COCO • i-H •© CO 1> T-H T-H i-H i-H CO CO I I I I RESULTS OF INBREEDING. 35 s < w s H M H s fc n O « 1*. !5 o ■ O Si In a « h H o « 0 n 3 P a o H fc W t-H 00 41 41 41 -H oooooh 0000000000 CD LO ■* ■* 4141 -H-H OS OS lO ■* lO iO iO IC COMMON "^OOt^CO CM CO lOtDHO 00 O CO ■<* IO l> IO i-H CM i-H CO>0 rh -O _ o> u o o3'fnr3 w CM > o MNHrt i I I I I I I I NNNO I I I I "# ■* •<* T^ I I I I Tt^ Tt** Tt^ Tf till TH Tf ^t* Tt* I I I I CO CO CO (TO CM CM 4i tH i i i i 10 0 10 10 I I I I I I I I T^l Tf^ Tt^ TH I I I I I I I I I— I I— I I— I 1— I I I I I I I i— 1 1— i co co ii ii i-H i— i io m ii ii OS OS OO CM CM Tt< 4* II II CM CM CM CM 36 CONNECTICUT EXPERIMENT STATION BULLETIN 207. length of ear is even more than it seems from this table because the variety contained plants which produced two ears of which the second is usually smaller than the first; whereas the inbred strains almost never produce more than one ear to a plant. The number of n6des per plant is reduced but as compared to height and length of ear this reduction is very much less. In the number of rows of grain on the cob there is a reduction in some lines and an increase in others. These tables show in the clearest manner that inbreeding has a greater effect on some characters than on others, and that segregation of characters has occurred. Perhaps the most noticeable effect of inbreeding as shown by these tables is the reduction in variability as brought out by the range and statistical constants. This reduction in variability is most apparent in the characters which are the least reduced by inbreeding — number of nodes and number of rows of grain on the ear — although the low variability is also apparent in height and length of ear. In variability, also, there is a difference between different lines. The variability in height and length of ear of the inbred strains is higher than it should be, owing to the fact that it was difficult to obtain a perfect stand of plants, on account of poor germina- tion of the seeds of the inbred strains. The aim was to have three plants in a hill. From four to eight seeds were planted as far as a limited supply of seed would permit, and later, thinned to three plants. In spite of this precaution it was extremely difficult to get anything like a perfect stand, so missing plants were replanted as soon as possible. These replants, owing to their late start, never entirely caught up with the other plants and are shorter in height and have smaller ears in consequence. It is unfortunate that this practice was followed because it is believed that much more reliable results would have been obtained otherwise. On the other hand missing plants introduce another source of error — that of unequal opportunity to grow. Because there was abundant seed of the variety, and it germinated well, practically complete stands of these plants were obtained. The reduction in variability is more apparent in the details of the structure of the plants and ears which cannot be expressed statistically. The beautiful uniformity of these plants in all characteristics at the present time is one of their most striking features. This can be seen fairly well in the accompanying photographs. (Plates I to V). RESULTS OF INBREEDING. 37 In view of this fact of great uniformity and constancy as a result of inbreeding one is astonished at the statement made recently by Stout ('16) in a discussion of the results obtained from inbreeding in maize by East and Hayes. Stout says (pp. 420-421) : ' "strains similar in homozygosity show widest variation indicative of spontaneous variation in natural vigor which is suggested that in such highly cultivated varieties such as corn extreme sporadic variations may be constantly occurring, a condition which is well sho.^n by the numerous and well-known results of the ear to row test." Several curious misconceptions are to be noted in this statement. In the first place, it has never been maintained by anyone to my knowledge that an equal number of generations of inbreeding produce an equal amount of homozgosity in different lines. Secondly, it has never been proposed that the degree of heterozygosity determined the form or structure of any organism, but that such a condition was accompanied by a stimulus to development which merely increased the expression of many hereditary factors. This stimulus is considered to be without any great effect in itself on variability. Granted that the inbred strains were equal in homozygosity at that time, that was no reason why they should be similar in vigor or in any other respect- — in fact the expectation is exactly the reverse of this. With regard to "spontaneous" and "sporadic" variation these inbred strains show unmistakably that there is practically no sporadic or spontaneous variation, that the indi- viduals making up an inbred strain are remarkably constant and uniform after some degree of homozygosity is obtained and that the diversity between different lines can be perfectly accounted for on the basis of segregation of characters. Also, in the following paragraphs in his paper Stout fails to see the distinction between crosses of diverse inbred lines and between crosses of non-inbred commercial varieties. Because Collins ('14) and Hayes ('14) failed to obtain increases in all crosses between commercial vari- eties of similar type Stout would question whether crossing in maize was ever beneficial. It is quite to be expected that there are many varieties already so widely crossed that further crossing does not result in greater heterozygosity, but may even reduce it. It is only in crosses between somewhat different varieties, like flint and dent (Jones and Hayes '17) or between varieties from 38 CONNECTICUT EXPERIMENT STATION BULLETIN 207. different geographical regions (Collins '10) that any great amount of heterosis in naturally widely crossed varieties is to be expected. Although there has been a striking reduction in size of plant, general vegetative vigor and productiveness in these inbred strains of maize, and in comparison with non-inbred varieties the inbred plants are more difficult to grow, emphasis must be put on the fact that the plants are normal and healthy. The monstrosities which are common in every field of maize, such as the occurrence of seeds in the tassels, anthers in the ears, dwarf plants, completely sterile plants, mosaic and albino plants and other similar anomalies never appear in these inbred strains. Furthermore, in the details of the size, shape, structure and position of the tassels, leaves, stalks and ears, these inbred strains show the most striking uni- formity. These minor details which characterize each of these groups of plants are difficult to describe but are perhaps the most noticeable feature about them. The stalks, the tassels or the ears of all of these four Learning strains if mixed together could be separated without the slightest difficulty by anyone familiar with them. Some of the differences which characterize the ears of these four strains are shown in Plate lb. It is to be noticed in this photograph that Nos. 1-7-1-2 and 1-7-1-1, which were originally from the same line, both have flat cobs. In one of them, however, it is colored, in the other uncolored. Other differences are to be seen in shape and color of seeds. The segregation of row number accompanied by a reduction in variability in these two strains is shown in Table 8 and Fig II. Data previous to the third generation are not available but since then a noticeable change in average row number has taken place without any selection one way or the other. The variability of each line has decreased at the same time. Whether the increase in variability, after the eighth generation, has any significance is not known. It is possibly due to the fact that both lines have become irregular in row number so that the correct determination of the row number has been rendered more difficult in the later generations. Also the number of plants grown in the generations from the 7th to the 10th are much too few to base accurate con- clusions upon. The sharp increase in average row number and decrease in variability in the 8th generation are probably due to the unusually favorable growing conditions of that year. KESULTS OF INBREEDING. 39 O T-H 00 CD lO OS OOxJH too • (M00 ON CD lO i-H > 6 1—1 ■H : 0 • 0 • -H-H COM 4141 : CO tH OS CO : 4]4i CO CD CO t-H +1 -H (NCO 1>00 ■H-H 0 co os co 4141 T« O OSTfH -H -H iO OS t-H CO T-H • CD 00 00O3 OSO r-H 0 • CO • (M 10 lo -H -W t- T-H -H -+H T-H OS (MCS OS • OO 000 M T-H OS IC i-H i-H HN 000 OIM i-H (M •CC • T— > t-H (M CO lO -ct~ 1 II II II II Genera- tions selfed. co c 3 **"* 1O1S CD CD 1>I> ooa D OSC S OO i-H t-H i-H i-H r-H i-H t > 0 u M j c c 1 0 ■> £ 3 C D OO D T-H T-H "S OS OS >-H i-H nJ « O 3 Ave. No. Rows of 1-7-1-1 Ave . No . Rows of 1-7-1-2 Generations Inbred Figure II. The reduction in variability and segregation of number of rows of grain on the ear in selfed strains of maize. East and Hayes ('12) have noted many characters which are isolated from maize by inbreeding. In addition to these, several other characters have been isolated in this and in other material. One of these characters is a constant difference in shade of color of the foliage — some are dark green, others are light, yellowish green. Some strains are lacking in root development and never stand upright throughout the season. Some have a single-stalked unbranched tassel, while others are profusely branched. Some strains have' peculiarly wrinkled or wavy leaves, particularly noticeable in the first leaves. Some strains produce a small pro- portion of connate seeds similar to those observed by Kempon ('13) in nearly every ear, while their occurrence has never been observed on other inbred lines derived from the same source. There are also marked differences in susceptibility to disease as will be shown later. These illustrations are sufficient to demonstrate beyond doubt that by far the greatest amount of the fluctuating variability found among ordinary cross-fertilized plants is due to the segrega- tion and re-combination of definite and constant hereditary factors. Many of these characters are seldom seen in continually cross-pollinated plants, and never are so many combined together. RESULTS OF INBREEDING. 41 This is due to the fact that they are recessive in nature and com- plex in mode of inheritance. The most significant feature about the characters which make their appearance in inbred strains is that none of them can be directly attributed to a loss of a physio- logical stimulation, although undoubtedly many of them may be modified by the vigor of the plant upon which they are borne. There is no one specific character common to all inbred strains but simply a general loss of vigor, a general loss of size and of productiveness accompanied by the appearance of specific char- acters more or less unfavorable to the plants' best development but these unfavorable characters are never all found in one inbred strain, nor is any one character common to all inbred strains. Probably the most common result of inbreeding in maize is a reduction in the amount of pollen produced. This becomes appar- ent in a smaller size of all parts of the tassel, in shrunken and abortive anthers which are often never released by the glumes, with a consequent reduction in the amount of pollen available for fertilization. A normal corn plant should produce, on the average, anywhere from lcc. to lOcc. or, in some cases, very much more pollen. I have made no actual measurements of the amounts pro- duced. Many of the inbred strains, however, now. produce only a small fraction of a cubic centimeter of pollen, and the production of this small amount is much affected by weather conditions, so that many strains, otherwise well developed and productive, are maintained with the utmost difficulty. It has been my experience that self -sterility in corn is due to ovule or pollen abortion. Whenever pollen is obtained it seems to be able to function. Failures to obtain seed after pollen is ap- plied are common, but are usually attributed to external factors. At least I know of no clear case where pollen is produced in which it fails to fertilize the ovules of plants which were capable of being fertilized by other pollen. Many cases of complete abortion of the pistillate part of the plant must occur, as many plants are lost through failure to set seed when good pollen has been applied. Just where the trouble lies is not always possible to detect. Un- doubtedly, many cases of complete abortion of either staminate or pistillate functions, or both, occur during inbreeding, and the plants are eliminated for that reason. Reduction in the amount of pollen produced is less serious than a reduction in the number of ovules, as a very small amount of 42 CONNECTICUT EXPERIMENT STATION BULLETIN 207. pollen suffices for fertilization when conditions are right. For that reason unconscious selection for good ovule production has been much more rigid than for pollen production. That is the reason, I believe, that more inbred strains now show a greater reduction in the staminate function than in the pistillate. A significant feature of the effect of inbreeding upon sterility is that some inbred strains are perfectly normal in their production of pollen, and the amount of pollen produced is only a little less than non-inbred plants, owing to the reduced vigor and size of the plants which produce the tassels. Out of about twenty-five inbred strains carried through at least seven generations, three of them are perfectly normal in the structure and function of their staminate parts. One of the Learning strains (No. 1-9) produces more pollen than many non-inbred varieties growing nearby. In every case, however, those plants which -produce the best devel- oped ears are the poorest producers of pollen, and those strains which produce abundant pollen have ears which are poorly de- veloped. In other words, inbreeding is bringing about a tendency for maize to change from a functionally monoecious plant to a functionally dioecious plant although, morphologically, both staminate and pistillate parts are still present. This is illustrated in Plates VI, a and b, where tassels and ears of four of the inbred strains are shown. Although no systematic selection has been practiced throughout the inbreeding experiment a great deal of selection upon many characters has been unavoidable as it is unavoidable in any in- breeding experiment. In maize, the difficulties of hand pollina- tion result in the selection of plants whose staminate and pistillate parts are matured synchronously. Any great differences in this respect, particularly towards proterandry, would render self- fertilization difficult or impossible, as pollen, according to Andro- nescu ('15) has very short viability, which fact my own experience confirms. Of course, all plants which are weak, sterile, diseased or in any way abnormal tend to become eliminated wherever these causes reduce the chance of obtaining seed. This uncon- scious selection becomes more rigid as reduction in vigor and pro- ductiveness increases in the later generations of inbreeding. The small amount of seed produced by hand pollination, under the most favorable circumstances necessitates the using of the best RESULTS OF INBREEDING. 43 ears obtained for planting in order to have enough plants upon which to make any fair observations. In every case inbreeding in maize has so far resulted in a reduc- tion in size, vigor and productiveness. Some thirty or forty inbred strains have been observed, many of which are additional to the ones reported previously. From the preceding statements in regard to the effect of in- breeding it can be said that this process produces types which differ in their power of development as follows: 1. Plants which cannot be perpetuated. 2. Plants which fail to complete normal development and can be propagated only with the greatest difficulty. 3. Plants which are perfectly normal but varying in the amount of growth they attain at maturity. These normal inbred plants, so far obtained in maize, are not as a rule as large, vigorous or productive as the original cross- fertilized plants. It is theoretically possible to obtain such plants, which cannot be reduced in vigor in a homozygous condition as will be explained later. There -is some evidence from the experi- ments of Darwin, that such plants have been obtained by in- breeding in other material, for example, in Ipomea and Mimulus. Selection will help to obtain these vigorous, unreduceable indi- viduals but may not be fully effective in doing so. More or less unconscious selection is unavoidable in any inbreeding experiment. These homozygous, normal, inbred strains, after the reduction in growth has ceased, are quite comparable to plants of a naturally self-fertilized species. Darwin found that self-pollination caused no reduction in vigor in Nicotiana, Pisum, Lathyrus, Phaseolus and other genera which are naturally self-fertilized to a large extent. Hayes and Jones ('17) have found similar results with the tomato. The only effect that inbreeding may have on such plants is merely to isolate pure lines, which are quite uniform among themselves, but may be diverse from one another, as shown by soy beans (Jones and Hayes ('17), but which show no reduction in vigor on continued artificial inbreeding. These results are perfectly in accord with Johannsen's genotype con- ception. 44 connecticut expeeiment station bulletin 207. The Approach to Complete Homozygosity. It now remains to be seen whether or not these inbred strains are reaching the limit of reduction. There are two ways of de- termining this, one is by growing two successive inbred generations side by side in the same year, the other is by crossing different plants within the same inbred strain. In Table 9 the results from two successive generations grown side by side in the same year are compared. On the whole, an additional year of inbreeding after the sixth produces very little change. In Table 10 are given the height, yield and length of ear of selfed and sib-crossed plants which were grown in 1917. In 1916, in each of the strains of which figures are given in the table, some plants were selfed and some were crossed by another plant within the same strain. Since all the plants grown that year in any one strain came from one individual of the preceding genera- tion, that generation is the significant one. In other words if the plant in that generation was homozygous, no increase of the sib-crossed plants over the selfed plants would be expected. The figures show that there is, on the whole, a slight increase in all the characters studied. The increase, however, is no greater in the cases where the common ancestor was inbred for seven genera- tions than in the cases where it was inbred nine generations. Shull ('11) compared sib-crosses with selfed plants in which the significant generation, as I understand it, was the fourth, and found that the crossed plants slightly excelled the selfed plants in height, number of rows on the ear and yield of grain. Similarly the Fi X Sibs exceeded Fi X self in yield, showing that in the* fourth generation complete homozygosis had not been attained. Whether or not complete homozygosis has been attained by some or all of the strains shown in Table 10 cannot be stated positively from the data given. In most cases the increase of the sib-crosses over the selfs is slight and probably of no significance as there are about an equal number of cases in which the reverse condition is shown. A few of the sib-crosses are, however, con- siderably greater than the selfs in all three characters and it may very well be that these strains have not attained the degree of homozygosity that the other strains have. More data are needed to establish this point with certainty as environmental factors which favored a certain plot in one character would also favor the other character as well. THE APPROACH TO COMPLETE HOMOZYGOSITY. 45 o m o p H « a o H * s H M fa H M * H CQ fa P9 O Q W « o S3 H i-l Q I- co •* -H -H -H -H -H -H -H CO co CO 01 1> oo t> lO «5 cp 1> Tf ■* Tfl -H -H tH -H -44 -H -H CO O C5 00 >C CD CM ON M H • Tt< 00 N i-l to f + + 1 T :T + •* lO o -* • 05 m oo CM O co o CM o CO • co CO 05 00 co IO ■ CO t« o oo CM co CM ^*1 • CO 05 CM t^ 05 o o o o o tO 00 05 C5 05 C3 C5 • Tf h in iji co ' CO i-l CM Tji ijl iO CM lO i-H IT3 lO CM CM »0 t- CM b- t-~ Tjl Tfl to to oi ■* i* m ■* (M -tfi CM i-i i-H T(< Tfl CM CM CM H t-h CO CO 00 1-1 l-l i-H i-H i-l i-H lO O) N N N CD to 46 CONNECTICUT EXPERIMENT STATION BULLETIN 207. H m o w fa- - ta 02 r/> 12; M frt < o fa H « ttl fa fa H o 15 fa EH o fa Eh fa (S Hi W H u h^ 14 <1 «! fa fa 'A < fa O M P a fc fa fa 15 ^ fa fa H •= fa H O H fa H fa 17) fa w !/J 0) o fa fe o c £ o 17) 3 & s o CD .a e § CD O -a fcn (3 CD fal Difference of sib crosses above selfed COOJOOltCOOOOlMHOMOlol OOOOOO^hOOt-i>-iOOOO + -H-rl-H-H-ff-H-H-H-H-ti-H-H-H-H-U comMMosiioonHMiiHtNnH +1 I+I++I+I+I+I+ m MtOCONO*O^COOS«)lOOCD OOOOOOOOO^OOOOO lO in -H-H-H-fi-H-H-H-H-H-H-fi-H-H-H-H ■*HOO)ONOINCi]CD01COt>.030 cDcDioio-*u3coNNco-*racorara Selfed strains tNCONcDCDlOCO'fcOOOMNCNiO OOOOOOOOOOOOOOO >n -H -n -H -H -H' -H -H -H -H -H -H -H -H -H -H CO^MIONMHiONOOCJiOHllo ir)cOmiOiOiOONNcD-*«in^M +3 S 03 fa u CD ft fa a '3 bfl O Difference of sib crosses above selfed iniooiTfiOHiios^ncoHccoo OOOOOOi-iOOOOOO^O o + . 1 ++ 1 1 1 + 1 + + + + + 1 + £ 1 ' CO o o OHOTfO!ONCD*OOii»0'0 (NMTfCOHHijico^-*cN(McMrtCO o TO CD .8 fa! o3 «2 1 BlcDHCo^onTfONOCOitJCSCO CMOlCOmcMNM^TliMNMiHOlCM CM a 03 "ft 'o fa M 'S W Difference of sib crosses above selfed t~TtJC32tOOq < 2 2 2 i-l heterozygosis and vegetative luxuriance. 47 The Effect of Heterozygosis on Vegetative Luxuriance. The most noticeable manifestation of heterosis in plants is a general increase in vegetative luxuriance. In maize this is par- ticularly noticeable in increased height of plant, diameter of stalk, root development, length of ear and productiveness of grain (see Plates III, V, VII, VIII, IX, X and XII). In crosses between inbred strains of maize the amount of heterosis shown is inversely proportional to the degree of relationship as shown in Table 11, Montgomery ('12) has obtained similar results. Some characters are much more affected by heterozygosis than others. In comparing Tables 12, 13 and 14 with Tables 15 and 16 it will be noticed that the yield of the crosses is increased 180 per cent., height is increased 27 per cent, and length of ear 29 per cent, over the average of their parental lines. On the other hand, the number of nodes per plant and number of rows of grain on the ear is increased only 6 and 5 per cent, respectively. In other words, heterozygosis does not increase the number of parts to anything like the extent that it increases the size of those parts. Those parts of the plants which are more or less indeterminate in size, like internodes, ears and seeds are augmented by crossing as the result of an increase in the rapidity and rate of cell division. The increase in size of parts is probably brought about by an increase in size of cells as well as an enormous increase in number of cells. Tupper and Bartlett ('16) have shown that gigas mutants in Oenothera have larger cells than the non-mutant type, so that a change in cell size may accompany a germinal change. From Table 11 it will also be seen that some first generation hybrids may even surpass the- original variety in yield, height or length of ear, although the comparison is rather unfair as the Learning variety was not acclimatized as were the inbred strains. The return of vigor realized in the first generation crosses is often enormous, and the same is true of crossing inbred strains derived from totally different types of maize as is shown in Table 17. Although there is an immediate and striking return to the vigorous condition of the non-inbred stock there is not a return in variability as shown in Tables 18, 19, 20, 21 and 22. The first generation crosses are no more variable than the inbred strains by which they are produced, in many cases less variable, and show striking differences when compared to the original stock. The coefficient of variability is entirely inadequate in bringing out 48 CONNECTICUT EXPERIMENT STATION BULLETIN 207. (X)io-* oo co -* i> oo i>- os co <— i co I ++ I + I + +++ +++ hOhq -h-h-h +:+:+;^ +;+:+: +;+:^ -h CO "0 IQ i-H>t>t~ t$< CO iO OS CO CO CO CO ■* iO iO lO •* id ■* CD lO ■* CO CO CO t — t— t— • • • • ^o-*< lOCOMH ONCD i> O Tt< . ... OS *-l CO : : : : i 7 + CO >0 o> C 9 o >> Hi + M lOOM CO CO 00 -H •* CO CO t> lO CO I +++ +++ t— t — t — co co co co i-H i— i oo o I I I l I i I i I i t^"* ^CH TH ■* CO iO I I I <-h . (M I I I OS CD rj< i i i * i i i i i i i i XXX xxx xxx xx; X; ^~ s I I I CO^cH «0 TH I I I I ■* t-- Xh CO i-H Ci I I I I I '^TJH'HCO *# C I I I I I rfiNNN rt< t^ W I> •* ■* l> I i i i ill i i i I TcH TcH OS CO ■<# ** OS CD ■* -^ •"*! I I I I III I I I I rM i— I (M TJH T)H 1-1 i i i rt-* CO rt TJH O O -H -H -H -H -H CO CO CN CO CO CO t^ © cn o —i oi ai oo co t^t^-^cO-^^-HOOOCO -H-H-H-H-H-H-H-H-H-H ONiijhoos^ooOS rtCliocO^INCBOSC) O'JlOOOlOONlONin H S3 O 00 IO CO CD CO ^ WJ N lO -U -H co c: -H -H N O N IO -H -H -H -H -H -H H OHO H N H ■* tf o h o OJ IH iH O 1H O O CO CO ^ O f-Hb-OiCOCOHHOCDt^Ci lO lO lO CO (O lO IQ lO O lo iO O lO iO ►J oc H S 02 3 £ > I 1 I? o v « f. OS CO • CO Tj< N o o ■ m cc to N O! rt OlM« . rH rH r-i i-H O) N O ■ Ol CO CO CO 00 O) CN • h rn CN N h h H'0-CMO<0«0 10 ON "-h O CN rH ■ OlOrtflCNOMOO"* iH rH o CO • OI h o iO • CN rH CN CN -C« 00 CO CO O CO ■ O CO CN CN K T(< t~ Tj( CO CN IO M IO rf< t^ CN t~ •*< ■* OS CD ^ H CN ■* CO ■-! CN CN t"~ rH . io f h S IO CO lO M IO CN IO tn CN l> Th N- ^* Ol CO Hi CO •H CN TH ■**! t^ — < CN CN CO CN CN "J) •* co CO H ^ IO CN >-H CN IO H ■* ti CN t^ r~ a co OS co r; X X X X X X X ■* M ■* CO IO >* «* ■* T(l TH CN ■* CN CN CN IO IO ^ ^i ^i N N CO CO CO M tH ■Hi ■*-*<■* COS M N f in CN CO i-H CN XXX CO CO CO IO IO IO ti t-i. ti CO CD CO CN CN OS OS CN CN CN CN COCOCONNt»NO)OCS HETEROZYGOSIS AND VEGETATIVE LUXURIANCE. 55 the beautiful uniformity of these crosses between inbred strains. In every respect each plant is a replica of the other. A collection of such vigorous and uniform maize plants in the field is a novel sight (see Plates IHb and Vb). Shull ('14) has pointed out that vigorous plants may be less susceptible to the effect of the environment than weaker types and that first generation hybrids, between uniform strains, may even show a reduction in variability. The results obtained show this quite noticeably. Particularly was this true of several FVs grown between their parental strains in a demonstration plot on rich low ground. During both seasons (1916— '17) when they were grown on this piece of ground, the weather was especially unfavorable when the plants were just starting, the ground being saturated with water most of the time. The germination in the selfed lines was extremely poor and many plants which did grow were stunted, and remained so throughout the season and never attained full height nor did they produce either tassels or ears. The variability of height, in these plants, was far greater than in many non-inbred varieties. Several plants, when killed by frost in the fall, were not over 30 inches tall while the average height of this strain is from 80 to 85 inches. The hybrids also had a poorer start than non-inbred varieties grown on the same ground on account of the small seed, but were able to overcome their handicap and in a few weeks were quite uniform. At the end of the season the difference in variability between the Fi on the one hand and the inbred strains and the varieties on the other was striking. These plants were not used in the statistical work given here. The crosses and parents which were used and which were apparently quite uniform show a slight reduction in variability, in the number of nodes and in height in the Fi's as compared with their parents as can be seen in Tables 18 and 20. As Shull also pointed out, the variability of some characters may be increased by heterosis. This is shown in number of rows on the ear. The inbred strains rarely or never produce a second ear. The vigorous hybrids almost always do, and as the data have been obtained by counting all the ears gathered from a plot, the variability of the crosses, as shown in Tables 19 and 21, consequently seems greater than it really is as the second ear on nearly every plant is smaller and contains a fewer number of rows. 56 CONNECTICUT EXPERIMENT STATION BULLETIN 207. Although reciprocal crosses are on the whole nearly equal in respect to the degree in which heterosis is shown, there is some evi- dence, from Table 12, that this is not always so. Observations from the crosses in the field showed clearly that those in which strain Number 1-6 was used as the female, were usually more vigorous and productive than the others. In Table 23 the yields of all the crosses and reciprocal crosses (from 1 to 4 of each) having the same parental races are averaged. An average of all those Table 23. Yield of reciprocal crosses among inbred strains of MAIZE. (All crosses grown 1916. Yield given as bushels per acre.) 1-9-1-2 d1 1-7-1-2 o" 1-7-1-1 a fa UJ 0 fa fcH n a CO Q O n is fc w H o rft r« fa H O O ■«, « r5 O r/7 M O Oh ■i Eh P O < ►J fa fa o z o is o H K fa w o W O w Ph fa a o in : -H -H -H id r- CO 3 ■* CO CM rH CM ,_, m CM O rH r-H CO 1-1 Cn rH < -H -H -H -H -H -H to co OS O0 ir y-l i> ci CO CO O CO CM CO CM CM C\ CM m oo oo OS oo co £ co t~ CO lO oo o lO CM CO o r-- CC T3 CM CO CD ir. ^ CO oc CO lO « CO IN "S (N r~ l> CM CO CO -t CD HH CO (M ■* lO oo 1 CC lO cc t^ CM ■* rH lO CO C lO CO r- co CO 00 ^H o o CM C to rH CM , CM CM Th *H CO CM -S a> CD XH o • i £ i S= o o o o o o cd CD CD CD O 111 >1 >> a +3 >1 5 is M M ci 15 M & 3 J Q CS 3 « E- •» "* OS QO O i-l CM CD -* CO co cm co co co IQ lO Th OS + o ■* ■* CD LO OS (N • i -3 0• 00 00 H NOlOOJMO i— I 00 t- 00 CO CD CO ^H CM CO CO CM i—i CM CO tH IC CD >"S ^ a ora3'3 S-l CD & X3 £> 66 CONNECTICUT EXPERIMENT STATION BULLETIN 207. was not equal quantities of functional pollen as the number of seeds given in Table 27 show. The great inequality of functional pollen may have been due to the fact that the pollen of the B strain was more moist and tended to aggregate into a flocculent mass while the pollen of the other was perfectly dry and each grain remained separated from the others. For this reason it was dif- ficult to measure the two lots of pollen equally and the dry pollen clustered about the fine lumps of moist pollen when the two kinds were mixed and was probably first to gain access to the stigmas. The difference between the two kinds of pollen was not due to any external differences, as far as could be seen, and indicate differences in the rate of maturing after shedding. Whatever may be the cause of the great difference in fertilizing power this does not effect the point under investigation. How- ever different the pollen may be, the seeds resulting from " yellow " pollen should be in the same ratio to the seeds resulting from the " white " pollen on one ear as the ratio of the same two kinds of seeds on the other ear within the limits of the error of random sampling if there is no selective fertilization one way or the other. And both these ratios should be the same as the third ratio obtained when this same mixture of pollen is used to produce seeds on a plant of a different variety of maize. Let us see what the figures given in Table 27 show. Of the reciprocal crosses and selfs the proportion, expressed as percent, is as follows: Seed color carried by pollen Yellow White Yellow White Type of Seeds Selfed Crossed Crossed Selfed Actual proportion obtained 98.490 : 1.510 :: 96.600 : 3.400 Closest perfect proportion 97.545 : 2.455 :: 97.545 : 2.455 Deviation 4- .945 -.945 -.945 ' +.945 The deviation from the closest perfect proportion is in favor of the selfed seeds. This theoretical ratio agrees very closely with the actual ratio obtained from the out-crossed seeds as shown in Table 27 although there is considerable difference in the results from the different ears. Letting S stand for selfed and C for crossed the probable error of the determination S . .6745 /(SHC) •- "; . S uc 1S ± sT~c v STC ' The fractlon sTc gives the percent of selfed seeds and the probable error is stated C as percent. Likewise the fraction = -^ gives the percent of b -7- V_' HETEROZYGOSIS AND ^SELECTIVE FERTILIZATION. 67 crossed seeds and the probable error is the same as for the percent of selfed seeds. This same experiment was repeated with about the same number of plants with the result of a similar excess of selfed seeds greater than would be expected from the probable error on the assumption that there is no selective fertilization. Does this mean that there is a selective fertilization in favor of a plant's own pollen and that the plant discriminates against foreign pollen even though the seeds resulting from that foreign pollen are greatly increased in size, weight, viability and the rate of growth of the ensuing plants? Unless there has been a constant error in classifying the seeds this seems to be the necessary conclusion to be drawn from the results so far given by maize. A sufficient number of plants will be grown from this seed to determine definately whether or not there has been any error in the separa- tion of the seeds so that this question can be answered with a high degree of certainty. In the meantime there is little doubt but that there is no great selective fertilization in favor of cross-pollination, if any, however much that cross-pollination may benefit the resulting seeds and the plants grown from them. If this is true crossing is without effect until the zygote is formed at the time of the union of the male and female nuclei. In a consideration of selective fertilization it should be remem- bered that there are two different conditions which may be included in the term selective fertilization. One may be said to be the selection of different germ-plasms; the other the selection of different cytoplasms. For example a heterozygous plant produces pollen grains with different germinal compositions but all enclosed in the same cytoplasm. On the other hand pollen from different plants may differ in the nature of the cytoplasm as well as in hereditary factors carried in the nuclear material. East and Park ('18) have demonstrated that in tobacco there is no selective fertilization between gametes coming from one plant although the pollen grains differ in factors which determine fertility or sterility of the ensuing plants. The case is quite similar to that of the shape of pollen grains in peas which may be either all round or all cylindrical according to the germinal composition of the sporophyte which produced them and not according to the factors which they carry. Where pollen grains differ both 68 CONNECTICUT? EXPERIMENT STATION BULLETIN 207. in the factors which they carry and in the plants from which they come, as is the case with these experiments with maize, the conditions are quite different. It would not be surprising that there should be selective fertilization in one case and not in the other. East and Park have shown that a tobacco plant which was self-sterile, pollinated with a mixture of its' own and pollen from another plant with which it was fertile, gave all crossed seeds — a maximum of selective fertilization. Darwin (" Cross and Self Fertilization ") found that there was a selective fertilization in favor of foreign pollen in different plants. Many of Darwin's experiments, however, were made in such a way as to be open to doubt whether or not he really did obtain such an effect. His experiments, in applying foreign pollen sometime after self-pollination had taken place, in which he obtained in some cases many or all apparently crossed progeny, are open to other interpretations. The purity of the plants pollinated was not known. External conditions influencing fertilization were not guarded against. Taken as they stand, however, his experiments with Mimulus, Iberis, Brassica, Raphi- nus, Allium and Primula do indicate that in these plants there may be a selective fertilization in favor of foreign pollen. It is to be expected that plants which show partial self-incompati- bility would show selective fertilization when a mixture of self and foreign pollen was applied. In maize, however, as mentioned before, the sterility shown is in the nature of pollen and ovule abortion, and whenever well formed pollen is produced it seems to be able to fertilize equally any plants if not too distinct in type. A distinction should be made, then, between self-fertile plants and self -sterile plants when dealing with selective fertilization. Hyde ('14) has shown clearly that in Drosophila both males and females of inbred lines are more productive of offspring when mated to an individual of a different line than when mated to one of their own. Both males and females, therefore, produce more functional gametes than are utilized when individuals of the same inbred lines are paired. Hence a female, impregnated with a mixture of two kinds of spermatozoa from the same and from different lines would produce more hybrid progeny than inbred progeny even if equal quantities of both types of sperma- tozoa were available for fertilization. In other words there would be selective fertilization in favor of cross-fertilization. LONGEVITY, HARDINESS AND VIABILITY. 69 Whether or not there may be a similar condition in other animals I do not know. Even in Drosophila, fertilization by the two types of sperm may take place equally, and a greater proportion of close-fertilized eggs, than cross-fertilized, fail to hatch, due to lesser vigor or lethal factors. In Hyde's experiments the type of fertilization had no marked effect on the number of eggs laid, only on the percentage which hatched. In maize, and possibly all plants which show no self-incompati- bility, the fact seems clear that crossing is wholly without effect until the fertilization process is completed. Although there is apparently no effect of crossing in maize until the zygote is formed, such an effect is apparent immediately afterwards. In addition to the increase in endosperm development there is also an increase in the vigor of the embryo. Whether or not the size of the embryo in the seed is increased has not been actually determined, other than by inspection, but it undoubtedly is, along with the endosperm. When crossed and selfed seeds from the same ear, grown on a plant which has been inbred previously for several generations, are planted a striking difference is soon apparent. The crossed seedlings appear from one to two days before the selfed seedlings and may be two or three inches above ground before any of the selfed plants begin to appear. (See Plate Xlb). From then on the superiority of the crossed over the selfed plants increases rapidly as shown by the curves in Figure III. The Effect of Heterozygosis upon Longevity, Hardiness and Viability. - An increased longevity, viability and endurance against un- favorable climatic conditions have been frequently noted in hybrids. Kolreuter and Wiegmann both mention this fact. Gartner in his book "Bastarderzeugung im Pflanzenreich" devotes considerable attention to this feature. Under the heading " Ausdauer und Lebenstenacitat der Bastardpflanzen" he makoo the following statements. " There is certainly no essential difference between annual and biennial plants and between these and perennials in regard to their longevity; for it is not seldom that different individuals of the same species have a longer life at times as, for example, Draba verna, which has annual and 70 CONNECTICUT EXPERIMENT STATION BULLETIN 207. biennial forms; the longevity of a plant furnishes thereby no specific differences but signifies at most a variability as Prof. W. D. I. Koch has shown. However, in hybrids this difference deserves special con- sideration. In most hybrids an increased longevity and greater endur- ance can be observed as compared with their parental races even if they come into bloom a year earlier. The union of a annual, herbaceous female plant with a perennial, shrubby species through hybridization does not shorten the life cycle of the forthcoming hybrid as the union of Hyoscyamus agrestis with niger, Nicotiani rus'tica with perennis, Calceolaria plantaginea with rugosa shows, and so also in reciprocal crosses when the perennial species furnishes the seed and the annual species supplies the pollen, as Nicotiana glauca with Langsdorfii, Dianthus caryophyllus with chinensis,, Malva sylvestris with Mauritiana or biennials with perennials and reciprocally as Digitalis purpurea with Ochroleuca or lutea and lutea with purpurea or ochroleuca with purpurea. From the union of two races of different longevity comes usually a hybrid into which the longer life of one or the other of its parent races is carried whether it comes from the male or female parent species." Many more instances are given by Gartner from his own ob- servations and those of others to enable him to reach the following conclusion : " These examples support the statement of Kolreuter's that the longer life of hybrid plants is to be counted among their usual properties." With regard to the resistance of hybrids to unfavorable weather conditions he goes on to say: " With their longevity stands, in the closest relation, the fairly common property of hybrids to withstand lower temperatures than their parental races without injury to their growth and vegetative life. Kol- reuter first observed that Lyciutn barbara-afrum in south Germany withstood the winter in the open field; although Lycium afrum must be wintered over, at least, in a cold frame. The cross of Nicotiana Tabaco- undulata, according to Sageret in France had an increased life, although in a protected place, in open field. W. Herbert reports that Rhododen- dron altaclarae, which is a hybrid union of R. pontica-cantawbiense 9 with the very sensitive Nepalense arboreum coccineum c?, has been grown in the open in England; also Robert Sweet confirms the same result by a hybrid crinum and many other hybrids of bulbous plants grown in open field whose parental species must be grown in the hothouse. " Lobelia syphilitica-cardinalis wintered over with a light covering in the winter of 1832-1833 with 5°F in open field. Lychnicucubalus albus and ruber lasted three years in open field although cucubalus viscosus in south Germany did not survive in open field. All hybrids of genus coccineum stood over the winter of 1842-1843 with 5°F. in the open, although the pure species seldom lives through our usual winters of 43° to 9.5° F. Prof. Wiegmann reports similar results. LONGEVITY, HARDINESS AND VIABILITY. 71 " Very frost sensitive species of Nicotiana and their hybrids did not withstand, under the same conditions, such low temperatures as the afore-mentioned plants; but we have flowered and carried over part of them wherever they were well covered with snow, for example, N. quadri-valvis glutinosa, rustica-quadrivalvis, these withstood 25° F. and yet have continued blooming although N. glutinosa,' quadrivalvis, panicu- late/,, Tabacum and rustica were already frozen by 32° F. Moreover other crosses of very sensitive and tender species of this genus as paniculata- Langsdorfii, vincaeflora-Langsdorfii, vincae-flora-quadrivalvis have been carried over in an active growing condition two to three years, and glauca-Langsdorfii three years in a cold house with 39° to 42°. The hybrid N. paniculatarustica-paniculata was kept over in a cold house in the cold winter of 1839-40 but its leaves were yellow. Among all the species of this genus the cross of N. suaveolenti-macrophylla showed itself to be the most hardy. On the 16th of October of its first year (1828) its top was frozen but it did not suffer from this, and 12 days later put out a new shoot from the root and its leaves lasted through the winter in a cold house in a fresh, green condition although the other species were yellow and this plant was the first to start into growth in the spring. The same endurance Sageret observed in Nicotiana suaveolenti-virginica. All these plants in the last year. of their vegetative life seemed to die off more as the result of the unfavorableness of the weather than of old Exceptions are noted by Gartner in that some species which were not resistant to cold did not give resistant hybrids. In many cases the hybrids were weak because of the distant re- lationship of the parental races. Sargent ('94) reports a remarkably vigorous and hardy hybrid tree supposed to be a cross of the tender English walnut, Juglans regia and the common butternut Juglans cinerea. He says: p. 434 "My attention was first called to the fact by observing that a tree which I had supposed was the so-called English walnut — Juglans regia, in the grounds connected with the Episcopal School of Harvard College at Cambridge, was not injured by the cold of the severest winters, although Juglans regia generally suffers from cold here — and rarely grows to a large size. This individual is really a noble tree; the trunk forks ab u five feet above the surface of the ground into limbs and girths, at the point where its diameter is smallest, fifteen feet and two inches. The divisions of the trunk spread slightly and form a wide, round-topped head of pendulous branches and. unusual symmetry and beauty, and probably sixty to seventy feet high." Heterosis is also shown in a resistance to bacterial and fungus diseases. Some of the inbred strains of maize are very susceptible 72 CONNECTICUT EXPERIMENT STATION BULLETIN 207. to the bacterial leaf-wilt and in some years at the end of the season all the plants of these strains appear as if they had been scorched by fire while other strains in adjoining rows are un- touched. Other strains have quite a large percentage of plants attacked by smut.* Crosses, however, of these susceptible strains with those which are not affected by these parasitic organisms are only slightly or not at all affected. Table 28. Susceptibility to smut ( Ustilago zeae) of a non-inbred VARIETY OF MAIZE, SEVERAL INBRED STRAINS DERIVED FROM THIS VARIETY AND THE FIRST AND SECOND GENERATION CROSSES BETWEEN THE MOST SUSCEPTIBLE AND THE LEAST SUSCEPTIBLE STRAINS. Percent of plants affected Total number of plants grown Total percent of plants affected Plot I Plot II Plot III 1 l_9_l-2-4-6-7-5 o' ' 2.17 8.79 0 ' .27 .35 10.16 0 2.48 1.75 .56 0 5.77 0 0 5.15 114 596 408 950 992 439 97 1.75 .34 1-7-1-2-2-9-2-1 .49 1-7-1-1-1-4-7-5 9.79 l_6_l_3_4_4-4_2 0 (1-6-1-3) X (1-7-1-1^.. . (1-6-1-3) X(l-7-l-l)F„.. . 2.28 5.15 In Table 28 are given the per cent, of plants affected by smut ( Ustilago zea, Beck. Ung.) of the original, non-inbred Learning variety of maize previously spoken of and four inbred strains derived from this variety by ten or eleven generations of self- pollination. Seed of the four inbred strains was planted in three rather widely separated plots in the same field in 1917. Two of the strains showed only a small infection by this parasite; one showed about 10 per cent infection and one had not a single plant affected in all three plots in a total of nearly one thousand plants. Since the differences which these four strains show are fairly con- sistent in the different places grown it can hardly be doubted but that segregation of susceptibility to parasitism has occurred in the inbreeding process. The first generation hybrid between the most resistant and the most susceptible strain was free from smut in one plot and but slightly affected in another. The second generation hybrid grown side by side with first generation showed LONGEVITY, HARDINESS AND VIABILITY. 73 considerably more infection although the number of plants grown was small. This is fairly good evidence that resistance to smut in maize tends to dominate in crosses between plants which differ in this respect. - Tisdale, according to L. R. Jones ('18) also finds that in flax disease resistance tends to be dominant although the hybrids are more or less intermediate in this respect and the method of inheritance is rather complex. Biff en ('12), on the other hand, concluded that the resistance to rust in wheat was recessive. Likewise, Weston ('18) states that maize and teosinte-maize hy- brids are extremely susceptible to a downy mildew (Peronospora Maydis, Rac.) in Java and other places, although teosinte (Euch- laena mexicana, Schrad.) is immune. Data from another source have been obtained from the garden radish (Raphanus sativus, L.). A white-rooted variety of radish was allowed to go to seed alongside a red-rooted radish. Seed collected from the white-rooted plants was sown thickly in a flat and when they came up it was seen that a number of the seedlings were crossed from their red -colored stems. The seedlings were quite badly attacked by the "damping-off" fungus and large numbers of them were killed, but a far less number of the crossed seedlings were affected as shown by the decay of the tissues at. the base of the stem. The figures obtained are given in Table 29. Table 29. Comparative susceptibility to " damping-opf of selfed and crossed radish seedlings. White Seedlings, Selfed Red Seedlings, Crossed Variety of Radish Number grown Number affected Percent affected Number grown Number affected Percent affected Short, white. . . Long, white. . . 349 76 142 28 40.7 36.8 30 7 4 0 13.3 0 Gernert ('17) reports a case of immunity to aphis attack .of teosinte-maize hybrids in which the maize parent was badly infested whereas the teosinte parent and the hybrid entirely escaped injury. Together with these manifestations of heterosis in its influence on hardiness there is an increase in the viability of crossed seeds as compared to selfed seeds from the same ears as shown in Table 74 CONNECTICUT EXPERIMENT STATION BULLETIN 207. Table 30. The effect of heterozygosis upon germination — a compar- ison OF crossed and selfed seeds from the same ears of maize Ml > ■ 3 o o s "pi o T3 TO X) CD TJ O TO CD (O TO -c CD O -a CD \t CD TO > TOO Eh Ot3 CD Pedigree number Pedigree number in m eg 13 CD CD c u - TO CD fi £'3 of female parent of male parent a m ■M O C " « 2"S m