Proceedings of the National Academy of Sciences, 5: 130-134. (1919)
Communicated by R. A. Harper, February 26, 1919

The common experience of horticulturists and plant breeders is that propagation by buds, cuttings, layering, etc. (asexual propagation) yields a comparatively uniform progeny, while propagation by seed (sexual reproduction) and especially that which involves mating of unlike parents whether of the same or of different species or races, is likely to give decided variation among progeny. On the other hand, common experience and practice recognizes the widespread occurrence of bud variations and the importance of utilizing them in developing new types of important commercial races, or in maintaining old races at a high standard, as is well illustrated by the recent studies (Shamel and others 1918) of bud variations in the citrus fruits.

In scientific and theoretical breeding, much attention has been given to the study of heredity in sexual reproduction. In many species this is the only method that can be utilized, and a knowledge of such heredity is of great practical as well as of theoretical interest. When, however, the question arises regarding the constancy of characters or of assumed factors of heredity, it seems very evident that critical studies of somatic variations, especially wherever it is possible to propagate vegetatively, are of fundamental importance.

To Darwin, bud variations in plants were evidences of the very indiscriminate variability that is everywhere present in organisms. Their broad significance and range were recognized by his conclusions that they include: (1) reversions to remote ancestral characters; (2) reversions (in hybrids) to the more immediate parental qualities; and (3) cases of real spontaneous change in hereditary composition of continuous as well as of discontinuous range. Darwin did not believe in fixed hereditary units.

These same types of bud variation are, in general, recognized by de Vries (1901). He attempts, however, to assign mutational value of discontinuous rank to the spontaneous somatic variations quite as he does to seed mutations. Yet he recognizes a wide variability and irregular hereditary performance in both. For example, he describes half races, middle races and eversporting varieties in the various steps in the development of varieties from pure species involving characters frequently concerned in bud sports and ascribes the series of changes to conditions of latency, semilatency, lability or activity of hereditary units (pangens). To many critics of the mutational doctrines it is difficult to recognize such spontaneous hereditary variations as discontinuous either from the facts or explanations presented by de Vries or by other investigators. We may note further that many bud variations, such as the development of variegated branches on pure green stems were considered by de Vries as progressive mutations.

In general, Cramer's (1907) classification of bud variations follows that of de Vries for seed mutants with, however, a greater emphasis on the operation of Mendelian segregations in certain groups. He recognizes, however, quite as did Darwin and de Vries, the occurrence of a large group of bud variations in which continuous and sporadic or eversporting variability is in evidence.

The present day Mendelian studies of the seed progenies from bud variations and of characters exhibiting such variations show as a rule decidedly mixed and non‑Mendelian results, or, at least, their interpretations involve subsidiary hypotheses. Some special tendencies in such interpretations may be noted: (1) the assumption that the transmission of certain characters is by the cytoplasm rather than by the nucleus; (2) the assumption that somatic variations are losses of hereditary factors accomplished by qualitative or segregative cell divisions, and (3) the claim that hereditary factors may themselves sporadically change, and the new factors come to immediate expression by dominance, or remain recessive, or exhibit various‑ influence as modifying factors. Thus in certain attempts to analyze the heredity of seed colors in variegated corn it is assumed that factors for variegated color can change reciprocally in a series of somatic divisions and that as many as ten 'multiple ailelomorphs' may be present, which, as Jennings (1917) remarks, "leap back and forth from one character to another in bewildering fashion."

A better understanding of the nature of the tissue complex in certain bud variations has been gained from the knowledge of chimeral structures. When a somewhat permanent somatic variation occurs in only a part of the cells or in a single cell of a growing point such a cell or cells may be so situated that the cell progeny form permanent layers giving periclinal chimeras, or sectors giving sectorial chimeras. There is also the possibility that irregular processes of development or the occurrence of repeated somatic variations during organogenesis may give complex mixtures or hyperchimeras. Anatomical proof of the existence of such chimeras was first presented by Baur (1909) and the experimental production or interspecific chimeras was demonstrated by Winkler (1907).

Numerous cases of albomarginate variegation are apparently of the periclinal type. The originial variation in such cases is partial, affecting a part of a growing point only. Further changes, such as return to pure green branches, may involve simply a mechanical, readjustment of the elements present in the growing points (Stout 1913). Chimeral association of cells differing sporadically but more or less permanently in fundamental hereditary qualities undoubtedly accounts for much of the irregularity seen in the seed progeny of bud sports. Various types of chimeras especially periclinal and hyperchimeras, without doubt grade imperceptibly into cases of ordinary differentiation in which the cells quite alike in fundamental hereditary qualities become differentiated through their relations as parts of the whole.

In general, our available knowledge regarding somatic variations indicates that they show a wide range of variability suggesting that hereditary elements or units are themselves variable even in a series of somatic cell‑divisions. The evidence is especially convincing, for here there is a most direct lineage of cell elements far more simple than that obtained in reproduction by seed progeny which involves the intricacies of periodic reduction and fertilization. Unquestionably the phenomena of bud variation involve the most fundamental questions of heredity. The intensive study of bud variants in successive generations propagated vegetatively should reveal definite facts regarding the nature, frequency, and permanence of spontaneous changes. Since 1911 the writer has studied bud variations in a variety of the variegated Coleus with these aims in mind. Over 1211 pedigreed plants have been grown, comprising fourteen‑ generations (two each year), all propagated by cuttings, the first of which were taken from two similar sister plants.

The frequent variations that have appeared range from sudden changes to gradual fluctuations, and these may first become evident in a part (even a small area) or the whole of either a leaf, a bud, or a plant. The characters studied have been those of leaf form and leaf coloration.

The somatic variations found in leaf coloration involved (1) gain or loss, increase and decrease of green and yellow, (2) reversals of the relative positions of the green and yellow in leaves, (3) increase and decrease of red pigmentation, and (4) changes in the distribution of the red pigmentation, especially that giving concentration in the epidermis of the upper surface of the leaves. Some of the variations involve very striking changes in epidermal pigmentation, superficially resembling chimeral relationships. During the first seven generations, sixteen distinct color patterns were obtained by bud variation and isolated by selection; fifteen of these were produced as marked sudden variations, and six of the types also occurred as fluctuating variations. One pattern has appeared only as a fluctuation. Between any of these types a wide range of intermediates was found from which many additional patterns might probably have been isolated. There has been reversion to parent patterns; colors that have been lost or thrown out have reappeared; yellow‑green patterns have given pure green sports and later the yellow has reappeared.

A calculation of the frequency of bud variations on the basis of the estimated number of buds that developed into branches shows clearly that decrease of red occurred with about twice the frequency as did increase of red; likewise decrease of yellow occurred about twice as often as increase of yellow. Among sister lines of clonal descent there were marked differences in the ratios of frequency for any one change in coloration. The most frequent change was loss of yellow with increase of green, for which the frequency ratio was 1:2960. The change occurring with least frequency was that of increase of epidermal red pigmentation to solid red for which the ratio was 1: 19,250.

Variations in leaf form were fully as striking as those of pigmentation. Deeply laciniate‑leaved forms arose in thirteen instances as fluctuations affecting an entire plant, and in one case as a decided bud variation. A striking feature of the laciniate character was the marked periodicity in its development; plants of the variety having, strongly laciniate leaves in winter produced entire leaves in summer, while the great bulk of the sister plants of other varieties produced only entire leaves. In general these variations in form are continuous and the extremes are in decided contrast with each other.

Selection for extremes and for intermediates has in every case given a progeny of marked constancy but with further fluctuations and sporadic variations about a new mode. The types thus arising are for purposes of propagation the equivalents of the 'Kleinarten' or 'biotypes' that commonly occur in species propagated by seed. In their bearing on the theories of continuous variation and the effects of selection, my results are quite identical with those obtained by Castle and Phillips (1914) in their study of color patterns in biparental reproduction in rats and interpreted as indicative of actual variation in the hereditary units. The results are also quite identical with those Jennings (1916) has obtained with Difflugia, by asexual propagation analogous to that I have used in Coleus.

It is quite clear that the changes seen in Coleus do not involve a permanent loss of definite hereditary units by vegetative segregation. There is also no opportunity for any such recombination of multiple modifying factors as is assumed to give similar variations in sexually reproduced progeny. As far as is known vegetative propagation gives the greatest possible degree of purity in cell lineage uncontaminated by the recombinations involved in the reduction and fusion accompanying sexual reproduction. The evidence is hence very conclusive that the hereditary complex and that individual units of the complex are subject to variations that become manifest either as sudden mutations or as fluctuating variations, and that any of these may perpetuate themselves.

The facts suggest strongly that the possibilities for the development of red, green and yellow may be present in all cells as metidentical characters (in the sense used by Detto, 1907). The total production, the distribution, and the concentration of the various chemical substances concerned, however, plainly involve interactions between different cells or groups of cells and are in this sense epigenetic. The explanation suggested by the production of patterns in colloids by the Liesegang precipitation phenomena, especially as applied by Gebhardt (1912) to the markings of butterfly wings and by Kuster (1912, 1917) to the development of many types of variegation in plants including Coleus, seems to apply to the production of color patterns in Coleus. On this view colored patterns may be considered as due to the formation of localized centers for the development, diffusion and concentration of pigments.

In Coleus both the (1) fundamental qualities (metidentical) and the (2) processes of cellular and tissue interaction immediately involved in the development of patterns exhibit spontaneous changes that are continuous in degree and are quite constant from the first or can be made so by selection.

Colored illustrations of the principal color patterns obtained, together with the presentation of data (for the first four years of the study) have already been presented (Stout 1915). With the facts and conclusions there given, the results since obtained are fully in agreement.