Regeneration

Vochting has also studied the regeneration of pieces of the liverwort, Lnmilaria vnlgaris. The results have been already partly given in the first chapter. If cross-pieces are taken from the thallus, each produces a new bud at its anterior or apical end (Fig. 9, A, A !). The new bud arises from the cut-surface, or very near it, from a group of cells of the midrib that lies nearer the under side (Fig. 9, A2). The bud gives rise to a new thallus that springs from a narrow base at its origin from the old piece. If a piece is cut longitudinally from the thallus along the old midrib, the new bud arises at the anterior end from the midrib (Fig. 9, B\ It comes either from the anterior cutsurface near the inner edge, or from the anterior end of the inner edge, and in some cases two new buds arise, one at each of these places. If the piece is removed from one side of the midrib it does not regenerate as quickly as when a part of the midrib is present, but when the new bud develops it arises from the anterior part of the inner edge (Fig. 9, B 1). If the piece is cut far out at one side, it may be a long time before the new bud arises. This difference in the rate of development of these pieces is explained by Vochting as due to the simpler character of the cells near the midrib.

If oblique pieces are cut off, with an anterior oblique cut-edge, as shown in Fig. 9, C, C1, the new bud arises along the anterior surface. If the piece includes a portion of the old midrib at its inner end, the new bud arises from this (Fig. 9, C), but if the piece lies to one side of the midrib, the new bud arises near the anterior end of the anterior oblique surface (Fig. 9, C1, C2) A number of experiments that were made in order to determine what part gravity and light may take in the regeneration gave nearly negative results. The regeneration appears to result largely from internal factors.

If a piece of the thallus is divided parallel to its surface, the two parts may each produce a new thallus, but this arises much more readily from the lower piece. If a piece of the latter is cut into small pieces no larger than half a cubic millimetre, and even much smaller, each may produce a new thallus. Vochting also studied the regeneration of parts having a limited growth. If a gemmiferous capsule is cut off, then split into two or four pieces, and these are placed on moist sand, it is found that new buds arise along the basal cut-edge. In order to show that this is

not due to the new part arising on the basal end because there is no other cut-surface, the apical part of some of the pieces was cut off. These pieces, with two free ends, produced new buds only on their basal ends. The sexual organs of lunularia are borne on the top of erect reproductive branches having a limited growth (Fig. 9, D\ which carry later the sporiferous branches. The branches have a stalk and a terminal disk. If pieces of the stalk are cut off they do not produce any new parts for a long time, but ultimately each produces from the basal cut-surface, or not far from the basal end, a new bud (Fig. El). If the disk is left attached to the piece, the result is the same as before (Fig. Dl\ If a twisted part of the stalk is used, new buds may develop at the base and also near the twisted region, as shown in Fig. 9, E 1. If pieces of the stalk are stuck into the sand, some with the apical end, others with the basal end in the sand, the former produce new buds at the upper basal end, the latter produce buds on the stalk just above the surface of the sand. Pieces that retain the old disk when stuck into the sand (Fig. 9, D) produce one or more buds along the stalk above the sand, often some distance above it. The part buried in the sand does not seem able to develop new buds, and as a result they are produced at the first region of the basal part of the stalk, where the conditions make it possible for buds to develop.

If the disk is cut entirely from the stalk and placed on moist sand, it produces adventitious buds in the region at which the stalk was removed. Buds are also produced at the bases of the rays that go off from the disk. They arise from the under side of the rays without regard to the position of the disk, i.e. whether it is turned upward or downward. If the rays are cut off they, produce new buds at the base (Fig. 9, F}, and if the outer tip of the ray is also cut off, the new bud still arises at the base, as shown in Fig. 9, Fl. These results on pieces with limited growth agree in every respect with those that have been obtained in flowering plants. Vochting thinks that the phenomenon is due in all cases to the limited growth of the parts. Goebel rejects this interpretation, and thinks that the results can be accounted for by the direction of the movement of formative or, at least, of building material. In favor of this view, he points out that in other liverworts the polarity is not shown in the same degree as in lunularia (according to Schostakowitsch), and also that in very old pieces of marchantia, as Vochting has shown, the polarity disappears. In the latter case the attractive action at the vegetative point, to which the building stuff is supposed to flow, is less strong ; and in longer pieces the influence of the apical region may not extend throughout the entire length of the thallus. In favor

of this interpretation he points out that in young prothallia of osmunda, adventitious shoots do not appear, but in older plants, that have become longer, these shoots may appear at the base, because this region is no longer influenced by the apex, and consequently it is possible for building material to accumulate at the basal end. It may be granted that Goebel's idea is possibly correct, viz. that the apex, or the apical end of a piece, may have some influence in preventing the development of shoots at the base, but it does not follow that this influence can be accounted for on the ground of a withdrawal of building stuff from the basal part. As I shall attempt to show in a later chapter, this influence may be of a different nature.

It has been found by Pringsheim and others that pieces of the stem of mosses may also produce new plants, and this holds even for pieces of the stalk of the sporophore and of the wall of the spore capsule (Fig. 10, A-D\ In this case, however, there is not produced a new moss plant directly from the end of the piece, but threads or protonemata grow out, as shown in Fig. 10, A, B, and from these new moss plants are formed in the same way as on the ordinary protonema. The threads that arise from the piece grow out from single cells in the middle part of the stem. These cells are less differentiated and are richer in protoplasm than are the other cells in the stem.

The prothallia of certain ferns are said by Goebel to regenerate if cut in two ; at least this is true for the part that contains the vegetative point. In a piece without the growing point, the cells are very little specialized, and the piece may remain alive ; yet it is incapable of producing a new growing point. Comparing this result with the power of regeneration possessed by lower animals, Goebel states l that since in a plant new organs may arise without the typical form of the plant being produced, "therefore, the completion of a leaf, for instance, that has been injured, would be of no use to the plant, while in animals that do not have a vegetative point, the loss of an organ is a permanent disadvantage in case the organ removed cannot be regenerated." The "explanation" of the difference in the two cases is supposed, apparently, by Goebel, to depend on the usefulness, or non-usefulness, of the regenerative act !

Brefeld has described several cases of regeneration in moulds. There is produced from the zygospore of Mucor mucedo a germinating tube that forms at its end a single sporangium. If the tube is destroyed or injured, a second one is formed from the zygospore, and if this is injured a third time, a new tube is produced. Each time the sporangium is smaller than in the preceding case. If the spore-bearing stalk of Coprinus stercorarius is cut off, the

end grows out and produces a new sporangium. If pieces of the stem are cut off and placed in a nourishing medium, they produce from the ends a new mycelium, and from this new erect hyphae may develop. In the former case, the cut-end regenerates the part removed in somewhat the same way that an animal regenerates at the cut-end ; in the latter, there is a return to the mycelium stage, as in FlG. 36. — After Goebel. Achimenes Haageana. A leaf-cutting of a plant in flower. The new plant, regenerating at base of leaf-stalk, proceeded at once to produce a flower.

the piece of the moss that produces a new protonema. If the mycelium and the protonema are looked upon as an embryonic stage in the formation of the sexual form, there is a return in these cases to an embryonic form or mode of development. One of the most remarkable and important discoveries in connection with the regeneration of plants is that the new individuals that develop from leaves cut off from certain plants differ according to the region of the old plant from which the leaf has been taken. Sachs discovered in 1893 that when the leaves of the begonia are taken from a plant in bloom, the adventitious buds that develop from the leaves very quickly produce new flowers. If the leaves are taken from a plant that has not yet produced flowers, the new plant that develops from the leaf does not produce flowers until after a much longer time. Goebel repeated the experiment with achimenes, and found that the new plants that develop from leaves from the flowering part of the stem (Fig. 36) produce flowers sooner than do the plants that develop from leaves from the base of the same plant. The former produce, as a rule, only one or two leaves and the flower stalk ; the latter, a large number of leaves.

Sachs explains these results as due to a flower-forming stuff that is supposed to be present in the leaves when the plant is about to blossom. This material is supposed to act on the new plant that develops from the leaves, and to bring it sooner to maturity. Goebel points out that the result may also be explained by the fact that the leaves in the flowering region may be poorer in food materials and, in consequence, the adventitious buds that they produce are weaker, and, as experience has shown in other cases, a weakening of the tissues brings about more quickly the formation of flowers. Nevertheless, Goebel inclines to Sachs' hypothesis of specific or formative stuffs, without, however, denying that there is also an inner polarity or "disposition" that also appears in the phenomena of regeneration. But Goebel seems to think that the phenomena of polarity "can most easily be brought under a common point of view by means of Sachs" assumption that there are different kinds of stuffs that go to make the different organs. In the normal life of the plant shootforming stuffs are carried to the vegetative points, while root-forming materials go to the growing ends of the roots. In consequence, when a piece is cut off and the flow of the formative stuffs is interrupted, the root-forming stuff will accumulate at the base of the piece and the shoot-forming stuffs at the apex. In the leaf the flow of all formative substances is toward the base of the leaf, and it is in this region that the new plants arise after the removal of the leaf." A confirmation of this point of view, Goebel believes, is furnished by the following cases.

Some monocotyledonous plants seldom set seed because the vegetative organs, the bulbs, tubers, etc., that reproduce the plant, exert a stronger attraction upon the building stuff than do the young seeds.1 Lindenmuth has shown in some of these forms that pieces of the stem produce, near the base, numerous bulblets, because the building stuff moves toward the base. In Hyacinthiis orientalis, on the other hand, bulblets are produced at 1 Examples of this are found in Lilium candidum, Lachenalia luteola.

the apical part of a piece of the flowering plant. In this plant the seeds ripen normally, presumably because of the migration of stuffs toward the developing seeds. The results in all these cases are due, Goebel thinks, to the direction of the flow of formative stuffs, and cannot be explained as connected in any way with the limited growth of the part. These cases, cited by Goebel, are not in my opinion altogether to the point ; and they fail also to establish convincingly the conclusion that Goebel draws from them. It may be granted that starch is stored up in certain parts of the plant, and if these parts are removed the starch may be stored up in other parts, as Vochting ('87) has shown; but that the movement of this starch to the base can account for the lack of development of the seeds in certain cases seems to me improbable, or, at least, far from being established by the cases cited. It may be granted that the presence of starch in a region may act on the organs there present and determine their fate. Vochting has shown in the potato that by removing the tubers the axial buds, especially in the basal leaves, become tuber-like bodies, but it should not be overlooked that the tubers themselves are formed from underground stolons, that arise in the same way as do those in the axils of the leaves. It would be erroneous, I think, to conclude from these cases of the effect of food stuffs on certain regions that there are formative stuffs for all the organs of the plant, and that these stuffs migrate in different directions and determine the nature of the part. Even the migration of such substances in definite directions in the tissue is itself in need of explanation, since it has been made highly probable by Vochting's experiments that this is not produced by agents outside of the plant. Furthermore, Vochting has shown that the tendency of starch to accumulate in the tubers and the formation of the tuber are separate phenomena.

This hypothesis of formative stuffs held by such able botanists as Sachs and Goebel demands nevertheless serious consideration, if for no other reason than that if it is true it offers quite a simple explanation of many phenomena of growth and of regeneration. We should, I think, distinguish between specific or formative stuffs and building or food stuffs. By specific stuffs is meant a special kind of substance which, being present in a part, determines the nature of the part. Sachs supposes, for instance, that a specific substance is made by the leaves of a plant which is transported to the vegetative, growing region (which has so far produced only leaves), and changes its growth so that flowers are produced. Goebel does not commit himself altogether to specific stuffs of this sort, but speaks also of building stuffs. By building stuff we may understand food material that is necessary for growth, and from which any part of the plant

may be made. Its presence in larger or smaller quantities may determine what a particular part shall become, but further than this it exerts no specific action. This means that the presence of a certain amount of food substance may determine what a given region shall produce, but it is not supposed that there are different kinds of food materials that correspond to each kind of structure. If there were such, they would not differ from specific substances, unless we wish to make subtle distinctions without any basis of fact to go upon.

Goebel points out that there is evidence to show that the greater or less quantity of food substance contained in a plant often determines the nature of its growth, as for instance the production of flowers when the food supply runs low and the production of foliage when the food supply is abundant. This difference may explain Sachs' experiment with begonia leaves ; and if so, there is no need for supposing specific flower stuffs to be made in the plant.

There is another point of view which has been, I think, too much neglected, viz. that the production of food stuffs is itself an expression of changes taking place in the living tissues, and if the structure is changed so that it no longer produces the same substances it may then lead to the development of different kinds of organs. The difference in the regeneration of an apical and a basal leaf of begonia may be due to some difference in the structure of the protoplasm. The greater or smaller amount of starch produced in these leaves may be only a measure of, and not a factor in, the result.

In this same connection another question needs to be discussed. It is assumed by several botanists that in a normal plant the latent shoots or buds along the stem do not develop so long as the terminal shoots are growing, because the latter use up all the food material that is carried to that region. If the terminal bud is destroyed the lateral shoots then burst forth, in consequence, it is assumed, of the excess of food stuff that now comes to them. I do not believe that the phenomena can be so easily explained. If a piece of a plant is cut off, the leaves removed, and the piece suspended in a moist chamber and kept in the dark, the lateral buds at the apex will begin to develop. If we assume that the piece cannot develop any new food substance in the dark, then it contains just the same amount as it did while a part of the plant, and yet that amount is ample for the development of the lateral buds. Moreover, only the more apical buds develop ; but if the piece is then cut in two, the apical buds of the basal piece, that had remained undeveloped, will now develop. How can this be explained by the amount of food substances in the piece ? If it is assumed that in the normal plant the food substances flow only to the growing points, and the buds are out of the main current and fail in consequence to develop, it can be shown that this

idea also fails to explain certain results. Vochting has found, for example, that if an incision is made below a bud and the piece containing the bud be lifted up somewhat from the rest of the piece, remaining attached only at its anterior end, the bud will begin to develop. In this case the conditions preclude an accumulation of food substances in the piece, and the bud is even farther removed than at first from the main current, yet it begins to develop.

We shall find, I think, that the idea of food stuffs fails to explain some of the simplest phenomena, and while it need not be denied that under certain conditions the presence or accumulation of food materials may produce certain definite results, yet such food stuffs seem to play a very subordinate part as compared with certain other internal or innate factors. THERE is a widespread belief amongst zoologists that a definite relation exists between the liability of an animal to injury and its power of regeneration. It is also supposed that those individual parts of an animal that are more exposed to accidental injury, or to the attacks of enemies, are the parts in which regeneration is best developed, and conversely, that those parts of the body that are rarely or never injured do not possess the power of regeneration.

Not only do we find this belief implied in many ways, but we find this point of view definitely taken by several eminent writers, and in some cases carried so far that the process of regeneration itself is supposed to be accounted for by the liability of the parts to injury. In order that it may not appear that I have exaggerated the widespread occurrence of this belief, a few examples may be cited. Reaumur in 1742 pointed out that regeneration is especially characteristic of those animals whose body is liable to be broken, or, as in the earthworm, subject to the attacks of enemies. Bonnet (1745) thought that such a connection exists as has just been stated, and that the animals that possess the power of regeneration have been endowed with germs set aside for this very purpose. He further believed that there would be in each animal that regenerates as many of these germs as the number of times that it is liable to be injured during its natural life. Darwin in his book on Animals and Plants under Domestication says : " In the case of those animals that may be bisected, or chopped into pieces, and of which every fragment will reproduce the whole, the power of regrowth must be diffused throughout the whole body. Nevertheless, there seems to be much truth in the view maintained by Professor Lessona1 that this capacity is generally a localized and special one serving to replace parts which are eminently liable to be lost in each particular animal. The most strik-

1 Delage and Giard give Lessona ('69) the credit for first stating that the phenomenon of regeneration is an adaptation to liability to injury; but Reaumur first suggested this idea in 1742, and Bonnet in 1745. Delage's interpretation, viz. that Lessona ascribed this to a prevoyance de la nature, has been denied by Lessona's biographer, Camerano (La Vita di M. Lessona, Acad. A', d. Torino, 2, XLV, 1896), and by Giard (Sur L'aulotomie Parasitaire, etc., Compt. Rendus de Seances de la Societe de Biologie, May, 1897).

ing case in favor of this view is that the terrestrial salamander, according to Lessona, cannot reproduce lost parts, whilst another species of the same genus, the aquatic salamander, has extraordinary powers of regrowth, as we have just seen ; and this animal is eminently liable to have its limbs, tail, eyes, and jaws bitten off by other tritons." Lang, referring to the brittleness of the tails of lizards, points out that this is a very useful character, since the bird of prey that has struck at a lizard gets hold of only the last part of the animal to disappear under cover ; the lizard escapes by breaking off its tail. The brittleness of the tail is, therefore, an adaptive character that has become fixed by long inheritance.

To this example may be added that of certain land snails in the Philippine Islands. The individuals of the genus helicarion live 'on trees in damp forests, often in great droves. They are very active, and creep with unusual swiftness over the stems and leaves of the trees. Semper has recorded that all the species observed by him have the remarkable power of breaking off the tail (foot) close behind the shell, if the tail is roughly grasped. A convulsive movement is made until the tail comes off, and the snail drops to the ground, where it is concealed by the leaves. Semper adds that in this way the snails often escaped from him and from his collectors, leaving nothing behind but their tails. The tail is said to be the most obvious part of the animal, and it is assumed that this is, therefore, the part that a reptile or bird would first attack.1 Lang states that in this case external influences have produced an extraordinarily well-developed sensitiveness in the animal, so that it reacts to the external stimulus by voluntarily throwing off the tail. It would be, of course, of small advantage to be able to throw off the tail unless the power of regenerating the lost organ existed, or was acquired at the same time as the extreme sensitiveness that brings about the reaction. Lang does not state, however, explicitly that he believes the regenerative power to have arisen through the exposure of the tail of the lizard and the tail of the snail to injury, although he thinks that the mechanism by means of which these parts are thrown off has been acquired in this way. Several other writers have, however, used these same cases to illustrate the supposed principle of liability to injury and power of regeneration.

Weismann in his book on The Germ Plasni has adopted the principle of a connection between regeneration and liability to injury and has carried it much farther than other writers. We can, therefore, most profitably make a careful examination of Weismann's 1 Whether, having once failed in this way to obtain the snail, the bird or lizard would not learn to make a frontal attack is not stated. Or shall we assume that the tail is all that is wanted?

position. His general idea may be gathered from the following quotation : 1 " The dissimilarity, moreover, as regards the power of regeneration in various members of tlie same species, also indicates that adaptation is an important factor in the process. In proteus, which in other respects possesses so slight a capacity for regeneration, the gills grow again rapidly when they have been cut off. In lizards again this power is confined to the tail, and the limbs cannot become restored. In these animals, however, the tail is obviously far more likely to become mutilated than are the limbs, which, as a matter of fact, are seldom lost, although individuals with stumps of legs are occasionally met with. The physiological importance of the tail of a lizard consists in the fact that it preserves the animal from total destruction, for pursuers will generally aim at the long trailing tail,2 and thus the animal often escapes, as the tail breaks off when it is firmly seized. It is, in fact, as Leydig was the first to point out, specially adapted for breaking off, the bodies of the caudal vertebrae from the seventh onward being provided with a special plane of fracture so that they easily break into two transversely. Now if this capability of fracture is provided for by a special arrangement and modification of the parts of the tail, we shall not be making too daring an inference if we regard the regenerative power of the tail as a special adaptation, produced by selection, of this particular part of the body, the frequent loss of wliich is in a certain measure provided for, and not as the outcome of an unknown ' regenerative power ' possessed by the entire animal.

This arrangement would not have been provided if the part had been of no, or of only slight, physiological importance, as is the case in snakes and chelonians, although these animals are as highly organized as lizards. The reason that the limbs of lizards are not replaced is, I believe, due to the fact that these animals are seldom seized by the leg, owing to their extremely rapid movements." Overlooking the numerous cases of the regeneration of internal organs that have been known for several years, and basing his conclusion on a small, unconvincing experiment of his own on the lungs of a few salamanders, Weismann concludes : " Hence there is no such thing as a general power of regeneration ; in each kind of animal this power is graduated according to the need of regeneration in the part under consideration ; that is to say, the degree in which it is present is mainly in proportion to the liability of the part to injury."

After arriving at this conclusion the following admission is a decided anticlimax : " The question, however, arises as to whether the capacity of each part for regeneration results from special process of adaptation, or whether regeneration occurs as the mere outcome — 1 The Germ Plasm. Translation by W. Newton Parker, 1893, PaSe IT6- 2 There are no facts that show that this statement is not entirely imaginary. T. H. M. which is to some extent unforeseen — of the physical nature of an animal. Some statements which have been made on this subject seem hardly to admit of any but the latter explanation." After showing that some newts confined in aquaria attacked each other, " and several times one of them seized another by the lower jaw, and tugged and bit at it so violently that it would have been torn off had I not separated the animals"1 and after referring to the regeneration of the stork's beak, Weismann concludes: " Such cases, the accuracy of which can scarcely be doubted, indicate that the capacity for regeneration does not depend only on the special adaptation of a particular organ, but that a general power also exists which belongs to the whole organism, and to a certain extent affects many and perhaps even all parts. By virtue of this power, moreover, simple organs can be replaced when they are not specially adapted for regeneration." The perplexity of the reader, as a result of this temporary vacillation on Weismann's part, is hardly set straight by the general conclusion that follows on the same page : " We are, therefore, led to infer that the general capacity of all parts for regeneration may have been acquired by selection in the lower and simpler forms, and that it gradually decreased in the course of phylogeny in correspondence with the increase in complexity .of organization ; but that it may, on the other hand, be increased by special selective processes in each stage of its degeneration, in the case of certain parts which are physiologically important and are at the same time frequently exposed to loss."

There are certain statements of facts in the same chapter that are incorrect, and the argument is so loose and vague that it is difficult to tell just what is really meant. As a misstatement of fact I may select the following case : It is stated that lumbriculus does not have the power of regenerating laterally if cut in two, and it is argued that a small animal of this form could rarely be injured at the side without cutting the animal completely in two. As a matter of fact, lumbriculus can regenerate laterally, and very perfectly, as any one can verify if he takes the trouble to perform the experiment ; but, of course, if the whole animal is split in two lengthwise the pieces die, or if a very long piece is split from one side the remaining piece usually disintegrates. If, however, the anterior end is split in two for a short distance, or if a piece is partially split in. two, the half remaining in contact with the rest of the piece completes itself laterally. The same result follows also in the earthworm.