Regeneration

tions, but suspended with the basal end uppermost, results that are in many respects similar to the last are obtained. Roots appear around the base of the piece, i.e. around the upper end, and the leafbuds that develop are those that stand nearest to the apical, at present the lower, end of the piece. These results seem to indicate that, in the main, the chief factors that determine the growth of the new part are internal ones ; and although internal factors do appear to be the dominating ones, since roots appear in both cases at the base and shoots at the apex, yet it would be wrong to conclude that gravity has no influence at all on the result. In fact, other experiments show that it does have an influence.

If an older branch (8-12 mm. in diameter) is cut off and hung up with its base upward, the result is somewhat different from that with younger branches. The roots appear along the entire length of the piece, as shown in Fig. 32, B; the largest are those near the base, and they decrease in size toward the apex of the piece. It is also noticeable that all the roots come from preexisting root-buds, and no adventitious roots are formed, even at the base. The leaf-buds that develop are those arising near the apex, as in the last experiments. They bend upward as they grow longer. A comparison of the results obtained from younger and older pieces may, at first, seem to show that the difference in their development is due to the greater amount of reserve food stuff in the older piece, and Vochting thinks it probable that this influence may account for the strength, length, and even for the number of roots that develop, but he believes that it is improbable that their mode of origin and their location can be so determined. Furthermore, the development of new roots around the base of the younger piece can hardly be explained as due to the absence of food stuff. The explanation of the production of a smaller number of roots in a young piece is that its tissues are less highly specialized, its buds less advanced, and the piece itself is in a lower stage of development. Another explanation must be found for the greater number of roots that develop in the older piece. This is due, as Vochting tries to show, in part to the influence of gravity on the piece.

Vochting's general conclusion is that " the force or forces that determine the polar differences in the piece are most evident and most energetic in very young twigs ; that this difference decreases with the age of the twig whose leaf-buds and root-buds become further developed. It is clear that the new roots of young twigs could appear in corresponding number and strength in exactly the same regions in which they grow out from pre-formed buds of a year-old twig. Since this does not occur, and since the roots appear only near the base of young twigsr the explanation must be that the innate polar forces

act more energetically in young twigs, and the buds that develop in the older twigs must arise in antagonism to the action of this force." The polar difference between apex and base is present, nevertheless, as Vochting's experiments show, even in quite old pieces. A series of experiments was carried out with the internodes of several plants in order to see if, in the absence of pre-formed buds, new buds FIG. 33. — After Vochting. A. Internodal piece of Begonia discolor. Apex upward. B. Same with apex downward. C. Internodal piece of Heterocentron diversifolium. Apex upward. D, E. Pieces of leaf of Heterocentron diversifolium. Apex downward. F. Same with apex upward. D, E, F. Same planted in earth.

would develop. The experiments were undertaken in order to ascertain whether the same polarity, exhibited by longer pieces, would be also found in internodal pieces. In most plants pieces of this kind do not produce new structures, but in Heterocentron diversifolium an internode produces roots at its basal end without regard to the position of the piece (Fig. 33, C). Leaves do not appear on these pieces. On the other hand internodes of Begonia discolor give the opposite result, as

shown in Fig. 33, A, B. In this case leaf-buds appear at the apex of the internodal piece (Fig. 33, A), even when the apical end is downward (Fig. 33, B). From the bases of the new shoots roots may then develop, as also shown in the figure (Fig. 33, B). Vochting concludes that the same polarity that is a characteristic feature of longer pieces is also present in internodal pieces. It is not necessary to separate completely portions of the stem in order to produce roots near one end and shoots near the other. If a ring, including the cambium layer, is cut from the piece, as indicated in Fig. 32, C, the part above and the part below act independently of each other, and each behaves as a separate piece. In various other ways the same result may be obtained, as by simply making an incision in the stem at one side, or by partially splitting off parts of the stem (Fig. 34, C).

If instead of a piece of the stem, a piece of a root is removed, the results are as follows. 1 It should be remembered that the basal end of a root is the part nearer the stem, the apex is the part nearer the apex of the root. If pieces of the root of the poplar, Populus dilatata, are suspended vertically (Fig. 32, D) in a moist chamber, a covering of new cells, a callus, appears over the cutends. From the basal callus numerous leaf-shoots may develop. Pieces of large roots may produce over a hundred of these shoots from a single basal callus. In some cases adventitious shoots may also arise from the side of the root near the basal end. Roots develop from the callus over the apical end ; less often from the sides near the end. If a similar piece of root is suspended with its apical end upward, the new shoots arise as before over the basal end, that is now turned downwards.

The leaves of some plants, as has long been known, are able to produce new plants. The begonias are especially well suited for experiments of this kind. A piece of the stalk of a leaf suspended in a moist atmosphere produces roots near its base. In most cases the opposite end of the stalk, i.e. the end nearest the leaf, putrefies and slowly dies toward the base. Near the base there may arise, before the breaking down of the piece has reached this point, leaf-buds that arise just above the first-formed roots. When these new shoots have reached a certain size they may produce their own roots at or near the base. If, however, a portion of the leaf is left attached to the leaf-stalk (Fig. 35, A), new roots arise near the basal end of the stalk, and later shoots grow out near the point of union of the leaf and its stalk at the point where the veins of the leaf come off. These shoots produce roots of their own near the base, and roots may also appear on the part of the leaf-stalk near its union with the lamina. If a

part of the mid-vein, or of any large vein of the leaf, is cut out, leaving a part of the lamina on each side (Fig. 35, B\ and the piece is suspended vertically, roots appear on the basal end of the vein, and in the same region one or more shoots arise. Leaves of heterocentron with the stalk attached, if kept in diffuse light, produce roots along the stalk, especially near the basal end, but shoots do not appear, even after five months (Fig. 35, C).

These experiments show that the leaves do not exhibit the same polar relations that are shown by pieces of the stem and root. Vochting points out that the results may be explained in either of two ways. The stem and the root have in general an unlimited growth with a vegetative point at the apex. The leaf has only a limited growth. Its cells form permanent tissue, hence the leaf does not produce a new plant from its outer part. The second possibility is this : the phenomenon is connected with the symmetrical relations that different structures possess. Stem and root are symmetrical in two or more directions, the leaf on the other hand is a flat structure with one plane of symmetry, and even symmetry in one plane may be absent. If the leaf could produce shoots at its apex and roots at its base, from the semilunar fibrovascular bundle of the leaf, then an individual (the leaf) with its single plane of symmetry would produce shoots and roots that are symmetrical in two planes. Such a result would be so anomalous that one may well doubt the possibility of its coming into existence.1

Later, Vochting attempted to see if the same relation found in the leaf would hold for other organs that have a limited growth. He found that such structures, as spines, for example, produce both shoots and roots near the base, as do leaves. These experiments of Vochting on the regeneration of pieces of the higher plants show that a piece possesses an innate polarity, or "force," as Vochting sometimes calls it (although he explicitly states that he does not use the word " force " in its strict, physical sense). It does not follow, of course, that external conditions may not also influence the regeneration, but in those experiments in which the pieces were freely suspended in a moist atmosphere, the external factors are as far as possible excluded, so that the effect of the innate tendencies are most clearly seen. In another series of experiments the influence of external conditions on the regeneration was especially

1 Vochting points out that a parallel case is found in certain conifers. In these there arise from a vertical many-sided main stem whorls of side branches that are symmetrical in one plane. These lateral branches, if cut off and planted, produce new roots and new branches, but the latter are always side-branches, like the parts from which they arise. They never produce a normal main axis. Nevertheless, although these branches cannot themselves produce a main shoot, a callus may be formed at the base of the piece, and from this a new main stem may arise.

studied. This analysis that Vochting has made of the problem of regeneration is in the highest degree instructive, since it shows how several factors, — some internal, others external, — take a hand in the result ; and it is only possible to unravel the problem by combining different experiments carried out in such a manner that one by one the different factors at work are separated. If a piece of a young stem of Salix iriminalis is suspended vertically in a moist atmosphere, with the lower end in water (for | of a centimetre), and the piece kept in the dark, the result is, in the main, the same as when similar pieces are suspended in moist air without coming into contact with water. Roots arise near the base, and shoots near the apex, without regard to which end is in the water. .

If the same experiment is repeated in ordinary air, i.e. air not saturated with water, the result is somewhat different. If the twig is suspended vertically with its apex upivard, roots soon appear on the basal end that is in the water, but no roots develop above the water. Small protuberances may appear above the water in the places at which roots would develop if the piece were surrounded by a moist atmosphere, but they do not break through the bark. If the piece is then covered by a jar containing air saturated with moisture, these protuberances may become roots. It is clear, therefore, that the dryness of the air has prevented their development.

If a similar twig is suspended (in the air) with its apex dowmvard, and the lower end in water, root protuberances appear, at first, only around the base, i.e. at the upper end. Under the water, at the apical end, small and weak roots may develop, or may even not appear at all. These results agree, in the main, with those in which the piece is surrounded by moist air, and give evidence of an inner polarity that is an important factor in the regeneration. The results show that in a piece with the basal end in water and the rest of the piece in the air the tendency to produce roots above the water is suppressed by the dryness of the air. In an inverted piece, however, with the apex in water, the innate tendency to produce roots at the basal end is strong enough to overcome the effect of the dryness of the air to suppress their development. The abundance of water absorbed by the apex of the piece makes the development of the roots possible under these conditions despite the dryness of the air.1

There is another factor connected with the submergence of the end of the stem in water that can be shown by putting a longer part of the end under the water. Neither roots, if it is a basal end, nor leaf-buds, if it is an apical end, appear on the deeper parts of the submerged end. This is due, in all probability, to the insufficiency 1 A piece suspended in ordinary air dries up without producing any new structures. of oxygen in the water, and as a result the buds are prevented from developing.

It can be shown that light has also an influence on the regeneration of pieces, and that it has a stronger influence on some plants than on others. In some plants roots develop only on that side of the stem that is less illuminated. In Lepismium radicans, for instance, adventitious roots are produced by the plant even in dry air. Pieces of the stem can produce roots on either the upper or the lower surface, according to which side is less illuminated. A piece of the stem of this plant that had been kept in the dark produced two roots, one above and one below, — one, therefore, opposed to the direction of the action of gravity, and the other in the direction of that action. Even in pieces of the willow, suspended in a moist atmosphere, roots develop better and over a greater length of the stem on the less illuminated side.

Although the experiments with pieces of young willow-twigs may seem to show that gravity is not a factor in regulating the development of the new parts, the results show in reality only that internal factors have a preponderating influence. By means of another series of experiments it can be shown that gravity does have an influence on the production of the new parts. It is evident that in order to test the action of gravity, pieces must be placed in different positions in relation to the vertical. It will be found, if this is done, that different results are obtained according to the angle that the piece makes with the vertical. If a piece is suspended in a moist atmosphere, with its apical end upward, the smaller the angle that the piece makes with the vertical so much the more are the leaf-buds that develop confined to the upper part of the piece, and so much the more do they develop from all sides of the upper end ; conversely, the greater the angle with the vertical, i.e. the more nearly horizontal the position of the piece, so much the more are the leaf-buds that develop found along the upper' side of the apical end (as well as around the end). If the piece is placed in a horizontal position, the leaf-buds develop not only around the apex, but they develop along the entire length of the upper surface, best, however, near the apical end.

If similar pieces are suspended in oblique positions, with the basal end upward, different results are obtained. In the preceding experiment the polarity of the piece and gravity act together, while in this experiment their action is opposed. Although there is a great amount of variability in the results, yet the action of gravity is found to have less influence on the result than has the inner polarity, and the influence of the latter is so much greater that the action of gravity is hardly noticeable.

The roots do not show as markedly the influence of gravity as do the leaf-buds, yet Vochting has found that the position in which they appear varies with the position of the piece with respect to the vertical. In the preceding cases the rudiments of the leaf-buds and of the roots were probably present in most cases, so that gravity only awakens them into activity. In other forms, as, for instance, in FlG. 34. — After Vochting. A. End of a piece of Heterocentron diversifolium. B. Piece of same bent and suspended " with concave-side upward."

Salix viminalls. Apex upward. A piece of the side has been lifted up and two wedges inserted. heterocentron, it is possible to show that gravity may even determine the production of new buds. If pieces of the end of a branch, including the growing point, are suspended vertically, some with the apical end upward, others with the basal end upward (Fig. 34, A), the former produce roots only around the base, but in the latter roots appear frequently, not only at the base, but even extending along the stem. They appear not only at the nodes, where pre-

formed rudiments may be present, but also in the internodes, where there are no rudiments of roots. Stems of heterocentron placed in a horizontal position produce a circle of roots around the base, and later, in several cases, roots from the under surface of the stem, both from the nodes and the internodes; but these roots are smaller than those at the base. Those around the base are often longer on the lower side than on the upper side. Vochting has also studied the regeneration of pieces of roots of the poplar and of the elm suspended horizontally in a moist chamber. A callus develops from the cambium region of the basal end, and from this a thick bunch of adventitious sprouts grows out. A weak callus may develop on the apical end also, from which a few roots develop. In other cases adventitious shoots are produced also from the apical callus, especially from the upper edge of the callus. The results are variable, but show that at times leaf-shoots may develop from the apical end of the root. It is also singular to find that, while pieces of the root produce new leaf-shoots very readily, yet they often fail to produce new roots, or produce only a few that arise from the apical callus or from the sides near that region. It is difficult to show that gravity has any influence on the result.

Vochting recognizes another sort of influence that determines the position of new organs on a piece. If a young, growing end of a stem of Heterocentron diversifolium is suspended by two threads in a horizontal position, the ends bend upward as a result of the negative geotropism of the piece. The new roots appear at the base of the piece, and also on the convex side of the bent part of the stem, as shown in Fig. 34, B. The same result can be obtained by forcibly bending a twig, and then tying the ends together, so that it remains in its bent position. If a piece of this sort is suspended in a moist atmosphere, with the bent inner concave side turned upward, the roots appear on the base and at the bend, especially on the under side, both from the nodes and internodes. If now in order to see if gravity takes any part in the result the next piece is suspended with the outer convex side of the bent part turned upward, it is found that many of the pieces produce roots only at the base, but others produce roots also at the bent portion of the stem, but they are fewer than in the last experiment. The roots arise for the most part on the under side of the arch, and only a few arise from the upper part. It is clear that gravity is also one of the factors in the result. Leafbuds arise in these pieces with the concave side turned upward only near the apex ; rarely one may develop on the lower part of the basal end. In pieces with the concave side turned dowmvard the leaf-buds arise for the most part at the apex, but sometimes they appear on the

upper part of the basal arm. The results are due to two factors, gravity and an inner "force" that is supposed to be the resultant of a growth phenomenon taking place in the bent portion. Vochting supposes that a process of growth takes place as a result of the bending ; " the plasma streams to this region, and a new development takes place here more easily." Vochting adds that this view will not explain the morphological character of the new organs, and that this must be due to quite other causes. The results may, I venture to suggest, find a simpler explanation as the result of the bending, disturbing the tensions of the protoplasm, causing the two arms of the piece to act as if they had been separated from each other. This idea is more fully developed in a later chapter.

Sachs has criticised Vochting's general conclusion in regard to the internal factors that determine the regeneration in a piece of the stem of a plant. He gives very little weight to the innate polarity of the piece, and attempts to explain the results as due to certain substances in the stem of such a sort that, accumulating in any region, they determine the kind of regeneration that takes place. Sachs also assumes that gravity acts on these substances in such a way that the root-forming substances flow downward and the shoot-forming substances flow upward. In a piece of a stem, the two formative substances contained in it accumulate at the two ends, and determine the kind of regeneration that takes place. It is evident that Sachs' hypothesis fails to explain the method of regeneration of an inverted piece suspended in a vertical position, since the roots appear at the upper end and the shoots at the lower end. Sachs explains this as the result of the previous action of gravity on the piece, while the piece was a part of the tree and stood in a vertical direction. He supposes the longer time that gravity has acted on the piece has determined its basi-apical directions, so that this influence is shown in the inverted piece, rather than the action of gravity on it in its new position. This conception involves quite a different idea from the original one of formative substances flowing in definite directions. Moreover, Vochting has met this interpretation by using the twigs of the weeping willow, that hang downward on the tree. If gravity has acted on these drooping twigs in the way that Sachs supposes it can act, then we should expect to find, if Sachs' view is correct, that roots would develop at the apical end of a piece of the twig, and leaves at the basal end, if the piece is hung vertically with its basal end (i.e.

the end originally nearer the trunk of the tree) upward. The regeneration of these pieces shows, however, that they behave in the same way as do pieces of twigs that have always stood vertically on the tree. There can be, therefore, no doubt that the distinction between base and apex is an expression of some innate quality of the plant itself. That an external factor, gravity, is also a factor in the regeneration of the pieces, is abundantly shown by the experiments of Vochting and others, but that innate factors are also at work cannot be doubted. We find evidence in many animals of a similar difference between the two ends of a piece, and we speak of this difference between the anterior and posterior ends of a piece as its polarity. What this polarity may be we do not know, and it is even doubtful whether we should be justified in speaking of it as a force in the sense that the difference in the ends of a magnet is the result of a magnetic force. The kind of polarity shown by animals and plants does not seem to correspond to any of the so-called forces with which the physicist has to deal, but a further discussion of this question will be deferred to a later chapter.

The preceding account of regeneration in some of the higher plants has shown that their usual method of regeneration is by means of latent buds that are present along the sides of the stem, or by means of adventitious buds that develop anew along the sides of the stem. In a few cases new buds may develop from the new tissue of the callus that forms over the cut-ends, but in such cases the new shoots, or the new roots, are much smaller in diameter than the end from which they arise, and usually several or many new shoots develop on the same callus. In these respects the regeneration of the higher plants is different from that of the higher animals, for, in the latter, the new part arises from the entire cut-surface. This difference is no doubt connected with differences in the normal method of growth in plants and in animals, and an explanation of the growth would, perhaps, also give an explanation of the mode of regeneration. The normal method of growth in higher plants takes place largely by the formation of lateral buds, as well as by terminal growth, and we find that regeneration takes place in most cases from the same lateral buds or from others of a similar kind that develop after the piece has been separated.

It is sometimes stated that the higher plants do not regenerate at the cut-ends, because they produce buds at the sides. The statement implies that there is some sort of antagonism between the regeneration of a bud at the end, and the development of buds at the side. It may be true that the development of a latent bud at the side might suppress the tendency to produce a bud at the end, if such a tendency exists ; but if we remove the lateral, pre-formed buds, new ones develop at the sides, and not at the end. That there need not be an antagonism between the formation of a bud, or of buds, at the end, and also at the sides, is shown in Vochting's experiments with the roots of the poplar. In these, leaf-shoots and root-shoots developed both from the callus over the cut-end, and at the side of the piece also. It has further been shown that, although a piece of the internode does

not produce new leaf-buds at the sides, neither does it regenerate a new apical bud at the end. A most interesting fact connected with the regeneration of the higher plants is, as has been pointed out, that even when a callus is formed over the cut-end, and new growth takes place from this callus, FlG. 35. — After Vochting. A. Leaf-stalk of Begonia rex with a portion of the lamina. Suspended with base upward. B. Piece of lamina of leaf of same. C. Leaf of Heterocentron diver sifo I turn. D. Leaf-stalk of Begonia discolor.

there is produced, not a single terminal bud, but a number of separate buds. The piece does not complete itself, but produces new buds, that make new branches. The explanation of this mode of regeneration in plants is not known. It appears to be connected with the production, by means of buds, of all the new structures. Why this should occur we do not know, and the only suggestion that offers itself is that the result may be in some way connected with the hard cell walls in plants that make difficult the organization of large areas into a new whole. As a result, the new development takes place in a small group of similar cells, that are sufficiently near together to organize themselves into a whole despite the interference met with in the cell walls.