Chapter IX: Part III: Tropism in Plants (6)

_Experiment 179._--The terminals of the galvanometer were suitably connected with the two diametrically opposite points A and B in the growing region of the root. Stimulus was now applied very near the point A, the various stimuli employed in different experiments being: (1) mechanical, (2) chemical, and (3) thermal. In every instance the excited point A becomes galvanometrically negative. This shows that the response of the root is in no way different from that of the shoot.

MECHANICAL RESPONSE TO INDIRECT STIMULUS.

Before describing the effect of indirect stimulus on the root, I shall recapitulate its effects on ordinary tissues. I have shown that the effect of indirect unilateral stimulus is to induce a movement away from stimulus. This was shown to be the case with the bud of _Crinum_ (p. 275) and the tendril of _Passiflora_ (p. 291). The mechanical and electric response to indirect stimulation in the shoot is shown in the diagrammatic representation (Fig. 170). I shall now proceed to describe the mechanical response induced by unilateral stimulation of the root tip. As the responding region of growth is at some distance from the tip, the stimulation is therefore indirect.

_Experiment 180._--I employed at first mechanical stimulus of moderate intensity by rubbing one side of the tip of the root of _Bindweed_; this induced a movement away from stimulus. Unilateral application of dilute acid gave rise to a similar response. Thermal stimulus of moderate intensity also induced responsive movement away from the stimulus (Fig. 171).

Darwin in his _Movements of Plants_ described experiments on the responsive behaviour of the tip of the radicle. He produced unilateral stimulation in three different ways, first by attaching minute fragments of cardboard to one side of the root-tip; this moderate and constant irritation was found to induce a convexity on the same side of the growing region, with the resulting negative movement, _i.e._, away from stimulus. His second method was chemical, one side of the tip being touched with silver nitrate; the third method of stimulation was a slanting cut. All these methods induced a movement away from stimulus.

ELECTRICAL RESPONSE TO INDIRECT STIMULATION.

The next investigation was for the determination of the electrical change induced in the growing region by application of unilateral stimulus at the root-tip.

_Experiment 181._--One of the two electrical connections with the galvanometer is made at one side of the growing region A, the other connection being made with the diametrically opposite point B. Unilateral stimulus was applied at the root tip _a_, of the bean plant and on the same side as A. I subjected the tip to various modes of unilateral stimulation. Mechanical stimulation was effected by emery-paper friction or by pin-prick; chemical stimulation was produced by application of dilute hydrochloric acid. Thermal stimulation was caused by the proximity of electrically heated platinum wire. In every case the response was by _induced galvanometric positivity at A_ (Fig. 171). This electrical variation took place within about ten seconds of the application of stimulus; the interval would obviously depend on the length of path to be traversed by the transmitted effect of indirect stimulation.

The galvanometric positivity at A indicated that there was induced at that point an increase of turgor and expansion, in consequence of which the organ would move away from stimulus. Thus both by the mechanical and electrical methods of investigation we arrive at an identical conclusion that the effects of unilateral stimulus at the tip of the root gives rise to a movement, by which the organ is moved away from the source of stimulus; since tropic movement towards stimulus is termed _positive_, this opposite response must be regarded as _negative_.

TABLE XXXVI.--EFFECT OF INDIRECT STIMULUS UNILATERALLY APPLIED AT THE ROOT-TIP.

+--------------------------------------------------------------+
|Effect at the proximal side A in the | Effect at the distal|
| growing region. | side B. |
+--------------------------------------+-----------------------+
|Galvanometric positivity, indicative | Negligible. |
| of increase of turgor and expansion. | |
|--------------------------------------------------------------|
|The corresponding tropic curvature is negative, _i.e._, a |
|movement away from stimulus. |
+--------------------------------------------------------------+

The root-tip when burrowing its way underground comes in contact with hard substances and moves away from the source of irritation. The irritability of the root-tip is generally regarded as being specially evolved for the advantage of the plant. But reference to experiments that, have been described shows that this reaction is not unique but exhibited by all plant organs, growing and non-growing. Indirect stimulus has been shown to give rise, in both shoot and root, to a _negative_ tropic curvature in contrast to the _positive_ curvature brought about by direct stimulation; the response of the root is therefore in no way different from that of vegetable tissues in general.

It will also be seen that an identical stimulus induces two opposite effects, according as the stimulus is applied at the tip or at the growing region itself. In the former case, the stimulus is indirect, and in the latter case it is direct. The results are in strict conformity with the laws of effects of direct and indirect stimulations that have been established regarding plant response in general (p. 231).

SUMMARY.

In the root, the responsive region is in the zone of growth. The tip of the root is separated from the region of response by a semi-conducting or non-conducting tissue.

Direct unilateral stimulus (applied at the region of growth) induces a positive curvature by the contraction of the proximal and expansion of the distal side.

The electrical response to direct unilateral stimulus is galvanometric negativity of the proximal, and galvanometric positivity of the distal side.

Indirect unilateral stimulus induces expansion of the proximal side resulting in negative curvature and movement away from stimulus.

The corresponding electric response induced is galvanometric positivity of the proximal side.

The responses of the root, to both direct and indirect stimulations, are precisely similar to those in the shoot. The assumption of specific irritability of the root as differing from that of the shoot, is without any justification.

XLII.--GEO-ELECTRIC RESPONSE OF ROOT

_By_

SIR J. C. BOSE,

_Assisted by_

SATYENDRA CHANDRA GUHA.

The effects of various stimuli, direct and indirect, on the response of the root have been described in the last chapter. These responsive reactions have been found to be in no way different from those of the shoot. But the shoot and the root exhibit under the stimulus of gravity, responsive movements which are diametrically opposite to each other. These opposite effects of an identical stimulus have been regarded as due to specific differences of irritability in the two organs, specially evolved for the advantage of the plant. The root is thus supposed to be characterised by "positive" and the shoot by "negative" geotropism.

As regards response to other forms of stimuli, the root has been shown to behave like the shoot. We have now to inquire whether the reaction of the root to gravitational stimulus is specifically different to that of the shoot.

The electric method of investigation described in the last chapter, holds out the possibility of discovering the character of the responsive reaction induced in the root by its displacement from vertical to horizontal position; we shall, moreover, be able to make an electrical exploration of the root-tip and the zone of growth, and thus determine the qualitative changes of response, induced in two regions of the root under the action of gravitational stimulus. For the detection of geotropic action in the shoot, electric contacts were made at two points diametrically opposite to each other. Displacement of the shoot from vertical to horizontal position induced excitatory change of galvanometric negativity at the upper side of the organ, demonstrating the effect of direct stimulation of that side; this excitatory reaction of the upper side finds independent mechanical expression in the induced contraction and concavity of that side of the organ.

I employ a similar electric method for detection of geotropic excitation of the root, responses to geotropic stimulus being taken at the root-tip and also at the zone of growth in which geotropic curvature is effected. I shall now proceed to give a detailed description of the characteristic electric responses of the tip and of the growing region.

The two diametrically opposite contacts at the tip will be distinguished as _a_ and _b_, the corresponding points higher up in the growing region being A and B. When the root is vertical the electric conditions of the two diametrically opposite points are practically the same. But when the root is rotated in a vertical plane through +90° a geo-electric response will be found to take place; the direction of the responsive current disappears when the root is brought back to the vertical. Rotation through -90° gives rise again to a responsive current, but its direction is found reversed.

GEO-ELECTRIC RESPONSE OF THE ROOT-TIP.

_Experiment 182._--I took the root of the bean plant and made two electric contacts with the diametrically opposite points, _a_ and _b_, of the root-tip at a distance of about 1·5 mm. from the extreme end. Owing to the very small size of the tip this is by no means an easy operation. Two platinum points tipped with kaolin paste are very carefully adjusted so as to make good electric contacts at the two opposite sides, without exerting undue pressure. For geotropic stimulation the root has to be laid horizontal, and as the root of the bean plant is somewhat long and limp, displacement from the vertical position is apt to cause a break of the electric contact. This is avoided by supporting the root from the top and also from the sides; for the latter purpose, I use paddings of cotton wool.

After due observance of these precautions the electric response obtained is found to be very definite; when the root is made horizontal, by rotation of the root through +90°, the point _a_ is above, and the responsive current is found to flow from _b_ to _a_, _the upper side of the tip_ becoming galvanometrically negative; when the root is brought back to the vertical, the responsive current disappears; rotation through -90° makes the point _b_ occupy the upper position, and the responsive current is from _a_ to _b_; the upper side thus exhibits in every case, an excitatory electric change of galvanometric negativity (Fig. 172). The root-tip thus exhibits the characteristic response to direct stimulation. Experiments carried out with 12 different specimens gave concordant results. The following table gives the absolute values of electro-motive force induced at the tip under geotropic stimulus.

TABLE XXXVII.--GEO-ELECTRIC RESPONSE OF THE ROOT TIP (_Vicia Faba_).

+--------------------------+
|Specimen.|Induced E. M. F.|
+---------+----------------+
|1 | 0·0005 volt. |
|2 | 0·0011 " |
|3 | 0·0010 " |
|4 | 0·0015 " |
+--------------------------+

ELECTRIC RESPONSE IN THE GROWING REGION.

_Experiment 183._--I next undertook an investigation on the electric variation induced in the growing region under the stimulus of gravity. The experimental difficulties are here greatly reduced, since the available area of contact for galvanometric connection is not so restricted as in the case of the root-tip. The specimen is securely mounted so that the root is vertical. It is next rotated in the vertical plane through +90°, so that the point A in the growing region occupied the upper position. The electric response in the growing region took place in a short time and was very distinct. The induced electric change at A was now galvanometric _positivity_ indicative of increase of _turgor and expansion_.

The series of experiments were carried out in the following order. The specimen was first rotated through +90° so that A was above. The responsive electric variation rendered it galvanometrically positive. The root was rotated back to neutral position when the current disappeared. The root was next rotated through -90° and the responsive current became reversed, the upper B becoming electro-positive (Fig. 173). The alternative rotations through +90° and -90° were carried out six times in succession with consistent results. The interval allowed between one stimulation and the next was determined by the period of complete recovery. Growing fatigue was found to increase this period; at first it was seven minutes, at the second repetition it was ten minutes, and at the third time it was prolonged to fifteen minutes.

I give below the series of electric responses induced by alternate rotations through +90° and -90°. The upper position was occupied by A in the odd series, and by B in the even series. In every case the upper side became galvanometrically positive.

TABLE XXXVIII.--GEO-ELECTRIC RESPONSE OF ROOT IN THE REGION OF GROWTH.

+---------------------------------------------------------------+
| Odd |Galvanometer deflection| Even |Galvanometer deflection|
|series.| A, positive. |series.| B, positive. |
+-------+-----------------------+-------+-----------------------+
| 1 | 20 divisions. | 2 | 18 divisions. |
| 3 | 16 " | 4 | 18 " |
| 5 | 10 " | 6 | 12 " |
+---------------------------------------------------------------+

ADDITIVE ACTION-CURRENT AT THE TIP AND THE GROWING REGION.

It has been shown that under geotropic stimulus the upper side of the tip, _a_, becomes galvanometrically negative, while the point A, higher up in the growing region, becomes galvanometrically positive. If now we make the two galvanometric connections with _a_ and A, the induced electric difference is increased, and the galvanometric response becomes enhanced.

_Experiment 184._--The root was at first held vertical, and two electric contacts made with _a_ and A. In this neutral position there is little or no current. But as soon as the root was laid horizontal, an electro-motive response was obtained which showed that _a_ was galvanometrically negative, and A galvanometrically positive (Fig. 173d). The induced electric response disappeared on restoration of the root to the vertical position. I give below the results of typical experiments with a vigorous specimen which gave strong electric response. It was possible to repeat the geotropic stimulation six times in succession, the results being perfectly consistent. The responses taken in succession exhibited slight fatigue, the first deflection being 140 divisions, and the sixth 115 divisions of the galvanometer scale.

TABLE XXXIX.--INDUCED E. M. F. VARIATION BETWEEN THE TIP AND THE GROWING REGION (_a_ NEGATIVE AND A POSITIVE).

+-------------------------------------------------------+
|Geotropic stimulation. | Resulting electric response.|
+------------------------+------------------------------+
|First stimulation | 140 divisions. |
|Second " | 130 " |
|Third " | 130 " |
|Fourth " | 123 " |
|Fifth " | 127 " |
|Sixth " | 115 " |
+-------------------------------------------------------+

The results of experiments 182 and 183 are summarised as follows:--

(1) the induced galvanometric negativity at root tip indicates
direct stimulation of the tip, and

(2) the induced galvanometric positivity of the growing region
shows that it is the effect of indirect stimulus that reaches it.

From these facts it will be seen that the tip perceives the stimulus and thus undergoes excitation, and that owing to the intervening tissue being a semi-conductor of excitation, it is the positive impulse that reaches the growing region and induces there an expansion and a convex curvature.

GEO-PERCEPTION AT THE ROOT TIP.

The results given above fully confirm Charles Darwin's discovery that it is the root tip that perceives the stimulus of gravity[37]; he found that removal of the tip abolished the geotropic response of the root. Objection has been raised about the shock-effect of operation itself being the cause of abolition of response. But subsequent observations have shown that Darwin's conclusions are in the main correct.

[37] "This view has been the subject of a considerable amount
of controversy. Wiesner denies the localisation of geotropic
sensitiveness. Czapek, on the other hand, supports Darwin's
theory. Recently Picard has attacked the problem in a new way
(and) concludes that not only the root tip but also the
entire growing zone is capable of perceiving gravitational
stimuli.... As both Picard's experimental method and his
interpretation are open to criticism, the author has repeated
his experiments with a more satisfactory apparatus. He finds
that in _Vicia Faba_, _Phaseolus multeflorus_ and _Lupinus
albus_, both apex and growing zone are geotropically
sensitive, the former being by far the more sensitive of the
two, and the curvature of the growing zone being without a
doubt largely induced by secondary stimuli transmitted from
the apical region. Charles Darwin's views were therefore in
the main correct."--Haberlandt--_Ibid_, p. 748.

The experiments which I have described on the geo-electric response of the root tip and of the growing region offer convincing proof of the perception of the stimulus at the tip, and the transmission of the effect of indirect stimulus to the growing region. These experiments exhibit in an identical _uninjured_ organ: the excitatory reaction at the upper side of the tip, the cessation of excitation, and the excitation of the opposite side of the tip, following the rotation of the organ through +90°, 0° and -90°. The effect at the growing zone is precisely the opposite to that at the tip, _i.e._, an expansive reaction which results from the effect of indirect stimulus, in contrast to the contractile reaction due to direct stimulation.

We may now proceed a step further and try to obtain some idea of the difference in the mechanics of geotropic stimulation of the shoot and of the root, to account for the different responses in the two organs. The reason of this difference lies in the fact that in the shoot the perceptive and responding region is one and the same; every cut-piece of stem exhibits the characteristic geotropic curvature. In the root the case is different; for the removal of the sensitive root-tip reduces or abolishes the geotropic action; the region of maximum geotropic perception is thus separated from that of response. It must be borne in mind _that this holds good only in the case of gravitational stimulus_, for the decapitated root still continues to respond to other forms of stimulation such as chemical or photic.

The cause of this difference in the reactions to geotropic and other stimuli lies in the fact that in the latter case, energy is supplied from outside. But in geotropism the force of gravity is by itself inoperative; it is only through the weight of the cell contents that the stimulus becomes effective. Want of recognition of this fundamental difference has led many observers in their far-fetched and sweeping attempt, to establish an identity of reaction of the root to geotropic and photic stimulations, in spite of facts which plainly contradict it. Thus the root moves away from the incident vertical line of gravity; but under light, the root very often moves towards the stimulus. The negative phototropic response of the root of _Sinapis_ is an exceptional phenomenon for which full explanation has been given in page 376.

We shall next consider whether the particular distribution of the falling starch-grains (which offers a rational explanation of geotropic stimulation) in the shoot and in the root, is capable of furnishing an explanation of the different geotropic responses in the two organs. In this connection, the results of investigation of Haberlandt and Nemec are highly suggestive. Haberlandt finds statoliths present in the responding region of the stem; the geotropic stimulation of the stem is therefore direct. Nemec's investigation on the distribution of statoliths in the root show, on the other hand, that it is the central portion of the root cap that contains the falling starch grains, and this would account for the indirect geotropic stimulation of the root.

The theory of statoliths is, however, not essential for the explanation of the opposite geotropic effects in the shoot and in the root. The observed fact, that the perceptive region in the root is separated from the responding region, is sufficient to explain the difference of geotropic action in the two organs. Through whatever means the stimulus of gravity may act, it is inevitable, from the fact that the stimulation of the shoot is direct and of the root indirect, that an identical stimulus should in two cases induce responsive reactions of opposite signs.

It will thus be seen that the postulation of two different irritabilities in the shoot and in the root is wholly unnecessary and unwarranted by facts. For the irritability of the root has been shown to be in no way different from that of other organs; an uniformity is thus found to exist in the reaction of all vegetable tissues.

SUMMARY.

On subjection of the tip of the root to the stimulus of gravity, the upper side exhibits excitatory reaction of galvanometric negativity. This shows that the root-tip undergoes direct stimulation.

The electric response in the growing region above the stimulated point of the root-tip is positive, indicative of increase of turgor and expansion. This is due to the effect of indirect stimulus.

The stimulus of gravity is perceived at the root-tip; it is the effect of indirect stimulus that is transmitted to the responding region of growth.

In contrast with the above is the fact that the growing region of the shoot is both sensitive and responsive to geotropic stimulus.

As the effects of direct and indirect stimulation on growth are antithetic, the responses of shoot and root to the direct and indirect stimulus must be of opposite signs.

There is no necessity for postulating two different irritabilities for the shoot and the root, since tissues in general exhibit positive or negative curvatures according as the stimulus is direct or indirect.

XLIII.--LOCALISATION OF GEO-PERCEPTIVE LAYER BY MEANS OF THE ELECTRIC PROBE

_By_

SIR J. C. BOSE,

_Assisted by_

SATYENDRA CHANDRA GUHA.

The obscurities which surround the phenomenon of geotropism arise: (1) from the invisibility of the stimulating agent, (2) from want of definite knowledge as to whether the fundamental reaction is contractile or expansive, and (3) from the peculiar characteristic that the stimulus is only effective when the _external_ force of gravity reacts _internally_ through the mass of contents of the sensitive cells.

The experiments that have been detailed in the foregoing chapters will have removed most of the difficulties. But beyond these is the question of that power possessed by plants of _perceiving_ geotropic stimulus by means of certain localised sense organs, which send out impulses in response to which neighbouring cells carry out the movement of orientation in a definite direction. Are the sensitive cells diffusely distributed in the organ or do they form a definite layer? Could we by the well established method of physiological response localise the sensitive cells in the interior of the organ? As the internal cells are not accessible, the problem would appear to be beyond the reach of experimental investigation.

It is true that post-mortem examination of sectioned tissues under the microscope enables us to form a probable hypothesis as regards the contents of certain cells causing geotropic irritation; we have thus the very illuminating theory of statoliths propounded by Noll, Haberlandt and Nemec. But for the clear understanding of the _physiological reaction_ which induces the orientating movement, it is necessary to get hold, as it were, of a single or a group of sensory cells _in situ_ and in a condition of fullest vital activity; to detect and follow by some subtle means the change induced in the perceptive organ and the irradiation of excitation to neighbouring cells, through the entire cycles of reaction, from the onset of geotropic stimulus to its cessation.

The idea of obtaining access to the unknown geo-perceptive cell in the interior of the organ for carrying out various physiological tests would appear to be very extravagant; yet I could not altogether give up the thought that the obscure problem of geotropic action might be attacked with some chance of success, by means of an electric probe which would explore the excitatory electric distribution in the interior of the organ. But the experimental difficulties which stood in the way were so great that for a long time I gave up any serious attempt to pursue the subject. And it is only when the present volume is going through the press that the very first experiments undertaken proved so highly successful that I am able to give a short account of the more important results, which cast a flood of light on the obscurities of geotropic phenomena. The new method has opened out, moreover, a very extensive range of investigation on the activities of cells in the interior of an organ, and enabled me to localise the conducting 'nerve' which transmits excitation in plants. These and other results will be given in the next volume.

METHOD OF EXPLORATION BY THE ELECTRIC PROBE.

The principle of the new method will be better understood if I first explained the steps of reasoning by which I was led to discover it. The experiments described in Chapter XL showed that the upper surface of a horizontally laid shoot exhibits sign of excitation by induced galvanometric negativity; that this was due to the stimulus of gravity was made clear by restoration of the plant-organ to the vertical position, when all signs of electric excitation disappeared. Now the skin of the organ on which the electrode was applied could not be the perceptive organ, for the removal of the epidermis did not abolish the geotropic action; the perceptive layer must therefore lie somewhere in the interior. As every side of a radial organ undergoes geotropic excitation, the geo-perceptive cells must therefore be disposed in a cylindrical layer, at some unknown depth from the surface. In a longitudinal section of the shoot, they would appear as two straight lines G and G´ (Fig. 174). In a vertical position the geo-perceptive layer will remain quiescent but rotation through +90° would initiate the excitatory reaction. Let us first centre our attention to the geo-perceptive layer G, which occupies the upper position. This sensitive layer perceives the stimulus and is therefore the focus of irritation; the state of excitation is, as we have seen, detected by induced galvanometric negativity, and the electric change would be most intense at the perceptive layer itself. As the power of transverse conduction is feeble, the excitation of the perceptive layer will irradiate into the neighbouring cells in radial directions with intensity diminishing with distance. Hence the intensity of responsive electric change will decline in both directions outwards and inwards.

The distribution of the excitatory change, initiated at the perceptive layer and irradiated in radial directions is represented by the depth of shading, the darkest shadow being on the perceptive layer. Had excitation been attended with change of light into shade, we would have witnessed the spectacle of a deep shadow (vanishing towards the edges) spreading over the different layers of cells during displacement of the organ from vertical to horizontal; the shadow would have disappeared on the restoration of the organ to the vertical position.

Different shades of excitation in different layers is, however, capable of discrimination by means of an insulated electric probe, which is gradually pushed into the organ from outside. It will at first encounter increasing excitatory change during its approach to the perceptive layer where the irritation will be at its maximum. The indicating galvanometer in connection with the probe will thus indicate increasing galvanometric negativity, which will reach a maximum value at the moment of contact of the probe with the perceptive layer.

It will be understood that the surface electric reaction under geotropic stimulus, which we hitherto obtained, would be relatively feeble compared to the response obtained with direct contact with the maximally excited perceptive layer. When the probe passes beyond the perceptive layer the electric indication of excitation will undergo decline and final abolition. The characteristic effects described above are to be found only under the action of gravitational stimulus; they will be absent when the organ is held in a vertical position and thus freed from geotropic excitation.

I have hitherto spoken of the excitatory effect of the upper layer; there must be some physiological reaction on the lower perceptive layer, though of a different character, represented diagrammatically by vertical shading. Had the physiological reaction on the lower side of a radial organ been the same as on the upper, geotropic curvature would have been an impossibility, for similar reactions on opposite sides would, by their antagonistic effects, have neutralised each other.

After this preliminary explanation, I shall give a detailed account of the experiments and results. It is to be borne in mind that the investigation I am going to describe presupposes no hypothesis of geotropic action. I start with the observed fact that an organ under the stimulus of gravity, exhibits responsive movement. I ascertain the nature of the underlying reaction by electric tests; I have, in my previous works, fully demonstrated that the excitatory contractile reaction is detected by electro-motive change of galvanometric negativity, and the opposite expansive reaction by a change of galvanometric positivity. With the electric probe I ascertain whether geotropic irritation is diffuse, or whether it is localised at any particular depth of the organ. I map out the contour lines of physiological reaction with its heights and depths of excitation.

I shall now proceed to describe the results of electric exploration into the interior of the organ. The trouble I foresaw, related to the irritation caused by the passage of the probe, and the after-effect of wound on variation of excitability.

THE ELECTRIC PROBE.

The wound-irritation is, however, reduced to a minimum by making the probe exceedingly thin. A fine platinum wire 0·06 mm. in diameter passes through a glass tubing drawn out into a fine capillary, and fused round one end of the platinum wire which protrudes very slightly beyond the point of fusion; the exploring electrode is thus insulated except at the protruded sharp point of the platinum wire. The length of the capillary is about 6 mm., just long enough to pass the experimental plant-organ transversely from one end to the other; the average diameter of the capillary is about 0·15 mm. The other end of the platinum wire comes out of the side of the tubing and is led to one terminal of the galvanometer, the other being connected with an indifferent point in the organ. The probe can be gradually pushed into the plant-organ by rotation of a screw head, one complete rotation causing a forward movement through 0·2 mm. (Fig. 175).

_Wound-reaction._--I have shown that a prick acts as a mechanical stimulus, and in normal excitable tissues induces an excitatory change of galvanometric negativity. This wound-reaction increases with the extent of the wound, and the suddenness with which it is inflicted. On account of the fineness of the probe, it insinuates itself into the tissue rather than make any marked rupture; the probe again is introduced very gradually; with these precautions the wound-reaction is found to be greatly reduced. The immediate effect of the prick is a negative deflection of the galvanometer, which declines and attains a steady value in the course of about 5 minutes.

_Effect of wound on excitability._--I have shewn (p. 81) that severe wound caused by transverse section induced a temporary abolition of irritability in _Mimosa_, but that the normal excitability was restored in the course of an hour. A prick from a thick pin was shown to depress temporarily the rate of growth, the normal rate being restored after an interval of 15 minutes (p. 202). In the case of geo-electric excitability, the depressing effect of the passage of the probe, I find, to disappear in the course of about 10 minutes.

For a choice of experimental material we have to find specimens which are not merely geotropically sensitive, but also exhibit large electric response under stimulus. In both these respects the shoot of _Bryophyllum_ and the flower stalk of _Nymphæa_ give good results.

ELECTRIC EXPLORATION FOR GEO-PERCEPTIVE LAYER BY MEANS OF THE PROBE.

_Experiment 185._--I shall now proceed to give a detailed account of the experiments. The first specimen employed was the shoot of _Bryophyllum_, one contact being made with the side of the stem, and the other with an indifferent point on the leaf which was always held vertical. In a particular experiment, the probe was introduced into the stem through 0·4 mm. and a feeble galvanometric negativity was induced as the wound-effect. After an interval of 5 minutes, this attained a steady value of -15 divisions. On the rotation of stem through +90°, the point A was above and a very much larger deflection of -82 divisions was obtained, being the result of summation of wound and geo-electric effects. On restoration of the plant to vertical position the geo-electric reaction disappeared, leaving the persistent wound reaction of -15 divisions unchanged. The true geo-electric reaction at a point 0·4 mm. inside the stem was thus -67 divisions which is the difference between -82 and -15 divisions. I obtained in this manner the excitatory reactions at different layers of the organ. The following table gives true values of geo-electric reaction at different layers of the stem as the probe entered it by steps of 0·4 mm.

TABLE XL.--SHOWING THE GEO-ELECTRIC REACTION AT DIFFERENT DEPTHS OF THE ORGAN (_Bryophyllum_).

+-------------------------------------------+
|Position of the| Geo-electric excitation |
|probe. |(galvanometric negativity).|
+---------------+---------------------------+
| Surface | 5 divisions. |
| 0·4 mm. | -20 " |
| 0·8 " | -24 " |
| 1·2 " | -22 " |
| 1·6 " | -18 " |
| 2·0 " | -14 " |
| 2·4 " | -10 " |
| 2·8 " | -5 " |
| 3·2 " | 0 " |
+-------------------------------------------+

The results given above, typical of many others, show that there is a definite layer in the tissue which undergoes maximum excitation under the stimulus of gravity, and that this excitation irradiates with diminishing intensity in radial directions inwards and outwards.

_The geo-perceptive layer may thus be experimentally localised by measuring the depth of intrusion of the probe for maximum deflection of galvanometric negativity._

_Localisation of geo-perceptive layer in_ Nymphæa: _Experiment 186._--I employed the same method for the determination of the perceptive layer of a different organ namely, that of the flower stalk of _Nymphæa_. The electric reaction in _Nymphæa_, even under the prevailing unfavourable condition of the season, was moderately strong, being about three times greater than in _Bryophyllum_. A dozen observations made with different specimens gave very consistent results of which the following may be taken as typical. The probe was in this case, as in the last, moved by steps of 0·4 mm. at a time. Other examples will be given later where readings were taken for successive steps of 0·2 mm.

TABLE XLI.--SHOWING THE DISTRIBUTION OF INDUCED GEO-ELECTRIC EXCITATION IN DIFFERENT LAYERS (_Nymphæa_).

+--------------------------------------------+
|Position of probe.|Galvanometric deflection.|
+------------------+-------------------------+
| Surface | 0 divisions. |
| 0·4 mm. | -16 " |
| 0·8 " | -42 " |
| 1·2 " | -20 " |
| 1·6 " | -10 " |
| 2·0 " | -2 " |
| 2·4 " | 0 " |
+--------------------------------------------+

It will be seen that as in _Bryophyllum_, so in _Nymphæa_, the geo-electric excitation increased at first with increasing depth of the tissue till at a depth of 0·8 mm. of the particular specimen the induced excitation attained a maximum value. The excitatory effect then declines till it vanished at a depth of 2·4 mm.

The depth of layer at which maximum excitation takes place varies to some extent, according to the thickness of the shoot. Thus while in a thin specimen of _Bryophyllum_ 3·6 mm. in diameter the geo-perceptive layer was found at a depth of 0·6 mm., it occurred at the greater depth of 0·8 mm. in a thicker specimen, 5 mm. in diameter. In _Nymphæa_ also the perceptive layer was found at a depth of 0·8 mm. in a thin and at a depth of 1·4 mm. in a thick specimen.

Having thus succeeded in localising the geo-perceptive layer by experimental means, it was now possible to examine the anatomical characteristics of the layer by examining it under the microscope. I also wished to find out from microscopic examination, the cause of certain differences noticed in the determinations of the perceptive layer in _Bryophyllum_ and in _Nymphæa_. In the former the probe always encountered the maximally excited geo-perceptive layer from whichever point of the surface it entered the organ; this indicated that the sensitive layer in _Bryophyllum_ was continuous round the axis. In _Nymphæa_, however, the probe occasionally missed the sensitive layer; but a new point of entry led to successful localisation of the perceptive layer; this was probably due to the particular layer not being continuous but interrupted by certain gaps.

MICROSCOPIC EXAMINATION OF THE MAXIMALLY EXCITED LAYER.

The specimens were taken out after the electric test, and the transverse sections made at the radial line of the passage of the probe. Thus in a particular experiment with _Bryophyllum_ the point of maximum geotropic excitation was found to be at a distance of 0·8 mm. from the surface. By means of the micrometer slide in the stage and the micrometer eye-piece, the internal layer 0·8 mm. from the surface was examined; the particular sensitive layer S was recognised as the _continuous_ 'starch sheath' or endodermis containing unusually large sized starch grains (Fig. 176). These often occurred in loosely cohering groups of 8 to 10 particles, and their appearance is very different from the small sized irregularly distributed grains in other cells.

Examination of the microscopic section of the flower stalk of _Nymphæa_ showed that the 'starch sheath' was not continuous but occurred in crescents above the vascular bundles which are separated from each other. The occasional failure of electric detection of the perceptive layer is thus due to the probe missing one of the crescents, which with intervening gaps, are arranged in a circle.

I give below a number of experimental determinations of the geo-perceptive layer in different specimens together with the micrometric measurement of the distance of the 'starch sheath' from the surface, the transverse section being made at the place where the probe entered the shoot. Eight different determinations are given, three for _Bryophyllum_ and five for _Nymphæa_.

TABLE XLII.--SHOWING THE POSITION OF THE GEO-PERCEPTIVE LAYER AND OF 'STARCH SHEATH' IN DIFFERENT SPECIMENS.

+-----------------------------------------------------------+
| Specimen. | Distance of | Distance of the |
| | geo-perceptive layer | starch sheath |
| | from surface. | from surface. |
| | (Method of | (Microscopic |
| | electric probe.) | measurement.) |
+---------------+----------------------+--------------------+
|_Bryophyllum_: | | |
| | (1) 0·6 mm. | 0·6 mm. |
| | (2) 0·8 " | 0·8 " |
| | (3) 0·8 " | 0·8 " |
| _Nymphæa_: | | |
| | (1) 0·6 " | 0·6 " |
| | (2) 0·8 " | 0·8 " |
| | (3) 0·8 " | 0·8 " |
| | (4) 1·0 " | 1·0 " |
| | (5) 1·4 " | 1·4 " |
+-----------------------------------------------------------+

Thus in all specimens examined, the experimentally determined geo-perceptive layer coincided with the 'starch sheath.' The theory of statoliths thus obtains strong support from an independent line of experimental investigation. The statolithic theory has been adversely criticised because in simpler organs the geotropic action takes place in the absence of statoliths. There is no doubt that the weight of the cell contents may in certain cases be effective in geotropic stimulation; it may nevertheless be true that "at a higher level of adaptation, the geotropically sensitive members of the plant-body are furnished with special geotropic sense-organs--a striking instance of anatomico-physiological division of labour."[38]

[38] Haberlandt--_Ibid_, p. 597.

In the instances of _Bryophyllum_ and _Nymphæa_ given above, the geo-perceptive layer localised by means of the electric probe is definitely found to be the endodermis containing large sized starch grains.

INFLUENCE OF SEASON ON GEO-ELECTRIC RESPONSE.

I shall now describe certain modifications in response, which result from the change of season and also from condition of high temperature. Physiological reactions, generally speaking, are much affected by different seasons; thus the seedlings of _Scirpus Kysoor_ exhibit a very rapid rate of growth of 3 mm. per hour in August, but a month later the growth-rate declines to only 1 mm. per hour. I find similar depression of growth with the advance of season in seedlings of _Zea Mays_, where a very rapid fall in growth takes place in the course of a fortnight. The intensity of geotropic responses, both mechanical and electrical, of _Tropæolum_ declines rapidly in the course of a month from February to March (p. 454). The flowers of _Nymphæa_ began to appear by the end of June when the flower stalks exhibited strong geo-electric response. But later in the season, by July and the beginning of August, the response underwent continuous decline, and by the end of August the response was nearly abolished.

Much time had to be spent in perfecting the apparatus, and it was not till the beginning of August that the investigations could be properly started; the responsive indications were, however, marked and definite, though relatively feeble compared to those obtained at the beginning of the season. The decline of the geo-electric response was to a certain extent also due to the prevailing high temperature.

_Effect of high temperature._--I shall in the next chapter describe experiments which show that geotropic response is diminished under rise of temperature. The specimens employed for localisation of geo-perceptive layer exhibited, as stated before, a decline of geo-electric response with the advance of the season. This may partly be due to unfavourable season, and partly to high temperature. In the middle of the season the responses were extremely feeble on warm days, but on cool mornings they became suddenly enhanced, to decline once more by the middle of the day. I could sometimes succeed in enhancing the sensitiveness by placing the specimen in a cold chamber. It thus appeared that certain internal change unfavourable for geo-perception takes place at high temperatures, and that the sensitive condition could sometimes be restored by artificial cooling. But later in the season, the internal change, whatever it may be, had proceeded too far, and artificial cooling did not restore the sensitiveness of the specimen. What are the physico-chemical concomitants which distinguish insensitive specimens, in which the electric indications had declined almost to the vanishing point?

TEST OF INSENSITIVE SPECIMENS.

I shall now describe the various physico-chemical concomitants which accompany the condition of relative insensibility. I have found three different tests: the electric, the geotropic, and the microscopic, by which the sensitive could be distinguished from the insensitive condition. The following tests were made on insensitive specimens.

_Electric test: Experiment 187._--By the end of August the geo-electric indications given by the probe had, as stated before, almost disappeared. The tonic condition of the specimen, _below par_, was independently revealed by the response to prick of the probe: this, in vigorous specimens, is by an electric response of galvanometric negativity. But the response to prick in sub-tonic specimens is very different. I find that when the physiological condition of the tissue falls _below par_, the sign of response undergoes a reversal into one of _galvanometric positivity_. The same reversal under condition of sub-tonicity was also shown to take place in growth, where under the stimulus of light a positive acceleration took place, instead of normal retardation of growth (p. 221). In the present investigation, the insensitive specimens were found to give abnormal positive electric response to the stimulus of prick made by the probe. The prick-effect in fact often gave me previous indication as to the suitability of the particular specimen for exhibition of geo-electric response.

_Test of geotropic reaction: Experiment 188._--I took four different specimens of _Bryophyllum_ and _Nymphæa_, and held them horizontal. These plant organs had, earlier in the season, exhibited very strong geotropic effect, the shoot curving up through 90° in the course of ten hours or less. But these specimens obtained later in the season exhibited very feeble curvature, which hardly amounted to 10 degrees, even after prolonged exposure to geotropic action for 24 hours.

_Test of microscopic examination._--I next made sections of _Bryophyllum_ and _Nymphæa_ and on examining them under the microscope discovered certain striking changes. A fortnight ago the group of large starch grains stained with iodine were the most striking feature of the starch sheath. But now these starch grains could not be found in any of the numerous specimens examined. The presence of the starch grains thus appears to be associated with the sensitiveness of the perceptive layer.

REACTION AT LOWER SIDE OF THE ORGAN.

There remains now the important question of the physiological change induced on the lower side of the horizontally laid shoot. The physiological reaction of two sides of the organ must be different, since the upper side exhibits contraction and the lower side expansion. It may be urged that the effect of one of the two sides might result from the passive yielding to the definite reaction induced on the opposite side. Investigation by the electric method enables us, however, to discriminate the two reactions from each other, since the electric response characteristic of the induced physiological change takes place in the organ, even under condition of restraint by which movement is prevented. We shall therefore investigate the geo-electrical reaction on the lower side of the securely held organ, and find out whether the induced electric change undergoes any variation in different layers from below upwards. There are two different ways in which the electric explorations of the lower side of the organ may be carried out. In the first method, the probe is introduced from below, and successive readings for geo-electric response taken as the probe enters the organ by successive steps. It is understood that the true geotropic effect is found from difference of galvanometer readings in vertical and horizontal positions. In the second method, the probe is introduced from above, and successive readings for the response taken for different positions of the probe as it enters the organ from the upper side and comes out ultimately at the lower side. This I shall call the METHOD OF TRANSVERSE PERFORATION. The intrusion of the probe on the upper side gives, as we have seen, increasing negative deflection of the galvanometer which reaches a maximum at the perceptive layer. Passage of the probe to still greater depths give deflections which decline to zero. But when the probe comes within the influence of the perceptive layer of the under side, the electric indication, as we shall presently find, undergoes a reversal.

ELECTRIC EXPLORATION OF THE LOWER SIDE OF THE ORGAN.

I shall first describe the results obtained from the first method, the probe entering the organ from the lower side.

_Experiment 189._--The investigation was carried out with the stem of _Bryophyllum_, and the flower stalk of _Nymphæa_. The probe was made to enter the organ through 0·4 mm. and the geo-electric effect found, on rotation of the flower stalk of _Nymphæa_ from the vertical to the horizontal, was a deflection of +6 divisions of the galvanometer. _The change induced at the lower side by geotropic stimulus is thus galvanometric positivity, indicative of enhancement of turgor, and, of expansion._ Intrusion of the probe through 0·6 mm. gave rise to an increased positive geo-electric response. That the sign of electric response depended on the relation of the side of the organ to the vertical lines of gravity was demonstrated by alternate rotation of the plant through +90° and -90°, the probe remaining at a definite position. Rotation through +90° brought A above, and rotation through -90° brought A below. When the probe was in the _up_ position the geo-electric response was negative, but when rotation through -90° brought it _below_, the response became positive. Thus with an identical contact in the plant, the electric response underwent reversal from negative to positive. This will be understood from the following table.

+--------------------------------------------------+
|Position of the | Galvanometer | Galvanometer |
| probe inside | deflection: | deflection: |
| the organ. | A _above_. | A _below_. |
+----------------+----------------+----------------+
| 0·4 mm. | -8 divisions. | +5 divisions. |
| 0·6 mm. | -16 " | +10 " |
+--------------------------------------------------+

It will thus be seen that physiological change induced at any point is modified by its relation to vertical lines of gravity. When the point is above, the induced change is _negative_, when below, the induced change is _positive_.

I shall next describe the variation of effect at different layers of the under side of the organ.

_Experiment 190._--A complete set of readings of the geo-electric reaction at different layers of the organ was taken, as the probe entered the lower side by successive steps of 0·2 mm. The following table gives the results obtained with a specimen of _Nymphæa_.

TABLE XLIII.--ELECTRIC EXPLORATION OF DIFFERENT LAYERS ON THE LOWER SIDE OF THE ORGAN (_Nymphæa_).

+-----------------------------+
|Position of the|Galvanometer |
| probe. | deflection. |
+---------------+-------------+
| Surface | 2 divisions.|
| 0·2 mm. | 4 " |
| 0·4 " | 8 " |
| 0·6 " |16 " |
| 0·8 " |20 " |
| 1·0 mm. |32 " |
| 1·2 |16 " |
| 1·4 " |12 " |
| 1·6 " | 4 " |
| 1·8 " | 0 " |
+-----------------------------+

It is thus seen that just as in the upper so also in the lower side, the electric variation undergoes at first an increase which attains a maximum; beyond this point the electric change undergoes a rapid decline. The induced electric change on the upper and lower sides are, however, different, galvanometric _negativity_ in one case and _positivity_ in the other.

The maximum galvanometric _negativity_ of the upper side was found to occur at the geo-perceptive layer. We may next inquire about the anatomical characteristic of the layer in the lower side of the organ which exhibits the maximum galvanometric _positivity_. Microscopic section of the specimen employed in the above experiment showed the particular layer to be the starch crescent which lies above the vascular bundle. Thus the same geotropic layer which when placed above shows the maximum galvanometric negativity, exhibits maximum positivity when placed below.

METHOD OF TRANSVERSE PERFORATION.

_Experiment 191._--I next carried out a complete exploration of the interior of the organ along the diameter. The probe started from the upper surface, and came out at the lower by successive steps of 0·2 mm., the corresponding geo-electric effects being observed at each step. It has to be borne in mind that the successive readings were obtained by rotation from vertical to +90° (A above); the rotation was never carried out in the negative direction through -90°. But the probe entering from above passed the central axis, and entered a region where the galvanometric indication was transformed from negative to positive. The following table gives the results obtained with the flower stalk of _Nymphæa_.

TABLE XLIV.--SHOWING THE INDUCED GEO-ELECTRIC DISTRIBUTION ACROSS THE FLOWER STALK OF _Nymphæa_ (diameter = 6·8 mm.)

+---------------------------------+
| Position of | Galvanometer |
| probe. | deflection. |
+--------------+------------------+
| Surface ... | - 10 divisions. |
| 0·2 mm. ... | - 26 " |
| 0·4 " ... | - 40 " |
| 0·6 " ... | - 50 " |
| 0·8 " ... | - 62 " |
| 1·0 " ... | - 72 " |
| 1·2 " ... | - 88 " |
| 1·4 " ... | -108 " |
| 1·6 " ... | - 72 " |
| 1·8 " ... | - 44 " |
| 2·0 " ... | - 30 " |
| 2·2 " ... | - 18 " |
| 2·4 " ... | - 10 " |
| 2·6 " ... | - 5 " |
| 2·8 " ... | - 2 " |
| 3·0 " ... | 0 " |
| 3·2 " ... | 0 " |
| 3·4 " ... | 0 " |
| 3·6 mm. ... | 0 divisions. |
| 3·8 " ... | 0 " |
| 4·0 " ... | 0 " |
| 4·2 " ... | + 2 " |
| 4·4 " ... | + 4 " |
| 4·6 " ... | + 5 " |
| 4·8 " ... | + 11 " |
| 5·0 " ... | + 22 " |
| 5·2 " ... | + 38 " |
| 5·4 " ... | + 46 " |
| 5·6 " ... | + 39 " |
| 5·8 " ... | + 32 " |
| 6·0 " ... | + 24 " |
| 6·2 " ... | + 18 " |
| 6·4 " ... | + 12 " |
| 6·6 " ... | + 6 " |
| 6·8 " ... | + 3 " |
+---------------------------------+

A curve constructed from the data given above is seen in figure 177. The diameter of the flower stalk was 6·8 mm. The negative geo-electric reaction is seen to undergo an increase till it attains a climax at the depth of 1·4 mm. It then undergoes a continuous diminution till it becomes zero at the depth of 3 mm.; this neutral zone extends through 1 mm. When the probe enters a depth of 4·2 mm. measured from the upper side, it enters a region affected by the perceptive layer situated on the under side, the opposite physiological reaction being indicated by induced electric change of galvanometric positivity. This positivity reaches a climax at a depth of 5·4 mm. measured from the upper side, and 1·4 mm. when measured from the lower side. The points of maximum positivity and negativity are situated symmetrically on the opposite sides of the organ. The electric variation of maximum positivity on the lower side is comparatively feeble, less than half the corresponding maximum negativity on the upper side. Microscopic section showed that the geo-perceptive layers were the same as the starch-crescents.