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

_By_

SIR J. C. BOSE,

_a.s.sisted 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 ma.s.s 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.

[Ill.u.s.tration: FIG. 174.--Diagrammatic representation of the geo-perceptive layer in unexcited vertical, and in excited horizontal position. (See text.).]

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 pa.s.ses 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 pa.s.sage of the probe, and the after-effect of wound on variation of excitability.

THE ELECTRIC PROBE.

[Ill.u.s.tration: FIG. 175.--The Electric Probe. Figure to the left represents one electric contact made with sepal of _Nymphaea_, and the other, with the flower-stalk by means of the probe; the included galvanometer is represented by a circle. Figure to the right an enlarged view of the probe.]

The wound-irritation is, however, reduced to a minimum by making the probe exceedingly thin. A fine platinum wire 006 mm. in diameter pa.s.ses through a gla.s.s 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 pa.s.s the experimental plant-organ transversely from one end to the other; the average diameter of the capillary is about 015 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 02 mm. (Fig. 175).

_Wound-reaction._--I have shown that a p.r.i.c.k 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 p.r.i.c.k 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 p.r.i.c.k 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 pa.s.sage 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 _Nymphaea_ 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 04 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 04 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 04 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.

04 mm.

-20 "

08 "

-24 "

12 "

-22 "

16 "

-18 "

20 "

-14 "

24 "

-10 "

28 "

-5 "

32 "

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_ Nymphaea: _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 _Nymphaea_. The electric reaction in _Nymphaea_, 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 04 mm. at a time. Other examples will be given later where readings were taken for successive steps of 02 mm.

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

+--------------------------------------------+

Position of probe.

Galvanometric deflection.

+------------------+-------------------------+

Surface

0 divisions.

04 mm.

-16 "

08 "

-42 "

12 "

-20 "

16 "

-10 "

20 "

-2 "

24 "

0 "

+--------------------------------------------+

It will be seen that as in _Bryophyllum_, so in _Nymphaea_, the geo-electric excitation increased at first with increasing depth of the tissue till at a depth of 08 mm. of the particular specimen the induced excitation attained a maximum value. The excitatory effect then declines till it vanished at a depth of 24 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_ 36 mm. in diameter the geo-perceptive layer was found at a depth of 06 mm., it occurred at the greater depth of 08 mm. in a thicker specimen, 5 mm. in diameter. In _Nymphaea_ also the perceptive layer was found at a depth of 08 mm. in a thin and at a depth of 14 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 _Nymphaea_. 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 _Nymphaea_, 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 pa.s.sage of the probe. Thus in a particular experiment with _Bryophyllum_ the point of maximum geotropic excitation was found to be at a distance of 08 mm. from the surface. By means of the micrometer slide in the stage and the micrometer eye-piece, the internal layer 08 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 _Nymphaea_ 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.

[Ill.u.s.tration: FIG. 176.--Transverse section showing continuous geo-perceptive layer S; enlarged view S' of cell of endodermis containing group of large starch grains. (_Bryophyllum_).]

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 _Nymphaea_.

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) 06 mm.

06 mm.

(2) 08 "

08 "

(3) 08 "

08 "

_Nymphaea_:

(1) 06 "

06 "

(2) 08 "

08 "

(3) 08 "

08 "

(4) 10 "

10 "

(5) 14 "

14 "

+-----------------------------------------------------------+

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 _Nymphaea_ 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.