Researches on Irritability of Plants

Fig. 90.—Effect of cold in inducing retardation and arrest of transmission : (xz) Normal record ; (2) Retardation due to slight cooling ; (3) Arrest of conduction brought about by intense cold; (4) Record of direct stimulation. of cold by means of cooled water or by means of small fragments of ice. Successive records were then taken at intervals of 20 minutes, which is more than sufficient for complete recovery from previous stimulation. Record 1 in fig. go gives the time-interval between the application of stimulus and response under normal conditions. There are 20°5 seconds spaces, each space representing ‘1 second. The latent period is ‘15 second. The true time of transmission is thus 19 second for 30 mm.; the transmission time

through Io mm. is therefore ‘63 second. The next record was taken when the intervening length of Io mm. in the petiole was moderately lowered in temperature by the application of cold water. This cooling should be com- menced immediately after the previous responsive fall of the leaf. This not only gives sufficient time for the localised cooling of the petiole but also avoids the excitatory disturb- ance of the pulvinus caused by sudden application of cold to the petiole. During the localised cooling of the petiole the leaf erects itself and becomes fully sensitive when the time arrives for the application of the next stimulus. Record 2 exhibits the effect of moderate cooling; the transmission period is now prolonged, the difference between the two records being a time-interval of ‘8 second. On the assump- tion that the effect of cooling had remained localised, it is seen that the lowering of temperature had prolonged the period of transmission through the 10 mm. of the petiole from °63 second to 1°43 second. The conductivity has thus been reduced by more than half.

In record 3 is seen the effect of further lowering of temperature by placing small fragments of ice on the strip of cloth. The excitatory impulse initiated by the maximal stimulus of induction-shock had hitherto been unfailingly transmitted. But under the action of intense cold the impulse is arrested. In order to show that the abolition is not due to the depression of motile excitability of the pulvinus, record 4 is taken of the effect of direct stimulation. An inspection of the record shows that the motile excitability has undergone no change. It is thus clear that the impulse initiated by the stimulus has been arrested by the physio- logical depression of conductivity induced by cold.

In connection with this subject I came across the interest- ing phenomenon of paralysis of conductivity as an after- effect of intense cooling. After obtaining the record of the block under local application of cold, the fragments of ice were removed and the cooled portion of the petiole allowed to regain the temperature of the room, which must have been.accomplished in the course of 20 minutes. After this, on taking the record of the transmitted effect of stimulus I found that the block of conduction was still persistent. The conducting-power of the benumbed tissue is thus paralysed for a period which generally lasts for about 45 minutes. I have, however, discovered the very suggestive fact that the lost conducting-power can be very quickly restored by subjecting the paralysed portion of the petiole to the action of tetanising electric shocks.

If in a nerve-and-muscle preparation a constant current be maintained in an intervening tract between the point of stimulation and the responding muscle, this current is found to act as a block to the passage of excitation. With moderate intensity of current the block of conduction is due to the depressing action of the anode. For demonstration of electrotonic block of nerve-conduction, an intensity of stimulus is found which is effective under normal conditions. But during the continuation of the blocking current the excitatory impulse due to the testing stimulus is found to be arrested. The transmission is, however, renewed on the stoppage of the current. It is of special interest to have thus at our disposal a physiological block which can be put ‘on’ and ‘ off’ at will and many times in succession. The alternate transmission and its arrest then affords a very striking demonstration of the excitatory character of the propagated effect.

I have found a similar block of conduction induced in the plant by electrotonus. Various forms of testing stimulus may be employed. It is, however, more satisfactory to employ a form of stimulus the intensity of which may be either gradu- ally increased or maintained constant. These requirements are fulfilled by thermic and electric modes of stimulation. I will now describe two typical experiments on electrotonic block, in Biophytum under thermal stimulation and in Mimosa under electric stimulation.

Biophytum.—In this plant we have a whorl of leaves bearing sensitive leaflets. Stimulus is applied on the stem by means of the electro-thermic stimulator. The intensity of stimulus is so graduated as to cause an excitatory impulse to traverse the petioles, effecting the fall of the leaflets in a centrifugal order. Selecting a leaf, electrotonic block is applied in the middle part of the petiole. When the anode A is to the left, the excitatory impulse is found ar- rested at A; when the current is reversed, the arrest is found to take place at the new anode A’ to the right. On the stoppage of the blocking current the excitatory impulse is observed to traverse the

whole length of the leaf. the electrotonic block. Mimosa.—In the next series Polarising circuit P inter- of experiments a different species Goer Ue eu eros : : secondary circuit s and of plant and a different testing- responding _ pulvinus. stimulus is selected. The employ- The polarising current t of lectrical a f acts as a block, whether Pee tical mode 0 ascending or descending. the difficulty of securing a stimulus the intensity of which may be either maintained constant or increased in a known manner. With the help of a sliding induc- tion-coil it is easy to arrive at an intensity of stimulus which is always effective in normal circumstances. The proximal of the two exciting electrodes was placed at a distance of 30 mm. from the primary pulvinus. Half-way between the point of excitation and the pulvinus were placed two polarising electrodes, 5 mm. apart, through which a constant current could be maintained, for the purpose of serving as a block (fig. 91). The first and uppermost

of the three records in the next diagram was taken without this block and with a stimulus intensity of 2. It will be seen (fig. 92) that excitation was transmitted as usual, the velocity of transmission in this case being 29 mm. per second. The specimen was exceptionally vigorous and the season the height of summer, which facts account for the high velocity.. The blocking current was next introduced. In order to prevent the excitation due to sudden make of

Fic. 92.—Record of effect of electrotonic block. Uppermost record normal; lowest record shows block of trans- mission of excitation; middle record shows restoration of conductivity on removal of block. current, the applied E.M.F. was gradually increased from zero to 2 volts, by means of a potential slide. During the passage of this constant current a second stimulus was applied of the same value as before; and it will be seen, from the lowest record, that there was no response, the transmission of excitation being effectively blocked. In order to show that this block would only persist during passage of the current, the latter was next reduced gradually to zero by manipulation of the potential slide. This was carefully done, to avoid the excitation due to sudden cessation of current. On again repeating stimulation, the block was found to be no longer operative (see intermediate record) and response due to transmitted excitation took place as usual.

plate. In the next record (fig. 93) a series of response-records to transmitted excitation were taken on a slower-moving plate. The testing stimulus was always the same, the difference being that the electrotonic block was ‘ off’ and Fic. 93.—Records of transmitted excitation with the block off and on. Arrest of transmitted excitation under electrotonic block at B, B. ‘on’ alternately. It will be noted that the excitation was invariably arrested whenever the block was applied at B, B.

Reference has been made to the inconclusive character of Pfeffer’s narcotisation experiment. The ineffectiveness of the. block, it was explained, might have been due to the thickness of the tissue preventing free access of the anesthetic to the conducting-elements in the interior. It occurred to me that the physiological block induced by a drug could be rendered more effective in two different ways: first, by the selection of a thin petiole in which access of the solution to the interior by absorption would be less difficult ; and, second, by the employment of strong toxic agents like

copper sulphate or potassium cyanide solutions. The choice of a strong poison was deemed advisable because the absorption of even a small quantity might in such a case prove effective in inducing depression or abolition of con- duction. Application of an anesthetic, like chloroform, has the drawback that the escaping vapour renders the motile organ insensitive. There is no such disadvantage in the employment of a non-volatile poison like copper sulphate, which by local application would affect the conductivity of the selected portion of petiole without modifying the motile sensibility of the pulvinus. The petiole of Biophytum was found to be very suitable for these experiments. Among the whorl of leaves, about half a dozen are found the leaflets of which are fairly sensitive. Graduated stimulus is applied to the stem by means of an electro-thermic stimulator. This latter consists, as explained in a previous chapter, of a V-shaped piece of platinum wire, through which an electric current of suitable intensity could be sent, the circuit being completed for a definite length of time by means of a metronome. The electric current is so adjusted as to give rise to a thermal shock without causing a burn. The effective intensity can be gradually increased by taking advantage of the additive effect of stimulus. Thus ina given case while the thermal shock was singly ineffective, it became minimally effective when repeated four times and maximally effec- tive when repeated eight times. The excitatory impulse originated at the stem, radiated subsequently to the leaves, where the progress of the excitatory waves was visually demonstrated by the serial fall of leaflets in a centrifugal order. It should be mentioned here that the excitation caused by thermal stimulus is most intense ; the block needs to be very perfect to arrest the conduction of such an excitation.

Arvest of conduction by copper sulphate solution.—After determining the value of an effective stimulus, poison was applied to the portion of petiole which is next to the stem. This was done by wrapping a strip of cloth 5 mm. in breadth round the petiole and soaking it with strong copper-sulphate solution. Warm solution was found to be more quickly absorbed than cold. Out of six sensitive leaves, two were subjected to the local action of poison. The selective and local arrest of the excitatory wave in the poisoned leaves would then afford a conclusive demonstration of the physio- logical character of the transmitted impulse. It should be remembered that the absorption of poison is likely to be a slow process. Hence the physiological block of conduc- tivity induced by poison will become increasingly effective with the duration of application. We may therefore expect the following sequence of events after the application of poison on a narrow zone of the petiole :—

(i) There will be no noticeable variation of conductivity at the beginning. (ii) After the lapse of a certain length of time the quantity of poisonous solution absorbed will be sufficient to induce a certain depression of conductivity. The minimal excitation which was formerly transmitted will now undergo an arrest. But the blocking action will not be sufficiently great to arrest maximal excitation. (ii) After a still longer interval, the depression of conductivity induced by poison will be very great. Evena maximal excitation will now be practically arrested.

(iv) This arrest will be due to the abolition of conduc- tivity in the localised poisoned zone. The conductivity of the petiole beyond the poisoned area, and the motile excitability of the leaflets will remain unaffected. I shall now describe experiments on the effect of poison on conductivity. The experiment was repeated with twelve different specimens of Biophytum, all of which gave similar results. The following experiment may be taken as representative of the rest. Among the whorl of leaves in the particular specimen of Biophytum there were six which were fairly sensitive. The additive effect of four successive thermal shocks applied on the stem was found to be sufficient to cause excitatory fall of the leaflets in all the six leaves.

Two out of the six leaves were subjected to the local action of poison in the manner previously described :— (x) The testing stimulus was applied 15 minutes after the application of poison. Excitatory impulse was found to traverse all the leaves. Absorption of the particular poison in the course of 15 minutes was too slight to induce any marked depression of conductivity. (2) The experiment was repeated after half an hour under the same stimulus as before—namely, the additive effect of four shocks. The excitatory impulse was found transmitted in the four normal, but arrested in the two poisoned leaves. The block in the two leaves was, however, forced by the application of a stronger stimulus due to the additive effect of six shocks.

(3) After two hours the conductivity of the poisoned portion of the petiole was found practically abolished in the two leaves. In these there was no transmitted effect, even under the maximal stimulus of sixteen additive shocks. The untreated leaves exhibited vigorous conduction, the leaflets undergoing their serial fall with great rapidity. (4) In order to show that the absence of the transmitted effect in the two leaves was due to local loss of conductivity of the treated area, and not to the loss of motile sensibility of the leaflets, stimulus was applied on the petiole beyond the poisoned zone. The leaflets exhibited their normal excitatory fall.

Mercuric chloride solution.—After obtaining the block of conduction by the action of copper sulphate, I tried the effect of various other poisons, some of which were found to be very virulent in their action. Mercuric chloride solution, for example, abolished the power of conduction in a much shorter time than copper sulphate. The only drawback in the application of mercuric chloride is that it exerts an excitatory action, the transmitted effect of which induces fall of the sensitive leaflets, which often remain persistently closed.

tatory action induced by this reagent ; its toxic power in abolishing conductivity is however very great. A strong solution is applied on a portion of the petiole in the usual manner, the leaflets remaining open and fully sensitive all the time. After an interval of an hour it is found that the effect of a strong thermal stimulus applied in the stem fails to be transmitted across the poisoned zone. The sensibility of the leaflets is however found unaffected. The leaflets in those petioles which have not been poisoned exhibit vigorous response to transmitted excitation.

I was next desirous of testing the effect of poison on a different species of plant and verifying the result by means of automatic records. The effect of copper sulphate solution in abolishing conductivity was not, as we saw, immediate ; it required time to have its toxic effect fully developed. Hence it appeared of much interest to test, by means of successive records, the progressive diminution of conducting power culminating in actual arrest. The specimen employed for these series of investigations was the petiole of Mimosa. Successive records of transmission-time were taken at intervals of 20 minutes, before and after subjecting an inter- mediate portion of the petiole to the action of poison. Effec- tive stimulus of the induction-shock was applied on the petiole, generally at a distance of 30 mm. from the responding pulvinus. The intensity of stimulus employed was maximal, being2units. The first record of the series gives the velocity of normal conduction; the second and the subsequent records exhibit progressive action of the toxic agent. This latter was applied on a strip of cloth 10 mm. wide, wrapped round the petiole midway between the point of stimulation and the responding pulvinus.

Copper sulphate solution—The normal record (I) in fig. 94 shows response to have taken place 27 spaces after the application of the stimulus, the interval between the successive dots being ‘1 second. The total time was there- fore 2°7 seconds. Subtracting from this the latent period ‘I5 second, we obtain 2°5 seconds as the actual time for transmission through 30 mm. The time of transmission through 10 mm. is therefore °83 second. Record 2 of the series shows the effect of application of copper sulphate solution for 20 minutes on a por- tion of petiole Io mm. in breadth. It is noticed that the transmission-time has been prolonged by ten spaces, 7.¢. by zr second. Assuming that the effect of poison was

Fic. 94.—Effect of copper sulphate solution in the retardation and final arrest of conduction: (1) Normal record; (2) Retardation caused by 20 minutes’ application; (3) Arrest caused by application for 40 minutes; (4) Record of direct stimulation. localised, it is seen that it had during 20 minutes’ application delayed transmission through 10 mm. from ‘83 second to 1°83 second. By the absorption of a small quantity of poison the conductivity has thus been reduced by more than 50 per cent.

Record 3 of the series was taken after a further period of 20 minutes. The transmitted effect is seen to be com- pletely blocked by the application of copper sulphate for 40 minutes. In order to show that the absence of response was due not to the abolition of motile excitability of pulvinus but to the block of conductivity of the petiole, a fourth record was taken under direct stimulation, which proves that the motile excitability of the leaf had remained unimpaired.

Mercuric chloride solution.—A series of records was next taken exhibiting the effect of mercuric chloride solution. The power of conduction was found arrested after a period of application so short as 10 minutes. The record obtained was very similar to that given in the next figure. Potassium cyanide solution.—I next took a series of records which exhibited the effect of strong solution of Fic. 95.—Abolition of conductivity by the action of potassium cyanide : (rt) Normal record; (2) Arrest of conduction after application for five minutes; (3) Record showing arrest of impulse due to very strong stimulus; (4) Record of direct stimulation.

potassium cyanide. Record 1, fig. 95, gives the normal transmission period. Record 2 was taken, as stated before, after allowing 20 minutes for recovery. The poisonous solution was however applied 15 minutes after the pre- vious record ; hence in the second record we see the effect of application of potassium cyanide for a period of 5 minutes only. The effect of this poison on the conductivity of petiole of Mimosa was so great that even with such a short application the transmission of excitation caused by maximal stimulus of 2 units was completely blocked.

Record 3 was taken with the secondary pushed close to the primary, the stimulus intensity being thus raised to 15 units. Even under this intense stimulation the conduction was found to be arrested. Record 4 was obtained under direct stimulation. The response shows that the sensi- bility of the pulvinus had undergone no change. It is thus clear that the abolition of response to indirect stimulation was solely due to the abolition of conductivity induced by the action of poison.

The arrest of the transmitted impulse in Mimosa by the physiological block induced by cold, by electrotonus, and by local application of poison, completely disproves the hydro-mechanical theory. The results of the various investigations that have been described lead on the other hand to the conclusion that the transmission of excitation in the plant is a process fundamentally similar to that which takes place in the animal, being in the one case as in the other a propagation of protoplasmic change.

Excitatory reaction is initiated in the petiole of various sensitive plants by the discriminative polar action of an electric current. Excitation is induced at the kathodic point at ‘make’ and at the anodic point at ‘ break.’ Transmission of such an excitatory impulse takes place in the absence of all mechanical disturbances. The excitatory nature of the impulse in plants is further demonstrated by the arrest of conduction brought about by various physiological blocks.

Local application of increasing cold retards and finally abolishes the conducting-power. Conductivity is for a time paralysed as an after-effect of application of cold. The lost conducting-power may, how- ever, be quickly restored by tetanising electric shocks. during the passage of the blocking current, the conducting- power being restored on the cessation of the current. Conductivity of a selected portion of the petiole may be abolished by local application of poison. The abolition of conducting-power takes place slowly under the action of copper sulphate and quickly under potassium cyanide.