Life Movements in Plants Vol 1/Chapter 5
V.—RESPONSE OF PETIOLE-PULVINUS PREPARATION OF MIMOSA PUDICA
By
Sir J. C. Bose,
Assisted by
Surendra Chandra Das, m.a.
The most suitable plant for researches on irritability of plants is Mimosa pudica, which can be obtained in all parts of the world. An impression unfortunataly prevails that the excitatory reaction of the plant can be obtained only in summer and under favourable circumstances; this has militated against its extensive use in physiological experiments, but the misgiving is without any foundation; for I found no difficulty in demonstrating even the most delicate experiments on Mimosa before the meeting of the American Association for the Advancement of Science held during Christmas of 1914. The prevailing outside temperature at the time was considerably below the freezing point. With foresight and care it should not be at all difficult to maintain in a hot-house a large number of these plants in a sensitive condition all the year round.
In order to remove the drawback connected with the supply of sufficient material, I commenced an investigation to find whether a detached leaf preparation could be made as effective for the study of irritability as the whole plant. Here we have at the central end of the leaf the pulvinus, which acts as the contractile organ; the conducting strand in the interior of the petiole, on the other hand, is the vehicle for transmission of excitation. The problem to be solved is the rendering of an isolated petiole-and-pulvinus of Mimosa as efficient for researches on irritability as the nerve-and-muscle preparation of a frog. On the success of this attempt depended the practical opening out of an extended field of physiological investigation which would be unhampered by any scarcity of experimental material.
In connection with this it is well to note the surprising difference in vegetative growth as exhibited by plants grown in soil and in pots. A pot-specimen of Mimosa produces relatively few leaves, but one grown in the open ground is extremely luxuriant. As an instance in point, I may state that for the last five months I have taken from a plant grown in a field about 20 leaves a day for experiment, without making any impression on it. A large box containing soil would be practically as good as the open ground, and the slower rate of growth in a colder climate could be easily made up by planting half a dozen specimens. The protection of the plants from inclemencies of weather can be ensured by means of a glass cover with simple heat-regulation by electric lamps, in place of an expensive green-house.
Returning to the question of the employment of an isolated leaf, which I shall designate as a petiole-pulvinus preparation, instead of the entire plant, the first attempts which I made proved unsuccessful. The cut leaf kept in water would sometimes exhibit very feeble response, at other times all signs of excitability appeared to be totally abolished. It was impossible to attempt an investigation on the effect of changing environment on excitability when the normal sensitiveness itself underwent so capricious a change.
These difficulties were ultimately overcome from knowledge derived through systematic investigation on the relative importance of the different parts of the motor apparatus, on the immediate and after-effect of section on the excitability of the leaf, and on the rate of decay of this excitability on isolation from the plant. The experience thus gained enabled me to secure long-continued and uniform sensibility under normal conditions. It was thus possible to study the physiological effects of changing external conditions by observing the responsive variation in the isolated petiole-pulvinus preparation. I propose to deal with the different aspects of the investigation in the following order:—
- The effect of wound or section in modification of normal excitability.
- The change of excitability after immersion in water.
- Quantitative determination of the rate of decay of excitability in an isolated preparation.
- Effect of amputation of the upper half of pulvinus.
- Effect of removal of the lower half.
- Influence of the weight of leaf on rapidity of responsive fall.
- The action of chemical agents.
- Effect of "fatigue" on response.
- The influence of constant electric current on recovery.
- The action of light and darkness on excitability.
The isolated petiole-pulvinus preparation is made by cutting out a portion of the stem bearing a single lateral leaf. The four diverging sub-petioles may also be cut off. In order to prevent rapid drying the specimen has to be kept in water. Preparations made in this way often appeared to have lost their sensibility. I was, however, able to trace this loss to two different factors: first, to the physiological depression due to injury caused by section, and, second, to the sudden increase of turgor brought on by excessive absorption of water. I shall now proceed to show that the loss of sensibility is not permanent, but is capable of restoration.
EFFECT OF WOUND OR SECTION IN MODIFICATION OF NORMAL EXCITABILITY.
In connection with the question of effect of injury, it is to be borne in mind that after each excitation the plant becomes temporarily irresponsive and that the excitability is fully restored after the completion of protoplasmic recovery. A cut or a section acts as a very intense stimulus, from the effect of which the recovery is very slow. If the stem be cut very near the leaf, the excitation of the pulvinus is very intense, and the consequent loss of excitability becomes more or less persistent. But if the stem be cut at a greater distance, the transmitted excitation is less intense, and the cut specimen recovers its excitability within a moderate time. I have also succeeded in reducing the excitatory depression by previously benumbing the tissue by physiological means. The isolated specimen can be made still more compact by cutting off the sub-petioles bearing the leaflets; the preparation now consists of a short length of stem of about 2 cm. and an equally short length of primary petiole, the motile pulvinus being at the junction of the two.
For the restoration of sensitiveness, and to meet working conditions, the lower end of the cut stem is mounted on a T-tube, with funnel-attachment and exit-tube, as shown in Fig. 30. The other two cut ends—of the stem and of the petiole—may be covered with moist cloth or may be closed with collodion flexile to prevent rapid evaporation and drying up of the specimen. A slight hydrostatic pressure maintains the specimen in a moderately turgid condition. A preparation thus made is
Fig. 30—The Resonant Recorder, with petiole-pulvinus preparation. (From a photograph.)
insensitive at the beginning, but if left undisturbed it slowly recovers its excitability. The history of the depression of excitability after shock of preparation and its gradual restoration is graphically illustrated by a series of records made by the plant (Fig. 31).
The petiole-pulvinus preparation thus made offers all facilities for experiment. Owing to its small size it can be easily manipulated; it can be enclosed in a small chamber and subjected to varying conditions of temperature and to the action of different vapours and gases. Drugs are easily absorbed at the cut end, and poison and its antidote can be successively applied through the funnel without any disturbance of the continuity of record. In fact, many experiments which would be impossible with the entire plant are quite practicable with the isolated leaf.
The arrangement for taking records of response is seen in Fig. 30, which is reproduced from a photograph of the actual apparatus. For recording the response and recovery of the leaf under stimulation, I use my Resonant Recorder fully described in the 'Philosophical Transactions' (1913). The petiole is attached to one arm of the horizontal lever. The writer, made of fine steel wire with a bent tip, is at right angles to the lever, and is maintained by electromagnetic means in a state of to-and-fro vibration, say, ten times in a second. The record, consisting of a series of dots, is free from errors arising from friction of continuous contact of the writer with the recording surface. The successive dots in the record at definite intervals of a tenth of a second also give the time-relations of the response curve.
On account of its small size, the petiole-pulvinus preparation offers great facilities for mounting in different ways suitable for special investigations. Ordinarily, the cut stem with its lower end enclosed in moist cloth is supported below. A very suitable form of stimulus is that of induction shock from a secondary coil, the intensity of which is capable of variation in the usual manner by adjusting the distance between the primary and the secondary coils. The motile pulvinus, P, may be excited directly. For investigations on velocity of transmission of excitation, stimulus is applied on the petiole at some distance from the pulvinus, by means of suitable electrodes. Excitation is now transmitted along the intervening length of petiole, the conducting power of which will be found appropriately modified under the action of chemical and other agents. In this normal method of mounting, the more excitable lower half of the pulvinus is below; excitatory reaction produces the fall of the petiole, gravity helping the movement. The preparation may, however, be mounted in the inverted position, with the more excitable lower half of the pulvinus facing upwards. The excitatory movement will now be the erection of the petiole, against gravity.
Under natural conditions the stem is fixed, and it is the petiole which moves under excitation. But a very interesting case presents itself when the petiole is fixed and the stem free. Here is presented the unusual spectacle of the plant or the stem "wagging" in response to excitation.
THE CHANGE OF EXCITABILITY AFTER IMMERSION IN WATER.
The isolated specimen can be kept alive for several days immersed in water. The excitability of the pulvinus, however, undergoes great depression, or even abolition, by the sudden change of turgor brought on by excessive absorption of water. The plant gradually accommodates itself to the changed condition, and the excitability is restored in a staircase manner from zero to a maximum.
In studying the action of a chemical solution on excitability, the solution may be applied through the cut end or directly on the pulvinus. The sudden variation of turgor, due to the liquid, always induces a depression, irrespective of the stimulating or the depressing action of the drug. The difficulty may be eliminated by previous long-continued application of water on the pulvinus and waiting till the attainment of uniform excitability which generally takes place in the course of about three hours. Subsequent application of a chemical solution gives rise to characteristic variation in the response.
QUANTITATIVE DETERMINATION OF THE RATE OF DECAY OF EXCITABILITY IN AN ISOLATED PREPARATION.
Variation of excitability after section: Experiment 23.—In order to test the history of the change of excitability resulting from the immediate and after-effect of section, I took an intact plant and fixed the upper half of the stem in a clamp. The response of a given leaf was now taken to the stimulus of an induction shock of 0.1 unit intensity, the unit chosen being that which causes a bare perception of shock in a human being. The specimen was vigorous and the response obtained was found to be a maximum. The stem bearing the leaf was cut at the moment marked in the record with a cross, and water was applied at the cut end. The effect of section was to cause the maximum fall of the leaf, with subsequent recovery. After this, successive responses to uniform stimuli at intervals of 15 minutes show, in (1) of Fig. 31, that a depression of excitability has been induced owing to the shock caused by section. In course of an hour, however, the excitability had been restored almost to its original value before the section. This was the case with a vigorous specimen, but with less vigorous ones a longer period of about three hours is required for restoration. In certain other cases the response after section exhibits alternate fatigue; that is to say, one response is large and the next feeble, and this alternation goes on for a length of time. The isolated specimen, generally speaking, attains a uniform sensibility after a few
Fig. 31—Variation of excitability after section. (1) Immediate effect; (2) variation of excitability in a second specimen during 50 hours: (a) response 4 hours after section; (b) response after 24 hours; (c) after 49 hours. Up-line of record represents responsive fall of the leaf, down-line indicates recovery from excitation.
hours, which is maintained, with very slight decline under constant external conditions, for about 24 hours. On the third day the fall of excitability is very rapid, and the sensibility declines to zero in about 50 hours after isolation [Fig. 31 (2)]. We may describe the whole cycle of change as follows: by the shock of operation the isolated preparation is rendered insensitive for nearly an hour, the excitability is then gradually restored almost to its normal value before operation. Under constant external conditions, this excitability remains fairly constant for about 24 hours after which depression sets in. The rate of fall of excitability becomes very rapid 40 hours after the operation, being finally abolished after the fiftieth hour. It is probable that in a colder climate the fall of excitability would be much slower. The most important outcome of this inquiry is the demonstration of the possibility of obtaining persistent and uniform sensibility in isolated preparations. On account of this, not only is the difficulty of supply of material entirely removed but a very high degree of accuracy secured for the investigation itself.
EFFECT OF AMPUTATION OF UPPER HALF OF PULVINUS.
Experiment 24.—The determination of the rôle played by different parts of the pulvinus in response and recovery is of much theoretical importance. Our knowledge on this subject is unfortunately very scanty. The generally accepted view is that on excitation "the actual downward curvature of the pulvinus is partly due to a contraction of the walls of the motor cells consequent upon the decrease of turgor, but is accentuated by expansion of the insensitive adaxial half of the pulvinus—which was strongly compressed in the unstimulated condition of the organ—and also by the weight of the leaf."[1] According to Pfeffer, after excitation of the organ, "the original condition of turgor is gradually reproduced in the lower half of the pulvinus, which expands, raising the leaf and producing compression of the upper half of the pulvinus, which aids in the rapid curvature of the stimulated pulvinus."[2]
It was held, then, that the rapidity of the fall of leaf under stimulus is materially aided (1) by the expansion of the upper half of the pulvinus, which is normally in a state of compression, and (2) by the weight of the leaf. So much for theory. The experimental evidence available regarding the relative importance of the upper and lower halves of the pulvinus is not very conclusive. Lindsay attempted to decide the question by his amputation experiments. He showed that when the upper half was removed the leaf carried out the response, but rigor set in when the lower half was amputated. Pfeffer's experiments on the subject, however, contradicted the above results. He found that "after the upper half of the pulvinus was carefully removed, no movement was produced by stimulation, whereas when the lower half is absent a weakened power of movement is retained." Pfeffer, however, adds, "since the operation undoubtedly affects the irritability, it is impossible to determine from such experiments the exact part played by the active contraction of the lower half of the pulvinus."[2]
The cause of uncertainty in this investigation is twofold. First, it arises from the unknown change in irritability consequent on amputation; and, secondly, from absence of any quantitative standard by which the effect of selective amputation of the pulvinus may be measured. As regards the first, I have been able to reduce the depressing action caused by injury to a minimum by benumbing the tissue before operation, through local application of cold, and also allowing the shock-effect to disappear after a rest of several hours. As regards the physiological gauge of efficiency of the motor mechanism, such a measure is afforded by the relation between a definite testing stimulus and the resulting response with its time-relations, which is secured by my Resonant Recorder with the standardised electrical stimulator.
In carrying out this investigation I first took the record of normal response of an intact leaf on a fast moving plate. A second record, with the same stimulus, was taken after the removal of the upper half of the pulvinus, having taken the necessary precautions that have been described. Comparison of the two records (Fig. 32) shows that the only
Fig. 32.—Effect of amputation of upper half of pulvinus. Upper record gives normal response before amputation, and the lower, response after amputation (Successive dots at intervals of 0.1 sec.). Apex-time 1.1 sec., in both.
difference between them is in the exhibition of slight diminution of excitability due to operation. But, as regards the latent period and the quickness of attaining maximum fall, there is no difference between the two records before and after the amputation of the upper half. The upper part of the pulvinus is thus seen practically to have little influence in hastening the fall.
EFFECT OF REMOVAL OF THE LOWER HALF.
Experiment 25.—The shock-effect caused by the amputation of the lower half was found to be very great, and it required a long period of rest before the upper half regained its excitability. The excitatory reaction of the upper half is by contraction, and the response is, therefore, the lifting of the petiole. Thus, in an intact specimen, excitation causes antagonistic reactions of the two halves. But the sensibility of the upper half is very feeble and the rate of its contractile movement, relatively speaking, very slow. The record of the response of the upper half of the pulvinus, seen in Fig. 33, was taken with an Oscillating Recorder, where the successive dots are at intervals of 1 sec.: the
Fig. 33.—Response after amputation of lower half of pulvinus. (Successive dots at intervals of a second; vertical lines mark minutes.) Apex-time, 40 secs.
imagnification employed was about five times greater than in recording the response of the lower half (Fig. 32). The intensity of stimulus to evoke response had also to be considerably increased. Taking into account the factors of magnification and the intensity of stimulus for effective response, the lower half I find to be about 80 times more sensitive than the upper. Thus, under feeble stimulus the upper half exerts practically no antagonistic reaction. The excitatory response of the upper half is also seen to be very sluggish.
INFLUENCE OF THE WEIGHT OF LEAF ON RAPIDITY OF RESPONSIVE FALL.
Experiment 26.—It is obvious that the mechanical moment exerted by the weight of the leaf must help its responsive fall under excitation. But the relative importance of the factors of active contraction of the lower half of the pulvinus and of the weight, in the rate of the responsive down-movement, still remains to be determined. A satisfactory way of solving the problem would lie in the study of the characteristics of response-records taken under three different conditions: (1) When the leaf is helped in its fall by its weight; (2) when the action of the weight is eliminated; and (3) when the fall has to be executed against an equivalent weight. An approximation to these conditions was made in the following manner. We may regard the mechanical moment to be principally due to the weight of the four sub-petioles applied at the end of the main petiole. In a given case these sub-petioles were cut off, and their weight found to be 0.5 grm. The main petiole was now attached to the right arm of the lever, and three successive records were taken: (1) With no weight attached to the petiole; (2) with 0.5 grm. attached to its end; and (3) With 0.5 grm. attached to the left arm of the lever at an equal distance from the fulcrum. In the first case, the fall due to the excitatory contraction will practically have little weight to help it; in the second case, it will be helped by a weight equivalent to those of the sub-petioles with their attached leaflets; and in the third case, the fall will be opposed by an equivalent weight. We find that in these three cases there is very little difference in the time taken by the leaf to complete the fall (Fig. 34).
It has been shown that the presence or absence of the
Fig. 34.—Effect of weight on rapidity of fall. N, without action of weight; W, with weight helping; and A, with weight opposing.
upper half of the pulvinus makes practically no difference in the period of fall; it is now seen that the weight exerts comparatively little effect. We are thus led to conclude that in determining the rapidity of fall, the factors of expansive force of the upper half of the pulvinus and the weight of the leaf are negligible compared to the active force of contraction exerted by the lower half of the pulvinus.
ACTION OF CHEMICAL AGENTS.
In connection with this subject it need hardly be said that the various experiments which I had previously carried out with the intact plant can also be repeated with the isolated preparation. I will only give here accounts of experiments which are entirely new.
The chemical solution may be applied directly to the pulvinus, or it may be absorbed through the cut end, the absorption being hastened by hydrostatic pressure. The normal record is taken after observing precautions which have already been mentioned. The reaction of a given chemical agent is demonstrated by the changed character of the record. The effect of the drug is found to depend not merely on its chemical nature, but also on the dose. There is another very important factor—that of the tonic condition of the tissue—which is found to modify the result. The influence of this will be realised from the account of an experiment to be given presently, where an
Fig. 35.—Stimulating action of hydrogen peroxide.
identical agent is shown to produce diametrically opposite effects on two specimens, one of which was in a normal, and the other in a sub-tonic, condition. The experiments described below relate to reactions of specimens in a normal condition.
Hydrogen Peroxide: Experiment 27.—This reagent in dilute solution exerts a stimulating action. Normal records, were taken after long-continued application of water on the pulvinus. The peroxide, as supplied by Messrs. Parke Davis & Co., was diluted to 1 per cent., and applied to the pulvinus; this gave rise to an enhancement of response. Re-application of water reduced the amplitude to the old normal value (Fig. 35).
Barium Chloride: Experiment 28.—The action of this agent is very characteristic, inducing great sluggishness in recovery. The preparation had been kept in 1-per cent. solution of this substance for two hours. After this the first response to a given test-stimulus was taken; the response was only moderate, and the recovery incomplete. The sluggishness was so great that the next stimulation, represented by a thick dot (Fig. 36), was ineffective. Tetanising electric shock at T, not only brought about
Fig. 36.—Incomplete recovery under the action of BaCl2 and transient restoration under tetanisation at T.
response, but removed for the time being the induced sluggishness. This is seen in the next two records, which were taken under the old test-stimulus. There is now an enhanced response and a complete recovery. Beneficial effect of tetanisation disappeared, however, on the cessation of stimulus. This is seen in the next two records which were taken after two hours. The amplitude of response was not only diminished, but the recovery also was incomplete.
Antagonistic actions of Alkali and Acid: Experiment 29.—Alkali and acid are known to exert antagonistic actions on the spontaneous beat of the heart; dilute solution of NaOH arrests the beat of the heart in systolic contraction, while dilute lactic acid arrests the beat in diastolic expansion. I have found identical antagonistic reactions in the pulsating tissue of Desmodium gyrans, the
Fig. 37.—Antagonistic action of alkali and acid. Arrest of response in cotraction under NaOH (↑), restoration and final arrest in expansion under actic acid (↑).
telegraph plant. It is very interesting to find that these agents also exert their characteristic effects on the response of Mimosa in a manner which is precisely the same. This is seen illustrated in Fig. 37, where the application of NaOH arrested the response in a contracted state; after this, the antagonistic effect of dilute lactic acid is seen first, in its power of restoring the excitability; its continued application, however, causes a second arrest, but this time in a state of relaxed expansion.
CuSO4 Solution.—This agent acts as a poison, causing a gradual diminution of amplitude of response, culminating in actual arrest at death. Certain poisons, again, exhibit another striking symptom at the moment of death, an account of which will be given in a separate paper.
EFFECT OF "FATIGUE" ON RESPONSE.
With Mimosa, after each excitation the recovery becomes complete after a resting period of about 15 min. With this interval of rest the successive responses for a given stimulus are equal, and are at their maximum.
Experiment 30.—When the resting interval is diminished the recovery becomes incomplete, and there is a
Fig. 38.—"Fatigue" induced by shortening intervening period of rest.
consequent diminution of amplitude of response. There is thus an increased fatigue with diminished period of rest. This is illustrated in Fig. 38, where the first two responses are at intervals of 15 min.; the resting interval was then reduced to 10 min., the response undergoing a marked diminution. Conversely, by increasing the resting interval, first to 12 and then to 15 min., the extent of fatigue was reduced and then abolished.
THE INFLUENCE OF CONSTANT ELECTRIC CURRENT ON RECOVERY.
Experiment 31.—From the above experiment it would appear that since the incompleteness of recovery induces fatigue, hastening of recovery would remove it. With this idea I tried various methods for quickening the recovery of the excited leaf. The application of a constant electric current was found to have the desired effect. Two electrodes for introduction of current were applied, one on the stem and the other on the petiole, at some distance from the pulvinus. In order to avoid the excitatory effect of sudden application, the applied current should be in-
Fig. 39.—Action of constant current in removal of fatigue by hastening recovery; N, curve of response in fatigued specimen; C, after passage of current.
creased gradually; this was secured by means of a potentiometer slide. In my experiment a current having an intensity of 1.4 micro-ampere was found to be effective. Responses at intervals of 10 min., as we have seen, exhibit marked fatigue. Two responses were recorded on a fast-moving plate, N before, and C after, the application of the current. It will be seen (Fig. 39) how the application of current has, by hastening the recovery, enhanced the amplitude of response and brought about a diminution of fatigue. In connection with this, I may state that the tonic condition is, in general, improved as an after-effect of the passage of current. This is seen in some cases by a slight increase in excitability; in others, where the responses had been irregular, the previous passage of a current tends to make the responses more uniform.
ACTION OF LIGHT AND DARKNESS ON EXCITABILITY.
In taking continuous records of responses I was struck by the marked change of excitability exhibited by the intact plant under variation of light. Thus the appearance of a cloud was quickly followed by an induced depression, and its disappearance by an equally quick restoration of excitability. This may be explained on the theory that certain explosive chemical compounds are built up by the photo-synthetic processes in green leaves, and that the intensity of response depends on the presence of these compounds. But the building up of a chemical compound must necessarily be a slow process, and it is difficult on the above hypothesis to connect the rapid variation of excitability with the production of a chemical compound, or its cessation, concomitant with changes in the incident light.
Experiment 32.—In order to find out whether photo-synthesis had any effect on excitability, I placed an intact plant in a dark room and obtained from it a long series of responses under uniform test-stimulus. While this was being done the green leaflets were alternately subjected to strong light and to darkness, care being taken that the pulvinus was shaded all the time. The alternate action of light and darkness on leaflets induced no variation in the uniformity of response. This shows that the observed variation of excitability in Mimosa under the alternate action of light and darkness is not attributable to the photo-synthetic processes.
I next took a petiole-pulvinus preparation from which the sub-petioles bearing the leaflets had been cut off, and placed it in a room illuminated by diffused daylight. The normal responses were taken, the temperature of the room being 30° C. The room was darkened by pulling down the blinds, and records were continued in darkness. The temperature of the room remained unchanged at 30° C. It will be seen from records given in Fig. 40, that in
Fig. 40.—Stimulating action of light, and depressing action of darkness. Horizontal line below represents period of darkness.
darkness there is a great depression of excitability. Blinds were next pulled up and the records now obtained exhibit the normal excitability under light. The sky had by this time become brighter, and this accounts for the slight enhancement of excitability. This experiment proves conclusively that light has a direct stimulating action on the pulvinus, independent of photo-synthesis.[3]
SUMMARY.
On isolation of a petiole-pulvinus preparation, the shock of operation is found to paralyse its sensibility. After suitable mounting the excitability is restored, and remains practically uniform for nearly 24 hours. After this a depression sets in, the rate of fall of excitability becomes rapid 40 hours after the operation, sensibility being finally abolished after the fiftieth hour.
Experiments carried out on the effect of weight, and the influence of selective amputation of the upper and lower halves of the pulvinus, show that in determining the rapidity of fall of leaf, the assumed factors of the expansive force of the upper half of the pulvinus and the weight of the leaf are negligible compared to the force of active contraction exerted by the lower half of the pulvinus. The excitability of the lower half is eighty times greater than that of the upper.
Chemical agents induce characteristic changes in excitability. Hydrogen peroxide acts as a stimulant. Barium chloride renders the recovery incomplete; but tetanisation temporarily removes the induced sluggishness. Acids and alkalis induce antagonistic reactions, abolition of excitability with alkali taking place in a contracted, and with acid in an expanded condition of the pulvinus.
The responses exhibit fatigue when the period of rest is diminished. The passage of constant current is found to remove the fatigue.
Response is enhanced on exposure to light, and diminished in darkness. Light is shown to exert a direct stimulating action on the pulvinus, independent of photo-synthesis.
- ↑ Haberlandt, 'Physiological Plant Anatomy,' 1914, p. 570. English Translation, Macmillan & Co.
- ↑ 2.0 2.1 Pfeffer—'Physiology of Plants,' vol. 3, pp. 75 and 76. English Translation, Clarendon Press.
- ↑ See also Bose and Das—'Physiological Investigations with Petiole-Pulvinus preparations of Mimosa pudica.' Proc. Roy. Soc. B. Vol. 89, 1916.