Plant Response as a Means of Physiological Investigation

Effect of direct application of stimulus on the growing region. — Many of the phenomena of growth-curvature are brought about, as we shall see, by means of the changes induced by external stimulus in the rate of growth, and there is much misconception as to whether the effect of stimulus is to enhance or to retard growth. I shall be able to show that this misconception is the result of the complexity of the problem, depending (i) on the tonic condition of the tissue, (2) on whether the stimulus is internal or external, and (3) on the point of application of stimulus. The fundamental effect of stimulus is, however, very definite.

question of great importance, namely, as to whether the effect Record of Responses of stimulus the usual contractile response alba to External Thermal (fig. 1 77). On the completion of reoriginal length. Hence the base-line of a series of these responses is horizontal. I shall now pass, by means of intermediate links, from this to the response of growing organs. And first I shall take the response of a style of Datura, in which, for want of a sufficient supply of internal energy, growth has come to a temporary stop. On applying thermal stimulus, at intervals of a minute, the first five responses are seen to be practically like those of the stationary style of Datura (fig. 178, cf. 177). But a portion of the stimulus applied is being absorbed and held latent in the organ, thus increasing the internal energy, or tonic condition. The result of this is seen in the renewal of growth at the sixth response. The stimulus now, therefore, finds bifurcated expression in maintaining response, and in renewing growth, as is seen by the trend downwards of the

hitherto horizontal base-line. This bifurcation causes the first contractile response of the now growing organ — that is the sixth response of the record — to be smaller than usual. But as the tonic condition is established, and the molecular mobility of the responding organ is increased, the contractile response becomes larger, and growth goes on at a certain steady rate. From this intermediate link we pass on to the case of response to stimulus of a style of Datura which is in a state of uniform growth (fig. 179). Here also we find that stimulus produces the normal contractile effect. All these clearly demonstrate that the response of growing organs is in no way different from that^of stationary organs. In a stationary organ, stimulation produces negative turgidity-variation, resulting in contraction. The same contraction is seen in growing organs, causing a temporary retardation of the rate of growth.

The retardation of growth which is caused in a growing organ by external stimulus Fig. 178. Photographic Record of Responses of Style of Datura alba in which Growth had come to a Temporary Stop The up curve shows contraction. As long as the base-line is horizontal, growth is seen to be at standstill. Renewal of growth at sixth response, after which growth-elongation is shown by the trend of the base-line downwards. may be exhibited in a somewhat different way. We may use the Method of Balanced Record, by which the normal rate of growth is made to appear as a neutral horizontal line. For the purpose of this experiment I took the growing peduncle of a Eucharis Lily. The balanced record (fig. 180) is here seen to be horizontal, save for minute autonomous oscillations about the neutral line. Stilation, in this case, was produced by tetanic electric shocks

organ, by means of non-polarisable electrodes, connected, one a little above, and the other a little below, the growing region. According to the conditions of the experiment, the balanced horizontal base-line will represent uniform growth under normal turgidity. An up curve will here represent a rate of growth below the normal, or a retardation ; a down curve, on the contrary, will represent a rate of growth above the normal, or an acceleration. When stimulus is applied a responsive negative turgidityvariation is induced, in consequence of which there is a temporary disturbance of the balanced growth-record. The curve thus produced exhibits the effect of stimulus, and recovery from that effect. It will be observed from the figure that on the application of tetanic electric shocks for two seconds, a responsive retardation ol

Fig. 179. Photographic Record of Response of Growing Style of Datura^ alba to External Stimulus Fig. 180. Balanced Record of Response in Growing Peduncle of Eucharis Lily to Electrical Stimulation Up curve here represents retardation of growth. First response is to stimulus of two seconds', the second to stimulus of three seconds', duration. growth was induced, as seen in the up curve, and that there was a recovery after an interval of nine minutes from the time of application of stimulus. It will also be noticed that during the process of recovery in this particular case, the rate

of growth is above the normal, which is due to the fact that the internal energy has been augmented by the absorption of the stimulus applied.1 A stimulus of longer duration, that is to say of three seconds, was next applied, and the responsive retardation had now a greater amplitude than before, the period of restoration being also longer, that is to say, sixteen minutes. • Similarities between motile and growth responses. — We have, then, in growth-response an exact parallel to the mechanical responses given by pulvinated or anisotropic organs. The following tabular statement will show the reason of this fundamental parallelism between responses whose modes of indication are so widely different :

Tabular Statement showing Comparative Effects of Stimulus in Pulvinated and Growing Organs (t>) Increased hydrostatic pressure : Increased rate of growth. In studying the mechanical response of plants we found that direct application of stimulus to the responding organ always produced a response characterised by the negative turgidity-variation, that is to say, a depression of the leaf. We also saw that internal energy, inducing positive turgidityvariation, caused the opposite response, that is to say, an erection of the leaf ; and that an increase of the internal energy

1 Other records showing the effect of external stimulus on growth will be found in Chapter XXXIV. of the organ was, in some cases, brought about by that increased suctional activity by which energy was transmitted, by means of water forced into the responding organ. This increased suctional activity was due in its turn to external stimulation of the roots. Or a similar transmission of energy to the responding organ might be the result of stimulation applied at a distance on the stem, in consequence of which a wave of positive turgidity-variation would travel towards the responding organ from the excited point. For the exhibition of this latter effect, however, it is necessary that the direct excitatory effect of stimulation should not be conducted to the organ. This condition is met if the point of stimulation be at a sufficient distance, or when the intervening tissue is not a good conductor of excitation. Again, the plant as a whole, it must be remembered, has its internal energy raised, as the after-effect of the absorption of stimulus from its surroundings.

Direct and indirect effects of stimulus and laws of growth. — In the phenomenon of growth-response we have a case which is exactly parallel. Direct stimulation of a growing organ always induces a negative turgidity-variation, with a concomitant responsive retardation of growth. Anything, on the other hand, which increases the internal energy, brings about the opposite effect, of positive turgidity-variation, with concomitant responsive increase in the rate of growth. This may be induced by a favourable rise of temperature, or by stimulating the root, and so increasing the ascent of sap. It may also be brought about by stimulating a distant point, and so causing a wave of positive turgidity-variation to be transmitted to the responding organ, the stimulated point being at a sufficient distance to prevent the direct effect of stimulus from reaching it. And, finally, the energy absorbed from external stimulus may, as an after-effect, increase the internal energy of the plant. The increase of internal energy, under all these different conditions, we shall for the sake of convenience designate as the indirect effect OF

(1) The response of a growing organ is the same as that of a stationary organ. Direct application of stimulus, inducing contraction, retards the rate of growth. (2) The effect of indirect stimulation is to increase the internal energy, and thus augment the rate of growth. The multiple response of growth is characterised by all the peculiarities seen, for instance, in the autonomous response of Desmodium. The rhythmic responses of growth exhibit periodic groupings.

External stimulus is found to renew growth in organs in which, owing to the deficit of internal energy, it had come to a temporary standstill. The increase of internal hydrostatic pressure, up to an optimum, increases the rate of growth. The effect of increased internal hydrostatic pressure is exhibited in the case of the leaf of Mimosa by an erectile response. In a growing organ the effect of increased turgidity is shown by growth-elongation. A drought-rigored Mimosa on being supplied with water responds by erection of the leaf; and similarly, a plant in which growth, owing to droughtrigor, has come to a standstill, responds, on being supplied with water, by renewed growth-elongation. When the root of Mimosa is supplied with ice-cold water, the responding leaf, owing to the consequent arrest of ascent of sap, becomes depressed. The increased suctional activity, agaki, which is caused by a supply of warm water to the root, induces an erectile response of the leaf. Similarly, corresponding variations in the rate of ascent of sap, brought about in a growing plant by the application of cold and warm water to the roots, cause respective depressions and accelerations of the rate of growth.

mitted to the growing region, and finds expression in the work of growth. A rise of temperature, up to the optimum, enhances the rate of growth. At a maximum temperature of about 440 C. growth is apparently arrested. This is not, however, due to any rigor or arrest of internal activity, but to the fact that in each pulsation of growth the constituent response and recovery are now equal. There is thus no resultant growth-elongation. At such a temperature the amplitude of pulsation is reduced, and the frequency increased, as in Desmodium.

Longitudinal tension has the effect, up to an optimum, of increasing the rate of growth. The effect of external stimulus on a growing, is precisely the same as on a stationary, organ, that is to say, a responsive contraction. On account of this contraction, and concomitant negative turgidity-variation, growth is retarded, as the direct effect of the action of external stimulus. External stimulus, however, when absorbed and held latent by the tissue, has the effect of increasing the internal energy of the plant. This indirect effect of stimulus causes acceleration of growth.

General consideration of difficulties of accurate determination of effects of temperature on growth— Four accurate methods: (i) Method of discontinuous observations— Accurate regulation of temperature by electrolytic rheostat— (2) Method of continuous observations — Thermo-crescent curve — Determination of the optimum point— (3) Method of balance — (4) Method of excitatory response — Translocation of the optimum point. Determinations of the effect of temperature on growth are usually carried out either by observing a single plant for several hours in succession, or by obtaining the average growth of various groups of plants, kept under different temperatures. In the latter case the individual peculiarities of different specimens cause them to give values which are more or less discrepant, but this fact is to some extent neutralised by taking the average of a large number. These methods, however, are all very laborious, and the results not highly consistent. Whichever method be adopted, we have to remember that in all cases in which observations stretching over periods of several hours are required, the growth of the plant will be liable to spontaneous variations, resulting from the periodicities impressed upon it by the changing conditions of its environment. It is, perhaps, owing to this fact that the results obtained by various authorities have been so widely divergent. For example, the optimum temperature for growth of Zea mats was found by Sachs to be 340 C. and by Koppen to be 30-2° C.

Clearly, the perfect method would be one in which we should be able to measure the effects of temperature alone on the growth of a particular plant, so that the varying factors of age and constitution, or tonic condition, should remain constant. And, further, it should be possible to carry out this determination of growth at different temperatures in a time so short that the spontaneous variations of the plant, if any, would be insignificant. Only by such a method could we hope to obtain results which would be reliable and consistent.

Keeping these considerations in view, I have been fortunate enough to be able to devise four distinct methods of determining the effect of temperature on the rate of growth, the perfection of which may be gauged from the fact that the results of all agree with and corroborate each other within a fraction of a degree. With some of these, the entire experiment on the different rates of growth at various temperatures, ranging from ordinary through optimum to maximum, can be carried out within about half an hour. The determination of any single cardinal point, such as the optimum, can always again be made within five minutes. The investigation thus gains by simplicity and experimental accuracy, and observations may be made on many different specimens within a very short period. I shall now proceed to describe these different methods.

(i) The method of discontinuous observations. — We shall first take the method by which rates of growth are recorded at different temperatures, say one degree apart. Some means of raising the temperature to exactly the required point, and maintaining it unchanged during the time of experiment, is an essential condition of all these investigations. This I have been able to accomplish in the following way. The electrical heating coil inside the plant chamber is put in connection with an external battery. The heat given out, and the consequent permanent rise of temperature in the chamber, depend on the intensity of the current that flows through the heating coil. This current again may be progressively regulated by the interposition of an electrolytic rheostat in the circuit, the resistance of which

can be subjected to gradual variation. The electrolytic rheostat consists of two semicircular troughs (fig. 181). By turning the handle, say to the right, the electrolytic resistance interposed is continuously increased, diminishing the current; and hence diminishing the temperature inside the chamber. Rotation of the handle in the opposite direction produces the opposite effect, that is to say, raises the temperature inside the chamber. Thus, by proper manipulation of the handle of the rheostat, the chamber can be raised to any temperature,

Fig. 181. Semicircular Electrolytic Rheostat intei posed in Heating Coil Current enters the first trough, filled with zinc sulphate solution, by the electrode, z', and is led to the second trough by the diagonal metallic connector, d d'. By turning the index-arm, 1, clockwise, the interposed resistance is increased, and the heating current thus diminished. Rotation in the opposite direction diminishes the resistance and increases the heating current.

which can then be maintained uniform for any length of time, by keeping the rheostatic resistance constant. From a previous experiment, the temperature-values of the position of the index in connection with the handle can be ascertained and marked. Thus by turning the handle to any given index number, say of 310 C.,the temperature of the chamber will be found to be raised permanently to that value. A delicate thermometer graduated in twentieths of degrees is placed in the chamber, and affords an independent indication of the

temperature thus attained. It is also necessary, for reasons to be fully explained in a subsequent chapter, that the specimen should not receive thermal radiation from the heating coil, as such radiation, I find, retards growth. A shield of mica, opaque to thermal radiation, is interposed between the heating coil and the plant, which is thus subjected only to the action of changes of temperature. For many of my experiments I selected specimens of the Crinum Lily, on account of its extreme regularity of growth, which is so uniform that on adjusting the record under balance, the external conditions being constant, the line of record remained horizontal for a period of certainly over an hour.

The rate of growth at the temperature of the room, say 300 C, is first taken on the recording drum, which is covered with paper divided into millimetres. The horizontal distance or abscissa represents time, which, with the particular speed of drum which I used was 6 mm. per minute. The ordinate represents growth-elongation, and as the growth-recorder, or crescograph, produces a magnification of 1,000 times, 1 mm. distance of the ordinate is equal to an actual growth of •001 mm. The ordinate corresponding to an abscissa of 6 mm. would thus be equivalent to a growth in thousandths of a millimetre per minute.

The rheostat handle is now turned to the index-number corresponding to the raising of the temperature of the chamber by 1 ° C. There is first a variable period of rise of temperature, after which a permanent degree is attained. During this preliminary stage, variation of temperature acts as a stimulus, giving rise to responsive contraction or retardation of growth. But after this transient disturbance, the growth attains a constant rate, characteristic of the given temperature. These peculiarities will be better understood on following the record given in fig. 182 — reduced here to half the original size — which was taken with a specimen of Crinum Lily, during ten minutes. The record during the first five minutes is for the temperature of 340 C. It will be seen that in two minutes the growth-elongation is fifteen divisions, and as each division

represents *ooi mm., the rate of growth is thus "0075 mrn. per minute. The rheostatic handle was afterwards turned to the mark 35° C. With the particular battery power used in this case, the permanent rate of rise to 350 C. was attained after a period of three minutes. It will be seen from the record that the stimulus of sudden variation of temperature caused a contractile twitch, after which growth proceeded at a very rapid rate during the variable period. But as soon as the temperature of the chamber had attained a permanent condition — i.e. 350 C. — the rate of growth became constant. The attainment of this constant rate was practically simultaneous with the attainment of the permanent temperature condition. The lag, in any case, if it existed, could not be more than fifteen seconds.

One curious and interesting fact to be fully explained later, which was noticed in the course of the experiments, was that the amount of contractile twitch went on increasing during the variable period, as the temperature was raised each time i° C, from 300 to 350 C, but after this point practically disappeared. The permanent rate of growth, then, at a temperature of 350 C, is, as will be seen from the figure, twenty-four divisions per two minutes, or 'OI2 mm. per minute. In this way, by taking successive records at different temperatures, I obtained the following rates of growth, in the cases of Crinum Lily and the peduncle of Crocus.

The dotted line represents the variable period of temperature ^change. Note the contractile twitch and transient highly accelerated growth which follows. The rate of growth became constant when the temperature became permanent at 350 C. (2) Method of continuous observations. — The method which I have just described gives us results which, though obtained at closely consecutive temperatures, are nevertheless discontinuous. There is, besides, some loss of time involved during the variable period, in addition to which there is the factor of transient stimulation during sudden changes of temperature. For these reasons I was anxious to perfect some method by which the curve of growth should afford a continuous means of obtaining the rate of growth at all temperatures. I also wished to eliminate from this record the preliminary disturbance caused by sudden change of temperature.

I was enabled to do this in practice by bringing about a gradual and continuous rise of temperature, instead of the former sudden variations by steps. This was effected by turning the handle of the electrolytic rheostat at a rate so graduated that the rise of temperature within the chamber was uniform. As it was necessary to complete the experiment within not too long a period, I found that a rise of i°C. per 2*5 minutes was sufficient to meet the requirements of the case. This means a rise of i° C. in fifteen seconds. An observer watches a delicate thermometer, which is placed in the plant chamber, with his hand on the handle of the rheostat. By means of this, and a chronometer beating seconds, he is able to regulate the uniform rise of temperature with the greatest nicety. Should the rate be too quick, it may be reduced by the slightest turn of the handle towards the increase of

resistance, or vice versa. After a little practice the process of regulation becomes almost instinctive. The record of growth is now taken continuously on the revolving drum, and the thermocrescent curve obtained under these conditions of continuous variation of temperature is seen to be extremely regular,giving data by which we may determine the rate of growth at any point of the curve (fig. 183). The revolving drum gave, as already said, a movement of the recording surface of 6 mm. per

Fig. 183. Thermo-crescent Curve of Growth in Crinum Lily under Continuously Increasing Temperature minute. That length of the abscissa would therefore represent one minute of time ; and since the rise of temperature was regulated, at i° C. of rise per 2-5 minutes, intervals of 15 mm. in the abscissa also represent i° C. in temperature. With the magnification used, 1 mm. of the ordinate represents a growth-elongation of a thousandth part of a mm. In order, therefore, to obtain from this curve the rate of growth at any given temperature, say at 340 C, we have to find the elonga-.