Chapter VIII: Part II: Growth and Its Responsive Variations (1)

X.--THE HIGH MAGNIFICATION CRESCOGRAPH FOR RESEARCHES ON GROWTH[S]

_By_

SIR J. C. BOSE,

_Assisted by_

GURUPRASANNA DAS, L.M.S.

In discussing the difficulties connected with investigations relating to longitudinal growth and its variations, special stress must be laid on the importance of maintaining external conditions absolutely constant. This constancy can only be maintained in practice for a short time. Lengthy periods of observation, moreover, introduce the uncertainty of complication arising from spontaneous variation of growth. The possibility of accurate investigation, therefore lies in reducing the period of the experiment to a few minutes during which we have to determine the normal rate of growth and its variation under a given changed condition. This would necessitate the devising of a method of very high magnification for record of the rate of growth.[S]

[S] A short account of my researches with the High Magnification Crescograph has been published in the _Proceedings_ of the Royal Society. I shall in the following Papers give a detailed account of my investigations on growth and on allied phenomena.

With auxanometers now in use, which give a magnification of about twenty times, it takes nearly four hours to determine the influence of changed condition in inducing variation of growth. It will be seen that if we succeeded in enhancing magnification from twenty to ten thousand times, the necessary period for experiment would be reduced from four hours to thirty seconds. The importance of securing a magnification of this order is sufficiently obvious.

The problem of high magnification was first solved by my Optical Lever.[T] The tip of the growing organ was attached to the short arm of a lever, the axis of which carried a small mirror; in this way it was possible to obtain a magnification of a thousand times. The magnified movement of growth was followed with a pen on a revolving drum. The record laboured under the disadvantage of not being automatic. This defect was overcome by the use of the photographic method which however entailed the inconvenience and discomfort of a dark room.

[T] BOSE--“Plant Response,” p 412.

I have, for the past six years, been working with a different method, which has now been brought to a great state of perfection. The problem to be solved was the devising of a direct method of high magnification and the automatic record of the magnified rate of growth.

METHOD OF HIGH MAGNIFICATION.

The magnification in my Crescograph is obtained by a compound system of two levers. The growing plant is attached to the short arm of a lever, the long arm of which is attached to the short arm of the second lever. If the magnification by the first lever be _m_, and that by the second, _n_, the resulting magnification would be _mn_.

The practical difficulties met with in carrying out this idea are very numerous. It will be understood that just as the imperceptible movement is highly magnified by the compound system of levers, the various errors and difficulties are likely to be magnified in the same proportion. The principal difficulties met with were due: (1) to the weight of the compound lever which exerted a great tension on the growing plant, (2) to the yielding of flexible connections by which the plant was attached to the first lever, and the first lever to the second, and (3) to the friction at the fulcrums.

_Weight of the Lever._--As the first lever is to exert a pull on the second, it has to be made rigid. The second lever serves as an index, and can therefore be made of fine glass fibre. The securing of rigidity of the first lever entails large cross section and consequent weight, which exerts considerable tension on the plant. Excessive tension greatly modifies growth; even the weight of the index used in self-recording auxanometers is found to modify the normal rate of growth. The weight of the levers introduces an additional difficulty in the increased friction at the fulcrums, on account of which there is an obstruction of the free movement of the recording arm of the lever. The conditions essential for overcoming these difficulties therefore are: (1) construction of a very light lever possessing sufficient rigidity, and (2) arranging the levers in such a way that the tension on the plant may be reduced to any extent, or even eliminated.

I found in _navaldum_, an alloy of aluminium, a light material possessing sufficient rigidity. The first lever is constructed out of a thin narrow sheet 25 cm. in length; it has, as explained before, to be fairly rigid in order to exert a pull on the second without undergoing any bending; this rigidity is secured by giving the thin narrow plate of the lever a T-shape. The first lever balances, to a certain extent, the second. Finer adjustments are made by means of an adjustable counterpoise B, at the end of the levers. By this means the tension on the plant can be greatly reduced; or a constant tension may be exerted by means of a weight T (Fig. 56). In my later type of the apparatus the plant connection is made to the right, instead of the left side of the first fulcrum. This gives certain practical advantages. The second lever is then made practically to balance the first, only a very slight weight being necessary for exact counterpoise. The reduction of total weight thus secured reduces materially the friction at the fulcrum with great enhancement of efficiency of the apparatus.

The second or the recording lever has a normal excursion through 8 cm. on the recording surface, which is a very thin sheet of glass 8×8 cm. coated with a layer of smoke. As the recording lever is about 40 cm. in length, the curvature in the record is slight, and practically negligible in the middle portion of 4 cm. The dimensions given allow a magnification of ten thousand times. A far more compact apparatus is made with 15 cm. length of levers. This gives a magnification of a thousand times.

AUTOMATIC RECORD OF THE RATE OF GROWTH.

Another great difficulty in obtaining an accurate record of the curve of growth arises from the friction of contact of the bent tip of the writing lever against the recording surface. This I was able to overcome by an oscillating device by which the contact, instead of being continuous, was made intermittent. The smoked glass plate, G, is made to oscillate, to and fro, at regular intervals of time, say one second. The bent tip of the recording lever comes periodically in contact with the glass plate during its extreme forward oscillation. The record would thus consist of a series of dots, the distance between successive dots representing magnified growth during a second.

The drawback in connection with the obtaining of record on the oscillating plate lies in the fact that if the plate approaches the recording point with anything like suddenness, then the stroke on the flexible lever causes an after-oscillation; the multiple dots, thus produced, spoil the record. In order to overcome this, a special contrivance is necessary, by which the speed of approach of the plate should be gradually reduced to zero at contact with the recording point. The rate of recession should, on the other hand, continuously increase from zero to maximum. The recording point will in this manner be gently pressed against the glass plate, marking the dot, and then gradually set free. It was only after strict observance of these conditions that the disturbing effect of after-vibration of the lever could be obviated.

This particular contrivance consists of an eccentric rod actuated by a rotating wheel. A cylindrical rod is supported eccentrically, so that semi-rotation of the eccentric causing a pull on the crank K (Fig. 57) pushes the plate carrier gradually forward. On the return movement of the eccentric, a light antagonistic spring makes the plate recede. The rate of the movement of the crank itself is further regulated by the device of the revolving wheel. This is released periodically by clockwork at intervals of one, two, five, ten, or fifteen seconds respectively, according to the requirements of the experiment. The complete apparatus is shown in figure 58.

_Connecting Links._--Another puzzling difficulty lay in the fact that the magnification actually obtained was sometimes very different from the calculated value. This unreliability I was able to trace to the defects inherent in thread connections, employed at first to attach the plant to the first lever, and the first lever to the second. These flexible connections were found to undergo a variable amount of elastic yield. Hence it became necessary to use nothing but rigid connections. The plant attachment, A, of triangular shape is made of a piece of _navaldum_; its knife-edge rests on a notch at the short arm of the lever, L. There are several notches at various distances from the fulcrum. It will be understood how the magnification can be modified by moving A, nearer or further from the fulcrum. The lower end of the attachment is bent in the form of a hook. The end of the leaf of the plant P, is doubled on itself and tied. The loop thus formed is then slipped over the hooked end of A.

The link, C, connecting L and L′, consists of a pin pointed at both ends, which rests on two conical agate cups fixed respectively to the upper and lower surfaces of the levers L and L′. This mode of frictionless linking is rigid and allows at the same time perfectly free movement of the levers.

_The fulcrum._--The most serious difficulty was in connection with frictionless support of the axes of the two levers. The horizontal axis was at first supported on jewel bearings, with fine screw adjustment for securing lateral support. Any slight variation from absolute adjustment made the bearing either too loose or too tight, preventing free play of the lever. When perfect adjustment was secured by any chance, the movement of the levers became jerky after a few days. This I afterwards discovered was due to the deposit of invisible particles of dust on the bearings. These difficulties forced me to work out a very perfect and at the same time a much simpler device. The lever now carries two vertical pin-points which are supported on conical agate cups. The axis of the lever passes through the points of support. The friction of support is thus reduced to a minimum. The levers are kept in place under the constant pressure of their own weight. The excursion of the end of the recording lever, which represents magnified movement of growth, was now found to be without jerk and quite uniform.

EXPERIMENTAL ADJUSTMENTS.

The soil in a flower pot is liable to be disturbed by irrigation, and the record thus vitiated by physical disturbance. This is obviated by wrapping a piece of cloth round the root imbedded in a small quantity of soil. The lower end of the plant is held securely by a clamp. In order to subject the plant to the action of gases and vapours, or to variation of temperature it is enclosed in a glass cylinder (V) with an inlet and an outlet pipe (Fig. 58). The chamber is maintained in a humid condition by means of a sponge soaked in water. Different gases, warm or cold water vapours, may thus be introduced into the plant chamber.

Any quick growing organ of a plant will be found suitable for experiment. In order to avoid all possible disturbing action of circumnutation, it is preferable to employ either radial organs, such as flower peduncles and buds of certain flowers, or the limp leaves of various species of grasses, and the pistils of flowers. It is also advisable to select specimens in which the growth is uniform. I append a representative list of various specimens in which, under favourable conditions of season and temperature, the rates of growth may be as high as those given below:--

Peduncle of _Zephyranthes_ 0.7 mm. per hour.
Leaf of grass 1.10 " " "
Pistil of _Hibiscus_ flower 1.20 " " "
Seedling of wheat 1.60 " " "
Flower bud of _Crinum_ 2.20 " " "
Seedling of _Scirpus Kysoor_ 3.00 " " "

The specimen employed for experiment may be an intact plant, rooted in a flower pot. It is, however, more convenient to employ cut specimens, the exposed end being wrapped in moist cloth. The shock-effect of section passes off after several hours, and the isolated organ renews its growth in a normal manner. Among various specimens I find _S. Kysoor_ to be very suitable for experiments on growth. The leaves are much stronger than those of wheat and different grasses, and can bear a considerable amount of pull without harm. Its rate of growth under favourable condition of season is considerable. Some specimens were found to have grown more than 8 cm. in the course of twenty-four hours, or more than 3 mm. per hour. This was during the rainy season in the month of August. But a month later the rate of growth fell to about 1 mm. per hour.

I shall now proceed to describe certain typical experiments which will show: (1) the extreme sensibility of the Crescograph; (2) its wide applicability in different investigations; and (3) its capability in determining with great precision the time-relations of responsive changes in the rate of growth. In describing these typical cases, I shall give detailed account of the experimental methods employed, and thus avoid repetition in accounts of subsequent experiments.

_Determination of the absolute rate of growth: Experiment 51._--For the determination of the absolute rate, I shall interpret the results of a record of growth obtained with a vigorous specimen _S. Kysoor_ on a stationary plate. The oscillation frequency of the plate was once in a second, and the magnification employed was ten thousand times. The magnified growth movement was so rapid that the record consists of a series of short dashes instead of dots (Fig. 59A). For securing regularity in the rate of growth, it is advisable that the plant should be kept in uniform darkness or in uniformly diffused light. So sensitive is the recorder that it shows a change of growth-rate due to the slight increase of illumination by the opening of an additional window. One-sided light, moreover, gives rise to disturbing phototropic curvature. With the precautions described the growth-rate in vigorous specimens is found to be very uniform.

After the completion of the first vertical series, the recording plate was moved 1 cm. to the left; the tip of the recorder was brought once more to the top by the micrometer screw, S, (Fig. 58), and the record taken once more after an interval of 15 minutes. The magnified growth for 4 seconds is 38 mm. in the first record; it is precisely the same in the record taken fifteen minutes after. The successive growth elongations at intervals of 1 second is practically the same throughout, being 9.5 mm. This uniformity in the spacings demonstrates not only the regularity of growth under constant conditions, but also the precision of the apparatus. It also shows that by keeping the external condition constant, the normal growth-rate could be maintained uniform for at least fifteen minutes. The magnified rate of growth is nearly 1 cm. per second, and since it is quite easy to measure 0.5 mm. the Crescograph enables us to magnify and record a length of 0.0005 mm. that is to say, the sixteenth part of a wave of red light. The absolute rate of growth, moreover, can be determined in a period as short as 0.05 of a second. These facts will give some idea of the great possibilities of the Crescograph for future investigations.

As the period of experiment is very greatly shortened by the method of high magnification, I shall, in the determination of the absolute rate of growth, adopt a second as the unit of time, and µ, or _micron_, as the unit of length,--the micron, being a millionth part of a metre or a thousandth part of a millimeter.

If _m_ be the magnifying power of the compound lever and _l_, the average distance between successive dots in mm. at intervals of _t_ seconds then:--

the rate of growth = _l_/_mt_ × 10^{3}µ per second.

In the record given _l_ = 9.5 mm.
_m_ = 10,000.
_t_ = 1 second.

Hence the rate of growth = 9.5/10,000 × 10^{3}µ per sec.
= 0.95µ per sec.

Having demonstrated the extreme sensitiveness and reliability of the apparatus, in quantitative determination, I shall next proceed to show its wide applicability for various researches relating to the influence of external agencies in modification of growth. For this two different methods are employed. In the first of these methods, the records are taken on a stationary plate: of these the record is at first taken under normal condition, the subsequent series being obtained under the given changed condition; the increase or diminution of intervals between successive dots, in the two series, at once demonstrates the stimulating or depressing nature of the changed condition.

In the second method, the record is taken on a plate moving at an uniform rate by clockwork. A curve is thus obtained, the ordinate representing growth elongation and the abscissa the time. The increment of length divided by the increment of time gives the absolute rate of growth at any part of the curve. As long as the growth is uniform, so long the slope of the curve remains constant. If a stimulating agency enhances the rate of growth, there is an immediate upward flexure in the curve; a depressing agent, on the other hand, lessens the slope of the curve.

I shall now give a few typical examples of the employment of the Crescograph for investigations on growth: the first example I shall take is the demonstration of the influence of variation of temperature.

_Stationary method: Experiment 52._--The records, given in Fig. 59_a_, were taken on a stationary plate. The specimen was _S. Kysoor_; the Crescographic magnification was two thousand times, and the successive dots at intervals of 5 seconds. The middle series, N, was at the temperature of the room. The next, C, was obtained with the temperature lowered by a few degrees. Finally H was taken when the plant-chamber was warmed. It will be seen how under cooling the spaces between successive dots have become shortened, showing the diminished rate of growth. Warming, on the other hand, caused a widening of intervals between successive dots, thus demonstrating an enhancement of the rate of growth.

Calculating from the data obtained from the figure we find:--

The absolute value of the normal rate 0.457µ per second.
Diminished rate under cold 0.101µ " "
Enhanced rate under warmth 0.737µ " "

_Moving plate method: Experiment 54._--This was carried out with a different specimen of _S. Kysoor_, the record being taken on a moving plate (Fig. 59_b_). The first part of the curve here represents the normal rate of growth. The plant was then subjected to moderate cooling, the subsequent curve with its diminished slope denotes the depression of growth. The question of influence of temperature will be treated in a subsequent Paper of the present series in much greater detail.

_Precaution against physical disturbance: Experiment 54._--There may be some misgiving about the employment of such high magnification: it may be thought that the accuracy of the record might be vitiated by physical disturbance, such as vibration. In physical experimentation far greater difficulties have, however, been overcome, and the problem of securing freedom from vibration is not at all formidable. The whole apparatus need only be placed on a heavy bracket screwed on the wall to ensure against mechanical disturbance. The extent to which this has been realized will be found from the inspection of the first part of the record in figure 60, taken on a moving plate. A thin dead twig was substituted for the growing plant, and the perfectly horizontal record not only demonstrated the absence of growth movement but also of all disturbance. There is an element of physical change, against which precautions have to be taken in experiments on variation of the rate of growth at different temperatures. In order to determine its character and extent, a record was taken with the dead twig, of the effect of raising the temperature of the plant-chamber through ten degrees. The record (Fig. 60) with a magnification of two thousand times shows that there is an expansion during the rise of temperature, and that the variable period lasted for a minute, after which there was a cessation of physical movement, the record becoming once more horizontal. The obvious precautions to be taken in such a case, is to wait for several minutes for the attainment of steady temperature. The movement caused by physical change abates in a short time whereas the change of rate of growth brought about by physiological reaction is persistent.

DETERMINATION OF LATENT PERIOD AND TIME-RELATIONS OF RESPONSE.

_Experiment 55._--In the determination of time-relations of responsive change in growth under external stimulus, I shall take the typical case of the effect of electric shock from a secondary coil of one second’s duration. Two electrodes were applied, one above and the other below the growing region of a bud of _Crinum_. The record was taken on a moving plate, the magnification employed being two thousand times, and successive dots made at intervals of two seconds. It was a matter of surprise to me to find that the growth of the plant was affected by an intensity of stimulus far below the limit of our own perception. As regards the relative sensitiveness of plant and animal, some of my experiments show that the leaf of _Mimosa pudica_ in a favourable condition responds to an electric stimulus which is one-tenth the minimum intensity that causes perception in a human being. For convenience I shall designate the intensity of electric shock that is barely perceptible to us as the unit shock. When an intensity of 0.25 unit was applied to the growing organ, it responded to it by a retardation of growth. Inspection of Fig. 61 shows that there is a flexure induced in the curve in response to stimulus, the flattening of the curve denoting retardation of growth. The latent period, in this case, is 6 seconds. The normal rate was restored after 5 minutes. The intensity of shock was next raised from 0.25 unit to one unit. The second record shows that the latent period is reduced to 4 seconds, and a relatively greater retardation of growth was induced by the action of the stronger stimulus. The recovery of the normal rate was effected after the longer period of 10 minutes. I took one more record, the intensity being three units. The latent period was now reduced to 1 second, and the induced retardation was so great as to effect a temporary arrest of growth.

TABLE X.--TIME-RELATIONS OF RESPONSIVE GROWTH-VARIATION UNDER ELECTRIC SHOCK (_Crinum_).

+------------+--------------+---------------+----------------+
|Intensity of|Latent period.| Normal rate. | Retarded rate. |
|stimulus. | | | |
+------------+--------------+---------------+----------------+
| 0.25 unit. | 6 seconds. |0.62 µ per sec.|0.49 µ per sec. |
| 1 " | 4 " |0.62 " |0.25 " |
| 3 " | 1 " |0.62 " |Temporary arrest|
| | | |of growth. |
+------------+--------------+---------------+----------------+

It is thus found that growth in plants is affected by an intensity of stimulus which is below human perception; that with increasing stimulus the latent period is diminished and the period of recovery increased; and that the induced retardation of growth increases continuously with the stimulus till at a critical intensity there is a temporary arrest of growth. I shall speak later of the effect induced by stimulus above this critical point.

_Experiment 56._--As a further example of the capability of the Crescograph, I shall give the record of a single pulse of growth obtained with the peduncle of _Zephyranthes Sulphurea_ (Fig. 62). The magnification employed was 10,000 times, the successive dots being at intervals of one second. It will be seen that the growth pulse commences with a sudden elongation, the maximum rate being 0.4 µ per sec. The pulse exhausts itself in 15 seconds, after which there is a partial recovery in the course of 13 seconds. The period of the complete pulse is 28 seconds. The resultant growth in each pulse is therefore the difference between elongation and recovery. Had a very highly magnifying arrangement not been used, the resulting rate would have appeared continuous. In other specimens, owing probably to greater frequency of pulsation and co-operation of numerous elements in growth, the rate appears to be practically uniform.

_Advantages of the Crescograph._--There is no existing method which enables us to detect and measure such infinitesimal movements and their time-relations. The only attempt made in measuring minute growth has been by observing the movement of a mark on a growing plant through a microscope. The magnification available in practice is about 250 times. The observation of the movement would itself be sufficiently fatiguing. But a simultaneous estimate of the time-relations of rapidly fluctuating changes would prove so bewildering, that accurate results from this method would be altogether impossible. A 1/12″ objective gives a linear enlargement of about 1,200 times. But the employment of this objective is impracticable in the measurement of growth elongation of an ordinary plant. With the Crescograph, on the other hand, we obtain a magnification which far surpasses the highest powers of a microscope, and it can be used for all plants. It does not merely detect growth but automatically records the rate of growth and its slightest fluctuation. The extreme shortness of time required for an experiment renders the study of the influence of a single factor at a time possible, the other conditions being kept constant. The Crescograph thus opens out a very extensive field of inquiry into the physiology of growth; and the discovery of several important phenomena mentioned in this Paper is to be ascribed to the extreme sensitiveness of the apparatus, and the accuracy of the method employed.

MAGNETIC AMPLIFICATION.

The magnification obtained with two levers was, as stated before, 10,000 times. It may be thought that further magnification is possible by a compound system of three levers. There is, however, a limit to the number of levers that may be employed with advantage, for the slight overweight of the last lever becomes multiplied and exerts very great tension on the plant, which interferes with the normal rate of its growth. The friction at the bearings also becomes added up by an increase in the number of levers, and this interferes with the uniformity of the movement of the last recording lever. For securing further magnification, additional material contact has, therefore, to be abandoned. I have recently been successful in devising an ideal method of magnification without contact. The movement of the lever of the Crescograph upsets a very delicately balanced magnetic system. The indicator is a reflected spot of light from a mirror carried by the deflected magnet. Taking a single lever with the lengths of two arms 125 mm. and 2.5 mm. respectively we obtain a magnification of 50 times. The magnetic system gives a further magnification of 20,000 the total magnification being thus a million times. This was verified by moving by means of a micrometer screw the short arm of the lever through 0.005 mm. The resulting deflection of the spot of light at a distance of 4 metres was found to be 5,000 mm. or a million times the movement of the short arm. It is not difficult to produce a further magnification of 50 times by attaching a second lever to the first. The total magnification would in this case be 50 million times.

A concrete idea of this will be obtained when we realise that by the Magnetic Crescograph a magnification can be obtained which is about 50,000 times greater than that produced by the highest power of a microscope. This order of magnification would lengthen a wave of sodium light to about 3,000 cm. I am not aware of any existing method by which it is possible to secure an amplification of this order of magnitude. The application of this will undoubtedly be of great help in many physical investigations, some of which I hope to complete in the near future.

Such an enormous magnification cannot be employed in ordinary investigations on growth, for the moving spot of light indicating rate of growth, passes like a flash across the screen. But it is of signal service in my investigations on growth by the Method of Balance, to be described in a future Paper. The principle of this method consists in making the spot of light, which is moving in response to growth, stationary, by subjecting the plant to a compensating movement downwards. The slightest variation caused by an external agent would make the spot of light move either to the right or to the left, according to the stimulating or depressing character of the agent. It will be understood, how extremely sensitive this method is for detection of the most minute variation in the normal rate of growth.

THE DEMONSTRATION CRESCOGRAPH.

Before proceeding with accounts of further investigations, I shall describe a form of Magnetic Crescograph with which I have been able to give before a large audience demonstration of a striking character on various phenomena of growth. The magnification obtained was so great that I had to take some trouble in reducing it. This was accomplished by the employment of a single, instead of a compound system of two levers. The reflected spot of light was thrown on a screen placed at a distance of 4 metres, and this gave a magnification of a million times; it is obvious that an increase of the distance of the screen to 8 metres would have given a magnification of 2 million times. As it was, even the lower magnification was far too great for use with quick growing plants like _Kysoor_. I, therefore, employed the slower growing flower bud of _Crinum_. It will be seen from Table X that the normal rate of growth of the lily is of the order of 0.0006 mm. per second. The normal excursion of the spot of light reflected from the Crescograph exhibiting growth was found to be 3 metres in five seconds or 60 cm. per second. This is a million times the actual rate of growth of the _Crinum_ bud. As it is easy to measure 5 mm. in the scale, it will be seen that with the Demonstration Crescograph it is possible to detect the growth of a plant for a period shorter than a hundredth part of a second.

_Experiment 57._--A scale 3 metres long divided into cm. is placed against the screen. A metronome beating half seconds is started at the moment when the spot of light transits across the zero division; the number of beats is counted till the index traverses the 300 cm. At the normal temperature of the room (30°C.), the index traversed 300 cm. in five seconds. The plant chamber was next cooled to 26°C. by the blowing in of cooled water vapour; the time taken by the spot of light to traverse the scale was now 20 seconds, _i.e._, the growth-rate was depressed to a fourth. Under continuous lowering of temperature the growth-rate became slowed down till at 21°C. there was an arrest of growth. Warm vapour was next introduced, gradually raising the temperature of the chamber to 35°C. The spot of light now rushed across the scale in a second and a half, _i.e._, the growth was enhanced to more than three times the normal rate. The entire series of the above experiments, on the effect of temperature on growth, was thus completed in the course of 15 minutes.

SUMMARY.

A description is given of the High Magnification Crescograph, which enables an automatic record of growth magnified ten thousand times. The absolute rate of growth can be easily determined from the data given in the record.

A magnification of a million times is obtained by the employment of Magnetic amplification. An increment of growth so minute as a millionth part of a mm. or 0.00000004 inch may thus be detected. It is also possible to detect the growth of a plant for a period shorter than a hundredth part of a second.

The influence of external conditions on variation of rate of growth is obtained by two methods of record. In STATIONARY METHOD, the increase or diminution of the distance between successive dots representing magnified rate of growth, demonstrates the stimulating or depressing nature of the changed condition.

In the second, or MOVING PLATE METHOD, a curve is obtained, the ordinate representing growth elongation, and the abscissa, time. A stimulating agent causes an upward flexure of the normal curve; a depressing agent, on the other hand, lessens the slope of the curve.

The action of external stimulus induces a variation of the rate of growth, the time relations of which are found from the automatic record of the growth. The latent period is shortened with the intensity of the stimulus. A responsive variation of growth is induced by an intensity of stimulus which is below human perception.

It is often possible to obtain record of the pulsatory nature of growth-elongation. Thus, with the growing peduncle of _Zephyranthes_, the growth pulse commences with a sudden elongation, the maximum rate being 0.0004 mm. per second. The pulse exhausts itself in 15 seconds, after which there is a partial recovery in course of 13 seconds, the period of complete pulse being 28 seconds. The resultant growth in each pulse is the difference between elongation and recovery.

The Magnetic Crescograph enables demonstration of principal phenomena of growth and its variation before a large audience.

XI.--EFFECT OF TEMPERATURE ON GROWTH

_By_

SIR J. C. BOSE,

_Assisted by_

SURENDRA CHUNDER DASS, M.A.

Accurate determination of the effect of temperature on growth presents many serious difficulties on account of numerous complicating factors. In nature, the upper part of the plant is exposed to the temperature of the air, while the root underground is at a very different temperature. Growth, we shall find, is modified to a certain extent by the ascent of sap. (See p. 189, _Expt. 70_.) The activity of this latter process is determined by the temperature to which the roots are subjected. The difficulty may be removed to a certain extent by placing the plant in a thermal chamber, with arrangement for regulating the temperature of the air. The air is a bad conductor of heat, and there is some uncertainty of the interior of the plant attaining the temperature of the surrounding air, unless the plant is long exposed to the definite and constant temperature of the plant chamber. Observation of the effects of different temperatures then becomes a prolonged process, with the possibility of vitiation of results by autonomous variation of growth. Reduction of the period of experiment by rapidly raising the temperature of the chamber introduces fresh difficulties; for a sudden variation of temperature often acts like an excitatory shock. This drawback may to some extent be obviated by ensuring a gradual change of temperature. This is by no means an easy process, for even with care the rise of temperature of the air cannot be made perfectly uniform, and any slight irregularity gives rise to sudden fluctuations in the magnified record of growth. Another difficulty arises from the radiation of heat-rays from the sides of the thermal chamber. These rays, I shall in a different Paper show, induce a retardation of growth. The effect of rise of temperature in acceleration of growth is thus antagonised by the action of thermal radiation. This trouble may be minimised by having the inner surface of the thermal chamber of bright polished metal, since the radiating power of a polished surface is relatively feeble.

The contrivance which I employ for ensuring a gradual rise of temperature, consists of a double-walled cylindrical metallic vessel; the plant is placed in the inner chamber, the walls of which are coated with electrically deposited silver and polished afterwards, and at the bottom of which there is a little water. The space between the inner and outer cylinder is filled with water, in which is immersed a coiled copper pipe. Hot water from a small boiler enters the inlet of the coiled pipe and passes through the outlet at the lower end. The water in the outer cylinder is thus gradually raised by flow of hot water in the coiled pipe. The rate of flow of hot water, on which the rate of rise of temperature depends, is regulated by a stop-cock. The air of the inner chamber in which the plant is placed, may thus be adjusted for a definite temperature. The small quantity of water in the inner chamber keeps its air in a humid condition, since dry hot air by causing dessication interferes with normal growth.

METHOD OF DISCONTINUOUS OBSERVATION.

_Experiment 58._--High magnification records are taken for successive periods of ten seconds, for selected temperatures, maintained constant during the particular observation. In figure 63 is given records of rate of growth obtained with a specimen of _Kysoor_ at certain selected temperatures. It will be seen that the rate of growth increases with the rise of temperature to an optimum, beyond which the growth-rate undergoes a depression. In the present case the optimum temperature is in the neighbourhood of 35°C.

METHOD OF CONTINUOUS OBSERVATION.

The method of observation that I have described above is not ideally perfect, but the best that could be devised under the circumstances. A very troublesome complication of pulsations in growth, arises at high temperatures, which render further record extremely difficult. Growth is undoubtedly a pulsatory phenomenon; but under favourable circumstances, these merge practically into a continuous average rate of elongation. At a high temperature the effect of certain disturbing factors comes into prominence. This may be due to some slight fluctuation in the temperature of the chamber, or to the effect of thermal radiation from the side of the chamber. This disturbing influence is most noticed at about 45°C, rendering the record of growth above this point a matter of great uncertainty. It will presently be shown that in plants immersed in water-bath growth is often found to persist even up to 57°C.

The only way of removing the complication arising from thermal radiation lies in varying the temperature condition of the plant, by direct contact with water at different temperatures. This procedure will also remove uncertainty regarding the body of the plant assuming the temperature of surrounding non-conducting air. The disturbing effect of sudden variation of temperature is also obviated by a more uniform regulation of rise of temperature. The inner cylinder containing the plant is filled with water; heat from gradually warmed water in the outer cylinder is conducted across the inner cylinder made of thin copper and raises the temperature of the water contained in the inner cylinder with great uniformity. A clock-hand goes round once in a minute; the experimenter, keeping his hand on the stop-cock, adjusts the rate of rise of water in the inner cylinder, so that there is a rise, say, of one-tenth of a degree every 6 seconds or of one degree every minute. The mass of water acts as a governor, and prevents any sudden fluctuations of temperature. The adoption of this particular device eliminated the erratic changes in the rate of growth that had hitherto proved so baffling.

The elongation recorded by the Crescograph will now be made up of (1) physical expansion, (2) expansion brought about by absorption of water, and (3) the pure acceleration of growth. The disentanglement of these different elements presented many difficulties. I was, however, able to find out the relative values of the first two factors in reference to the elongation of growth. This was done by carrying out a preliminary experiment with a specimen of plant in which growth had been completed. It was raised through 20°C in temperature, records being taken both at the beginning and at the end. This was for obtaining a measure of the physical change due to temperature, and also of the change brought about by absorption of water. I should state here that for the method of continuous record of growth which I contemplated, the record had to be taken for about 18 minutes. The magnification had to be lowered to 250 times to keep the record within the plate. With this magnification, the fully grown specimen did not show in the record a change even of 1 mm. in length in 18 minutes, while the growing plant under similar circumstances exhibited an elongation of 100 mm. or more. In records taken with low magnification, the effect of physical change is quite negligible.

DETERMINATION OF THE CARDINAL POINTS OF GROWTH.

The cardinal points of growth are not the same in different plants; they are modified in the same species by the climate to which the plants are habituated; the results obtained in the tropics may thus be different from those obtained in colder climates. At the time of the experiment, the prevailing temperature at Calcutta in day time was about 30°C.

_Temperature minimum: Experiment 59._--For the determination of the minimum, I took a specimen of _S. Kysoor_, and subjected it to a continuous lowering of temperature, by regular flow of ice-cold water in the outer vessel of the plant-chamber. Record was taken on a moving plate for every degree fall of temperature; growth was found to be continuously depressed, till an arrest of growth took place at 22°C. (Fig. 64).

The arrested growth was feebly revived at 23°C., after which with further rise of temperature there was increased acceleration. The optimum point was reached at about 34°C. In some plants the optimum is reached at about 28°C., and the rate remains constant for the next 10 degrees or more.

_Temperature maximum: Experiment 60._--For the determination of the maximum, the temperature was raised much higher. At 55°C. growth was found to be greatly retarded with practical arrest at 58°C. At 60°C. there occurred a sudden spasmodic contraction (Fig. 65), which I have shown elsewhere to be the spasm of death. This mechanical spasm at 60°C. is also strikingly shown by various pulvinated organs. An electric spasm of galvanometric negativity, and a sudden diminution of electrical resistance also take place at the critical temperature of 60°C.[U]

[U] BOSE--“Plant Response,” p. 168; “Comparative Electro-Physiology,” p. 202, p. 546.

I have described the immediate effect at the critical point. Long maintenance at a temperature few degrees below 60°C. will no doubt be attended with the death of the organ. Fatigue is also found to lower the death-point.

THE THERMO-CRESCENT CURVE.

_Experiment 66._--I was next desirous of devising a method by which an automatic and continuous record of the plant should enable us to obtain a curve, which would give the rate of growth at any temperature, from the arrested growth at the minimum to a temperature as high as 40°C. In order to eliminate the elements of spontaneous variation, the entire record had to be completed within a reasonable length of time, say about 18 minutes for a rise of as many degrees in temperature. This gives a rate of rise of 1°C. for one minute. Separate experiments showed that at this rate of _continuous_ rise of temperature there is practically no lag in the temperature assumed by thin specimens of plants. For observation during a limited range I use the slower rate of rise at 1°C. per two minutes. But the result obtained by slower rise was found not to differ from that obtained with one degree rise per minute. The curve of growth is taken on a moving plate, which travels 5 mm. per minute. Successive dots are made by the recording lever at intervals of a minute during which the rise of temperature is 1°C. A _Thermo-crescent Curve_ is thus obtained, the ordinate of which represents increment of growth, and the abscissa, the time. As the temperature is made to rise one degree per minute, the abscissa also represents rise of temperature (Fig. 66). The vertical distance between two successive dots thus gives increment of growth in one minute for 1 degree rise of temperature from T to T′. If _l_ represents this length, _t_ the interval of time (here 60 sec.), and _m_ the magnifying power of the recorder, then the rate of growth for the mean temperature (T+T′)/2 is found from the formula: rate of growth at (T+T′)/2 = _l_/(_m × t × 60_)10^{3}µ per sec.

TABLE XI.--RATE OF GROWTH FOR DIFFERENT TEMPERATURES.

+-------------+----------------+-------------+----------------+
| Temperature.| Growth. | Temperature.| Growth. |
+-------------+----------------+-------------+----------------+
| 22°C | 0.00 µ per sec.| 31°C | 0.45 µ per sec.|
| 23°C | 0.02 µ " " | 32°C | 0.60 µ " " |
| 24°C | 0.04 µ " " | 33°C | 0.80 µ " " |
| 25°C | 0.06 µ " " | 34°C | 0.92 µ " " |
| 26°C | 0.08 µ " " | 35°C | 0.84 µ " " |
| 27°C | 0.12 µ " " | 36°C | 0.64 µ " " |
| 28°C | 0.16 µ " " | 37°C | 0.48 µ " " |
| 29°C | 0.22 µ " " | 38°C | 0.30 µ " " |
| 30°C | 0.32 µ " " | 39°C | 0.16 µ " " |
+-------------+----------------+-------------+----------------+

I give in figure 67 a curve showing the relation between temperature and growth.

It will thus be seen that, in the course of an experiment lasting about twenty minutes, data have been obtained which enable us to determine the rates of growth through a wide range of temperature. We have likewise been able by the first method to make very accurate determinations of the temperature maximum and minimum. In short, by adopting the methods described, the cardinal points of growth and the rate of growth at any temperature, may be determined with a precision unattainable by the older methods, of averages or of prolonged observation.

SUMMARY.

Temperature induces variation in the rate of growth. In accurate determination of the growth, the disturbing effect of radiation of heat has not been eliminated.

A continuous record of growth under uniform rise of temperature gives the Thermo-crescent curve, from which the rate of growth at any temperature may be deduced.

Different plant-tissues exhibit characteristic differences in their cardinal points of growth. In _Kysoor_, growth is arrested at the temperature minimum of 22°C. The optimum temperature is at 34°C., after which growth-rate declines and becomes completely arrested at 58°C. At 60°C. there is a sudden spasmodic contraction of death.

In other plants the cardinal points are different. In some plants the optimum growth is attained at 28°C. and remains constant up to 38°C.

XII.--THE EFFECT OF CHEMICAL AGENTS ON GROWTH

_By_

SIR J. C. BOSE,

_Assisted by_

GURUPRASANNA DAS.

Chemical agents are found to exert characteristic actions on growth. The method of investigation sketched here opens out an extended field of investigation. The effect of a chemical substance, I find, to be modified by (1) the strength of the solution, (2) the duration of application, and (3) the condition of the tissue. A poisonous substance in minute doses is often found to exert a stimulating action. Too long continued action of a stimulant, on the other hand, exerts a depressing effect. The influence of the tonic condition is shown by the fact that while a given dilution of a poisonous substance kills a weak specimen, the same poisonous solution, applied to a vigorous specimen, actually stimulates and enhances the rate of the growth. I give below descriptions of a few typical reactions.

The reagent, when in a liquid form, is locally applied on the growing organ. The records, taken before and after the application, exhibit the stimulatory or depressing character of the reagent. A different method of application of the reagent is employed for plants with extended region of growth. The specimen is then enclosed in a glass cylinder, with inlet and outlet pipes. The cylinder is first filled with water, and the normal rate of growth recorded. This rate remains constant for several hours; but prevention of access of air for too long a time affects the normal growth. After obtaining normal record, water charged with the giving chemical agent is passed into the cylinder; and the subsequent record shows the characteristic effect of the reagent. The introduction of a gas into the chamber offers no difficulty.

EFFECT OF STIMULANTS.

_Hydrogen Peroxide: Experiment 62._--This reagent, as supplied by Messrs. Parke Davis & Co., was diluted to 1 per cent. and applied to the growing plant. Its stimulating action on growth is demonstrated in the right hand record of Fig. 68_a_, where the rate of growth is seen enhanced two and a half times the normal rate.

_Ammonia: Experiment 63._--The immediate effect of dilute vapour of this reagent is an enhancement of growth, seen in the middle record of Fig. 68_b_, where the rate is seen to be double the normal. Continued action, however, caused a depression; the third record of this series shows this, where the reduction is three-fourths of the normal rate.

EFFECT OF ANÆSTHETICS.

_Ether: Experiment 64._--In Fig. 68_c_, the records exhibit the effect of introduction of ether vapour into the plant chamber, and its recovery after the removal of the vapour. Ether is seen to depress the rate of growth to a little more than a third of the normal rate. The recovery is seen to be nearly complete half an hour after the removal of the vapour.

_Carbonic Acid: Experiment 65._--The action of this gas is very remarkable. The plant was immersed in water and normal record taken; the plant chamber was now filled with water, charged with carbonic acid gas. This induced a very marked acceleration of growth (Fig. 69). In a seedling of Onion, the increase was found to be two and a half times. In the flower bud of _Crinum_, the rate was found enhanced threefold from the normal 0.25 µ to 0.75 µ per second. After this preliminary enhancement, there was a depression of growth within 15 minutes of the application, the rate being now reduced to 0.15 µ per second. These effects were found to take place equally in light or in darkness.

ACTION OF DIFFERENT GASES.

_Coal Gas: Experiment 66._--Coal gas induces a depression. It is curious that subjection to the action of this gas does not produce so evil an effect as one would expect. The introduction of the gas had reduced the growth-rate to more than half; but there was a recovery half an hour after the introduction of fresh air.

_Sulphuretted Hydrogen: Experiment 67._--This gas not only exerts a depressing effect, but its after-effect is also very persistent. The plant experimented on was very vigorous and its rate of growth was depressed to half by subjection to the action of the gas for a short time. The record taken half an hour after the introduction of fresh air did not exhibit any recovery.

ACTION OF POISONS.

_Ammonium Sulphide: Experiment 68._--This reagent in dilute solution retards growth, and in stronger solution acts as a poison. The following results were obtained with a wheat seedling under different strengths of solution:--

Normal rate 0.30 µ per sec.
0.5 per cent. solution 0.15 µ " "
2.0 " " " 0.08 µ " "

_Copper Sulphate: Experiment 69._--The effect of a solution of this reagent is far more depressing than the last. One per cent. solution acting for a short time depressed the rate from 0.45 µ to 0.13 µ per sec. Long continued action of the poisonous solution kills the plant.

SUMMARY.

The effect of a chemical agent is modified by the strength of the solution, the duration of application and the tonic condition of the tissue.

Dilute solution of hydrogen peroxide induces an acceleration of growth.

The action of dilute vapour of ammonia is a preliminary enhancement followed by depression of growth.

Ether vapour depresses the rate of growth. On the removal of the vapour there is a recovery of the normal rate.

The effect of carbonic acid is a great enhancement of the rate of growth; after this preliminary action, growth undergoes a decline. The effect described takes place equally in light or in darkness.

Coal gas induces a depression of the rate of growth from which there is a recovery after the removal of the gas. The action of sulphuretted hydrogen is far more toxic, the after-effect being very persistent.

Solution of ammonium sulphide induces increasing retardation of growth, with the strength of the solution. Copper sulphate solution acts as a toxic agent, retarding the rate of growth and ultimately killing the plant.

XIII.--EFFECT OF VARIATION OF TURGOR AND OF TENSION ON GROWTH

_By_

SIR J. C. BOSE.

The movements of leaves of sensitive plants are caused by variation of turgor in the pulvinus induced by stimulus. The down movement or _negative_ response of _Mimosa_ is caused by a diminution or negative variation of turgor, while the erection or _positive_ response is brought about by an increase, or positive variation of turgor.

We shall now investigate the change induced in a growing organ in the rate of growth by variation of turgor. Turgor may be increased by enhancing the rate of ascent of sap or by an artificial increase of internal hydrostatic pressure. A diminution of turgor may, on the other hand, be produced by withdrawal of water through plasmolysis. In order to maintain a constant terminology I shall designate an increase, as the positive, and a diminution, the negative variation of turgor.

RESPONSE TO POSITIVE VARIATION OF TURGOR.