Water-cultures with Clay Suspensions and with Nutrient Solutions

Wheat, buckwheat and rice were grown in cylinders on a clay suspension as the only source of mineral constituents. As nitrogen sources some ammonium nitrate and some nitric acid were added (the latter in order to lower the pH of the suspension from 7,4 to 5,3). When sufficient clay was present plants could be grown from seed to seed in this nutrient medium without renewing it. Wheat grown on the clay suspension with ammonium nitrate without nitric acid got yellow-striped leaves and a very sickly appearance. After some weeks the plants recovered by and by owing to the fact that during the growth the pH was lowered by the plants themselves. By wrapping the glass-cylinders in corrugated cardboard and kitchenpaper the mean maximum-temperature of the nutrient medium in the unheated glass-house was 4 to 5° C. lower than that of the air; the mean minimum-temperature about 2° C. higher. (The mean daily range of the watertemperature was 6°,6 C. less than that of the airtemperature). In paraffined and unparaffined new cylinders of resistant glass, each containing 2,1 liters of distilled water and 40 mgs of ammonium nitrate, wheatplants were grown. The air-dry grains weighed 57 mgs, the total air-dry weight of the plants, harvested at the age of 86 days was 98 mgs per plant from the paraffined and 121 mgs from the unparaffined cylinders. Wheat, buckwheat and rice were grown on clay suspension in which the quantities of clay were dosed in the proportion 1 : 3 : 9 : 27; for wheat the yield of dry matter of each series was about 2,7 times as much as of each preceding series. The proportional weights of the air-dry matter of the superterranean parts were: for wheat: 1 : 2,78 : 7,26 : 19,16 for rice : 1 : 3,01 : 7,77 ; 21,95 The air-dry weight of the superterranean parts of the 9 and 27 series showed for the wheatplants, which were grown from a pure strain, a far smaller standard error than for the buckwheat and the rice, where this was not the case. When growing plants on clay suspension, the clay particles did not adhere to the roots of wheat and rice, as they did to the roots of buckwheat. In the clay suspension and the fine earth the available components were determined according to Von Sigmond’s method. Especially K20, Na20, Fe203, P205 and Si02 were present in the clay particles in a far higher rate than in the fine earth. The same components were determined in the harvested plants for the roots and shoots separately, and the quantities of mineral components per plant were compared with the quantities of available components present in the given quantity of clay suspension. The method of Von Sigmond for determining the available quantities of phosphoric acid and bases proved in the clay suspension to give results rather too low than too high for phosphoric acid and potash. This results from the fact that wheat which had withered entirely some time before the grains were ripe, on account of deficiency of these two constituents, was found to contain more phosphoric acid and potash than resulted from Von Sigmond’s method in the same quantity of suspension. From the same quantity of suspension rice absorbed far more mineral components than wheat. For lime the quantity was not yet twice, for silicic acid, sodium and iron it was three times as much; for magnesium four times and for manganese even twelve times the quantity absorbed by a wheat plant. Though the wheat exhausted the suspension, as far as potash and phosphoric acid are concerned, yet the rice was able to absorb about 25 % more potash and about 40 % more phosphoric acid than the wheat had done. This greater absorption may be ascribed to the fact that the temperature of the nutrient medium for the rice was always at least 5° C higher than for the wheat, which probably has resulted in a greater solubility and as a consequence a faster and larger absorption of the mineral matter. It would be worth while to investigate whether extraction according to Von Sigmond's method would yield larger quantities of available components at a higher temperature than at room temperature. For trials concerning the influence of various salts or other components on plants, a clay suspension in some cases may be preferred to the usual nutrient solutions, as the nutrient components are not present in higher but in lower concentration than in the soil solution, whilst in the nutrient solutions used in most experiments described in literature this concentration is far much higher than in most soils. Besides the presence of the clay makes the circumstances far more comparable with those in the soil, while these circumstances can be constantly controlled much more effectively than with experiments in soil. So may for instance the effect of brackish water on plants probably be studied better in a clay suspension than in a nutrient solution of salts. In the first place, while no soluble salts besides those of the brackish water are added and in the second place while in the suspension part of the Na from the seawater will be exchanged for Ca, just as is the case with soil, while all the Cl of the sea-water remains in solution. The composition of a solution of nutrient salts, to which seawater is added, will show much difference with a clay suspension to which the same quantity of sea-water has been added, while the latter may be sooner compared with a brackish soil as regards the prevailing conditions. CHAPTER III. The influence of Auxiliary Elements in Nutrient Solutions on the Development of Plants. During the preliminary tests in 1930 a couple of wheat plants were grown on the solution after Knop and on that after Pryanishnikov. The solutions were made of tapwater and salts of the pure quality for pharmaceutical use. Difficulties caused by chlorosis which after some time appeared, especially in consequence of the use of tapwater, were set right by reducing the pH under 5,5 with some 0,1 n. acid. The wheat developed reasonably well, but the vaginae, laminae and later the stalks lacked the firmness which marks the wheat grown on soil. The ears were impotent to undo themselves from the vaginae of the uppermost foliage leaves of the stalks: their tops stuck, which than were bent while the stalks grew on; consequently they had to be loosened by hand (cf. plate III, fig. 1). The plants grown on these two different solutions diverged little in size and number of ears and were alike in the limpness of their bodies. In the experiments with wheat, buckwheat and rice of 1931 recorded in chapter II plants were grown on nutrient solution by the side of those grown on suspension. The solution after Pryanishnikov was used with and without addition of compounds of boron, manganese and fluorine. To each cylinder of 2 liters were added: H3BOs 2 mgs (= 0,36 mg B) CaF2 2 „ (= 0,97 ,, F) MnCl2 4 H20 .... 4 „ (= 1,15 „ Mn) The buckwheatplants to which the auxiliary elements were added when they were put in the cylinders, were the first to show a marked difference. Before long the new internodes of the plants without the auxiliary elements became very short, leaves and stems grew red and fleshy; the plants did not flower and made very short lateral branches. On the other hand those plants with auxiliary elements developed normally and produced ripe fruit. At the time of the harvest the buckwheatplants without auxiliary elements reached a height of about 10 cms, with a few lateral branches of at most 4 cms long; the lateral branches broke off easily at the stem. The air-dry superterranean parts weighed 1,12 grs per plant, the roots 0,07 gr; the quantity of water transpired was 410 cms3 per plant or calculated on a gram of air-dry matter 344 cms3. The buckwheat grown with auxiliary elements was at the time of the harvesting about a meter high with three lateral branches; about one hundred of ripe fruit were produced per plant with a fair average weight; the air-dry superterranean parts weighed 5,63 grs per plant, the roots 0,48 gr; the quantity of water transpired was 1560 cms3 or calculated per gram of air-dry material 255 cms. One half of the wheatplants on the nutrient solution after Pryanishnikov got the addition of the auxiliary elements after 27 days, when they had been in the solution for 17 days. During the first weeks after this addition no marked difference in growth between the plants with and without auxiliary elements was visible, both series developing normally and producing a strong number of shoots, but the former plants were firmer than the latter at the time the ears had to be formed. Of the plants without auxiliary elements the oldest shoots did not develop, the stalks remained short and limp and no ear appeared (cf. plate III, fig. 2, no. 28). The plants with auxiliary elements had long and solid stalks; after 77 days the ears appeared; the plants looked very sound (cf. plate 111, fig. 2, no. 29). The oldest shoots of the plants without auxiliary elements became yellow, wilted and withered; new shoots developed in their place, which neither produced an ear. The plants were harvested after 114 days; those without auxiliary elements did not bear a single well-developed ear; the number of shoots per plant was on an average 30; when the shoots were pulled to pieces a few contained a half-developed, empty ear. The plants with auxiliary elements each bore five ears with average weight of 5,5 grs per plant (the grains were not yet fully ripe) and six shoots which had not formed ears; the number of grains per plant was on an average 158. The dried plants without auxiliary elements could easily be crushed, because they were practically void of any solid matter. The quantity of water transpired for the formation of 1 gram of air-dry matter was abnormally high with the plants grown without auxiliary elements, as may be seen from table 13. The quantity of water transpired for the formation of a gram of air-dry matter with the plants grown in a nutrient solution with auxiliary elements is about the same as with those grown in the clay-solution; this figure on the contrary is with the plants grown without auxiliary elements about 2% times as high. Maze, who grew maize on nutrient solutions with a great number of auxiliary elements among which zinc and manganese were always present, whereas one of the others was in turn left out, found a quantity of water transpired varying from 161 to 195 cms3 per gram of dry matter.x) An explanation of the exceedingly high quantity of water transpired per gram of air-dry matter by the plants grown without auxiliary elements may be found in table 14. It is evident, that the higher weight of the plants grown with auxiliary elements is especially due to the ears and the stems of the fertile stalks. On the contrary the weight of the laminae, to which the transpiration has chiefly to be ascribed, is higher with the plants grown without auxiliary elements, so that we may take for granted that their surface too was larger. 2) The laminae of the plants grown without auxiliary elements however withered sooner than the other ones; so the transpiring surface of both groups at a given time may have been equal. Though transpiration and leaf-surface were about equal, the formation of organic matter was evidently depressed in a high degree by the deficiency of auxiliary elements. When the riceplants had been for three days in the nutrient solution they received at the age of 12 days the same quantities of auxiliary elements as the wheat and the buckwheat. This amount of auxiliary elements however turned out to be harmful; the plants with auxiliary elements developed fewer lateral shoots and consequently fewer leaves; the roots got a slightly yellowish brown colour and the oldest leaves withered. This stagnancy in growth is demonstrated most clearly in the quantity of water transpired: till the age of 61 days the transpiration per plant was 540 cms3, without auxiliary elements 710 cms3. In the long run however the auxiliary elements worked more favourably: the plants with auxiliary elements overtook the others and left them behind. This is also demonstrated by the quantities of water transpired: from the 61st day till the 124th day the transpiration per plant with auxiliary elements was 2700, without 1700 cms3. As a consequense of the effect of the auxiliary elements, at first harmful, later beneficient, the differences between both series of riceplants were less marked than with the wheat and the buckwheat, and the differences among the plants of the same series were greater. The favourable effect of the auxiliary elements was demonstrated best in the fructification. The riceplants without auxiliary elements were not limp as was the case with the wheatplants; the stalks were reasonably well developed and solid and so were the vaginae; both had a normal weight. From this description it follows that the rice needed less auxiliary elements than buckwheat and wheat, and that the quantity added was already slightly poisonous for these 12- days-old riceplants. A similar effect did not show itself with the buckwheatplants which also received the auxiliary elements immediately after being put in the cylinders, nor with the wheatplants, which however were then already 27 days old and so had considerably more roots than at the moment of transplantation and could probably endure more. In the literature on the investigations of the beneficient or harmful effect of auxiliary elements we find that special attention has been paid to the concentration of the auxiliary elements in the nutrient solution and the good or bad effect of this concentration always has been mentioned. Whether a certain solution is harmful or beneficient probably does not exclusively depend, however, on the concentration. The absorption of the auxiliary elements which are present in very low concentrations also will depend on the quantity of nutrient solution: the more solution, the more absorption. We may take for granted that the whole quantity of nutrient solution comes in contact with the roots because through the difference in day- and night-temperatures currents are always caused in the solution. In the experiments mentioned in this paper the solutions were moreover stirred every two or three days in order to give them the opportunity to absorb oxygen; by so doing the whole solution may in any case be con- sidered to have come in contact with the roots. The effect, be it harmful or not and the dose desirable for the plant will moreover depend on the development of the plant. Just as the dose of strongly active chemical substances used for the medical treatment of animals or men is given more or less proportional to the weight of the body, so with the plants a similar proportion will probably exist between the desirable dose and the quantity of dry matter of the plant or perhaps the total active surface of the roots. Moreover a smaller quantity of certain elements will apparently suffice during the vegetative period than in the period of the development of the organs of propagation. Did not the wheatplants, which developed a plentiful number of shoots and leaves with the slight quantities of auxiliary elements found in the usual „pure” salts, fail to produce any flowers? With the small quantities of the auxiliary elements present in the salts used, the riceplants could however not only make their leaves, but also produce a small number of grains; the effect of a bigger quantity of the auxiliary elements resulted from the much greater number of grains produced by the plants, which were grown with the addition of auxiliary elements. As of the three testplants grown, rice showed the least need of auxiliary elements, and as in the literature, as far as could be ascertained, rice did not occur among the plants for which the necessity of auxiliary elements had been established, the authoress resolved to try to grow rice entirely without auxiliary elements. At the same time could be examined, whether the presence of a smaller or greater quantity of auxiliary elements in the grains had a marked influence on the development of the plants. The grains harvested from the riceplants cultivated on the nutrient solutions with and without auxiliary elements afforded a good opportunity for this purpose. As it was only her intention to investigate how far rice would grow without any auxiliary elements, and the measure to which auxiliary elements generally were useful, those elements had to be excluded as far as possible from some plants. To other plants those elements which had proved beneficial should be added, but in such quantities that a harmful effect was not to be feared. In chapter I is mentioned how Maze proved in 1914 the indispensability of manganese and zinc for maize and his description in detail of the symptoms of their deficiency. In 1919 he proved the indispensability of boron for maize too. From Maze’s paper the most favourable dose of manganese and zinc cannot be concluded, as he added 20 mgs of MnCl2 and ZnCl2 per liter; through the presence however of 2 grs of calcium carbonate per liter the manganese and the zinc both were nearly entirely precipitated. The boron was given as 4 mgs of borax per liter. Warington1) proved the indispensability of boron for a great number of plant species; in her experiments a concentration of 1 : 12.500.000 gave the best results with barley grown 92 days; with barley grown 71 days a concentration of 1 : 100.000.000 proved still more favourable. The symptoms of boron-deficiency with Vicia Faba are fully described by her in another publication. 2) s’Jacob described the same symptoms as Warington when growing Vicia Faba without boron. 3) Brenchley 4) and Warington experimented on a large scale on the symptoms of boron-deficiency and the degree of indispensability of boron for different plant-species. With Vicia Faba the apices continually died in default of boron and numeral lateral shoots were formed, which for the greater part died too. Many other plantspecies began to die at the apex of the shoot and failed to flower; the roots were short and stunted. The necessity of boron always became more manifest at the approach of the flowering period. For some plant-species boron appeared to have a favourable effect, but not to be indispensable; for some no effect could be established. Swanback1) proved the indispensability of boron for tobaccoplants; two parts per million of boric acid in the nutrient solution gave an optimum growth. M e s 2) describes the symptoms of boron-deficiency with tobacco. Johnston and Dore3) proved the indispensability of boron for tomato-plants. In default of boron the conducting tissues in the stem broke down, and they were able to prove that in consequence the total of sugars and starch was larger in the leaves and stems of the plants deficient in boron than in those of normal plants. Johnston and Fisher4) continued the investigations of the necessity of boron for tomatoplants and found that the plants had to be supplied with boron throughout the growing and fruiting-period; two weeks after the boronsupply was stopped the plants ceased growing and the fruit were covered with black spots; 0,5 mg. of boron per liter of the nutrient medium kept the tomatoes healthy. Schmucker1) proved the indispensability of boron for the germination of the pollen-grains of Nymphaea zanzibarensis Casp. The indispensability of manganese for garden peas was proved by Mac Hargue. 2) In accordance with Maze’s description of his maize experiments he notes as a first effect of manganese deficiency that the young leaves get a yellowish instead of a normal green colour; soy beans and cow peas showed the same symptoms as garden peas. Samuel and Piper3) found that the grey speck disease of oats was caused by manganese deficiency. In nutrient solutions without manganese oats showed symptoms which were similar to those of the grey speck disease in the field; the plants died in the seedling stage unless manganese was supplied. 0,25 mg of manganese per liter sufficed to prevent the disease. Miller 4) proved the necessity of manganese for tomatoes, tobacco, cabbage, wheat, oats and maize. For these plants too the most striking symptom of the manganese deficiency was the chlorosis. The indispensability of zinc which was proved by Maze for maize, was investigated by Sommer and Lipman1) and by Sommer2)3) for a number of plants. These investigations were carried out with special precautions. In order to prevent dust falling on the plants they were cultivated in a small glass-house built within a larger one; the ventilators were provided with an apparatus to prevent the dust from entering; the cemented floor was kept dustfree. The utmost care was taken to use only the purest salts available and vessels which did not yield any trace of soluble components that might interfere in the experiments. In 2) was proved the necessity of zinc for sunflowers and barley; in 3) for wheat, buckwheat, broad beans and red kidney beans. Especially flowering and fructification failed in default of zinc; wheat and barley died already in the first growingperiod when zinc was lacking. The symptoms mentioned by Maze when zinc was lacking (cf. p. 40) were not described by Sommer and Lipman. Sommer proved that copper too is an essential for plant growth. 4) Just as the investigation described above with zinc, this too was made with the utmost care. All the plants received besides the principal elements traces of Mn, Zn, Al, I, F, Na, Cl and B and partly Cu, partly not. The plants without copper showed less growth after two weeks then those with copper, those without copper did not yield any fruit and bore hardly any flowers. For sunflowers, tomatoes and flax the necessity of copper was evident; 0,06 mg of copper per liter was sufficient. The quantity of copper is to be dosed carefully as Sommer found that 0,25 mg of Cu per liter already stopped the growth of the roots. From the literature cited it appears that a considerable number of plants have been proved not to be able to fructify, in many cases even not to develop their vegetative parts in case of a total absence of boron, manganese, zinc and copper and that the presence of aluminium, fluorine and iodine is beneficial, if perhaps not necessary to plant development. Consequently these elements as well as sodium and chlorine (because they are always present in irrigation water and in every soil solution in comparatively considerable quantities) were added as auxiliary elements. The potassium phosphate used for the nutrient solution was „according to Sorensen” and the other salts were „pro analysi'’ in original packing ofSchering-Kahlbaum. The salts used for preparing the solution with auxiliary elements were of the usual pure quality, as for my purpose there could be no objection to traces of other elements than those mentioned in the solution with auxiliary elements. The water was distilled from a tinned copper still with tinned tube and gathered in demijohns. The formula of the nutrient solution was similar to that used by Sommer,1) the quantity of phosphor taken was a little higher in relation to the other elements and the solution was almost twice as weak in order to come up to natural circumstances. For the solution with auxiliary elements the formula given by Sommer in the same publication was used as a guide. A solution containing the auxiliary elements was made, from which, if necessary. the desired quantity could be added to the cylinders with nutrient solution. Both formulae are given in table 16. From the riceplants grown the year before on a nutrient solution without addition of auxiliary elements 13 grains, each weighing 15 till 20 mgs and together weighing 232 mgs were selected; the remaining grains, which weighed less, were rejected. Of the grains harvested from the plants grown with auxiliary elements also 13 grains were selected, each weighing 15 till 20 mgs and together weighing 234 mgs. These grains were washed, soaked and laid down to germinate as has been described. All grains germinated. After three days one sixth of the distilled water was exchanged for the nutrient solution to give the seedlings an opportunity to absorb the principal elements as soon as possible. After 7 days the seedlings which had all one leaf consisting of vagina and lamina and 4 to 5 roots, were put in the cylinders. Eight cylinders contained the nutrient solution of table 16 only; in four of them the seedlings of the poor grains were put, and in four those of the normal grains. To four other cylinders was added 1 cm3 of the solution with auxiliary elements, consequently the addition of each element came to as many gammae as the solution contained mgs per liter. After 22 days another cm3 was added, after 28 days three cm3 and after 37 days 5 cm3. The traces of auxiliary elements which the young seedlings received at the start can in no way have been harmful. After 17 days the influence of the additional auxiliary elements was already visible. After 20 days the eight plants without auxiliary elements had three laminae, the four plants with auxiliary elements had already four, the third lamina being much longer than with the plants without auxiliary elements. The plants were altogether more vigorous and the colour of the leaves was of a bright green, whilst the plants without auxiliary elements still retained the more yellowish green colour of very young rice seedlings; the roots of the plants with auxiliary elements were much longer than those of the others. After 26 days the three series were well distinguishable. The plants from the poor grains had three laminae each of the fourth lamina 3 till 6 cms were visible; only the first lamina had a light but still normally green colour, the next two were yellowish; they had only four white roots longer than 3 cms (the roots which developed first) and about seven brownish roots, shorter than 3 cms, which had not yet reached the nutrient solution. The plants from the normal grains without auxiliary elements had three full-grown laminae; the fourth showed a length of from 8 till 18 cms; the first and second had a light but still normal colour; the third and fourth were yellowish; they had 5 white roots, longer than 3 cms and about six shorter ones, mostly of a brownish colour. The plants grown from the normal grains with auxiliary elements had five laminae, all of a bright green colour, about 10 roots longer than 3 cms and about 5 short ones; all the roots were white. The longest roots of these plants were about 15 cms long, while those from all the plants without auxiliary elements only 7 cms. After 31 days the first lateral shoot became visible in the axilla of the fifth leaf (with the third lamina) of the plants with auxiliary elements; four days later the second lateral shoot appeared in the axilla of the next leaf. The plants without auxiliary elements made no lateral shoot, but showed many symptoms of deterioration; the fourth and the fifth lamina were yellow with light yellow-green vertical stripes; the plants from the poor grains had still less green in their leaves than those of the normal grains. Some of the leaves had got brown spots and were withering. The length of the roots was about the same as after 26 days; those shorter than 3 cms had slightly increased in number; many of them had grown dark brown and were drying up or getting mouldy. After 45 days the plants from the poor grains were on the point of dying; those from the normal grains without auxiliary elements were not looking so bad as those from the poor grains; the difference was however small; those grown with auxiliary elements were healthy and vigorous. Three of each series of plants without auxiliary elements were photographed, described, dried and weighed. The differences between the plants of the same series were rather considerable. Probably the quantity of auxiliary elements which the different grains of one series contained in themselves had not been exactly the same and these differences between the grains were all the more obvious now that the auxiliary elements were entirely lacking in the nutrient medium. The plants of the series grown with auxiliary elements closely resembled each other; they had the same number of leaves at the main shoot; all four had two lateral shoots, with the same number of leaves for each shoot; both length and vigour of shoots and roots were for all four plants the same. From left to right in fig. 3 of plate III nos 1 till 3 had been grown from the poor grains and nos 5 till 7 from the normal grains, both without auxiliary elements; the middle plant had been grown with them. The differences between the three series are obvious and are stated clearly by the data in table 17. Details of the appearance of the three series are shown in plate IV fig. 1, where the second, the seventh and the fourth plant of the foregoing photograph have been taken together. The laminae of the largest plant have been partly cut off as they covered the smaller plants. The difference in root-development should be especially noticed: the largest plant counted 63 healthy white roots; of the other plants no roots had reached the nutrient solution except the four of five oldest. It should be noted how the length of the subsequent vaginae and laminae continues to grow in the plants with auxiliary elements and remains the same in the other plants. Length of vaginae: plant 2: 3 —4 —5% — 4% — 5%— 4 cms „ 7:4 5% — 5% — 8 „ 4: 3 — 7 y2—11 —14 —21 — 17 — 27 cms (The probable cause of the smaller length of the sixth vagina of plant 4 will be discussed later). Length of laminae: plant 2:3 — 13 — 16 — ? — ? — ? cms „ 7: 3 — 14 — 16% — 16 — 19 — 18 „ „ 4:3—14 — 23 —29 —38 —45 —48 —56 cms (The lengths of the three youngest laminae of plant 2 could not be measured in consequence of their crinkling and shrivelling). As the plants grown without auxiliary elements developed, their chlorosis became more obvious, especially with those grown from the poor grains; moreover these chlorotic leaves were very thin and so were the nerves, which was the cause of their crinkling and shrivelling. The chlorosis of these plants was caused only by a deficiency of manganese: the pH of the nutrient solution after harvesting ranged between 4,6 (plant 3) and 4,9 (plant 6), so lack of iron caused by a too high pH could not be thought of. Besides the fourth plant of both series had got a trace of manganese after 28 days; after 37 days the new leaf of the plant from the normal grain had a green colour and two young white and longer roots had been formed (the plant from the poor grain reacted in the same way but only many days afterwards, because its condition was so much worse). The plant recovered by and by, but never got a quite healthy appearance. Of the plants described above it is clear that riceplants — just as other plants, for which this was investigated — cannot grow without auxiliary elements in their nutrient medium. The less the quantity of auxiliary elements in the grain, the earlier and the more striking are the symptoms of deficiency. Besides the fact that without auxiliary elements riceplants cannot develop, it had to be proved that with these elements a prosperous growth and fructification can be obtained. Consequently the cultivation of the three remaining plants grown with auxiliary elements was continued. In order to keep the plants in good health the pH had to be controlled. When nitrogen is given as nitrate only, the pH rises as soon as the growth becomes vigorous. As was observed above, the pH of the nutrient solution which at the start was 4,4 had risen in the cylinder with the most vigorous plant without auxiliary elements in 38 days only to 4,9. The plants grown with auxiliary elements suddenly showed chlorosis after 33 days (when they had stood 26 days in their nutrient solution); the sixth lamina of the main shoot and the first lamina of the first lateral shoot of all four plants which had just appeared at this moment, were yellow; with bromocresol purple the pH with three of the plants was determined at 5,9, with one plant it was still higher. With 5 cms3 of 0,1 normal nitric acid the pH was brought down from 5,9 to 4,4. Three days later the influence of the changed pH was already perceptible as the parts of the laminae which became visible were green again. The short time of the chlorosis left its mark on the plants: every following vagina was some cms longer than its predecessor, but the sixth vagina only of all four plants remained some cms shorter than the fifth, (cf. p. 99). Every time when the transpired water had to be replaced the pH was tested and when it was too high, it was brought down