UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 4, No. 14, pp. 413-444, 12 figures in text January 24, 1924 INFLUENCE OF REACTION ON INTER-RELA- TIONS BETWEEN THE PLANT AND ITS CULTURE MEDIUM BY • J. J. THERON (tontribution from the Laboratory of Plant Nutrition, College of Agriculture) I. INFLUENCE OF THE REACTION OF THE MEDIUM UPON THE PLANT Introduction* The reaction of the substrate in which the roots of plants develop is of obvious importance to the life of the plants. Earlier plant physi- ologists have neglected this factor, and it was not until recently as a result of studies on the intensity of the acidity of the soil solution of certain soils, on the one hand, and the relative resistance of different varieties of plants to alkaline conditions in certain types of 'alkali soils,' on the other, that the significance of this factor was fully realized. Since the preliminary studies of Pantanelli 26 and Hoagland, ir ' sev- eral investigators have attacked the problem. The practical, as well as theoretical importance of a more thorough understanding of the influ- ence of the reaction of the culture medium on the growth and meta- bolism of plants seemed to warrant the investigation here described. The object was twofold: (1) a study of the effect of various con- centrations of hydrogen ions on the external appearance and growth of the more common agricultural plants; (2) the effect of the reaction on the metabolism of these plants. * The writer wishes to acknowledge his indebtedness to Professor D. E. Hoagland for advice and kindly suggestions during the course of the investigation. 414 / niversity of California Publications in Agricultural Sciences [Vol.4 For obvious reasons, it was impossible to employ more than a few types of plants to accomplish these aims; hence plants were selected which were adapted to the methods of experimentation, and which may be considered as representative of the majority of field crops. These were alfalfa. (Medicago sativa), cotton (Gossypium hcrbaccum, Durango variety), cucumbers (Cucumus sativa. White Spine variety), Bermuda grass (Cyanodon dactylon), corn (Zea mais. White Dent field corn), barley {Hordcam vvlgare, Beldi variety), and peas (Pisum sativum, Canada field), the latter two being the principal ones used in the study of the inter-relations between the metabolism of the plant and the reaction of the culture solution. Owing to the complexity of the soil and the reactions taking place therein and because of the many complicating factors which enter when sand cultures are used, solution cultures were employed exclusively. Experimental Baker's analyzed salts and the ordinary distilled water of the laboratory were used in making all culture and stock solutions. The stock solutions were those used regularly in this laboratory. Table 1 gives the weights of salts added to 18 liters of water to make up those solutions. TABLE 1 Weights of Salts Dissolved in 18 Liters of the Distilled Water to Make up the Stock Solutions Solution I Solution II Solution III KN0 3 MgSO, 1200 grams 679 grams Ca(N0 3 ) 8 : 1805 grams KH,P() 4 : 900 grams In table 2 is given the composition of the culture solution used throughout in this investigation (except where otherwise stated). This solution was made by adding 80 c.c. of solution I, 40 c.c. of solu- tion II, 480 c.c. of solution III. and 24 grams of XaNO.. to 44 liters of water. TABLE 2 Composition of Culture Solution Expressed as Equivalents, per Liter K no 3 H;PO ( Ca Mg so, Na Pn .0052 .0087 0040 .0011 .0007 0007 .0064 4 9 Iron was supplied in the form of ferric tartrate, one cubic centi- meter of a 0.5 per cent solution being used per liter of culture solution. 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 415 Tn figure 1, the titration curve of the solution is reproduced. This was obtained colorimetrieally. By interpolation the amounts of acid or alkali to be added to eleven liters of the solution to obtain any desired P H within the useful range can be found from this graph. 45 40 35 30 25 Ctty h OH" 20- 15 1 1 L pH of Solution 50 60 riar. i 7.C 60 90 In an effort to keep the composition of the solution as constant as possible over the entire range of reactions used, the concentrations of Ca and Mg were kept low, and were regulated by the amount of 416 University of California Publications in Agricultural Sciences [Vol. 4 calcium which will remain in solution at P H 8.0. A precipitate usually occurred at P H 8.5 and often at 8.0 after a few days. A comparatively high concentration of phosphate was used, on the other hand, in order to increase the buffer effect of the solution. Unfortunately, the buffer effect varies over different ranges of reactions. This serious defect may be partly remedied by the addition of an acid with a dissociation constant of about 4.5 and a base with a con- stant of about 5.5. The only non-toxic acids having the desired con- stant are organic acids, e.g., citric acid. Owing to the danger of excessive bacterial growth in solutions containing organic matter, however, these non-toxic, organic acids cannot be used satisfactorily (Salter and Mcllvaine 27 ). Ammonium hydroxide may be used to supplement the buffer effect of the phosphate at P H 8 to P H 10, but the advantages to be gained here are small and the presence of the ammo- nium ion may introduce complicating factors. Growth in the culture solution was very satisfactory if changes were made weekly. Sulfuric acid and sodium hydroxide were used to regulate the P H values of the solutions. Measurements of the reac- tion were made by the indicator method of Clark and Lubs. 13 Frequent use was also made of a Ilildebrand-type hydrogen electrode. The plants were germinated between sheets of wet paper toweling and the usual methods of solution culture technique followed. At first properly covered Mason jars of 950 c.c. capacity were used as containers. In each jar, three plants were grown, ten jars being employed for each P H tested. The plants were grown in series of solutions having the following initial values: 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 8.5, and 9.0. Since the reaction changed very rapidly in the direction of neutrality, the solutions were renewed every second day. These frequent renewals, however, did not prevent the reactions of the solutions from being changed consid- erably during the later stages of growth. The maximum changes in reactions are tabulated in table 3. The plants were grown from 3 to 4 weeks, within which time sufficient growth was made to determine ;it which reactions they were affected adversely. TABLE 3 Maximum and Minimum Values of the Reactions at Time of Change Initial Ph of Series 4.0 4.5 5.0 6.0 7.0 S.O 8.5 Maximum and minimum Ph at time of change 4.1-4.4 4.6-5.0 5.0-5.2 6.0-6.1 7.0-6.9 8.0-7.9 8.4-8.0 1024] Theron: Inter-relations Between the Plant and Its Culture Medium 417 We assume naturally that the slightly increased concentrations of Na ions and S0 4 ions used in regulating the P H values of the different solutions have no effect on the plant, and that all differences in the external characteristics are caused directly or indirectly by the activi- ties of the hydrogen or hydroxyl ions. The influence of the different reactions was determined by the relative weights of plants grown in the different solutions, the length and appearance of the roots, and the height and color of the tops. The general effect of excessive acidity is very characteristic, and is the same for all the plants used in the experiment. If the culture solution is injuriously acid, the roots thicken and soon become a dull white in color which is easily distinguishable from the silky white appearance of normal roots. Depending upon the degree of acidity, the roots may stop growing in length entirely or may grow only sloAvly. In the latter case, they become knobby, because of the excessive development of laterals which penetrate the outer layers of the root with apparent difficulty. Lateral roots may develop to within a few millimeters from the growing tip. If the injury is not too severe the roots recover very rapidly when placed in a more favorable solution. The tops of the plants show a marked stimulation in growth and general vigor, as a rule, when compared with the plants grown in a more favorable solution. The stimulation, however, is of short dura- tion and after two weeks they begin to lag behind. Similar results were obtained by Hixon. 14 An injurious alkalinity of the culture solution is very readily recognized by a yellowish discoloration of the roots. In extreme cases, the roots become gelatinous and soon disintegrate. At first the tops showed no differences in size and vigor as a result of injury to the roots when compared with the tops of plants growing in a more favorable solution. After two to three Aveeks, however, a decided stunting was noticeable, and chlorosis of the new leaves set in. Chlorosis is generally ascribed to the lack of available iron. This was probably the main cause of the chlorosis of those plants grown in the alkaline series. That excessive concentrations of hydroxyl ions, however, may cause chlorosis directly seems certain from the following considerations. A distinct test for iron could be demonstrated in a solution kept at P H 8.5 even after chlorotic plants had been growing in it for a week. Cucumbers and alfalfa will show chlorosis at P H 7 within two weeks; at this reaction neither barley nor peas show any chlorosis 418 University of California Publications in Agricultural Sciences [Vol.4 even after nearly two months' growth. By this time, one would expect the supply of iron stored in the seeds of the latter plants to be depleted. Gile and Carrero 8 found that ferric tartrate supplied the necessary iron to plants grown in solutions which they thought to be alkaline. -i r "i r 1 r 100 60 40 zo G-reenWts of Tops in &rs- ph of Solution. 30 40 30 60 Fig. 2 7.0 5.0 10 It may be objected that the iron is not translocated from the roots to the tops in the case of the plants growing in an alkaline solution, and hence the plants are nevertheless suffering from a lack of iron. The reaction of the root juices, expressed after freezing, indicates. 19L'4] Theron: Inter-relations Between the Plant and Its Culture Mediuvi 419 however, that it is hardly possible for the increased concentration of the hydroxy! ions to interfere with the translocation. All the plants grown on the acid side of P H 6.0 were deep green; above this reaction the color gradually became paler green, merging into complete chlorosis at the higher P H values. It is apparent that the plant is influenced strongly by the reserve store of food material in the seed. Great care must be taken in making any deductions from the experiments in which the plants have been grown for a short period of time only. A much more thorough study of the problem lias been made using the technique described below. The results of the experiments just discussed are therefore summarized in table 4 without further detail here. TABLE 4 Effect of Acidity and Alkalinity on Growth of Various Plants Plant Alfalfa Cotton Cucumbers Barley Bermuda grass Ph injuriously acid 4.2-4.5 4.2-4.5 4 2-4 5 4.2-4 5 4.2 Ph at which optimum growth takes place 4.8-6.0 5 0-7 4.8-6 4.5-7.0 4.5-8.0 Ph injuriously alkaline 7 S I) 7 S '.) o Remarks Very sensitive Fairly resistant \ i n sensitive Resistant Highly resistant All the varieties of plants tested, except the Bermuda grass, were affected adversely by approximately the same intensity of acidity. Alfalfa and cucumbers were affected much more severely, a fact which is correlated with their greater sensitiveness to alkaline conditions. In all cases, the best growth was made when the reaction of the culture solution was between P H 5 and P H 6. It may be of interest here to note that Fred and Davenport' 1 found the critical point for the growth of alfalfa bacteria to be at P H 4.9. This reaction is well within any possible critical range for the host plant. The use of the technique described above involves an excessive amount of labor and errors are unavoidable. At best, we are unable to control the reactions of the solutions satisfactorily. The advantages of the technique evolved later and described below will at once be evident. Whereas with the former technique, 30 plants were grown in ten different jars at every P H value in the experiment, all 30 plants were now grown in one three-gallon (eleven liter) earthenware crock. The 420 University of California Publications in Agricultural Sciences [Vol.4 plants were supported as before in perforations on a cork sheet made by binding together three 12" x 4" x %" cork slabs with two strips of wood nailed on the edges. To prevent lateral movements, small pieces of wood were nailed on the underside of the overlapping corners. Slabs of wood %" thick serve the purpose even better, since the plants can be supported more firmly in them. These slabs must be soaked thoroughly in hot pure paraffin so as to prevent the absorption of water. By growing all thirty plants in this large volume of solu- tion, the effect of the inherent variability of the plants is minimized and most of the experimental errors are eliminated. The roots can 40 30k' Z0 w Lengths R g f 0T5 BARLtY -Grown 44 Days in Cm CORN • - 2.6 ■ PEAS - • 27 ■ dH of Solution 40 30 60 7.0 SO 90 Fig. 3 be inspected readily and the P H of the solution can be adjusted con- veniently, rapidly, and as frequently as desired. The P H is adjusted by withdrawing 5 c.c. samples of the solution and determining the reaction colorimetrically. The amounts of acid or alkali which must be added to bring the P H to the original value are read off from figure 1, and the required quantities added to the solution. Figure 1 applies strictly to only the fresh solution. Within a week, however, the composition of the solution did not change sufficiently to invalidate the method. The solutions were changed every week and the P H adjusted twice a day, i.e., in the morning and evening. During the later stages of growth, this becomes necessary more frequently in the case of plants growing at the reactions P H 4.0-5.0 and P H S. 0-9.0. Over these ranges, the buffer effect of the solution is relatively small and the power of the plant to change the P H of the solution is increased iscc figs. 4 and 5). All the plants experimented with showed a tend- ency to change the P H of the solutions to a value between P H (>.'_' and 6.8. ' 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 421 Because of the lack of time, it was not possible to subject all the plants used in the former experiments to these better controlled methods. This was done, however, with four widely different types of plants, namely, barley, peas, cucumbers, and corn. 60 60 40 20 Cc.ty * pM of Solution ■ i 60 40 ZD 30 40 30 £0 Fig. 4 9.0 In table 5, one experiment with peas is summarized. In the first column are given the desired reactions of the solution, in the second the highest and lowest P H values reached during the course of the experiment, and in the seventh the number of cubic centimeters of normal hydrogen (sulphuric acid), or normal hydroxide ions (sodium hydroxide), added during the entire period of growth to replace that neutralized by the plants. 422 University of California Publications in Agricultural Sciences [Vol. 4 In figure 2, the green weights of the tops of 30 plants of the four types are plotted against the P H of each series (see column 1, table 5), and in figure 3, the length of the roots. Since neither the change of P H nor the change in the total molality of acid or base with time is an arithmetic function, it is impossible to calculate an average P H . The true average P H values differ only by a small amount from the desired P H such that the given curves are not greatly different from the curves which would be obtained if the true average reactions were used. The differences are within the limits of the experimental error. TABLE 5 Summary of a Typical Experiment with Peas Desired Pnof Maxi- mum range of Ph Days grown No. of plants Green weight of tops gms. Length of roots cms. C.C. N/1 Reagent neutralized Remarks series Acid Alkali 3.9 3.9-4 25 30 82.8 28 14.8 Roots severely injured 4.5 4.5-4.7 25 30 86.2 35 8.8 Very slight injury to roots 5.0 5.0-5.2 25 30 95.3 40 8.9 Best growth 6.0 6 6 1 25 30 94.2 40 7.0 Best growth 7.0 7.0-6.9 25 30 81.1 38 8.0 8.0 8.0-7.9 25 30 59.4 25 60.0 Tops slightly chlorotie 8.5 8.5-8.3 25 30 27.6 20 41.0 Roots badly injured. Tops chlorotie The juices of the plants were needed for other experiments, so the dry weights were not determined. For the present purpose, the green weights of the tops give a reliable criterion of the general vigor and size of the plants. The differences in the weights of the barley and pea plants can hardly be considered as significant in themselves on account of the inherent variability of the plants. If the observa- tions on the other effects are taken into consideration, however, it becomes evident that the small differences in weight are true expres- sions of the effect of the corresponding reactions on the growth of the plants. The maximum changes in Ph brought about by all four types were the same as that given for peas in column 2, table ."), excepl in the case of corn, grown in the alkaline solution, where the reaction fre- quently reached the P H 8.1. Since tour widely different types of plants were used, the curves may be considered as a definite measure of the effect of the reaction of the culture medium on the growth of 1924] Therein: Inter-relations Between the Plant and Its Culture Medium 423 most agricultural plants as indicated by the yield. They show unmis- takably that the optimum range of the reaction for the propagation of these plants in solution cultures is between P H 4.5 and 6.0, and agree substantially with the results found with the earlier method of experimentation (see table 4). In figure 4, the amounts of normal acid or alkali neutralized by 30 plants during the first 25 days of Fig. 5 growth is represented graphically for each type. These curves are not strictly comparable, since the plants were grown at different times of the year. The amounts of acid or alkali neutralized within a definite period of time depend largely upon the rapidity of growth. Although the curves are onry of a qualitative significance, they are very expressive of the power of the plant to overcome any unfavor- able acidity or alkalinity, especially the latter. This power is of obvious importance to the plant and must form an integral part of any study of acid or alkali resistant crops, either in the soil or in 424 University of California Publications in Agricultural Sciences [Yo\.i solution culture. Under natural conditions, the plant has to contend with the reaction of the medium in which its roots are immersed or imbedded from the time of germination to maturity. If the medium is sufficiently highly buffered or is continually renewed, such that little or no change of reaction is brought about under the influence pHof Sap CO 50 BARLCY M pH of Solution 40 50 60 Fig-. 6 7.0 flO Fie. of the plant, the ability to overcome any unfavorable reaction is cor- rectly expressed by these curves. From a purely theoretical point of view, however, this ability may be determined at different reactions for plants treated similarly up to the time of experimentation, so that the vigor and internal mechanism of all the plants will as nearly ]<»24| Theron: Inter-relations Between the Plant and Its Culture Medium 42") as possible be the same when subjected to the different acidities or alkalinities. These must be such that the plant mechanism will not be injured or altered materially during: the period of experimentation. Five sets of 25 barley plants . each were grown in earthenware crocks of 7 1 /-) liter capacity. All the solutions had a reaction of P H 6.8, and were changed weekly. When plants were four weeks advanced, the sets were transferred to solutions having the reactions 4.0, 5.0, 6.0, 7.0, and 8.0, and these were kept as constant as possible for four days by the addition of N/5 acid or alkali. It was assumed that the sets of plants were not affected materially by the differences in reactions within this period of time. In figure 5, the amounts of N/5 acid or alkali neutralized are plotted against the desired P H as before. Unfortunately the number of determinations made are insufficient to permit of the smoothing out of the curves. Their general shape, however, is obvious. On the alkaline side of P H 6.8, the ability to neutralize excessive concentra- tions of hydroxyl ions increases very rapidly with the increase in P H and probably does not reach a maximum even at P H 8.0. On the acid side, however, the increase is less rapid and reaches a maximum between P H 4.0 and 5.0. Influence of Factors other than the Reaction The plants were grown in the open during the summer months and in a heated greenhouse during winter. In the course of the investigation, it became evident that plants grown at different seasons show slight differences in their resistance to the effect of the reaction. This is most probably due to the differences in the rate of growth under different atmospheric conditions. The influence of the composition of the culture solution on the effect of the reaction was not determined, as only one solution was used throughout the investigation. It is highly improbable, however, that the composition of the solution, within wide limits, is a factor in any of the divers phases of this study. The results obtained by Salter and Mellvaine 27 and those obtained by the writer seem to substantiate this assumption. The amounts of water transpired by plants from solutions of different reactions were found to be the same within the limits of the experimental error. 426 University of California Publications in Agricultural Sciences [Vol. 4 Discussion The conclusion reached by earlier workers 2 ' 4 - 22 was that the H ion was more toxic than the OH ion to plants growing in solution cultures. Their results, however, are untenable because they failed to distinguish between potential and actual acidity or alkalinity. The ability of the plant to change the reaction of the nutrient medium was likewise overlooked. In a series of papers, Hoagland 15, 16, 17> 1S has called attention to both these factors and showed that the OH ion is much more toxic to barley seedlings in solution culture than the H ion. An OH ion concentration greater than P H 8.2 was distinctly injurious, whereas an H ion concentration of P n 5.0 was found to be favorable to growth and to cause no injury. Similar results were obtained by Duggar 5 using various types of solutions and growing the plants under the most diverse environmental conditions. One of the most complete and satisfactory studies on this problem is that of Salter and Mcllvaine. 27 These investigators experimented with corn, wheat, soybeans, and alfalfa, growing the plants at seven different II ion concentrations. The plants were grown for relatively short periods of time and the solution changed once every four days. A distinct maxi- mum in the growth of the plants was found at P H 5-P H 6. At a neutral reaction, decided decreases in the yields could be demonstrated. We have already called attention to the advisability of growin-a- the plants for a considerable length of time so as to overcome the influence of the food supply stored in the seed. Only in this way is it possible to obtain a true measure of the effect of the reaction of the solution. The growth periods employed by these investigators were undoubtedly too short. On the other hand, the variations in reaction caused by young plants are relatively small, so that plants grown in accordance with the technique they employed will give more reliable results if the experiment is discontinued after two weeks than if the plants are grown for a longer period of time. Our results agree substantially with those of these investigators. Hixon 14 found a distinct minimum in the development of young plants as measured by the growth in length of the roots and tops at P H 5 for Pisum and P H 6 for most other plants. This minimum point is interpreted as that of greatest efficiency and the point of normal growth. We have been able to confirm his results in part. A decided stimulation occurred at acidities which injured the plants definitely 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 427 later on. No stimulation was noticed in the tops of plants grown in alkaline solutions. The roots were occasionally longer than those of the plants grown at P H 5 or P H 6. A glance at figures 4 and 5 is .sufficient to make evident the import- ance of controlling the reaction of the solution under investigation. Fig. 8 428 University of California Publications in Agricultural Sciences [Vol. 4 Changes of solution every fourth or fifth day are obviously insufficient to maintain the P H constant even approximately, when the plants are three to four weeks advanced. With small volumes of solutions supporting relatively large numbers of plants grown at P H 5, this becomes increasingly difficult. In an investigation by McCall and Haag, 23 this point seems to be lost sight of completely. From their investigations, it appears that wheat plants grow best at reactions between P H 3 and P H 4. It is very plain, however, that the reactions of the solutions in the neighborhood of the roots must have been very different from what they were assumed to be. It is not strange that the solution with the highest buffer effect gave the poorest growth. In culture solutions, the diffusion of solutes is relatively rapid and as a rule the reaction around the roots is the same as that in the bulk of the solution. If, however, the free diffusion is interfered with, such as often happens among the roots in the upper few inches of the solution, the reaction may be very different in this region from what it is in the bulk of the solution. Over the ranges of low buffer effect, a difference of 0.5 P H can occasionally be demonstrated under such conditions. In soils, the diffusion is infinitely slower and the reaction of the solution in contact with the absorbing roots will be determined solely by the ability of the plant to overcome the buffer effect of the soil complex in its immediate vicinity. Considering the power of growing plants to regulate the Ph value of the culture medium, the conclusion is inevitable that the direct effect of the actual reaction of most soils can hardly be a factor in the complex which determines the growth of the plant in that soil, provided the plant has the. ability to establish itself firmly. In this connection the work of Joffe 10 with alfalfa is very elucidating. The results obtained with solution cultures agree well with those of this investigator using soils acidified artificially. From the determination of the reactions of numerous acid soils reported by Gillespie," and Sharp and Hoagland, 28 it is apparent that the reaction of the majority of these soils can have little or no direct effect on the growth of plants. The infertility of acid soils can usually be ascribed to causes other than the H ion concentra- tion. The solubility of aluminum in the slightly acid soil solutions of these soils is undoubtedly responsible for some of the phenomena attributed formerly to the acidity of the soil. 1 ' 12, "■ 25, 29 The soil solution of many alkali soils has a highly alkaline reaction, which tends to prevent the young plants from germinating or develop- 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 429 ing. Germinating seeds have a remarkable ability to change the reaction of the alkaline medium in which they are immersed, in the direction of neutrality, so that the P H value around the seeds may be made favorable to germination. The ability of the seedling to Fig. 9 regulate the reaction is comparatively small and hence the young roots may be unable to penetrate beyond the regions of the favorable reaction brought about by the seed. If the soil solution has both a high P H value and a high concentration of salts, the seedlings Avill natnrallv be unable to survive. 430 University of California Publications in Agricultural Scioices [Vol.4 Effect of Reaction of Culture Solution on the Reaction and Buffer Effect of the Plant Juices The plants from the experiments described above were frozen immediately after they were harvested. This was done in a cold room kept at 12° F., from which they were only removed as they were needed. The plant juices were obtained by grinding the frozen mass, thawing this rapidly in a warm room, and then expressing the sap by hand through a few thicknesses of cheesecloth. All determinations were made as soon as possible after the frozen ground material was thawed out. THE H-ION CONCENTRATION OF THE SAP The reaction of the juices of the roots and tops, obtained in the above way, was measured by means of a Hildebrand hydrogen elec- trode. Difficulty was experienced in making the measurements as reduction of N0 3 ions apparently took place on the electrode. This was especially true in the case of the juices from those plants grown at the acid reactions. This difficulty was obviated to some extent by leaving the NaNO : . out of the culture solution during the last week of the experiments. In figures 6 and 7, the reactions of the tops and roots of cucumber, barley, pea, and corn plants grown at different reactions are repre- sented graphically. The reactions of the sap expressed from the tops were not influenced by the reaction of the culture solution, the varia- tions in reaction being within the limits of the experimental error. On the other hand, the reactions of the root juices are decided ly changed by the reaction of the solution.* It is plain, however, that the reactions of the roots are very different from the reactions of the solutions, except when these are between P H 6 and P H 7. Whether the reaction of the root juices is influenced according to any definite rule by the reaction of the solution, as may be suggested by the curve for pea roots, it is impossible to say at present, owing to the relatively large experimental error involved in these measurements. Truog and Meacham, 31 after studying the effect of additions of lime to a soil, concluded that the reaction of the soil can influence the reaction of the sap expressed from the tops of the plants. It seems obvious, however, that the differences in the reactions from the limed and unlimed plots * Compare Bryan, O. O, Effect of different reactions on the growth and nodule formation of Soy beans. Soil Science, vol. 12, no. 4, pp. 271-2S7 (1922). 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 431 are within the experimental error, apart from the fact that many other factors enter in the case of plants growing in limed and un limed acid soils. Fig. 10 432 rniver.siti/ of California Publications in Agricultural Sciences [Vol.4 THE BUFFER EFFECT OF THE SAP The juice obtained from the plants in the way described was titrated electrometrically (after an equal volume of water had been added) with N/20 acid and alkali. The P H value was invariably increased by about one-tenth of a magnitude of the dilution. Fig. 11 In figures 8 and 9 the lit ration curves for barley tops and roots, respectively, are given. The curves are represented as if 25 c.c. of undiluted sap had been titrated with N/10 reagents. The correspond- ing curves for peas are given iii figures 10 and 11. 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 433 Hempel 13 has shown that the buffer effect of plant juices is mainly due to the organic acids and salts of these acids contained in the plant system. It appears from figure 8 that the reaction of the nutrient solu- tion has influenced the concentration of those acids with dissociation constants less than 10" very markedly in the tops of barley plants although the reaction of the expressed sap is apparently unchanged. Fig. 12 In the roots the buffer effect is also influenced. Here, however, only those acids with a dissociation constant higher than 10~ 6 are affected. In the case of the pea plants, neither the reaction nor the buffer effect of the sap expressed from the tops was influenced by the reaction of the nutrient medium. The roots on the other hand were affected similarly to the barley roots. These plants were grown in a green- house during the winter. In figure 12, the results of a similar experiment with peas grown in the open in summer are given. In this experiment, the reaction of 434 University of California Publications in Agricultural Sciences [Vol. 4 the sap expressed from the tops was unchanged, but the buffer effect was influenced by the reaction of the culture solution. The effect, however, is the reverse of what it was in the case of the tops of the barley plants, and in both instances was only noticeable in the con- centration of those acids with a dissociation constant lower than 10~ 6 . Unfortunately, it was not possible to pursue this line of investiga- tion with additional plants and under the different atmospheric con- ditions. It seems, however, that a thorough study along these lines will throw considerable light on the salt metabolism of plants. Summary 1. The influence of the reaction of the culture medium on the growth and metabolism of the common agricultural plants was stiidied by growing typical plants in solution cultures at different reactions. 2. After experimenting with several different methods, a technique was devised by which the reaction of the solution could be conveniently controlled. Particular attention was given to the constant mainten- ance of the desired hydrogen-ion concentration during the experi- mental periods. 3. Plants grown in solution cultures have an optimum growth reaction at P H 4.5 to P H 6. 4. The reaction of the juice expressed from the tops of the plants was not influenced by the reaction of the culture medium, whereas the reaction of the juices expresesd from the roots was modified considerably. 5. The buffer effect of both the roots and the tops may be influ- enced by the reaction of the culture solution. In the tops, the acid reserve is affected and in the roots, the alkali reserve. 6. Observations were made on the ability of the growing plant to chanffe the reaction of either acid or alkaline culture solutions. 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 436 LITERATURE CITED i Abbott, J. B. Connor, S. D., and Smallet, H. E. 1913. Soil acidity, nitrification and the toxicity of soluble salts of aluminum. Indiana Agr. Exp. Sta. Bull. 170, pp. 329-374. - Breazeale, J. P., and LeClerc, J. A. 1912. The growth of wheat seedlings as affected by acid or alkaline con- ditions. U. S. Dept. Agr., Bur. Chem., Bull. 149. s Clark. W. M., and Lubs, H.A. 1917. The colorimetric determination of hydrogen ion concentration and its application in bacteriology. Jour. Bact., vol. 2, pp. 1-34, 109-136, 191-236. •» Dachxowski, Alfred 1914. The effects of acid and alkaline solutions upon the water relations and the metabolism of plants. Amer. Jour. Bot. vol. 1, no. 8, pp. 412-440. , = Duggar, B. M. 1920. H-ion concentration and the composition of nutrient solution in relation to the growth of seed plants. Ann. Missouri Bot. Garden, vol. 7, no. 1, pp. 1-49. e Fred, E. B., and Davenport, Audry 1918. The influence of reaction on nitrogen-assimilating bacteria. Jour. Agr. Res., vol. 14, no. 8, pp. 317-336. 7 Fred, E. B., and Loomis, N. E. 1917. Influence of hydrogen ion concentration of medium on the reproduc- tion of alfalfa bacteria. Jour. Bact., vol. 2, no. 6, pp. 629-633. s Gile, P. L., and Carrero, J. O. 1916. Assimilation of iron by rice from certain nutrient solutions. Jour. Agr. Res., vol. 7, no. 12, pp. 503-528. 9 Gillespie, L. J. 1916. The reaction of the soil and measurements of hydrogen ion concen- tration. Jour. Wash. Acad. Sci., vol. 6, no. 1, pp. 7-16. io Gillespie, L. J., and Hurst, L. A. 1918. Hydrogen-ion concentration — soil type — common potato scab. Soil Sci., vol. 6. pp. 219-236. " Haas. A. R. C 1920. Studies on the reaction of plant juices. Soil Sci., vol. 9, no. 5, pp. 341-368. i- Hartwell. B. L., and Pember, F. R. 1918. The presence of aluminum as a reason for the difference in the effect of so-called acid soil on barley and rye. Soil Sci., vol. 6, no. 4, pp. 259-277. i s Hempel, Jenny 1917. Buffer processes in the metabolism of succulent plants. Compt. Rend. Lab. Carlsberg, t. 13, no. 1, p. 130. uHixon, R. X. 1920. The effect of the reaction of the nutrient solution on germination and the first stages of plant growth. Med. K. Veten. Nobel Inst., Band 4, no. 9, pp. 1-28. is Hoagland, T). R. 1917. The effect of the hydrogen and hydroxyl ion concentration on the growth of barley seedlings. Soil Sci., vol. 3, no. 6, pp. 547-560. 436 University of California Publications in Agricultural Sciences [Vol. 4 is Hoagland, D. B. 1918. The relation of the plant to the reaction of the nutrient solution. Science, n. s., vol. 48, no. 1243, pp. 422-425. 1 " HOAGLAND, D. B. 1919. Relation of the nutrient solution to the composition and reaction of cell sap of barley. Bot. Gaz., vol. 68, no. 4, pp. 297-304. is Hoaglaxd, D. R. 1919. Belation of the concentration and reaction of the nutrient medium to the growth and absorption of the plant. Jour. Agr. Bes., vol. 18, no. 2, pp. 73-117. m JOPFE, J. S. 192(i. The influence of the soil reaction on the growth of alfalfa. Soil Sei., vol. 10, no. 4, pp. 301-307. 2° Jones, L. H., and Shive, J. W. 1921. Effect of ammonium sulphate upon plants in nutrient solutions sup- plied with ferric phosphate and ferrous sulphate as sources of iron. Jour. Agr. Bes., vol. 21, no. 10, pp. 701-728. -i Kappex, H. 1920. Ueber die aziditatsformen des Bodens und ihre pflanzenphysiologische Bedeutung. Landw. Vers. Stat., vol. 96, pp. 277-307. -- Loew, r. A. 1903. The toxic effect of H and OH ions on seedlings of Indian corn. Science, n. s., vol. 18, no. 453, pp. 304-308. 23 McCall, A. G. and Haag, J. B. 1921. The relation of the hydrogen ion concentration of nutrient solu- tions to growth and chlorosis of wheat plants. Soil Sei., vol. 12, no. 1, pp. 69-77. 24 Mirasol, J. J. 1920. Aluminum as a factor in soil acidity. Soil Sei., vol. 10, no. 3, pp. 153-218. 25 MlYAKE, K. 1916. The toxie action of the soluble aluminum salts upon the growth of the rice plant. Jour. Biol. Chem., vol. 25, no. 1, pp. 23-28. 2« Paxtanelli, E. 1915. Ueber Ionen Aufnahme. Jahrb. Wiss. Bot. (Pringsheini), Band 5C, pp. 689-733. 2" Salter, B. M., and McIlvaixe, T. C. 1920. Effect of reaction of solution on germination of seeds ami growth of seedlings. Jour. Agr. lies., vol. 19, no. 2, pp. 73-95. 2s Sharp, L. T. and Hoagland, D. B. . Acidity and absorption in soils as measured by the hydrogen elec- trode. Jour. Agr. Bes., vol. 7, no. 3, pp. 123-145. 2n Stoklasa, J. 1918. Ueber den Einfluss des Aluniinnniions ant' die Keimung des Samens und ilie Entwickelung der rflanzen. Biochem. Ztschl - ., vol. B9, pp. 137-223. SO Texjog, E. 1918. Soil Acidity: I. Its relation to the growth of plants. Soil Sei., vol. 5, no. 3, pp. 169-195. ■" TRUOG, E., and MEACHAM, M. B. 1919. Soil acidity: II. Its relation to the acidity of the plant juice. Soil Sei., vol. 7, no. 6, pp. 469-474. 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 43/ II. POSSIBLE MECHANISM OF THE PLANT'S INFLUENCE ON THE REACTION OF THE CULTURE SOLUTION Recent studies on the absorption of inorganic ions by plants as well as the large amount of work done on the problem of the antagon- ism between ions and the physiological balance in culture solutions have thrown some light on the mechanism by which the plant obtains its inorganic elements. The importance of a more thorough knowl- edge of this process is undisputed. Unfortunately investigations on this problem are hampered by our meager knowledge of the true nature of solutions and the methods of analysis at our disposal. In considering the absorption of any ion, account must be taken of the activities of that ion inside and outside of the membrane effective in absorption. We are at present unable to determine the activity of any ion in a system as complex as a complete culture solu- tion except that of the H ion, which can usually be determined with sufficient accuracy. Since the activity and the total molal concentration of the H ion are conveniently and rapidly determined and since we have every reason to believe that the H and OH ions are absorbed, fundamentally, in the same way as any other positive or negative ion, we have here a very efficient means of studying this problem. In Part I of this investigation, a series of experiments were described which were concerned mainly with the effect of the reaction of a culture solution on the growth and metabolism of several types of plants. In the present paper, some preliminary experiments are described which are concerned with the effect of the growing plant on the composition and especially on the reaction of culture solutions. Pantanelli 8 observed that plants always changed the reaction of a single salt solution in the direction of neutrality except when (NH 4 ) 2 S0 4 was the solute. In a solution of this salt, the reaction remained at the initial value, namely P H 5. Similar results were obtained by Hoagland 4 with barley plants. Later work shows that solutions of (NH 4 ) CI, K.,S0 4 and some other salts behave similarly to (NH 4 ) 2 S0 4 . The reaction may even change appreciably toward a higher acidity especially when the plants have not been previously grown in a complete culture solution. When complete culture solu- tions were used, the reaction was invariably changed toward neutral- 438 University of California Publications in Agricultural Sciences [Vol.4 ity. These results with complete culture solutions were confirmed by Duggar 2 and several later workers for different types of plants and solutions. Jones and Shive 5 found that the reactions of 20 repre- sentative solutions of the Tottingham series were changed toward neutrality. When, however, (NH 4 ) 2 S0 4 was substituted for KNO. in these solutions, the reactions remained practically constant at the original value, namely P H 4.8. In the present investigation, all the plants experimented with invariably changed the reaction of the complete culture solution toward some point between P H 6.5 and P H 6.9, irrespective of what the original concentration might have been (see Part I). The exact mechanism by which the plant changes the reaction of a solution has not been established. In a uni-salt solution, this may he ascribed to ionic exchanges, and on the alkaline side, the OH ions are partly neutralized by carbon dioxide excreted from the roots. In a complete culture solution, the problem becomes more complex. It will thus be of advantage to tabulate the different methods which a growing plant conceivably might have at its disposal for changing the reaction of the solution. The decrease of H ion concentration would be accomplished: 1. By neutralizing II ions by OH ions derived from some base excreted by the roots or from dead root cells. 2. By absorbing H ions and simultaneously replacing these by some other positive ion. 3. By absorbing an anion and excreting OH ions simultaneously. 4. By absorbing H ions and an equivalent amount of some negative ion. 5. By absorbing an anion and excreting simultaneously another anion which forms an acid with a lower degree of dissociation or an acid which is volatile under the conditions. The H ion concentration is increased: (a) If OH ions are neutralized by II ions derived from some acid excreted by the plant or from dead root cells. (6) If OH ions are absorbed but simultaneously replaced by some other anion. (c) If OH ions and an equivalent amount of a cation are absorbed simultaneously. (<7) If a positive ion is absorbed and replaced by H ions. Excretions by the roots are confined to the acid HOO.r (or C0 2 ) and, under certain conditions, small amount of cations, notably 1924] Theron: Inter-relations Between the Plant and Its Culture Medium 43(» calcium. The quantities of the latter are, however, insufficient to account for more than a very small part of the power of the plant to change the reaction of the solution. The increase in the P H value must thus be accounted for by methods 3, 4, or 5. The decrease in the P n value of a culture solution might take place by any or all of the methods outlined under a, b, c, and d. In some cases the P H value of the solution is decreased to aboiit P H 3.2, as frequently happens in a uni-salt solution of K 2 S0 4 , for example. Since the concentration of the OH ions is very small at this reaction, it is possible that method <1 is chiefly involved. Method c, however, can- not be excluded from consideration. The plant has a very efficient means at its disposal for reducing the alkalinity of a solution in that it normally excretes relatively large amounts of C0 2 (method a). This, however, is not the only mechanism involved as is apparent from the results of the following experiment. Corn plants growing in a complete culture solution maintained at P H 8.5, as was described in Part I, neutralized within one week 0.0257 equivalents of alkali. When the solution was analyzed only 0.0185 equivalents of C0 2 were found. Hence approximately one-fifth of the alkali added must have been neutralized by methods c and