C E 3RARV RePrmted from THE BOTANICAL GAZETTE, 42: 127-134, August, 1906 W/f/Uj 3A *&* *>"* (I IAGRICI ON THE IMPORTANCE OF PHYSIOLOGICALLY BALANCED SOLUTIONS FOR PLANTS.1 ^. I. MARINE PLANTS. W. J. V. OSTERHOUT. CALr RINGER demonstrated that animal tissues live longer in a solut of NaCl to which a small amount of KC1 and CaCl2 is added than in a solution of NaCl alone. Various explanations of this fact were given by different investigators, all of whom, however, agreed upon the essential point that KC1 and CaCl2 are essential for the mainte- nance of life. HOWELL assumed that CaCl2 is the stimulus for the heart beat, while NaCl is an indifferent substance, necessary only for the mainte- nance of osmotic pressure. Similarly RINGER concluded that Ca is the stimulus for the systole, while K is necessary for the diastole of the heart beat. HERBST made experiments on the influence of the composition of the sea water on sea urchin eggs, eliminating in each successive experiment a different constituent of the sea water. He found that the eggs would not develop in any solution which did not contain all the salts of the sea water. From this he concluded that each of the salts found in sea water is necessary for the development of the egg. LOEB called this view in question as the result of his experiments on Fundulus. He found that this marine fish cannot live in a pure NaCl solution of the same osmotic pressure as the sea water, but that it can live indefinitely in a mixture of NaCl, KC1, and CaCl2, in the same proportions in which these salts are contained in sea water. The fish can also live indefinitely in distilled water. This proves that it does not need any of the three salts mentioned for the mainte- nance of its life, and that the Ca and K are only required to overcome the poisonous effects which would be produced by the NaCl if it alone were present in the solution (at the above mentioned concen- tration). 1 I wish here to express my sincere thanks to Professor LOEB, who kindly placed the facilities of his laboratory at my disposal and assisted me in every way during these investigations. 127] [Botanical Gazette, vol. 42 128 BOTANICAL GAZETTE [AUGUST It is noteworthy that the Ca and K, which are added to inhibit the toxic effect of NaCl, are themselves poisonous at the concentra- tion at which they are here employed. These antagonistic effects of Ca and K toward a pure NaCl solu- tion were illustrated still more strikingly in experiments on the egg of Fundulus. The newly fertilized eggs of this fish develop equally well in sea water and in distilled water, but die in a pure m/2 NaCl solution without forming an embryo. If, however, a small but defi- nite amount of a salt with a bivalent kation, even of such poisonous salts as BaCl2, ZnSO4, and Pb(CH3-COO)2, is added, the eggs will produce embryos. From these and similar observations LOEB was led to formulate his conception of the necessity of physiologically balanced salt solutions, in which are inhibited or counteracted the toxic effects which each constituent would have if it alone were present in the solution. The blood, the sea water, and to a large extent RINGER'S solution, are such physiologically balanced salt solutions. The observations of HERBST, as well as those of RINGER, are easily explained on this basis. The fact that the elimination of any one constituent from the sea water makes the solution unfit to sustain life does not prove that the eliminated substance is needed by the animal for any purpose other than to counteract the poisonous action of some other constit- uent of the solution. Botanists have not thus far made use of these conclusions, for the obvious reason that facts similar to those mentioned above have not been observed in plants. I have recently made a number of experi- ments which show that there exist in plants phenomena similar to those observed by LOEB on Fundulus and other marine animals. The species of marine plants chosen for investigation may be divided into two groups: Group i comprises plants which can live a long time in distilled water. It includes the following: BLUE-GREEN ALGAE, Lyngbya aestuarii; GREEN ALGAE, Enteromorpha Hopkirkii; FLOWERING PLANTS, Ruppia maritima. Group 2 is composed of plants which quickly die in distilled water. It includes the following: GREEN ALGAE, Enteromorpha intestinalis; BROWN ALGAE, Ectocarpus confervoides ; RED ALGAE, I9o6] 6 STERH OUT— BALANCED SOLUTIONS 129 Ptilota filicina, Pterosiphonia bipinnata, Iridaea laminarioides, Sarcophyllis pygmaea, Nitophyllum multilobum, Porphyra naiadum, Porphyra perforata, Gelidium sp., Gymnogongrus linearis, Gigartina mammillosa.2 If plants of either group be placed in a solution of pure sodium chlorid (isotonic with sea water), they die in a short timer- -This might be attributed to the lack of certain salts which are necessary for their metabolism, rather than to the toxicity of the sodium chlorid. In the case of the plants of Group i there can be no doubt on this point, for these plants live a long time in distilled water. If we add pure sodium chlorid to the distilled water it kills them in a very short time. An inspection of the tables will show that these plants in their behavior toward sodium chlorid and other salts, closely agree with those of Group 2, which can live but a short time in dis- tilled water. Sodium chlorid is certainly toxic to the first group, and there can be little doubt that it is so to the second group as well. The plants of the first group were found in a ditch in a salt marsh through which the tide ebbs and flows; there is always a foot or so of water even at low tide. The salt content of the water fluctuates around a mean of approximately 2 . 3 per cent. The plants of the second group were collected at the entrance to San Francisco Bay, where the salt content of the water fluctuates about a mean which is probably not far from 2.7 per cent. The only exceptions are Enteromorpha intestinalis and Ectocarpus con- jervoides, which came from wharves in the bay, where the mean salt content is about 2.3 per cent. All the plants used in the experiments were transferred from the sea water directly to distilled water. After rinsing in this they were placed in glass dishes, each containing 2oocc of the solution to be tested. The dishes were then covered with glass plates to exclude dust and check evaporation. Only a small amount of material was placed in each dish. The temperature during the experiments did not vary far from 18° C. Artificial sea water was prepared3 according to VAN 'T HOFF'S 2 The determinations were kindly made by Professor SETCHELL. 3 The water used was distilled in glass only and the first part of the distillate rejected. The purity of each salt was carefully tested before using. 130 BOTANICAL GAZETTE [AUGUST formula4 as follows: iooocc NaCl, 3^/8; y8cc MgCl2, 3^/8; 38CC MgSO4, 3^/8; 22CC KC1, 3^/8; iocc CaCla, 3w/8.s This closely approximates the bay water. The plants thrive almost as well in it as in sea water, especially when a very little NaHCO3 or KHCO3 is added to produce a neutral or faintly alka- line reaction. A series of solutions was tried, beginning with pure NaCl 3^/8 and adding to it in turn MgCl2, KC1, and CaCl2, either singly or in combination, in the proportions given above. These salts were also used in pure solutions of the same concentration at which they exist in the artificial sea water described above. It should be said that little difficulty was experienced in deter- mining the death point with sufficient precision. The color reactions and the microscopic appearance of the cells allowed this to be done with sufficient accuracy, so that the results were not in doubt on this account. The results of the experiments are set forth in the tables. The figures represent the average of four parallel series carried on simul- taneously. A control series was also carried on in which each solu- tion was made faintly alkaline by the addition of NaHCO3, KHCO3, or Ca(OH)2. This had a beneficial effect during the first two or three days of the experiment, but the final results were practically the same as in the other series. From a consideration of the results for Group i we may draw the following conclusions. i. The plants die much sooner in a pure sodium chlorid solution (isotonic with sea water) than in distilled water. The poisonous effect of the NaCl largely disappears if we add a little CaCl2 (iocc CaCl2 3^/8 to iooocc NaCl 3^/8) ; in this mixture the plants live nearly as long is in distilled water. Addition of KC1 to this mix- ture enables them to live longer than in distilled water. Further addition of MgCl2 and MgSO4 enables them to live practically as long as in sea water. 4 VAN'T HOFF, J. H., Physical chemistry in the service of the sciences 101. Univ. of Chicago Press, 1903. s This corresponds approximately to the proportion of Ca in the sea water of the bay. I9o6] OSTERHOUT— BALANCED SOLUTIONS TABLE I. DURATION OF LIFE IN DAYS. GROUP i GROUP 2 CULTURE SOLUTION. Lyngbya aestuarii E n t e r o- morpha Hopkirkii Ruppia maritima Ptilota filicina Pterosi- phonia — bipinnata Tridaea Lominar- ioidcs Sea water (total salts 2.7%) 95 150 + 150 + II 24^ 24 Artificial sea water: 1000 c NaCl sm/S 78 ' MgCl2 38 ' MgS04 " 90 150 + 150 + io| 24^ 23 22 ' KC1 10 ' CaCl2 Distilled water 30 80 ! , 2* Tap water 32 + 36 8s 2| oi - 2 IO NaCl 3^/8 22 O 15 0 23 Ij y 2 4 1000 cc NaCl " ) 10 " CaCl2 " J 29 23 65 4 6 5 1000 " NaCl " ) 22 " KC1 " £ 35 32 88 3^ 10 9 10 " CaCl2 " ) 1000 " NaCl " ) 78 " MgCl2 " [ 29 23 45 3 6 6 10 " CaCl2 ) 1000 " NaCl " ) 78 " MgCl2 " ( 25 13* 30 2 4 4 22 " KC1 " ) 1000 " NaCl " ) 22 " KC1 " ] 23 13* 23 I 2 5 1000 " NaCl " ) 78 " MgCl2 " \ 22| !3i 25 I* 2 2 1000 " Dist. H2O ) 78 " MgCl2 « J * 16} •9 I 2 2* 1000 " Dist. H2O I 38 " MgS04 « \ »7i 13 23 I 2 2 1000 " Dist. H2O ) 22 " KC1 " J 21 •31 56 I .1 s* 1000 " Dist. H2O ) 10 " CaCl2 " \ 26 + .4 58 a* 5 2 132 BOTANICAL GAZETTE [AUGUST TABLE II. DURATION OF LIFE IN DAYS. GROUP 2. rt &^ «J a rt el B 5 1 1 CULTURE SOLUTION. Enteromo intestin &fc 22 KCI r 0.50 o o 1000 NaCl ) O. C2 O . I 78 MgCla J MgCla 0.50 o o KCI 0.50 o o CaCla 4ee 810 • j j ' EQUISETUM The spores of Equisetum retain their vitality for only a few days. The fruiting cones were brought into the laboratory and allowed to stand for a day or two. The freshly shed spores were then placed on the surfaces of solutions in covered glass dishes. They germinate rapidly and in a few days produce prothallia of fair size. The aver- age results of four series of experiments are shown in Table VI and fig-4- FLOWERING PLANTS The most extensive series of experiments was made with wheat. The variety selected is known as Early Genesee. The percentage 1907] OSTERHOUT— BALANCED SOLUTIONS 265 of germination is very high and the growth is vigorous from the start. The plan first tried was that of carefully placing the seeds on the surface of the solutions so that they float. This worked well with O Q 0 FIG. 3. — Growth of gem- mae of Lunularia in various 3w/8o solutions during 150 days. The quantities are stated in cubic centimeters, and the gain in length of thallus in per cent. I, CaCl2, gain 810. 2, NaCl 1000 + MgCl2 78 + CaCla 10, gain 1000. 3, dilute arti- ficial sea water, NaCl = ap. 3w/8o, gain 1204. 4, NaCl 1000 + KC1 22, gain o. 5, NaCl 1000 + MgCl2 78, gain 4. 6, NaCl 1000 + MgCl2 78 + KC1 22, gain 8. 266 BOTANICAL GAZETTE [OCTOBER wheat and other small seeds during the first stages of germination; but if the experiments are to be carried beyond this stage, the seed- lings must be supported so that the leaves do not come into contact with the solution. After some trials the following device was hit upon which answers the purpose admirably. A strip of filter paper is folded lengthwise and one of the folds turned back as shown in fig. 5. The seeds are placed in the trough thus formed and the whole strip is then bent into a circle and placed in a tumbler previ- ously filled with solution. The strip should be of such length that when placed in the top of the tumbler the ends just meet and so form a stiff collar which just fits inside the top of the tumblers and which will FIG. 4. — Development of spores of Equisetum in various 3^/160 solutions during 50 days. Quanti- ties are stated in cubic centi- meters; the gain in length of thallus exclusive of rhizoids is stated in per cent. I, distilled water, gain 1760. 2, dilute arti- ficial sea water, NaCl = ap. 3w/i6o, gain 1500. 3, NaCl 1000 + KC1 22 -f Cada 10, gain 1500. 4, NaCl iooo+CaCl2 10, gain 980. 5, CaCl2, gain 700. 6, NaCl, gain o. not slip down. A large number of these collars may be prepared, filled with seeds, bent into circles, and secured by ordinary paper-clips placed on the overlapping ends. They may be piled in trays until wanted. It is then only necessary to remove the clips and set the collars in glasses previously filled with solutions. In some cases, especially where larger glasses are employed, a strip of paper of double thickness may be used ; this makes a stiff er 1907] OSTERHOUT— BALANCED SOLUTIONS 267 collar. It is then advisable to perforate the bottom of the seed trough by means of a tracing wheel such as is used for patterns. This allows the roots to penetrate the paper freely and without delay. Care should be taken that the solution does not cover the seeds. The paper must be spread open at the top so as to allow the air to come into direct contact with the seeds. The micropyle should be in contact with the moist filter paper. Careful experiments were made to determine whether the filter paper exerted any influence on the solution (by absorption, etc., or by concentration of the solution about the see'd as the result of evaporation) which might affect the results, but no such influence TABLE VI. EQUISETUM All quantities given are cubic centimeters of 3^/160 solutions. GROWTH IN 50 DAYS j CULTURE SOLUTION length of thallus in mm. Per cent, increase in length of thallus, exclusive of rhizoids Aggregate length of rhizoids per thallus in mm. Dilute artificial sea water: 1000 NaCl 78 MgCl2 38 MgSO4 0.8o I^OO 8.1 22 KC1 o 10 CaCl2 Distilled water o . 93 1760 9.0 NaCl O.OtC A 1 \J\4 o y o 1000 NaCl o o. 54 980 "v^ 4.7 10 CaCl2 *T * / 1000 NaCl ) 22 KC1 [• 10 CaCl2 ) 0.80 1500 5-2 1000 NaCl ) 78 MgCl2 [ o-93 1760 9.0 10 CaCl2 ) 1600 NaCl ) 78 MgCl r 0.07 40 O 22 KC1 2 ) 1000 NaCl I o o 22 KC1 J • ' 1000 NaCl I 0.07 40 o 78 MgCl2 ) • / MeCU o.oc o o KC1 . ... 0 o o CaCl2 0.40 700 3 • 2 / 268 BOTANICAL GAZETTE [OCTOBER could be detected. The solutions were renewed from time to time and the concentration ascertained by occasional titration. It should be said that in general the growth of roots (or any parts in direct contact with the solution) furnishes a much better criterion of the effect of solutions than the aerial portions of the plant. In certain solutions which are so poisonous that the roots cannot develop, the leaves may grow fairly well for a time. In these cases the poisonous solutes are apparently filtered out by the tissues of the seed as the solution passes through them on its way to the leaf. For this reason the figures for the growth of roots only are here given. The results are shown in Table VII and figs. 6 and 7, which give the average of five series of ex- periments. Each number represents average measurements of at least four or five hundred seeds. This is necessary in order to do away with the individual variation so common in seeds. FIG. 5. — Sectional view of wall of tumb- ler and seed sup- ported by folded filter paper; p, paper; s, seed; t, tumbler; •w, water line. TABLE VII. WHEAT All quantities given are cubic centimeters of 3^/25 solutions. CULTURE SOLUTION Dilute artificial sea water: 1000 NaCl 78 MgCl, 38 MgS04 22 KC1 io CaCl2 Distilled water. . . . NaCl... 1000 NaCl io CaCl2 1000 NaCl 22 KC1 io CaCl2 ' 1000 NaCl 78 MgCla io CaCl2 MgCl2.. KC1.... CmCl... GROWTH IN 40 DAYS Aggregate length of roots per plant in mm. 360 59 254 324 327 68 70 1907] OSTERHOUT— BALANCED SOLUTIONS 269 A similar though less extensive series of experiments was carried out with flax, alfalfa, red-beet, and radish seeds. Another series was made by placing pieces of the fresh- water aquatics, Zannichellia and Potamogeton, in solutions, or in the case of Lemna, by allowing the plants to float on the sur- face. The results in all these cases were similar to those given above. It is thought desirable to see how cuttings would behave under simi- lar treatment. Cut- tings (about nine inches in length) of Tradescantia and Tropaeolum were placed upright in bottles, the lower three inches of the plant being submerged TABLE VIII. CUTTINGS All quantities given are cubic centimeters of 3^/32 solutions. FIG. 6. — Growth of roots of wheat in various 3^/25 solutions. Quantities are stated in cubic centimeters, and the aggregate length of roots in millimeters. I, NaCl 1000 + KC1 22 + CaCl2 10, length 324. 2, NaCl 1000 + CaCl2 10, length 254. 3, CaCl2, length 70. 4, NaCl, length 59. GROWTH IN 10 DAYS UULTURE bOLUTION Tradescantia Tropaeolum Dilute artificial sea water: 1000 NaCl 78 MgCl2 ?8 MgSO4 . Long roots Long roots 22 KC1 10 CaCl2 Distilled water Very long roots Very long roots NaCl No roots No roots 1000 NaCl I Short roots Short roots ioCad2 ) 1000 NaCl ) 22 KC1 [ Medium length roots Medium length roots 10 CaCl2 ) 270 BOTANICAL GAZETTE [OCTOBER in the solution. Absorbent cotton was packed in the neck of the bottle to exclude dirt and hinder evaporation. The results were similar to those described above, as will be seen from Table VIII. Finally the question was raised whether the tissues of the stem and leaf, if brought into direct contact with the solution, would behave like the root. To answer this, sections of considerable (but uni- form) thickness were cut with a microtome and placed in the solutions. The results appear in Table IX. The results described in this paper are in all essentials in striking agreement with those obtained from the study of marine plants, as well as from the study of marine and fresh-water animals as referred to in the first part of this paper. This agreement shows that the principle of balanced solutions is of general validity.3 The application of this prin- ciple to soil and river water4 and to nutrient solutions, I hope to take up in a subsequent paper. 3 LOEW and his pupils have shown that calcium antagonizes magnesium (cf. Bull. No. 18, Div. Veg. Phys. and Path. U. S. Dept. Agric. 1899). See also the antago- nistic effects noted by KEARNEY and CAMERON (Report No. 71, U. S. Dept. Agric. 1902) in their studies on the salts of alkali soils. The method employed by them (observation of the root-tip only) is so different from mine that I have not attempted to compare the results. 4 In the first part of this paper I have referred to the composition of tap water, but it seems advisable to defer the discussion of this point. FIG. 7. — Growth of roots of wheat for 40 days. I, in dilute artificial sea water (NaCl = ap. 3^/25), aggregate length of roots 360""". 2, in distilled water, aggregate length of roots 74omm. 1907] OSTERHOUT— BALANCED SOLUTIONS 271 TABLE IX. SECTIONS All quantities given are cubic centimeters of 3^/32 solutions. DURATION OF LIFE IN DAYS CULTURE SOLUTION Red beet: Cross-sections of root Mesembry- anthemum : Cross-sections of leaf Tradescantia : Cross-sections of stem Tropaeolum: Cross-sections .aLleaf Dilute artificial sea water: 1000 NaCl . 78 MgCl3 38 MgSO,, •2C 16 10 2C O j-»j-£o\-^4 22 KC1 j o y •j 10 CaCl2 Distilled water •7C 18 2O •72 NaCl •JJ 18 12 O 14. 1000 NaCl 10 CaCla ! " 25 12 18 xiT 20 1000 NaCl I 22 KC1 10 je 10 22 10 CaCl2 , i o j y For the sake of clearness it seems desirable to call attention to the distinction between balanced solutions and ordinary nutrient solu- tions. A nutrient solution may be used in such dilute form that none of its components could exert any toxic action even if the other con- stituents were removed. In this case there are no poisonous effects to be inhibited and consequently no balancing of the solution is required. Our only concern is to supply all the substances needed for nutrition, irrespective of any balancing action, and so form a complete nutrient solution. If we increase the concentration of this solution, however, we soon reach the point where some or all of the components begin to exert their individual toxic effects, whereupon it may become necessary to inhibit these effects by proper adjustment of the relative propor- tions of the substances present or by the addition of other substances. The substances added to produce a balance do not necessarily have a nutritive value. For example, LOEBS was able to balance certain solutions by adding zinc, cobalt, aluminum, etc.6 s Am. Jour. Physiology 6:411-433. 1902. 6 To make clear this distinction between balanced and nutrient solutions is more necessary, since LOEW and Aso (Bull. Coll. Agr. Tokyo Imp. University 7:395. 1907) confuse the two kinds of solutions. Their criticisms are wholly based on this mis- conception and do not affect the matter as I have presented it. The distinction between nutrient and balanced solutions is due to LOEB, who has explained it clearly in his Dynamik der Lebenserscheinungen 115-120. 272 BOTANICAL GAZETTE [OCTOBER In general we may know when the solution is properly balanced by comparing its effects with those of pure1 distilled water. In a properly balanced solution we expect the organism to live approxi- mately as long as in distilled water, and while it will not grow so fast (on account of the osmotic pressure), the ultimate development reached should be comparable with that attained in distilled water.8 Why all these effects are so, we are not at present prepared to say in detail. LOEB has gone farther than any other in the explanation of these phenomena, referring them to the effects, of salts and ions on proteids9. According to his conception any metal must be poisonous when it alone is present in the solution, for it will enter the proteids and 'drive out other metals in accordance with the law of mass action. This will of course alter the properties of the proteids and so cause disturbances in function. The only way to prevent this is to main- tain a proper balance between the various metals in the solution. It may be pointed out that an analogy exists between the effects described here and various reactions in which proteids are in- volved. Antagonism between Na and Ca, for example, is seen in the clotting of blood, which is hindered by Na and favored by Ca. The thing of chief importance is the agreement in behavior of such a great diversity of plants with the fresh-water and marine animals already studied. Thereby is brought to light a new point of similarity between animals and plants which is fundamental in character and which must be taken into consideration in attempting to formulate a theory of living matter. UNIVERSITY OF CALIFORNIA 7 Water twice distilled from glass, the first third of the distillate being rejected is usually regarded as pure. But such water may be quite poisonous if any part of the apparatus, including stoppers, be new. The longer the apparatus is used the less poisonous the water becomes, until it finally ceases to be toxic. 8 Higher concentrations excepted. 9 See references in the first part of this paper, BOT. GAZ. 42: 134. 1906 14 DAY USE RETURN TO DESK FROM WHICH BORROWED ffS&y This book is due on the last date stamped below, or on the date to which renewed. Renewed books are subject to immediate recall. 1SS3 LD 21-50m-12,'61 (C4796slO)476 General Library University of California Berkeley