INTERACTIVE EFFECTS OF COUMARIN, DITHIOOXAMIDE, ASCORBIC ACID ANT) AUXINS UPON THE EARLY DEVELOPMENT OF ROOTS By Jacques Marie Alamercery A TIiESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1952 INTERACTIVE EFFECTS OF COUMARIN, DITHIOQXAMIDE, ASCORBIC ACID AND AUXINS UPON THE EARLY DEVELOPMENT OF ROOTS cques Marie Alamercery aN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1952 Approved Jacques M. Alamercery The interactive effects of dithiooxamide and of ascorbic acid upon the development of roots and its inhibition by coumarin and by the "auxins" 2,/+-D, indole-acetic acid and naphthalene-acetic acid have been investigated in Petri dishes under controlled conditions of light and of temperature, with cucumber for root-elongation and lettuce for germi­ nation percentage. Treatments of dithiooxamide which were stimulative in light were strongly inhibitive in darkness for the elongation of cucumber roots; no stimulation has been observed in darkness; the inhibitions induced by higher concentrations of dithiooxamide, whether in light or in darkness, were not considerable before the third day after that germination had started. Dithiooxamide was able to increase the early low rates of let­ tuce germination in light at 29°G«, or in darkness at 25°C.. Ascorbic acid alone did not produce any significant effects on the development of roots at. the concentrations which were used (below 2,000 ppm). Ascorbic acid was able to reinforce synergistically or to reduce the inhibitions of cucumber root elongation induced by dithiooxamide, depending upon the conditions of illumination, the procedure of testing (simultaneous or successive applications of the chemicals) and the order and the length of the treatments, in the case of successive applications of the chemicals. Both dithiooxamide and ascorbic acid were able to reduce the inhi­ bition of cucumber root elongation induced by coumarin, in light only in the case of dithiooxamide, in both light and darkness in the case of ascorbic acid. A similar reduction of the inhibition of lettuce germina­ tion induced by coumarin was produced by both chemicals, in light arid -2Jacques M. Alamercery darkness by dithiooxamide and in light only by ascorbic acid in the case of simultaneous applications with coumarin throughout the experiments; for both chemicals, it took place in darkness only, when they were sepa­ rately applied after a presoaking of the seeds in a solution of coumarin alone in darkness (case of an artificial dormancy)# Ascorbic acid wa.s more potent in the case of cucumber, dithiooxamide was more potent in the case of lettuce. Both dithiooxamide and ascorbic acid were able to reduce the inhi­ bitions of cucumber root elongation induced by the "auxins" 2,4-D, indoleacetic acid, and naphthalene-acetic acid, both in light and in darkness,, In every case, ascorbic acid was more potent than dithiooxamide. As a tentative interpretation of the interactive effects of dithiooxa­ mide, ascorbic acid, and coumarin, a natural biological system is outlined in which -SH groups and coumarin derivatives may be independently inter­ related with the metabolism of Vitamin C in plants, directly or indirectly through the metabolism of -SH enzymes. Two phases of this system appear likely to be interfered with by the "auxins". ACZB OWLEDGl-iEKTS The author wishes to express his indebtedness and sincere appreciation to Dr. C. L. Hamner for his guidance, encouragement and never failing help which made this investigation possible. Thanks are due to Dr. H. B. Tulcey for his advices and sug­ gestions which are reflected in the presentation of this manuscript. Appreciation is due to Dr. H. M. Sell, Dr. G. Steinhauer, and Dr. L. M. Turk for their advice. Thanks are due also to the Rockefeller Foundation whose finan­ cial help made this work possible. TABLE- OF CONTENTS Page INTRODUCTION....................................... ................. 1 MATERIAL AND METHODS........... A. B. C. Root Elongation Tests....................... Germination Tests....................................... ....... Environmental Conditions. 4 4 8 10 FART I : DITHIOOXAMIDE, GROWTH-REGULATING ACTIVITY, ANTAGONISM TO COUMARIN ........................... 13 I. Introduction................................................ .14 .15 A. Review of Literature.................. B. Problem. ...... .....20 II.Experimental Results................. 22 A. Preliminary Experiments with old Marketer Cucumber and Wheat seeds. ................. 22 3. Experiments with a new sample of Marketer Cucumber seeds................. .25 C. Experiments with Burpee Hybrid Cucumber seeds..... ....27 D. Experiments with lettuce seeds....,............ 31 III. Discussion............. ...37 A. The Conditions of the Experiments...... 37 3. Interpretation of the Results.,.,................ ......42 IV. Sunmary .................. PART II : ASCORBIC ACID, ANTAGONISM TO COUMARIN, INTERACTIONS .WITH DITHIOOXAMIDE........ I. Introduction. ..... A. Review of Literature............... B. Problem.................. 45 47 47 .47 57 II. Exoerimental Results........................ .59 A. Effects of Ascorbic Acid on Root Elongation...........,59 B. Antagonism of Cournarin and Ascorbic Acid. ........ .60 C. Interactions of Ascorbic Acid and Dithiooxamide....... 66 D. Interactions of some Chemicals related to Ascorbic Acid or bo Dithiooxamide......... 78 Page ..... .82 III. Discussion. A. The Conditions of the Expea’iments.,......... ..........82 B. Interpretation of the Results...,..,............. .......8? IV. Summary. ............ .91 PART III : THE ANTAGONISMS OF DITHIOOXAMIDE AMD ASCORBIC ACID TO THE AUXINS.............. ..... ..........................93 I . Introduction ........... A. Review of Literature 3» Problem. ...... II. 94 ...........................«. 95 102 ..... .103 Experimental Results. A. Reduction of the inhibitory Effects of 2,4-D....... ...104 B. Reduction of the inhibitory Effects of Indole-Acetic Acid................... ...... .. ............. .......... .108 C. Reduction of the inhibitory Effects of NaphthaleneAcetic Acid.......... 110 III. Discussion. ..... IV. Summary......... GENERAL DISCUSSION. GENERAL S U M M A R Y 112 .115 ............. 116 ....... 122 LITERATURE CITED APPENDIX Detailed Results : I to XI General Results : 1-A to 42-B Formulas INTRODUCTION For the past ten years, investigations in the field of plant-growth-regulators have yielded spectacular results, chiefly because of the outstanding properties of a group of synthetic substances, of which 2, 4-dichlorophenoxyacetic acid (2, 4“D) is the best representative, however, up to the present time, little is known concerning the physiological processes which account for this chemical control of growth; also little is known regarding the mechanism by which indole-3-acetic acid (IAA) induces similar but less striking effects. During the same time, a less spectacular but nevertheless steady advance was achieved in studies dealing with the influence upon plant-growth of the chemicals containing an unsaturated lactone ring; many of those, such as coumarin and some of its derivatives are able (143) both to promote or to inhibit growth at very low concentrations. Meanwhile, it was found that many of these compounds were naturally occurring substances, and some evidences of a relationship between them and compounds containing -SH groups was established. It is only recently that Van Overbeek (153) Put forward the idea that an accumulation of coumarin derivatives in plant tissues might result from the applications of the "auxins", 2, 4-D, naphthalene-acetic acid (NAA), IAA, and account for at least part of their growth-regulating activity. The initial scope of this work was to investigate the antagonism between some sulfhydryl derivatives and some unsaturated lactones (chiefly coumarin) which induced inhibitions of plant growth, and then to test the possibility that similar antagonisms exist between sulfhydryl derivatives and the "auxins". However, after the discovery of the outstanding properties of dithiooxamide (DTO), theoretical considerations led to think that the effects of DTO might be the result of an interference with the metabolism of ascorbic acid (ASA), (part l). Therefore, the interactions of ASA with coumarin and with DTO were then extensively studied (part II). This provided another tool for testing the analogies between coumarin and auxins by the investigation of the inter­ actions of AS a , as well as of DTO, with 2, 4~D, MA a , and IAA, (part III). Since the conditions of light were known to be important factors for the growth activity of coumarin, and for the amount and distribution of ASA and of IAA in plant tissues, the great majority of this experimental work has been carried out in varied conditions of illumination. 4ATJ2RIALS AMD METHODS A simple and rapid quantitative method was desirable, to determine the effects upon growth of a number of solutions of chemicals. The cucumber test, as used by Ready et al. (125) and germination tests seemed to have both qualities, and were extensively used in this laboratory for screening out a great number of chemicals suspected of having herbicidal properties. Seeds of cucumber (Cucumis sativus, var. Marketer and Burpee Hybrid) and lettuce (Lactuca sativa, var. Grand Rapids), were used. The seeds were placed in Petri dishes impregnated with a solution of the chemical under investigation. The percentage of germination of lettuce seeds, and the length of the main root of cucumber seeds were found to be good indicators of plant response. A few experiments using the length of the longest root of wheat seeds were also performed. Throughout the experiments, Petri dishes 100 m m in diameter and, except for the very first experiments, filter paper Mo 501 from A. H. Sargent and Co. were used. A. Root Elongation Tests. The first tests were made with seeds of the marketer variety of cucumbers, which had been in the laboratory for several years, and were quite satisfactory. However, other samples of the same variety were damaged and unreliable; therefore, the variety Burpee Hybrid was used in later experiments, the seeds of which were uniform and gave constant results. For the few tests conducted with wheat seeds (Triticum vulgare) the variety Henry was used. The age of this material is unknown. 1. Tests using constant solutions. In these tests, the seeds remained in the original solution until completion of the experiment, that is until the meas­ urements were taken. The first tests were conducted in tap water, by placing fifteen seeds in a solution of five milliliters of chemical in tap water in a Petri dish with one layer of filter-paper, and allowing them to germinate and grow several days in the laboratory without any special precaution for temperature (fluctuating rather widely around 25° C.) or light. In these first experiments, it was observed (Appendices 1-A, 1-3, l-'-C, and 1-D) that the normal fluctuations of the rate of germination made it difficult to compare quantitatively the effects of the various treatments: the differences had to be large in order to be significant and it also appeared that variations from test to test might be due to variations in the composition of tap water. Therefore, distilled water was then used for all the solutions and, in an effort to make the test more accurate, the number of seeds for each treatment was increased to 48 (three Petri dishes with sixteen seeds each). Care was taken that the seeds were' distributed on the filter-paper uniformly. 6 All Petri dishes had been previously cleaned together, first by soaking overnight in an activated charcoal suspension, then rinsed and washed with soap, then rinsed again with tap water and then with distilled water. Because of this uniform cleaning, each set of the three Petri dishes was considered as a unit. After the seeds had been allowed to germinate and grow for a given length of time, the length of each primary root was measured to the closest millimeter with a standard college ruler, and recorded. The measurements of treatments to be compared were always performed in such a way that there was no significant difference in growth during the time of measuring(Usually from 3 to 5 sets of three Petri dishes could be measured in one hour of time). In an attempt to account for an unavoidable proportion of seeds germinating later than the bulk of the seeds, 'which would induce wide fluctuations in root length, it was decided to consider only the average of the thirty highest figures out of the 43 root lengths recorded for each treatment. It was observed that only small variations from dish to dish occurred within a same treat­ ment in most experiments. If one Petri dish would give an average exceedingly different from the two others in the same set, it was eliminated, and an average based on the 20 highest figures of the two other Petri dishes was used. The significance of the differences between averages of two treatments was statistically tested by the t test when it was questionable (116). Detailed data and statistical analyses of a few such experiments have been given (Appendices with roman figures) so that the accuracy of the test can be evaluated. Usually, prob­ ability higher than 0.99 and 0.95 were obtained for a difference between treatments of 10;c and 1% respectively. The same technique was used in tests with wheat seeds, but experimentation with this material was soon discontinued because of frequent fungus infestation. All experiments reported here are fungus-free. 2.Tests using transfers from a solution to another solution. Because some of the effects observed in the experiments conducted according-:,to the technique previously decribed might have been caused by a chemical reaction within the solution, making part of one chemical unavailable to the plant, a new technique was designed in which seeds were first germinated in a solution of one of the chemicals under investigation, and then rinsed in distilled water and transferred to a solution of another chemical; in this way, the two chemicals could not react together before getting into the plant. For each treatment, three Petri dishes containing 20 seeds each received a solution of the first chemical (pretreatment). After a given time, all the Petri aishes receiving a same pretreatment were gathered and 16 of the best germinaued seeds from each dish were selected and all put together in the same cheesecloth, rinsed in distilled water according to a standardized procedure, and then transferred to Petri dishes containing the solution of the second chemical (post-treatment), on. the basis of three Petri dishes containing 16 seeds each for each post-treatment. Therefore, for all seeds receiving a same pretreatraent, the post-treatraent 'was assumed to be the only different factor. A similar procedure was used for all other pretreatments in the same experiment. All the solutions were made of distilled water, all the operations of rinsing and transferring were done in the controlled room and in light, so that for experiments reported as performed in continuous darkness, and interruption of the dark conditions could not be avoided. This interruption never exceed 20 minutes. The roots were then measured and averaged in the way previously described. B. Germination Tests All the germination tests were done with black lettuce seeds, variety Grand Rapids. ‘ Two samples were used; the first one i^as two years old, whereas the second one was received in June 1951. In all these experiments, distilled water was used. In order to avoid the floating of the small lettuce seeds over the solution, three layers of filter-paper 'were used instead of one only. The procedure was first to select a number of lots of 50 9 or 100 seeds each, then to place the three filter-papers in every dish and to moisten them 'with five milliliters of the solutions under investigation. The seeds were then rapidly scatter­ ed all over in the dish, and immediately put under the environ­ mental conditions to be applied. In the tests conducted in continuous or alternate illum­ ination the germinated seeds 'were removed from the dishes every 24 hours, counted and the percentage of germination up to this day was computed. For the tests in darkness, no counting of seeds was done until the end ox the first dark period in order to prevent illumination for even a very short period, (because lettuce seeds treated with coumarin appear to be sensitive to a very short period of illumination), after this time, the Petri dishes were moved to an illuminated table and removal and counting of the germ­ inated seeds were then performed in the way previously described. An experiment was also conducted according to the method of pretreatment used by hutile (112). A number of seeds were soaked for 24 hours in a coumarin solution (25 ppm) rinsed in distilled water and dried for 4 days in an oven at 43° C., all these operations being done in perfect darkness. After 17 days in a dark drawer in the laboratory, they were put in lots of $0 seeds each and then germinated according to the usual process in various solutions. G. Environmental Conditions. Except for those in which Marketer cucumber seeds were used, all the experiments were conducted in a special room where light and temperature conditions were controlled. Temperature was maintained at 25° G., and there were certainly not more than 1° C. fluctuations. Unless otherwise stated, all the experiments were carried out on a bench receiving from 130 in the center to 85 foot-candles on the edges of a light which was 70% fluorescent and 30% incandescent. Because of this light, the actual temperature on the bench was 26° C.; Petri dishes were so grouped that all treatments received about the same intensity of light. For experiments in alternate illumination, the Petri dishes were placed on the same illuminated bench, but were covered twelve hours .a day with dark paper and cardboard. For experiments in darkness, the Petri dishes were placed in a drawer and covered with cardboard and dark paper immediately after the solutions were poured into the dishes. For most experiments, treatments were simultaneously applied both in light and d a r k n e s s i n these cases, we prepared only one solution for each treatment, 'which was then poured into several sets of three Petri dishes to be placed in the various environmental con­ ditions. The often observed differences of the responses in light and darkness were then a proof that the results were not due to indidental mistakes in the making of the solutions, since the same solution would induce, different responses according to the . environmental conditions. II The experiments with Marketer cucumber seeds were done in the laboratory without any special attention to light and temperature conditions. For those of these tests which were performed in continuous light, the illumination by night was provided by a few fluorescent tubes giving an intensity of about 300 foot-candles. There was no control of moisture, but it can be assumed that the atmosphere in the Petri dishes was pretty close to sat­ uration; distilled water was added when necessary in order to keep the filter-papers in a uniformly moist condition. In the experiments with coumarin and/or DTO, it was observed that the addition of these chemicals had practically no influence on the pH, which would not be changed by more than 0.2 by the highest concentration of either one. For a number of either one. For a number of experiments with ASA, it was necessary to use solutions of low pH in order to have about the same acidity in all the treatments. Accordingly, several Sorensen’s buffer solutions were tried, which gave strong inhibitions of growth (see for instance the difference between controls of experiments 24-A and 24-B). The simplest and least harmful way to get the desired pH was to put a drop of dilute sulfuric acid (chemical grade), or 0.05 ml., for each 500 ml. of distilled water. Such a solution was then used for the making of all the chemical solutions of the test and for the controls, (see for instance experiments 24-A and 24-C for comparison of the controls). d Vi The pH of the solutions were thus adjusted as follows: Distilled water 6.1 Acid water 3.5 ASA 250 ppm 3.5 ASA 500 ppm 3.4 ASA 1000 ppm 3.25 ASA 2000 ppm 3.1 The other chemicals did not induce any significant change in pH at the concentrations which were used. PART I DITHIOOXAMIDE GROWTH-REGULATING ACTIVITY ANTAGONISM TO COUMARIN 14 I. INTRODUCTION In the course of routine experiments carried out in this laboratory, an investigation of the phytocidal activity of coumarin derivatives was started. Because these substances have an un­ saturated lactone structure which may react with -SH groups, it was an attractive idea to investigate also some sulfbydryl derivatives; therefore a few of those were selected which, for chemical reasons, appeared most likely to exhibit growth-regulating properties. Out of ten such chemicals, the screening tests showed that only thio-beta-naphthol and, chiefly, dithiooxamide (DTO) were most promising. Accordingly, the investigation became centered around DTO which was suspected of being a possible antagonist to coumarin. A. Review of Literature 1. Unsaturated Lactones Effects on Plant-growth KLebs (83) seems to have been the first worker to report upon the effect of coumarin application to plants, when he observed its inhibitive activity for the growth of Algae. Even though Cameron (28),in 1910, reported a similar effect of coumarin and of daphnetin for the growth of wheat, it was not until 1943 that the work of Kuhn et al. (87) definitely called the attention of plant students 15 ■upon unsaturated lactones. These workers demonstrated the inhibitions of germination (cress seeds) and pollen tube development (Antirrhinium) caused by coumarin, and showed that parasorbic acid (synthetic or extracted from malt) had a similar activity. The same year, Veldstra et al. (154) confirmed and extended these results to a few other unsaturated lactones, and seme benzo-coumarins. Since then, compounds of this series have received increasing attention from plant scientists, chiefly as regards their effects on germination and root development. Nutile (112) investigated the artificial dormancy induced by coumarin in lettuce seeds and the influence of light thereupon, pointing to its likeness with natural dormancy. Similar effects were later shown by Weintraub (163) to be also produced by a number of other chemicals, including a few unsaturated lactones, but to a much lesser degree. Cornman (39, 40), treating onions and lily roots with saturated aqueous solutions of coumarin, produced within a few hours a blocking of alii, mitoses resembling the effect of colchicine, whereas parasorbic acid only slowed down mitosis, causing an accumulation of metaphases in onion roots. Similar activity of protoanemonin has been reported by Erickson et al. (51). Haynes et al. (74) synthesized several alpha-beta-unsaturated lactones which also inhibited the germination of cress, and stressed their outstanding light-absorption capacity. Ciferri et al. (36) indicated that seeds of various species would become photosensitive after treatment with coumarin. Accordingly Lavollay et al. (92), aesculin (6-glucoxy-7-hydroxy-coumarin) presents growth-regulating properties resembling those of IAA in their effects upon germinating peas. Audus et al. (8) observed 16 a significant herbicidal activity of coumarin, and Audus (6), working with pea and cress, found the first species to be more sensitive to this chemical which, at high concentrations, hastened maturation processes in root meristems. Dicoumarol, the natural substance responsible for the sweetclover hemorrhagic disease (29), has been investigated by Marx et al. (101), and found to be a powerful inhibitor of germination, about three times as effective as coumarin itself, but similar investigations of a number of coumarin derivatives failed to show any good correlation betweeen prothrombin inhibition and blastokolin activity. As for scopoletin, a coumarin derivative widely occurring in plant tissues, which can be determined by fluormetric procedures, Best (15, 16, 17) showed that in tobacco plants its concentration falls off during the rapid growth stages, and reaches its maximum at time of maturity. In plants infested with spotted wilt it occurs at greater concentration than in healthy plants, though with the same relative distribution. Andreae et al. (3, 4) detected simil­ arly the blue fluorescence of scopoletin in tubers and leaves of potatoes infested with leafroll virus, whereas healthy plants did not seem to accumulate any of it. Goodwin et al. (59) observed its fluorescence in five days old Avena roots, and estimated its concentration to be about 10 ppm; this amount varied in different parts of the roots, and since it possesses growth-inhibiting properties, (61), they suggested that its relatively lower con­ centrations in the most actively growing parts of the roots might be of biological significance. 17 Scopoletin is not the only coumarin derivative to be present in plants, and lists of natural1y occurring coumarins and unsat­ urated lactones have been given by Goodwin et al. (60) and Geismann (56). Goodwin et al. (61), having further investigated the influence of coumarin derivatives upon root development in Avena, and found only a few of those to be strong inhibitors. The latest known advances in the study of unsaturated lactones have been made in this laboratory, with the discovery of Hamner et al. (67) that beta-methyl-umbelliferone was a selective growth-inhibitor for higher plants and fungi, and with the finding of Alamercery et al. (2) and Hamner et al. (68) of the selective inhibition of chlorophyll by a tetronic acid derivatives. b. Interactions with sulfhydryl groups. The first clue regarding the mechanism of action of unsaturated lactones was not to be found in the botanical field. Aside of their effects upon plant growth, it had been known for some time that a number of them possess bacteriostatic properties. In 1944, Cavallito et al. (32) discovered that cystein was able to anta­ gonize the inhibitions of bacteria], growth induced by some of them. Similar antagonistic capacity of cysteine was also demonstrated for the growth-regulating activity of hexeno-lactone by Hauschka et al. (71) and the bacteriostatic activity of a number of alpha-beta-unsaturated ketone by Geiger et al. (55)» Then, Cavallito et al. (33) gave evidences for the occurrence of a reaction between the -SH groups and the unsaturated lactone ring, probably of the type of the reaction studied by PosneE (120) in 1902. 18 Even before these results were known, Nutlle (112) observed that thiourea was able to break the artifical dormancy induced by coumarin in lettuce seeds (as well as the natural dormancy). A few other evidences of interaction of sulfhydryl compounds with unsaturated lactones were then given, Lavollay et al. (93) reported an antagonism of thiourea to the inhibition of root development induced in barley seeds by coumarin. Thimann et al. (143), using Avena coleoptile and pea stem as plant material, found that protoanemonin and coumarin were able to promote or to inhibit growth according to the concentration used, and that some dithiols, and chiefly 2, 3-dimercaptopropanol (BAL), were able to prevent their inhibitive effects. Higher concentrations of BAL were inhibitory. Since they had previously shown (141, 142) that various inhibitors of -SH enzymes (arsenite, indo-acetate, para-chloro-mercurybenzoate) inhibited the growth of the same plant materials, they concluded that the two unsaturated lactones were probably able to attack the essential -SH groups of some enzymes. 2. Sulfhydryl Compounds : Effects on plant-growth. It is well known that -SH groups are very important for biological processes. Barron (10) has recently given an excellent review of the "Thiol groups of biological importance". They seem to be essential for many enzymes. Hammett (65, 66) claims that the -SH groups of amino- and nucleic-acids accelerate the rate of increase in the number of cells, and that their partial oxidation retards the production of new cells. 19 It is therefore not surprising that some sulfhydryl derivatives exert some influence upon plant growth. The chemical first and most often tested seems to be thiourea, which has been found in free state in Laburnum (84). Thompson et al. (148, 149) discovered that it was outstanding for breaking the dormancy of lettuce seeds. Allyl-thiourea, thiocyanate, thio-semicarbazide, thioacetamide were less effective; the presence of sulfur was essential for the activity of these compounds. Similarly, thiourea has been reported to hasten and increase the germination and sprouting of Gladiolus (132), but reduces that of several alpine plants (54). Thompson et al. (147) found that it was able to counteract the detrimental effect of too high a temperature for the gemination of lettuce. No abnormality resulted of its use. Working with endives seeds in similar conditions, he also demonstrated (146) a similar improvement of germination; but he observed that, in some cases, presoaking with water alone and then drying was almost as beneficial as the same treatment with a solution of thiourea which, however, was much more effective in most cases. Tukey et al. (150) succeeded also in breaking dormancy of peach seeds, with the same substance, but observed some physiological changes: dwarf seedlings, short internodes, abnormal leaves. Other sulfhydryl compounds have also been tested for their plant-growth activity. Out of a large number of those, Brian et al. (24) found that only thioacetic acid and methyl dithiocarbamate were very toxic to wheat seeds. Dithiobiuret (49) seems to exert various effects upon gemination and root growth. 20 Both gluthathione (42) and cysteine (43) have been reported by Davis to promote healing of wounds, and glutathione (64) has been found to shorten the dormancy of potato tubersj many other chemicals containing -SH groups are quoted by Avery et al. (9) as having the same property. B. Problem A common feature of most sulfhydryl compounds exhibiting some growth-regulating activity is the presence of an aminoor an imino-group close to the -SH group, (for instance thiourea, dithiocarbamates, cysteine, dithiobiuret, glutathione...). On the other hand, BAL, of which we have previously mentioned the growth activity and the antagonism to coumarin and protoanemonin (143), is a powerful enzyme inhibitor (162), which seems to be due to the disposition of the two -SH groups on two adjacent carbon atoms. Dithiooxamide combines these two important struct­ ural features, two -SH groups on adjacent carbon atoms, each one flanked by an imino-group, as shown by itsmore reactive tautomeric formula (see appendix, formula) This chemical has never been extensively tested for biological activity. It came out unnoticed from screening tests for rodenticidal (46) goitrogentic (81), antivesicant (144), bacteriostatic (96, 128) activity; similarly, it was placed in the class of lowest activity after Thompson et al. (145) 's gigantic tests for herbicidal properties. In spite of these unfortunate precedents, and because its chemical structure allowed to expect that it would exhibit both growth-regulating properties and antagonism to unsaturated lactones, it was decided to study it more thoroughly from these two viewpoints. 22 II. EXPERIMENTAL RESULTS A. Preliminary Experiments with old Marketer Cucumber and VJheat seeds. 1. Screening out the most active chemicals. As previously indicated the first tests with Marketer seeds were performed in tap water and under laboratory conditions. The results are given in tables I and II. Only a few of its derivatives, besides coumarin itself, appeared to be strongly effective in inhibiting root-elongation. Germination apparently takes place, but very soon root-elongation ceases and a bulbous enlargment becomes conspicuous (Fig. 2, 3, 4). At the lower concentrations, after a few days of a retarded elongation, chiefly the main root becomes watery and decayed, sometimes dries out, even though the lateral roots can be unaffected and even sometimes extremely developed. Two sulfhydryl derivatives, thio-beta-naphthol and chiefly dithiooxamide, were the most effective for inhibition of root elongation. Both compounds were found to produce similar effects on root-development of Alaska peas and Yellow Globe radish, but experiments were continued only with cucumber seeds which are most sensitive. The growth of the seedling was retarded, but no formative effect or toxic symptom other than reduced growth was observed. TABLE I PERCENT GERMINATION AND GROWTH RATE OP MARKETER CUCUMBER SEEDS SOAKED IN TAP WATER SOLUTIONS OE VARIOUS COUMARIN COMPOUNDS Name of Compound. Growth Rate mm. Percent Percent of Cone. Germination Control in 3 days 3 days 3 days PPa. 5 97 94 23 10 90 86 21 50 70 80 20 100 35 48 12 200 30 4l 10 500 28 ^7 11 1000 4 33 8 100 90 4o 10 100 0 0 0 6,7-Dime thoxy- coumari n, 3-Ethylcarboxylate 6,7-Dimethoxy-couraarin, 3“Car­ boxyl ic acid 6,7-Dimethoxy-coumarin 100 42 24 6 100 85 8 2 100 51 12 3 8-Methoxy-coumarin, 3-Carboxylic acid S-Methoxy-coumarin 100 6l 16 4 100 91 16 4 7-Methoxy-coumarin, 3-Ethylcarhoxylate 7-Hydroxy-coumarin, 3-Ethylcarhoxylate 2,4,5-Trihydroxy Benzaldehyde 100 58 20 5 100 61 4o 10 100 68 12 3 7-Hydroxy coumarin, 3-Carhoxylic acid Benzyl-Beta-Methyl-Umbelliferone 100 88 20 5 100 100 83 21 5,7-Eibydroxy 4-Methyl coumarin 100 100 41 10 5,7-Nibydroxy-dimethyl-coumarin 100 100 100 25 97 100 25 Coumarin 7-n-Propyloxy, coumarin, 3 carboxylic acid 7-n-Propyloxy coumarin Control — TABLE II PERCENT GERMINATION AND GROWTH RATE OF MARKETER CUCUMBER SEEDS SOAKED IN TAP WATER SOLUTIONS OF VARIOUS SULFHYDRYL CHEMICALS Name of Compound 2 - Mercaptobenzothiazole B - Dithiodiglycol 2 - Thiobarbituric Acid Thioacetamide Thio - p - Naphthol 2 - Mercaptoethanol Sodium Thiocyanate Dithiooxamide Acid Thioglycollic (Mercaptoacetic Acid) Thiourea Control — Growth rate mm. Cone. Percent of Percent Control in Germination 3 days ppm 3 days 3 days 50 9s SO 20 100 50 45 97 0 100 15 25 100 46 32 8 50 95 80 20 100 50 32 8 50 97 100 25 100 49 4o 10 50 17 8 2 100 0 0 0 50 100 i4o 35 100 50 80 20 50 97 100 25 100 45 32 8 50 0 0 0 100 0 0 0 50 97 80 20 100 48 60 15 50 92 40 10 100 48 32 8 97 100 25 23 2. Effects of DTO on root elongation. a. Old Marketer cucumber seeds. with the same sample of old Marketer cucumber seeds which was used in the screening experiments, a study of the effects of various concentrations of DTO on elongation of roots was conducted. Several experiments were run, according to the technique of constant distilled water solutions. The detailed results are given in appendices I, II, 3-A, 3-B, 3“C, and 3-D. Significant promotions of root elongation were observed at low concentrations, whereas inhibition took place above 20 ppm. It was noted, in the case of strong inhibitions, that they could not be detected by eyes before the third day. A curve where elongation (in percent of the controls) is plotted against concentration is given in Fig. 1. Each point represents the average of the responses in the several experiments reported. It should be indicated here that the roots of most seedlings grown in DTO solutions had less root hairs, slightly less lateral roots, and were a little thicker anfl more ligneous than roots from water controls. b. Y/heat seeds. A similar investigation was carried out with wheat seeds, the results of which can be found in appendices 2-B, 2-C, 2-D and 2-E. No indication of growth promotion was ever observed; a less abrupt curve of growth inhibition has been deduced from these data and plotted on the same graph than the corresponding curve for cucumber seeds (Fig. 1). The different responses of the two species are evident. jigure ly» Effects of distilled water solutions of Dithiooxamide (DTO) on the elongation of the roots of Marketer cucumber and of wheat (var,Hemy). Semi-logarithmic scale. 24 3. Antagonism of DTO to Coumarin. a. Old. Marketer cucumber seeds. Old seeds from the same sample of Marketer cucumbers, were used under similar conditions (constant solution technique), to investigate the effects of various mixtures of DTO and coumarin. The first experiment, reported in appendix III was carried out in tap water, and with only one petri dish. After 3 days, the roots of the seeds receiving a mixed solution of coumarin at 150 ppm and DTO at 10 ppm, were clearly longer than those of the seeds receiving only the same concentration of coumarin. A similar experiment was then run in distilled water, using the technique previously described (constant solutions) with three Petri dishes for each treatment. The roots were measured on the fourth day, and it can be seen in appendix IV and in Fig. 2, 3, 4 how strikingly the inhibitions of root elongation caused by 100 and 150 ppm of coumarin were reduced by low concentrations of DTO. This experiment was repeated with only the concentration of 150 ppm of coumarin and the same concentrations of DTO. Similar results were obtained and were as conspicuous; no picture or measurement was recorded this time. The same experiment was tried in continuous darkness, under similar conditions but no conspicuous difference was observed between seeds receiving coumarin alone and those receiving coumarin and DTO. Marketer cucumber seedlings grown in mixtures of Coumarin and DitMooxamide for five days. A. B. C. D. E. Coumarin 150 ppm. Coumarin 150 ppm plus DitMooxamide 1 ppm. Coumarin 15O ppm plus DitMooxamide 5 PI®1* Coumarin 150 ppm plus DitMooxamide 7*5 PP®» Coumarin 150 ppm plus DitMooxamide 10 ppm. Marketer cucumber seedlings grown in mixtures of Coumarin and DitMooxamide for five days. A. B. C. D. E. Coumarin Coumarin Coumarin Coumarin Coumarin 100 100 100 100 100 ppm. ppm ppm ppm ppm plus plus plus plus DitMooxamide DitMooxamide DitMooxamide DitMooxamide 1 ppm. 5 PP®» 7.5 ppm. 10 ppm. Marketer cucumber seedlings grown in mixtures of Coumarin and DitMooxamide for twelve days. A. B. C. Coumarin 150 ppm. Coumarin 150 ppm plus DitMooxamide7*5 PI01* Coumarin 150 ppm plus DitMooxamide10 ppm. 25 At the same time, with the same sample of seeds and under the same conditions, an experiment was run to test the possibility of an antagonism of thio-beta-naphthol to the inhibitory activity of coumarin, but no positive result was obtained. It was then decided to investigate more extensively the properties of DTO. b. Wheat seeds. Similar experiments were conducted with wheat seeds. However, in the Petri dishes containing a coumarin solution, heavy in­ festations of fungi occurred. Therefore these experiments were discontinued. B. Experiments with a new sample of Marketer cucumber seeds. After our first supply of Marketer cucumber seeds was exhausted a new sample was ordered. The new shipment was less uniform and was different in appearance; many seeds had to be rejected because they were cracked or damaged. A few experiments were carried out in a similar line of investigation, and it soon appeared that the new seeds did not respond in the same way (appendices 4-A and 4-B). Attempts to repeat the results previously obtained with mixture of coumarin and DTO gave erratic and less striking results. Because we had been also using a newer sample of DTO, we thought that a change in the chemical might be responsible for these changes in the results. But a test reported in appendix V showed that both old and new samples of the chemical induced similarly erratic results. Some indications of antagonism by DTO at 3*3 ppm were questionable. 26 Since winter was over, and the conditions of light and temperature of the laboratory were much changed, it was thought that environmental factors might be involved. Accordingly, it was decided to conduct the next tests in environmental conditions as well controlled as possible. A special room was then used, as indicated on page 10. However, these new experimental conditions did not change the results appreciably much, as can be seen in appendices 5-A, 5-B, and 5-C. Even though there were some indications of antagonism, in continuous or alternate illumination, this was not consistent and was irregular. From these tests and other tests in which no measu­ rements were taken, it could be concluded that DTO acted more slowly, and was antagonistic only to lower concentrations of coumarin than it was in the tests with the older seeds. After several days, the inhibited main roots of the seeds placed in coumarin alone were not much shorter than in the solutions con­ taining also DTO. Only after about 6 days, when the main roots of the seeds in the coumarin solution ceased growing and started decaying, the main roots of the seeds in the mixed solutions were in better condition. Little or no decay was observed, and growth continued for a few more days, giving then a difference in length. This was true only of seeds put in continuous or alternate illumination; in darkness, such beneficial effects of DTO were not observed, apparently because the decay of the seeds receiving coumarin alone was not as rapid as in light. The reduction of inhibition of coumarin by DTO at 1 ppm in darkness (new sample), shown in appendix V, seems to have been incidental. 27 Because of a seed factor obviously involved, as suggested by the changes in the response to DTO and by the difference in age and appearance of the two samples, it was decided to investigate another variety. A Burpee Hybrid variety was chosen which has given satisfaction in every respect. C. Experiments with Burpee Hybrid cucumber seeds. 1. Antagonism of DTO to Coumarin a. Tests using constant solutions. All experiments were conducted in the controlled room. Mixtures of chemicals were tested according to the technique previously described. The aspect of the roots and seedlings grown in solutions of coumarin alone or mixed with DTO was similar to that exhibited by the last sample of Marketer cucumber seeds, except that Burpee seeds in light showed a better response to higher concentrations of DTO, as can be seen in the appendices 6-A and 6-B. There seemed also to be a maximum concentration of coumarin above which the antagonism failed to appear. Experiment 6-B is especially interesting because it demonstrates clearly the importance of the conditions of illumination: DTO did not reduce at all the inhibition of root elongation induced by coumarin in darkness. Since the same solutions were used for each treatment irrespective of the conditions of light or darkness, the same solution induced a positive response to DTO in light, and no response at all in darkness, which rules out the possibility of an error in the making of the solutions. b. Tests using transfers. Since Cavallito et al. (33) have shown that unsaturated lactones and sulfhydryl compounds can react together, it was thought that the antagonism between coumarin and DTO might be due to a chemical reaction taking place in the solution and making part of the coumarin unavailable to the seeds receiving mixtures of the two chemicals. We therefore conducted several experiments in which the technique of transfer, previously described, was used, in order to prevent a contact of the chemicals outside of the seeds. A preliminary experiment (7-A) conducted in both light and dark conditions, indicated that treating first with DTO and then transferring to coumarin was more promising than the other way, from coumarin to DTO. A second one (7-B) showed that concentrations of coumarin higher than 150 ppm seemed to inhibit root elongation irreversibly; in the same experiment, a slight antagonism was observed in darkness. However this can have been incidentally due to a low figure for the control set (transfer from water to coumarin) since this is the only case where some inhibition was observed in darkness, as shown by several tests (7"A, 7-B, 8-A and 8-b) and that it was slight (but significant with a probability of 0.99). Concentrations tested for antagonism in darkness ranged from 5 to 75 ppm for coumarin, and from 25 to 300 ppm for DTO. Important reductions of inhibition were observed in light during the experiment 8-A, which was confirmed by three other experiments performed in light only (9-A, $-B and 9-0) with, 29 each time, similar success demonstrating beyond any doubt the antagonistic action of a pretreatment of 300 ppm of DTO for the inhibitions of root elongation caused by coumarin afterwards. The data given in 8-A, 9-A, 9-B and 9-C have been averaged and put in table III so that a comparison of the reductions of in­ hibition at various concentrations of coumarin in light can be made. It is specially interesting to compare them to the promotion of growth (see below) induced by the pretreatment of 300 ppm of DTO over the controls grown in distilled water throughout the experiments; it can be seen that the reduction of the inhibitions caused by coumarin is much larger (both in absolute and in relative values) than the corresponding growth-promotive effect of DTO. 2. Effects of DTO on the elongation of roots. a. Constant solutions: Inhibition as a function of time. Because it had been previously observed that the inhibitions of root elongation induced by higher concentrations of DTO were not visible before the third day, an experiment was run in order to determine precisely how soon they become considerable. A concentration of 200 ppm of DTO was used. Burpee cucumber seeds were germinated in the usual way and the root lengths measured after, 2, 3 , 4, and 5 days (appendices 10-A and VI). No formal measurement was taken after the first day, but a ^ew seeds were measured at the time of several transfers, which fluctuated between 4 and 6 mm in light, and were never longer than 1 mm in darkness (the difference was conspicuous). The results are given in the form TABLE III EFFECTS OF SUCCESSIVE APPLICATIONS OF DITHIOOXAMIDE (DTO) AND OF COUMARIN IN DISTILLED WATER SOLUTIONS ON THE ELONGATION OF BURPEE HYBRID CUCUMBER ROOTS GROWN UNDER CONTROLLED CONDITIONS IN LIGHT FOR FIVE DAYS. TRANSFER AFTER 24 HOURS. Treatment General Average averages Average Difference % of growth mu,________________ mm.________ in mm.______(base: water) Pretreatment DTO Distilled 300 ppm Water Po st- treatment Coumarin 8-A 80 ppm 9-a 9-B 9-C _ 41.6 52.1 38.2 40.1 44.0 3.9 47.6 52.2 109.7 45.3 64.5 19.2 53.7 76.5 142.4 50.7 72.5 21.8 60.1 86.0 143.0 59.2 84.7 25.5 70.2 100.5 143.0 84.3 97.9 13.6 100.0 116.1 116.1 8-A 9-A 9-3 9-c 40.5 43.8 48.9 48.1 61.2 68.1 66.3 62.3 Coumarin 20 ppm 8-A 9-A 9-B 49.4 47.1 56.7 49.7 65.4 68.1 78.2 78.3 65.4 80.5 53.1 89.O Coumarin 10 ppm 8-A 9-A 9-B 9-C 8-A Distilled Water 9-A (Controls) 9-B 9-c $ Increase produced by DTO over the check (coumarin alone) 41.9 41.0 37.4 Coumarin 40 ppm 9-c Distilled DTO Water 300 ppm DTO Distilled 300 ppm Water — ' mm 80.0 90.9 81.9 95.7 100.3 97.7 30 of several curves where average root length is plotted against time for DTO and controls in distilled water in both light and darkness (Fig. 5)> which shows that the growth inhibitory action of DTO becomes considerable only after the end of the second day. However the difference between controls and seeds treated with DTO are significant at the 0.99 level as soon as the end of the second day, in both cases of light and darkness (appendix VI). Of special interest is the change in the ratio of the length in light to the length in darkness for both controls and treated seeds; it fluctuated between 4 and 6 after the first day, but was already significantly smaller than 1 at the end of the second day (appendix VI). b. Transfers; Influence of the conditions of illumination. (1) promotion of growth in light. In the previously reported experiments 9-A, 9-B and 9-C, conducted in light a promotion of root elongation was observed as a result of the application of a pretreatment of DTO at a concentration of 300 ppm during 24 hours. High statistical significance (probability of 0,99) of the differences between the averages of the treated seeds and of the controls were found in each case (appendix VII). Similar data have been extracted from several experiments which will be reported in part II, and in which the only difference was that the post-treatments were performed in water at pH 3*5 instead of distilled water, as indicated on page 12. Similar statistical comparison with their I 9 a irne. ; :m o o m r s e 5®“* Eilongation of; Burpee Hybrid cucumber iroots in distilled ■ :- j jwater and Dijbhiooxamide (DTO) at 200 ppm, in light and in darkness at various* intervals of time* i I :I :■ j . . . - i - 31 controls (appendix VIII) yielded the same high significance in each case (0.99). It should be noted that the Burpee Hybrid used in the latter se­ ries of experiments were not from the same shipment than those of the first series. The data from the six experiments demonstrating the promo­ tion of root elongation have been gathered in table]V and averaged. (2) Inhibition of growth in darkness. No promotion of growth was ever observed in darkness as a result of a pretreatment of DTO. This is demonstrated by experiments 8-B, 25-A, 25-B, 26-A, 26-B, and 27-A, most of them with post-treatments in water at pH 3.5. The concentrations covered a range from 25 to 300 ppm. Concentra­ tions as low as 25 ppm were tested because the concentration of 300 ppm which promotes root elongation in light gives a strong inhibition in dar­ kness. Since no difference was observed between the controls and the seeds receiving a pretreatment of 25, 37.5, 50, or 75 ppm of DTO in darkness during 24 hours, it seems impossible that any growth stimulation may occur at a lower concentration. From the experiments 26-A, 26-B, and 27-A, a table has been extracted, which shows the difference between the effects of pretreatments with various concentrations of DTO in light or in dar­ kness: concentrations which promote root elongation in light, inhibit it strongly in darkness (table V). D. Experiments with Lettuce seeds In order to test the possibility of DTO and coumarin it seemed that be exhibited the investigation that the antagonistic relationship only by of some cucumber seedlings, other plant species TABLE IV STIMULATION OF THE ELONGATION OF BURPEE HYBRID CUCUMBER ROOTS BY A PRETREATMENT (24 HOURS) OF DITHIOOXAMIDE (DTO), UNDER CONTROLLED CONDITIONS IN LIGHT. Root-1ength averages mm.______________ General averages mm. Pretreatment Distilled Water DTO 300 ppm Distilled Water DTO 300 ppm Average $Increase in root-1 ength. of treated seeds over Water controls. Post^-^treatme^ 1st sample Distilled water acidified at pH 3.5 25-B 26-A 26-E 89.7 93.0 86.7 100.3 109.9 98.3 89.8 102.8 14.5 2nd sample Distilled water 9-A 9-B 9-C 80.0 90.9 8I .9 95.7 100.3 97*7 84.3 97.9 16.1 TABLE V EFFECTS OF A ERETREATMENT (24 HOURS) OF DITHIOOXAMIDE (DTO) IN DISTILLED WATER SOLUTIONS ON THE ELONGATION OF BURFEE HYBRID CUCUMBER ROOTS GROWN UNDER CONTROLLED CONDITIONS IN LIGHT OR IN DARKNESS FOR 5 DAYS. Transfer after 2*+ hours. Treatment averages mm. General averages mm. Post-treatment Distilled Water acidified at p H 3.5 _____ Light Darkness Pretreatment 100.1 103.3 102.4 Distilled water 26-A 26-E 27-A DTO 375 PPm 26-A 26-B 27-A - — 103.0 26-A 26-B 27-A — 104.4 94.8 DTO 75 PP® DTO 150 ppm DTO 300 ppm 93.0 86.7 - 26-A 26-B 27-A 97.1 94.2 - 26-A 26-B 27-A 109.9 98.3 Distilled Water acidified at pH_ 3 *5_ Light Darkness 89.8 103.0 - 87.6 81.3 — 72.8 67.3 — 101.9 99.6 95.6 81*.4. 104.1 70.3 32 might be of interest. The antagonism between coumarin and thiourea reported by Nutile (112) for germination of lettuce seeds suggested that lettuce might respond to DTO and to coumarin in a way similar to cucumber. 1. Preliminary tests with old seeds. The first experiments were run with a limited sample of Grand Rapids lettuce seeds which were more than two years old at the time of the tests. Mixed solutions of DTO and coumarin were used, and their effects compared with those given by coumarin alone or distilled water solutions, according to the previously described technique of germination. It was found in preliminary experiments that as little as 5 or 10 ppm of DTO could produce a considerable reduction of the inhibition of germination induced by 25 ppm of coumarin, and that this antagonism was strongly influenced by the conditions of light (appendix ll-A). Because of the small number of seeds available, an experiment using only one replicate (100 seeds) for each treatment was conducted to investigate the influence of the duration of an initial dark period upon this antagonism. Each series inclu ded one control in distilled water, one treatment with coumarin alone, and two treatments where coumarin was mixed with 5 and 10 ppm respectively of DTO. All the seven series investigated were run at the same time, in the same environmental conditions of light (70% fluorescent and 33 30% incandescent) and temperature (26° C. on the illuminated bench, 25° C. in darkness)• Fresh solutions were added on the fourth day, then distilled water was used to keep the paper moist. After one series had received a given initial period of darkness, it was moved to the illuminated bench, where it was receiving then an alternate illumination (12 hours of darkness, 12 hours of light). Detailed results are given in appendix 11-B and have been presented in form of graphs where the percent germination is plotted versus time; in these graphs, only one mixture of coumarin (25 ppm) and DTO (10 ppm) has been compared to the corresponding curves for water and for coumarin (25 ppm) alone (Fig. 6). 2. Experiments with younger seeds. a. Antagonism of DTO to Coumarin. Similar experiments were sonducted with a shipment of younger seeds. But, in this case, there was no antagonism to the inhibition caused by coumarin from concentrations of DTO lower than 50 ppm (appendix 12-A). The optimum response took place at 100 ppm, because at higher concentrations, some seeds exhibited an abnormal germination in which cotyledons developed prior to radicle emergence. Some of these experiments were conducted on two benches, one being the bench previously used which received a light of composition: fluorescence 70%, incandescence 30%; the other one received an equal total intensity of light, but was 50% fluorescent, 50% incan­ descent. On this one a temperature of 29-30° C. was induced by the Pig. 6 Effects of simultaneous applications of Coumarin and Dithiooxamide (DTO) on the germination of lettuce seeds in distilled water solutions under various conditions of illumination. A. Continuous illumination. B, C, D, E, F, G-, - Alternate illumination after an increa­ singly long dark period (B, none; C, 12 hours; D, 2h hours; E, hS hours; F, 72 hours; G-, 96 hours). >2.0 C; t&8 J44 >92 21(3 22?C 2 64 joo ia 72 as l m .e 12.0 . in. -I f-44 : >• /68 •I , h: i : J9SL- 2/€>. ...'.40 J 2 6 4 . o u r s lerL.^ L ~ I: / 1 ^ I j C ount aj r l n X 5 p p m ‘ ; :-r Coum a.rln 2 5 p p m + D T 0 !0 34 increased proportion of incandescent light. It was observed in this case (12-B) that the germination was reduced, as compared with the other bench, except for the seeds receiving high concentrations of DTO (200 ppm). The antagonism between coumarin and DTO was still evident, both the check receiving coumarin only and the treatment with a mixture of coumarin and DTO exhibiting less germination; because the reduction was considerable chiefly for the check, a relatively better antagonism was observed than on the other bench. A much smaller antagonism was exhibited in darkness than in either light in these experiments (12-A and 12-B). Since it is well known that lettuce germination is reduced by high temperatures, it was thought that the difference of 3-4° 0. between the two benches might explain the difference in the results. However, it was also possible that the compostion of light had some influence. In order to eliminate the factor of temperature, a sheet of plain, ordinary window glass was interposed between the second bench and its lights, just below the incandescent bulbs and the fluores­ cent tubes. This was enough to cut down the heat radiating from the bulbs, and bring down the temperature to 26-27° C. so that the only difference between the two benches was now only the composition of lights (and a difference of temperature less than 1° C.). Under such conditions, a comparative experiment was run in continuous illumination, with and without a previous dark period of 96 hours. Three replicates were used for each treatment. The seeds were from the same sample which had been used fro the experiments 12-A and 12-B four months before. 35 The results are given in appendix 13, and have been averaged in table VI for convenience of comparison. Within each series, the antagonism effect of DTO upon the inhibition of germination induced by coumarin was evident. Some influence of light composition was visible only in the treatments receiving coumarin, and only in the case of no former dark period. The influence of temperature can be appreciated by comparing the germination of the controls on the second bench with that of the controls in experiment 14-A. It can be seen also that, in the present experiment, germination after 96 hours was lower in darkness than in continuous illumination for the controls and for the treatments receiving coumarin alone, but was about the same for all the treatments receiving DTO. In this experiment, the antagonism functioned in darkness about as well as in light. b. Promotion of germination by DTO. In the preceeding experiments, it was repeatedly observed that, when the germination of the water controls was reduced by either darkness (12-A, 12-B, and 13) or high temperature (12-A, 12-B), a treatment of DTO alone at 100 ppm (13-A) or at 200 ppm (12-A and 12-B) was able to restore the germinating ability completely. Especially interesting was the case of high temperature in light. In order to ascertain the restoration or germinating ability by DTO in light at high temperature, an experiment was rsn on the second bench (without the sheet of glass) in three replicates, TABLE VI EFFECTS OF SIMULTANEOUS APPLICATIONS OF COUMARIN AND DITKIOOXAMIDE (DTO) ON THE GERMINATION OF NEW LETTUCE SEEDS UNDER CONTROLLED CONDITIONS. PERCENT GERMINATION AT VARIOUS INTERVALS OF TIME IN HOURS, AVERAGE OF 3 REPLICATES OF 100 SEEDS EACH. Composition of light 70$ Fluorescent + 30$ Incandescent 50$ Fluorescent Illumination No dark period continuous Illumination No dark period continuous Illumination 96 hours darkness then continuous Illumination 4 50$ Incandescent 96 hours darkness then continuous Illumination Time (hours) 2k U8 72 96 120 96 120 lUU 168 2k 48 72 96 120 96 120 lUU 168 Distilled water 7^ 82 87 90 91 50 82 89 89 68 78 83 85 86 58 89 95 95 DTO 100 ppm 77 85 87 89 90 83 91 92 92 7^ 76 78 82 8U 85 91 92 92 Coumarin 25 ppm 10 29 U7 63 7^ 5 5 11 21 0 8 10 18 22 7 12 15 22 Coumarin 25 ppm ♦ DTO 100 ppm 63 78 82 sU 85 77 78 82 83 38 63 69 73 76 75 78 79 80 in which germination in distilled water and germination in a solution of 200 ppm of DTO were compared. The results are given in appendix 14-A, showing clearly the improved germination in the solution of DTO. YJhen not in combination with coumarin, 200 ppm of DTO did not seem to induce much abnormal germination of lettuce seeds. c. Abolition of artificial dormancy by DTP. Nutile (112) has shown that it is possible to throw lettuce seeds into artificial dormancy by a presoaking of 24 hours in a solution of 25 ppm of coumarin in absolute darkness. Such an experiment was conducted (see technique page 9) and the seeds were then germinated in solutions of DTO at various concentrations in darkness, continuous or alternate illumination, and' in water. As shown in 14-B only the seeds germinated in darkness exhibited improvement from the use of DTO, and this improvement vanished after 24 hours in light. This is in agreement with the findings of Nutile relative to the abolition of artificial dormancy by use of thiourea. 37 III. DISCUSSION The preceding experiments suggest that DTO has a definite growthregulating activity, and that this chemical is able to reduce the inhibitions of root development induced by coumarin. As already pointed out, the beneficial effect of DTO in the complex treatments (DTO and coumarin) as compared with the check (coumarin alone), is much greater than the increase in length of the check (DTO alone) over the water controls, as can be seen in table III. Since the reduction of the inhibition due to coumarin is greater than the simple promotion of growth, it can be spoken of a true antagonism of DTO to the effects of coumarin. Before attempting to interpret the physiological meaning of these phenomena, it seems interesting to emphasize first the strong influence of the factors of both seed and environment. A. The conditions of the experiments 1. Seed factors It is remarkable that, for both Marketer cucumber seeds and Grand Rapids lettuce seeds, the older seeds appeared to be the most sensitive to the antagonistic power of DTO against inhibitions induced by coumarin. In the case of Marketer seeds, this antagonism was effective against high concentrations of coumarin able to 38 suppress completely the elongation of the roots, in sharp contrast with the younger seeds for which DTO was ineffective against such concentrations. In the case of older lettuce seeds, l/lO as much DTO was required to counteract the effects of the same concentration of coumarin than it was for the younger seeds (5-10 ppm, or 50-100 ppm respectively). However it is possible that the age of the seeds in not the only factor responsible for these differences, and the previous story of the mother plants might also effect the seed properties. 2. Environmental factors. a. ?fater. The differancesin the results of Marketer cucumber seeds germination in solutions of DTO show that purity of the water used for making the solutions is important: the use of tap water in the experiments reported in table II is the only possible explanation for a strong inhibition of germination which was never observed with solutions made of distilled water. The same explan­ ation holds true for the wide variations of the first experiments, as exemplified by the tests 1-A and 1-B. From table IV, it can be seen easily that the pH of the solutions do not influence much the length of the cucumber roots, at least between 3.5 and 6 .1 . b. Illumination That the conditions of light are of capital importance is visible from experiments with cucumber and lettuce seeds. Whereas 39 the antagonism between coumarin and DTO functions in light only for cucumber, it works also in darkness for lettuce, although it seems less consistent in the latter case when the chemicals are mixed together. For lettuce, in the experiment by the procedure of pretreatment, the antagonism worked in darkness only, not in light. However, it seems that in the case of lettuce two phenomena are involved. In the case of mixed chemicals in which the seeds are soaked during the full length of the test, it is a matter of toxicity only in light (which breaks dormancy), and of toxicity added to a dormancy in darkness; whereas, in the case of a pre­ treatment, there is nothing to be antagonized in light since dormancy is destroyed, and in darkness just the dormancy remains, as indicated by Nutile (112). This matter appears more complex, from the fact that both chemicals individually induce different responses according to the donditions of light. Coumarin is more toxic in darkness than in light for lettuce seeds, which may be due to the existence of an artificial dromancy in darkness (112). However, in all ex­ periments where both alternate and continuous illuminations were tested, coumarin also induced more inhibition in continuous than in alternate illumination! In the case of DTO, the promotion of elongation of cucumber roots took place in light only; but in darkness, DTO improved the early germination of lettuce seeds, which could not occur in light because the germination of the controls was faster (see for instance appendix 13-A). 40 c. Time of application of light or chemicals. The opposite responses of cucumber and lettuce to the variations in the conditions of illumination might be due to, in part, the fact that they differ also according to the stage of development of the seedlings at which the chemicals were applied. As for cucumber seeds, it is remarkable that a soaking of the seeds in DTO solutions continuously throughout the test induced about the same inhibition of elongation in both cases of light or darkness, whereas a pretreatment of 24 hours with 300 ppm of DTO induced a promotion of elongation in light, and a strong inhibition in darkness. In the continuous treatment, the inhibition does not become considerable before the third day; for both control and treated seeds, root elongation starts faster in light, then becomes slower than in darkness after the first day. This seems to indicate that the conditions of illum­ ination are specially important during the first day of germination. Because cotyledons do not emerge from the seed coat before the middle of the second day, it appears probable that photosynthesis is not concerned with this higher early rate of elongation in light. As for lettuce seeds, the length of the first dark period appears important for the manifestation of the antagonism between coumarin and DTO. Increasing it depressed the antagonism, as shown in Fig. 6, which is due to the fact that the curve of germination of the seeds in the mixture travels faster towards the right than 41 does the curve of germination of the seeds in the check solution (coumarin alone). One day added to the length of the first dark period induced a delay of one day and a half in the mixture between D and E, E and F, and F and G, whereas the delay is of one day only for the check (see Fig 6 ). However the disparition of the antagonism observed after a darkperiod of 96 hours was never observed with the younger seeds, which may be due to the ihigher concentrations of DTO used in this case, and to the fact that, they were moved to continuous illumination instead of alternate illumination. d. Temperature. Its importance has been investigated with lettuce seeds only. It had been known for a long time that the germination of lettuce drops abruptly above the optimum of 25° U.. Thompson has shown (146, 147) that thiourea was able to counteract this detrimental effect of too high a temperature, DTO has the same ability (14-A); however, the concentration of DTO effective in this test was 200 ppm, which is much lower than the requirement for thiourea (0 .5% or 5,000 ppm). Apartial inactivation by heat of an enzyme necessary for germination and its protection by DTO and thiourea might constitute a tentative interpretation of these facts. 42 B. Interpretation of the Results Because general effects upon the growth of the whole seedling have been investigated, it is difficult to deterinine what phsiological processes are more specifically involved in the phenomena previously described. The exact chemical structure of thioamides such as thiourea and DTO is still disputed by organic chemists. A structure of zwitterion seems to be most probable presently, which is compatible with the great ability of thiourea (34) and thiomides in general (133) to react like thiols possessing the -SH group. As previously indicated, DTO would be in this casepossess two such groups on adjacent carbon atoms. For DuBois et al. (48), studying the in­ activations of some enzymes by thioureas, it seemed most likely that the -SH groups of these substances were involved in these biological reactions. It is therefore reasonable to think that DTO has the properties of a dithiol. The recent investigations performed with BAL have shown that dithiols are outstanding in three respects at least (12): (a) they are strong, although sluggish oxidation-reduction systems, rapidly oxidized in the presence of a number of cata­ lysts, of which copper and iron-porphyrin are the most powerful; (b) they combine with a number of heavy metals, forming complex compounds usually insoluble; (c) in the presence of oxygen, they can destroy iron-por­ phyrin compound* (haemin, oxy-haemoglobin) by opening the por­ phyrin ring. 43 These chemical properties enable the dithiols to inactivate many enzymes; Webb at al. (162) found that, among the most sensitive, are: polyphenol oxidase (a copper enzyme), carbonic anhydrase, catalase and peroxidase(which has an iron-porphyrin prosthetic group), and they concluded that BAL may be considered as a potent inhibitor of metal containing enzymes Barron et al. (13) confirmed and extended these results to other dithiols, adding that the oxidation products of dithiols seem to be able of inhibiting -SH enzymes. Evidences of the effects of thiols and thioamides upon polyphenol oxidase and peroxidase have been dontributed. DuBois et al. (48) have reported that several thioureas are able to inhibit tyrosinase activity in rat tissues. Jaques (80) observed that several thioureas may inactivate potato phenol oxidase. According to Randall (123), thiourea and other thiols do not inactivate animal peroxidase, but rather serve as a substrate for it, and their ability to reduce hydrogen peroxide is increased by per­ oxidase. Among the metal-containing enzymes not tested by the students of BAL, ascorbic acid (ASA) oxidase, a copper enzyme, is prominent in cucumber seedlings. If we recall that both polyphenol oxidase (109) and peroxidase (139)(140) are able to oxidize ASA indirectly, it comes out that dithiols may attack the three most important enzymes which control the metabolism or ASA in plant tissues. ASA possesses an unsaturated lactone structure (see appendix, formula), and is widely occurring in plant tissues,where it may assume a role of redox system. The possibilitythen is that one 44 effect of the substances containing -SH groups, and specially of dithiol and thioamides, be an upset of ASA metabolism. Coumarin might interfer with this mechanism by its own unsaturated lactone ring. However, other biological processes may also be affected by the unsaturated lactones and be sensitive to sulfhydryl compounds at the same time. For instance, since it has been shown (33) that unsaturated lactones can react with natural *SH groups such as those of cystein, the -SH enzymes might be inactivated by a sub­ stance like coumarin. Toennies (151) has shown that thioamides also may immobilize the -SH groups of cystein; so the natural -SH groups might be sensitive to both coumarin and DTO. The antagonism of these substances would then be explained by the fact that mercaptide forming substances and reducing agents, such as ASA, may in certain cases reactivate the natural sulf­ hydryl groups, as indicated by Barron (101). It is remarkable that in either one of the previously suggested mechanisms ASA seems to be entitled to play a key role on the inhibitory effects of coumarin and of DTO and on their antagonism. 45 IV. SUMMARY The effects and interactions of coumarin derivatives and sulfhydryl compounds upon germination of lettuce seeds and elongation of the main root of cucumber seedlings have been investigated in Petri dishes, and under controlled conditions of light and temp­ erature . (1) Several substituted coumarins, besides coumarin itself, have been found to inhibit root growth of cucumber, with an accompanying bulbous enlargment of the hypocotyl. (2) Chiefly two sulfhydryl compounds, dithiooxamide and thiobeta-naphthol, were effective in inhibiting root-growth of cucumber. (3) Dithiooxamide has been found to promote root growth of cucumber in light, but not in darkness. To a promotion of root grov/th induced by a solution of dithiooxamide (300 ppm} applied as a pretreatment of 24 hours in light, corresponded a strong inhib­ ition of root-growth by the same pretreatment applied in darknessA treatment of 200 ppm continued for five days produced a strong inhibition of boot-growth in both light and darkness, which became apparent only after the third day. (4} Dithiooxamide was able to reduce considerably the inhibition of root growth mf cucumber induced by coumarin in light, but not in darkness. (5) Dithiooxamide was able to reduce considerably the inhibition of germination of Grand Rapids lettuce seeds induced by coumarin both in light and darkness. 46 (6) Dithiooxamide ivas able to increase considerably the germination of lettuce seeds at 29 °~ 30° C. in light and at 25° C. in darkness. (7) These results are discussed relatively to the age of the seeds, the conditions of light, and the stage of development at which the chemicals or the conditions of light were applied. A possible biochemical relationship between unsaturated lactone and -SH groups is suggested. PART II ASCORBIC ACID ANTAGONISM TO COUMARIN INTERACTIONS WITH DITHIOOXAMIDE 47 I. INTRODUCTION The experiments reported in part I, which establish the growth regulator properties of DTU and its antagonism to the inhibitions of root development induced by coumarin, thus const­ itute another evidence for an interaction of unsaturated lactones with dithiols and thioamides. Looking for the biochemical reasons of this growth-regulating activity and this antagonism, it has been suggested that two mechanisms of physiological relationship chiefly might be involved, in both of which a key role of regulation could be played by ASA. Because these two hypotheses were both of a highly theo­ retical character, a thorough search of the literature dealing with ASA, its effects upon plant-growth, and its physiological relationships with the natural -SH groups and with unsaturated lactones was achieved, before going into experimental work for testing the ability of ASA to interact with DTO and coumarin. A. Review of Literature 1. Ascorbic Acid: Effects on Plant-Growth a. General growth In 1935, simultaneously Havas (72) and Hansen (70) reported about the effects of ASA upon plant growth, The first worker, study­ ing the germination and early development of wheat, did not observe 43 any stimulation of germination by ASA but found an increase and an acceleration of the growth of the seedling, amounting to 25-30% of the shoot weight, and 50% of the root weight after 12-13 days. High concentrations were inhibitory, He observed also that ASA was able to increase the dry weight of tomato plants by 20%, but at the same time decreased the fruiting. Hansen reported similar results with peas. Davies et al. (44), in 1937, made a thorough study of the effects of ASA on various growth phases of several plant species. For generation of buds and roots in willow branches, ASA was beneficial, more than IAA during the first few days, less after the first week. ASA also promoted the germination and the elongation of roots for oats, cress, and mustard seeds in Pfeffer's inorganic nutrient solution. It did not induce bending in the decapitated or the normal oat plant. Applied to tomato petiole, it produced a general growth stimul­ ation with a considerable increase in dry weight, but no local nastic response. Bonner et al. (21) found that ASA was able to improve the development of pea embryos. This was not true of all pea varieties, which explains the negative results of Kogl et al. (85) working with another variety of the same species. Bonner observed bn excellent correlation between the ASA content of different vari­ eties of peas and their response to applications of ASA: the lesser ability to synthesize ASA naturally, the better response to an addition of this vitamin. He also emphasized two of the main difficulties encountered in investigation dealing with ASA: first, the extreme variability of the responses to ASA applications, due to great variations in ASA content induced in plant tissues by the plant and the environmental (see below) factors, and, second, difficulty in obtaining materials deficient in natural Vitimin C in order to get a response from ASA addition. This probably accounts for many failures to induce plant materials to respond.to ASA applications. Borgstrom (23) has reported that ASA promotes the growth of Allium Cepa dnd Allium fistulosum: Onchatschek (113) observed that it increased the growth of eighteen species of mixotrophic algae, chiefly when they were growing in autotrophic conditions. Dennison (45) observed a general increase in growth of tobacco treated with ASA. As for Ulva Lactuca, contradictory results were contributed by Tore Levring (95) and by Harald Kylin (89). Here again, a matter of environmental conditions was involved (namely: the composition of the artificial sea-water used for growing the algae). Raadts et al. (122) found that dehydro-ascorbic acid stimulates the growth of Avena coleoptile. Wetmore et al. (165) observed that the incorporation of ASA in agar blocks receiving natural auxins from foxtail would increase the curvature of Avena coleoptile. Virtannen et al. (155), working with cotyledonfree peas, found that ASA would give a growth comparable to that of normal plants if the nitrogen was supplied in nitrate form. Running (88) found ASA to be about as effective as IAA and thiamin in producing cambial activity in decapitated bean plants and sun­ flowers. 50 b. Germination. A number of workers have investigated the effects of ASA upon germination and root development. After the negative results of Havas (72) with wheat, we have already cited' the positive findings of Davies et al. (44). Bonner et al. (22) did not find ASA to be essential for the growth of isolated pea roots in a mineaal nutrient solution containing sucrose, vitamin B and Nicotinic Acid. Cooper (38) has reported that A6a is very effective in inducing the germination of pollen grains of Carica papaya. However, Addicott (1) found it to be inactive on the pollen of Tropaeolum and of Milla.. and so did Wang (160) for the pollen of Lotus Corniculatus. Germination of rye and barley was accel­ erated by ASA, according to von Euler et al. (156). Also Rugge (127) found that- the germination ability of barley c ould be incre­ ased by addition of ASA, especially for old seeds with a diminished germinative power. c. Environmatit and plant factors. That the age of the seeds influences the response of plants to applications of ASA is only an example of the many factors which interfer with the effects of ASA upon plant growth. Those are probably important because they change the amount of ASA which is normally present in the tissues, as shown by Bonner (21) for several varieties of peas. It is well known that light, temper­ ature, nutrition, trace elements are important in this respect, and the literature dealing with their influence upon ASA content is considerable. An accurate investigation of some of these factors has recently been performed by Somers et al. (135)} who have also 51 listed some of the most important works on this subject (136). It is generally admitted that ASA content is higher in plants grown in light than in plants grwon in shade; Clark (37) has been one of the first workers to remark that gradients of chloro­ phyll and ASA concentration in plant tissues are similar. However, he recognized also that light was not necessary for ASA synthesis, which has been condirmed (62). The precursor of ASA is still un­ known, although there are someindications that hexose sugars might constitute one of the steps of its synthesis (98). Speak­ ing of light, it should be recalled here that ASA solutions deteriorate more rapidly in light (5), chiefly if riboflavin is present (75). 2. Interactions between Ascorbic Acid and Sulfhydryl groups. The protection of ASA by natural compounds possessing -SH groups (glutathione, cysteine), is among the most conspicuous and, seemingly, the most important processes involved in the meta­ bolism of ASA. It seems that such mechanisms may function direct­ ly by chemicll reaction with oxidizing agents able to attack ASA, or indirectly, through influence on the enzymes which catalyse the oxidation-reduction reactions of the ASA-dehydroascorbic acid system. Evidences of a direct protection of ASA by synthetic compounds with an —SH group can also be found in the literature, as well as of a working together of natural -SH groups with Vitamin C in some metabolic processes. a. Physiological protection of ASA by -SH groups* The first evidence of a connection between the metabolism of ASA and -SH groups appears to have been provided by Pfankuch (118) who reported in 1934 that cysteine was able to reduce dehydroascorbic acid back to ASA in the pressed juice from potato, probably by an enzymatic process. Then Mawson (102) indicated a protection of ASA by glutathione, cysteine, and cystine in animal tissues. Hopkins et al. (76) were apparently the first workers to study in detail the protection of ASA by glutathione in plant tissues. They demonstrated the existence of an enzyme able to oxidize ASA, the oxidation of which was pre­ vented by glutathione. They found that glutathione was able to afford the same protection both in the presence of the enzyme of of copper and also tb reduce the oxidized ASA in presence of the enzyme. They furthermore observed that the enzyme would not oxidise glutathione when ASA was not present, but that, in presence of the system ASA, enzyme and glutathione, the last substance was oxidized at the same rate at which ASA would have been oxidized, had glutathione not been present. A protection of ASA by glutathione has also been demonstrated by Barron et al. (11) in animal tissues. Crook et al. (41) discovered that 9 species of plants at least contain an enzyme able to catalyze the reduction of dehydro­ ascorbic acid by reduced glutathione (dehydroascorbic acid reductase) which would thus proteet ASA from oxidation by atmos­ pheric oxygen. Synthetic -SH derivatives, such as sodium diethyldithiocarbamate (103, 137) in small concentrations may inhibit the activity of ASA oxidase from cucumbers, therefore they protect ASA from oxidation. The same chemical, thiourea and H 2S markedly 53 inhibit the activity of soybean ASA oxidase which would induce a reversible oxidation of ASA to the stage dehydroascorbic acid only, according to Rangnekar et al, (124), who also indicated that the enzyme practically developed to a maximum in 4$ hours, then remained constant in the cotyledons, but decreased in the sproutslr Thiourea was also found to cause an extreme reduction in the amount of (reduced) ASA in plasma and tissues of rabbits (82). Thiouracil was less effective, when given high, but not lethal, amounts of thiourea,rabbits could live for a long period of time with an extremely low concentration of plasma-ASA without development of scurvy symptoms. The previous works show that ASA and sulfhydryl groups are interrelated in plant and animal physiology. However the nature of this relationship is still far from being perfectly clear. It is the opinion of Barron (10) that, most probably, the protection of ASA by glutathione is an indirect one, by combination with copper for instance, although he does not completely rule out the possibility of a direct mechanism. Certainly also, indirect protection by interference with enzyme systems do exist. * Mapson et al. (99) have recently reported that a complex system involving coenzyme II, glutathione and Ascorbic acid functions in plant tissues for the stranport of hydrogen, in which dehydroascorbic acid is reduced by glutathione independently of the reductase. 54 b. Direct protection of ASA by synthetic -SH compounds* ' I A few examples of direct protection by thiol derivatives exist. Arcus et al. (5), after ascertaining the rapid deterior­ ation of ASA in the presence of ultra-violet light observed that glutathione may protect ASA when the two chemicals were mixed toget­ her, or when a glutathione solution was interposed as a screen between thelight and the ASA solution. In 1943, Drake et al. (47) showed that by iodine titration and by change in optical activity of the mixture that ASA may form complex or addition aompounds with glutathione, cysteine and thioglycolic acid, on a mole to mole basis. Yusuke Sumiki et al. (166) reported that thiourea, thioglycolic acid and reductic acid were fairly effective agents for the stabilization of Vitamin C in solution. Ghoten Inagaki (35) found that thiourea was a very good stabilizer of ASA and Uampbell et al. (30) reported that it was true also of glutathione and sodium ehtyl dithiocarbamate beside thiourea. c. Various associations of ASA with -SH groups^ There are a few known facts which strongly suggest that ASA and -SH groups might be associated in various metabolic processes. As early as 1938, Giri (57) investigated the inhibiton of phosphatase activity (in sprouted soybean) brought about by a complex system ASA-copper. The additon of glutathione,cysteine, cystine, H2 S and some other reducing agents annuled this inhibition. Similar mechanism was then demonstrated by the same author for the animal 55 phosphatases (58), and he suggested that ASA and glutathione may contribute the two parts (hydrolysis and synthesis) of a system controlling the metabolism of phosphorus. Pett (117) has shown that, in potatoes, glutathione and ASA contents undergo parallel variations, both rising sharply with sprouting and then declining simultaneously. Kretsovitch et al. (86) demonstrated that similar changes occur in germinating seeds (wheat, rye, and corn). Hopkins et al. (77) for glutathione in a number of seeds, and Cailleau et al. (26)(27) for ASA in wheat and peas confirmed that the amount of these substances, from nil at the start of germination, rises sharply during the earliest stages of the seedling development. Virtanen et al. (155) found that both ASA and several thiol derivatives (glutathione, cysteine) enabled cotyledonfree peas to use the nitrogen from nitrates and grow almost as well as intact plants; they believed that the -SH compounds reduced dehydroascorbic acid formed and thus allowed the dotyledon-free embryo to use more effectively the small amount of Vitamin C which it contains, and which, they thought, functions as a donor of hydrogen. It has been recently reported by Carruthers (31) that ASA oxidase, the leading enzyme for the phsiological oxidation of AS&, presents a catalytic wave characteristic of -SH groups in polarographic determinations. It should be recalled here, as stated 56 by Barron (10) that: "When the active -SB groups of thiol enzymes are abolished, either by oxidation oh by mercaptide formation, they may, under certain conditions, be regenerated with complete restorationof the enzyme activity by the addition of reducing agents (ASA, Hg S, cyanide) or of mercaptide forming substances". Thus, the all important -SH enzymes constitute another common ground of action for ASA and -SH compounds, where those have most opportunities to compete and interact with each other. 3. Interactions between ASA and Unsaturated Lactones. Whereas it was possible to find many cases of interactions of ASA with sulfhydryl compounds, it is more difficult to gather indications of an interrelationship between unsaturated lactones and ASA. Weintraub (163) found that, among many other chemicals, ASA was, like coumarin, able to inhibit the germination of lettuce seeds and this inhibition was also photosensitive. Buston et al. (25), investigating the effects of several unsaturated lactones upon seed germination, did not find any antagonistic action of ASA for the inhibitions induced by hexeno-lactone. Yforks in animal phsiology have indicated an antagonism between the effects of dicomarol and Vitamin C. Working with rabbits (115), rats (14) and Guinea pigs (138) Link et al. gave several evidences pointing to the existence of an antagonism between ASA and the sweet-clover hemorrhagic disease induced by dicoumarolj repeated applications of ASA may reduce the symptoms 57 of the disease; animals suffering with scurvy are more affected by applications of dicoumarol than healthy one§, and in certain cases dicoumarol applications induced a temporary excretion of Vitamin C. Martin et al. (100) confirmed part of these works. Another indication of a possible relationship, is provided by several works dealing with scopoletin, another coumarin der­ ivative. As previously indicated, scopoletin (4) does not accum­ ulate in healthy potato tubers but is apparently built up in case of leafroll virus infection(3)> and is also associated with spotted wilt of tobacco (1$). It has been found by Smith et al. (134) that potatoes infested with leafroll virus have also an excessive content of ASA; this has been confirmed by Newton (111) and used as a basis for a method of detection of diseased plants, Further investigation by Andreae et al. (4) led them to discover that potato tubers metabolize scopoletin with production of an unstable blue intermediary product. The reaction, can be accelerated by H2 02 and inhibited by a few substances including high toncentrations of ASA. There is there­ fore a possibility that the accumulation of scopoletin in the diseased tissue be due to a blocking of its metabolism caused by the build up of ASA. B. Problem The preceding review of literature has procured facts which demonstrate the importance of ASA as a natural growth-regulator for plants; it has revealed substantial evidences indicating that 58 ASA and natural -SH groups constitute a complex metabolic system widely occurring in plant tissues, and very likely to be sensitive to the applications of synthetic thiol derivatives. Some works also suggest the existence of a connection between the metabolism of coumarin derivatives and ASA. These evidences are consistent enough to substantiate the hypothesis that ASA might be able to interfer with the growth regulating activity of the chemicals previously studied (coumarin, DTO), such as was suggested in the discussion of the first part of this work. The purpose of the experiments now reported was to investigate the effects of ASA upon the inhibitions of root elongation and of germination caused by coumarin and by DTO. This investigation has been extended to sane chemicals closely related to ASA, and to thiourea which resembles DTO. II. EXPERIMENTAL RESULTS A. Effects of Ascorbic Acid on Root Elongation A few experiments were performed in tap-water with Marketer cucumber and wheat seeds to test the effect of ASA on root elong­ ation. But the results were not consistent and varied widely from test to test and even within & same test (appendices 1-A, 1-B, 1-C, and 1-D). No special experiment has been run in distilled water for the study of the effects of ASA alone on roots, but from the checks used in many experiments for purpose of compari­ son, some conclusions can be drawn regarding the effects of ASA on root elongation. As a rule, no considerable effect was pro­ duced by a concentration of ASA lower than 2,000 ppm. According to the time and the technique ef application (constant solution, pretreatment or post-treatment), according to seed factors such as variety and environmental factors, such as air-composition or moisture content which were not controlled, slight variations occurred, seldom higher than 10^ of the length of the controls, some times as a promotion, some times as an inhibition. For instance, in experiment 37-C, ASA concentrations up to 2,000 ppm were not inhibitive and even slightly growth-promotive (500-1,000 ppm), in lightj but, in experiment 32-B in light, 250 ppm of ASA were already inhibitivej in experiment 24-D in light, growth-inhibition was not apparent before a concentration of 1,000 ppm was reached. Slight promotions of growth were observed several times, both in light (experiments 4-B, 26-A, 24-C) and in darkness (24-C). Therefore, in many cases, checks with ASA alone were run, in order to determine accurately what was the contri­ bution of ASA in the. results of the complex treatments. As for the aspects of the roots grown in solutions containing only ASA, they were more twisted, and had generally more root- hairs and more lateral roots than the controls. B. Antagonism of Ascorbic Acid and Coumarin 1. Preliminary Experiments with Marketer Cucumbers. The first experiments were donducted with the idea that, if ASA was to exhibit any antagonism to coumarin such as did DTO, the mixture of DTO and ASA might give an antagonism stronger than either chemical alone. Therefore, in experiments 4-A, 4-B, and 5-A, seeds grown in mixtures containing coumarin and DTO or ASA or both chemicals were compared to seeds grown in coumarin alone. Wo attention was paid to the pH of the solutions at that time, and the Marketer seeds used were from the second sample which did not respond to DTO as well as the first and older sample. In the tree experiments, clear evidences for-an antagonism of ASA to the effects of coumarin were obtained. In spite of some Variations from test to test, it was clear also that this antag­ onism functioned vs well in darkness as it did in light. It can be seen in 4-B that the slightly promotive effect of ASA alone was much smaller than the difference between the root length in 61 the complex treatment (coumarin plus ASA) and the check (coumarin only). The roots from the complex treatment were sometimes twice as long as the roots from the check, and more than half of the inhibition waused by coumarin was suppressed (67$ in the case of seeds placed in alternate illumination in experiment 5-A). As for the addition of the antagonisms of DTO and of ASA to coumarin, the failure of the seeds to exhibit the antagonisms of DTO throws a doubt on the results obtained with mixtures of the three chemicals. Only in the treatments in coutinuous illumination of experiment 4-A did DTO reinforce the effects of ASA. In all the other cases, DTO depressed them, or even suppressed them complete­ ly. Special attention will be devoted elsewhere to the interactions of ASA with DTO. 2. Experiments with Burpee Hybrid Cucumbers a. Constant Solutions In all the experiments now reported, all the solutions were made using acid water at pH 3*5, so that only the treatments receiving 1,000 ppm or 2,000 ppm of ASA had a slightly different pH (respectively 3.25 and 3.1 as indicated on p.12). The addition of coumarin did not change the pH. Two experiments were conducted, in which the seeds were from different samples of Burpee Hybrid cucumbers. Photographs were taken after six days of germination in experiment 15-B (Fig. 7, 8, 9, 10) and, in each case, measure­ ments were taken which are reported in 15-A and 15-B. Fig. 7. Burpee Hybrid cucumber seedlings grown in mixtures of Coumarin and Ascorbic Acid for six days in light. 1. 2. 3. 4. Fig. 8. Coumarin 75 Coumarin 75 Coumarin 75 Coumarin 75 ppm. 250 ppm. ppm plus Ascorbic Acid 500 ppm. ppm plus Ascorbic Acid ppm plus Ascorbic Acid 1,000 ppm. Burpee Hybrid cucumber seedlings grown in mixtures of Coumarin and Ascorbic Acid for six days in darkness. 1. 2. 3* 4. Fig. 10. 150 ppm. 150 ppm plus Ascorbic Acid 250 ppm. 150 ppm plus Ascorbic Acid 500 ppm. 150 ppm plus Ascorbic Acid 1,000 ppm. Burpee Hybrid cucumber seedlings grown in mixtures of Coumarin and Ascorbic Acid for six days in light. 1. 2. 3. 4. Fig. 9 . Coumarin Coumarin Coumarin Coumarin Coumarin 150 Coumarin 150 Coumarin 150 Coumarin 150 ppm, ppm plus Ascorbic Acid 250 ppm. ppm plus Ascorbic Acid 500 ppm. ppm plus Ascorbic Acid 1,000 ppm. Burpee Hybrid cucumber seedlings grown in mixtures of Coumarin and Ascorbic Acid for six days in darkness. 1. Coumarin 75 ppm. 2 . Coumarin 75 ppm plus Ascorbic Acid 3. 4. 250 ppm. 500 ppm. Coumarin 75 ppm plus Ascorbic Acid Coumarin 75 ppm plus Ascorbic Acid 1,000 ppm. As can be seen from the pictures and from the data, the antagonism was considerable under all conditions of illumination; no promotion of elongation by ASA alone was observed, and the concentration of 1,000 ppm of ASA, which was slightly inhibitory by itself, gave the better response for the antagonism to coumarin (15-B). Like in the case of Marketer seeds, it was common to observe suppression of more than 60% of the inhibition caused by coumarin (73$ in 15~B). Twice the roots of the treated seeds were very close to three times as long as those of the check receiving coumarin only (15-A, in alternate illumination and 15-B in light with 150 ppm of coumarin). b. Transfers In order to test the possiblity that the antagonism observed in the experiments conducted in constant solutions be due to a chemical reaction occurring in the solution and making part of the coumarin unavailable to the seeds, the technique of transfers was used in the experiments now reported. The pH was not the same throughout the tests; the solutions of coumarin and their controls were made of distilled water, whereas acid water at pH 3.5 was used for the solutions of ASA and their controls, so, for all the seeds in each test, the pH was the same at a given time, but used to be similarly changed at the time of the transfer from the pretreatments to the post-treatments. Because it had been found in part I that transfers from DTO to coumarin used to make the antagonism of DTO to coumarin more manifest than transfers from coumarin to. DTO, the first experiment 63 was conducted by applying first ASA f o r 24 or 48 hours, then trans­ ferring to coumarin solutions. This did not give any evidence of antagonism (16-A). Therefore another experiment was run, in which the other way of transferring was used (from coumarin to ASA). Only in the case of a pfcetreatment for 24 hours in light did we observe a strong reduction of the inhibition caused by coumarin (17-A). Two other tests were performed in a similar way and with the same results. A new sample of seeds from the same origin was used in experiment 18-A. (1). Slight antagonism in darkness. The results of experiments 17-A, 17-B, and 18-A all agreed to indicate that ASA is able to slightly reduce the inhibition caused by coumarin in darkness; on a range of concentrations going from 37*5 ppm to 150 ppm for coumarin, and from 125 ppm to 1,000 ppm for ASA, it was impossible to uncover an antagonism comparable to the antagonism in light. However, many evidences of a slighter antagonism can be found in these tests. The best result was ob­ tained in 17-A with a pretreatment of 48 hours: the root length in the complex treatment was 17$ longer than the roots receiving only coumarin. (2). Strong antagonism in light. The same experiments showed that, in continuous illumination, ASA gives a considerable reduction of the inhibition of root-elongation caused by a pretreatment of coumarin for 24 hours; the same does not seem to take place if the pretreatment is applied for 64 48 hours, as shown in 17-A. In both 17-A and 17-B where a post-treatment of 500 ppm of ASA was slightly but significantly inhibitory, the complex treat­ ment gave a slight promotion of grwwth over the controls receiving no chemical: The inhibition of elongation induced by the pretreatment of coumarin was not only completely suppressed, it was even reverted into a promotion. This was observed four times (three replicates of 17-B, once in 17-A), but was statistically signifi­ cant only twice (see appendix IK) at the level of probability 0.95. In 18-A, the effects of various concentrations of ASA on the inhibitions caused by several concentrations of coumarin are given. Several times, the roots in the dishes receiving the complex treatment, were 30% longer than in the check, and in this experiment also complete suppressions of the root inhibition were observed. The results of experiments 17-A, 17-B, and 18-A in light have been put together and averaged in table VII. 3. Experiments with Lettuce Seeds a. Prelimina.r»y Experiments with old Lettuce Seeds. Since DTO was able to reduce the inhibitions of lettuce germination induced by coumarin, it was thought that ASA might be able to do this also. Accordingly, experiments similar to those which have already been reported in the case of DTO (part I) were run with ASA. Due to the different pH, fungi and molds developed frequently in the solutions, chiefly those of ASA. TABLE VII EFFECTS OF SUCCESSIVE APPLICATIONS OF COUMARIN IN DISTILLED WATER SOLUTIONS AND OF ASCORBIC ACID (ASA) IN ACID WATER SOLUTIONS AT pH 3 .5 ON THE ELONGATION OF BURPEE HYBRID CUCUMBER ROOTS GROWN UNDER CONTROLLED CONDITIONS IN LIGHT FOR 5 DAYS Average root-1ength (mm.) Pretreatments (24 hours) Distilled water 17-A 17-B 18-A Average Coumarin 75 Ppm 17-A 17-B(1) 17-B(2) 17-B(3) 18-A Average Coumarin 150 ppm 17-A 17-B 18-A Average Coumarin 300 ppm Average Po st-treatment s Acid ASA ASA Water 250 ppm 500 ppm 83.6 75*5 78.1 80.2 70.9 90.8 93.2 84.9 80.1 72.1 68.7 68.8 65.5 69.4 69.5 79.9 77*0 ■73*8 6s.4 ?6*9 52.6 - 60.2 59*4 17-A 17-B 18-A — 43.7 Post-treatments Acid ASA ASA Water 250 ppm 500 ppm 100 80.6 Percent increase produced "by ASA over the checks (coumarin alone) Post-treatments Acid ASA ASA Water 250 ppm 500 ppm 92.0 94*3 100 92.0 94.3 90.6 io5*3 100 112.4 130.6 94.6 100 - 135*1 64.9 100 122.1 126.0 344 m t ■- — — 53*4 - 43.7 92.3 85.2 86.3 88.3 Percent average root-length (Water controls: 100) 53-4 8 ■SH GROUPS ASCORBIC ACID Metabolism Direct * Stabilization » Inhibition of Metabolism \ -SH * Polyphenol Oxidase and Tyrosine + ENZYMES Figure 11 Suggested interrelationships between coumarin, -SH groups and ascorbic acid„ ^Derivatives IV. SUMMARY The effects of ascorbic acid on the inhibitions of root devel­ opment induced by solutions of coumarin and of dithiooxamide have been investigated under controlled conditions of light and temperature in Fetri dishes. (1) Ascorbic Acid has been found to exert no significant effect upon the elongation of cucumber roots at the concentrations used in this work (below 2,000 ppm). (2) Ascorbic Acid was able to overcome the inhibitions of cucumber root development induced by solutions of coumarin both in light or in darkness; it was equally potent in either case when the chemicals were simultaneously applied throughout the tests; it was more potent in light than in darkness when the seeds were successively treated with a solution of coumarin first, and then with a solution of ascorbic acid. (3) Ascorbic acid was able to overcome the inhibitions of lettuce germination induced by coumarin; this took place in light only and not in darkness when the chemicals were simultaneously applied through­ out the test; it occurred only in darkness when the seeds were first soaked in a coumarin solution for 2k hours in darkness, and then germinated in ascorbic acid solutions (breaking of artificial dor­ mancy) . (4) Ascorbic Acid, simultaneously applied with dithiooxamide throughout the tests, reduced the inhibition of cucumber root devel- 92 opment induced by dithiooxamide in light, but increased it in darkness. (5) Ascorbic acid, applied as a post-treatment after a pretreat­ ment of dithiooxamide, increased the inhibition of cucumber root elongation induced by dithiooxamide in darkness, and changed into inhibition the stimulative effects of dithiooxamide in light. (6) Ascorbic acid, applied as a pretreatment for at least 48 hours before a post-treatment of dithiooxamide was able to reduce signifi­ cantly the inhibition of cucumber root elongation induced by dithio­ oxamide both in light and in darkness. (7) These interactive effects of ascorbic acid were always much more considerable than the slight stimulations or inhibitions of cucumber root elongation which were sometimes induced by ascorbic acid alone in solution, so that it can be truly spoken of antagonisms or synergisms of ascorbic acid with coumarin or with dithiooxamide. (8) Gluco-ascorbic acid and d-iso-ascorbic acid exhibited inter­ active effects similar to those of ascorbic acid upon the inhibition of cucumber root elongation induced by dithiooxamide. 3-(alpha-iminoehhyl)-5-methyl tetronic acid increased this the inhibition approx­ imately in proportion of its own inhibitory effects. Addition of ascorbic acid to solutions of thiourea produced a synergistically increased inhibition of cucumber root elongation in either light or or darkness. No antagonism was observed. (9) These results are discussed relatively to the conditions of illumination and to the time and the order of application of the chem­ icals. A complex biochemical system of interrelationships between the metabolism of Vitamin C in plants and -SH groups on the first hand and coumarin derivatives on the other hand is suggested. PART III THE ANTAGONISMS OF DITHIOOXAMIDE AND ASCORBIC AC ID TO THE AUXINS I. INTRODUCTION For several years, plant physiologists have wondered about the physiological changes responsible for the spectacular effects caused in plants by the natural hormone IAA and by some synthetic growthregulators which are now extensively used in agriculture and the objects of an important business. It was a logical idea to try to tie them up with some naturally occurring plant growth substances. Coumarin derivatives and other unsaturated lactones have outstanding growth properties, and are of wide occurrence in plant tissues. Recent discoveries may allow to think that auxins induce deep changes in the natural distribution of these substances, which may then account for their ability to control plant growth mechanisms, as has been actually suggested by Van Overbeek in a recent paper (153)• We should indicate at this point that it is commonly considered, (but not proven) that all auxins actupon plants in some fundamentally similar way. In the further progress of this work, it will be spoken of the auxins as of a class of substances presenting the same general physiological activity. If Van Overbeek*s idea is right, and since the data of the two first parts of this work indicate that ASA and DTO are interferring with the metabolism of coumarin, it seems that these two substances might also be able to influence the still mysterious fate of the so-called "auxins" inside of the plant tissues. 95 Therefore, after showing how this concept of interrelationship between natural unsaturated lactones and auxins became progressively elaborated, and what few experimental works support Van Overbeek's attractive hypothesis, the following review of literature has been extended to the facts suggesting a similar connection between auxins on the first hand, and thiol groups and ASA on the other hand. A. Review of Literature 1. The Relationships between Unsaturated Lactones and Auxins The possibility of an interrelationship between unsaturated lactones has apparently not been seriously considered before 1943. This year, Veldstra et al. (154), after reporting an antagonism of coumarin to NAA in the pea test, advanced the idea of a connection between unsaturated lactones and the natural auxins a and b which can readily assume a saturated lactone structure. This might in turn give an unsaturated lactone ring which, in the case of auxin a would be the same as that of hexenolactone (parascorbic acid) and coumarin, both of which are physiologically active. Following his wwn observations that the phytostatic action of coumarin closely resembles the effects produced by 2,4-D, (6, 8), Audus, in an extensive review of the field of plant growth-regulators activity, (7) suggested that unsaturated lactones and auxins may act on a same metabolic process, namely the dehydrogenases (-SH enzymes). Larsen (90), in the course of an investigation of IAA precursors in plants, observed that mixtures of unsaturated lactones and synthetic 96 indole-3-acetaldehyde give the same curvatures in the Avena test than the neutral growth-substance present in certain plant extracts. Out of these lactones only parasorbic acid give a slight response in the Avena test. From these data, Larsen suggested that a double system of growth controlling substances may exist in plants, of which the auxins would constitute the growth-promoting complex, and the unsaturated lactones, the inhibiting part. Moewus (107) in 1949, reported an antagonism of coumarin and heterauxin. In the inhibitory zone of IAA, the effects of the two inhibitors were add­ itive. Buston et al. (25) also reported an antagonism of dl-hexenolactone to IAA in the Avena test, and to natural auxins contained in plant extracts, although it did not exhibit any auxin activity ( the d-idomer, parasorbic acid has some according to Larsen). The antagonism to auxins was overcome by beta-alanine. Van Overbeek et al. (152) observed an antagonism of transcinnamic acid to auxin in the pea stem test; cis-cinnamic acid exhibited auxin acitivity. These properties of the cinnamic acids are of interest because they are structurally related to coumarin (see appendix, formulas). The works previously cited afford only indications of a orelationship which is deduced from theoretical considerations, from the observations of similar effects upon plants, or, more convinc­ ingly, of direct antagonisms or interactions. Fortunately, a direct evidence of a connection between a synthetic auxin, 2,4-D, and a natural coumarin derivative, scopoletin, has been recently contributed by Fults et al. ( ) who discovered 97 that 2,4-D induced accumulation of scopoletin in several plants, and suggested that an excess of this toxic substance might be the cause of the phytotoxic effects of 2,4-D. Van Overbeek then gave its present form to this hypothesis. Bringing together several facts, such as the natural occurrence of small amounts of scopoletin in many plant tissues, and its toxicity, the resemblance of the phytocidal symptoms of coumarin and of these of 2,4-D, the similar herbicidal selectivity of the later and of beta-methyl-unbelliferone (another coumarin derivative) he suggested that 2,4-D induced a deep change in the metabolism of plants, as indicated by the works of Hamner, Sell and other workers of Michigan State College (67, 97, 108, 130), resulting in the accumulation of toxic coumarin derivatives. 2. The Relationships between Thiols, ASA and the Auxins. Most of the works dealing with ASA have been deboted to its influence on physiological processes rather than to the external effects produced by its application to plants, which is quite logical since, due to its presence in all of th em, plant tissues do not respond very clearly to external treatments with ASA in every case. Besides, its determination in vivo being relatively easy provides a useful tool for detecting metabolic changes. a. Distribution of Auxins, Vitamin C and -SH groups in Plants. Soon after the discovery of its growth-regulating properties, Clark (.37; investigated ASA distribution in Avena Coleoptile and compared it with those of auxin and chlorophyll. He found that the 1 98 tip was .richer than the base in ASA (reduced form), and the opposite for dehydroascorbic acid (oxidized form of ASA); there seemed to be a correlation between chlorophyll and ASA content in Avena tissues from seedlings grown in light. He observed also that extracts from basal sections of coleoptile would oxidize ASA mor than extracts from apical sections, the same occurs for the destruction gradient of auxin the coleoptile. These observations have been donfirmed by several workers. Reid (126) found a correlation between dry w&ight, ASA content and activity of the cells in cowpea seedlings. Shaw et al. (131) found the highest concentrations of ASA in the *most actively growing parts of broad bean seedlings. As precedently indicated, it is well known that the amount of ASA, usually nil at the start of germination, rises sharply during the early most active stages of development of the seedling, then drops off.: rapidly. The occurrence of the highest concentrations of ASA in the most actively growing parts of plants has been also observed by Lebrec (94)j who furthermore showed that it is not due to a greater stability of ASA in these tissues, but to a greater capacity of younger tissues for synthesizing ASA. As pointed out by Clark, the gradient of ASA concentration in plant tissues suggestively resembles the corresponding gradient of auxin. It is interesting to recall here the already cited works of Pett (7) and Kretsovitch et al. (86), and others who have shown that the amount of natural -SH groups in young seedlings undergoes variations quite similar to those of ASA. Thus, ASA and -SH groups are concen­ trated in acitvely growing tissues, the same as for the natural auxins. 99 However, it should be pointed out that the similarities of amount and distribution of ASA, IAA and -SH groups in plant tissues do not allow any positive conclusion regarding an interaction bet­ ween them. It would probably be wiser to consider a complex of growth substances, working together, as suggested by Kunning (88) after his study of the similar stimulation of cambial activity induced by IAA, ASA-and other growth-factors in decapitated bean and sunflower. b. Interactions of ASA and thiols with auxina. Immediate evidences of interactions of ASA and of sulfhydryl derivatives with auxins have been contributed by two kinds of works: observation of antagonism of these chemicals to the effects of auxins, and study of the inactivations by one group of chemicals of the enzymes controlling the metaboiim of the other group of substances. Zopf (167) observed that the toxic effects of NAA were antagonized by a mixture of Eitamin B^_ and C; however he did not determine exactly what part Vitamin C played in this antagonism. Podesva (119) reported that the toxic effects of naphtyl-octanoic acid upon radish were decreased by addition of thiourea, and completely suppressed by a mixture of thiourea and vitamin C. In contrast, Dykyj et al. (50) did not find any decrease in NAA acitvity from the presence of either thiourea or ASA in "extensive vegetation experiments". Bonner (19) investigating the effects of amino-acids upon the growth of excised sections of Avena Coleoptile in sucrose medium found that cysteine was outstanding for the inhibition of the growth due to IAA. 100 Speaking of cysteine, we may mention here that Hansch et al. (69), in a recent paper, advanced the hypothesis that auxins may attack the cysteil group of proteins fey an "ortho-reaction", which seems highly theoretical. Recent work by Mitchell et al. (106) indicates that applic­ ation of para-chlorophenoxyacetic acid just before harvest preserves a relatively high content of Vitamin C in bean pods; however, since this is accompanied by a better storage quality, the higher ASA content may just be the result of this improved keeping quality. Similar effect of 2,4-D for Red McLure potatoes was observed by Fults et al. (53)* West et al. (164) observed that the inhibitions of oxygen uptake induced by 2,4-D in the roots of lupine was sign­ ificantly reduced only by ASA out of a number of chemicals, from what they deduced that ASA oxidase is attacked by 2,4-D. The effects of auxins upon ASA oxidase have been the object of several recent investigations, together with other enzymes systems likely to be attacked, or to be interfered with by auxins. 7/agenknecht (157) in 1947 did not find any interaction between ASA oxidase and auxins. Negative results were also obtained by Mitchell et al. (105), but these workers observed that IAA did not induce the same effects as other auxins on the respiration of bean roots. Respiration was stimulated by IAA, but depressed by NAA and by 2,4-D at high concentrations; since Wagenknecht (158) found that bean roots contained an enzyme capable of oxidizing IAA which was inactivated by diethyl-dithiocarbamate, they tested 101 a mixture of diethyl-dithiocarbamate and IAA at high concentration and found it to "behave similarly to the other auxins. Then Wagen- knecht et_ al. (159) demonstrated that in crude bean-leaf and beanroot juices, the oxygen uptake was stimulated by the presence of ASA, and that the stimulation was significantly reduced by addition of auxins, although they had no inhibitive effect on oxygen up­ take in the absence of ASA (except for NAA and 2 , k - D t in the only case of juice from roots). Similar observations were made by Miller et al. (104) with crude juice expressed from barley seedlings. Finally Newcomb (110) demonstrated recently that IAA at low concen­ trations stimulates the ASA oxidase activity of tobacco pith cells (up to 1900 ■%) and inhibits it at higher concentrations. These effects had some bearing upon cell growth and respiration. There are also indications that ASA and -SH compounds may interfer with auxin metabolism through a mechanism involving polyphenol-oxidases. Wetmore et_ al. (165) found that NAA checked the growth of shoot apices of foxtails, on agar.blocks. This inhibition was somewhat reduced by phenylthiourea, diethyl-dithiocarbamate and, more effectively, by ASA. A polyphenol oxidase present at the cut surface seems to oxidize the auxin in the medium as well as that which diffuese from the apex. Similar results were obtained with fern apices; curvatures of Avena coleptile could be obtained by auxin diffusion technique from foxtails*shoots only when ASA was incorporated in the agar block. For Larsen (91), these results may indicate that ASA interfers with an oxidative step in the conversion of the precursor tryptophane to IAA. 102 The enzyme concerned in this process would be a polyphenol oxidase, of which we already know that it cqn indirectly oxidize ASA (139), which has even been suggested to be part of itscoenzyme fraction (18, 129). B. Problem. A few evidences supporting Van Overbeek's attractive hypothesis can be found in the literature pertaining to plant-growth-substances. Since this hypothesis assumes that auxin phytotoxicity is the result of a disturbance of metabolism which produces an accumulation of toxic coumarin derivatives, it was reasoned that chemicals able to reduce the inhibitory effects of coumarin should also overcome those of the auxins, if van Overbeek's idea was right. It has been demonstrated in the two first parts of this work that DTO and ASA were able to overcome the inhibitions produced by coumarin. A few examples of antagonistic interactions of thiols and of ASA with auxins have also been found in the literature, as well as some indications that auxins may be able to affect ASA oxidase; it seems also that thiols and ASA may interfer with the normal metabolism of IAA, and the transformation of neutral to active natural auxin in plant tissues. It was therefore an attractive idea to investigate the effects of DTO and of ASA, both antagonistiets of coumarin, upon the inhib­ itions of root-elongation induced by the auxins in cucumber seedlings. II. EXPERIMENTAL RESULTS Only the three most important so-called "auxins" presently known have been investigated, namely: 2 ,4- dichiorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and alpha-naphthaleneacetic acid (NAA). Because 2,4-D produces the most spectacular effects on plants, is the most important commercially, and has actually been shown to induce accumulation of ASA and of coumarin derivatives (scopoletin) in plant tissues, this work was first and most extensively devoted to its interactions with DTO and with ASA. The natural growthregulator, IAA, was then investigated; the last tests were devoted to a rapid study of NAA to show that the same results could be ex­ tended from 2,4-D and IAA to this chemical. Only concentrations of the auxins giving moderate inhibitions were tested, so that the physiological processes of growth were not irreversibly suppressed, and could therefore be restored by the antagonist substances under investigation, if they were to be effective. In all the experiments now reported, a techniqua of mixed constant solutions has been used as it has been previously described. 104 A. Reduction of the Inhi~bitory Effects of 2,4-D 1. The Effects of Dithiooxamide a. Experiments with Marketer Cucumber Seeds* The first experiments were conducted with Marketer cucumber seeds and the same troubles were encountered as in the case of the antagonism of DTO to coumarin in Part I of this work. Only low concentrations of DTO were used in this experiment, because they were believed to be specially effective at that time. The experiments were carried out in the laboratory, without any attention to the conditions of light or temperature. In a preliminary test (33-A), only two Petri dishes with ten seeds from the old sample each were used. Slight indications of an antagonism were observed at the concentration of 0.1 ppm of 2,4-D. Experiment 33-3, performed in the usual way with measurement of the seeds after 4 and 7 days, confirmed these results. However, in experiment 'fa-k designed for a preliminary inves­ tigation of the interaction of ASA with 2,4-D and of its possible synergism with the antagonism of DTO to 2,4-D, the later antagonism was questionable; two sets of checks receiving 2,4-D alone had been used in this test, and they gave different results, so that, according to one or the other, this antagonism did or did not function. Because one of the checks had been readied at the beginning of the setting up of the experiment, whereas the other one had been prepared at the end, just before the lights were turned off, a difference in 105 the illumination during the very earliest stages of germination was tentatively made responsible of this discrepancy. Therefore in the next tests run in alternate illumination, two replicates were started: one in the morning, the other just before turning the lights off. In 35-A, performed -with seeds from the old sample, both replicates gave evidences of antagonism of DTO to the inhibition induced by 2,4-D. But in the experiments 35-B, 35-C, 36-A and 36-B, where new seeds were used, erratic results were obtained. The antagonism was sometimes visible in alternate illumination, slightly in darkness, never in continuous light. b. Experiments with Burpee Hybrid Cucumber Seeds. The failure of the precedent tests to give consistent results was blamed upon the poor quality of the new sample of seeds. Therefore, the investigation was carried over with seeds from the variety Burpee Hybrids, in the controlled conditions of the special room previously described. All the solutions were made of acid water at pH 3*5« A first experiment (37-A) with this new material gave good evid­ ences that the antagonism functions as well in continuous illumination, in alternate illumination or in continuous darkness. The higher the concentration of DTO, the better the antagonism, which suggested that part of the failure of the previous experiments with Marketer seeds may have been due to the use of too low concentrations of DTO. 106 Two other experiments were run later with an other sample of seeds from the same origin, and higher concentrations of DTO were tested, both in continuous light and in continuous darkness, (37-B and 37-C), and with the same success. A concentration of 100 ppm of DTO was found to be most effective to antagonize the effects of 2,4-D, in all case*. As can be seen in 37-B, the better antagonistic effects were obtained at concentrations of DTO which were slightly toxic by themselves; the concentrations which promoted the root elongation were less effective for the antagonism. The best reduction of inhibition was given by100 ppmof DTO in experiment 37-C in light, exactly 50% o f the inhibition was suppressed. Inhibition caused by 2,4-D (o.l ppm)................... 32.3 mm Inhibition caused by 2,4-D (0.1 ppm) plus DTO(100 ppm).16.1 mm 2. The Effects of Ascorbic Acid. a. Experiments with Marketer Cucumber Seeds. The preliminary experiment (34-A) has already been mentioned and the discrepancy between the two sets of checks has been report­ ed (pagel04). However, clear indications of an antagonism of ASA to the inhibition induced by 2,4-D can be found within each series receiving mixtures of DTO, and 2,4-D with (treatments) or without (checks) ASA. In another experiments (35-A), the seeds of the older sample gave further evidences for the existence of this antagonism. 107 Erratic results were obtained in experiments 35-B, 35-C, 36-A and 36-B 'where new Marketer seeds were used. Once only (35-B) the antagonism was apparent as well in darkness as in light, but three times (35-C, 36-A and 36-B) it did not function in darkness. Opposite trends were manifest in continuous or alternate illamination for experiments 36-A and 36-B. b. Experiments with Burpee Hybrid Cucumber Seeds. In the controlled conditions of the special rood previously described, the investigation of the antagonism of ASA and 2,4-D was carried over with Burpee seeds. All the following experiments were done at pH 3»5» From the three experiments 37-A, 37-B, and 37-C, it can be seen that ASA reduced the inhibition induced by 2,4-D as well in darkness as in continuous or alternate illumination. From 37-B and 37-C, in which a check was run for each concentration of ASA tested, it is evident that the difference in root length is much larger between check (2,4-D alone) and complex treatment than between check (ASA alone) and controls. The best results were obtained for a concentration of 1,000 ppm of ASA in light (37-C), as follows: Effect of 2,4-D (0.1ppm)............................... -32.3 mm Effect of ASA (1,000 ppm)............................+6.9 mm Sum of these effects.................... ........ -25.4 mm (-Inhibition ) (^Promotion) Effect of 2,4-D (0.1ppm)plus ASA (1,000 ppm)............ -11.3 mm so that the addition of ASA (l,ooo ppm) to the solution of 2,4-D resulted in the loss of almost two thirds of its inhibitory power. 108 B. Reduction of the Inhibitory Effects of Indole-Acetic Acid. 1. The Effects of Dithioaxamide The interactions of DTO and IAA have been investigated along two lines of research: the effects of low concentrations of IAA upon the inhibitions caused by high concentrations of DTO, and the effects of DTO upon the inhibitions caused by higher concentrations of IAA. In all the following experiments, seedsof the Variety Burpee Hybrid were grown in mixed constatn solutions of the chemicals in the special room where light and temperature were automatically controlled. a. Indole Acetic Acid against ~the Inhibitions caused by Dithiooxamide. Two experiments were conduceted where it was tried to overcome with IAA the inhibition induced by a high concentration of DTO. In the first one, 3&-B, concentrations of IAA ranging from 0.1 ppm to 2 ppm were used; they exhibited no.antagonistic effect to the inhibition or toot elongation caused by 200 ppm of DTO. On the contrary, the trend was rather of an increased toxicity of the mixture over the check receiving DTO only. Another test was run to ascertain that a higher concentration of IAA would not produce the desired antagonism, but here also (38-D), it was found that the inhibitions induced by IAA (5 ppm) alone and DTO (150 ppm) alone were additive rather than antagonistic, in light and there was no interaction in darkness. 109 b. Dithiooxamide against the Inhibitions caused by Indole Acetic Acid An antagonism of DTO to the inhibitions induced by IAA was repeatedly observed, which is apparently quite similar to the antagonism of DTO against 2,4-D. In the case of IAA, the most effective concentrations of DTO for antagonism were low. In experiments, 38-A and 38-C, 20 ppm of DTO gave the best reductions of inhibition, in all cases of continuous or alternate illumination and of darkness. Only one concentration of IAA was used, 5 ppm, which gave moderate inhibitions. The best results were obtained in light in 38-C, where the roots grown in the mixture were about twice as long as the roots receiving IAA alone, which corresponded also to a suppression ofhalf of the inhibition induced by IAA, as indicated below: Inhibition caused by IAA (5 ppm) alone........... ....48.5 mm Inhibition caused by IAA (5 ppm) plus DTO (20ppm)....... 23.0 mm In two other experiments, in which another sample of Burpee seeds was used as material, the best results were obtained with about the same concentration of DTO (10-20 ppm). In 39-A, all the solutions were made of acid water at pH 3*5 > two concentrations of IAA (5-10 ppm) were used; in 39-B, where the solutions were made of distilled water like in the previous experiments, two concent­ rations of IAA were also used (2, 5 ppm); in both cases, the best results were obtained with 5 PPm of IAA, and 10 or 20 ppm of DTO. Here agin, the antagonism worked about as well in darkness as in light. 110 2. The Effects of Ascorbic Acid Two experiments (40-A and 40-B) were performed with mixtures of IAA and ASA in light and in darkness. In both cases, clear reductions of the inhibitions induced by IAA alone were observed, specially in light. In experiment 40-A for instance, in light 2 ppm of IAA induced 45# of the root length to be inhibited (.42.8 mm), which were reduced to 14# (12.8 mm) by addition of 750 ppm of ASA, that is: 70# of the inhibition was overcome by ASA. In darkness, less striking results were obtained. However, in experiment 40-B, the roots from seeds grown in mixtures of ASA (750 ppm) and IAA (10 ppm) were more than one and a half times as long as those from seeds grown in IAA alone (44*6 and 29.3 mm respectively). The results were less coherent than in light, and no sharp optimum of ASA concentrations was visible, whereas the most consistently effective concentration in light was 750 ppm of ASA. B. Reduction of the Inhibitory Effects of Naphthalene-Acetic Acid 1. The Effects of Dithiooxamide From the experiments 41-A, 41-B, and 42-A, evidences were obtained that DTO may reduce the inhibitions of root-elongation induced by NAA, provided these inhibitions are moderate. This was especially visible in experiment 41-A, in light in which distilled water solutions were used. The reduced root growth obtained from seeds treated with NAA was more improved Ill by addition of ASA when ihe inhibitory treatment was of 0.1 ppm of NAA than when it was of 0.3 ppm or 1.0 ppm of NAA. In the same experiment in darkness, DTO did not produce any beneficial effect. However in experiments 41-B and 42-A performed with acid water solutions, good evidences were obtained that in darkness also DTO may reduce the inhibition of cucumber root elongation induced by NAA at lower concentrations (0.1 and 0.05 ppm). In experiment 42-A in light it was also clear that DTO could not overcome significantly the inhibition caused by 0.25 ppm of NAA, but was effective in the ease of a treatment of 0.1 ppm of MAA. In all cases the optimum concentration of DTO appeared to be around 10-20 ppm. ■* 2. Effects of Ascorbic Acid. ASA was observed to be more potent than DTO to overcome the inhibition of NAA, which is similar to the results obtained in the cases of coumarin and of the other auxins. This was observed as well in darkness as in light (42-A and 42-B). In experiment 42-A, 0.1 ppm of NAA alone induced an inhibition of cucumber root elongation which was 36.3% the total root- length (30.8 mm out of 84.9 mm) in light; this was reduced to 6.5% (5.5 mm) by the addition of 1,000 ppm of ASA, which was a suppression of more than 80% of the total inhibition. In darkness in the same experiment, 0.05 ppm of NAA gave an inhibition of growth which was 43.8% of the total root length (42.2 mm out of 96.4 mm); this was reduced to 9*6% (9«3 mm) by addition of 1,000 ppm of ASA, so that more than 75% of the inhibitions used by the auxin was sup­ pressed. III. DISCUSSION The objective of the experiments reported in the precedent pages was to investigate the effects of DTO and of ASA on the inhibitions of root elongation induced by the auxins. The results show that these inhibitions were significantly reduced by the addition of both substances to the solutions of the auxins, in a way which was similar to the antagonisms that they opposed also to the effects of coumarin. Such similarity was expected on the grounds of Van Overbeek's hypothesis that at least part of the toxic effects of the auxims are caused by metabolic changes which result in the accumulation of toxic coumarin derivatives: coumarin antagonists were therefore expected to be also antagonistic to the auxins. The only important difference between the two kinds of antag­ onisms is that DTO seems to reduce the inhibition induced by coumarin only in light whereas it antagonizes the auxins as well in darkness as in light. In all cases, for the auxins as for coumarin, it was observed that ASA was a more potent antagonist than DTO, as shown by the technique of constant solutions of mixtures of chemicals, the only one used in part III. Although the general results of these experiments bring forth good evidences in favor of Van Overbeek's theory, it does not seem H3 essential to consider an intermediary poisoning "by coumarin in order to understand how the auxins can arrest growth and he antagonized by DTO and ASA. There are evidences that the auxins may directly affect the enzymes controlling ASA metabolism, such as ASA oxidase (110, 159) chiefly and also polyphenol oxidase (165). Quite a few evidences point to the importance of polyphenol oxidase for ASA metabolism (123) and for respiration (20); ASA oxidase also seems to be important for respiration (l6l, 110); ASA oxidation-reduction system, besides a certain importance in respiratory processes (78), may play a key role as a bridge between oxidative and glycolitic processes through its influence on phosphatase activity (57* 79)• That these effects of auxin or others may induce an accumul­ ation of coumarin derivatives is possible, although only few evidences support it; by themselves, however, they afford an explanation quite sufficient for the growth inhibitory effects of auxins and their an­ tagonism by DTO and by ASA. An addition of ASA would for instance supplement the natural Vitamin C immobilized by oxidase inactivation, and thus allow the terminal oxidation to take place, whereas the dithiols might interfer directly with the inactivation of the enzymes by the auxins and thus slow down their effects. Such a mechanism is most likely to function in cucumber seedlings which are known to be especially rich in ASA oxidase. It may be temporarily concluded that probably both indirect and direct mechanisms may account for the inhibitions of root elongation caused by the auxins, and their overcoming by DTO and ASA. I Because coumarin is involved in this matter and therefore it is impossible not to refer to the data disclosed in the first parts of this work, a general discussion will now he presented, in an attempt to tie together all the interactions investigated in this work. IV. SU'MMAJtil The effects of dithiooxamide and of ascorbic acid upon the inhib­ itions of cucumber root-elongation induced by the "auxins" 2,4-D, indole-acetic acid and naphthalene-acetic acid have been investiagetd in Petri dishes under controlled conditions of light and temper­ ature. In every case, both in light and in darkness, dithiooxamide and ascorbic acid were able to reduce the inhibitions induced by the auxins, and sometimes to suppress them almost completely. In every case it was observed that ascorbic acid was more eff­ ective than dithiooxamide in overcoming these inhibitions. A possible mechanism for an action of the "auxins" upon the metabolism of Vitamin C in plants is suggested. GENERAL DISCUSSION The various interactions occurring between several natural or synthetic substances which possess outstanding plant-growth-regul\ ating properties have been experimentally studied, and their meaning separately interpreted in the three parts of this work. At the end of this investigation, it seems worthy to bring together all these data, and to coordinate them in a synthetic discussion, where the results obtained in the first and the third parts should become / integrated in the scheme elaborated at the end of part II, which appears to be the very knot at which the physiological effects of the substances tested are entagled. Experimentally, ASA is the heart of the interactions observed since it antagonizes the effects of coumarin, those of the auxins, and antagonizes or reinforces those of DTO according to the circum­ stances . Therefore the complex system is centered around the metabolism of ASA, which, applied alone, does not affect much root develop­ ment. Thiols compounds and sulfhydryl groups may int erfer with its metabolism in two ways: by attacking the oxidawes enzymes which control its oxidation-reduction-activity, or by stabilizing it dir­ ectly; ASA, in return, may reactivate the -SH enzymes which may be inactivated by the thiols (formation of dissulfide) or the unsaturated lactones, and so constitute another pole of the system directly connected with the three other constituents of the system: -SH groups, ASA and coumarin. A last process connects ASA to the coumarin derivatives, the normal metabolism of which can be blocked by high concentrations of ASA. (Fig. 11) The antagonism of DTO to the inhibition -• induced by coumarin may enter into the scheme in two ways. One would be a direct pro­ tection of the -SH enzymes from the inactivation by coumarin; the second one, would indirectly function through an intermediate upset of the metabolism of ASA which would for instance accumulate and arreet the poisonous metabolism of coumarin, or protect the -SH enzymes. In the present status of our knowledge, both mechanisms should be held for equally probable. Analogies between the antagonisms of DTO and of ASA to coumarin are arguments for the indirect mechanism (such as the general similarity of the effects of DTO and of ASA upon the lettuce germination inhibited by coumarin, the lesser antagonism of ASA to coumarin in the transfer experiments in dark­ ness resembling the failure of DTO to antagonize coumarin in dark­ ness in case of cucumbers); the fact also that light conditions affect the interaction of DTO with ASA would make possible to under­ stand why the antagonism of DTO to coumarin does not function in darkness, because the intermediate step involving ASA would thus be changed; the greater potency of ASA for all the antagonisms would also suggest that ASA acts more directly than does DTO. However, the direct mechanism has the advantage of explaining why coumarin is toxic, besides its simplicity and its great likelihood of actual existence. 119 As for the intergration of the results obtained with the auxins into the scheme of interactions previously described, some diff­ iculties arise, cheifly as regards the accumulation of coumarin derivatives. The direct mechanism suggested in part III and by which the auxin would affect the oxidases controlling ASA metaboilsm fits well into the system. By blocking these oxidases, the auxins may be able to affect the plant respiration, and the metabolism of ASA, which are important for the growth processes; addition of ASA to the solution would then supplement the ASA previously immobilized and addition of DTO would directly interfer with the attack oi' the oxidases by the auxins. At first, the accumulation of coumarin deriva t ives induced by the auxins seems easy to understand. One can imagine that the auxins, by blocking the oxidases, may induce an i mmobilization and an accuiiulation of ASA (which been actually observed twice, 53, 106), which in turn would inhibit coumarin derivatives normal metabolism (4) and therefore cause them to accumulate. According to VSn Overbeek, those would then be toxic to plants. Auxin toxicity would thus be the indirect result of ASA accumulat ion, which is difficult to be conciliated with the antagonisms between auxins and ASA which have been observed in the present investigations, when more ASA was supplied to the plants. It would then become necessary to suppose that ASA is afele to induce two opposite effects at the same tim e (accumulation of coumarin derivatives, and counteraction of t h eir toxic effects, 120 such as protection of -SH enzymes), and to assume that, according to the level of its concentration (increased indirectly hy addition of DTO, directly "by addition of ASA) one or the other becomes proeminent. This seems to he quite improbable. However, it may be questionable that the accumulated coumarin derivative be toxic, if their accumulation is due to the mechanism previously imagined, because this mechanism implies a suppression of their natural metabolism and therefore of their utilization, rather than an increased manufacture: it is doubtful that they may be harmful if they remain unused. The antagonistic effects of ASA and of DTO in this case might be explained by the direct mechanism suggested in Part III. It is also possible that the accumulation of ASA induced by the auxins is not large enough to inhibit the metabolism of coumarin deri­ vatives and cause their accumulation. In this case, the accumulations of coumarin derivatives and of ASA produced by the auxins would be independant; the accumulated coumarin derivatives would be toxic until an increased amount of ASA (directly supplied by addition of ASA or indirectly by addition of DTO) suppresses their metabolism or counteracts their effects by one of the mechanisms previously included in the.general scheme of interactions. The auxins appear therefore to interact with the substances previous­ ly coordinated in the complex natural system by two main processes. One involves an attack of the enzymes (oxidases) controlling ASA metabolism; the second one, which is an accumulation of coumarin derivatives, is largely unknown, but there is a possibility that it be a consequence of of the metabolic upset caused by the first one. 121 These two lines of auxin activity are included in the definitive scheme presented in Fig. 12. It should be emphasized at this point that this scheme does not pretend to account for all the growth regulating properties of the substanced investigated, but is only a coordination of those processes which are most likely responsible for their multiple interactions reported in the present work. Beyond the hypothesis and the suggestions which are the only tools of the human mind for a better understanding of complex natural processes, some facts should remain from this work: ascorbid acid and dithiooxamide exert upon root development very complex interac­ tive effects (synergistic or antagonistic according to the circumstances of the experiment) and both substances antagonize the similar inhibitions of root growth induced either by coumarin or by the so-called "auxins" 2,4-D, indole-acetic acid and naphthaleneacetic acid. AUXINS ASCORBIC ACID Metabolism Direct Inhibition of Metabolism Polyphenol Oxidase COUMARIN and Tyrosine -----*■ Derivatives * Stabilization * Figaro 12 Suggested interrelationships between coumarin, —SH groups ascorbic acid and the auxins* ■ GENERAL SUMMARY am The interactive effects of dithiooxamide and of ascorbic acid upon the development of roots and its inhibition by coumarin and by the "auxins" 2,U_B, indole-acetic acid and naphthalene-acetic acid have been investigated in Petri dishes under controlled conditions of light and of temperature, with cucumber for root-elongation and lettuce for germination percentage. (1) Treatments of dithiooxamide which were stimulative in light were strongly inhibitive in darkness for the elongation of cucumber roots; no stimulation was observed in darkness; the inhibitions induced by dithiooxamide, whether in light or in darkness, were not considerable before the third day after germination was started. Dithiooxamide was able to increase the early low rates of lettuce germ­ ination in light at 29°C. or in darkness at 25° C.. Ascorbic Acid alone did not produce any significant effects on the development of roots at the concentration which were used (below 2,000 ppm). (2) Ascorbic acid was able to reinforce synergistically or to reduce the inhibitions of cucumber root elongation induced by dithio­ oxamide depending upon the conditions of illumination, the procedure of testing (simultaneous or successive applications of the chemicals) and the order and the length of the treatments, in the case of successive applications. (3) Both dithiooxamide and ascorbic acid were able to reduce the inhibitions of cucumber root elongation induced by coumarin, in light only in the case of dithiooxamide, in both light and darkness in the case of ascorbic acid. A similar redaction of the inhibition of lettuce germination induced by coumarin was produced by these chemicals, in light and darkness with dithiooxamide, in light only with ascorbic acid when they were simultaneously applied with coumarin throughout the experiment; with both chemicals, it took place only in darkness when they were separately applied after a presoaking in a solution of coumarin alone in darkness (case of an artificial dormancy). Ascorbic acid was more potent in the ca.se of cucumber, dithiooxamide was more potent in the case of lettuce. (4) Both dithiooxamide and ascorbic acid were able to reduce the inhibitions of cucumber root-elongation induced by the "auxins" 2,4-D, indole-acetic acid and naphthalene-acetic acid, both in light and in darkness. In every case, ascorbic acid was more potent than dithiooxam­ ide. 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Tusuke Sumiki, and Hiroshi Hayashi. J. Agr. Chem. Soc., Japan 23:1-5. 1949. (Chem. Abstr. 44:7943. 1950). Zcpf, L. C. The growth effects of thiamin chloride, ascorbic acid and phytohormones on Belladonna and Ricinus. J. Am. P h a m . Assoc. 29:437-496. 1940. Appendix I_. Effects of distilled water solutions of Dithiooxamide (DTO) on the elongation of Marketer cucumber roots grown under laboratory conditions for 4 days. 2/1^/1951 Water (control) 60 78 51 66 7? 6k 63 65 5k 71 69 56 72 70 73 68 58 8k 62 73 7k 73 57 67 60 60 62 67 55 57 Total (30) Average 1959 65.3 Sy2 129.665 S.S. 1742 t (treatme water control) Root-length expressed in mm. DTO DTO 1 Ppn 5 PPm 66 70 79 77 87 87 90 75 65 73 56 77 Ik 73 68 79 68 69 66 63 72 59 58 79 68 63 66 63 66 73 67 77 81 59 7-6 7* 79 77 70 72 83 87 66 56 78 55 66 7*+ 58 76 65 7^ 56 58 68 76 62 74 72 55 DTO 10 ppm 71 56 71 62 67 53 56 75 53 62 66 60 85 71 61 67 78 70 76 58 60 57 79 61 5? 54 65 74 7^ 70 . 2072 69.1 1^5.038 1932 1.25 Not Significant 21^8 71.6 156.250 _____ 2453________ 2.87 Highly Significant ..... 1965 . 65.5 Appendix II. Effects of distilled water solutions of Dithioxamide (DTO) on the elongation of Marketer cucumber roots grown under laboratory conditions for 4 days. 3/27/1951 Water (control) 77 78 7^ 55 63 60 59 93 50 76 75 82 63 68 65 65 62 55 58 81 71 71 66 90 75 78 71 70 70 62 Root-1ength expressed im mm. DTO DTO DTO DTO 2.5 ppm 10 ppm 1 PPm 5 PPm 85 92 85 79 72 90 76 75 84 82 80 92 80 78 73 93 96 so 95 79 64 86 63 75 64 84 76 65 64 82 72 72 62 58 78 73 90 92 76 73 81 88 84 76 77 77 7* 77 70 92 79 73 70 76 83 83 66 92 77 77 91 67 87 71 84 66 72 59 61 70 70 57 69 73 59 87 88 86 83 77 82 76 78 83 76 90 75 71 88 70 78 71 82 80 96 107 94 82 86 75 68 90 72 87 102 64 72 87 81 79 71 89 84 65 73 91 64 90 72 55 Total (30)_20S3 ______ 2390 Average 69.4 79.7 Sy2 147.b07 S*S. 2977 t (water controltreatment) 192.152 1 7 lf~ 4.4 Highly Significant 2460 82.0 204.1l4 239U 5.06 Highly Significant 2315 77.2 2215 73.8 DTO 20 p p A 66 71 73 77 63 85 77 62 60 71*65 81 69 73 70 69 70 7^ 69 66 92 73 64 72 83 67 60 73 76 2l4g ' 71.£ 182.491___ 166.523_________ 3850 2982 ^ . 2.785 1.68________ Highly Hot SignifSignificant icant Appendix III. Effects of simultaneous applications of Coumarin (COU) and Dithioxamide (DTO) in distilled water solutions on the elongation of Marketer cucumber roots grown under laboratory conditions for 4 days, concentrations expressed in ppm. 1/10/1951 Root-length expressed in mm. Water 3S 31 15 32 24 2k 35 33 39 35 45 37 Total cou 150 8 6 8 6 8.5 7.5 7.5 8 - 388 59.5 No. of seeds 12 8 Average 32.3 7.* cou 150 cou 150 cou 150 & DTO 0.1 & DTO 1 & DTO 10 6.5 7 8.5 7 10.5 5 8 7.5 9 7 7 10 8 8.5 8.5 8 6 8 8 7 11 96.5 12 8.0 — 8.5 10 13.5 8 8 5.5 15 12.5 19 14 - - - - 69.5 114.0 9 10 7.7 11.4 Appendix IV* Effects of simultaneous applications of Coumarin (COU) and Dithioxamide (DTO) in distilled water solutions on the elongation, of Marketer cucumber roots grown under laboratory conditions for 4 days, concentrations expressed in ppm. 1/ 28/1951 Water 71 7S 71 76 70 64 78 75 so 72 75 65 88 73 78 75 70 63 82 74 59 66 68 59 55 56 66 73 55 55 Total (30) 2090 Average 69.7 Root-iength expressed in mm. COU 100 COU 100 COU 100 COU 100 & DTO 1 & DTO 7.5 & DTO 5 42 i4 30 39 11 26 35 29 14 27 15 27 24 10 9 31 21 21 11 37 10 16 19 29 14 11 29 30 l4 36 15 23 11 11 29 29 i4 21 28 25 8 18 28 26 14 18 38 27 24 8 31 27 23 13 31 23 12 21 17 25 l4 26 29 17 20 11 30 13 28 25 9 13 28 10 32 9 12 14 35 29 8 36 19 27 14 22 31 27 22 34 25 25 23 35 27 17 24 21 19 39 21 21 20 32 24 20 12 35 l4 24 12 IS l4 24 l4 17 11 17 25 13 4o6 13.5 546 IS.2 907 30.2 779 26.0 COU 100 & DTO 10 42 31 30 30 33 38 34 32 36 30 32 32 31 2J 34 31 36 36 34 30 42 43 33 28 27 29 32 34 33 30 990 33.O Appendix IV. (Continued) Root-length. expressed in mm. cou 150 6 6 6 7 9 13 9 8 6 7 7 10 5 6 6 . 5 7 10 7 5 10 8 8 6 6 8 6 7 7 6 Total (30) Average 216 7.2 COU 150 & DTO 1 13 n 20 13 7 7 8 13 10 17 n 8 10 13 14 12 9 8 8 9 16 22 7 10 12 7 12 13 7 7 COU 150 & DTO 5 27 29 19 21 13 13 15 10 8 7 8 19 19 23 18 24 25 10 17 7 39 21 17 17 19 9 11 8 6 7 33*+ 486 11.1 l6.2 cou 150 & DTO 7.5 ^7 36 27 3^ 25 21 20 18 14 15 20 24 26 27 19 22 l4 12 17 11 19 12 23 21 19 16 16 11 10 631 21.0 COU 3 & DTO 23 31 17 19 24 23 9 19 15 8 9 13 27 20 25 4 14 l4 13 15 18 i4 30 30 18 22 17 23 16 8 557 18.6 Appendix V. Effects of simultaneous applications of Coumarin (COU) and old or new Dithiooxamid® (DTO) in distilled water solutions on the elongation of Marketer cucumber roots grown under laboratory conditions for 5 days, concentrations expressed in ppm. 5/26/1951 Root-1 ength. expressed in mm. Darkness COU 100 COU 100 COU 100 & old DTO 1 & new DTO 1 Root-length expressed in ram. Alternate Illumination ' COU 100 COTJ 100 COU 100 & old DTO 3.75 & new DTO 3.75 68 40 88 69 to) 29 27 27 17 32 39 23 23 17 Total 23^ Ho. of seeds 9 Average 26.0 94 55 42 52 39 39 32 33 26 31 22 4o 22 25 27 30 23 62 24 64 20 24 M? 32 28 26 39 32________ 281 6k) 347 11 31.5 10 10 28.1 6 k 0 41 32 28 21 24 21 24 21 20 300 10 .. 30.0, 22 24 24 20 32 31 30 25 17 92 91 89 76 66 77 72 7M 39 7k? 10 ,7k-5 ... . 17 16 25 16 28 18 30 23 21 19 23 31 21 21 20 14 17 12 26 19 21_______ 225 .239 11 9 25-0 21.7 20 17 22 11 25 20 17 15 20 l4 181 199 10 10 19.9 18.1 57 25 38 31 20 29 26 3*+ 20 280 9 31.1 99 69 68 35 48 ^5 58 3^ 27 21 1£ 521 11 h j.k General Results: Illumination Alternate Darkness Control COU 100 29.0 .23^2 . _ Average of root-length of 20 seeds expressed in mm. Old Sample of DTO New sample of DTO COU 100 COU 100 COU 100 COU 100 COU 100 COU 100 COU 100 & DTO 1 & & & & DTO 1 & & DTO 3.7 DTO 7.5 DTO 15 DTO 3.7 DTO 7.5 27.6 46.0 26.0 21.7 29.9 52.2 23.6 19.0 20.0 20.7 19.0 40.0 24.4 19.2 COU 100 & DTO 15 22.5 18.9 Appendix VI. Effects of distilled water solutions of Dithiooxamide (DTO) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 48 hours. 8/4/1951 Water 32 33 27 29 32 27 29 32 33 31 30 25 31 31 28 36 34 28 29 27 28 27 29 30 26 34 31 28 29 26 Total (30) Average Sy2 S.S. t (comparison with water in light) 892 29.7 26.736 2 ii Root-length expressed in mm. Light Darkness DTO 200 ppm Water DTO 200 ppm 26 29 29 28 28 38 33 27 27 24 30 33 24 26 37 28 33 23 28 25 27 32 23 25 26 32 30 28 24 29 28 26 36 26 27 32 25 27 25 26 33 27 29 23 25 24 32 25 25 25 31 24 23 27 22 23 29 38 31 23 30 34 29 26 35 27 33 29 27 27 31 27 24 29 27 29 23 33 24 32 25 30 23 25 24 23 33 28 23 23 786 26.2 20.792 199 953 . 31.8 ... 30.549 276 5.07 Highly 2.8 Highly Significant Significant _ 763 25.4 — - Appendix VII. Stimulation of the elongation of Burpee Hybrid cucumber roots induced by an application of Dithiooxamide (DTO) in distilled water solution for 24 hours followed by a transfer to distilled water for 4 days in continuous illumination, concentrations expressed in ppm. 82 7^ 62 102 62 97 85 82 56 96 99 7S s7 66 103 64 92 65 59 64 66 79 119 69 103 82 108 72 52 65 Total (TO) 2TO0 Average 79.7 Sy2 S.S. t 97 98 97 79 88 77 99 88 77 108 96 99 100 87 78 108 96 82 117 116 108 114 99 97 122 112 84 80 82 86 2871 95.7 SO 79 77 96 88 82 117 109 105 88 79 123 107 90 92 85 75 ill 91 82 84 79 99 85 85 118 91 86 73 71 96 93 92 116 98 95 102 97 113 97 90 117 105 90 101 124 109 110 102 88 125 93 93 98 96 105 93 91 88 96 2727______ 3013 90.9 100. U 91 88 83 92 106 92 7? 64 90 66 76 71* 96 91 94 88 84 62 91 78 80 68 78 88 91 87 73 65 72 71 0 1 O' Root-length expressed in mm. Test 9-A Test Test 9-B Water Water Water DTO 300 DTO 300 then then then then then water water water water water DTO 300 then water 10*484 io4 102 99 103 93 . 103 100 98 109 90 83 110 107 94 103 101 94 107 103 96 110 92 81 98 97 88 96 81 2458______ 29TO 81.9______ 97.7 199.412 279.767 253.671 305.683 204.946 288.198 1*1.021 8.865______________ 5.589_______ 3.98 2.97 6*23 Highly Significant Highly Significant Highly Significant Appendix VIII. Stimulation of the elongation of Burpee Hybrid cucumber roots induced by an application of Dithiooxamide (DTO) in distilled 'water (DW) solutions for 24 hours followed by a transfer to acid water at pH 3*5 (F) f o r 4 days in continuous illumination, concentrations expressed in ppm. Root-length expressed in mm. Test 23-B Test 26-A Test 26-3 DW then P DTO 300 DW DTO 300 DW DTO 300 then P then P then P then P then P SO " " 6 r 1 103 115 75 115 88 101 92 91 99 113 88 92 80 so 105 73 121 127 130 91 115 91 134 96 107 115 99 77 82 106 103 87 95 91 96 105 72 103 99 71 106 66 111 103 91 63 92 89 100 90 96 95 88 90 ill 94 96 90 88 114 120 88 79 93 114 86 106 76 107 95 106 126 80 98 92 87 104 111 107 79 89 125 88 96 106 93 107 97 100 82 98 113 87 73 89 130 87 95 97 io4 94 111 98 107 79 102 94 121 80 107 75 102 106 95 71 73 93 104 102 89 99 99 103 124 104 114 103 85 93 94 86 111 92 127 89 101 120 94 96 77 77 104 92 114 97 103 77 108 101 110 126 77 93 96 106 109 86 99 67 126 106 85 72 92 95 104 81 106 66 115 73 66 65 89 103 93 87 Total (30) Average Sy2 3.S. t 2692 89.7 3009 100.3 248.064 304.093 ................ . JL752 3.31 Highly Significant 2791 93.0 3293. 109.,8 2602 86.7 2948 98.3 291 .488 268.483 365.661 230.856 13.026 . . .6*27.4 „ 4736 ' 4.09 Highly Significant Highly Significant Appendix IX. Effects of successive applications of Coumarin (COU) in distilled water (DAT) solutions and AscorMc Acid (ASA) in acid water solutions at pH 3*5 (2) °n the elongation of Burpee Bybrid cucumber roots grown under controlled conditions for 5 days in continuous illumination, concentrations expressed in ppn. Root-length expressed in mm. Test 17-A Test 17-B DW COU 75 then DW COU 75 then COU 75 then then P ASA 500 then P ASA 500 ASA 500 106 93 89 69 93 92 86 106 106 91 81 100 103 91 93 90 80 66 106 91 108 85 75 75 71 88 100 96 80 70 90 66 72 89 67 3.25 s4 100 55 77 124 101 66 96 65 62 106 96 87 84 62 100 79 75 112 SO 96 78 97 108 92 101 101 78 98 101 81 100 85 98 86 78 70 95 92 80 80 79 91 94 80 86 80 95 81 85 78 73 75 64 96 89 70 7^ 110 SO 85 73 73 62 101 94 70 72 124 92 76 98 117 80 78 70 95 91 84 68 86 96 95 98 85 90 91 79 78 86 so 89 70 86 62 95 91 73 98 76 72 95 79 100 103 75 75 91 SO 61 70 72 85 Total (30) Average .2509 83.6 27S9 92.3 Sy2 217.295 261.309 S.S. . J3,,iS9„..„... t (treatmentwater control) Significant 2407 80.2 2648 88,3_. 236.339 199.005 2,603. _..5.,88X_ 2.59 Significant 2584 86.3 226.470 3.902 1.82 Not Significant Appendix X. Effects of successive applications of Ascorbic Acid (ASA) in acid water solutions at pH 3*5 (P) and Dithiooxamide (DTO) in distilled water solutions (DW) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days in dark­ ness, concentrations expressed in ppm. Root-1ength expressed in mm. Test 28-B Test 28-A Testi 28-B P then P then ASA 250 ASA 250 P then ASA 250 DTO 300 then DTO 300 then then DTO 300 (2) DTO 300 DTO 300 DTO 300 (1? Total (30) Average Sy2 S.s. t 68 63 61 64 56 51 51 47 81 67 58 72 58 55 62 53 55 52 62 74 56 51 57 50 50 59 57 59 55 54 75 66 62 84 62 68 64 72 65 74 72 63 72 70 82 68 69 60 66 81 67 73 62 79 S3 79 81 68 62 63 1758 58. b 2112 70.4 91 70 61 76 70 70 66 60 56 54 52 67 56 55 66 63 58 63 58 65 59 59 77 66 59 66 63 55 50 52 79 74 69 75 72 67 79 72 70 75 60 68 73 96 75 77 74 74 84 82 69 68 68 67 64 75 75 64 79 69 1883 2193 62.8____ 73«1 104.784 150.224 120.369 161.723 3,304 ~ 3.594 6.05 5*05 Highly Significant Highly Significant 60 57 54 53 50 50 63 50 47 69 68 62 62 47 60 46 49 69 63 67 61 62 57 54. 49 56 73 50 48 46 75 67 66 81 72 72 76 70 66 68 67 72 65 65 80 66 74 71 68 66 65 81 79 73 84 76 73 7? 64 65 1702______ 2137 56.7 71.2 98.386 153.169 8.13 Highly Significant Appendix XI, Effects of successive applications of Ascorbic Acid (ASA) in acid water solutions at pH 3*5 (P) and Dithiooxamide (DTO) in distilled water solutions (DW) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days in continuous illumination, concentrations expressed in ppm. Root-length expressed in mm. Transfer after Transfer after 72 hours 48 hours Test 25-B Test 25-B P then ASA 500 P then ASA 500 DTO 300 then DTO 300 then DTO 300 DTO 300 53 65 60 51 67 61 57 50 54 55 52 53 57 60 61 52 55 67 54 54 61 59 53 63 60 60 52 69 65 56 68 68 73 59 62 72 60 64 57 59 . 58 57 63 76 67 64 61 58 64 76 73 65 78 88 69 59 67 70 82 64 Total (30) 175£> 2001~ Average ____ 57.9_____ b6.7 Sy2 S.S. t 101.274 155.245 2.596______ 5.09 Highly Significant 87 69 71 68 77 67 65 78 68 69 74 70 64 68 75 64 76 65 65 65 7r 74 83 80 65 71 73 65 63 64 76 69 73 71 81 72 72 70 76 74 83 so 85 73 81 71 74 70 73 71 75 78 80 SO 73 77 75 82 85 82 2117 70.6 2282 76.1 150.501 174.244 1.771______ 3.85 Highly Significant Transfer after 72 hours Test 28-A P then ASA 500 DTO 300 then DTO 300 71 63 61 71 63 67 78 77 75 79 65 69 66 63 7? ■ 64 74 64 62 61 79 68 98 74 66 65 66 73 64 61 72 75 87 83 92 75 77 70 68 71 89 78 73 85 76 72 81 68 65 76 75 70 87 81 69 68 7? 74 69 77 2085______ 2281 69.5______ 76.0 146.665 174.845 3.171________ 3.40 Highly Significant Appendix 1. Effects of tap water solutions of Coumarin (COU) and Ascorbic Acid (ASA) on the elongation of Marketer cucumber and wheat roots (var. Henry) grown under laboratory conditions. Concentrations expressed in ppm. A. 1/14/1951 Treatments Marketer cucumbers - Length of the test: Jdays Water ASA 10 ASA 100 g ASA 1000 Humber of seeds 10 10 Average root length (mm.) 23.3 25.S 23.9 34.0 COU .03 COU .1 COU .3 COU 1 Treatments 13 COUJ. Number of seeds 10 12 12 10 l4 Average root length (mm.) 26.2 24.1 25.S 25.9 24.7 Wheat - Length of the test: 3 days B. 1/1S/1951 Treatments Yfeter ASA 10 ASA 100 ASA 1000 Number of seeds 13 12 12 11 Average root length (mm.) 32.3 35.2 30.2 23.O c. 2/3/1951 Treatments Wheat - Length of the test: 3 days Water ASA 10 ASA 100 ASA 500 ASA 1000 Number of seeds 30 30 30 30 30 Average root length (mm.) 23.2 21.S 31.8 36.1 36.5 D. 2/3/1951 Treatments Marketer cucumbers - Length of the test: 4 days Water ASA 10 ASA 100 ASA 500 ASA 1000 Number of seeds 30 30 30 30 30 Average root length (mm.) 40.7 49.0 33.2 41.6 45.2 Appendix 2. Effects of distilled water solutions of Ascorbic Acid (ASA) and Dithiooxamide (DTO) on the elongation of wheat roots (var. Henry) grown under laboratory conditions for 3 days* Concentrations expressed in ppm. Average of root length of 30 seeds expressed in mm. A. 2/7/1951 Treatments Average Length. (mm_Q Percent of controls Water ASA 10 ASA 100 ASA 500 ASA 1000 48.3 33.8 53-g 4l,0 35.9 100.0 111.4 111.4 84.9 74.3 B. 2/15/1951 Treatments Water DTP 20 DTP 66 DTO 200 Average length (mm.) 57.6_______ 46^8_______ 42.4 32.7 Percent of controls________ 100.0_______ 81.2_______ 73.6_______ 56.8 0. 3/3/1951 Treatments Average length (mm.) Percent of controls Water .53-3 DTO 1 48.7 46.1 91.4 S6.5 . 100.0 DTO 9 DTO 10 43.6 _ 81.8 D. 3/7/1951 Treatments Average length (mm.) Percent of controls Water 49.6 100.0 DTO .1 51.0 102.8 DTO .5 48.3 97.4 Water 54.0 100.0 DTO .01 50.0 92.6 DTO .05 52.8 97.S E. 3/12/1951 Treatments Average length (mm.) Percent of controls Appendix 2.. Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) in distilled water solutions on the elongation of Marketer cucumber roots grown under laboratory conditions for 4 days. Concentrations expressed in ppm. Average of 30 root lengths expressed in mm. A. 2/24/1951 Treatments Average length Percent of control B. 3/3/1951 DTO 25 DTO 50 S3.3 73.S 60.1 100.0 88.6 72.1 Treatments Water DTO 50 Average length Percent of control 73.1 100.0 Treatments c. 3/7/1951 Water DTO 200 59.0 43.6 32.9 80.7 59.6 45.0 Water DTO .1 DTO .5 65.9 64.3 66.2 100.0 97.6 100.5 Average length Percent of control DTO 100 D. 4/2/1951 Treatments Average length Percent of control Water DTO 1 DTO 2.5 DTO 5 COU 2 COU 5 COU 10 75.5 76*7 79*0 75*7 71.7 70.4 58.7 100.0 101 .6 104.6 100.3 95.0 93*2 77.7 COU 10 & DTO 5 COU 5 & DTO 1 Average length 56.8 70.2 Percent of control 75.2 93.0 Treatments cou 5 & DTO 5 cou 2 & DTO 1 63.8 feb.7 68.2 67.6 78.3 84.5 88.3 90.3 89.5 103.7 cou 5 & DTO 2.5 cou 2 & DTO 2.5 COU 2 & DTO 5 Appendix 4, Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) and/or Ascorbic acid (ASA) in distilled water solutions on the elongation of Marketer cucumber roots groim under laboratory conditions for 7 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 4/23/1951 cou 100 & DTO 7.5 & ASA 500 33*6 31.9 79.9 93.0 Alternate 41.3 41.3 92.6 54.1 Darkness 36 .S 32.9 42.2 38.6 COU Conditions of Illumination 100 Continuous COU 100 COU 100 & DTO 7 .5 & ASA 500 B. 5/16/1951 COU 100 & ASA 500 COU 100 & DTO 7.5 & ASA 500 Water ASA 100 Continuous 27.4 23.4 54.6 43.7 SO.4 S5-3 Alternate (starting with light) 26.3 25.9 45.9 25.6 114.1 130.1 Alternate (starting with darkness) 30*6 25.7 50.7 22.2 105.9 Decay 20.S 19.3 34.4 21.9 Darkness COU COU 100 & DTO 7 .5 Conditions of Illumination 500 Decay Decay Appendix 5. Effects of simultaneous applications of Coumarin (COU and Ascorbic Acid (ASA) and/or Dithiooxamide (DTO) in distilled water solutions on the elongation of Marketer cucumber roots grown tinder controlled conditions. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 6/1/1951 Length of the test: 6 days, » COU cou 100 100 & DTO 3.3 Conditions of Illumination Water Continuous 103.S 42.9 41.4 72.3 53.5 Alternate* 108.1 33.6 49.7 84.4 47.8 Darkness 109.1 33.9 27.8 62.7 37.2 B. 6/16/1951 COU 100 & ASA 500 COU 100 & DTO 3.3 & ASA 500 Length of the test: 5 days. Conditions of Illumination COU 100 Water 80.1 Continuous Alternate 102.5 110.4 Darkness c. 12 /26/1951 COU 100 & DTO 7.5 41.9 40.6 47.2 40.6 35.7 32.5 Length of the test; 5,toys. cou 37.5 cou 37.5 & DTO 50 & DTO 150 COU Conditions of Illumination COU Continuous 41.9 49.6 40.1 24.8 32.5 Darkness 31.1 25.9 24.1 14.5 19.1 37*5 COU 37-5 & DTO 15 75 cou 75 cou 75 cou 75 & DTO & DTO 50 '& DTO 150 31.0 27.6 19.5 11.9 12.0 15 ... ♦Composition of light: 50$ Fluorescent 50% Incandescent . - Appendix 6. Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) in distilled water solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 7/13/1951 Conditions of Illumination Length of the test: 6 days. ■ cou 75 cou 75 cou 75 cou 75 & DTO .8 & DTO 2.5 & DTO 7.5 & DTO 22.5 cou 75 Continuous 31.6 37*7 36.0 36.5 36.0 Alternate 35.0 35*0 33*7 39*^ 37.1 Darkness 28.!♦ 29.6 31.6 30.7 29.1 cou 150 cou 150 cou 150 cou 150 & DTO .8 & DTO 2.5 & DTO 7*5 & DTO 22.5 13.6 cou 150 Continuous 15.8 18.0 i4.6 16.9 Alternate 13.*+ 17.4 17.1 11.6 - Darkness 19.3 16.5 15.4 15.1 - Length of the ibest:-7 days. B. 9/28/1951 Conditions cou of 75 Illumination cou 75 cou 75 & & DTO 1 DTO 3.3 Continuous^ 35.2 31.8 39*9 Alternate^ 39.2 36.2 35.2 Darkness 38*7 33*0 38.3 cou 75 COU 75 & DTO 10 COU 75 & ' DTO 25 COU 75 & DTO 75 36.0 36.4 39*2 5^.9 42.8 51.2 50.1 35*6 35.1 32.5 39.6 ♦Average of 20 root lengths ♦♦Composition of light: 50$ Fluorescent 50$ Incandescent & DTO 150 Appendix 7» Effects of successive applications of Dithiooxamide (DTO) and Coumarin (COU) in distilled water solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppn. Average of 30 root-lengths expressed in mm. Transfer after 24 hours. A. 10/14/1951 Pre-treatment s Post-treatments DTO 300 COU 75 Water COU 75 COU 75 . Water ... DTO 150 In light 42.0 45.1 64.1 52.5 In darkness 25.0 24.1 56.6 45.2 B. 10/30/1951 Transfer after 24 hours. In light Pre-treatments: Post-treatments: Water COU 150 COU 37.5 17.2 4l.l In darkness DTO 300 COU 150 COU 37.5 IS.2 53.4 Water COU 37.5 DTO 75 COU 37.5 DTO 150 COU 37.5 DTO 300 COU 37.5 36.7 38.4 40.5 40.2 In light 32.7 COU 75 50.8 COU 150 33*5 O Water COU 150 1—1 on Pre-treatments Po st-1reatment s 8 0 Transfer after 4g hours. cou 75 54.8 Appendix 8 . Effects of successive applications of Dithiooxamide (DTO) and Coumarin (COU) in distilled water solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 11/17/1951 Po st-treatments Pre-treatments (24 hours) Water DTO 300 DTO 150 In light COU 10 COU 20 COU HO 65.H H9 .H H0.5 In darkness COU 5 COU 10 COU 20 84.1 71.4 5H .5 DTO 75 80.5 65.H 61.2 70.4 67.5 H9.7 77.0 69.O 51.4 B. 1/29/1952 Post-treatments In darkness Water Pre-treatments (24 hours) Water DTO 25 DTO 50 87.3 88.2 81.8 Appendix 2.. Effects of successive applications of Dithiooxamide (DTO) and Coumarin (COU) in distilled water solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions in continuous illumination for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 12/6/1951 Pre-treatments (24 hours) Post-treatments COU 20 COU 40 Water COU 80 Water 79.7 47.1 43.8 41.9 DTO 300 95.7 68.1 64.0 4l.6 B. 12/27/1951 Post-treatments Pre-treatments (24 hours) Water DTO 300 Water COU 20 cou 4o COU 80 90.9 56.7 48.9 4l.O 100.4 78.2 66.3 52.1 c. 1/14/1952 Post-treatments COU 20 COU 40 Pre-treatments (24 hours) Water COU 10 Water 81.9 53.1 49.7 48.1 37.4 DTO 300 97.7 89.O 7S.3 62.3 38.2 COU 80 Appendix 10. Effects of distilled water solutions of Dithiooxamide (DTO) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions at various intervals of time. Average of 30 root-lengths expressed in mm. A. S/4/1951 Illumination Continuous Treatments Water DTO 200 ppm. Darkness Water DTO 200 ppm. After 48 Hours After 72 Hours After 96 Hours After 120 Hours 29.7 54.7 67.O 77.6 26.2 34.0 3S.3 44.2 31.S 59.6 85.8 102.8 25.4 34.9 40.7 47.0 Appendix 11. Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) in distilled water solutions on the germination of old lettuce seeds under controlled conditions. Concentrations expressed in ppm. Percent germination A. 6/2/1951 cou 25 COU 25 & DTO 5 COU 25 & DTO 10 Darkness 4 days Alternate illumination 4 days 66 Continuous illumination 4 days 20 64 65 Darkness 4 days then alternate illumination 7 days *+3 77 83 1 2 6 74 B. 6/20/1951 Treatments Water cou 25 COU 25 & DTO 5 COU 25 & DTO 10 Percent germination at various intervals of time in hours. 48 24 72 144 192 9b 120 168 216 82 86 72 87 87 87 87 87 87 l4 16 5 43 33 47 53 15 53 1 66 82 82 52 52 72 45 79 12 85 60 66 66 72 35 85 79 Alternate starting with light Water cou 25 COU 25 & DTO 5 COU 25 & DTO 10 72 9 11 17 85 35 77 79 87 47 81 81 88 55 81 82 91 76 82 82 91 77 83 83 91 81 85 83 91 81 87 84 Alternate starting with 12 hours darkness Water cou 25 COU 25 & DTO 5 COU 25 & DTO 10 73 0 1 4 76 29 73 68 7S 48 S3 7s 84 52 85 80 86 60 87 81 86 71 88 81 86 81 88 84 87 85 91 85 Illumination Continuous 240 87 8^ 85 Appendix 11» B. (Continued) Illumination Treatments Alternate starting with 48 hours darkness Water COU 25 COU 25 & DTO 5 COU 25 & DTO 10 Alternate starting with 72 hours darkness Water Alternate starting with 96 hours darkness Water Percent germination at various intervals of time in hours 24 % 72 ' 96 1 20 l44 168 192 2l6~ 57 2 0 _L 79 3 12 22 81 86 87 87 4b 64 67 81 86 74 71 75 7s 79 21 81 81 82 82 83 47 46 88 71 cou 25 0 COU 25 & DTO 5 COU 25 & DTO 10 1 73 7 6 1 7 53 59 cou 25 1 COU 25 & DTO 5 CPU 25 & DTO 10 3 _1 87 88 88 64 75 68 78 70 ll 66 21 73 4 6 86 26 86 45 35 _1 28 27 W 88 80 88 71 82 74 21 21 21 87 58 36 67 87 36 88 79 36 21 21 72 Appendix 12. Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) in distilled water solutions on the elongation of new lettuce seeds under controlled conditions. Concentrations expressed in ppm. A. 7/20/1951 Percent germination at various intervals of time in hours Alternate light and dark Continuous light 50$ Fluorescent - 50$ Incandescent 50$ Fluorescent - 50$ Incandescent Treatments Water DTO 200 cou 25 cou 25 & DTO 25 cou & cou & 48 6s 48 81 94 15 23 72 82 94 18 25 96 0 1 24 74 89 8 IS — 18 25 2 1 2 1 37 27 51 36 52 39 12 17 17 62 74 48 48 48 5s 78 84 (1) (2) 24 46 74 0 0 0 0 I2 65 84 0 0 (1) (2) 1 0 2 1 2 1 0 7 11 44 25 DTO 50 . 25 DTO 100 . 96 .. 65 0 1 120 65 - 120 96 hours dark then alternate light 18 25 96 30 78 2 2 120 31 83 2 2 53 39 54 39 3 5 3 5 75 — — 5 6 87 — _ 12 13 - B. 7/2S/1952 Illumination Treatments Continuous Water light: DTO 200 COU 25 (1) 50$ Fluorescent 50$ Incandescent (2) cou 25 & DTO 100 (1) (2) 24 86 82 4 2 72 70 Percent germination at various intervals of time in hours i44 48 120 168 192 .72 ... 96 216 91 91 91 85 91 21 16 66 43 75 87 10 16 30 55 77 84 S3 84 84 84 85 240 Appendix 12. B. (Continued) Illumination Continuous light: 50$ Fluorescent 50$ Incandescent Dark (96 hours) then continuous light: 50$ Fluorescent 50$ Incandescent 24 56 Treatments Water DTO 200 cou 25 20 0 (2) 0 (1 ) cou 25 & DTO 100 (1) 17 (2) 37 Percent germination at various intervals of time in hours ikk 16>2 2l 6 4g 120 192 72 96 26 & 73 22 22 22 2 2 12 1 36 5 13 53 4 l4 2 50 32 52 0 27 59 69 61 72 62 95 3 (2) 31 76 3 4 DTO 100 (1) (2) 6 6 9 Water DTO 200 cou 25 (1 ) 51 52 54 67 5 cou 25 & 6 67 70 79 22 79 95 7 7 20 14 240 25 64 72 72 72 12 13 4l 17 59 27 72 63 35 24 52 36 69 59 7? 24 ?. Appendix 1 Effects of simultaneous applications of Coumarin (COU) and Dithiooxamide (DTO) in distilled water solutions on the elongation of new lettuce seeds under controlled conditions. Concentrations expressed in ppm. A. 12/16/1951 Percent germination at various intervals of time in hours 96 hours dark, then continuous light Continuous light 50/5 Fluorescent 70$ Fluorescent 50$ Fluorescent 70$ Fluorescent 30$ Incandescent 50% Incandescent 50% Incandescent 30$ Incandescent 24 42 72 96 120 - 24 75 77 21 25 21 22 90 2b 26 87 92 90 a 90 94 92 22 91 72 65 26 S3 21 87 22 89 22 2b 2iL 2l 70 24 77 25 78 21 25 9 9 12 10 23 23 42 29 39 42 52. W 65 51 12 63 22 70 21 78 2b 75 42 72 9b 120- 96 120 144 83 72 zi 78 87 21 22 S3 32 24 24 85 86 75 Sb a 76 77 87 Zl 78 78 90 Zl 82 i4 5 _i 2 12 6 -I 10 42 69 LL 63 51 75 20 S9 57 79 22 73 162 - 96 120 50 42 52 50 84 75 86 82 91 85 22 89 91 85 22 58 63 sit 58 87 Sb 21 79 24 92 80 82 24 8? 83 29 91 22 90 92 21 90 93 21 36 11 15. 18 22 12 2 4 8 5 3 4 62 81 68 81 81 77 144 . 162 Treatments Water 1 2 3 Average 7k DTO 100 1 2 3 Average 79 67 cou 25 1 2 3 Average cou 25 & 1 DTO 100 Average 2 3 26 2b 22 52 29 90 a 74 56 66 72 25 74 0 0 0 59 74 87 78 22 24 29 78 §2. 85 1 24 44 in 38 90 85 24 31 9 8b 76 91 92 8 2 _1 11 5 11 70 81 81 78 75 85 81 82 89 39 91 94 21 91 94 21 95 95 88 94 21 88 94 21 87 94 22 92 85 87 81 85 91 92 18 13 2L. 21 5 2 -I 7 7 15 8 18 11 11 12 15 10 21 31 22 76 85 81 S3 70 78 Zi 75 73 80 SI 78 73 81 81 79 71*81 81 80 92 Appendix l^UA* Effects of applications of Dithiooxamide (DTO) on the germination of new lettuce s&eeds under controlled conditions at 29°C. in distilled water solutions. . A. S/4/1951 Percent germination at various intervals of time ________________ in hours______________________ Distilled water____________ DTP200ppm. 24 48 72 24 48 72 31 "P"' 61 69 77 24 60 71 71 81 86 4l 6S Zi Z5. 24 88 33 6l 69 72 81 87 Replicates Tl T (2) (3) Average Appendix l4-3. Effects of applications of Dithiooxamide (DTO) after a presoaking in Coumarin (COU 25 ppm) for 24 hours in darkness, on the germination of new lettuce seeds, under controlled conditions in distilled water solutions. Concentrations expressed in ppm. B. 8 /21/1951 Treatments Water 24 (l) 0 (2) 2 DTO 50 (1) 0 (2) 0 DTO 100 (1) 4 (2) 0 DTO 200 (1) 0 (2) 0 Percent germination at various intervals of time in h o u r s ______________ In continuous In alternate 96 hours darkness illumination illumination then continuous illumination 4s 72 96 120 24 4s 72 96 • .. 96. 120 52 42 28 34 28 42 30 4o 80 88 76 76 72 76 6b 64 74 72 80 82 0 4 0 0 0 0 0 0 78 82 78 76 72 76 46 50 86 92 86 88 84 88 72 72 82 SO 48 38 56 72 82 82 76 66 76 80 88 86 82 88 80 7^ Appendix 15. Effects of simultaneous applications of Ascorbic Acid (ASA) and Coumarin (COU) in acid water solutions at pH 3*5 on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentrations expressed in ppm. Average of 30 rootlengths expressed in mm. Length of the test: 4 days. A. 7/17/1951 Illumination Continuous* Alternate* Darkness Water cou 75 68.8 78.1 86.6 32.8 3^.0 29.6 cou 75 & ASA 500 51+.2 58.7 47.9 cou 150 15.9 13.8 13.1 Length of the test: 5 days. B. 3/8/1952 Illumination Continuous Darkness Water Si.5 92.2 ASA 250 78.8 93.7 ASA 500 78.3 89.3 ASA 1000 65.*+ 81.7 Illumination cou 75 cou 75 & ASA 250 5^.5 50.2 cou 75 & ASA 500 65.9 1+S.5 cou 75 & ASA 1000 68.3 55.5 cou 150 & ASA 250 24.9 23.3 cou 150 & ASA 500 38.0 28.3 cou 150 & ASA 1000 1+3.2 33.9 Continuous Darkness Illumination Continuous Darkness 32.1+ 28.5 cou 150 13.5 13.0 ♦Light compositions: 50$ Fluorescent, 50$ Incandescent cou 150 & ASA 500 35.5 37*7 28.3 Appendix 16. Effects of successive applications of AscorMc Acid (ASA) in acid water solutions at pH 3.5 (P) "and Coumarin (COU) in distilled water (DW) solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 2/20/1952 Post-treatraents Pre - treatments 24 hours P In light In darkness ASA 500 Pre - treatments 48 hours P ASA 500 DW cou 4o cou 150 83.6 48.9 19.7 78.7 40.7 21.4 07.6 56.3 29.9 74.3 51.8 33-1 DW COU 4o cou 150 104.7 35.1 13.4 100.3 38.1 l4.0 101.1 40.4 25.9 S7.9 44.0 28.6 Appendix 17. Effects of successive applications of Coumarin (COU) in distilled water solutions (DVf) and Ascorbic Acid (ASA) in acid water solutions at pH 3.5 (P) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 2/16/1952 Post-treatments In light P ASA 500 In darkness P ASA 500 Pre-treatments 24 hours COU 150 DW COU 75 68.7 58.6 S3 . 6 92.3 81.2 75.5 107.3 92.4 67.8 71.s 54.7 58.6 Pre-treatments 48 hours DW COU 150 COU 75. 60.0 35.6 99.7 85.I 58.8 37.8 98.8 84.1 48.9 57.^ 79.7 73.5 B. 2/27/1952 Post-treatments DW In light In darkness P ASA 250 ASA 500 P ASA 250 80.2 78.1 70.9 __ _________Pre-treatment3 24 hours cou 75 COU 75 COU 37*5 cou 75 (2) _ (3) 68.8 69.4 65-5 77.0 73.8 79.9 85.2 — 88.3 86.3 79.^ 82.6 67.5 71.9 cou 150 — — 48.2 54.1 cou 300 43.7 53.^ 55.1 Appendix IS. Effects of successive applications of Couinarin (COU) in distilled water solutions (DW) and Ascorbic Acid (ASA) in acid water solutions at pH 3*5 (P) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentrations expressed in ppm. Average rootlength of 30 seeds expressed in mm. a. b/h/i 952 Po st-1 reatment s DW In light In darkness Pre-treatments (2*f hours) COU 150 COU 37.5 COU 75 P ASA 500 ASA 1000 ASA 2000 90.8 93-S 95.6 85-3 97.7 92.9 93-5 81.1 81.1 80.7 P ASA 500 ASA 1000 96.0 95.7 91.6 S*M 83.4 87.4 69.5 94.7 60.2 79-5 COU 300 U7.I 78.6 65.8 49.4 Appendix 19« Effects of simultaneous applications of Coumarin (COU) and Ascorbic Acid (ASA) in distilled water solutions on the germination of old lettuce seeds under controlled conditions. Concentrations expressed in ppm. Percent germination Alternate illumination 4 days A. 5/28/1951 Continuous illumination 4 days 88 23 21 53 Water cou 25 COU 25 & ASA 250 COU 25 & ASA 500 Darkness 4 days then alternate illumination 5 days 81 9 51 72 Darkness 4 days 64 5 85 40 32 8 6 50 B. 6/13/1951 Illumination Continuous 36 Water 82 86 87 88 7 7 38 53 88 63 88 2 84 5 86 cou 25 cou 25 & ASA 250 7 11 22 4o 59 76 87 88 4 16 33 49 .75 77 81 84 84 cou 25 & ASA 500 64 8 90 25 93 51 93 93 cou 25 cou 25 & 54 68 93 74 93 78 93 87 ASA 250 15 27 3s 60 7^ 7* so 81 13 3^ 56 71 80 80 84 85 Water Alternate starting with light Percent germination at various intervals of time in hours 84 108 60 204 132 . 156 iso 228 Treatments cou 25 & ASA 500 Appendix 19-B. (Continued) Percent germination at various intervals of time in hours 204 228 84 108 132 60 156.. ... 180 76 Illumination Treatments 82 17 88 45 90 67 90 Alternate starting with 12 hours darkness Water COU 2f> cou 25 & ASA 250 cou 25 & ASA 500 70 90 74 91 77 91 83 83 27 36 38 52 69 83 86 88 21 4i 59 7^ 80 80 82 82 Water cou 25 cou 25 & ASA 250 cou 25 & ASA 500 SO 0 87 33 90 63 90 74 90 92 92 78 81 85 92 88 2 29 42 46 b3 72 77 80 0 70 59 70 73. .73 73 73 77 l 88 43 88 65 89 76 90 79 90 82 90 88 1 29 62 75 82 85 88 1 31 62 71 .. 73. _ 77 82 51 85 11 18 27 85 35 45 64 85 44 79 ... 77 85 49 86 84 85 bl 87 87 85 65 88 87 91 5 7 11 93 22 27 42 93 52 45 69 93 93 66 59 81 67 71 Alternate starting with 24 hours darkness Alternate starting with US hours darkness . Water cou 25 cou 25 & ASA 250 cou 25 & ASA 500 Alternate starting with 72 hours darkness Water cou 25 COU 25 & ASA 250 COU 25 & ASA 500 Alternate starting with 96 hours darkness Water cou 25 COU 25 & ASA 250 COU 25 & ASA 500 3 0 1 .... 69 2 1 2 . 91 84 Appendix 20. Effects of simultaneous applications of Couraarin (COU) and Ascorbic Acid (ASA) in acid water solutions at pH 3.5 on the germination of new lettuce seeds under controlled conditions. Concentrations expressed in ppm. A. 7/17/1951 _____ Percent germination at various intervals of time In hours____________________ Continuous illumination Alternate illumination 96 hours dark, then ______ continuousillumination cou 25 COU 25 & ASA 250 COU 25 & ASA 500 24 0 k 11 4s 5 17 29 72 6' 23 34 B. S/lS/l951 Illumination Continuous cou 25 & ASA 500 cou 25 & ASA 2000 k2 61 24 11 10 19 120 53 49 70 4s 22 21 27 96 2 0 4 72 . 96 _ 86 60 90 69 SS 53 120 2 29 26 i44 6 51 57 Percent germination at various intervals of time in hours Light: Light: 7C$ Fluorescent and 50$ Fluorescent and 30$ Incandescent___________ 50$ Incandescent i44 4s 24 24 4s 120 96 120 72 72 96> Treatments cou 25 96 46 (X) (2 ) 0 2 10 10 34 30 kk (1 ) (2) 2 6 36 28 76 72 SS (1 ) (2 ) 0 0 26 32 32 62 38 — 46 52 4s 54 2 0 6 0 8 2 0 0 8 6 32 24 0 0 0 0 8 4 — — 14 14 i44 24 4o mm Appendix 20-B. (Continued) Illumination Treatments (1 ) (2 ) 0 0 10 is 36 3* 54 38 (1) (2 ) 4 80 86 84 0 44 44 cou 25 & ASA 2000 (1) (2) 0 0 30 28 42 42 cou 25 Alternate 56 4o 0 0 8 0 22 22 26 0 0 30 42 70 58 80 68 0 0 12 8 16 18 44 cou 25 & ASA 500 (1 ) (2 ) 0 6 4 14 b 6 (1 ) (2 ) 2 6 4 6 6 10 cou 25 & ASA 2000 (1 ) (2 ) 4 0 4 8 0 14 cou 25 96 hours dark>ness then alternate Percent germination at various intervals of time in hours_____ Light: Light: 70$ Fluorescent and 50$ Fluorescent and 50$ Incandescent 30$ Incandescent 2k 48 120 96 2k 48 72 96 . 120 72 cou 25 & ASA 500 30 44 1^4 46 64 ' Appendix 21. Effects of simultaneous applications of Coumarin (COU) and Ascorbic Acid (ASA) in acid water solutions at pH 3.5 on the germination of new lettuce seeds under controlled conditions. Concentrations expressed in ppm. A. 2/2/1952 ____________ Percent germination at various intervals of time in hours 96 hours dark. then continuous light Continuous light 70$ Fluorescent 50$ Fluorescent 50$ Fluorescent JOfj Fluorescent 30# Incandescent 50$ Incandescent 50$ Incandescent 30$ Incandescent 144 24 48 72 96 24 48 72 120 144 96 96 120 96 Treatments Water (1) (2) (3) Average ASA 500 (l) (2) (3) cou 25 (1 ) (2) (3) Average cou 25 (l) & ASA 500 (2) (3) Average 66 69 77 78 62. 62. 71 72 37 53 £ S3 88 79 2| 86 % 78 79 84 80 80 72 12 75 ?° 42 5i 88 92 si 89 4> 42 58 49 80 88 80 73 2 3 6 2 3 6 80 88 86 85 21 29 30 27 87 48 43 5k 79 92 78 2i 87 59 O. 92 78 21 61 77 71 12. 73 0 0 0 0 0 25 2 0 1 14 19 37 33 22 31 0 0 0 0 0 0 1 0 5 0 4 3 1 0 65 57 55 71 74 11 26 1 0 _0 1 0 JL 5 16 16 1 13 59 74 0 71 71 7^ 60 82 73 UL 68 Average 45 66 79 83 84 82 51 49 55 52 6a 70 23 % 5? 72 55 45 42 33 v ... 11 .... 4' 2 0 10 1 2 8 0 1 2 28 13 2 1 2 1 JL 4 11 33, -5. 1 9 24 3 9 4 2 5 33 ~4 2 3 6 T T 8 3 3 3 3 JL 21 U. _o _0. 16 _8 3 24 2 2 11 Appendix 21. (Continued) B. 3/11/1952 Percent germination at various intervals of time in hours Continuous illumination (70$ Fluorescent. 30$ Incandescent) 72 96 120 i*S 168 48 24 Treatments Water (l) (2) (3) 53. 5? 75 75 76 75 SI S5 S6 §¥ (1) (2) (3) 3^ 14 50 33 71 57 84 71 SO 7^ 21 82 SI 79 21 iPf (1) (2) 0 0 0 2 3 6 7 IQ (3) _0 0 _0 1 2 T 0 0 0 0 5 3 33 9 28 23 Average ASA 500 Average cou 25 Average cou 25 & ASA 500 Average (1) (2) (3) 56 52 -* 27 24 30. 27 51 34 11 9 16 17 12. 17 53 35 46 45 bS 58 51 61 98 b8 68 71 81 77 n 77 Appendix 22. Effects of applications of Ascorbic Acid (ASA) in acid water solutions at pH 3»5» after a presoaking in Coumarin (COU) (25 ppm) in distilled water solutions for 24 hours in darkness, on the germination of new lettuce seeds under controlled conditions. Concentrations expressed in ppm. A. S /2 1 /1 9 5 1 _____________ Percent germination at various intervals of time in hours________ Continuous illumination Alternate illumination 96 hours darkness, then alternate 24 48 24 120 48 120 96 illumination 96 72 72 96 120 Treatments Water (1) (2) 0 2 26 50 76 88 0 0 82 64 90 70 62 44 84 82 ASA 200 (1) (2) 2 0 52 12 86 74 0 0 7b 64 86 78 40 70 68 82 ASA 650 (1) (2) 6 0 3^ 24 72 72 78 90 0 0 74 80 76 88 64 52 86 7* ASA 2000 (1) (2) 0 0 6 0 12 2 28 12 0 0 4o 40 48 54 80 76 82 80 44 20 64 60 64 62 Appendix 23. Effects of simultaneous applications of Dithiooxamide (DTO) and Ascorbic Acid (ASA) in distilled water solutions on the elongation of Marketer cucumber roots grown under controlled contitions. Concentrations expressed in ppm. A. 5/31/1951 Illumination Length of the test; 4 days. Average of 20 root-lengths expressed in mm. Water - DTO 200 - DTO 200 & ASA 250 - DTO 200 & ASA 500 Continuous 6 7 .0 34.9 54.3 - Alternate 87.3 37.7 4 0 .2 46.4 Darkness 66.4 34.5 26.8 29.5 6 Continuous 67.9 2 1 .0 2 8 .1 27.3 Alternate 64.7 26.3 22.3 2 2 .6 Darkless 66.3 23.7 22.4 19.7 01 0 0 8 0 8° Illumination Length of the test: 4 days. Average of 30 root-lengths expressed in mm. Water - DTO 200 - DTO 200 & ASA 250 - DTO : B. 6/5/1951 c . 6/ 16/1951 Illumination Length of the test: 5 days. Average of 30 root-lengths expressed in mm. Water - DTO 100 - DTO 100 & ASA 250 - DTO 100 & ASA 500 Continuous 75.4 54.1 70.9 68.7 Alternate 95.3 5 S .6 74.4 72.4 1 0 2 .7 4 3 .7 38.5 31.2 Darkness Appendix 24. Effects of simultaneous applications of Dithiooxamide (DTO) and Ascorbic Acid (ASA) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 6/26/1951 Illumination Water Distilled water solutions DTO 100 DTO 100 DTO 100 & & ASA 250 ASA 100 DTO 100 & ASA 500 Continuous 87.7 69.1 88.2 98.4 85.0 Alternate 93-3 70.5 79-9 94.9 84.9 Darkness 114.8 60. S 57.^ 49.1 38.1 B. 7/2/1951 Buffered solutions, pH 4.5 (Sorensen) Water DTO 100 Illumination DTO 100 & ASA 100 DTO 100 & ASA 250 DTO 100 & ASA 500 Continuous 43.8 42.2 53-9 56.3 51.9 Alternate 50.3 51.3 b4.8 66.1 66.6 Darkness 51*3 50.8 48.2 52.0 43.9 Appendix 24 (Continued) C. 7/1^/1951 Acid water solutions, pH 3*5 Water ASA 250 DTO 200 Illumination DTO 200 & ASA 100 DTO 200 & ASA 250 DTO 200 & ASA 300 Continuous 77-4 85.3 44.5 62.6 61.3 65.9 Alternate 89-9 98.1 49.7 57.8 69.4 62.8 Darkness 98.1 107.2 52.5 47.0 38.5 35.1 ASA 1000 ASA 2000 D. 4/5/1952 Acid water solutions, PH 3.5 Water ASA 125 ASA 250 ASA 500 In Continuous Illumination Controls ♦ DTO 200 93.0 32.7 92.S 33-8 90.8 39-5 93.3 49.2 80.1 41.5 ..65.5 31.1 In Darkness Controls + DTO 200 98.S 33.4 40.2 96.6 33.6 93.5 33.5 84.5 24.7 47.5 19.5 Appendix 25. Effects of successive applications of Ascorbic Acid (ASA)in acid water solutions at pH 3.5 (P) and Dithiooxamide (DTO) in distilled water (DW) solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. Group I A. 9/18/1951 In light In darkness p ASA 500 DTO 300 DTO 300 Transfer after 24 hours Transfer after 48 hours 81.5 .93.0 46.7 60.5 45.8 65.1 100.2 78.9 47.7 Transfer after 24 hours Transfer after 48 hours 113.9 123.3 53-7 67.4 50.8 72.9 121.2 126.0 91.4 77.3 46.9 46.2 «• Group I Pretreatments Post-treatments In darkness Group II DTO 300 P ASA 500 DW B. 10/20/1951 In light DW P Pretreatments Post-treatments P DW DW DTO 300 ASA 500 DTO 300 Transfer after 48 hours Transfer after 72 hours 106.1 Transfer after 48 hours Transfer after 72 hours — — 59.0 67-5 110.3______________ 68.3____ 105.9_________ 83.0 57-9 70.6 102.1 66.7 76.1 Group II Pretreatments Post-treatments DW P DTO 300 ASA 500 P ASA 500 In light Transfer after 8 hours Transfer after 24 horn's - 39.7 78.2 91.S 89.6 100.3 89.4 81.1 In darkness Transfer after 8 hours Transfer after 24 hours 93.8 95.0 73.8 67.5 54.9 48.6 102.2 Appendix 2b. Effects of successive applications of Dithiooxamide (DTO) in distilled water (DW) solutions and Ascorbic Acid (ASA) in acid water solutions at pH 3.5 (P) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 10/11/1951 Pretreatments Post-treatments In light In darkness DTO 150 ASA 400 DW ASA 400 P P DTO 300 P ASA 400 Transfer after 8 hours Transfer after 24 hours 90.2 93.0 91*2 69-1 97-1 57.9 93.4 109.8 79.9 b2.4 Transfer after 8 hours Transfer after 24 hours 96.9 100.1 85.2 86.8 87.6 52.S b9.6 72.8 45.9 4i.o B. 10/20/1951 Illumination In light In darkness Po st-treatments P ASA 100 ASA 250 ASA 400 DW 86.7 85.I 82.2 75-3 P . ASA 250 103.3 98.4 Pretreatments 24 hours DTO 75 DTO 150 DTO 300 . 94.2 98.3 94.3 78.7 93*6 83.1 104.4 65.6 81.3 58.3 67-9 48.3 Pretreatments 8 hours DW DTO 300 9b.6 98.3 81.7 72.9 Appendix 27. Effects of successive applications of Dithiooxamide (DTO) in distilled water (DW) solutions and Ascorbic Acid (ASA) in acid water solutions at pH 3*5 (P) the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 11/20/1951 Pretreatments (24 hours) Post-treatments In darkness P DW ASA 250 100.7 102.4 DTO 37.5 P ASA 250 103.0 S3 .4 DTO 75 P ASA 250 94.8 72.O B. 2/9/1952 Pretreatments (48 hours) Post-treatments In light P DW ASA 250 104.1 96.3 93.9 DTO 100 P ASA 250 87.8 DTO 200 P ASA 250 96.8 73.6 c. 11/1/1951 Pretreatments (24 hours) Post-treatments Light then darkness Darkness then light P DW ASA 250 99.0 87.8 DTO 300 P ASA 250 76.6 SO.5 78.7 78.4 D. 5/12/1952 Pretreatments (24 hours) Po st-1reatment s In light In darkness DW P 80.6 P 91.0 57.8 DTO 300 ASA 25 ASA 50 86.3 59.^ 88.0 59*7 ASA 100 91.8 57.3 52.2 b5.5 Appendix 28. Effects of successive applications of Ascorbic Acid (ASA) in acid water solutions at pH 3*5 (P) an) on the elongation of Burpee hybrid cucumber roots grown under controlled conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 12/15/1951 Water ASA 500 TU 600 TU 600 & TU 600 & TU 600 & ASA 125 ASA 250 ASA 500 In light 74.2 8 3 .S 50.4 35.4 40.0 33.6 In darkness 79.1 76.9 52.3 26.3 29.7 26.1 ASA 25 ASA 50 ASA 125 ASA 250 ASA 500 B. 2/9/1952 Water Control 80.1 79*0 77.1 77-5 7 2 .2 6 9.6 + TU 600 46.1 47.3 45.5 41.4 3 8 .2 2 6 .6 Control 102 .6* 105.0 1 00.8 96.5 94.8 99.0 49.2 49.3 46.1 38.1 33.2 In light In darkness ♦ TU 600 51.8 ♦Average of 20 root-lengths Appendix 33. Effects of simultaneous applications of 2,4-D and Dithioox­ amide (DTo T in distilled -water solutions on the elongation of Marketer cucumber roots grown under laboratory conditions. Concentrations expressed in ppm. A. 4/2/1951 Length of the test: 5 days. 2,4-D‘0.1 2,4-D 0.1 2,4-D 0.1 2,4-D 0.5 2,4d 0.5 2,4-D 0.5 ________ * DTO 2.5 * DTP 5 ; _________ * DTO 2.5 + DTO 5 Number of seeds Average length (ram) 17 29.0 17 17 18 18 18 34.1 36.8 11.3 13.4 13.2 B. 4/9/1951 Average of 30 root-lengths expressed in mm. ~27fcrro.i 2^ £ d 0 . 1"" 2,4-D O'.1 "2,4-D 0 .1 + DTO 5 ♦ DTO 7.5 + DTO 10 After 4 days 23.8 36.7 39.^ 37.7 After 7 days 24.4 48.5 44.0 45.7 Appendix ^4. Effects of simultaneous applications of 2,4— D, Ditliioox;— amide (DTO-)" and Ascorbic Acid (ASA) in distilled water solutions o n the elongation of Marketer cucumber roots grown under laboratory c o n ­ ditions for 4 days. Concentrations expressed in ppm. Average of 2 0 root-lengths expressed in mm. A. 4/15/1951 2,4-D 0.1 31.4 2,4-D 0.1 4 DTO 2.5 36.7 2,4-D 0.1 + DTO 5 39.1 2,4-D 0.1 + ASA 25 37.5 2,4-D 0.1 DTO 2.5 + ASA 25 39-0 2,4-D 0.1 4 DTO 5 4 ASA 25 41.0 2,4-D 0.1 + dto 7*5 2,4-D 0.1 4 DTO 7.5 + ASA 23 3S.0 41.0 2,4-D 0.1 f DTO 10 2,4-D 0.1 *- DTO 10 » ASA 25 2,4-D 0.1 4 ASA 100 36.3 2,4-B 0.1 4 DTO 2.5 ♦ ASA 100 35.6 2,4-D 0.1 + DTO 5 r ASA 100 39.3 2,4-D 0.1 4 DTO 7.5 4 ASA 100 42.6 2,4-D 0.1 4 DTO 10 v ASA 100 2,4-D 0.1 + ASA 250 2,4-D 0.1 42.8 37.2 2,4-B 0.1 + DTO 2.5 + ASA 250 40.0 2,4—D 0.1 + DTO 5 t ASA 250 2,4— D 0.1 4 DTO 5 r ASA 5 0 0 49.0 47.2 2,4-D 0.1 4 DTO 7.5 4 ASA 250 2,4-D 0 . 1 4 DTO 7.5 4 ASA 5 0 0 50.1 4b.S 2,4-D 0.1 4 DTO 10 4 ASA 250 2,4-D 0 . 1 4 DTO IO ASA 5 0 0 Appendix 35. Effects of simultaneous applications of 2,4-D, Dithiooxamide (DTO) and Ascorbic Acid (ASA) in distilled water solutions on the elongation of Marketer cucumber roots grown under laboratory conditions for 4 days. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 4/21/1951 Illumination 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 DTO 7.5 ASA 250 ASA 250 DTO 7*5 Alternate (started with light) 33-7 41.0 51.7 52.3 Alternate (started with darkness) 31.9 38.2 39.2 48.4 B. 4/23/1951 Illumination 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 DTO 7.5 ASA 250 DTO 7.5 ASA 250 Water Continuous 44.6 42.0 5^.5 56.7 62.6 Alternate 42.4 41.9 51.0 44.6 72.9 Darkness 31.8 33-6 44.0 34.3 66.7 C. 5/7/1951 Illumination 2,4-D 0.1 2,4-D 0.1 2,4—D 0.1 2,4-D 0.1 DTO 7-5 ASA 250 ASA 250 DTO 7.5 Water ASA 250 Continuous 55.2 53-^ 60.3 67.O 67-7 78.5 Alternate (started with light) 38.8 49.4 46.2 47.1 72.7 78.1 Alternate (started with darkness) 37.2 45.5 43.1 49.0 7^.3 73-9 Darkness 38.5 37-1 38.9 39.6 82.5 82.3 Appendix 36. Effects of simultaneous applications of 2,4-D, Dithiooxamide (DTO) and Ascorbic Acid (ASA) in distilled water solutions on the elongatim of Marketer cucumber roots grown for 4 days under laboratory conditions. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 5/21/1951 Illumination Water ASA 500 2,4-D 0.1 2,4-D 0.1 ASA 500 ASA 500 DTO 7.5 2,4-D 0.1 2,4wD 0.1 DTO 7.5 Continuous 71.0 79-3 52.s 55.5 67.1 63.0 Alternate (started with light) S3-3 - 82.5 53.S 51.9 45.1 48.9 Alternate (started with darkness) 78.9 S9 .O 45.5 52.5 54.8 57-6 Darkness 90.1 87-2 42.3 32.9 42.3 3^.5 . B. 6/S/1951 Illumination Water 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 2,4-D 0.1 DTO 7.5 ASA 250 ASA 250 DTO 7.5 Continuous 53.9 27.7 25.4 30.1 30.6 Alternate 66.2 21.1 28.7 35-1 30.2 Darkness 65.g 27.5 31.9 26.5 25.7 Appendix 37 • Effects of simultaneous applications~of 2,4-D, Dithiooxamide (DTO) and Ascorbic Acid (ASA) in acid water solutions (pH 3.5) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 8/9/1951 Length of the test: 4 days. Water 2 ,4 4 b o.i Illumination 2,44b 0.1 DTO 3.75 2,4-D 0.1 DTO 7.5 2,4-D Oil ASA 500 Continuous 6 3 .6 35 .6 44.3 46.7 54.4 Alternate 82.6 34 .2 38.3 4 7 .2 54.6 Darkness 84.2 3^-5 38.7 45-5 44.0 B. 2/23/1952 Length of the test: 5 days.• Water In light In Darkness DTO 10 DTO 30 DTO 100 ASA 250 ASA 500 Controls 75.8 8 5 .8 80.1 6b.2 77-4 78.5 4 2,4-D 0.1 49.6 5 0 .2 56 .0 ■61.7 58.1 64.0 Controls 92.9 81.9 68.1 ' 6 0 .5 85.5 77 .8 + 2,4-D 0.1 37.8 46.3 46.9 55-1 4 7 .4 6 1 .2 Appendix 37• (Continued) c. 3/1/1952 Length of the test: 5 days. Water In light DTP 100 DTP 300 ASA 500 ASA 1000 ASA 2000 Controls 82*7 JS.6 28.9 89.9 89.6 85*3 * 2,^-D 0.1 50.4 66.6 3^-8 68.1 71*^ 68.7 88.9 60.0 33-5 85.5 80.0 Ul.8 35.4 52.8 31.O 50.7 61.9 40.8 Controls In darkness + 2,4-D 0.1 Appendix 38. Effects of simultaneous applications of Indole Acetic Acid (IAA) and Dithiooxamide (DTO) in distilled water solutions on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions. Concentration expressed in ppm. Average of 30 rootlengths. A. 7/20/1951 Length of the test: 4 days • In light 35.7 38.9 IAA 5 DTO 8 41.4 In darkness 27.7 33.5 27.8 IAA 5 IAA 5 DTO 2 IAA 5 DTO 20 IAA 5 DTO 50 46.6 42.9 41.6 40.2 DTO 200 IAA. 1 DTO 200 IAA 2 B. 7/27/1951 Length of the test: 4 days • In light i+9.0 43.7 DTO 200 IAA .3 48.6 In darkness 49.6 50.3 52.4 DTO 200 DTO 200 IAA .1 50.9 42.6 49.7 44.4 c. 8/17/1951 Length of the test: 4 days,» Water Illumination Continuous Alternate Darkness 74.9 89.q 88.4 IAA 5 26.4* 25.3 20.0 IAA 5 DTO 3,3 34.7* 37-6 24.5 IAA 5 DTO 20 51.9 40.2 36.4 D. 9/13/1951 Length of the test: 4 days.> Water DTO 150 IAA 5 In light 73.1 *+9.2 52.7 40.3 In darkness 88.0* 48.4 33.8 34.4 *Average of 20 root-1engths. DTO 150 IAA 3 IAA 5 DTO 100 33.2* 36.2 34.1 Appendix 39« Effects of simultaneous applications of Indole Acetic Acid (IAA) and Dithiooxamide (DTO) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Conceru trations expressed in ppm. Average of 30 root lenths expressed in mm. A. V9/1952* Acid water solutions at pH 3*5 IAA 5 IAA 5 DTO 1 IAA 5 DTO 5 IAA 5 DTO 20 IAA 5 DTO 75 37.3 39-s 41,7 62.7 41.6 38.3 27.g 34.6 34.6 3S.5 29.8 29.5 IAA 10 IAA 10 DTO 1 IAA 10 DTO 6 IAA 10 DTO 20 IAA 10 DTO 75 IAA 10 DTO 150 In light 30.5 33.2 39-7 37.4 36.1 36.7 In Darkness 24.9 21.6 32.8 ' 27.8 24.5 In light In darkness - )' "•Temperature 21°C. B. 4/17/1952 Distilled water solutions Water IAA 2 IAA 2 DTO 10 IAA 2 DTO 20 IAA 2 DTO 40 si .9 51.7 60.5 55.6 48.9 100.5 3S.9 55-0 52.1 42.2 IAA 5 IAA 5 DTO 10 IAA 5 DTO 20 IAA 5 DTO 40 In light 33*1 42.9 4g.9 42.3 In darkness 25.1 42.1 37.6 35.^ In light In darkness IAA 5 DTO 150 Appendix 40. Effects of simultaneous applications of Indole Acetic Acid (IAA)”~and Ascorbic Acid (ASA) in acid water solutions (pH 3*5) on the elongation of Burpee Hybrid cucumber roots grown under control­ led conditions for 5 days. Concentrations expressed in ppm. Average of 30 root-lengths. A. 4 /24/1952 Water IAA 2 IAA 2 ASA 100 IAA 2 ASA 250 IAA 2 ASA 750 In light 94.2 51.4 54.1 73.6 SI.4 69.S In darkness 86.1 45.3 55-4 55.3 52.4 52.1 IAA. 5 IAA. 5 ASA 100 IAA 5 ASA 250 IAA 5 ASA 750 In light In darkness IAA 2 ASA 1500 IAA 5' ASA 1500 37-7 71.3 67.8 6S .7 62. S 37-0 44.0 39.7 43.6 38.1 IAA 10 IAA 10 ASA 100 IAA 10 ASA 250 IAA 10 ASA 750 5S.3 65.9 70.2 58.4 4o .5 37.O* 44.6 39.s B. 5/14/1952 Water In light 77*7 In darkness 92.7 - 29.3 ♦Average of 20 root-lengths. IAA 10 ASA 1500 Appendix 4l. Effects of simultaneous applications of Naphthalene Acetic Acid (NAA) and Dithiooxamide (DTO) on the elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Concentra­ tions expressed in ppm. Average of 30 root lengths expressed in mm, A. 4/25/1952 Distilled water solutions .* NAA. .1 + NAA. •3 + NAA 1.0 Water DTO 5 DTO 20 DTO 100 46.2 37.2 30.2 53*1 48.0 35*6 61.0 46.6 34.1 43.6* 41.6 30.5 In darkness NAA .1 -v NAA •3 + NAA 1.0 Water DTO 5 41.0 29.5 23.5 4?. 5 29.0 22.6 DTO 20 DTO 100 41.8 31.4 25.8 41.4 32.3 26.0 B. 5/15/1952 Acid water solutions (pH 3*5) Illumin­ ation Water Darkness 90.6 NAA .1 NAA .1 DTO 1 NAA .1 DTO 2.5 NAA .1 DTO 7.5 NAA .1 DTO 20 43.0 48.1 47.2 48.1 52.6 *Average of 20 root-lengths. Appendix 42. Effects of simultaneous applications of naphthalene Acetic Acid (NAA) and Dithiooxamide (DTO) or Ascorbic Acid (ASA) in acid water solutions (pH 3*5) on tk® elongation of Burpee Hybrid cucumber roots grown under controlled conditions for 5 days. Con­ centrations expressed in ppm. Average of 30 root-lengths expressed in mm. A. 5/5/1952 Water In light + NAA .1 -r NAA .25 DTO 5 DTO 10 DTO 20 DTO 40 54.1 ^5.9 53*7 42.2 52.1 51.6 64.4 50.2 59.5 42.3 42.2 54.2 51.1 64.2 42.6 62.7 49.8 60.8 47.6 55.2 ASA 250 ASA 500 ASA 1000 4 NAA .1 4 NAA .25 65.2 62.1 62.5 66.4 1 3 .b 71.0 In 4 NAA .1 darkness 4 NAA .05 55.1 76.S 72.5 S3-3 69.2 27.1 NAA 2 NAA 2 ASA 100 NAA 2 ASA 250 26.5 34.2 In + NAA .1 darkness 4 NAA .05 In light Water Controls 84.9 96.4 B. 5/14/1952 Illumina­ tion In darkness Water 92.7 25.O NAA 2 NAA 2 ASA 750 . ASA 1500 41.0 40.2 FORMULAS H 9N - C = N H ch2 - c h — ch2oh H N = C — C=NH I II SH SH SH I I SH SH. Thiourea Dithiooxamide 2 ,3-Dimercaptopropanol iH II c— C = C — C - C — CHoOH II I I II 0 OHOH H OH n O■ | 0 -- 1 I I c— C = C - C — CHo II I I I 3 0 H H3 C 1-Ascorbic Acid H H A A I II I C HC. 3- (alpha-Iminoethyl)-5-Methyl Tetronic Acid H HC \XC/ \0/ HC 0=0 H AI IIA CH f i NH HO-C C £ \ C/ O\ / H H A A I II I H3 C - O — C CH I 0=0 HO— C v A A I II I V „/ C HO— C O H HC-NH2 C=0 C H Umbelliferone HC C \C /OV H Coumarin H Scopoletin A A I II I VJ HC HO— C C CH O C=0 I HO Tyrosine Caffeic Acid (3,4-Dihydroxycinnamic Acid) CH 0=C ERRATA Page 11. Line 14 from the top :omit " For a number of Page 42. Line 11 from the top :omit " be Page 56. Line 5 from the bottom : read " dicoumarol " dicomarol ", Page 60. Line 6 Page 66. Second Page 85. Line 4 either one ", "instead of instead of " only it was applied Page 86. Line 5 from the top :read " the same processes " instead of " the processes ", Page 95. Line 9 from the bottom : read " parasorbic acid " instead of " parascorbic acid 11. Page 98. Line 7 from the top :read " of auxin in the coleoptile instead of " of auxin the coleoptile ", "