THE USE OF 1J.-PHTHALIMIDO-2, 6 -DIMETHYLPYRIMIDINE AS A PLANT GROWTH REGULATOR By Paul Joseph Rood A THESIS 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 Year 1953 ProQuest Number: 10008417 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008417 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENT \ I wish to express my sincerest appreciation to Dr. Charles L. Hamner, for his thoughtful guidance, enthusiastic interest, and kindly suggestions. I also wish to express my gratitude to Dr. H. B. Tukey and the entire staff and graduate students of the Department of Horticulture for their help in all phases of this work. X wish to thank Professor C. D. Ball of the Chemistry department for making laboratory facilities available, and Dr. W. D. Baton for aid with the statistical analyseso 1 appreciate the chemicals received from Dr. L. H. Goodhue of the Phillips Petroleum Corporation and the phthalamic acid derivatives received from the Naugatuck Division of the United States Rubber Company. Lastly, I am very deeply indebted to my brothers for typing the manuscript, and to my wife Gerry, for her assistance throughout« Paul Rood TABLE OP CONTENTS INTRODUCTION ........................................ REVIEW OF LITERATURE ........ ...................... EXPERIMENTS ON THE SETTING OP TOMATO FRUITS.......... Greenhouse Experiment* ............ .. Field Experiments* » • • ........ * • • * * * • • * Experiments with the Pall Crop of Ten Greenhouse Tomato Varieties * * * * * * * * * * * * * * * * « EXPERIMENTS ON THE SETTING OP MUSKMELON AND CUCUMBER FRUITS * ♦ . . ........................ . Experiment with Muskmelons • * * * • * • « • « • * • Experiment with Cucumbers* * * • • • • • * ........ EXPERIMENTS ON THE ABSCISSION OP COLEUS PETIOLES • * • Lanolin Application* v * * * * * * * * * * * * * * * Spray Application* • • • « • * • * • • • • • • • • • Defoliation* « • • • * • • • • « * • ........ • • * EXPERIMENTS ON THE THINNING OP TREE FRUITS AT BLOSSOM T I M E ........ ............................. Duchess of Oldenberg Apples* * e * o * o * ........ Bartlett Pears * * * * * * * * * * * * * * « * * o * peaches* • • • • « • • * • • * * * * * * * * * * * * EXPERIMENTS ON THE DELAYING OP THE PREHARVEST DROP OP A P P L E S .................... ............... EXPERIMENTS ON THE ROOTING OP CUTTINGS . * .......... Succulent Cuttings of Coleus * ........ * * * o * o Succulent Cuttings of Tomato * • * • • • • • • • • • Hardwood Cuttings of Willow* • * * • ........ . Hardwood Cuttings of Apple • • • • • • • • « • • * * Magnolia Cuttings* • • * • • • • • • • • • • * • • • Evergreen Cuttings of Boxwood* * * q q « * * * o * o Evergreen Cuttings of Arbor Vitae.......... .. EXPERIMENTS ON THE RELATION OP CHEMICAL STRUCTURE AND THE BIOLOGICAL ACTIVITY OP Ij.-PHTHALIMIDO2, 6-DIMETHYL PYRIMIDINE ............................ TABLE OF CONTENTS CONT# Pag© Inhibition of Root Growth bj Different Concentrations of i|.-phthalimido-2,6 dimethylpyrimidine » « • • # • • • • . # . • « • • An Experiment Testing the Interaction of il-Phthalimi do-2,6 -dimethylpyrimidine with thiamin# A Comparison of the Effect of Several Phthalic Acid Derivatives Upon the Growth of ............ . . Cucumber Roots A Comparison of the Formative Effects of Leaves of Cranberry Beans and John Baer Tomatoes Produced by Several phthalic Acid Derivatives, • . o # # # # # . * . . , . . # # . . The Effect of Several Phthalic Acid Derivatives Upon the Rate of Respiration of Several Plant Tissues# # . ............. o o # # . o # # . . # o 79 8 ij. Q$ 93 96 THE CHANGES IN THE CHEMICAL COMPOSITION OF CRANBERRY BEANS SPRAYED WITH 1+-PHTHALIMID02,6 -DIMETHYLPYRIMIDINE 106 EXPERIMENTS SUGGESTING THE MODE OF ACTION OF ip-PHTHALIMI DO-2, 6 -DIMETHYL PYRIMIDINE UPON THE PHYSIOLOGY OF P L A N T S ............. 113 The Interacting Effects of Indoleacetic Acid and lj.-Phthalimido-2,6 -dimethylpyrimidine Upon the Growth of Cucumber Roots . . « • • • • • . . » The Inhibition of the Translocation of Auxins by Ip-Phthalimido-2 ,6 -dimethylpyrimidine in the Petioles of Coleus Plants. The Inhibition of Apical Dominance by Treating the Apical Shoot of Coleus Plants with Ip-phthalimido-2 ,6 -dimethylpyrimidine • • • • • • • lli^. 117 118 SUMMARY................ 123 LITERATURE C I T E D .............................. 12£ LIST OP TABLES Table I II III IV V VI VII VIII IX X Page Effectiveness of lp-phthalimido-2,6 -dimethyl­ pyrimidine in stimulating the parthenocarpic development of non-pollinated flowers of tomato plants grown in the greenhouse .................................. Yield of early tomatoes as affected by fruit setting sprays applied at blossom time . 20 Characteristics of the fruit ripening early in the season which was set with growth regulators • • • • ............ • 22 Effect of setting the lower hands of John Baer tomato plants upon the yield of fruit throughout the season, • • • . . . . . . o . . 2k The total yield of fruit produced by ten greenhouse tomato varieties as affected by weekly treatment with three fruit setting chemicals or a foliar fertilizer spray . * • * 26 The yield of early fruits produced by ten greenhouse tomato varieties as affected by weekly treatment with three fruit setting chemicals or a foliar fertilizer spray • . • « 27 The average number of fruits set per plant on ten greenhouse varieties as affected by weekly treatment with three growth regulators or a foliar fertilizer spray* * 0 « 28 The average weight per fruit of tomatoes from ten greenhouse varieties as affected by weekly treatment with three growth regulators or a foliar fertilizer spray. • . * 29 The percentage of graded fruit exhibiting blotchy ripening from ten greenhouse tomato varieties as affected by weekly treatments with three growth regulators and a foliar fertilizer spray 30 The percentage of fruit with blossom end rot from ten greenhouse tomato varieties as affected by weekly treatment with three growth regulators and a foliar fertilizer spray........................................ 31 LIST OP TABLES CONT Table Page XI The abscission rate of debladed coleus petioles as influenced by growth regulators applied in lanolin to the tips of the p e t i o l e s.................................... k3 XII Abscission rate of debladed coleus petioles as influenced by sprays of growth regulators • lj.$ XIII Abscission rate of debladed coleus petioles as Influenced by sprays of growth regulators e lj-6 XIV The effectiveness of growth regulators in thinning the fruits of Duchess apple trees » o 53 XV The effectiveness of i4.-phthalimido-2 ,6 dimethylpyrimidine and N-l naphthyl phthalamic acid sprays applied at full bloom in thinning the fruits of Bartlett pear trees • » « » * • » • * • • • ' • » « » • $6 XVI The effectiveness of sprays of i|.-phthaliraido-2 ,6 -dimethylpyrimidine and phthalamic acid derivatives in thinning peaches • • * s a 57 XVII The effectiveness in delaying the preharvest drop of Duchess apples on limbs sprayed with growth regulators just before dropping began« « « « « • » * « .............. 60 XVIII The number and maximum length of roots on coleus cuttings 28 days after treatment with growth regulators • o « » » » o » o « o o 65 XIX The average number and length of roots of tomato cuttings 11 days after treatment with different concentrations of l|.-phthalimido-2 ,6 -dimethylpyrimidine • • • » • 67 XX The effect of soaking willow cuttings in solutions of growth regulators on their rooting as measured llj. days after treatment* » 70 XXI The effect of growth regulators on the callusing of McIntosh apple cuttings • * « o o 72 XXII The percentage of boxwood cuttings which rooted and the average number and length of roots per cutting as affected by treatment with two growth regulators • o a « « » o » « o 7J4. LIST OP TABLES CONT. Table XXIII XXIV XXV XXVI XXVII x x v in XXIX XXX XXXI XXXII XXXIII Page The percentage of arbor vitae cuttings which rooted and the average number and length of roots per cutting as affected by treatment with growth regulators* * • « • 76 The effect of varying concentrations of growth regulators upon the growth of cucumber and wheat roots * • ..........• « * 83 The interaction of if-phthalimido-2,6dimethylpyrimidine and thiamin on the growth of cucumber roots * • • * * * « « » « 86 The approximate pH of the growth regulator solutions used in these experiments* * * * * 90 The comparative effect of varying concen­ trations of compounds structurally related to phthalic acid upon the growth in length of roots of cucumber seedlings » « « « • « » 91 The rates of respiration of cucumber seedlings 2 if hours after germinating and growing in 250 ppm solutions of phthalic acid derivatives a * * * * * * * * * * * * * 100 The rates of respiration of bean, tomato, and oat leaves sprayed with 1000 ppm of phthalimide or phthalamic acid derivatives * 101 The composition of cranberry bean plants six days after spraying with 1000 ppm of if-phthalimido-2 ,6 -dimethylpyrimidine » * * o 110 The composition of cranberry bean plants six days after spraying with 1000 ppm of lf-phthallmi do-2 ,6 -dimethylpyrimidine « * * « 111 The interaction of if-phthalimido-2,6dimethylpyriraidine and indoleacetic acid on the growth of cucumber roots* • « « « • » 116 The translocation rate of auxins applied to the tips of debladed coleus petioles as affected by the application of if-phthalimido2 ,6 -dimethylpyrimidine in lanolin to the center of the petioles as indicated by the rate of abscission * * o * o « « * * * * o 119 LIST OP TABLES CONT* Table XXXIV Page The decrease In the dominance of the apical shoot over the growth of the lateral shoots of coleus plants achieved by smearing a lanolin paste containing 1000 ppm of lf-phthalimIdo-2 ,6 -dimethylpyrimidine upon the apical shoot* * • • • • • • • • • • • • • • 121 LIST OP FIGURES Figure 1 2 3 Ij. 5 6 7 8 9 Page A tomato plant with fruit set with 100 ppm of l^-phthalimido-2 ,6 -dimethylpyrimidine • « • 13 Views of fruits and undeveloped ovules of tomatoes set with li.-phthalimido-2 ,6 dimethylpyrimldine. • . ........ 15 Minimum night temperature during the blossoming period of tomatoes at East Lansing, 195>2 .................... 18 Price of 8 quart cartons of Michigan No. 1 tomatoes on Benton Harbor Market in 1952. • • 18 Longitudinal sections of Burpee Hybrid cucumber fruits showing parthenocarpic fruits and normal fruits with seeds » « • o • 37 The delay In the abscission or debladed petioles of coleus plants resulting from a spray with 1000 ppm of 4 -phthalimido2 ,6 -dimethylpyrimidine. i|.7 The rooting response of willow, arbor vitae, and boxwood cuttings after treatment with !|.-phthalImido-2 ,6 -dimethylpyrimidine. . . . . 68 The structural formulas of compounds related to ]+-phthalimido-2 ,6 -dimethylpyrimidine • » o 89 The Inhibition of root growth as affected by varying concentrations of growth regulators « 82 The formative effects of cranberry bean, tomato, Bartlett pear, and peach leaves caused by sprays of l4-phthalimido-2 ,6 dimethylpyrimidine or phthalamic acid derivatives 95 INTRODUCTION The isolation of indoleacetic acid, and the discovery that it was one of the most important natural occurring auxins regulating the growth of plants has stimulated research on a large variety of chemical compounds which exhibit similar activity. Slight changes in the structure of these compounds frequently alter the physiological responses of plants or the agricultural uses for the com­ pound. One of these growth regulating chemicals, 2,^-dichlorophenoxyacetic acid is so much more active than the natural auxin that It Is used extensively in weed control. An even more effective growth regulator for the control of woody perennials was developed by substituting an additional chlorine atom in the number five position of the ring making 2,i|.,5“trichlorophenoxyacetic acid. By omitting the chlorine atom in the ortho position of 2 ,[}.-dichlorophenoxyacetie acid still another compound, para-chlorophenoxyacetic acid was synthesized which was less toxic and capable of inducing parthenocarpic development of tomato flowers. Naphthaleneacetic acid is used in thinning tree fruits soon after blossom time, and in delaying the preharvest drop of apples In the fall. Other compounds will Increase the color of fruit and hasten its ripening. Indolebutyric 2 acid, and many of these other compounds are useful in the rooting of cuttings. More recently many other compounds with quite different structures have been found to show growth regulating activity. Some of these such as maleie hydrazide, the benzoic acid derivatives, and trans cinnamic acid have been called anti-auxins. Maleic hydrazide is used in preventing the sprouting of carrots, onions, and potatoes. It influ­ ences the development of the seed stalk of celery and causes a delay in the blossoming of raspberries. It has also been used as an inhibitor of plant growth and as a weed killer. Trans cinnamic acid and the benzoic acids have been used chiefly in physiological investigations. A multitude of practical applications have been found for plant growth regulators in many phases of agriculture; however, the biochemical mechanism by which these compounds affect the physiology of the plant is not entirely clear. Studies with growth regulators have frequently yielded valuable fundamental knowledge of normal plant metabolism. It is very difficult to predict just what the structure of a compound should be for it to be most effective for a certain purpose, but by empirically testing different compounds, new growth regulators can be found. The investigations described in this thesis were con­ ducted to explore many of the practical applications as well as the fundamental physiological effects of a new plant growth regulator, ip-phthalimido-2 ,6 -dimethylpyrimidine. 4 same as those given a preliminary test by Hoffmann and Smith and were synthesized and furnished by Naugatuck Division of the United States Rubber Company with which they are associated. Phthalic acid was tested by Thompson (9 I4-) in the gigantic screening program of the U« S. Army Chemical Corps and classified by him as one of the least promising compounds. If the activity of lp-phthalimido-2,6 -dimethylprimidine is primarily due to the pyrimidine nucleus, it is one of the first synthetic compounds of this type to be a growth regulator. The review of literature relevant to the varied horti­ cultural practices or physiological problems in which these growth regulators were used will be discussed with the appropriate experiment* EXPERIMENTS ON THE SETTING OP TOMATO FRUITS Review of Literature The first plant which was stimulated with a synthetic growth regulator to develop parthenocarpic fruit was the tomato. This was achieved by Gustafson (30) in 1935>* and since that time many different compounds have been tested (31» 90, 10?) on many different plants. This early work has been reviewed by Gustafson (32) and Avery (6 ). More recently Hoffmann and Smith (3 8 ) have reported that a group of N-aryl phthalamic acids are capable of setting fruit on Bonny Best tomato plants. The concentrations required to set fruit, to inhibit fruit set, and to produce formative effects on the leaves varied with the substituents on the aryl group. The minimum concentration which was effective in setting fruit was 63 ppm. They also report that the N-aryl phthalimides also behave similarly, but do not mention any specifically or give any data. The compound used in these experiments, l^-phthalimido-2 ,6 dimethylpyrimidine belongs to this class of chemical compounds. The ability of growth regulators to set tomatoes has afforded a solution to two practical problems. One is the setting of greenhouse tomatoes during the cloudy winter months, and the second is the setting of tomatoes in the 6 field when the night temperatures are low. Tomatoes grown in the greenhouse during the winter in some areas do not receive enough sunlight to develop normally. Burk (16) found that the stigmas of Bonny Best tomatoes grown under low light intensities and short days, protruded beyond the stamens making self pollination difficult, while the pistils of tomatoes receiving more light were about the same height as the stamens. Howlett (3 9 ) confirmed this and in another paper (i+0 ) reported that morphologically normal pollen produced by tomato plants low in carbohydrates failed to germinate. Pollen produced by plants still more deficient in carbohydrate frequently degenerated during its develop­ ment. This degeneration usually occurred following the liberation of the microspore from the tetrad but sometimes occurred later during its maturation up to the time of the mitotic division of its nucleus. Howlett utilized the classification of Kraus and Krabill (1|_8) who had found that tomato plants grown on soils high in nitrogen, or under conditions unfavorable for photosynthesis, tended to make rapid vegetative growth, be spindly, and did not set fruit well. Plants growing in warm, humid, greenhouses in cloudy winter weather often grow this way. Withrow (105) has shown that the yield of greenhouse tomatoes can be increased by supplying additional light to the young seedlings. Wittwer (106) observed that the low light intensity during the setting and development of the fall crop of greenhouse tomatoes was accompanied by a much lower yield as compared 7 with the yield of the spring crop of tomatoes grown when the light intensity was greater. The last three investigators mentioned and many, others (6 5 , 6 8 , 7 7 * 8 8 * 1 1 2 ) have attempted to increase the set and yield of greenhouse tomatoes with growth regulators* This has proven practical and para-chlorophenoxyacetic acid and B-naphthoxyacetic acid have been found to be th© best compounds# The fruit set with these compounds fre­ quently softens prematurely, has a green pulp around the seeds which may give the fruit a scalded or blotchy appear­ ance to the skin which also takes on a glossy, water soaked appearance which decreases its market value (IjJ., lj.2 )* The influence of various normal metabolites and envir­ onmental conditions, upon the ability of para-chlorophenoxyacetic acid to set excised flowers of a male sterile line of John Baer tomatoes was studied by Leopold and Scott (52). They found asparagine and glutamine gave the greatest increase in set and that adenine, guanine, thio­ urea, urea, pyridoxin© (vitamin B^), and thiamin (vitamin B~l) increased the number of flowers which set in about th© order in which they are mentioned. It is noteworthy that thiamin, which has a pyrimidine nucleus, ranks low on this list, so the activity of l^-phthalimido-2 ,6 -dimethylpyrimi­ dine in setting fruit is probably not due to the similarity of its structure with that of thiamin. They also found that sugars, the organic acids of the dicarboxylic acid cycle, ascorbic acid, glutathione, amino acids, and inorganic 8 sources of nitrogen also increased fruit set* The importance of an adequate supply of nitrogen containing compounds is evident* The optimum temperature in these experiments was about 68 degrees Fahrenheit with very poor fruit set occurring at 50 degrees. They report that abscissing flowers are still capable of being set* They also state that the failure of some flowers to develop which do not absciss is due to the lack of nutritive materials and not to a lack of sufficient auxin. The methods employed in this study are somewhat arbitrary and the small number of flowers used in each experiment probably do not give too reliable estimates* Still this is the most complete study of its kind and the trends indicated are probably valid. Xn another study Zalik, Hobbs, and Leopold (111) reported that tomato fruits set by applying para-chlorophenoxyacetic acid to the joint or abscission zone of the pedicel, were puffy, having large grooves in the carpel wall. Xf the night temperature is below 58 degrees Fahrenheit tomatoes will not set well. This has been the experience of Wittwer (108) in Michigan, of Went and Cosper (102) in California, and of Went (101) under the controlled conditions of his air conditioned greenhouses. Smith and Cochran (83) studied the effect of temperature on the germination and tube growth of tomato pollen. They found the optimum temperature for pollen germination was between 70 and 85 degrees Fahrenheit and that pollen tube growth was best at 70 degrees and very poor at $0 degrees* Even under optimum 9 conditions th© pollen tube required about two and a half to three days to grow the length of the style, and If it was too cold for good growth the pollen tube never reached the ovule* In a recent study, Osborne (?0) reports that low temperatures from 50 to 63 degrees Fahrenheit are capable of inducing parthenocarpie development of unpollinated tomato flowers* This may have been a factor in some of the early fruit setting experiments* Greenhouse Experiment Methods and Materials This experiment was conducted to determine if lj.-phthalimido-2 ,6 -dimethylpyrimidine was capable of setting parthenocarpic tomatoes of higher quality than those produced by the other growth regulators* Early in November of 19519 Rutgers tomato seeds were planted in a flat* They were transplanted to three-inch pots and later to six-inch pots* During January while the plants grew from five Inches to a foot high, they received additional light from fluorescent lights used on a neigh­ boring bench* This amounted to about six hours in addition to the normal day length of eleven hours. This was a longer period of light per day, for more days, at a later stage of development of the plant, but a lower light intensity than that used by Withrow (103)* The plants were growing in a soil composed of two parts 10 of Hillsdale loam, one part of a raw peat, and one part of sand. This soil mixture had a pH of 7*1* Using the active Spurway methods (8 6 ), the nitrogen tested 80 pounds per acre, the phosphorus tested 13 pounds per acre, and potassium tested 381+. pounds per acre. The pH was near optimum, however the soil lacked sufficient nitrogen, and was severely deficient in enought phosphorus for optimum plant growth. The amount of potassium was more than sufficient for good growth. About a half a gram of mono-calcium phos­ phate was placed In holes in the soil of each pot at inter­ vals throughout the growing season to correct this deficiency. Ammonium nitrate was dissolved in water and the plants were watered with this solution periodically. On one occasion some of the solution was accidentally applied to the lower leaves whieh developed necrotic spots but the plants outgrew this in a couple of weeks. In the middle of February the plants were transplanted to twelve-inch pots and tied to stakes. The plants were trained up on strings when they outgrew the stakes and the suckers were kept pruned out. As the plants grew, they became pot bound and fertilizers were applied as described earlier. The plants sometimes showed nitrogen and phosphorus deficiency symptoms, although tissue tests for these elements indicated a moderate supply at all times. In order to prevent normal self-pollination and fruit set the flowers of the first four hands were emasculated just before sinthe sis during March and April. Every three or four days, the flowers were dipped in solutions of 11 1 0 , 2 $, 5 0 , or 100 pyrimidine* ppm of ip-phthalimido-2 ,6 -dimethyl- With few exceptions, all of the hands on a plant received the same treatment* The flowers of one group of plants used as controls were emasculated but not set* Fresh solutions were carefully made up at least every two weeks to avoid any natural decomposition* All of the compounds used in these experiments, including i4.-phtb.alimido-2 ,6 -dimethylpyrimidine are crystalline solids in their pure state at room temperature* They were dried in a desiccator over calcium chloride, for at least 214- hours before weighing as recommended by G-ortner (29) who found the failure to do this could easily result in errors of 25 percent because of water of crystallization* Enough distilled water was added to make a stock solution of 1000 ppm and this was shaken until the compound went Into solution* The density of water at room temperature was corrected for, so it was felt that the concentration of the solution was accurate to three significant numbers on a weight per weight basis* The solutions of other concen­ trations were made by diluting the stock solutions* All of the glassware was kept very clean by soaking it in activated charcoal for a day or longer to absorb any growth regulator present, as described by Lucas and Hamner (5^-) and then carefully washed with soap and water and rinsed with distilled water 0 The tomato plants receiving the different treatments were randomized on the bench* The dates of emasculation 12 and the dates of dipping to set the fruit were recorded on a tag as shown in Figure 1. When the fruit matured its weight was recorded, it was cut open and inspected for seeds, or abnormalities, and the number of days between emasculation and ripening of the fruit was recorded* Results The If-phthalimi do-2, 6 -dimethylpyrimidine did induce the parthenocarpic development of tomato fruits* The 100 ppm concentration was superior to all others and the 5>0 ppm concentration was about half as effective as 100 ppm in causing parthenocarpic development on tomatoes as shown in Table X* The 25 ppm concentration was capable of setting fruit, however, 10 ppm was too low a concentration for effective fruit set* The fruit set with Ij.-phthalimido- 2 ,6 -dimethylpyrimidine required approximately as much time to develop to a red tipe condition as did the control fruit* The parthenocarpic fruits were just as large as the pollin­ ated fruit of the control treatment and had excellent color* The fruits were of normal shape except that some were slightly pointed at the blossom end and the style persisted on some as is shown in Figure 2* The dipping of the flowers in this solution seemed to make more of them absciss as shown in Table I* The 50 ppm treatments appeared to delay the process of abscission however* These factors are of little importance in tomato growing but indicate that the compound might be useful in thinning tree fruits# 13 figure 1 * A tomato plant with fruit set w i t h 100 p p m of ij.-rbthalim5.do-2 ,6 - d i m e t h y l o y r i m t d in © * 01 d d 0 O *S *H O « H T O4h •H O -P d © **o TO O bO $H TO d -P 0 0 d h d bD 0 TO TO TO d •* TO H h e o «h p. H O > S Oh TO d <1 P d 0 0 43 0 TO A, OJ rH CM CM rl pH I>a P\A § P O TO H •H 0 ••H n w 0 0 . .rH s>a bO CO X3 S ^ o rH d P «d ❖ * CO o -d* * TO tH TO TO TO d d^J g 0 0 TS > a © •rl •H *H d d TO73 TO P d -p d p0 ©no XI 0 p £ O P TO vO a d P 0 d rH C"1A C*— U\ \A \A TO O s «H 0 d »O 6 t 0 d C ?S$H «H rH d P <*-1 d TO P p o d d •H 0 © d 6 p pH »H 0 a s 0 •H *rl d d W TO TO P £ -P 0 O CO o cr\ vO CM t1-d- pH O gg o0 O *H d •H ® of ^•phthallml do 6-dlmethylpyrimidine Lower lefts Side view of a parthenocarpic fruit showing most extreme pointedness of blossom end resulting from setting with 100 ppm of 4*phbhallmldo<»2t&'» dlmethylpyrimt dine Lower rights Blossom end of a partheno* oarpio fruit set with 50 ppm of it~phbhalimld0~2 »6 ~dlmebhylpyrimldlme showing the persisting style which frequently occurs 16 There were many abnormal fruits produced by all of the treatments which were not included in the data above. They were small, weighing less than 65 grams, which is too small to have commercial value. They required more than 85 days to ripen after emasculation and setting, and they were puffy, with large grooves in the carpel walls, and frequently had small, brown masses of tissue the size of aborted ovules. Since there was a break in the frequency distribution curve between these and the normal fruit, with regards to all £hree classifications, the data of the ab­ normal fruit were eliminated which was necessary before an analysis of variance could be calculated. The poor nutrition may have delayed the development of some of the fruit until it was corrected as suggested by Leopold (52). Perhaps enough of the growth regulator was applied to the abscission zone of the pedicel which may have been slightly separated during dipping to produce the puffy fruit characteristics described by Zalik (111). The abnormal fruit of this experiment closely resembled those pictured by him. Field Experiments Methods and Materials Several flats of John Baer tomato plants were planted April 11* 1952. They were transplanted to four-inch pots during the last week of April and grown in the greenhouse. On May 30 brie hundred of the best plants were selected for planting in the field. They all had the flower buds of the 17 first hand well developed and some flower buds were open at this time* The plants were planted in a single row three and a half feet apart. The one hundred plants were divided into five replications. Each replication contained five plots which included four plants each. The soil varied from a Hillsdale sandy loam at the upper end of the row to a silt loam at the lower end* ence between replications. This was the major differ­ The plants were watered with a starter solution of "Take Hold1* soon after planting* The hands with open blossoms were sprayed on June 6, 9i and 12* All of the first hand and the second hand of some plants were included in these sprays* The weather was warm for a period so no treatments were applied until June 18 and 22, at which time the flowers were treated by dipping them in the solutions* The minimum daily tempera­ ture as recorded at the hydrogeologic station a half mile away is shown in Figure 3» The five treatments used were 100, 25>0 , and 500 ppm of ip-phthalimido-2,6-dimethylpyrimidine, one treatment was sprayed with 30 ppm of para-chlorophenoxyacetic acid, which is the recommendation (1 0 7 ) made to tomato growers, and distilled water was sprayed on the flowers of the control treatment* The sprayer used in this and all subsequent experiments consisted of an ordinary knapsack sprayer with an air pump* This was used only as an air reservoir and the spray solution was kept in a 125 ml erlenmeyer flask fitted with a cork and glass tubing like a wash bottle used in a Minimum Night Temperature 1952 « 0. %* o. ma oII wN CM o O •O July During the Blossoming Period of Tomatoes at East Lansing- 1952. figure 9, <0 o August is CM M tO o> X o CD PC IO to CM o o rH *> O-» « 19 chemistry laboratory, A fine spray nozzle was attached to the outlet, and a stopcock was attached at the inlet to the air hose so that the sprayer could be turned off. All the parts of the nozzle could be easily cleaned and a clean flask could be used for each different compound. The first ripe fruits were picked on July 21, and at two-day intervals until August 8 . The fruit of the lower hands which had been sprayed were picked by then. They were the only ones which were parthenocarpic. August 8 was the day which the price of tomatoes broke on the Benton Harbor, Michigan, market which is the largest fresh fruit market in the world. The price dropped from about three dollars to less than a dollar and a half as can be seen in Pi gure 3• These early fruits were weighed individually, graded according to the grade laws of the United States Department of Agriculture (97) and cut open and inspected for the presence of seeds or abnormalities. The fruits ripening after August 8 were picked at four-day intervals until the middle of September when they were picked about once a week. This was done to determine the effect of setting the fruit of the first hands upon the later yield of the plants. Results There was a striking increase in the amount of fruit produced early in the season on the plants set with l4.-ph.thalimido-2,6 -dimethylpyrimidine as can be seen in Table XI. The 20 CM vO • g p p I £ X! o o p © g T$ t© O fl ^•rl UN O ' CO r UN UN o CM d UN d * CM rH • • • • O O rH © ON CO d - © • UN rH ON iH CM X * I bO XJ © © © © *rH G O g <4h p p 5 Xf *SH o •H P< u \ CM •eH ip» rH © UN rH © ON ON d o IN - CO IN • UN ON O • ON O ' ON • O si _p .d p i d g P P o o G I—I © rrH UN CO o CM rH CM 0 d C— CM • ON ON b • i—1 © X* I M M W P W < Eh *tn G O Sh ro -P f? o x* *A G O rH O ' -P o rH © i—I rH © > © rH © rH ON CO 0 CO UN o d CM o rH t— r 0 CM O ' CM • CM « rH O ' o 0 rH +» O Eh ~rf* rH -P G H ON CM I O ' I— CM I ON CM rH ON I C^CM • bO G d I rH ON P © © P © X* © P G © £ TO P G © £ P © © G P © © G P -p © o rH *H o UN © xJ (—1 © 0 p G © £ p © © G 43 0 t>s> •"3 rH G •“3 ■3 l“D rH 2 h> bO G < CM CM 0 vO G © © $ P © CM O 0 co © © xJ rH © f t f t rH © P O p i—1 © P O P o o G © © 0 O ' G © © Si © P P © P bO G •rH -P © © p bD G •H P © © P bO G •H P © © P G O P CO 1 d i—1 P © El © o © > G © CM CO • ON rH G O f>5 <«-( O O ' UN 0 © •H G © 0 © P* sO rH CO IN 0 O ' 1—1 CO IN 0 p ( h *h © o si © § p p OOP 0 > © G G O vO ON sO d # • p O P X* rH © rH tH on P • CO * p G O Cm • o p xJ H © *H ON P m m P • P • rH i—1 © G P V-3 O EH G O H © G P « I O Eh • CO • p 21 500 ppm treatment produced a quantity of fruit earliest* The 250 ppm treatment produced the largest amount of early fruit, but the peak of its early production was somewhat later. The 100 ppm treatment also increased the amount of early fruit, and the peak of its early production occurred after that of the 250 ppm treatment. The greatest differ­ ence between treatments occurred early in the season in July so the data were divided to show this difference. The fruit set with this compound were of as high grade as the check fruit, as shown in Table XXI. Cracks and a little blossom end rot, both of which were partially due to the irrigation practices were the major causes for tomatoes not being U.S. No. 1« The set- fruits were also somewhat larger than the check fruit. The higher the con­ centration of the spray, the larger the percentage of parthenocarpic fruit. Although the 250 and 500 ppm treat­ ments had many more early fruits, there was a highly significant decrease in the actual number of fruits with seeds, which seems to indicate that these sprays decreased the amount of viable pollen, or the amount of fertilization, or hindered the development of fertilized ovules. At no time was the yield of plants treated with 30 ppm of parachlorophenoxyacetic acid significantly different than that . of the control treatment. The plants of 250 and 500 ppm treatments were slightly smaller at the middle of the picking season than the plants of the other treatments. This was primarily due to the fact that the plants of the 21 500 ppm treatment produced a quantity of fruit earliest* The 250 ppm treatment produced the largest amount of early fruit, but the peak of its early production was somewhat later. The 100 ppm treatment also increased the amount of early fruit, and the peak of its early production occurred after that of the 2$0 ppm treatment* The greatest differ­ ence between treatments occurred early in the season in July so the data were divided to show this difference* The fruit set with this compound were of as high grade as the control fruit, as shown in Table III* Cracks and a little blossom end rot, both of which were partially due to the irrigation practices were the major causes for tomatoes not being U.S* No* 1* The set fruits were also somewhat larger than the control fruit* The higher the concentra­ tion of the spray, the larger the percentage of parthenocarpic fruit. Although the 2$0 and $00 ppm treatments had many more early fruits, there was a highly significant decrease in the actual number of fruits with seeds, which seems to indicate that these sprays decreased the amount of viable pollen, or the amount of fertilization, or hindered the development of fertilized ovules. At no time was the yield of plants treated with 30 ppm of para-chlorophenoxyacetic acid significantly different than that of the control treatment* The plants of 2$0 and $00 ppm treatments were slightly smaller at the middle of the picking season than the plants of the other treatments. This was primarily due to the fact that the plants of the control treatments were 22 © •H 43 £ © o £ © O 43 O tH £ £ £ © £ © x j s~» P 43 £ O Ica co cm cm © & £ © 4£ 3 43 r to O § 43 43 o £ £ © © £ ■d S 43 rH H © s© £L 50 £ 50Oh O © •H *H *H £ £ W n ,d £ 43 & rH 43 • £ O 43 © *H O £ • H £ © CO £4 tu • •P ,5£0 •H £ © *H & © «rH 50£ ©£ £ *H © > » > 0 O . 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P 2 CO *0 & £ O ft po-4 *H •H PH CM CM ft ,£ o 3 I •H CM P ii n CM £ cJ •a rH rH <*> 0 0 «OH > ^* bO 0 0 P P m8 0 0 0 > £ ft bo © J * 0 f t i>» 0 © P 0 S> 0 ft 0P P *s g *P 0 0 ££ S ft -P P 0 hb<£ 0 f 0t•H pft g rC 0 £ •H > in £ 0 £ *H > A 0 £ M rH cd 2 0 £ J P CA 0 0 f t EH £ > ^ -P p p 0 0 0 ^ rO rQ b3 bo bO £ *£H *H£ |.^ *P 0 0 Q 0 0h 0 01 Eh Eh CO E •H P 27 , ra >> 0uo Pt •0 Ht f f 0 CCS > £> ccs VO THE YIELD OP EARLY FRUITS PRODUCED BY TEN GREENHOUSE TOMATO VARIETIES AS AFFECTED BY WEEKLY TREATMENT WITH THREE FRUIT SETTING CHEMICALS OR A FOLIAR FERTILIZER SPRAY rH -4vo • o H to o02 & to • o ON 02• p o ft O2 rOH 6ft 0 0 •H ft P rH o o• rH 02• o o o• o ON NO rH • o CO to CO • o CV rH to CO 02• o-* o i—! CO CO vo rH O rH CO t vo o • o to• o • • 02• vo to o• rH NO o« rH 0o 2 t • o sfcv o ft S 3 o o o p o r a © p •H © roP© 2 O *fH t P © 2 o ■a 02• CO On • • O O O tO nO (H ^ rH CV C VO « • • rH ® J, 2 'OH Cn « 0i •EH-rP)* >X5 P Pi 0 o A-p *4rj . P I Pi ftvO I ^ CO v o CO pH 02• i—1 txO •PH f 0t § O P&4 3 P0 £o PP •uHI > » 33 tpo P §3 w* •fSt' a J0 CO P t o to • > P i=> © P HD h 3 ft, 1 m • o £ 3 o 02 02 ft 0 ■ 3 •H p . f? 525 ON ON • o 8 O bO 5 O ft r° 0 P ■3 CO • O •H X CO § rH £ P 0 •PH J> rH 2 Si VO VO 0 P A0 P £ ■8 A0 P O ' 02 o to o 0 I tP uO P u ! > cd p P 0 P cd 0t f e-* P 0 Ch 0 o t i o cd w PP 0 0 bO cdpI p S Ps» 0 f 0 P •H p t P ft § ► a0 p ft JP •H btO<0H 0f Pft § *H •O P P P 0 0 0 0 0 0 ^ ^ P P +0 0 0 P P P bO bO bO A A A p p P0 Q W 0 0 0 0 CO E E Eh iH h h 28 >* © g u £a I> c •P tiO © o cv u i s o s Pi CO ci£ cv §s P o on vD UN CV CV ON O'' ON on on cv on CH. cv O to CV 0\ o cv NO rH rH un CV ON UN Ht ON $ ON ON UN §• 3- O ON >» CV rH © o on o\ o O ON ON -t ON cv . CO |L o g P h p< o o « H *H O X I P .U N to cv cv h^nO J> rH ? & V © O N 'fcg. UN 0\ UN ON rH O S3 *n Ph o o on on UN CV pH ON ON cv on cv UN ON ON CV UN Oi, to ON Ch- CV O ON M > »D P* H ® H MS I g to o ON on on CV cv vO ON SO ON to cv ON ON NO '4- ON UN ON O ON M l fe,1"1 fV vo P. 1 «* ■Hfcv CO o s § on cv s £• ON ON £> ON UN ON o tiO p p P *H O P o o © -p m © p «H 0 1© g CD o Pi 'p •iH © *P U M % S3 P © -O CO P •H u o o m p© IS3 & g «e p CO 3 u e S3 a XI a CO tx<3 P O u cc- JS § •§ o rH o p •H X o •H X ON £3 UN *s © p **H CV rH UN IS © S fc» g eo CV UN -p P •O P © S* © X ON © © S Eh"d © © © O to © m u p © © P © > © w © g © -p 5h © © p © *> © P © T* p P -P © p P £ P © $ g P P -P vH © © W) Cm 5h «H F M m •rl P g 'O P P P © © © © © © > s s HP p p © © XI M X) ttO WJ tVJ p P p •H •H •n ? P -p P CD © © © • © © © CO Eh Eh Eh 29 THE AVERAGE WEIGHT PER FRUIT OF TOMATOES FROM TEN GREENHOUSE VARIETIES AS AFFECTED BY WEEKLY TREATMENT WITH THREE GROWTH REGULATORS OR A FOLIAR FERTILIZER SPRAY ^ 05 -p & rH c\j .Us © s O c\j cn c\j tQ ON to tto O rH Z> NO tO 1> to rH 3 OS > i> eo ov P.< c CO CV rH i n O' Ii 01 o <2 P cv NO ON n o 1= o rH k §3o $ 03 & ° O *H P < oG o a c - O £ O> Nc 0 n^ rH rH rH rH rH m in d'g.R "i o s a, rH O n in ON rH on V •o I -H < L ) O «H C O *H -P P. rH © tO <©. -poo ° N £3 £ ©r PQ i cv s s a s X l _ 'tf?. O NO g © »H © rH a 3 » to ON > -p © •H Pi p ro h •H |h * r f a & a xf*g Pi S'S _g_g ■go -h o fc.' PkvTO o. I •* "t CV -p 9 CO o cn rH I —I *n 3 cv o NO PI NO rH cn ON cn m on to to 01 t© cO cO g -P © © Pi l-l oi P -p g £> 3 cn rH rH 9 o 0 ti no c n t0rH o to On O rH ? cO •H -p IH-p -P g f> © £ % £ Pi On O rH o -p -H G J • tut) l> £ *1-1 £ •H o ■p ra po © •H © -©P © §13 ■©S P © r‘ C5 P3 E*4 £ •© -p •g pCO £ w & CO -p c© a ©© © © © O rH © •H PH CO CV XI O •H cv Pi © r© s sh; • ■p -p -p © bO © Pi P PH a 'o g © C p§ =< © -P Pi o bo £ •H O Pi O © H0 3 3 rH CV m s A © a »rl > M SCO 13 8 CO -p £ © X cO © o A n 2 ♦H £ rH > g h • -P m • © W Eh fi -P to © tn -P w © Eh 30 ■8 * 8 P P W> X I © gj tiO P w ts > US CV cn •rt fn © ® © > O . CV *4 !>» A «* 01 * 0 HH g .. A ^ P O CV in cv &2 0 cn in in vO £> -41 cn M0 cn m cn in -<*• to "4- o cn cn cv cv to rH -4* cn m cv m cv cn cv «H cn cn J o t—i p cv cn & no © p © © -4" p m ax as r-i © & 2 O *H £ G © O © r-i a* © s » a & 0 -o H •rJO xi fttm b in O m cn C- vO vo cn cn in § to -4“ \o 3 1 1 1 a P P © © N O *5 p o X o © XI © £ o © c © O in to *•4- <8 vO cv cn H cn -4 cv O CV m cn o cn Hjfr cn cn o c v m £3 CQ a ■P'D ft to cv -4“ in ir­ on v\ cv cv -ct cn ao u H o c*4 © rQ -p © p $ © p 05 PJ S) aO 3 M & £© © p © f£ 05 © X> o rH O .3 pk xt o *H s x cn CV c 3 in a in no' G *3 Pi ■©3 > H cv m 'O fl A© A© G P > G P > © © cn vD P P rH X! © o 3 p p CPX G ra § O 01 P Op © &Q G ®S PP< cp rH O P-t © Pi O S © «sf © ffi Xi £ G PI O ro © G © > P P P ©P © o 1 g © Pi © <3lJ £ P Pi © u <£ »I P f l © o ?H ■d hfl •H W X to s © Pi © ax © tiO Pi © © © «“* 43 u E-t o rH o o O O - £ o c*\ CV D- rH rH G>- sO $m rH sO CV *«OH rH e0 o M O g CO *H P« a> CV O C*~\ £> O# O different PQ O U~\ to O Ph s ^a> -p o o O £S3 R 3 P I < L > 8 from each other at the *01% level as tested by CV c4 O O G> o o O V \ ~ U 5 {§ © i3, rE £ (D rO P 'S. w <0 3 > 3 0) £ •H > -P H X CO Ph •P *V hours long, and only the earlier flowers were used to avoid the possibility of natural parthenocarpic fruit# Results No conclusions can be reached from this experiment concerning the ability of Ip-phthalimido-2,6-dimethylpyrimidine to cause the parthenocarpic development of cucumbers because over one-third of the fruits of each treatment, including the control treatment were parthenocarpic. A parthenocarpic and a normal cucumber are shown in Figure ij.. The parentage of the Burpee Hybrid is not published however there are English strains which are naturally partheno­ carpic and one of these might be a parent of the seed and explain the large number of naturally parthenocarpic fruit. 37 F i g u r e ii* Two p a r t h e n o c a r p i c Burpee H y b r i d c u c u m b e r s on the left and a normal cu c u m b e r cn the right# R e p r e s e n t a t i v e ovules from each tyre are shown dir e c t l y above the cucumbers. EXPERIMENTS ON THE ABSCISSION OF COLEUS PETIOLES Review of Literature The abscission of various organs is a wide-spread phenomenon of plants. The abscission of leaves, especially of deciduous plants in the fall, the abscission of flowers and immature fruits of plants, the abscission of ripe fruit, and the abscission of branches of some plants are very common occurrences# Although abscission is frequently accompanied by some cell division and the formation of a primary protective layer, the primary process of abscission usually Involves a dissolution of the intercellular substance and a mechanical breaking of the vascular tissue* The biochemists (15>) have chosen to call the intercellular pectic substance protopectin, to distinguish it from the other pectic materials. This galacturonic acid polymer which makes up the bulk of the intercellular substance in young cells probably has a definite orientation and the manner of crosslinkage and arrangement of calcium and methyl groups may be entirely changed in any extraction process. The enzyme which attacks protopectin yielding soluble pectin is known as protopectinase and an adequate chemical characterization of it has not been carried out (13)• Because of this, the 39 best available method of studying the effect of growth regulators on the process of abscission is by studying the rate of abscission* La Rue (50) was the first man to study the effect of auxin on the abscission of petioles* Ke found that by applying a lanolin paste containing 50 ppm of indoleacetic acid to the tip of debladed petioles of coleus plants kept in the dark, the petioles stayed on two and a half times longer than the controls. Beal and Whiting (10) reported that the abscission of stems of the four-o*clock plant, Mirabilis jalape, could be inhibited by applying indole­ acetic acid in lanolin to the cut surface of a branch* The delaying of the preharvest drop of apples with sprays of growth regulators is another example which will be discussed later. The physiological processes involved in abscission are not well known* In recent years there has been consider­ able objection to the theory that abscission is the result of cell divisions and growth of an abscission layer which pushes off the organ (28). The division of cells may or may net accompany abscission. More emphasis has been placed upon the breakdown of the intercellular substance and the biochemical changes responsible for this* Since most chemical changes in living organisms are accomplished by enzymes, many attempts have been made to isolate or identify protopectinase. Kertesz (45) and others have been unable to find an enzyme, and suggest that perhaps just the potassium ko ion, or sunlight may be the catalyst involved® Whatever the mechanism may be, indoleacetic acid has been found to inhibit abscission, and ethylene and other compounds have been found to stimulate it® Upon these facts, the hypothesis has been advanced that it is the balance of auxin and ethylene which governs abscission® Ethylene is a normal product of plant metabolism, whose production is increased under anaerobic, conditions® In ripening fruits it is known to accelerate its own production, and to accelerate the rate of respiration, which makes the conditions more anaerobic if the fruit is kept In a closed place as fruits are® Hall (3k-) found that ethyl alcohol, pectin and pentoses were very good substrates for an ethylene producing enzyme which he extracted from yeast® Ethyl alcohol, a two-carbon molecule, is one of the non-volatile end products which pectins and pentoses might be broken down to during anaerobic respiration® Ethylene Is a vola­ tile, two-carbon gas which apparently can come from ethyl alcohol® One must remember however that this is an endo- thermic reaction requiring about 77 kilo-calories per mole. Hall (33) found that in the presence of indoleacetic acid his enzyme produced little ethylene while in the absence of indoleacetic acid larger amounts of ethylene were produced. Thimann (91) and Bonner (13) and others have suggested that indoleacetic acid is necessary in organic acid metabolism, a process in aerobic respiration® Leaves and flowers are known to produce indoleacetic acid. The Ifl hypothesis which has been proposed is that when the supply of auxin to the abscission zone is decreased, and the respiration becomes more anaerobic, ethylene is evolved in the tissue which further stimulates its respiration and the utilization of pectic substances, and pentoses which are responsible for holding the cells together. Neely ejb al (66) found the activity of pectin methoxylase was nearly doubled in stems and leaves of red kidney bean plants treated with 2,lj--dichlorophenoxyacetic acid0 Perhaps this indicates that the auxins affect directly the activity of enzymes responsible for the splitting off of methoxy groups from pectin. The influence of l4.-phthalimido-2,6-dimethylpyrimidine upon the abscission of debladed petioles of coleus plants was studied in a series of experiments on potted plants in the greenhouse. The abscission of debladed petioles of coleus is quite precise, with the lower, older petioles abscissing earlier. The growth regulators were applied in lanolin, in the manner used by La Rue, and as a spray, the common commercial method,, Lanolin Application Methods and Materials Cuttings of the Christmas Gem variety of Coleus blumei L. were made in January. The lateral shoots which develop in the axils of leaves were cut off to remove that source of k2 auxin. Forty-six plants were graded for size and the blade of one leaf at each of the lower nodes was cut off. The tips of the petioles of the different lots of plants were then covered with a lanolin paste containing 100 ppm, 2^0 ppm, 500 ppm, 750 ppm, and 2j?00 ppm of l4-phthalimido-2 ,6 dimethylpyrimidine• A pure lanolin paste was used as a control and 100 ppm of naphthaleneacetic acid was used for comparison with a standard growth regulator. The growth regulators were dissolved in a small quantity of ethyl alcohol before mixing into warmed lanolin. The concen­ trations stated above were made up as weight of dry growth regulator per weight of lanolin. The amount of lanolin paste applied per petiole varied somewhat. The potted plants were laid out in a latin square design on the green­ house bench with each of the seven treatments replicated seven times except the 2^00 ppm treatment which contained only three plants. The number of petioles which did not absciss with slight pressure to break the vascular bundles, was recorded once a day. Results Only the 100 ppm treatment of naphthaleneacetic acid caused a highly significant decrease in the rate of abscission, while the highest concentration of l4-phthalimido-2,6-dimethylpyrimidine appeared to increase the rate of abscission as can be seen in Table XI. This is not significant and might possibly be due to a higher concentration of alcohol which according to Hall could be converted to ethylene. 43 THE ABSCISSION RATE OF DEBLADED COLEUS PETIOLES AS INFLUENCED BY GROWTH APPLIED IN LANOLIN TO THE TIPS OF THE P E T IO L E S REGULATORS Pi Pi O O to d P as •H © U P Jh © P at UN CVJ © 0 U \ • UN C 'UN • UN CM O ' • CM • O ' • O H Pi © P* -P at IQ -P a at H a © © B 0 UN • UN 0 UN • UN •H P CM • UN rl CO C-— • CM UN O • CM -P g Pi P I nO O 9i o UN CO I 0 43 +3 C— UN • UN js* vO vO U N UN O• O' O• • • UN -d" rH rH e •H g P P o UN CM 0 UN 0 UN r— UN 0 c*N O • UN CM O ' a CM O ' O • CM 1 C— UN • UN CO CM • UN rH C *A -d * co CM « CM rH C'•rH aS S © IQ © pH O •H P © a O Pi p d © •H Tt d © © p£ © bD d •H P C^- CO UN • CM0 UN UN O ' O ' UN rH 0 A COA 0 -d* -d- -d* © © P Pi IOp 1—I © > © O Pi bO at p p I © TJ d «h © o at g M4 43 O P P iH 43 P O pi© O S SH Pi © rQ © © Pi © •H Pi © p d Pi S P P O O (3 at -Bp at •H * P © TJ bO d © © •H a o -P © © S £ • n -P IQ Tt O •H f*N CM d •H Tt i Pi Pi P< l» O Pi UN UN IN- CM UN rH C—, UN -d- COA 0- U'A A A A UN UN -d* CM 1 —1 -P d o o H UN P © © > O bO d © Pi *H Pi © +3 d P at O <»H © tn at Pi -P CO CM -d" vD c*N v O vO O' i-3 i+4 Spray Application Methods and Materials The lj.-phthalimido-2,6-dimethylpyrimidine was applied in aqueous solution as sprays in two similar experiments* The entire plant was sprayed with the knapsack sprayer with the attachment allowing a clean flask to be used for each compound* The plants used in these experiments had been used previously, however, the same treatments were applied to the plants in both experiments and considerable time was allowed to elapse between the experiments* None of the treatments caused any formative effects or morphological changes. Re sult s Both of these experiments showed the same trend* Tables XXX and XXII and Figure 5 show that only the 1000 ppm concentration of Ip-phthalimido-2,6-dimethylpyrimidine and the 10 ppm of naphthaleneacetic acid caused a significant decrease in the rates of abscission, and there was no significant difference between these concentrations of these chemicals* Defoliation The results of the first experiment with the high concentration of l^-phthalimido-2,6-dimethylpyrimidine applied in lanolin seemed to indicate that this compound might be useful as a defoliant* Salts and acids of 3*6-endoxohexa- k$ g ft ft O O o "bO 0 ft ft P W o ft 73 t>s1 A CM CA • 6 H g Ph O P CO Pft PQ I s O Js O o (d P iw rH P. © rft 43 ft ft ft S © •H CM IP 0 < -P ft © ft © (ft S ft ft -p •H O O 0 ft d CM XA 0 CA NO CA 0 fA d o 0 CA vO P • rH •H ti ft © © 43 © A i © t* bO ft ft*H © O 43 •ft ^ O ■P *H rft * P ^ p, ft ft© O ft O H O O 0 d o o 0 d o o 0 d p co 0 CA ft cq d 0 CA © 43 ft O < f t ft rH © > © i —I c © bO 43 a. ft © ft ft © ft 0 CM ft ft ft ft 4> n ft © 43 ft •H © © © A! -p ft 43 rft bO ft o CM r• CA •H ft o •H S o d •H ft ft I so•v o 43 H <1 CA g ft co cO P « P o O Eh M < H CO CO p o CO PQ d -P © 43 M CO o d rH P- S3 S5 d • IP O, O *H ft l-M £ ft ft © P PQ W P d d © *H 43 13 PQ P CO rH S ft 6 © >H PQ p o o © •H ft 183 EH o o TJ s ft a © tS Ph p* to p o o •H O CO H o o ft -p ft o o O O 0 vO CO o O nO 0 P d 0 O O 0 d CA CA fA CM 43 ft P* bO . ft CO 09 ft *rl ft © XA -P ft43 ft O 5 x a a p rH © S O O • vO SO rH • XA XA O • A— C— # CA O -=h • rH Cl P 43 © £ g PI CQ <*: pq Eh CQ CQ PQ PS Pi O O EH H <*J Eh p PQ B pHc ! 3 pq CQ PS tp w m pi Eh O IS oo PS § P «© P P PQ P Oh O PQ Eh 55 SE5 o H w CQ M O CQ ffi Xl -P o o • v£> rH A• -d“ ca CM • -=h CA CM • -d* CA CM • CM -P a) 09 43 £ o © © 43 •H -p © § w •H rH Ti -P p d © rd 43 c o fckO d •H a •H © E © Ci 09 © rH O •H P © P Ch E S rH O 23 © © © E s •H p. nd p 1 vO o •> X A C\J I © pi PQ bD u d © iH O Ci 43 d O O o o • vO CM CA • XA rH A• CA h ± XA • CA AA— • rH Ci VL XA +3 © © > •© p bO d © Cl iH Ci © 43 d 43 © O H O ) |Xj © Ci 43 « CO * o cA P H CA A— CA CA CM rH Pi 1+7 VP :-r d a3 P-rH tn P of

st1 O X d -p <15 at o m at g ai d p. •H C\ P m 4 \A d U r** o 4.8 hydrophthalic acid, were reported by Tischler, Bates, and Quimba (93) to be defoliants for cotton, beans, hydrangeas and other woody plants and to be herbicides. Pridham (70) found disodium-3>6-endoxohexahydrophthalate effective in defoliating about 80 different species of nursery stock. Coleus plants sprayed with a solution containing 1000 ppm of l4--phthaliraido-2 ,6-dimethylpyrimidine and 10,000 ppm of ammonium thiocyanate, a defoliant used on cotton, developed large necrotic areas on the leaves and a drooping of the leaves, but the leaves did not absciss. The N-l naphthyl phthalamic acid used in thinning pears caused serious defoliation when applied at full bloom. The l^-phthalimido- 2, 6-dimethylpyrimidine was ineffective as a defoliant on tomatoes, beans, cucumbers, coleus, apples, pears, peaches or cherries. EXPERIMENTS ON THE THINNING OP TREE FRUITS AT BLOSSOM TIME Review of Literature The thinning of the overly abundant number of flowers or fruits which fruit trees frequently have is primarily a process of increasing the abscission of flowers or fruits. Growth regulators have been experimented with and have been used with varying degrees of success for several years for this purpose. Burkholder and McCown (17) were the first to notice that sprays of naphthaleneacetic acid and naphthaletamide would reduce the fruit set of apples. An entirely satisfactory method of thinning peaches has not yet been found (lj.9* 85) • The degree of thinning resulting from sprays of growth regulators is frequently greatly influenced by tree vigor, variety, time of application, environmental conditions, and it is very hard to make predictions or recommendations concerning their use. Stebbins (8 7 ) and other workers have found that about 20 ppm of naphthalene­ acetic acid is the proper concentration for Duchess. The only phthalamic acid derivatives known to have been tested are N-phenylphthalimide, which Detar (20) reported ineffect­ ive in setting fruit or thinning Bartlett pears, and N-l naphthyl phthalamic acid. Chadwick, Miller and Erskine (18) reported that the latter compound successfully eliminated 5o the flowers of Norway Maple, Acer platanoides, but caused a moderate twisting of leaves* The flowers of purple crabapple, Malus purpurea, and horse chestnut, Aescuius hippocastanum, were eliminated with 1|22 ppm with only a little foliage injury on the crabapple but considerable Injury if the horse chestnut was sprayed at full bloom. The use of the common growth regulators in thinning fruit trees is summarized by Batjer and Hoffman (8 ). Purpose and G-eneral Methods Experiments were conducted to determine if i|-phthalimido-2 ,6 -dimethylpyrimidine and related phthalamic acid derivatives would be effective in the thinning of apples, pears, or peaches* All of the sprays were applied with the knapsack sprayer with the attachment which allowed a different flask to be used for each treatment* The nozzle was very simple and was rinsed between treatments* The N-l naphthyl phthalamic acid and the N-2 chlorophenyl phthalamic acid used In these experiments is not soluble in water, but is soluble in water as a diethanol amine salt* The diethanol amine salt was prepared In this and all other experiments by making a paste of the powders of the phthal­ amic acid derivatives with an equal weight of diethanol amine which is a viscous liquido Each treatment was applied to a limb on three different trees which were located next to each other on the horti- 5i cultural farm at East Lansing, Michigan, in the spring of 1952• The ordinary fungicide and insecticide sprays were applied to control diseases* Although the thinning and stop-drop sprays were con­ ducted on a very small scale, large differences are necessary for a compound to be useful commercially and experiments of this size should detect such differences* The number of flowers or fruits on the limbs at the time of spraying was recorded and the number of these which had not abscissed after the June drop, and had developed into normal fruit was counted the last week in June* This data was converted to percentages, the averages of which are recorded in the tables of this section. Since percent­ ages are not normally distributed, the percentages were transformed to the angle equal to the arc sine of the square root of the percentage as suggested by Snedecor (814.) and analyzed by analysis of variance* Duchess of Oldenburg Apples Methods Sprays of 100 ppm, 500 ppm, and 1000 ppm of Ip-phthalimido-2 ,6 -dimethylpyrimidine; 100 ppm, 500 ppm, 1000 ppm, and 5000 ppm of N-l naphthyl phthalamic acid; and 20 ppm of naphthaleneacetic acid were applied to limbs of Duchess trees during the middle of the day on May 5* The maximum number of flowers were probably in bloom earlier that morning. The average number of flowers per spur (5®05) on the limbs 52 used for the different treatments, was calculated and there was no significant difference at the beginning of the exper­ iment* Entirely different 3.imbs on the same trees were sprayed on June Ip or 30 days after full bloom to determine if sprays during the normal June drop would increase this drop. Only high concentrations, 1000 ppm and 2000 ppm of the diethanol amine salts of N-l naphthyl phthalamic acid, which had been effective on pears, and N-2 chlorophenyl phthalamic were used because only a limited supply of ip-phthalimido-2 ,6 dimethylpyrimidine was left and this type of experiment requires several gallons. Results Prom Table XIV it can be seen that the small number of trees and the large variation between trees make it impossible to draw any definite conclusions from this experiment. The apparent Increase in the number of fruits remaining on the limbs sprayed on June Ip with N-l naphthyl phthalamic acid, and the lower concentration of N-2 chlorophenyl phthalamic as compared with the control limbs is probably an artifact because more vigorous, higher limbs were chosen for the later spray treatments while the control limbs were the same as those used for the earlier spraying and were somewhat lower. The higher concentration of N-2 chlorophenyl phthal­ amic acid did appear to thin with only a slight amount of leaf injury. 53 TABLE XIV T H E E F F E C T IV E N E S S O F GROWTH REGULATORS I N T H IN N IN G TH E F R U IT S OF DUCHESS A P P L E TR E E S A v e ra g e p e r c e n t o f num ber o f f r u i t s o o n lim b s w h ic h d i d a b s c is s b y th e e n d T re a tm e n t S p ra y e d M ay 5 a t F u ll B lo o m C o n tro l 20 ppm 100 ppm th e r ig in a lly not o f June 2 *6 N a p h th a le n e a c e tic 3.5 a c id 5-8 2 .6 5 0 0 ppm 1000 ppm 100 ppm 500 ppm 1 *5 1000 pp m 4-.Q 5000 ppm 3.4- No 2.4N -l n a p h th y l s ig n ific a n t p h th a la m ic d iffe r e n c e S p ra y e d June 3o5 a c id b e tw e e n t r e a tm e n ts 4- D u r i n g June D ro p 2 1 .8 C o n tro l 1000 ppm 2000 ppm 1000 ppm 2 0 0 0 ppm N -l n a p h th y l p h th a la m ic a c id US. k* 45.3* N -2 c h lo r o p h e n y l p h th a la m ic a c id 3 2 .8 1 1 .9 * * ^ H i g h l y s i g n i f i c a n t l y h i g h e r a t 1% l e v e l t h a n c o n t r o l * * * V e r y n e a r l y s i g n i f i c a n t l y l o w e r a t 5% l e v e l t h a n c o n t r o l 5k- Bartlett Pears Methods On May the day of full bloom, sprays of 100 ppm, 500 ppm and 1000 ppm of [(.-phthalimido-2 ,6 -dimethylpyrimidine and 100 ppm and 2000 ppm of N-l naphthyl phthalamic acid were applied to limbs of bartlett pear trees of medium vigor. There was no significant difference in the average number of flowers (7 .2 0 ) per spur on the limbs used for different tre atment s• Results The l|-phthalimico-2,6 -dimethylpyrimidine was ineffect­ ive in thinning. The 2000 ppm concentration of N-l naphthyl phthalamic acid caused a severe epinasty as shown in Figure 9o It inhibited further increase in the size of leaves which made these leaves appear smaller when compared with the leaves on the control limbs and many of the leaves abscissed. Many of these leaves had black necrotic areas. However, this may not be due entirely to the treatment since there was some pear psylla out then. During the fourth week after spraying at the calyx stage of development, the fruits receiving this treatment, abscissed at a rapid rate. On May 20 about half (average of I4.8 percent) of the fruits had abscissed from the treated limbs whereas the normal drop had not yet started on the other limes. The 100 ppm concentra­ tion of the same compound did not cause the severe effects on the leaves and resulted in much more nearly the desired 55 amount of thinning as shown in Table XV. peaches Methods Sprays of 100 ppm, $00 ppm and 1000 ppm of l4.-phth.alimido-2,6 -dimethylpyrimidine and 100 ppm and 1000 ppm of N-l naphthyl phthalamic acid were applied to different limbs on three peach trees on the evening of May 3, 1952, which was about two days after the peak of full bloom. The trees used were in an old block of mixed varieties so that in order to get trees near to each other and of approximately equal vigor, trees of three different varieties, a South Haven, a Salberta, and a Shipper Late Red, were used. On June I4., or 35 days after full bloom, a second group of limbs on the same trees were sprayed with the following solutions: 1000 ppm of Lp-phthalimido-2 ,6 -dimethylpyrimidine, 1000 ppm and 2000 ppm of N-l naphthyl phthalamic acid and 1000 ppm and 2000 ppm of N-2 chlorophenyl phthalamic acid. Results ■ The N-2 chlorophenyl phthalamic acid appeared to cause a yellowing and curling of leaves about a week after spraying, as shown in Figure 9* Although this compound shows- the * most promise of the compounds tested for the thinning of peaches, the injury to leaves makes its use inadvisable. The large variation between the trees and the small number of limbs used make it impossible to draw any definite con­ clusions from this experiment. 56 TABLE XV T H E E F F E C T IV E N E S S O F l^ - P H T H A L IM ID O - 2 ,6 - D IM E T H Y L P Y R IM ID r N E A N D N - l N A PH TH Y L P H TH A LA M IC A C ID SPRAYS A P P L IE D A T F U L L BLOOM I N T H IN N IN G T H E F H U IT S O F B A R T L E T T PEAR TR EES A v e ra g e p e rc e n ta g e o f f lo w e r s w h ic h d i d n o t a b s c is s b y l a s t w e e k in June T re a tm e n t C o n tro l 15 100 ppm i j . - p h t h . a l i m i d o - 2 , 6 - 4 im e t h y l p y r i m i d i n e 500 pp m 23 1000 pp m 14 100 ppm N -l n a p h t h y l ;p h t h a l am i c a c i d 7+ i* 2 0 0 0 ppm + A lm o s t 20 s ig n ific a n tly ■ ^ S ig n ific a n tly le s s le s s th a n th a n c o n tro l c o n tro l at at %% l e v e l 5% l e v e l TABLE XVI T H E E F F E C T IV E N E S S O F SPRAYS OF t ( .- P H T H A L IM ID Q - 2 ,6 - D IM E T H Y L P Y R I M I D I N E AN D P H TH A LA M IC A C ID D E R IV A T IV E S I N T H IN N IN G PEACHES A v e ra g e p e rc e n ta g e o f f r u i t s p r e s e n t a t tim e o f s p r a y in g w h ic h d i d n o t a b s c is s b y J u l y T re a tm e n t L im b s S p ra y e d M ay 3* About F u ll B lo o m C o n tro l 36 16 100 ppm 500 p pm 7 1000 ppm 23 100 ppm 1000 ppm No I p - p h t h a l i m i d o - 2 , 6 - d im e t h y l p y r i m i d in e N -l n a p h th y l p h th a la m ic 8 s ig n ific a n t L im b s 21 a c id d iffe r e n c e S p ra y e d Jun e 4, b e tw e e n or tre a tm e n ts 35 D ays A f t e r F u l l B lo o m ko C o n tro l 1000 ppm [|_ -p h th a l i m i do - 2 , 6 -d im e t h y l p y r i m i d i ne 22 1000 ppm N -l 26 2000 ppm 1000 ppm n a p h th y l p h th a la m ic a c id 1*0 N -2 c h lo r o p h e n y l p h th a la m ic a c id 2 0 0 0 ppm No s ig n ific a n t 18 13 d iffe r e n c e b e tw e e n tre a tm e n ts EXPERIMENTS ON THE DELAYING OP THE PREHARVEST DROP OP APPLES Review of Literature One practical application of the effect of growth regulators upon the process of abscission is the use of growth regulators to delay the preharvest drop of fruits* In 19399 Gardner., Marth, and Batjer (26) reported for the first time, using naphthaleneacetic acid to delay the dropping of apples* Since that time it has been found to be quite effective on many varieties of apples, especially the early varieties, on pears, and on some varieties of oranges, grapefruits, and apricots* Batjer and Thompson (9) found that 10 ppm of 2 ,lp-dichlorophenoxyacetic acid was more effective than naphthaleneacetic acid in delaying the drop of Winesap apples and that the best time to apply the 2 ,l}_-dIchlorophenoxyacetic acid was between 20 and 30 days before the dropping begins. They found 2,l{.-dichlorophen- oxyacetic acid to be entirely ineffective on the Duchess variety* Napthaleneacetic acid sprays used to delay dropping of apples are usually applied within a week before or at the time dropping begins. Recently 2,1^,5-trichlorophenoxy- propIonic acid, sold under the trade name of "Color Set” has been found to be quite effective in delaying the dropping* The use of growth regulators In relation to the production 58 of tree fruits has been reviewed by Gardner (2 5 ) and Pearse (71)• Methods and Materials A small scale experiment was conducted to determine if l|.-phthalimido-2 ,6 -dime thylpyrimi dine or a few deriva­ tives of 2,l|.-dichlorophenoxyacetic acid which Hamner (35) had observed to be less toxic in his trials would be effective in the delaying of the preharvest drop# Duchess of Oldenberg is an early maturing variety of apples on which fruit drop is a problem# There were only three trees and only a limited amount of chemicals available so differ­ ent limbs were used for the different treatments# All of the treatments were applied to each tree, so the three different trees could be considered replications# One of the trees was less vigorous as could be seen from its response to the thinning and stop drop sprays, the circum­ ference of its trunk, and growth of its terminal shoots0 This tree also had a heavier crop. All the trees, however, appeared very healthy and, except for a little scab early in the season, diseases were well controlled® Concentrations of 100 ppm, 500 ppm, and 1000 ppm of 4 -phthal imi do-2 ,6 -dimethylpyrimidine, 10 ppm, 25 ppm and 100 ppm of 2 ,2 ,2 -trichloroethyl-2 *,Ij.T-hichlorophenoxyace­ tate, and 10 ppm, 25 ppm, and 100 ppm of (2 ,i|_-dichloro­ phenoxy )- cc -hy dr oxypr op ionic acid were used. The l4.-phth.al- imido-2 ,6 -dimethylpyrimidine is water soluble, the second 59 compound came in a solution which did not form a good solution or emulsion with water even when some ethyl alcohol and some synthetic detergent were added* By shaking vigorously before spraying a fairly good emulsion was obtained, but a perfectly uniform distribution of the compound on the leaves was probably not attained* The third compound was readily soluble in water as a diethanol amine salt* The compounds were applied with a knapsack type sprayer with the attachment which allowed a different flask to be used for each different chemical* The sprays were applied on August lif, 1952, at mid-day when the temperature was about 85° F* Batjer (6 ) obtained the best response to naphthaleneacetic acid stop-drop sprays when they were applied at temperatures above 80° F* When the limbs were sprayed the seeds of the apples were turning brown, and some apples were falling* A moderate rain occurred 38 hours after spraying* Results There was no apparent decrease in the dropping of the fruit and on August 20, 1952, six days after spraying, the records of Table XVII were taken, which also show no significant difference between treatments* 60 TABLE XVII THE EFFECTIVENESS IN DELAYING THE PREHARVEST DROP OF DUCHESS APPLES ON LIMBS SPRAYED WITH GROWTH REGULATORS JUST BEFORE DROPPING BEGAN tre a tm e n t A v e ra g e p e rc e n ta g e o f a p p le s on a t t im e o f s p r a y in g w h ic h d i d n o t d ro p i n s i x days 6k C o n tro l ij.-p h th a lim id o -2 ,6 -d im e th y lp y r im id in e 100 ppm 500 ppm 51 1000 ppm 53 10 ppm 2 , 2 ,2 -tr ic h lo r o e th y l-2 ,l|.-d ic h lo r o p h e n o x y a c e ta te ppm 10 ppm 71 B - ( 2 , [ | . - d i c h l o r o p h e n o x y ) cc - h y d r o x y p r o p io n ic a c id 2 5 ppm 100 38 60 2 5 ppm 100 57 ppm No significant difference between treatments us 61*. 71 EXPERIMENTS ON THE ROOTING OP CUTTINGS Review of Literature One of the first uses found for growth regulating chemicals was as an aid in the rooting of cuttings. In 1929 9 Went (98) found that a non-specific, heat resisting substance could be extracted from leaves or germinating barley which, when applied to cuttings, promoted the development of new roots. One of the most active ingred­ ients of Went* s preparations was probably indole-3-acetic acid which Thimann and Went (93) found to aid in the rooting of cuttings in 1934* During the next decade a huge number of different chemicals, primarily different derivatives and formulations of aryloxy-alkyl-carboxylic acids and aryl-alkyl-carboxylic acids, were tried on a large variety of different species under different conditions. This work has been condensed and collected by Thimann and Behnke (92), Avery and Johnson (6) and Pearse (71). Succulent cuttings of adaptable species are usually quite easy to root naturally, but applications of growth substances frequently increase the number of roots and speed of rooting, and may alter the type of roots formed* Dormant hardwood cuttings, greenwood cuttings and evergreen cuttings are frequently harder to root. Rooting is usually 62 preceeded by the formation of callus tissue around the wound surface* The root primordia are initiated from the pericycle but may arise from surrounding tissues* The roots may emerge through the callus tissue, or through the basal portion of the cutting* Growth regulators have commonly been applied to cuttings by dipping the cuttings in solutions, by smearing on a lanolin paste, or by dusting with talc containing the regulator* The physiological mechanism by which growth regulators stimulate the rooting of cuttings is not clearly understood* Stuart (89) analyzed different parts of cuttings of young bean plants and Alexander (5) analyzed detopped beaii plants whose cut surfaces were treated with indoleacetic acid. Both men.found carbohydrate and nitrogenous food materials were translocated to the treated area* These materials might aid in the growth of roots* General Methods Several preliminary experiments were conducted to determine if ij-phthalimido-2,6-dimethylpyrimidine which acted as a growth regulator in other processes was effective in the rooting of cuttings* Two different species each, of succulent, hardwood and evergreen cuttings were included in the experiments* The growth regulators were applied by soaking the cuttings in various concentrations of solutions because the concentration of growth regulator applied could 63 b© more accurately known and its entry into the cutting was more certain than when it is applied in a dust or in lanolin. Two control treatments were utilized, one in which the cuttings were dipped in distilled water and another in which they were dipped in a solution of 10 ppm of naphthaleneacetic acid, a standard growth regulator frequently used in the rooting of cuttings. Succulent Cuttings of Coleus Methods and Materials Cuttings were taken from the main shoots of 38 Christmaa Gem variety Coleus blumei L. plants on March 2, 1952. These plants had been used previously in the experiment on the rate of abscission of leaf petioles as influenced by growth regulators applied in lanolin to the tip of the petiole. Any effects of the previous experiment were mini­ mized by allowing several weeks between experiments, by using only new shoot growth above where the petioles had been, and using the same chemical at the same or a slightly higher concentration in the cutting experiment. The plants appeared normal in every respect at the time of the experi­ ment. Seven cuttings, each were placed in beakers contain­ ing the solutions of distilled water used as a control, 10 ppm naphthaleneacetic acid, 10 ppm, 25 ppm, 75 ppm, 250 ppm, and 500 ppm of i4.-ph.thalimi do-2 ,6 -dimethylpyrimi­ dine for three-quarters of an hour. contained only three cuttings. The last treatment The cuttings were placed 614. in flats of washed sand, on the greenhouse bench, and watered with tap water. The lower leaves of the cuttings wilted at times, and lost some color as food materials were withdrawn from them,, On March 30* 1952, the cuttings were taken out and the total number of roots and the length of the longest root was recorded* Results The optimum concentration of lp-phthalImido-2, 6 -dimethyl pyrimidine in this experiment appears to be between 75 ppm and 2^0 ppm* The 10 ppm treatment appears to have stimu­ lated the growth of roots. The proper concentration of lp-phthalimi*lo-2 ,6 -dimethylpyrimidine significantly increased the number and the length of the longest root of the coleus cuttings in this experiment, as is shown in Table XVXIX* Succulent Cuttings of Tomato Methods and Materials Succulent cuttings were taken from the suckers of the Rutgers variety of tomato plants used in the greenhouse fruit setting experiment. It was over two months since any treatment had been applied to the plants, and it was applied then as a dip of only the flowers* The cuttings were dipped in distilled water used as a control, 10 ppm, 50 ppm, and 100 ppm solution of l4.-phthali.mido-2 ,6 -dimethylpyrimidine and were rooted in vermiculite, an expanded mica* The roots 65 e © bO O © U e § TJ insignificant a bO PA « PA •H © o +3 +» 9 * S -bO P© 4* 9 CA • PA O • PA PA -d- PA • PA PA • PA O •rl Vj V h V h 0 O9 <9 o o • A- X A— rH • rco PA o • PA 31.2 t—1 • CM CM NO 0 EH i © rH xi 0 _ o 0 •H O P «H © -P U 9 P rH £ & O 0 ft ft 9 O ©© a o o o o Js P 9 o o s? © Vi d o © © 0 d g i bO © ft sO © P ft & 0 © © O > 0o © PIPy CM I© o 9 •H d 0 -H •H 0 © rH © 9 © 0 *> © 0 © © © P © & p* K„ P p 1A d «h rH *H © 0 x! ^ ft P P bOP bO © 9 © 9 •rl 0 *H P hH P P-t -d* 0 ft ft O rH © *H © X ©X 0 ft ft 1A CM 0 ft ft 1A C— g ft ft O 1A CM © Vi © 0 ft ft O O U\ CO © &h © Eh other treatment d •H o © o •rt P © o © © 0 © ■d b* X I -P x s p 66 were counted and measured after 11 days* Results As shown in Table XXX the small number of cuttings (Lp.) used and the large difference between cuttings make it impossible to draw any conclusions from this experiment# However, the low concentrations of l4.-phthalimido-2,6-dimethylpyrimidine seemed to stimulate root growth as they did in the coleus experiment# Hardwood Cuttings of Willow Methods and Materials Cuttings of willow Salix blanca L* were taken on April 18, just before the buds burst. The cuttings ranged from 3 /8 to l/2 inch in diameter and were graded so that each treatment contained similar sizes of cuttings* The cuttings were placed in beakers containing distilled water used as a check, $0 ppm naphthaleneacetic acid, and $0 ppm and $00 ppm solutions of i|.-phthalimido-2,6-dimethylpyrimidine for 13 hours. They were then placed in vermiculite, in flats in a shaded portion of the greenhouse on the floor# Results The $0 ppm concentration of naphthaleneacetic acid and the $00 ppm of Ij.-phthalimido-2,6-dimethylpyrimidine greatly retarded the opening of the leaf buds on cuttings as can be seen in Figure 6. The same treatments caused a significant 67 TABLE XIX THE AVERAGE NUMBER AND LENGTH OP ROOTS OF TOMATO CUTTINGS 11 DAYS AFTER TREATMENT WITH DIFFERENT CONCENTRATIONS OF 4-PHTHALIMIDO-2, 6-DIMETHYLPYRIMIDINE Treatment Control Average number of roots per cutting Average length of roots per cutting 31.0 U -5 .7 ppm 28 .6 5 3 .5 f>0 ppm 20.1 5 9 .2 100 ppm 21.1 5 0 .6 10 No significant difference between treatments Figure 6* Top - The effect on rooting and inhibition of bud development after dipping willow cuttings in 50 ppm and 5>00 Ppro of i4.-phthalimido - 2, 6-dimethylpyrimi dine , 50 ppm of naphthaleneacetic acid or distilled water. Bottom The effect of dipping arbor vitae cuttings shown on the left, and common boxwood cuttings shown on the right, upon rooting. 68 69 decrease in the number of cuttings which rooted and a highly significant inhibition of both the number and length of roots as is shown in Table XX and Figure 6* The inhibition of the development of the leaf buds indicates that the lj.-phthalimido-2,6-dimethylpyrimidine is translocated in the vascular system, at least for short distances® Hardwood Guttings of Apple Methods and Materials Cuttirigs were taken from dormant McIntosh apple shoots. Gare was taken to make angular cuts slightly above and below the nodes. They were placed in beakers containing distilled water used as a control, ppm, 250 ppm, and 500 ppm of 4.-phthalimido-2,6-dimethylpyrimidine for ten hours. The cuttings were then placed in vermiculite In flats in a shaded area on the floor of the greenhouse. The temperature was probably frequently below optimum for rooting. Apple cuttings are usually made in the fall and winter and allowed to callus before being set out. This experi­ ment was not anticipated in time so dormant shoots taken for scions were used although they were not callused. This is important because apple Is considered to be difficult to root tinder ideal conditions. The cuttings did not root before the hot days of summer made the greenhouse so hot that the leaves and wood of the cuttings dried out and died. However, some of the cuttings did callus. 70 G 0 P. ^ bO W G P *H O 43 O P g g O G 0 d g 3 G • -d- bo w G P »H O P © M o p as G 3 G O © *h > O sO • 1A vO • cA O -d• -d* i— 1 • C"rH vO -d- • CM \A • rH rH ♦ CM rH Q m 0 bO 0 P G © 0 tiO ^ H TS P 0 P P G o o o o G G © *r* -dvO 09 TO rsO - d ov 0 0 O 0 co 3 09 ^ O* 0 g H t>5-P 09 H r H *H •P rH H »H d *H & d O § O 0 cniA •H i rH TJ •H O 0 O •H P 0 O 0 © G H 0 d p d p» 0 Is; 0 P THE r• -d* »H G 0 P EFFECT OF SOAKING WILLOW CUTTINGS THEIR ROOTING AS MEASURED IN SOLUTIONS OF GROWTH REGULATORS 11*. DAYS AFTER TREATMENT ON rCj -P to G 0 rH p 0 £3 B 0 0 G Eh rH O G p G o o tU) •H d t d •* CM 1 0 d G * H *H s rf •H *H rH & 0 *H d G p l» d a P 1 fi p p o o o UN 1A fa P G CD ^3 CO O t*D O -P fi a fa a-p •H G © t» d p 0 6 O d bOrH O O CJ1A • G •H • fa o \A 71 R e s u lts A ll o f th e g ro w th r e g u la to r s s ig n ific a n t r e d u c tio n c a llu s e d c o m p a re d t o T a b le as XXX* The 50 in th e th e caused c a llu s in g o th e r A ll th e th e tre a tm e n ts le a f buds and th e re d u c e d * The th e h ig h s iz e c o n tro l te m p e ra tu re s of th e le s s except seen in \4 iic h in h ib itio n th e th e n o t have su m m e r w e r e w h ic h o f ij.-p h th a lim id o -2 ,6 - d e la y e d le a v e s c o u ld of can be s ig n ific a n tly g r e a tly a h ig h ly c u ttin g s as tre a tm e n ts , e x p e r im e n t caused num ber o f ppm c o n c e n t r a t i o n d im e th y lp y r im id in e th a n used c o n tr o l* o p e n in g of th e d e v e lo p w as c o n tin u e d fo r d id been of b e g in n in g to k ill th e c u ttin g s * Magnolia Cuttings A fe w m a lle t s o u la n g e a n a * used as ty p e c u ttin g s Some w e r e a c o n tr o l, and soaked 12 None e ffe c ts on th e E v e rg re e n le a v e s d is tille d About 70 c u ttin g s m ade w h ile g ro w th h a d th e y s ta rte d * w h ic h c u ttin g s ro o te d , but caused d e v e lo p e d * o f B oxw ood and M a te r ia ls o f boxw ood, w e re w a te r ppm o f 4 " P ^ t h a l i m i d o - th e s e C u ttin g s M e th o d s new of in 5 0 0 ppm o f i ^ - p h t h a l i m i d o - 2 , 6 - d i m e t h y l p y r i m i d i n e fo r m a tiv e w e re h o u rs 500 som e i n 2 ,6 -d im e th y lp y r im id in e * th e w e r e m ade o f M a g n o lia s till B uxus q u ite T h ey w e re s e m p e r v ir e n s d o rm a n t b e f o r e g ra d e d so t h a t L *, any s im ila r 72 TABLE XXI THE EFFECT OF GROWTH REGULATORS ON THE CALLUSING OF MCINTOSH APPLE CUTTINGS Percent of cuttings callused three months after treatment Treatment 16 •35*55* Control 50 ppm Naphthalene acetic acid 25 ppm ii.-phthalimido-2,6-dimethylpyrimidine ■35* 9 250 ppm 2 500 ppm 2 •5H*H i g h l y s i g n i f i c a n t l y o r p r o b a b ility i s .0 1 of d if f e r e n t th a n o th e r g e t t in g c h i s q u a re l e s s tre a tm e n ts t h a n 6«61j. •35*Significantly higher than other treatments except control or probability of getting chi square less than 3®8^4- is . 0 5 73 size of cuttings were included in each treatment* The treatments consisted of a 12 hour soak in solutions of distilled water used as a check, 50 ppm of naphthaleneacetic acid, 50 ppm and 500 ppm of l|-phthalimido-2,6-dimethylpyriraidine* The cuttings were then rooted in vermic- ulite, in flats placed in a shaded place on the floor of the greenhouse* The number of cuttings rooting, the number of roots per cutting, and length of each root was determined* Those cuttings which had not rooted at the first time were placed back in the vermiculite and allowed to root if they would* This was done a third time also* The data on the average number and length of roots per cutting was obtained from the first observation only because the roots were sometimes injured in counting, and a comparison of the number and length of roots of cuttings which had differing amounts of time to grow would not be too valid especially in such a small population* Results The SO ppm of ip-phthalimido-2,6-dimethylpyrimidine was the best concentration tried and that concentration was approximately as effective as the same concentration of naphthaleneacetic acid at the time of the first observation as can be seen in Table XXIX and Figure 6* Both of these treatments are highly significantly better than the check when tested by chi square. This treatment was highly significantly better than naphthaleneacetic acid and the 7k id © d +3 d g © O bO © B o d © © © -p -p d o o fl a ^ © co d rl O r l -P © O bO Q O Is;Eh < 4 <4 W) o © ( j d © o o O' d tH d © O d pq cb pq Pq g 04 fE5£j EH cq > cbo sg § §Eh pq W W Eh Eh H S <4 Eh 125 Q _ pq g Eh Eh O <4 o pq H 04 P4 Eh H StH m s ® ;s p £ «4 Eh pq CO EH Cb o is;Pq H Ph Eh Ph Eh <4 P <4 -P d § © d © g o d © d •H p «M d rO © § § ■P «H © 0+3 bO o -P ~d" vO cm CM O CM O > O «© © bO d •rH +3 +3 d O d d O 4h © d 43 d O £ 1A sO CO CM ca oa O d © © bo+3 © o 43 O d d © o d © PH © © d d 43 © d +3 d o £ © d O +3 is d 43 o CM CO CM CO CM CA sO 3 rA -d rd* CM CO C M n0 bOd © +3 ^ rH © +3 d © d •H "d •H s d •H o © o •H 43 © O © © d © rH © d +3 d p. © *25 pti O 04 pq *3 Cb Eh Eh +3 d © fi +3 (4 © d Eh © rH O d +3 d O O g p. Pu o u\ d P* »T* O l>3 d d *H 43 s © *H g rH *H © d d I 43 sO d *» PHCM f ~d* e Pr pu o IA s Pr Pr O O XA ©© d O © d d d +3 © 2 d +3 o O 43 w *H ©d S3 © o © S PiH rH >43 © •H rH d rd P-+3 Or d d O CO <4 cO 121O pq Q O 04 § Oh rH H +3 © +3 bO d •H © © o © £ d +3 © d O © > © £5 d © P d a dbO © O S3 <*! o © O *H P O 60 O P © Pi 3 Pi o © Vi -d •V* o• vO rH CA O » • o -d O O n |> s P © ,d pH Pi S3 o P fi Si •H CO -d 1 T\ vO c^- CA S3 O © Pi © S3 © *H P Pi S3 © © O 60 Pi S3 © *H P S3 ,S3 © 60 Pi •H © S i Vh Vi o* 60 TS © m © Pi O Ph p Vi Pi P > •H © O © CO is © IA © p © Vi p erf Is 8 © © d Pi o p jS© r©l •H •H *H r-H'feS. >o Pi Vl u J2; -O Pi © Pi P © S P« | 60 C WC P »H ©OP •H U •H © P a o © O © M P £ S3 P o ca pH r— -d © *H i —iS i S i p Pi § © o C \ J CA Vi s ? *a P o rH O o pH © T3 © e■ p© © 60 O S3 T3 TJ •rH O •H tH © o •H P © O © © S3 © rH © Si P si 0j P a © s p© © Pi EH © is; rH o Pi p S3 O o e sx Cb o u\ e •H Pi >> (X 1 rH o t>» •H P s© i —1»H © T3 PsO« 'O d ^3 PiC\*J l -d 6 Cb a o 1A © pq w Vi p © g S 3b S^ o o IA 77 treatment® The cuttings treated with naphthaleneacetic acid developed many thin roots while the cuttings of the other treatments and check developed fewer thicker roots® E X P E R IM E N T S ON T H E R E L A T IO N OF C H E M IC A L STRUC TUR E AND T H E B IO L O G T C A L A C T I V I T Y OF i f - P H T H A L IM ID O - 2 ,6 - D IM E T H Y L P Y R IM ID IN E Intro duction One of the fundamental problems currently receiving a great deal of attention is the structural requirements of compounds which are necessary for them to be active in living organisms* This problem is frequently related to studies on the biochemical mechanism of a reaction because most reactions in living organisms are catalyzed by enzymes, and if a chemical compound has the proper structure and physical characteristics associated with this structure, it may act as an inhibitor, or stimulant for an enzyme system# If the enzyme system involved Is known, it can some­ times be isolated and the effects of the chemicals on it can be studied In vitro* Frequently experiments done in vitro do not correlate well with experiments done with intact living tissue. Because of this, or because the enzyme system Is unknown, biological tests have been found to be very useful# 79 In h ib itio n o f o f Root G ro w th of ro o ts fo r th e and G ra n t o f am ount of T h is s y n th e tic m e d ia fa ir ly in v o lv e s fo u n d to m e th o d h a s s in c e th e u n ifo r m s u p p ly a v e ry a c tiv e ly a q u a n tita tiv e th e le n g th w as o f th e re s p o n s e and w h e a t, c o n c e n tr a tio n s s tu d ie d to F ifte e n No* 1 a fa ir ly of s in g le of of ta p r e a d ily b io a s s a y in seeds a w a te r in no c o n ta in g ro w th * I t th e tip , o b ta in e d ro o t w ith g ro w th r e q u ir in g fo r g r o w in g m e r is t e m is a c id th e fo o d m a t e r ia l and a c c u ra te a d v a n ta g e s c o ty le d o n s m e a s u re filte r of ro o t b y m e a s u r in g a n in e x p e n s iv e th e ppm , 0 *1 d e te r m in e w e re paper in fiv e 250 ppm , ppm , ro o ts th e fro m th e c u c u m b e r, a d ic o ty le d o n o u s p la n t, e ffe c tiv e d is tr ib u te d a p e tri to a w id e 1 .0 500 to be ppm , ppm , P h illip s of The of th is filte r a s o lu tio n te s te d . 10 ra n g e upon a p ie c e d is h * m illilite r s ppm , 7 5 0 ppm , im ld o -2 ,6 -d im e th y lp y r im id in e r e c e iv e d of ra n g e o f lj.-p h th a lim Ic L o -2 ,6 -d im e th y lp y r ir a id in e c o n c e n t r a t i o n w h ic h w as u s e d w e re and M a t e r ia ls a m o n o c o t y le d o n o u s seeds m o is te n e d w i t h 100 g e r m in a tio n r u le r * The o f be th e M e th o d s p la n t, th e 2 ,4 -d ic h lo r o p h e n o x y a c e tic and m e tr ic (7 6 ) o f L ite r a tu r e cucum ber seeds s o lu tio n * a C o n c e n tr a tio n s Ij.-p h th a lim i d o -2 , 6 - d im e th y l p y r im id in e R e v ie w R eady by D iffe r e n t The 2 $ ppm , com pound* o f W h a tm a n p a p e r w as of a c e r ta in c o n c e n tr a tio n s 50 ppm , 7 5 ppm , a n d 1 0 0 0 ppm o f 4 - p k t h a l - a n d a n o t h e r c o m p o u n d w h ic h w a s P e tr o le u m C om pany a t th e sam e 80 tim e * The la tte r p o w d e r w h ic h w a te r* X ts re fe rre d d is h e s te s tin g each th e th e cucum ber f o r m u la fiv e w e re U s u a lly C ra c k e d , w a s n o m o ld g r o w t h lin g s th e seeds on th e of or th e so as to tre a tm e n ts . d id The not c a lc u la tio n of is so s o lu b le it ij-7 5 * w ill e th y lp y r im id in e d is h e s or w heat in be Pour p e tri r e p lic a tio n s in u s in g r e p lic a tio n s e x p e r im e n t a ll s m a ll of and th e th e b e in g re c o rd s g e r m in a te th e s e g re w in fo r ro o t of of or fro m th e o f th e of one in to th e th e d is h due t o F a rm w h e a t* c o v e re d p e t r i fiv e days* cucum ber fiv e of seed­ ro o ts each of O ne tre a t­ w as g ro w th w h ile r e p lic a tio n s th e in s te a d s e e d s ,in c lu d in g g ro w w e l l w e r e e x p e r im e n ts * seeds w e re th e second r e p lic a t io n a ll seeds and added to g e th e r * v a r ia n c e ta k e n th e s h r iv e le d e x a c tly le n g th s e e d lin g s or of w as e x p e r im e n ts * w e re m e a s u re d put e x p e r im e n ts e x p e r im e n t w i t h th e s e bench th e th e s e o b ta in e d th e p r im a r y and of about 80 p e rc e n t and w as m e a s u re d b e f o r e m e a s u re m e n ts w e re w h ic h th e s e e d lin g s r e p lic a tio n , m e a s u re d in in in la b o r a to r y w e re m e a s u re d w heat m e n t, w as u s e d g e r m in a te d le n g th g iv e n as th e G o ld e n B a n n e r w h e a t , T h e re th e in b ro k e n , D e p a rtm e n t, Then 6- d i m used C ro p s d is h e s used p e tri seed used B u rp e e H y b r id * The Xt a n d b ro w n c o m p o u n d n u m b e r ij.75* e x p e r im e n t w i t h g e r m in a te d * w as n o t seeds w e re w h ile cucum ber a lu m p y y e l l o w a p p e a r hom ogeneous* i ^ - p h t h a l i m i d o - 2, seeds, n o t used* w as m a n u fa c tu r e r * s n u m b e r, c o n c e n tr a tio n The th e by th e cucum ber not c h e m ic a l to of d id s u b s ta n c e in c lu d e d th e in of fe w th e 81 R e s u lts In g e n e r a l, pounds caused a lth o u g h one r e s u lte d in ro o ts 75 and a ls o in c r e a s in g a n in c r e a s e d an in s ig n if ic a n t 2S> ppm a n d d ra w n fro m a v e ra g e w h ic h a re th e show n i n show n i n n e c e s s a ry on th e fo r X X IV v e r tic a l a x is 0 .1 11 cm m a r k th e ppm w h i c h w a s 12 on F ig u r e s h o u ld b e 11 tr a tio n w as p lo t t e d q u e n tly done c u rv e ( 9^-) in fo r w e re 8* th e a v e ra g e th e w heat and th e 13 on a lo g a r ith m ic assays to th e can be d iffe r ­ le n g th to The of be th e id e n tic a l The w as o m it t e d , 12* The s c a le w h ic h o b ta in to such e s tim a te d , s im ila r th e a so concen­ is fre ­ s m o o th e r i f a v e ra g e th e c o n d itio n s , lig h t a m o n th w as about h a lf te s te d o f its concen­ o f ro o ts of o th e r o f i4. - p h t h a l i m i d o - In lo s t s o lu tio n absence w h ic h h a d b e e n l e f t about A th e le n g th e th y lp y r im id in e have know n. in a p p r o x im a te 2 , 6- d i m fo r is c u rv e s fa c to rs , to s e e d lin g , check* s c a le g ro w th fo u n d w e re le a s t as of • g ro w n u n d e r th e le n g th e x p e r im e n t* so s h o u ld b e a o f w heat com pound c u rv e s th e th e 8 th e of each sam e a s le n g th in fo r a d ju s te d com ­ g ro w th P e tr o le u m ro o ts th e fo r th e These about b io lo g ic a l By r e fe re n c e tr a tio n in a lo n g w it h s ig n ific a n c e cucum ber and w h eat ro o ts at of th e s e e th y lp y r im id in e in c r e a s e F ig u r e le n g th T a b le 6- d i m P h illip s show ed a n i n s i g n i f i c a n t of o f ro o t In c r e a s e £ 0 ppm o f as s c a le s In h ib itio n ppm o f i | . - p h t h a l i m i d o - 2 , cucum ber ro o ts ence c o n c e n tr a tio n s in in th e th is a c tiv ity . g re e n h o u s e w ay an d 82 Length in centimetere of eheat roots. ^ ^ * CD C- © IQ THE INHIBITION OP ROOT OROiTH AS AFFECTED BY VARYING CONCENTRATIONS OP GROWTH 2 ^ 3 U e •H pq Eh o P P w o ptf 55 o d o bD d 5 o d bO d P aJ d Is >* 0 Eh bO ad 0 12 < Eh O' rH O d o *H B P P P 0 d 43 d 0 o d o o B P P B P P P P P P P P P P p o d p d o u •S.D. ,S.D. > ao CM GO d •H id g GO CM * rH r— vO -d* £ © - C — OH C — OH 0 Phthallo Acid Phthallalde N Phenyl Phthallalde H-l Naphthyl Phthalaalc A d d o OH Cl C -N H-2 Chloro phenyl N-4 Chloro phenyl Phthalealc A d d Phthalaalc Acid Cl 90 TABLE XXVI T H E A P P R O X IM A T E pH OP T H E GROWTH REGULATOR S O L U T IO N S U S E D I N TH E S E E X P E R IM E N T S A p p r o x i m a t e pH a t 1000 ppm Com pound 4 - P h t h a l i m i d o - 2 , 6- d im e t h y l p y r i m i d i n e 4 .1 P h th a lic Ij-oO a c id P h t h a l i m i de 4 *7 P h illip s 5 .2 D i e th a n o l N -l c o m p o u n d 1^.75 a m in e n a p h th y l 9 .3 p h th a la m ic 9 .2 a c id N -2 c h l o r o p h e n y l p h t h a l a m i c a c id 9 .2 N -4 a c id 9 .7 c h lo r o p h e n y l p h th a la m ic Th.e p h t h a l a m i c a c id s w e re used as d le th a n o l a m in e s a lts 91 n m 0 g t £ f M CL f t a O T3 UN o o O o pH CO o vO "LA cA CO vO CM O sO CO GO A- vO CA OJ CM CM o- o o rH rH CM £ H g ft ft O UN CM g ft ft 43 O O O rH O O CA C— CA rH CA CA C V J CO CA GO GO CO UN UN CO UN O pH O UN CM CM rH 43 fctO •H g ft rH ft w O O £ 0 £ £ H •H tJ 43 £ O O n O O -d i —I CA CO CA CA vO xO CM xO O rH rH g ft ft O O O O rH CO CA UN CM rH -d rH rH UN CA rH CM CA CM A- UN UN CA UN O O rH UN CO vO O 1—1 CO 0 rH C^ CA 1—1 CA O rH vO CM C— CA UN O O rH -d O rH O -d rH O -d rH CO CM H 1 1 O rH rH vO CO vO C— CO CM rH £ H a < eh g ft 0 UN g ft CL O UN CA rH H © £ • f t 1r l xO TJ •k»H O & £ © CM g I »H g 0 O Tf o ft rs ^ •H ft g H •H © T* *H rH O ft 43 p £ O U o rH Xl a J -P 43 © -i g O CCS •H rH 05 6 -P £ Ph rH f i d 43 OUN ftA- 43 _ d P®H'O m B ftO •H ft rH g rH O 43 •H © •£* Ph £ Ph © £ •H s 3 rH O £ CCS 43 43 © •rH Q H ccS 43 43 43 ft rH 43 -P 43 ft ci tJ £ *H O rH Cd 1 Tf rH *H O £ CCS © 43 O ftfH O £ ft 3 O pH rH OS 43 43 O 43 43 CM ft ■ 52! rH »H © £ cd © 43 O ft*H O g ft 3 O rH rH CCj 43 43 O 43 ,43 -d ft l Sz; L*S«D. at $% level o 92 w ith th e check tre a tm e n ts fo r s ig n ific a n c e n e c e s s a ry as 100. w e re The kept d iffe r e n c e s in p r o p o rtio n ® R e s u lts Som e g e n e r a l T a b le som e X X V II® cases, e lo n g a tio n w e re tre n d s a re P h th a lim id e , p h th a lic of a c id e v id e n t P h illip s p ro d u c e d The l4 . - p h t h a l i m i d o - 2, 6- d i m b ra n c h w ith ro o ts , s e e d lin g s ® ro o ts and w e re T h is fr e q u e n tly s e e d lin g s show ed tre a te d ta p e re d c o ty l a th in b ra n c h ro o ts . 250 c o n ic a l lin g s n o rm a l w ith w ith The ro o ts of p h th a la m ic shape w ith except fro m w ith th o s e th e s e e d lin g s of concave s id e s . p h th a lic a c id so m ew h at lo n g e r ® th e h a d fe w e r c o n tro l g e o tr o p is m * a c id s The The c o m p o u n d I 4.75 t e n d e n c ie s . had ro o ts la r g e r o n ly a c id s a p p e a ra n c e . s e e d lin g a c id s th e l4 .-p b .t h a l- s iz e a p p e a ra n c e w it h th e in e th y lp y r im id in e e x tre m e , th e i-n th a n of P e tr o le u m p h th a la m ic r a p id ly ro o ts a n e g a tiv e as of p h th a la m ic a r a t-ta ile d not s tr in g -lik e ppm o f tre a te d th e th a n P h illip s a lth o u g h d ia m e te r to th e m e x h ib ite d tre a te d s im ila r , s e e d lin g s about gave s h o rte r d a ta s tim u la tio n w h i c h w e r e m u ch m o r e t o x i c im id o -2 ,6 -d im e th y lp y r im id in e . tre a te d th e c o m p o u n d 1+75* a cucum ber ro o ts w h ile in h ib ito r s fro m of d e v e lo p e d The r o o t s w hose th e a fe w tre a te d of and p h th a lim id e The hypo- s h o rt w ith a th e s tu b b y seed­ appear 93 A Comparison of the Formative Effects of Leaves of Cranberry Beans and John Baer Tomatoes Produced by Several Phthalic Acid Derivatives Review of Literature The formative effects and curvatures of leaves and stems of tomato and bean plants have been used by Hitchcock and Zimmerman (37) to obtain a quantitative measure of the response of plants to growth regulators* Methods and Materials Cranberry bean plants were planted in a soil mixture of Hillsdale loam, sand and peat in four-inch pots in the greenhouse on April 11, 1952* Eight days later all but the two most vigorous plants in each pot were removed* On April 24 the plants were graded for uniformity and different groups of four pots per group were sprayed with 1000 ppm of 4 -phthalimido-2 ,6 -dimethylpyrimidine, phthalic acid, phthalimide, Phillips petroleum compound 47$, N-phenyl phthalimide, diethanol amine, N-l naphthyl phthalamic acid, N —2 chlorophenyl phthalamic acid, and N-4 chlorophenyl phthalamic acid. The plants were removed from the room while being sprayed, and the knapsack sprayer with the attachment which allowed a different flask to be used for each compound was used* A group of John Baer tomato seedlings were transplanted to a similar soil on May 4 and were sprayed with the same treatments when they reached a height of eight to twelve inches a 914 - Results A few days after treatment, the young trifoliate leaves of the bean plants sprayed with lp-phthalimido-2,6dimethylpyrimidine and those sprayed with N-l naphthyl phthalamic acid developed an epinastic twisting and a mottled dark green color as shown in Figure 9* The formative effects produced by both compounds appeared identical# The trifoliate leaves of the plants sprayed with N-2 chloro­ phenyl phthalamic acid also developed an epinasty and also necrotic yellow and brown areas along their margins* The other treatments produced no noticeable formative effects* The plants flowered and the beans were allowed to mature* They were then dissected and inspected for abnormalities* The pods, ovules, cotyledons and embryos of the beans of all the treatments appeared normal# Another group of cranberry beans which were sprayed at an earlier stage of development did not develop many flowers and the apical shoot lost much of its dominance* Apparently the stage of development at the time of spraying influences the response obtained* The only compound producing formative effects on the tomato leaves was N-l naphthyl phthalamic acid* It pro­ duced an inhibition of leaf growth, and an epinasty of the young leaves as shown in Figure 9* The other compounds did not produce noticeable formative effects when applied at a concentration of 1000 ppm, however, younger plants might have been more sensitive* Figure 9* Top Formative effects of cranberry bean leaves on plants sprayed with 1000 ppm of distilled water, N-l naphthyl phthalamic acid, ]+-phthalimi do-2,6-dimethylpyrimidine, and N-q. chlorophenyl phthalamic acido Bottom Upper row: Formative effects of John Baer tomato leaves produced by spraying with 1000 ppm of N-l naphthyl phthalamic acid. Middle row: Epinasty of Bartless pear leaves sprayed with 2000 ppm of N-l naphthyl phthalamic acid. Lower row: Epinasty of peach leaves sprayed with 2000 ppm of N-2 chlorophenyl phthalamic acid* The leaf on the left of each picture represents the check treatment and other treatments producing no formative effects* 96 The l4.~phthalimid.0 -2 ,6 -dimethylpyrimidine did not cause formative effects in the other experiments which included coleus, cucumber and muskmelon plants, and apple, pear, cherry, and peach trees. Concentrations from $00 ppm to 2000 ppm of N-l naphthyl phthalamic acid did cause severe ©pinasty of young pear and apple leaves when they were applied as a spray during full bloom. Peach leaves sprayed with 1000 ppm or 2000 ppm of N - 2 chlorophenyl phthalamic acid as shown in Figure 9 also developed an epinasty0 The Effect of Several Phthalic Acid Derivatives Upon the Rate of Respiration of Several Plant Tissues Review of Literature The influence of various growth regulating substances upon the respiratory rates of different plant tissues, homogenates, and extracts have been studied by many workersa Kelly and Avery (1^3) found that in general low concentra­ tions of 2,i|.-dichlorophenoxyacetic acid stimulated respir­ ation while high concentrations inhibited it* They found that lower concentrations of 2 ,l4.-dichlorophenoxyacetic acid were required to cause a stimulation of respiration in the stems of peas which are dicots, than in ceoleopliles of oats, which are monocots. This is in accord with the common observation that dicots are much more sensitive to herbicidal sprays of this compound than monocots. In a later work ()|J|) they found that young tissue was much more responsive than older tissue, which only shows an inhibition of respir- 97 at ion as increasing concentrations of growth regulators are used0 Mitchell, Burris and Riker (60) and Miller and Burris (59) have found that benzoic acid and salicylic acids affect respiration of sliees of tomato stems, and of cell-free enzymes of barley in the same way as many of the auxins* All of these chemicals inhibited the respiration of the tissues which were used* Methods and Materials The rate of respiration as indicated by the uptake of oxygen by plant tissues was determined with a standard Warburg apparatus as described by TJmbreit, Burris, and Stauffer (96)* Cucumber seeds were germinated in petri dishes with five milliliters of a 2?0 ppm solution of the following compounds: l|-phthalimido-2,6-dimethylpyrimidine, phthalimide, phthalic acid, Phillips Petroleum compound i|-75* diethanol amine, N-l naphthyl phthalamic acid, N-2 chlorophenyl ph thalamic acid, and N-Ij. chlorophenyl ph thalamic acid* Twenty-four hours after the cucumber seeds were moistened, when the tap root is approximately one—half inch long, the seeds were rinsed in distilled water to remove any fungi, six uniform seedlings were selected and put in the Warburg flasks, and the rate of respiration was determined. The amount of oxygen consumed in consecutive 15 minute intervals was recorded, to observe if the process continued at a fairly uniform rate. The data for the experiments with cucumber seedlings is expressed as microliters of oxygen 98 consumed by the six seedlings per* iiour, The meristematic regions or a seedling respire so much more rapidly and comprise such, a small portion of* the total weight of the seedling as compared to the large cotyledons and seed coat that it was felt that this was a less biased way of express­ ing the data than on the conventional dry weight or fresh weight basis* The rat© of respiration of the top young leaf of the tomato plants was determined* Different plants had been sprayed with a 1000 ppm solution of the following compounds: J4.-phthalimido-2,6-dimethylpyrimidine, phthalimide, N-l naphthyl phthalamic acid, N-2 chlorophenyl phthalamic acid and control* Since the Warburg apparatus is not designed to accomodate as much tissue as a whole leaf and since a single leaf is not a very reliable sample, duplicate one-tenth gram (fresh weight) samples obtained by slicing several leaves and immersing them immediately in cold buffer solution were used* Xn July some cranberry beans were planted in pots as described in the previous experiment on the formative effects* When the primary leaves were from two to three inches in width, before the trifoliates expanded, different pots of plants were sprayed with 1000 ppm of the following solutions: lj.-phthalimido~2,6-dimethylpyrimidine, phthalimide, N-l naphthyl phthalamic acid, N-2 chlorophenyl phthalamic acid, and N-lj. chlorophenyl phthalamic acid* The rates of respiration of duplicate one-tenth gram samples taken by slicing the primary leaves of the plants receiving the 99 different treatment were determined forty hours after the plants were sprayed# Some Mohawk oats were grown in pots of vermiculite at the same time and sprayed, when the first leaf was about three inches long and the third leaf was not yet unrolled, with the same compounds and concentration used in the respiration studies on the bean leaves* The rate of respir­ ation was determined on duplicate two-tenths gram samples obtained from sections from the middle of the outer, lower leaves of the plants of the different treatments 6i|. hours after the plants were sprayed* Results Xn all of the respiration studies difficulty was experienced in selecting uniform samples of six cucumber seedlings or small amounts of leaf tissue* The 4-phthal- imido-2,6-dimethylpyrimidine and the phthalimide generally caused a slight increase in the rate of respiration, while the N-l naphthyl phthalamic acid, N-2 chlorophenyl phthalamic acid, and chlorophenyl phthalamic acid usually caused very definite increases in the respiratory rates of the different plant materials as shown in Tables XXVIII and XXIX* This is in accordance with the observation that an increase in the toxicity of growth regulators is frequently associated with an increased rate of respiration (74-* 81)* There certainly Is not enough evidence to establish any causal relationship however# 100 TABLE XXVIII THE RATES OP RESPIRATION OP CU CUMBER SEEDLINGS 2lj. HOURS APTER GERMINATING AND GROWING IN 2$0 p p m SOLUTIONS OP PHTHALIC ACID DERIVATIVES Treatment Microliters of oxygen consumed per hour by six cucumber seeds Control 108 i|.-phthalimido-2 ,6 -dimethylpyrimidine III4. phthalimide 112 Phthalic acid 103 Phillips Petroleum compound 14-75 116 Diethanol amine 122 N-l naphthyl phthalamic acid 138 N-2 chlorophenyl phthalamic acid 108 N —ij. chlorophenyl phthalamic acid 128 L.S.D. at 5% level 12 101 w © o w Eh © © Q X! W a S3 P CO > 03 03 CO Eh bO CO > AJ !> S < H fd Q o o l>* Eh H P 43 § O s 8) Eh i— 1 P © © 4> O O •H •H *tH •H •H P ■P 43 bO •H P CjO AJ C— 4s! P c8 P O S >s © Ft > P c8 © © rP 1 —1 43 © P o rP bO *H P d o rH O CO v£> t*— O vO r- d co PA aO rH AJ d rH CO CO d CO AJ CA d CA vO PA AJ d vO AJ O d GO d d AJ d •H O © d •rH O © d •rH o © o *H o •H o •H © rH © 4S © i—1 © 43 41 P bO •H •H 03 03 O O 53 ^ O sO rH H d d XA CO © P •H d P t? o 03 © © o o PH H *4 o O o 03 P © -P •ri H O Ft O •H s © P m © •H Eh © P O •H -P t-f d P O O p O *H -P © Ft *H Pj © © Fh *d B •H p . i— i 1>S 43 p 43 © b £ W W Eh O AJ rH ro CD &0 O CO pq Eh PA i— 1 rH P PQ Ph P -P Of O o o o H • > XA rH rH © rH Ph X 08 08 o Ph P« 03 rH •H d 1 vO •\ CM 1 O d •H B •H rH © O xs 43 +> 43 Ph 1. P P O O d © iH © 43 43 41 Ph P- rP 43 a 43 rH 1—1 43 P b © 43 b p © 43 a O a o rH © d *rl B •H rH © 41 43 41 43 43 P © P P P O rH 41 O i— 1 I S 23 AJ 1 *2! rH © > © © rH i—1 rH © p o rH 41 o d I Is XA rH 43 © • Q • 00 • id 43 © • P• W • P 102 The stimulation of respiration resulting from treat­ ment with l|.-phthalimido-2,6 -dimethylpyrimidine would be expected to utilize more of the sugar and fatty acid unless their synthesis was stimulated at a greater rate than their respiration. A decrease in sugars and fatty acid was observed in bean plants analyzed for these constituents0 The respiratory quotient of cucumber seedlings treated with l+-phthalimi do-2,6-dime thylpyrimi dine was 1®29 and of untreated seedlings was 1 ®214.0 These differences are well within the limits of experimental error and are not significantly different® Discussion The experiments which have been conducted quite clearly demonstrate that 4-phthalimi do-2, 6-dime thylpyrimi dine is a new growth regulator which is capable of inducing parthenocarpic development of tomatoes, of stimulating the rooting of cuttings and causing morphological and chemical changes in plants. This is of special interest because thestructure of this compound is quite differentthan that of the auxins although many of the plant responses are similar® Koepfli, Thimann and Went (lf7) have stated that for a compound to have activity as an auxin it should at least have: 1* a ring system as a nucleus 2* a double bond in the ring i 3. a side chain 103 !}.• a carboxyl group or & structure readily convertible to a carboxyl group on this side chain at least one carbon atom removed from the ring 5* a particular space relationship between the ring and the carboxyl group It can be seen from Figure 7 that lj.-phthalimido-2,6dime thylpyrimi dine would not fulfill any of the last three requirements for activity* Even if the imide linkage were hydrolyzed the compound should have no activity according to this theory* From its structure it can be seen that ij.-phth.alimido2, 6-dime thylpyrimi dine could be renamed N-l|. (2,6-dimethyl pyrimidine) phthalimide. The great similarity of the formative effect of bean leaves produced by if-phthalimido2, 6-dime thylpyrimi dine and N-l naphthyl phthalamic acid, the fact that both compounds cause a reversal of polarity or a negative geotropism in the roots of bean and cucumber seedlings (35* 59)* and the fact that l^-phthalimido-2,6dimethylpyr imi dine is capable of inducing par theno carpi c development of tomatoes as well as the N-aryl phthalamic acids and N-aryl phthalimides all suggest that it may be the phthalimide portion of ij.-phthalimi do-2, 6-dime thylpyrimi dine which is primarily responsible for its activity* The difference in activity of N-l naphthyl phthalamic acid, N-2 chlorophenyl phthalamic acid in the respiration studies, the experiments on formative effects, the experiments with the cucumber seedlings, and the experiments of Hoffmann 10 ^ and Smith (37) all indicate that the aryl group and the manner in which it is substituted influences the degree of activity of the compound although not the type of activity* These differences are not removed by expressing the con­ centrations in moles instead of parts per million* It is the structure of the phthalamic acid or phth&limid© portion of all of these compounds, which they have in common, but the different aromatic nuclei (benzene, naphthalene, and pyrimidine) to which the phthalic acid derivatives are attached may be responsible for the differ­ ences in toxicity* Veldstra (98) has proposed a theory that the balance between the lipophylic and hydrophilic properties of organic compounds is important in determining their physiological activity* Many modern workers (91) have doubted that action of growth regulators Is purely a surface phenomenon, but perhaps these qualities are impor­ tant in the entry, or translocation of the compound within the plant, or in the movement of the compound into the cells* The phthalamic acids might be considered ortho-substituted benzoic acids, and fit into the schemes of Hansch, Muir and Metzenberg (36)* They and Poster, McRai and Bonner (2\\) have suggested that the auxins are attached at two positions to a protein* One is through a salt linkage with the carboxyl group and the other linkage, perhaps with a sulfhydryl group, occurs at the ortho position of the ben­ zene ring* Phthalamic acid or a hydrolyzed phthalimide might react at its carbonyl group and their electropositive amide group* The substituents or the character of the 105 other aromatic ring might influence the charge at the amide group* A theory such as this affords an explanation of the apparent activity of ij.-phthalimi do-2, 6-dime thyl­ pyrimi dine as an anti auxin, since it and the endogenous auxin may be competing for the same points for attachment* Since there is hardly enough consistent evidence to support the two-point attachment theory, and the experiments described herein yield little information concerning this theory, any suggestions such as these are entirely specu­ lative* The stimulation of the growth of cucumber roots caused by phthalimide and in some cases by Phillips Petroleum compound ij.75 is interesting* Allen and Skoog (3) have reported that N-phenyl succinimide, especially, and N-para-* chlorophenyl succinimide, and N-2, If-dichlorophenyl succin­ imide stimulated the growth of the roots and shoots of Henry wheat and the root growth of Scarlet Globe radish seedlings, in an experiment similar to the experiments with cucumber seedlings. Bonner and Bandurski (lLj.) have suggested that many antiauxins cause a stimulation of root growth of seedlings* If phthalimide proves capable of stimulating the growth of seedlings of other species besides cucumber, It might be beneficial to incorporate it in the material used for pelleting seeds# THE CHANGES TN THE CHEMICAL COMPOSITION OF CRANBERRY BEANS SPRAYED WITH Ip-PHTHALIMIDO-2, 6-DIMETHYL PYRIMIDINE R e v ie w The r e s u ltin g ant in changes fro m th a t in th e y in d ic a te p la n t of tr a n s lo c a tio n In c r e a s e fo r or have been d e c re a s e liv e s to c k c h e m ic a l tre a tm e n t w ith o f th e th e th e o f L ite r a tu r e ho w c o m p o s itio n o f p l a n t s g ro w th th e a lte r e d , in th e is d ir e c tio n w ith in c o n s titu e n ts o r h u m an b e i n g s a re n o rm a l m e ta b o lic or o f m a te r ia ls r e g u la to r s a ls o th e p ro c e s s e s and e x te n t p la n t. o f n u tr itio n a l v e ry im p o r t­ Any v a lu e im p o r ta n t. In general, the treatment of responsive plants with the auxin-like growth regulators, causes a decrease In the carbohydrate reserves of the plant, and frequently an increase in nitrogenous compounds (61). Stuart (8 9 ) found that the total amount of starch, reducing sugar and sucrose in cuttings of kidney bean plants decreased when treated with 100 ppm of indoleacetic acid and that the amount of nitrogen increased. These trends were true for the different organs however there was considerable translocation from leaves and the upper portion of the stem to the base of the hypocotyl. Mitchell (61) found that kidney bean plants treated with naphthalene ace tic acid or naphthalene acetimide had less total sugar, starch, and dextrin in the roots and hypocotyls. The tumorous stems of his treated plants 107 contained mope nitrogen, most of* which, was In water soluble forms* Tn a later work (614.) he reported that the rate of starch hydrolysis In bean leaves was Increased after they were sprayed with naphthalene ace tic acid. Hamner, Sell, and others have extensively studied the physiological responses of the red kidney bean plant to treatments with 2 ,l|.-dichlorophenoxyaeetic acid and have found that it caused a decrease in the amount of the differ­ ent carbohydrate fractions in the stems (79) o The amount of protein In these stems was approximately doubled and the proportion of the different amino acids making up this protein was altered* The differences in the leaves and roots were not great except that the non-reducing sugar in the treated leaves and roots was completely depleted (9 9 )• The amount of thiamin, riboflavin, nicotinic acid, panto­ thenic acid and carotene In the stems and leaves of red kidney beans were also changed (5 3 )* Similar changes resulting from treatment with 2,lp-dichlorophenoxyacetic acid have also been reported for dandelion (7 5 )9 bindweed (8 2 ), tomato (6 2 ), wheat (2 3 ), annual morning glory (6 1 ), and wild garlic (l±6 )® Methods and Materials Cranberry bean seeds were planted In a uniform soil mixture in five-inch pots. The plants were thinned to two per pot and the pots of plants then graded by eye so as to insure that plants of about the same range of morphological 108 development were Included in each treatment and replication# Two replications of plants were sprayed with an aqueous solution of 4-phthalimi do-2,6-dime thylpyrimi dine when the first trifoliate leaf was well expanded and the second tri­ foliate was about one-half inch in length# The plants were about nine inches high, with no flowers visible# The sprayed trifoliate leaves developed an epinastic twisting and mottled appearance about three days after spraying, and did not continue to expand appreciably* The plants were harvested by cutting the stems just above the soil, six days after treatment, and promptly taken to the laboratory* There the plants were divided into the primary leaf blades, trifoliate leaf blades, and the remainder which included the stems and petioles, which is here referred to as stems* The fresh weight was obtained and the samples for each analysis were promptly killed# Total sugar and reducing sugar were determined as described in the Official Methods of Analysis of the Association of Official Agricultural Chemists in Sections 6#i|.8, 22*33* 29*36, 29*39, 41 #13 (69). Total nitrogen was determined by the macro Kjeldahl method described in the same book under 2*22* The free fatty acid and unsaponi- fiable material was determined as suggested by Dill (21)* Results There was slightly less reducing sugar in the stems and less total sugar in the trifoliate leaves of the treated 109 plants than In the same organs of the control plants as shown in Tables XXX and XXXT. Since the value for sucrose is determined by subtracting reducing sugar from total sugar any inaccuracies in either determination would influence this value, and since there is so little sucrose in young bean plants only quite large differences should be regarded as important* There was only about half as much fatty acid in the treated plants as in the control plants, and a comparison of the averages indicates that this decrease occurs mostly in the primary leaves and secondly in the stems. There appears to be no great difference in the amount of unsaponifiable material in the treated and control plants. Although this fraction includes many compounds whose metabolism is not well understood, the unsaponifiable material is not commonly regarded as a readily available reserve food material* The difference in Kjeildahl nitrogen is insignificant statistically, although there appears to be a slight increase in the percent of the nitrogen in the treated trifoliate and primary leaves; however, this difference might be due to a difference in water content of the leaves, and because of the decrease in size of the leaves this is not a real increase in the amount of nitrogen in a leaf* The increased rate of respiration of bean leaves treated with the same concentration of i4.-phtb.alimi do-2 ,6 dimethylpyrimidine, would be expected to result in a decrease 110 ■3 P O Eh O' d W IT H CM S 0 CQ O' CO at cq d vO CA XA CM o CM CM » XA • O AO CM P d A0 © O ' CM o O d sO 0 CO rH O XA 0 N0 rH rH sO O ' CA A 0 CO vO XA 0 d XA cA d i— 1 rH CA 0 rH THE COMPOSITION 1000 OP CRANBERRY BEAN PLANTS SIX DAYS AFTER SPRAYING PPM OP 1 4 .-P H T H A L IM ID O -2 , 6-DIMETHYL P Y R IM ID IN E d 0 cA 0 CM 0 p aS 0 0 •SH 0 PM CM 0 d o o 0 SO o o 0 d CM CM 0 o O CM CA 1— 1 CO 0 A- CO 0 % 0 P at O 0 as OJ rH a O C M rH • O o o• o o cA CM sO CM d 0 CA C O a 0 0 0 O ' a XA CM O XA CO CM 0 rH rH 0 > 0 09 0 0 o 0 & at S .0 •H o 0 3- sO d CM O © o Ph O' CM XA XA CO O • 0 XA cA 0 GO O XA © P a) TO TO 0 0 P at •H rH O <4H •H Pi co rH o O XA i—1 CA O -r± zr 0 0 0 o o i —I XA 0 o rH rH XA CO CA rH 0 0 CM NO 1—1 vO CM 0 o P § O •H < 0 EH I) 0 0 at Pi at bO 0 TO bO 0 TO bO 0 ♦H at P O Eh O 3 d 0 0 c 0 0 Pi 0 <0 <0 0 «tH to d o Pi ^ 0 ,0 F*'-' C O fO d •H o at at •H <0 rH •H at 0 *H O Pi PLO P P 0 (in at p to at S3 S s 0 bO O Ph P •H 0 rH 3at d i—I 0 •o 0 •H 0 •H 0 ^ P XA O CM Pi • pLvO © d 0 K is Pi'-' o P rd bO •H 0 £ 43 TO 0 0 fc TO rH ■s •H .W 111 & 0 P CQ sO c— • ao xO • CO C'~* s P t>0 d n W a 0 rH 0 P O EH o d ■O 0 Ph 0 O pH o d CQ T* ♦H O 0 P P 0 fin 0 rH P 0 •H •rH Pi d 0 bO o Ph P •H PI i— 1 P O a 0 0 T3 (— 1 0 *r-» P Id ra 112 in sugars, fatty acids, and other carbohydrate fractions as these analyses indicate if the capacity to synthesize these materials was not increased so as to overcome the increased rate of breakdown during respiration# This is apparently what happened# ^These alterations in the composition of plants are similar to those caused by many other growth regulators such as 2,ip-dichlorophenoxyacetic acid, indoleacetic acid and others# The decreases in all of the major constituents which were determined indicate that the food value of these plants was not increased in these respects for animals#. The analyses are valuable in that they indicate some of the changes which have occurred in the treated plants# EXPERIMENTS SUGGESTING THE MODE OP ACTION OP lj.-PHTHALIMIDO2,6-DIMETHYLPYRIMIDINE UPON THE PHYSIOLOGY OP PLANTS Intro due tion Th© manner In which £|.-phthalimido-2, 6-dimethyl pyrimidine affects the growth of plants was suggested by three experi­ ments# In all of these experiments, lj.-phthalimido-2,6-di- methylpyrimidine seemed to lessen or antagonize the main effect of auxin or to interfere with its translocation whether th© auxin was of natural or synthetic origin# According to the theory of Went and Thiraann (103) and the large amount of substantiating evidence and experience which is in accord with this theory, auxin is responsible for correlating and controlling many different phases of the morphological development and growth of plants. Auxin is thought to be synthesized In the apical meristematic regions of stems and in leaves, from natural occuring substrates, and to travel in a morphologically downward direction with a naturally occurring polar gradient. The supply of auxin from leaves Is thought to be an Important factor in pre­ venting their abscission (80). Physiologically high con­ centrations of auxin are probably responsible for inhibiting the development of the lateral buds of shoots and allowing the apical bud to maintain its dominance# The concentration of auxin which is physiologically active In root growth is Ilk m uch lo w e r is s till th a n of r e s p o n s ib le a r tific ia lly in h ib it th a t a n d g ro w th fo r s u p p lie d g ro w th . of th e ro o t g ro u n d p o r t io n , g ro w th , c o n c e n tr a tio n s A u x in b ra n c h above a ls o a lth o u g h of p r o b a b ly E ffe c ts G ro w th of M e th o d s w h ic h c h e m ic a lly endogenous lin g s s u p p ly g e n e r a lly a d d itio n s of a u x in w i t h i n ro o t A s m a ll fa c to r ia l if-p h th a lim id o upon th e in h ib itio n g ro w th in flu e n c e th e ro o ts of (1 ) c o n tr o l, p y r im id in e , (3 ) th e of is s e e d lin g s * 25 tre a tm e n ts a c id , w e re s e e d lin g s had bench fiv e fo r added to r e p lic a te d g ro w n i n days th e th e y th e seed­ p r e v io u s 2 5 0 ppm a d e c id e d cucum ber r o o ts * to d e te r m in e a n y m a jo r upon th e if e ffe c t g ro w th of c o m b in a tio n s of p h t h a l i m i d o —2 , 6 —d i m e t h y l — ppm o f i n d o l e a c e t i c w e re of fo u r o f i|.-p h th a lim id o -2 , 6 - d im e th y lp y r im id in e in d o le a c e tic o f in ta c t causes had The n o rm a l know n t h a t g ro w th a c id th e F ro m c o n d u c te d in d o le a c e tic 2f>0 ppm o f to (1 ), it e th y lp y r im id in e cucum ber (2 ) of e x p e r im e n t w as -2 ,6 - d i m and Upon th e ro o ts o f ^ -p h th a lim id o -2 ,6 -d im e th y lp y r im id in e c o m p le te A c id o f h e te r o a u x in , a c id , th e cu c u m b er s e e d lin g s but not in itia tio n and M a t e r ia ls in d o le -3 -a e e tic in h ib its e x p e r im e n t w i t h th e C u cu m b er R o o ts exogenous is a ll s e v e r e ly o f In d o le a c e tic U .-P h th a lim id o -2 ,6 -d im e th y l p y r im id in e or It ro o ts * The In te r a c tin g A r tific ia l m ost a u x in g o v e rn s but in fo u r p e tri w e re th e a c id , p lu s p e tri d is h e s d is h e s m e a s u re d * and (4 ) 250 ppm 2 5 ppm o f d is h e s . each. on th e The A fte r fo u r th e la b o r a to r y 115 Results Either the l|.-phthalimido-2,6-dimethylpyrimidine or the indoleacetic acid when applied alone caused an inhi­ bition of growth but when the two were applied at the same time the amount of inhibition was not additive. Statis­ tically this was a significant interaction as shown in Table XXXII# This result might be interpreted in many ways* It could mean that ip-phthalimido-2,6-dimethylpyrimidine Inhibits the enzymatic synthesis of the endogenous supply of Indoleacetic acid from tryptophane* It might mean that it stimulated the destruction of indoleacetic acid probably by the indoleacetic acid oxidase system* These results also might indicate that the two compounds are involved in two different steps of a series of reactions so that if one step is inhibited the latter step does not occur at its usual rate so Its inhibition is not added. Another possi­ bility is that l4.-ph.thalimi do-2 ,6 -dimethylpyrirai dine inter­ feres with the translocation of the endogenous supply of auxin coming from the root tip to the region of elongation, or the translocation of free auxin arising from the liber­ ation of bound auxin in the cotyledons to the region of elongation* 116 TABLE XXXII THE INTERACTION OP 1+-PHTHALIMIDO-2, 6-DIMETHYLPYRIMIDINE AND INDOLEACETIC ACID ON THE GROWTH OP CUCUMBER ROOTS Treatment Control Average length of roots in cm i*h Indoleacetic acid 1*5 250 ppm i|.-phth al imi do- 2,6 -dime thyl pyrimi dine I1-.6 25 ppm 250 ppm Indoleacetic acid plus l4.-phthalimi do-2,6-dime thylpyriraidine l.ij- 25 ppm Interaction significant at 5$ level 1X7 The Inhibition of the Translocation of Auxins by ij.~Phthalimi do-2,6-dimethylpyrimidine in the Petioles of Coleus Plants Methods and Materials An experiment was conducted to determine if ij.-phthalimido-2,6-dimethylpyrimidine interfered with the trans­ location of indoleacetic acid or naphthaleneacetic acid* Thirty-two Christmas Gem coleus plants were graded for uni­ formity and divided into four treatments of eight plants each. The blades of half of the leaves were cut off, as was done in the previous studies of abscission. A lanolin paste containing 1000 ppm of indoleacetic acid was placed on the tips of the petioles of two of the treatments of plants and a paste containing 100 ppm of naphthaleneacetic acid was placed on the tips of the petioles of the other two treatments of plants. The plants of one group treated with indoleacetic acid, and one group treated with naphthaleneacetic acid were treated with a ring of lanolin paste con­ taining 1000 ppm of i|-phthalimido-2,6-dimethylpyrimidine applied around the petioles between the abscission zone and the tip. A ring of lanolin containing no growth regulators was applied to the other two groups of plants which served as controls. The rate of abscission of the petioles was used as an index of the amount of inhibition of auxin translocation because auxin usually delays the abscission of coleus petioles (26)* 118 Results The petioles with a ring of i^-phthalimido-2,6-dimethyl­ pyrimidine between the supply of auxin and the abscission zone did absciss earlier than the petioles with no ij.-phthalimido-2,6-dimethylpyrimidine between the supply of auxin and the abscission zone as shown in Table XXXITI* This result might be interpreted as indicating that 1^-phthalimido-2,6-dimethylpyrimidine inhibited or interfered with the translocation of indoleacetic acid, or of naphthaleneacetic acid. Both of these auxins responded similarly. These results do not eliminate the possibility that ip-phthalimido-2,6-dimethylpyrimidine and the auxins act independently upon the abscission zone* A similar experiment using cucumber plants in pots in the greenhouse was conducted. The leaves were not cut off but were left intact to supply a physiologically normal concentration of auxin. A ring of lanolin containing 1000 ppm of i|.-phthalimi do-2,6-dimethylpyrimidine failed to make the leaves absciss. This may be due to the fact that cucumbers do not have a pectolytic enzyme in the abscission zone as reported by Bell (10)* The Inhibition of Apical Dominance by Treating the Apical Shoot of Coleus Plants with il-Phthalimi do-2,6 -dime thylpyrimi dine Methods and Materials An experiment was conducted to determine if the down— 119 I © TJ d *H *H rH o ©o o o _ H © § 2 o O f§H P<.d p p «H H rd -p -P © op,© d d O di c od co I © *d •H h f l *IH CS *rl O 6 XJ nj o©© •h -P Td _ rH © _ rH ® »H b M B •H £ S © P^rd o P X ! *H O P © g P p © P © d d *H rH d » O T3 «H O PvO P I -v o d o O r) rl tfi H - d C M © o *H -P © P © > < o d- CO • CA cA 1—1 Eh O JM pq PQ W Eh d P H d d o P o © •H d P cd •H i —I © X> O © P Pi rH rH © P © © P © •H •H w £ © d •H SB at the same time is l®8l o -d V « -d d O © -P p d 1952. 127 25. Gardner, E. E. Growth, substances in relation to the production of tree fruits. Plant Growth Substances, pp. 207-224. Madison: University of Wisconsin Press# 26. Gardner, P. $3. and W. 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