STUDIES ON FOLIAR ABSCISSION OF CAULIFLOWER AND CABBAGE IN STORAGE, WITH SPECIAL REFERENCE TO THE EFFECTS OF CERTAIN GROWTH REGULATING SUBSTANCES By SHU-HSIEN LEE 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 1948 ProQuest Number: 10008362 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 10008362 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 48106- 1346 ACKNOWLEDGMENTS The writer wishes to express his appreciation to Dr. R. L. Carolus for his effective guidance throughout this investigation. He also wishes to express appreciation to Dr. H. B. Tukey, Dr. R. E. Marshall, Dr. E. H. Lucas, Dr. S. H. Wittwer, and other members of the Department of Horticulture, and to Dr. F. L. Wynd, of the Department of Botany and Plant Pathology, for their advice and cooperation. 203045 CONTENTS Page INTRODUCTION ........................................ 1 REVIEW OF LITERATURE Anatomical Nature of the Abscission Formation ...... 2 Relation of Chemical Changes to Abscission Formation ......................................... 4 External Factors Associated with Abscission ..6 Formation.................... The Effect of Growth Regulating Substances in Modifying Abscission Formation.............. 8 EXPERIMENTATION The Effect of Post-harvest Treatment with Growth Regulating Substances on Foliar Abscission General Methods ........................... Effect of Different Timesof Treatment ....... 12 14 Effect of Different Formulations of the Growth Regulating Substances ........ 20 Effect of Different Methods of Application .... 24 Effect of Temperature ...... 28 Effect of Trimming .......................... 30 The Effect of Field Treatment with Growth Regu­ lating Substances on Foliar Abscission Material and Methods ......................... Results and Discussion ................. iii 36 37 Pag© Anatomical Changes in Relation to Foliar Abscission ........................ 45 Anatomy of the Abscission Zone ................ 4-5 Leaf Order in Relation toAbscission Formation .. 46 Material and Methods Biochemical Changes in Relation to Foliar Abscis­ sion Sugars ............. 58 Dry Matter ........ 64 Ascorbic Acid ...... 68 Respiration .............................. Catalase Activity ....... 71 75 GENERAL DISCUSSION The Role of Plant Hormone in Relation to Abscis­ sion Formation ........ 86 The Transport of Growth Regulating Substance in Relation to Abscission Formation .... 89 Biochemical and Physiological Changes in Relation to Abscission Formation. CONCLUSION ........... .................. LITERATURE CITED .............. -....................... iv 94 99 102 LIST OF TABLES Table I. Page Effect of methyl ester of naphthaleneacetic acid on the foliar abscission In cauliflower• 17 II• Per cent loss in weight of cauliflower in storage, following treatment with different concentrations of the methyl ester of naphthaleneaeetlc acid.............................. III. IV* Vw VI* VII* VIII* DC* X* XI. XII. 19 Per cent loss in weight of cabbage in storage following treatment with different concentra­ tions of the methyl ester of naphthaleneacetic acid........................................ 19 Effect of different formulations of naphtha­ leneacetic acid and 2,4-diehlorophenoxyaeetic acid on the color, abscission, and elongation of the inflorescence ofcauliflower........... 22 The effect of point of application of the growth regulating substances on leaf fall of stored cauliflower........................ 26 Time required for the initiation of abscission in cabbage stored at varioustemperatures 29 The effect of leaf blade tissue on abscission formation of cauliflower (expressed as the per cent of leaves that dropped per head)......... 32 The influence of methods of trimming on weight loss in cauliflower..................... 33 Gell size in midrib tissues of cauliflower, taken one week after field treatment with 2,4-dlehlorophenoxyacetic acid at 250p.p.m...* 40 Number of fallen leaves per head in cabbage, following field spray application with 2,4dlchlorophenoxyacetic acid and naphthalene­ acetic acid................ 42 The effect of field treatment on the per cent of leaf fall of cauliflower in storage........ 43 Leaf order in relation to percentage of separation in leaf abscission of cabbage, 80 days after treatment...................... 56 v Table XIII. XIV. XV.. XVI. XVII. XVIII. XIX. XX(a). XX(b). XXI. Page Leaf order in relation to percentage of se­ paration of leaf abscission in cauliflower, 67 days after storage for treated and 32 days after storage for check............... 56 Leaf drop in cauliflower used for sugars and dry matter analyses.............. 62 The effect of methyl ester of naphthaleneacetic acid on the sugar content of stored cauliflower................ 63 The effect of methyl ester of naphthalene­ acetic acid on the dry matter content of stored cauliflower.............. 66 The effect of methyl ester of naphthaleneacetic acid on the ascorbie acid content of stored cauliflower................ 70 The effect of methyl ester of naphthalene­ acetic acid on the rate of respiration of cauliflower................... ............ 73 The relation between the volume of oxygen released and the time of mixing the extract of the leaf blade of cauliflower with hydro­ gen peroxide......... ...*• 30 The catalase activity of stored cauliflower following treatment with methyl ester of naphthaleneacetic acid (expressed as amount of 02 released at various timeintervals).... The catalase activity of untreated cauli­ flower during storage...................... 81 82 The effect of leaf order on the catalase activity of untreated cauliflower........... vi 83 LIST OF FIGURES Figure 1, 2. 3* 4. 5• 6* 7« 8• 9. 10, Page Effect of methyl ester of naphthaleneacetic acid on the foliar abscission of cauliflower 33 days after treatment.................... 18^ Tumor tissue on the cut surface of the stem of cabbage, three weeks after treatment with methyl ester of naphthaleneacetic acid...... 23 Effect of method of application and formula­ tion of 2,4-dlehlorophenoxyacetic acid on foliar abscission of cauliflower, 55 days after treatment............................ 27 The effect of the methyl ester of naphthalene­ acetic acid on retarding the foliar abscission of cauliflower with leaf blades removed 34 The effect of the methyl ester of naphthalene­ acetic acid on retarding the foliar abscission of cauliflower with leaf blades attached 35 Cauliflower, one week after field treatment, the leaves were sprayed on the under surface and bend upward after treatment............. 41 Cauliflower, one week after treatment, 250 p.p.m. of 2,4-dichlorophenoxyacetic acid was sprayed on the upper surface of the leaves which bend downward after treatment,... 41 Retarding effect of field treatment on the foliar abscission of cauliflower, 45 days after storage ............................ 44 Diagrams of the formation of foliar abscis­ sion at various stages of development in cabbage, a, b, c, d, and in cauliflower, e, 6» ................................. 51 Foliar abscission of cabbage, showing the meristematic layer near the upper side of a midrib.......... 52 vii j Figure 11. 12. 13* 14. 15* 16. 17 • 18. 19. 20. 21. 22. Page Foliar abscission of cabbage, showing the separation layer.......... 52 Foliar abscission of cauliflower, showing the meristematle layer near the upper side of a midrib. ............................ 53 Foliar abscission of cauliflower, showing the separation layer ........... 53 Foliar abscission of cauliflower, showing the separation layer near a vascularbundle. 54 Foliar abscission of cauliflower, showing the breaking down of a vascular bundle 54 Foliar abscission of cabbage, showing the protective layer on the exposed surface of a leaf scar after abscission formation...... 55 Leaf order in relation to percentage of separation of leaf abscission in cabbage, 80 days after treatment ..... 57 Leaf order in relation to percentage of separation of leaf abscission in cauli­ flower ..... 57 Effect of treatment with methyl ester of naphthaleneacetic acid on the sugar content of eauliflower in storage................. 64 Effect of treatment with methyl ester of naphthaleneacetic acid on the dry matter content of cauliflower in storage ........ 66 Effect of methyl ester of naphthalene­ acetic acid on the rate of respiration in cauliflower.......... 74 Effect of methyl ester of naphthaleneacetic acid on the ascorbic acid content of stored cauliflower.............................. 74 viii Figure 23* 24. 25* 26 • Page Effect of methyl ester of naphthaleneacetic acid on the catalase activity of stored cauliflower ......................... 84 The relation between catalase activity and the leaf order on untreated cauliflower 85 Diagram illustrating the theoretical rela­ tionship between the change of leaf order and the change of percentage of separation in cabbage........................... 93 Diagram illustrating the theoretical rela­ tionship between the change of leaf order and the change of percentage of separation in cauliflower.......... 93 ix INTRODUCTION During storage, premature leaf fall in cauli­ flower, and to some extent in cabbage, will frequently reduce the maketability of these crops. In connection with a storage study with cauliflower, some preliminary work conducted by Carolus, et a l . (1947) indicated that the abscission of the jacket leaves surrounding the head could be delayed by treatment with methyl ester of v of Miabllls .jalana the starch in the abscission cells A decreases as abscission progresses, until it is very materially reduced or entirely disappears. It was con­ sidered to be used as a source of energy for the separa­ tion of cells during their growth* In a study of leaf abscission of Valencia orange, Scott, et al. (194?) found that the nodal zone is roughly indicated by starch accumulation, and that intercellular space distribution is not clearly defined until the Imminence of leaf fall. This chemical changes of cell wall in the abscis­ sion zone might result from or cause the physiological change of other parts of the cells. Cytoplasm, nuclei, and nucleoli according to Lloyd (1916) bear evidence of greater physiological activity, and are alive and normal when separation is achieved* He also claimed that there is meanwhile no loss of turgor^however, Livingston (1947) points out in citrus leaves that pressure and tension of the abscission zone might be involved in this process* -«• 6 ** External Factors Associated with. Abscission Formation As mentioned before, the formation of abscission layers of leaves as well as of flowers and fruits is not merely a mechanical rupture, but Is a result of physio­ logical activities which might cause the chemical change of the cell walls in the abscission layer* Any condition, internal or external, which might retard or accelerate the rate of physiological activity, would naturally affect the abscission formation* Studying the effect of growth substanees on the abscission layer in Coleus leaves, layers (194-0) reported that abscission could be accelerated by a number of exter­ nal factors such as high or low light intensity, high or low water supply, high or low temperature, low concentra­ tions of anesthetics, toxic concentrations of acids and salts, ethylene gas, and wounding or complete removal of the blades. Sampson (1913) in Coleus Blumel» found that leaf fall was accelerated by treatment with ethylene, amputation of the blade, and by allowing the soil to be­ come dry and then suddenly applying an excess of water* Kendall (1918) claimed that the abscission of flowers and fruits in Solanaceae could be accelerated by nicotine vapors, injury of floral organs, sudden rise in tempera­ ture, and even changes in soil conditions* - 7 In addition, Hoffman (1940) indicated that an application of nitrogen-carrying fertilizer made under conditions which would permit the trees to obtain exces­ sive amounts of nitrates during the latter half of the growing season increases the pre-harvest drop of McIntosh apples. Heinicke, Reuther, and Cain (1942) found that an excessive pre-harvest drop of McIntosh apple was associated with incipient stages of boron deficiency which may not be severe enough to cause cork or drought spot. In a study of apple flower and fruit abscission, MacDanlels (1936) suggested that the rate at which the abscission zone is cut across is probably influenced by the carbohydratenltrogen relationship within the tissue. When there is excessive amount of carbohydrate as compared to nitrogen, more woody tissues are formed so that the abscission zone is not so easily cut across. All these external factors, which are able to in­ fluence the abscission formation, possibly could affect the synthesis of the growth substances being associated with this process• Since growth substances are believed to be synthesized in the leaf blade (Avery, 1935)» its removal should hasten leaf drop. LaRue (1936) pointed out that the petiole of Coleus from which the blade has been removed will fall within a few days. He also found that the leaf blade is necessary to prevent abscission formation in the leaves of Betula alba var* papyrlfera Spach., and Alnus incana Moench. If the leaf blade was entirely removed, leaving only the petioles, the latter fall in most plants within a few days. Furthermore, in Morus bombvcls Koidz., Cudranla triloba Hee., Ficus earlca L., Thea sinensis L., Sallx vlminalls L., and Ginkgo blloba L., Okabe (1940a) re­ ported that if the leaf blades are removed it appears that the growth promoting substance in the petioles of the leaves goes on decreasing and that the growth-inhibit­ ing substance Increases day by day until the petioles fall off. Myers (1940) mentioned in Coleus leaves that parti­ ally debladed petioles do not fall as soon as petioles from which the entire blade has been removed. Actively expending portions of the blade are more effective in checking abscission than the mature parts. The Effect of Growth Regulating Substances in Modifying Abscission Formation From the standpoint of the phenomenon of abscis­ sion, the physiological activity of the organ that will subsequently fall is of greater significance than that of the remaining parts of the plant • It has long been known that the premature drop of fruit is due to the lacking of certain growth hormones which are produced by the ferti­ lized ovule and translocated downward to the abscission zone of the pedicel. Similarly, the falling of a blade- less leaf is considered to be due to the absence of certain growth hormones which $re synthesized in the leaf blade and transported downward to the abscission zone of the petiole. As concluded by LaRue (1936), the abscission always result­ ed when the development of the abscission layers was not inhibited by some substances produced in the leaf blades, and the abscission layers did not need any special stimu­ lus for their development. Based on this consideration, many synthetic growth regulating substances have been recognized to replace the function of the naturally produced phytohormones. Some of them have been used’ commercially to prevent the pre-harvest drop of fruits by great number of workers. Gardner, et al. (1939), Hitchcock and Zimmerman (1941), Tukey and Hamner (1945). used naphthaleneacetic acid; and Batjer and Thompson (1946), Harley, et al. (1946) and many others used 2,4-dichlorophenoxyacetic acid to prevent the pre­ harvest drop of apples. Davey (1942) reported that spray­ ing with naphthaleneacetic acid, and naphthaleneacetamlde reduced the fruit drop of Bartlett pears 50 per cent. Hasse and Davey (1942) also used naphthaleneacetic acid and naphthaleneacetamlde to prevent the fruit drop of apricot and peach. Recently, Stewart and Klotz (1947) used 2,4-dichlorophenoxyacetic acid to prevent the fruit drop of oranges• - 10 Beal and Whiting (1945) reported that the pints Mlrabills Jalapa L. decapitated and treated with a 2 per cent lndoleacetic lanolin mixture on the cut surface of the stem showed continued growth of the internodes, and the entire absence of abscission or of an abscission zane at the bases of the internodes. LaRue (1936) and Efyers (1940) found that auxin-containing substances, when ap­ plied to the cut end of the petiole, have the ability to delay abscission in Coleus. Gardner and Cooper (1943) have tested 156 organic compounds for the activity to de­ lay petiole drop. None of them, however, showed suffi­ cient activity for practical usage. The prevention of leaf abscission appears to require a much higher concen­ tration of applied growth substance than the prevention of mature fruit drop. This result, according to Gardner and Cooper (1943), may be related to the differences in the types of abscission involved. The effectiveness of the growth regulating sub­ stances on the retardation of abscission formation varies with the substances used and the plant organs treated. It also depends on the maturity of the leaves and fruits (Batjer and Thompson, 1946), (Hesse and Davey, 1942), (Batjer and Moon, 1945), the temperature at which the treatment was made (Batjer and Moon, 1945), the formula­ tions and methods of application (Hoffman, et al., 1942), 11 (Southwick, 1942), (Tukey and Hamner, 1945), (Ennis and Boyd, 1946), and even varies with different trees of the same variety and with different branches of the same tree (Hitchcock and Zimmerman, 1941) • Generally, any condition that influences the phytohormone production in the plants, might influence the effectiveness of the growth regulating substances used. Mineral nutrition, light, temperature, age of the plant, position of the plant organ, as pointed out by Gustafson (1946), should be considered in connection with the application of the growth regulating substances for this purpose. Externally, man can probably do nothing to modify the position or the structure of abscission, but he has done much to retard or accelerate this process. - 12 EXPERIMENTATION The Effect of Post Harvest Treatment with Growth Regulating Substances on Foliar Abscission General Methods The cauliflower and cabbage used in testing the effects of time of treatment, method of application, and the formulation of the growth regulating substances on ab­ scission were procured from the local market; while the cauliflower (Snowball X) and cabbage (Resistant Detroit) used for the test of the effect of trimming and,tempera­ ture on abscission were grown on the college farm in 1947, under ordinary culture practices. The cabbage and cauliflower were treated immediate­ ly after harvest or after shipment, and then stored at a temperature of approximately 32° F. at a relative humidity of 85 per cent . Each head was stored in ah open kraft paper bag. The methyl ester of both 2,4-dichlorophenoxyacetic acid (2,4-D), and a-naphthaleneacetic acid (NA) were first sprayed on shredded paper which was then placed loosely around the cabbage or cauliflower heads• The concentration of the methyl ester of either 2,4-D or NA is expressed as ml11Igrah of the liquid per head contained in the shredded paper. The sodium salt of either 2,4-D or NA was sprayed 13 with a hand sprayer directly on the leaves of the plants to be treated. The concentration of the sodium salt is ex­ pressed as parts per million (p.p.m.) in aqueous solution and the amount of the solution used is expressed as millil­ iters for each head. In certain experiments a soluble plasite material (Geon X-31), capable of filming, was mix­ ed with the growth regulating substance in order to local­ ize and concentrate the effect of treatment. The sodium salts of NA and 2,4-D were used in this way mixed with 50 per cent Geon containing 56$ solids. Fresh weight loss was determined at various storage intervals. The falling leaves were counted periodically during the storage period. The per cent of leaf drop per head was calculated on the basis of the total number of leaves per head. Usually a cabbage head contained 40 to 50 leaves (excluding those shorter than three inches), and a cauliflower head contained 25 to 30 leaves. Obtained from Goodrich Chemical Co., Cleveland, Ohio Effect of Different Times of Treatment Fresh cabbage and cauliflower shipped from Arizona and procured on the local market were used in this experi­ ment. The cabbage and cauliflower were treated four heads t for each treatment with three different concentrations of the methyl efiter of NA, at 50, 100, and 200 mgs. per head. In order to determine the effect of time of treatment on abscission, cauliflower heads were also treated in the same manner immediately after harvest in Texas and then shipped to East Lansing.* The fresh weight and number of dropped leaves were recorded weekly. Results and Discussion Time required for abscission— -Fifty per cent of the leaves dropped in untreated cauliflower head in 30 days, while in the treated heads 80 days storage were required to cause the same percentage of leaves to fall. The shredded paper placed either on top or under the heads was equally effective in delaying abscission. Observation indicated that abscission progressed from the inner (younger) to the outer (older) leaves of the treated cauliflower, and from the outer to the inner leaves in the untreated heads. * Heads were collected and treated by Mr. S.B.Apple, Extension Horticulturist, Weslaco, Texas. - 15 In order to study the effect of high humidity on abscission, a few heads treated with 100 mgs. of NA were loosely wrapped with pliofilm. Wrapping of treated heads delayed abscission of 50 per cent of the leaves for 150 days, or 60 days longer than the unwrapped heads. It was observed that the retarding effect of the methyl ester of NA on the abscission of cabbage was not as pronounced as in cauliflower. Unlike cauliflower, which usually held its leaves for only a few weeks, the untreated cabbage held their leaves for more than three months with­ out any dropped leaf. Loss in weight— -Before the formation of abscission, only slight differences in the rate of weight loss were found between the treated and untreated heads. After leaf fall, loss of weight was higher in the untreated than in the treated lots (Table 2). This was due to the fact that tie fallen leaves withered rapidly. The weight loss in cabbage was similar to that of cauliflower (Table 3)• Since the loss in fresh weight during storage is mainly due to transpiration, the rate of loss, therefore, varied with the exposed surface area on the one hand, and the permeability of the epidermis on the other . In cabbsge where the leaves fold closely on one another and were not trimmed, the rate of fresh weight loss was less than that 16 of cauliflower, where the leaves were more exposed and usually trimmed* This higher rate of weight loss in cauliflower might accelerate abscission formation, and is probably one reason why cabbage can be stored for a longer time than cauliflower. - 17 TABLE I. Effect of the Methyl Ester of Naphthaleneacetic Acid on Foliar Abscission in Gauliflower (Average.of five heads). Treatment Days required for 50 per cent of leaves to drop Treatment after shipment Treatment before shipment 50 mg. NA per head 80 — _ 100 mg. NA per head 84 95 200 mg. NA per head 92 103 Check 32 45 * In storage at 32°F. 18 Figure 1, Effect of methyl ester of naphthaleneacetic acid on the abscission of cauliflower, 33 days after treatment* E, directly sprayed with NA; B, check; D, treated with 100 mg. of methyl ester of NA. - 19 TABUS II. ...... Per Cent Loss In Weight of Cauliflower In Storage, Following Treatment with Different Concentrations of the Methyl Ester of Naphthaleneacetic Acid (Average of four heads). After Shipment Before Shipment Days Per Cent Fresh after Weight Loss Treat­ NA NA ment 100 mg. 200 mg . Check Per Cent Fresh Days Weight Loss after Treat­ NA NA** ment 100 mg. 200 mg. Cheek 16 1.9 2.4 3.3 10 4.1 0.0 5.4 31 7.1 9.2 10.5 19 6.2 0.5 7.7 44 11.5 13.2 18.9 38 11.8 1.3 14.9 61 16.3 17.4 24.9 59 18.1 3.0 23.8 80 20.1 20.8 31.6 95 26.3 5.4 34.3 93 23 .4 22.7 — 113 34.1 8.9 44.4 ** Wrapped in pliofilm. TABLE III. Per Cent Loss in Weight of Cabbage in Storage Following Treatment with Different Concentra­ tions of the Methyl Ester of Naphthaleneacetic Acid (Average of four heads). After Shipment Per Cent Fresh Days after Weight Loss Treat­ NA NA ment 100 mg. 200 mg • Check Before Shipment Per Cent Fresh Days Weight Loss after Treat­ NA* NA ment 100 mg. 200 mg. Check 16 1.1 1.5 2.8 10 2.9 0.2 2.4 31 44 61 80 6.6 10.2 13.8 16.6 7.4 9.8 4.4 93 18.7 19.9 16.3 21.6 25.9 28.7 0.3 0.6 2.3 4.8 3.6 11.3 15.0 17.6 19 38 59 95 113 * Wrapped in pliofilm. 8.3 11.6 15.8 19.4 7.7 7.5 11.1 15.9 20.4 - 20 Effect of Different Formulations of the Growth Regulating Substances Both the methyl ester and the sodium salt of both 2,4-D and NA were used to determine their effectiveness In retardlns abscission formation, inflorescence elongation, and leaf color change. Also, the sodium salt of 2,4-D mixed with a 50 per cent solution of Geon (X-31) was used. Five heads of locally purchased cauliflower were used for each treatment. Results and Discussion From the data in Table 4, it appears that both the methyl ester and the sodium salt of 2,4-D delayed the drop of 50 per cent of the leaves for longer than 100 days. On the other hand, the sodium salt of NA at the concentra­ tion used had little effect on leaf fall. Elongation of the cauliflower inflorescence was indueed by all treatments. There was no elongation of the inflorescence in the untreated cauliflower, even after all the leaves had fallen. Apparently, there was little rela­ tionship between the abscission formation and the elonga­ tion of the inflorescence• The methyl ester forms of either NA or 2,4-D m r e found to be more effective in inducing elongation of the inflorescence than their sodium salts, and 2,4-D was more 21 effective than NA. The color of the leaves was also Influ­ enced by the treatment. With either NA or 2,4-D, the sodium salt was more effective in lessening the chlorophyll eontent in the leaves than the methyl ester forms. The leaves of the untreated cauliflower did not lose color up to the time of leaf fall. The presence of chlorophyll seemed to have little relation to abscission formation. In both cabbage and cauliflower, after treatment with methyl ester of NA, tumor tissue was formed on the cute surface of the stem after a few weeks storage (Figure 2) • These tumor tissues were usually found on the exposed sur­ face near the cambium and pericyele regions, and occasion­ ally on the pith. Anatomically, the cells of this tumor tissue were particularly larger and the cell walls were weaker than that in normal tissue. This tissue originated from the reversion of the subepidermis of the exposed surface to a merlstematic state, from which the tumor tissue was differ­ entiated. However, it has also been observed that the re­ version of a secondary tissue into a merlstematic state could result from high humidity. - 22 TABLE IV, Effect of Different Formulations of Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid on the Color, Abscission, and Elongation of Inflorescence of Cauliflower. ' Treatment (amount per head) Color of Outer Leaves ME*of NA 100 mg. on paper Green SS**of NA 5 mg. on leaf Greenish yellow Days until -50% Leaf Drop > 100 55 Days for the Initiation of Elongation after Treatment 56 No elongation ME of 2,4-D 50 mg. on. paper Green >100 48 S3 of 2,4-D 5 mg. on leaf Yellow >100 95 SS of 2,4-D in Geon, 5 mg. on leaf Yellow >100 95 Check Green ♦ME, methyl ester **SS, Sodium Salt 40 No elongation 23 Figure 2. Tumor tissue on the cut surface of the stem of cabbage, three weeks after treatment with methyl ester of naphthaleneacetic acid; x ca. 100. - 24 Effect of Different Methods of Application This experiment was conducted to study the dire etion of transport of growth substances in cauliflower tissue. The sodium salts of either 2,4-D or NA at 500 p.p.m. to Geon mixture were used to retard the abscission formation. Five different treatments were set up as follows: 1) NA was applied on the cut ends of all the midribs, 2) NA on the cut end of the stem, 3) 2,4-D on the cut ends of all the midribs, 4) 2,4-D on the cut end of the stem, 5) 2,4-D applied only on the cut ends of the midribs on one side of a cauliflower head. Four heads were selected for each treatment, ahd the leaf fall was recorded at ten day intervals. Results and Discussion The Sodium salt of NA mixed with Geon, when applied V either on the cut ends of the midribs or the stems, was found to have little effect on the retardation of abscis­ sion formation. However, the application of 2,4-D at the same concentration and Tinder the same conditions was very effective. It is interesting that the retarding effect on the foliar abscission of cauliflower was obtained only when 2,4-D in Geon was applied on the cut ends of the midribs. 25 When it was applied on the cut ends of the stems, only the outer most whorl of the leaves was affected* It appears that the transport of 2,4-D in stored cauliflower tissue is polar, that is, its predominant move­ ment is downward* This conclusion differs from the obser­ vations of Hitchcock; and Zimmerman (1935) and Ferrl (1945) who reported that growth substances move both upward and downward in the transpiration stream, and is probably due to the difference in the physiological activity of Intact and excised plants. Not only did the 2,4-D move downward vertically, but it also moved horizontally in the stem* When the 2,4-D mixed with Geon was applied on the cut ends of the trimmed leaves on one side of a cauliflower, the abscission of the leaves on the other side were retarded (Figure 3)* 26 TABLE V . The Effect of Point of Application of Growth Regulating Substances on Leaf Fall of Stored Cauliflower (Average of five heads)• Per Cent Leaf Fall Days after Treatment Check NA on Cut End of Midrib NA on Cut 2,4-D on Cut End End of of Midrib Stem 2,4-D on Cut End of Stem 23 7.2 10.8 9.1 0 0 30 29.7 35.1 46.6 0 0 40 73.8 59.4 73.9 0 0 48 92.8 79.5 83.6 0 6.9 56 100 .0 89.2 90.9 0 20.4 69 — 100.0 100.0 0 53.0 82 — 0 64.1 99 — 0 79.5 — Figure 3. Effect of method of application and formulation of 2,4-dichlorophenoxy~ acetic acid on foliar abscission of cauliflower, 55 days after treatment. Left to right, 1) methyl ester of 2,4-D, 100 rag. per head; 2) sodium salt of 2,4-D in Geon solution, on cut end of stem only; 3) sodium salt of 2,4-D in Geon solution, on cut ends of midribs on only one side of a head(T); 4) check. 27 - - 28 Effect of Temperature Five heads of freshly harvested cabbage, Resistant Detroit, were treated with the methyl ester of NA (100 mg. per head), and five left untreated as check. The cabbage was then held at approximately 32° F. (cold storage), and 70° F. (room temperature). The number of dropped leaves was recorded every 48 hours until all the leaves had fallen. Results and Discussion The methyl ester of NA retarded the leaf drop at both room temperature (70° F.) and cold temperature (32° F.) (Table 6) • Since the higher temperature would increase the rate of transpiration, respiration, enzymic activity and probably many other metabolic functions, the higher tempera­ ture would be expected to accelerate the abscission formation. The data in Table 5# at room temperatures, indicates that the abscission of the untreated cabbage occurred within one week, while treatment with NA delayed the abscission from three to four weeks. Under low temperatures, the treated cabbage could be stored for two to three months without any leaf fall. Similar results were also obtained with cauli­ flower, however, the time required for abscission was much shorter than that in cabbage at both room and cold tempera­ tures • «. 29 *■» TABLE VI. Time Required for the Initiation of Abscission in Cabbage Stored at Various Temperatures (Average of five heads). Treatment Temperature (Sept .27,1947) 32°F - 70°F Days required for initiation of abscission HA, 100 mg. per head 63 Check 21 HA, 100 mg. per head 25 Check 6 « Difference in days between treated and check 42 19 «t 30 •* Effect of Trimming In order to study the effect of the leaf* blade on the formation of foliar abscission,“cauliflower (Snowball X) was used* There were four treatments: 1) leaves trimmed blade attached, 2) leaves untrlmmed blade attached* 3) leaves trimmed blade removed, 4) leaves untrimmed blade removed. Eaeh treatment comprised ten uniform heads treated with methyl ester of HA (100 mgs* per head), and ten con­ trols* The number of dropped leaves was recorded weekly. Results and Discussion Regardless of trimming treatment, the leaves of the untreated lots began to drop after two weeks storage. Treatment with HA delayed abscission in all tests* Leaf blades were found to have little effect on the leaf fall In both trimmed and untrimmed cauliflower* One hundred days after harvest, only ten per cent of the leaves in treated lots had dropped* In the untreat­ ed lots, all the leaves had completely fallen after 40 da# storage* Although LaRue (1916), Hitchcock and Zimmerman (1935), and. Myers (1940) considered that the growth hormone were synthesized in the leaf blade, in this study with 31 stored cauliflower the blade had little Inhibiting effect on abseission. Since the loss of weight is mainly due to the loss of water through the transpiration process, the larger the exposed surface area, the more water will be transpired* Consequently, the cauliflower with leaf blade attached lost weight faster than heads in which the blade had been re­ moved. Generally, the fresh weight loss was found to be higher in the treated, and somewhat lower in the untreated lots • 4 32 TABLE VII. ...... Treatment Check Treated NA 100 mg. per head * The Effect of Leaf Blade Tissue on Abscission Formation of Cauliflower (Expressed as the per cent of leaves that dropped per head)• Days after harvest Leaf trimmed Blade Blade attached removed Leaf untrimmed Blade attached Blade removed 18 4.4 1.0 8.9 10.0 25 38.2 25.7 41.0 50.0 33 67 *2 56.7 73.5 72.6 39 89.4 81.5 89.0 84.9 46 96.2 95.2 96.8 92.4 55 100.0 100.0 100.0 100.0 7.4 7.7 6.1 89 102 — 18.5 16.5 10.7 116* mm mm 27.7 38.4 20.4 The cauliflower was not markable after 116 days storage. TABLE VIII. The Influence of Methods of Trimming on Weight Loss of Cauliflower (Average of five heads}• Condition Treatment Original Weight per Head (gm.) Untrimmed Blade attached NA 100 mg. per head 1676 1216 27 .5 Check 1745 1151 34.0 NA 100 mg. per head 1277 1101 13.8 Check 1197 1034 13.6 NA 100 mg. per head 1756 1649 6.1 Check 1732 1636 5.5 NA 100 mg. per head 1413 1352 4.32 Check 1478 1415 4.2 Untrlmmed Blade removed Trimmed Blade Attached Trimmed Blade removed Weight per Head 31 Days after Harvest (gm.) per Cent L088 Of .Weight Figure foliar ment. 4. Effect of the methyl ester abscission of cauliflower with 5) treated, and 6) check. of naphthaleneacetic acid on retarding leaf blade removed, 45 days after treat­ 34 Figure 5* Effect of the methyl ester of naphthaleneacetic foliar abscission of cauliflower with leaf blade attached, treatment. 7) treated, and 8) check. acid on retarding 45 days after — 36 The Effect of Field Treatment with Growth Regulating Substances on Foliar Abscission Material and Methods In this experiment, cabbage, Wisconsin Hollander No. 8, and cauliflower, Snowball A, grown under ordinary cultural practices were used. Treatment of cabbage— The sodium salt of a-naphthaleneacetic acid and 2,4-dlehlorophenoxyacetic acid were used at three different concentrations: p.p.m. 100, 250 and 500 Eaeh plant was sprayed with approximately 10 ml. of the solution with a hand sprayer. In order to determine the most effective interval between application and harvest with respect to the retardation of abscission formation, two dates of harvest, one day and seven days after treat­ ment, were chosen. There were twelve treatments and two checks of five plants each arranged in randomized blo&s, with four replications. After harvest, the cabbage was stored at 34° F. and one hundred days after harvest all the heads were examined and the number and percentages of leaf fall were recorded. Treatment of cauliflower— As in the case of cabbage, the sodium salt of NA and 2,4-D in aqueous solu­ tion were applied in three different concentrations. 100, 250 and 500 p.p.m. In addition, the combination 37 of sodium salt of NA and 2,4-D (both in 250 p.p.m. and with 1:1 ratio) was used. An average of 15 ml. of each solution was sprayed on each of ten plants. Results and Discussion Cabbage— Treatment with the sodium salt of 2,4-D before harvest was found to have an effect in retarding the leaf fall of the stored produet. The 2,4-D treatments within the limit of concentrations used were more effec­ tive than the NA treatments • One hundred days after har­ vest, about one half of the leaves had fallen from the heads that were left untreated or treated with NA. On the other hand, those treated with 2,4-D on an average, had lost approximately only one leaf per head. From the data in Table 10, it appears that there were great differences in the retarding effect of the sodium salt of NA and 2,4-D on abscission formation. However, the methyl ester of both NA and 2,4-D, as found in previous experiments, had almost comparable effects. Cauliflower— The effects of growth regulating substances on foliar abscission of cauliflower were more pronounced than in cabbage. After 80 days in storage, 100 per cent of the leaves of the untreated lots had fallen. The sodium salt of 2,4-D was found to have a much greater retarding effect on foliar abscission than the sodium salt of NA. - 38 There was little difference between the untreated and those treated with NA in all concentrations used; fifty per cent of the leaves had fallen after fifty days storage. No difference was found between the effectiveness of the three concentrations used. From the standpoint of market­ ability, ordinary cauliflower could be stored for about two weeks, while those sprayed with sodium salt of 2,4-D might be stored up to 100 days without any leaf drop. The treat­ ment with the combination of 2,4-D and NA had the same effect as using 2,4-D alone. When sprayed with the sodium salt of 2,4-D at 100 p.p.m. one to seven days before harvest, cauliflower as well as cabbage can be stored without leaf fall for at least three times as long as untreated lots • Another ad­ vantage of field spraying which does not obtain with post harvest treatment is the uniform effect on the retardation of abscission in all the leaves. This uniform effeet is probably due to the transport of the growth regulating substances within the tissues of the growing plants. Leaves of growing cauliflower that had been sprayed on the upper surfaces with NA or 2,4-D were found to bend downward; that is, the chemicals induced a negative curva­ ture of the leaves. The degree of curvature was found to be more pronounced in those treated with 2,4-D than in those treated with NA, and also generally proportional • 39 — to the concentration of the growth substances used. At the same time, if the growth substances were sprayed on the lower side of the leaves, the leaves turned upward due to the same type of effect. FJrom an anatomical point of view, this effect was caused by an increase in the size of the epidermal and subfepidermal cells of the midribs and not to an Increase in cell number (Table IX) • The ratio between the diameters of the upper and lower epidermal cells was found to be higher in the treated midribs than in the untreated ones. In this experiment, the young undeveloped leaves were more sensitive to treatment than older leaves. It was probably due to the fact that their cells had not obtained the maxi­ mum size at the time of treatment. - 40 «•-. TABLE IX* ...... Treatment Cell Size in Midrib Tissue of Cauliflower, Taken One Week after Field Treatment with 2,4-Dichlorophenoxyacetic Acid at 250 p.p.m. (Average of five midribs). Average Length of Epidermal Cells Upper Lower Upper tu) Lower M Average Diameter of Subepidermal Cells* Upper Upper Lower Lower (a) On) Treated 23.1 21.1 1.09 34.7 39.5 0.88 Check 22.7 27.3 0.83 36.5 42.8 0.85 * The term, subepidermal cells, used here to indicate the five tiers of cells next to the epidermis; most of them are collenchyma cells. - 41 - Figure 6• Cauliflower (Snowball A) one week after treatment; left, 250 p.p.m. NA; right, 250 p.p.m. 2,4-D; the leaves were sprayed on the under surface and bend upward after treat­ ment • Figure 7. Cauliflower (Snowball A) one week after treatment; the 2,4-D, 250 p.p.m. was sprayed on the upper surface of the leaves which bend downward after treatment. - 42 TABLE X. Number of Fallen Leaves Per Head In Cabbage Following Field Spray Application with 2,4Dichlorophenoxyacetic Acid and Naphthaleneacetic Acid, After 106 Days Storage. (Each replication on average of five heads). Replication Treatment I II Average * III IV Harvested One Day after Treatment' NA,100 p.p.m. 18.0 20.1 18.2 21.2 19.3 NA,250 p.p.m. 20.6 20.2 21.6 16.4 19.7 NA,500 p.p.m. 25.6 21.4 19.4 21.2 21.9 2,4-D,100 p.p.m. 0 3.6 2.0 0 1.4 2,4-D,250 p.p.m# 0 2.8 0 0 0.7 2,4-D,500 p.p.m. 1.8 2.6 3.0 1.4 2.2 20.5 21.4 21.1 23.6 21.7 Check Harvest Seven Days after Treatment NA,100 p.p.m. 10.2 13.4 15.6 11.8 12.8 NA,250 p.p.m. 10.8 10.0 11.7 16.0 12.1 NA,500 p.p.m. 12.0 11.8 13.2 14.5 12 .9 2,4-D,100 p.p.m. 0 0 0 0 0 2,4-D,250 p.p.m. 0 1.2 0 0 0.3 2,4-D,500 p.p.m. 3.0 4.5 0 0 1.9 19.6 25.9 20.2 21.7 Check * 21.8 The total number of leaves per head is 47*6 ± 4.4, excluding the leaves which are shorter than three inches • -43- C^• PJ o © X! o PC cm CM The Effect of Field Treatment with Naphthaleneacetic Acid and 2,4-Diehlorophenoxyacetic Acid on the Per Gent of Leaf Fall of Cauliflower in Storage. (Average of five heads for each treatment). O *4 • ua u\ • ru\ UA • O cs- 00 • CM 00 NO • o CM CA . NO C'A O' . -4 UA NO • rH ON ■4 . ON £> o . o o rH o 1 1 • . o o rH 1 XI. •d © p © © p > 2> £ o 43 •x © P © rH xi © © > © 43 P © P P! © P 55 O H O •H1 P p o o P O O © lArl « ^1 Q © 1 43 -4 - P x *H CM © h O O UA CM • s N # P • P PC o O O o o O o P -p 1 P O % * O © a> a> iH NO .o oo CA On Xi © 3£ S3 Hft« Si rH UA © >)ONH c d. H P & *H © -4 CM -P P © Xi 43 3 e-* © n . OA -4 H TABLE 43 •s P •d p © © © % © © '©H ©© h, © © •o X i O- CM ». ■. rH nO (V H P P OO o o £ 0 £O TJ X) © © p p © m © © 1 t © © © _ H (M +J «© pc +- * Figure 8. Retarding effect of field treatment on foliar abscission 45 days after treatment. 9) sprayed with sodium salt of 2,4-D, 100 sodium salt of NA, 100 p.p.m.; 15) check. of cauliflower p.p.m.; 10) - 45 Anatomical Changes in Relation to Foliar Abscission Material and Methods The plant material for sectioning was taken from cabbage and cauliflower which had been treated with the methyl ester of NA,100 mg. per head, and from untreated lots • Samples were obtained from the second outer whorl of leaves of both cabbage and cauliflower. The tissues were first fixed in FAA (Formalin, acetic acid and alcohol) killing solution. Dehydration was carried out in an alco­ hol series, and finally the tissue was embedded in paraf­ fin. Sections were made with a microtone at 20 u thickness and stained with safralne 0 (aqueous solution) and light green (alcoholic solution)• Since the abscission formation varies greatly with the position of the stem on which the leaves are borne, the abscission structure of every fifth leaf taken from outer (older) to inner (younger) whorls was also studied, in order to find the relationship between the leaf order and the development of the abscission. Tissues for this purpose were prepared by free hand section. The length of the separation layer of the abscission zone was mea­ sured by a micrometer. Anatomy of the Abscission Zone The abscission of both cabbage and cauliflower leaves resulted from two processes, growth of a 46 «• meristematic layer, followed by dissolution of certain lamellae in the separation layer. However, in the upper half of the petiole, the meristematic layer was normally found preceding separation, whereas in the lower half of the petiole, especially in cauliflower, this was not the case • The meristematic layer was differentiated at a very early stage after harvest• Two weeks after storage five to ten layers of meristematic cells could be seen in a meristematic layer at the base of the petiole (Figures 10 and 12) • These cells were characterized by small size, well defined protoplasts and embryonic nature. In cabbage these cells were oblong in shape, arranged in parallel, and normally they were well developed, especially near the upper epidermis; in the petiole of cauliflower, on the other hand, the cells were isodiametric in shape and were not so well developed. In both cabbage and cauliflower, the differentia­ tion of the meristematic layer generally was initiated near the upper epidermis of the petiole and became less well developed toward the lower epidermis • This was followed by the formation of the separation layer, which also began from the upper epidermis and progressed toward the lower epidermis. However, the separation layer in the lower side started to form after the separation layer in the upper side had developed about half way across the petiole, even though the meristematic layer was lacking in the lower side (Figure 9). From an anatomical point of view, the difference in foliar abscission between cabbage and cauliflower was found to be the position of the separation layer in rela­ tion to the meristematic layer. In cabbage, the separation layer always occurred five to ten tiers of parenchymatous cells apart from the outer side of the meristematic layer. In other words, it was formed from the secondary meristem (Figures 9 and 11). On the other hand, the separation layer in the petiole of cauliflower always occurred within the meristematic layer itself; that is, it developed from one or two tiers of the meristematic layer or it was formed from the primary meristem (Figures 9, 13 and 14) . The separation layer of cabbage and cauliflower was not formed smoothly across the petiole. The direction of this layer was sometimes modified by the vascular bundles or other mechanical elements and left a sunken leaf scar after leaf fall. After the formation of the separation layer was complete, the whole leaf was supported only by the vascular bundles which were then ruptured mechanically (Figures 14 and 15). So the separation of this tissue was not the result of the dissolution of the middle lamellae or a physiological disintegration but was due to a mechani­ cal force. 48 After the falling of the leaf, a protective layer wae differentiated on the exposed surface of the leaf scar* In the case of cabbage, the cells of the meristematic layer could still be seen near the exposed surface after leaf fit11 (Figure 16). Delay of abscission formation by growth regulating substances may be due to retardation of either the differ­ entiation of the meristematic layer or the weakening of cell walls in the separation layer. The meristematic layer was apparently more clearly defined and well developed in leaf petioles from treated heads than in the petioles from untreated heads* Leaf Order in Relation to Abscission Formation Under natural conditions, in both cabbage and cauliflower, the outer (older) leaves drop first and, sub­ sequently the inner (younger) leaves fall* In many in­ stances, the abscission of the outer leaves had been com­ pletely formed and the leaves had dropped, while the inner leaves were still attached* In mature cauliflower with few leaves (usually less than 30) and no very young leaves, the abscission was found even in the innermost leaves after one and a half months of storage* However, in cabbage, which contained many whorls of younger leaves, usually abscission of the outer twenty leaves occurred after three months of storage, and the inner leaves remained intact for a considerably longer length of time* - 49 The above relation between leaf order and abscission was drastically altered by post harvest treatment with grow­ th regulating substances, With treatment, the leaves of the Intermediate whorls in the case of cabbage, and of the inner­ most whorls in the case of cauliflower, dropped first and the outer whorls last. This inverted relationship possibly is both of theoretical interest and of practical importance. This phenomenon may be caused by the lack of an appreciable upward transport of the growth regulating substances in the plant tissues. Treatment with 2,4-D only on the cut surface of the stem, which did not delay the abscission formation of the inner younger leaves, indicated that this is probably the case. With treated cabbage, the abscission of the inter­ mediate whorls of leaves were always formed first. Treat­ ment with the growth regulating substances could only delay the abscission in outer leaves which came in contact with the chemicals, but apparently did not affect the inner younger leaves. From the data in Table XII, the "percent- age of separation"* of the outer five leaves of the un­ treated cabbage heads was much higher than that of the 25th leaf. On the other hand, in the treated heads, the The term, "percentage of separation”, is used here to Indicate the depth of the separation layer of the ab­ scission zone of the petiole (in longisection) in re­ lation to the thickness of the petiole; i.e., _ .. Depth of the separation layer Y 100 Percentage of separation • m ITT x Total thickness of the petiole — 50 "percentage of separation" of the 15th leaf was higher than both the first and the 25th leaves* The time required for abscission of the 30th leaf was approximately the same in both treated and untreated lots. In treated cauliflower, the relationship between the abscission formation and leaf order was basically the same as that in cabbage. The outer five leaves of a trested cauliflower, 67 days after harvest, showed no separation on the abscission zone, while the leaves in the 20th position indicated 80 per cent separation. It was also observed that with the pre-harvest field treatment with 2,4-D, there was no abscission to be noted during a storage period of more than 100 days. This was probably due to a more ■uniform and rapid transport of the applied chemicals into every leaf of growing plants in com­ parison with stored plants. 51 - ft**!*ainGSwSSSv? (A ax E d ax o *\ *>-P •H r—I ax d a> 01 *. <0 ? ax > o d rH -P Cm 0) ax c5 •H cO HI d c o CtJ rt ♦H cx O 3 ax •H XX ax c -P d •rl•H O cO ax -P ax X> (0 cS V ’£ ox d •*» ax cO CO "d 6 > •rl r-1 * »v ♦> O o x> Cm > •» S • # s «H X> ox ax O § *H 4-> p E - 1 1 *vax a> rC ■a a> *H • rl bO ft ft cO ax ax d d o OX a 0) I o a S', -P iH d «oH ft ft <1) ax o> 0) d a Iu o >» d bOH ) c d> » H < e ) a Xd l d« a o o o bO-p ***rt 0) «ax • c fl d d flen $ft O &H -chP ) • ox ft 0> 0• )0 «H - 52 - Figure 10. Foliar abscission of cabbage, showing the meristematic layer near the upper side of the midrib; x ca. 100. Figure 11. Foliar abscission of cabbage, showing the separation layer; x ca. 100. V ■\ *x >-'V/ '“'N v , sjS.• •‘V / 5 . ; • ' ' ■ ■•gy: f.a ■••*,*. *"3' **Tj-,p^v /*.• \ r* v iv*-1 Figure 12• Foliar abscission of cauliflower, •bowing the merle ter sialic layer near the upper •id© of the midrib; * oo* 100* Figure 15* Foliar abscission of cauliflower, •bowing the separation layer; x ca* 100* - 5* - Figure 1A. Foliar abscission of cauliflower, showing the separation layer near a vascular bundle; x ca. 100. Figure 15. Foliar abscission of cauliflower, showing the breaking down of a vascular bundle; x ca. 100. - 55 - Figure 16. Foliar abscission of cabbage, showing the protective layer on the exposed surface of the leaf scar after abscission formation. 56 TABLE XIX. Leaf Order in Relation to Percentage of Separa­ tion of Leaf Abscission in Cabbage, 80 days after Treatment (Average of five heads). Leaf Order Treated with ME^ of NA, 100 mg./head Check 1 (per cent) 16.9 (per cent) 100.0 5 45.9 100.0 10 46.7 83.2 15 65.9 66.4 20 22.1 18.7 25 10.5 5.2 3° 0.0 0.0 TABLE XIII. Leaf Order Leaf Order in Relation to Percentage of Separation of Leaf Abscission in Cauli­ flower, 67 Days after Storage for Treated, and 32 Days after Storage for Check (Average of five heads). Treated with ME of NA, 100 mg./head Check (per cent) (per cent) 1 0.0 100.0 5 0.0 82.5 10 4.0 37.3 15 63.2 26.8 20 77.3 12.3 25 — 0.0 - 5 7r- 100 TREATED 80 CHECK a. ui 4 0 20 10 20 30 40 50 LEAF ORDER Figure 17. Leaf order in relation to percentage of separation of leaf abscission in cabbage, 80 • days after treatment with methyl ester of naphthaleneacetic acid* 100 80 B 60 ui 4 0 ft. 20 20 25 LEAF ORDER Figure 18. Leaf order in relation to percentage of separation of leaf abscission in cauliflower, treated with methyl ester of naphthaleneacetic acid. a 59 Biochemical Changes in Relation to Foliar Abscission Sugars Material and Methods Cauliflower plants, variety Snowball X, were harvest­ ed on Oct. 28, 1947, and placed in cold storage immediately* Forty heads of uniform size and good quality were selected. Twenty of them were treated on Oct. 29 with 100 mg. of the methyl ester of NA. The other twenty heads served as con­ trols • Analyses of total sugars, reducing sugar, and dry matter were made at weekly Intervals from Nov. 3 to Dec. 22, 1947* at which time nearly all the leaves of the untreated lots bad fallen. The method of sugar analysis followed was the one that is used in the Agricultural Chemistry Labora­ tory, Michigan Agriculture Experiment Station.# The proce­ dure is as follows: Each cauliflower sample was separated into three portions, the ”head"**, the leaf blade, and the midrib. Twenty grams samples of each portion were weighed out for * *« Mrs. Dorothy R. Waldron assisted in carrying out the analysis• The word "head” is used here, for the convenience of description, to indicate the edible part of the cauli­ flower from which the inflorescence develops; and the compact edible radical leaves as a whole of cabbage. It is not a morphological term, however. - 59 each, determination and then transferred to a blender re­ ceptacle to whieh 0.5 gram of GaCOj and 100 ml. of 95 per cent ethy 1 alcohol were added • The blender was operated for three minutes. The blended material was then filtered and made up to 200 ml. with 80 per cent alcohol, in a volu­ metric flask. Fifty ml. aliquots were transferred to beakers and condensed to a small volume on a steam bath. Lead acetate solution was added to the evaporated material, which after precipitation was filtered and the excess lead was removed with potassium oxalate. The filtrate was made up to 50 ml.; a 25 ml. aliquot was measured for the estima­ tion of reducing sugar, the other 25 ml. were hydrolysed with HC1 for the determination of total sugars • Reducing and total sugars were determined by the precipitation of cuprous oxide according to the official A.0 .A.C* method (1940). Resultsuand Discussion The amount of both reducing and total sugars con­ tent varied not only with the length of the storage period, but also with the different tissues (Table 15 and Figure 20). In the "head" tissue, total sugars were much higher than reducing sugar in both the treated and untreated cauli­ flower. The ”heads” which had been treated with the methyl ester of NA contained less sugars than the untreated ones. * The formation of foliar abscission had little influence on — 60 «• the change of sugars, especially the total sugars. Fifty five days after harvest, the reducing sugar content of the untreated heads was over twice as high as that found in the treated heads and total sugars were about 20 per cent higher. The decrease in the sugar content in leaf blade tis­ sue was most rapid in the early stage of storage just after harvest and before abscission formation. The difference be­ tween total and reducing sugars in the leaf blade was much less than that in the “head” tissue, indicating the presence of very little invert sugar* After three weeks storage, 50 per eent of the leaves had dropped in the untreated lots, whereas no leaf fall occurred in the treated lots. The leaf blade of both treated and untreated cauliflower at that time contained less than 0.2 per cent of either total or reducing sugars. The sugar content in the midrib was more closely related to abscission formation. The difference between total and reducing sugars was quite uniform throughout the stored period. Treatment with NA retarded the loss of both total and reducing sugars, especially after 50 per cent of the leaves of the untreated lots had fallen. When all the leaves of both treated and untreated heads had fallen, there was only a slight difference in sugar content between the treated and the untreated lots. 61 During storage, sugars decreased more rapidly in the midrib and blade tissues than in the "head* tissue* Treatment with the methyl ester of NA retarded the loss of sugars in the midrib for a time, and finally had little effect • 62 «* TABLE XIV • Leaf Drop in Cauliflower used for Sugars and Dry Matter Analyses (Average of five heads)• Date Days after Harvest Per Cent Leaf Drop Untreated Treated (NA, 100 mg ./head) Nov. 18 21 10.0 0 Nov. 24 2? 22.6 0 Dee. 1 34 32.1 0 Dee • 8 41 71.8 0 Dec. 15 48 92.6 0 Dec • 22 55 100.0 8.5 Jan. 4 68 — 31.4 Jan. 17 81 77.3 Jan. 24 88 96.9 Jan. 31 95 — 100.0 - 63 TABLE XV • The Effect of Methyl Ester of Naphthaleneacetic Acid on the Sugar Content of Stored Cauliflower. (Expressed on fresh weight basis, average of duplicate determinations). Date Sampled "Head” Midrib Treated Check Leaf Blade Treated Check Treated Cheek Reducing Sugar(% ) Nob. 3 1.13 1.53 2.74 2.94 1.14 1.39 Nov. 12 1.06 1.34 2.10 1.76 0.43 0.52 Nov. 19 1.03 1.18 1.31 1.12 0.01 0.01 Nov. 26 0.91 1.14 1.75 0.54 0.09 0.01 Dec. 10 0 .6? 1.42 0.94 0.65 0.25 0.23 Dec. 22 0.58 1.43 0.55 0.72 — — Total Sugar (%) Nov. 3 3.04 2.58 3.22 3.36 1.52 1.49 Nov. 12 2.29 2.64 2.25 1.94 0.52 0.59 Nov. 19 2.30 2.45 1.42 1.28 0.08 0.08 Nov. 26 2.33 2.62 1.92 0.64 0.41 0.06 Dec. 10 2.30 2.71 1.11 0.73 0.32 0.30 Dec. 22 2.32 2.76 0.62 .86 ammm » 64 -T O T A L SUGAR.TREATED LF. BLADE 1-6 • »> >’ .CHECK ‘ REDUCI NG S U G A R . T R E A T E D .CHECK 3-6r midrib 3.2 Id 24 3.2 '"head 2.8 2.4 2.0 16 12 22 26 NOV. DEC- Figure 19i Effect of treatment with methyl ester of naphthaleneacetic acid on the sugar content of cauliflower in storage. The vertical line bisects the graph at the time when the untreated plants had dropped 10% of their leaves, whereas the treated lots had dropped none. Dry Matter Per cent dry matter was determined on ten grams samples from the same cauliflower tissues used in the sugar analysis. The samples were oven dried at 100° 0. for 24 hours, and the results were expressed on the fresh weightbasis. Results and Discussion The dry matter in both leaf blade and midrib was somewhat higher in the untreated cauliflower than in the treated lots, and was only slightly different in the "head” tissue (Table 16). It varied more in the leaf blade than in the other two tissues, especially after the formation of abscission. Probably the increase in dry matter result­ ing from storage that was found in both treated and un­ treated leaf blade tissue was due to the loss of water through transpiration, while the slight decrease in dry matter in both "head” and midrib tissues was due to a de­ crease in sugar from respiration. With the formation of abscission, the dry matter content of the leaf blade increased, whereas there was no appreciable effect on the "head" tissue. - 66 TABLE XVI • The Effect of Methyl Ester of Naphthaleneacetic Acid on the Dry Matter Content of Stored Cauliflower (Expressed on fresh weight basis, average of duplicate deter­ minations ). Date Sampled .. . "Head" Treated Leaf Blade Midrib Check Vo V. Treated Cheek sr % 8.48 13.65 13.07 7.23 12.01 13.60 8.39 9 .20 Nov. 12 9.02 9 6.91 Nov. 19 8.84 8 .45 6.66 6.88 13.06 33 .53 Nov. 26 8.81 8 .45 6.31 6.79 14.89 16.17 Dec • 10 9.15 8 .77 6.33 7.19 14.59 16.45 Dec. 22 7.26 7 .90 5.11 6.47 e Nov, 3 CM Check o Treated % 9.27 — -- 67 -j 16 ■leaf blade C* UJ CHECK. TREATED; 26' 3 Nov. / 10 22 DEC. Figure 20. Effect of treatment with methyl ester of naphthaleneacetic acid on the dry matter content of cauliflower in storage. Vertical line indicates the time when the untreated plants had dropped 10% of their leaves, whereas the treated lots had dropped none. - 68 - Ascorbic Acid Material and Methods Forty heads of cauliflower were selected on Jan. 16; twenty were treated with methyl ester of NA in order to retard the abscission formation, and the other twenty served as controls . Analyses were made at weekly intervals from Jan. 27 until practically all the leaves had fallen in the untreated lots on Feb. 26. The method of analysis used by Lucas (1944) was followed. Three different sections, the "head", the leaf blade, and the midrib, of the cauliflower were sampled. The tissues were collected from four heads for each deter­ mination. 200 ml. of 2 % metaphosphoric acid were placed in blender container with 40 gr. of tissue. The blender was operated for 3 minutes. The extractant was first fil­ tered through cheese cloth into a suction flask and then transferred to a centrifuge tube and centrifuger for 10 minutes. The supernatant aliquot was used for titratbon. Two m l . of the extractant were titrated with a 0.02 per cent solution of sodium 2,6-dichlorobenzenoneindophenol to a faint pink end point. Results and Discussion The ascorbic acid content was found to be the highest in the. leaf blade, low in the "head", and still lower In the midrib (Table 17) • After the first month of storage the ascorbic acid content declined in all tissues • The rate of loss in the ascorbic acid was lower in both "head" and midrib tissues than in the leaf blade. On Feb# 26, after 51 days in storage, both "head" and midrib tissues contained about 40 mg# per 100 grams of fresh weight, indicating a loss of two thirds of the ascorbic acid in the "head", and about one half in the midrib. There was no appreciable difference between treated and un­ treated tissues of head and midrib# However, in the leaf blade, the loss of ascorbic acid occurred early in the storage period, with the untreated lots showing the greater loss# Since the treatment with NA retarded the foliar ab­ scission, the leaf blade of the treated heads lost less ascorbic acid than that of the untreated cauliflower (Table 17). - 70 TABLES XVII. The Effect of Methyl Ester of Naphthaleneacetic Acid on the Ascorbic Acid Content of Stored Cauliflower (Average of duplicate determinations)• Ascorbic Acid, mgs. per 100 gms. Fresh Weight Date Sampled "Head*' Midrib Leaf Blade Treated Check Treated Check Treated Check Jan. 27 115*8 106.1 70.8 68.4 159.6 158.6 Feb. 3 113.7 111.1 70.7 67.2 147.3 124.8 Feb. 12 100.4 107.4 72.8 67.8 143.6 129.8 Feb. 19 81.6 78.3 69.7 62.7 105.6 96.0 Feb. 26 46.2 42.0 41.7 37.8 101.7 69.6 71 Respiration Material and Methods Two uniform cauliflower heads, one treated with methyl ester of NA (100 mg. per head), and the other left untreated were placed into two desiccators which were set up as respiratory chambers, at a temperature of approximately 70° F • The respiratory rate was determined by the method of Haller and Rose (1932) before and after treatment at varying intervals • The sealed desiccators which served as respira­ tory chambers, were connected by tubing to two closed litergraduated cylinders containing Og • The CO2 given off by the respiration of the cauliflower heads was absorbed by KQH solution, and the Og used was replaced by the Og in the cylinder. At the end of a run, 24 hours after operation, the KOH solution in the funnel on the bottom of the desicca­ tors was drained into a flask for titration. then added again for another determination. Fresh KOH was The KOH was titrated against 2N H^SO^. to the phenothalein end point and then to the methyl orange end point and the amount of CO2 computed from the difference between the two end points. In one experiment, two smaller heads (average 790 grams per head) were used, while in a second experiment, two larger heads (average 1500 grams per head) were used to compare the difference of respiratory activity between various sizes of heads as well as the effect of the methyl ester of NA. 72 Results and Discussion The rate of respiration of cauliflower was found to be slightly increased by the treatment with methyl ester of HA* Ten days after completion of the respiration deter­ mination, all the leaves of the untreated heads dropped, whereas the leaves of the treated heads remained intact. The fluctuation of the respiratory rate during the course of this experiment was considered to be more closely related to environmental temperature change rather than the effect of treatment • The smaller cauliflower heads bad higher rate of respiration per unit of weight than the larger heads* The heads used in experiment I were about one half the weight of those used In experiment II, whereas the rate of respiration of the heads in experiment I was about twice as that found in experiment II* TABLE XYIII • The Effect of Methyl Ester of Naphthaleneacetic Acid on the Rate of Respiration of Cauliflower. Experiment I Days after Treatment Experiment II Mg. CO2 per Kg. Days after Plant Meterial Treatment per Hour Mg. CO2 per Kg. Plant Material per Hour Treated Treated Check Check 1 58.5 52.2 1 88.3 90.6 2 54.9 44.2 2 102.1 108.9 5 41.5 41.9 10 75.5 68.2 7 43.9 32.5 12 105.9 90.2 10 42.3 37.4 15 73.3 65.3 100 EXP.I . 80 u . UJ K 2 released at various time intervals). Oxygen Released in ml e 40 60 100 120 240 300 ISO sec. see. sec • 806 sec • sec • sec. - 10 20 see.* see. • HHeadM Tissue Jan. 27 .60 .75 1.30 1.75 2.55 2.90 3.90 4.65 5.25 Feb. 3 .55 .75 1.25 1.70 2.50 2.85 3.80 4.50 5.10 Feb • 12 .55 .80 1.30 1.70 2.50 2.85 3.70 4.35 4.90 Feb. 19 .50 .70 1.2 1.65 2.40 2.65 3.65 4.30 4.80 Feb. 26 .50 .70 1.15 1.60 2.4 2.75 3.6 4.30 4.85 Midrib Tissue Jan. 27 .60 .8 1.15 1.35 1.75 1.85 1.90 2.10 2.10 Feb. 3 .40 .6 .90 1.20 1.55 1.65 1.75 1.85 1.85 Feb. 19 .3 .5 .75 1.0 1.25 1.45 1.55 1.6 1.6 Feb • 12 .45 .65 .95 1.15 1.4 1.50 1.55 1.6 1.65 Feb. 26 •2 .3 .45 .7 1.0 1.1 1.25 1.35 1.4 Leaf Blade Tissue 1.0 1.8 2.6 3.6 4.4 4.6 5.4 5.6 5.8 Feb. 3 .9 1.4 2.2 2.6 3.4 3.6 4.0 4.2 4.4 Feb • 12 .8 1.2 1.6 2.0 2.4 2.6 2.8 3.0 3.0 Feb • 19 .4 .7 1.0 1.4 2.0 2.3 2.6 2.8 2.8 Feb. 26 .4 .7 1.0 1.2 1.4 1.6 1.8 1.8 1.8 Jan. 27 # Sec• — Seconds• - 82 TABLE! xx(b) Date Sampled The Catalase Activity of Untreated Cauliflower During Storage (Expressed as amount of O2 re­ leased at various time Intervals) • 10 20 sec.* sec. :_-:' ------Oxygen Released in ml 40 60 100 120 300 180 240 sec • sec. sec • sec • sec • sec • see. “Head” Tissue Jan* 27 •6 •8 1.2 1.7 2.4 2.8 3.8 4.5 5.2 Feb. 3 .6 •8 1.2 1*7 2.6 2.9 3.9 4.6 5.1 Feb. 12 .6 *9 1.3 1*7 2.5 3.0 3.85 4.6 5.1 Feb. 19 .4 .6 1.1 1*3 2 *5 2.7 3.65 4.3 4.8 Feb. 26 *5 .7 1.2 1.6 2.55 2.8 3.75 4.3 4.7 Midrib Tissue Jan. 27 .6 •8 1.1 1.4 1.9 1.9 2.1 2.2 2.2 Feb. 3 .4 .6 1.0 1.3 1.6 1.7 1.9 2.0 2.0 Feb. 12 .4 .6 .8 1.2 1.5 1.6 1.7 1.8 1.8 Feb. 19 .2 .4 .6 .9 1.2 1.3 1.5 1.6 1.6 Feb. 26 .2 .3 .5 .7 1.0 1.0 1.15 1.2 1.2 Leaf Blade Tissue 1.4 2.0 3.6 4.2 5.2 5.6 6.2 6.6 6.6 Feb. 3 .8 1.2 2.0 2.6 3.4 3.6 4.1 4.4 4.5 Feb. 12 .6 1.1 1.6 1.8 2.4 2.6 2.9 3.1 3.2 Feb. 19 .4 .6 1.0 1.4 1.8 2.0 2.3 2.5 2.7 Feb. 26 .4 .6 1.0 1.3 1.5 • 1.8 1.9 2.0 2.2 Jan. 27 * sec• — Seconds• -83 - TABLE XXZ • The Effect of Leaf Order on the Catalase Ac­ tivity of Untreated Cauliflower (Expressed as amount of ©2 released at various time intervals). Lot Leaf No. Order Oxygen Released in ml 10 20 40 60 100 180 120 300 240 sec • sec • sea * sec sec • sec. sec • sec. sec. 0 Leaf Blade Tissue* I II 1-5 .9 1.4 2.1 2.7 3.4 4.0 4.9 5.5 6.1 6-10 .8 1.3 1.9 2.4 3.1 3.6 4.4 4.8 5.3 11-15 .6 1.0 1.6 2.1 2.5 2.8 3.2 3*5 3.7 16-20 .6 1.0 1.5 1.8 2.3 2.5 3.0 3.2 3.5 21-25 1-5 .6 1.6 1.4 2.0 .5 1.1 .8 1.7 2.2 2.2 2.6 2.6 2.7 3.3 2.8 3.8 2.9 4.2 6-10 .5 ..8 1.2 1.6 2.1 2.4 3.0 3.5 3.9 11-15 .4 .7 1.1 1.4 1.7 2.0 2.4 2.7 3.0 16-20 .5 1.1 1.3 1.6 1.8 2.2 2.4 2.6 21-25 .4 1.0 1.2 1.4 1.6 1.9 2.1 2.2 .7 .6 Midrib Tissue** 1-5 .3 .4 .6 .8 1.1 1.2 1.4 1.4 1.5 6-10 .3 .4 .7 .9 1.2 1.3 1.4 1.5 1.5 11-15 .3 .5 .9 1.1 1.3 1.4 1.5 1.6 1.6 16-20 .4 .6 1.0 1.2 1.4 1.5 1.6 1.7 1.7 21-25 .4 .6 1.0 1.2 1.4 1.5 1.6 1.7 1.7 1-5 .3 .4 .7 .8 1.0 1.1 1.3 1.4 144 6-10 .3 .5 .7 .8 1.0 1.1 1.2 1.3 1.4 11-15 .3 .5 .7 .8 1.0 1.1 1.2 1.3 1.3 16-20 .3 .4 .6 .7 .9 1.0 1.1 1.2 1.3 21-25 .3 .4 .5 .6 .7 .8 1.0 1.1 1-2 I II * ** 15 ©as. of plant material in 100 ml. water. 40 gms• of plant material in 100 ml• water• 3 TREATED CHECK ,J A N . 2 7 FEB.3 2 26 M ID R I B 0 TR EAT ED JA N. 2 7 CHECK CATALASE A C T I V I T Y , ML. 04 R E L E A S D 6 5 -FEB. 3 4 > 3 F E B . 12 ^ F E B .1 9 2 F E B .2 6 lea f blade o -J AN .2 7 FEB.3 FEB. 12 ■FEB. 19 'FEB-26 TREATED 5 CHECK 4 3 2 HE AD 50 100 150 TIM E 200 250 300 IN SE CO ND Figure 23. Effect of methyl ester of naphthaleneacetic acid on the catalase activity of stored cauliflower. ML. Ox RELEASED 85 so 100 ISO soo TIME IN SECONO eso 300* Figure 24. The relation between catalase activity and the leaf order of untreated cauliflower leaf blades, (Expressed in ml, of Og released per second). -86DISCUSSION The Role of Plant Hormone in Relation to Abscission Formation Leaf fall has been generally recognized as being associated with a lack of sufficient growth hormone to inhibit abscission formation. Application of synthetic growth regulating substances augment or substitute for the dwindling supply of phytohormones that exist in the plant tissues. Since the phytohormone Is a product of synthesis through physiological activity, any external condition or treatment that would alter physiological activity might in­ fluence the effectiveness of the applied growth substances. Maturity of the plant, the amount of the leaf blade attach­ ed to the midrib, temperature and humidity of the storage are possible external factors which be practically important. The hormone content of different leaves is not the same. As found by Avery (1935) in Nicotlana leaves, the concentration of the hormone Is roughly inversely propor­ tional to the age of the leaf. In Morus alba. Okabe (1940a), using the Avena method to test the effective­ ness of the growth substance, found that negative curva­ tures result from treatment of young leaves, and posi­ tive curvatures developed from treatment of mature leaves. In the rosette leaves of Solidago, Goodwin (1937) reported that the total amount of auxin diffusing a leaf is very small in the early stage of development, but in­ creases to a maximum, which is almost coincident with the most rapid growth period of the leaf, and then falls off with approaching maturity. Under natural conditions, therefore, older leaves always drop first, and the younger leaves later. This phenomenon Is very significant from a practical point of view. In both cabbage and cauli­ flower, the leaves of the older mature heads, if other conditions are equal, drop earlier than the younger im­ mature leaves. Apparently, the application of growth regulating substances is more effective for the older leaves in which the auxin content is lower than for the younger leaves in whleh the auxin content is higher. Nearly all positional differences in effective­ ness resulting from the application of growth substances, as reported by Hitchcock and Zimmerman (1941) in apples as well as the results of the present study, are caused by or related to the degree of maturity of the plant organ. Since the growth hormone is believed to be syn­ thesized in the leaf blade, and probably is associated with photosyntheses, deblading should hasten leaf drop­ ping. This was true in the case of Intact plants, but -88was not true in the case of excised plants, such as cabbage and cauliflower in storage. The results of the trimming experiment indicate little effect of the leaf blade on leaf falling. This conclusion is probably due to the dif­ ference of metabolic activity between the growing plants and the stored crop. Under cold storage in the absence of light, the leaf blades are probably unable to synthesize the growth hormone so abscission could not be affected by the leaf blades. To a certain extent, the length of the midrib is probably not a major factor in connection with the abscis­ sion formation. With respect to abscission, the role of the trimmed midrib (reduced about one half), was the same as that of the untrimmed one. But observations have also been made that if the midrib was cut too short (less than one inch in length), the leaf would fall much earlier, even in those treated with a growth regulating substance. This observation fonfirms the assumption of Gardner and Cooper (1943) that the action of the growth substances depends on or is conditioned by the presence of a neces­ sary second substance or substances already present in the plant tissue• -89The Transport of Growth Regulating Substance in Relation to Abscission Formation As a result of the present study, the transport or movement of the growth regulating substances in the stored cauliflower is polar, that is, from the tip toward the base in the leaf as well as in stem. Not only can the growth regulating substances move downward vertically, but they also move horizontally In the stem. This phenomenon had been discussed by many investigators. Avery (1935) studied the movement of auxin in the Nicotiana leaf and found that it moves from the tip toward the base of the leaf, and from the lateral vein into the midrib. This polar auxin transport in plant tissues, according to Glark (1938), is not caused by the electrical polarity but by entirely different mechanisms, as shown by the different effects of light and gravity. In Morus alba, Thea sinensis, Ginkgo biloba, amd many other asiatic woody plants, Okabe (1940a) found that movement of growth-promoting substances in the petioles seems to be basipetally while that of the growth inhibit­ ing substances has no polarity of movement. But according to the results obtained by Hitchcock and Zimmerman (1935) and Ferri (1945) the synthetic growth suhstances can be absorbed from the soil by plants and transported upward, as are mineral salts. -90The apparent divergence of these results is pro­ bably due to differences in the physiological activity of intact and excised plants and of natural and synthetic growth substances. It is general known that natural growth hormones move or diffuse normally in a polar manner, whereas the synthetic growth substances may be transported upward in the growing plants • But this upward movement did not occur in the case of stored plants, such as the cauliflower used in this study. Since the upward movement of the applied growth sub­ stances was affected by conditions influencing the rate of transpiration, Hitchcock and Zimmerman (1935) claimed that the movement occurred in the transpiration stream. If, as in the stored cauliflower and cabbage, the transpiration rate decreased, the movement of the applied growth substance would naturally be slowed down, so that no retarding ef­ fect on the abscission would be found when a growth sub­ stance, such as 2,4-dichlorophenoxyacetic acid, was ap­ plied only on the cut end of the stem. But It did not prove that the downward movement also occurred in the tran­ spiration stream. The retarding effect of the growth substance which was applied only on the cut end of the midrib did not necessarily mean that the growth substance itself moved in a polar manner. Probably, the retardation M 91 ** of the abscission formation is a secondary effect of the growth substance used, and was not caused by the direct contact with the abscission layer* The polar movement and local effect of the growth regulating substance on the stored cabbage and cauliflower are probably the main reason for the Minverted relationship1' between leaf order and the development of the abscis­ sion here discovered. Under natural conditions, the formation of foliar abscission of untreated plants is mainly controlled by the maturity of the leaves. The older the leaves, the earlier the abscission will be formed; in other words, the degree of abscission is primarily governed by one factor, the gradient of maturity. The foliar abscission of the heads treated post harvest, however, is probably governed by two factors, the maturity of the leaves and the effectiveness of the growth regulating substance. Based on this theoretical consideration, the rela­ tionship between the abscission formation and leaf order can be illustrated by the diagrams in Figures 25 and 26. In the case of cauliflower, in which there are no very young leaves at the time of harvest, the percentage of separation is directly proportional to the increase of the leaf order on the post harvest treated heads, and in- " 92 ■ versely proportional to the increase of the leaf order on the untreated heads• If, for example, the outer leaves of a cauliflower, which had been treated with NA after harvest, show 40 per cent of separation. It ean be expected that the innermost leaves might have completely abscissed. On the other hand, in the untreated cauliflower, the innermost leaves might have no abscission at all. The differences between the relationships occuring in treated and untreated cauliflower were not evident at either the early stage or the final stage of the storage life (see Figures 25 and 26). In the case of cabbage, in which there are many young immature leaves at the time of harvest, the percent­ age of separation of the abscission on the untreated heads, as in cauliflower, is also inversely proportional to the increase of the leaf order. On the post-harvest treated cabbage, however, the intermediate leaves always have higher percentage of separation than the outer and inner leaves. In this work post-harvest treatment with the growth regulating substance only retard the abseisslon formation of the 20 outermost leaves. There was little difference between the treated and untreated cabbage in the percentage of separation of the inner young leaves. From the diagram for cabbage, Figure 25, the maximum point of the separation curve at a given time is - 93 loo oc 60 Cl uj z . ui 40 o 20 20 30 40 50 L E A F ORDER Figure 25* Diagram illustrating the theoretical rela­ tionship between the change of leaf order and the change of percentage of separation in cabbage, cf. figure 17. treated, check. 100 80 a. u 40 20 20 25 LEAF ORDER Figure 26. Diagram illustrating the theoretical rela­ tionship between the change of leaf order and the change of percentage of separation in cauliflowerm cf* figure 18, --- treated,------ check. taken as the intersection of the theoretical eurve of maturity at which the foliar abscission will naturally be formed, and the eurve of the effectiveness of the growth regulating substance* As time advances, the curves move upward until all the leaves fall* It must be remembered, however, that the time required for a given percentage of separation of the outer leaves of the treated cauliflower or cabbage is much longer than that for the untreated heads• Since these relationships of abscission formation and leaf order in either cabbage or cauliflower are not observed in pre-harvest treated plants, it is probably that the relationship is due to the slow and polar trans­ port of the applied growth substances in the plant tissue. Biochemical and Physiological Changes in Relation to Abscission Formation Microchemically, the nature of the abscission zone is greatly changed in the course of development of abscission formation* However, the present study indi­ cates that this change has little effect on the nutritive value of the edible product• The variation of total sugar, reducing sugar, dry matter, and ascorbic acid between treated and untreated cauliflower, as found in this study, is considered mainly - 95 to be the result of the abscission rather than the cause of It • The slightly higher sugar content of the untreated cauliflower whead” may be due to the decreased sugar con­ sumption taking place in the leaves after abscission, but it is not true in the case of dry matter and ascorbic acid* Generally, the content of these substances varies more in the leaf, especially the leaf blade, than in the "head” . It is thought that this variation is caused by the second­ ary effect of the abscission and is not the direct effect of the growth regulating substance. As Indicated before, the growth regulating sub­ stances may have a pronounced effect on the bio-chemical transformation of the growing plant, and may have compara­ tively little effect on the stored plant. have investigated this problem* Many workers Alexander (1938) found a movement of carbohydrate toward the apical swelling and an accumulation of starch below it after treatment with lndole-3-acetie acid on bean plants* Mitchell, Kraus, and Whitehead (1940) showed that naphthaleneacetic acid sprayed upon the leaves of kidney bean plants hastened the hydrolysis of starch and dextrins in the leaf. Bausor (1942), in an experiment using indoleacetic or b-naphthaleneacetic afcid in lanolin on tomato cuttings, reported that amylolysis follows treatment of tissues with a growth substance even in the presence of high carbohydrate supply • -96Miller (1933), treating dormant potato tubers with sulphur compounds, found that the chemicals increased the sucrose content and respiration. As a result of this change, the maturity of the plant would naturally be hastened, as was found by Wittwer and Murneek (1946) who reported that snap beans sprayed with 2,4-D and several other substances in the flowering stage, matured earlier. On the other hand, many instances have also been found in which growth regulating substances can retard the physiological activity. The use of methyl ester of naph- thaleneacetie acid for inhibiting sprouting of potato tubers has been found by Denny (1942, 1945), Guthrie (1933, 1939) and many others. Recently stromme and Hamner (1948) also i found that bean plants sprayed with solution of 2,4-D at a non-herbicidal concentration delayed maturity. The retarda­ tion of abscission formation is but one of these effects. These apparently contradictory results are probably related to the potency of the growth regulating substances used and the metabolic activity of the plant organs treated. In the case of stored plants such as cabbage and cauliflower the chemical and physiological changes following treatment with growth regulating substances, is not as great as that observed in growing plants in the field. Catalase activity was altered by the treatment in this work. Generally catalase activity is more closely associated with the metabolism of the plant than the other chemical changes• The ability to decompose hydrogen per­ oxide, or the reaction of catalase activity, as pointed out by Heinike (1924), is a more sensitive measure of the metabolic status of the tissue than the ordinary chemical analyses. Ranjan and Mallik (1931) found that catalase activity is correlated with the monosaccharides, and is influenced more by hexose formation than by the actual amount of hexose present. One of the main physiological effects of the growth substance is the mobilization of material toward the region of vigorous growth and a redistribution of storage material in the plant. After treatment of bean plants with indol-3-acetie aeid, Alexander (1938) found a movement of carbohydrates toward the apleal swelling. This mobilization seemed to be effected through the ac­ tion of the growth substances on the enzymatic system. It was found by Miller (1933), that sulphur compounds may break the dormancy of potato tubers, and will result in an increase in catalase and peroxidase activity as well as respiration. Apparently, low concentration of the growth substance may retard the maturity, catalase activi­ ty, hydrolysis of starch and, to a certain extent, respir­ ation of the treated plants; whereas, in higher concentra­ tion, the reverse may be true. - 98 As a result of the present study, the catalase activity was inhibited by the action of the growth regulat­ ing substance, especially in the leaf blades of caullflowsr. However, there was little effect on the ’’head” tissues, which were probably of little importance in connection with abscission, in comparison with the leaf organ that subsequently abscissed. The relationship between the leaf order and ab­ scission is probably related to the catalase activity of the leaves* The outer (older) leaves have higher catalase activity and thus they will drop earlier; the inner (younge^ leaves have lower catalase activity, and thus they will drop later* The mechanism of the retarding of foliar abscission is probably due to the growth regulating substance which may inactivate certain enzymatic systems which are required for the formation of the abscission* CONCLUSIONS Leaf abscission formation in cabbage and cauliflower stored at temperatures from 34° to 70°F. was markedly retard­ ed by treatment with <*-naphthaleneacetic and 2,4-dichlorophemoxyacetic acid compounds• The methyl ester forms of these two chemicals induced some elongation of the cauli­ flower inflorescence during storage, while their sodium salts tended to reduce the chlorophyll content of the leaves. Low storage temperature at a high humidity also delayed ■ abscission formation. Results of experiments indicated that the stimulus of the growth regulating substances was transported vertical­ ly from the tip of the leaves or stems downward and also horizontally from one side to the other, but did not move upward. Leaf blade tissue had little effect in delaying abscission of the stored cauliflower. Treatment with the methyl ester of c(-naphthaleneacetic acid at a concentration of 100 p.p.m. can retard the leaf fall of both trimmed and untrimmed cauliflower with leaves either debladed or left intact. Pre-harvest treatment of cauliflower or cabbage with the sodium salt of 2,4-dichlorophenoxyacetic acid at a con­ centration of 100 p.p.m. applied one or seven days prior to harvest more effectively and uniformly retarded foliar abscis -100sion during subsequent storage than any post-harvest treat­ ment. However, the sodium salt of naphthaleneaeetie acid, when applied by the same method and in the same concentra­ tion, had little effect. Anatomically, leaf fall in both cabbage and cauli­ flower was shown to be due to a weakening of the leaf attach­ ment caused by the differentiation of a meristematic layer, followed by the dissolution of the middle lamellae in the separation layer and the mechanical rupture of the vascular bundle. Formation of a separation layer on the lower side of the petiole usually did not follow the differentiation of a meristematic layer. Differences in foliar abscission between cabbage and cauliflower were in the cell shape in the meristematic layer and the position of the separation layer. Delay in the development of abscission formation through treatment with growth regulating substances m§iy be due to either the impediment of differentiation of the meristematic layer or the retardation of cell wall weaken­ ing of the separation layer. With untreated cabbage and cauliflower it was found that the outer leaves always drop before the inner leaves. In the case of heads treated following harvest, the inner leaves In cauliflower or the intermediate leaves in cabbage dropped first; however, in storage following pre-harvest treatment, little difference was found In the time of abscission between the older and the younger leaves• -101Sugars, dry matter, and ascorbic acid were generally found to vary more in the leaf blade than in the "head” tissue of cauliflower. The differences in the content of sugars, dry matter and ascorbic acid observed between treati ed and untreated cauliflower tissues are considered to be the result or secondary effect of foliar.abscission and are not the cause. Not only did the catalase activity of cauliflower decrease with increase in the length of the storage, but also it decreased from outer (older) leaves to inner (young­ er) leaves. Also treatment with the methyl ester of naph- thaleneacetic acid decreased the intensity of catalase activity, especially in the leaf blade. Apparently the partial inactivation of catalase activity by treatment is a possible cause for the delay in abscission formation. — 102 — LITERATURE CITED Alexander, T.R. 1938* Carbohydrates of bean plants after treatment with indol-3-aeetle acid. PI. Physiol. 13: 845-858. Association of Official Agricultural Chemists• Official and tentative methods of analysis. 1940. Washington, D.C. Avery, G.S.,Jr. 1935. Differential distribution of a phytohormone in the developing leaf of Nleotlana. and its relation to polarized growth. Bull. Torrey Bot. Club. 62: 313-330. Batjer, L.P. 1942. Temperature in relation to effective­ ness of pre-harvest drop spray on apples. Proc. Amer. Soc. Hort. Scl. 40: 45-48. , and Moon, H.H. 1945. Effects of naphthaleneacetic acid sprays on maturity of apples. Proc• Amer. Soc. Hort. Sci. 46: 113-117* _______ , and Marth, P.C. 1941. Further studies with sprays in controlling pre-harvest drop of apples, Proc. Amer. Soc. Hort. Sci. 38: 111-116. _______ , and Thompson, A.H. 1946. rophenoxyacetic acid sprays pre-harvest drop of several Proe. Amer. Soc. Hort. Sci. Effects of 2,4-dichloin controlling the apple varieties. 47: 35-38. Bausor, S.C. 1942. Effects of growth substances on reserve starch. Bot. G-az. 104: 115-121. Beal, J.M., and Whiting, A.G. 1945. Effeet of indoleacetic acid in inhibiting stem abscission in Mirabilis .1alana • Bot • Gaz • 106: 420-431 • Carolus, R.L., Lee, S.H., and Vandemark, J.S. 1947. Effect of the methyl ester of d-naphthaleneacetic acid on the storage life of cauliflower. Proc • Amer • Soc • Hort. Scl. 49: 367-369. Clark, W.G. 1938. Electrical polarity and auxin transport. PI. Physiol. 13: 529-552. 103 Davey, A.E. 1942. Experiments with sprays in the control of pre-harvest drop of Bartlett pears in California, proc. Amer. Soe. Hort. Sci. 40: 49-53. Benny, P.E. 1942. The use of methyl ester of d-naphthaleneacetic acid for inhibiting sprouting of potato tubers, and an estimate of the amount of chemical retained by tubers. Boyce Thompson Inst. Gontrib. 12: 387-403. _______ 9 1945. Further tests of the use of the methyl ester of d-naphthaleneacetic acid for inhibiting the sprouting of potato tubers. Ibid. 14: 15-20. _______ * and Guthrie, J.D. 1942. Effect of the vapor of the methyl ester of d-naphthaleneacetic acid on the sprouting and the sugar content of potato tubers. Ibid. 12: 253-268. Dickson, G.H. 1939* Some factors affecting the dropping of McIntosh apples. Scl. Agr• 19: 712-721. Ennis, W*B.,Jr., and Boyd, F.T. 1946. The response of kid­ ney bean and soybean plants to aqueous spray appli­ cations of 2,4-diehlorophenoxyacetic acid with and without earbowax. Bot. Gaz. 107: 552-559. Ferri, M.G. 1945. Preliminary observation on the translo­ cation of synthetic growth substances. Boyce Thompson Inst. Contrlb. 14: 51-68. Feher, D. 1925* Untersuehungen fiber den abfall die Fruehte einiger Holzpflanzen. Ber. Beut. Bot. Ges. 43: 52-61 . Gustafson, F.G. 1946. Influence of external and internal factors on growth hormone in green plants. PI. Physiol. 21: 49-62. Gardner, F.E., Marth, P.O., and Batjer, L.P. 1939. Spray with plant growth substances for control of the pre-harvest drop of apples • Proc. Amer. Soe • Hort • Sci. 37: 415-428. Gerhardt, F., and Allmendinger, D.F. 1946. The influence of d-naphthaleneacetic acid spray on the maturity and storage physiology of apples, pears, and sweet cherry • J . Agr. Res. 13: 189-206. 104 Goodwin, F.G-. 1937* The role of auxin in leaf development in SolIdago species. Amer. J. Bot. 24: 43-51. Guthrie, J.D* 1939. Inhibition of the growth of buds of potato tubers with the vapor of the methyl ester of naphthaleneaeetie acid. Contrib. Boyce Thompson Inst. 5; 83-94. Hamner, C .L., Gartner, J .B., and 0 *Rourke, F .L. 1948 . A non-toxic plastic coating to improve the keeping quality of cut foliage. Mich. Agr. Exp. Sta. Quart. Bull. 30: 268-271. Hamner, K.C., and Kraus, E.J. 1937. Histological reactions of bean plants to growth promoting substances• Bot. Gaz. 98: 735-807. Haller, M.H., and Rose, D.H. 1932. Apparatus for deter­ mination of COo and Oo of respiration. Science. 75: 439-440. Hesse, C.O., and Davey, A.E. 1942. Experiments with sprays in the control of fruit drop of apricot and peach. Proc. Amer. Hort. Sci. 40: 55-62. Helnicke, A.J., Reuther, W«, and Cain, J.G. 1942. Influen® of boron application on pre-harvest drop of McIntosh apples. Proc. Soc. Hort. Sci. 40: 31-34. Helnicke, A.J. 1924. Catalase activity in dormant apple twigs: its relation to the condition of the tissue, respiration and other factors. N.Y. (Cornell) Agr. Exp. Sta. Mem. 74. Hitchcock, A.E., and Zimmerman, P.W. 1935. Absorption and movement of synthetic growth substances from soil as indicated by the responses of aerial parts. Contrib. Boyce Thompson Inst. 7: 447-476. , and . 1941. The use of naphthaleneaeetie acid and its derivatives for preventing fruit drop of apple. Proc. Amer. Soc. Hort. Sci. 38: 104-110. Hodgson, R.W. 1918. An account of the modes of foliar ab­ scission in Citrus. Univ. Calif. Pub. (Bot.) 6 : 417-428. Hoffman, M.B. 1940. The pre-harvest drop of mature McIntosh apples as influenced by application of nitrogen carrying fertilizers • Proc• Amer • Soc• Hort• Sci• 37: 438-442. - 105 Kendall, J.N. 1918* Abscission of flowers and fruits in the Solanaceae with special reference to Nicotlana. Univ. Calif. Pub. (Bot.) 5: 347-428. Knott, J.E. 1926. Catalase in relation to growth and to other changes in plant tissue. N.Y. (Cornell) Agr. Exp. Sta. Mem. 106. LaRue, C.D. 1936. petioles. The effeet of auxin on abscission of Proc. Nat. Avad. Sci. 22: 254-259. Lloyd, F.E. 1916. Abselssion in Mlrabilis .lalapa. Gaz. 61: 213-230. Bot. Livingston, G.A., and Addicott, F.T. 1947. Meehanical factors in foliar abscission of citrus. Amer. J. Bot. 34: 586. (Abstract). Lucas, E.H. 1944. Determining ascorbic acid in large numbers of plant samples. Ind. and Eng. Chem. 16: 649-652. MacDanlels, L.H. 1936. Some anatomical aspects of apple flower and fruit abscission. Proc. Amer. Soc. Hort. Sci. 34: 122-129. McCown, M. 1939* Abscission of flowers and fruits of the apple. Proc. Amer. Soc. Hort. Sci. 36: 320. (Abstract)• _______ • 1943. Anatomieal and chemical aspects of ab­ scission of fruits of the apples. Bot. Gaz. 105: 212-220 . Miller, L.P. 1933* Effeet of Sulphur compounds in break­ ing the dormancy of potato tubers and in inducing changes in the enzyme activities of the treated tubers. Contrib. Boyce Thompson Inst. 5: 29-81. Mitchell, J.W., Kraus, E.J., and Whitehead, M.R. 1940. Starch hydrolysis in bean leaves following spray­ ing with alpha naphthalene acetic acid emulsion. Bot. Gaz. 102: 97-104. Myers, R.M. 1940. Effect of growth substances on the ab­ scission layer in leaves of Coleus. Bot. Gaz. 102: 323-338. Okabe, Y. 1940a. Growth-promoting and growth-inhibiting substances in the petiole. Bot. Mag. (Tokyo) 54: 357-365• (in Japanese with English summary). 106 « 1940b • On the distribution of growth promoting and growth inhibiting substances in Morus alba L.. and Cassia ocoidentalis L. Bot, Mag, (Tolsyoj-40: 453-461 • (In Japanese with English summary), Penfound, W.T., and Mihyard, V. 1947. Relation on light intensity to effect of 2,4-dichlorophenoxyacetic acid on water hyacinth and kidney bean plants. Bot. Gaz. 109: 231-234. Ranjan, S., and Mallik, A.K. 1931. A study of the catalase reaction, with special references to respiration. New Phytol. 30: 355-381. Rice, E.L. 1948. Absorption and translocation of ammonium 2,4-diehlorophenoxyacetate by bean plants. Bot. Gaz. 109: 301-314. Sampson, H.C. 1918. Chemical changes accompanying abscis­ sion in Coleus Blumei. Bot. Gaz. 66: 32-53. Scott, F.M., Schroeder, M«, and TurrelL , F.M. 1947. Leaf abscission in the Valencia orange and suberisatlon of the Internal surface cf the Valencia and other leaves. Amer. J. Bot. : 589. (Abstract). Smith, F.G. 1948. The effect of 2,4-dlehlorophenoxyacetIc acid on the respiratory metabolism of bean stem tissue. Plant Physiol. 23: 70-83. Stewart, W.S., and Klotz, L.F. 1947* Some effects 6f 2 ,4dlchlorophenoxyaeetie acid on fruit drop and morphology of oranges • Bot • Gaz • 109: 150-162 • Stromme, E.R., and Hamner, C.L. 1948. Delayed maturity of bean plants sprayed with solution of 2,4-dichlorophenoxyacetic acid of non-herbicldal concentra­ tion. Science 10:: 2772: 170-171. Sweeney, B.M., and Thimann, K.V. 1942. The effects of auxin on protoplasmic streaming. Jour. Gen. Physiol. 25: 841-854. Thimann, K.V. 1934. Studies on the growth hormone of plants VI. The distribution of the growth sub­ stance in plant tissues. Jour.Gen. Physiol. 18: 23-24. Thompson, B.F. 1945. Tissue responses to physiologically active substances. Bot. Rev. 11: 593-610. 107, Tukey, H.B., and Hamner, C.L. 1945. Retardation of pre­ harvest drop of apples through aerosol application of growth regulating substance. Proc. Amer. Soc. Hort. Scl. 46: 102-108. Vyvyan, M.O. 1946. Experiments with growth substance sprays for reduction of pre-harvest drop of fruit. Jour. Pomol. Hort. Sci. 22: 11-37. Weaver, R.J., and DeRose, H.R. 1946. Abscission and trans­ location of 2,4-dichlorophenoxyacetie acid. Bot. Gaz. 107: 509-521. Wittwer, S.H., and Murneek, A.E. 1946. Further investiga­ tions on the value of *hormone” sprays and dusts for green bush snap beans • Proc • Amer • Soc • Hort • Sci. 47: 285-293. Zimmerman, P.W., and Hitchcock, A.E. 1937. Comparative effectiveness of acids, esters and salts as growth substances and methods of evaluating them. Con­ trib. Boyce Thompson Inst. 8: 337-350. and Wilcoxon, F. 1939. Responses of plants to growth substances applied as solution and as vapors. Ibid. 10: 363-376.