EFFECT OF iNDOLE-BUTYRiC ACID SOLUTEONS ON THE ROOTING CF DORMANT \X’OODY CUTTINGS Thesis for 1126 Degree of M. S. MICHLGAN STATE COLLEGE Bizabetb VanMaren 1939 R” 94 k. k u I ‘-\: . .. ah -‘VV. .. ‘r tor-i |IVO 4 O. L .uli. :4 - lislianniiii- f; - EFFECT OF INDOLE-BUTYHIC ACID SOLUTIONS ON THE ROOTING OF DOREANT WOODY CUTTINGS by Elizabeth YanMaren Submitted in partial fulfilment of the requirements for the degree of Master of Science in the Graduate School, Michigan State College, Department of Botany June 1939 TABLE OF CONTENTS Page Introduction 1 Review of literature 3 Methods 5 Tables and discussion of data 11 General conclusions 27 Summary 31 BibliOgraphy - 33 121494 .H... :i\L'IlnIH,I.-r'lirll I‘ f . y o Table I. II. III. IV. VI. VII. VIII. IX. XI. LIST OF TABLES Names and dilutions of growth substances used. Rooting taken Rooting taken Rooting tings Rooting taken Rooting taken Rooting tings Rooting taken Rooting taken Rooting taken Rooting taken response of Selix cuttings in November. response of Thuja cuttings in November. response of §yringa_cut- taken in November. response of Salix cuttings in December. response of Thuja cuttings in December. 8 , r sponse of Svringa cut- take n in December. response of Halus cuttings in December. response of Salig cuttings in January. restonse of Thuja cuttings in January. response of Thuja cuttings in early Apr . Page 12 1’4 16 LIST OF FIGURES Figure l. Salix cuttings (January) showing roots at the end of eleven days. 2. Salix cuttings (January) showing roots at the end of eleven days. 3. Salix cuttings (January) showing roots at the end of eleven days. 4. Salix cuttings (January) showing roots at the end of eleven days. 5. Salix cuttings (January) showing roots at the end of five weeks. 6. Salix cuttings (January) showing roots at the end of five weeks. Thuna cuttings (December) showing roots sixteen weeks after Auxilin treatment. Thu a cuttings (December) showing roots sixteen weeks after indole-butyric acid treatment. Untreated Thuja cuttings (December) show- ing callus at the end of sixteen weeks. Plate II II III III IV IV ACKNOWLEDGMENT The writer is grateful to Dr. R. P. Hibbard, Mr. H. C. Beeskow and Dr. E. A. Bessey for aid in this study and preparation of the manuscript. 4. M. 1 . ifl.¢£..llo..! .- flpi‘! Il.?!1 |.. -1- INTRODUCTION Plant hormones and synthetic growth substances, such as indole-butyric acid, indole-acetic acid, and naphthalene acetic acid, have attracted much attention during the past few years. While various effects which they may have on plants have been studied, one of the most important appli- cations is in the initiation of roots on cuttings. Several commercial preparations of these synthetic substances have been placed on the market and quite widely advertised. This has created considerable interest for people engaged in plant propagation. Rooting of dormant cuttings of most species of woody plants has never been very successful. The usual practice followed with hardwood cuttings taken in the late fall or early winter is to bury them in sand in a well drained place and keep them cold, so that callusing may take place. The following spring they are removed and set in propagat- ing boxes to allow roots to develop. This method is successful with several species, but with others, such treatment will not cause the cuttings to strike root. The term dormancy is used in this paper to apply to the entire period of inactivity which plants undergo dur- ing the winter months. It is known that cuttings of many woody plants will resume their activity after a short period of cold weather, if they are brought into a green— house and kept well watered. However, when kept out-of- doors, these same plants remain inactive until spring. The peach is a particularly good.example of this. A few warm days in winter will cause the buds to become active, and during the next cold period they are frozen, causing serious damage. Such observations would lead one to suppose that environment is mainly responsible for dorman- cy. However, some other plants (e.g., lilies and tulips) become dormant during the summer, and do not renew their growth until fall or the following spring. This would in- dicate a hereditary cause. Still other causes have been suggested for the dormancy of some seeds. When all of these things are considered, it seems reasonable to think of dormancy as resulting from a combination of environ- mental and hereditary factors. It has been suggested by some workers that the lack of success in rooting cuttings during their dormant per- iod might be due to a lack of a necessary hormone in the twig when the plant is inactive (2). The experiments des- cribed in this paper were started with the idea that if treatment with synthetic growth substances could be made to induce the rooting of dormant cuttings, such a method would be distinctly advantageous. At the time when these experiments were started, most work favored the use of in- dole—butyric acid for the rooting of cuttings. For that reason and because of the fact that most of the market preparations were solutions of indole—butyric acid in alco- hol, it was decided to use different concentrations of in- dole-butyric acid as the test solutions. Some of the re- cent work (22) shows better results from the use of indole- acetic acid. REVIEW OF LITERATURE As early as the middle of the eighteenth century, the presence of a substance in plants, which was capable of in- itiating callus and root growth, was postulated by du Mon- ceau. Sachs also presented a theory that plants contain a root forming substance which moved toward the base of the stems. He considered that when "an obstacle for further downward movement" such as a ring or girdle about the stem existed, this substance gathered above the wound and start— ed the formation of roots at that place. The "growth en- zymes" of Beijerinck's publications were closely related (26). The works of Boysen Jensen (1914), Went (1926), and van der Lek (1925) were influential in bringing the hor— mone concept into studies of root formation in recent times. During the last ten years the amount of literature concerning phytohormones and their effects on plants has increased very rapidly. In this country, Zimmerman, Hitchcock, and other workers at the Boyce Thompson Insti- tute have been instrumental in starting the work on root initiation in cuttings. Phytohormcnes or synthetic growth substances are, by no means, the panacea for all of the difficulties of the plant propagator. It seems evident that such a growth substance is not the limiting factor in cuttings made from some plants (8, 22). A theory has been presented, principally by Went (25) and Cooper (9), that the absence of some other hormone-like factor in the plant is responsi- ble for the lack of rooting. This substance, which they call "caline" or "rhizocaline", when present, is stimulated by the action of auxins, and the cuttings root successfully. It has been found that the treatment with synthetic growth substances will make some cuttings root while un- treated cuttings kept under the same conditions fail to form any roots. In other cases, however, no treatment so far discovered will induce the rooting response desired. One explanation that has been offered is that some plants in themselves, have plenty of the auxin present, and the treatment will not be advantageous in any way; the others have the auxin present in insufficient quantity, and they benefit by the treatment. That a growth hormone is not present in the tissues during the dormant season has been postulated by Avery and others (2). They also state that the centers for the production of this hormone are in the terminal buds. These workers have attempted to show that the supply of growth hormone increases during the time when the terminal buds are swelling, and reaches the maximum just prior to the period of most rapid expansion of the current season's shoots (2, 12). Evidence seems to indicate that the auxin is formed from some inactive precursor, the chemistry of which is not thoroughly understood (21, 26). Some workers have obtained results showing that the age of the tree from which the cutting is taken is an im- portant factor. They also hold that the position of the cutting on the parent tree (whether it is terminal or lateral, and whether it comes from the basal or apical portion of the tree) affects greatly the rooting response of the cutting (22). In cuttings which invariably fail to root, auxin supply is probably not the limiting factor, and it has been suggested that other factors, such as supply of car— bohydrate, vitamin B1’ bios, or some other hormone-like substance may be insufficient (5, 18, 9, 25). Treatment with growth substances has been found to inhibit the growth and development of buds. In many plants the development of the terminal bud will inhibit the growth of lateral buds, and if the terminal bud is re- moved, one or more of the lateral buds will develop. ”mind-l... . VHHI...§ -6- Thimann (20) has found that the inhibition of the develop- ment of lateral buds in Egggg seedlings by pure auxin is not compensated by growth elsewhere, and involves a de- crease in dry weight. Also the extent of the inhibition is not dependent on the distance between the bud and the point of application of the auxin. Thimann and Skoog (23) have found that this inhibition is not due to injury. That auxin is present in larger amounts during the period just preCeding most rapid shoot development has been previously stated (2, 12). It would seem more rea- sonable then, to expect that the addition of auxin to a cutting would cause more rapid development of the buds. Since just the opposite is true, the problem of explana_ tion becomes very difficult. It seems that the same sub- stance can'both inhibit and promote bud development, and the conditions under which each of these reactions takes place is a question still to be solved. METHODS The cuttings used throughout the experiment repre- sent four species, Syringe vulgaris (lilac), Egggg pumila (apple), Thuia occidentalgg (arbor vitae), and Salix babylonica (weeping willow). Of these, the first two are considered difficult or impossible to propagate from cuttings. The arbor vitae is not so difficult to root, while willow roots very readily under most condi- tions. The cuttings were taken periodically during the dor- mant season of 1937-1938 and during the early part of the 1938-1939 dormant season. An interval of three weeks was usually allowed between dates of sampling, in an attemp to include any changes which might occur during the period commonly spoken of as dormancy. All of the cuttings were taken from branches at the basal part of mature plants, only the terminal parts of the preceding season's growth being used. On the willow, this meant using the pendant twigs, while on the other species the position of the cutting on the parent plant would have been upright or somewhat horizontal. The cut was made close below a node and at an angle. Each cut- ting was approximately ten centimeters long. The cuttings considered here were not treated with wax or any such material to out down transpiration. Al- so when leaves developed they were allowed to remain on the cutting. Checks were run on one set to determine whether or not the removal of several buds, before these had an opportunity to develop, would be beneficial to the rooting, since such treatment would reduce the sur- face for transpiration. The results were not considered indicative of difficulties of that nature, so other sets -g- were all run on cuttings whose buds were not removed. All of the solutions used were forms of indole- butyric acid. One is sold under the trade name of "Auxi— lin”. This material is already in solution form and the dilutions used were suggested by the direction sheet fur— nished with the preparation. The second material goes under the trade name, "Hormodin A“, and is also in solu— tion in alcohol. Again the directions accompanying the bottle were considered in making the dilutions. Pure crystals of indole—butyric acid were obtained from the Eastman Kodak Co. for use in another set. Because of the insolubility of these crystals in water, they were first put in solution in ethyl alcohol. For each pre- paration 0.5 grams of the indole-butyric acid crystals were dissolved in 20 cc. of 95% ethyl alcohol, and then 5 cc. of glycerin were added. This was further diluted by adding a measured quantity of the mixture to a defi- nite quantity of water. To avoid impurities found in ordinary tap water, dilutions of all three of the mater- ials were made with distilled water. Table I gives the concentratiOns of each dilution. Fresh solutions were prepared each time any cuttings were to be treated. The cuttings were placed in beakers of the solution so that approximately the lower two cen- timeters of each cutting was immersed. They remained TABLE I TRADE HAHES AND CDNCENTRATIGNS OF GROWTH SUBSTANCES USED Solution Concentration Auxilin A 40 mg. per L. Auxilin B 80 mg. per L. Hormodin A 20 mg. per L. Hormodin B #0 mg. per L. Indole-butyric acid A 20 mg. per L. Indole-butyric acid B 40 mg. per L. in the solutions for twenty-four hours, during which time they were kept at room temperature and away from direct sunlight. The controls were immersed to the same dis— tance in distilled water and kept under identical condi- tions. At the end of the twenty-four hour treatment, all of the cuttings were removed from the solutions, rinsed in tap water and placed in the rooting medium. This root- ing medium was made by mixing two parts (by volume) of sand with one part of peat. It was placed in standard size greenhouse flats, and watered thoroughly before planting. The cuttings were placed in the flats at an angle <1f about 45°. Though the spacing varied somewhat with tire type of cutting, most of them were placed about one 1IM3h apart in rows that were about three inches apart. -10- Such spacing was, of course, impossible in the case of the Ihgla_cuttings because of their large spreading tops. These were placed at sufficient distance to prevent crowd- ing. The flats were placed on the bench in a greenhouse and, during the first two weeks or more after planting, they were kept covered with newspapers to prevent rapid drying out of the soil and to keep the air about the cut- tings very moist. The relative humidity in the greenhouse varied from about 50% to 80% and the temperature was so re— gulated that there was a day and night variation which, during the 1937-1938 season was about 65° F. for night and between 75° and 80° F. in the daytime. During the 1938-1939 season the temperature was more constant, re- maining mostly between 65° and 70° F. At intervals during the test period the soil was loosened around one or two of the cuttings in order that they might be removed and checked for callus or root de- velopment and then replaced without injury. Aside from such inspection the cuttings were not disturbed until time for making the final count. That time differed with the species under consideration. The time for each set is given in the tables following. In the species where no rooting results were obtained, the cuttings -11... were left in the rooting medium until the whole shoot was definitely dead. Whenever any cuttings in any set died, they were removed and recorded without disturbing any of the others. TABLES AND DISCUSSION OF DATA The following tables show the results from the treat- ment of nearly 2000 cuttings. Since the weather during October and early November of 1938 was unseasonably warm, the results given in tables III to IV" epresent work with cuttings in the very early part of the dormant season, or possibly before all of the changes usually considered as accompanying dormancy had occurred. Tables V to VIII re- present results from the experimental work with cuttings taken in late November and early December of 1937 when the trees were very definitely inactive. Tables IX and X express results for the month of January -- about the middle of the dormant season. Table XI is the record of a group of Ihglg cuttings taken in early April 1938 when dormancy had probably been overcome. No tables are given for the latter part of the dormant season but the results of the experiments are discussed following table X. TABLE II ROOTING RESPONSE OF SALIX CUTTINGS TAKEN IN NOVEMBER* No. Treatment Dead; Dead; LiVing; treated not rooted rooted rooted no. % no. % no. % no Auxilin A 13 32.5 27 67.5 o 0.0 #0 Hormodin A 10 25.0 30 75.0 0 0.0 #0 Control 3 7.5 A 10.0 33 82.5** * Cuttings were removed from rooting medium after 6 1/2 weeks. ** These living and rooted cuttings were in poor condi- tion when removed. The treatment of willow cuttings during the very early part of the dormant season was not beneficial. The temperature preceding the collection of this set had been warm, with only a few frosty nights to initiate the changes which accompany dormancy. The leaves of the cur- rent season were dry and dead but many of them were still clinging to the twigs when they were brought in. The buds on these cuttings did not develop repidly as was common for the willows treated later in the season. Very minute leaves did develop on most of the cuttings, but on some even the swelling of the buds was never noticeable. Soon after being set, the treated cuttings began to show signs of death, and this observation was substantiated by -13.. the fact that the roots which did form were very few and small, in contrast to the abundant root systems formed when the cuttings were taken later in the season. All of the treated plants were dead when they were re- moved from the rooting medium at the end of the six and one- half weeks period. Of the controls, a large percentage were still alive, and though their root systems were rela- tively small, in conparison with those formed on cuttings taken at other times, it seems reasonable to believe that practically all of them would have survived if they had been transplanted and kept in favorable conditions. These facts would appear to indicate some injurious effect due to the treatment. The same concentration of solutions at other times did not show any harmful effects, so it is doubtful that the solutions were too strong, unless the cutting might be considered much more sensitive to the addition of growth substance at the period when the plant was first becoming inactive. To apply the facts previously mentioned concerning the small quantity or lack of auxins in the dormant buds of many woody plants, it would seem that the added supply of synthetic growth substance should be of greater value then than at any other time of the year. Judging from the above data, however, this cannot be the case. A smaller proportion of the controls survived at -14- this time than at any other time during the winter months. Such results would imply that November is a very poor time to start willow cuttings. There would be no advantage in having started them this early, since their own slowness to develop roots would allow cuttings taken at a later time to surpass them before the time would come for trans— planting to out-of-doors conditions. Just what physiolog- ical conditions are responsible for such slow development is a question which would involve many factors and be difficult to answer completely without very thorough analysis of the material at frequent intervals during these changes. TABLE III ROOTING RESPONSE OF THUJA CUTTINGS TA KEN IN NOVEMBER* No. Treatment Dead; Living; Living; treated not rooted callused rooted no. % no. % no. % 25 Auxilin A 20 80.0 0 0 5 20.0 50 Auxilin B As 96.0 0 0 2 4.0 25 Hormodin A 22 88.0 0 0 3 12.0 50 Hormodin B 44 88.0 0 0 6 12.0 50 Control 34 68.0 7 14.0 9 18.0 * Cuttings were removed from the rooting medium after 14 weeks. Arbor vitae cuttings, taken from the trees in Novem- ber, showed poorer rooting response when treated with the indole-butyric acid solutions than when untreated. Those which had rooted, especially the controls, had vigorous root systems and the tops were bright green and showing new growth at the tips, when the plants were removed from the rooting medium in February. Fourteen percent of the control plants had formed abundant callus at the cut sur- face, and several of these probably would have rooted if they had been allowed to remain for a longer time in the rooting medium. It is worthy of notice that none of the treated specimens which had failed to root, showed any indication of callus. The tops of all of these non—root- ed, hormone—treated specimens, were dead when the records were taken. The growth substance, again, seemed to show an in- . jurious effect when used on Arbor vitae cuttings taken in the very early dormant season. While some of the con- trols rooted very slowly, the percentage of rooting was sufficiently higher than that on the treated cuttings, to be significantly in favor of the non-treated groups. TABLE Iv ROOTING RESPONSE OF SYRINGA currrwcs TAKEN IN NOVEMBER* No. Treatment Dead; Dead; Living; treated not rooted callused rooted I no. oz; no. at no. 9% 50 Auxilin A A7 94.0 2 u.0 1 2.0 50 Auxilin B 46 92.0 4 8.0 0 0.0 25 Auxilin 20** 22 88.0 1 v.0 2 8.0 25 Auxilin 160** 22 88.0 1 v.0 2 8.0 50 Control u7 94.0 3 6.0 0 0.0 ‘ Records were taken at the end of fourteen weeks. ** Auxilin 20 represents a solution containing 20 mg. per liter and Auxilin 160 contains 160 mg. per liter of indole—butyric acid. These concentrations of the Auxilin were not used in the other series. In the lilac cuttings taken during November, root- ing seemed very difficult, as it was during all of the dormant season. Here the untreated cuttings showed no rooting at all after fourteen weeks, while the variously treated groups showed from two to eight percent rooting. The number callused but not rooted was about the same in both the treated and control groups. In practically all cases these calluses were turning dark brown and soften— ing and the cutting appeared to be dead. Therefore it is unlikely that a longer period in the rooting medium -17- would have appreciably altered the percentage of rooting. These callused cuttings are not comparable to the callus— ed arbor vitae cuttings mentioned in the preceding table, since in those the callus appeared healthy, and the plant still would have a chance of rooting successfully. TABLE V ROOTING RESPOs- SE OF fiALIX CUTTIIJGS TAKEN IN DECEnEBER* N0. Treatment Dead; Living; treated not rooted rooted no. % no. % lO Auxilin A H 40.0 6 60.0 10 Auxilin B 3 30.0 7 70.0 10 Hormodin A 4 40.0 6 60.0 10 Hormodin B 2 20.0 8 80.0 10 Indole-but. A 3 30.0 7 70.0 10 Indole~but. B 5 50.0 5 50.0 . 20 Control 8 40.0 12 60.0 * Records taken after cuttings had been in rooting me— dium 13% weeks. er than those involved in most of the other tables. The samples represented in the above data are small- The fact that the controls showed approximately the same a- mount of rooting as any of the treated cuttings would inc dicate a lack of noticeable effect from the treatment. -13- This is in contrast to the results obtained on the Novem- ber cuttings where the indole-butyric acid showed an in— jurious effect. Again, in contrast to the samples taken earlier in the dormant season (November) the December cuttings which rooted, all survived for more than three months and probably would have suffered no mortality in a longer time. The length of the roots on these success- fully rooted Specimens was short, even at the end of the thirteen and one—half weeks. TABLE VI ROOTING RESPONSE OF IHUJA CUTTINGS TAKEN IN DECEMBER* No. Treatment Dead; Living; Living; treated not rooted callused rooted no. % no. % no. % 10 Auxilin A 3 30.0 3 30.0 4 40.0 10 Auxilin B 4 40.0 0 0.0 6 60.0 10 Hormodin A 6 60.0 1 10.0 3 30.0 10 Hormodin s 6 60.0 2 20.0 2 20.0 10 Indole-but. A 9 90.0 0 0.0 1 10.0 10 Indole-but. B 6 60.0 0 0.0 4 40.0 20 Control 15 75.0 5 25.0 0 0.0 * Records taken at the end of 13% weeks. For the cedar cuttings taken in December, the controls had formed no roots after thirteen and one-half weeks in the rooting medium, while some cuttings in each of the treat— ed groups had rooted successfully. These percentages of successful rooting varied considerably in the different groups, and since each sample was rather small, the varia- tions cannot be highly significant. Nevertheless, it might be noted that the highest percentage of rooting occurred where the strongest concentration of indole- butyric acid was used; namely, in the Auxilin B which re- presents 80 milligrams of indole-butyric acid per liter. The treatments with 40 milligrams per liter also show a better average of rooting response than the treatment with 20 milligrams per liter. Twenty-five percent of the controls had formed callus when the cuttings were removed. Had all of these formed roots later, they still would not have brought the per- centage of successful rooting as high as that for most of the treated groups. Since in three of the treated groups there was also a fair percentage of callus formation, one might expect a slight increase in the number of plants rooted, if they had been allowed to remain longer in the rooting medium. The mortality rate was high, both in treated groups and control. To the propagator this would mean handling a large number of cuttings which would never make salable plants. For practical purposes, then, December could not -20... be considered a good month to take cuttings of arbor vitae. TABLE VII ROOTING RESPONSE OF HERINQA CUTTIJGS TAKEN IN DECEHEER* No. Treatment Dead; Dead; Living; treated not rooted callused rooted non % no. fl n04 % 10 Auxilin A 9 90.0 0 0.0 1 10.0 10 Auxilin B 8 80.0 2 20.0 0 0.0 10 Hormodin A 9 90.0 1 10.0 0 0.0 10 Hormodin B 9 90.0 1 10.0 0 0.0 10 Indole-but. A 9 90.0 1 10.0 0 0.0 10 Indole-but. B 8 80.0 2 20.0 0 0.0 20 Control 20 100.0 0 0.0 0 0.0 ed any rooting. butyric acid per liter, in the form sold as Auxilin. * Records taken at the end of 19 weeks. Only one cutting in the lot taken in December show- It was one treated with 20 mg. of indole- The callus which formed in a small percentage of the other specimens had, in nearly all cases, become soft and show- ed no evidence of being able to root if given more time. The leaves on these cuttings developed to a small extent but soon became dry and dead, and, when the cuttings were removed, there remained no buds which showed any indica- tion of life. -21- TABLE VIII ROOTIIC FBSPCNSE OF lfl;§§,CUTTINGS TAKEN IN EECEIEER* No. Treatment Dead; Dead; treated . not rooted callused no. % no. % 10 Auxilin A 0 0.0 10 100.0 10 Auxilin B O 0.0 10 100.0 10 Hormodin A 2 20.0 8 80.0 10 Hormodin B 1 10.0 9 90.0 10 Indole—but. A. 0 0.0 10 100.0 10 Indole-but. B l 5.0 9 90.0 20 Control 1 5.0 19 95.0 *Records taken after 19 weeks. Nearly all of the apple cuttings formed callus, but this had already softened and become dark-colored when the cuttings were removed from the sand—peat mixture. No roots formed on any of these cuttings and all of the cut- tings were dead when the records were taken. The work of Cooper (8) and others is in agreement with these results, for they too, found it impossible to root apple cuttings. In accordance with this fact apple stems are supposed to lack "rhizocaline" according to their theory. Whether this is sufficient explanation is questionable. In view of the work done it would appear reasonable that not the auxin supply, but the supply of -22.. some other substance or group of substances, may be the limiting factor in the failure of apple cuttings to root. TABLE IX ROOTING RESPONSE OF SALIE CUTTINGS TAKEN IN JANUARY* No. Treatment Dead; Living; treated not rooted rooted no. % no. % 20 Auxilin A 0 0.0 20 100.0 20 Auxilin B O 0.0 20 100.0 20 Hormodin A 0 0.0 20 100.0 20 Hormodin B 0 0.0 20 100.0 20 Indole-but. A 0 0.0 20 100.0 20 Indole—but. B 0 0.0 20 100.0 20 Control 0 0.0 20 100.0 * Records taken at the end of 1% weeks. In this series of cuttings rooting and survival was 100%. The roots develOped a few days earlier in those which had been treated with any of the concentrations of the synthetic hormone solutions. These plants were replaced in the rooting medium after the records were taken to determine what effect a longer time might have. Within about three weeks (after the treatment) the control plants had developed enough so that little or no difference was discernible between Edi-K1437 :1... 4 "H il. I I .2}. quantity and length of roots on the controls and on the treated specimens. This would show little or no advan« tags in the use of auxins, for the few days' difference in speed of rooting would not be valuable and would be overcome as the plants were left longer. Five cuttings from each treatment of this series were placed in water instead of the sand-peat mixture. This did not alter rooting percentages and therefore all of the results were brought into the one table. TABLE X ROOTING RESPONSE OF TEQQA_CUTTINGS TAKEN IN JANUARY* No. 4 *Treatment Dead; Living; treated not rooted rooted no. % no . ‘fb 20 Auxilin A 14 70.0 6 30.0 20 Auxilin B 15 75.0 5 25.0 20 Hormodin A 15 75.0 5 25.0 20 Hormodin B 16 80.0 4 20.0 20 Indole—but . A 18 90.0 2 10.0 20 Indole-but. B 19 95.0 1 5.0 20 Control 19 95.0 1 5.0 * Records taken at the end of 9 weeks. The arbor vitae cuttings taken in January showed a very loW'percentage of rooting. A small number (five 9 -. “10"” -2u_ from each treatment) were placed in a water medium, to facilitate observation of the time when roots first start- ed.. In the January series no callus formation was noted on the non-rooted specimens when the records were taken. This is quite different from the results shown by the earlier series, where a fair percentage had formed callus. At this time of the season then, the rooting, if it were to take place at all, did occur rapidly. In January the plants out—of-doors have been exposed to many very cold weeks, and it is likely that the dormancy is easily broken in the arbor vitae when that has occurred. ROOTING RESPONSE OF SXEINQA CUTTINGS TAKEN IN JANUARY. In the same manner as was used for the willow and arbor vitae, a series of one hundred-forty lilac cuttings were treated with the different concentrations of the auxin solutions. One group of twenty cuttings was kept as controls. From both the treated and control groups there was no rooting development after ten weeks. Neither was there any noticeable callus formation on any of the samples. The buds on the cuttings deveIOped some- what, but the leaves which were formed, soon dried, in spite of sufficient quantities of water being present I -E‘iii.« . -25- in the soil at all times. At this time of the year, the cuttings need something more than just added growth sub- stance if any roots at all are to be formed. ROOTING RESPONSE OF MALUS CUTTINGS TAKEN IN JANUARY. One hundred-forty apple cuttings were taken in Janu- ary and divided into groups for the various treatment and the control in the same manner as used for cedar in table X. At the end of ten weeks, when the records were taken not a single cutting had formed any roots. Again there was callus on nearly every one, but this had‘become dark and soft and thecutting was dead. The buds had made only a very slight development when the cuttings were first set in the rooting medium. With apple, in January as well as all other times in the dormant season, it was impossible to induce root— ing by the addition of indole-butyric acid solutions. ROOTING RESPONSE OF SALLX,CUTTINGS TAKEN IN FEBRUARY Groups of ten willow cuttings were treated with each of the six solutions used in January (see table IX). At the end of two weeks in the treated and the control lots every cutting had formed roots. Like the January cuttings, -26- those which were treated developed roots a few days earlier, but this advantage was soon overcome. ROOTING RESPONDE OF TRUJA, EALUE, AND SYRIUGA CUTTINGS TAKEN IN FEBRUARY The time in February when the cuttings were taken had been preceded by a period of a few days warm rain. Each series of seventy cuttings was divided into groups of ten, and these groups treated with the different con- centrations of auxin mentioned in table IX for the treat- ment of willow. Records were taken at the end of twelve weeks. At that time all of the cuttings were dead and no roots had formed on any of them. This indicates an inability of any of these three species to root if out in February. TABLE XI ROOTING RESPONSE OF‘IEQJA,CUTTINGS TAKEN IN EARLY APRIL* No. Treatment Dead; Living; treated not rooted rooted no. % no. % so Auxilin A A6 92.0 n 8.0 50 Hormodin A 46 92.0 A 8.0 50 Indole-but. A 47 94.0 3 6.0 50 Control 46 92.0 4 8.0 -27- *Records taken after 15 weeks. These cuttings represent a group taken very soon after the dormant season was over. The mortality was very high on this group. The tops began to turn brown within a few weeks and when the records were taken they were completely dead. Callus was not noticeable on these dead cuttings, and it is doubtful if they remained alive long enough for any such development to occur. If the auxin supply is the limiting factor in the rooting of arbor vitae, and the supply of natural auxin is more a- bundant in the spring than at any other time, then this series should have rooted better than those taken in De- cember and January. The conclusion from such an experi- ment might be that the arbor vitae lacks some other sub- stance which would be conducive to rooting when taken in the winter and early spring. GENERAL CONCLUSIONS On the whole, the treatment of cuttings of apple, lilac, arbor vitae, and willow with solutions of in- dole-butyric acid in an attempt to induce rooting dur- ing the dormant season, seemed quite unsuccessful. In a few cases slight differences-could be observed, but . . ”crab“ -28.. these differences were so small that they would be of little value in suggesting some cause for the failure of such.cuttings to root during the winter months. {illow cuttings taken very early in the dormant sea- son were very difficult to root, and the synthetic hor- mone used.seemed to have an injurious effect under the con- ditions of these experiments. It appeared to inhibit root formation and favor a higher mortality rate among the root- ed plants. Rooting occurred in a larger percentage of the cuttings taken later in the winter, and the toxic effect of the indole-butyric acid disappeared. In the January and February groups, where all of the cuttings developed roots, the treatments made roots form more rapidly and more abundantly. On remaining about three weeks longer in the rooting media, however, the controls surpassed the treated plants in number, size, and vigor of roots. (See figures 5 and 6.) The small amount of time gained by the use of the hormone solutions in these cases would probably be of negligible value. Similar results show- ing that the later rooting tends to minimize the effects brought about by treatment, have been shown also by the experiments of other workers (ll). Arbor vitae cuttings taken at any time during the dormant season give very poor rooting response. On the large samples taken during the early part of the 1938- 1939 dormant season, the percentage of cuttings which formed roots was smaller in the treated sets than in the control sets. While this difference was not great, it was consistent for all treatments and would indicate an injurious effect due to soaking the bases of the cuttings in the indole-butyric acid solutions. Through December and January, cuttings taken at any time showed some bene- fit from being treated with the synthetic hormone solu- tions. However the mortality of‘both treated and untreat- ed groups of cuttings taken during these months was very high. For this reason anyone interested in propagation of large numbers of plants would find the slight advan— tage due to the treatment of questionable value. Cut- tings which were taken at the latter part of the dormant season (February) gave no indication of rooting. If some substance, or group of substances is responsible for the successful rooting at other times, then this substance must have been lacking during the latter part of the dor- mant season. From the data of these experiments, the evidence indicates that auxin supply may be insufficient in arbor vitae, but that this may not be the only limit- ing factor. What these other factors may be is not known at the present time. Lilac cuttings taken at any time during the dormant season showed negligible rooting response. Lilac is con- -30- sidered very difficult to root at any time, and for this reason, that type of propagation is little practiced. Callus forms in many cases, but does not indicate that roots will necessarily follow. Apples, according to the results of this study, would be placed among the plants which are impossible to propagate from cuttings during the dormant season. In practically all cases, callus formed but soon became dark and soft, and no roots deveIOped. Whether the poor rooting results, particularly with lilac and apple, may be due to a lack of some substance in the cutting itself is a question under debate at the present time, there seems to be some evidence to support such a theory, and it might help to explain why rooting is usually much more successful on cuttings taken during the spring and early summer, for such a substance would 'probably be more abundant during the seasons of more rapid growth. There was a small amount of bud inhibition on the treated samples of some of the sets. On cuttings taken during the later part of the dormant season the buds, particularly on lilac, developed very rapidly. This would mean that the dormancy was readily broken at that time. The rapid transpiration caused by this relative— ly large leaf area undoubtedly had some effect on the -31.. Inortality rate, but the soil was kept moist at all times so that the cuttings could take in water to the full ex— tent of their own ability. That some other factor was of greater importance in their failure to root eems conclu- sive. Since all of these cuttings were taken from mature plants, there might be some explanation of their failure to root, according to the work of Thimann and Delisle (22). Their work on difficult plants shows that better rooting results from using the cuttings taken from very young trees. They also have found some correlation between the position of the cutting on the parent plant and its ability to root. These results favor the cuttings taken at the basal part of the tree, and the cuttings used in the experiments described in this paper were from the basal parts of the plants. SUMEARY l. Cuttings of willow, arbor vitae, lilac, and apple were made at approximately three week periods dur- ing the dormant season. These were treated with indole— butyric acid solutions of different concentrations. 2. During the early part of the dormant season (Nov- ember) treatment with indole-butyrio acid showed an in- jurious effect on willow and cedar. 3. Treatment of willow cuttings later in the dormant season caused more rapid rooting responSe. This effect was overcome when the cuttings were left about three to five weeks in the rooting medium. 4. Arbor vitae cuttings showed very low percentage of rooting at any time in the dormant season. There is some evidence that treatment aided root formation in the low degree that it occurred. 5. Lilac cuttings were not induced to form roots in any appreciable number of cases. Cuttings taken late in the dormant season leafed out very rapidly, but died with- out having formed either callus or roots. 6. Apple cuttings formed no roots at any time in the dormant season. Callus was usually formed, but soon be— came soft and dark colored. 7. Treatment with indole—butyric acid solutions can- not be recommended as of any practical value in aiding root development on cuttings taken during the dormant season in the Species of plants used in this series of experiments. 1' i l -mn-hfi -33.. BIBLIOGRAPHY 1. Avery, George S. Growth hormones in plants. Ohio Journ. Sci. 31; 317—332. 1937. 2. Avery, George 8., Paul R. Burkholder, and H. B. Creigh- ton. Production and distribution of growth hormone in shoots of Aesculus and I.'alus, and its probable role in stimulating camoidm activity. Am. J. Bot. 24: 51-58. 1937. 3. Bonner, J. The action of th we plant growth hormone. J. Gen. Physiol. _1: 63- 76. 1933. 4. Borthwick, H. A., K. C. Hamner, and a. W. Parker. His- tological and microchemical studies of the reactions of tomato plants to indole-acetic acid. Bot. Gaz. 9§; ”91-519. 1937. 5. Carlson, Margery C. liicrochemical studies of rooting and non-rooting rose cuttings. Bot. Gaz. _1; eu-so. 1929. 6. Cooper, wm. C. Effect on root formation of retreating cuttings with growth substances. Sci. _1: 39C. 1938. 7. Cooper, Wm. C. Hormones in relation to root formation on stem cuttings. Pl. Physiol. 19: 789-794. 1935. 8. Cooper, Wm. C. Hormones and root formation. Bot. Gaz. 99; 599— 614. 1938. 9. Cooper, Wm. C. Transport of root—forming hormone in woody cuttings. 91. Physiol. ll! 779-793. 1936. 10. Curtis, Otis F. Stimulation of root growth in cuttings by treatment with chemical compounds. Cornell Univ. Ag. Exp. Sta. Memoir _&: 1918. 11. Davies, W., G. A. Atkins, and P. C. B. Hudson. The effect of ascorbic acid and certain indole deriva— tives on the re: :eneration and germination of plants. Ann. Bot. n.s. i; 329- 351. 1937. 12. Goodwin, R. H. The role of auxin in leaf development in Solidago sp. Am. J. Bot. g3: 43-50. 1937. 13. in. 15. 16. 17. 18. 190 20. 21. 22. 23. -34- Hitchcock, A. E., and P. W. Zimmerman. Effect of Growth Substances on the Rooting Response of Cuttings. Boyce Thomp. Inst. Cont. §, 63-79. 1936. Kraus, E. J., Nellie A. Brown, and K. C. Hamner. His- tolcgical reactions of bean plants to indole-acetic acid. Bot. Gaz. 9s: 37o—uao. 1936. Loeb, Jacques. Influence of the leaf ‘pon root forma- tion and geotropic curvature in the stem of Bryophyl- lug_galycinum and the possibility of a hormone theory of these processes. Bot. Gaz. 63: 25.50. 1917. McCallum, Wm. B. Regeneration in plants. Bot. Gaz. .9Q: 97-120; 2u1—263. 1905. Pearse, H. L. Experiments with growth-controlling substances. I. The reaction of leafless woody cuttings to treatment with root-forming substances. Ann. Bot. n.s. g; 227—236. 1938. Schneider, Chas. L. The interdependence of auxin and sugar for growth. Am J. Bot. g5: 258-270. 1938. Snow, Albert G. Jr. Use of indole-butyric acid to stimulate the rooting of dormant aspen cuttings. Jour. For. 36; 6. 1938. Thimann, K. V. On the nature of inhibition caused by auxin. Am. J. Bot. g9: 407-412. 1937. Thimann, K. V. and James Bonner. Plant growth hor- mones. Physiol. Rev. lg; 524-548. 1938. Thimann, K. V. and A. L. Delisle. Vegetative prOpaga- tion of difficult plants. J. Arnold Arboretum g9: 116-137. 1939. Thimann, K. V. and F. Skoog. Studies of the growth hormone of plants. The inhibiting action on bud de— velopment. Proc. Nat. Acad. Sci. 195'71u-716. 1933. Went, F. W. Auxin, the plant growth hormone. Bot. Rev. 1;‘162—182. 1935. Went, F. W. Specific factors other than auxin affect- ing growth and root formation. P1. Physiol..l3: 55-80. 1938. Went, F. W. and K. V. Thimann, Phytohormones. New ----I~‘1 -35.. York:- Macmillan Co. 1937. 27. Zimmerman, P. W., Wm. Crocker, and A. E. Hitchcock. Initiation and stimulation of roots from exposure of plants to carbon monoxide gas. Boyce Thompson Inst. cont 0 5: 1-17 o 1933 o 28. Zimmerman, P. W. and A. E. Hitchcock. Effect of growth substances on the rooting response of cuttings. Boyce Thompson Inst. Cont. g; 63-79. 1936. Figure Figure Figure Figure Figure Figure l. 6. I -30- 'EYPLANATIONS OF FIGURES Salix cuttings (January) showing roots at the end of eleven days. Group on the left was treated with Auxilin, #0 mg. per liter. Group on the right was treated with Hor- modin, 20 mg. per liter. Salix cuttings (January) showing roots at the end of eleven days. Group on the left was treated with indole-butyric acid, 20 mg. per liter. Group on the right was un- treated. Salix cuttings (January) showing roots at the end of eleven days. The group on the left was treated with Auxilin, $0 mg. per liter. The group on the right was treated with Hormodin, #0 mg. per liter. Salix cuttings (January) showing roots at the end of eleven days. The group on the left was treated with pure indole-butyric acid, #0 mg. per liter. The group on the right was untreated. Salix cuttings (January) showing roots at the end of five weeks. The first group on the left was treated with Auxilin, 40 mg. per liter. The second group was treated with Hormodin, 20 mg. per liter. The third group was treated with pure indole—butyric acid, 20 mg. per liter. The group on the right was untreated. Salix cuttings (January) showing roots at the end of five weeks. The first group on the left was treated with Auxilin, SO mg. per liter. The second group was treated with Hormodin, 40 mg. per liter. The third group was treated with pure indole-buty- ric acid, #0 mg. per liter. The group on the right was untreated. Zhula cuttings (December) showing roots six— teen weeks after Auxilin treatment (80 mg. per liter). See Table VI. Figure 8. Thuja cuttings (December) showing roots six- teen weeks after indole—butyric acid treatment ( 40 mg. per liter). See Table VI. Figure 9. Thuja cuttings (December) showing callus but no roots. These were untreated and had been in the rooting medium sixtee. weeks. lulu See Table VI. Plate I Ficure l Figure 2 Plato II Figure 3 Figure 4 Plate III Figure 6 Plate IV Figure 7 Figure 8 Plate V '1‘. v c’ ‘ MICHIGAN STATE UNIVERSITY LIBRARIES E o I 3 77 6374 I I 3 1293