-,^,^-y-4^rv y :'J- \ m '■^^^ i*fj _^3 OforttcU Utttweraitg Slihtarg Mliata, ^tta ^nrtt Cornell University Library QK 746.C98 Stimuation of root growth in cuttings b 3 1924 024 759 361 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924024759361 S'VvCr: ^i^iJil^ii^::^^i|';^§i^»fe^^ "-■ J,'"/-'//';; ■ •),""„ .; ;Reprinted from Memplf No. .14, August, 191^ of Cornell Univeraty Ajpricuttiual ?■ ,--v '^^;..'fc,?■ feS'' STIMULATION OF ROOT GROWTH IN CUTTINGS BY TREATMENT WITH CHEMICAL COMPOUNDS A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY^^^ OTIS F.^CURTIS JUNE, 1916 ( Reprinted from Memoir No. 14, August, 1918, of Cornell University Agricultural Experiment Station) CONTENTS JPAGB Part I. Effect of inorganic compounds on root growth ■. 78 Effect of nutrient solutions 78 Influence of treatment with potassium permanganate 79 Reasons of stimulation 88 Possible effect on food supply 88 . Relation to rest period 88 Effect on rest period of twig as a whole 88 Effect on rest period of basal part 91 Stimulation of root growth in twigs that have passed the rest period 92 True rest shown only by buds of woody cuttings 93 Possibility that a treatment affecting the rest period may at the same time affect root growth 94 Effect of treatment on correlation between tops and roots 95 Effect of treatment on respiratory activity 97 Effect of treatment on growth of microorganisms 100 Comparison of the effect of potassium permanganate with that of other manganese compounds, and also with that of iron, of aluminium, of boron, and of phosphorus 104 Experiments with herbaceous cuttings 104 Experiments with woody cuttings 110 Continuous treatment 110 Limited treatment 110 Discussion of stimulation by compounds other than potassium permanganate Ill Manganese Ill Iron and aluminium 114 Sulfur 114 Boric acid 115 Phosphates 115 Part II. Organic nutrition of cuttings 116 Review'of literature 116 ■^iffect of limited treatment with sugar solutions on immature twigs 117 Treatment with different concentrations 117 Treatment for one day, for two days, and for twelve days 118 Influence of solutions of sugar for one, five, and fourteen days, respectively, in an incubator 1 20 Effect of continuous treatment with sugar solutions 121 Effect of sugar alone on immature twigs -121 Effect of sugar with manganese dioxide, boric acid, or ferric chloride 122 Effect of Umited treatment with sugar solutions on maturer cuttings 123 Forcible injection of sugar solutions 125 Discussion 126 Part III. General discussion with reference to practices commonly followed by green- house and nursery men 129 Summary 133 Acknowledgment 134 Bibliography 135 71 STIMULATION OF ROOT GROWTH IN CUTTINGS BY TREATMENT WITH CHEMICAL COMPOUNDS STIMULATION OF ROOT GROWTH IN CUTTINGS BY TREATMENT WITH CHEMICAL COMPOUNDS ^ Otis F. Curtis Increasing numbers of plants must of necessity be propagated vege- tatively, either because of the difficulties involved in propagation by seed or because many valuable forms do not reproduce true to seed. A large proportion of such plants are started as cuttings, which when placed under suitable conditions will develop into complete plants. In spite of the very general use and importance of this method of propagation, nq adequate investigations have thus far been made concerning the prin- ciples or the factors involved. When the importance of propagating plants by cuttings is considered, the value of any treatment that will stimulate root formation in cuttings is obvious. Many special methods of treatment have been suggested. For the most part these are based on practical experience, yet the reasons given for the practices described are often directly conflicting or are not well founded on physiological facts so far as these are known. It would seem, therefore, that some definitely directed research in the physiology of root formation in cuttings would be of value in this field. During some investigations to determine the effect of various chemical compounds on the rest period of woody plants, conducted in the Labo- ratory of Plant Physiology at Cornell University, it was found that when twigs of Ligustrum ovalifolium Hassk. were treated for a short time with a solution of potassium permanganate, roots developed to a greater extent than on the checks or on twigs treated with other compounds. These, results suggested the possibility of an investigation concerning the effect of various chemical compounds on the root growth of cuttings, and this has been the primary purpose of the present investigation. No experi- ments have been conducted to compare directly the value of different methods in common practice among propagators, as those methods cover 1 Contribution from the Laboratory of Plant Physiology, Cornell University. 75 76 Otis F. Curtis chiefly the moisture, temperature, and light relations, as well as the effect of the time and manner of making the cuttings. Some of those practices, however, have been briefly discussed in the latter part of this paper, in so far as they are related to the present investigation. In the experiments conducted by the writer, the cuttings, taken under different conditions as stated for each experiment, were subjected either to a limited or to a continuous treatment with the various compounds used. For limited treatment the time ranged for the most part from one to two days. The cuttings were then rinsed and placed in flasks or in sand. For continuous treatment the cuttings were placed in flasks containing the various compounds dissolved in distilled water, tap water, or full nutrient solution. A few tests were made in which the compounds were applied to sand in pots or flats. and the cuttings were placed directly in these, but no very decided results have thus far been obtained. For the limited treatments 10.0 cubic centimeters of solution was used unless otherwise stated. These treatments were administered either in tall glass cylinders, in large test tubes, in flasks, or in tumblers. The cuttings that were left in liquid media were placed in Erlenmeyer flasks of resistance glass, 250 cubic centimeters in capacity. The flasks were carefully cleaned and then covered with black paper. In the majority of cases ten twigs were placed in each container, and the con- tainers were set in trays on benches in the greenhouse. No special pre- cautions were taken for controlling the temperature and the relative humidity. The cuttings in sand were on a greenhouse bench with no bottom heat. In comparing the relative values of the several treatments, the average total root length per twig was taken as the best criterion. The number of roots and the average length of the individual roots were also determined in some cases, but these figures were not consistent. For example, in a given culture one twig might have a large number of short roots while another had a small number of long roots, and therefore, according to the number of roots, the two would be quite distinctive. The total root length per twig, however, proved to be fairly constant. The total green and dry weights per culture were determined in a few instances. Stimulation of Root Growth in Cuttings 77 In choosing a form with which to experiment, there were several points that had to be considered, as follows: 1. Large numbers of cuttings must be available. 2. The cuttings must be uniform with respect to size, shape, age, position on parent form from which they were taken, and conditions under which they were grown. Otherwise they would be likely to root unevenly. 3. Uniformity in rooting was a very important factor, for without uniformity there was found to be a large probable error which would necessitate the using of great numbers of twigs for each treatment, thereby complicating and increasing the mechanical labor involved. In view of the fact that in these experiments over twelve thousand cuttings of Ligustrum were actually used in addition to four thousand cuttings of other species, it is obvious that the problem would be rendered more difficult if it were necessary to increase the number of cuttings as much as five to ten times. 4. It was important that cuttings should be used which would root in as short a time as possible. The field of research is new, and if one were forced to wait two months or more for results on preliminary tests the experiments would be long drawn out or it would be too late to obtain cuttings for a second set. The writer experienced this trouble for three consecutive years, even with comparatively quick-rooting Ligustrum. On those occasions the December freezes had killed the twigs on the bushes at about the time when the first set of cuttings was showing results. Furthermore, the immature condition of the twigs rendered impossible an earlier beginning of the preliminary experiments. The use of hardy forms would not solve this difficulty, for, tho freezing would not kill the twigs, it would bring them out of the resting condition, which would be detrimental to rooting. 5. It was necessary to choose a form that would root readily. Com- parative results could thus be obtained which would allow for manipulation to determine more nearly the optimum, treatment. Ligustrum ovalifolium Hassk. most nearly fitted the requirements outlined, and therefore it was used in the majority of the experiments. The following forms also were used: Cydonia oblonga Mill., Ribes Houghtonianum Jancz., Pyrus malus Linn., Prunus cerasifera Ehrh., Kerria japonica DC, Evonymus europaea Linn., Berberis Thunbergii DC, 78 Otis F. Cuhtis Diervilla sp., Populus nigra var. italica Du Roi, Spiraea Vanhouttei Zabel, Forsyihia sp., three species of Salix, Iresine Herbstii Hook, f., and Lycopersicum esculentum Mill. PART I. EFFECT OF INORGANIC COMPOUNDS ON ROOT GROWTH EFFECT OF NUTRIENT SOLUTIONS In order to be able to follow more closely the development of the roots, it was necessary to grow the cuttings in liquid media. To determine whether a nutrient solution might increase root growth, various strengths of Knop's and Crone's solutions were employed, as indicated in table 1. Cuttings of Ligustrum ovalifolium were taken from two hedges which had been growing under somewhat different conditions. The twigs from one hedge (Column B in table 1) soon produced a strong growth of tops, resulting in such a large increase in transpiration surface and TABLE 1. Effect of Nutrient Solutions on Root Growth in Woody Cuttings (Continuous treatment from November 21, 1914, to January 8, 1915. Ten twigs to a culture) Solution Average total length of roots per twig (millimeters) Number of twigs rooted Number of twigs rooted Crone's solution,* 0.5 per cent . 1 per cent 0.01 per cent Knop's solution,! 0.5 per cent . 1 per cent 0.01 per cent Modified Knop's solution, |0.5 per cent. . . 1 per cent . . 0.01 per cent. "Tap water .' . Distilled water Dead 110±26.4 251±23.8 Dead 72±27.5 260±28.5 218±26.8 203±37.5 10 7 10 10 10 Dead Dead 5 Dead Dead 4 Dead Dead 3 3 * Crone's solution contained salts in the following proportions: KNO3, 4; CaS04, 2: MgSOi, 2; Fea(P04)!, 1; Cai(P0()2, 1. t Knop's solution contained salts in the following proportions: CaCNOs)!, 1 ; KNOs, 0.25; MgSOj, 0.25: KH2POJ, 0.25; Fe2(P04)j, 0.06. , t The modified Knop's solution was similar to the preceding but contained five times the proportion of iron phosphate. In both cases iron phosphate was used in place of iron chloride. Stimulation of Root Growth in Cuttings 79 in loss of water that the flasks containing them became dry. The twigs in some of the cultures had produced roots. Such cases were noted but no measurements were taken. The twigs from the other hedge (Column A in the table) remained dormant for some time and the buds were just starting at the time when the roots were measured. It is shown fairly clearly in table 1 that nutrient solutions of the strengths used in culture work with seedlings are distinctly injurious to woody cuttings. This injurious effect of nutrient solutions, especially at the higher concentrations — 0.1 per cent or above — was further proved by subsequent experiments in which similar results were obtained. In four experiments, sixteen out of a total of twenty cultures showed distinct retardation of growth on the addition of nutrient solutions. Even single nutrient salts, such as CaCU, NaH2P04, KNO3, and KH2PO4, in very dilute solutions, showed marked retardation of the root growth. No very extensive experiments with nutrient solutions were attempted, since the few results obtained' showed clearly that such treatments tend to be .more injurious than beneficial to woody cuttings. A few experiments with herbaceous cuttings indicated that these are less injured by con- centrations up to 0.2 per cent than are woody forms. The root development in woody cuttings is usually lessened by an increase in concentration of the nutrient solution, as shown by the figures in table 1. This is somewhat comparable to the effect of high concen- trations of soil or nutrient solutions on seedlings observed by a number of investigators — PoUe (1910), Harris (19i4), Stiles (1915), Brenchley (1916), and many others. The writer has found, however, that woody cuttings are much less tolerant of the stronger concentrations than are seedlings. A discussion of the causes for increase or decrease of root growth resulting from a change in concentration of nutrient solutions, and reports of further experiments, are reserved for a later paper. INFLUENCE OP TREATMENT WITH POTASSIUM PERMANGANATE As already stated, it was found that twigs of Ligustrum ovalifolium developed more extensive roots when treated with potassium permanganate than when treated with a number of other compounds. Potassium permanganate was therefore tried in the first of these experiments. In 80 Otis F. Curtis order to determine the limiting concentrations, as well as the optimum time of treatment, single lots of ten cuttings each of Ligustrum ovalijoUum were treated as indicated in table 2: TABLE 2. Influence of Concentrations and of Duration of Treatment with KMn04 on Cuttings of Ligustrum ovalifolium (Cuttings taken on November 29, 1913) Cuttings grown in solutions in flasks, or treated and placed in tap water. Roots measured January 14, 1914 Solutions used and time of treatment Average total length of roots per twig (millimeters) Average total length of roots relative to check as unity Average number of roots to the twig Average length per root (milli- meters) Check. . 88.4±18.7 1.00±0.21 8.8 10 Treatment 24 hours KMn04, 2 per cent .... 361.4±31.9 4.09±0.36 15.2 23 8 317.0±15.0 3.59±0.17 13.9 22 8 0.5 per cent. 276.0±36.5 3.12±0.41 10.8 25 6 Treatment 5 days KMnO,, 0.50 per cent 234.0±31.2 2.65±0.35 9.6 24.3 0.25 per cent 217.0±30.6 2.45±0.35 11.8 18.5 0.10 per cent 60.0±11.0 0.68±0.12 4.3 14.2 0.05 per cent 26.0±13.9 0.29±0.16 3.2 7.9 Continuous treatment KMn04, 0.10 per cent 294.0±25.0 3.33±0.28 12.6 23.4 0.05 per cent 183.0±14.8 2.07d=0.17 13.3 13.8 0.01 per cent 145.0±14.5 1.64±0.17 9.1 15.8 0.005 per cent 176.0±16.6 1.99±0.19 10.2 17.1 0.001 per cent 115.0±21.3 1.30±0.24 8.5 13.5 0.0001 percent 44.0±11.5 0.50±0.13 4.1 10.8 Stimulation of Root Growth in Cuttings Table 2 {concluded) 81 Cuttings treated and placed in sane January 19, 191^ . Roots measured Solutions used and time of treatment Average total length of roots per twig (millimeters) Average total length of roots relative to check as unity Average number of roots to the twig Average length per root (milli- meters) Check 149.0 13.0 11.4 Check 143.0 11.2 12.8 Average of checks 146.0±12.1 l.OOiO.08 12.1 12.1 Treatment 24 hours 183.0±18.5 1.25±0.13 9.8 18.2 352.0±15.0 2.41±0.10 17.3 20.4 0.5 per cent 211. 0± 7.2 1.45±0.05 14.0 15 Treatment 5 days ■>-- , ■ ■■'■■ • : ~''* KMnOi, 0.50 per cent 287.0±20.6 1.97±0.14 11.4 25/2 0.25 per cent 212. 0± 6.6 1.45±0.05 10.6 20.0 0.10 per cent 199.0=t22,l 1.36±0.15 12.0 16.6 0.05 per cent 111.0=tl2.5 0.76±0.08 8.0 15.3 As shown by table 2 and also by figure 1, potassium permanganate causes a marked stimulation of root growth in Ligustrum in the 24-hours, 5-days, and continuous treatments. With but one exception the maximum stimulation occurred in the strongest solutions, indicating that higher concentrations might give even better results.. Subsequent experiments have shown, however, that concentrations close to the optimum were used in the 24-hours and continuous treatments. The optima in these two gave results of 4.09 ±0.36 and 3.33 ±0.28, respectively, relative to the check as unity. The difference between the check and the best treatment is over eight and a half times the probable error. The cuttings placed in the sand, as shown by the same table, indicated a lesser degree 82 Otis F.- Curtis of stimulation, but the optimum results show a root growth 2.41 ±0.10 and 1.97±0.l4 times that of the control. Tho but single cultures were used, and there were only five twigs in the sand cultures — except in the check, in which the number was sixteen — yet the results were unexpectedly consistent, showing a continuous change with change in concentration with one unimportant exception in which the culture having a slight increase in concentration had a somewhat lower root length. In four cases the weakest concentrations gave a root length shorter than Fig. 1. LIGUSTRUM CUTTINGS AS AFFECTED BY TREATMENT WITH POTASSIUM PERMAN- GANATE Upper row; Cuttings treated for twenty-four hours in 1-per-cent KMnOi; the five at the left then Vlaced in sand, the remainder in tap water Lower row: Check. Cuttings left for twenty-four hours in tap water; the five at the left then placed in sand, the remainder in tap water that in the check. These dilutions were so great and the results so similar to those of the check that the latter may indicate merely a probable variation between separate checks. The stimulation here obtained has been fully confirmed by later ex- periments in which duplicate and triplicate cultures were made, and by others in which one hundred or more twigs were used in each treatment. This table was given in preference to the others, as the single experiment covers a wider range of concentrations and lengths of treatment than any one subsequent experiment. Stimulation of Root Growth in Cuttings 83 In addition to the experiment just reported, Ligustrum was used in nine other experiments in which the cuttings were treated with potassium permanganate. In these experiments, some of which are described later in this paper, there were fifty-two treatments with potassium permanganate at different concentrations, usually in duplicate, and for different lengths of time. All these fifty-two treatments, with the exception of three in which the concentrations were too high, showed stimulation of root growth above that obtained in the checks. Since Ligustrum was so clearly benefited by the treatment, a few ex- periments were made to determine whether or not other species would be similarly affected. Some of these experiments were started in the latter part of winter, which proved to be too late for successful rooting. Cuttings of Ribes, Cydonia, and Berberis formed roots and the results seemed to indicate some stimulation, but there was such wide variation between duplicate cultures that no definite conclusions could be drawn. Treatment with potassium permanganate stimulated root growth in cuttings of a number of woody plants, as is shown in table 3. With several forms the treatment not only resulted in an increase in root length per twig, but also caused a larger proportion of cuttings to take root. The results obtained with Prunus cerasifera are shown in figures 2 and 3. All the forms here reported can be fairly readily propagated by hardwood cuttings if these are taken at the proper time of year and if sufficient precautions are used in setting and handling them. It is apparent, however, that if the cuttings are taken late in the season and placed in the sand, with no especial precautions regarding the supply of bottom heat, moisture, light, and temperature, a treatment with potassium permanganate results in a marked improvement in root pro- duction in respect both to the aggregate length and dry weight of the roots of a given twig and to the proportion of twigs forming roots. In addition to the experiments reported in table 3, an experiment with Ligustrum shows very clearly this improvement under adverse con- ditions. Cuttings were taken very late in the season — March 16, 1917. Contrary to the usual practice, the base of each twig was cut square across with rather duU pruning shears and no clean diagonal cut was made with a sharp knife. The twigs were treated as indicated in table 4 and then placed in sand. Because of the lateness of the season and the rather careless treatment, the check twigs failed to develop roots as freely as M Otis F. Curtis & h O Ph > H a M Q O < ^ a -<1 H > -■1 < K P4 t/J & „ le t; m m PL, S ^- E o ^ « m t3 fe ;«g Uh ^ H n p "^ a i;' ^ &w c; „ ?1 u o 3 -< O fe a S ^ >^ > h o jllBo on puB si^ooj [ ( - i • ll I I pasnofi'BO ^nq s^ooj ou q^iM sSiAix pa^ooi s3iM^ JO igqran^ (sm'Bja) am) jad ■51 bis 3 |i U^ M^ h-* < H CD I w II I I :| rtja O w ea ■^ S S k^ V CO CO CO I to H PQ a " 1 ^> ■3 2 o S 03 a "■" o 8 S J3d sSUi) JO jsquitifi^ ^ fM Mlla 1 ■£ 3113 P u O 0) 1 s Stimulation of Root Growth in Cuttings 85 o 1 « O 1 ^ II CO « « »o m "M t- 1 lO 1 (N 1 CM 1! 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(N w CM F-l,H S 5 1 III aSBiSAv ? g § 8 8 8 to CM 8 Ih ■ d o o d OS d '* Tt 1 « ■* >co oo oo o oq oq lOO oo H & ^""■i S puB X sajn^[no oo do do do d 00 d rjioo OOO Tff^ e! " 1 !z; WCD cooi S g* Z fe 8 ■■ 6 : 1| .a 1. ;? : ftH o u "o ■3g. ■§ T3 H § fel : g d ^ a gP. e 1 1 o 1 1 1 ■9o << 11 It o d O n if d 9 si 6° 8 d O G 1 1 * £ •S 9 s ^f^ s 1^ S ti fi e B M M « M M 90 Otis F. Curtis table and by figures 5 and 6, the treatment with potassium permanganate markedly increased root growth but had little or no effect on the tops. In the cultures with 0.01 and 0.001 molecular potassium permanganate, the roots were visible about ten days earher than they appeared in the checks. This might seem to have been due to an effect on the rest period. If this were the case, however, the treatment had not affected the whole Fig. 5. RELATION OF STIMULATION TO REST PERIOD Upper row: Cuttings taken early in rest period, October 15, 1915. Twigs at left, check twigs, kept continuouslyiu distilled water until December 27, then placed in tap water. Twigs at right kept in O.OI mol. KMnOi until December 27, then placed in tap water. The buds are ju8t.starting to grow in both lots, showing ■approximately equal growth, while the root growth is very much greater in the treated twigs Lower row: Cuttings taken at end of rest period, on Decembers. Twigs at left, check twigs, left for twenty-four hours in distilled water, then transferred to flasks containing distilled water. Twigs at right, treated for twenty-four hours in 0.1 mol. KMn04, then transferred to fiasks containing distilled water. The shoots are well formed in both sets, while the roots have not grown so far as in the twigs shown above. Both roots and tops showed better growth in the treated twigs twig, for, tho a few buds were started by December 18, these were equally developed on all the twigs whether treated or not. Altho these few buds opened by December 18, most of the buds did not grow until about the first of March. The equal development of the buds indicates that the treatment has no effect on the resting condition of the whole twig. Effect on rest period of basal part. — The only possibility remaining, so far as an effect on the rest period is concerned, is that the treatment Stimulation of Root Growth in Cuttings 91 may have started growth in the basal part, leaving the tops dormant, for in some instances the roots of the treated twigs started from three to ten days earlier than those of the checks. This difference in time of starting, however, is insufficient to account for the great difference in growth. As shown by table 5, the roots of the treated twigs when they had been visible less than two weeks (on November 29) showed growth more than three times that in the checks when the roots of the latter had been visible for at least three weeks (on December 18). Furthermore, as a result of the exposure on November 29, when the cultures were being photographed, the roots in the permanganate cultures Were partly broken and dried, so that when measured the second time many of the roots first formed were completely rotted or the ends had died back and new branches were starting. The checks were not injured for no roots had started at that time. In spite of this injury to the treated twigs, the growth was much better in these than in the checks and continued better as long as the cultures were kept. This great difference in root length at the time of the final measurements, which were taken more than twelve weeks after growth had commenced, cannot be explained on the ground that the treated twigs had a start of ten days. Stimulation of root growth in twigs that have passed the rest period. — The following experiment^ furnish additional proof that the stimulation of root growth by potassium permanganate has nothing to do with the rest period. Cuttings of Ligustrum were taken on December 8, 1915. The bushes from which they were taken had been twice subjected to several days of freezing weather, and, as is brought out later, these twigs had passed out of the resting condition. One hundred and fifty twigs were treated for twenty-four hours in water, and the same number in 0.1 molecular potassium permanganate. After being rinsed, duplicate lots of ten twigs from each treatment were placed in distilled water in a cool room at a temperature of from 5° to 10° C, thirty twigs of each treatment were placed in sand in a somewhat warmer, shaded greenhouse at from 10° to 15° C, arid one hundred twigs of each treatment were placed in sand in a still warmer, unshaded greenhouse at from 18° to 22° C. The buds of the twigs in the warmest house started almost immediately after the cuttings were set out. They were not in the resting condition, and the treated and the untreated twigs started equally; but by the 92 Otis F. Cuetis last of January, when this lot was taken up, the twigs treated with potas- sium permanganate showed accelerated top growth. No actual measure- ments were taken of this lot, but the root growth was fully twice that of the checks. The buds of the lot placed in sand in the shaded house opened soon after these. There was no apparent difference in time of starting between the treated and the untreated twigs, but at the end of about ten days those treated with potassium permanganate clearly showed increased top growth. Similar results were obtained with the cuttings placed in water in the cool room. Results obtained from experiments conducted in the spring of 1917 were very similar to those just described. On cuttings treated either continuously or for a limited time with potassium permanganate, an increased growth of roots ranging from 2.17 to 11 93 times that in the checks was produced. Results following the placing of the cuttings in sand after treatment for twenty-four hours in 0.1 molecular potassium permanganate are shown in figure 4 (page 87). The buds on all the twigs in these experiments opened very soon after the twigs were set out, and even before the roots started. For this reason the stimulation cannot be explained as due to an effect which may bring the basal, part of the twig out of the resting condition and leave the tops dormant. These twigs apparently had completely passed the rest period, and therefore any stimulation obtained cannot have been due to an effect on the rest period. Furthermore, it has been shown by Howard (1915 b), and by other investigators, that, so far as the rest period is concerned, treatments which will exert a stimulating effect if applied during the rest period will have no effect, or may even produce a retarding effect, if appUed at or near the end of that period. ' True rest shown only by buds of woody cuttings. — The effect of stimulation of root growth is evidently independent of the rest period, as already stated. So likewise are callus formation and root development, which proceed in untreated twigs whose buds are dormant. The buds of cuttmgs taken on October 15 (fig. 5) did not start general growth until about March 1, approximately four months after the cuttings were taken, while the buds of twigs taken on December 8 started almost immediately. Those taken on December 8 were well started by December 22, only two weeks after the cuttings were made and more than two months earlier than those taken on October 15. The roots in the untreated twigs, however. Stimulation of Root Growth in Cuttings 93 developed at approximately the same rate whether the twigs were taken before or after the end of the rest period. In both cases the roots were about equally developed forty-five days after the cuttings were made. Very sim lar results were obtained with cuttings of Prunus cerasifera, Comus stolonifera Michx., and Evonymus europaea, and with regard to callus formation in Pyrus malus. Cuttings of Evonymiis taken in the latter part of September had a strong root system developed by March 1, yet the buds did not break until May 1.; while the buds on cuttings taken from the same bush on December 29 started growth within ten days, but as no root system was developed a large number of the twigs withered. Root and callus forma- tion in cuttings, therefore, appear to be independent of the rest period, as stated above. There is a possibihty that the wound shock at the cut end brings that part out of the rest period; Howard (1915 b) and other investigators have found that such a stimulus may shorten the rest period. But in some cases the roots develop at some distance from this- wounded tissue, and therefore this explanation is insufficient. The cut of course exposes the adjacent tissue to better aeration, resulting in a greater oxygen supply as well as a more rapid loss of carbon dioxide, which might serve to start growth in the dormant cambium. When the twig is immersed in water any inhibiting substances may be washed- out of the tissues or the ready access of oxygen may oxidize them. Such an explanation is not entirely satisfactory, however, for if the twig is kept in a moist chamber, not only will both cut ends develop a callus but also cork cells will be formed at the lenticels, indicating cellular activity in regions far from the wounded part; yet in no cases do the buds develop, even when submerged in water or when situated very close to the cut end. Simon (1906) has shown that root and callus formation proceed when the buds are in a resting state. Howard, in his work on shortening the rest period of cut twigs, makes no mention of any effect on their rooting ability. In a report on experiments with several species of potted woody plants (Howard, 1915 b), he states that treatments which stimulated the tops had no effect on root growth. He does not say, however, how he determined this, and he gives no data. Possibility that a treatment affecting the rest period may at the same tim£ affect root growth — On the other hand, the fact that the stimulation obtained was independent of an effect on the rest period is no proof 94 Otis F. Curtis that treatments which would affect the rest period might not also have an effect on root growth. In order to test this point, Ligustrum twigs were taken on November 13 and tied in bundles of twenty twigs each. These bundles were suspended in water for two hours, at the depths and temperatures indicated in table 6, and were then placed in sand in the cutting bench. It is to be noted that at each temperature the top TABLE 6. Effect of Warm-Bath Treatment on Dormancy and Root Growth (Treatment lasuing two hours; (a), from 2s to 3 centimeters at base immersed; (c), entire twig immersed. Twenty twigs per culture) Treatment Total root length per twig (milli- meters) Order of greatest root growth Total top length per twig (miUi- meters) Order of greatest top growth 1 (a) Check 20° C 1 (c) Check 20° C 2 (a) 35° C 2 (c) 35° C 3 (a) 40° C 3 (c) 40°C 4 (a) 45° C 4 (c) 45° C 10± 3.8 6± 3.4 38± 8.9 20± 6.1 55±10.8 35d= 7.6 2± 0.4 6±2.3 4±1.6 23±3.8 20db3.4 29±4.1 28±4.6 3±1.1 growth, as well as the root growth, was better in those twigs of which the bases only were immersed. The order of greatest growth of roots follows almost identically that of the greatest growth of tops. The results indicate that some treatments which start or increase growth in the entire twig may correspondingly increase root growth. It does not seem possible, however, to start growth at the base of the twig by the warm-bath treat- ment and leave the tops dormant. Very similar results as to root growth were obtained with twigs taken on September 10 and treated in the same manner. No notes as to the effect on top growth, however, were made at that time. Effect of treatment on correlation between tops and roots Another possibility that has suggested itself is that the treatment may have brought about a change in correlation between tops and roots, an effect that might be due to a divergence of food movement from tops Stimulation of Root Growth in Cuttings 95 / to roots. The tables already presented show that this is not the case with the permanganate treatment. In the experiment recorded in table 5 (page 89) there is practically no difference between top growth in the treated twigs and in the checks, and yet there is a very great difference in root development (fig. 5, page 90). The results obtained in the warm-bath treatment (table 6), and in four cases in which the twigs were treated with potassium permanganate after the rest period, showed that both top and root growth were better in the treated twigs than in the checks (fig. 5). This proves clearly that in these cases the roots did not develop at the expense of the tops. It is to be noted here that if the treatment is applied early in the rest period it generally has little or no effect on top growth even for some time after the buds start; while if it is applied after the rest period, tho the buds may start equally the shoots of the treated twigs usually soon surpass those of the checks in development, so that the length and the dry weight of both the tops and the roots exceed those of the checks. With the stronger concentrations, however, a point is apparently reached where there is still stimulation of root growth but at the same time a distinct retardation of top growth. This is well illustrated in table 10 (pages 106-7), which shows that the stronger solutions of both potassium permanganate (KMn04) and ferric chloride (FeCls) increased root growth but clearly decreased top growth. Weaker solutions stimulated both tops and roots. Accompanying the increased growth of tops and roots there is at first a lessened dry weight of the stem. After continued growth, however, the stems become heavier, probably due to the increased photosynthetic activity of the larger tops. On the other hand, F^aivre (1871) has shown that in some cases the tops may develop at the expense of the roots. He found that after the buds had formed shoots there was insufficient food left in the stem for root formation. The rooting process seems, as a rule, to be slower than growth of tops, so that if the tops are in a condition to grow they will develop faster than the roots and deplete the food supply, or else, as suffi- cient water for the increased transpiration is not supphed, the shoots will die, causing the twigs to wither at the same time. As stated earlier, cuttings of various species were taken in late autumn before the end of the rest period and were set out in the greenhouse benches. The tops of most of these remained dormant until March or later, while the roots — or the callus in the case of the twigs of Pyrus — proceeded to develop 96 Otis F. Cuetis vigorously. The buds on the twigs of the same forms taken in January started to grow within two weeks after the cuttings were set out. In the majority of such cases the young shoots, as well as the cutting itself, soon withered and died from lack of water. With Ligustrum, however, root development was so rapid that the majority of the twigs remained ahve. Whether this is a matter of food distribution or merely a water relation is not clearly shown by these experiments. Effect of treatment on respiratory activity The explanations of stimulation of root growth which have thus far been discussed have been shown to be insufficient to fully account for the increased growth resulting when cuttings are treated with potassium permanganate. A more probable explanation appears to be that the manganese increases the rate of respiration in the treated twigs or causes more complete oxidation, thereby preventing the accumulation of inhibiting or toxic products of catabolism. It has long been recognized that good aeration is essential for root growth either of seedlings or of cuttings, and especially of the latter. De Saussure (1804), early in the nineteenth century, pointed out the fact that oxygen is necessary for root growth of seedlings. He grew chestnut seedlings in flasks containing air, nitrogen, hydrogen, and carbon dioxide, respectively. The carbon dioxide was distinctly injurious to the roots, while the other gases did not allow for growth and the roots soon died. It is commonly understood that one of the chief reasons for the practice of soil drainage is to provide better aeration. Stoklasaand Ernest (1908) have shown that poor aeration of root systems leads to the formation of compounds such as acetic and formic acids within the roots, which may result in their death. Harris (1914) and a number of other investigators have shown that the growth of roots and the depth to which they penetrate are closely correlated with the water content of the soil and the height of the water table. Harris, as well as several other investigators, states that this is probably due to the fact that a high water content excludes oxygen and the lack of oxygen limits root development. There is apparently an even greater need for good aeration in soils in which cuttings are placed, than exists for seedlings. This fact has been Stimulation of Root Growth in Cuttings 97 recognized for some time. Sorauer (1895) lays strong emphasis on the need of aeration, especially for herbaceous cuttings. Kiister (1903) states that cuttings always callus more readily in a moist atmosphere than in water, tho he considers transpiration, as well as oxygen supply, a determining factor. Klebs (1903) found that cuttings of Salix pentandra develop roots only at the cut end of the twig, not thruout its length as do cuttings of Salix alba vitellina pendula (Spath.) ; however, if the cork layer is removed at a point some distance from the cut end, roots will develop in or near this region. He explains this on the theory that water is the determining factor. He suggests that on removal of the impervious cork layer, water enters the twig, and the roots then develop. He does not reaUze, ap- parently, that when the twig is immersed the tissues must be practically saturated — certaialy of greater water content than those of the basal end when the cutting is suspended in moist air, yet under the latter con- dition roots wUl develop freely. The more probable explanation is that the removal of the cork allows for better aeration, thus increasing the oxygen supply at that point as weU as supplying an outlet for the carbon dioxide produced in respiration. The work of Appleman (1916) is sug- gestive in this connection. He found that tubers of Solanum tuberosum were caused to sprout when the skins were removed. The results he explains as due to increased permeability to oxygen. It is very generally known that when potassium permanganate comes in contact with organic matter, manganese dioxide is precipitated and oxygen is liberated. Some of the most recent work on this line is that of Bunzell and Hasselbring (1917), who found that various organic com- pounds — • glucose, alcohol, and others — will decompose potassium permanganate with the formation of manganese dioxide and in some cases a straw-colored solution containing manganese and giving strong oxidase reactions. The cuttings treated with potassium permanganate were very much blackened by this precipitate of oxide of manganese, which clings firmly to the surface of the twig. Sections of the twig showed the oxide de- posited |in the xylem cells for a distance of from one to four millimeters from the cut end. They showed also a sUght penetration into the cortex cells thruout the length of the immersed part. 98 Otis F. Curtis Most manganese compounds are active oxygMi carriers or are easily changed to such. This is especially true of potassium permanganate and manganese dioxide. The latter, which is precipitated on and in the twigs as just described, will almost instantly blue guaiacum or redden aloin and decompose hydrogen peroxide. It can therefore act as an oxidase, a peroxidase, or a catalase. Bunzell and Hasselbring (1917), as stated above, have found that various organic compounds — glycerin, tyrosine, ^ peptone, glucose, and others — will decompose potassium permanganate, producing a precipitate of manganese dioxide and also a solution containing some manganese which will give oxidase, peroxidase, and catalase reactions. Whatever stimulation was obtained in these experiments was probably due chiefly to this oxide adhering to the walls or precipitated within the cells. It is probable that it was not due to the permanganate itself, as this seemed very toxic to the roots of seedlings or of herbaceous cuttings. In such cases the roots were formed when the purple color was still present in solution. With the woody cuttings, how- ever, all the purple color had disappeared before the roots started, and the solution contained only the dark brown or black oxides of manganese. In continuous treatments the solution may have contained also some manganese available for oxidizing, as found by Bunzell and Hasselbring. In those cases in which the treatment lasted for only a limited time, the twigs were thoroly rinsed before being placed in the final medium, and therefore no permanganate was carried over. It has been shown by several investigators that manganese is closely associated with oxidation reactions occurring in plants. Bertrand (1897) found it to be an important constituent of the oxidizing enzyme laccase. When a sample of laccase was not very active, its activity was increased by adding a small quantity of manganese sulfate. Schreiner, Sullivan, and Reid (1910) found that the presence of manganese markedly increased oxidation by soils and by plant roots. This occurs, however, only in neutral or alkaline soils (Skinner and Reid, 1916). McHargue (1914) found manganese to be especially abundant in the seed coats of various seeds next to the cotyledons, and suggested that it played an important r61e in respiratory activities during germination. He found that the oxidase activity of different parts of seeds, tubers, roots, and stems varied directly with the manganese content. Kastle (1910:122-131) cites sev- Stimulation of Root Growth in Cuttings 99 eral instances in which investigators have found that manganese is im- portant in the action of a number of oxidases. It would therefore seem reasonable to explain the marked stimulation obtained with treatment by potassium permanganate as due to the effect of the manganese dioxide, deposited on and in the twig, on the respiratory activity of the cutting, either by directly hastening respiration or by causing more complete oxidation and thereby preventing the accumula- tion of partially oxidized, toxic, or inhibiting products of catabohsm. Such an effect occurring within the tissues would be very similar to that which may occur in the external medium in the presence of microorgan- isms, as discussed later. The lack of stimulation with certain forms, notably Salix, may be explained on the theory that the oxygen supply does not become a limiting factor with such forms, and therefore, as the effect of the manganese is merely to increase oxidation, no increased growth is to be expected. The fact that Cannon and Free (1917) found Salix to be peculiar in that its roots were uninjured by lack of oxygen or excess of carbon dioxide, lends weight to this explanation. Effect of treatment on growth of microorganisms The increased growth of roots when cuttings are treated with potassium permanganate cannot be due to any direct effect in keeping the solution sterile and free from microorganisms, altho in experiments with Pyrus malus, Evonymus europaea, and Forsythia sp., and also in other experi- ments not here reported, the cuttings treated with permanganate remained alive and vigorous for a much longer period without root formation than did the checks. Pyrus twigs remained in good condition for from six to ten months, while most of the check twigs were rotting after from one to three months. On close examination the treated twigs appeared to be heavily coated with a slimy growth of fungi and bacteria, yet they were*aUve and healthy. The cultures were certainly not sterile; in fact, in one experiment several of the cultures that showed the greatest root growth apparently had a growth of microorganisms even greater than that in the cultures not treated with permanganate. As shown in table 7, the twigs in the culture with 0.004 molecular cane sugar were slimy to a slight extent on December 18, and those in the culture with 0.004 molecular cane sugar plus . 004 molecular potassium permanganate were ]00 Otis F. Curtis f S S -a ffl I o .53 ■^ .a pq < I o O 12; o S 3 E-i '-' o pC :.a ^ g I s ^^ a i I I I e op V a«22 ■fe ° o ^ v -a .9-5 tn <-ri o lis o p |1 ~.a o K 3 5 a 03 « > o -e -SS'S 3 M-S > (M CD OCO WO 00 (N li30 *-■ pd -t^ ^ ^1 o ♦J 0010 00 o -H S OCO 13 a s w «*-< pd 1! o CD CM 00 <3 -H CD d C5 OtH I>CD ■I IN 00 (NO CDTO Stimulation of Root Growth in Cuttings 101 Fig. 6. EFFECT OF TREATMENT WITH POTASSIUM PERMANGANATE ON ROOT GROWTH AS CORRELATED WITH ITS EFFECT ON GROWTH OF MICROORGANISMS Upper row; Lower row: Cuttings grown in 0.004 mol. cane sugar Cuttings grown in 0.004 mol. cane sugar with 0.004 mol. KMn04 102 Otis F. Curtis much more slimy, with a mat of growth around the stem; yet the roots of the latter, were much better developed. When measured later, on March 11, the twigs treated with permanganate and sugar had a root growth 3.39 times that of the twigs treated with sugar alone (fig. 6), which were noticeably less sUmy; and the former had a growth 1.94 times that of the plants not supplied with sugar, the stems of which were not sUmy to the touch. It would seem, then, that the growth of microorgan- isms does not of itself injure the cuttings, but that for some reason they become weakened and die, following which fungous growth sets in, attacking the dead twigs. It, is very probable that the presence of soluble organic material allows for a much increased growth of microorganisms,- which would result in an increase of carbon dioxide and various toxic compounds and a decrease of oxygen. This increase of carbon dioxide has been found to occur in soils wher^ green crops are plowed under or in ground that has been heavily manured (Boussingault and Lewy, 1852, and Lau, 1906). Kidd (1914) found that the carbon-dioxide content of the soil was very much increased when clipped grass was buried in a pit beneath it; after seven months the soil air of such a pit contained 8 per cent of carbon dioxide As stated earlier, De Saussure (1804) found that an increased carbon- dioxide content retarded root growth in seedlings. Boehm (1874), Chapin (1902), and Cannon and Free (1917) also have reported retardation of root growth in the presence of carbon dioxide. It would seem very probable that the increased carbon-dioxide content resulting from the action of microorganisms on the organic material in the medium is injurious to cuttings. Kidd (1914) has shown that carbon dioxide has a retarding effect on respiration, and that this effect is enhanced by a decrease in oxygen content. Microorganisms growing in a medium containing organic matter not only increase the carbon-dioxide content but also decrease the percentage of oxygen. This decreased oxygen supply, coupled with an increased carbon-dioxide content, would tend to retard respiration or to retard the further oxidation of toxic products, thereby causing the death of the twigs, which later might be attacked by saprophytic forms or might be so altered as to be easily attacked by semiparasites. Furthermore, Chapin (1902) observed that the roots of plants are more resistant to a high carbon-dioxide content than are the tops. This Stimulation of Root Growth in Cuttings 103 may partially explain the fact that parts of tops, such as cuttings without roots, are more quickly injured in a soil with a high organic and carbon- dioxide content or a low oxygen content than are rooted cuttings or seed- lings. It is certainly true that cuttings are less tolerant of poor aeration. Kidd (1914) found that a rise in temperature lessened the inhibiting effect of carbon dioxide. This might partially explain the beneficial effect of bottom heat, which is discussed later. In a few preliminary experiments with cuttings inclosed in chambers with increased oxygen content and in chambers with increased carbon- dioxide content, respectively, the cuttings in the increased carbon-dioxide content were distinctly injured, while those in the increased oxygen con- tent remained healthy much longer than did cuttings in normal inclosed air. In none of these preliminary experiments did any of the Ligustrum cuttings remain healthy long enough to form roots. Some very suggestive results, however, were obtained by placing twigs under a suction pump, reducing the pressure by suction, and then replacing the air with carbon dioxide or oxygen. Results from such treatment are illustrated in table 8 : TABLE 8. Effect on Root Growth when the Gas in Twigs op Lioustrum is Replaced with Oxygen or Carbon Dioxide (Cuttings taken on March 29, measured on June 2, 1917. Ten twigs to the culture) Num- ber of cultures used Roots Tops Treatment Average total length per twig (milUmeters) Relative to check as unity Average total length per twig (millimeters) Relative to check as unity Check, untreated Oxygen Carbon dioxide* 2 4 2 77.0±18.4 76.6±15.5 1.5± 0.8 1.00±0.24 0.99±0.20 0.02±0.01 101.6±3.7 105.6±2.8 76.8±4.7 1.00±0.04 1.04±0.03 0.75±0.05 *0nly one twig in each culture waa rooted; the others had very weak calluses or none at all. The twigs were placed under suction for twenty minutes and then allowed to stand in the gas at atmospheric pressure for sixteen hours. They were then removed and placed in flasks of tap water. The single injection of carbon-dioxide gas distinctly retarded both root and top development. 104 Otis F. Curtis The injurious effects resulting from the presence of organic matter may not be hmited to those directly correlated with increased carbon- dioxide or decreased oxygen content. It is very probable that there are certain toxic, partially oxidized substances formed which are directly injurious. In fact, evidences of fermentation and putrefaction were shown by tests for alcohol and by the odor of the solution. Aside from the direct effect of increased carbon-dioxide and decreased oxygen on the respiration of the cuttings, the same condition would tend to increase the production of these partially oxidized toxic substances in the medium. Stimulation by manganese may not be directly due to its effect on respira- tion of the cutting, as discussed in the preceding pages, but it may be indi- rectly due to the reduction of toxicity as a result of more complete oxidation carried on either by the cutting or by the microorganisms, or possibly in the solution independent of either. Loew (Loew and Sawa, 1902-03) has suggested a similar role for manganese — that it serves to carry on oxida- tion to completion, thereby preventing the accumulation of toxic, par- tially oxidized by-products. COMPARISON OF THE EFFECT OF POTASSIUM PERMANGANATE WITH THAT OF OTHER MANGANESE COMPOUNDS, AND ALSO WITH THAT OF IRON, OF ALUMINIUM, OF BORON, AND OF PHOSPHORUS Since manganese dioxide seemed to be one of the active principles in stimulating root growth in the experiments thus far cited, experiments were made to determine whether this compound added directly to the medium would have a similar effect. At the same time other compounds were used which, according to the literature available, had under certain conditions stimulated root growth in seedlings, or which, as in the case of iron, were supposed to have some connection with oxidizing enzymes. Experiments with herbaceous cuttings The first experiment was made with tomato cuttings, since these root very quickly and would give an idea as to the approximate concentra- tions to be used. The stems were not of uniform size but care was taken to distribute them equally. Duplicate cultures of five cuttings each were set up. The roots were measured six days after the cuttings were started, with the results shown in table 9: Stimulation of Root Growth in Cuttings 105 TABLE 9. CoMPAEiBON of the Effect of Various Stimulants on the Root Growth OF Tomato Cuttings (Duration of experiment, July 31 to August 6. Five cuttings to the culture) Solution Total root length per cutting (in millimeters) Average Order of growth Cul- ture 1 ■Cul- ture 2 1 Check — Distilled water Check — Tap water 49 178 822 969 60 679 629 589 922 1,095 1,307 917 618 664 637 925' 461 33 "776 546 80 715 587 688 1,034 1,015 1,070 "680 571 354 1,071 16 41± 6.0 178± 32.9 799±104.4 757± 75.9 70 697± 7.8 608± 67.2 638± 65.6 978±145.4 1,055± 91.3 1,189± 82.4 917±144.2 649± 60.1 617± 52.9 496± 60.3 925±160.4 766±305.5 8± 3.3 17 15 8 MnOj, 0.1 mol 6 4 Mn02, 0.01 mol 8 5 6 KMn04, 0.001 mol.* MnSOi, 0.002 mol 7 MnS04, 0.0005 mol the solution 16 (roots mostly- above solution, tips brown) 9 8 MnSO., 0.00002 mol 9 NaHzPOi, 0.05 mol Dead 10 NaHzPOi, 0.01 mol 13 11 NaHzPOi, 0.002 mol 11 \'?. H.BO3, 0.001 mol 3 13 H,BOs, 0.0001 mol 2 14 HsBOs, 0.00001 mol.. . . 1 IS FeCls, 0.001 mol.*. Ifi FeCIs, 0.0001 mol 5 17 CaClz, 0.01 mol 10 18 CaClj, 0.005 mol 12 19 CaCh, 0.001 mol 14 20 CaClz, 0.005 mol., and HaBOi, 0.0005 mol 4 21 NaHzPO., 0.01 mol., and MnOi, 0.01 mol 7 22 FeCL, 0.001 mol., and KMnO,, 0.001 mol. . The few roots pres- ent were dead * All stronger solutions showed no growth. The results were more consistent than was to have been expected from the small number of stems used and their lack of perfect uniformity. The four treatments showing the best growth all contained boric acid. Ferric chloride stood fifth, while the three containing manganese dioxide stood next in order followed by manganese sulfate in its weakest solution. 106 Otis F. 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