ROOT INITIATION AND DEVELOPMENT IN ,AIR-LAYEIIED PINE AND SPRUCE Thai: for II“ Dunn of pk. D. MICHIGAN STAYE UNIVERSITY James Roger Feucht 1 9 6 0 LIBRARY Michigan State University This is to certify that the thesis entitled Root Initiation and Development in Air-layered Pine and Spruce presented by James Roger Feucht has been accepted towards fulfillment of the requirements for M degree in W i com 14/ /‘? glitz; Major professor ROOT INITIATION AND DEVELOPMENT IN AIR-LAYERED PINE AND SPRUCE By JAMES ROGER FEUCHT AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1960 gm a re wax; a“ M \x a0 “\ \mes ROGER FEUCHT ABSTRACT ‘«“‘\ A 4 At,/\~ Exploratory experiments were conducted in the 1958 growing season to establish the value of nine different growth- regulators for the induction of root initials in air-layered branches of seven to 12-year-old trees of flies: gla_uc_a and Hui sylvestris. From the results of the exploratory experiments growth-regulators were selected for use in further investigations conducted in the 1959 growing season. Air-layers were prepared on the lower, middle and top whorls of 15 ( trees of each species at the beginning of April, May, June, July and August, 1959. The air-layers at each of the three whorls on gm g_la__uca were injected with aqueous solutions of one of the following treatments: 0. 5 ppm 2, 4, 5-Tri- chlorophenoxyacetic acid, 100 ppm naphthaleneacetic acid, lOinpm 4-thianaph- theneacetic acid, 1. 000 ppm indolebutyric acid and distilled water, and Bing sylvestris air-layers were injected with 1 ppm 2, 4-dichlorophenoxyacetic acid, 100 ppm naphthaleneacetic acid, indolebutyric acid, and 4-thianaphthaleneacetic acid, 100 ppm indoleproprionic acid and distilled water. Results 100 days after treatment showed that there were no significant differences in rooting betWeen treatments, or between positions of the air- layers on the trees. A significantly greater number of Eigea glfla stems rooted, however, when air-layers were applied at the beginning of May, little rooting occurred when applied in July. Mean root length was significantly greater at the end of the June treat- ments when compared with April, July and August. 7f -__m 3 —-- -“;-w 3 3.}: . l"" \\‘ '\ . .L.l|h-" 12;” "it. u .«~‘ AF , ,‘ p- l- n» \i- " xx .4! -'.': J ‘ . ' . 3.1:: fl I _ ‘v i ‘ , .'.‘ l, jl‘) I Ii ' ’3’ ‘ 1.. .1 '9: 7 ' .’ /’ J‘s f 2,. "fi bi '\\\ 3,0 WES ROGER FEUCHT ABSTRACT - 2 The number of rooted stems and the mean root length were correlative with the mean minimum and maximum air temperatures which might suggest that temperatures above 80’F occurring at the beginning of a treatment hindered root initiation and increased the incidence of stem death, but that root length g was increased by gradually rising daily mean temperatures and hindered by g temperatures which prevailed below 60'F. i Anatomical descriptions of typical stem pieces were included. Examinations of sections cut through the wounded area of M gE._u_ca i and _P_ir_1_u_s sylvestris stems showed that root initials arose from secondary phleom rays of tissue proliferations produced by the vascular cambium. Root initials in stems of £2313 sylvestris invariably occurred at the apex of a knob-shaped tissue proliferation. Anatomical examinations of dead stems of w gl_a_uca showed that no meristematic activity had occurred at or near the wounded area and it was suggested that high temperatures at the beginning of the treatment period may have prevented the formation of a protective periderm over the cut sur- face, causing stem death. Survival of rooted air-layers of flia 8.13% planted in September was 100 percent after 70 days in contrast with 52 percent in July when observed after 130 days. Eleven tables and nine figures were included. no "Wot— .a-v... ROOT INITIATION AND DEVELOPMENT IN AIR-LAYERED PINE AND SPRUCE By JAMES ROGER FEUCHT A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture l 960 ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Dr. Donald P. Watson, F. L. S. O'Rourke, to members of the guidance committee, Drs. C. L. Hamner, A. L. Kenworthy, L. W. Mericle, F. B. Widmoyer, and J. W. Wright for their helpful suggestions and criticisms, and to Drs. J. D. Downes and S. K. Ries for their valuable assistance in statistical analysis. Appreciation is also extended to Mr. Max Gruner and Mr. and Mrs. Stylie Ferris for so generously providing the plant materials, and to the author's wife, Beverly, for her untiring assistance in the mechanics of air-layering and typing. a0 TABLE OF CONTENTS Page INTRODUCTION ..................... I g _ REVIEW OF LITERATURE ................ 2 Natural Layering in Conifers and the History of Air-layering 2 1 Influence of Physiological Age on Rooting ....... 7 I] Effect of Season of Year on Rooting . . . . . ..... 13 2 Rooting Responses as Influenced by Growth- Regulator Appli- [ cations . . . ................. 16 The Effect of Genetic Variation on Rooting Capacity . . 20 The Formation and Development of Adventitious Roots from the Standpoint of Anatomy and Histochemistry . . . . 21 EXPERIMENTAL METHODS AND MATERIALS ........ 30 Exploratory ..................... 30 g 1959 Growing Season ................. 35 Temperature Studies ...... . .......... 37 Methods of Anatomical Study ............. 38 Planting of Rooted Air-layers ............. 40 RESULTS ......................... 41 Exploratory ..................... 41 Temperature Studies ................. 52 1959 Experiments ................... 55 Rooting ..................... 55 —.._ ————__ -m “Irma i TABLE OF CONTENTS CONT'D Tissue Proliferations. . . ........ - . . . Injuries t_o Air-layered Stems . ......... Anatomical Stu—dies ............ Suwivalgjm Air-layers . . . . ...... DISCUSSION OF RESULTS ................. SUMMARY . . ...................... ANNOTATED BIBLIOGRAPHY . . . ............ 57 61 61 75 77 87 91 Figure LIST OF FIGURES Page Preparation and treatment of air-layers . . . . . . 32 Rooting and wound proliferation of Picea glauca and Pinus sylvestris air-layers . . . . . . . . . . . 49 Comparative anatomy of Picea glauca and Pinus sylves- tris stems in radialview . . . . . . . . . . . . 65 Comparative anatomy of Picea glauca and Pinus sylve - tris stems in transverse view . . ..... . . . 66 Photomicrographs showing the anatomical nature of tissue proliferations. . . . . . . . . . . . . . 70 Photomicrographs showing some of the stages of root initiation and development in Picea glauc . . . . . 72 Photomicrographs showing some of the stages of root initiation and development in Pinus sylvestri . . . 73 Total number of rooted air—layers of Picea glauca in comparison with five-day maximum and minimum air°temperature means . . . . . . . ..... 79 Mean root length of Picea glauca in comparison with five-day maximum and minimum air-temperature means... ....... ..........81 A.“. _. 9 Table I. VII. V In. XI. LIST OF TABLES Coefficient of Stem Death, Total Survived and Number Rooted of Picea glauca Air-layers 70 Days After Treatment in a 60°F Greenhouse . . ....... . Rooting Results of Picea glauca Air-layers, July 1958. . Rooting of Pinus sylvestris Air-layers, July 1958. . . . . Number of Pinus sylvestris Stems Exhibiting Root-like Tissue Proliferations and Tissue Proliferations of Brachyblast Wounds in the August to November and August to June Treatments. . . . . . . . . . . . . Temperature in Proximity of, and Inside Air-layers. . . Rooting Results (After 100 Days) of Picea glauca' Air- layers.. ..... Tissue Proliferation (After 100 Days) of Picea glauca Air-1ayers.. ...... Tissue Proliferation (After 100 Days) of Pinus sylvestris Air-layers. . ..... . . . . . ..... . . . Proliferations of Brachyblast Wounds and the Total Num- ber of Stems with Knob- shaped Proliferations at the Girdle Area in Pinus sylvestri . . . . . . . . . . . Injury and Stem Death of Air-layered Picea glauca after lOODays. ...... ............. Survival of Rooted Picea glauca Air-layers Planted in 'I‘woDifferentMedia. . . . . . . . . . . . 51 54 56 58 60 62 63 76 INTRODUCTION Vegetative propagation of woody and herbaceous plants is used primarily for the perpetuation of individuals selected for desirable Char- acteristics. Propagation by vegetative means is used most commonly by nurserymen and foresters and is often the sole means of producing plants with some of the most useful, aesthetic, and economic features. Many species of plants are propagated vegetatively by cuttage and graftage; others are reproduced but with great difficulty by either method. The lack of Clonal material among many genera can be attributed (to a great extent) to the absence of a suitable means of vegetative propagation. As a result of these difficulties Clonal populations which do exist in these genera are few in number and consist of selections from only a small representation of the species. Rooting of cuttings and successful graft unions are rare in numerous species of Pius and Pic—ea. Interest in the process of air-layering has stimulated investigation into the feasibility of this propagation method of plants which do not produce roots easily. The present investigation was conducted to determine the feasibility of air-layering as a method of reproducing two species of coni- fers, to screen the effects of growth regulators on root initiation and develop- ment, to study inherent and environmental factors affecting rooting and to examine the anatomical abberations exhibited in stems that were air-layered. REVIEW OF LITERATURE Natural Layering in Conifers and the History of Air-layering Few species of coniferous plants reproduce naturally by vegetative means, but drooping branches of T_hu_ja occidentalis growing on the limestone cliffs and shallow-soiled uplands of Mackinac Island, Michigan, have been observed to root when covered with soil and humus (Potzger, 1937). Success- ful layering of branches has also been reported in iies balsamea, A_bie_s lasiocarpa, Cryptomeria japonica, Juniperus pachyphloea, Pic—ea canadensis, w excelsa, PM mariana, Pic—ea sitchensis, Pin—us chihuahuana, ms Potzger has also noted that natural rooting by layering may occur in m- ts_ug, Sequoia, M and TU—Inlfll, but with the exception of M occidentalis the occurrence of natural layering in these species is not common. Cooper (1911) found that natural layering of conifers is more common in regions of higher altitude and latitude. He cited M balsamea and M sitchensis as the most commonly layered conifers in these regions and described the layering habit of w sitchensis, Ting mertensiana and Egg heterophylla (Cooper, 1931). Natural branch layering of ma mariana in sites where the soil is shallow has been described by Fuller (1913) who also observed that a single Parent tree might give rise to a circular colony of young vegetatively propa- gated trees when its lower branches were gradually covered by soil. Lemberg (1933) reported that in many instances Pinus sylvestris par- ‘- tially buried by sand dunes on the coast of Finland will layer and form cir- |v cular "colonies" of rooted branches. A method of inducing roots known as "air—layering", "Chinese layering", "marcottage", "mossing—off', or "propagation from gootes", has been used by the Chinese for over 2000 years (Thakurta, I940; Mergen, I955). The manufacture of plastic films has increased the use of the air-layering method within the last 12 years because of its properties of high gaseous exchange and low water permeability. I '1 The first to use plastic films for air-layering was Colonel William Grove of Laurel, Florida in 1947 (Grove, 1947). Grove experimented with 1/ two types of plastic films, "Pliofilm" and "Vitafilm"_ on the Lychee, Litchi chinensis. Pliofilm disintegrated before roots could form, but the heavier Vitafilm was found to be suitable. Both films have similar properties in that they prevent water evaporation, but permit the passage of gases through the film (Grove, 1947). The film currently of widespread use in air-layering 2/ is called polyethylene , and sold under various trade names. All polyethylene has the properties of very low transmission of water vapor and high gas per- meability (Wyman, 1952)- l/ ‘ Manufactured by Goodyear Tire and Rubber Company, Inc. , Akron, Ohio. 2/ P°1Yethylene is a high polymer of ethylene developed by the Imperial Chemi- cal Industries Ltd. of England and licensed for manufacture in the United States in 1 943 by E. L DuPont de Neumours and Company. l I I .5 L‘rm Mow-1‘ . with the use of polyethylene—wrapped air-layers. Creech (I950) successfully rooted the Rhododendron hybrid "America" Of 18 air-layers made, 11 rooted and grew after being severed from the source plant and placed in soil. Rooting of Magnolia grandiflora, A_Ce_r palmatum and selected hybrid tea roses was obtained through air—layering by Hanger e_t a_l. (1954). In i nearly all of the stems which were rooted, however, considerably injury to the current growth was observed. A study of air-layered Queen Elizabeth roses in which black and white colored polyethylene films were used to prevent moisture loss showed that this rose variety produced numerous roots six weeks after the initial air- layering, but no differences in rooting occurred between black and white polyethylene wrapped stems (Ching e_t 31: , 1956). Abraham (1956) experimented with the air-layering process on cashew, Anacandium occidentale at the Central Cashewnut Research Station, Madras, India and reported that an average of 75 percent rooting can be obtained with this species throughout the ’year. Certain species of Artocarpus were rooted by marcottage by Fabello (1934) and this method of propagation is widely used on many tropical and SUb-tropical fruits, principally the mango, Mangifera iniica L. , and the avocado, Persea gratissima Gaertn. , (Fielden, 1936; Singh, 1953; San Pedro, 1935). u. Limbs of the guava, Psidium guajava Linn. , more than 1/2 inch in diameter were rooted in 3 to 5 weeks, using the air-layering methods Ruehle, I948). Rao _e_t§_l_. (1952) attempted air-layering on Diospyros k_3_k_1 Linn., no rooting occurred. Wyman (I 952) attempted air-layering on 250 plant species represent- ing 58 genera of "hard—to—root" woody plants, including some gymnosperm s, and successfully rooted 140 species. Species ofPiLus and flea were not included. In 1952, Mergen air-layered branches of _Pl_l'll_l_S_ e_l_li__ottii Engelm. pre- pared by completely removing a ring bark and cambium 1/4 inch in width treating with Hormodin N0. 3‘l‘/and wrapping with moist Sphagnum enclosed in a tight wrap of polyethylene film. Although no rooting was recorded 5 1/2 months after treatment, his later studies showed that rooting of Pin—us elliottii by air-layering was more successful than by cuttings (Mergen, 1955). Hoekstra (1957) conducted additional studies of this same species and reported rooting as high as 93. 6 percent using six-year-old trees. Exten- sive studies conducted by Mergen and Cutting (1958) using air—layered Mt £183 and £i_c_e_a pungens resulted in 15 and 3 percent rooting, respectively. Less extensive studies of air-layered stems of M palustris were per- formed by Johansen and Kraus (1958) in which 9 stems of the 15 air-layered stems produced roots. 1 / ———————— A comm ercial preparation of Merck and Company, Rahway, New Jersey. ~ s I I ’m We?“ —u._.. Hitt (I955) attempted air-layering on Pinus resinosa, Pinus banksiana, Pinus strobus and Pinus sylvestris. N0 rooting results were given. Pinus echinata and Pinus taeda were rooted by Zak (1959) using a unique method of air-layering. Two and one—half—year-old plants growing in pots were placed horizontally so that the stems were resting in notches of a wooden frame filled with moist Sphagnum. An aluminum cover was then placed over the frame for 53 days. Eight stems of E_ng echinata and six off. ta_edg out of ten stems each rooted using this method. Zak also used DuPont synthetic sponges in the place of Sphagnum with some rooting response. The roots were not ableto penetrate the sponge material and, therefore, Zak suggested using cup-shaped sponges to prevent the retard- ing of root growth. In Sumatra, Lasschuitt (1950) air-layered Eng merkusii and reported that eight months after applying the air-layers, seven of the 75 treated branches. had produced roots. Frolich (1957) air-layeredPigag a_bi_es_, E a_bie_s pygnaea, _P_. 3% nidiformis, 3 pungens glauca, Larix decidua, I; leptoleptis, Abies concolor Chameacyparis Lawsoniana and Eng sylvestris in addition to ten Angio— Sperms including the genera "Illig, Populus, Quercus, fl, [_Jinls, Rhamnus, m§ and Carpinus. All of the Angiosperms rooted well, usually higher than 60 Percent, and many 100 percent. The coniferous species rooted from 20 to 80 Percent, with the exception of Pinus sylvestris which did not root at all. EXtensive literature reviews concerning factors affecting rooting of cuttings haVe been compiled by Priestley and Swingle (1929), Swingle (1940) and Nienstaedt (1958) which indicated that these studies have been more frequent than those with air-layering, yet factors having influence on the rooting of cuttings have also been found to play a role in the rooting of air-layers. For this reason some of the findings must not be overlooked. Influence of Physiological Age on Rooting The term "physiological aging" has been used to refer to an inherent change of an organism which brings it closer to senescence (Robbins, 1957). According to Schaffalitsky de Muckadell (1959) this change is associated with meristematic aging. She states that "Generally speaking the annual shoot is rightly considered the youngest part of a tree. It is important to notice that in studies of ontogenetic development, the situation is quite the contrary. The annual shoot should be looked upon as the latest developed, or even the 'oldest' part of the plant. " Physiological aging is apparent in most plants, but is particularly pro— nounced in plants which exhibit a wide variety of leaf—shapes. Difficulties arise. however, in determining whether the change in leaf shape is caused by "meristematic aging" or environmental influences. Molish (1915) orig— inated the term "topophysis", which he describes as a condition in which shoots exhibit individuality as related to position on the tree. This effect l l i l N .. ‘0 t . ‘ 'lhas been EXhibited on cuttings of Araucaria excelsa where cuttings from a 6 first order terminals develop into upright plants and those from second order branches develop into single "thread-like" horizontal shoots (Vbchting, 1904). Seeliger (1924) has referred to the effect of aging on plants as "zyklophysis" (cyclophysis) and considered cyclophysis to be identical with topophysis. Schaffalitsky de Muckadell (1959), however, rejected Seeliger's view and differentiated between topophysis and cyclophysis in that cyclophysis was a definite effect of age of the meristems and topophysis, an effect of shoot individuality as proposed earlier‘ by Molish (1915). A condition in a tree or its ramets which was caused by earlier effects of the environment has been termed "periphysis" by Blisgen and Mllnch (195.7). The adult form of Hedera fl is conditioned by cyclophysis and periphysis because the condition arises only in old age and everywhere on the old plant where it is exposed to full light eliminating the Change in leaf form being due to topophysis (Busgen and Mllnch, 1957). Rooting ability in plants also appears to depend upon the cyclophysis and periphysis phenomena. Delisle (1942) using lateral and terminal cut— ti138‘s of the current season's wood taken from two-, four- and over four- Year-old trees of m strobus, found that the cuttings from two—year-old trees rooted only with the aid of an auxin and those from trees over four years old did not root at all. Further studies using the brachyblast It _ ' ,lfascicles 0r short-shoots) showed only three to five percent rooting when the short-shoots were taken from older trees (over four-year-old) and up to 74 percent rooting when taken from younger trees. There was no re- ported difference in rooting between terminal and lateral shoots. Doran and Holdsworth (1940), however, obtained 70 percent rooting of ng strobus cuttings from a 33-year-old tree when the cuttings were taken from lower branches and treated with an aqueous solution of 200 mg per liter indolebutyric acid. No rooting was observed from cuttings taken near the top of the 33- year-old tree. The effect of the position of the cutting on the plant was also observed in a remarkable experiment by Toole (1948) in which stem cuttings of Albizzia julibrissin Durazz. originating directly from roots, rooted 100 per- cent, while no rooting occurred when the cuttings were obtained from stems originating distal to the roots. Toole suggested that the ease of rooting increases as cuttings are taken from nearer the root system due to a greater concentration of root-promoting substances in the root than in the aerial parts of a plant. Gardner (1929) studied the relationship between tree age and the root- ing of cuttings from one- to twelve-year—old trees of EELS m, _P_. communis, « Lnus m, P_ mahaleb, E cerasifera, 3 persica, Ulmus americana, 2' Pum ila, @- saccharum, A. dasycarpum, Ilflt m, Robinia pseudoacacia, m Speciosa, Amorpha fruticosa, Pinus strobus, 3 _r_esinosa, E. taeda, /’A\ .4 l1. §YlV€StTiS, PM. zilLes, M occidentalis and Taxodium distichum. Nearly all of the species rooted faster and in greater abundance when one- year-old trees were used as a source of cutting material. Less rooting occurred the older the parent tree. Outstanding examples of the age phenome- non as observed in Gardner's experiment are listed as percent rooting for one-, two-, and three-year-old trees, respectively: Engs sylvestris, 77, 8 and 0 percent; Pinus strobus, 98, 51 and 12 percent; and Hex opaca, 100, 64 and 47 percent. Rooting investigations of hard-to-root plants by Thimann and Delisle (1939) led the investigators to conclude that the . . . most important factor. . . " in rooting plants is the age of the ortet. Bodmangt 11: (1952) also found that the age of the ortet is an impor- tant factor in rooting Pseudotsuga taxifolia gl_agn cuttings and even affects the survival of out-planted rooted cuttings. Rooting of cuttings from five-, 12-, 17- and 42-year-old trees was 16, 25, 30 and 0 percent respectively, while survival was 43, 29 and 43 percent, respectively. Hoekstra (1957), after applying air-layers to the distal tips of the branches in the upper one-third of crown in six- and 23-year-old Pius elliottii, found that rooting occurred in 12 weeks in both age classes. Six- Year-old trees produced rooting up to 93. 6 percent and 23-year-old trees rooted 80 P6rcent. McCullock (1943) made cuttings using one-, two- and three-year-old wood from a 50-year—old Pseudotsuga menziesii gla_uca and observed rooting from one-year—old wood only despite chemical treatments. Johansen and Kraus (1958) reported that nine out of the fifteen air- layers applied to a 52-year-old£i_n_u_s paulustris rooted. All fifteen air-layers Were applied to the crown region of the tree. Deuber (1940) experimented with cuttings ofgic_ea 51%, EE ms, 2. resinosa. E. bungeana Zuce. , g. densiflora Sieb. and Zuce. and Ts_wg2 canadensis L. Carriere. Five-year-old Ea—big cuttings rooted more readily, in general, than cuttings from 26- and 40-year—old trees, however, the season of the year in which the cuttings were made influenced the effect of age. Cuttings of Pigs strobus from juvenile ages, especially from seed- lings two to four years old, rooted to a greater extent and more consistently than cuttings from older trees. Some rooting did occur, however, from trees 5. 7. 15, 18, 25, 30, 40 and 60 years old. Lateral cuttings rooted "more abundantly" than terminal cuttings. No rooting occurred in the limited trials 0f M resinosa. Ten-year-old P_inu_s bungeana cuttings rooted 52. 5 percent, lS-year-oldErgs densiflora, 25. 7 percent and four-year-old Egg canadensis, up to 65 percent. Grace (1939) investigated the difference in rooting response of cuttings taken from upper and lower regions of 18-year—old Pigs} a_bie_s and observed that 75 Percent of the cuttings rooted when taken from the lower branches and 43 percent rooted from branches taken near the top of the tree. Position, however, had no significant effect on the number of roots produced per cut- ting. Tsunahide and Ogasawara (1957), on the other hand, found that cuttings of Metasequoia glyptostrobeides from lower branches formed a larger number of roots and longer roots per cutting. Effects of position of a given whorl on rooting were investigated by Farrar and GraCe (1942) using cuttings from lower branches of El a_bie_s. Cuttings were classified into six types, as follows: Type 1, first order ter- minals which averaged 127 mm in length: Type 2, second order terminals averaging 81 mm in length; Type 3, second order large laterals averaging 93 mm in length; Type 4, second order small laterals averaging 71 mm in length; Type 5, third order laterals averaging 59 mm in length; and Type 6, the distal part of Type 1, averaging 79 mm in length. They concluded that the position of the cutting on the branch had "little" effect if the cuttings were made six to nine cm long. Sixty- seven percent of the first order terminals rooted but when made shorter than six cm, only 32 percent rooted. When a "heel" of older wood was left on cuttings of P_icEt ties, root- ing was inhibited (Deuber and Farrar, 1940). Farrar and Grace (1942), how- ever, found in later experiments that heel cuttings appeared to favor root length When placed in a peat medium and survival and rooting was higher with heel cuttings than with simple cuttings when used in sand. No explanation was given for this effect of age of wood and media type. Effect of Season of Year on Rooting The importance of the month of collection of cuttings has long been recognized by plant propagators whether working with gymnosperms or with angiosperm s. Farrar and Grace (1941) collected cuttings of m _aEg at 24 inter- vals throughout a year making seven collections when new growth was forming, six at semi-monthly intervals from July to September, four duringOctober and seven at monthly intervals during the period of winter to April. Rooting success gradually decreased during the summer months, and increased to a high of 80 percent in September and October. Cuttings stored over winter or taken in April and May did not respond well. Results also indicated that cut- tings taken just as the buds are opening rooted more readily than cuttings taken before and immediately after bud expansion. Deuber and Farrar (1940), using a total of 3200 cuttings of E3 abis, collected at monthly intervals. reported that rooting increased from October to a high in December and de- creased rooting occurred in January. In contrast, a study of the seasonal variation in the natural rooting capacity of M a_big and _P_ sitchensis (Larsen, 1955) Showed that rooting was highest in June and July, lowest in September, with a gradual increase in rooting occurring on cuttings made during the winter ' months. Differences in results between Deuber and Farrar and the work of L'511'8611 might be attributed to the climatic differences between Ottawa, Canada and Denmark, and to the climatic differences in the years during which the work was carried out. Doran e_t_a_l. (1940) found that Pinus strobus cuttings yielded 70 percent rooting when taken in March. Systematic studies of cuttings from P_iffii alias and Pinus strobus made by Deuber (1940) clearly showed that dormant stems collected in mid—winter season root more readily than at any other season. Deuber termed the period of best root production in E523 ab_ies from October to December as the "grand period of root generation", while m Eggs cuttings rooted moist abundantly when collected in December, January and February. Cuttings of Pseudotsuga menziesii glgga and Picea sitchensis were found to root best when taken in December through February (Griffith, 1940). Childers and Snyder (1957), using 16-year—old pistillate trees of Ilflf w 'Arden' and 'Cumberland' and 40—year-old staminate trees 'Old Hale and Hearty', collected cuttings on the first and fifteenth day of each month from August 1 to November 15 with variable results. 'Arden' was found to be the eaSiest to root and no significant differences in seasonal effects on rooting were observed. 'Old Hale and Hearty' rooted highest when cuttings were col- lected in September and November. 'Cumberland', the most difficult to root, r"Quad highest when cuttings were obtained on September‘l with very low rooting occurring when collected on November 15. It was interesting to ob- serve that cuttings Collected from the oldest parent, 'Old Hale and Hearty', exhibited a higher rooting capacity than the cuttings from the younger cultivar, ' Cumberland'. Recent interest in the practice of air-layering of conifers stimulated Mergen (1955) and Hoekstra (1957) to investigate the seasonal variation in rooting of air-layered Egg elliottii. Mergen, in a preliminary experiment, showed that rooting would occur when air-layers were applied to l/4-inch girdled stems in October, May and August. No significant differences were obtained in the number of branches rooted, but August treatments yielded more roots per marcotte and in a much shorter time than the October and May treatments. Hoekstra (1957) concluded that July was a better month than September to apply air-layers on w elliottii growing in Lake City, Florida. He con- sidered the gradual decrease in daily mean temperatures to be an important factor in the reduction of rooting capacity in the September treatments. Recent air-layering studies conducted by Mergen and Cutting (1958) ‘18ng @ abgies and E pungens growing in New York, demonstrated that rooting was highest when the marcotte was applied in April before active shoot elongation had taken place. Johansen and Kraus (1958) were successful in rooting air-layeredErgig m stems which were applied in October. Only 15 air-layers were attempted, however, and no seasonal variation study was conducted. Rooting Responses as Influenced by Growth-regulator Applications Within the past two decades plant propagators have become keenly in- terested in the use of growth-promoting substances for root induction and growth in cuttings. As a result, there are large quantities of literature concerning the effects of chemical substances on the formation of adventitious roots under greatly varied conditions and on many plant species and varieties. Experiments conducted with coniferous species concern, for the most part, attempts to determine the value of various growth regulators in the root- ing of cuttings. Some workers in this field have concluded that for the parti- cular species they are studying, growth-regulators are of no significant value, yet others using the same species, will conclude that growth-regulators are Of great value in stimulating adventitious roots. Deuber (1940), using indoleacetic acid at four mg per gram of talc, Hormodin No. 1 and No. 3, and indolebutyric acid at one, two and four mgs per gram of talc, concluded that on M strobus cuttings ". . . there were relatively few instances in which the chemical treatments materially increased the rooting response." Delisle (1942), on the other hand, reported the Egg Sim cuttings root "very little" without chemical treatments. Although Delisle used indolebutyric acid for treatments, as did Deuber, Delisle increased the concentration three-fold, which may explain the differences in conclusions betWeen the two authors. Another rooting study of Pinus strobus by Doran (1940) , _.——,—L. r...“ ,7-_;.1 Am Ti' showed that 70 percent rooting was obtained from a 33-year-old tree using wood from lower branches and treated with indolebutyric acid at 200 mg per liter for five hours. Treatment in the aqueous solution for 24 hours prevented root formation. Grace (1940) treated cuttings of P_ice_a fies with talc preparations containing indolylacetic acid and napthylacetic acid each at 0, 1000 and 5000 ppm, and combined with 0 and 10 percent cane sugar and ethyl mercuric phosphate at 0 and 50 ppm. Indolylacetic acid at 1000 ppm increased rooting from 10 to 42. 5 percent, but at 5000 ppm reduced rooting significantly. Naphthylacetic acid re- duced rooting at both concentrations when compared with control cuttings. Mean root length was increased only by indolylacetic acid at l, 000 ppm. Farrar and Grace (1940) found that Egg gtflllg and Egga w cut- tings received no benefit from applications of indoleacetic acid. Kirkpatrick (1940) lists 18 species of broad-leafed evergreens and 61 Species of conifers which root more readily when treated with indolebutyric aCid at concentrations ranging from 10 to 80 mg per liter of water. Kirkpatrick cOrieluded that ". . . there is no question but that indolebutyric acid is helpful and beneficial in inducing roots. " Two hundred mg per liter of water of indolebutyric acid was found to be Very beneficial in rooting M s_tro___bus by Doran (1946). Doran pointed out that the benefit may be influenced by individuality of trees in their rooting capa- City even though all of the trees used were of the same age. In addition to Pinus strobus the rooting ability of cuttings from other five-needled pines was studied by Deuber (1942), includinngg monticola, E. parviflora, E flexilis, P_ koraiensis, E: _p_e_t_1gg, 3 cembra and P_ lgrgbg- fli. Indolebutyric acid at 2 to 200 mg per gram of talc increased rooting in E strobus, f: monticola andi. CM, but no rooting occurred in the other species. Eigg strobus rooted as high as 60 percent on 15-year-old trees with 2 mg per gram of talc. Only 5. 5 percent rooting was obtained with the cut- tings of mg monticola and 30 percent with P_ cembra. The low rooting response obtained with E_n_g monticola was attributed to using trees 56 years old. The effects of indolebutyric acid, indoleacetic acid and alpha naphthoxy- acetic acid on the rooting of Pseudotsuga menziesii w and Bic—ea sitchensis were investigated by Griffith (1940) by soaking the basal ends of the cuttings in aqueous solutions of the growth-regulators for 24, 48 and 72 hours. The solution concentrations were 0, 6. 35, 12. 5, 25. 0, 37. 5, 50. 0, 75. O, 100. 0, 125. 0, 150. 0 and 200. 0 ppm. Results obtained 160 days after treatment showed that indolebutyric acid at 25. 0 ppm gave 100 percent rooting on P_ice_a sitchensis and 80 percent rooting on Psuedotsuga menziesii g_lau_ca when collected in February. Bodm an (1952), on the other hand, found that the best rooting on Pseudotsuga W M was obtained with 50 ppm indolebutyric acid. Komissarov (1938) treated cuttings ofPligs sylvestris with beta-indoly- 1aCetic acid at a concentration of 100 ppm. No rooting occurred in the first attempts but a repeated experiment yielded 70 percent rooting with cuttings obtained from three-year-old trees treated in the same manner as in the pre- vious experiment. Komissarov concluded that chemical treatment is of great importance in rooting this species. The comparative activity of the stimulation of roots in cuttings was in- vestigated by Hitchcock and Zimmerman (1939). Aqueous solutions of 1 to 80 mg per liter of water produced essentially the same effects as talc preparations at the concentration range of 0. 5 to 50 mg per gram of talc. Potassium salts of indoleacetic, indolebutyric and naphthaleneacetic acids were more effective than the acids; of these, the salt of indolebutyric acid was more effective than indoleacetic acid and naphthaleneacetic acid on most of the plants tested. A later study by Hitchcock and Zimmerman (1940) indicated that-mixtures of in- dolebutyric acid and naphthaleneacetic acid were more effective than individual substances as characterized by more uniform rooting, a greater number of roots and a greater rooting percentage. Stoutemyer and O'Rourke (1945) obtained significant increases in rooting Percentage by spraying 2, 4, 5-trichlorophenoxyaceticacid and its sodium salt 011 Cuttings of w sempervirens handsworthii, Il_e_x crenata convexa, g vomitoria, Emil amurense and _1_.._. ibolium. Effective concentration depending upon the Species. In a later paper Stoutemyer (1954) found that 2, 4-Dichlorophenoxyacetic acxd and 2, 4, S-T were veryeffective in root promotion at a concentration as low as 0. 5 ppm. Bud inhibition, however, may result with the use of 2, 4-D. No bud inhibition was observed with application of 2, 4, 5-T to the cut ends of numerous species of cuttings. The Effect of Genetic Variation on Rooting Capacity Genetic variation between individual plants of a given species or between clones has been found to have a marked effect on the rooting capacity of some plants. Deuber (1940) observed highly significant variations in rooting ability in one collection of cuttings from E_ce_a a_big in which rooting of cuttings taken from various individual trees ranged from 0 to 100 percent. Similar variation of rooting was observed in hybrid seedlings of mi attenuradiata in an experi- ment conducted by Duffield and Liddicoet (1949). The variation of rooting was attributed solely to genetic differences between seedlings. A range of rooting of cuttings from 3 to 55 percent between 15 clones of POBUIUS deltoides were recorded by Cunningham (1953). Hybrid seedlings of E- deltoides showed rooting up to 97. 5 percent. Snow (1941) tested the rooting responses of 24 clones of E rubrum and reported a rooting range of 17. 5 to 97. 5 percent between the clones. Gregory and Van Overbeek (1945) found that the red-flowered Hibiscus I'OSa- sinensis rooted readily, yet a white-flowered selection of this variety rooted with difficulty. In 1942, O'Rourke found marked differences in rooting capacity and survival of cuttings taken from 13 clones of Robinia pseudoacacia. Scions were gxrafted onto seedling rootstocks and after growing in the field for one year cutt ings were collected and their bases placed in a sandy loam soil. After eight months, the number of survivors of each clone was recorded. A 1‘al'lg'e of 0 percent to 50 percent survival was obtained. The average length 0f growth was also recorded in this experiment and the data indicated that the percent survival paralleled the average vigor. Those clones which resulted in a higher survival also attained a greater length of growth. The physiological significance of inherent rooting capacities is not Understood at the present time, but Mirov (1941), in investigations which did n0t involve rooting, has shown that the concentration of naturally occurring gTOWth regulators in the stems of Pinus ponderosa and Pinus torreyana par- allels the vigor of the plant and is a hereditary trait. The Formation and Development of Adventitious Roots from the Standpoint of Anatomy and Histochemistry Adventitious root formation may be induced in many types of plants as evidenCed by the preceding review of the factors that affect rooting. The effect Of these factors differ with the species of plant tested. The type of tissues in- V01V€d in the initiation and subsequent development of adventitious roots vary With the Species and its anatomical characteristics. COImard and Zimmerman (1931) studied the origin of adventive roots in cuttings of Portulaca oleracea L. and found that the pericycle, cambium and ray c ells all take part in root formation, but that the root initials arise only from " ray" cells adjacent to primary vascular bundles. Roots were well differentiated before emerging from the stem and appeared to dissolve inter- fer ing cells of the cortex and epidermis. Adventitious roots of Vaccinum corymbosum were shown by Mahlstede and Watson (1952) to originate from the phloem region and usually immediately adjaCent to a xylem ray. Roots were found most commonly in association with a bUd gap. A mechanical inhibition of root emergence from the stem caused the developing r00ts to bend in the cortex before emerging through the epider- mis. Mahlstede and Watson suggested that the repression of root development Was cansed by the presence of a dense ring of pericyclic fibers or was the re- sult of a thick cuticle which was equal in thickness to the subtending epidermal layer. Jiminez (1937) reported that Ceiba pentandra and Sandoricum koetjape Cuttings formed roots arising from newly formed phellogen cells and Egg orrelis Cuttings developed roots which originated from "callus" or from preformed meristematic areas. Studies by Carlson (1929, 1933) using stemcuttings of Dorothy Perkins and American Pillar roses showed that roots originate from parenchymatous cells of the secondary phloem, but may occasionally arise from the cambial area, Pericycle, epidermis and cortex, and bud- gap parenchyma. Anatomical studies of the origin of roots in stem cuttings of red and black raspberries by Sudds (1935) showed that most of the adventitious roots aro se from near the protoxylem points at the periphery of the pith. Stangler (1949) found that roots invariably originated in the pericycle 0f Chrysanthemum morifolium Bailey, Dianthus caryophyllus L. and Rosa 3% Rehd. In Dianthus there was a band of thiclewalled fibers external to the 130th of root origin which prevented roots from developing vertically through . the Cortical region and therefore curved downward, emerging at the base of the cuttings_ No mechanical hinderance of root development was observed in W and R__°Sa- Adventitious roots were found to be initiated in woody stems of Acanthus mOnta \- nus T. Anders, (Taylor, 1926), Forsythia suspensa (Swartley, 1943) and SL118 Sp. , M sp. and film—S sp. (Corbett, 1897). In addition to roots aris- ing from the cambium, Sandison (1934) observed pre-existing root initials in an area between the cambium and pericycle of Lonicera japonica stems. Sandison reported that adventitious roots will arise from the cambium in this species only when the tissues surrounding the cambium are wounded. Van Tieghem and Douliot (1888), after extensive studies of the origin of roots in many vascular plants, concluded that adventitious roots which occur naturally in young stems are initiated only in the pericycle. Older stems may contain adventive roots which arise in the phloem parenchyma and later in the cambium. Stages in the initiation and subsequent development of adventitious roots from cuttings of Ta_xus cuspidata were observed by Hiller (1951). Roots which emerge from near the base of the cuttings were initiated from the rays in the secondary phloem and roots which emerged more distal to the basal end of the cuttings were initiated in secondary phloem ray cells and the surrounding Parenchyma. Priestley and Swingle (1920) emphasized that young stems in general form roots from the pericycle and in older stems from cambial cells, but in eithEI‘ Case the roots are intimately associated with the rays and involve more than One layer of cells. This concept was supported in a later study by Datta and Majundar (1943) of several dicotyledonous plants. Ray-cell association with adventitious roots was found by Bannan (1941) in an anatomical study of m occidentalis L. stems which had rooted by natural layering. Roots originated adventitiously from "outer tissues" and invariably were continuous with xylem rays composed of parenchyma cells only. The association of adventitious root formation with rays was also noted in Species of Populus and Silil‘ by Trecul (1846') and van der Lek (1924) in Begonia maCUIata and E semperflorens (Smith, 1936) and in Tropaeolum _rgfls L. (Smith, 1942)~ Adventitious roots in Populus and Sa_lix species originated from near the cambium and at the extremity of convergent rays, or in some instances, from a single ray (Trecul, 1846). In the more succulent species, Begnia and Tropaeolum, roots arose from the interfascicular cambium and occasionally in the fa scicular cambium at the edge of a vascular bundle (Smith, 1936, 1942). Delisle (1942) described the sequence in the initiation and development of roots arising from stem cuttings offlg stLbus L. In tissues from both " Young" and "old‘ trees, the first apparent change in cellular activity near the exposed end of the cutting was the formation of a periderm from sub- epidermal cells and "extensive activation" of the cortical and pericyclic cells. Cortical cells generally acquired a binuclear condition after periderm forma- tio“: While cambial cells became active and produced considerable parenchyma es well as xylem and phloem. Cambial activity did not proceed equally in a radial direction throughout the circumference of the cuttings. More active areas produced "cushions" of cells which were located at the ends of a congerie 0f rays and adjacent to leaf traces. These "cushions" were shown to eventually develop into adventitious roots. Delisle also observed roots arising from the parenchyma of leaf traces. Auxin treatment caused tracheal cells of the pro— liferated xylem to form smaller but numerous bordered pits on both the tangential and radial walls. Adventitious roots of Cotoneaster dammeri arise only from one of the two groups of parenchymatous cells in the divided bud gap and is an apparent resumption of the activity of the parenchyma cells (Wolfe, 1934). Wolfe also found in this same species that vascular elements of young roots differentiate very early in their development and that the roots are fully differentiated into a cortex and central vascular cylinder by the time that emergency from the stem ta kes place. E sau (1953) concluded that, in general, if the organ is young the adven— titiou s primordium is initiated by a group of cells near the periphery of the vascular tissues, and if the organ is older, the origin of adventitious root primordial are located more closely to the vascular cambium. Younger stems fOI‘m roots more commonly from interfascicular parenchyma and older stems from vascular rays. Callus development in relation to root initiation and development was StUdied by Swingle (1929) with Malus sp. and Salis sp. cuttings. Swingle con- cluded that rooting and callusing are two distinct processes and that no rela- tionship exists between callus formation and root initiation and development. Taler (1926) also concluded that callus tissues have no part in the formation Of roots, in a study of Acanthus montanus cuttings. Snyder (1954) on the other hand, reported that . . in some instances a cambium develops within the callus tissue. . . " from which root initials may arise. Some confusion of the term "callus" is apparent in the literature. Swingle (1929) defines callus as a form of "tissue hypertrophy", which sometimes re- SUHS in large masses of parenchyma. Sledge (1930) referred to wood prolifer— ations as "callus2, and Hiller (1951) called proliferations of the xylem, phloem and cortex, a "callus" structure. Bloch (1941) characterized the tissues which proliferate during the healing of wounds as "wound callus", and parenchyma proliferations which are not organi zed into a definite tissue as " cell proliferations". After extensive studies of a large number of pteridophytes and represen- tat ive S of both monocotyledonous and dicotyledonous plants, La Rue (1937) con- eluded that pteridophytes do not develop callus on wounds, only a few mono- COtyledonous plants form callus; sclerophyllous and succulent species, in general, tend to form periderm only, and most dicots form callus upon wound- ing unless they possess specialized structures for vegetative reproduction. The histological responses of stem cuttings to applications of growth- regtllfilliors were examined by Beal (1951) using Phaseolus vulgaris L. A three Percent concentration of indoleacetic acid in lanolin paste applied to the base of bean cuttings caused tumor-like outgrowths and the formation of adventitious 1‘COtS l 10 to 120 hours after treatment. Cells of the epidermis and pericycle reSPOTICIed less actively than other tissues. Parenchyma of the cortex enlarged many diameters when compared to non-treated stems and cells near the endo- dermis became meristematic, differentiating into phloem and xylem elements and 1aI‘ge parenchyma cells. Some cells remained meristematic and formed 1'00t histogens. Near the surface of application the cells of the rays adjacent to the xYlem proliferated and differentiated into a confused mass of tracheids. Rays 0f proliferated phloem often gave rise to root histogens. Struckmeyer (1951) conducted experiments on the effects of one percent 2, 4-dichlorophenoxyacetic acid (2, 4-D) on the stem anatomy of Lycopersicon escul entum, Coleus blumatus and a species of Xanthium, Dracena and Philo- dendron- Struckmeyer found that 2, 4-D stimulated rooting activity in all of the Sp ecies except the Philodendron sp. and rooting was usually preceded by heavier tissue proliferations than in untreated cuttings. Rooting occurred rapidly in Lycopersicon, Xanthium and Coleus, but in Dracena, rooting did n0t occur until a large mass of proliferated vascular tissue had formed. The influence of the carbohydrate and nitrogen content on the rooting 0f cuttings of Lycopersicon esculentum and Tradescantia virginiana was studied by Starring (1923) employing the iodine-potassium iodide (IKI) test for starch, Benedict's solution for a test of sugars, and diphenylamine-sulfuric acid test for nitrates on stem tissues of each species. The amount of carbohydrates and nitrogen in the plants was superficially controlled before making the cuttings by increasing or decreasing the nitrogen in a fertilizer and increasing or de- creasing the duration of natural light. Very little rooting occurred in stems which Were Observed to have a low carbohydrate and high nitrogen content. The great- est amount of roots were produced when stems contained a high nitrogen and high carbOhydrate level; however, longer and slightly fewer roots were produced with a high carbohydrate and low nitrogen content. Carlson(l929) conducted microchemical tests on rooted and non-rooted cuttings of Dorothy Perkins and American pillar roses. The Dorothy Perkins rose I‘OOIed more readily than the American pillar rose and results of iodine- Potassium iodide tests for starch showed that the former variety contained a greater quantity of reserve starch in the stem tissues. Brandon (1939), on the other hand, showed that the content of starch in 42 rose varieties including those used by Carlson (1929), did not parallel the ease of rooting. Brandon conducted the study from November to July, inclusive, and Carlson in March and April. EXPERIMENTAL METHODS AND MATERIALS Exploratory Exploratory experiments to determine the effects of nine growth- regulators on the rooting of air—layeredgifl _g_la_u_c_a and w sylvestris were conducted between February 23, 1958 and June 10, 1959. Five six—year-old plants offligg M1 were planted in 12-inch clay pots and grown in a 60°F greenhouse. When the buds were beginning to ex- pand, treatments were prepared and applied by the following methods: Twenty-five 50- gram lots of air-dried, Sphagnum were placed into individual polyethylene bags and to each bag 1, 000 grams of water was added. The bag was sealed and allowed to stand at room temperature for 24 hours to ensure complete saturation of the Sphagnum by the water. At the end of this period 600 grams of water was extracted by squeezing from each lot yielding a moist, but not saturated, rooting medium. After water extraction five grams of 100, l, 000 and 5, 000 ppm each of one of the following growth-regulators prepared in talc by standard alcohol evaporation was added to the Sphagnum and mixed thoroughly: 2, 4—dichlorophenoxyacetic acid (2, 4-D), 2, 4, 5-Trichlorophenoxy— acetic acid (2, 4, 5-T), alpha-naphthaleneacetic acid (NAA), indolebutyric acid (IBA), gibberellic acid (GA), beta—naphthoxyacetic acid (RNA), indole-3-proprio- nic acid (IPA) and indole-3-acetic acid (IAA)l,/ making a total of 25 different treatments. — For convenience, abbreviations of the growth-regulators will be used hereafter. Air-layers were prepared by selecting first order terminals of lateral branches, removing the leaves from five inches of the one-year-old stems be- ginning leaf removal three to four inches distal to the terminal bud and remov- ing a ring of bark two mm wide from around the stem near the center of the leaf-free area‘using a tool designed for this purpose (Figure ‘1). Fifteen grams of the treated Sphagnum were immediately applied around the wound 1/ and wrapped tightly with a 4" x 5" x 0. 0015" sheet of polyethylene film‘ secured at each end with plastic covered wire. The polyethylene was overlapped at least one-third of the circumference of the Sphagnum mass to reduce the a- mount of evaporation of water from within the air-layers. Temair-layers per treatment were applied to the trees at random, pre- cautions being made that each tree contained at least one air-layer of each treatment. Superficial observat'nns of survival of the air-layers were made at periodic intervals and 70 days after treatment the branches containing the air-layers were cut from the trees by severing the stem immediately proximal to the location of the air-layer. By removing the polyethylene film and care- fully removing the sphagnum, rooting, wound, proliferation and injuries were observed. To determine the seasonal effect on the rooting response of @ glauca and P_H'ES. sylvestris, studies were conducted at the Max Gruner Tree Farm located in Shiawassee county near Perry, Michigan. Air-layers were applied 1 -/Cut from polyethylene film obtained from the Bakelite Plastics Company, Boundbrook, New Jersey. E ‘1‘ Figure 1. Preparation and treatment of air-layers. Aa - assembled girdling tool; Ab - aluminum cover removed to show arrangement of one pair of the razor blades; B - leaves removed from stem; C - cutting bark with tool; D - ring of bark removed (arrow); B - application of Sphagnum and plastic; F - treatment by injection with a hypodermic syringe. on April 30, 1958 to nine-year-old Pinus sylvestris trees and to nine- to twelve-year-old _P_icia gEru—ca trees on May 7, 1958. Two hundred and thirty air-layers were prepared for each species, 115 per species on one—year-old wood and 115 on two-year-old wood. Sphagnum was prepared by soaking in water for 24 hours and extracting the water at the end of this period with a hand-operated wringer. The extracted sphagnum was then mixed thoroughly by repeated tumbling and weighed into individual lots of 20 grams. The pre- weighed sphagnum lots were then wrapped in the similar sheets of polyethylene which were used previously and stored in a polyethylene-lined box. The branches were prepared by removing the leaves from five— to six-inches of stem, wounded by making a v-shaped notch on the abaxial side of the stem completely removing a piece of bark one-quarter inch wide at its widest point, and wrapping the wound with the sphagnum as previously described (Figure 1). Each air-layer was treated with a solution of growth- regulator or with water by injecting with a hypodermic syringe five ml of an aqueous solution through the polyethylene film into the sphagnum (Figure l). The following is a list of the growth-regulators used: _Pin_us sylvestris, 2, 4-D, 2,4, S-T, 1AA, NAA, BNA, IPA and IBA at the rate of 1, 100 and l, 000 ppm, and onffli M l and 100 ppm of 2, 4-D and 2,4, 5-T and 1, 100 and l, 000 ppm of IAA. NAA, BNA, IPA, IBA and a new chemical, 4-trinaphtheneacetic 1/ acid (4TNA)_. Distilled water was used for comparative treatments. Ten air- 1 -/Supplied through the courtesy of the Upjohn Company, Kalamazoo, Michigan. 34. layers were prepared for each treatment, five on one-year~old wood and five on two-year-old wood for each species and applied at random over six trees of each species. Buds in both species were beginning to elongate at the time of treatment. Amount of rooting, wound proliferation and type of injuries were recorded 75 and 90 days after treatment in Pic—ea gWI—ia and fl sylvestris, respectively. Marcots were again applied July 16, 1958 on w gla_uca and July 24, 1958 on EELS sylvestris in the same manner as before, but differing in that wounding was accomplished by removing a complete ring of bark two mm wide in P_icia gl_auca, and three mm wide in Pius sylvestris, using the girdling tool described previously. Only one-year-old wood was treated in each species and the treatments used were as follows: in Pyle: w 0. 5 and 1 ppm 2, 4-D, 2,4, S-T; l and 100 ppm NAA, BNA and 4TNA; and 1, 100 and 1, 000 ppm IBA, IAA and IPA; inms sylvestri , 0. 5, 1 and 100 ppm 2, 4-D and 2, 4, 5-T; and 1,100 and l, 000 ppm IBA, NAA, BNA, 4TNA, IPA and LAA. Control treat- ments in each species consisted of distilled water as before. The amount of rooting, wound-tissue proliferation and injury was re- corded 75 and 90 days after treatment of fife—a M3 and Pm sylvestris,re- spectively. On August 4, 1958, eighteen lZ-year-old Picea glauca trees were air- layered applying ten air-layers per tree. Treatments included 30 each of 100 ppm BNA, 1, 000 ppm IBA, 1,000 ppm 1AA, 100 ppm NAA, 100 ppm 4TNA and dis- tilled water. 35. Similar treatments were made on twenty-five eight-year-old Pinus sylvestris trees August 14, 1958 treating with 1, 000 ppm NAA, IAA and IPA, 100 ppm of 2, 4-D, IBA and 2, 4, 5-T and distilled water. Thirty air-layers were made for each treatment, totaling 240 air-layers, ten per tree with ten additional air-layers on the 25th tree to be used for periodic observations. Fifteen air-layers of each treatment and of each species were removed 75 and 90 days after treatment in Picea glauca and Pinus sylvestris, respectively. The remaining air-layers were left on the trees over winter and removed on June 1, 1959. Data were recorded in the same manner as for the July experi- ment. 1959 Growing Season To obtain more information concerning the effect of the time of year on rooting, and to determine the effect of position of the air-layers on the trees, experiments were conducted during the 1959 growing season. Air- layers were applied on the first of April, May, June, July and August, using eight-year-old Pinus sylvestris and nine- to twelve-year-old Picea glauca trees growing on a game preserVe in Shiawassee county, near Perry, Michigan. Each month fifteen trees of both species were air-layered applying the air- layers to branches. of the lower, middle and top whorls designated as position one, two and three, respectively. To each of the three positions 1 ppm 2, 4-D, 100 ppm NAA, IBA and 4TNA; l, 000 ppm IPA; and distilled water were applied 36. “3.3.193? sylvestris; 0. 5 ppm 2, 4, 5-T; 100 ppm NAA and 4TNA; 1,000 ppm IPA and distilled water were applied to Ei_ce_a_ gl_aE. The sphagnum and air- layers were prepared as in the earlier studies, girdling the one-year-old stems two and three mm in width on 29331 gla_uc_a andflifl sylvestri , respec- pectively. The treatments were injected into the sphagnum with a hypodermic syringe as previously, using only two ml per air—layer. All air-layers were removed for observation 100 days after treat- ment and the trees were labeled for future observations. After carefully removing the sphagnum from around the stems of each marcot, the number of roots, root length in mm, degree of wound-tissue pro- liferation, and observations of injuries and unusual callus formations were recorded. The degree of wound-tissue proliferation was scaled from one to seven, one indicating no proliferation and seven indicating maximum tissue proliferation. The ratings were first determined by observing random samples of the April 1 treatments and combined with observations made in previous ex- periments, thus establishing a standard for each of the seven values. The rooting data was analyzed by first rating the rooting values obtained according to the number of roots produced per stem as follows: no roots - a value of one: one to five roots, inclusive - three; six to ten roots — five; 11 to 15 roots - seven; 16 to 20 roots - nine; 21 to 25 roots - 11; 26 or more roots - 13. All data were transformed to the square root of x + 1 as described by Goulden (1952) and subjected to analysis of variance. *1 I a}. Q . Temperature Studies To determine the temperature differences between the sphagnum medium and the surrounding air medium, thermocouples made of copper constantan wire were inserted into the sphagnum media of three air-layers located on the southeast, north and west exposures of a. six-year—old Biff} glauca tree. The tree was placed in an unprotected area to simulate field con- ditions. Two thermocouples were used to obtain temperatures of the surround- ing air, one housed in standard U. 8. Weather Shelter and the other attached to the tree and unprotected. Temperatures were recorded by a Honeywell- Brown temperature recorderlénce each hour from 12:00 noon until 4:00 p. m. on September 6, 1958 and from 9:00 a. m. to 4:00p. m. on September 7 and 8, 1958. A similar experiment was conducted for 48 hours starting on November 11, 1958. Six thermocouples were placed in the media of air-layers of which three were located on the southeast and three on the northwest exposure of the tree. Two thermocouples were inserted so that the tip of the wires were lo- cated immediately under the polyethylene film, two midway between the stem and polyethylene film and two at the stem surface. Outside air temperatures were recorded from thermocouples placed as in the first study. Tempera- ‘ tures were recorded once each four hours throughout the two-day period. 1/ . " Honeywell-Brown Instruments, Minneapolis, Minnesota. P!" -u— w—V— 9, on dn" ”1” ’ ‘1 .’,.-.. l l l (- . 1 'fl— - ‘.I ‘ 3'.“ '1’ ‘ 090~K ..'.. ..‘ '20": . 1 O 38. Methods of Anatomical Study After the examination of the marcots as described in the previous air-layering experiments of 1958 and 1959, stem pieces exhibiting character- istics observed in each chemical treatment and in the two species were im- mediately placed in a killing and fixing solution consisting of 70 percent ethyl alcohol, formalin and glacial acetic acid (FAA). Dehydration was accomplished with a tertiary butyl alcohol series and the specimens were embedded in Par- lodinl/by gradually adding small pieces of the celloidin material to a 1:1 ether- alcohol solution after placing the sample jars in an oven at 55° C for 24 hours or more between each addition of Parlodion. To prevent the corks from being forced from the bottles by the pressure produced from ether—alcohol vapori- zation, the corked bottles were tightly clamped between two sheets of heavy gauge steel with eight 1/4-inch bolts. After the Parlodion was thickened to a "stringy" consistency, the infiltrated sample and a surrounding mass of the Parlodion medium was poured into a paper mold placed over a hard-wood block. The specimen was then quickly moved into a position suitable for sec- tioning and the entire block and mold submerged into absolute chloroform for 12 hours or more to ensure complete hardening of the Parlodion. A surface of the specimen was exposed with a sharp razor blade into a solution of two parts 95 percent ethyl alcohol and one part glycerine for 12 hours for softening. 1 —/Parlodion is the trade name for a purified pyroxylin manufactured by Mallinckrodt Chemical Works, Philadelphia, Pennsylvania. Som e specimens were sectioned longitudinally or transversely with a Slid' - . - mg m 1 Qrotome and others with a rotary microtome at 10 to 20 microns in thic - , , kne S S - Sections were stained with safranln and fast green, as outlined l by Johansen ( 1 940) and mounted in Piccolyte—{3n microscope slides. Free hand sectiOnS and sections made with a freezing microtome at 50 microns Were also prepared from fresh material obtained after harvesting and observ- ing the marcots applied in the 1959 experiments. These preparations were stained with fast green or Delafield's hematoxylin and fast green, accom- plishing clehydration with an ethyl alcohol series. Stained sections were mounted in Piccolyte as previously. All slides prepared were examined microscopically to study the pre- Sence 0f root primordia, origin of roots, characteristics of the tissues and Other Observations which might be peculiar to a given treatment, or an un- treated marcot. To determine what changes in cell growth occurred after girdling and treating the stems, detailed camera lucida drawings were made of non- girdled Stems for comparison. Particular attention was given to those marcots which had not rooted after the period of treatment to determine whether root pri- mOrdia existed and whether the lack of root development was due to anatomi- cal barriers, such as fiber bundles, or from injury resulting. from chemical treatment or girdling. " Piccolyte is the trade name for a pure resin product of the General Biological Supply Company. Planting of Rooted Air-Layers Ro . . . . Oted stems of both Picea glauca and Pinus sylvestris obtained from the e lor xp atQI‘y experiments and not selected for anatomical study, were Planted in t1"tree-inch clay pots containing a soil mixture consisting of three Parts sa Tidy loam soil and one part peat, and placed out-of- doors in a par- tially Shaded area The number of roots present at the time of planting and the survival. after one or more months, was recorded for each stem. Rooted stems of M w obtained from the 1959 experiments, not used in anatomical studies, were planted in shredded sphagnum in three- inch c: lay Dots and fed periodically with an aqueous solution of a 4-8-8 ferti- lizer. P\ecords were maintained in the same manner as in the previous Stu . dy Wlth soil -planted material. Z. If], . s lllllfi. .l'irl! ultilt ., 5 RESULTS Exploratory Experiments with six-year-old greenhouse- grown plants of Picea glauca conducted February 23 through May 3, 1958 resulted in the rooting of two of the ten stems treated with 100 ppm NAA and one stem each of those treated with five additional substances (Table I). Twenty-five days after treatment, the current growth of many of the marcots wilted, and after three to five days the stem and subtending leaves became dry. During this same period, a yellowing of the leaves on the one- year-old stems occurred above the wound, followed by browning and eventual leaf abscission. One week after leaf abscission, the stems became brittle and were then considered dead. Later, one stem exhibited the same symptoms as those which were observed initially. Calculations based on the weighted average of the number of surviv- ing stems 45 days after treatment and every eighth day thereafter until har- vest, showed that although death occurred in all treatments, 2, 4-D, 2, 4, S-T and GA treated stems resulted in the greatest coefficient of non-living stems when compared with the remaining treatments (Table I). An examination of the girdled area of the dead twigs showed that in the GA treatments the stem tissue was killed below and above the wound, but in the remaining treatments, death of stem tissue occurred only distal to the girdled area. .fiwmo Eoum no 39H .33on m 3865 moszS wchmoHoE X - - - - - - m mm .s o our .520 v mo .0 o oo .o o m we .N. H - - - <3 Ha so .> o oo .w o H. co .0 o - - - HRH m me .e 0 mo 4. H m em .0 o - - - H m mm .5 N - - - <.Sm .300 .02 .>.3m .300 .02 Show .300 .02 ooom coo H 00 H o Em Samoa. AoHS EQE conmbnoocou ‘I'I‘Iullllu|||ull.llllllllllul| Lllql ccocbmonh you mucmHnH oHv omzoceoouw mace m :H EoEumoHH < mafia on 95.37.34 8:me 3on we couoom .8952 one oo>H>H=m H38. .238 59m .3 EoHocuHmoU ,8: H M1232. Proliferation of stem tissue occurred along the upper rim of the girdled wound in all of the stems which were alive 70 days after treatment. No dif- ferences in the degree of proliferation between treatments could be detected. The use of talc was an unsatisfactory method of treatment as it was difficult to control the quantity applied and even the application of the growth regulator. Aqueous injection with a hypodermic syringe was found to be a superior method of applying the growth-regulators. It was possible to observe the diffusion of a one percent aqueous solution of safranin 0 in preweighed sphag- num of a known moisture content. After four hours, a 5 m1 injection had stained 90 percent of the sphagnum and after 12 hours all of the sphagnum was stained. Two-ml injections stained the same quantity of sphagnum in 24 hours. Field experiments on both Picea glauca and Pinus sylvestris in May, using five m1 aqueous injection of the treatments and wounding with a v-shaped notch on the abaxial side of the stem resulted in no rooted stems in any treat- ment of Pinus sylvestris and in Picea glauca, - one stem each of 100 ppm NAA and of 100 ppm 4TNA rooted when applied to one-year-old stems. Treatments of 1000 ppm 2, 4, 5-T, NAA, BNA and 4TNA resulted in death of the stems above the wounded area in Picea glauca. Since leaves became chlorotic in 100 ppm 2. 4-D and 2, 4, S-T treatment of the same species, these treatments were eliminated from further experimentation. Stunting of new growth and a slight yellowing occurred in the l, 000 ppm , I . .» ' _ q... l ) I f .. . - '1 "P l _ ' - 44. LAW S‘T treatments in Pinus sylvestris. Treatments of 1, 000 ppm 2, 4-D of the same species caused tip curling of the current growth and considerable stunting of the leaves. These treatments were therefore eliminated from further study. No proliferation of the stem tissue was observed in the wounded area in either species. In all of the treatments, the notched area in the stem was completely healed over and, except for the absence of leaf bases in the case of Picea glauca and brachyblasts in Pinus sylvestris, appeared like an un- wounded stem. Air-layers applied in July to one-year-old stems of Picea glauca resulted in five stems rooted when treated with l, 000 ppm IBA, three stems rooted when treated with 0. 5 ppm 2, 4, S-T and one anleO ppm NAA (Table 11). Two stems rooted in the 1, 000 ppm 1AA treatments and no rooting occurred in this species when treated with one ppm IPA and 100 ppm [AA and IPA. All of the remaining treatments and the control resulted in the rooting of one stem each (Table II). The average number of roots produced per rooted stem and the average length of the roots varied considerably between treatments (Table 11). Of the treatments in which only one stem rooted, one ppm 1AA resulted in nine roots, one ppm IBA - three roots, and the remainder averaged l. 5 roots per stem and of the treatments which yielded three rooted stems, O. 5 ppm 2, 4, S-T aver- aged 6. 3 roots per rooted stem (Table II). The degree of tissue proliferation in the living stems of Picea glauca _'=fi< .3635 2839a E topmoHuE mm mEBm 9 Same .8 >55: We 3383 85258.5 Eob “BEE?” w .w m .H - - w .3 - - - AEEV cum—m: boom .w>< - o .o o .N - - w .m - - - Eoum emuoou E38 .0: .w>< oooH - N H a a m __. .. .. touoon 9:03 .02 o .NH - - AEEV 59.3 uoou .w>< o H - - Eoum oouoou ooH \muoou .o: .m>< H5308 2.8% .02 I O O v—a 0') —1 H ‘I- * o.NH AEEvfimgHuooH .m>< o .N 53m @808 H .o \muoou .o: .w>< nouoou mEoum .02 I u—i 0 -—¢ CO -—4 -—1 O —« .3 a a. HEEEPwECoS a}. H m .0 89m umuoon m .o \muoou .oe .m>< - - - - - - - H m Uouoou mEoum dz .2 - - - - - - - - HEEV 598208 .m>< H - - - - - - - - 8on H3300.“ o .o \muoou .oc .m>< H - - - - - - - - H588 286 .oz CID Heme on: <3 f: <75 .22 <9 <25. 9: Feed 5325880 EoEHmonh \“ mama me “33x ooEmem "mmoH 33. .EoEuaouh you mEoum cop. .muo>mH-.HH< 8:me $05 We 333% 958% HH min—«PH. -W' ./ smiled but no differences were apparent among treatments. Stem death occurred in all treatments of this same species 20 to 35 days after applying the air-layers and at the end of 70 days, 65. 5 percent of the air-layers were dead. There were no differences in survival as a result of treatments. Rooting in Pinus sylvestris occurred in three stems treated with 1, 000 ppm IPA and two stems treated with one ppm 2, 4-D and 100 ppm IBA, BNA and IPA. One ppm 2, 4, 5-T and BNA, IBA and LAA were similar in their action to the use of distilled water, rooting one stem each. No rooting occurred in three remaining treatments (Table III). Proliferation of tissues above the wound was observed in many treatments (Figure 2), and varied according to the moisture content of the sphagnum medium. Some water had evaporated from sphagnum in many of the air-layers as a result of exposure when grasshoppers chewed holes in the polyethylene. Sphagnum surrounding rooted stems also tended to become dry as a result of water uptake by the newly-formed roots. In those air-layers in which the sphagnum was nearly dry no proliferation occurred, but in those which had moist, and in some instances extremely wet sphagnum, the proliferation was abundant. Extremely wet sphagnum occurred in air-layers which, when wrapped with polyethylene, were not secured sufficiently with theplastic-coated wires, thus allowing water entry between the P01yethy1ene and the stem during heavy rains. Stems having proliferated parenchyma were also larger in diameter than comparable stems not exhibiting proliferations. 47. .mucoEHumaxo 2626.3 E 35865 ma PST: Ho omswoon HoosHEOa I If“! I i [ll rill II I - - 306. 9:788 .02 m - AEEV EwaoHuoou .w>< o - - 8.8m H3308 2:: n.” E33 .0: .m>¢ . .. .. oouoou mEoum .oZ OO Ode I no HCSN O OOF'I l N .H .H vain oo'o o. 0 ON DC) I r-d O --1 392 9:758 .02 3:5 5.?me Soon .w>< Emum cocoon 2: >308 .oc .m>< p.808 mEmum .02 .l I om u-I—Jco or» Hider co co 03—3—4 oo :5in o lOv—1 hide) Ndfl" o cc: 6661 CO 6600 v—t 305 9:738 .02 AEEV newcoH goon .m>< 83m cocoon o .H \muoou .o: .w>< nouoou 955m 62 l‘ waive o urn—iv N GO oo'N N i-4—4CO N COD 0 Helm oo oo oo oo 66—1 moo aide o I 0 to F4 30on 9:788 .02 AEEvfiwaoH Soon .m>< 53m cocoon m .o \muoou .o: .w>< - - - - - - - o 0 H6808 28$ .02 oo CSCSO N 00 0'60 - - - - .. - - - .050ch 9:782 .02 AEEVfiwaoH Hoou .m>< - - - - - - - - 53m cocoon o .o \muoou .o: .m>< H - - - - - - - - c908 953m .02 000 wide: I I I I I I I I 353 ON: <3 5: 320 coo: ooUNH o .H H+ 0 .3 m .oH+ w .3 m .3 + m .3: o .3 o .$ o .3 He20 .E .m oouHH o .w + o 4% m .m + m .ew m .3 + m .moH o .3 o .3 o .3 He$0 .E .m 8;: m .m + m .3 o .m + o .8 m .3 + m .3 o .5. o .2. 0 .me .820 . .E .m come 32 K .Snanmm m .m + m .3. o 4. + o .3 m .m + m .ow o .2. o .me o .me 20:20 335QO .E d 8:. o .H. + o .3. m .o + m .3 o .o + o .ow o .3. o .3. o .3. £0220 coyoteom .E d 85 o .m + m .3. o .m + o .2. m .m + o .3 m .2. m .2. m .3 3:06 umuufimom .E d comm o .m + o .3. o .o + o .2. o .m + o .3 o .3. o .Hs 0 .He moaoHo wouozmom .E d oouH o 4. + m .2. o .oH+ m .8 m .w + o .3 m .2. m .9. m .9. £520 umuotmom coo: conuH wmoH 6 HBEBQom EMOE =NOE HHMQE mflHmuHHO Ufimwuso OUHWHSO 582.60 a . Bosses m . so: .59 a . E .0 E .0 E .0 ouawonxm .2 9:5on .3 ouzwonxm .m .m .322 nouoououacb nouoououm :oHpHHEoO wEHouooom .Ho Essmmcam :56on HH< oEmSO 3m 252. use 0:5 moHaaoooEHoFH. Ho :oHumooq muozmHLE 032: can do .CHEerm E ouzuwuanoF > mdmrd. 1959 Experiments Rooting After 100 days of treatment there was no significant influence of growth- regulators or of the position of the girdled branch in the rooting ofP‘ifia gla_uca and M sylvestris air-layers applied at the beginning of April, May, June, July and August. ' Significant differences in the rooting capacity of individual Mt lauca trees treated April 1 were observed. Out of 15 trees treated, on one tree six air-layers rooted, on two trees five rooted, on two trees four rooted, on four trees one rooted, and on six trees no air-layers rooted. Treatments applied toPi_nus sylvestris on July 1 also showed a significant difference in rooting capa- city of individual trees as follows: on one tree seven of 18 air-layers rooted, on one tree two rooted, on four trees one rooted, and on nine trees none rooted. An analysis of the effect of the time of year on the rooting of Pic—ea glLuca showed that when air-layers were applied on May 1, a significantly greater num- ber of stems rooted at the end of the lOO-day period than those applied on the first day of April, June, July and August, and that although the rooting differ- ence between April and June treatment was not significant, applications of air- layers at the beginning of these months resulted in a significantly greater number of rooted stems than those applied July 1 and August 1 (Table VI). Time of appli- cation did not significantly influence the number of roots per rooted stem. The tOtal number of air-layered Pinus sylvestris stems rooted after 100 days of L I _, w"- :“ I : F l s 0- 56. TABLE VI Rooting Results (After 100 Days) of Picea glauca Air-layers Date of Application Treatment in ppm (45 stems each) Monthly and . Dist. 0. 5 100 1000 100 Mean TYPe Of Observation H20 2’ 4' 5-T NAA IBA 4TNA (225 stems only) April 1 Tota 1 rooted 6 5 6 6 5 5. 6* % I‘OOted 13.33 11.11 13.33 13. 33 11.11 12.44 A Vg~ no. roots/ I'Qoted stem 4. 33 2. 40 2. 16 2. 33 3. 20 2. 82 AVE. root length (mm) 9. 73 10. 58 12. 76 11. 38 9. 75 10. 84"” May 1 Total rooted 5 6 8 8 l4 8. 2” % rooted 11.11 13. 33 17.77 17.77 31.11 18.21 Avg. no. roots/ ' 1— ooted stem 2. 40 2. 33 5. 50 2. 50 4. l4 3. 60 Avg. root length (mm) 9. 58 16. 28 16. 41 , ll. 20 13. 71 13. 43’“ June 1 Total rooted 5 2 9 8 5 5. 8* % rooted 11.11 4.44 20. 00 17. 77 11.11 12. 88 AV g. no. roots/ rooted stem 3. 80 5. 50 4. 22 4. 50 5. 00 4. 60 Avg. root length (mm) 24. 21 21. 09 16. 86 22. 94 19. 70 20. 96M 1&3 To tal rooted 0 1 0 1 1 o. 6* 9?: ‘rooted 0. 00 2. 44 o. 00 2. 44 2. 44 1. 33 Vg. no. roots/ rooted stem 0. 00 l. 00 0. 00 6. 00 2. 00 3. 00 vg. root length (mm) 0. 00 l. 00 0. 00 8. 83 10. 50 4. 06M 3% tom rooted 1 2 3 5 4 3. 0* 9% rooted 2. 44 4. 44 6. 66 11. 11 8. 88 6. 66 Avg. no. roots/ rooted stem 1. 00 6. 50 6. 00 l. 75 3. 50 3. 75 Avg. root length (mm) 2. 00 8. 30 5. 94 3. 75 2. 92 4. 58*“ _"Differences significant at the 5% level. “Differences significant at the 1% level. Multiple Range Tests Number of Roots Root Length (mm) Expected F: 1%, 14.15; 5%, 5. 84 1%, 14.15; 5% 5. 84 Observed F: 11. 12 20. 34 Monthly mean: April May June July August April May June July August 5._6 8. 2 5._8 0. 6 3. 0 10. 84 13. 43 20. 96 4. 06 4. 58 M3 Any tWO means not underscored by the same line are significantly different. Any tWO means underscored by the same line are not significantly different. 35 1.11 101/.” «1' ‘ “V. : ,,, . -\ A . "\\ 7 31¢ . 57. “ treatment when applied on July 1 was 13; on June 1, five; August 1, four, and no ‘ rooting occurred at the end of 100 days when air-layers were applied on the first of April and May. Because of the effect of individual trees in the July treatments, there was no significant effect of time of year on the rooting of this species. Mean root length was not significantly influenced by chemical treat- ! ments or by the position of the air-layer on the tree within any given lOO-day period. Air-layers applied to this species on the first day of June, however, resulted in a significantly greater mean root length at the end of 100 days of treatment than those applied in April, May, July and August. The mean root 1 length was significantly greater when air-layers were applied April 1, and May 1 than on the July and August applications (Table VI). ( Because of the low rooting response of fly sylvestri , no analysis I was made of the mean root length between monthly treatments. Tissue Proliferations The degree of tissue proliferation at the wounded area in P_i_c_ea gla_uca, as indicated by numerical ratings of one to seven did not vary significantly within four of the five lOO-day treatment periods. Indolebutyric acid treatments caused a significantly greater amount of tissue proliferation than 2, 4, 5-T, NAA, 4TNA and control treatments applied AUEUSt 1. but in the April, May, June and July treatment periods no significant differences between treatments were obtained (Table VII). TABLE VII Tissue Proliferation (After 100 Days) of Picea glauca Air-layers Average of Numerical Ratings—1! (45 stems each) Date Of Treatment (ppm) Monthly Application _ Mean Dlst. 0. 5 100 1000 100 (225 Stems) H20 2. 4. 5-T NAA IBA 4TNA AprLl l 2.13 2. 24 2. 11 2. 06 2. 13. 2.13" May 1 l. 91 2. 00 2. 02 l. 78 2.17 l. 97* June 1 2. 02 1. 57 2. 35 2. 24 1. 75 1. 98* July 1 1.17 l. 20 l. 04 l. 60 l. 28 1. 26* August 1 l. 54 l. 64 l. 95 2. 35* 1. 87 l. 87* ‘Difference significant at the 5% level. / V - Numerical ratings were given according to a standard of values from 1 to 7; a value of 1 indicated no proliferation with increasing values indicating a given quantity of tissue proliferation at the wounded area. Multiple Range Test Expected F: 1%, 14.15; 5%, 5.84 Observed F: 5%, 10. 97 April May June July August Monthly mean: 2. l3 1. 97 l. 98 1. 26 L53 Note: Any two means not underscored by the same line are significantly different. Any two means underscored by the same line are not significantly different. A monthly mean of tissue proliferation ratings obtained at the end of the April 1 treatment period was significantly greater than the monthly means of May, June, July and August treatments, and the May, June and August monthly means were significantly greater than that of the July treat- ment period (Table VII). Proliferations of wound tissue in stems of m; sylvestris were greater, on the average, than those of Pie: gla_uca, but did not vary signifi— cantly between any two monthly means, nor was there a significant difference between treatments (Table VIII). A pronounced difference in the general appearance .of the wound proliferations was observed between E sylvestris and M glica. In M proliferations were generally smooth-surfaced and evenly distributed in the wounded area, but in many stems of M sylvestris, regardless of Chemical treatment, root-like projections from wound proliferations were observed. These observations compared with those of the exploratory ex- periments (Page 48). Treatments applied to M in April resulted in 142 (52- 58 percent) of the stems with root-like proliferations; in May, 154 (57. 03 Percent); June, 204 (75. 55 percent); July, 166 (61. 48 percent) and in August, 144 (53. 33 percent) of the stems had root-like outgrowths of proliferated tissues. Tissue proliferations from wounds which resulted from removal of the brachyblasts in the air-layered region occurred in 2, 4-D, NAA and 4TNA __. i .‘ ,"‘.,‘V - o'u ' l' \ - - . a'1‘ C" ‘ - fl 1 \, ‘~ . " . ‘ . 1 a0 \ 60. I l 1 . I - — TABLE VIII Tissue Proliferation (After 100 Days) of Pinus sylvestris Air-layers Average of Numerical Ratingsl/ (45 Stems Each) Monthly Ea“:- Of . Treatment (ppm) Mean pp lemon Dist. 1. o 100 100 1000 100 (270 Stem 5) H20 2, 4-D NAA IBA IPA 4TNA April 1 2. 42 2. 53 3.02 2.40 2. 54 2. 97 2. 66 May 1 2. 97 3. 17 3. 17 3. 02 2. 91 3. 15 3. 06 June 1 3.15 3.17 2. 88 3. 08 2. 88 3. 3,3 3. 08 J uly 1 2. 77 2. 88 2. 57 2. 91 2. 75 3. 00 2. 81 August 1 2. 80 3.04 2. 68 2. 80 2. 80 2. 80 2. 82 — NUmerical ratings were given according to a standard of values from 1 to 7. A value 0f 1 indicated no proliferation with increasing values indicating a given quantity of ”35118 proliferation at the wounded area. treatments and the frequency was significantly greater in NAA treatments of April, May and June than with 2, 4-D and 4TNA treatments in these same per- iods. The lowest frequency of brachyblast~wound proliferation occurred in the treatments applied in July (Table IX). Injuries to Air-layered Stems The leaves on some air-layered M flaLuca stems frequently became yellow, gradually turned brown and dehisced. Yellowed leaves and dead stems were observed in all treatments at nearly the same frequency, but variations occurred between times of application. The greatest frequency of stem death was observed in the air-layers applied June 1, but the greatest frequency of air~layers with yellowed leaves occurred in those applied July 1 (Table X). The lowest total number of dead stems was observed in the August treatments With two stems dead, followed by 32 dead stems in the treatments applied April 1. Anatomical Studies Studies of transverse and longitudinal sections of non-air-layered Picea 3% and Pinus sylvestris stems from one-year-old shoots at the beginning of Illne showed structural differences between the two species. Current growth at this time was still in the elongation stage; Pinus sylvestris averaging approx- imately 10 cm in length, and Picea glauca averaging approximately six cm in length. «3 mm 2 o o : m o a 333. on: : o o o m o o H 3% «ON a..." a o o E a o 2 2i 42 3 cu o o 2 m o a .32 a: 3. S o c on a o 2 Ea... a 59m 2020 .2: <3 «E .22 n3. .N our 25:88:83 353m 020 2: 82 on: a: o 4 .85 858:3... uoamcm hoax HEB. a . . 5:5 manm 00 35:22 A5 3 36:5on0. We 630 :38. 35:22 mnofimhouzoum “$330me 525 .8852 0809 Ebmozwm main 5 no.5. 66.50 65 Hm 32228:on noamsm -nocv— 2:3 953m 00 Honfisz “Sch. 9: use "#0583 umwfin>zomnm «o mqofluuouzoum 0a 255. 1.1.1.- it . 11. 85111.11 1’1 TABLE X Injury and Stem Death of Air-layered Picea glauca After 100 Days Total of 225 Stems Each Date of Application No. Dead Stems No. Stems with Yellow Total Injured Leaves April I 32 23 55 May 1 103 9 112 June 1 117 22 139 July 1 29 140 169 August 1 2 68 h 70 Stems of Picea glauca were characterized by a definite layer of epider- mal cells with heavily cutinized walls. These cells were rectangular in shape being two or three times as long radially as transversely. Beneath the epidermis was an irregular layer one to several cells deep consisting of collenchyma. The periderm from seven to nine cells' in depth was composed of large parenchyma- tous cells with slight suberization. At frequent intervals on the surface of the stem were concave longitudinal bands consisting of lamellar collenchyma. The cortical region comprized almost one-third of the stem, and was made up of irregularly-shaped, isodiametric parenchyma cells many of which contained tannin deposits. Large resin canals were present in the cortex and were sur- rounded by one to several layers of relatively thick—walled secretory cells containing dense cytoplasm. The pericyclic and phloem regions were difficult to delimit because they coalesce. Only occasional bundles of pericyclic fibers were in evidence. A region of cambial cells clearly separated the phloem from the xylem, and consisted of lanceolate-shaped tracheids with numerous bordered pits on their radial walls. At relatively even intervals throughout the xylem were radially arranged uniseriate rays consisting entirely of parenchyma cells. Ver- t'iCaI parenchyma was largely confined to discrete areas along the perimeter of the growth ring. The pith was composed of large thin walled cells, and cham- bered by means of layers of simply pitted sclerenchymatous cells (Figures 3 and 4). r. e_t... a...“ . Ar. "1 -- _- IE' " ‘- Pmus snvssrms :§:3..,._r ._ a... PHLOEM' XYLEu MTH N w G E R L M B u A C EPIKNMIS YOUNG PERIDERI CORTEX PERICYCLIC REGION \ SECTIONS ,mu&. PICEA GLAUCA RADIAL Semi-diagramatic. Approx. 40 X. Figure 3. Comparative anatomy of Picea glauca and Pinus sylvestris stems in radial view. L‘ J. , ('1. 1r- MINM ‘1 «E h’ '1" PICEA GLAUCA TRANSVERSE SECTIONS Figure 4. Comparative anatomy of Picea transverse View. Semi-diagramatic. EPIDERMIS , COLLENCHYMA ’ CELLS .X ‘5; . "’ ‘35?“ >2 vouns PERIDERM CORTEX CAMBIAL REGION .. .33.... PERICYCLIC REGION ‘1‘ 1gp”? {. r {0’ {W ( PHLOEM "' """ " XYLEM PITH Pmus suvasrms glauca and Pinus sylvestris stems in Approx. 40 X. Stems of Pinus sylvestris were characterized by a single row of epider- mal cells nearly isodiametric in shape and covered with a thin cuticle. The young periderm consisted of a slightly suberized layer two to four cells deep. Beneath the periderm was deep cortex consisting of irregularly- shaped paren- chyma cells, many containing tannin. Large evenly- spaced resin canals were found in the cortex and were bordered by secretory cells containing dense cyto— plasm. The pericyclic region was not well defined in films, having cells about the same shape as in the cortex, but in general, slightly smaller in size. Phloem tissue consisted almost entirely of sieve cells with occasional phloem parenchyma. Phloem rays were heavily cytoplasmic and contained prominent nuclei. Xylem and phloem were clearly delimited by the cambium. Xylem tissue was character- ized by lanceolate tracheids with numerous bordered pits on the radial walls and radially arranged rays consisting of densely cytoplasmic parenchyma bordered on their transverse walls with tracheid-like cells having half-bordered pits on . the wall surface adjacent to ray parenchyma. This arrangement of tracheids gave the ray the appearance of being multiseriated, but in most stems, uniseriate secondary rays dominated. Resin canals, smaller in diameter than those of the cor- tex were relatively evenly dispersed, bordered by densely cytoplasmic cells, and often found in association with a secondary ray. Primary rays were conspicuous in this species, consisting of comparatively large parenchyma cells which were indistinguishable from cells of the pith. The tracheids adjacent to the pith did not contain circular bordered pits but instead, f'llll... I}? . , Iran-luff}. a... ’1 1‘ l ; flatly oval— shaped bordered pits. No vertical parenchyma existed in the xylem «1‘ of this species (Figures 3 and 4). The outstanding differences observed between stems of Picea and Pinus are summarized below: ; Picea Pinus p—a I Collenchyma layer beneath epider- 1. No collenchyma layer beneath epider- mis. mis. N o 2. Bands of lamellar collenchyma in Vertical collenchyma bands absent. vertical grooves on the surface of the stem. l . 3. Vertical parenchyma at the peri- 3. No vertical parenchyma in the xylem. : meter of a growth ring in the xy- lem. 4. Heavy tannin deposits in cortex. 4. Tannin deposits in cortex approxima- tely one-third less in quantity when compared with Picea. 5. Occasional fiber groups in the peri- 5. No fibers present in the pericyclic cycle. regions. 6. Primary xylem rays not conspicu- 6. Conspicuous primary xylem rays. ous. 7. Uniseriate secondary rays com- 7. Secondary rays bordered on the radial posed of parenchyma only. walls of ray parenchyma with tracheid— like cells. Uniseriate rays present, rarely multiseriate. After wounding the stems of both Picea glauca and Pinus sylvestris by removing a ring of bark, the sequence of anatomical changes which took place at and near the wound were as follows: approximately one mm above the upper rim of the girdled area, a meristematic zone was formed from dedifferentiated ~. Parenchyma cells of the cortex, existing phellogen and phloem. The new peri- derm was continuous with that of the non-wounded stem area and parenchyma cells formed by periclinal and anticlinal divisions of the cambium (Figure 5). "Two meristems, therefore, simultaneously took part in the production of wound proliferation - a meristem produced by the dedifferentiation and the existing cambium. The wound phellogen continued to divide, developing parenchymatous tissue basipetally and a phellem acropetally. As the phellem developed, the cortex and phloem tissues most proximal to the girdle rim began to break up and eventually became sloughed-off (Figure 5). The cambium increased in activity first producing loosely arranged iso- diametric parenchyma cells followed centripetally by shortened but lanceolate tracheids and a well defined phloem tissue centrifugally. Numerous secondary rays were also produced by the cambium, following an irregular course in the wound xylem and less irregularly in the phloem. The tracheids of the wound xylem were characterized by the presence of numerous, large bordered pits on all wall surfaces and in many instances with more than one longitudinal row of these pits on each wall (Figure 5). w sylvestris stems produced a greater amount of wound prolifera- tion than M M, and it often took the form of knob-shaped proliferations which were identified as areas of greater meristematic activity at the ends of primary rays and closely paralleled secondary rays of the xylem. The knob- shaped proliferations consisted of a well defined phloem developed centrifugally don N .mwfimoZNm mafia E Eugxueesoa - m .mm X .floéoamzofi 8583 335:0; £033 xofioo .868 - om ”mo—smut ego? no.5 uofiuoH Emotion - om 2:on 8:2» aooE voHehw a mo EC moan: Eon“ nofimnomzond U583 - < .m 95me 71. fr om a cambium that was continuous with the vascular cambium of the non- Proliferated stem area, a very irregularly arranged mass of tracheids inter- Sp ersed centripetally with occasional parenchyma cells and a wedge-shaped area of parenchyma tissue adjacent to the end of a primary ray. Root initials arose from the ends of phloem rays of wound tissues in Iooth species. In firms, however, the initials were invariably found at or near the apex of a knob-shaped tissue proliferation described above. The initials in both species were first evident as bulbous-shaped masses of cells which were densely cytoplasmic and contained prominent nuclei (Figures 6 and 7). Further development of the embryonic root was characterized by cell elonga- tion basipetally and continued cell divisions acropetally, pushing into the cortex of the stem (Figures 6 and 7). No dissolution and only light crushing of inter- fering cells of the cortex was observed in either species as the root developed. At the time of emergence of the root from the stem in both E and P_icga, a well defined plerome was present. A definite calyptrogen, dermatogen and periblem were not distinguishable, but appeared to be a relatively homo- geneous group of cells (Figures 6 and 7). Root emergence in both species was accomplished by a rupture of the periderm and epidermis (Figures 6 and 7). A well developed adventitious root contained a central core of undifferentiated tissue and centrifugally, primary vascular strands, a pericycle, a prominent endodermis, thick cortex and a suberized epidermis (Figure 6). 3..."... » J ,4 Figure 6. Transverse sections of air-layered Picea glauca stems. A - root initial arising from phloem ray (arrow) X 55; B - root histogen (arrow) projecting further into the cortical region X 75; C - root emerging through periderm (pe) X 75; D - base of a well developed root; ep — epidermis, c - cortex, en - endo- dermis, p — pericycle, v - yOtmg vascular tissue, u — undifferentiated tissue X 55. Figure 7. Transverse sections of air-layered Pinus sylvestris stems. A - root initial (arrow) arising from a phloem ray at the apex of a tissue proliferation X 55; B - further elongation and differentiation of a root histogen X 75; C - emergence of root histogen through periderm (pe) X 75; D - young root (non-median) after emergence from stem, (r) root- tip X 75. Proliferations from wounds made by removing the brachyblasts from the stems of M sylvestris were similar in structure to the knob- shaped Proliferations produced in the wounded area, but differed in the relative amounts of wound tissues produced and were not associated with vascular rays. In com- parison, the brachyblast wound proliferations consisted of a thicker periderm and thinner cortex, phloem and xylem than the proliferations of the girdle wound. Wound xylem and phloem was produced by diVisions of vascular cam- bium of the brachyblasts and the periderm and cortex from dedifferentiated cells of the surrounding cortical areas and existing periderm. Anatomical studies of longitudinal sections cut through the girdled area of dead M M stems were conducted in an effort to determine whether girdling deeper than the cambium was the cause of stem death. After detailed observations of sections from six randomly selected stems, one showed the presence of cuts from girdling in the xylem of the previous seasons' growth. Similar cuts, however, were observed in living stems of this species. Further observations of the sections from dead stems showed that no periderm had formed over the wound and the vascular cambium had ceased dividing as evidenced by the amount of new xylem produced when compared with living stems of the same treat- ment period. Survival of Rooted Air-layers Because of the low rooting response obtained in mi} sylvestri , most of the rOOted stems were used in the anatomical study and not planted for further 81'0th A sufficient number of rooted stems was obtained from P_iciea gl_a_uca treatI'I‘Ients to enable a study of the survival of rooted air-layers when planted in soil or in shredded sphagnum. A period of at least 30 days after planting was found necessary before sur- vival Counts could be made because indications that some stems would not survive were not apparent from visible symptoms for approximately 30 days; when cool air temperatures and frequent cloudiness prevailed, a period of at least 45 days after planting was necessary in order to make survival counts. Because of this variation in time requirement, final counts of survival were not made until Novem- her 2 1. 1959, giving a period of 70 days for the last planting of the rooted Pic—ea % air-layers which were removed from the source plants on September 10, 1959. Twenty of the stems rooted in the exploratory experiments were planted in a Soil mixture of which seven survived. Survival of rooted air-layers planted in Shredded sphagnum July 10, August 10 and September 10 was 52 percent, 91 percel‘it and 100 percent, respectively (Table XI). Periodic examinations of the amount of root growth of the planted stems ShOWed that lateral roots form within one week after planting and after one month the length of the roots which were present at the time of planting were more than four times greater in length. After three months a very extensive root system w . . . . as Present, but no new roots originating from the stem were observed (Figure 2). TABLE XI Survival of Rooted Air-layers of Picea glauca Planted in Two Different Media Date Planted October 9, 1958 July 10. 1959 AugUSt 10, 1959 September 10, 1959 _ Number Number Percent Medium Planted Survived Survival Nov. 21, 1959 Soil mix 20 7 35. 0 Shredded Sphagnum 24 15 52. 0 Shredded Sphagnum 36 33 91. 0 Shredded Sphagnum 27 27 100. 0 f /’ I “I \ l 1 o l s l l l \ 1 l - . A - . o . v 1 . l 1,: ill _ l 77. DISCUSSION OF RESULTS The results of the exploratory experiments conducted in 1958 and the periodic seasonal studies of 1959 have clearly shown that the initiation and development of adventitious roots in nine- to lZ-year—old Picea glauca and seven- to eight-year-old Pinus sylvestris trees is physiologically possible by the air- layered method and that no significant benefit was achieved with the use of any of nine growth-regulators in the promoting of root initiation and development. No conclusions could be given for this failure to respond to Chem icals in the rooting of the two species, but it might be noted that, to the knowledge of the author, the only previous rooting of these species was observed by F arrar and Grace (1926) and Kirkpatrick (1940) with cuttings offiigejt gLa_uca_1 and Komissarov (1938) and Hitt (1955) with cuttings ofP_in_u_s sylvestris. The yol-‘11ger plants used by these investigators suggested that the seven- to lZ-year- 01d tI'ees were too old for favorable response to a growth-regulating stimulus. SuDDOrt for this reasoning was given by Deuber (1940) and Thimann and Delisle (1 93 9) in which rooting of cuttings from Mahdi trees reduced sharply as the age of the source plants increased. Rooting, however. was found to vary significantly with the time of appli— cation of the air—layers in 31331 g_la_uca and in both species, some individual trees were significantly greater in rooting response than others. This response indiefitted that physiological and inherent factors had a greater influence on the rooting of these two species than exogenously applied growth-regulators. In A order t0 secure convincing evidence that individual trees rooted significantly greater than others in these experiments, further air-layering of propagules WOUId be necessary. Wright e_t a_l. (1958) have pointed out that ". . . . the genetic variation attributable to species hybridization, geographic variation and individual tree variation are not separable in work done to date on material of unknown pro- venaIICe. " The seed-grown trees used in these experiments were of unknown origin and probably represented progeny from more than one provenance. The significant differences in number of roots and of root lengths ob- served between the five lOO-day treatment periods of gigs} glLuca is closely correlative with temperature fluctuations recorded by the local United States weather Bureau Station during these periods. In April treatments of 3123 % when effects of individual trees were encountered, 28 out of 225 air- 1ayel‘s rooted. 1f the effect of individual trees was removed, there was a total of 15 rooted. The highest rooting (41 ‘of 225 stems) resulted from the air-layers applied on May 1 and removed August 1. At the time of application in May, air temper- atures did not exceed 60°F and remained above 41°F, followed by gradually in- Crea Sing temperatures reaching a high of 89°F at the end of June. The lowest I.ooting response occurred when air-layers were applied July 1 at a time when the rhaximum air temperature reached 88°F followed by a 34-day period with a mean maximum temperature of 82. 9'F (Figure 8). The greatest number of in ' . Jured stems also occurred among those treated in July. .cmeozz .wEmcwq .mfiomou 255m Abies; .m .3 am: 2.: 50.8 cog—3030.. .wcmoE ouzImuodEBLIm 53555 new EDEIme >me-o>c 52> :omCmQEoo 5 822m «.85 I0 983:-me @908 we comics: :38. .w v.59...» mIIEoIz HonEmSoZ H3800 umnEBQom Iwzws< 35a 2:; >22 Ivar. \\\ \XNoCdxxg >\/\< yd. . villi. .V\~N\<\.\ X\\V\XV~; om .. . .. , 47.9. ._.,..,,.H. 3.... .9.. 9.. II : . , .y. N... .. .., _ . XV...,...._ I \ I . Kg, .\ Y , \I \ .m I . II . . I Ix L m IK .I b > x 9 H. ow / I \ (a ow .w a . .I . m II I. I \/I .. m I n m. I II I I \II \ II\ a o 00 I .. I I. R I .\ Ll on I. I. I ~ _ e I \ u H I. I I > a I > \ o I. I e I x I a I .I x m 0 I \ I x z > a y \ I x m x I \I / x I \I I an P I.\ x I a < I I III < 3 0w J — \ I\ m a N. I x \ < co 5 a a < d m I e s I \ u. \ I > , , > .I 2: Eng: IE :82 on w. 02 1 > ow I < . EIIEIISE =32 \- , 7 I, m..'. Studies of the temperature in the air-layers showed that when the air- teml>3‘eir'a.ttire in the proximity of an air-layer reached 79. 5°F, the sphagnum medium directly exposed to the sun was 105. 5°F (Table V). It is probable, therefore, that high temperatures of 105. 5°F and higher occurred in some of the air - layers applied at the beginning of July and might have accounted for the low I‘ooting response and high number of injured stems. Higher rooting responses and Comparatively little stem injury in other treatment periods were likely a result of more favorable temperatures at the time of treatment. Observations of longitudinal sections through the girdled area of dead Stem S showed that no meristematic activity in the proximity of the wound had occurred. Temperature extremes occurring at the time of, or shortly after, W0“riding may have prevented the formation of a protective periderm layer near the Wound surface by killing the functional cambium and preventing dedifferentia— tion of parenchyma in the cortex and phloem. The failure to correlate stem death with Possible injury from girdling too deeply, substantiates this reasoning. An influence of temperature on root length was suggested from a compar- ison 0f root length with air-temperatures during the treatment periods. Cool temperatures (60 to 75° F) seemed to favor rapid root growth after initiation. Air-layers applied on June 1 resulted in a significantly greater mean root length than those applied the first of April, July and August. The maximum mean temperatures averaged 77. 5°F in June, and increased to an average of 84' F until harVe st (Figure 9). The lowest mean root length was observed at the end of the If" y' dwwEozz _mecmq .muaoaou smousm .8583 .m .2 $2 2: Eob omamIzuImUs .memoE unamquEoILIm EIIEIEE new :52:me 3.652% :33 cemimanu E 8:me mesa .8 5mm“: Sea :32 .o 95mg 93:22 HonEmSoZ yonoIoO HmnEmEom 3:91... .33. 9:: .922 2.2: , h l 1 I 2 I 2 on I . I. \ I. \II \ ”<~ _— \s K > ON , I F u \ I ~ 0* II _ III I. I. W I ’ _ —— \l s ”Ham m I _ I I s \ I I m m I II > m . I \ II x. I.\ w m on I _ I; N .l I. I . . om m 1 : I I > a I > \ n I . . e I x I a I Z \ 1 w I\ r a ../ x I \I a I a I . m. cm. a (x.— \\ II a .x a (a ¢ u \ . \I 0* V — \\ ‘4 < 00 m m , _ a on m _ \ 9 ( u a I, x. s om _ I\ on NI EzEIJIE :82 m . W . > > a so , ./ < a 8 5:555 :82 82. JUIY treatment period. At the beginning of this period the maximum mean tem- perature was 89° F, and continued to reach 80°F or above through the months Of 1111)? and August. The low mean root length resulting from the August treat- ments might be attributed to the steady decline in temperatures beginning one month after treatment (Figure 9). Although the time required for root initiation was difficult to observe in theSe experiments, evidence was found from anatomical observations that roots initiate relatively early in the treatment period in 13:21 W Additional evidence of early initiation of roots was observed in the exploratory studies condUCted in a greenhouse when well developed roots were present as early as five Weeks after the initial application of the air-layers. These comparisons suggested a critical period for root initiation with re S[Dect to air temperature soon after treatment. Temperatures reaching above 80- F or below 60°F tended to hinder root initiation and development, the mean root length at the end of the treatment period was less. Factors other than tree age, inherent capacities, and temperature, un- doubtedly influenced root initiation and development. These other factors were the physiological condition of the stem at the time of initial treatment and dur- ing the treatment period, the amount of air space in the sphagnum medium, and the nutritional status of the plants. The anatomical studies have shown that root initials arose from secondary rays of the phloem which were produced by divisions of ray initials of the cambium. “ was reasonable, therefore, to presume that root initiation occurred more readily during the period of rapid cambial activity and when food reserves in the Stem were present in sufficient quantity to support the formation of root initial 3. Both of these conditions were ideal at the beginning of May in _P_icia M (Kienholz, 1934). Increased moisture and subsequent decreased oxygen in the sphagnum medium as a result of heavy rains may have contributed to a reduction of root initiation and development in both species. In this respect the use of poly- ethylene films in air-layering was not completely satisfactory without a prac- tical method for sealing the film against the stem and thus preventing the pene- tration of water. The position of the air-layered branch with respect to the rank of the whorl on the trees had no significant effect on rooting and callusing of Pi_ce_a % and ms. sylvestris. Yet studies of Albizzia by Toole (1948), P_inu_s m by Doran and Holdsworth (1940), $132 a_bie_s by Grace (1939) and others haVe shown that cuttings taken from lower branches root more readily than those from positions higher on the tree. Reasons for rooting in some stems and not in others on the same tree and Within the same treatment period are difficult to discern. The possible fact01's influencing this variability of rooting are postulated as follows: Be- c . . . . . auSe treatments were applied on all Sides of the trees, variations in the length 0 - . f tll‘ne that the air-layers were exposed to rays of the sun would also vary, 1.- . '1"! Inn-I.— O ww- - I ssh-Kg 1 | ‘1 1 ; ‘ , 1 V , ‘ II' thus causing higher temperatures in the sphagnum medium of some and lower temperatures in others. Thermocouple recordings support this reasoning (Table V). In addition to temperature differences, the degree of stem vigor and the rate of carbohydrate assimilation could be expected to vary with the length of time that the stems were directly exposed to the sun. Anatomical changes at and near the wound surface of both species were Similar to those observed by Hiller (1951) from TaiS cuspidata cuttings and by Beal (1 951) from several species of gymnosperms. The initiation and develop- ment of adventitious roots from knob- shaped proliferations of the vascular tissues in % sylvestris is unique to this study and has not been observed in £22 51313? and not believed to have been observed previously in other species. The irregular arrangement of tracheids of the wound xylem and the pres- ence of bordered pits on all of their wall surfaces in w sylvestris (Figure 5) was observed in Titis cuspidata stems by Hiller (1951). This distortion of xylem in % was observec both in air-layers treated with growth-regulators and in those treated with distilled water. The appearance of bordered pits on the tangential as well as the radial walls of the tracheids when observed from a longitudinal section of the stem may have been a result of cell distortion occurring in such a manner that cell walls Which WOtild normally be in a radial plane were twisted into the tangential plane. occasiollal groups of multiseriate bordered pits, however, cannot be explained on . . . . . the ba SIS of cellular distortion. A pos31ble cause of this phenomenon was suggested from the work by Jeffrey (1917) who postulated that aberrant tissues, formed as a result of wounding, may be a temporary reversion expressing cell CharaCteristics of ancestral plant groups. A comparison of the tracheids of wound xylem in m sylvestris with tracheids of the fossil genus Erepinus as described by Jeffrey shows a remarkable resemblance. The proliferation of tissues from wounds caused by removal of dwarf shoots in Eng sylvestris was the most pronounced in treatments of NAA, 4TNA l and 2» 4 - D. This suggests that these growth-regulators are more active in stimu- lating meristematic activity than IBA and IPA. Anatomical examinations of the prOIiferations showed that the meristematic activity was limited for the most part ‘30 periderm formation and there were no indications of root initiation. As a result of the relatively small number of rooted stems obtained, an adeqUate comparison between survival of rooted air-layers planted in sphagnum and in soil could not be made. The data obtained nevertheless suggest the impor- tance 0f time of planting for the survival of rooted air-layers. All of the rooted stem 8 of Pifla w survived when planted on September 10, 1959 in contrast to 52 perCent survival of those planted July 10, 1959 (Table XI). Air temperatures at planting time in September reached a high of 84° F and decreased gradually during this mOnth, while temperatures at the planting time in July reached 82" F, and the daily mean maximum continued to reach above80°F for 60 days. The cooler temperatures of the September planting may have favored lower transpiration of the plants until a sufficient root system had developed. The stems planted in this same month, however. also had a greater mean root length at the time of planting than those of the July and August plantings and a consider- able ianUence on survival may have resulted from the amount of root- surface present- On the basis of the results from these experiments, the use of air- layering to propagate Pifia gla_uca and EM sylvestris commercially on a large Scale is not recommended. For the perpetuation of desirable clones, rare S‘pecimens and experimental plant material, which is not readily propa- gated by other vegetative methods, air-layering has considerable merit. SUMMARY Vegetative propagation of woody and herbaceous plants is used primar— ily f0]? the perpetuation of individuals selected for desirable characteristics. Many Species of plants are propagated vegetatively by cuttage and graftage; others are reproduced, but with great difficulty, by either method. Interest in the process of air-layering has stimulated some investigations into the feasibility of this method for propagating plants which do not readily PTOdUCe roots. Promising results have been obtained from air-layering experi- ments with several deciduous shrubs and shade trees of the temperate zone (Creech, 1950; Wyman, 1952; Hangere_ta_l. , 1954; Ching_e_t a_l. , 1956). Many tr0pica1 plants are almost exclusively propagated by air-layerage, principally the mango, Mang‘fera ildiia, avocado, P_er_se_a gratissima and the Lychee, % chinensis (San Pedro 1935; Fielden 1936; Grove 1947i Singh 1953). Investi- gatiOns by Lasschuitt (1950), Mergen (1952, 1955), Frolich (1957) and Hoekstra (1957) have shown that rooting by air-layerage is possible in several coniferous SPECieS. On the basis of the results obtained by some of these investigations, the Present study was conducted to determine the feasibility of air-layering and some 0f the factors affecting the rooting offlic_e_a_i W and w sylvestris. Exploratory experiments were conducted in the 1958 growing season to establish the value of nine different growth-regulators for the induction of root initials in air-layered branches of seven- to lZ—year-old trees “EC—8? M and Pinus —\ Sleestris. In March 1958 air-layers were applied toP_icEi g_l':iu_ca plants growing in a greenhouse and treated with talc—carried growth-regulators. Little rooting occurred in these treatments, and the method of applying the growth-regulators was fOUnd to be unsatisfactory. All treatments were thereafter injected into the P01yethy1ene-wrapped sphagnum in aqueous form with a hypodermic syringe. Experiments using field-grown trees of Egg gl_afii andflnuj sylvestris were conducted in May, July and August, 1958. Stems of the May treatments were Wounded with a v-shaped notch on the abaxial side and those in July and Aug-us: by removing a complete ring of bark two to three mm wide. Some of the treatments applied in July rooted in both species; up to 50 percent rooting occurred in 1, 000 ppm indolebutyric acid treatments ofP£e_a gflia and 30 per- cent rooting from indOIeproprionic acid treatments Ofm sylvestris. No root- ing re Sulted from the May treatments, and little rooting resulted from air-layers applied in August, the latter occurring only in §i_ce_a glau_ca._ From the results of the exploratory experiments growth-regulators were selected for use in further investigations conducted in the 1959 growing season. Ail“layers were prepared on the lower, middle and top whorls of 15 trees of eaCh SDecies at the beginning of April, May, June, July and August, 1959. The air‘layers at each whorl on M w were injected with aqueous solutions of One of the following treatments: 0. 5 ppm 2, 4, S-trichlorophenoxyacetic acid, 100 ppm naphthaleneacetic acid, 100 ppm 4-thianaphtheneacetic acid, 1,000 ppm ind . . . . . . . olebutyric ac1d and distilled water and on Pinus sylvestris air-layers were -£ injected with 1 ppm 2, 4-dichlorophenoxyacetic acid, 100 ppm naphthaleneacetic acid, indolebutyric acid and 4-thianaphtheneacetic acid, 1, 000 ppm indolepro- Prionic acid and distilled water. Results 100 days after treatment showed that there were no significant differences between treatments or between positions of the air-layers on the trees. A significantly greater number of $263 gla_uc_a stems rooted, however, when air —1ayers were applied at the beginning of May, little rooting occurred When applied in July. Mean root length was significantly greater at the end of the June treat- ments when compared with April, July and August. The number of rooted stems and the mean root length were correlative with the mean minimum and maximum air temperatures, which might suggest that temperatures above 80°F occurring at the beginning of a treatment hindered r00t initiation and increased the incidence of stem death, but that root length was inc reased by gradually rising daily mean temperatures and hindered by temperatures which prevailed below 60° F. Outstanding differences in stem anatomy between M andms were observed. Pifia stems were characterized by a layer of collenchyma directly beneath the epidermis and in vertical grooves on the surface of stem, vertical pare‘nchyma at the perimeter of the growth rings in the xylem, groups of peri- Cyclic fibers, parenchymatous secondary rays and inconspicuous primary rays. Pm& sterns, on the other hand, did not have a collenchyma layer beneath the epiderm is and bands of pericyclic fibers, but were characterized by conspi- cuous primary rays and tracheid-like cells bordering the radial walls of secondary ray parenchyma. Anatomical examinations of sections cut through the wounded area of Picea glauca and Plni sylvestris stems showed that root initials arose from secondary phloem rays of tissue proliferations produced by the vascular cam- bium - Root initials in stems of 2111.13. sylvestris invariably occurred at the apex Of a knob-shaped tissue proliferation. Anatomical examinations of dead stems of Pita glflcg showed that no meri Stematic activity had occurred at or near the wounded area, and it was sugge Sted that high temperatures at the beginning of the treatment period may have prevented the formation of a protective periderm over the cut surface causing stem death. Survival of rooted air-layers of w w planted in September was 100 Percent after 70 days in contrast with 52 percent in July when observed after 130 days. U “I , _ w;-l"_.“, l r “.7 4’51 3.":- | n . ‘. a” ”wdr 4' — t a >1 3‘ lo {I 5‘ Lu- 9- .a t '0 ‘ 1 a 91. ANNOTATED BlBLIOG RAPl-IY Abraham , P. 1956. Air-layering is a success with cashew. Indian Farming, Jan. pp. 26-27. Bannan_ M. W. 1934. Origin and cellular character of xylem rays in gymno- sperms. Bot. Gaz. 96: 260-281. 1941. Vascular rays and adventitious root formation in ’I_‘hi_i_]_a occidentalis L. Amer. Jour. BOt. 28: 451-463. BartOIOrne, N. S. 1935. 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