m" r l w W I ‘ \ M mm k “W M WW \ IL‘ I _'(.0—3 1010 U)\IU1 GROWTH OF WHITE PINE SEEDLINGS IN RELATION TO AVAILABLE CARBOHYDRATES Thesis for the Degree of M- S. MlCHlGAN STATE COLLEGE Charles E. Darrell 1940 A “1&1? 7 . .Jhgauuflfiwuvu...) , GROWTH OF WHITE PINE SEEDLINGS IN RELATION TO AVAILABLE CARBOHYDPATES Thesis for degree of Master of Science Michigan State College 07 }~ Charles E. Darrell 1940 THESIS 486 ml 2 1 GROWTH OF WHITE PINE SEEDLINGS IN RELATION TO AVAILABLE CARBOHYDRATES by Charles E. Darrell A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Forestry 1940 ACKNOWLEDGMENT The author wishes to take this Opportunity to ex- press his apnreciation to Professor M. E. Deters for the unlimited COOperation and assistance given by him in the performance of this work, and to Professors R. P. Hibbard and H. C. Peeskow for their advice and assistance and for making available the eouipment of the Botany Department laboratories. CONTENTS Introduction ..................................... 1 History .......................................... Field procedure .................................. Laboratory procedure ............................. Carbohydrate determination .................... SOIUble OP redu01ng Sugars 000000.0..000...0.00 (OQOCDCDQN) Total dextrins, starches, and hemicelluloses .. Determination of reducing sugars .............. 10 Results .......................................... 15 Carbohydrates ................................. 15 Height growth ................................. 15 Total growth .................................. 17 Summary and conslusions .......................... 23 Bibliography ..................................... 25 LiSt Of Tables and Figures 00.000.00.0000000000000 i TABLES AND FIGURES Table I. Carbohydrate content of seedlings of different ages and grades ................... 11 Table II. Carbohydrate content in grams, height growth and total growth in inches of shoots of white pine seedlings of different ages and grades .................................. 12 Figure 1. Mean weight of sugars and polysac- charides per tree for grade B stock one to four years old............................... 19 Figure 2. Current annual height growth for stock of various grades ..................... 20 Figure 3. Relation of height growth to total available carbohydrates ..................... 21 Figure 4. Relation of total growth to total available carbohydrates ..................... 22 INTRODUCTION Some forest tree seedlings, when planted in the field, become established rapidly and grow vigorously. Other seedlings fail to become established or grow very slowly despite reasonably favorable growing conditions. Nurserymen therefore often classify planting stock into several grades, attempting to cull the inferior, low vig— or stock-fthose which would not survive if planted in the field. The criteria commonly used in culling prac- tices are: size of stock, top-root ratio, length and color of leaves and general appearance. Seedlings from a dominant position in the seedbed usually deve10p as select stock while suppressed seedlings or those grown very densely are second grade or cull stock. While the external characteristics of seedlings are of great value in determining the quality of the stock, there must be a physiological basis for the differences which exist. Since stored food reserves have an impor- tant influence upon plant growth, it was considered that the available carbohydrate content of tree seedlings might provide a satisfactory measure of growth follow- ing transplanting. The study was made using white pine seedlings of from one to four years of age and of three quality classes except for the one year seedlings in which only two quality classes were obtainable. HISTORY Research in carbohydrate content of trees and their influence on various tree organs and physiological activity is generally limited to a few species and has been in progress a comparatively short time. Especially is this true where forest trees are concerned. This latter condition can be attributed to the fact that for- estry as a science is of recent date, particularly in America. Graber, et al.,(8), in their work on organic food reserves in relation to the growth of alfalfa and other perennial herbaceous plants conclude that l. Stor- age of organic food reserves in roots takes place dur- ing the fall dormancy period, 2. Some loss of food re- serves in roots occurs during winter and before growth starts in snring, and 5. The maturity and.amounts of top growth, and the longevity of the alfalfa plants were very generally associated with a high content of carbo- hydrate and nitrogen reserves in the root. McCarty(16), working with Elymus ambiguus, Vasey and Scribn., and Muhlengergia gracilis, (Nutt.)Hitch., states, "In general, the trend of carbohydrates are from low concentration in the early part of the growing 5. season to high concentration, the maximum occurring during the declining phase of growth. The greatest change is in the starch fraction, while there are only slight changes in the reducing sugars. There is a de- cline in the hemicellulose concentration during time of flowering and develOpment of fruit." Seasonal march of carbohydrates was found to be in inverse ratio to the rate of growth of the herbage. Mitra(17), in his work on two year old seed- lings of apple, concluded that total carbohydrates in- crease rapidly in stem and roots at the close of the growing period in August, September, and October. .From October through December there is a gradual decrease, followed by a rapid decrease in January, February and March, when the minimum is reached. The translocation sugars, glucose and maltose, are most abundant during the dormant season, increasing rapidly in stems during the early spring. This increase in sugars during the winter, fol- lowed by a decrease in spring and early summer, was also noted in conifers by Dixon and Atkins(55,in‘£igea‘ggng- densis,L., Linnaea borealis, Gronov., and Pyrola rotun- dafolia, L., by Lewis and Tutt1e(14), in some western evergreens by Gail(6), in apple spurs by Hooker(9), and in Spruce by Goldsmith and Harris(7). Preston and Phil- lips(24) state that "There is no great increase in the 4. content of sugars in stems and roots, except in spring as the buds unfold," and "The maximum for total carbo- hydrate reserves for deciduous leaved trees is at the time of leaf fall in autumn, whereas the maximum is at the opening of buds in the spring for persistent leaved trees." Abbott(l) describes what he terms a "rest per- iod" in apple and peach trees occurring between July and October in which there is no response to growing conditions. He points out that accumulation of carbo- hydrates and the beginning of the rest period are as- sociated phenomena. In their treatise on The Physiology of the Ap- £13, Butler, Smith, and Curry(4) state that the supply of carbohydrate material needed by the growing parts is mainly furnished by the hydrolysis of the saccharase stored in the small roots. This statement is not sub- stantiated by others who assume that the necessary sug- ars are furnished by hydrolysis of starch. Traub(25) found for 2-5 year portions of apple twigs that the minimum in reserve carbohydrates is reached during April and May, just before inception of growth. Hemicelluloses may be regarded as a reserve carbohydrate supply, Murneek(21). They occur primarily as thickenings of the cell wall, and are made up chief- ly of dextrosans, mannans, galactans and pentosans. Hemicelluloses may be hydrolyzed, at least in part, though probably not completely, to the correSponding sugars. This is substantiated by Jones and Bradlee's(lO) work on maple trees. They state that sucrose and hex- ose increases and starch decreases during the winter sea- son are always associated with a decrease in hemicell- ulose content. In evergreens, roots and stems are not the only places of starch storage. Miyake(20) points out that starch in evergreen leaves, generally Speaking, begins to decrease in November, reaching its minimum during January and the beginning of February, and increases again from the end of February. The starch content of evergreen leaves is generally more abundant in spring than in late summer or early autumn. Langlet(12) concluded from extensive experi- menting that the sugar content of one year pine seed- lings as well as needles of older plants are low in the fall, high during the winter, and low again in the Spring. The disappearance of the soluble carbohydrates in the spring is probably due either to their reconver- sion to insoluble carbohydrates, to their rapid util- ization in early spring growth, or both, Meyer(17). In general, then, reserve or stored carbohy- drates in perennial plants are in greatest amounts in roots, stems, and leaves in early fall. They decrease gradually until inception of growth in Spring then rapidly until a minimum is reached just before the be- ginning of rapid photosynthetic activity. Although there are periodic fluctuations in amounts of both su- gars and polysaccharides, the greatest changes occur in the latter. Hemicellulose as well as starch is a source of sugars in plants. These are drawn upon even though suf- ficient quantities of starch are present. In conifers, carbohydrates are stored in the needles as well as in roots and stems. FIELD PROCEDURE White pine, Pinus strobus, L., of 1-0, 2-0, 5-0, and 4-0 stock grown in the Michigan State College for- est nursery was selected and used for the experiment. All seedlings were lifted on April 15, 1959, before active spring growth started. Each age group was divided into three grades: A, select; B, average; and C, poor. The grade in which a seedling fell was deter- mined by size of stem, amount of foliage and relative vigor as determined by general appearances. Twenty trees of each grade were lifted, care be- ing taken to save as much of the root system as pos- sible. Each seedling was tagged immediately upon lift- ing and placed in wet burlap to prevent drying of root hairs. Ten trees of each grade were healed-in and plant- ed the following day, April 16, 1939 in a well prepared nursery bed. The soil was well-drained, gravelly, sandy loam. Rows were one foot apart and seedlings within the rows were set at ten-inch intervals to allow sufficient space for growth. Throughout the growing season the~ bed was watered frequently to maintain favorable soil moisture content. These methods of spacing and watering eliminated any important effects from.competition. Weeds and grass were kept out of the area during the growing season. 8. Measurements of the season‘s growth were made to the nearest one-tenth inch after the 1959 growing season had ended. Two separate sets of measurements were taken: 1) Height growth —-growth of the terminal shoot and Q) Total growth--sum of growth of all ter- minal and lateral shoots. LABORATORY PROCEDURE On the day that the trees were lifted, April 15, 1959, ten seedlings of each grade were prepared for laboratory analysis. They were carefully washed to re- move all dust and dirt, cut into small pieces to facil- itate drying, and placed in an electric oven. The tem- perature was kept at 100°C for half an hour and then dronped to 65°C until complete drying of samples. The high initial heat was used to stOp all enzymatic action. The samples were dried to a constant weight after having been in the oven for 26 days. After the seedlings were dried and weighed, weights being taken to the nearest miligram, they were ground to a fine powder. In each grade three separate samples of the ground mater- ial were taken for carbohydrate determination. Carbohydrate determination. The powdered seedling samples were placed in 400cc. of 80% alcohol and boiled in a reflux condenser 9. for one hour. The liquid was decanted and the process repeated. In order to separate the soluble from the in- soluble carbohydrates, the liquid and residue were poured into a folded filter paper, allowed to filter, and washed thoroughly with warm 80% alcohol. The filtrate then contained all the soluble matter such as the mono- and di-saccharides, lipoidal material, mineral matter, etc. The residue contained the soluble and insoluble starches, dextrines, and hemicellulose besides lignin, cellulose, complex proteinaceous matter and other complex substan- ces. Soluble or reducing sugars. The filtrate was transferred to a large, evapor- ating dish and evaporated over a steam bath until the odor of alcohol was no longer perceptible. The remain- der was then treated for removal of lipoid material which would interfere with the carbohydrate analysis. An ex- cess of Horne's lead subacetate was added, the precipi- tate removed by filtering, and excess lead subacetate removed with sodium acid phosphate and filtering. The filtrate was then placed in a 500cc. volumetric flask and.made up to volume. All material, when not being used, was kept in a refrigerator. Total dextrins, starches, and hemicelluloses. The residue of the original extraction was spread on an evaporating dish to dry off all alcohol. 10. The residue and original filter paper were placed in 400cc. of 2%% sulphuric acid and boiled in a reflux con- denser for 2% hours. After cooling, the solution was neutralized with sodium hydroxide and was ready for de- termination of its reducing power. Determination of reducing sugars. Reducing sugars in each case were determined by the Bertrand method. 11. «amp mafiacmmmuamm * ¢O.¢m mb.¢a mm.m mowo. ammo. mmvm. 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