RESEARCH BULLETIN NO. 8. UNIVERSITY OF MISSOURI COLLEGE OF AGRICULTURE AGRICULTURAL EXPERIMENT STATION THE KILLING OF PLANT TISSUE BY LOW TEMPERATURE COLUMBIA, MISSOURI December, 1913 LI Di\ni^ * UNIVERSITY OF MISSOURI COLLEGE OF AGRICULTURE Agricultural Experiment Station BOARD OF CONTROL. THE CURATORS OF THE UNIVERSITY OF MISSOURI. EXECUTIVE BOARD OF THE UNIVERSITY. THOMAS J. WORNALL, Chairman. Liberty. SAM SPARROW. Kansas City. J. C. PARRISH Vandalia. ADVISORY COUNCIL. THE MISSOURI STATE BOARD OF AGRICULTURE. OFFICERS OF THE STATION. THE PRESIDENT OF THE UNIVERSITY. F. B. MUMFORD, M. S., Director, Animal Husbandry. J. W. Connaway. D.V.S., M.D.. Veterinary Science. Frederick Dunlap, F. E., Forestry. C. H. Eckles, M.S., Dairy Husbandry. 'W. L. Howard, Pti.D., Horticulture. C. B. Hutchison, M.S. A., Agronomy. M. F. Miller, M.S. A., Agronomy. G. M. Reed, Ph.D., Botany. E. A. Trowbridge, B.S.A., Animal Hus- bandry. P. F. Trowbridge, Ph.D., Agricultural Chem- istry. J. C. Whitten, Ph.D., Horticulture. H. O. AUison. M.S. A., Apjmal Husbandry. H. L. Kempster, B.S.A., Poultry Hus- bandry. A. J. Meyer, Assistant to Director. L. S. Backus, D.V.M., Veterinary Science. T. R. Douglass, B.S.A.. Agronomy. J. B. Gingery. D.V.M., Veterinary Science. Howard Hackedom. B.S.A., Animal Hus- bandry. J. C. Hackleman, M.A., Agronomy. L. D. Haigh, Ph.D., Agricultiu-al Chem- istry. Leonard Haseman, Ph.D., Entomology. O. R. Johnson, M.A., Farm Management. H. F. Major, B.S.A., Landscape Gardening. C. R. Moulton, Ph.D., Agricultural Chem- istry. L. 8. Palmer. Ph.D., Dairy Chemistry. L. G. Rinkle. M.S. A.. Dairy Husbandry. L. A. Weaver. B.S.A., Animal Husbandry. P. L. Gainey, M.S., Assistant, Botany. R. R. Hudelson, B.S.A., Assistant, Agron- omy. C. A. LeClair, M.S., Assistant, Agronomy. E. C. Pegg, M.S., Assistant, Forestry. C. E. Brashear, B.S.A.. Assistant. Animal Husbandry. C. E. DeardorlT, B.S.A.. Assistant in Soil Survey. A. J. Durant. B.S.A.. Research Assistant, Veterinary Science. A. R. Evans. B.S.A., Assistant, Agronomy. W. E. Foard, B.S.A., Assistant, Farm Man- agement. J. F. Hamilton, Assistant, Veterinary Sci- ence. Elmer H. Hughes, B.S.A., Assistant, Ani- mal Husbandry. F. Z. Hutton. (1) B.S.A., Assistant, Soil Survey, M. A. R. Kelley, B.S. In M.E.. Assistant. Agronomy. E. W. Knobel, B.S. A., Assistant in Soil Sur- vey. H. H. Krusekopf, B.S. A., Assistant in Soil Survey. T. C. Reed, B.S. A., Assistant, Dairy Hus- bandry. W. Regan, B.S. A., Assistant, Dairy Hus- bandry. Helman Rosenthal, B.A., Assistant, Agri- cultural Chemistry. O. C. Smith, A.B., Assistant, Agricultural Chemistry. A. C. Stanton. B.S. A.. Assistant, Dairy Hus- bandry. E. R. Spence, B.S. A., Assistant, Veterinary Science. A. T. Sweet, (1) A.B., Assistant, Soil Survey. Boleslaus Szymoniak. B.S. A.. Assistant, Horticulture. Thomas J. Talbert, B.S. A., Assistant, Ento- mology. B. W. Tillman. (1) B.S.A., Assistant. Soil Survey. T. T. Tucker, B.S. A., Assistant, Veterinary Science. E. E. Vanatta, M.S. A., Assistant, Agricul- tural Chemistry. E. S. Vanatta. (1) B.S. A.. Assistant, Soil Survey. W. I. Watkins, B.S. A., Assistant in Soil Sur- vey. C. C. Wiggans, A.M., Assistant, Horticul- ture. C. A. Webster, B.S. A., Assistant. Poultry Husbandry. George Reeder, (1) Dir. Weather Bureau. Flora G. Ernst, A.M., (1) Seed Testing La- boratory. J. G. Babb, M.A., Secretary. R. B. Price, B.S., Treasurer. R. H. Gray, Accountant. T. D. Stanford, Clerk. Vallye Boyce, A.B., Stenographer. J. F. Barham, Photographer. Arthur Rhys, Herdsman, Animal Husbandry. C. M. Pollock. Herdsman. Dairy Husbandry. (1) In the service of the U. S. Department of Agriculture. CONTENTS PAGE Summary 143 Review of Literature on Freezing 149 Effect of Sap Density on Temperature , 155 Experiments on Seedlings of Zea Mays 158 Kjeklahl-Gumming Method for Estimation of Nitrogen 184 Other Features that Influence the Freezing to Death of Plants. . 187 Efifect of Previous Exposure to Temperature Slightly Above Kill- ing Temperature 223 Relation of Low Temperature to Peach Growing 256 Varieties with the longest Rest Periods 269 Effect of Vigor of Trees on Rest Periods 272 Breeding Varieties Hardy under Missouri Conditions 288 Killing of Apples 293 Killing of Cherries and Plums 302 Acknowledgments 303 Bibliography 305 (141) THE KILLING OF PLANT TISSUE BY LOW TEMPERATURE. W. H. Chandler. Summary. 1 . "^he term sap density, as often used in this publication, refers, not to specific gravity, but to molar concentration. These sap den- sities have been determined by the freezing point method, making use of the fact that the molecular weight in grams of any non-elec- trolyte lowers the freezing point 1.86^ C. The sap density is gen- erally given in terms of depression, meaning the number of degrees Centigrade that the freezing point is lower than the freezing point of water. 2. By the eutectic point is meant the temperature at which the substance in solution crystallizes out. At that temperature there would be at the same time ice, crystals of the solute, and un- frozen solutions. 3. There are several forms of injury from cold, some of them purely mechanical, such as tearing of tissue due to tension developed at low temperature, or evaporation from the surface when the con- ducting tissue is frozen so as to prevent the movement of water to that tissue, and killing as a result of long continued exposure to low temperature. The term freezing to death, however, is applied here only to a very specific set of phenomena. With all plant tissues, when a certain temperature is reached very shortly after thawing, it will be found that the tissue has taken on a brown, water-soaked appearance, and evaporation from that tissue is much more rapid than from living tissue. These are characteristics of plant tissue frozen to death. 4. Results of many investigations have shown that during freezing (which may or may not result in freezing to death), ice forms in the tissue, generally not in the cells but in the intercellular spaces, the water moving out of the cells to form crystals in these spaces. The most commonly accepted theory is that killing from cold results from the withdrawal of water from the protoplasm. The amount of water loss necessary to result in death varies with the dilTerent l)lants and different tissues. (Pages 147-155). (143) 144 Missouri Agr. Exp. Sta. Research Bulletin No. 8 5. In the experiments described in this bulletin, the killing temperature of plant tissue that kills at relatively high temperature has been reduced whenever the sap density of the tissue has been increased. (Pages 155-187). 6. In addition to ripe apples and pears, and the leaves of Agave Americana observed by Miiller-Thurgau and Molisch, leaves of let- tuce kill at a slightly lower temperature if they are thawed slowly than if thawed rapidly. In case of all other tissues tested by this station or by others, including unripe apples and pears, there is no indication that the rate of thawing has anything to go with the amount of killing at a given temperature. (Pages 187-l'94). 7. Rapid wilting of tissue has not generally increased the re- sistance of plants to low temperature over that of unwilted tissue with a dry surface. However, tissue with a wet surface killed worse at a given temperature than did tissue with no moisture on the sur- face. (Pages 194-198). 8. Slow wilting or partial withholding of water through a long period increased the resistance of tissue to low temperature. (Pages 198-199). 9. In case of hardy winter buds and wood, a rapid decline in temperature greatly increased the severity of injury from a given low temperature. (Pages 199-218). 10. There seems to be no constant relation between the rate of growth of plant tissue and resistance to low temperature. Young leaves of fruit trees kill at a higher temperature than do old, mature leaves, while the young leaves of lettuce withstand a lower tempera- ture than do the older leaves. (Pages 218-222). 11. Previous exposure to low temperature above that at which the tissue kills seems to increase the resistance of tissue to low tem- perature. (Pages 223-224). 12. The most important feature afTecting the hardiness of plant tissue is maturity, that is, the condition of resistance that the plants reach during the winter dormant period. Maturity in the case of cambium may be intimately associated with the process of drying out. However, this can not be true at least of cortex of win- ter twigs. There is little difference between the moisture content of unfrozen cortex in seasons when it is very tender and seasons when it is hardy. The wood at the base of the trunk and at the crotches of all rapidly growing branches seems to reach a condition of maturity in early winter more slowly than does most other tissue. (Page 224). Killing of Plant Tissue by Low Temperature 145 13. Of the tissue above ground during periods when the most complete maturity is reached, the most tender parts are the pith cells and the fruit buds. During periods of rapid growth there is little difference in hardiness of the different tissues. The tissue which is most tender at all seasons of the year is the root. There is much less difference, however, in the killing temperature of roots in summer and winter than between the killing temperature of twigs or other wood in summer and winter. (Pages 224-239). 14. Roots of the French crab used as stock seem to be more tender than roots which come from scions of an average variety of apple (Pages 239-243). 15. Marianna plum roots are certainly more hardy than Myro- bolan roots, and Mahaleb cherry roots seem slightly more hardy than Mazzard roots. (Pages 243-252). 16. That part of the root system nearest the surface and the largest, oldest roots are more resistant to cold than are small roots further from the surface. (Pages 233-252). 17. Pollen of the apple will withstand much lower temperatures than will any other tissue of the flower when in full bloom. (Page 253). 18. Scales of peach buds do not serve to protect them from low temperature. Buds frozen in the laboratory with the scales removed were slightly more resistant to low temperature than were buds with the scales not removed. (Pages 254-256). 19. The killing of wood of peach trees from freezing is one of the most important determining factors in peach growing. Little can be done to influence the amount of killing except to have the trees start into winter in proper condition of maturity. The weakest growing trees, however, do not generally reach this condition of ma- turity in the most satisfactory manner. Trees one or two years in the orchard, or old weak trees, are most liable to succumb to effects of low temperature. Pruning the trees severely following a winter when the wood has been killed, altho apparently in the best condition of maturity, seems to reduce the amount of killing. However, such pruning following winters when the wood has been killed on account of its not having reached the proper condition of maturity in the fall, generally due to the presence of wet weather following a drought the season before, is liable to result in greater loss than if no pruning were done. (Pages 256-266). 20. The hardiness of peach buds when in fully dormant condi- tion seems to be greatly increased by continuous low temperature preceding the date at which the temperature goes low enough to kill. This capacity to withstand hnv Icuipcratures seems likely to bi' due 146 Missouri Agr. Exp. Sta. Research Bulletin No. 8 to the slow fall in temperature under such conditions rather than to hardiness developed as the result of exposure to low temperature. (Pages 266-269). 21. In the peach growing district of South Missouri and Arkan- sas, and probably other similar climates, the most important factor influencing the loss of peaches from low temperatures in winter is keeping the buds from starting into growth during warm periods in winter. In that section the best means of accomplishing this end is prolonging the growth of the trees in autumn, either by heavy pruning or by fertilizing with nitrogen the spring before. Some varieties of peaches have a much longer rest period than othei" varie- ties and therefore are started into growth more slowly by warm peri- ods in winter. (Pages 269-283). 22. The killing temperature of peach blossoms when the tree is just coming into full bloom, under Missouri conditions, seems to vary from about 22° F. to 25 or 26° F. After the blossoms are old enough that they are probably pollinated, and from that time on until the peaches are as large as one-half inch in diameter, at least, they continue to become more tender until they will withstand but very few degrees below the freezing point, the seeds of young peaches killing at a higher temperature than other peach tissue. (Pages 283- 286). 23. So far the investigations at this station indicate that early varieties of peaches are not started into growth more readily by warm periods in winter than are later varieties. Some of the very early varieties of the Chinese Cling group are the most slowly started into growth in early winter and bloom as late as any of the varieties. How- ever, after blooming time these early peaches grow much more rap- idly and are much more liable to be killed by a freeze after the fruit is set. (Pages 286-293). 24. Killing of wood of the apple is of considerable importance in some apple growing sections. Among the most common injuries are root killing, crown rot, crotch injury, sun scald, trunk killing and killing back of top and branches. (Pages 293-297). 25. Killing of apple buds from low temperatures is not common but has been observed. (Pages 297-298). 26. The blossoms and young fruit of the apple will not generally withstand as low temperature as will the blossoms or young fruit of equal age of the peach. (Pages 298-302). 27. While the killing of cherry and plum buds is less common than the killing of buds of the peach, such killing is often to be ob- served in some sections. The young fruit of the Wild Goose plum is among the most resistant to late frosts in spring. (Pages 302-303). Killing of Plant Tissue by Low Temperature 147 STUDIES ON THE KILLING OF TISSUES OF HORTICULTURAL PLANTS BY LOW TEMPERATURE The work to be reported in this paper was begun during the season of 1904-05. At that time the studies concerned only the effect of certain cultural methods on the hardiness of the fruit buds of the peach under climatic conditions that prevail in the southern half of Missouri. Later they were extended to include the possible effect that a large amount of potassium or other mineral elements in the soil might have on the ability of the peach fruit buds to with- stand,-;old. In taking up this problem it is necessary to distinguish between some of the various phases of injury from cold. Various writers have mentioned observations on frost cracks, that is, a formation of cracks in the wood of the tree during freezing weather. Caspary^ showed that the formation of these cracks is due to a greater contrac- tion of the tissue of the tree tangentially than radially. Later, Miiller-Thurgau^ made a very careful study of these cracks and confirmed Caspary's opinion, but showed that this great contraction tangentially is due rather largely to the shrinking of the cells of the medullary rays. These medullary rays extend in lines from the center out, and are made up of rather thin walled cells separated no- where by very rigid tissue. These rays separate wider strips of rigid tissue extending in a wedge shape from the surface to the center with no lines of more pliable tissue crossing them. Then where the cells of the medullary rays contract on the passage of water into the intercellular spaces to form ice crystals, shrinking toward the center is limited to the rate of shrinking of the strong wedges of rigid tissue, while radially there is the shrinking of the rigid tissue and the more rapid shrinking of the medullary ray tissue. There seems to be another type of injury' to the wood of trees, especially the small twigs, due apparently to the fact that during a long cold period much moisture will be lost from these twigs that can not be replaced because of the frozen condition of the conducting tissue. Thus death will result from the great loss of water from the twigs by evaporation. Killing of this kind seems to be worse in regions with prevailing strong winds and continuous low winter tem- perature. Thus Allen* finds a rather direct correlation between the 'Dot. Zelt. Vol. 13. pp. 449-62. 473-82. 489-500 (Blbl. No. 17); Hot. Zolt. Vol. 15, pp 329-35. 343-50. 361-71 (Bibl. No. 10). 2Landw. Jahrb. Vol. 15. p. 4.\3. 1HH6 (Hlhl. No. 78). 3A. Nolison, Wyo. Agr. Exp. Sta. Hul. 15. 1893. (Blbl. No. 81). «Maator"8 Thesis. Iowa Agr. Exp. Sta. (Blbl. No. 2). 148 Missouri Agr. Exp. Sta. Research Bulletin No. 8 hardiness of the different varieties of apples and the rate at which water will be evaporated from their twigs. Another form of injury, at least an injury that has been attribut- ed to the effect of low temperature, results in the formation of dead areas on the bark of the tree trunk, especially near the top of the ground or in the crotches formed by the branches. Such injury has been studied by Goeppert\ Sorauer^, Grossenbacher' and others. Grossenbacher finds this injury greater on the side of the tree next to the prevailing wind, indicating that the great evaporation from the bark during a long period when it is frozen, and especi^ally the tearing due to the bending of the tree when the bark is unci er high tension, may have something to do with this form of injury. This may not be a diflferent form of injury from direct freezing to death which will be discussed later. Sachs observed that the foliage of certain plants wilted following exposure to a temperature above the freezing point. He and also Miiller-Thurgau* conclude that this wilting was not due directly to the effect of cold, but indirectly to the inability of the roots to take up moisture at so low a temperature to replace the evaporation from the leaves. According to Molisch^, plants continuously exposed to a temperature too low for normal metabolism, but above the freezing point, will eventually die. Under these conditions, death ensues more slowly than where plants are killed by a sudden freeze. The plants gradually turn yellow and die, or dark colored dead spots are formed on the foliage. Miiller-Thurgau^ limits the term freezing to death ("erfrieren") to the most common phenomena to which we have reference in speak- ing of killing from cold. It is death of the tissue following, directly, the lowering of the temperature below the freezing point, with the accompanying formation of ice crystals. When plant tissue is thus frozen to death in the case of growing plants, the foliage in prac- tically all cases has a wilted or limp appearance. Pronounced color changes take place. Thus in most cases the green color due to the chlorophyll is lost, and the tissue takes on a brownish watery color. Plant cells containing coloring matter give up this coloring matter to the adjoining cells of the liquid in the intercellular spaces. Other 'Ueber die Warmeentwlckelung In dem Pflanzen, etc. Book, 1830. (Blbl. No. 44). 'Landw. Jahrb. Vol. 35. pp. 465-525. 1906. (Bibl. No. 105). »N. Y. Geneva. Agr. Exp. Sta. Tech. Bui. 23. (Bibl. No. 50) ; N. Y. Geneva, Agr. Exp. Sta. Tech. Bui. 12, (Blbl. No. 51). . p. 4r,3, l.sso. (Hlbl. No. 78). *Ueher die WttriiuHjntwlckolunK, I'tc. Hook. 1830. (Hlbl. No. 44). *Landw. Jahrl),, Vol. 35, pp. 4(i0-fi25. IVMW. (Hlhl. No. I0r>). ^Mera. d. I'Acad. Hoy. Scl.. Parl.s, 1737, pp. 27a--'98. (Illbl. No. 30). 150 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Goeppert^ was among the first to make a careful study of killing from cold. He observed the formation of ice within the cells, and also in the intercellular spaces. Sachs' found that it was almost always in the latter. Miiller-Thurgau' in his very excellent studies found that when the ice crystals were found within the cells, it was due to very rapid freezing such as Goeppert used, and when the temperature was low- ered very slowly, ice crystals were seldom found within the cell. MOller-Thurgau proved that ice formation within the tissue is neces- sary to freezing to death. It is well known that a liquid ma"'' often be supercooled several degrees below the freezing point bel'ore ice formation begins. He observed that this often occurs in cooling plant tissue in the laboratory, and when pieces of tissue (potato) were supercooled, if they could be warmed without ice formation they were not injured, while if ice formed they would kill at a higher temperature than that to which they were supercooled. Voigt- lander measuring his temperatures with more delicate apparatus, has proved this perhaps more conclusively. When he supercooled tissue to four or five degrees centigrade below the point at which it would kill when ice formed, if the temperature could be raised to above the freezing point without ice formation, killing never occurred. It was held by many scientists, at the time of Miiller-Thurgau 's first work, as well as by a large majority of practical observers, that death was due not directly to low temperature, but to rapid thawing. Goeppert was of the opinion, however, from his studies, that the killing was a direct result of freezing and that death actually occurred before the thawing began. Sachs, from some experiments with plants immersed in cold water to thaw, after freezing, held that the amount of killing of the plants at any given temperature was determined by the rate of thawing. Miiller-Thurgau showed that the method used by Sachs of thawing plants in cold water was not a method of slow thawing, but rather a very rapid thawing since a layer of ice crystals would form on the outside of the tissue, giving off heat that would thaw the tissue very rapidly. In fact the tissue thawed in cold water much more rapidly than in the air at room temperature. Miiller-Thurgau using a large number of plants, thawing them from the same temperature, some rapidly and some more slowly, lUeber die Warmeentwlckelimg, etc. Book. 1830. (Blbl. No. 44). »Ber. u. d. Ver. d. Kon. Sachs. Gesell. d. Wlss. zu Leipzig, 1860, Vol. 12, pp. 1-50. (Bibl. No. 94). 'Landw. Jahrb. Vol. 16. p. 453, 188er. (Blbl. No. 78). Killing of Plant Tissue by Low Temperature 151 was never able to detect any difference in the amount of killing when thawed rapidly or slowly, except in the case of the fruit of the apple and pear. Molisch\ following the work of Miiller-Thurgau, tried also slow and rapid thawing from the same temperature with a very large number of plants, and found that in nearly all cases the rate of thawing had nothing to do with the killing. In the case of the fruit of the apple and pear, and the leaves of Agave Americana, the slow thawing gave less injury, but even with these, a slightly lower tem- perature than that at which they kill with rapid thawing, would kill them, regardless of the rate of thawing. Miiller-Thurgau observed carefully the freezing of tissue under the microscope and found that ice was very seldom formed within the cell, but usually ice crystals formed outside the cell in the intercellular spaces and continued to increase in length as the temperature went lower, the water passing from the cell into the intercellular spaces increasing the length of the crystals. By placing plant tissue frozen to various temperatures in 100 cc. of water carefully insulated, and noting the temperatures to which the water was lowered, excluding the losses of heat for warming up the plant tissue, correcting for the heat of the beaker, etc., making use of the fact that eighty gram calories are required to melt one gram of ice, Miiller-Thurgau^ was able to determine, apparently with some accuracy, the percentage of the plant water that is frozen into ice at various temperatures. Only plant tissue with a deter- mined moisture content was used. With the apple he gives the fol- lowing percentages of water frozen out at given temperatures: at -4.5°, 63.8 per cent of the water was frozen; at -13°, 74.4 per cent of the water was frozen; at -15.2°, 79.2 per cent of the water was frozen He also attempted to measure the percentage of the water frozen out of woody tissue by means of frost cracks. His method was to freeze a section of a young tree trunk until a frost crack of a certain width was formed. On thawing of the tissue this crack would close. His next step was to dry the section of tree trunk until a frost crack of the same width was formed. He assumed that the percentage of water loss necessary to form this crack is equal to the percentage taken from the cell during freezing sufficient to form an ice crack of the same width. Molisch studied with great care, under the microscope, the freezing of various plant tissues, observing the same i)hen()nicna de- 'UntorBucluing Ubor daa. etc. 1897. book. (BIbl. No. 76). 'Landw. Jahrb. Vol. !», p. 4r,-A. 1880. (IJIbl. No. 78). 152 Missouri Agr. Exp. Sta. Research Bulletin No. 8 scribed by Miiller-Thurgau with reference to the freezing of the cell water in the intercellular spaces, rather than within the cells. Both Miiller-Thurgau and Molisch hold the view that freezing to death results from the rapid withdrawal of water from the cells to form these ice crystals in the intercellular spaces. Matruchot and MolHard^ observed that water is extruded from the nuclei of plants that have been frozen, dried or subjected to the action of solutions of high osmotic concentration. Gorke has recently offered an interesting theory as to the cause of death by freezing. He found that when the plant sap is frozen, certain proteids may be precipitated out and apparently those plants that are most easily killed by freezing have their proteids precipitated out at the highest temperature. Thus begonia, which is very easily killed, had its proteids precipitated at -3° while sap from pine needles required a temperature of -40° to precipitate any proteids. Gorke^ assumes then that killing from cold may be due to the precipitation of the proteids, and accounts for this precipitation by the greater concentration of the salts in the sap as water is removed to form ice crystals. It is well known that certain proteids can be precipitated out by increasing the concentration of salts, especially zinc sulphate and ammonium sulphate. Gorke made up solutions of albumen with zinc sulphate and found that after freezing to -20° there was a large precipitation of proteids. Lidforss^ working with the wintergreen plants of South Swed- en, has found that with most of them at least during cold weather, the starch is almost entirely changed to sugar, though on the return of warm weather starch may be again deposited in the cell. He assumes that this sugar is formed during cold weather as a means of protecting the plant against freezing by lowering the freezing point of its sap. He was able also to increase the resistance to low temperature of the leaves of wintergreen plants and the roots of Zea Mays by keeping them for a time immersed in 5 to 10 per cent sugar solutions. Schaffnit^, following the work of Gorke, found that the pro- teids of rye grown in the open at low temperatures are not readily precipitated by freezing, while the same temperature will readily precipitate proteids from sap of rye grown in the greenhouse at much iCompt. Rend. Acad. Scl. Paris, Vol. 132 (1901) pp. 495-8. (Bibl. No. 71a). JLand. Vorsuchis. Vol. 65, p. 149. 1906. (Bibl. No. 47). 'Lunds Unlversitets Arssk., Vol. 2. No. 13, 1907 (Bibl. No. 62); Bot. Centlb., Vol. 68 No. 2, p. 33. (Bibl. No. 63). ^Mitt. Kaiser Wllhelm Inst. Landw. Bromberg. Vol. 3, No. 2, pp. 93-115, (Bibl. No. 98); Zoits. f. AUg. Phys., Vol. 12, pp. 323-36. (Bibl. No. 99). Killing of Plant Tissue by Low Tempeelature 153 higher temperatures. He also found that he could prevent the pre- cipitation of proteids from this sap of greenhouse rye by adding to it small quantities of sugar. He concludes then that the formation of sugar in wintergreen plants described by Lidforss may be the means of protecting the plants against precipitation of proteids. However, Schaffnit concludes that precipitation of proteids is the only way by which loss of water during freezing kills plant tissue. Maximow^ has recently published three very interesting papers covering work in freezing sections of plants, mainly red cabbage and Tradescantia discolor. Thin sections of tissue from the upper side of the leaves were frozen in solutions of various strengths of both organic and inorganic substances after they had stood for varying lengths of time in the solutions. He found remarkable protection to be ex- erted by both organic and inorganic substances whenever their eutectic point (the temperature at which they crystallize out, giving mixtures of solute crystals with the ice crystals) does not lie too near the freezing point and whenever the substance is not excessively toxic. He used strengths varying from -, to 2N of glucose and glycerine, and -5- to 2N of methyl and ethyl alcohol, and mannite. Of inorganic substances he used solutions with strengths of O.IN to 2N of sodium chloride, potassium chloride, calcium chloride, sodium nitrate, potassium nitrate, calcium nitrate, sodium acetate, potassium acetate, calcium acetate, sodium lactate, potassium lac- tate, sodium oxalate, and potassium oxalate, also magnesium nitrate, magnesium chloride, ammonium nitrate, ammonium chloride, sodium sulphate, and potassium sulphate. According to Maximow, mannite, sodium sulphate, potassium sulphate, potassium nitrate, and sodium oxalate show little protec- tion because of their high eutectic point; and magnesium chloride, magnesium nitrate, and ammonium chloride because of their toxicity, while calcium chloride and calcium nitrate show reduced protection because of their toxicity. All other solutions, however, showed great protection that was very uniform for the same osmotic con- centration. Sometimes a temperature as low as -32° did not kill all of the cells of the red cabbage. Probably the most interesting re- sult of Maximow 's work was the observation that when the sections were immersed in these solutions and immediately frozen, as much protection was exerted as when they had been permitted to remain in the solutions for twenty-four hours or longer. The tender Trad- escantia cells immersed in expressed sap of the red cabbage and ini- 'licr. der Doutach. Bot. GeseU.. Vol. 30. pp. 62-06, 203-306. 604-16. (HIbl. No. 73). 154 Missouri Agr. Exp. Sta. Research Bulletin No. 8 mediately frozen, would actually withstand more cold than the hardier red cabbage when frozen in winter. Maximow concludes that the part of the cell which is injured when exposed to low temper- ature is the plasma membrane, and that as long as a film of water was kept in contact with this membrane, death was not likely to occur His theory then would not be greatly different from that of Miiller- Thurgau and Molisch, that withdrawal of water kills, except that in Maximow 's opinion killing following the withdrawal of water seems to be limited to the plasma membrane. If Maximow 's work is verified by further experimenting, using other plants, it is certainly a very interesting contribution toward determining just what freezing to death of plant tissue is. Mez^ studied the effect of supercooling upon plant tissue. He finds that where ice formation begins at once on reaching the freezing point, the killing is not so great as where there is supercooling when large masses of ice are formed rapidly after crystallization begins. By use of the thermo-couple he studied the fall of temperature in the plant, using stems of Impatiens to determine the eutectic points of the sap solutes. At each of these points there will be a halting in the temperature fall due to the heat given off on crystallization. From this work he concludes that when a temperature of -6° is reached, all solutes will crystallize out. He thinks this should dis- prove the theory of Miiller-Thurgau and Molisch, since there should be complete loss of water at this temperature and the plant should never survive a lower temperature if loss of water from the cell is the cause of death. He holds that the heat liberated by the crystallizing of the solutes and the formation of ice, will after the cells are insulated by the ice mass, aid in keeping the temperature of the cell above that of the surroundings. He holds, therefore, that each plant has its specific minimum point at which death occurs due to the direct effect of the cold, and that if supercooling takes place, large amounts of heat are lost before the cells are insulated by the ice mass and therefore this specific minimum will be more quickly reached. The work of Miiller-Thurgau^ and of Voigtlander^, (a pupil of Mez) where plants supercooled to below the killing temperature remained alive if ice did not form, certainly refutes the theory of Mez. If further evidence were needed, the protective action of organic and inorganic substances shown in Maximow 's work certainly proves the fallacy of Mez's conclusion. Even his conclusion that the sap solute iPlora, Vol. 94, p. 89. 1905. (Blbl. No. 74). 'Landw. Jahrb. Vol. 15, p. 453, 1886-. (Bibl. No. 78). 'Beitr. z. Biol, der Pfl. Cohn. Vol. 9, 1909. pp. 359-414. (Bibl. No. 110), FIGURE KILLED AT I. — FREEZING APPARATUS FOR PLANTS THAT A TEMPERATURE NOT LOWER THAN -12 TO -15 C. 1. Space in which salt and ice mixture was placed; 2. Chamber in which plants were frozen: 3. Lid which covered freezing chamber: 4. Wire leading to small electric fan beneath hardware bottom on which plants were frozen. (See page 156.) cloth FIGURE II. — APPARATUS FOR FREEZING TISSUE QUIRED LOWER THAN -12 TO -15 C. TO KILL. THAT RE- 1. Galvanized iron cylinder to the bottom of which the twigs con- taining buds were fastened; 2 and 3. Galvanized iron cylinders placed one within the other with space between their walls for freezing mixture; 4. Cylinder to receive 3, allowing space for freezing mixture around 3; 5. Insulating box filled with sawdust; 6. Wheel operated by electric motor and belt in order to keep (1) turning continuously while freezing; 7. Lid for (2). (See page 157.) FIGURE III. — APPARATUS FOR EXPRESSING PLANT JUICE. The two blocks between which the plant tissues were pressed are shown at (1) and (2). The juice escaped through a hole (3) in block (1). The plant tissue was placed between the blocks (1) and (2), which in turn were placed between the pieces of movable 4"x4" pieces which are drawn together by bench-screws. (See page 158.) Killing of Plant Tissue by Low Temperature 155 must all be crystallized out at -6° C. can not be true since sugars would remain in solution at lower temperatures than that. Further, when we have evaporated the cortex sap of peach twigs in winter condition to one-sixth or one-eighth of its volume without permitting the temperature to go above 50° C. ice would not form when the temperature was lowered to -22° C. though many of the buds would be killed at that temperature. Mez seems also to ignore the force of imbibition which would tend to hold the water in the protoplasm even after the sap solute may be crystallized out. The fact, however, that when great supercooling takes place, plants are more liable to be killed, is of interest and is, of course, associated with the fact which will* be discussed later that rapid cooling is more injurious to plant tissue than is slow cooling. EFFECT OF SAP DENSITY ON KILLING TEMPERATURE. If the theory of Miiller-Thurgau and Molisch be true (even as it is modified by Maximow) it would seem that some plants might be hardy because the plasma membrane has the property of with- standing great loss of water. Some might be relatively hardy be- cause of a property by which sufficient water to protect the plasma membrane at low temperatures is prevented from freezing, and some might be relatively hardy on account of the presence of both condi- tions. It is generally considered that after the effect of the sap solute in holding water unfrozen is exhausted, there is still left the force of imbibition. The relative importance of these two forces, however, is not determined. Disregarding the force of imbibition (which, however, may be the more important), it would appear to be true that if the sap density (by sap density is meant not specific gravity but molar concentration of the sap; that is, the number of gram molecules of the sap solute in one thousand grams of water) were doubled, then at any given temperature below the freezing point, but above the eutectic point of the solute, twice as much water would be held un- frozen to protect the protoplasm. With this idea in mind, experiments were started in September, 1908, to determine whether or not an increase in the sap density would lower the killing temperature. Seedlings of corn, cowpeas, garden peas, tomatoes, squash, cabbage and lettuce were grown in sand and watered with varying strengths of potassium chloride and ammonium chloride at first — later magnesium chloride, sodium chloride and sodium nitrate were also used — while check plants were grown under similar conditions 2 156 Missouri Agr. Exp. Sta. Research Bulletin No. 8 except that they were watered only with distilled water. The plants were permitted to grow only as long as they would make good growth in the sand, probably about the time the food supply of the seed was becoming exhausted. Some of the plants of each set were then frozen while others had the sap expressed for osmotic strength (freezing point) determination by the use of a Beckmann apparatus. As a measure of the osmotic strength the term depression will be used in the tables, meaning of course the number of degrees centigrade be- low zero at which, with no supercooling, ice formation begins in the sap. Method of Freezing. At first an effort was made to grow the plants in the greenhouse and expose them to outside temperatures to determine the killing temperature. However, this was soon found to be unsatisfactory and the plants were frozen in a chamber sur- rounded by a freezing mixture made of salt and ice. It is evident that the temperature throughout such a chamber would not be uni- form so long as it were falling and great care was necessary to secure as uniform a temperature as possible. The apparatus shown in Fig. 1 was used. In the lower part was an electric fan; the upper part was the chamber in which the plants were frozen. An effort was made to keep the temperature uniform within this chamber by the operation of the electric fan just beneath the hardware cloth shelf on which the plants were frozen. Careful tests showed that the temperature throughout this chamber was always uniform on the same level though sometimes it would vary slightly in different levels. Fearing, however, that this would not always be true, the plants on freezing were always placed, not only on the same level, but at the same distance from the galvanized iron wall of the chamber. In this way it was practically impossible that plants in the freezer would not all be exposed to the same temperature, and consistent results were secured. In addition to being sure that the plants were at a uniform tem- perature, it was necessary to control very carefully the rate of fall of the temperature, since rapid falling of temperature very greatly increases the killing. For this reason it was practically impossible to secure results that would be sufficiently accurate so that one freez- ing could be compared with another, except where differences were wide; that is, the plants to be compared must be frozen at the same time. However, when the differences are large it is possible to make a fairly accurate estimate of the relative hardiness of the plants frozen at different times, if great care is taken to duplicate as nearly as possible, the rates of temperature fall. It was found possible to Killing of Plant Tissue by Low Temperature 157 lower the temperature of all the plants together to a point that would probably kill the most tender, and after removing these to lower it further. The rate of fall would thus be the same for all down to the temperature at which the most tender were removed. The thermometer used in the earlier years of freezing was a pentane thermometer graduated to one-half degrees. The zero point was far enough above the bulb so that when the thermometer was inserted through a cork at the top of the jacket of the freezing chamber, the bulb would be on the same level with the plants. Later, special mercury thermometers graduated to low temperature were used. These were standardized by the makers. However, new thermometers were checked by those used with previous work, and also, checked from time to time, with standard thermometers of the Columbia Branch of the United States Weather Bureau. No effort was made to read the thermometers to closer than one-half degrees. Plants on being removed from the freezer were always examined to see if the tissue were frozen stiff. In freezing buds and woody tissue that killed at a temperature lower than -15°, the apparatus shown in figure 2 was used. The twigs or pieces of tissue and the thermometer were fastened to the inner cylinder which was filled with cotton. This was set in a cylinder enough larger to leave a surrounding space of about three-fourths of an inch. The second cylinder was about six inches taller than the inner one, and was set with a one and one- half inch space between them. There was about two inches of space between the walls of this third cylinder and those of the one in which this was placed. The fourth cylinder was well insulated by being packed in dry saw dust. Ice and salt were first packed loosely and then firmly in the space between the fourth and third cylinder. In this way the temperature of the twigs could be lowered generally to -17° C. When it was necessary to secure a lower temperature, the space between the third and second cylinders was packed loosely and later firmly with salt and ice. In this way the temperature of the air surrounding the inner cylinder could be gradually lowered at the rate of two to three degrees an hour after the freezing point was reached. By packing salt and ice to the top of the second cylinder the temperature from top to bottom of the inner c>linder would vary but little. However, the freezing tissue to be compared and the thermometer bulb were always kept the same distance from the bot- tom of the cylinder. Since these cylinders were of galvanized iron and would conduct heat rapidly, it would seem probable that the tem- perature aroimd the central cylinder would not vary. However, fearing that there might be some such variation in temperature, the 158 Missouri Agr. Exp. Sta. Research Bulletin No. 8 central cylinder with the twigs or woody tissue and the thermometer on its surface was kept slowly revolving by means of a small electric motor. During the freezing, this was stopped only for thermometer readings which were taken generally every fifteen minutes. Method of Determining Freezing Point of Sap. The density of sap was determined by means of a Beckmann Freezing Point Appa- ratus. The tissue was ground in an ordinary food grinder, using the knife that grinds the finest, and the sap was expressed with the appa- ratus shown in Figure 3. The large block with the hole in the center is of sugar maple which will not split readily. The smaller block is of the same material and is made to fit in the depression in the large block leaving about one-eighth of an inch surrounding space. The ground tissue was wrapped in a clean piece of eight or ten ounce duck and put in the depression in the large block, the small block put against it and the two pressed together between pieces of 4x4 lumber drawn together by a pair of bench screws as shown in the figure. The sap could be expressed very quickly. With succulent plants loss by evaporation in all cases was negligible. In the case of leaves of peaches it required a considerable length of time for the sap to exude. In all cases before plants with different treatments were frozen or before they were ground for expressing sap, they were, after being pulled, kept with the roots in a glass of water until they became apparently turgid, since wilting sometimes seems to reduce slightly the killing temperature, and would appreciably affect the sap density determinations. EXPERIMENTS WITH SEEDLINGS OF ZEA MAYS A large number of corn seedlings were grown and frozen. The following table gives the date of freezing, the solution with which the material was watered, the temperature to which the plants were subjected, the percentage killed and percentage partly killed, and the freezing point of the sap. The freezing point is given as depression, meaning the number of degrees below zero, centigrade, at which ice begins to form in the sap, assuming no supercooling. Killing of Plant Tissue by Low Temperature 159 Table 1. Showing Effect of Watering With Mineral Solu- tions ON Sap Density and Hardiness in Zea Mays Plants. Watered With Potassium Chloride (.0804 N).. ... . .. Potassium Chloride (.0402 N)..^ ...... Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) I'otassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N; Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Date Temper- ature Num- ber Plants Percent- age Killed Per- centage Killed and Partly Killed Depres- sion Dec. 12, '08 -3 6 33.3 83.3 1.10 Dec. 12, '08 -3 6 16.6 100 1.06 Dec. 12, '08 -3 6 33.3 100 .94 Dec. 12,'08 -3 6 50 100 1.19 Dec. 15,'08 -3 6 33.3 33.3 1.265 Dec. 15,'08 -3 5 40 60 .94 Dec. 15, '08 -3 4 0 0 1.16 Dec. 15,'08 -3 6 100 100 .875 Dec. 16,'08 -3.25 7 42.8 71.7 1.26 Dec. 16,'08 -3.25 7 14.3 57.1 .94 Dec. 16,'08 -3.25 6 50 100 1.16 Dec. 16,'08 -3.25 9 33.3 88.9 .875 Jan. 9,'09 -7 13 46.2 61.5 1.315 Jan. 9,'09 -7 11 63.6 81.8 1.195 Jan. 9,'09 -7 9 77.8 100 1.005 Jan. 9, '09 -7 12 66.6 100 .935 Jan. 9,'09 -6.5 11 27.3 54.5 1.315 Jan. 9,'09 -6.5 13 61.5 84.6 1.195 Jan. 9, '09 -6.5 10 80 90 1 . 005 Jan. 9,'09 -6.5 14 50 85.7 .935 Jan. 9, '09 -7.5 13 61.5 76.9 1.315 Jan. 9, '09 -7.5 12 75 83.3 1.195 Jan. 9, '09 -7.5 8 87.5 100 1 . 005 Jan. 9,'09 -7.5 12 91.0 91.6 .935 Jan. 19,'0<> -^ 1^ 16.6 50 1.315 i6o Missouri Agr. Exp. Sta. Research Bulletin No. 8 Watered With Potassium Chloride (.0402 N) _. .. Ammonium Chloride (.0804 N) _. .. Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) Potassium Chloride (.0402 N) _. .. Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N) _. ... Potassium Chloride (.0402 N) _. .. Ammonium Chloride (.0804 N) Ammonium Chloride (.0402 N) Potassium Chloride (.0804 N)...... ... Potassium Chloride (.0402 N) _. .. Ammonium Chloride (.0804 N) _. . . Ammonium Chloride (.0402 N) Date Temper- ature Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Jan. Feb. Feb. Feb. Feb. 19,'09 19,'09 19,'09 19,'09 19,'09 19,'09 19,'09 19,'09 19/09 19,'09 19,'09 21, '09 21, '09 21, '09 21, '09 21, '09 21,'09 21,'09 21,'09 2,'09 2,'09 2,'09 2,'09 -3 -3 -3 -6.5 -6.5 -6.5 -6.5 -6 -6 -6 -6 -4.5 -4.5 -4.5 -4.5 -4 -4 -4 -4 -5 -5 -5 -5 Num ber Plants Percent- age Killed 16 17 11 20 16 11 18 13 12 11 12 21 19 15 19 16 17 17 16 10 9 11 13 Potassium Chloride (.0804N), average.. Potassium Chloride (.0402 N), average.. Ammonium Chloride (.0804N), average Ammonium Chloride (.0402N), average 18.8 11.8 27.3 25 25 66.6 11.1 7.7 0 0 0 61.9 68.4 86.7 57.9 0 17.6 17.6 37.5 0 11.1 0 30.8 29.6 34.3 42.6 46.3 Per- centage Killed and Partly Killed 43.8 41.3 54.5 40 37.5 66.6 16.7 15.4 0 0 0 66.6 68.4 93 63.2 12.5 23.5 23.5 50 50 66.7 54.5 38.5 51.3 58.9 64.1 65.8 Depres- sion Killing of Plant Tissue by Low Temperature i6i It will be seen from these tables that by taking an average of a large number of these freezings, the percentage of killing is uniformly lower when the depression is increased. Shaded Zea Mays seedlings were watered with .0804 N potas- sium chloride, and others with water with results as follows: Table 2. Showing Effect of Watering With Mineral Solu- tions ON Sap Density and Hardiness of Shaded Zea Mays Plants. Watered With Date Tem- pera- ture Number of Plants Percent- age Killed Percent- age Killed and Partly Killed Depres- sion Potassium Chloride (.0804 N) Water Potassium Chloride (.0804 N) Water Potassium Chloride (.0804 N) Water ....... Potassium Chloride (.0804 N) Water Potassium Chloride (.0804 N) Water Feb. 2, '09 Feb. 2, '09 Feb. 2, '09 Feb. 2, '09 Feb. 2, '09 Feb. 2, '09 Feb. 12, '09 Feb. 12, '09 Feb. 12, '09 Feb. 12, '09 -3 -3 -5 -5 -5 -5 -5 -5 -4.5 -4.5 18 17 17 14 14 15 8 7 8 9 5.6 17.6 41.2 78.6 64.3 80 12.5 57.1 50 100 34.7 66.7 5.6 17.6 58.8 78.6 85.7 80 50 71.4 75 100 55.0 69.5 .93 .835 .93 .835 .93 .833 1.22 .65 1.22 .653 Potassium Chloride (.0804N), average Water, average 1.046 761 The following table gives results of freezing cowpea seedlings that had been watered with solutions containing 6.03% normal potassium chloride, sodium chloride, magnesium chloride, ammonium chloride, sodium nitrate, and distilled water. 1 62 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 3. Effect of Watering With Mineral Solutions on Sap Density and Hardiness of Cowpeas. Watered With Potassium Chloride. . . Sodium Chloride Magnesium Chloride. Ammonium Chloride.. Sodium Nitrate Distilled Water Potassium Chloride. . . Sodium Chloride Magnesium Chloride.. Ammonium Chloride.. Sodium Nitrate Distilled Water Potassium Chloride. . . Sodium Chloride Magnesium Chloride.. Ammonium Chloride.. Sodium Nitrate Distilled Water Potassium Chloride. . . Sodium Chloride Magnesium Chloride.. Date June June June June June June June June June June June June July July July July July July July July July Tem- pera- ture Ammonium Chloride... July July July July July July July July July July July July July July July Sodium Nitrate. Distilled Water Potassium Chloride. . . Sodium Chloride Magnesium Chloride.. Ammonium Chloride.. Sodium Nitrate Distilled Water Potassium Chloride. . . Sodium Chloride Magnesium Chloride.. Ammonium Chloride.. Sodium Nitrate Distilled Water 29,'ll 29,'ll 29,'ll 29, '11 29,'ll 29,'ll 29,'ll 29,'ll 29,'ll 29,'ll 29,'ll 29,'ll 8/11 8,'ll 8/11 8/11 8/11 8/11 9/11 9/11 9/11 9/11 9/11 9/11 20/11 20/11 20/11 20/11 20/11 20/11 21/11 21/11 21/11 21/11 21/11 21/11 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3 -3 -3 -3 -3 -3 -2, -2, -2. -2, -2. -2, -2, Number Leaves 75 75 75 75 75 75 75 -2.75 -2.75 -2.75 -2.75 -2.75 -2.75 -2 . 75 -2.75 -2.75 -2.75 -2.75 35 35 33 33 33 40 30 36 36 29 24 36 21 22 30 12 12 30 12 12 29 12 12 30 12 12 12 12 12 30 12 12 13 12 12 30 Percent- age Killed Potassium Chloride, average. Sodium Chloride, average. . . . Magnesium Chloride, average Ammonium Chloride, average Sodium Nitrate, average Distilled Water, average 45.71 28.57 69.69 3.03 9.09 77.50 56.66 30.55 66.66 58.62 70.83 91.66 33.33 36.36 85.33 83.33 0.0 90.00 100.00 66.66 93.10 83.33 0.0 76.66 16.66 8.33 58.33 16.66 58.33 80.00 8.33 0.00 15.39 8.33 16.66 86.66 43.45 28.41 64.75 33.05 25.82 83.73 Percent- age Killed and Partly Killed 57.14 48.57 100.00 15.15 33.33 77.50 70.00 38.88 77.77 75.86 79.16 97.22 42.85 54.54 100.00 91.66 41.66 100.00 100.00 66.66 100.00 100.00 58.33 83.33 58.33 33.33 75.00 50.00 83.33 93.33 16.66 8.33 30.77 16.66 16.66 93.33 57.49 41.72 80.59 58.22 52.08 90.78 De- pres- sion 1.05 1.065 .975 1.035 1.05 .825 1.05 1.065 .975 1.036 1.05 .825 1.13 1.17 1.00 1.155 1.23 .78 1.13 1.17 1.00 1.155 1.23 .78 1.13 1.17 1.00 1.155 1.23 .78 1.13 1.17 1.00 1.155 1.23 .78 1.10 1.135 .991 1.115 1.17 .795 Killing of Plant Tissue by Low Temperature 163 It may be said, however, that the solutions all reduced the growth of the plants, as indicated by the following weights: Average weight of plants watered with Potassium Chloride 2.27 grams Average weight of plants watered with Sodium Chloride 1 . 89 grams Average weight of plants watered with Magnesium Chloride 2.33 grams Average weight of plants watered with Ammonium Chloride 1 . 78 grams Average weight of plants watered with Sodium Nitrate 1 . 68 grams Average weight of plants watered with Distilled Water 2 . 63 grams The percentage of killing will thus be seen to be as much in proportion to growth as in inverse proportion to depression. Corn seedlings were also grown where water was withheld, being watered only when it was necessary to keep them from dying. The following table gives the results: Table 4. Showing Effect of Withholding Water on Sap Den- sity AND Hardiness of Zea Mays. Percent- Percent- age Treatment Date Tem- Number age Killed De- pera- Plants Killed and pres- ture Partly Killed sion Well watered Feb. 2, '09 -4 13 84.6 92.3 .785 W'ater withheld Feb. 2, '09 -4 10 0.0 40. 1.07 Well watered Feb. 2, '09 -5 13 61.5 74.6 .835 Water withheld Feb. 2, '09 -5 5 20.0 80.0 1.07 Well watered Feb. 2, '09 -5.5 12 ■66.7 100.0 .835 Water withheld Feb. 2, '09 -5.5 6 16.7 50.0 1.07 Well watered Feb. 12, '09 -4.5 10 60.0 60.0 .71 Water withheld Feb. 12, '09 -4.5 9 44.5 68.19 44.5 81.72 1.085 Well watered, average. . .791 Water partially withhel d, average. . 20.27 53.60 1.074 It will be seen again that withholding water increased the sap density (depression) and lowered the killing temperature. It also reduced the rate of growth and probably the size of the cells, so we can not conclude with certainty that the greater hardiness is due only to the greater sap density. Tomatoes were grown in the same way except that there were three lots — some well watered, others watered only when it was necessary to keep them from dying, and some others grown outside at a temperature considerably lower than that in the greenhouse. The folUnving table gives the results: 164 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 5. Showing Effect of Condition of Growth on Sap Density and Hardiness of Tomatoes. Treatment Out of doors.. . . Greenhouse, wet Greenhouse, dry Out of doors.. . . Greenhouse, wet Greenhouse, dry Out of doors.. . . Greenhouse, wet Greenhouse, dry Out of doors. . .. Greenhouse, wet Greenhouse, dry Date Apr. 29,'ll Apr. 29,' 11 Apr. 29,'ll May 2, '11 May 2,'ll May 2, '11 May 4,'ll May 4,'ll May 4,'ll May 6,'ll May 6,'ll May 6,'ll Tem- pera- ture -2 -2 -2 -2 -2 -2 -2.5 -2.5 -2.5 -2.5 -2.5 -2.5 Number of Plants 4 4 4 4 4 4 4 4 4 4 4 4 Result of Freezing All dead Leaves all dead; stems slightly injured Uninjured except very young leaves All dead (larger and stockier) Leaves dead; lower stems alive Only few leaves killed Foliage and upper one- third stems killed. . . . All killed Leaves killed; stems un- injured Leaves killed; stems un- injured Leaves dead; upper one- third stems dead Foliage and growing tips of three plants dead; one plant un- injured De- pres- sion 0.73 0.84 1.16 .73 0.84 1.16 0.73 0.84 1.16 .73 0.84 1.16 Contrary to what might be expected, those tomato plants grown in the greenhouse but watered sparingly were more hardy than those grown outside; also the depression was greater. The results in this table again indicate that as the depression is lowered, plants are made more hardy. Cabbage, kale and turnips were each grown in the greenhouse some watered well and others with water withheld except when it was necessary to keep the plants alive, while others were grown out of doors. The following table give results, and depressions for these plants. Killing of Plant Tissue by Low Temperature 165 Table 6. Showing Influence of Condition of Growth on Sap Density and Hardiness. Treatment CABBAGE Out of doors Greenhouse, Greenhouse, Out of doors Greenhouse, Greenhouse, Out of doors Greenhouse, Greenhouse, Out of doors Greenhouse, Greenhouse, Out of doors Greenhouse, Greenhouse, Out of doors Greenhouse, Greenhouse, dry. wet. dry. wet. dry. wet. dry. wet. dry. wet. dry. wet. Date Tem- pera- ture Number Percent- of Leaves age Killed Percent- age Killed and Partly Killed Nov. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. Dec. 2,'ll 2, '11 2,'ll 4,'ll 4,'ll 4,'ll 17,'ll 17, '11 17,-11 9,'ll 9,'ll 9,'ll 9,'ll 9,'ll 9,'ll 13,'ll 13, '11 13, '11 -5.5 -5.5 -5.5 -6 -6 -6 -6.5 -6.5 -6.5 -5 -5 -5 -5 -5 -5 -4 -4 -4 Out of doors, average Greenhouse, dry; average. Greenhouse, wet; average. 3 0 0 3 0 0 3 33.4 33.4 3 0 0 3 0 0 3 66.7 66.7 4 0 0 5 100 100 5 100 100 4 0 0 4 100 100 4 100 100 4 0 0 4 100 100 4 100 100 3 0 0 3 0 0 2 100 100 0 50 83.3 0 50 83.3 De- pres- sion 18 90 99 TURNIPS Out of doors Greenhouse, dry Greenhouse, wet Out of doors Greenhouse, dry Greenhouse, wet Nov. 2, '11 Nov. 2,'ll Nov. 2, '11 Nov. 4,'ll Nov. 4,'ll Nov. 4, '11 -5.5 -5.5 -5.5 -6 -6 -6 3 3 3 3 3 3 0 100 100 0 100 100 100 100 100 0 100 100 KALE Out of doors, coldframe Out of doors, bed Greenhouse, dry Greenhouse, wet Dec. 8, '11 Dec. 8,'ll Dec. 8, '11 Dec. 8, '11 -6.5 -6.5 -6.5 -6.5 2 3 3 3 50 0 100 100 50 0 100 100 .... LETTUCE Out of doors Greenhouse Out of doors Greenhouse Out of doors Greenhouse Mar. 9,'13 Mar. 9/13 Mar. 29,' 13 Mar. 29,' 13 Apr. 30,'13 Apr. 30,'13 -3.5 -3.5 -5 -5 -3.5 -3.5 8 9 18 16 24 32 0 83.3 0 68.7 0 18 0.0 56.6 25 100.0 27.7 93.7 3.^.3 48 .900 .867 .900 .867 .920 .740 Average, lettuce out . Treatment Date Tem- pera- ture Number of Plants Per- Per- cent- •"cent- age 'age -Total All Leaf ' Killed Area Killed 0.0 16.3 24.0 74.0 0.0 36.6 80.0 95.0 15.4 63.4 33.5 71.01 30.9 48.61 De- pres- sion Early Harvest apple twigs and leaves... . iiui. onautu j uijr x, x^ Pear twigs and | | leaves iShaded. . . . June 28/13 Pear twigs and ' leaves Pear twigs and leaves ^i.-a.^^^^. . . . j uxn, ^u, x^ Pear twigs and I ! leaves Not shaded June 28/13 Not shaded July 1/13 Shaded.... June 28/13 I Not shaded June 28/13 Shaded.... June 28/13 -5.5 -4 -4 -5 -5 46 25 28 25 26 2.252 Average, Shaded, excluding pears Average, Not shaded, excluding pears. .985 1.173 In practically all cases the cortex, cambium and sap wood of the twigs were injured rather severely, and in all cases the injury was worse with the shaded twigs. In the case of the pear twigs and leaves sufficient sap for depression determina- tion could not be secured. Material Treatment Date Tem- pera- ture Number of Plants Per- cent- age All Killed Per- cent- age Total Leaf Area Killed De- pres- sion Labrusca grape leaves , Labrusca grape leaves , Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Labrusca grape leaves Shaded 38 hours Not shaded Shaded 38 hours. Not shaded Shaded 22 hours.. Not shaded Shaded 22 hours.. Not shaded Shaded 22 hours.. Not shaded Shaded 22 hours.. Not shaded July 8.'13 July 8,'13 July 8,'13 July 8.'13 July 10,'13 July 10,'13 July 10,'13 July 10,'13 July 11, '13 July 11, '13 July 11,'13 July 11/13 -3 -3 -4.5 -4.5 -3.5 -3.5 -4.5 -4.5 -4 -4 -5.5 -5.5 18 17 22 17 10 10 10 10 10 10 10 10 Average, shaded. ... Average, not shaded. 27.2 35.3 100. 76.4 0.0 0.0 30.0 0.0 10.0 0.0 60.0 40.0 34.53 25.29 54.2 58.8 100. 94.1 2.5 0.0 30.0 15.0 25.0 17.5 80.0 52.5 48.61 39.65 .695 .733 .695 .733 .755 .920 .755 .920 .835 1.085 .835 1.085 .761 .912 Killing of Plant Tissue by Low Temperature 179 While the dififerences are not large, it will be seen that the sap density of the shaded plants is uniformly lower and the killing greater. Ohlweiler^ at the Missouri Botanical Garden seemed to find some relation between the density of the sap of different plant species and their resistance to cold. This is true especially in the case of the different species of magnolia, where the leaf structure of species with dense sap and of those with dilute sap is similar, so there would not be this influence involved to modify the results. From the beginning of these experiments, observations have been made in autumn as to plants killed by the various early frosts and freezing point determinations were made from leaves of these to see if there is to be found any relation between hardiness and sap density. In the following table the plants are listed as nearly as could be determined according to hardiness, the most tender first, though it is certain that almost any of the plants could be changed two or three places in the succession and be as accurately placed in order of hardiness. The depressions are also given. The leaves for the depressions were all taken in the morning as soon as the dew was off so they would be equally turgid. Table 11. Showing the Relative Hardiness of Growing Plants Compared With Their Relative Sap Density. Plant Morning-Glory (Ipomoea purpurea) Coleus (C. Blumei) Sweet Potato (Ipomoea Batatas) Moon Vine (Ipomoea Bona-Nox) Watermelon (Citrullus vulgaris) Cantaloupe (Cucumis Melo) Cucumber (Cucumis sativus) Caladium (Colocasia antiquorum) Pumpkin (Cucurbita Pepo) Tomato (Lycopersicum esculentum) Lantana (L. Camara) Dahlia (Dahlia variabilis) Blue Salvia (Salvia patens) Red Salvia (Salvia splendens) Rose Geranium (Pelargonium graveolens) (jcranium f F'elargonium Hortorum) Eggplant (Solanum Melongcna) Alternanthera (Telanthcra versicolor) Periwinkle ( Vinca major) Ageratum (A. conyzoides) Chard (Beta vulgaris var. Cycla) Celery (Apium graveolens) '23d. Aiil. Kpt. Mo. Hot. Card. 1912, pi). lOI-.U. (Ull)!. H7) Depression .920 .428 .96 .863 .882 .588 .585 .745 .785 .832 .962 .711 1.025 .765 1.835 1.075 .805 1.058 1.20 1.055 .805 1.442 i8o Missouri Agr. Exp. Sta. Research Bulletin- No. 8 Plant Gaillardia (G. pulchella) Chrysanthemum (C. Sinense) Sedum spectabile Dandelion (Taraxacum officinale) Dock (Rumex crispus) Verbena (V . hybrida) Hollyhock (Althaea rosea) Horse-radish (Cochlearia Armoracia) California Poppy (Eschscholzia Californica) , Clover (Trifolium pratense) Violet (Viola odorata) Cabbage (Brassica oleracea) Plantain (Plantago major) Strawberry (Fragaria Chiloensis) Depression .803 1.955 .575 .975 .997 1.093 1.130 1.125 1.198 1.290 1.225 1.115 1.380 1.865 All these depressions were determined as late in autumn as it was possible to secure healthy tissue. While it is evident that other factors than sap density influence the hardiness, yet it seems true that more of the very tender plants are found among those with slight depressions, and a majority of the most hardy among those with greater density. It is possible that if a larger list of plants were obtained, this might not be true. All of these tests have been made with succulent plants that kill at a temperature a few degrees below the freezing point. It is true that some rather hardy plants like cabbage, kale, lettuce and garden peas have been influenced in their killing temperature as great- ly as have plants like tomatoes and cowpeas that kill at a tempera- ture but slightly below the freezing point. However, in case of win- ter wood, and buds that have developed great resistance to cold by some sort of change, the problem would perhaps be difi"erent. This experiment started with the idea that it might be possible to increase the hardiness of buds of the peach in winter by increasing the sap density through the use of fertilizers. Accordingly, plots of peach trees at Dixon, Missouri, were fertilized with potassium chloride at the rate of about 500 pounds to the acre, in the springs of 1907, 1908, and 1909. Plots receiving potassium chloride at the rate of a little more than 500 pounds to the acre during the seasons of 1906, 1907, 1908, and 1909, were located in the orchard of the Ozark Orchard Company at Goodman, Missouri; and plots receiving 480 pounds of potassium chloride to the acre, beginning March, 1910, were located with the Ozark Fruit Farm Company, Brandsville Missouri. Potassium was used in these experiments because some experiments indicate that it is more readily taken into the cell to become an osmotically active agent than most other mineral nutri- Killing of Plant Tissue by Low Temperature i8i ents. In these orchards we have had opportunity to observe the results following cold periods that killed all the buds, cold periods that killed nearly all of them, and cold periods that left a fair crop, but in no case has there been any apparent effect on hardiness re- sulting from the use of potassium as a fertilizer. Twigs were se- cured from these plots in winter, spring, and summer and the density of the sap of the cortex determined by expressing the sap, and deter- mining its freezing point, and no difference in the density could be detected between the plots fertilized with potassium and those re- ceiving no fertilizer. These determinations could not be made with sap from buds because it could not be secured in sufficient quantities. If it were possible to increase the sap density by accumulation in the cell of potassium or other materials applied to the soil, it could possibly affect the killing temperature of the bloom or young fruits or young growth in spring, even if it should have no effect on the killing temperature of the buds in their dormant condition. C. Dussere^ observed apparently a slightly greater hardiness of young grape shoots on vines that had been fertilized with potassium than on vines that were not so fertilized. However, analyses failed to show the presence of more potassium in the wood of those vines receiving potassium fertilizer than in the wood of those that had not received fertilizer. It may be said that on our peach plots we have had occasion to observe the effect of spring frosts on the bloom, as well as the effect of winter cold on the buds and have failed to observe any dif- ference due to application of potassium to the soil. Briefly summerizing all our results with the relation of sap den- sity to hardiness, we are safe in concluding that, at least in case of plants not in a resting condition, a large amount of dissolved material either in the sap within the cell or in a solution surrounding the cell, will protect the cell from injury due to low temperature, to some ex- tent at least. The protection where plants take the material into the cell seems to be much less than where the material in solution surrounds the cell, if the work of Maximow is confirmed. The practical means of increasing hardiness seem to be very limited. Withholding water from plants in a plant bed \\ill check the growth and thus increase the sap density and the hardiness. It was thought possible that with such plants as cabbage which take up potassium salts readily, watering in the plant lied with a solution of such salts would tend to increase their hardiness. However, cab- bage and tobacco plants growing in a hotbed soil watered for eighteen »Bul. Soc. Scl. Nat. 6. sor 48 (1912) No. 176. pp. 393. (IJibl. No. 31). i82 Missouri Agr. Exp. Sta. Research Bulletin No. 8 days with a solution of potassium chloride ranging from 4.02 per cent normal at first to 16.08 per cent normal toward the last showed no increase in either sap density or hardiness over that of plants not so treated. It is only in sand cultures that we have been able to increase the sap density of plants by watering them with solutions. If freezing to death results from too complete withdrawal of water from the protoplasm, then, even with winter buds and wood, if the sap density could be increased with some material that stays in solution at temperatures lower than that at which the tissue kills, it would hold more water in solution and thus lower the temperature at which a killing degree of desiccation is reached. Miiller-Thurgau's measurements showed that a very large percentage of the water in plant tissue is in the form of ice. In case of winter resting tissue very little water can be left unfrozen at a killing temperature. If freezing to death of plant tissue results from desiccation, then very small quantities of water must be sufficient to protect the protoplasm in case of the more hardy plants. Some writers hold that since some plants continue to live after nearly all the water is frozen out, desic- cation of the protoplasm can not be the explanation of death from low temperatures. Others seem to hold the opinion that the pro- tective action of the sap solute ends at the freezing point of the sap. It should be remembered that so long as the temperature is above theeutectic pointof anyof thesap solute, there will be some water pre- sent in the liquid state. The number of degrees which the killing tem- perature is lowered by an increase in sap density should be greater with plants that kill at the lower temperatures. Thus in case of a solution containing a gram molecule of a non-electrolyte in a liter of water, the freezing point is -1.86° C. In case of a solution with one-half of a gram molecule to a liter of water, the freezing point would be -0.93° C. The following figures would evidently be approxi- mately true: Fraction of water un- Fraction of water un- Temperature frozen, gram-mole- frozen, one-half a cule, In a liter of gram-molecule, in a water liter of water -0.930 c. all all -1.86»C. all 1/2 -3.72" C. 1/2 1/4 -7.44" C. 1/4 1/8 -14.880 C. 1/8 1/16 -29.760 c. 1/16 1/32 Killing of Plant Tissue by Low Temperature 183 This is, of course, assuming that the eutectic point of the solu- tion is below these temperatures. It also assumes that the freezing point is lowered exactly in proportion to the molar concentration of the solution; that is, if there be four gram molecules in a liter of water, the freezing point should be four times -1.86° C. This is probably not quite true but the difference is so slight that it may be ignored here. It will thus be seen that with a solution in which there is a gram molecule to a liter of water of a solute that stays in solution below -29.76° C, there would be as much water unfrozen at -14.88° C, as there would be at -29.76° C, if the molar concentra- tion is only half as great. Maximow concludes that the protective action of the material he used was greater than could be explained by the depression of the freezing point of the sap. He may not have considered the above facts. Another fact should be considered. With the plants Maxi- mow used, possibly a large percentage of the sap solute crystallized out at temperatures considerably above the minimum temperatures that he used. Maximow used as a protective substance a solution of equal density with the plant sap and with a low eutectic point, then at a temperature of say -10° C, it would be considerably more dense than the plant sap from which crystallization had taken place. While theoretically the protective action of increased den- sity should be greater with plants that kill at a rather low tempera- ture like winter buds and wood, we have found no way to demonstrate such protective action with such tissue. The only means of increas- ing the sap density of such tissue that suggested itself to us was by applying fertilizers like potassium chloride to the soil and this method has not had that effect. From the experience of Maximow and experience here with cabbage and other plants where the salts in solution have increased the resistance to cold as much as it was increased by organic com- pounds, and in some cases more, it seems that one is safe in conclud- ing that killing from cold is more likely a mechanical injury due to the withdrawal of water from the protoplasmic membrane than an injury resulting from the precipitation of proteids as suggested by Gorke, since an increase in the percentage of any mineral salt should tend to hasten the precipitation of proteids rather than reduce it. It seemed that one good test of the theory of Gorke^ would be to find whether or not a lower temperature is required to precipitate proteids from the sap of twigs as they increase in hardiness during 'Landw. Vorauchs. Vol. 65. p. 149, 190G. (Hlbl. No 17). 184 Missouri Agr. Exp. Sta. Research Bulletin No. 8 early winter. It is known that from the time the leaves fall for at least one or one and one-half months, the cortex as well as the winter buds and the sap wood of deciduous trees (at least of peaches) con- tinue to increase in hardiness. This Station made an effort to de- termine whether this increase in hardiness might not be due to such a change in the composition of proteids that they would tend to remain in solution at lower temperatures. Consequently twigs were gath- ered on the dates shown in the following table, beginning before leaf fall. Large quantities of cortex sap were expressed. After pouring back and forth to insure a uniform solution, one-half of this sap was frozen at the temperatures shown in the table, a temperature that would at that time kill the cortex and practically all other tissue of the twig. The other half was kept in an ice box until the freezing of the other sap was complete, then both were taken to the Department of Agricultural Chemistry for proteid analysis, the frozen sap being thawed on the filter except when otherwise stated in the tables. The analysis was by the following plan, kindly furnished by the Depart- ment of Agricultural Chemistry: KJELDAHL-GUMMING METHOD FOR ESTIMATION OF NITROGEN. "Weigh out accurately 4-6 grams of the sap by difference, place in 500 c. c. Kjeldahl flask, add 0.7 gram of mercury and 25 c. c. of sulphuric acid, heat for a few minutes until frothing ceases, add 7 grams of potassium sulphate and digest over a flame until clear, cool; wash down the neck of the flask with distilled water, heat again one hour until water is expelled. Cool and dilute with water until flask is 2 3 full, add a piece of zinc and a piece of paraffin and 80 c. c. of alkaline solution containing 500 grams potassium sulphide and 18 kilos sodium hydroxide in 40 liters of the solution. Distil into standard hydrochloric acid and titrate back with standard ammonium hydrox- ide, using cochineal as indicator". The factor 6.25 was used in re- ducing nitrogen reading to proteid readings. Twigs of apple, peach, pear and plum were used with results as shown in the following table: Killing of Plant Tissue by Low Temperature l8: Table 12. Showing Percentage of Proteids in Frozen and Unfrozen Sap from Tissues of Apple, Pear, Peach and Plum. Tem- Average per Average per cent Kind of Sap Date pera- cent proteids. proteids. Sap ture Sap unfrozen frozen Wealthy apple twigs Sept. 4,'ll -17 0.397 0.435 Wealthy apple twigs Oct. 2, '11 - 9.5 0.556 0.544 Elberta peach twigs Sept. 4,'ll -17 1.488 1.894 Elberta peach twigs Oct. 2, '11 - 9.5 2.644 2.550 Elberta peach twigs Oct. 16, '11 - 9.5 2.550 2.531 Elberta peach roots Oct. 16,'ll - 9.5 1.563 1.563 Elberta peach twigs Nov. 30,'ll - 9 2.050 2.100 Chabot plum twigs Sept. 4,'ll -17 0.651 0.694 Chabot plum twigs Oct. 2, '11 - 9.5 0.819 0.800 Kieffer pear twigs Sept. 4,'ll -17 0.144 0.031 Kieffer pear twigs Oct. 2, '11 - 9.5 0.406 0.375 Kieffer pear twigs Oct. 16,'ll - 9.5 0.288 0.288 Kieffer pear roots Oct. 16,'ll - 9.5 0.181 0.206 Kieffer pear twigs Nov. 30,' 11 - 9 0.350 0.375 In no case does there seem to be any conclusive indications that proteids were precipitated by temperatures low enough to kill the plant tissue. Since there was no precipitation from the sap of tender twigs in early autumn, sap was not taken from twigs later in the season. Cabbage and other succulent plants grown in the greenhouse, some well watered, some under dry conditions and others grown out of doors, also well watered but under conditions where they would withstand lower temperatures than would those grown in the green- house, had each an abundance of sap expressed, half of it being frozen to a temperature that would kill those well watered in the greenhouse, but would not kill those grown out of doors. Samples of each of these frozen and unfrozen were analyzed for proteids, with the re- sults shown in the following table: i86 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 13. Showing Percentage of Proteids in Frozen and Unfrozen Sap From Succulent Plants. Kind of Sap Cabbage greenhouse, dry soil Cabbage, greenhouse Cabbage, out-of-doors Cabbage sap, outside Cabbage, inside dry soil. . . Cabbage, inside Tomato sap, greenhouse. . . Tomato sap, greenhouse, dry soil Tomato sap, outside Pea sap, inside Pea sap, outside Lettuce, sap inside Lettuce sap, outside Kale, greenhouse Kale, outside Date Apr. 8,'12 Apr. 8,'12 Apr. 8, '12 Nov. 28,'ll Oct. 28,'ll Oct. 28,'ll Dec. l,'ll Dec. 1/11 Dec. l,'ll Apr. 27, '12 Apr. 27, '12 Apr. 27,'12 Apr. 27,'12 Dec. 16,'ll Dec. 16,'ll Tem- pera- ture -3 -3 -3 -5 -5 -5 -3.7 -3.7 -3.7 -5 -5 -5 -5 -6.2 -6.2 Average per cent proteids. Sap frozen. 0.975 0.781 1.406 1.675 1.144 0.919 0.594 1.313 0.700 2.069 3.088 0.519 0.269 1.744 1.963 Average per cent proteids. Sap unfrozen. 1.069 0.838 1.438 1.856 1.281 0.956 0.438 1.444 0.713 2.413 1.625 0.613 0.263 1.831 1.994 In case of cabbage, kale, lettuce and peas there is some evidence of precipitation in case of the more tender greenhouse plants, but it is possible that the differences are within the range of experimental error. In March, 1913, apple roots that had been growing in the green- house for sixty days had sap expressed, half of it frozen to a tempera- ture that would kill them, the other half not frozen. At the same time sap was taken from roots that had been kept in cold storage at a temperature seldom varying from 32°F., with the following results : Table 14. Showing Percentage of Proteids in Frozen and Unfrozen Sap From Apple Roots. Material Treatment Date Percent- age Nitrogen Apple"stock, cold storage Not frozen.. Frozen Not frozen.. Frozen Mar. 14,'13 Mar. 14,'13 Mar. 14,'13 Mar. 14,'13 0 325 Apple stock, cold storage Apple stock, ^greenhouse 0.325 0 592 Apple stock, greenhouse 0 548 Killing of Plant Tissue by Low Temperature 187 In case of the greenhouse grown apple stock there seems to be slight evidence that proteids were precipitated by the low tempera- ture. However, the difference is probably within the range of error. In these cases great care was taken to prevent any change in the soluble proteid content of the sap before analysis began. The unfrozen sap was immediately started for analysis, after being ex- pressed. In all cases the frozen sap v;as thawed on filter paper so that little change could take place after the thawing. From the above tables, we are safe in concluding that in the case of the cortex of twigs from fruit trees in autumn or early winter, precipitation of proteids plays no part in the killing since tempera- tures that will kill all the cortex will precipitate no proteids. In the case of some succulent growing plants there seems some evidence that a killing temperature will precipitate a slight percentage of the soluble proteids present, but since increasing the amount of salts present that are supposed to precipitate the proteids increases in- stead of decreasing the resistance to low temperature, we are probably safe in concluding that precipitation of proteids does not explain freezing to death. OTHER FEATURES THAT INFLUENCE THE FREEZING TO DEATH OF PLANTS. Rate of Thawing. The effect of the rate of thawing on the killing from cold has been discussed previously with reference to the works of Miiller-Thurgau,' Molisch,^ and Sachs.^ However, it is not out of place to discuss it briefly as one influence to be dealt with in the killing of certain plants from cold. As mentioned before, Miiller-Thurgau and Molisch found only mature fruits and leaves of Agava Americana to be affected by the rate of thawing. Prac- tically all plants used in freezing experiments to be reported in this paper have been tested with reference to the effect of rapid thawing, particularly because of the effect such thawing would have on the results with other experiments. The following table gives the re- sults with peach fruit buds that were thawed slowly and rapidly: 'Landw. Jahrb. Vol. I.""), p. 4.5.3, 1886. (Blbl. No. 78). 'UnttTsuchutiK liber das Erfrlorcn, otc. Hook, 1H. (Hlbl. No. 88). 'Jahrb. f. Wis.s. Bot. Vol. 52. 1013. pp. 4n7-.'>n0. (lUbl. No. 121). 200 Missouri Agr. Exp. Sta. Research Bulletin No. 8 20" F. to 30° F. below zero with but slight injury where the tempera- ture fall is not so rapid. No fruit buds have been found on the Experiment Station grounds that can not be killed by a temperature secured by salt and ice when in the most dormant state, by very rapid fall. The following is a list of the freezings in our laboratory, with the results: Table 20. Showing Effect of Slow and Rapid Temperature Fall on Freezing to Death of Plant tissue. Kind of Buds Rice's Seedling peach. . . Yellow Swan peach .... Early Richmond cherry, Rice's Seedling peach. . Yellow Swan peach. . . . Early Richmond cherry Dyehouse cherry Rice's Seedling peach. . . Elberta peach Jonathan apple Montmorency cherry. . . Lombard plum Hills Chili peach Rice's Seedling peach. . Early Richmond cherry Elberta peach Hills Chili peach Rice's Seedling peach. . . Rice's Seedling peach.. . Elberta peach Jonathan apple Montmorency cherry. . . Chabot plum Date Num- ber Buds Percent- age Killed an. 25, '13 80 an. 25, '13 64 Jan. 25, '13 100 Feb. IS, '13 117 Feb. 15, '13 95 Feb. 15, '13 100 Feb. 15. '13 83 Mar. Mar. Mar. Mar. Mar. Feb. Feb. Feb. Slowly to -19.5" C. 15 28 16 Slowly to -20" C. 10.3 30.5 45.0 22.9 Slowly to -18.5" C. 42.3 97.7 34.3 14.1 12.9 Slowly to -15.5" C. 17.3 10.6 29.8 Slowly to -18" C. 15, '13 104 15, '13 104 15,'13 35 15, '13 85 15,'13 112 22, '13 63 22, '13 104 22,'13 94 Mar. 8, '131 66 Mar. 8,'13 120 Mar. 8,'13| 70 Mar. Mar. Mar. Mar. Mar. 30.3 20.8 21.4 Num- ber Buds Percent- age Killed Rapidlyto-17.7'> C. 55 86 90 96.4 100.0 97.7 Rapidly to -19" C. 68 64 89 90 100.0 100.0 98.8 97.6 Rapidly to -12" C. 101 86 20 87 99 37.6 61.6 70.0 17.2 47.3 Rapidly to -10" C. 49 81 73 10.2 4.9 54.8 Rapidlyto-13.5° C. 92 71 82 Slowly to -18« C. Rapidly tc 22, '13 138 44.2 154 22, '13 100 88.0 85 22, '13 34 64.7 33 22, '13 176 58.5 184 22, '13 236 78.3 183 70.6 76.0 54.9 51.9 92.9 75.7 62.5 86.8 Killing of Plant Tissue by Low Temperature 201 Kind of Buds Date Num- ber Buds Percent- age Killed Rice's Seedling peach Elberta peach Jonathan apple Montmorency cherry Chabot plum Elberta peach Elberta peach Elberta peach Slowly to -I6.50 C. Mar. 24,'13i 145 Mar. 24,' 13 Mar. 24,' 13 Mar. 24,'13 Mar. 24,'13 170 39 166 191 57.0 82.9 46.2 76.5 100.0 Slowly to -16" C. Nov. 3,'10 382 Slowly to -I6.50 C. 77.8 Dec. 7, '11 150 Dec. 7, '11 210 1.3 Num- ber Buds Percent- age Killed Rapidlyto-11.5° C. 158 i 53.2 86 I 75.5 40 70.0 189 1 59.2 176 I 73.8 Rapidly to-12.5»C. 188 69.1 Rapidly to -16" C. 200 100.0 Slowly to -16.20 C. jRapidlyto-16.5°C. 14.8 150 96.6 Table 20a. Showing Effect of Slow and Rapid Temperature Fall on Freezing to Death of Plant Tissue. Kind of Buds Manner of Freezing Montmorency cherry ^Slowly to -20" C. . Montmorency cherry Rapidly to -20° C. Early Richmond cherry Slowly to -20" C Early Richmond cherry I Rapidly to -20" C. Date Mar. 2, '12 Feb. 29,'12 Mar. 9,'12 Mar. 14, '12 Num- ber Buds 163 130 297 263 Percent- age Killed 3.0 96.0 5.0 98.0 Table 20b. Showing Effect of Slow and Rapid Temperature Fall on Freezing to Death of Plant Tissue. Kind of Buds Date Ben Davis apple I Jan. Ben Davis apple Jan. Ben I^avis apple iF"eb. Ben Davis apple. Jonathan apple. . Feb. Jan. Early Richmond cherry J^n- Early Richmond cherry 'Jan. Montmorency cherry ili*"- Early Richmond cherry Feb. Montmorency cherry JFcb. Cherry .Feb. 18,'12 19,'12 2, "12 3, '12 18,'12 18,'12 19,'12 19,'12 9,'12 10,'12 15, '12 Tem- pera- ture Time -20.5 2 hrs -20.5 2 hrs -20 VAhrs... -20 2K hrs... -20.5 2 hrs -21 IHhrs... -20.5 2 hrs -20.5 IK hrs... -20.5 IH hrs... -20.5 \H hrs... -20.5 12 hrs.... I(slowly) Rapidly, -f 20» C. to -17" C. in 45 minutes Jonathan apple JFeb. 20,'13 Vermont Beauty pear |Fcb. 20, '13 Chabot plum iFcb. 20,'13 Num- ber Buds 40 47 60 82 35 272 200 178 252 195 132 20 25 28 Percent- age Killed 50.0 36.2 0.0 2.44 48.6 98.6 100.0 87.0 100.0 94.8 38.0 100.0 1 00 . 0 100.0 202 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 20c. Showing Effect of Slow and Rapid Temperature Fall on Freezing to Death of Plant Tissue. Material Date Time Tem- pera- ture Results Elberta peach twigs Rice's Seedling peach twigs. . Jonathan apple twigs Chabot plum twigs Montmorency cherry twigs Elberta peach twigs Rice's Seedling peach twigs Jonathan apple twigs Chabot plum twigs Montmorency cherry twigs Elberta peach twigs Elberta peach twigs Rice's Seedling peach twigs Rice's Seedling peach twigs Jonathan apple twigs Jonathan apple twigs Montmorency cherry twigs Montmorency cherry twigs Chabot plum twigs Chabot plum twigs Mar. Mar. Mar. Mar, Mar. Mar. Mar. Mar. Mar. Mar. Mar. 21,'13 21, '13 21, '13 21,'13 21, '13 21,'13 21, '13 21, '13 21, '13 21, '13 22,'13 Mar. 22,'13 Mar. Mar. Mar. Mar. Mar. Mar, Mar. Mar. 22,'13 22,'13 22,'13 22, '13 22, '13 22, '13 22, '13 22, '13 Hi hrs. 734 hrs. 7M hrs. 7M hrs. 714 hrs. 1% hrs. 1% hrs. 1% hrs. l^A hrs. 1% hrs. 1 hr. 7H hrs. 1 hr. . . . 7H hrs. 1 hr. . . . 7M hrs. 1 hr. IVi hrs. 1 hr, 7H hrs, -18 -18 -18 -18 -18 -13.5 -13.5 -13.5 -13.5 -13.5 -11.5 -16.5 -11.5 -16.5 -11.5 -16.5 -11.5 -16.5 -11.5 -16.5 Sap wood and pith onlyinjuredregions. Rice's Seedling showed the least in- jury. Othersinjured equally. Killing about the same for twigs whose tem- perature fell rapidly to -13.5° and those whose temperature fell slowly to -I80. (Buds injured worst in rapidly frozen ones). Pith and sap wood injured. Pith and sap wood injured. Very slight browning in cortex; none in cambium. No difference in in- jury between rapid and slow. Pith and sap wood slightly injured in both. Pith injured in both cases. No other tissues in- jured. Pith injured in both cases. Pith and sap wood injured equally in both cases. With these twigs it will be seen that the killing temperature of rapidly frozen twigs was four and a half degrees higher than that of the more slowly frozen twigs, and even then the buds of the rapidly frozen twigs killed the worst. In rapid freezing it required from one to one and three-fourths hours to reach a temperature of -20° C. In slow freezing it required from seven to ten hours to reach the same temperature. Many young fruits and succulent plants were also frozen slowly and rapidly Killing of Plant Tissue by Low Temperature 203 but there was so little apparent difference between the results that the data are not given. The killing temperature lies so near the freezing point that possibly the slowly frozen tissue kills badly be- cause it is exposed to temperatures around the killing point longer. This tender tissue was exposed to the minimum temperature for from twenty to thirty minutes. It will be seen that the rate of temperature fall with winter twigs and buds exerts the greatest influence on the extent of killing at a given temperature of any feature we have so far discussed. And in the case of very forward, rather tender fruit buds, the rate of temperature fall exerts great influence. Thus on March 24, 1913, when all buds, especially of peaches, plums and cherries, had made much growth, a temperature of -11.5° C. killed as many buds with rapid temperature fall as a temperature of -16.5° C. with a slower temperature fall. Tests were made to see whether the rapid temperature fall that does the most harm is in the early part of the ice forming state, or in the later part. The following table gives the results with peach buds frozen slowly one-half way down to the killling temperature and rapidly the remainder of the way, and others frozen rapidly one- half the way down and slowly the remainder of the way, and others slowly all the way down : 204 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 21. Showing the Relative Effect on the Resistance to Low Temperature of Rapid Temperature Fall Toward the Beginning and Toward the End of the Freezing Period. Kind of Buds Manner of Freezing Date Number of Buds Percent- age Killed Elberta peach Elberta peach Elberta peach Elberta peach Elberta peach Elberta peach Elberta Montmorency cherry. . . Montmorency cherry. . . Montmorency cherry. . . Montmorency cherry. . . Early Richmond cherry Early Richmond cherry Early Richmond cherry Early Richmond cherry Dyehouse cherry Dyehouse cherry Dyehouse cherry Slow to -12; fast -12 to-16 Fast to -12; slow -12 to -16 Slow to -17. 5 Fast to -16 Slow to -12; fast to -16 Fast to -12; slow to -16 Medium to -12; fast to-16 Fast to -12; slow to -20 Slow to -12; fast to -20 Fast to -20 Slow to -20 Slow to -12; fast to -20 Fast to -12; slow to -20 Slow to -20 Fast to -20 Slow to -12; fast to -20 Fast to -12; slow to -20 Fast to -20 Dec. 20/11 Dec. Dec. Dec. Dec. Dec. Dec. Feb. Feb. Feb. Mar. 20,'l 20 8 8 8 13 24 27 27 2 Mar. 5 Mar. Mar. Mar. Mar. Mar. Mar. 7 9 14 16 19 '1 '13 '1 '1 •1 '12 '12 '12 '12 '12 '12 '12 '12 '12 '12 22, '12 135 77 129 135 113 135 155 142 136 130 163 291 283 297 263 184 200 150 3.7 71.4 6.2 98.5 3.5 29.0 52.3 75.0 15.4 96.0 3.0 14.0 83.0 5.0 98.0 56.0 99.0 95.0 It will be seen that rapid falling in the early part of the freezing period down to -12° C, does more harm than rapid fall in the latter part of the period, from -12° C. to the killing temperature. This rapid freezing probably has considerable to do with the amount of killing at times in nature, though just how much it is difficult to tell. In this investigation it was not possible to cause, the temperature to fall more slowly than the most rapid fall to be observed naturally in the climate of this station. Yet there are probably times when on sunny, cold days the temperature of some tissue may rise to near the freezing point due to the absorption of the heat by the dark color of the bark. In this case when the sun is off the twigs, the tempera- Killing of Plant Tissue by Low Temperature 205 ture will fall very rapidly. Since rapid temperature fall near the freez- ing point seems to be more harmful than rapid temperature fall near the killing temperature, it would seem certain that greater killing should thus result. It does not seem impossible that "sun scald" of apple trees may be explained in this way. This rapid fall of temperature may also be a feature to be con- sidered in heating an orchard. Thus any one who has worked with orchard heaters knows that if on a still night a few of the heaters go out, the temperature will immediately fall to about that which would prevail without the heaters. In this case it is possible that the tissue may kill worse than if the heaters had not been there, since the blossoms or fruit would freeze very rapidly. We can not be certain of this, however, for results with rapid freezing of blossoms at this station have not been uniform enough to be conclusive. Maturity and Hardiness. Probably the greatest factor in deter- mining the amount of cold that can be withstood by trees and shrubs that live through the winter is a condition of maturity. Emerson^ has studied the question of maturity of fruit and other trees in Ne- braska and has found that the varieties most hardy in wood are those that mature early. Where growth can be checked early in the season, as by a gross feeding cover crop like millet, the trees will also withstand more cold. Selby^ made a study of the injury to fruit trees and ornamentals by the severe freeze early in the winter of 1903-4, and attributes the severe injury to the fact that the trees grew late in the fall on account of a very wet period following a period of dry weather. Eustace^ reports a study of the effect of the same winter on fruit trees and describes similar conditions. It seems in this case also the great injury is due to the trees' having grown late in autumn. Winkler^ found that the resistance of native trees of Germany is least in May, June, July, and August, and gradually increases during September, October, November and December, and is great- est in January, as measured by laboratory freezings. In summer the fruit buds, for example in August after they can be easily detected, may be killed by a temperature of -9° to -10° C. or somewhat lower on some years. At this time there is little dilTer- ence between the hardiness of the buds, the wood, and even the foli- age, though the foliage kills slightly the worst; while in winter fruit 'Nebraska Akp. Kxp. Sta. Bui. 70. 1003. (nibl. No. 33): Nc-h. Arp. Exp. Sta. Iliil. 02. 190G. (Blbl. No. 34); Net). Akp. Kxi). Ht;i. .\iil. ICpt. No. 10, lOOli, pp. 101-10. (Hlbl. No. 3.')). ><)hIo Agr. Exp. Sta. Hul. 102. lod.s. dUbl. No. 101). •Now York ((ienova) Agr. Kxp. Sta. Hul. -'(.0. lW)r>. (IHbl. No. 38). •Jahrb. f. WIss. Hot. Vol. Oli. 1013, pp. 4(17-50(1. (Hlbl. No. 121). 2o6 Missouri Agr. Exp. Sta. Research Bulletin No. 8 buds have been known to survive temperatures of -30° C. and lower, and the tree will survive, under favorable conditions, considerably lower temperature than that. In fact Macoun^ cites an instance where a Pyrus baccata — Pyrus Malus hybrid — has withstood for five years a climate whose temperature frequently falls to -50° F., and in 1909 it fell twice to -59° F. At the beginning of winter, as observed by the authors above, the tree tissue generally — whether it is buds, wood, cambium or cortex — will stand less cold than later in the winter. Observation at this station indicates that at least some tissue increases in hardiness rather rapidly for a short time following leaf fall. The following table gives the temperature and result of freezing peach fruit buds, beginning in summer when they are first plainly to be observed and continuing until January: Table 22. Showing the Relative Hardiness of Fruit Buds at Various Seasons of the Year. Kind of Buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Elberta peach buds Oldmixon peach buds. . . . Oldmixon peach buds . . . . Oldmixon peach buds. . . . Oldmixon peach buds. . . . Late Duchess apple buds Jonathan apple buds. . . . Jonathan apple buds. . . . Jonathan apple buds. ... Date July July Sept. Sept. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Dec. Dec. Dec. Dec. Dec. Dec. Jan. Jan. Feb. Aug. Nov. Nov. Dec. July July Nov. Jan. 15,'13 16,'13 15, '11 27, '10 I.'IO l.'ll 14,'ll 12, '09 17,'ll 18,'ll 18,'ll 1,'09 1,'09 6,'ll 6,'ll 14,'09 18,'09 8,'13 13, '10 23, '10 23, '10 26,'09 26,'09 6,'09 15, '13 15,'13 4,'ll 18,'12 Tem- pera- ture - 6 - 5 - 9 -15 -16 - 9 -12.5 -13.5 -14.5 -12.3 -13.3 -22 -21 -14.7 -16 -20.5 -22 -20 -20 -19 - 8 -18 -19 -22 - 6 - 5 -12 -20.5 ,5 ,5 -19.5 Number of Buds 23 47 186 245 382 104 188 198 133 100 150 385 343 225 278 190 608 105 290 168 162 160 229 290 20 53 68 35 Percent- age Killed 78.2 100.0 64.6 39.0 77.8 60.5 69.1 1.5 100.0 91.0 99.3 75.3 84.2 40.0 56.5 74. 48. 51.0 97.0 63.1 93.2 81.2 15.3 93.1 70.0 73.6 48.5 58.6 iProcs. Soc. for Hort. Science, 1912, p. 65. (Bibl. No. 69). Killing of Plant Tissue by Low Temperature 207 The following table gives the result of freezing twigs: Table 23. Showing Killing Temperature of Twig Tissue at Different Seasons. Variety PEACHES Elberta Elberta Young Champion. Champion. Belle of Georgia five years old . Elberta Elberta, one year old. Seedling peach, one year old . Elberta Elberta. Elberta. Elberta . Elberta . Elberta . Elberta. Elberta. Results July 6,'13 July 17, '13 July 29,'12 Aug. 5,'12 Aug. 29,'12 Sept. 24,'12 Oct. 5,'12 Oct. 8,'12 Oct. 14,'ll Oct. 19, '11 Oct. 21, '11 Oct. 26,'ll Nov. l,'ll Nov. 2, '11 Nov. 16,'12 Dec. 6,'12 Seedling, one year old iDec. 18, '12 Seedling, one year old Dec. 19, '12 Elberta. Dec. 7,' 12 i:iberta Jan. 11, '13 Rice's Seedling Jan. 25, '12 Elberta 'Mar. 2 1, "13 Rice's Seedling Mar. 21, '13 Elberta lApr. 5. '13 6 5 5 - 5 - 5 - 9.5 - 5 I - 7.5] - 9 - 9 - 9 -IS -15 -16.5 -18 -16 -20 -19.3 -18 -18 -12.5 13 twigs. Cambium dead in 5; cambium and cortex in 8. 20 twigs. Cambium and cortex dead in all. Twigs 10" long. All tissue injured in terminal 6"; slight injury to cortex only at base of tree. Bark and cambium killed in all stems. Bark, cambium and sap wood killed in all twigs. Bark, cambium, and outer portion sap wood killed. Cambium and cortex injured except at base of one twig. Only injury to sap wood was in terminal part of one twig. Twigs injured in cambium and cortex region. 24 twigs. 100% dead. Cambium only killed. 38 twigs. No injury. 46 twigs. 73.9% dead. Cambium only killed. 39 twigs. Killing confined largely to cambium. 23 twigs. 60.6% killed. Cambium only injured. 51 twigs. 84.3% killed. Cambium only injured. Slight injury in cortex. Cambium uninjured. Cortex injured in all; cambium un- injured; pith killed and sap wood occasionally showed injury. Injured slightly in cortex and pith; cambium and wood sap uninjun-d. Injury confined to pith and cortex. Injury confined to shoulder below bud and pith region. Pith (lead, oiIrt tissues uninjured. Cortex injured slightly; cambium entirely killed. Injury to sap wood and pith. Injury to sap wood and [)ith less than with Elberta. Wood uninjured. 2o8 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Tem- Variety Date pera- Results ture APPLE TWIGS Jonathan twigs May 17,'13 - 4 Cortex and epidermis hardiest in a given part. Near base twigs hardier than near tip. Jonathan twigs May 24,'13 - 4 Slight injury near terminal part. Jonathan twigs June 7,'13 - 5 Cambium injured throughout all twigs. In terminal 3 in. injury extended to cortex, sap wood and pith. Terminal leaflets more se- verely injured than remaining ones. Ben Davis, Severe injury in cambium, cortex seven years old June 26,'13 - 4 and sap wood. Young Early Harvest. June 27,'13 - 4 Very slight injury in cambium near terminal. Early Harvest June 27,'13 - 5 Cambium, cortex, sap wood and pith injured. More severe near terminal. Ben Davis, Cambium and cortex severely in- five years old July 2, '13 - 4.5 jured. Sap wood injured. Late Duchess July July 15, '13 - 6 4 twigs; no injury. 20 twigs; no injury. Jonathan twigs 16,'13 -5 Young apple July 27,'12 - 5 All tissue injured 4" back from ^ ^^ — ^^ *-— ^^ f* — ^^ •■•»»••»■ terminal. No tissue injured far- ther back than 7" from terminal. Cortex injured most and cambium next. Jonathan Aug. 15,'12 - 5 Cortex and cambium injured slightly in all samples. Sap wood and pith not injured. Only slight difference between younger and older parts. Ben Davis Oct. Oct. Oct. Nov. 9,'12 13,'12 16,'12 4, '11 - 8 - 9.5 -15 -12.5 No parts killed. No parts killed. Cortex killed; cambium uninjured. 23 twigs. No injury. Ben Davis Jonathan Jonathan Jonathan, *» * J- Slightly injured in cortex. Cambium one year old Nov. 16,'ll -15 uninjured. Jonathan, 38.5 inches of twigs; 67.5% killed one year old Nov. 27,'ll -20 in all tissues. Ben Davis, 40.5 inches of twigs; 14.8% killed one year old Nov. 27,'ll -20 in all tissues. Jonathan, 158 inches of twigs; 34.1% killed one year old Nov. 29,'ll -19.4 in all tissues. Ben Davis, one year old Nov. 29,'ll -19.4 No injury. Ben Davis, 80 inches of twigs; 7.5% killed in one year old Nov. 29,'ll -21 all tissues. Gano, 38 inches of twigs; 7.8% killed in two years old Nov. 29,'ll -21 ail tissues. Jonathan, 136 inches of twigs; 59.8% killed in one year old Dec. 5,'ll -20 all tissues. Gano, 113 inches of twigs; 15% killed in two years old Dec. 11, '11 -20.7 all tissues. Killing of Plant Tissue by Low Temperature 209 Tem- Variety Date pera- ture Results PLUM TWIGS Mature plum tree. . . . July 30,'12 - 5 Cambium, pith and cortex injured throughout. Sap wood slightly injured throughout. Marianna Oct. 9,'12 - 8 Very slight injury. Marianna, New shoots all killed only in one year old Nov. 8,'ll -11 spots at basal ends. Marianna, Injury in cortex and pith. Sap one year old Dec. 18,'12 -16.5 wood and cambium uninjured. Marianna, Injury confined to pith and cortex. one year old Dec. 19,'12 -18 slight injury to pith. Other tissues uninjured. Marianna Jan. 11, '13 -20 Slight injury to pith. Other tissues uninjured. Eustace^ often observed the greatest injury to peach trees to be just above the snow line. Following the winter of 1904 Green and Ballou^ reported that an Ohio fruit grower was able to save his trees by banking the bodies with manure, thus keeping the tempera- ture of the trunks near the ground from going as low as that to which the remainder of the tree was exposed. To test this point at the Missouri Experiment Station, sections of tissue from young and old trees were taken at different points along the trunk beginning near the ground and continuing upward into the branches. These were collected and frozen at intervals throughout the year beginning in September and ending the follow- ing July. The results are shown in the following tables: •New York (Geneva) Agr. Exp. Sta. Bui. 269, 1905. (BIbl. No. 38). 'Ohio Agr. Exp. Sta. Bui. 157, 1894. (Bibl. No. 48). 210 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 24. Showing Relative Hardiness of Different Tissue. Tem- Location Variety Date pera- ture of Tissue Results Ben Davis Sections taken at Current sea- apple tree intervals from son's twigs sections Sept. 4,' 12 -5 near the ground entirely free to the twigs from injury. In the older wood injury was slight and confined 1 to cambium and outside part of sap wood. The lowest part of trunk had cambium more severely injured than parts higher up. Section^ from eight ■ Sections taken at Injury most year old Improved intervals from severe in cam- Janet apple tree. . . Sept. 11,'12 -6 near the ground to the twigs. bium region. Younger stems less browned than older. Sap wood injured in 50% of stems, great- est injury on most rapidly thickening side of stem. Sections from five Sections taken at Older wood year old Improved intervals from from near Janet apple tree.. .. Sept. 12,'12 -6 near the ground to the twigs ground decid- edly more brown than younger wood of twigs. Cor- tex showed first injury and greatest. Cambium in- jury slight. Sap wood injury con- fined to older wood. Ben Davis apple, Crown (highest seven years old, point where roots All sections 2 in. in dia. at attach 6 in. below from lowest crown June 26,'13 - -5 surface) to highest in- Same, 2 in. in dia June 26,'13 -5 6 in. above crown (surface of soil) jured in cam- bium. All Killing of Plant Tissue by Low Temperature 211 Variety Same, l^i in. in dia. at crown Same, l?s in. in dia.. Same, IH in. in dia. Same, 1 }4 in- in dia. Same, 1 in. in dia. Same, Same, ^'y in. in dia. 3^ in. in dia. Same, J^ in. in dia. Same, J^ in. in dia. Same, }^ in. in dia June 26,'13 June 26,'13 June 26,'13 June 26,'13 June 26,'13 June 26,'13 June 26,'13 Ben Davis apple, five years old, 3 in. in dia. ... Same, 2 3^ in in. . dia Same, 2% in. in dia Same, 2 in. in dia Same, 1 J^ in. in dia Same, 1 in. in dia Same, 1 in. in dia... Same, % in. in dia. July 2,'13 July 2,'13 July 2,-13 July 2,'13 July 2,'13 July 2,'13 July 2, '13 July 2,'13 July 2,'13 July 2,'13 July 2, "13 -5 -5 -5 -5 -4 -5 -5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 -4.5 Location of Tissue 12 in. above crown. 18 in. above crown. 24 in. above crown. 36 in. above crown. 48 in. above crown. 60 in. above crown, 72 in. above crown. Crown 6 in. above crown. . 12 in. above crown. 18 in. above crown. 24 in. above crown. 36 in. above crown. 48 in. above crown, 60 in. above crown 72 in. above crown, 84 in. above crown. 96 in. above crown Results above two feet also in- jured in cor- tex. All sec- tions above four feet in- jured in cam- bium, cortex and sap wood. The crown was hardiest and the high- er up from the crown showed the more injury. Cambium slightly injured. Cambium slightly in- jured. Cambium in- jured. Cambium se- severely injured. Cambium se- verely injured cortex slight- ly injured. Cambium se- verely injured cortex slight- ly injured. Cambium and cortex severe- ly injured. Cambium and cortex severe- ly injured; sap wood slightly in- jured. Cambium and cortex severe- ly injured; sap wood slightly in- jured. Cambium and cortex severe- ly injured; sap wood slightly in- jured. Cambium, cor- tex and sap wood severely injured. 212 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Variety Early Harvest apple, 10 in. in dia.. Same, 7 in. in dia Same, 3 in. in dia Same, 1 in. in dia Same, ^ to J4 in. in dia. Jonathan apple, four years old . . . . , Same, four years old Same, four years old Same, four years old Same, four years old Same, four years old Same, four years old Same, four years old Same, four years old Jonathan apple, four years old, 2 J^ in. in dia Same, 2}^ in. in dia Same, 1% in. in dia Same, 1 in. in dia. . . Same, Ha in. in dia Same, .8 in. in dia.. , Same, .5 in. in dia.. . Same, J^ in. in dia. . Same, }4 in. in dia. . . Same, i\ in. in dia Sept. 28,'12 Sept. 28, '12 Sept. 28, '12 Sept. 28, '12 Sept. 28,'12 Oct. Oct. Oct. Oct. Oct. Oct. Oct. Oct. Oct. 19,'12 19,'12 19,'12 19, '12 19,'12 19,'12 19,'12 19,'12 19,'12 Oct. 23,'12 Oct. 23,'12 Oct. 23, '12 Oct. 23/12 Oct. 23, '12 -9 Oct. 23, '12 -9 Oct. 23, '12 -9 Oct. 23, '12 -9 Oct. 23, '12 -9 Oct. 23, '121 -6 -6 -6 -6 -6 -9 -9 -9 -9 Near ground 48 in. above ground 96 in. above ground 12 ft. above ground 15 ft. above ground (includes this year's growth) At ground. . 12 in. above 24 in. above 36 in. above 48 in. above 60 in. above 72 in. above 84 in. above 96 in. above ground ground ground ground ground ground ground ground Results At ground 12 in. above ground 12 in. above ground 36 in. above ground; 24 in. above ground; 48 in. above ground | 60 in. above ground 72 in. above ground 84 in. above ground 96 in. above ground (Last 4 new growth) Buds largely killed, bark and cambium taken near ground se- verely in- jured. The very young- est wood showed little or no injury. In the wood near the ground, the browning was uniformly dis- tributed. In wood higher up the brown was irregular and in spots. In the young wood, cam- bium appear- ed normal — only cortex injured. No injury No injury. No injury. No injury. No injury. No injury. No injury. No injury. No injury. All wood, ex- cept 34 in- in dia. or small- er, injured. The most s e ve rely browned sec- tion was 2 ft. aboveground, at the crotch of a rapidly growing branch. The section at the ground was less injured than the one 1 foot above. Cortex, cam- bium and sap wood injured. Killing of Plant Tissue by Low Temperature 213 Variety Jonathan apple, four years old, Jonathan apple, four years old Jonathan apple, four years old Jonathan'apple, four years old Jonathan apple, five years old. (Thickest part of trunk, 3 in. in diameter) Same Same Same Jonathan apple, five years old. (Thickest part of trunk, 3 in. in dia.) Same Same Jonathan apple, five yearsold. (Thickest part of trunk, 3 in. in dia.) Same Nov. 14/12 Nov. 14,'12 Nov. 14,'12 Nov.44,'12 Jan. 15, '13 Jan. 15, '13 Jan. 15/13 Jan. 15/13 Jan. 15, '13 Jan. 15, '13 Jan. 15, '13 Jan. Jan. 20,'13 20,'13 Same Jan. 20,'13 Jonathan apple, five yearsold. (Thickest part of trunk, 3 in. in diamcterj ijan. 20, '13 -12 -12 -12 -12 -20 -20 -20 -20 -20 -20 -20 ■15, ■15, -15.5 -15.5 Location of Tissue At ground. 6 in. above ground 12 in.' above ground 18 in. above ground At crown , 3 in. above crown At level of ground. 3 in. above ground 6 in. above ground . 12 in. above ground 18 in. above ground At crown 3. in. above crown At ground. 3 in. above ground Results Most severely injured. Cam- bium cortex and sap wood injured. Cambium in- jured. Cor- tex slightly injured. Cambium in- jured. Cortex slightly in- jured. Cambium in- jured. Cor- tex slightly injured. All tissue in- jured severe- ly. All tissue in- jured. Slight injury in bark; none in cambium; sap wood in- jured. Injury in same tissue as above, but not quite so severe. Same as above. Same as above. No injury in cortex or cam- bium; sap and heart wood slightly in- jured. All tissue in- jured severe- All tissue in- jured slightly less than above. Injury in cor- tex region. Slight injury in cortex region. 214 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Variety Location of Tissue Results Same Jan. 20/13 Same Jonathan apple, five years old. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Same. Jan. 20,'13 Mar. 25/13 Mar. 25/13 Mar. 25/13 Mar. 25/13 Mar. 25/13 Mar. 25/13 Mar. 25/13 Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. 25/13 25/13 25/13 25/13 25/13 25/13 25/13 25/13 -15.5 6 in. above ground. -15.5 12 in. above ground -12.5 At crown. -20 -12.5 -20 -12.5 Mar. 25/13 -20 -12.5 -20 -12.5 -20 -12.5 -20 -12.5 -20 -12.5 -20 At crown. 3 in. above crown. 3 in. above crown. 6 in. above crown., 6 in. above crown. 3 in. above ground. 3 in. above ground. 6 in. above ground. 6 in. above ground. 9 in. above ground. 9 in. above ground. 12 in. above ground 12 in. above ground 18 in. above ground 18 in. above ground Very slight in- jury in cor- tex region. No injury. Severe injury in cortex, cambium and sap wood. Very severe in- jury in cor- tex, cambium, and sap wood. Injury in cor- tex, cambium and sap wood less severe than at crown Less severe in- jury than at crown. Very slight in- jury in cortex and cambium. Less severe in- jury than above in same regions. No injury. Very slight in- jury in cor- tex. No injury. No injury. No injury. No injury. No injury. No injury. No injury. No injury. It will be seen that the part of the trunk that most slowly de- velops hardiness on approach of winter is that near the surface of the ground and near the junction of rapidly growing limbs. All the tissue at the lower part of the tree is more tender in early winter than is the upper portion. Of course this might not be true on other seasons. The autumn of 1912, however, was a normal one, the wood apparently going into winter in a well ripened condition. In June and early July the wood in the upper portion of the tree is most tender. It is also interesting to note that in June and July when the Killing of Plant Tissue by Low Temperature 215 tissue is generally most tender, the tissue near the base of the tree is most hardy. Selby^ observed that the great tenderness in the early part of the winter is probably due to a greater moisture content. He ob- served that the cambium in winter during the time when plants are very hardy, is in a much dried out condition and in normal seasons has to some extent reached this condition by the time of the early freezes, but in seasons like that one preceding the winter of 1903-4 it is still in a somewhat succulent condition when the early freeze comes. It is well known that seeds in a dry condition will withstand very much lower temperature than when they have absorbed moist- ure. Thus Schaffnit^ reduced the germination percentage of wheat from 100 to 40 by soaking it in water for eight hours at room tem- perature. In twigs it is probable that a dry condition is essential to the hardiness of the cambium. Shutt^ and also Allen^ seem to find a relation between moisture content and hardiness of apple twigs. However, it does not seem that the increase in hardiness of other tissue than cambium, at least of cortex, during early winter can be explained by a decreasing moisture content. During the winter of 1912-13, beginning November, twigs of apple, peach, plum and cherry were scraped, the cortex ground, weighed carefully, evaporated to dryness in a water bath (to which later glycerine was added to raise the boiling point in the water jacket and thus raise the temperature), and weighed at intervals of two to three days until a constant weight was reached. Samples were taken again in January and again in May. No samples were taken when the tissue was frozen, since then the percentage of mois- ture would be smaller. The evaporated moisture could not be re- placed from below. The following table gives the results: lOhio Agr. Exp.Sta. Bui. 192, 1908. (Bibl. No. 101). 2Mitt. Kaiser Wilhelm Inst. Laxidw. zu Bromberg. VoL 3, No. 2, pp. 93-113, 1910. (Bibl No. 98). 'Procs. and Trans. Roy. Soc. Canada, ser. 2, Vol. 9. pp. 149-163. (Bibl. No. 104.) 4Master'8 Thesis, Iowa Agr. Exp. 8ta. (Bibl. No. 2). 2i6 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 25. Showing the Moisture Content of the Cortex in November, January and May. Material Date Weight Fresh Sample. Grams Weight Dry Sample. Grams Per- cent- age Water Average Percent- age Water Jonathan apple, entire twig. Jonathan apple, entire twig. Elberta peach, entire twig.... Elberta peach, entire twig.... Jonathan apple, buds, bark and cambium Jonathan apple, buds bark and cambium Elberta peach, buds, bark and cambium Elberta peach, buds, bark and cambium Jonathan apple, buds bark and cambium Jonathan apple, buds, bark and cambium Elberta peach, buds, bark and cambium Elberta peach, buds, bark and cambium Jonathan apple, entire twig. Jonathan apple, entire twig. Elberta peach, entire twig.... Elberta peach, entire twig.... Nov. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Jan. Jan. Jan. Jan. May May May May 15, '12 15, '12. 15, '12 15, '12 27,'12 27, '12 27,'12 27,'12 11, '13 11, '13 11, '13 11, '13 3, '13 3,'13 3,'13 3, '13 36.45 35.82 26.10 28.84 30.05 29.95 30.15 30.00 25.00 25.00 25.00 25.00 20.00 20.00 20.00 20.00 17.15 16.75 12.35 13.85 13.60 13.40 13.25 13.05 12.25 12.10 10.65 11.10 8.96 8.80 9.15 9.00 52.95 53.52 52.61 51.98 54.70 55.60 56.00 56.50 51.00 51.60 57.40 55.60 55.50 56.80 54.25 55.00 53.24 52.33 55.2 56.3 51.3 56.5 56.15 54.63 It will be seen that there Is no constant difference in moisture content of the twig cortex from November to May. The difference in the hardiness of the cortex can not be accounted for by the differ- ence in the moisture content, but must be accounted for in some other way. The suggestion is sometimes made that a greater sap density of the twig and other tissue during winter might account for this greater hardiness. It was not possible with our apparatus, at least, to secure sap from the sap wood. The cortex, however, shows this increase in hardiness to a slightly greater extent than does the sap wood. The following table shows the cortex sap density of apple and peach twigs at various seasons of the year, through a period of three years: Killing of Plant Tissue by Low Temperature 217 Table 26. Showing Sap Density of Apple and Peach Twigs Throughout the Year as Measured by the Freezing Point Depression. Date Elberta peach Jonathan apple Gano apple Depression Depression Depression January February March April 1.902 1.841 1.758 1.765 1.055 1.263 1.252 1.652 1.748 1.743 1.765 1.694* 2.054 2.170 No data 1.055 0.915 1.415 1.500 1.623 1.605 1.892 1.924 2.016 1.630 No data 1.616 .949 May June 1.085 1.289 July August September October November December 1.469 1.570 1.690 1.728 1.665 No data *Only one depression taken in December. It will be seen that while the sap density of the cortex of winter twigs is much greater than that of early summer twigs, yet it is not appreciably greater than that of twigs in late September and October, when the tissue is still considerably more tender than in December and January. Some may suspect that the low sap density of the early summer twigs may be due to their young and somewhat succu- lent condition. It may be said, however, that the density of the cortex of these young twigs is generally greater than that of any other tree tissue except the leaves. (Data to be published in another bulletin ) . 1 1 would seem certain then that while a part of the increased hardiness of tree tissue in winter may possibly be accounted for by the greater sap density, not all of it can; certainly not the greater hardiness of December tissue over that of October. I n the case of plants killing at as low temperature as those at which winter twigs kill, it seems possible that the sap solute, if it remains in solution, may tend to keep a small amount of water unfrozen and thus protect the protoplasm to some extent. If this should be true, the eutectic point of the sap solute would play a very import- ant part in determining the amount iA killing. Some efTorts were made to determine whether or not there may be changes in the sap solute as winter comes on that give it a lower eutectic point. Just at the time of leaf fall or slightly before, twigs had the cortex scraped 2i8 Missouri Agr. Exp. Sta. Research Bulletin No. 8 from them and the sap expressed in large enough quantities that it could be evaporated down to one-fourth to one-eighth of its volume and leave enough for freezing point determinations with a Beckmann thermometer. The evaporating was done in a dry oven where the temperature never was above 50° C. The evaporating was done in broad, shallow dishes and was generally accomplished in one day. There was apparently no fermentation. In all cases sap taken in October or early November would be thick and gummy long before it could be concentrated to one-sixth of its original volume. It was noticed that it was very difficult indeed to filter the sap from the twigs in autumn or very early winter, while sap taken in December or Jan- uary or later, filtered much more easily and could be concentrated to one-sixth to one-eight of its volume. In this case it would stay in solution at temperatures as low as could be secured with salt and ice; that is, temperatures low enough to kill many peach buds, indi- cating that there is certainly a probability that at least a part of the sap solute remains in solution at a temperature low enough to hold water unfrozen to protect the protoplasm. Of course in the earlier season the solidifying of the liquid may be due to colloidal substances in large quantities, and it is entirely possible that the solute had just as low an eutectic point. It was not possible to determine the eutectic point by keeping temperature records since no apparatus was availa- ble other than the Beckmann thermometer which could not be used without changing the setting several times, for such low temperature. It would seem highly probable that, except in the case of cam- bium, the additional hardiness acquired by the different tissues of the tree as they pass into winter, is a change in the protoplasm such that it can withstand the great loss of water rather than a change in the percentage of moisture or in sap density. It is also possible that changes in the sap solute that lower its eutectic point may occur and that these may increase the resistance to cold by holding water unfrozen to protect the protoplasm from too complete desiccation at lower temperatures. Rate of Growth and Hardiness. At the time of most rapid growth of deciduous plants in early summer they are generally most tender. Whether this is because they are furthest from the con- dition of maturity they acquire in autumn and early winter, or be- cause of the very low sap density at this time, it is not easy to say. In some cases the young tissue is most hardy. Thus Goeppert found young leaves more hardy than older ones. Apelt^ found the >Cohn. Beitrage z. Biol. d. Pfl. VoL's. p. 215. (Bibl. No. 3). Killing of Plant Tissue by Low Temperature 219 young outer ends of potato shoots to withstand lower temperatures than would the older basal portion. Rein^ found young onion leaves more hardy than older ones. Winkler- found young one-year-old needles of evergreens more hardy than older ones. Shumacher^ found young yeast cells more hardy than older ones. Fisher^ found newly formed colloids to regain their normal characteristics after being exposed to a low temperature that would irreversibly change older colloids. On the other hand Bartetzko° found that young cultures of Aspergillus niger would not withstand as low temperatures as would older ones. It seemed worth while to make some freezings to determine whether or not plant tissues making rapid growth are generally frozen to death at higher temperatures than are tissues growing more slowly. Leaves of various plants were used, leaves that were certainly full grown — in case of those from fruit trees — and leaves that had apparently ceased growing, in case of plants like lettuce, cabbage, kale, etc., were frozen at the same time with young rapidly growing leaves from near the growing tips of the stem. The following table gives the results: iZcits. f. Naturw. Vol. 80 (1908) p. 1. (Blbl. No. 92). 2Jahrb. f. Wiss. Bot. Vol. 52, 1913, pp. 467-506. (Bibl. No. 121). 'Sitzungsber der Math. Phys. Klasse d. Wiener Akad. d. Wiss. Alt. 1, 1874. (Bibl. No. 103). - will exj-lain the greater 222 Missouri Agr. Exp. Sta. Research Bulletin No. 8 hardiness of the older leaves. In fact the following table, giving results where twigs with young leaves were placed in cane sugar and glycerine solutions, indicates that increasing the sap density of the young leaves to that of the old leaves will increase their hardiness to nearly that of the old leaves. Table 28. Showing Relative Hardiness of Young and Old Apple Leaves and of Young Leaves that had Absorbed Glycerine and Cane Sugar. Age of Leaf Old.... Young, Young. Young. Old Old.... Young. Young. Young. Young. Old.... Old.... Young. Young. Young. Young. Treatment Water 30 hrs Water 30 hrs... . Cane sugar 3 hrs (10%)......... 10 %Glycerine 3 hrs Fresh Water Fresh Water Cane Sugar Glycerine Fresh Water Fresh Water Cane sugar Glycerine Date July 8 July 8 July 8 July July July July July July July July July July July July July 12 12 12 12 12 12 12 12 12 12 12 12 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 '13 Tem- pera- ture -4 -4 -4 -4 -4 -4 -4 -4 -4 -7 -7 -7 -7 -7 -7 Num- Per- Per- ber cent- cent- of age age Leaves all Surface Killed Killed 20 0.0 55.00 26 50.0 82.70 18 11.1 34.90 26 0.0 5.70 20 0.0 17.50 20 0.0 28.70 19 26.3 48.70 21 71.4 91.70 22 27.2 48.90 22 22.7 32.90 20 35.0 46.20 20 80.0 85.00 22 77.7 94.30 22 100.0 100.00 19 57.9 89 . 40 27 44.1 69.40 De- pres- sion 1.480 1.340 1.818 3.400 1^546 i '. 230 1.792 3.400 i '. 540 i '. 230 1.792 3.400 In case of lettuce, however, the depression for old leaves was 0.575, and for young leaves 0.520, and the young leaves are the most resistant to the low temperatures. It is possible that the waxy or oily covering on the surface of the young lettuce leaves increased their resistance to low temperatures. In our experience leaves and fruits dipped in glycerine or paraffine have been uniformly more resistant than have tissues not so treated. Killing of Plant Tissue by Low Temperature 223 EFFECT UPON HARDINESS OF PREVIOUS EXPOSURE TO TEMPERATURE SLIGHTLY ABOVE THE KILLING TEMPERATURE. Closely related to the questions of the relation of maturity to hardiness, and the relation of the rate of growth to hardiness, is the relation of exposure to low temperature, above that at which the plants may kill, to hardiness. In fact these problems are so intertwined that it is difhcult, if not impossible, to separate them. Thus in case of the greater hardiness of roots kept in cold storage as compared with those kept in warmer places, unquestionably maturity plays a large part but it is not impossible that exposure to cold also had its effect. However, by referring to Table 14 it will be seen that there was little difference among the killing of roots kept in cold storage at a temperature of 31° F., those kept frozen up in the soil, and those kept at a higher temperature in our basement storage room. The relation of exposure to cold to hardiness of winter buds and wood may also be confused with the rate of temperature fall. This problem will be discussed for peach buds in a later part of this paper. In case of some succulent plants, however, the temperature at which they grew must exert an influence on their hardiness. Thus when cabbage, kale and lettuce were grown out of doors in late autumn or early winter, their hardiness was increased over those grown in the greenhouse more than can be explained by the increased sap density. At least their hardiness was increased more than the same increase in sap density brought about by any other means would in- crease it. When these plants were grown out of doors in early spring or late autumn, it required a much lower temperature to kill them than was required in June or July. On the other hand, plants like tomatoes or cowpeas are influenced in hardiness but slightly by the temperature at which they grow. Goeppert^ found little increase in hardiness due to continuous exposure to low temperature with tender tropical plants, but there was such adaptation with more resistant plants. Apelt- found that potatoes kept at a temperature of 22.5° C. four to seven weeks were killed at -2.14° C. while potatoes kept at 0° C. for the same length of time killed at -3.08° C. Rein' found that a rather large list of very tender plants kept at a temperature of 8° (' were not apprccia- 'Uebor die WlirmeentwIckcIuriR In dom ITlanzoii. otc, book. 18.10. (Ulhl. No. J J). »f 'ohn's ntltraj?o z. Hlol. d. ITl. Vol. 0, 1007, p. 215. (BIbl. No. 3). •Zolts. f. Naturw. Vol. HO. 1908. p. 1. (lUI)l. No. 02^. 224 Missouri Agr. Exp. Sta. Research Bulletin No. 8 bly hardier than when kept at a temperature of 20° C, while more resistant plants were considerably more hardy when kept at a low temperature. Fisher^ found that it required a lower temperature to change the nature of colloids like starch paste that had been kept at low temperatures than to change the nature of colloids kept at a high temperature. Relative Hardiness of Different Tissues at Different Seasons of the Year. When trees are in a rapidly growing condition, appar- ently the most tender part of the wood tissue is the cambium and the young cortex, and sap wood cells. However, in winter after the wood has reached its greatest maturity, this is not the case. In fact when severe cold comes, the first tissue to kill seems to be the pith in the case of young twigs, and there will be browning in the sap wood and part of the cortex. In case of the cortex the browning is generally worse in the outer or older cells. This was observed by Eustace^ on peach trees following the winter of 1903-04. We have often observed the same in artificial freezings we have made, as well as on peach trees badly injured in winter. Peach trees so injured that the sap wood seemed practically all browned have, under favora- ble conditions, had the cambium form new layers of sap wood sur- rounding this wood. This injured wood soon becomes entirely dead and the tree depends on the new sap wood formed for conductive tissue. We have also observed dead areas of bark following the win- ter of 1911-12 when underneath there was healthy new bark and healthy cambium. In these cases, at least, the cortex was more tender than the cambium. The fruit buds of the peach in late summer during the grow- ing season are generally about as hardy as the cortex and cambium, or sap wood of the twigs, though perhaps slightly less hardy than the same tissues in older wood; while in winter under normal conditions, at least with peaches, the fruit buds are generally somewhat less hardy than any of the wood tissue, with the possible exception of the pith cells. However, in the case of a cold wave that comes on very grad- ually, say during a period of two or three weeks with a very cold night at the end, some of the buds may survive a temperature low enough to kill the sap wood badly. Thus following the winter of 1904-5, when the temperature at Columbia went to -25° C, after several weeks of very cold weather, nearly all of the peach trees had a few live buds left while the wood was very badly damaged ; and in the case of peaches in New York following the winter of 1903-04, iBeitr. BioL der Pfl. Vol. 10, pp. 133-234, 1911. (Bibl. No. 40). 2New York (Geneva) Agr. Exp. Sta. Bui. 269, 1905. (Bibl. No. 38). Killing of Plant Tissue by Low Temperature 225 Eustace observed that the trees may be badly injured and yet enough fruit buds left for a full crop of fruit. This last, however, was the condition when the tissue of the tree had not reached the proper maturity before the cold period came. The fruit buds seem, some- times at least, to reach about their maximum condition of maturity more quickly than wood tissues, especially that near the base of the tree. Tables 23 and 24 give some information as to the relative hardiness of other tissues, and the following table gives the result of some additional freezings where buds were frozen in comparison with other tissues: Table 29. Showing Relative Hardiness of Different Tissue, Including Buds, at Different Seasons of the Year. Variety Elberta peach twigs.. . July 15, '13 -6 Elberta peach twigs.. . EJberta peach twigs, buds and sections of wood Champion peach twigs 10 in. long July 29,'12> -5 July 16,'13 July 28,'13 Belle of Georgia peach tree, five years old; sections of trunk and limbs Aug. 29,'12 Elberta peach tree, buds and twigs and sections of trunk and limbs ^ept. 14, '12 Elberta peach twigs [ j (one year oldj Oct. 5, '12 -5 -5 -5 -5 Elberta peach twigs.. . Nov. l.'ll, -9 I 1 Elberta peach twigs.. . Dec. 7, '12 -16 Results 23 buds, 78.3% killed. 10 twigs 7 injured in cambium. 3 in cam- bium and cortex. 50 buds, 100% killed. 20 twigs, all injured in cambium and cortex. 37 buds, 100% killed. Roots, dead in cambium and cortex. Wood just above ground, severely in- jured in cortex and cambium. 4 feet from ground, same as above. This years' growth, same as above. Sap wood and pith of youngest portions dead. 95% of leaf surface dead. Cortex injured 6 in. back of terminal. Cambium, pith and sap wood in- jured to 8 in. back of terminal. Buds, 100% killed. Young sap wood and cambium killed in all sections. Least injury to bark was in three year old wood. Bark, cambium and sap wood killed in twigs. Buds 100% injured. Bark cam- bium and outer portion of sap wood injured in all cases. Buds 90. 91% dead. Cambium and cortex injured cxccjit at ba.-^e of one twig. Sap wood injured only in terminal part tuig. 104 buds; 63' t dead. .>:.^ twigs; 60.6% killed in cambium, other tissue not injured. Injury confineith rigion. Buds prop- er, uninjured. 226 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Variety Elberta peach twigs. Rice's Seedling peach twigs . . Late Duchess apple twigs Jonathan apple twigs. Jonathan apple twigs. Young apple twigs, variety not given . . Young apple twigs, variety not given. Jonathan apple twigs and wood from three-year old Jon- athan tree Jonathan apple stems from }s in. to 1 in. in diameter Dyehouse cherry young and old wood Cherry twigs Date Tem- pera- ture Mar. 23,'13 July 15,'13 July 16,'13 July 16,'13 July 27, '12 July 28,'13 -18 Mar. 23, '13! -18 -6 -5 -5 July 27,'12; -5 -5 Aug. 15, "12 -5 July 28,'13 -5 Aug. 13, '12' -6 Results 100 buds, 88% killed. Injury to wood confined in all cases to sap wood and pith. 138 buds, 44.2% killed. Injury to wood confined in all cases to sap wood and pith. Injury less than to Elberta. 20 buds, 70% killed. 4 twigs, no injury. 29 buds, 86.2% killed. 9 twigs, no injury. 24 buds, 58.3% killed. 11 twigs, no injury. 12 buds, 100% killed. Cortex browned in places, especially around the buds. Cambium dead in region of annual ring and ter- minal of year's growth. Pith dead in region of annual ring. Leaves practically all injured. Bark killed about 7 in. back from ter- minal. Cambium killed about 7 in. back from terminal. Sap wood killed 5 to 6 in. back from termin- al. Pith killed 4 to 5 in. back from terminal. 44 buds, 70% dead. Roots, cam- bium and cortex injured through- out. Wood, sections just above ground and 3 feet above dead in cambium and cortex. This year's growth injured in cambium and cortex in older parts, and all tis- sue dead at tips. Cortex and cambium injured slight- ly in all samples. Sap wood and pith not injured. No marked difference between the different diameters. 40 buds, 100% killed. 25 out of 36 buds killed. Some buds at base of new twigs and on spurs on old wood alive. Leaves all partially injured; only 25% entirely killed. Youngest wood injured worst. Slight injury in cambium and cortex. No injury to sap wood ol two year twigs or older. By referring to this table and to Tables 22, 23 and 24, it will be seen that the fruit buds are in all seasons apparently less hardy than any other tissue, except the pith and the tissue at the base of the buds, and in early winter the wood at the surface of the soil. Killing of Plant Tissue by Low Temperature 22'J The most tender tissue of the tree is in the roots. Thus in case of the tree frozen July 2, 1913 (Table 24) when the tissue above ground is most tender, the roots were injured in the cambium at -3.5° C, and severely injured in cambium, cortex and sap wood at -4.5° C. Goeppert' found that the roots of hardy plants kill at a tem- perature of from -10° to -15°. A study of the killing temperatures of roots of various fruit trees has been made at this station. In the summer there is not so large a difference between the killing tempera- ture of roots and other tissue. However, the roots do not seem to develop as great maturity as the tissue above ground. The following table shows the results with freezing the roots of trees in summer and winter seasons: Table 30. Showing Killing Temperature of Apple, Peach, Plum and Pear Roots. Kinds of Roots Two-year Ben Davis apple roots Seedling two-year old apple stock roots... . Two-year Ben Davis apple roots Seedling apple stock roots Seedling apple stock roots Two-year Ben Davis apple roots Two-year Ben Davis apple roots Seedling apple stock roots from basement since December 20th Seedling apple stock rootsfrom basement since December 20th Apple stock buried 5 in. below surface outside since Jan. 8th June 23, '13 June 24,'13 June 25, '13 Aug. 20,'12 Aug. 22, '12 Dec. 7, '12 Dec. 7, '12 Jan. 13, '13 Jan. 17, '13 -5 -5 -3 -6 -5 -6.5 -4 -10 Results Cambium only injured in larger roots. Cortex also injured in roots Y\ in. in diameter. In 2 out of 4 cases, crown uninjured. In other 2 cases there was slight injury to cambium at crown. No injury at all No injury in roots above }/i in. in diameter. Wood just above crown was injured in cambium. Cortex and cambium region all dead. Cortex and cambium injured, but less severely than above. Cortex, cambium and wood in- jured. One small root injured. Injury confined to ( ortt'x region. Injury slight and confined to cortex region. One root browned and the otlur not injured Mar. 8, '13 Ueberdlo Wiirniui-ntwIcktlunK In (It-m ITIaiizon. etc.. book. is;<(). (lUbl. No. 14). 228 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Kinds of Roots Results Apple stock buried 5 in. below surface outside since Jan. 8th Two year Ben Davis apple roots Mar. 24,'13 -10 Mar. 8, '13 -14 Seedling peach roots... Seedling peach roots... Elberta peach roots... Oct. 14/11 Elberta peach roots... Oct. 18, '11 Elberta peach roots. . . Seedling peach roots.. . Dec. 7, '12 Seedling peach roots... Dec. 7, '12 Marianna plum root... June 25, '13 June 25, '13 -3 Tune 25, '13 -9.5 -5.5 Oct. 19,'ll' -4.5 -6.5 -4 -5 Marianna plum roots. June 26, '13, -3 Marianna plum roots.. Marianna plum roots.. Kieffer pear roots Kieffer pear roots Kieffer pear roots Kieffer pear roots Dec. 7, '12 Dec. 7. '12 Oct. Oct. 10,'ll 14. '11 Oct. 19,'ll Mar. 27, '13 -4 -6.5 -5.5 -9.5 -4.5 -10 All roots browned No injury in first 2 inches; cam- bium injury throughout remain- der. Cortex showed next greatest injury, and in smaller roots sap wood also injured. Entire system injured in cambium and sap wood. Injury slightly less in crown. All roots very severely injured in cambium and cortex and portion of sap wood. Crown as severely in- jured as terminal roots. Stem just above ground injured in cortex and cambium. 5 roots; 100% dead. 13 inches of root length; 100% dead. 301^ inches of root length; 100% injured, smaller roots injured worst. Cortex, cambium and wood injured. Slight injury. Largest root injured severely in cortex. No apparent difference between crown and remainder of root system. Crown 1 in. in diameter shows slight injury in cambium; 3-10 in. down cortex injured also; sap wood also injured towards tips. Slight injury in cortex. Cortex, cambium and sap wood in- jured. 141^ inches; 65.5% dead. 5 roots; 100% dead. Twigs at same temperature, cambium only killed. 33}^ inches; 100% injured. Kill- ing more severe in younger roots some distance from the trunk than in larger ones. Injury grading from none in crown to injury of all tissues where di- ameter of root was not greater than 3-10 inch. It will be seen that the killing temperature of the roots varies from about -3° C. in summer when most tender to about -12° C. in late winter with rather rapid freezing. The roots are certainly as hardy in March as in January. Thus they are later in becoming tender in spring than are twigs. They are still very tender in autumn Killing of Plant Tissue by Low Temperature 229 when tissue above ground has begun to increase rapidly in hardiness. This may be because the soil is still too cold for growth well up into March, generally, and continues warm late in autumn. The following table gives the result of freezing young apple roots (stock) kept in cold storage at a temperature of 31° to 32° F., in the earth frozen up where the temperature varied from the freez- ing point to 39° F., and others kept in greenhouse conditions whereby they started into growth, and others kept in basement storage room at a temperature varying from 4° C. to 15° C. from January 8, 1913 to February 16, 1913, the date of freezing. Table 31. Showing Relative Resistance to Low Tempera- tures OF Apple Roots Kept in Dormant Condition AS Compared with Those in a Grow- ing Condition. Kind of Root Tem- pera- ture Number of Roots Greenhouse Frozen soil Basement Storage Room Cold Stor- age Crown cut diameter 1^0 in Second cut diameter -6 2 No injury. . No injury No injury No injury Min Third cut diameter iin -6 -6 2 2 No injury. . A. Cortex and cam- bium brown. B. Cambium No iniur\- No injury No injury Fourth cut diameter ys in Crown cut diameter 1^0 in -6 -7.5 2 2 brown All tissues injured .... A. Cortex and cambium No injury No injury No injury No injury No injury No injury Second cut diameter Kin -7.5 2 brown. B. Cambium brown A. Cortex and cambium br^in -7.5 2 All tissues injured. . . . Cambium brown.. . B. No injury. Cambium brown. B. No injury. No injury Crown cut diameter u) in -9 2 All tissues injured No injury No injury ACambium injured. B. No injury. Second cut diameter Min -9 2 All injured.. No injury ACambium injured. BCambium and cortex injured. ACambium injured. BCambium and cortex injured. Third cut diameter iin -9 2 All injured.. Cambium injured. All injured All injured Fourth cut diameter Vz in -9 2 All injured. Cambium and cortex injured. All injured All injured Stored Dec. 20th. The second, third and fourth cuts are sections of the stock of equal length below the crown. Killing of Plant Tissue by Low Temperature 231 Table 31a. Showing Relative Resistance to Low Tempera- tures OF Apple Roots Kept in Dormant Condition as Compared with Those in a Grow- ing Condition. Kind of Root Crown cut diameter i% to >4 in Lower cut diameter J^ to 1^6 in _ Crown cut diameter fe to >4 in ,••.•■■■.• Lower cut 14 to i\ in. in diameter Crown cut diameter i\ to Min Lower cut diameter 34 to 3 10 in. Crown cut diameter 1^6 to k in Lower cut diameter 34 to A in Crown cut diameter i^g to Kin Lower cut diameter 34 to A in Tem- pera- ture Num- ber Roots -4 2 -4 2 -6 2 -6 2 -8 2 -8 2 -10 2 -10 2 -14 2 -14 2 Outside No injury. No injury. No injury. No injury. No injury. No injury. No injury. One root brown; other not injured. All browned. . . All browned. . . Greenhouse No injury. . . . No injury. . . . No injury. . . . No injury. . . . One root very brown; other slightly. Both roots brown in cor- tex and cam- bium. Stored January 8, 1913. 232 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 31b. Showing Relative Resistance to Low Tempera- tures OF Apple Roots Kept in Dormant Condition AS Compared with Those in a Grow- ing Condition. Largest Tem- Where Stored Diameter Length pera- ture. Results. Outside .3 in 9+ in... -9 No injury in first 6 in. from top. Cambium and cortex slightly injured in remainder. Outside .3 in 9+ in... -9 Cambium injured slightly in last 7 inches. Outside .3 in 8+ in... -9 Cambium injured slightly in last 7 in; cortex also injured in last 5 in; sap wood injured only at terminal. Outside .3 in 8+ in... -9 Cambium injured slightly in last 7 in; cortex injured in last 5 in; sap wood injured at terminal. a) Cold Storage .3 in 7 in -9 No injury in first 5 in; slight in- jury in cortex and cambium in remainder. Cold storage.. . .25 in 9 in -9 No injury in first 6 inches. Cam- bium injured in remainder. Cold Storage.. .35 in 8 in -9 Cambium injury throughout. Cold storage.. . .3 in 7 in -9 Slight injury in cambium through- out. b)Greenhouse. . .3 in 12 in -9 Slight injury in cambium throughout. Greenhouse. . . . .25 in 10 in -9 Very slight injury in cambium in first 5 inches. Cambium and cortex injury throughout the re- mainder. a) In cold storage since January 12, 1913. b) In greenhouse since January 12, 1913. Table 31c. Showing Relative Resistance to Low Tempera- tures OF Apple Roots Kept in Dormant Condition as Compared with Those in a Grow- ing Condition. Tem- Where Stored pera- ture Results Greenhouse since March 29, 1913 -7.5 Injured in cortex, cambium and sap wood throughout. Cold storage since April 1, 1913. . -7.5 Cambium injured throughout entire root. Cortex showed injury only in the terminal 3 to 5 inches. No injury in sap wood. Basement store room since De- cember, 1912 -7.5 Slight injury. Killing of Plant Tissue by Low Temperature 233 It will be seen that there is little difference between the killing temperature of those in storage and those in a storage room at 10 to 37° F. higher temperature and those kept out in the soil. However, those that were in a growing condition were less hardy but with nothing like the difference that would be observed in the case of twigs kept under similar conditions. The reason the roots kept in the basement store room were more hardy than we should expect, is possibly because of their being kept in a somewhat dry condition. In the case of young peach roots, those kept in cold storage showed a greater hardiness than those kept in the soil outside. Some growth may have taken place in the roots kept out in the soil. The following table gives results of the freezing of peach roots: Table 32. Showing Relative Resistance to Low Tempera- ture OF Year-Old Seedling Peach Roots Growing and in a Thoroughly Dormant Condition. Date of Freezing, March 22, 1913. Location Tem- Where Stored of Root Diameter Length pera- ture Results Outside since January 12, 1913 Top even with surface . 65 in 14 in.... -9 No injury in first 2 in. be- low the surface of the soil. Cor- tex, cambium injured in next 4 in. All tis- Outside since sues injured January 12, in remainder. 1913 2 inches below low surface.... .35 in 12 in.... -9 Cambium and cortex injured throughout. Sap wood Outside since slightly injured January 12, in last 3 in. 1913 4 inches be- low surface.... .3 in 12 in.... -9 Cortex and cam- bium injured s e \' e r e 1 y throughout. Sap wood and pith in last 8 inches. 234 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Where Stored Outside since January 12, 1913 Cold storage. . . since January 12, 1913 Cold Storage since January 12, 1913 Cold storage. . . since January 12, 1913 Cold storage. . . since January 12, 1913.... Greenhouse since January 12. 1913.... Greenhouse since January 12, 1913..., Greenhouse since January 12, 1913 Location of Root 6 inches be- low surface.. Top even with surface 2 inches below surface 4 inches be- low surface. 6 inches be- low surface.. Top even with surface 3 inches below surface Diameter .3 in 3 inches below surface ,7 in. ,3 in. .35 in. .3 in. .7i in. Length 10 in..., 11 in. 9 in. 8 in. 6 in. 11 in.. ,55 in. ,5 in. Tem- pera- ture -9 -9 -9 -9 15 in. -9 Results Cambium and cortex injured throughout. Sap wood and pith in last 6 inches. Very slight in- jury of cam- bium through- out. Cortex slightly in last 2 inches. Very slight in- jury of cam- bium through- out. Cortex in- jured in last 3 in. Cambium and cortex injured throughout. Pith injury last 3 inches Cambium and cortex slightly injured throughout. (Injury in all cases very much less than in those kept outside). Very severe in- jury through- out in cam- bium and cor- tex. Pith and wood less se- verely injured. Very severe in- jury in all tissues Very severe in- jury in all tis- sues. Killing of Plant Tissue by Low Temperature 235 The following table gives the results of freezing Marianna plum roots : Table 33. Showing Relative Resistance to Low Tempera- ture OF Year-Old Seedling Plum Roots Grow- ing and in a Thoroughly Dormant Condition. Date of Freezing, March 22, 1913. Where Stored Location of Root Diameter Length Tem- pera- ture Results Outside Top even with surface .8 in 16 in.... -9 Very slight in- jury of cam- bium in first 10 in. Corte.x and cambium in- jured in re- Outside 3.5 inches be- low surface.... .3 in 12 in.... -9 mainder. Cambium and Outside 4 inches below surface .3 in 14 in.... -9 cortex injury throughout. Cambium and Outside 4 inches below surface . 25 in 6 in... . -9 cortex injury throughout. Cambium and cortex injury Cold storage. . . Top even with surface .8 in. .. 9 in.... -9 throughout. Pith injured slightly in last 4 inches. Very slight in- jury in cambi- um in first 7 in. Cortex and cambium in- Cold Storage. . . 7 inches below surface .35 in 8 in.... -9 jury in remain- der. Slight injury of cambium and cortex through- Cold storage. . . 7 inches below surface .35 in 7 in.... -9 out. Slight injury of cambium and cortex through- Greenhouse.. . . Top even with surface .7 in 7 in.... -9 out. Very severe in- jury of cortex and cambium t li ro u n ho u t. Pitli and sap wood injurrd. 236 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Location Tem- Where stored of Root Diameter Length pera- ture Results Greenhouse. . . . 7 inches below surface .45 in 10 in.... -9 Very severe in- jury of cortex and cambium throughout. Pith and sap wood injury. Greenhouse. . . . 7 inches below surface .35 in 3 in.... -9 Very severe in- jury of all tis- sue. In the root system of trees growing out of doors there is great difference in the relative hardiness. The crown of the tree — that is, the part of the root just beneath the ground — wull withstand con- siderably lower temperatures than parts of the root lower down, and the small ends of the roots kill more easily than the larger parts. In fact as the roots extend away from the crown they become more and more tender and apparently this tenderness is greater on those roots that extend downward into the soil. Goff thinks that in Wisconsin the ends of the roots may be killed during every winter. The following table presents data covering this point. The Angers quince roots were frozen on January 25, 1913; the seedling peach roots on March 22, 1913 and the balance on March 24, 1913. See also Table 36 for the same kind of data on plum and cherry roots. Table 34. Showing Relative Hardiness of Various Parts of THE Root System of Fruit Trees. Kind of Root Location of Root Largest Diameter Length Tem- pera- ture Results Two-year Kieffer pear on Japan stock Top even with surface 1 in 16 in.... -10 No injury in first inch. Cam- bium injury throughout remainder. Cor- tex injury last 13 inches. Sap wood in last 9 in. Killing of Plant Tissue by Low Temperature 237 Kind of Root Two-year Kieffer pear on Japan stock Two-year Kieffer pear on Japan stock Two-year Kieffer pear on Japan stock Kieffer pear roots Kieffer pear roots Kieffer pear roots Kieffer pear roots Location of Root Largest Diameter I Length Tem- pera- ture Results 4 inches below surface 4 inches below surface 4 inches below surface Top even with surface . 5 in. 12 in.... .45 in. 10 in. -10 -10 .3 in. 6 in. 4.5 in below surface 6 inches below surface 1.2 in. 15 in. -10 . 6 in. 12 in. -10 -10 .3 inches be- j low surface. . .4 in.. 12 in.... -10 ,3 in. 8 in. -10 Cambium and cortex injury throughout. Sap wood in- jury in last 8 inches. Cambium, cor- tex and sap wood injured. throughout. Cortex and cam- bium injured severely throughout; sap wood in last 5 inches. No injury in first 8 inches; slight injury in cortex and cambium in re- mainder. No injury in first 2 inches; cambium and cortex injury in remainder; pith injury in last 5 inches. No injury in first 3 inches; cambium and cortex and sap wood injury in remainder. Cambium and cortrx injury throughout. Sap wood in- jury in last 4 ii jury in inches.. 238 Missouri Agr. Exp. Sta, Research Bulletin No. 8 Kind of Root Two-year Ben Davis apple roots Two-year Ben Davis apple roots Two-year Ben Davis apple roots Two-year Ben Davis apple roots Location of Root Two-year Ben Davis apple roots Two-year Ben Davis apple roots Two-year Ben Davis apple roots Two-year Ben Davis Apple roots Top even with surface 6 inches below surface 6 inches below surface Largest Diameter Length .8 in. ,25 in. Tem- pera- ture 12 in.. 10 in. 6 inches below surface 6 inches below surface Top even with surface 25 in. ,2 in..., ,2 in. ,8 in. 5 inches below surface 9 inches below surface 8 in. -10 -10 -10 6 in. 12 in. 14 in. -10 -10 ,25 in. ,35 in. 8 in. 8 in. -10 -10 -10 Results No injury in first 2 in. from top. Cambium injury in re- mainder. Cor- tex injury in last 3 inches. Slight injury in cambium throughout. Cortex injury in last 2 inches. Cortex and cambium in- jury through- out. Pith in- jury in last 5 inches. All tissues in- jured through- out. Cortex and cam- bium injury throughout. Sap wood in- jury in last 6 inches. Cambium in- jury through- out. Cortex injury in last 6 in; sap wood in last 5 inches. Very slight in- jury in cortex and cambium in last 2 inches. Cortex and cam bium injury throughout Sap wood in- jury in last 5 inches. Killing of Plant Tissue by Low Temperature 239 Kind of Root Location of Root Two-year Ben Davis apple roots One-year French apple seedlings One-year French apple seedlings 11 inches be- low surface.. ,3 in.... 6 in.... -10 One-year French apple seedlings One-year Japan pear seedlings. . . One-year Japan pear seedlings. . . One-year Japan pear seedlings. . . Angers Quince roots .4 in.... ,4 in... . 12 in.... 11 in... -10 -10 35 in... 8 in.... I -10 .4 in.... 11 in.... -10 .4 in.... 10.5 in... -10 35 in... 0 in.... -10 5 in. -7 Results Cortex and cam- bium injury throughout. No injury in sap wood. Cambium in- jury in last 11 in; cortex in- jury in last 6 in; pith injury in last 2 inches. Cambium in- jury through- out; cortex in- jury in last 10 in; sap wood injury in last 4 inches. Cambium slight- ly injured throughout; cortex injury in last 3 inches. Cambium in- jury through- out; cortex in- jury in last 7 inches sap wood injury in last 3 inches. Cambium in- jury through- out; cortex in- jury in last 8 inches; sap wood injury in last 2 inches. Cambium in- jury through- out; cortex in- jury in last 7 inches; s a p wood injury in last 4 inches. Cortex slightly injured. 240 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Kind of Root Angers Quince roots Angers Quince roots Angers Quince roots Angers Quince roots Angers Quince roots Angers Quince root Angers Quince roots Seedling Peach roots Seedling Peach roots , Seedling Peach roots Seedling Peach roots Location of Root Largest Diameter Top even with surface 2 inches below surface 4 inches below surface . 25 in.. .18 in.. .12 in.. . 5 in.... . 25 in.., .18 in.. , 12 in.., .65 in... Length ,35 in... 12 in Tem- pera- ture 14 in. -9 -9 -9 ,3 in... 6 inches below surface , 3 in 12 in. 10 in. -9 Results Cortex injured. Cortex and cam- bium injured. Cortex and cam- bium injured. Cortex injured. Cortex injured. Cortex and cam- bium injured. Cortex and cam- bium injured. No injury in first 2 inches from top; cor- tex and cam- bium slightly injured in next 4 in; all tissues injured in re- mainder. Cambium and cortex injury throughout. Sap wood slightly injured in last 3 inches. Injury very slight nearest crown. Cambium and cortex injured severely throughout. Sap wood and pith injury in last 8 in. Cambium and cortex injury throughout. Sap wood and pith injury in last 6 inches. Four-year old Elberta peach roots subjected on October 19, 1911, to a temperature of -4° C, showed both live and dead tissue intermingled. The younger roots lying some distance from the trunk of the tree showed the tissue to be all dead, killing worse than the larger roots near the trunk. Killing of Plant Tissue by Low Temperature 241 These roots were in all cases kept during the time preceding the freezing in such a position that all parts must have been exposed to practically the same temperature, so it is not possible that the diminished hardiness of the parts furthest from the crown could have been caused by their being exposed to a higher temperature during the period preceding the freezing. It probably represents the most rapidly growing tissue, but at times of freezing the tissue had not been growing for at least three months. It also represents tissue that under normal conditions is not so liable to be exposed to low temperatures so in the evolution of the plant it would not be so necessary for it to develop hardiness. Of great interest, practically, is the hardiness of various stocks of fruit trees. Through the courtesy of Mr. E. S. Welch of Shenan- doah, Iowa, this station was able to study various stocks. In the case of apple trees worked on French crabs, a considerable number of trees were furnished that had rooted from the scions, as well as from the stock, thus permitting a comparison between these scion roots and the roots from the stock. The trees were received Decem- ber 20, 1912 and were heeled-in in the shade. The roots were thus at a temperature near the freezing point from the time they were re- ceived until they were frozen. The following table gives the results: 242 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 35. Showing Relative Hardiness of Stock and Scion Roots. Date Freezing Feb. 18,'13 Feb. 18,'13 Feb. 20,'13 Feb. 20,'13 Feb. 20,'13 Feb. 20, '13 -15 Feb. 20, '13 -IS Feb. 20, '13 -15 Feb. 20, '13 -15 Feb. 20, '13 -15 Feb. 20, '13 -15 Mar. 3, '13 -15 Mar. 3, '13 -15 Mar. 3,'13 Mar. 3,'13 Mar. 3,'13 Mar. 3,'13 Mar. 3,'13 Mar. 3,'13 -11 -11 -15 -15 -15 -15 -15 -15 -15 -15 -15 Material and Results Two-year old Ben Davis apple trees. 6 inches. Scion root diameter i\ in. uninjured for first Cortex and cambium injury in remainder. Similar root from stock dia. ui in-, uninjured for first 7 inches. Cortex and cambium injury in remainder. A. Scion root, largest dia. ^g in., length 14 in. No injury whatever. B. Scion root, coming from scion 2 inches below A. Largest dia. J^ in; length 12 in. No injury in first 5 inches; last 7 inches were slightly browned. C. Scion root, 2 inches lower than B. Largest dia. i^e in; length 12 in. No injury in first 6 inches; slight injury in last 6 inches. D. Stock root, 1 inch below C. Largest dia. % in; length 10 in. Severe injury throughout. E. Stock root, 2 inches below D. Largest dia. J^ in; length 10 in. Very severely injured throughout. F. Stock root, 3^ inch below E. Largest dia. % in; length 12 inches. Severe injury throughout. G. and H. Arise at same point as F. Each 14 in. in dia. at largest point; both 12 in. long. Severe injury in both. Main root. Largest dia. % in. No injury to scion part. Injury to cortex in stock part. Fibrous roots under i^g in. dia. Injury on those from stock and not on those from scion. A. Scion root 4 inches below ground. Largest dia. J^ in; length 10 in. No injury in first 5 inches. Remainder very slightly browned. B. Stock root attached 2 inches below A. Largest dia. J^ in; length 14 in. Injury throughout entire length. C. Stock root 2 inches below B. Largest dia. 3^ in; length 10. in . Injury in first 4 inches slight; remainder well browned. D. Stock root same size as C. Attached at same portion of main root. Injury somewhat more severe than C. Main root. Scion part, dia. % in., shows no browning. Stock part, dia. 3^ in. is slightly browned. Two-year old Wealthy apple trees A. Scion root. Largest dia. 3/2 in; length 12 in. No injury in first 7 inches; remainder slightly injured. B. Continuation of main root. Largest dia. 3-8 in; length 8 in. No injury in first 3 inches; last 5 inches slightly in- jured. C. Stock root. Largest dia. 3^ in; length 12 in. Arises nearly opposite Root A. mainder slightly injured. A and B. No injury in first 6 inches; re- No marked difference between Killing of Plant Tissue by Low Temperature 243 In most cases those roots that came from scions were more hardy than those of the same size coming from the stock, indicating that the French seedling roots are less hardy than roots of a variety like Ben Davis. This station has been unable to compare the hardiness of scion roots from hardy varieties like Fameuse with those from very tender varieties like Jonathan. A comparison similar to the foregoing was made with different stock of plums and cherries using the Myrobolan and Marianna varieties of plums and Mazzard and Mahaleb varieties of cherries. The following table gives the results: Table 36. Showing Relative Hardiness of Mazzard and Ma- haleb Cherry Stock and Marianna and Myrobolan Plum Stock. Date of Freezing, January 25, 1913. Location Largest Tem- Kind of Root of Root Diameter Length pera- ture Results Mazzard cherry roots. . Min -7 No injury. Mazzard cherry roots. . Min -7 No injury. Mazzard cherry roots.. i\ in -7 No injury. Mazzard cherry roots.. Hin -7 Slight injury in cortex. Mazzard cherry roots.. Min -9 No injury. Mazzard cherry roots. . Hin -9 No injury. Mazzard cherry roots. . A in -9 Slight browning in cortex. Mazzard cherry roots. . Hin -9 Cortex and cam- bium injured. Mahaleb cherry roots. . Min -7 No injury. Mahaleb cherry roots. . Jiin -7 No injury. Mahaleb cherry roots.. i\ in -7 Cortex injured. Mahaleb cherry roots.. ^In -7 Cortex injured. Mahaleb cherry roots.. Hin -9 Cortex injured. Mahaleb cherry roots.. Kin -9 Cortex and cam- bium injured. Mahaleb cherry roots.. i^« in -9 Cortex and cam- bium injured. Mahaleb cherry roots. . ^in -9 Cortex and cam- bium injured. 244 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Date of Freezing, March 18, 1913. Location Largest Tem- Kind of Root of Root Diameter Length pera- ture Results Mazzard cherry roots. . 3 inches below surface 1.1 in.... 7 in.... -10 Cambium and cortex injured throughout. Least severe Mazzard near crown. cherry roots. . 3.5 inches be- low surface. . . .3 in.... 5 in.... -10 Cambium and cortex injured throughout. Least severe Mazzard near crown. cherry roots. . 4 inches below surface .3 in.... 7 in.... -10 Cambium and cortex injured throughout. More severe Mazzard than above. cherry roots. . 4.5 inches be- low surface.... .3 in.... 12 in.... -10 Cambium and cortex injured severely Mazzard thro u g h o u t cherry roots. . 5 inches below surface .45 in... 6 in.... -10 Very severe in- iury in cam- bium and cor- tex through- Mazzard out. cherry roots. . 5. 5 inches be- low surface.... .4 in 10 in.... -10 Very severe in- jury in cam- bium and cor- tex through- out. Mazzard cherry roots. . 5^ inches be- low surface.... .28 in... 28 in.... -10 Cortex and cambium in- jured severely. Pith injured slightly. Mazzard cherry roots. . 6\4: in. below surface .15 in... 8 in.... -10 All regions in- jured. Mazzard cherry roots. . 6% inches be- low surface.... 2 in 6 in.... -10 AH regions in- jured. Mazzard cherry roots. . 9}4 in. below surface 1 jn -10 All regions in- jured. Killing of Plant Tissue by Low Temperature 245 Location Largest Tem- Kind of Root of Root Diameter Length , pera- ture Results Mazzard cherry roots. . 2 inches below surface 1 in 8 in.... -10 Cortex and cam- bium injury throughout. Least injury near crown. Pith killed last Mazzard 5 inches. cherry roots. . 3 inches below surface .3 in... . 6 in.... -10 All tissues se- Mazzard verely injured. cherry roots. . 4 inches below surface .3 in.... 10 in.... -10 Cambium and cortex injured severely first 3 inches of root. All tissues injured in re- Mazzard mainder. cherry roots. . 4 inches below surface . 5 in.... 10 in.... -10 Cambium and cortex injury throughout. Pith injury last Mazzard 5 inches. cherry roots. . 4J^ inches be- low surface.... .45 in... 14 in... . -10 Cambium and cortex injured very severely throughout. Pith injured in Mazzard last 12 inches. cherry roots. . 6H inches be- low surface.... .4 in.... 8 in.... -10 Cambium and cortex injured very severely throughout. Pith injured in Mazzard last 3 inches. cherry roots. . 7H inches be- low surface. . .2 in.... 6 in... . -10 All tissues in- jured through- Mazzard out. cherry roots. . 9 inches below surface .15 ill... 6 in.... -10 All tissues in- jured through- Mazzard out. cherry roots. . 10|/2 inches be- low surface.... .2 in.... 4-6 in.. -10 .\11 tissues very severely in- Mazzard jured. cherry roots. . 6 inches below surface .15 in... •^ in.... -10 .Ml tissues very se verily in- jured. 246 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Kind of Root Mahaleb cherry roots. Mahaleb cherry roots. Mahaleb cherry roots. Mahaleb cherry roots. Mahaleb cherry roots. Mahaleb cherry roots. . Mahaleb cherry roots. . Mahaleb cherry roots. Mazzard cherry roots. . Location of Root 2 inches below surface 8 inches below surface 10 inches be- low surface..., 6 inches below surface Largest Diameter 2 inches below surface. . . . 6 inches below surface 10 inches be- low surface.. 9J^ inches be- low surface.. Date of F At surface. . . . 1 in. ,2 in.... .35 in.,. 1 in. 1 in.... .4 in.... .35 in. , 2 in... Length Tem- pera- ture 13 in.... 4 in.... 4 in.... 4 in. 14 in. reezing, Ma 1.1 in.... 6 in. 2 in.... 3 in.... rch 21, 1 11 in.... -10 -10 -10 -10 -10 -10 -10 -10 913. -10 Results No injury in first 6 in. Very slight injury in cambium in next 3 in. Cam- bium and cor- tex injury in remainder. Cambium and cortex injury throughout. Cambium and cortex very slightly i n - jured. Cambium and cortex injured severely. No injury in first 4 inches. Cambium and cortex injured very slightly throughout remainder. Cambium and cortex injury throughout. Slight injury in cambium and cortex. Cambium and cortex injured. Cambium in- jured through- out. Cortex in jury in last 8 in. More severe near terminal; least injury near crown. Killing of Plant Tissue by Low Temperature 247 Kind of Root Mazzard cherry roots. Mazzard cherry roots. Mazzard cherry roots. Mazzard cherry roots. Mazzard cherry roots. Mazzard cherry roots. Mazzard cherry roots. Location of Root Largest Diameter Mazzard cherry roots. Mazzard cherry roots. 3 inches below surface 3 inches below surface 3H inches be- low surface.. 5 inches below surface 10 inches be- low surface.. At surface. . . 2 inches below surface 2 inches below surface lYi inches be- low surface... Length ,45 in... 8 in ,3 in... . .4 in... .3 in.. .3 in. 1.1 in. 5 in.. 4 in. 6 in. 10 in. 8 in. 13 in... 10 in... .45 in... 8 in Tem- pera- ture -10 -10 -10 -10 -10 -10 -10 ,5 in. 5 in. -10 -10 Results Cambium i n - jured through- out. Cortex in- jured in last 5 in. Least in- jury nearest crown. Cambiumslight- ly injured throughout. Cambium and cortex injury very slight. Cambium and cortex injured throughout. Pith injured in last 3 inches. Cambium and cortex severely injured throughout. Pith injured less severely. Cambium i n - jury through- out. Cortex and cambium in last 8 in. Cortex, cam- bium and pith in last 5 inches. Cambium i n - jury through- out. Cortex and cambium in last 8 in. Cortex, cam- Ilium and pith in last 5 inches. Cortex and cam- hiuin injury throughout. Cortex and cam- bium injury throughout. 248 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Kind of Root Mazzard cherry roots. . Mazzard cherry roots. , Mahaleb cherry roots. Mahaleb cherry roots. . Mahaleb cherry roots. . Mahaleb cherry roots. Mahaleb cherry roots. Mahaleb cherry roots. Location of Root Largest Diameter 3 inches below surface 7 inches below surface Date of F 1 inch below surface .... 2 inches below surface , 3 in.. 25 in.., reezing, M 1 in.... .15 in. Length Tem- pera- ture 5 in... 5 in. arch 21,1 10 in... 6 in.... 3 inches below surface 4 inches below surface 5 inches below surface 1 inch below surface ,4 in... .4 in... .3 in. 1 in.... 8 in.... -10 -10 913. -10 -10 -10 6 in.... -10 4 in. 14 in. -10 -10 Results Cortex and cam- bium severely injured throughout. Pith injury in last 4 in. Cortex and cam- bium injury throughout. Pith injury in last 3 inches. No injury in 5 in. nearest crown. Cortex injury slight in remainder. Cambium slight- ly injured in first 2 in. Cam- bium and cor- tex in remain- der. Cambium and cortex injury throughout. Very slight nearest crown. Pith injury in last 2 inches. Cambium and cortex injury slight in first 2 in.; more severe in next 2 in. Cambium, cor- tex and pith in- jury in termi- nal 2 inches. Cambium and cortex slightly injured. No injury in highest 7 in. Very slight injury in cam- bium and cor- tex of remain- der. Killing of Plant Tissue by Low Temperature 249 Location Largest Tem- Kind of Root of Root Diameter Length pera- ture Results Mahaleb cherry roots. . 4 inches below surface .3 in.... 8 in.... -10 No injury in first 4 in.; slight injury in cam- bium and cor- tex in remain- Mahaleb der. cherry roots. . 4 inches below surface .45 in... 10 in.... -10 No injury in first 3 inches; cambium and cortex injury in remainder; injury becom- ing more severe Mahaleb near terminal. cherry roots. . 4 inches below surface .5 in.... 5 in.... -10 No injury first inch. Cambium and cortex in- jured in re- Mahaleb mainder. cherry roots.. 7 inches below surface .3 in.... 3 in.... -10 Slight injury in cambium and cortex. Date of F reezing, M arch 18, 1 913. Myrobolan plum roots.... 2 inches below surface .8 in.... 7.5 in -10 Cortex injured first 5 in.; cor- tex and cam- bium injured Myrobolan next 2 3^ inches. plum roots.. . 3 inches below surface .4 in.... 9.5 in. -10 Cortex and cam- bium injured in all. rith in- jured in last Myrobolan 53-2 inches. plum roots.. . 6 inches below surface .2 in.... 6 in.... -10 All regions in- jured, less se- vere near point Myrobolan of attachiiunt. plum roots.. . 9 inches below surface .3 in.... 3 in.... -10 All regions in- jured, less se- verely near point of at- Myrobolan tachment. plum roots.. . 12 inches be- low surface. . .25 in... 3 in... . -10 All regions very severely i n - jured. 250 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Kind of Root Location of Root Largest Diameter Length Tem- pera- ture Results Myrobolan plum roots.. Myrobolan plum roots.. Myrobolan plum roots.. Myrobolan plum roots. Myrobolan plum roots. Myrobolan plum roots. Myrobolan plum roots. Myrobolan plum roots. Marianna plum roots. Marianna plum roots. 14 inches be- low surface.... 2 inches below surface 2^ inches be- low surface.. 3 % inches be- low surface... 4J^ inches be- low surface... 4^ inches be- low surface.... 11 inches be- low surface.... 11 inches be- low surface Date of F 7 inches below surface 9 inches below surface 1 in.... .7 in. .3 in.... . 2 in... . .3 in . 2 in .35 in... .15 in... reezing, M 1 in .6 in 6 in. 12 in. 6 in... -10 -10 -10 8 in... 10 in... 9 in.... 3 in 3 in arch 20, 1 2 in... . 12 in.... .-10 -10 -10 -10 -10 913. -10 -10 All regions very severely i n - jured. Cortex injured slightly in first 3 in.;cortexand cambium i n - jured slightly next 6 in.; pith injured slightly and cortex and cambium s e - verely in last 3 inches. Cortex injury in first inch. Cor- tex and cam- bium injury in remainder, severe near ter- minal. All tissues in- jured. Injury most severe near terminal. All tissues in- jured. Injury most severe near terminal. Same as above. Same as above. Same as above. No injury no- ticeable. Slight injury to cambium in first 6 inches; all tissue in- jured in re- mainder. Killing of Plant Tissue by Low Temper.\ture 251 Kind of Root Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots. Marianna plum roots Location of Root 9 inches below surface 9 inches below surface 9 inches below surface 9 inches below surface 12 inches be- low surface.... 9 inches below surface Date of F 1 inch below surface Largest Diameter . 55 in... 5 in. 5 in.... .45 in.. . 2 in... Length 6 in. 12 in... 5 in 10 in... 6 in. 4 in... . 10 in.... reezing, M 1 in. 2 inches below surface 2 }^ inches be- low surface... 2 inches below surface 6 inches below surface 4 in. arch 24, 1 14 in... 8 in. Tem- pera- ture -10 -10 -10 -10 -10 -10 913. -10 -10 3 in... . 2 in .... , 35 in... li) in. 8 in. 14 in. ■10 •10 -10 Results Cambium i n - jured first 3 inches. C a m- bium and cor- tex in remain- der. Cambium i n - jured first 3 inches. Cortex and cambium next 2 inches; cortex, cam- bium and pith in remainder. Cambium and cortex injured very slightly throughout. All tissues in- jured. All tissues in- jured. All tissues in- jured. No injury to first 5 inches; slight injury t o cambium next 3 inches; slight injury to cortex in re- mainder. Slight injury in cortex and and cambium throughout. More severe towards tip. Same as above. Same as above. Caml)ium and cortex injured throughout. Morf severe last 8 in. Pith last 4 in. 252 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Location Largest Tem- Kind of Root of Root Diameter Length pera- ture Results Marianna plum roots.. . 6J^ inches be- low surface.... .15 in... 6 in.... -10 Cambium and cortex injured Marianna throughout. plum roots.. . 8J^ inches be- low surface.... .15 in... 4 in.... -10 Cambium and cortex injured Marianna throughout. plum roots.. . lOJ^ inches be- low surface.... .45 in... 3 in.... -10 Cambium and cortex slightly Myrobolan injured. plum roots.. . 1 inch below surface .8 in.... 12 in... -10 Cortex, cam- bium and pith injured slightly throughout. More severe further from Myrobolan crown. plum roots.. . 3 inches below surface .3 in.... 10 in.... -10 Cambium and cortex injured severely throughout ; pith injured Myrobolan slightly. plum roots.. . 4H inches be- low surface.... .3 in.... 8 in.... -10 Same as above. Myrobolan plum roots.. . 5 inches below surface .25 in... 9 in.... -10 All tissues se- verely injured. Myrobolan plum roots.. . At surface. . . . .8 in.... 10 in.... -10 Cambium, cor- tex and pith in- jured in all. More severely Myrobolan near tip. plum roots.. . 3 inches below surface .35 in... 8 in.... -10 Cambium and cortex severe- ly injured throughout. Pith injured in Myrobolan last 5 inches. plum roots.. . 5 inches below surface .2 in.... 4 in.... -10 Cambium and cortex severe- ly injured throughout. Pith injured Myrobolan slightly. plum roots.. . 7 inches below 1 surface ■ .2 in.... 4 in.... -10 All tissues se- verely injured. Killing of Plant Tissue by Low Temperature 253 Freezing to Death of Pollen. The killing of the bloom and young fruit of the peach, apple and some other fruits will be dis- cussed later in this paper. However, it is perhaps not out of place to discuss here the killing of pollen. Schaffnit^ exposed the pollen of a number of species of plants to a temperature of -17° C. for eight hours with no apparent harmful effects, while Sandsten^ apparently reduced the germination percentage of pear, plum, cherry and peach pollen by exposing for six hours to a temperature of 1.5°. At this station pollen of the Jonathan and Cillago apples was frozen with the result shown in Table 37. The pollen was frozen for eighteen hours and then germinated in a 10% sugar solution. Table 37. Showing Effect of Low Temperature on Germinat- ing Power of Pollen Grains. Kind of Pollen Date Tem- pera- ture Time at Minimum Percentage Percentage Germination Germination Unfrozen Frozen onathan apple /onathan apple pollen dried Cillago apple Cillago apple Apr. 24/13 May 29,'13 May 10,'13 May 10,'13 -3 -13 -8 -12.5 45 min... . 60 min 30 min.. . . 30 mill.. . . 84.0 25.0 (estimate) 66.66 66.66 33.0 20.0 (estimate) 25.0 00.0 Thus the pollen will still germinate after exposure to as low temperature as -8° C., and dried pollen at as low a temperature as -13, a much lower temperature than the other flower parts will with- stand. Discussion of the killing temperature of other flower tissues will be found in a later part of this paper. Rest Period of Plants. Closely associated with the cjuestion of maturity in the fall is that of maintaining a condition of maturity in late winter. Plants that have started into growth in spring soon reach a condition whereby they are as tender as they were in early fall, and sometimes even more so. Under conditions such as prevail in the southern half of Missouri, where growth may continue latr in autumn, and where in January and early Februar>-. there are likely to be da>'s warm enough for coiisidcrablo growth to take place, it has 'Mitt. Kalsor Wllhclm Inst. Landw HroinlMTK. Vol. :i. No. 2. pp. iKMl.1. IIUO. (Hll)l. No. OS). -Wis. Agr. Exp. .Stu. Ucacarch Hul », pp liW-5 (HIbl. No. 07). 254 Missouri Agr. Exp. Sta. Research Bulletin No. 8 been found that with the peach, the winter rest period of the fruit buds is an important factor in influencing the amount of killing from cold. By the rest period is meant the period during which the buds will not respond in growth to favorable temperature conditions with- out special treatment. This rest period is shorter with some varie- ties than with others, and with all varieties tried at this station the rest period is prolonged later into the winter if the tree makes a vig- orous growth and continues growing rather late in the season. They will not, therefore, respond so readily to warm periods that may come in late December or January. Winter Protection of Buds. The buds of trees in winter are covered with bud scales. Some hold that the insulation formed by these scales will keep the buds from reaching a temperature as low as that of the surrounding air. Wiegand^ however, found that when a thermometer bulb was carefully inserted in large buds there was no great difference between the rapidity of the fall of tempera- ture on such a thermometer and one with a naked bulb. He, there- fore, holds that the bud scales offer little protection in the way of holding heat in the buds, but that their protection is against evapora- tion. To test this matter at this station the scales were removed from buds of peaches in winter before freezing in the freezer pre- viously described with the results shown in Table 38. iBot. Gaz. Vol. 41, pp. 373-424. (BibL No. 117). Killing of Plant Tissue by Low Temperature 2^ :>:> Table 38. Showing Effect of Removing Scales From the Buds Before Freezing on Their Resistance to Very Low Temperature. Variety Elberta Elberta Elberta Elberta, wilted .... Elberta water-soaked. . . . Elberta, normally turgid. Elberta, wilted. ... Elberta, normally turgid.. Elberta, wilted .... Elberta, normally turgid. , Elberta, water-soaked Elberta, normally turgid . , Elberta, water-soaked Elberta, normally turgid . . 01dm" 01dm 01dm 01dm 01dm 01dm 01dm 01dm 01dm Oldm Oldm Oldm Oldm ixon i.xon ixon ixon ixon ixon ixon ixon ixon ixon ixon ixon ixon Bell's Oct., slowly... Hell's Oct Bell's Oct Bell's Oct., rapidly... Bell's Oct Bell's Oct., slowly.. . Bell's Oct., rapidly... iiell's Oct fiencral Lee, slowly.. General Lee. Date of Freezing Nov. Nov. Feb. Dec. 12 12 23 15 Dec. 15 Dec. Dec. Dec. Dec. 15 23 23 24 Dec. 24 Dec. Dec. 27 27 Dec. 28 Dec. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Nov. Dec. Dec. Jan. Jan. Mar. Feb. Feb. Feb. Feb. Feb. Feb. Feb. Dec. Feb. Feb. 28 26 26 26 26 26 26 26 26 6 6 14 15 23 7 8 15 16 17 18 19 6 26 26 '09 '09 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 '09 '09 '09 •09 '09 '09 '09 '09 '09 '09 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 '10 •lOi Tem- pera- ture -15 -15 -18 -20.2 -15 -15 -21.5 -16 -20.5 -20.5 -19.7 -19.7 -20.5 -20.5 -16 -16 -21 -21 -14 -14 -21 -21 -22.5 -18.5 -21 -21 -10 -20 -20.5 -20 -20.5 -20 -18 -18 -21 -20.3 -21 Number of Buds. Scales Off Total number of buds. . . . Average percentage killerl. 149 96 117 79 39 89 59 56 70 75 60 57 69 54 100 98 80 79 150 86 144 74 196 50 185 218 156 210 298 267 200 180 121 76 69 233 71 4430 Number of Buds. Scales On 65 133 168 83 35 74 57 59 79 75 77 48 72 56 87 83 137 92 151 96 198 76 132 290 223 262 140 107 239 353 249 200 195 245 100 220 111 5078 Percent- age Killed. Scales Off 2.6 2.0 47.8 59. 64. 46. 45. 55. 74. 52. 93. 57. 94. 79. 29. 18.4 6.2 2.5 14.6 17.4 81.2 64.8 15.7 40. 96.6 75.6 57. 33.8 46.6 100. 100. 100. 17. 82. 66. 8. 11. ,3 .9 5 1 Percent- age Killed. Scales On 51.0 1.5 1.5 63.6 81. 71. 75. 100. 76. 100. 32. 19. 75. 45. 71. 74.7 73.5 13.8 17.4 62.2 47.9 98.9 96. 54.5 93.1 99.5 97.7 57.1 83.1 63.6 97.4 100. 98. 40.5 93.0 45. 13.8 19.8 68.5 256 Missouri Agr. Exp. Sta. Research Bulletin No. 8 It will be seen that not only in this case were the scales no protect- ion against the cold, but in actual fact those buds that had the scales removed had very uniformly a smaller percentage killed than those still protected by the bud scales. This is at least true during the most dormant condition in winter. Toward spring when the buds have swelled considerably, during warm days, there seems to be practi- cally no difference between the percentage killed with the scales removed and those with the scales on. This seems to demonstrate conclusively that, at least in the case of peaches, the bud scales do not serve to protect the bud from cold, but as Wiegand points out, the bud scales serve only to protect the bud from loss of water by evaporation. RELATION OF LOW TEMPERATURE TO PEACH GROWING. Peach Wood Killing. As mentioned previously, on some years the peach wood is badly injured. Generally this injury follows very severe cold winters with lower temperatures than are necessary to kill the buds. However, on some years the wood has been reported badly injured even when a fair set of bloom followed. It is difficult to say just what weather conditions favor winter killing of peach wood in all cases. In the case of young trees, one to three years old, Emerson^ found that in Nebraska they are more likely to survive the average winter if forced to mature their wood early in autumn, by using gross feeding cover crops as previously mentioned, though older trees were not benefited by such a practice. Eustace- reports that following the severe winter of 1903-04 in New York, peach trees more than seven years of age were killed worse than peach trees about seven years of age or younger, except trees one year in the orchard which killed the worst. Green^ and Ballou, following the same winter, observed that old trees were killed worse than young trees and that trees kept in a good condition of vigor by use of stable manure, mulch, etc., were in better condition than trees that had been permitted to become weak in their growth. This may have been partly because of the dry weather in early summer followed by wet weather in late summer and autumn. The old trees would have been checked in growth more by the drought and thrown into more succu- lent growth by the later rains, while the more vigorous young trees would be better able to secure sufficient water and tend to continue >Neb. Agr. Exp. sta. Bui. 79, 1903. (BlbL No. 33). 2N. Y. (Geneva) Exp. Sta. BiU. 269. 1905. (BibL No. 38). 'Ohio Agr. Exp. Sta. Bui. 157. 1894. (Blbl. No. 48). Killing of Plant Tissue by Low Temperature 257 their growth through the drought, and be just starting into their normal dormant period when the wet weather began. They would, not therefore, be so easily pushed into new growth as if they had passed through a drying out period. At least observation indicates that the weak tree which ceases growth early in the season is most readily pushed into spring-like growth in autumn. It is probably also true that the young tree would offer less resistance to the movement of sap upward in spring. It would thus be able to secure sufficient water supply to prevent drying out until a new layer of sapwood is formed. The old tree would have a greater leaf surface to evaporate moisture and would offer more resistance to the movement of sap. It would, therefore, seem possible that the young tree might recover from the freeze better, though its injury had been greater than that of the older tree. In the peach orchards on the Horticultural grounds and at Brandsville and other points in South Missouri, following the severe winter of 1904-05 and again following the severe winter of 1911-12, the trees in a healthy, vigorous condition withstood the low tempera- ture better than trees in a weak condition of growth. At Brands- ville and other extreme southern Missouri points, following the freeze of 1911-12, recovery was much more satisfactory from trees that had been fertilized with nitrate of soda than with trees that had not been so fertilized. This was even true with one year old trees which is contrary to the experience of Emerson,^ but it should be noted that the soil is more conducive to early maturity and the growing season longer in southern Missouri than in Nebraska. As to the best means of securing recovery of trees from winter injury, a great deal depends upon the nature of the injury. Thus following the winter of 1899, W^hitten- reports that trees very severely pruned, leaving the branches only a few feet long, recovered much better than trees not headed so severely. Following the severe win- ter of 1904-05 trees rather severely headed-in recovered better than trees that had only one-third or one-half of the previous season's wood cut off. However, Eustace' found that this severe heading back of the trees in New York, following the winter of 1903-04, was very harmful to old trees, though apparently beneficial to young trees. Moderate pruning gave much better results. «Neb. Agr. Exp. Sta. nul. 79. 1903: (BIbl. No. 33) and Nob. .\gr. Exp. Stn. .\r\l. Kpt. No. 19. 1906, pp. 101-10 (Hibl. No, ri.'j). »Mo. Agr. Exp. Htn. Hul. n.-i, 1902. (Blbl. No. 115). 'N. Y. (Geneva) Exp. Sta. Bui. um). 190.5. (Blbl. No. 3S). 258 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Waite^ reports that very severe pruning of trees in Michigan and other parts of the North, following the same winter, did not give as good results as light pruning — removing about one-third to one- half of the one year old wood, — and in Missouri, both in the Experi- ment Station orchard and in orchards at Brandsville and Hollister of southern Missouri, old trees that were severely cut back following the severe winter of 1911-12 did not recover as well as old trees with even no cutting back. The amount of cutting that seemed to have been the most desirable, following either the winter of 1908-09 or 1904-05, in Missouri, was in a large number of cases fatal in 1911-12. At Brandsville a large block of trees that had reached a height of something like fifteen feet had their branches cut back to stubs four or five feet long, and a large percentage of the trees died, after making some slight growth in the spring, while trees with no pruning or very light pruning recovered much better. Just what the difference is it is hard to say, except that pre- ceding the winter of 1911-12, growth conditions were much the same as those described by Waite, Selby and Eustace for Michigan, Ohio and New York preceding the winter of 1903-04; that is, dry weather in the early part of the season, followed by very wet weather causing a late succulent growth. In deciding how much pruning to give a tree following a severe winter, one must consider the kind of injury. If the lower part of the tree is very severely injured, as it will be when the tree is forced into late succulent growth following conditions that seriously check its growth earlier, the pruning should probably be such as to remove not more than one-half of the one-year-old wood, while if the injury is distributed throughout the tree, and is not so severe that the cortex and cambium are entirely killed at any point, it would seem highly probable that more severe cutting back — say into two- year-old wood — would be desirable. Such cutting back is often beneficial to the trees even when they do not need it to help them recover from severe freezes. However, very severe cutting back, such as Whitten describes, is probably not the most desirable follow- ing any kind of a winter since much less cutting will give entirely satisfactory results and a better tree with more fruit buds for the following season 's crop. Cutting back to induce recovery from win- ter injury has always been more successful at this station on young trees than on old ones and on trees kept vigorous by severe pruning during previous years than on less vigorous trees. In fact if trees are old and neglected, severe cutting back all in one year should always be avoided. >U. S. Dept. Agr. 13. P. I. Bill. 51. pp. 15-19. (Bibl. No. 111). Killing of Plant Tissue by Low Temperature 259 Eustace reports that trees pruned in January, shortly after a severe freeze, gave much better results than trees pruned in March, probably because a large amount of drying out had been avoided by reducing the top. At this station better results have been secured by the early pruning. Fertilizing the soil with a nitrogenous fer- tilizer, as nitrate of soda or ammonium sulphate, has, at Brandsville, Missouri, resulted in much more satisfactory recovery from winter injury. Where the fertilizer was applied both the year before and the year following the freeze, very slight permanent injury was to be found when surrounding unfertilized trees suffered great injury. Hardiest Varieties in Wood. The effect of the condition of growth on the hardiness of peach wood is so great that it is difficult to reach an accurate conclusion as to which are the hardiest varieties. Observations must be on a large number of trees, and through a large enough number of years to include many different seasonal conditions. Hedrick,^ basing his opinion on replies secured from New York growers, places Crosby, Hills Chili, Stevens Rareripe, Gold Drop and Elberta as most hardy in wood; and from replies from Michigan growers. Hills Chili, Crosby, Gold Drop, Kalamazoo, and Bernard. Elberta, Smock and Salway, considered hardy in New York, were considered tender in Michigan. Wager, Jaques Rareripe, Carman, Belle of Georgia, and Hale 's Early were considered above the average in hardiness. Eustace^ found in New York, fol- lowing the winter of 1903-04, that Stevens Rareripe, Elberta, Thur- ber and Salway showed little or no wood injury, while Chinese C ling was the most seriously injured. R. F. Howard, formerly of the Nebraska Experiment Station, states in a letter that RusselP is one of the most hardy in wood. At the Missouri Experiment Station there is a peach orchard that has been exposed to the severe winters of 1898-99, 1904-05 and 1911-12. These trees have had such different treatment that they necessarily went into winter in conditions not equally favorable to withstanding the low temperature. We arc, therefore, not able to place the varieties as to hardiness with any degree of accuracy. However, it can be said with certainty that, although Elberta is very tender in hud, its wood is exceeded in its ability to recover from such winters by very few varieties. At least this is true of any but one- or two-year-old trees. Hills Chili, Salway, Bernard and Gold Drop have also proved hardy. Chinese Cling has been prol)al)ly most •Procs. Westorn N. Y. Hort. Soc. 1908. p. 180. (Blbl. No. an). «N. y. (Gonova) Kxp. .Sta. Hul. 2tl15 an acre, so before one would be justified in recommending such a method, it should be tried under orchard conditions for a long enough term of years to be sure that, say in twelve or fifteen years, enough peaches would be saved to justify the expense. There would unquestionably be some years when no benefit would be received in return for the whitewashing. Killing Temperature of Peach Blossoms. A considerable amount of effort has been made to determine, under average condi- tions, the temperature at which the blossoms of peaches are killed. Unquestionably there is a considerable difference in the killing tem- perature of bloom in different years. The killing temperatures in- dicated by laboratory experiments will be given in the last part of this paper (Table 51.) Here the temperature at which bloom and young fruit has been killed in the orchard, and the condition which favor the smallest amount of killing will be discussed. During the spring of 1908 a freeze came on April 3rd when the temperature went to 24° F. at Columbia. Phenological notes taken that spring show that the first bloom on peach trees ranged from March 25th to March 30th, and full bloom ranged from April 5th to April 8th. The following table gives the percentage of bloom killed by this freeze: 10 284 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 46. Showing the Percentage of Peach Bloom Killed ON THE Basal End and the Outer End of Twigs by the Freeze of April 3, 1908, When the Temperature Went to 24° F. Basal hal: of twigs End half of twigs Variety Number Buds Percentage Killed Number Buds Percentage Killed Oldmixon 159 426 648 374 121 163 95 623 503 354 251 579 483 256 129 106 171 151 1850 395 8367 70.44 11.03 15.12 19.78 38. 52.1 5.26 46.70 14.25 45.48 23.1 32.29 26.70 22.22 85.2 14.1 12.2 5.9 11. 11.1 363 598 828 603 205 143 155 553 492 413 186 529 431 192 72 109 111 216 980 507 8257 84.39 Oldmixon 41.97 Oldmixon 67.23 Elberta 44.92 Elberta 69.2 Elberta 71.3 Crawford's Earlv 25.16 Ortiz 55.50 Sneed 65.44 Sneed 53.75 Sneed 50.5 Kalamazoo 54.23 Family Favorite 63.80 Family Favorite Golden Gate 66.14 50. Dewev Cliner 73.4 Dewev Cline 17.1 Elberta Seedling Elberta Seedline 73. 16.3 Connet 43.1 Total No buds Averap'p nerc&nta.C'e killed 27.24 53.68 Of course many of these blooms were not yet fully open and a much larger percentage of the flowers were fully open on the outer half of the twigs than on the basal half, as the following table will show: Table 47. Showing Percentage of Bloom Fully Open on the Basal Half and the Outer Half of Twigs BY April 4, 1908 Basal Half Outer half Variety Number Buds Percentage Unopened Number Buds Percentage Unopened Elberta Seedling Elberta Seedling Connet 151 1850 '395 174 251 13.2 22.7 65.8 16.0 6.7 216 980 507 252 186 0.0 60.2 0.0 Sneed Elberta 0.4 0.0 Killing of Plant Tissue by Low Temperature 28^ Not nearly so large a percentage of unopened as open bloom was killed, as the following table will show: Table 48. Showing Percentage of Open and Unopened Bloom Killed by the Freeze of April 4, 1908 Buds Open Buds U nopened Variety Number Buds Percentage Killed Number Buds Percentage Killed Oldmixon Free Oldmixon Free Elberta Elberta 253 326 347 309 69.9 25.4 24.4 51.13 30 241 85 26 36.6 15.3 7.1 1.1 It will be seen that enough fully open bloom was uninjured at this temperature, 24° F., for a good crop if the tree had a heavy set of bloom. In fact a minimum thermometer that checked with those of the U. S. Weather Bureau registered a temperature in the Missouri Experiment Station orchard of 23° F., so in some years at least, peach bloom may be expected to withstand a temperature that low. Those three tables bringing out the facts, that the bloom just before the petals open will withstand lower temperatures than fully open flowers; that the flowers open more rapidly toward the tips of the twigs, and that therefore flowers on the outer half of the twigs are less likely to survive a spring frost coming before the bloom is fully open, suggest that the tree should be pruned to a sufficiently open head that the leaves at the base of the twigs will not be shaded off before fruit buds are formed. The greater hardiness of unopened buds is apparently not due to the protection of the petals. In freezing 59 unopened blooms to a temperature of -3° C, 11.8 per cent of the pistils were killed while of 50 unopened bloom with the petals and stamens removed, no pistils were killed. On April 30, 1908, the temperature fell to 28° F. in Columbia. The calyx tube was just breaking from the young fruit. The follow- ing table gives the percentage of fruits kilkil at Columbia: 286 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 49. Showing Percentage of Young Fruits From Which THE Calyx Tube Has Just Fallen Killed by a Temperature of 28° F. April 30, 1908 Variety Number Fruits Percentage Killed Kalamazoo Globe 749 279 248 301 300 442 411 454 26.3 22.5 Elberta 7.6 Elberta .33 Elberta 5.6 Sneed 85.0 Alexander 76.8 Bonanza 5.94 It will be seen that large percentages of the fruits were killed only on early varieties like Sneed and Alexander that had reached a larger size. In the southern portion of Missouri where the fruit was larger, more was killed than at Columbia, though the tempera- ture was higher, Koshkonong reporting 32° F. Of course in the lower land where most was killed, the temperature was naturally lower. It is apparently certain then that under average seasonal conditions, the older the fruit at the time of the freeze, the more easily it kills. It is possible that this would not be true in years when the bloom is pushed out very rapidly by exceptionally warm weather and when continuous cool weather follows the setting of the fruit. There is a rather general opinion that just when the calyx tube falls, the fruit is left more susceptible to cold because it has lost the insulation furnished by the calyx tube and requires time for ad- justment. The fact that pistils of unopened flowers are not more easily killed when the external flower parts are removed, and the fact that buds of the peach were not more easily killed by freezing when the scales were entirely removed, would indicate that with the slow falling of the temperature that prevails under natural conditions, the insulation amounts to but little. It seems certainly true, from the experience of the season of 1908, that peach fruits are more tender a week or more after the calyx falls than say one day after it falls. In examining, the fruits that were killed in the southern portion of the State in that year, in very many cases no injury was found to the flesh of the fruit, but the seeds were killed. Where the rate of fall of temperature is not too rapid under laboratory conditions, it is generally the seeds that are killed at the highest temperature. In fact from a large number of observations upon the results of Killing of Plant Tissue by Low Temperature 287 freezing peaches in the laboratory, the tissue kills in the following order, beginning with the most tender: the veins surrounding the seed, the kernel, the flesh. When the peach has reached considera- ble size, the woody covering surrounding the kernel is most hardy. The greater tenderness of the seed may be accounted for by the difference in sap density. Thus the freezing point depression for the seed kernels, when they are large enough to separate from the seed practically, was found to be 0.765° C, while that for the same fruit with the kernels excluded was 1.075°. At Koshkonong and at Goodman in 1908, following the freeze of April 30th, the fruit was injured least on the young vigorously growing trees. Thus at Koshkonong in the orchard of Mr. \V. C. Paynter, weak trees in various parts of the orchard and trees on rocky, uncultivated portions had all the fruit killed, while vigorous trees further down the hill where the temperature must have been lower, as well as further up the hill, bore a full crop. In the case of killing of buds in winter, whether they have been started into growth or not, and in the case of killing of blossoms in spring in the southern part of Missouri, in some years the Elberta is one of the most tender varieties. Yet where it is the young fruit that is killed, the Elberta seems to be one of the most hardy varieties, at least other varieties were killed to a larger extent than Elberta in that year. Early varieties where the fruit was large at the time of the freeze uniformly killed worse than later ones. In Missouri, at least, early varieties do not bloom earlier than late varieties like Salway. In fact in ex- treme South Missouri such early Chinese Cling varieties as Sneed, Victor and Carman, on account of their longer rest period actually bloom later than late varieties like Elberta or Crawford's Late or Heath Cling, yet from the time of pollination there is much more rapid growth of the young fruit of the early varieties. Thus when the calyx tube is dropping from the Elberta peaches, the young Sneed will be much larger. As to means of handling the trees to avoid injur>- at blooming time or after fruit is set, there is not so much that can be done except that, as mentioned above, in the most southern portions of the peach belt it is possible, by increasing the vigor of the tree, to cause the blooming to be later and, therefore, the young fruit at any given tinu' after the bloom falls, to be smaller when the frost might come. This, of course, would not apply further North. Vigorous, trees may also have their fruit killed to a smaller extent because, in their tendency to make wootl growth, the truit develops apparently more slowly, at least fruit is practically alwaj's later in ripening on \ ig- 288 Missouri Agr. Exp. Sta. Research Bulletin No. 8 orous trees than on weakly growing trees. The whitewashing might also in some years have some effect on the amount of killing at bloom- ing time, though it would probably have very slight effect on the amount of killing of young fruit after the bloom has fallen. BREEDING VARIETIES HARDY UNDER SOUTH MISSOURI CONDITIONS The most important ultimate means of reducing the amount of injury to both fruit buds and blossoms from low temperatures is by plant breeding. From what has been said above, for this southern region the varieties that would seem the most promising to use in breeding for hardiness would be some of the Chinese Cling group. With but few exceptions, the varieties of this group are more hardy than the average peaches, not only for this southern peach belt, but fortunately for the northern section also. Elberta, however, is a marked exception. It may be said further that the Chinese Cling comes nearly enough true to seed that it has been very useful in securing new varieties with size and shippping qualities desired. The quality, however, is rather low and the color poor so it must be crossed with something that will give color, — preferably yellow, — and quality. It seems highly probable that desirable hardy varieties of long rest period could be secured by crossing this strain with some high quality yellow fleshed peach like Fitzgerald, etc. However, the Elberta, the most promising example of this crossing, has certainly been a failure so far as hardiness is concerned. The Gold Drop and Lemon Free, being peaches of yellow flesh, fair quality and very hardy in bud for northern or southern climates, and rather hardy in wood, is promising, though so far we have been unable in the first cross to secure yellow flesh. The Hills Chili group may be among the hardiest varieties in both wood and bud for south- ern as well as for northern climates, but the quality is poor and re- sistance to rot so slight that it is a question whether they will be de- sirable for use in developing new hardy varieties for market condi- tions. Reference to Table 41 will show that the Green Twig varie- ties have as long rest periods as those of the Chinese Cling group. In addition to this they are hardy because their pale color reflects the sunlight instead of absorbing it;^ yet their small size and indiffer- ent quality, together with the fact that in all crosses with Purple Twig varieties they have taken the typical purple color, would seem 'J. C. Whitten, Mo. Agr. Exp. Sta. Bui. 38, 1897. (Bibl. No. 114). J. C. Whitten, Das Verhaltnis der Farbc, etc.. 1902. (Bibl. No. 117). Killing of Plant Tissue by Low Temperature 289 to eliminate them as promising material for breeding hardy varieties of desirable quality. Hardiness of Seedlings. With reference to the opinion some people have that seedlings are, for some unaccountable reason, more hardy than budded fruits, it may be said that in the season of 1911 the Missouri Experiment Station was able to secure the percentage of buds killed by two different freezes, one before any buds had swelled and one after some swelling, from seedlings of a number of common varieties, the most important being seedlings of Chinese Cling, General Lee, Elberta and Family Favorite of the Chinese Cling group, Lewis, Early Michigan and Hills Chili of the Hills Chili group; and of the Snow, one of the hardy Green Twig varieties. The following table gives the results: 290 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Table 50. Showing the Relative Hardiness of Seedlings of Various Varieties of Peach as Indicated by Percentage of Buds Killed by a Temperature of -8" F. January 5, 1911, and by a Temperature of +6° F. Feb. 23, 1911. Percentage Percentage Percentage Number of of of Buds Variety of Buds Buds Alive Buds Killed Killed After Both Jan. 5, 1911 Feb. 23, 1911 Freezes Elberta 32 96.8 0.0 3.2 Elberta 89 79.8 7.8 12.4 Elberta 57 93. 5.4 1.6 Elberta 100 96. 4. 0.0 Elberta 133 66.9 9. 24.1 Elberta 36 100. 0.0 0.0 Elberta 107 80.4 12.2 7.4 Elberta 72 103 41.7 77.6 51.4 16.6 6 9 Elberta 5.8 Elberta 110 60 79.1 85. 17.3 13.5 3 6 Elberta 1.5 Elberta 28 44 60 100 96.4 88.7 66.3 84.0 0.0 2.3 20.0 10.0 3 6 Elberta 10 0 Elberta 13 7 Elberta 6.0 Elberta 38 52 54 94 78.9 71.1 98.2 61.7 2.6 9.6 1.8 8.5 18 5 Elberta 19 3 Elberta 0 0 Elberta 29.8 Elberta 9 55 100.0 81.8 0.0 10.9 0 0 Elberta 73.0 Elberta 26 88.4 11.6 0.0 Thurber 66 63 52 90 75.7 46.0 94.2 75.5 18.2 33.4 5.8 18.1 5 1 Thurber 20 6 Thurber 0 0 Thurber 6.4 Thurber 75 136 58 80 73.3 69.8 60.3 41.2 8.0 13.3 37.9 20.0 18 7 Thurber 16 9 Thurber 1 8 Thurber 38.8 Thurber 112 101 110 63.4 75.2 57.3 25.0 9.9 10.0 11 6 Thurber 14 9 Thurber 42.7 Thurber 32 128 56 71.9 71.1 57.1 6.2 6.2 42.1 21 9 Thurber 3 9 Thurber 10.8 Thurber 83 122 86 160 174 143 108 49.3 31.9 70.9 28.1 65.5 68.5 68.5 33.8 51.7 20.9 28.8 18.4 25.2 28.7 16 9 Thurber 16 4 Thurber 8 2 Thurber 43 1 Thurber 16 1 Thurber 6.3 Thurber 2.8 Thurber 63 82.5 12.7 4.8 Thurber 130 73.1 20.8 6.1 Thurber 150 67.3 22.0 10.7 Thurber 106 78.3 17.9 3.8 Killing of Plant Tissue by Low Temperature 291 Percentage Percentage Percentage Number of of of Buds Variety of Buds Buds Alive Buds Killed Killed After Both Jan. 5, 1911 Feb. 23, 1911 Freezes Thurber 48 77.1 18.8 4.1 Thurber 118 84.7 13.6 1.7 Thurber 120 56.6 28.4 15.0 Thurber 78 97.4 2.6 0.0 Thurber 188 75.0 22.3 2.7 Thurber 133 92.5 6.0 1.5 Thurber 134 70.9 15.6 3.5 Thurber 152 82.9 15.1 2.0 Family Favorite 116 82.7 16.4 0.9 Family Favorite 106 75.4 14.2 10.4 Family Favorite 72 86.2 6.9 10.5 Familv Favorite 86 74.4 15.1 10.5 Family Favorite 75 86.6 10.7 2.7 Family Favorite 108 59.3 32.4 8.3 Family Favorite 97 40.2 36.0 23.8 Family Favorite 107 74.8 15.9 9.3 Family Favorite 103 95.2 3.9 0.9 P'amiiy Favorite 100 94.0 3.0 3.0 Family Favorite 44 86.3 3.7 10.0 Family Favorite 185 48.1 44.3 7.6 Family Favorite 124 77.4 20.2 2.4 Family Favorite 98 79.5 19.4 1.1 Family Favorite 134 66.4 27.6 6.0 Family Favorite 122 72.9 24.6 2.5 Family Favorite 91 84.6 12.1 3.3 Family Favorite 132 72.7 25.8 1.5 Family Favorite 142 64.8 34.4 0.8 Family Favorite 145 79.3 20.0 0.7 Family Favorite 161 45.3 38.5 17.0 Family Favorite 133 73.7 24.8 1.5 Family Favorite 101 92.2 5.9 1.9 Family Favorite 154 70.1 28.5 1.4 Family Favorite 107 79.4 18.7 1.9 Family Favorite 124 86.3 12.9 0.8 Snow 281 33.8 51.6 14.6 Snow 170 32.4 61.7 5.9 Snow 290 145 265 231 224 232 298 226 161 34.5 40.7 35.6 33.7 40.0 43.5 25.1 28.7 74.5 58.6 43.4 57.8 57.8 58.0 54.3 61.4 60.0 24.9 6.9 Snow 15.9 Snow 6.6 Snow 8.5 Snow 2.0 Snow 2.2 Snow 13.5 Snow 11.3 Hills Chili 0.6 Hills Chili 118 77 133 153 105 83.0 79.2 77.4 67.3 50.4 16.1 19.5 19.6 32.0 44.7 0.8 Hills Chili 1.3 Hills Chili 3.0 Hills Chili 0.7 Hills Chili 14.9 Hills Chili 136 61.8 30.8 7.4 Hills Chili 136 111 135 55.1 54.0 57.0 40.4 34 . 3 31.1 4.5 Hills Chili 11.7 HilU Ciiili 11.9 292 Missouri Agr. Exp. Sta. Research Bulletin No. 8 i Variety Hills Chili Hills Chili Hills Chili Hills Chili Hills Chili ...... Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Early Michigan. Lewis Lewis Lewis Lewis Lewis Lewis Lewis General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . General Lee. . . . Chinese Cling. . , Chinese Cling. . . Number Buds 155 154 89 233 127 153 181 94 93 81 86 112 67 184 71 57 81 74 78 75 118 63 92 94 75 111 115 181 152 132 124 231 130 229 153 139 100 88 109 108 108 112 170 131 115 105 101 114 109 100 102 131 154 149 Percentage of Killed Jan. 5, 1911 53.5 55.2 77.5 49.8 51.1 91.0 47.0 57.4 48.3 39.5 55.8 62.5 43.2 27.2 40.8 33.3 61.6 37.8 42.3 90.6 31.3 50.8 38.0 57.0 82.7 65.8 40.9 43.0 51.9 73.5 62.1 37.2 50.0 65.9 73.2 35.2 47.0 58.0 86.2 71.3 38.9 44.7 22.4 60.3 63.4 32.4 39.6 77.2 75.3 78.0 50.0 77.1 63.6 62.4 Percentage of Killed Feb. 23, 1911 42.0 36.3 20.2 45.5 44.2 9.0 26.5 16.0 42.0 12.3 32.5 12.5 38.8 47.3 23.9 54.4 29.6 35.1 47.4 8.0 18.7 39.7 38.0 38.3 13.3 30.6 31.3 45.3 38.2 19.7 29.0 7.4 16.1 22.2 22.2 30.9 30.0 38.6 9.2 19.4 40.7 15.3 40.0 21.4 15.7 10.5 17.8 20.2 12.8 7.0 28.5 22.9 35.0 14.8 Percentage of Buds Alive After Both Freezes 4.5 8.5 3.5 4.7 4.7 0.0 26.5 26.6 9.7 48.2 11.7 25.0 18.0 25.5 35.3 12.3 7.8 27.1 10.3 8.6 50.0 9.5 22.0 4.7 4.0 3.6 27, 11. 9. 6. 8. 55.4 30.0 11.9 4.6 23.9 23.0 3.4 4.0 9.3 20.4 40.0 37.6 18.3 20.9 57.1 42.6 2.6 11.9 15.0 21.5 0.0 1.4 22.8 7 ,9 ,8 .9 Killing of Plant Tissue by Low Temperature 293 Variety Number Buds Percentage of Killed Jan. 5, 1911 Percentage of Killed Feb. 23, 1911 Percentage of Buds Alive After both Freezes Chinese Cling Chinese Cling Chinese Cling Chinese Cling Chinese Cling Chinese Cling Chinese Cling 118 204 64 123 121 176 219 37.2 33.3 84.4 54.5 39.6 34.6 34.2 28.1 20.0 12.5 32.5 38.1 28.4 13.2 34.7 46.7 3.1 13.0 21.3 37.0 51.6 Elberta, average 10.92 Thurber, average 11.5 Family Favorite, average Snow, average 1 5.4 8.7 Hills Chili, average 8.3 Early Michigan, average Lewis, average 19.9 10.4 General Lee, average 21.6 Chinese Cling, average 23.5 It should be said that the varieties of the Hills Chili group are usually less vigorous growing trees, and since these seedlings were very closely planted, trees of the Hills Chili group would suffer more from shade in summer and thus have a larger percentage of buds not fully developed that are killed during even a mild winter. It will be seen that as a general thing those varieties which are most hardy have given seedlings that are also most hardy. By referring to this table and to Table 39 it will be seen also that these seedlings were generally not more hardy than the parents, though of course in the case of Elberta — a cross between the rather hardy Chinese Cling and Crawford's Early, and tender like Crawford 's Early — some of the seedlings would tend to be more hardy than Elberta. KILLING OF APPLES Killing of the Roots. Killing of roots was not discussed for peaches because so far as can be learned, the killing of peach roots does not vary greatly from the killing of apple or of other fruit-tree roots. The killing of apple roots is probably a more common phe- nomenon than the killing of peach roots because peach roots are not so much more tender than apple roots as peach wood or peach fruit buds are more tender than apple wood or api^K- fruit buds. On ac- count of the greater tenderness of wood and fruii buds, the peach tree 294 Missouri Agr. Exp. Sta. Research Bulletin No. 8 is not grown commercially in as severe climates as those in which apples are sometimes grown, yet killing of roots of peach trees is observed in fairly important commercial peach districts.^ Macoun^ reports that killing of apple roots is fairly common in the northern portion of the apple growing section and says that the root killing they have had in Canada has been when apples were worked on seedlings of southern forms, and that since the crab has been used as stock, the killing has been much less. Emerson^ at the Nebraska Experiment Station planted apple trees in boxes two feet square and eighteen inches deep, each box hav- ing twenty-five young roots. The boxes were left out of doors about the middle of December, the soil having different percentages of moisture. The trees were examined on February 25. In an un- protected box containing 10.4 per cent of moisture, twenty roots were killed, only five remaining uninjured. Another unprotected box containing 15.2 per cent of moisture had nineteen killed and six injured. In a box containing 19.8 per cent of moisture, three were killed, ten injured and twelve uninjured. A box covered with straw mulch and containing 16 per cent of moisture had none of the roots killed, and only seven injured. A box covered occasionally with snow and containing 15.8 per cent of moisture had seven dead, eight in- jured and ten uninjured. No roots were injured in a box stored in a cool, dry cave, though it contained but 10 per cent of moisture. It is shown from this experiment that the low moisture content in itself did not do the harm, but the low moisture associated with low temperature. It does not seem, however, that one is justified in concluding, as Macoun apparently does, that plants at any given temperature kill worse in media with as small as 10 per cent of mois- ture than in media with 20 per cent of moisture. From experience here it would seem likely that the reverse is true. Careful tempera- ture records would probably indicate that the temperature was lower in the boxes containing the smaller moisture content, and a dry soil will freeze more deeply than will a moist soil. Thus a mulch or sod or cover crop that tends to prevent evaporation from the soil will prevent its freezing so deeply and thus prevent injury to the roots by keeping the temperature higher. Craig^ studied root killing in Iowa for the winter of 1898-99. The injury was worst in light dry soils not protected by a cover crop iStone. Mass. (Hatch) Exp. Sta. Rpt. 1911, p. 66. (BibL No. 107). Green, W. J. and Ballou, F. H., Ohio Agr. Exp. Sta. Bui. 157, 1894. (Bibl. No. 48). ^Canada Exp. Farms, Rpt. 1907-8, pp. 110-16. (Bibl. No. 68). sNebraslca Agr. Exp. Sta. Bui. 79. 1903.- (Bibl. No. 33). *Iowa Agr. Exp. Sta. Bui. 44, 1900. (Bibl. No. 25). Killing of Plant Tissue by Low Temperature 295 In case of light soils like the "loess" he found that deep planting helps to reduce the danger from root killing. Such soils would of course freeze more deeply and the temperature of the roots would be lower. He had little opportunity to observe directly the differ- ence in hardiness of different stocks, except in one case where Shield Crab was used as stock, three-year nursery trees of Jonathan, Whit- ney, Grimes and Willow came through the winter in good condition, though nursery trees generally were badly injured. In examining injured trees he found that the stock at the union of stock and scion was often killed while immediately adjoining scion tissue was uninjured. He found scion roots to be more resistant than stock roots. This is in accordance with experience here in freezing roots in the laboratory. We have also found that the root system becomes more tender as it gets further from the crown. Emerson and others have observed that roots of trees are killed at a much higher tempera- ture than are other tissues. This has been our experience. Results with freezing roots of orchard trees will be found in Tables 30, 31, 32, 33, and 34. In case of one-year-old roots of the French crab used as stock by most of the nurserymen, about 5° C.to -8° C. is as low a tempera- ture as they can be depended upon to withstand with no injury. Of 90 Jonathan apple grafts with French crab stock frozen toatempera- ture of -9° C. or lower and then planted in soil in the greenhouse, none lived. Four out of ten lived after being frozen to a tempera- ture of -8° C; three out of ten after a temperature of -7° C; six out of ten after a temperature of -6° C, and practically all grew after a temperature of -5° C. These roots had not been exposed to a high temperature during the winter preceding the freezing, which was done from March 2, to March 18, 1911. According to Craig and also Stone, when the roots are frozen, the results do not show to the inexperienced at once in spring. The trees will often bloom and usually the leaves will partially expand before the injury begins to show clearly. If only a part of the root system is killed, only certain connected branches will show the cfTects of the root killing. Injury to Apple Wood. Macoun' lists a number of forms of winter injury to wood of fruit trees, especialh' llu' apple, as follows: Bark splitting, which he says usually occurs when growth has continued late in autumn and an early summer has prt'\entO(l the soil from freezing. 'Canada Exp. Farms Kpt. 1907-8, pp llo-Ki. (Ilihl No. OS). 296 Missouri Agr. Exp. Sta. Research Bulletin No. 8 Trunk-splitting, sun scald, a killing of the bark on the south or southwest side of the tree which he reports very serious in north- ern and eastern Ontario and in the Province of Quebec. Killing-back which he says results from inherent tenderness of the variety, or from insufficient maturity of the wood. Crotch-injury, a killing of the cortex and perhaps other tissue in crotches of the limbs. Macoun, Morse, and Grossenbacher have studied this form of injury, and have found it to follow severe winters. Macoun and Morse attribute this injury to the lodging of ice in the crotch, while Grossenbacher attributes it to tearing caused by ten- sions developed by the shrinking of the tissue during its frozen con- dition. Grossenbacher observed this form of injury most commonly in the crotch formed by vigorously growing secondary branches. Black-heart, a condition which follows the killing of the sap wood when the cambium is left alive to form new wood outside the killed area. It is troublesome to nurserymen in northern sections. Trunk-injury or body injury, a killing of the older parts of the tree above the snow line. Macoun thinks this injury may be due to loss of water while the tree is frozen. Grossenbacher^ describes a similar injury as Crown Rot, and attributes it to tearing from ten- sion in the tree when frozen, and to a loss of water while frozen. He seemed to find it worse on the side of the tree trunk next to the prevailing winds. The experience of the Missouri Experiment Station where the wood of the body of the tree, especially at the base of the tree trunk and at the crotches, becomes hardy more slowly in autumn in some years at least than does the tissue of the secondary branches or even the twigs (see Table 24) may have some bearing in connection with some of the above forms of injury, especially crotch injury and body injury, or crown rot. It seems possible that those forms of injury are merely direct freezing to death and that such injuries are more commonly found at the base of the trunk, or at the crotch, because on such years these are the most tender points. Grossenbacher thawed the bark of a tree with warm water and worked the tree backward and forward when the temperature was -26° C. on January 8. In March practically all of the bark was found to be dead, showing apparently the characteristic browning of direct freezing to death. It would seem doubtful if this condition of com- plete death of the tissue with the browning would be found so soon >New York (Geneva) Agr. Exp. Sta. Tech. Bui. 23, 1912. (Bibl. No. 50). New York (Geneva) Agr. Exp. Sta. Tech. Bui. 12. 1909. (Blbl. No. 51). Killing of Plant Tissue by Low Temperature 297 if it were merely a mechanical injury such as tearing loose of the bark. In this case it seems highly probable that the killing resulted from rapid temperature fall. The thawed tissue would fall to the temperature from which it was thawed, very rapidly, and from ex- perience here with rapid freezing (see Table 20) it would seem proba- ble that this tissue was killed by direct freezing the same as buds would be on account of this rapid lowering of the temperature. Sun scald is generally thought to result from the effects of the heat gathered by the dark tissue that is in the direct sunlight. Since Macoun reports sun scald as being serious in climates as far north as the Province of Quebec, it would seem doubtful if there would be enough heat gathered on the sunny side of a tree trunk in winter there to raise the tissue to a temperature high enough for growth to take place. From experience here where the rate of temperature fall makes such a large difference in the killing, it seems highly prob- able that, at least in northern sections, sun scald may not result from the tissues starting into growth, but it more likely results from the temperature being raised to near the freezing point on sunny days during cold weather and dropping very rapidly to the tempera- ture of the air as soon as the sun is off that part of the tree trunk. As to varieties most hardy in wood under extreme conditions, Macoun^ reports an examination of about 700 varieties in which he found that summer and fall apples were generally more hardy in wood than late winter varieties, probably because their wood reaches a condition of maturity earlier. In his experience some of the most hardy varieties were: Canada Baldwin, Winter Rose, Ontario, Stockton and Mcintosh, all apples of northern origin. Oldenburg was cited by him as being one of the hardiest in wood. The group of apples which Hedrick lists as hardy for the coldest part of the state of New York are: of summer apples, Yellow Trans- I)arcnt, Tetofsky, Oldenburg, Red Astrachan, and Lowland Rasp- berry; and of fall and winter apples, Wealthy, Hibernal, and Fa- mcuse groups. Among the varieties that would not withstand the cold in the northern districts were: of summer apples, Early Harvest; and of fall and winter apples, Baldwin, Black Gilliflower, Jonathan, Rome, Winesap, and in the very coldest region, even the Northern Spy group. The Killing of Apple Buds. The buds of apples will withstand much lower temperature than will the buds of peaches or of even plums and cherries, and Macoun reports the killing of buds of apples 'Canada Exp. Frt. Farms. Kpt. 1900. pp. 291-92. (IJlbl. No. 70). 298 Missouri Agr. Exp. Sta. Research Bulletin No. 8 even in a very dormant condition. Whipple^ describes a considera- ble amount of killing he has observed in Montana. When apple buds are killed it is not necessarily true that all parts of the buds are killed; generally only the flower parts, so that the buds will open in the spring into a whorl of leaves and for this reason the fact that any killing occurred may not be observed. He found also that in some cases not even all the flower parts were killed, but on opening, various malformations were to be observed; thus in some the pistils were entirely absent and in some both stamens and pistils. In some cases seedless apples were developed from such flowers. Under Missouri conditions, especially in the Ozark region in the thinner soil, we have observed a considerable amount of killing by the freezes of December 9 and 29, 1909, when the temperature went to -5° F. and -8° F. respectively. During the same year buds of the York Imperial, as well as Jonathan, that had been largely killed by the same freeze, were sent in from points in Illinois. In most cases, however, the entire bud was killed. The Jonathan is the one variety in which a considerable amount of such injury has been observed. In low places where the cold air may settle and re- sult in a very low temperature, all of the Jonathan buds were killed, even on healthy trees. However, in the Missouri Experiment Sta- tion orchard following a temperature of -20° F. on January 7, 1912 not a large percentage of the buds were killed. Of 200 Jonathan buds counted, 38 per cent were found to have been killed, and of the same number of Ben Davis buds counted, none were found dead. Of course some buds in which the flower parts were injured may have been overlooked. However, a good crop was secured in that year. In this orchard the trees had been kept in a healthy condition, while practically all the orchards observed, and all the orchards from which twigs were sent, were neglected orchards where the trees had made a weak growth the summer before and had set their fruit buds very early, probably pushing them too far early in the season before this freeze. Killing of Apple Bloom. Killing of the flowers is a common form of injury to apples resulting from low temperatures, at least under Missouri conditions. An effort was made to determine what is approximately the killing temperature of the full bloom of apples. The following table gives the results of freezings with apple, peach and other fruits, just before the bloom opens, in full bloom, just after the bloom falls, and when the fruit is as large as it has been when it 'Montana Agr. Exp. Sta. Bui. 91, 1912. (Bibl. No. 113). Killing of Plant Tissue by Low Temperature 299 was killed in Missouri. The material was generally kept at the mini- mum for thirty minutes. The temperature fall was as slow as it could be made with the freezer used. Table 51. Giving Results of Artificial Freezing of Fruit Blossoms Material Apple, Ben Davis Peach, Elberta Seedling Pear, Kieffer Plum, Wild Goose Cherry Apple, Stannard Peach, Elberta Seedling Pear, Kieffer Plum, Wild Goose Cherry Apple, Ben Davis Peach, Rareripe Apple, Jonathan Apple, Jonathan Apple, Jonathan Cherry, English Morello Cherry, English Morello Apple, Rome Beauty... Peach, Hiley Cherry, Montmorency.. Apple, Jonathan Peach, Early Bernard. . Peach, Gold Drop Cherry, Dyehouse Plum, Wild Goose Pear, Kieffer Apple, Jonathan Peach, Lewis Cherry, Dyehouse Condition of Flowers When Frozen Not fully open Fully open. . . . Fully open Fully open. . . . Not fully open Fully open.. . . Fully open.. . . Fully open.. . . Fully open. . . . Fully open.. . . Fully open. . . . Petals falling. . Not fully open Fully open.. . . Petals falling.. Fully open. . . . Petals falling. . Fully open. . . . Young fruit . . . Fully open. . . . Young fruit Young fruit. . . Young fruit . . . Young fruit . . . Young fruit . . . Young fruit . . . Young fruit . . . Young fruit. . . Young fruit . . . Date Apr. Apr. Apr. Apr. Apr. Apr. Apr. Apr. Apr. lApr. 'Apr. Apr. Apr. Apr. Apr. Apr. Apr. May May I May May May May May iMay May May May IMay 14,'ll 14,'ll 14,'ll 14,'n 14, '11 15,'ll 15, '11 15, '11 15, '11 15, '11 15, '11 28,'13 28,'13 28,'13 28, '13 28, '13 28,'13 1,'13 1,'13 1,'13 10,'13 10,'13 10,'13 10,'13 10,'13 10,'13 17, '13 17, '13 17. '13 Tem- pera- ture 27.5 27.5 27.5 27.5 27.5 27.5 27.5 27.5 27.5 27.5 27.5 26.6 26.6 26.6 26.6 26.6 26.6 24.8 24.8 24.8 26.6 26.6 26.6 26.6 26.6 26.6 24.8 24.8 24.8 Num- ber Blossoms 87 66 40 60 97 45 87 70 50 91 61 86 100 78 120 130 107 83 76 40 53 74 65 44 93 32 19 23 IS Percent- age Killed 31.0 6.1 0.0 0.0 50.5 40.0 60.9 32.8 0.0 58.2 22.9 2.5 8.0 56.4 18.0 48.5 67.1 40.9 1.3 52.5 71.7 94.6 75.4 70.4 16.1 78.1 68.4 43.5 66.6 These artificial freezings are not accurate in determining the minimum temperature which the bloom, etc., will withstand, since it is not possible to duplicate in the freezer the rate of temperature fall, etc., outside, nor is it possible to make two freezings exactly duplicate each other. However, it is safe to conclude from this table that the unopened flowers are slightly more hard\' than the fully open ones, and that the fruits are slightly more tender than the flowers. The difference with apples, jutlging from these freezings and from our oliservations on freezings outside, is not so great as with peaches. 11 300 Missouri Agr. Exp. Sta. Research Bulletin No. 8 These results indicate that peaches, either in blossom or young fruit, will withstand slightly lower temperatures than will apples. The greater hardiness of the peach bloom is probably under-empha- sized in this table. In the year 1911 the Elberta Seedling peach tree came into full bloom on April 12, while the Ben Davis apple came into full bloom on April 18 to 22, and the Stannard apple on April 20. It is almost certain then that the peach blossoms listed as fully open were older than the apple blossoms so listed and had been pol- linated and had started rapid growth, while the apples had not. In 1913 the Early Bernard peach came into full bloom on April 11, and the Hiley and Rareripe on April 16-17, while Jonathan apple came into full bloom April 24, and the Rome on April 29. Here then the young fruits of the peach were older than were the young apple fruits used. From results of freezes as seen in the orchard, we are con- vinced that at times, at least, bloom and young fruit of peaches will withstand lower temperatures than will apple bloom or young fruit. The freeze of April 30, 1908, when the temperature in Columbia went to 28° F., killed only the percentages of young peach fruit shown in Table 49, and an excellent crop was secured from the peach trees the summer of 1908, while practically all the apple fruit was killed ; yet the peach fruit had been set longer. The peach trees in that year came into full bloom from April 5 to 7, while varieties of apples like Ben Davis, Jonathan and Grimes, fruit of which was all killed in this orchard, came into bloom from April 15 to 17. The Ingram apple in that year came into full bloom on April 22 to 24 and had some bloom left when the freeze came. It had a fair crop of apples left, and the Ralls, which came into full bloom from April 23 to 25 and had a considerable amount of open bloom when the freeze came, had very few fruits or blooms killed. If peach blooms or fruits are more often killed in spring than that of the apple, it is because peaches usually bloom considerably earlier. In avoiding a loss from low temperatures in spring, the most important factor is late blooming. Some varieties like Ingram and Ralls bloom so much later than ordinary varieties that, except in very few sections of the State, they are practically never killed by spring frosts. Other varieties that apparently are seldom killed under Missouri conditions are Benoni and Mother, according to in- formation furnished by Mr. F. W. Faurot, formerly of the Missouri State Fruit Experiment Station at Mountain Grove, Missouri. Growers in southern Illinois also report Benoni as being rather safe from frosts. Rome Beauty on many years blooms enough later than other varieties to escape^injury. Whether in the southern portion Killing of Plant Tissue by Low Temperature 301 of the apple growing region this blooming in spring can be caused to be delayed by prolonging the rest period, as in the case of peaches, has not been studied. Mr. Faurot however, tells of an instance in which cultivated trees were three days later in coming into bloom than uncultivated trees of the same variety, with like treatment in every other way. The fact that the buds on well kept Jonathan orchards on good soil were not killed to such a large extent by the freezes of December, 1909, as were the buds on trees in neglected orchards that had gone dormant much earlier the season before, seems to indicate that the buds in early winter, at least, are kept dormant later by keeping the tree vigorous. An opinion is held by some fruit growers and horticulturists that if the tree is in a vigorous condition the bloom or young fruit will withstand lower temperatures because of the healthier condi- tion of the bloom or young fruit. Three Gano apple trees on the Station grounds have been left unsprayed and uncultivated and are in a weak condition, one of them in a very weak condition. At times bloom or young fruit from these have been frozen along with bloom or young fruit from well kept Gano trees. These weak trees and the vigorous trees in Columbia bloomed at the same time. The following table gives the results: Table 52. Showing Relative Hardiness of Bloom and Fruit From Weak and From Vigorous Gano Apple Trees Material Vigorous sprayed tree Weak unsprayed tree Vigorous sprayed tree Weak unsprayed tree Vigorous sprayed tree. ... Weak unsprayed tree Very weak unsprayed tree Vigorous sprayed tree (fruit ■'h in. in dia.) Very weak unsprayed tree (fruit }4 in. in dia.) 1,'12 1,'12 1,'12 1,'12 4,'12 4,'12 4,'12 May 18,'12 May 18,'12 Tem- Num- pera- ber ture Frozen -3 28 -3 16 -5 50 -5 40 -2 52 -2 56 -2 46 -4 98 -4 79 Percent- age Killed 71.4 87.5 100.0 95.0 21.1 19.7 32.6 81.9 83.5 It will thus be seen that there is no constant difference in the hardiness of the fruit from the weak and the vigorous tree. Casual observers may mistake loss of crop from cool weather at blooming 302 Missouri Agr. Exp. Sta. Research Bulletin No. 8 time, poor pollination, or other reasons, for injury from freezing. It is certainly true that a poor set of fruit is often attributed to frosts when a careful observation following the frost would have shown that no bloom or fruits were killed. KILLING OF CHERRIES AND PLUMS Killing of various tissues of other fruits has not been extensively studied at this Station, except as described in the early part of this paper. As to killing of buds, Macoun^ states that under Canadian conditions, especially in the Province of Quebec when away from the protection of a body of open water, European and Japanese plums, and cherries are injured more or less every winter. He lists Mount Royal and Raynes as new varieties very hardy in bud. Japanese plum fruit buds have killed in Missouri only after cold periods fol- lowing sufficient warm weather to start them into growth. In case of killing by spring freezes, the Wild Goose plum is very resistant to low temperature as will be seen by reference to Table 51. On April 24, 1910, when all other fruits in the Station orchard, ex- cept Ingram and Ralls apples which were just in bloom, were killed by a temperature of 27° F. Wild Goose plums were uninjured though they had reached a diameter of 3-16 of an inch. We are not prepared to say, however, that the young fruit of the Wild Goose plum is not more hardy than the bloom. In the case of cherries observations have been made on the kill- ing of the Early Richmond cherry buds to a large extent, and other varieties to a small extent, especially by cold periods following warm weather. On January 7, 1912, the following percentages of fully dormant cherry buds were killed by a temperature of -20° F. : Early Richmond, of 200 buds counted, 52.5 per cent were killed; Mont- morency, of 150 buds counted, 12 per cent were killed; Dyehouse, of 175 buds counted, 11.4 per cent were killed. Macoun holds that fruit buds of cherry, peach and Japanese and European plum kill more easily than do the buds of the apple and the pear because they have less protection from evaporation. Goff^ found a larger percentage of cherry buds to survive a tempera- ture of -273/2° F- in Wisconsin in February, 1899, in the central por- tion of the tree than on the ends of the outer branches. He concludes that this results because those near the center of the tree were par- tially protected from drying winds. It does not seem probable >Canada Exp. Farms, Rpt. 1907-8.-pp. 110-16. (Bibl. No. 68). neth An. Rpt. Wis. Agr. Exp. Sta. pp. 283-88, 1899. (Blbl.iNo. 46). Killing of Plant Tissue by Low Temperature 303 that evaporation affects the killing of buds unless it be indirectly by the drying out of the twigs. Cherry buds are more tender than apple buds when exposed to sudden freezes like that at Columbia, Missouri, January 7, 1912. They are also more tender when frozen in the laboratory under conditions such that buds with the scales removed kill no worse than normal buds. Evaporation could cer- tainly play no part there. The buds at the ends of peach twigs are generally more tender than those near the base of the twigs or on spurs along the branches, but buds on the ends of twigs down in the tree are as tender as any. It is probable that the buds that Goff found more hardy toward the center of the tree were formed earlier in summer and were more mature when the freeze came. One of the most promising means of avoiding loss from low temperatures is by orchard heating, burning oil, coal or other material. In some sections this practice is followed profitably, and it is recom- mended in others. This subject is being studied at the Missouri Experiment Station by Dr. W. L. Howard. In the work here re- ported, no data has been gathered that is of value in a study of or- chard heating, unless the results with freezing bloom and young fruit may be of some value in determining the temperature at which heaters should be lighted. Data on the killing temperature of the bloom of fruit to be protected is very essential in orchard heating, otherwise the heaters will too often be lighted and large expense incurred when it is unnecessary, or perhaps sometimes they may not be lighted when it is necessary, and loss will be incurred. A study of orchard heating, like a study of the value of whitewashing as a means of preventing killing from cold, should be carried on through a large number of years since heating involves an expense certainly not less than an average of $10 or $12 per acre a year, besides a large initial investment, and the mere fact that the crop can be saved by heating is no positive indication that in a period of twelve or fifteen years enough crops will be saved, in spite of accidents inherent in the method of healing, to pay for the cost of heating. ACKNOWLEDGEMENTS This work was begun under the direction of Dr. J. C. Whitten of the Department of Horticulture of the Missouri Agricultural Exper- iment Station, and advice has been received from him throughout the work. Advice has also been received from Professor \V. L. Howard of the Department of Horticulture, Professors B. M. Duggar, C. Stuart Gager, and G. M. Reed of the Department of Botany; 304 Missouri Agr. Exp. Sta. Research Bulletin No. 8 and Dr. Hermann Schlundt of the Department of Chemistry of the University of Missouri has been repeatedly called upon for informa- tion where the work in this bulletin required knowledge of physical chemistry. Mr. A. J. Heinicke and other advanced students, who have from time to time been connected with the Department, have assisted in the laboratory in securing the data here published. All chemical analyses have been made under the direction of Professor P. F. Trowbridge of the Department of Agricultural Chemistry. Professor U. P. Hedrick of the Geneva, New York, Experiment Station and Professor H. P. Stuckey of the Georgia Agricultural Experiment Station, furnished twigs used in some of the freezings reported in this bulletin. Professor Hedrick and also Professor R. A. Emerson of Lincoln, Nebraska, were kind enough to furnish publications that were not available here, as well as repeatedly to give advice. 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Bui. 22, pp. 17-21. 1899. 120 .Wilson, W. M., Frosts in New York, N. Y. (Cornell) Exp. Sta. Bui. 316 1912. 121. Winkler, H., Ueber den Einfluss der Aussenbedingungen auf die Kalte- resistenz ausdauernder Gewachse. Jahrb. f. Wiss. Bot. Vol. 52, 1913, pp. 467-506. 122. Woodbury, C. G. and Wellington, J. W., Orchard Heating. Purdue Agr. Exp. Sta. Bui. 154. llG GENERA'ERATURE CURVES FOR GENEVA, NEW YORK, COVERI WES ARE F — wj M i>4 (^ ^7~ — -— -— Kj KJ 1*1 ^ r- ■"* >>* K* V, MO Feb.'au.'ll Feb. '11 "kTkTTJ ONONG, MO S» ^ - O, ^ Ci ^TS^^M ff^ 5 i 5 K pob.'07 Jan. '11 F«b •11 Dec. '11 Jan. '12 I>t<:-'n Jan.'lfi NG A SIMILAR PERIOD FROM Dec 08 Jan. '00 — *w OO Kj »\ Feb '09 GE^fEVA,N.Y. Doc.'OO Jtn.'io i*!j.'l(> M TEMPERATURE CURVES FOR GENEVA, NEW YORK. COVERI COLUMBIA. MO 10 JaiJ.'H ■ Feb. '11 J*a.'12 KOSHKONONG, MO "i3 j; ^ S - 1^ -o Ci rT? .'10 J»n.'ll F«b.'ll ^"■■11 Jan.MC LLY TO THE DATES I OR MAXIMUM AND AT WHICH BUDS WERE Kl LOWER CURVES FOR MIN COLUMBIA. MO 4x oo^i 5: S ^ U - ^ -«^ =3 :2 ti t; bS vj Dec. '07 Jiin.'OS Feb. '08 A£F Dec. '07 *^ Jan. '08 Feb. SUCH KILLING OCCURRED. ALSO MAXIMUM AND MINIMU Dec. '09 Jsin.'lO Feb.'lC KOSHKONOKO. MO *^ ^^ Q: Dec' 861 il IS 74 70 62 58 S4 SO 46 42 Si 54 30 2i, 22. li 14 10 6 '2 -2 -6 -10 -14 -IS -21 -2i> -3C .Sf£ Deo. '09 Jan. '10 Feb. MO De VLLY TO THE DATES FOR MAXIMUM AND AT WHICH BUDS LOWER CURVES WERE KILLED FOR MINIMUM N FEBRUARY WHEr TEMPERATURES. *^oo;i 5: g ^ ^ -3 ^ ^ o5 =5 1! t; ts Dec. '07 Ja.n.'08 K3 Feb. '08 Dec. '08 Jaa.' KOSHKONONO. MO ' ^ o ^ 00 Doc. '07 Jan. '08 "eb. t^ -j-o;:5KtJ^*- «*' D*c. '08 Jan.'( ^E SEASON BEGINNING DECEMBER 1, AND EXTENDING GENER^J JANUARY, 1908 TO FEBRUARY, 1910. UPPER CURVES ARE F A., MO Jan. '06 Feb. '06 Dec. '06 Ji^- ^ VJ VJ 04 £v - y> -o «*i — a — Feb.'C SHKONONQ. MO Jan. '06 Feb.'Oe Dtfc.'Oe Jfcn. 07 Fob. '07 /IBIA AND KOSHKONONG DURING THE YEARS 1901 TO 1912. AND INCLUDING T BIA, MO Jan. '04 Jan. '05 Feb. '06 COLUMflJ Dec. '04 Dec. '05 NONQ, MO i "r- — >J >^-> ^^n^.^ot--- t»4 ~) -^ Dflc ■^ — VJKJOJ^ — — Vj>o>J , — 04 Jan. '05 F«ib.'05 r CHART SHOWING MAXIMUM AND MINIMUM TEMPERATURE CURVES AT COLUT '01 Dec. Jan. '02 KOSHtfONONO -^ 313 MO S^^'^S-^^Si '-- -03 Feb. '03 KO.S I IKONONG, MO ^ C r-- •- ff^ O ^ o* 66 63 58 54 50 46 42 Zl J4 Xh XI \t 14 10 f +2 -2 -6 -10 -14 -/8 -22 •26 -JO f ., ;'• ■' ' ' Ml,., ' ■ , 1 , 1 , ^1 If .11 <• ft / coLvy Dec. '03 w^:^ J\:^:>:!^^*^o»^3- I}0c.*o^ J»n. '03 Ftb.'Oa NG ''^ 2798