A SEARCH FOR FACTORS DETERMINING WINTER HARDINESS IN ALFALFA ProQuest Number: 10008383 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008383 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 A SEARCH FOR FACTORS DETERMINING- WINTER HARDINESS IN ALFALFA By Clive R* Megee A THESIS PRESENTED TO THE FACULTY OF MICHIGAN STATE COLLEGE OF AGRICULTURE AND APPLIED SICNECE IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY East Lansing 19 3 5 ACKNOWLEDGMENTS The writer wishee to express his appreciation to Dr* C* E* Millar, Dr* G* J* Bouyoucos and Dr* C* H* Spurway of the Soils Department, Professor H. C* Rather of the Farm Crops Department, and Dr* R* P. Hibbard of the Botany Department for their helpful suggestions dur­ ing the course of the investigation of this problem and in the preparation of this paper* 102357 A SEARCH FOR FACTORS DETERMINING WINTER HARDINESS IN ALFALFA INTRODUCTION In the northern part of the United States winter kill­ ing of alfalfa is often prevalent and the seeding of winter hardy strains is of paramount importance in maintaining satisfactory stands* Field tests to determine the relative winter hardiness of the various strains of alfalfa have con­ tributed much toward enabling Michigan to become the second highest state in the acreage of alfalfa in the United States* The hardy strains of alfalfa, such as the Hardigan and Grimm are usually able to withstand low temperatures without winter killing while the non-hardy strains, such as the Arizona Common and Hairy Peruvian often are killed the first winter* There are many strains occupying an intermediate position of winter hardiness between these extremes* During the past 15 years the writer (14) has conducted many field tests to determine the relative winter hardiness of the various strains of alfalfa of the United States and many foreign countries* In order for a field test to reach a high degree of dependability it is necessary that it extend over a period of from 4 to 8 years and this involves a serious delay as well as considerable expense in conducting the test* While such tests have been of much value in determining the 2- relative winter hardiness of the various strains of alfalfa, they have in no way explained the phenomenon of winter hardi­ ness* Increase in colloid content and osmotic pressure of root sap, and decrease in moisture content of alfalfa tissue have been suggested as an explanation for winter hardiness* Should it be possible to locate some one or more factors definitely correlated with winter hardiness that could read­ ily be determined in the laboratory, this problem of the agronomist would be greatly simplified* The object of this paper is to present data on the rela­ tionship between winter hardiness of alfalfa and various properties of alfalfa root tissue as measured by electrical conductivity, moisture equivalent, swelling, heat of wetting of oven dry tissue, respiration, freezing point depression, moisture content and the rate of loss of moisture* Should any of the above factors prove to be directly correlated with winter hardiness, the fact would be an important step in determining and possibly explaining the phenomenon of winter hardiness* It is generally recognized that damage to plants may result from winter injury due to several causes, such as low temperature, heaving of soil and plants by the building up of layers of ice in the soil, suffocation under an ice crust, and the desiccation of plant tissues due to dry winds* In -3- this paper only the first form, winter injury due to low temperature, is considered. In the experiments here re­ ported, the other three forms of winter injury were almost, if not entirely, absent* REVIEW OF LITERATURE No attempt will be made to review the voluminous literature on the subject of winter hardiness, Harvey (12) gives a very complete bibliography on the low temperature relations of plants. Chandler (5), Dexter (6), Graber (10), Gortner (9), Harvey (11), Maximov (13), Newton (15) and Rosa (16) not only give their views but also have reviewed the work of other investigators. Dexter (6) upon mention­ ing the work of other investigators states; “It may be briefly stated that most, if not all, of the theories of winter hardiness are built around the idea of the water relation of the plants and that structural, osmotic, or colloidal protection from water withdrawal or ice forma­ tion form the basis of explanation for individual or varie­ tal differences in hardiness". MATERIAL USED Three strains of alfalfa of known winter hardiness; the Hardigan, Utah Common and Arizona Common, were selected for these experiments from among a large number of strains representing most of the seed producing sections of the world# Field tests of these strains for winter hardiness were previously conducted by the writer over a period of eight years# 1929# Plots seeded in 1931 were continued until Other plots seeded in 1922, 1923 and 1924 were con­ tinued for periods ranging from three to five years# Table 1 shows the comparative winter hardiness of these three strains under Michigan conditions# Table 1* Comparative winter hardiness as indicated by yield of Hardigan, Utah Common and Arizona Common alfalfas at Fast Lansing, Michigan# Yield per acre 100 100 * t 100 100 100 * 100 t 100 CO 1922 1923 1924 1925 1926 1927 1928 1o 1* 1* H „ co Yields of hay expressed in per cent of Hardigan yields Ave. * Ave. Tons 100 4.8 Hardigan 100 Utah Common 93.7 82.9 90.7 65.9 50.2 47.0 64.6 57.6 69.1 Arizona Common 63.3 10.8 19*6 11.7 21.9 0.0 0.0 0.0 15.9 3.3 0.8 The Hardigan is a variegated strain of alfalfa developed at the Michigan Agricultural Experiment Station and is very winter hardy under Michigan conditions# The Utah Common is a strain of only medium winter hardiness and has been grown for many years in the alfalfa seed producing section of the Uinta -5- Basin of Utah* The Arizona Common is from the Yuma Valley of southwestern Arizona and is one of the least winter hardy strains of alfalfa* It has been observed that alfalfa plants sometimes live through the winter entirely devoid of leaves* In view of this observation the material used in the laboratory tests consisted of the root and a portion of the crown, the top being removed just above the lower crown buds* Although containing a portion of the crown, this material is referred to as the root* The roots used were of the first season*s growth and were grown on a Hillsdale sandy loam soil* from both fertilized and unfertilized plots* Roots were used The amounts of superphosphate and muriate of potash necessary to apply to the soil in order to have a good supply of available phos­ phorus and potassium present as determined by the Spurway Method (17) were found to be equivalent to the nutrients in 3400 pounds per acre application of an 0-8-24 fertilizer* These materials were applied to a series of plots on May 29 and worked in as the soil was prepared for seeding which was done June 8. On September 4 a like application was made as a top dressing on another series of plots* On the same date an application of 500 pounds per acre of nitrate of soda as a top dressing was made on a third series of plots* The -6- fertilized plots were 25 x 300 feet and the varieties of alfalfa were seeded in quadruplicate plots 25 x 330 feet running across the fertilized and unfertilized plots* This arrangement provided roots of both hardy and non­ hardy alfalfas grown on fertilized and unfertilized soil* -7EXPERIMENTAL RESULTS THE RELATION BETWEEN WINTER HARDINESS AND THE LIBERATION OF ELECTROLYTES BY ALFALFA ROOT TISSUE EXPOSED TO LOW TEMPERATURE • It is a matter of frequent observation that as the liv­ ing protoplasm of a cell is injured it becomes more permeable and the electrolytes pass out of the cell* As the injury be­ comes more intense the permeability increases, and additional electrolytes are liberated* To determine the amount of elec­ trolytes liberated when alfalfa roots were submitted to low temperature, electrical conductivity determinations were made on water extract of roots of hardy and non-hardy alfalfas collected from fertilized and unfertilized plots* The method suggested by Dexter (6) was used. Extreme care must be exercised in preparing alfalfa field material for electrical conductivity tests* Hardigan, though one of the most uniform strains of alfalfa, is not a pure line selection owing to its method of pollination* Con­ sequently, some variations will be found in the winter hardi­ ness of different individual plants* soil cannot be secured for field work. Also an entirely uniform To eliminate these variations in so far as possible, plants were selected from various parts of the plot and after thorough washing and removal of the external moisture, the roots were cut into three-fourths inch pieces and the pieces thoroughly mixed so that the ten gram sample used would represent the plot as a whole * -8- The samples were then placed in Pyrex glass tubes and the tubes held in alcohol slush in a cold chamber* The alcohol slush was found to be very efficient in maintaining the material at a constant temperature, variations seldom exceeding one degree* Samples taken in October and November were frozen at -9° 0 for four hours, but as the season ad­ vanced it was found necessary to freeze later samplings for a longer period, or at a lower temperature* At the end of the freezing period 75 c.c* of distilled water were added and the tubes placed in a water bath maintained at 2° 0* Exosmosis was allowed to proceed for twenty hours, at the end of which time conductivity readings were made by means of a Wheatstone bridge* The Wheatstone bridge readings are expressed in specific conductivity (x 106) in reciprocal ohms (mhos) per gram of dry matter* Before making the read­ ing each tube was inverted three times to secure an equal distribution of the electrolytes through the solution* Students method was applied to data from triplicate samples and in most instances differences greater than 8 per cent were found to be significant* Electrical conductivity tests were made on samples of roots taken October 16, 1933, from the Hardigan and Arizona Common varieties from plots receiving no fertilizer treat­ ment and from plots receiving each of the fertilizer treat­ ments* Ho varietal or plot differences were found. Hardening -9- had not developed to a point where a distinction could be made between hardy and non-hardy alfalfas* On November 23, 1933, the test was repeated* The roots were frozen four hours at -9° 0 and 20 hours were allowed for exosmosis* Table 2* Effect of freezing on the liberation of electro­ lytes from roots of hardy and non-hardy alfalfas grown on fertilized and unfertilized soil* Test made November 23, 1933* Fertilizer Treatment Portion Root Used Hardigan Arizona Common Difference Per Cent mhos mhos (Upper 5 inches 37*0 53.7 45.1 (Lower&Fibrous 61.5 70.5 14.6 0—8—24 (Upper 5 inches 50.5 64.5 27.7 Sept.4 ILower&Fibrous 75.1 77.5 3.1 None The results, presented in Table 2, show that the Hardigan roots were much more resistant to low temperature than were those of the Arizona Common* Resistance to damage from low temperature developed most rapidly in the upper portion of the tap root while it was delayed at lower depths and in the small lateral and hair roots. Heavy applications of an 0-8-24 fertilizer on September 4 caused resistance to damage by cold to develop slowly* Ho difference was found in the average -10- air-dried weight of hardy and non-hardy alfalfa roots, consequently the weight of the root is not a factor in winter hardiness. In contrast to the results presented above, no differ­ ences were found in the winter hardiness of the alfalfa roots from the plots receiving no fertilizer, the roots from plots receiving fertilizer on May 29, and the roots from plots receiving nitrate of soda on September 4. The electrical conductivity tests were repeated on samples taken December 16, and December 18* The relation­ ship of Hardigan to Arizona Common and the effects of dif­ ferent soil treatments had not changed* Somewhat more resistance to liberation of solubles through freezing had developed, but just how much could not be determined because the roots dug December 16 were frozen for fifteen hours at -9° C and those dug December 18 were frozen for six hours at -15° C. Another lot of Hardigan roots was dug January 15, the object of this test being to determine the relative winter hardiness of the roots from plots receiving no fertilizer treatment with those from the plots receiving 2400 pounds of 0-8-24 on September 4, and also to determine the relative amounts of electrolytes liberated when the roots were not cut, cut into 3/4 inch lengths, into 3/8 inch lengths, and when ground. The roots were frozen 15 hours at -9° C and -11- 20 hours were allowed for exosmosis at 2° 0* After the determinations were made the samples were boiled for 15 minutes, 20 hours allowed for exosmosis at 2° 0, and the electrical conductivity redetermined. Table 3. Part of Roots Used The effect of freezing upon the liberation of electrolytes from roots of hardy alfalfa when grown on fertilized and unfertilized soil and when tap and fibrous roots were cut at differ­ ent lengths and when ground. Test made Janu­ ary 15, 1934. Size of Pieces Ho soil treatment & Frozen Frozen Boiled Fertilized Sept.4 Frozen Dif- fo Difference f erence Frozen & Frozen Boiled Boiled mhos mhos mhos mhos Tap Uncut 25.2 121.3 30.5 135.0 21.0 3.0 Tap 3/4 in. 28.7 137.2 34.7 139.5 20.9 1.6 Tap 3/8 in. 34.3 143.6 41.2 144.5 20.1 0.6 Tap Ground 52.8 151.5 60.5 153.2 14.5 1.7 Fibrous 3/4 in. 49.0 151.6 54 .7 155.2 U.l 2.3 Fibrous Ground 63.4 156.2 68.6 158.0 8.2 1.1 The results in Tables 2 and 3 show that the roots from plots receiving an 0-8-24 fertilizer on September 4 hardened less rapidly during November than the roots from the other plots, but during late December and January they hardened more rapidly* The injurious effect of low temperature upon alfalfa roots receiving fertilizer during September would be -12- determined largely upon whether the soil temperature was low during the fall or whether it was low during the winter* During 1933-1934 the soil temperature was not low until after January 15 and all plots of Hardigan came through with very little, if any injury. None of the Arizona plots had been injured to any appreciable extent by January 15, but winterkilled 99$ by spring. The test ran January 15 showed that all plots of alfalfa had made a considerable increase in winter hardiness since November 23* This in­ crease was greater than is shown by comparing the figures in Tables 2 and 3, since the roots dug November 25 were frozen four hours at -9° 0, while those dug January 15 were frozen 15 hours at -9° 0 and the latter liberated less electrolytes. Data in Table 3 also show that grinding tap roots and grinding and cutting fibrous roots failed to bring out dif­ ferences in winter hardiness by the electrical conductivity method to the extent that was brought out with uncut roots and with tap roots cut in lengths of 3/4 inch and 3/8 inch. The results secured after boiling show that the greater liber­ ation of electrolytes from the roots from plots receiving fertiliser on September 4 was due to less hardening and not to an excess of salt stored in the roots. The results secured when the tests were repeated during the season 1934-35 were the same, except that the amount of electrolytes released -13- after 'boiling was somewhat more variable, likely due to the extreme drought of the summer of 1934* This fact did not change the relationship of one variety to another or of one plot treatment to another in comparison with the results of 1933-1934♦ The electrical conductivity test was found to be valuable in determining the relative winter hardiness of different alfalfas. -14- PART IAL CHEMICAL ANALYSIS OF HARDY AND NON-HARDY ALFALFA ROOTS A chemical analysis* of the roots was made to determine whether there was a relationship between winter hardiness and the chemical composition of the roots# The results of the chemical analysis are shown in Table 4# Table 4# A comparison of partial chemical analysis of roots of hardy and non-hardy alfalfas# Treatment Variety 1° Hardigan Arizona C# Hardigan Arizona 0# Hardigan Arizona C* None None 0-3-24 May 29 0-8-24 Sept.4 7 #39 7.26 7.15 7.22 7.22 7.04 Ave rage 1* Ash CT HgO Protein **! Roots dug October 16, 1934 * Crude Fiber N #F #E# C H 0 * t * 15.60 14.99 14.57 14.63 15.16 14.29 2.11 0.66 2.61 3.32 2.04 2.63 2.80 0.77 0.63 0.48 0.69 0.39 16.68 18.04 14.34 17.02 14.60 17.33 57.56 56.33 59.99 58.69 59.70 58.15 74.24 74.37 74.35 75.71 74.30 75.48 14.37 2.58 0.60 16.33 58.40 74.74 Roots dug December 20, 1934 Hardigan Utah C. Arizona C. Hardigan ) Utah C. ) Arizona C.) Hardigan Arizona C. None None None 0-8-24 May 29 0-8-24 Sept.4 Average 5.27 6.56 5.92 5.63 5.98 6.03 5.46 5.76 19.08 17.71 16.34 17.81 17.50 15.71 17.98 16.41 2.33 2.25 2.17 2.87 2.53 2.63 3.16 2.85 1.76 1.59 1.47 1.32 0.97 1.32 1.99 1.28 23.39 21.19 25.68 18.30 19.02 21.25 19.74 23.84 48.27 50.70 43 .42 54.57 53.65 53.41 51.67 49.86 71.66 71.89 74.10 72^37 72.67 74.66 71.41 73.70 17.31 2.59 1.46 21.55 51.32 72.80 •The writer appreciates the assistance of the Chemistry Section of the Michigan Agricultural Experiment Station in determining the chemical analysis# -15- The chemical analysis of the alfalfa roots afforded no positive indication concerning factors causing winter hardiness. There are, however, some consistent differ­ ences in the protein and crude fiber content of the Hardi­ gan and Arizona Common roots. On December 20, the date when a certain degree of winter hardiness had developed, the protein content of the Hardigan was higher than that of the Arizona Common and the increase in protein content at this date over that of October 16 was greater in the case of the Hardigan than for the Arizona Common. The crude fiber content of the Hardigan alfalfa was lower in all cases than that of the Arizona Common at both dates of sampling and the crude fiber content of Arizona Common in­ creased markedly over that of the Hardigan between the two dates. The sum of the protein and nitrogen free extract content of Hardigan was slightly but consistently greater than that of the Arizona Common. THE MOISTURE CONTENT OF THE ROOTS OF HARDY AND NON-HARDY ALFALFAS The moisture content of many winter hardy plants de­ creases as the fall and winter seasons advance. In order to determine the relationship between winter hardiness and moisture content, the amount of moisture in the roots of Hardigan and Arizona Common strains of alfalfa was determined -16— at intervals during the fall and winter months. trically controlled oven maintained at 90° this purpose* Table 5* 0 An elec­ was used for The results are presented in Tahle 5* The moisture content of the roots of hardy and non-hardy alfalfas from October to February. Date Hoots Dug 1934-35 Per Cent Moisture Hardigan Arizona Common Oct. 24 66.7 67*0 Hov. 19 60.0 61.3 Dec* 22 62.9 63.0 Jan. 1 62*4 62.3 Jan. 21 64.0 64.0 Feb. 68*4 68.5 Feb. 20 68.2 66.7 Growing 76.4 76.8 Average 66*1 66.2 6 The results show that no varietal differences in the moisture content of the roots existed neither during the fall nor winter months nor when the roots were in a grow­ ing condition. Hardened roots of winter hardy strains of alfalfa were low in moisture, as were the roots of the non­ hardy alfalfas. Consequently, there is no significant cor­ relation between winter hardiness and moisture content* -17THE HATE OF LOSS .OF MOISTURE FROM THE ROOTS OF HARDY AHD HOH-HARDY ALFALFAS Protection against water withdrawal has been advanced as of importance in preventing winter injury. In case this is true the Hardigan should release its moisture more slowly at low temperatures than should the less winter hardy Ari­ zona Common. The relative loss of moisture of the roots of "both the Hardigan and Arizona Common was determined. Hardened living roots were dug February 30, and slowly reduced in mois­ ture content in desiccators held at 3° C and at -9° C. The samples were weighed daily and the daily loss of moisture cal­ culated. The results are shown in Table Table The rate of loss of moisture from roots of hardy and non-hardy alf alfas held at 3 ° C and -9° C « 1 . . Held a1i 2° C. Held, at -9° C. Total Loss ner day % Total loss *lo Loss -msr day % Hardi­ Ari­ Hardi­ Ari­ Hardi­ Ari­ Hardi­ Ari­ gan zona C. zona C. gan gan zona C. zona C. gan 21.0 14.0 14.0 21.0 23.0 18.3 18.2 23.0 CO m o H Ho. days 6 6 3 33.8 32.4 10.8 11.4 25.6 27.0 11.6 8.8 3 48.2 45.8 14.4 13.4 35.6 38.8 10.0 11.8 4 59.0 57.4 10.8 11.6 43.8 48.0 8.2 9.2 5 62.4 62.6 3.4 5.2 47.6 52.4 3.8 4.4 6 64.0 65.4 1.6 2.8 48.6 53.4 1.0 1.0 7 64.8 66 .2 0.8 0.8 60.0 62.4 11.4 9.0 8 65.0 66*4 0.2 0.2 63.0 63.8 3.0 1.4 9 65.4 66.8 0.4 0 .4 65.0 64.6 2.0 0.8 10 65.6 66.8 0.2 0.0 66.8 65.2 1.8 0.6 11 66.0 67.4 0.4 0.6 67.4 66.0 0.6 0.8 13 68.2 69.2 2.2 1.8 68.6 66*0 1.2 0.0 -18On the eighth day samples of roots of both Hardigan and Arizona Common were removed from the desiccator held at 3° C. The roots were transplanted into flower pots filled with sandy soil and held at favorable conditions for growth. Growth was resumed, although 65 per cent of the original weight of the roots had been lost due to the loss of moisture. The results show that both hardy and non-hardy alfalfa roots can withstand a heavy loss of moisture without being severely injured. The results also show that there is no consistent difference between hardy and non-hardy alfalfas in either the rate of loss of moisture or total moisture lost, and that the loss of moisture was not correlated with winter hardiness. THE RELATIVE RATE OF RESPIRATION OF ROOTS OF HARDY AHD HOH-HARDY ALFALFAS Respiration is the process of energy release in the living cell. The end products of aerobic respiration are CO3 and water and the intensity of respiration may be de­ termined by measuring the amount of COg liberated over a given period of time. Plants become dormant as the fall and winter seasons advance and the process of energy re­ lease slows down. In order to determine the relationship between winter hardiness and the rate of energy release, the relative rate of respiration of hardy and non-hardy alfalfas was deter­ mined. The roots were placed in 500 c.c. Erlemeyer flasks -19- fitted with rubber stoppers and in-take and out-let tubes. The tubes were kept stoppered except when connected to the train for carbon dioxide determinations. set up as follows: The train was air pump, potassium hydroxide solution, ascarite, phosphoric anhydride, concentrated sulphuric acid, sample, distilled water, potassium hydroxide* cate twenty gram samples were used. checked repeatedly for leaks. Dupli­ The apparatus was Each sample was connected into the train for five minutes, a previous test having shown that the air was completely changed in two minutes* The results are shown in Table 7* Table 7, The relative rate of respiration of hardy and non-hardy alfalfa roots held at different tem­ peratures. Condition of Hoots Temperature Centigrade Average Daily Liberation of COg Hardigan Arizona Common Grams Grams Hot hardened 21° 0.1151 0.1185 Hot hardened 10° 0*0163 0.0157 Hot hardened 2° 0*0139 0.0129 Hardened 15° 0.0400 0.0384 Hardened 2° 0.0145 0.0140 The results show no difference in the rate of respirtion between roots of hardy and non-hardy alfalfas. -20THE RELATION BETWEEN WINTER HARDINESS AND THE PHYSICO-CHEMICAL BEHAVIOR OF THE COLLOIDAL MATERIAL IN ALFALFA ROOTS The great importance of colloidal materials in the func­ tioning of living matter immediately raises the question of the relationship between winter hardiness and the physico­ chemical nature of the colloidal material in hardy and non­ hardy alfalfas* Moisture equivalent (3), swelling (4), and heat of wetting (2 ) afford good indices of physico-chemical differences of colloidal materials* The above mentioned indices were used to study the relationship between winter hardiness and the nature of the colloidal materials present in hardy and non-hardy alfalfas* Roots of the hardy and non-hardy alfalfas from plots receiving the various fertilizer treatments were dug at dates ranging from October 21, to December 17* These roots were thoroughly washed, dried at air temperature, and ground to a very fine meal* Each sample was thoroughly mixed to fa^- cilitate sampling* THE MOISTURE EQUIVALENT OF COLLOIDAL MATERIAL OF THE ROOTS OF HARDY AND NON-HARDY ALFALFAS The moisture equivalent was determined as follows: A Buchner funnel fitted with a filter paper was filled with ground roots and the funnel tapped gently twenty times to secure a uniform settling of the material* The funnel was then placed in a beaker of water for four hours and the -21- excess water removed by means of suction filtration for fifteen minutes* The material was then removed from the filter, weighed, and placed in an electrically controlled oven held at ninety degrees Centigrade for sixteen hours* The material was then weighed and the amount of water adsorbed was calculated* Table 8 * Table 8 * The results are presented in The moisture equivalent of the colloidal material in roots of hardy and non-hardy alfalfas* Fertilizer Treatment Date Kind Applied *34 Date Hoots Dug 1934 Moisture Equivalent Arizona Utah Hardigan Common Common per cent 200 -- 199 0-8-24 May 29 187 -- 180 6 0-8-24 Sept. 4 201 -- 194 6 Nitrate of Soda Sept. 4 207 -- 198 219 --- 222 May 29 221 --- 215 --- 263 261 265 None Nov* 6 Nov. Nov. 1 ca 6 1 CD -- O 182 Nov. ct * per cent May 29 21 0 per cent 172 Nov. 24 None Nov. 24 0-8-24 Dec. 17 None Dec. 17 0-8-24 May 29 266 262 252 Dec. 17 0-8-24 Sept. 4 256 264 269 Dec. 17 Nitrate of Soda Sept. 4 260 265 259 — - -22- The results show a marked change from late October into December in the nature of the colloidal material present in the roots of both hardy and non-hardy alfalfas# Winter hardiness is not correlated with this change in the nature of the colloidal material, since the change takes place to the same extent in both hardy and non-hardy alfalfas# THE SWELLING OF COLLOIDAL MATERIAL IN THE ROOTS OF HARDY AND NON-HARDY ALFALFAS A 5 gram sample of material was weighed out and placed in a 50 c.c ♦ graduated cylinder which was then filled with distilled water to the 50 c.c. mark. The material was stirred to secure uniform wetting, shaken, and enough water added to again fill to the 50 c.c. mark. Care was exercised in securing cylinders of the same diameter and calibration# The cylinders were then placed in a chamber maintained at 10° C for 16 hours, at the end of which time readings were made. - Table 9. 23 < The swelling of the colloidal material of the roots of hardy and non-hardy alfalfas during the fall and early winter months* Fertilizer Treatment Date Kind Applied *34 Swelling Utah Hardigan Common Arizona Common c *c. c.c* c.c. Sept. 4 24.0 — 24.5 --- 27.0 -— 26.5 Nov* 6 None Nov* 6 0-8-24 May 29 26.5 --- 27.0 Nov. 6 0-8-24 Sept. 4 27.0 --- 27.0 Nov* 6 Nitrate of Soda Sept. 4 27.5 --- 33.5 --- 33.0 May 29 33.0 --- 31.0 --- 34.0 34.0 34.0 w 1 21 CD Oct, 0 1 Date Roots Dug 1934 — 26.0 Nov. 24 None Nov. 24 0-8-24 Dec* 17 None Dec* 17 0-8-24 May 29 33.5 34*0 33.0 Dec. 17 0-8-24 Sept* 4 35.0 36.0 35.0 Dec. 17 Nitrate of Soda Sept. 4 34*0 33.0 35.0 34.1 34.2 34.3 Average Roots Dug Dec • 17 The results show a change from October 21 to December 17 in the physico-chemical nature of the colloidal material pre­ sent in the roots* This change is shown by the increase in the swelling of the colloidal material. The seasonal change -24— from October 31 to December 17 was the same for the nonhardy as for the hardy alfalfas. did not influence the change. Fertilizer treatments These results support those secured by the moisture equivalent index, in that winter hardiness is not a direct result of certain physico-chemi­ cal changes in the colloidal material of the alfalfa roots. THE HEAT OF WETTING OF COLLOIDAL MATERIAL IN THE ROOTS OF HARDY AND NON-HARDY ALFALFAS A 20 gram sample of air-dried material was placed in a wide glass tube and allowed to dry in an electrically heated oven at a temperature of 90° C for 16 hours. The tube was then removed from the oven, closed tightly with a rubber stopper, and placed on the desk close to the calori­ meter, a liter of distilled water, and another sample of material, so that all samples and equipment would reach the same temperature. 100 After a uniform temperature was established c.c. of water was placed in the calorimeter and the heat of wetting of the plant material ascertained (2). The tube was handled with a heavy cloth so that heat would not be imparted to the material. Great care was taken to have the calorimeter, the water, and the material at the same tempera­ ture which was that of the room. -25- Table 10. Date Roots Dug *34 Nov. 1 Nov. 1 The heat of wetting of colloidal material in the roots of hardy and non-hardy alfalfas. Fertilizer Treatment Date Kind Applied *34 None 0-8-24 Dec. 17 None Dec. 17 0-8-24 Heat of Wetting Arizona Hardigan Common Calories Calories 100.0 101.8 103.0 100.0 131.0 131.2 129.3 128.1 --- May 29 --- May 29 A change in the physico-chemical nature of the colloid­ al material from November 1 to December 17 was shown by the increase in the heat of wetting. both hardy and non-hardy alfalfas. The change was similar for These results support those secured by both moisture equivalent and swelling de­ terminations. Neither moisture equivalent, swelling, nor heat of wetting were found to be an index of winter hardi­ ness in alfalfa. THE SOLUBLE MATERIAL IN ROOTS OF HARDY AND NON-HARDY ALFALFAS AS SHOWN BY THE FREEZING POINT METHOD The concentration of the material in solution in the plant cell influences substantially the intake, outgo, and translocation of materials in plants. The freezing point method was used to determine the relation between winter hardiness and the quantity of soluble -36material in roots of hardy and non—hardy alfalfas# A 5 gram sample of air-dried root material was placed in the freezing tube and 20 c.c* of distilled water was added* The contents of the tube was stirred thoroughly and al­ lowed to stand for twenty minutes* The freezing point was then determined (l), making use of a freezing bath with a temperature of -2.5° C. The contents of the tube was super­ cooled about one degree before solidification was induced. Table 11. Date Roots Dug 1934 Oct• 21 Freezing point depression of roots of hardy and non-hardy alfalfas during October, November and December. Fertilizer Treatment Date Ap­ Kind N-P-K plied »34 0-8-24 Sept. 4 --- - Freezing Point Depression Utah Arizona Hardigan Common Common -°0 — °0 —°C 0.56 --- 0.56 0.61 - — 0.60 Nov. 6 None Nov. 6 0-8-24 May 29 0.60 — Nov. 6 0-8-24 Sept. 4 0.62 ----- 0.61 Nov. 6 Nitrate Soda Sept. 4 0*66 — — 0.63 0.64 — — 0.63 0.68 --- 0.67 0.52 0.53 0.53 , — 0.57 Nov. 24 None Nov. 24 0-8-34 Dec. 17 None Dec. 17 0-8-24 May 29 0.71 0.70 0.69 Dec. 17 0-8-24 Sept* 4 0.67 0.68 0.66 Dec. 17 Nitrate Soda Sept. 4 0.68 0.67 0.67 May 29 --- -27- The soluble material in a small number of undried samples was determined by the freezing point method. Immediately upon digging, the roots were washed and ground after removing the external moisture. minations were made without addition of water. The deter­ The freez­ ing point depression of the Hardigan roots averaged -1.56°C and of the Arizona Common -1.50° C when the roots were dug December 17. The results showed little difference in the concentration of soluble material in the roots of hardy and non-hardy alfalfas when measured by the freezing point method. This method cannot be used, therefore, to measure the winter hardiness of strains of alfalfa. DISCUSSION The relative winter hardiness of the various strains of alfalfa has been quite successfully determined by means of field tests. In order to insure a high degree of ac­ curacy, the field tests must extend over a long period of time. A simple laboratory test for predicting winter hardi­ ness would aid the agronomist in determining the relative winter hardiness of the different strains of alfalfa and also assist in explaining the phenomenon of winter hardiness. In this paper are presented data from comparative studies of roots of hardy and non-hardy strains of alfalfa including -28- determination, by electrical conductivity, of soluble material liberated through submission to low temperatures; chemical composition; moisture equivalent, swelling, and heat of wetting of finely ground root material; amount and rate of loss of moisture; and respiration* These are fac­ tors which it was thought might in part determine or explain the phenomenon of winter hardiness* The electrical conductivity method was found useful in measuring the relative degree of hardening which had taken place in the alfalfa roots at the time the tests were con­ ducted* Ho differences in hardening between hardy and non- hardy alfalfa were found on October 16, however, marked dif­ ferences were found on November 23, and at later dates* The varietal differences agreed with the previously conducted field tests* Hardening was found to develop most rapidly in the upper portion of the tap roots, while it was delayed in the lower portion and in the small lateral and hair roots* A heavy application of an 0-8-24 fertilizer on September 4 caused hardening to develop much more slowly during November and December* Even though the electrical conductivity method was of value in determining the relative winter hardiness of dif­ ferent lots of alfalfa, it did not offer an explanation of the phenomenon of winter hardiness* -29- Three hypotheses may he suggested to account for the different degrees of winter hardiness found in the differ­ ent strains of alfalfa. !• These hypotheses are as follows: Winter hardiness may he caused hy a physico-chemical difference in the roots of hardy and non-hardy alfalfas, due to the condition of the material (whether colloidal, in solution, etc*) and composition (whether present as sugar, starch, and protein), and the structure of the tissue present* Factors such as heat of wetting, swelling, moisture equivalent, freezing point depression, chemical analysis, and the amount and rate of loss of moisture afford good indices of physico-chemical differences* These indices showed that there were no physico-chemical differences present in the roots of hardy and non-hardy alfalfas, therefore, affording no support for this hypothesis* 2* Winter hardiness may be caused by bio-chemical or functional differences brought about by the secretion of sub­ stances such as enzymes and hormones, and these differences reflected in the energy release in the cell protoplasm* The rate of respiration was used as an index of energy release and was found to be the same for both hardy and non-hardy al­ falfas, The work of Dexter (8 ) and Tysdal (19) support these results* The acceptance of this hypothesis is not justified* 3 . Winter hardiness is an hereditary factor transmitted from generation to generation* This hypothesis has not been -30worked out in detail by the geneticists and plant breeders* However, the behavior of succeeding generations of Hardigan, G-rimm, Utah Common, Arizona Common, etc*, in transmitting different degrees of winter hardiness affords sufficient evidence to warrant the acceptance of this hypothesis* COHCLUSIGHS A search was made to find factors that could be used in the laboratory to predict the relative winter hardiness of alfalfas* The results showed that the relative degree of injury of alfalfa, roots by low temperature was indicated by electrical conductivity and in this manner the relative winter hardiness of different lots of alfalfas was determined* Ho direct relationship was found to exist between winter hardiness and heat of wetting, swelling, moisture equivalent, freezing point, chemical composition, respiration and amount and rate of loss of moisture in roots of hardy and non-hardy alfalfas* Heredity is the most plausible explanation of the phe­ nomenon of winter hardiness* For the time being it is necessary to adhere to field tests aided by electrical conductivity tests for determining the relative winter hardiness of different lots of alfalfas. 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