GUSTAFSON-RIBOFLAVIN SYNTHESIS

5. GUSTAFSON, F. G. Synthesis of B-vitamins by ex-cised parts of white lupine seedlings grown insterile culture. Arch. Biochem. Biophys. 52: 190-196. 1954.

6. LEOPOLD, A. C. Auxins and Plant Growth. Pp. 1-354.Univ. California Press, Berkeley, California. 1955.

7. SKOOG, F., SCHNEIDER, C. L., and MALAN, S. Inter-action of auxins in growth and inhibition. Amer.Jour. Bot. 35: 782-787. 1942.

8. THIMANN, K. V. and BONNER, W. D., JR. Experi-ments on the growth and inhibition of isolatedplant parts. The action of several enzyme inhibi-tors on the growth of Avena coleoptile and onPisum internodes. Amer. Jour. Bot. 36: 214-221.1949.

9. WHITE, P. R. A Handbook of Plant Tissue Culture.Pp. 1-277. Ronald Press Company, New York.1943.

THE INFLUENCE OF RATE OF COOLING AND WINTER COVER ON THEWINTER SURVIVAL OF LADINO CLOVER AND ALFALFA"12

MILTON A. SPRAGUEDEPARTMENT OF FARM CROPS, NEW JERSEY AGRICULTURAL EXPERIMENT STATION,

NEW BRUNSWICK, NEW JERSEY

The loss of perennial forage legumes during thewinter season continues to be a major problem. Muchis still unknown concerning the winter climates towhich plants are exposed and their relationship todamage from low temperatures.

This study deals with winter temperature varia-tions of the air near the surface of the soil and ofladino clover stolons where they are covered withgrass or snow and where uncovered. In addition, theinjury to alfalfa and ladino clover plants exposed tosimilar temperature variations was determined in thelaboratory. An interpretation of the results fromthese two studies offers a better understanding of therelationship between fall stubble, surface mulches, andsnow cover and winter survival.

METHODS AND PROCEDURELadino clover (Trifolium repens L.) and alfalfa

(Medicago sativa L.) were used as test plants be-cause of their prominence in agriculture. Stolons ofladino clover used in the laboratory studies wereselected for uniformity and good vigor. They weretaken from the field less than 36 hours before testingand were trimmed to 10 cm in length from the tip-end bud. Each sample consisted of 8 stolons. Threereplicates were used in all treatments. Cold hardenedplant materials consisted of 2 clones of ladino cloverpreviously selected for differences in cold hardinessplus field run ladino clover from Oregon certified seed.

Alfalfa used in laboratory freezing studies wasmostly seedling material of the Buffalo variety sownAugust 20, 1951. Plants were dug from the field inmid-winter, 1952, washed in cold water and trimmedto root lengths of 10 cm and stem lengths of 2.5 cm.Eight roots made up each sample with 3 replicates foreach treatment.

All plant samples were dug from the field, washed,trimmed and immediately inserted into large test

1 Received April 22, 1955.2 Paper of the Journal Series, New Jersey Agricul-

tural Experiment Station, Rutgers University, the StateUniversity of New Jersey, Department of Farm Crops.New Brunswick, New Jersey.

tubes. They were then stored in a cool room at 34 to360 F for 24 hours before applying freezing treat-ments. After freezing all samples were stored at360 F for 24 hours before being planted in quartzsand in the greenhouse. The growths made during 21days were used as indices of the amount of injury.

EXPERIMENT 1: During January, 1951, tubes con-taining ladino clover stolons from the cold hardyclone (No. 5) and from the less hardy clone (No. 3),were taken from the cool room and placed in an alco-hol bath at 360 F. The temperature of the bath wasthen lowered at a rate of 20 F per hour. Successivesamples were later moved from the cool room to thealcohol bath when the temperatures had reached 30,25, 20, and 150 F. All samples were removed fromthe alcohol bath when the stolons had reached 100 Fexcept one set which was put in at 360 F and removedwhen the temperature had reached 8° F. An addi-tional group of samples was not frozen.

EXPERIMENT 2: Buffalo alfalfa plants were re-moved from the field on January 3, 1952, stored at340 F and subjected to the same freezing treatmentsas the ladino clover in experiment 1. Additional sam-ples were exposed to slightly lower temperatures(50 F) in consideration of greater cold hardiness ofalfalfa.

EXPERIMENT 3: The principle treatments in ex-periments 1 and 2 were repeated with alfalfa andladino clover during February, 1952. Additional tem-perature ranges were included to test plant tolerancesto low temperature more completely. The alcoholbath was cooled at the same 20 F per hour rate from360 F. Replicate samples of ladino clover and ofalfalfa were removed from the cool room at intervalsand put into the freezing bath when it had reached36, 20, 15, and 100 F. Samples were removed whenthe bath had reached 15, 10, 5, and 00 F.

Samples which were put into the alcohol bath be-tween 36 and 300 F and lowered to the temperatureat which the samples were removed at the rate of20 F per hour are referred to as receiving the "slowfreezing" treatment. By comparison, those samplesremoved from the cool room storage at 36 or 340 F

447

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

and put into the alcohol bath when it was 15, 10 or

50 F are referred to as being "fast frozen."FIELD TEMPERATURE OBSERVATIONS: Air tempera-

tures were determined during January and February,1953, 1954 and 1955 using shielded copper constantanthermocouples and a recording potentiometer. In1954 and 1955 plant temperatures also were deter-mined with very small thermocouples made from No.30 B. & S. guage wire for insertion into the tissue.The tissues were punctured at an oblique angle witha small needle and the thermocouple inserted. Tem-peratures were recorded continuously night and day,on some occasions, and during critical periods on

others.

RESULTS AND DISCUSSIONLABORATORY FREEZING STUDIES: Fast freezing con-

sistently inflicted more injury to cold-hardened alfalfaplants and ladino clover stolons than did slow freez-ing. In two other similar experiments not reportedthe results were the same. Stolons, roots and crowns

which were cooled slowly withstood lower tempera-tures for a longer period of time without dying thantissues cooled rapidly. This comparison was consist-ent with both clones of ladino clover but the injuriessustained were less with the hardy than with the lesshardy clone (table I).

In experiment 2, the injury to over-winteringparts of cold-hardened alfalfa was less when theplants were cooled slowly to 50 F than when cooledrapidly to 100 F. Further, those plants cooled slowlywere exposed to freezing temperatures more than 4times longer than those cooled rapidly (table II,fig 1). Accordingly, the time of exposure to belowfreezing temperatures did not appear to be critical inthese trials.

In experiment 3, the alfalfa roots and crowns ex-

hibited greater tolerance of low temperature than theladino clover stolons (table III). In general, slowcooling permitted both plants to survive approxi-mately 50 F colder temperatures than did fast cool-ing. Alfalfa plants cooled rapidly to 50 F were allkilled; whereas those cooled slowly to the same tem-

TABLE IIRECOVERY GROWTH OF BUFFALO ALFALFA PLANTS DURING3 WKS IN THE GREENHOUSE FOLLOWING COLD TREAT-MENTS OF DIFFERENT RATES OF COOLING. ALL SAMPLESWERE STORED OVERNIGHT IN A COLD ROOM AT 340 F

BEFORE AND AFTER FREEZING

MINIMUM TIME IN AVERAGE RECOVERY GROWTHTEMP FREEZING RATE OF DRY WT ROOTS

REACHED BATH COOLING PRODUCED ALIVE

OF hr °F/hr gm %Not frozen 0 ... 1.76 100.0

10 12 2.0 0.67 97.510 10 2.4 0.76 100.010 7.5 3.2 0.74 91.310 5 4.8 0.43 66.210 2.5 9.6 0.15 50.05 14.5 2.0 0.59 100.05 5 5.8 0.05 1.65 2.5 11.8 0.08 2.1

perature made fair recovery. Similarly, ladino stolonscooled rapidly to 100 F were killed and those cooledslowly to the same temperature made good recovery.

The exact interval of time during which any ofthese plant tissues were below a given temperaturewas not determined. However, subsequent plant tem-perature measurements indicated that above 250 Fequilibrium to within one degree of the temperatureof the alcohol bath was reached within a few minutesafter a sample was inserted. With samples put intothe alcohol bath at 200 F or lower, plant tempera-tures dropped rapidly to 270 F and presented ap-proximately a straight line cooling curve thereafter.Accordingly, the principles of fast and slow coolingset forth in this paper remain intact.

During January and February, 1953, small thermo-couples were inserted into 4 roots and 4 crown budsof alfalfa and their temperatures recorded at 30-sec-ond intervals while the plants were cooling. Sampleswhich were cooled slowly from 36 to 100 F exhibitedfrom 4 to 60 F of undercooling. A freezing point of26.5 to 27° F was observed. All plants lived. Noundercooling was detected in samples cooled rapidly

BLE IRECOVERY GROWTH OF LADINO CLOVER STOLONS DURING 21 DAYS IN THE GREENHOUSE FOLLOWING

COLD TREATMENTS OF DIFFERENT RATES OF COOLING. ALL SAMPLES WERE STOREDOVERNIGHT IN A COLD ROOM AT 360 F BEFORE AND AFTEER FREEZING

RECOVERY GROWTHMINIMUM TIME IN AVERAGE

TEMP FREEZING RATE OF DRY WT PRODUCED STOLONS ALIVEREACHED BATH COOLING

CLONE 3 CLONE 5 CLONE 3 CLONE 5

° F hr ° F/hr gm gm % 0

Not frozen 0 ... 1.06 0.88 87.5 85.410 13 2.0 0.53 .66 61.0 76.910 10 2.6 .73 .68 68.7 81.510 7.5 3.5 .37 .55 19.0 68.510 5 5.2 .05 .31 11.2 61.010 2.5 10.4 .00 .08 1.3 17.38 14 2.0 .59 .76 54.7 61.7

448

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

449SPRAGUE-WINTER SURVIVAL OF FORAGE LEGUMES

TABLE IIIRECOVERY GROWTH OF LADINO CLOVER AND BUFFALOALFALFA DURING 3 WKS IN THE GREENHOUSE FOLLOWINGCOLD TREATMENTS OF DIFFERENT RATES OF COOLING. ALLSAMPLES WERE STORED OVERNIGHT IN A COLD ROOM AT

360 F BEFORE AND AFTER FREEZING

DRY WT OF TOPMINIMUM TIME IN AVERAGE GROWTH PRODUCED

TEMP FREEZING RATE OFREACHED BATH COOLING LADINO ALFALFA

CLOVER

0F hr 0F/hr gm gm

Not frozen 0 ... 0.98 1.5115 10.5 2.0 ... 0.9310 13 2.0 0.28 0.545 15.5 2.0 0.03 0.450 18 2.0 0.00 0.00

15 2.5 8.4 0.13 0.5210 2.5 10.4 0.00 0.145 2.5 12.4 0.00 0.00

to 100 F, and the freezing point was indistinct. Allplants died.

These observations were repeated with Grimmalfalfa, African alfalfa, and ladino clover stolons in asimilar manner during January, 1954. In these trials,the temperature of the tissues was determined at15-second intervals and of the cooling bath and airinside the large test tubes every 3 minutes while slowcooling was taking place. Although the temperaturesof the African alfalfa and ladino clover tissues werewithin 0.50 F of those of the surrounding air above270 F, this relationship at no time existed at tempera-tures between the apparent freezing point and 100 F.The temperature differences between the air and thetissues were greatest (6 to 70 F) about an hour afterthe maximum point of undercooling was reached (fig2). As the temperatures of the less hardy legume

FIG. 2. Temperature differences between plant tis-sues and the cooling medium (air) under conditions ofslow cooling through the freezing point.

tissues again began to drop after undercooling theydid not rapidly approximate those of the surroundingair as in the case of the more cold hardy Grimmalfalfa. Rather the tissues cooled slowly and con-tinued several degrees warmer than the air inside thetubes for several hours. This relationship was ob-served in numerous tests with alfalfa roots and ladinoclover stolons. Differences were less under conditionsof fast cooling than with slow cooling.

The Grimm variety of alfalfa is well known for itscold hardy characteristics; whereas, African alfalfahas consistently shown less winter-hardiness. Thelarge differences between the curves of these two (fig2) suggest that the water in the live tissues of thesevarieties may be in different phases.

Resistance to cold injury of plants has frequentlybeen associated with greater bound water content andosmotic concentration of the cell sap (1). The dataobtained in this study tend to support these associa-tions. It is suggested that the apparent freezing point

FIG. 1. Recovery growth of alfalfa following freezing treatments of (left to right) no freezing, slow and fastcooling. Experiment 2. January, 1952.

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

PLANT PHYSIOLOGY

of about 270 F observed in these trials is that of ad-hered water free of most colloidal bonds. Further,the apparent lag in temperature fall of cooled tissue,is a response to heat of crystalization from progres-sive freezing within the tissue and varies in propor-tion to the rate of freezing. Each successive amountof water removed from the cell by freezing increasesthe concentration of the remaining cell sap and in-creases the bond strengths of the remaining boundwater in proportion to the amount of water lost. Thisconcept recognizes no single freezing point but pre-sumes that crystalization under conditions of slowcooling is a continuous process of freezing freedwater. Under conditions of rapid cooling water maybe unable to move out of the cell at a sufficientlyrapid rate to prevent lethal ice formation within thecell (3).

CLIMATIC OBSERVATIONS: Fluctuations in air tem-perature near the surface of the soil are of greatermagnitude and are more rapid than at the standardheight of 4.5 feet at which the U. S. Weather Bureautemperatures are obtained. This is accounted for bythe ground surface representing the source heated by

PMJANUARY 28a,U155

FIG. 3. Characteristic daily temperature fluctuations

of air and clover stolons, without snow cover, (luringmid-winter at New Brunswick, New Jersey. 1955.

DEGREESF.

ao-

40-

30_

2O0

10

.{j

ZO 29 30 31JANUARY

MAXIMUM

MINIMUM

' AIR 4 1/2 FT.

SrOLON UNDER MULCH

I STOLON UNCOVERED

OVERCAST SNOW IINOW1 2 3FEBRUARY

FIG. 4. Daily maximum, minimum and mean tem-peratures of air and c!over stolons in the field underconditions of no cover, a grass mulch, and fresh snow.New Briinswick, New Jersey. 1955.

incoming radiation during the day and, by outgoingradiation, the cold source during the night (2). Inwinter, the stolon level in the field (0 to 1.5 cm abovethe soil surface) may be within a cover of grassstubble or snow or may be uncovered. Daily tem-perature fluctuations of both air and of plant tissuesobserved at New Brunswick, during January andFebruary, 1953, 1954, and 1955, indicate that stolontemperatures reach greater extremes than air tem-peratures and temperatures within a cover are farmore stable than where no cover exists (fig 3). Onclear days air and plant temperature changes at thestolon level were much greater and more rapid wherethere was no cover than under snow or a grass stubble(fig 4). Further, on frequent occasions, the uncoveredstolons cooled from 50 to 100 F in approximately 5hours, a range and rate approximating that foundinjurious in laboratory tests. On cloudy days andnights stolon temperatures more closely approximatedair temperatures.

On some clear days uncovered stolons remainedabove freezing for 4 to 5 hours and reached maximaof 45 to 550 F. On the same days the maximum airtemperatures at 137 cm above the ground were from22 to 250 F, and the temperatures of stolons beneatha 5-cm grass mulch seldom exceeded 300 F. On clearwinter nights the temperatures of uncovered stolonscooled more rapidly than, and reached minimum tem-peratures from 10 to 150 F lower than, stolons be-neath a mulch (fig 4).

The surface of a grass or a snow cover absorbsradiant energy during the day and also insulates theplants beneath to av'oid high maximum temperatures.Similarly these surfaces are cooled by outgoing radia-tion at night but the insulating properties of a grassor snow cover decreases cooling of the living plantmaterial beneath it. Plant temperatures beneath aslittle as 4 cm of snow were more stable than anyothers observed (fig 4).

In plots where these temperature observations

I*1450

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

SPRAGUE-WINTER SURVIVAL OF FORAGE LEGUMES

were made, ladino clover over-wintering in a 10-cmstubble of orchardgrass made good growth in spring,whereas nearly all of the stolons not covered with amulch were dead.

SUMMARYIn laboratory studies cold-hardened ladino clover

stolons and alfalfa roots and crowns were injured lessseverely when cooled slowly (20 F/hr) from 36 to 10and 50 F than when cooled rapidly (7 to 100 F/hr)to the same temperature. Undercooling of 4 to 60 Fwas observed with slow cooling and a freezing pointof 270 F was observed. Undercooling was not de-tected under conditions of a rapid drop in tempera-ture and the freezing point was indistinct.

Variations in mid-winter plant temperatures in thefield on clear davs and nights were in excess of thoseemployed in the laboratory and the rate of coolingapproached that found lethal. On cloudy days andnights stolon temperatures were more stable. Highestdaily maximum and lowest daily minimum plant tem-peratures existed where no grass or snow cover was

present to shield the clover stolons. Daily maximumtemperatures of clover stolons frequently exceeded450 F while air temperatures 137 cm above the soilremained from 22 to 250 F. Growth in spring indi-cated that ladino clover stolons overwinterin(g in agrass stubble survived well but nearly all were killedwhere no cover was present.

Acknowledgment is due Dr. E. R. Biel and Prof.A. V. Havens of the Department of MIeteorology forcooperation in microclimate observations.

LITERATURE CITED1. LEVITT, J. Frost Killing and Hardiness of Plants.

Pp. 125-126. Burgess Publ. Company, Minneapo-lis, Minnesota. 1941.

2. SPRAGUE, V. G., NEUBERGER, HANS, ORGELL, W. H.,and DODD, A. V. Air temperature distribution inthe microclimate layer. Agron. Jour. 46: 105-108.1954.

3. STUCKEY, IRENE H. and CURTIS, OTIS F. Ice forma-tion and the death of plant cells by freezing.Plant Physiol. 13: 815-833. 1938.

IRON, THE LIMITING ELEMENT IN A CHLOROSIS: PART I.AVAILABILITY AND UTILIZATION OF IRON DEPEND-

ENT UPON NUTRITION AND PLANT SPECIES1,2

J. C. BROWN AND R. S. HOLMESSOIL AND WATER CONSERVATION RESEARCH BRANCH, AGRICULTURAL RESEARCH SERVICE,

U. S. DEPARTMENT OF AGRICULTURE, BELTSVILLE, MARYLAND

Iron is the limiting element in a chlorosis whichdevelops in plants grown on some naturally calcareoussoils. This chlorosis is caused by the failure of thesusceptible plants to absorb iron from the soil or, insome cases, to utilize the iron which is absorbed (1,2, 3, 8, 9, 10). The causative factors which act inthe soil and plant to inactivate iron are not clearlyunderstood, although several concepts relative to thisproblem have appeared in the literature (7, 11, 4,14). Hewitt (8) reported a chlorosis in plants, typi-cal of iron deficiency, which developed upon supply-ing a given level of cobalt, copper, zinc, nickel, chro-mium, manganese, or lead ions to sand cultures.Willis and Piland (16) found that copper affected theabsorption and utilization of iron in corn and cotton,and Reuther et al (12) observed the same thing incitrus.

In this investigation, copper affected the absorp-tion and utilization of iron in corn, but it had noaffect in other plant species. When grown on cal-careous soil plant species also differed in their sus-ceptibility to an iron-deficiency chlorosis. Results re-ported in this paper show that iron metabolism differsbetween plant species and varieties and that these dif-ferences are dependent upon the growth media of theplant.

1Received May 6, 1955.2 This work was supported in part by funds contrib-

uted by the Atomic Energy Commission.

METHODS, MATERIALS AND RESULTSABSORPTION OF IRON BY SEVERAL PLANT SPECIES

DIFFERING IN THEIR SUSCEPTIBILITY TO CHLOROSIS:In a previous study (5) plants were grouped accord-ing to their susceptibility to iron or copper deficiencyas follows: 1) Those plants which developed an iron-deficiency chlorosis when grown on a naturally cal-careous soil, but did not develop copper-deficiencysymptoms when grown on an organic soil low inavailable copper; 2) plants which developed copper-deficiency symptoms when grown on the organic soilbut did not develop iron-deficiency chlorosis on thecalcareous soil; 3) plants which sho*ed retardedgrowth, slight deficiency symptoms in some cases,when grown on either the cafcareous or organic soils;and 4) plants which showed no chlorosis or deficiencysymptoms when grown on either soil. A plant ivasselected to represent each of these groups respec-tively: 1) PI-54619-5-1 soybeans, Glycine Max, 2)Thatcher spring wheat, Triticum sativum, 3) Minne-sota Hybrid No. 800 corn, Zea Mays, and 4) Hawkeyesoybeans, Glycine Max). These four plants wereused to study the absorption and translocation ofiron (Fe55-59) as affected by two soils and two mix-tures of these soils: 1) 3.2 kg calcareous soil, pH 7.9,2) 1.A kg calcareous soil and 0.8 kg organic soil, pH7.5, 3) 0.8 kg calcareous and 1.0 kg organic soil, pH7.2, 4) 1.8 kg organic soil, pH 6.0, 5) 1.8 kg organicsoil plus 0.37 gm CUSO4 * 5 H20. The calcareous soil

451

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.