Multiple Freezing Points Test for Viability in the ... · variables such as moisture content, sample size, thermocouple placement, and cooling rate on freezing curves was analyzed

Plant Physiol. (1969) 44, 37-44

Multiple Freezing Points as a Test for Viability of Plant Stemsin the Determination of Frost Hardiness-R. C. McLeester2, C. J. Weiser, and T. C. Hall3

Department of Horticultural Science, University of Minnesota, St. Paul, Minnesota 55101

Received July 11, 1968.

Abstract. A technique is presented for a simple, rapid, and reliable means of determiningthe viability of plant tissue subjected to freezing temperatures. Freezing curves of excisedstems of Cornas stolonifera Michx., and several other genera were studied. Tissue temperaturewas recorded during freezing of plant stem sections. The heat of crystallization deflected theresultant freezing curves at points where tissue froze. Living stem sections of all generastudied revealed 2 freezing points, while dead tissue exhibited only 1. The influence ofvariables such as moisture content, sample size, thermocouple placement, and cooling rate on

freezing curves was analyzed. Stem samples wrapped in moisture-proof film with a thermo-couple inserted into the pith were frozen to a predetermined test temperature, thawed, andsubjected to a second freezing cycle. The presence or absence of 2 freezing points in thesecond freezing cycle was used as a criterion for establishing viability. The results were

immediately available and identical to results from regrowth tests which took about 20 days.

It is difficult to determine whether many planttissues are dead or alive following exposure tofreezing temperatures (18). Techniques for deter-mining viabilitv bv such criteria as regrowth (11,20). vital staining (10, 19, 23), fluorescence (8),electrolyte (3, 26) or metabolite leakage (9, 23),metabolic activity (14, 23), plasmolysis-deplasmolysis(19, 21) or macroscopic symptoms of damage (3,12, 25), are frequently time-consuming and in somecases unreliable.A plot of tissue temperature as a function of time

produces curves which are deflected by the heat ofwater crystallization at points where plant tissuesfreeze ('13). Studies have indicated that living planttissues usually exhibit 2 (12), or more freezingpoints (7). while dead tissues exhibit onlv 1. In1937 Luvet and Gehenio (12) reviewed early- in-vestigationis of this phenomenon by Maximov,Zacharowa, and Walter and Weismann. All work-ers verified that double freezing points were char-acteristic of manv herbaceous plant tissues. Aokiet al. (1). and more recently Hudson and Idle (7),and Hatakeyama and Kato (5), further describedthe miiultiple freezing points of plant tissues. Theyfound that seasonal differences in freezing curves didnot provide a reliable means of predicting resistanceof plant tissues to freezing stress. Luyet andGehenio (12) stiggested that the absence of multiplefreezing points in dead tissues might be used as a

1 Scientific Journal Series Paper No. 6627, MinnesotaAgricultural Experiment Station. This research was sup-ported in part by a grant from the Louis W. and MaudHill Family Foundation.

2, 3 Present Address: Horticulture Department, Uni-versity of WNisconsin, Madison, Wisconsin 53706.

criterion for determining viability. Unltil now, re-search has not been conducted to evaluate this possi-bility.

The influence of tissue hvdration, sample size.thermocouple placement, and cooling rates on thefreezing curves of excised dogwood stems wasevaluated. Based on these studies. a standardfreezing curve viability test was developed and testedon 11 other genera.

Materials and Methods

Experimental samples were stem sections fromcurrent season's growrth of a single clone of red-osierdogwood (Corni us stolonifera Michx.). Stems wereobtained from plants grown in the field, greenhouse,or growth chamber. During the course of the study,700 to 800 individual freezing curve viability testswvere conducted. Data reported are typical modelcurves of individual tests.

Temperatuire Recording. The temperature of ex-cised stem sections during freezing was measuredwith 26 gauge chromel-constantan thermocouples in-serted 1 centimeter into the pith. Temperatureswere recorded on an adjustable span Barber-Colemanmultipoint recording potentiometer (Model 8461-2700-000-75). A chart speed of 122 cm per hr wasused with a 15.6 sec interval between successivetemperature recordings. The span was adjusted sothat 28 cm was equal to 180.

Samizple Preparation. For each test, uniform1-year old twig sections 5 cm in length and about8 mm in diameter were collected from a single plant.Each twig was tightly w-rapped in a 3 X 10 cmpiece of Saran and secured with cellophane tape.

37

Dow

nloaded from https://academ

ic.oup.com/plphys/article/44/1/37/6090423 by guest on 23 July 2021

38PLANT PI1'YSL)LOGX

A tiernliCcou)le xas. theln inlserte(l thironigi the Sarallinto p)itll at the ctut encd of tile twig. Individual twigsectionl.s w-ere placed ill sci)lrate precoole(l ( 1°)thei-illos bottles. A\botut '3) cimi of tile tlermllocoui)lelead was placed inside the thlrilmos bottle to rediiceheat loss Vz'ta the wire (lurilig freezing. Bottles werelai(d on their side during freezing to minimiilize tileeffects of tenilperatnre strattificatioI.

FrcciZvn Cvcles. For tile llr(liillss t'Lest, sail)leswN-ere frozeln to a test tenilpr-attire wlile t\w-ig teil-peratnlre was recordled. thien tile twig Wa thllawed.To test for- freezing inlnrlvsaulpies were snlbiectedto a secon(1 freezing cycle. If a curve ind(icatillnviablilitv resulted. it was evi(leilt tllat tile tisslne Wascoi(l acclinillte(d to a low-er tClil)era-tilre tilall tha;t ofthe first cvcle. By fr-eeznllg Illl( sachilng si Illiles. tosuccessively lower test tenlilperaturse, tile killin- pointco0ild le (deternliledl.

Sn1percoolilng WaIIsC always associated witil tile firstfreezilg point FT) ), tlil- thait point was eLsv toidlelltifv. Tlhe seconId( freez-ng point (jF.), (al ll(Otlgllnot as (listinct. couli lI e reco(lllize(l aLs (lefecctionof the freezin- cuirve cIlue to tile secol(i p)erio(ld ofcrvstali ization. Si ltitg(ifdwvi tile curve revealedtile secoldi freeziigjpoilnt to le ill tile vicinity of tileiilflection poilnt of the signlloi(l portioll of tIle cirvefollowing tlhe F-1, plateatu.

I i(ability Evalutations. A fter tile secoid(I freezilngcycle, thermocouples were reilloved and twigs wereslowlyv thawed. Thawed twigs were pliaced in aglass chamlber Nvith a wN-atel- saturated atmosphlere atroomi temperature. After 4 (lays, the twigs w-ereexamllilled for dliscolorationl anild l)reakdlownl of in-jtired lark, and their viahilit -v status was suibjectivelyratedi. Tlle bark was slice(i to exanllille wliethierinternlal discoloration of tile cortex, pllioelll or cam-biunii areas ilad Cccurrd(l. -fter- 20 days ill tileltiuilidl cilalaber, the twigs. \\w'ere score(l for callus oradventitiotis root grow-tll. (iroowthl wvas tile iltinliatecriterioll of viahiiitv.

7Tissic SieC and Hviratio 0. Silice tile tests de-peild tipon tile freezing of tissue filids, tile totalIiloistiure ill tissue is ilillportant. Variation ill tilesliape of the freezing curve.s was inlvestigated fortw\igs of differelit leulgtll 'ndl diallleters, and alsofor twigs of the salne size blilt fr-om11 elvirollnilentairegillles wxilich gave rise to (lifferelit levels of tissuenlloistlire.

.A coniilarisonl was nlladle bletweell () Sallld)eS frolmi2 group)s of Corinius stoloniifcerai plilats. One groupxvas sub-irrigated for 3 d(ays prior to saill)ling tomlaintain the soil nloisture niear fiel(d cal)acitv; tileother- group was not wvateredl. -Moisture lo s (llirillgthe freezing cycles was los ldeteriiniiie(d in 3 ul-wrapped aIld 3 Saran wr-rapped salmlles froni eachlgroup by weighilng each salllple before aand after the2 freezing cycles. After tile secoind cycle tlle steillswere dried to constalnt weight at 370 to dleteriillethe total moisture contelit of samlpies froml the 2

environmental regimes. Altiouglh Iiiglier dryingtenlperatures remove greater amotunts of moisture.

70 alpllarently reilmoved free cell wIter relsonlsilbl(for the mlaoIjOrchanges lioted in tile freezing clrves.

Highl levels of tissue hlvdrationl were obtained bvs-.oakin- iiilifol-ril twig, sectionls in (listiltlled water.Twigs collecte(d in jalnuar frolil a hlard lalailt iivtile field( weret soake(l fol 0, 2. 24. cr 48 llr lbeforefreezillng tests. rissu.e.l llloizturl-e w-. (leterili+e(l aslpreviousl (ldscrii)c(i.

Thecrmiiocouiple I'lactmciiici. Tlhe illfluien,ce of tlher-moconlIlp lplacell.elt oil (letectioin of freezing9 culrlVes

-sv(li (l lbv aIttachlin- 3 tilermocoulIlc, to a siin-letwig sectioll Stibljecte(d to a fr-eezillng cycle. Olietiluctioll Was inser'td 1 0 cimi ilito tile pitil; allotiletw1as fastened tiglltly to tile bllrk \withl tefloni tale:tile til rd was lpoIsitiMonde(l about 1 cmi fromll tile ieof tile twilg, witil cellpllane tape.

Coolinq Rates. Ill tile 700 to 8(0H individnalfr-eezillgy tes.s.; conl(diucte(l, tile rate of cooling varie(frolll .3 to 400 per lhr. ( Fol example. a fr-eezer-cilest temlilerattiure of -()0 giave a co(olillg rate ofsOmlle'300 per h1r fol a samliple in a preclilledI bottle.Tile infiltencc of fr-eezin,- rate i.s illlustrated h)v coill-paing curves fr-oni a sinigle dlo-(woo(l ti il cliwas c(ole(l at ; 11(1 ))220 per hr ini slcces-v e ftreezing-cycles.

Tile relationslhillp lbetweell tile suliercooilIn lpio tand tile lenlgthl of tile fir.-t freeziing plateau was (le-terimiilledi fol- (6 tlilforill tN-ig samlilles froill a sinlgicplant frozen tiniderl idcliltical coil(litioils.

Determillationcs were illade of the freezilng curvaof uniformll twigs frozeIn in tilerillos bottles whichwere eitlile- l)reclilled ( 4') oi ait rooni tenlilperature2- 0 ).Standard I'Proccd(nl^-e for- the Hfarduels.s l-e-st. T[lie

stanldard hlardlilleSiS test procedure whilic evolved iIItile course of tllis sttudly conisisted of wrapplilnIiliforlil twig sapllle atbot 5 cmi long in Saranl toretice mllOilsture loss., ilsertinlg a thlermlocotilde 1 cullilltO the pithl ait thie cut eild of twig and recording0,sallmple telllperatulre wlile subjectillg to 2 succe.sivefreezill, cycles at a coolilg- rate of about 300 per hlr.

bI the first freezinig cycle. a lprecililleci thlcrmiosblottle containing the samlpllle was pllacedi ina ( deelpfreeze aIt constalt templilerature. Sailipie temiperattirewa; recor(ledl during freezinig. \N'leln a predleter-iililled test temlperature was reached, tile twig ail(iattachled thlermilocouple were removed fronii the thier-illos andi thlala ed slovlv in air at 40. S.anples were

placedi aigaill illtO prechilled thlerlmos bo)ttiles alldl lpitilnto tile deel) fr-eeze for- tile seconll( freezing cycle.Tile temlperature was recorded as sa(lll)es wvere re-frozenl to alpproxilmlately -8°. This templl)era1tuire \V1slow eilougil to reveal tile douible freezingg point whlenit occurredi. In no case (lid tile illiiilill tempillera-ttiu-e in tile secoil(i freeziilg cycle exceed the minimum11111test templerature of tile first cycle. The curvesl oftile first anld second freezing cycles xwere conimpared.The presence of a second freezing poinlt itl the secondlfreeziilg cycle indicated that the twig wvas alive:absence of this freezing point indicated it wvas dead.

Freceing Curves of Oilier Gcenera. The freezing

3X

Dow



MCLEESTER ET AL.-MULTIPLE FREEZING POINT V'IABILITY TEST

curve viability test was also applied to excised twigsof Acer sacchlarinitmn L., Samilbutcuts puibens Michx..Ginkgo biloba L., Loniicera Morrowii Gray, Phila-delphus inodoruis L., Betutla papyrifcra -Marsh., For-sythia suispensa Vahl., Sorbus amiiericania Marsh..Pyrus miialins, Rhododenidron Mollis hlbrid and stemsections of Dianithuis caryophylluis. In Acer, Ginikgo.Betuila, and Rhododenidroni stems it was not possibleto push the junction into the pith. For twigs ofthese genera, a small hole was drille(l in the centerof the cut end permlittinig insertion of the thermiio-couple.

Results

The typical freezing curves of li-ing and deadtissues are shown in figure 1. In A. an activelygrowing twig was killed wlhen frozen to a test tem-perature (TT) of -3.9o. In B, a -similar twigsection from the same branch w%as not killed at atest temperature of -2.4'. The first and secondfreezing points (FP, and FP.,) were rea(lily ap-parent in both A and B during the first freezingcycle. In the second freezing cycle. however, thedead sample (A) had only 1 freezinlg point whilethe living sample (B) had 2. A single freezingpoint, followed by a smooth hyperbolic coolinig curve.was typical of all dead plant tissues studied.

The water content of twigs varies considerablywith cultural conditions and growtll status of fieldplants. For example, twigs (8 nmmi diamieter) froman actively growing dogwood plant had a moisturecontent of 70 % (w/w). Dornmanit twigs of thesame diameter collected from the same plant in

mid-winter contained about 40 % moisture. Whendormant twig sections were allowed to imbibe waterthere was a considerable increase in FP1 plateaulength correlated with the increase in moisture con-tent. Twigs soaked for 48 hr contained 70 % water(the unsoaked control had 41 %) and showed a verylong FP1 plateau which masked the FP..

The freezing curves for samples grown in eitherdry or saturated soil also gave variation in the lengthof the FP1 plateaus which were correlated withmoisture content. (Fig 2; compare curves A and Cand B and D). These curves illustrate 3 additionalpoints. First, the FP.,s in the first freezing cyclewere less distinct for samples with a high moisturecontent. Second, the unwrapped samples (A and B)lost considerable nmoisture (10 %) during the firstfreezing cycle. Third, moisture loss during the firstfreezing cycle (Curves A and B) accentuated thedistinctness of FP.s in the second freezing cycle.Thus, Sarai was necessary to reduce moisture lossand increase the reproducibility of the curves.

A parallel study using shorter (2.5 rather than5 cm) twig sections revealed that sample length hadlittle effect on the freezing curves of Saran wrappedsamples. Unwrapped samples lost 18 to 20 % waterin the first freezing cycle. When twig samples ofvarying length and diameter were dried at 370 in aforced air oven it became apparent that most of thewater was lost from the cut ends of the twigs.

Twig diameter greatly influenced the FP1. Fig-ure 3 shows the freezing curves of stem twigs ofvarious diameters. A difference of nearly 20 in theapparent FP1s for twigs of 4, 6, or 8 mm diameterwas detected. Twvigs larger than 8 mm in diameter

Lli~~~~~~~~~~~~~~~~~~~~~L~ 2

FIRSTFREEZINGCYCLE(LiVD FP

cr a_2

FIRST FREEZING CYCLE (LIVING) FIRST FREEZING CYCLE (LIVING)

5 _...... SECOND FREEZING CYCLE (DEAD)

-5 _ SECOND FREEZING CYCLE (LIVING)

-6 -6.

5 10 15 20 Min. 2 4 6 8 9mTIME 20 Mln. TIME

FIG. 1. The freezing curves of living and dead stem sections of Cornuiis stolontifera collected from a single activelygrowing branch. A) Sample killed at the test temperature (TT) of -3.99. B) Sample uninjured at the TT of-2.40. FP, and FP, are the first and second freezing points respectively.

39

Dow



PLANT PHYSIOLOGY

0

-2

L -4

.5

-6

FPP ........ -1

t~~~.. * . @. .D

/*_FRTFREEZING CYCLE lLIVING) **.v

...,,SECOND FREEZING CYCLE (LIVING) */ TWIG WRAPPED N SARAN

/MOSTURE CONTENT560(WS W) -4

J* MOISTURE LOSS AFTER FIRST FREEZING; CYCLE 2-4TOTAL MOISTURE LOSS IN BOT4 FREEZING CYCLES 2-_ 5

:~~~~~~~~~~~~~~~~~~~~~4 6 144 Mir.

rIME

|- SECONDFREEZING

NOT WR4APPED INSARAN*MlOISTURE CONTt:NT47%(W,W)*

/ MOISTURE.LOSS AFTER rlRST FREE21t.G CYCLE ',C

TOTAL MOISTURE LOSS IN aoTW FREEZING CYCLESt61_* W~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~p

2 -: S 10 Min.7 IN1E

FIG. 2. Water loss from uninjured stem sections of Cornus stolonifera during 2 freezing cycles. Twigs were col-lected from plants grown in dry soil (B and D), or in water saturated soil (A and C); and either wvrapped inSaran (C and D), or not wrapped (A and B).

the double freezing point were dependent in largepart on the moisture content of the sample. Slowrates of cooling gave the best resolution of the FP2for samples with low moisture content, while rapidfreezing was best for more hydrated samples. Fig-ure 4 shows the freeziing curves of a twig with low

FIG. 3. The freezing curves of stem sections ofCornus stolonifera 5 cm in length and 8, 6, or 4 mm indiameter.

gave FP1 plateaus similar to the 8 mm sample. Theapparent depression in the freezing point of thesmaller samples is due to excessive heat loss inrelation to the latent heat produced. The 4 mm

sample had no real plateau.Accurate recording of temperature of the tissue

,depended upon the placement of the thermocouple.A thermocouple placed in the pith gave better reso-lution of the FP2 than a thermocouple pressed tightlyagainst the bark. Temperature curves recorded bya thermocouple positioned in the air 1 cm from thetwig revealed that the heat produced by freezingsamples warmed the ambient atmosphere in thethermos. Thus accurate measurements were not.possible on more than 1 twig per thermos bottle.

The freezing rates which gave best resolution of

-1

LL

D-2

Lw - 30-

uJ

H-4

5°/hr.COOLING RATE

A

5 10 15 Min.TIME

FIG. 4. The freezing curves of a Corntus stoloniferastem section at coolinig rates of 50 and 220 per hr.MIoisture content of sample 47 %.

a.. -A

v4 -5-

.C

0,

Jr- I

't3,Q -4

5

FIV (LIVINT FREEZING CYCLEF 1- . . . . . . . . . . .

SECOND FREEZING CYCLE (LIVING)

NOT WRAPPFD IN SARAN

MO.STURE CONTENT 56-1.(WIW)

MOISTURE LOSS AFTCR FIR5T FRCEZING CYCLE 10'!.

TO'AL 110,STURE LOSS I- BOTH FREEZING CYCLES 18'.

2 0 n.

40

-FIRST FREEZING CICLE

........sEc,7ND FREFZIW.. -IC.E .L'111G'

TWIG WRAPPED IN SArlAN

MGISTURE CONTENT 47-1.

WASTURE LOSS -IFTER 'IRST FREEZ;IG CYCLE 141,,.

TllT.L MOISTUR. LOSS "I BoT. FREEZING CICLES 1.4.

T.,

Dow



MCLEESTER ET AL.-MULTIPLE FREEZING POINT VIABILITY TEST

moisture content (47 %) frozen in successive freez-ing cycles at 50 and 220 per hr respectively. At50 per hr, the FP2 was well defined. while at 220per hr it was less distinct.

The influence of moisture content is furtherillustrated by comparing the lack of resolution of theFP.2 in the first freezing cycle (Curve A. fig 2)with the distinct FP2 in the second cycle after 10 %water loss. Good resolution of FP2 was often pos-sible with Cornuts twigs of high moisture content(64 %) at high rates of cooling (400 per hr). Thechoice of cooling rate is limited because fast ratesmav, in themselves, cause injury (25). However,actively growiing Cornus stolonifera twigs could befrozen at rates up to 400 per hr withlout injurv fromfast freezing. Dormant winter hardy twigs werefrozen at rates of 2{)0° per hr without injury, butrates of 5000 per hr caused damage. Although itwas difficult to select a single rate of cooling thatgave good resolution of the FP2 at all times of theyear, 300 per hr was a reasonable compromise forCorntis stolonif era.

Precooling the thermos bottles had the advantageof shortening the time required for the test whilereducing the cooling rate at the time of freezing.Figure _5 shows that the cooling rate betw-een 00 andthe supercooling point was about 40 per hr for atwig in a precooled thermos, and about 240 per hr intwigs from a thermos initially at room temperature.Precooling reduced the time required for this freezingcycle from 3 to 2 hr.

Mir. 3-60 90 2. 150

TIN'E

FIG. 5. The freezing curv-es of stem sections ofCoruims stoloniifera frozen in thermos bottles initially atroom temperature (A) or precooled (B).

There are wide and unpredictable variations inthe supercooling of uniform samples frozen under thesame conditions. Samples with only slight super-cooling had FP1 plateaus which were proportionatelylonger than those for samples which supercooledmore. Table I illustrates the variation in super-cooling of 6 dogwood twig samples of essentiallythe same size and weight, collected from a singlebranch. The samples vere frozen at the same timeunder similar conditions. In the 6 samples super-

cooling varied from less than 10 to almost 70, andthe FP1 plateau varied from about 6 to 26 min in

Table I. The Degree of Suipercoolintg and Duratiott ofthe First Freezing Point Plateau for 6 UniformCornus stolonifera Samnples Collected From aSingle Branch and Frozent Simultaneously

UnLder Simiiilar Contditions

Length ofSupercooling first freezing point plateau

deg w1 i;-0.55 26.6-3.70 13.6-5.65 8.7

5.95 8.4-6.60 7.5-6.95 5.9

length. Without ice-seeding. these randonm varia-tions in supercooling could not be controlled. Varia-tions in supercooling and natural differences in water

content of samples and insulating efficiency of

thermos bottles make it difficult to duplicate curves.

btut have no effect on the validity of the techniquesas a viability test.

Living twigs from dogwood and the other 11genera studied (see Mlaterials and Methods) alwayshad at least 2 freezing points, while dead twigsexhibited only 1. Figure 6 shows the curves ex-

hibited bv twigs of Forsythia suspensa, Pyrus inaluts,Diantthus caryophyllits, and Sorbus amiiericania. Sim-ilar curves were produced from tests on the othergenera. Uninjured Cornttts stolonifera stem sectionshad strong potential for regrowtth and showed callusgrowth in 20 days during all times of the year.

There wA-as over 98 % agreement between theresults of the freezing curve viability test and theregrowth test in the 700 to 800 samlples evaluated.There was about 90 % agreemiient betweein the sub-jective visual viability rating and the regrowth andfreezing curve tests. Thle visual ratilng presentedsome problems with hardy samples becauise somle

twigs which had been killedl stayed green and gave

the appearance of being alive.Table II summarizes studies which compare the

3 methods for determininig viability; The high de-gree of agreement between the 3 methods is appareint.The results of the freezing curve viability test forfro3t hardiness w-ere available within 1 day. Thisfeature combined w-ith the absolute nature of theresults, the high degree of qualitative reproducibility%and the small amount of tissue required, are themajor advantages of the technique. The informationon supercooling and freezing point depression repre-

sented by the freezing curves is also useful in many

hardiness studies.

Discussionand Conclusions

A double freezing point was found to be charac-teristic of freezing curves of living stem sections in12 genera. This substantiates the original observa-

41

Dow



FP . .. ..

- Forsythia suspensa \

- FIRST FREEZING CYCLE (LlIVING) \

-*- . SECOND FREEZING CYCLE (DEAD)\

5 10TI ME

PLANT PHYSIOLOGY

o0

0

-1

L

D _2a:

0-

WLJ -5__

15 20 Min.

-e

[ ~~~~FP,":t~~~~' \

Dianthus caryophyllus

- FIRST FREEZING CYCLE (LIVING)

..000SECONDFREEZING CYCLE (DEAD)

I I I a

5 10TI ME

FIG. 6. Freezing curves of stem sections of severalgenera. Conditions for the tests were: Forsythia sus-pensa. Diameter 9 mm, water content of sample 42 %,cooling rate 100/hr. Pyrus mialus. Diameter 6 mm,water content of sample 49 %, cooling rate 150/hr.Dianthus caryophyllus. Diameter 4 mm, water contentof sample 86 %, cooling rate 150/hr. Sorbus americana.Diameter 10 mm, water content of sample 75 %, coolingrate 180/hr.

tion of this phenomenon by Maximov in 1914 (16)on petioles of Tussilago farfara.

Several theories have been proposed to explaindouble freezing points., Luyet and Gehenio (12)believed that the first freezing point was that ofextracellular fluid, and the second either intracellularfluid after its extraction from the cell by rapidosmosis, or vacuolar sap. They noted the absenceof a pronounced first plateau in relatively dry tissue,and its prominence in tissues allowed to imbibe water.

Hudson and Idle (7) studying potato petioles,agreed with Luyet and Gehenio that the first freezingpoint was due to extracellular fluid, but they sug-

gested that the second freezing point was caused bya sudden leakage of inorganic solutes from the cellsafter the first freezing point had been reached. Theypostulate that released solutes cause extracellularthawing by lowering the freezing point, and thatsubsequent freezing points result from refreezing of

thawed extracellular water. They st;tte that the

salting out of proteins, whiclh mliglht occur if purewater were lost from the protoplast. may be avoidedby the frost-induced leakage of solutes from the cells.

Blochi et al. (2) agree that the first freezingpoint is due to crystallization of free extracellularfluids. However. thev feel that the second freezingpoint represents a depressed freezing point of waterstructurally bound in a gel network. These investi-gations were performed on the adductor muscle ofclam and oni non-living model systems. such asaqueous gels of polyacrilic acid anid polyvinyl alcoholwhich do have double freezing points. The firstfreezing cycle of these gels destroys their abilitv tobind water anid eliminiates the FP. in subsequentfreezing cycles.

Based on work -\ith spruce needles, Salt and

Kaku (21) have suggested that the 2 points repre-sent ice cry\stallization in 2 different nmajor tissues

42

*c01-

-1

wr -2

a -3w

u -4

-5

-6

-7

-c0I

15 20Min.

Dow



'MCLEESTER ET AL.-MIULTIPLE FREEZING POINT V"IABILITY TEST

Table II. A Comiiparisoni of V'iability Evaluation byt Freezing Cnrvcs. Visual E.ramnination, antd RegrowthThe hardiness tests were run on Corunis stoloziferra twTigs collected from the field on: (1) September 4th. (2)

Januarv 4th. and (3) April 14th.

Double freezingpoint present

in secondfreezing cycle'

1 2

Barkdiscolorationobserved2

3 1 2Callus growth3

3 1 2

yes yes yes n10 no no

yes yes ... no no

no yes ... yes no

no yes ... yes no ...

no yes yes yes no no

... yes yes ... no no

... yes no ... no yes/no

... yes no ... n10 yes

... yes no ... no yes

... yes no ... no yes

yes yes vesyes vesno yes

no yes ...

no yes yes

... yes yes

... yes no

... yes no

... yes no

yes no

Evaluated after 2 to 6 hr.2 Evaluated after 4 to 7 days.

Evaluated after 20 days.I Control.

(stele and spongy mesoplhyll). However, dogwNoodtwigs with all bark removed have 2 distinct freezingpoints, as do saml)les of homiogeneous tissues fromstorage organs (13).

While the cause of the seconid freezing point is

not known, its presence or absence provides a conl-venient means of determining viability providinggcertain factors are controlled. The Imlost imnport !ntconsideration was the prevention of moisture lossfrom the sample. This was readily aiccomplished bvtightly wrapping samples in a moisture proof filmsuch as Saran. The control of moisture loss isimportant since water content can influence the coldresistance of plant tissues. Sun (24) has slhownthat reducing the moisture content of freshly germi-nated excised pea embryos to 27 % permitted themto survive -1960 wThile more highly hydrated emil-

bryos were killed at much higher temperatures.Modlibowska (17) found that water sl)rinkled on

excised apple blossoms prior to a 1.5 hr frest at-2.9° made them more susceptible to frot injury.She suggested that the highler water content ofsprinkled blossoms suppressed supercooling and in-creased injury, while low-er mnoisture content favoredstupercooling.

In experiments withl dogwood, the exposure ofuniprotected samples to the desiccating effects of lowtemperatures during the first freezing cycle removedas much as 10 % of their nmoisure and lowered thesecond freezing point plateau (fig 2). Luvet andGehenio (12) found that most plant tissues were notinjured until they were cooled below the plateau ofthe second freezing point. Desiccation-induced low-ering of the second freezing point could increasecold resistance and make the hardiness test invalid.

A high moisture content often prevented resolu-tion of the second freezing point. Controlled drying

betw een the first an(d Second freezing cycle may

provide a means of accenituating tlle FP., in thesecond cvcle without alterinlg the resistance of the

samlple to the coldl stress administered in the firstcycle.

The placemlienit of the temlperatulre ;enior is ini-portant. In these tests. insertionl of the thermo-couple in the pith provided the best resolution oftlle second freezing point. Care should also be takento re(luce the conductioni of heat fronm the sampleto the cold freezer -via the thermocouple leads. Aconsiderable length of wvire (about 30 cImi for 26gauge) should be placed inside the thernmos. Theuse of finer thermocouple Nvire cani also help re(dticeheat transfer.

Modlibowvska ('17) presented freezing curves ofaplple blossoms in which the recorded air tempera-ture was apparently higher than tissue temperatureduring cooling. The air temperature was recordeed

with 26 gauge junctions and the blossom1i tenmperatuirewith 40 gauge. These anomalous results may bedue to a greater heat feed-back to the junctioni ofthe large thermiocouple. MIarshall observed a cvl-inder of ice aroulnd a thermistor probe inserted intoa pear fruit subjected to freezing (15). This isprobably due partially to heat drain fronm the fruit tothe colder ambient air via the thermistor lead.

Sample size for a particular plant material shouldbe determined in preliminary tests. Excessively longor large diameter samples often produced indistinctfreezing points because freezing coukl start at a

considerable distalnce fronm the senisor. In our stud-ie;, large samples usually did not stipercool as much

as small samples.Cooling rates should also be determined by pre-

testing. Rates between 30 and 400 per lhr were usedsuccessfull in these tests. Resolution of the second

Test temp

deg+ 14-6-12-16-20-24-28-32-60-80

3

43

Dow



44 ~~~~~~~~PLANTPHYSIOLOGYfreezinig point depended upon thle combination of

freezing rate and moisture content of the sample.Fast cooling rates were useful for detecting second

freezing points in samples with high moisture levels.

Care should be exercised in the choice of coolingrates. Hatakeyama (4) was able to demonstrate

double freezing points in highly hydrated leaves of

Bu.rus microphylla; however, the high rates of cool-

ing (3O0001O0000 per hr) injured tissues. Havis (6)found that Rhododendron leaves with hiigh moisture

conitent were injured at about -~40o when frozen at

about 30 per hr, but at about 180 when they were

frozen more rapidly.

Living stem sections of the 12 genera tested had

2 freezing points. Stem sections which had been

killed by freezing had only 1 freezing point. This

well known phenomenon provides the basis for a new

and simple viability test which may have widespreaduse in biological research.

Literature Cited

1. AOKI., K., E. ASAHINA, AND I. TERUMOTO. 1953.

Analysis of the freezing process of living organ-

isms. IX. The relation between the shape of the

freezing curve and the frost hardiness. Low.

Temp. Sci. 10: 69-80.

2. BLOCH, R., D. H. WALTERS, AND W. KUHN. 1963.

Structurally caused freezing point depression of

biological tissues. J. Gen. Physiol. 46: 605-15.

3). DEXTER, S. T., W. E. TOTTINGHAM, AND L. F.

GRABER. 1932. Investigations of the hardiness of

plants by measurement of electrical conductivity.Plant Phiysiol. 7: 63-78.

4. HATAKEYA-MA, I. 1961. Studies on the freezing of

living anid dead tissues of plants, with specialreference to the colloidally bound water in livingstate. 'Mem. Coll. Sci. Ulniv. Kyoto B. 28: 401-

29.

5.HATAKEVAMIA, I. AND J. KATO. 1965. Studies on

the water relation of Butxis leaves. Planta 65:

259-68.

6. HAVIS, J. R. 1964. Freezing of Rhododentdrontleaves. Am. Soc. Hort. Sci. 84: 570-74.

7. H-iUDSON, M. A. AND D. B. IDLE. 1962. The for-

mation of ice in plant tissues. Planta 57: 718-30.

8. KRASAVTSEV, 0. A. 1962. Fluorescence of forest

plant cells in the frozen state. Planit Physiol.U.S.S.R. 9: 3159-67.

9. LAPINs, K. 1961. Artificial freezing of 1-year-oldshoots of apple varieties. Can. J. Plant Sci. 41:

381-93.

10. LEVITT, J. 1957. The moment of frost injury.Protoplasma 48: 289-302.

1 1. Li, P. H., C. J. WEISER, AND R. VAN HUYSTEE.1965. Changes in metabolites of red-osier dogwoodduring cold acclimation. Am. Soc. Hort. Sci. 86:723-29.

12. LuYET, B. J. AND P. M. GEHENIO. 1937. Thedouble freezing point of living tissues. Biody-namica 30: 1-23.

13. LuYET, B. J. AND P. M. GEHENIO. 1940. Life andDeath at Low Temperatures. Biodynamica, Nor-mandy, Missouri. p 341.

14. MACIRVING, R. M. AND F. 0. LANPHEAR. 1967.Environmental control of cold hardiness in woodyplants. Plant Physiol. 42: 1191-96.

15. MARSHALL, D. C. 1961. The freezing of planttissue. Australian J. Biol. Sci. 14: 368-90.

16. MlAXIMOV, M. A. 1914. Experimentelle und kri-tische Untersuchungen fiber das Gefrieren und Er-frieren der Pflanzen. Jahrb. Wiss. Botan. 53:327-420.

17. MODLIBOWSKA, I. 1962. Some factors affectingsupercooling of fruit blossoms. J. Hort. Sci. 87:249-61.

18. PARKER, J. 1953. Criteria of life: Some methodsof measuring viability. Am. Scientist 41: 614-18.

19. SAKAI,, A. 1962. Studies on the frost hardiness ofwoody plants. I. The causal relation betweensugar content and frost hardiness. Inst. LowTemp. Sci. 11: 1-40.

20. SAKAi, A. 1965. Survival of plant tissue at super-low temperatures. III. Relation between effectiveprefreezinig temperatures and the degree of frosthardiness. Planit Physiol. 40: 882-87.

21. SALT, R. W. AND S. KAKU. 1967. Ice nucleationand propagation in spruce needles. Can. J. Botany45: 1335-46.

22. SCARTH, G. W. 1941. Dehydration injury and re-sistance. Plant Physiol. 16: 171-78.

23. SIMINOVITCH, D., H. THERRIEN, F. GFELLER, ANDB. RHEAUME. 1964. The quantitative estimationof frost injury and resistance in black locust, al-falfa, and wheat tissues by determination of aminoacids and other ninhydrin-reacting substances re-leased after thawing. Can. J. Botany 42: 637-49.

24. SUN, C. N. 1958. The survival of excised peaseedlings after drying and freezing in liquid ni-trogen. Botan. Gaz. 119: 234-36.

25. WHITE, W. C. AND C. J. WEISER. 1964. The re-lation of tissue desiccation, extreme cold, and rapidtemperature fluctuations to winter injury of Amnier-ican arborvitiae. Am. Soc. Hort. Sci. 85: 1554-63.

26. WILNEIR, J. 1959. Note on ani electrolytic pro-cedure for differentiating betwveen frost injury ofroots and shoots in woody plants. Can. J. PlantSci. 39. 512-13.

44

Dow



Documents

Multiple Freezing Points Test for Viability in the ... · variables such as moisture content, sample size, thermocouple placement, and cooling rate on freezing curves was analyzed