Plant Physiol. (1974) 53, 705-708

Freezing of Nonwoody Plant TissuesI. EFFECT OF RATE OF COOLING ON DAMAGE TO FROZEN BEET ROOT SECTIONS' 2, 3

Received for publication September 25, 1973 and in revised form December 26, 1973

BERNARD J. FINKLE, E. SA B. PEREIRA, AND M. S. BROWNAgricultural Research Service, United States Department of Agriculture, Western Regional Research Labora-tory, Berkeley, California 94710

ABSTRACT

Small cylinders of red beet (Beta vulgaris) root were frozenat various rates. Ultraslow cooling at 0.2 C per hour to -4 Cproduced little damage, as determined by leakage of pigmentand electrolytes, and softening. All of these increased at fasterrates of cooling or at lower temperatures. Cooling at the ultra-slow rate appears to induce extracellular freezing, resulting ina protective dehydration of the cell contents.

with the requirements of large pieces of tissue, encourage theformation of extracellular ice with consequent partial dehydra-tion of the protoplast. These are conditions that can preservethe frozen cell (10), and appear also to contribute to the natu-ral process of freeze-hardening (7, 8). This paper reports ex-plorations of rate of cooling and its effect on the degree ofdamage sustained by frozen cylindrical sections of red beetroot.

During the process known as "cold-hardening" an appro-priate regimen in the cooling of plants has the effect of pre-venting tissue damage during subsequent freezing (9). Not allplants, however, and not all tissues of any given plant will nor-mally undergo cold-hardening. The vegetative tissues of manygarden vegetables, for example, and the fruits of hardy varie-ties of trees generally do not become freeze-resistant. Theysuffer cellular damage when subjected to freezing weather, orwhen frozen for the commercial market.A recent review, while contrasting winter freezing of plants

in cold climates with freezing as a method of food preservation,emphasized factors in the freezing process whose study wouldindicate methods for reducing damage to edible tissues ofplants (4).

Cooling rate emerges as one such factor whose control mightprove particularly favorable in preserving multicellular piecesof tissue. At one extreme, the benefits that can be derived fromvery fast freezing of single cells or thin sections of tissue-namely, preservation of tissue structure and viability-are de-nied to relatively large pieces of plant tissue and whole plantorgans. The larger pieces, with their high thermal resistance,can be cooled only at rates that are orders of magnitude slowerthan those at which the viability of unicellular organismsor thin tissue sections can be preserved. At the other ex-treme, however, slow cooling has also been cited as diminishingfreezing damage to tissues. Such rates, technically compatible

'This work was partially supported by the American FrozenFood Institute.

2 Portions of the content of this article were communicated to theXIII International Congress of Refrigeration (5).

3 Portions of this work were submitted in partial fulfillment ofthe requirements for the degree of Master of Science in Food Sci-ence, University of California, Berkeley, by E. Sa B. P.

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FIG. 1. Freezing apparatus. The thickness of plastic foam insula-tion in the beaker determined the rate of heat loss to the cold alcoholbath.

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FINKLE, PEREIRA, AND BROWN

5 10 15

Plant Physiol. Vol. 53, 1974

20 25

ELAPSED TIME (Hours)FIG. 2. Cooling and freezing curves of beet cylinders. Supercooling at C was of variable degree and duration. Freezing r-ates were calculated

between A (ceginning of freezing) and B (-3 C).

Table 1. Effects of Coolinig onI Beet Cyliinders

Leaching of Compressibility2

Experiment Coolinl- Rate (C lhr)l or :\ethod -

l'igment EIlectrolytes Control FrozennTot. frozen) Foe

8.2 (±2.3)1 16.0 (±1.4) 0.32 (±0.07) 0.43 (±0.06)51.2 (±2.8) 72.9 (±1.8) 0.32 (±4-0.07) 1.57 (±0.15)12.2 (±4-2.6) 20.4 (±2.7) 0.37 (±O.06) 0.68 (±0.12)48.2 (±4-2.6) 72.8 (±2. 3 ) 0.37 (±0.C6) 1.73 (+0.33)77.3 (±1.3) 94.8 (±1.5) 0.31 (±0.03) 2.26 (±0.07)75.3 (1. 3) 83.6 (±4.9) 0.33 (±0.02) 1.68 (±0.21)67.5 (±fi2.7) 83.4 (±2.3) 0.35 (±O0.01) 1.28 (+0..10)74.9 (±1.4) 88.4 (+1.7) 0.37 (±O.C6) 1.14 (40.05)72.8 (±1.5)

'VMeasureJ between beginning of freezing and -3 C. Final temperature -4 C, except for Experiments II

attained the temperature of the freezing medium.2 Maximum force of 400 g.

3 SE

4Beets for experiment 1I not from same lot as those for I and lII.5 Immersion in Fluorinert at -4 C.Immersion in Fluorinert at -5.8 C.Dichlorodifluoromethane, b.p. -29.8 C.

MATERIALS AND METHODS

Red beet root (Beta vulgaris) tissue obtained from the com-mercial market was chosen as experimental material because ofits year-round availability, ease of handling, and its betaninpigment, useful as an indicator of cellular damage. Cylindricalsections were removed with a 5-mm cork borer, inserted radiallyalong the equatorial portion of the root. The cylinders weretrimmed to 20 mm length and then washed in running tap waterfor 2 to 3 hr prior to use. After this washing, the cylinderscould be kept in water in the refrigerator for more than 24 hrwithout measurable leaching of pigment.To obtain slow rates of cooling, the tissue cylinders were

frozen in the air space of a chamber held within a refrigeratedalcohol bath (Fig. 1). Rate of cooling was adjusted by varyingthe bath temperature and the thickness of a plastic foam insu-lator between the samples and the chamber wall. Ultr<slowrates of cooling, of the order of 0.2 C/hr. were obtained witha bath temperature of -8 C and an insulator thickness of ap-proximately 1 cm. Slow rates, near 3 C/hr, were obtained byremoving the insulator.

and Ill in which samples

Faster cooling rates were obtained by direct immersion ofthe samples into powdered solid carbon dioxide, dichlorodi-fluoromethane (R-12), or liquid nitrogen. The final tempera-tures achieved by the latter treatments however, were consid-erably lower than those attained during the slower modes offreezing. Hence, an alternative method of direct immersionrapid cooling was later adopted, making use of a perfluorocar-bon liquid (Fluorinert FC-77, 3M Co.4) in a refrigerated bath.Temperature at the geometric center of one of the cylinders was

measured with a 24-gauge hypodermic needle thermistor probeconnected to an electronic thermometer (Yellow Springs InstrL-ment Co.). This temperature was recorded on a 13-cm stripchart recorder (Varian G-14) calibrated for the temper2tuirerange of +1 to-6C.Each frozen cylinder was thawed by direct immersion in

Reference to a company or product name does not imply ap-proval or recommendation of the product by the United States De-partment of Agriculture to the exclusion of others that may besuitable.

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FREEZING RATE AND TISSUE DAMAGE

about 80 ml of water at 18 C for 1 hr, after which the solutionwas diluted to 100 ml for measurement of leached pigment andelectrolytes. Pigment content of the liquid was determined byspectrophotometric measurement at the absorption maximumof 535 nm. Electrolyte concentration was determined by mea-suring the electrical conductivity of the solution.

Softening of the cylinders, due primarily to loss of cell tur-gor, was measured with an Instron Universal Testing Machine.Compressibility recorded here was the reduction in cylinderdiameter when pressure was applied at the rate of 5 mm/minto a maximum force of 400 g. This did not produce a signifi-cant loss of pigment or electrolytes.

Following these measurements, each cylinder was groundwith a mortar and pestle and extracted with its original aque-

ous solution. The suspension was filtered through a coarsefritted glass filter under vacuum. The filtrate was a clear redsolution, and the residue on the filter contained little or no visi-ble red pigment. Pigment and electrolyte composition of thissolution was determined as before, and the amount of each re-leased during and after thawing was expressed as a percentageof the total amount in the final extract.

RESULTS

Recordings of internal temperature during freezing of beetcylinders are presented in Figure 2. The cooling rates listed inTable I were calculated between the beginning of the freezingplateau and -3 C (Fig. 2, A and B). Duration and degree of

A

B

CFIG. 3. Beet cylinder (5-mm diam X 2 cm) after ultraslow cooling. Melting of external ice (A) revealed shrunken cylindei of tissue (B) that

regained original dimensions after 1 hr soak in water at room temperature (C).

Plant Physiol. Vol. 53, 1974 707

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FINKLE, PEREIRA, AND BROWN

supercooling prior to freezing were variable (Fig. 2, brokenline at C) and did not appear to affect freezing damage. Super-cooling could be terminated by mechanical shock or by inocu-lation with ice crystals.The effects of slow and ultraslow cooling upon the condi-

tion of beet cylinders after thawing and comparison with sev-eral rapid cooling procedures are presented in Table I. Eachvalue is the average from at least six cylinders. These data indi-cate a significant effect of the rate of freezing to -4 C on thecharacteristics of the thawed tissue. Ultraslow cooling (0.16C/hr) produced little change in any of the properties measuredcompared to control samples. In contrast, a rate of 3.3 C/hrproduced almost as much leaching and softening as did freez-ing to much lower temperatures with solid carbon dioxide,R-12, Fluorinert, or liquid nitrogen.

Another noteworthy difference was observed between cylin-ders cooled at the ultraslow rate and those cooled at the moredamaging rates of 3.3 C/hr or greater. Under the ultraslowcooling regime, a characteristic mode of water migration (i.e.,tissue dehydration) took place. Figure 3 illustrates the migra-tion of water from internal regions of the beet cylinders to theexterior, with attendant contraction of the beet tissue. Thiswater then froze as an encasing layer of ice (Fig. 3A). Thawingof this ice layer revealed a shrunken section of tissue (Fig. 3B),which rehydrated to its original size and cylindrical shapewhen immersed in water for about 1 hr (Fig. 3C). These cylin-ders, as indicated in Table I, were damaged little by the freez-ing treatment. More rapidly cooled cylinders displayed no suchencrustation of ice nor other signs of water migration awayfrom the cells, and they were extensively damaged upon thaw-ing.

DISCUSSION

The data for red table beet root clearly indicate the superior-ity of ultraslow cooling for decreasing damage during freezingto -4 C. Similar results were also reported from this labora-tory for other plant tissues and organs (12). Golovkin (6) de-scribed a stepwise adaptation of apples to withstand freezing athigh subzero temperatures. However, these reports indicatedtemperature thresholds of -4 to -8 C, below which damageincreased. With the beet cylinders, we likewise observed, inpreliminary experiments, the appearance of tissue damage atjust a few degrees below -4 C even when the cooling rate wasultraslow. Thus an ultraslow cooling rate simply lowers thetemperature of freezing damage by a few degrees.

This displacement of the damaging temperature may be theresult of slow cellular dehydration prior to freezing. There isabundant information that as the rate of cooling is decreased,especially in the absence of supercooling, there is an increasedtendency toward extracellular freezing (1, 7, 10,11). This cryo-dehydration has been indicated as a factor in preventing freez-ing damage to plant tissues, although dehydration in itself, de-pending on its degree and on the methodology employed, hasbeen shown to display either positive or negative effects (4). Acorrelation, or even integral connection, between cryodehydra-tion and avoidance of freezing damage under the present ultra-

slow conditions of freezing is supported by the observed migra-tion of cellular water toward the outside of the tissue cylinderwhere it then froze as the shell of ice illustrated in Figure 3.Thus under ultraslow cooling (0.16 C/hr) where water migra-tion out of the cells was encouraged so that a large amount ofextracellular freezing occurred, injury to the samples was buta fraction of the damage observed when similar tissue wascooled at a 20-fold faster (but still rather slow) rate of 3 C/hr.Dehydration is often a part of the natural freeze-hardeningprocess.

There was little significant difference in leaching betweensamples cooled at 3 C/hr and at the much greater rates estab-lished by direct immersion in liquid fluorocarbons (-4 to-30 C), powdered carbon dioxide (-79 C), or liquid nitrogen(-196 C). Cryodehydration was not observed with any of thesetreatments, and damage was almost complete. A more ambig-uous pattern of freezing injury was displayed by compressibil-ity data. Fast cooling rates resulted in intermediate compres-sibility, between that from ultraslow and slow cooling. Com-pressibility possibly reflects complex effects of cooling rate onthe degree of damage to the cell wall structure, in addition to ef-fects on the protoplast and membranes.

In the course of this work, small temperature spikes wereobserved in some of the freezing curves between -2 and-4 C. Details of their occurrence and significance are reportedin two other publications (2, 3).

Acknowledgments-WVe thank- R. N. Sayre and F. W. Reuter for their help-ful suggestions, and L. V. Richards for technical assistance.

LITERATURE CITED

1. ASAHINA, E. 1956. The freezing process of plant cell. Contr. Inst. Low Temp.Sci. 10: 83-126.

2. BROWN, M. S., E. SA B. PEREIRA, AND B. J. FINIKLE. 1974. Freezing of non-woody plant tissues. II. Cell damage and the fine structure of freezingcurves. Plant Physiol. 53: 709-711.

3. BROw-N, M. S. AND F. W. REUTER. 1974. Freezing of nonwoody plant tissues.III. Videotape micrography and the correlation between individual cellularfreezing events and temperature changes in the surrounding tissue. Cryo-biology. In press.

4. FINKLE, B. J. 1971. Freezing preservation. In: A. C. Hulme, ed., The Bio-chemistry of Fruits and Their Products. Academic Press, London. pp. 653-686.

5. FI-NKLE, B. J., E. SA B. PEREIRA, AND M. S. Bitow:N-. 1973. Prev-ention offreeze-damage to vegetable and fruit tissues by ultra-slow cooling. Proc.XIIIth Int. Congr. Refrigeration 3: 479-483.

6. GOLOVsKIN, N. A. 1966. Storage of food products at tempeiatures close to thecryoscopic point. Proc. 2nd In. Congress of Food Sci. Technol. pp. 161-169.

7. HATAKEYA-MA, I. 1961. Studies on the freezing of living and dead tissues ofplants, with special reference to the colloidally bound water in living state.Mem. Coll. Sci. Univ. of Kyoto Ser. B 28: 401-429.

8. LEVITT,J. 1941. Frost Killing and Hardiness of Plants. Burgess, MIinneapolis.9. LEvITT, J. 1972. Responses of Plants to Environmental Stresses. Academic

Press, New York.10. SAKAI, A., K. OTSUKA, AN-D S. YOSHIDA. 1968. Mechanism of survival inplant

cells at super low temperatures by rapid cooling and rewarlling. Crvo-biology 4:165-173.

11. SAMYGIN, G. A. AND D. SH. BLIADZE. 1969. Microscopic study of ice formationin lemon and tea plant tissues. Soviet Plant Pliysiol. 16: 761-764.

12. SAYSRE, R. N.,MI. S. BROW'N, B.J. FIN`KLE AND E. SA B. PEREIRA. 1973. Pre--ention of freeze-damage to whole fiuits andl vegetables bv ultra-slowcoolilg. PIoc. XIlIthInt. Congr. Refrigeration 3: 485-589.

708 Plant Physiol. Vol. 53, 1974

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