An improved conductivity method for the ...

An improved conductivity method for the measurement of frost hardiness M. I. N. ZHANGA N D J. H. M. WILLISON' Biology Department, Dalhousie University, Hal*,

N.S., Canada B3H 4JI

Can. J. Bot. Downloaded from www.nrcresearchpress.com by 192.95.27.218 on 06/05/13 For personal use only.

Received June 2, 1986 ZHANG,M. I. N., and WILLISON, J. H. M. 1987. An improved conductivity method for the measurement of frost hardiness. Can. J. Bot. 65: 710-715. A suspension culture of Bromus inermis Leyss was grown under a variety of conditions to provide a range of frost hardiness. The growth conditions involved both low-temperature treatment and the addition of abscisic acid to the culture medium. Frost hardiness (LT,,, i.e., subzero temperature causing 50% mortality) was estimated by both fluorescein diacetate vital staining and by a conventional method involving estimation of ion leakage from frost-damaged cells by measurement of the conductivity of a bathing solution. It was found that the latter method always underestimated the frost hardiness by comparison with the former. Evidence that the vital staining estimates were reliable is presented. A subsequent time-course study of ion leakage from frost-stressed cultures showed that leakage increased with time of immersion in deionized water, and that the extent of the difference was dependent upon the growth conditions and the minimum temperature experienced. Analysis of the experimental data showed that this ion leakage differential can be used to estimate LT,, reliably by comparison with the vital staining estimates, and it is proposed that the protocol might be useful as an improved standard method in testing the frost hardiness of plant tissues. The physiological basis for this test is discussed.

J. H. M. 1987. An improved conductivity method for the measurement of frost hardiness. ZHANG,M. I. N., et WILLISON, Can. J. Bot. 65 : 710-715. Une suspension de cellules de Bromus inermis Leyss a CtC cultivCe sous diverses conditions d'endurcissement au gel. Les conditions de croissance comportaient un traitement 2 basse tempCrature et l'addition d'acide abscisique au milieu de culture. La rksistance au gel (TL,,, i.e., la temperature sous zCro causant 50% de mortalit&)fut CvaluCe par deux mCthodes differentes : la coloration vitale avec le diacCtate de fluorescCine et la conductivitC Clectrique de la solution, mesurant la sortie des ions des cellules, suite aux dommages causCs par le gel. L'on trouvC que cette demibre mCthode, comparativement B la premzre, sousestimait l'endurcissement au gel. Une preuve de la fiabilitC de la coloration vitale est prCsentCe. Une Ctude subsCquente des temps de sortie des ions, chez les cellules ayant subi le gel, a montrC que la perte d'ions augmentait avec le temps d'immersion dans l'eau dCsionisCe, et que l'importance de la diffkrence dCpendait des conditions de croissance et de la tempkrature minimale subie. L'analyse des risultats a rev616 que cette difference de sortie d'ions peut Ctre utiliske de faqon fiable pour la TL,, en comparaison avec les valeurs estimCes de la coloration vitale. L'on propose que le protocole pourrait Ctre utile en tant que mCthode uniformisCe et amCliorCe dans les essais d'endurcissement au gel des tissus vCgCtaux. La base physiologique de la mCthode est discutCe. [Traduit par la revue]

Introduction Various methods exist for estimating the frost hardiness of plant varieties. The most widely used and reliable is the regrowth test in which batches of plants are frost stressed before transfer to environments suitable for promoting regrowth. Frost hardiness is then defined by LT5,, the freezing temperature at which 50% of the plants are killed. The method has the disadvantages that it is slow, expensive, and of limited applicability in estimating the frost hardiness of organs. Various alternative a ~ ~ r o a c h have e s been derived. the most promising of which is -the conductivity method, first outlined by Dexter et al. (1932). This estimates freezing injury by measuring the electrolyte efflux from the tissue immediately after thawing (see Levitt 1980, pp. 134- 135, 256-258). While this method predicts the hardiness rank of plant tissues, neither the precise testing protocol nor the reliability of the method in predicting LT50 are well established (for comparison, see Sukumaran and Weiser 1972; Yoshida and Uemura 1984; Uemura and Yoshida 1984; Pearce 1980; and the comments of Levitt 1980, pp. 134- 135). The present study has been directed toward improving both the testing protocol and understanding of the physiological basis of the conductivity test for frost hardiness. For this investigation, we selected a plant cell suspension culture which frost hardens to different extents in response to 'Author to whom all correspondence should be addressed. Printed in Canada I Imprim6 au Canada

cold treatment or treatment with abscisic acid (Chen and Gusta 1983), thus providing us with a suitable range of test materials. It was presumed that the large surface area of this experimental system would provide ideal conditions for ion leakage into the test medium, thereby maximizing the sensitivity of the test. Additionally, it was realized that an independent assay of frost damage could be obtained by measuring cell viability using vital staining, allowing comparison to be made with the conductivity assay.

Materials and methods Cell culture A bromegrass (Bromus inermis Leyss) cell suspension culture was obtained from Dr. L. V. Gusta, University of Saskatchewan (for details, see Chen and Gusta 1983). In our laboratory, it was maintained in 25 mL of Eriksson's liquid medium (Gamborg and Wetter 1975) in 125 mL Erlenmeyer flasks, gently shaken (100 rpm) at 20°C with a 12-h photoperiod and weekly subculture in fresh medium. Frost-hardening treatments Four levels of frost hardiness of the culture were obtained by growing the culture in the dark for 7 days with the following conditions: (i) 2OoC, frost hardy to -9°C (see Results and discussion); (ii) 20°C, with the addition of 7.5 x lo-, M abscisic acid (ABA) (Sigma Chemical Co., St. Louis, U.S.A.) to the culture medium (see Chen and Gusta 1983), frost hardy to -25°C; (iii) 2"C, frost hardy to - 15°C; (iv) 2OC, with the addition of 7.5 x lo-, M ABA to the culture medium, frost hardy to -21 "C.

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FIG. 1. Bright-field micrograph of the culture, frost hardened by treatment 20°C ABA, after freezing to -27S°C. Fluorescein is present; frost-plasmolyzed cells are indicated by arrows. Bar = 100 pm. FIG. 2. FD-fluorescence micrograph, corresponding with Fig. 1, which reveals the living, stained cells (arrows). Comparison with Fig. 1 demonstrates that frost-plasmolyzed cells are dead. Bar 100 = pm. TABLE1. Frost hardiness of the cell culture after the various treatments measured by the fluorescein diacetate method (FD) and the conductivity method (CON) LT,o,"C Treatments 20°C 20°C 2°C 2°C

+ ABA + ABA

By FD -9 -25 - 15 -21

By CON -2.6 -11 -7.2 -9

Frost-hardiness tests Conductivity method Fresh cultures were harvested and filtered through a double layer of absorbant paper (Kimwipes, Kimberly-Clark Corp., Toronto). They were then washed in deionized water (150 mL/5 g tissue), the water was discarded, and the tissue divided among 2 x 15 cm test tubes (0.5 g per tube). Two drops of fresh deionized water was then added to each test tube. These tubes were then partly immersed in a synthesized hydrocarbon liquid cryostatic bath (HAAKE F3-K cryostat with PG20 temperature programmer, HAAKE Mess-Technik GmbH, Dieselstrasse 6, Karlsruhe 41, Federal Republic of Germany). As an experimental control, some tubes were not immersed but were kept at 4°C in a refrigerator. Freezing was initiated by inoculating cultures with fine ice crystals at -2OC. Tubes were then sealed with Parafilm and held at -3OC overnight (14 h) to permit the completion of the development of extracellular ice. Subsequently a cooling rate of 4.8"C/h was used consistently in further frost stressing the cultures. At any desired subzero temperature, samples were taken and thawed at 4°C in a refrigerator. After the last sample had thawed (i.e., in the case of an experimental series stressed to -5, -10, - 15, -20, and -25"C, this would be about 5 h after the -5°C sample had been

placed in the refrigerator and 1 h after the -25°C sample had been placed there), tubes were transfered to a 5OC cold room where 15 mL of precooled (5°C) deionized water was added. After 1 h the conductivity of the water was read at this temperature by a conductance meter (YSI model 35, Yellow Springs Instrument Co., Inc., Yellow Springs, OH, U.S.A.). Conductivities were again measured after leaving the tubes for 18 h. The tubes were then autoclaved and the conductivity was read again at 5OC. The conductivity of the water in a tube before autoclaving was calculated as a percentage of that after autoclaving, and this ratio is defined as percent leakage (% leakage) (Flint et al. 1967; Pearce 1980). Flint et al. (1967) and Pearce (1980) have proposed that frost hardiness can then be defined in terms of 50% damage,

where %leakage, is the experimental freeze-treated measurement and %leakagec is that of the 4°C control. For each experimental treatment, %leakage, was separately calculated for two samples (i.e., the contents of two test tubes). In the Results and discussion section, the reported data are averages of these two measurements. Deviations between these two measurements fell within the range 0.06- 1.6% in all cases. Fluorescein diacetate vital staining Cultures were collected, washed, frozen, and thawed as for the conductivity method given above. Samples were then stained with 0.05 % (w/v) fluorescein diacetate (FD) (Sigma Chemical Co., St. Louis, U.S.A.) for 10 min in centrifuge tubes and washed twice with water by resuspension after centrifugation at approximately 100 X g. Cell viability was determined by the ability of cells to esterify FD (Widholm 1972; Nag and Street 1975) which gave bright yellowgreen fluorescence using exciter filter BG12 and barrier filter 53/44 in a Ziess large fluorescence microscope (Zeiss, Oberkochen, F.R.G.). By this method, LT,, is defined as the temperature at which 50% of


CAN. I. BOT. VOL. 65, 1987

FIG.3. Relationship between %leakage, minimum temperature experienced by the culture (temperature, "C ), and time after immersion of the thawed culture in water ( 0 ,1 h; A , 18 h). The data points represent means of two estimates; deviations in %leakage were always smaller than the height of the symbols marking data points. the cells were stained. Since many samples had to be checked in a relatively brief time period, the liveldead cell ratios were visually estimated. Occasional checks using micrographic records showed this method of estimation was reliable.

Results and discussion In this experiment, comparison has been made between frost hardiness measured by an established conductivity method (Flint et al. 1967; Pearce 1980) and that measured by FD vital staining. The results are shown in Table 1. Although there is a good correlation (r = 0.98) between these two sets of LT,, (i.e, the rankings are the same), in each case measurement by the FD method indicates substantially greater frost hardiness than measurement by the conductivity method.

It would be useful to know which of the above sets of estimates is the more reliable as a measurement of frost hardiness. The fluorescein diacetate method estimates the proportion of cells which retain the capacity to display esterase activity, and therefore tests for metabolic viability on a cell-to-cell basis. The conductivity method, by contrast, estimates the average percentage damage of the cell population, based on the assumption that the greater the efflux of ions the greater is the average freezing injury to the plasma membranes of the cells tested (Palta et al. 1977; and see Levitt 1980, pp. 256-258). Thus, these two methods reveal different characters of the frost-damaged cells. Microscopical examination of the cells before and after freezing provided evidence which supported Widholm's

ZHANG AND WILLISON


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(a)

2O0C

FIG.4. Differential %leakage (DPL) compared with freezing pretreatment for each of the four culture conditions. On each graph, the subzero temperature corresponding with half maximum DPL (%DPL,,,) is indicated. This value closely corresponds with LT,, estimated by the FD method (see Fig. 5).

(1972) interpretation of the basis of the FD test. Cells which took up FD after a freezing treatment retained their prefreezing morphology, whereas unstained cells had no cellular contents or exhibited permanent pseudoplasmolysis (Figs. 1 and 2), both of which seem reliable indicators of cell death. Futhermore, our FD results are comparable with those obtained by Chen and Gusta (1983) on the same culture using both triphenyl tetrazolium chloride (TTC) reduction and FD fluorescence viability tests, and their test data also correlated well with a regrowth test. Similarly, the comparability of FD test and regrowth test results has been demonstrated for spring wheat, winter wheat, and this bromegrass cell culture (Chen and Gusta 1982). This consistency indicates the reliability of the FD test. We can therefore conclude that the present con-

ductivity method is relatively unreliable and tends to underestimate frost hardiness. To improve understanding of the phenomenon of ion leakage following cellular frost damage, a time-course study of conductivity after thawing was initiated. The data in Table 2 show that while LT,, estimated by the FD test is constant with time of post-thaw immersion in deionized water, the conductivity test data indicate apparent, but clearly unreal, decreases in frost hardiness with length of immersion period. These apparent changes in frost hardiness result from increases in %leakage with time of immersion in deionized water (Fig. 3). The increase in leakage is not simply time dependent, however, but varies according to both the freezing pretreatment and the culture conditions (compare Figs. 3a-3d). The differ-

CAN. J. BOT. \'OL. 65, 1987

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TABLE2. Frost hardiness of bromegrass cell culture measured at 1 and 18 h after the immersion of the thawed culture in deionized water

Treatment

Time (in H,O), h

20°C 20°C


By FD

1 18 1 18 1 18 1 18

+ ABA

2 "C 2°C

By CON

+ ABA

LT5,,

by

FD

method ( O C ) ,

X

FIG.5. Relationship between LTso estimates by EDPL,,, and by FD vital staining for eight cultures having different hardiness (the four described in this paper together with four others). NOTE:The regression line (slope, 1.07) is significantly different from zero regression line at P < 0.001, and is close to (and not significantly different from; P > 0.5) a regression line of 1.00. The intercept is not significantly different from zero (P > 0.5). Thus X is not significantly different from Y. ence between leakage after 18 h in deionized water and leakage after 1 h in deionized water can be termed "differential %leakageM(DPL). When DPL was plotted against freezing pretreatment for each culture condition (Figs. 4a-4d), it was found that the curves had a characteristic form. DPL is highest at the highest subfreezing temperature and declines as cells experience deeper subfreezing temperature. This decline in DPL with subfreezing temperature is more rapid in less hardy cell cultures (cf. Figs. 4a and 4b). Given the relationship between DPL and pretreatment, we considered it possible that this phenomenon could be useful in estimating frost hardiness. It was noted that subfreezing treatment corresponding with half the maximum DPL (?hDPL,,,) was similar to LT,, estimates by the FD method (cf. Figs. 4a-4d with Table 2). To test for reliability of the correlation, a regression analysis was performed using data from eight different culture conditions (the four described here together with four others in another experiment). A graphical representation is presented in Fig. 5. No significant difference was found between LT,, estimated by the FD method and the subfreezing

temperature corresponding with EDPL,,,; thus, the latter can be considered to be a conductivity method of estimating LT,,, at least for the bromegrass culture used here. At present it is possible to provide only tentative proposals for the physiological basis of this test, but the following is a working hypothesis. Frost-killed cells, upon thawing, leak electrolytes rapidly, while thawed living cells leak electrolytes slowly, having intact plasma membranes. Thus, as more cells are damaged, so the difference in leakage in relation to immersion period will decline. If the maximum difference (DPL,,,) corresponds with 100% living cells, then half this difference may correspond with 50% living cells, which is the basis for the FD test. The hypothesis above is insufficient, however, to explain all the data obtained. The DPL for controls (4°C) was always found to be smaller than that for nondamaging subfreezing treatments (see Figs. 4a-4d), even though the 4°C controls survived maximally. Presumably, the freezing process increases membrane permeability by some means, such as by damaging active transport systems as proposed by Palta et al. (1977). We conclude that, for this bromegrass culture, LTS0can be estimated by the conductivity method using ?hDPL,,,. Further experimentation is required for a full understanding of the physiological basis of this test. Furthermore, the test may need refinement before it can be used as the sole indicator of LT,, in whole plant tissues. We are continuing with experimental work to these ends.

Acknowledgements The authors are grateful for the help of Marshall Greenwell and Nancy Francis of the Atlantic Regional Laboratory of the National Research Council of Canada, Halifax, for supplying facilities to maintain the cell culture. The cryostat was obtained under DSS contract OSC83-00415 and we are grateful to Dr. Michio Suzuki for permission to use it in this experiment. We are also grateful to Veronica Klein for photographic assistance. This work was supported by an NSERC operating grant A0507 to J.H.M.W. CHEN, P. M., and GUSTA, L. V. 1982. Cold acclimation of wheat and smooth bromegrass cell suspensions. Can. J. Bot. 60: 1207- 1211.

CHEN,T. H. H., and GUSTA,L. V. 1983. Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiol. 73: 71-75.

DEXTER, S. T., TOTTINGHAM, W. E., and GRABER, L. F. 1932. Investigations of hardiness of plants by measurement of electrical conductivity. Plant Physiol. 7: 63 -78. FLINT,H. L., BOYCE, B. R., and BEATTIE, D. J. 1967. Index of injury-a useful expression of freezing injury to plant tissues as determined by the electrolytic method. Can. J. Plant Sci. 47: 229 -230. GAMBORG, 0 . L., and WETTER, L. R. 1975. Plant tissue culture methods. National Research Council of Canada, Prairie Regional Laboratory, Saskatoon. LEVITT,J. 1980. Responses of plants to environmental stresses. Vol. 1. Chilling, freezing, and high temperature stresses. Academic Press, New York. NAG,K. K., and STREET, H. E. 1975. Freeze preservation of cultured plant cells. I. The pretreatment phase. Physiol. Plant. 34: 254-260. PALTA, J. P., LEV~TT,J., and STADELMANN, E. J. 1977. Freezing injury in onion bulbs. I. Evaluation of the conductivity method and ion and sugar efflux from injured cells. Plant Physiol. 60: 393 -397.

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PEARCE, R. S. 1980. Relative hardiness to freezing of laminae, roots and tillers of tall fescue. New Phytol. 84: 449-463. SUKUMARAN, N. P., and WEISER,C. J. 1972. An excised leaflet test for evaluating potato frost tolerance. HortScience, 7: 467-468. UEMURA,M., and YOSHIDA,S. 1984. Involvement of plasma membrane alterations in cold acclimation of winter rye seedlings (Secale cereale L. cv. Puma). Plant Physiol. 75: 818-826.

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WIDHOLM, J. M. 1972. The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47: 189- 194. YOSHIDA, S., and UEMURA, M. 1984. Protein and lipid compositions of isolated plasma membranes from orchard grass (Dactylis glomerata L.) and changes during cold acclimation. Plant Physiol. 75: 31 -37.