Aug 6, 1987 - ... of Horticulture Science (N.T., C.S.) and Crop Development Center (L.V.G.), University of ... nitrogen and the quantity of liquid water in apple bud sections ... center. When the desired prefreezing temperature was reached,.
Plant Physiol. (1988) 87, 201-205 0032-0889/88/87/0201/05/$01 .00/0
Freezing of Water in Dormant Vegetative Apple Buds in Relation to Cryopreservation1 Received for publication August 6, 1987 and in revised form February 2, 1988
NANCY TYLER, CECIL STUSHNOFF*, AND LARRY V. GUSTA Department of Horticulture Science (N.T., C.S.) and Crop Development Center (L.V.G.), University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO ABSTRACT Various empirical prefreezing protocols have been used to facilitate cryopreservation of dormant buds from woody plants. The objective of this research was to determine the quantity of water remaining in liquid phase, under different prefreezing conditions using pulsed nuclear magnetic resonance spectroscopy of dormant apple (Malus domestica Mill.) buds from three cultivars. During prefreezing, the quantity of water remaining in the liquid phase was less at -40C< - 30°C< - 20°C for all cultivars tested. The prefreezing temperature had a greater influence on reducing the quantity of liquid water than the duration of prefreezing. Prefreezing to -40°C for 24 hours was optimal for 'Patterson' and 'McIntosh,' the hardiest cultivars, compared to - 30°C for 24 hours with 'Red Delicious.' Cryopreservation of dormant apple buds depends upon the quantity of liquid water during prefreezing, prior to immersion in liquid nitrogen, and upon the cultivar.
Vegetative, dormant apple (Malus domestica Mill.) buds can be cryopreserved by cooling frozen tissue from -20 to - 50°C (14, 16) prior to immersion in liquid nitrogen. The prefreezing treatment dehydrates the cells, thus preventing large intracellular ice crystals from forming when the tissue is immersed in liquid nitrogen. Tyler and Stushnoff (our unpublished data) found a higher percent survival of apple buds following cryopreservation if the buds were cooled slowly to - 30°C and held isothermal for 24 h prior to immersion in liquid nitrogen. For certain cultivars, it was necessary to artificially dehydrate the tissue at 0°C prior to cryopreservation. These results suggest that only a certain amount of intracellular liquid water can be tolerated in cells prior to immersion in liquid nitrogen. Also, in these tissues, freezing is not in equilibrium, even at slow cooling rates (10°C/h), and a period of time is required for water to escape to extracellular sites of ice crystallization. Chen et al. (6), using NMR spectroscopy, found a strong positive correlation between percent liquid water at 40°C in Catharanthus roseus cells and percent survival following storage in liquid nitrogen. In the present study, NMR pulse spectroscopy was used to determine the relationship between survival in liquid nitrogen and the quantity of liquid water in apple bud sections at given prefreeze temperatures. The loss of liquid water due to crystallization as a function of time was quantified to determine the effects of dehydration on cryopreservation. -
MATERIALS AND METHODS Plant Materials. Dormant twigs with vegetative apple (Malus domestica Mill.) buds of cv 'Patterson' and cv 'Rescue' were Supported by Agriculture Canada, Plant Gene Resources. 201
collected in December 1984 and in February and March 1985 from the field plots of the Department of Horticulture Science, University of Saskatchewan, Saskatoon, Saskatchewan. The cvs 'McIntosh' and 'Red Delicious' were obtained from H. A. Quamme, Agriculture Canada, Summerland, B.C. Samples were stored in sealed plastic bags at - 3°C. NMR Spectroscopy. Liquid water content as a function of temperature was determined from - 4 to - 50°C with a Brukes Mini Spec p 20 spectrometer as described previously (8). Briefly, the method involved using a series of 900 pulses separated by 3 s to prevent saturation. The free induction decay after the second and each succeeding pulse was monitored 20 As after the pulse center. When the desired prefreezing temperature was reached, samples were transferred from the freezer to the NMR sample chamber maintained at the same temperature. After the quantity of liquid water was determined by NMR spectroscopy, the samples were returned to the freezer and maintained at the same temperature. After storage for 24 to 96 h, samples were transferred back to the NMR spectrometer to determine the quantity of liquid water. Five samples were used for each cultivar and each temperature. A sample consisted of the bud and a small portion of cortical tissue. Moisture content was determined grav-
imetrically. Viability Following Cryopreservation. Bud-section samples consisting of a 2.5 cm stem section containing a vegetative bud developed during the previous season were placed in plastic cryovials and cooled 2°C/h to - 20, - 30, or - 40°C. Upon cooling to selected prefreezing temperatures, the buds were either immersed directly in liquid nitrogen or held 24 or 96 h prior to immersion in liquid nitrogen. Samples were removed after 24 h in liquid nitrogen and placed at 2°C to thaw. Survival of the buds was determined by the oxidative browning method. Effect of Dehydration on Freezing H20. Twigs of McIntosh, Red Delicious, and Patterson collected in February 1985 were cut into bud-sections approximately 2.5 cm in length containing a vegetative bud and were dehydrated at 2°C for 3 d. Sections of cortical tissue containing a bud from nondehydrated twigs and from dehydrated bud-pieces were placed in capped NMR tubes. Samples were placed at - 4°C in a chamber for 30 min and cooled at 2°C/h to - 50°C. Freezing was initiated as previously described. The quantity of liquid water at -4, -5, -7.5, -10, - 20, -25, - 30, - 35, - 40, and - 50°C was determined by transferring the sample from the cooling chamber to the NMR spectrometer maintained at the same temperature. Determinations were made on three samples for each cultivar and each treatment. The moisture content for each sample was determined gravimetrically. Effect of Prefreeze Temperature on Cryopreservation. Budsections of Rescue, collected in December 1984, and Patterson, collected in December 1984 and March 1985, were placed in plastic cryovials and cooled 2°C/h to - 16°C and thereafter at
202
Plant Physiol. Vol. 87, 1988
TYLER ET AL.
10°C/h to the desired temperature. Upon cooling to either 20 - 30°C, samples were either placed in liquid nitrogen or held at -20 or - 30°C for 24 h and then placed in liquid nitrogen. Another group of samples was cooled at - 300C, held at this temperature for 24 h, and then warmed back to - 20, - 10, or 0°C for 30 min. Samples warmed to - 20 or - 10°C were either placed directly into liquid nitrogen or recooled to - 30°C and then were placed in liquid nitrogen. Samples warmed from - 30 to 0°C were recooled to 30°C and then placed in liquid nitrogen or were held 24 h at this temperature and then placed in liquid nitrogen. Survival after storage in liquid nitrogen was evaluated by oxidative browning as described previously. Statistical Analysis. Analyses of variance were conducted for each variable in each experiment. Survival percentages were analyzed using arcsine transformations and detransformed for presentation. -
LIQUID WATER (g/g dry wt)
or
0.1
.
0.4
0.3
0.2
(a)
{
-
20C
-30&C -40°C
-
RESULTS There was a significant cultivar x prefreezing temperature x duration of prefreezing interaction for survival. Optimum cryopreservation conditions were obtained when the buds were cooled 2°C/h to -30 or - 40°C and held at these temperatures for 24 h prior to immersion in liquid nitrogen. Patterson and McIntosh buds held at - 40°C for 96 h prior to immersion in liquid nitrogen had lower bud survival than buds held only 24 h. Buds prefrozen to 20 or 30°C and placed directly in liquid nitrogen had the lowest survival rate for all cultivars. Survival increased substantially when the samples were held at -20 or - 30°C for 24 h. This was also the case for Patterson and McIntosh at 40°C, but not for Red Delicious. Buds of Patterson, the hardiest cultivar (17), prefrozen to - 20°C, and held at this temperature for 24 h, had a survival rate of 85% compared to only 10% survival for McIntosh and Red Delicious (Table I). The quantity of liquid water in the apple buds decreased significantly as the prefreezing temperature was lowered but not when the duration was increased from 24 to 96 h (Fig. 1). Patterson had less liquid water at the onset of freezing and after 24 and 96 h at - 20, - 30, and - 40°C compared to McIntosh and Red Delicious. However, the quantity of liquid water, expressed on a percentage basis, was highest in Patterson at - 20°C. The quantity of liquid water in McIntosh was similar to the quantity in Red Delicious buds at 20, 30, and - 40°C at the onset of prefreezing. Holding the samples isothermal at - 20, - 30, and 40°C for an additional 96 h resulted in a decrease in liquid water, but primarily within the first day (Table II). The freezing of water was not in equilibrium with cooling (2°C/h) within this temperature interval. -
-
-
-
-
-
Table
)
(
C
+
)
~~~~~~~~~96h
+
FIG. 1. Boxplot of liquid water content as a function of (a) prefreezing temperature and (b) duration of prefreezing interval. The median = +,
the lower and upper hinges are the left and right edges of the box, and the 95% confidence interval = ( ) (18).
Dehydration of apple bud sections at 0°C decreased the total water content by as much as 55 to 60% of the original field level. At subzero temperatures, the dehydrated bud sections had less freezable water over the entire temperature range of - 10 to - 50°C (Fig. 2-4). The difference in liquid water between dehydrated and control samples was greatest at the warmer temperatures; this difference gradually diminished as the temperature was lowered, but the dehydrated buds had less liquid water at all temperatures tested. For each cultivar, there was a significant hydration level x temperature interaction. The freezing
for both the controls and the dehydrated samples of McIntosh and Red Delicious were similar, while the values for Patterson were lower. Dehydrated samples of Patterson could not be nucleated until 20°C in contrast to 10°C for McIntosh and Red Delicious (Fig. 2-4). Plots of the amount of liquid water versus temperature as hyperboles were described by Gusta et al. (8) and Anderson et al. (1). Liquid water (LT) versus temperature (T[°C]) exhibits the following relationship (8): LT = ([L k]IATmT7) + k. The plot of LT versus -1lT is linear with slope (L0 -k)AT,,. and intercept k, when Lo is the liquid water at 0°C, AT,, is the average melting point depression, and k is the unfreezable water. There was no apparent difference in k for the three cultivars tested. However, dehydrated bud sections had lower values for k than the control sections. The values for k obtained from dehydrated samples were comparable to those of citrus leaves (1).
curves
-
-
-
Effect of Duration and Prefreezing Temperature on the Survival of Apple Buds Cryopreserved in Liquid Nitrogen Survival Duration Temperature McIntosh Red Delicious Patterson ha %0 °C 0 24 96 0 24 96 0 24 96
-30
-40
Time samples
were
Oh
24h 96F
+
(q_+
I.
-20
a
(b)
15 85 45 40 100 90 50
0 10 10 0 80 66 65
100 76
100 81
0 10 19
0 85
60 63 66 75
LSD = 16% held under prefreezing conditions prior to immersion in liquid phase liquid nitrogen.
203
FREEZING OF WATER IN DORMANT APPLE BUDS Table II. Quantity of Liquid Water for Three Apple Cultivars Maintained at Three Prefreezing Temperatures for 0, 24, and 96 h Liquid Water Patterson McIntosh Red Delicious Mean ± SD h °C H2Olg dry wt -20 0 0.39 (52)a 0.41 (42) 0.41 + .01 0.42 (43) 24 0.34 (45) 0.38 (38) 0.36 (37) 0.36 ± .02 96 0.33 (45) 0.35 (36) 0.34 (35) 0.36 + .01 -30 0 0.29 (28) 0.33 (25) 0.30 (24) 0.30 ± .02 24 0.23 (21) 0.26 (20) 0.24 (20) 0.24 ± .02 96 0.23 (21) 0.25 (19) 0.24 (19) 0.24 + .01 -40 0 0.17 (18) 0.22 (24) 0.20 (21) 0.20 ± .03 24 0.14 (15) 0.20 (22) 0.19 (19) 0.17 ± .03 96 0.12 (13) 0.17 (18) 0.16 (16) 0.15 ± .03 a Values in parentheses indicate the quantity of liquid water at subzero temperatures as a percent of the total water.
Control samples for the three cultivars had a similar melting point depression, approximately 3.7 osmolar, as determined from the freezing curve. Dehydrated McIntosh and Red Delicious bud sections had a melting point depression of approximately 9.5 osmolar, whereas Patterson sections had a value of 11.2 osmolar. This agrees with the degree of supercooling exhibited by these samples. The survival of Rescue and Patterson buds frozen and/or warmed to various temperatures before storage in liquid nitrogen is shown
in Table III. Buds of both cultivars cooled slowly to - 20°C and then stored in liquid nitrogen either did not survive this treatment or had a low survival rate. However, survival of the buds increased if the samples were stored at - 20°C for 24 h prior to storage in liquid nitrogen, as was shown earlier. As in the previous study, there was also a high recovery rate of buds prefrozen to - 30°C and stored for 24 h prior to storage in liquid nitrogen. If buds were frozen and stored at - 30°C for 24 h and warmed to - 10°C, then stored in liquid nitrogen, there was a low recovery rate. Buds held at - 30°C for 24 h, warmed to - 10°C for 30 min, and then cooled to - 30°C had the same survival rate as buds cooled to - 30°C. The survival rate of buds cooled to - 30°C for 24 h and warmed to - 20°C prior to storage in liquid
-10
T
(°C)
FIG. 2. Amount of liquid water, L-AgH2O/g dry wt) in dehydrated and control buds of Red Delicious apple cooled through a series of subzero temperatures.
-20
-30
-40
-50
T (0C) FIG. 3. Amount of liquid water, LAgH20/g dry wt) in dehydrated and control buds of McIntosh apple cooled through a series of subzero temperatures.
TYLER ET AL.
204
Plant Physiol. Vol. 87, 1988
DISCUSSION The survival of apple buds stored in liquid nitrogen was dependent mostly on the temperature to which the samples were cooled prior to immersion in liquid nitrogen, and only slightly on duration of prefreezing exposure prior to immersion in liquid nitrogen. Samples cooled at 2°C/h to -30 or -40°C and held at either temperature for 24 h prior to storage in liquid nitrogen had the highest recovery rate. Previously, Tyler and Stushnoff (unpublished data) working with 15 apple cultivars found the highest survival was obtained when samples were held at 30°C for 24 h prior to immersion in liquid nitrogen. Sakai (15) found very cold-hardy tissue could tolerate warmer prefreezing temperatures better than less hardy tissues. Cryopreservation was optimal for Catharanthus cells (6), wheat cultures (7), and strawberry meristems (10) prefrozen quickly (approximately 1°C/min) to - 40°C. Katano et al. (11) reported dormant apple shoot tips survived cryopreservation when prefrozen quickly (0.5°C/min) to - 10 to - 70°C. McIntosh and Red Delicious did not survive storage in liquid nitrogen if prefrozen 2°C/h to 20 or 30°C (N Tyler, C Stushnoff, unpublished data). However, if samples were cooled at 2°C/h and then held for 24 h at either -20 or 30°C, survival increased and the liquid water content decreased; this suggests an association between unfrozen water and cell survival during slow freezing prior to storage in liquid nitrogen. Harrison et al. (9) suggested that approximately 70% of the freezable water must be frozen in hardy dogwood stems before they can survive immersion in liquid nitrogen. However, in this study it was found that holding the tissue at either 20, 30, or 40°C for 96 h compared to 24 h prior to immersion in liquid nitrogen sometimes resulted in a decrease in bud section survival while the liquid water remained the same (Tables I and II). Red Delicious buds prefrozen to - 30°C had the same quantity of liquid water but lower viability after 96 h compared to 24 h of prefreezing. Therefore, with Red Delicious there is not a simple relationship between liquid water content and survival of buds after storage in liquid nitrogen. A substantial amount of water in the apple buds remained unfrozen at temperatures lower than 20°C. In contrast, nearly all the freezable water in red-osier dogwood stems was frozen at - 20°C. Only 0.25 to 0.30 g liquid water/g dry weight remained at - 20°C (2) or - 40°C (9). In contrast, in this study, apple bud sections had a higher liquid water content at -20°C (0.39-0.42 g water/g dry weight), and also, in these samples, water continued to freeze as the samples were cooled to 40°C. The liquid water content at -40°C was 0.17 to 0.22 g water/g dry weight. The amount of liquid water from -20 to 40°C was higher than -
-
-
-
-
T (0) FIG. 4. Amount of liquid water, LAgH20/g dry wt) in dehydrated and control buds of Patterson apple cooled through a series of subzero temperatures.
nitrogen was higher than buds cooled directly to - 20°C and then stored in liquid nitrogen. Patterson buds held at - 30°C for 24 h, warmed to - 20°C for 30 min, and then cooled to - 30°C prior to storage in liquid nitrogen had a higher survival rate than buds cooled to - 30°C, but not held for 24 h before storage in liquid nitrogen. The enhanced survival of buds held at - 30°C was lost if the buds were warmed to 0°C and then cooled back to - 30°C prior to storage in liquid nitrogen. However, if the buds were held at 30°C for 24 h after being thawed at 0°C the enhanced survival was recovered. -
-
-
-
-
Table III. Effect of Prefreezing Conditions Prior to Immersion in Liquid Nitrogen on the Survival of Dormant Vegetative Apple Buds Survival Prefreezing Regime
Rescuea
Pattersona
Pattersonb
°C, (time) N 0 dc 15 e 0d -20, -196 - 20 (24 h), -196 85 b 40 b 76 b 40 d 45 c 45 b -30,-196 - 30 (24 h), -196 95 a 85 a 100 a 20 c 0d 10 f -30 (24 h), - 10, -196 - 30 (24 h), - 10, -30, -196 40 d 35 c 55 b - 30 (24 h), -20, -196 25 c 60 c 50 b 80 a 30 c 65 c -30 (24 h), -20, -30, -196 - 30 (24 h), 0, - 30, - 196 40 b 35 c - 30 (24 h), 0, - 30(24 h), - 196 80 a b a Scions collected March 1985. c Within columns, values followed Scions collected December 1984. by the same letter are not significantly different at the 5% level.
-
FREEZING OF WATER IN DORMANT APPLE BUDS reported for either Solanum (4) or cereals (8). However, these values are in agreement with the values reported for citrus leaves which have rigid cell walls (1). If the apple bud sections were held 24 to 96 h at - 20, - 30, or - 40°C, the liquid water content approached the values obtained for cereal crowns. Patterson, the most cold hardy of the cultivars, had the least amount of liquid water at each prefreezing temperature. Gusta et al. (8) found no relationship between hardiness and the nonfreezable water of hardy or tender winter wheat cultivars at - 40C. Generally, survival in liquid nitrogen was highest when the tissue had 20% or less liquid water at the prefreezing temperature (Table II). Patterson bud sections held at - 20°C for 1 d had over 45% liquid water and a survival rate of 85 % following liquid nitrogen storage. It is unlikely that the intracellular water effluxed to extracellular ice when the tissue was immersed in liquid nitrogen. Therefore, either intracellular ice formed that was noninjurious to the cells, or the intracellular water underwent a slow transition. The prefreezing temperature must be low enough to dehydrate cells to allow formation of small, noninjurious intracellular ice crystals or formation of glass transitions. Prefreezing to - 30 and - 40°C resulted in less liquid water and an increase in survival for Patterson and McIntosh as compared to sections prefrozen to - 20°C. However, if the prefreezing temperature is too low, injury may occur prior to storage in liquid nitrogen. For example, survival of Red Delicious did not improve at - 400C, probably due to a combination of temperature and dehydration. Survival in liquid nitrogen decreased for several apple cultivars when prefrozen to - 400C as compared to - 300C (N Tyler, C Stushnoff, unpublished data). This study demonstrated that the amount of liquid water in buds at subzero temperatures is dependent on both temperature and time. It has been assumed that cold-hardy tissues have increased permeability to water efflux. However, even though buds were held at - 40°C for 15 h and then cooled slowly (2°C/h), freezing was not in equilibrium with the temperature. Tissue had to be held isothermally for at least 24 h before freezing was in equilibrium with temperature. In cereal crowns which have pliable cell walls, near ideal freezing behavior is observed (8). However, in citrus leaves, which have rigid cell walls, pressure potential plays an important role in cell water relationships at freezing temperatures (1). Dehydration of the bud sections reduced the total amount of water present by 60% and reduced the quantity of liquid water at subzero temperatures. A reduction in water content has led to increases in cold hardiness in several woody plants (3, 5, 1214) and in our research to increased survival following immersion in liquid nitrogen. In our work, samples were prefrozen to - 300C for 24 h. If the differences in liquid water between dehydrated and control samples, as determined in the present study, were maintained after the tissue was held at - 300C for 24 h, formation of innocuous intracellular ice crystals would be more probable in the dehydrated tissue. Dehydrated samples had approximately 50% less liquid water than controls at the onset of freezing. Why a difference in liquid water persisted during freezing from - 10 to - 500C is unclear. Dehydration may have resulted in partial collapse of the cell which in turn may have allowed water to escape faster. The dehydration process may have affected the pliability of the cell wall allowing it to collapse during freezing. However, this appears unlikely from the freezing and thawing results presented
205
in Table III. The survival of bud sections cooled to - 300C, warmed to 0°C, and then cooled to - 30°C was not greater than sections cooled to - 30°C and then stored in liquid nitrogen. Not all of the cells may have dehydrated at the same rate. This would result in an underestimation of the water in the tissue. Dehydrated buds of Red Delicious and McIntosh did not freeze until the temperature was lowered to - 10 to - 12°C. Attempts to nucleate the samples at warmer temperatures with ice crystals were unsuccessful. The dehydrated Patterson buds did not freeze until the samples were cooled to - 200C. Since the bulk of the water had been removed by dehydration, these supercooled sections were not injured when freezing occurred at these lower temperatures. These results suggest that intracellular freezing did not occur when freezing was initiated. The cell solute concentration would have increased from approximately 2 m osmolar for hydrated samples to approximately 5 m osmolar in the dehydrated samples. This increased cell solute concentration could have protected the supercooled cells from freezing intracellularly at - 10 to - 200C. Successful cryopreservation of apple buds is dependent, in part, upon the quantity of liquid water at the temperature to which the tissue is cooled prior to immersion in liquid nitrogen. LITERATURE CITED 1. ANDERSON JA, LV GUSTA, DW BUCHANAN, MJ BURKE 1983 Freezing of water in citrus leaves. J Am Soc Hortic Sci 108(3): 397-400 2. BURKE MJ, RG BRYANT, CJ WEISER 1974 Nuclear magnetic resonance of water in cold acclimating red-osier dogwood stem. Plant Physiol 54: 392398 3. CHEN PM, PH Li, CJ WEISER 1975 Induction of frost hardiness in red-osier dogwood stems by water stress. HortScience 10(4): 372-374 4. CHEN PM, MJ BURKE, PH Li 1976 The frost hardiness of several Solanum species in relation to the freezing of water, melting point depression and tissue water content. Bot Gaz 137: 313-317 5. CHEN PM, PH Li, MJ BURKE 1977 Induction of frost hardiness in stem cortical tissues of Cornus stolonifera Michx by water stress. I. Unfrozen water in cortical tissues and water status in plants and soil. Plant Physiol 59: 236239 6. CHEN THH, KK KARTHA, F CONSTABEL, LV GUSTA 1984 Freezing characteristics of cultured Catharanthus roseus (L.) G. Don Cells treated with dimethylsulfoxide and sorbitol in relation to cryopreservation. Plant Physiol 75: 720-725 7. CHEN THH, KK KARTHA, LV GUSTA 1985 Cryopreservation of wheat suspension culture and regenerable callus. Plant Cell Tissue Organ Culture 4: 101-109 8. GUSTA LV, MJ BURKE, A KAPOOR 1975 Determination of unfrozen water in winter cereals at subfreezing temperatures. Plant Physiol 56: 707-709 9. HARRISON LC, CJ WEISER, MJ BURKE 1978 Freezing of water in red-osier dogwood stems in relation to cold hardiness. Plant Physiol 62: 899-901 10. KARTHA KK, NL LEUNG, K PAHL 1980 Cryopreservation of strawberry meristems and mass propagation of plantlets. J Am Soc Hortic Sci 105(4): 481484 11. KATANO M, A ISHIHARA, A SAKAI 1983 Survival of dormant apple shoot tips after immersion in liquid nitrogen. HortScience 18(5): 707-708 12. Li PH, CJ WEISER 1971 Increasing cold resistance of stem sections of Cornus stolonifera by artificial dehydration. Cryobiology 8: 108-111 13. LuMis GP, RA MECHLENGURG, KC SINK 1972 Factors influencing winter hardiness of flower buds and stems of evergreen azaleas. J Am Soc Hortic Sci 97(1): 124-127 14. SAKAI A 1960 Survival of the twig of woody plants at -196°C. Nature 185: 393-394 15. SAKAI A 1965 Survival of plant tissue at super-low temperatures. III. Relation between effective prefreezing tempeatures and the degree of frost hardiness. Plant Physiol 40: 882-887 16. SAKAI A, Y NISHIYAMA 1978 Cryopreservation of winter vegetative buds of hardy fruit trees in liquid nitrogen. HortScience 12(3): 225-227 17. STRANG JG, C STUSHNOFF 1975 A classification of hardy North American apple cultivars based on hardiness zones. Fruit Var J 29(4): 78-108 18. VELLEMAN PF, DC HOAGLIN 1981 Applications, Basics and Computing of Exploratory Data Analysis. Boston, Duxbury Press
