CRYOBIOLOGY

13, 243-247

( 1976 )

Ice Tolerance

of Cold-Hardened GEORGE

United

‘Valencia’

Orange

Wood

YELENOSKY

States Department of Agriculture, Agricultural Research 2120 Camden Road, Orlando, Florida 32803

Service,

of mixed fertilizer including 6 cm3 of 12-6-6 liquid fertilizer per plant. Routine greenhouse conditions were maintained with 35°C maximum days and 15.6” minimum nights. Uniform plants about 80 cm high and 0.7 cm in stem diameter, 8 cm above soil level, were selected for tests. Plants were cold-hardened in a 4 x 3 x 1.8 m walk-in controlled-environmental chamber with a mylar barrier having 86% input wattage of cool-white fluorescent lighting and 14% incandescent. Air temperatures were controllable +-0.3”C, and relative humidity was +5%. Standard coldhardening was 2 weeks of 21.1” for 12-hr days and 10°C at night, followed by 2 weeks of 15.6”C day and 4.4”C night. Light intensity at the top of the plants was maintained at 200 peinsteins/m’/sec measured with a Lambda Ll-185 meter.l Air was circulated at 60 m/min, shown by a hotwire anemometer. Relative humidity at 60* 5% was maintained by automatic steam injection. Freeze tests were in a 3 x 3 x 1.8 m chamber. All tests were conducted in the dark, with relative humidity maintained at MATERIALS AND METHODS 50 -C 5%. Plants were cooled about 4”C/hr All tests were with 16- to 20-month-old to different temps. To inoculate plants with potted Valencia orange seedlings. Seeds ice crystals, they were sprayed with a mist were germinated in a greenhouse, and seed- of precooled, distilled water from a preslings were transplanted 3 months later into surized hand sprayer cooled to 0°C. Ice cans 15.4 cm in diameter and 16.7 cm high. 1 Mention of a trademark name or a proprietary The soil was a mixture of 1 part sand, 2 parts vermiculite, 7 parts peat, and a trace product does not constitute a guarantee or war-

Severe freeze damage in citrus trees occurs only after ice forms in the tissues. This is true in some other major crops. Some crops are injured by low temperatures in the absence of ice (8). The presence of ice in citrus is shown by exotherms and watersoaking in the leaves (10, 13). Supercooling delays and sometimes prevents freeze damage in citrus. The injury that follows nucleation is a result of many factors ( 1, 5, 6, 9). In the general classification of plant resistance to freeze injury, cold-hardy plants have the capacity to harden-off and tolerate ice for hours at very low temperature. Commercially grown citrus trees are not very cold-hardy. The capacity of ‘Valencia’ orange (Citrus sinensis ( L. ) Osbeck) plants to coldharden and tolerate ice in this study adds new information on ice tolerance of citrus wood and supports existing information on citrus leaves ( 11, 13). The study is part of continuing work on citrus cold-hardiness to screen cultivars and improve cold protection practices.

Received

March

ranty of the product by the USDA, imply its approval to the exclusion ducts that may also be suitable.

3, 1975. 243

Copyright All rights

1976 by Academic Press, o9 reprhiuction in any form

Inc. reserved.

and does not of other pro-

244

GEORGE TABLE

YELENOSKY

were rated by the percentage of foliage killed and dieback of the main stem. In other tests, the onset of freezing and rate of ice spread in the wood were determined by exotherms. Sensors were six copper-constantan 36gauge thermocouples held against the main stem by plastic clips about IO cm apart. Thermocouple leads were connected to digital multimeters with a resolution of lpV/digit. Rated accuracy was +l digit with less than 2 set settling time. Microvolts x 0.025 equaled “C. Freeze curve profiles were recorded simultaneously on 0 to 100 mV variable-speed, strip-chart recorders connected to the multimeters. These tests started at 1.7”C for 1 hr, followed by a 55”C/hr decrease until freezing was identified by heat release. Nucleating temperatures were maintained for different durations after the onset of freezing in the wood. Thawing rates were about lO”C/hr and were not a significant variable in preliminary trials. In citrus groves, such rates are not unusual immediately after sunrise.

1

Foliage-Kill (% F) and Stem-Dieback (% S) on l&year-old Valencia Orange Seedlings Inoculated with Ice at Different Temperatures, Maintained at Inoculating Temperatures for 3 hr DC

Unhardened %F

-2.2 -3.3 -4.4 -5.5 -6.7

58 * 13h 92 f 4 100 100 100

Cold-hardened* %S

%F

4zt2 15 f 5 32 z!z 4 55 f 7 100

0 0 16f5 48 f 12 100

%S

0 0 0 4zt2 16 f 9

o Two weeks of 21.1” days and 10” nights, followed by 2 weeks of 15.6” days and 4.4” nights. “MeanfSE.

covered the plants within 5 min after spraying. This method during preliminary trials induced ice formation in the plants within 10 min after spraying. Ice in the stems was detected by heat of crystallization, shown by embedded thermocouples shielded from external ice, and by extensive watersoaking in the leaves. Ice-covered plants were maintained for 3 hr at inoculating temperatures. At the end of test periods and before thawing, leaf disks were taken for determining O2 uptake activity. Standard Warburg methods with a differential respirometer measured activity as ~1 02/mg N determined by micro-Kjeldahl 1 procedures. Plants were kept at room temperature (23°C) for 3 hr after freeze tests and then returned to greenhouse conditions for observation of injury for 5 weeks. Plants

RESULTS

AND

DISCUSSION

Table 1 shows that in tests with plants inoculated with ice at different temperatures, similar damage resulted for hardened plants at -5.5”C, and for unhardened plants at -2.2”C. This 3.3”C difference as a result of cold-hardening is a significant increase in the ice tolerance of Valencia orange. Hardened wood tolerated ice con-

TABLE

2

Oxygen Uptake of Leaves on Valencia Orange before and after Inoculation with Ice at Different Temperatures, Maintained at Inoculating Temperatures for 3 hr “C

Prefreeze Unhardened

-2.2 -3.3 -4.4 -5.5 -6.7 QMean

f

61 60 62 60 58 SE of ~1 OJmg

Cold-hardened

f 1.3a f 1.5 f 1.9 f 1.6 f 0.9 N/hr

56 64 58 58 59 at 39°C.

f f i i f

0.3 0.6 0.6 1.1 0.7

Unhardened

14 f 2.0 5 f 0.7 0 0 0

Cold-hardened

52 47 40 31 19

f f zt f f

0.8 0.9 0.3 1.1 1.0

ICE TABLE

TOLERANCE

OF ORANGE

3

The Mean Percentage of Foliage-Kill (SF) and Stem-Dieback (o/,S) in Valencia Orange after Different Durations of Ice at Mean Nucleating Temperatures D~tu&on

Hardeninga

10

uh 11

I:,

--

“C

-6.0 -6.2 -

11

%F

%S

100 1

16 0

-

-

-7.0

7s

0

30

uh h

-6.5 -6.2

100 87

39 0

60

uh h

-6.0 -6.9

100 100

86 8

90

uh h

-6.S -7.3

100 loo

96 10

a uh = greenhouse maintained. h = 2 weeks of 21.1”C, 12 hr days and 10°C nights, followed by 2 weeks of 15.6”C days and 4.4”C nights.

ditions for :3 hr without injury at temperatures 2 to 3°C lower than for unhardened wood. Some wood and more than 50% of the leaves were killed on unhardened plants at --2.2”C, whereas the wood of cold-hardened plants first showed injury at -5.5”C and the leaves at -4.4”C. In cold-hardier citrus types with more cold-hardening than we used, citrus leaves tolerated ice at -5.5”C without injury (11). Also, ice tolerance was related to membrane changes, which some researchers associate with increased phospholipids in leaves of cold-hardy citrus cultivars (4). In this study, membrane damage to Valencia leaves was shown by reduced 02 uptake activity in leaf disks (Table 2). After 3 hr of -2.2”C, activity for hardened leaves was 93% of that found before the freeze, in contrast to 23% for unhardened leaves. Respiratory activity continued in hardened leaves after -6.7”C for 3 hr. Unhardened leaves ceased to function after -4.4”C. From -2.2”C to -4.4”C, activity in hardened leaves was reduced by about 5% per 1°C decrease in test temperature. Below

WOOD

245

-4.4”C, the reduced rate was as much as 22% per 1°C decrease. Reduced O2 uptake activity for hardened leaves after -2.2”C and -3.3”C tests suggests incipient injury, because injury was not visible until -4.4”C (Table 1). It has been shown that both photosynthesis and respiration are affected by freezing between -4.4”C and -9.4”C in several citrus varieties, and -17.8”C for 4 hr completely destroys the photosynthetic system in Valencia leaves ( 12). Both mitochondrial cristae and chloroplasts are injured in frozen citrus leaves (14), and other electron microscope studies of freezing in woody plants show that freeze injury to mitochondria follows mechanical disruption of the endoplasmic reticulum (3). The longer ice persists in tissues, the more critical it becomes to plant survival. This was found for Valencia wood in this were study, where wood temperatures maintained from 10 to 90 min after nucleation. Hardening was much more effective in the wood than in the leaves in offsetting the damage from extended ice periods at relatively low temperatures. For example, stems of cold-hardened Valencia orange plants at -6.2”C withstood ice for 30 min with no visible injury. Unhardened stems were injured within 10 min, regardless of ice forming at slightly higher temperatures (Table 3). Exposure for 14 hr at -6.5”C was enough to cause almost 100% kill of unhardened plants, whereas the same duration of a still lower temp, -7.3”C, resulted in 100% foliage kill but only 10% stem dieback of hardened plants. The critical duration for 100% foliage kill was within 10 min at -6.O”C for unhardened plants and about 30 min at -6.2”C for hardened plants. The relatively quick injury to unhardened Valencia-orange wood within 10 min after the onset of freezing in the mainstems is also shown in other citrus cultivars ( 10). As in citrus, ice in many unhardened plants becomes increasingly injurious as tempera-

2-16

GEORGE

tures decrease. For example, alfalfa, sugarbeets, and peas tolerated extensive ice formation in the tissues, when temperatures did not go below -5°C (2). Frost on unhardened citrus leaves at -5°C is lethal ( 11). This is one reason why temperatures of citrus trees in relation to atmospheric dew point are important during natural freezes. Slower rates of ice spread in the wood were another result of cold-hardening in this study. Rate of linear ice spread averaged I5 t 2 mmlsec in hardened wood at -6.9” *0.2”C. In unhardened wood, the rate of spread was twice as fast, 30 + 5 mm/set, at a slightly higher temperature of -6.2” -I 0.3”C. A slow rate of ice spread is important in minimizing freeze damage caused by tissue rupture and membrane penetration. The slowest rate of ice spread in hardened Valencia wood was 3.6 mm/ set at -5.5”C. At -5.2”C, rates of ice spread were estimated by others to be faster than 2.5 mm/set in lemon tree branchlets (7). Rates slower than 3.6 mm/ set would be expected where strong supercooling did not exist, as in this study. Very little is known about differences in supercooling in citrus groves, but supercooling may influence freeze damage variability. Supercooling resulted in no visible injury to unhardened plants at -2.2”C for 3 hr. At temperatures Iower than -22°C supercooling did not persist long enough to prevent injury, but appeared to delay freezing long enough to reduce the extent of injury. In this study, supercooling was ineffective in reducing injury to Valencia orange at -6.7”C for 3 hr. Faster rates of ice spread resulted with lower nucleating temperatures in hardened wood (T = 0.8474), but this was not as evident in unhardened plants (r = 0.1323). The capacity of Valencia orange wood to cold-harden is small in comparison to that of plants considered cold hardy (1, 5). However, a 2 to 3°C added protection, as a resuIt of hardening, is meaningful in

YELENOSKY

conservation of energy and cheaper cold protection. By one classification of hardiness, Valencia wood would be slightly hardy and the leaves tender (6). SUMMARY

Seedlings of Valencia orange were coldhardened in environmental chambers and inoculated with ice at different temperatures during controlled freezes. Hardening resulted in more than a 3°C decrease in temperature for injury to the wood and a 2°C decrease for injury to the leaves. The main stems of hardened plants withstood ice without injury for periods three times as long as those for unhardened plants. Hardening also reduced by one-half the rate of linear ice spread in the wood and maintained oxygen uptake activity under conditions metabolically destructive to leaves of unhardened plants. REFERENCES 1. Alden, J., and Hermann, R. K. Aspects of the cold-hardiness mechanism in plants. Rot. Reu. 37, 37-142 ( 1971). 2. Cary, J. W., and Mayland, H. F. Factors influencing freezing of supercooled water in tender plants. Agron. J. 62, 715-719 (1970). 3. Krasavtsev, 0. A., and Tutkevich, G. I. Electron microscope investigation of freezing and frost-death in woody plants. Soviet Plant Physid. 17, 317321 ( 1970). 4. Kuiper, P. J. C. Surface-active chemicals, membrane permeability, and resistance to freezing. Proc. 1st Intern. Citrus Symp. 2, 593-595 ( 1969). 5. Levitt, J. Winter hardiness in pIants, p. 495563. In H. T. Meryman (ed.), Cryobiology. Academic Press, New York ( 1966). 6. Levitt, J., and Dear, J. The role of membrane proteins in freezing injury and resistance, In “Ciba Foundation Symposium on the Frozen Cell.” G. E. W. Wohtenholme and M. O’Connor (Eds.), pp. 149-174. Churchill, London 1970. 7. Lucas, J. W. Subcooling and ice nucleation in lemons. Plant Physiol. 29, 246-251

(1954). 8. Mayland, chilling

H. F., and Gary, J. W. Frost and injury to growing plants, A&. Agron. 22, 203-234 (1970).

ICE

TOLERANCE

9. Weiser, C. J. Cold resistance and injury in Science 169, 1269-1278 woody plants. (1970). 10. Yelenosky, G., and Horanic, G. Subcooling in wood of citrus seedlings. Cyobiology 5, 281-283 ( 1969). 11. Young, R. Cold hardening in citrus seedlings as related to artificial hardening conditions. J. An%. Sot. Hoti. Sci. 94, 612-614 (1969).

OF ORANGE

247

WOOD

12. Young, R. Effect thetic system

of freezing on the photosynin citrus. Proc. 1st Intern.

Citrus Symp. 2, 553-558 (1969). 13. Young, R., and Peynado, A. Freezing and water-soaking in citrus leaves. J. Am. Hart. Sci. 91, 157-162 (1967). 14. Young, R., and Yelenosky, G. Effect of freezing on cell ultrastructure in citrus tissues. Cryobiology 10, 531 (Abstr.) (1973).