70
FLORIDA STATE HORTICULTURAL SOCIETY, 1963
COLD TOLERANCE AND FREEZING POINT OF
CITRUS SEEDLINGS1
L. K. Jackson and J. F. Gerber2
Cooper et al. (2) and Young (13) have shown
that the cold tolerance of citrus is related to en
vironmental conditions prior to the exposure of
the plant to freezing temperatures.
eratures
and
short
Cool temp
photoperiod brought
about
dormancy and reduced cambial activity of citrus.
Nijjar and Sites (7), working with controlled
climate chambers, found that a 60° F. light per
iod of 16 hours and 38° F. dark period increased
cold tolerance. According to Reese (8), the most
effective preconditioning for citrus consisted of
a 12-hour light period during which the tem
perature was maintained at 78° F. and a dark
period of 38° F. Plants exposed
ditions were usually able to tolerate
tures better than plants which had
jected to such treatments.
Many (2, 4, 6, 9, 12) workers
to these con
cold tempera
not been sub
have investi
gated the nature of the changes which occur in
plants due to preconditioning whereby cold tol
erance is obtained. Their findings were: (a)
there is a discernible change in the protoplasm
in the plant, (b) dormancy is induced or growth
retarded, (c) dehydration may occur with sub
sequent changes in the morphology of the cells,
i.e., the cells tend to become smaller and cell
walls thicker. Vasil'yev has reported (12) that
the osmotic pressure of the vacuole contents in
creases in lemons and mandarins. Levitt (4) in
dicated that ice formation within the cell, as
occurs in nature, was always fatal. Plants which
were able to tolerate freezing temperatures did
so by allowing ice formation only in the spaces
between the cells.
Research with tree crops has shown that po
tassium may induce cold tolerance in certain
species. Sharpe et al. (11) observed that 'Moore'
pecans supplied with high levels of potassium
were more cold tolerant than were trees supplied
with lower levels of this element. Brown and
Potter (1) showed that a deficiency of potassium
in tung trees brought about more pronounced
damage following a freeze. Reese (8) investilWork
done
as
partial
fulfillment
University of Florida, December, 1962.
of
M.S.A.
Degree
2Assistant in Fruit Crops, currently Assistant in Horti
culture, Florida Agricultural Extension Service, and As
sistant Climatologist, Department of Fruit Crops, Florida
Agricultural Experiment Station, Gainesville.
Fi1™da A»ricultural Experiment Stations Journal Series
■No. 1773.
gated the effect of potassium upon cold tolerance
of seedlings of trifoliate orange and sour orange.
His results indicated that potassium did not in
duce statistically significant changes in the cold
tolerance of these 2 varieties. Field observations
made by Smith and Rasmussen (10), following
the 1957-58 freeze in Florida, showed that high
potassium may not cause citrus to be more hardy.
One of the difficult aspects in measuring cold
tolerance of plants is the necessity to sacrifice
at least a portion of the plants during the freez
ing. Hendershott (3) has reported that the
freezing point of 'Pineapple' orange leaves is 20
dz 0.5° F. Young and Peynado (14) also reported freezing temperatures of citrus leaves and
plants with a considerably wider range in values.
Marshall and Padfield (5) investigated the freez
ing point of pear fruit and found that deter
minations made in the laboratory agreed with field
observations.
Since cold tolerance is one of the important
aspects in determining distribution of citrus,
more information is needed concerning the plant's
mechanism for becoming tolerant of freezing
temperatures. This paper deals with the nature
of citrus cold tolerance through the regulation
of potassium nutrition and the determination of
the freezing points of both intact 'Pineapple'
orange seedlings and detached leaves.
Materials and Methods
'Pineapple' orange seedlings, approximately
1-year-old, were used in the experiment. The
plants were potted in 4-inch plastic pots con
taining a medium of 2 parts No. 5 mesh acid
washed granite gravel and 1 part horticultural
perlite so that nutritional variables could be
carefully controlled. The plants were grown in
a greenhouse and were preconditioned in con
trolled climate chambers described by Nijjar
and Sites (7) and by Reese (8). Prior to the
preconditioning period, the nutritional level of
all plants was the same.
The preconditioning period in the controlled
climate chambers consisted of a 12-hour photoperiod with a day temperature of 78° F. and a
night temperature of 38° F. During this period
of 5 weeks, the plants were fertilized on alter
nate days with 50 ml. each of modified Hoagland's
solutions. Potassium was supplied in the solution
JACKSON AND GERBER: COLD TOLERANCE OF SEEDLINGS
71
as rates of 0, 8, 32, and 128 parts per million.
which received no potassium showed deficiency
The experiment was designed as a split plot in
symptoms.
order that cold tolerance and potassium nutri
tional
effects
could
be
Each chamber contained
1
After the plants had been removed from the
simultaneously.
freezing chamber for 15 days they were ranked
replicate, and since
according to a method described by Nijjar and
studied
only 2 chambers were available, it was necessary
Sites (7) and by Reese (8). This rating was used
to repeat the entire process twice in order to
to determine percent survival and damage index.
obtain 4 replicates.
Each replicate contained 168
After the preconditioning and establishment of
different levels of potassium in the foliage, the
plants were removed from the controlled climate
chambers to the freezing chamber.
The freezing
chamber was precooled to 35° P., and after the
plants were in place, the temperature was reduced
at the rate of 2° F. per hour. When the desired
minimum temperature had been reached, the
temperature was increased at the rate of 2° F.
per hour until 35° F. was again obtained. At
this
time, the
The determination of the freezing point of de
tached leaves that was made in the laboratory
plants.
plants
were
removed
to
a
con
trolled climate chamber at 38° F. for 12 hours
with a gradual warming to 78° F. Three mini
mum freezing temperature treatments were used.
These were 17° F., 19.5° F., and 22° F. During
the course of the experiment it was discovered
that the mixing of air within the freezing cham
ber was insufficient to insure that the tempera
tures of the leaves and of the air was the same. "
Therefore, thermocouples were placed on the
leaves, and leaf temperatures were measured
rather than air temperatures.
It was necessary
to reduce the air temperature in the chamber
approximately 2° F. lower than the anticipated
minimum temperature in order to bring the leaf
temperatures to the desired values. Leaf tempera
tures could not be maintained with great pre
cision in the freezing chamber.
The best control
that was available was approximately ±1°
F.
In order to determine the minimum tempera
ture to which the plants were to be subjected,
leaves from preconditioned plants were removed
and frozen in the laboratory. These results indi
cated that the freezing temperature for the leaves
was 19.5° F. which was used as an indication of
the killing temperature of the plant.
was performed by the use of a small freezing
chamber.
This chamber consisted of an alcohol-
ice bath that was maintained at —16.6° F.
bath was placed in a
tube was inserted into the bath that was large
enough to accept the leaf.
A small bead ther
mistor was attached at the midrib of the leaf
with a piece
resistance
of masking tape.
in
the
thermistor,
The change in
which
was
pro
portional to temperature, was measured by the
use of a Wheatstone bridge circuit.
The bridge
output was fed into a Leeds & Northrup micromicroammeter amplifier and into a 1 milliampere
Esterline-Angus recorder. In this manner, it was
possible to measure and record simultaneously the
temperature of the leaf.
Results and Discussion
In order to determine the mean freezing point
and the standard deviation, 165 detached citrus
leaves
were
frozen.
The
mean
freezing
point
obtained was 19.5° F. with a standard deviation
of 1.08° F.
This value was used as the killing
temperature of the leaves.
Accordingly, it was
further reasoned that 19.5° zb 2° F. should kill
95 percent of the leaves found in the lot of plants
used.
This value is in agreement with Hender-
shott's
(3)
'Pineapple'
value of 20
orange
zt
leaves.
0.5°
F. for mature
The
variability
en
countered in the freezing point may have been
due to random variation in the plant material
or to the lack of refinement and precision in the
method of determination of the freezing point.
However, the results of the freezing point de
terminations did agree with results obtained in
the freeze chamber.
The potassium nutritional status of the plants
was determined by performing foliar analyses
This
Dewar flask, and a test
to 22°
±
served.
At 19.5°
±
ob
plete,
killed to the soil line.
and the potassium
though the method was not precise and exact, it
content
determined.
potassium
Plants
contained
0.59
17°
damage was
1° P. defoliation was com
for potassium. A total of 50 leaves were selected
ppm
at
When plants were subjected
F. virtually no
from each potassium treatment prior to freezing
supplied with 0
and
1°
zb
1°
F.
the
plants
were
This indicated that even
did provide very useful data in terms of lethal
percent foliar potassium, 8 ppm potassium, 0.70
limits to which the plant could be subjected.
percent, 32 ppm potassium, 0.89 percent, and
128 ppm potassium, 1.60 percent.
The plants
this way it may be possible to select a random
sample of leaves from
In
a citrus grove and de-
72
FLORIDA STATE HORTICULTURAL SOCIETY, 1963
termine the cold tolerance of plants at any time
without sacrificing or exposing the entire plant.
Freezing
points
of
leaves
were
determined
from plants that had been subjected to different
potassium
Table
1.
levels.
From
proximately 1°
These
the
values
standard
are
shown
deviation
of
in
ap
F., potassium levels did not in
fluence the mean
The effect of the
freezing points
examined and is
freezing points of the leaves.
preconditioning treatment upon
of detached leaves was also
shown in Table II. Whereas
leaves from preconditioned plants froze at slightly
lower temperatures than leaves from plants which
were actively growing, the mean variance and
the standard deviation were so large that no
statistically significant differences were observed.
Reese (8) and Nijjar and Sites (7) have shown
that preconditioning may have a statistically
significant effect upon the killing and recovery of
citrus seedlings. In view of this it is surprising
that differences could not be obtained in the
freezing point of detached leaves. However, the
rate of cooling in the freezing chamber was ex
tremely rapid, approximately 450° F. per hour.
Various workers (4, 9, 12) have shown that ice
formation within the cell itself is always lethal
under conditions approaching natural freezing.
The rapid rate of freezing which was used in the
laboratory may have prevented water from pass
ing out of the vacuole into the spaces between the
cells. Thus, freezing inside the cells may have
occurred.
The effect of potassium nutritional levels upon
freeze survival and damage index of the plants
is shown in Table III. There were no statistically
significant differences in either survival or dam
age due to varying potassium levels. This is in
agreement with the work of Reese
(8)
but is
not in general agreement with concensus among
growers.
The influence of freezing temperatures upon
the percent survival and damage index of Tineapple' orange seedling is shown in Table IV. Both
damage and survival were significantly affected by
the temperature to which the plant was subjected.
It is noteworthy that even though potassium
may induce cold hardiness in plants other than
citrus, neither the work of Reese (8) nor this
work has indicated any increase in cold tolerance
due to potassium levels. This was true both for
TABLE I.—Mean1 subcooling and freezing points of detached leaves supplied
with varying levels of potassium.
Potass ium
Subcooling Point
ppm
o F.
0
8
32
° F.
16.9°
19.^°
18.5°
17.4°
18.1°
16.2°
17.9°
128
Freezing Point
19.6°
110 leaf samples.
TABLE
I I.--Mean freezing point of dormant and actively growing
'Pineapple' orange.
Dormant Group
Freezing Point ° F.
Mean
Standard Deviation
t value
t value (a) 5%
]20 leaf samples.
18.63
0.94
1.50
2.23
leaves of
Active Group
Freezing Point ° F.
.20.03
JACKSON AND GERBER: COLD TOLERANCE OF SEEDLINGS
TABLE
III.
Influence of potassium levels supplied to the plant upon per
cent survival
and damage
0 ppm K
Damage
Index
32 ppm K
128 ppm K
11.8
32.6
27.6
SIGo
17.6
38.8
32.0
N.S.
N.S.
IVo — Influence of exposure to low temperatures upon percent survival
and damage
index.
19.5° F.
17.00 F.
Damage
index.
8 ppm K
12.7
% Survival
TABLE
73
1.0
15.0
4.60
35.90
Index
% Survival
**Significant at 0.01
22.0° F.
SIGo
70.00
100.00
level.
detached leaves and for intact plants.
On this
basis, it would be reasonable to assume that po
tassium nutritional status per se does not affect
cold tolerance of small citrus seedlings. This was
especially true for the freezing point of detached
leaves.
32
Temperatures to which plants were exposed
exerted a very pronounced and strong influence
upon the survival
and
damage
which was
ob
served. It appears as if variability of the freez
ing point of the plant and killing points are not
as great as might have been expected.
During
the preliminary work in this investigation, when
plants were frozen in the freezing chamber with
Freezing point
out leaf temperature measurement, great vari
ability in apparent cold tolerance was
tered.
air
Subcooling point
encoun
However, during the investigation of the
temperature
records
measured .at
various
points within the chamber, it was discovered that
air temperatures were highly variable and that
poor temperature uniformity was obtained. When
additional mixing of the air was introduced and
leaf temperatures were measured instead of air
temperatures,
it
was
found
that
point of the leaves was uniform.
the
freezing
The agreement
between the killing point of the leaves in the
chamber
of
detached
leaves appears to be surprisingly good.
and
It would
seem logical
the
freezing
on this basis
point
to
assume that the
2
freezing point at which damage to the leaf would
occur could be
determined by
removing leaves
and freezing them in the laboratory.
3
4
TIME (min.)
Figure
I.—Typical
<
3 detached
'Pineapple'
FLORIDA STATE HORTICULTURAL SOCIETY, 1963
74
that heat was being released from an exothermic
32
process similar to the initial freezing.
Conclusions and Summary
28
The
freezing
point
of
detached
'Pineapple'
orange leaves was determined in the laboratory
24
by the use of a freezing chamber.
Freezing point
of these leaves was 19.5° F. with a standard de
20
2
First freezing point
First subcooling point
16
viation
of
approximately
plants
of
'Pineapple'
1°
F.
orange
When
seedlings
frozen in the freezing chamber at 22°
intact
were
F. very
little damage occurred and all plants survived.
When frozen at 19.5°
dz
1° F. defoliation was
complete with some loss of plants, and when froz
Hi
en at 17.5° F. defoliation was complete and most
12
plants were killed
Second freezing
point
Second subcooiing
point
to the soil line.
This
indi
cated that for 'Pineapple* orange seedlings the
lethal or killing temperature could be determined
by freezing
detached leaves
in
the laboratory.
The effect of potassium nutritional status upon
the freezing temperature and the killing of 'Pine
apple' orange leaves and seedlings was investi
gated.
Potassium nutritional status was found
to have no effect upon the killing and freezing
2
3
4
points.
On this basis, it was concluded that the
potassium nutritional status does not significantly
TIME (rnin.)
influence the cold tolerance of 'Pineapple' orange
s
seedlings.
2.--A cool ing cur
LITERATURE CITED
A typical cooling and freezing curve for de
tached leaves is shown in Figure 1. As is noted
the leaf cooled considerably below the freezing
point before freezing occurred. This phenomenon
is called subcooling, and while it was observed in
the freezing chamber in the laboratory, it did
not occur in the large freeze chamber with in
tact plants. It is doubtful if subcooling occurs
with leaves in nature. After the leaf had been
frozen the refreezing point was always consider
ably higher than the initial freezing point. For
example, leaves which froze at 19.5° F. initially,
commonly refroze at 27° F. This represents a
fundamental change in the internal structure of
the leaf. The value of 27° F. was the freezing
point of the entire leaf, cell wall, and vacuole
content mixture and does not represent a change
in the killing temperatures since the leaf had
already been killed. In no cases in the laboratory
were observations made of leaves which had been
frozen and
were not killed.
In the laboratory
freezing, leaves commonly exhibit a double freez
ing point as may be observed in Figure 2. An
explanation of this secondary point can not be
given at this time. However, it does indicate
1.
Brown, R. T. and G. F. Potter.
1949. Relation of
fertilizers to cold injury of tung trees occurring at Lucedale, Mississippi, in March, 1948.
Proc. Amer. Soc. Hort.
Sci. 53: 109-113.
2.
Cooper, W. C, B. S. Gorton, and S. Tayloe. 1954.
Freezing tests with small trees and detached leaves of
grapefruit. Proc. Amer. Soc. Hort. Sci. 63:167-172.
3.
Hendershott, C. H.
1961.
The response of orange
trees and fruits to freezing temperatures. Proc. Amer. Soc.
Hort.
4.
Sci.
80:247-254.
Levitt, J.
1956.
The Hardiness of Plants.
Academic
Press Inc. New York, New York.
5.
Marshall, D. C, and C. A. S. Padfield.
1962. The
freezing point of pears. Jour. Hort. Sci. 37:106-114.
6. Maximov, N. A. 1929. Internal factors of frost and
drought resistance in plants.
Protoplasma. 7:259-291.
7. Nijjar, G. S., and J. W. Sites. 1959. Some effects of
day length and temperature on cold hardiness. Proc. Fla.
State Hort. Soc. 72:106-109.
8. Reese, R. L. 1961. The relation of levels of potassium
to
the
cold-hardiness
of
citrus
seedlings.
M.S.A.
thesis,
Department of Fruit Crops, University of Florida.
9. Scarth, G. W., and J. Levitt. 1937. The frost-harden
ing mechanism of plant cells.
U.S.D.A. Exp. Sta. Rec.
77:312.
10. Smith, P. F., and G. K. Rasmussen. 1958. Relation
of fertilizer to winter injury of citrus trees.
Proc. Fla.
State Hort. Soc. 71: 170-175.
11.
Sharpe, R. H., G. H. Blackmon, and N. Gammon,
Jr. 1954. Relation of potash and phosphate to cold injury
of Moore pecans. Better Crops with Plant Food. 38(1) :1718, 48-49.
12.
Vasil'yev,
I. M.
1950.
Wintering: of Plants.
and Roger, Inc., Washington, D. C.
13. Young, R. H.
1961. Influence
intensity, and temperature on growth,
Royer
of day length, light
dormancy, and coldhardiness of Red Blush grapefruit trees. Proc. Amer. Soc.
Hort. Sci. 78:174-180.
14. Young, R. H., and A. Peynado. 1962. Growth and
cold-hardiness of citrus and related species when exposed
to different night temperatures.
Proc. Amer. Soc. Hort.
Sci.
81:238-243.