Effects of Cooling Rate on Seeds Exposed to Liquid Nitrogen

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Effects of Cooling Rate on Seeds Exposed to Liquid Nitrogen
Plant Physiol. (1989) 90, 1478-1485
0032-0889/89/90/1 478/08/$01 .00/0
Received for publication September 6, 1988
and in revised form April 4, 1989
Effects of Cooling Rate on Seeds Exposed to Liquid
Nitrogen Temperatures
Christina W. Vertucci
U.S. Department of Agriculture, Agricultural Research Service, National Seed Storage Laboratory,
Ft. Collins, Colorado 80523
ABSTRACT
The effect of cooling rate on seeds was studied by hydrating
pea (Pisum sativum), soybean (Glycine max), and sunflower
(Hehlanthus annuus) seeds to different levels and then cooling
them to -1900C at rates ranging from 10C/minute to 7000C/
minute. When seeds were moist enough to have freezable water
(> 0.25 gram H20/gram dry weight), rapid cooling rates were
optimal for maintaining seed vigor. If the seeds were cooled while
at intermediate moisture levels (0.12 to 0.20 gram H20 per gram
dry weight), there appeared to be no effect of cooling rate on
seedling vigor. When seeds were very dry (< 0.08 gram H20 per
gram dry weight), cooling rate had no effect on pea, but rapid
cooling rates had a marked detrimental effect on soybean and
sunflower germination. Glass transitions, detected by differential
scanning calorimetry, were observed at all moisture contents in
sunflower and soybean cotyledons that were cooled rapidly. In
pea, glasses were detectable when cotyledons with high moisture
levels were cooled rapidly. The nature of the glasses changed
with moisture content. It is suggested that, at high moisture
contents, glasses were formed in the aqueous phase, as well as
the lipid phase if tissues had high oil contents, and this had
beneficial effects on the survival of seeds at low temperatures.
At low moisture contents, glasses were observed to form in the
lipid phase, and this was associated with detrimental effects on
seed viability.
The rate at which hydrated biological samples are cooled
to subfreezing temperatures has a great effect on their subsequent viability (3, 10, 11). Most tissues exhibit a biphasic
response to cooling rate in which they are severely damaged
if cooled too slowly or too rapidly (3, 10, 1). The optimum
rate is tissue dependent and is perhaps a function of the
permeability of the plasmalemma to water (10, 1). Optimum
rates range from about 3YC/h for whole plant tissues to about
2000C/min for red blood cells (10, 11).
In partially hydrated systems such as seeds, cooling rate has
dramatic effects on tissue survival during exposure to low
temperature. Rapid cooling of lettuce seeds, for example, can
protect seeds from freezing injury (12), whereas rapid cooling
of sesame seeds can have detrimental effects ( 14). The purpose
of this paper is to explore further the nature of cooling effects
on seeds in relation to the level of hydration.
and sunflower (Helianthus annuus, cv No. 452, Sigco Research, Inc.) were used in germination and DSC' studies.
Moisture contents in seeds were controlled by either storing
seeds in various relative humidity chambers or adding known
quantities of water to weighed samples ( 15). Moisture contents
are expressed as g/g, dry weights being determined after seeds
had been heated at 95°C for 5 d. Moisture contents studied
ranged from about 0.02 to 0.4 g/g.
Whole Seed Experiments
To determine the effects of cooling rate on the viability of
seeds at 15 different moisture levels, seeds, equilibrated to
given water contents, were sealed in plastic cryovials and
cooled to liquid nitrogen temperatures at a variety of rates.
Six cooling rates were achieved by embedding cryovials in a
series of insulated materials similar, in principle, to those used
by Diaper (2): (a) seeds wrapped in Parafilm and immersed
directly into liquid nitrogen, (b) cryovials immersed in liquid
nitrogen, (c) cryovials immersed in liquid nitrogen vapor, (d)
cryovials in two padded envelopes and immersed in liquid
nitrogen vapor, (e) cryovials in five padded envelopes and
immersed in liquid nitrogen vapor, and (f cryovials in an
unevacuated dewar flask immersed in liquid nitrogen vapor.
To monitor the cooling rate, thermocouples were embedded
in seeds treated similarly and the change of temperature with
time was measured. Cooling rates were determined as the
slope of the cooling curve between -10 and - 140'C.
After exposure for 16 h at -190'C, seeds were warmed on
the bench for 2 h. They were then rolled in germination paper,
watered, and incubated for 96 h at 250C. Seed vigor is expressed as the germination index: radicle length after 96 h x
percent germination. Each treatment consisted of 25 seeds.
Experiments with soybean and pea seeds were repeated twice
and experiments with sunflower were repeated once.
MATERIALS AND METHODS
Differential Scanning Calorimetry
To determine how the rate of cooling affected the thermal
behavior of the seed tissue, 20 mg slices of the cotyledons
were loaded into aluminum sample pans and cooled to
-150C in a Perkin Elmer DSC-4 at a variety of rates. The
effect of moisture content on the thermal behavior of seeds
was studied using soybean and pea cotyledons, hydrated as
described above and cooled at 1, 10, and 200'C/min. All
samples were heated at 10C/min with the warming thermo-
Seeds from soybean (Glycine max, cv Williams'82, Dewine
Seed Co.), pea (Pisum sativum, cv. Alaska, Burpee Seed Co.),
' Abbreviations: DSC, differential scanning calorimetry; g/g, g
H20/g dry weight.
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1 479
EFFECTS OF COOLING RATE ON SEED VIABILITY
grams recorded. After the DSC measurements, the pans were
punctured and dry weights were determined.
The thermal behavior of lipids extracted from soybean and
sunflower seeds was determined by similar methods. The lipid
fraction was extracted with a chloroform:methanol (2:1) solution. The solvent was then evaporated off. Sample sizes for
the lipid experiments ranged between 5 and 9 mg extract.
RESULTS
A series of cooling rates ranging from VC to 700'C/min
were achieved for whole seed experiments as shown in Figure
1. These rates varied slightly with species and water content
especially when cooling from 22 to -10C. Samples were
warmed at about 12'C/min before running germination tests.
Cooling rate had a variable effect on seed vigor dependent
upon seed moisture content and species. Fifteen moisture
treatments were studied in three different species (Tables IIII). Cooling of sunflower and soybean seeds at any moisture
level at 700'C/min had detrimental effects on seed survival
(Tables II and III). In contrast, pea seeds were notably resistant
to damage due to rapid cooling (Table I).
At high moisture contents, all three species were damaged
when exposed to - 190TC. When pea seeds with moisture
contents between 0.36 and 0.41 g/g were cooled at rates of
40'C/min and faster, germination was improved over seeds
that were cooled at slower rates (Table I). Similarly, rapidly
cooled (40-200C/min) soybean seeds with moistures between 0.29 and 0.39 g/g germinated better than their slowly
cooled counterparts (Table II). The same trend was also
observed for sunflower seeds with moisture contents between
0.16 and 0.20 g/g (Table III). In most cases, however, rapid
cooling of high moisture seeds was not effective at maintaining
seed vigor at control (uncooled) levels (Tables I-III). Freezing
sunflower seeds at moisture levels higher than 0.21 g/g killed
seeds regardless of cooling rates (Table III). Moisture levels
higher than 0.41 g/g in pea or 0.39 g/g in soybean were not
studied.
At intermediate moisture levels, cooling rates between 1
and 200C/min had little effect on the viability of pea, soybean, or sunflower seeds (Tables I-III). Germination of pea
seeds at moistures of 0.31 g/g or less was not different than
the untreated controls (Table I). When soybean seeds were
cooled to - 190C at 0.26 g/g moisture, germination was not
affected by the cooling rate; however, it was lower than
uncooled controls (Table II). There was no effect of cooling
observed in soybean seeds at 0.20 and 0.22 g/g or in sunflower
seeds at 0.11 and 0.09 g/g (Tables II and III).
The effect of cooling rate on the germination of seeds at
low moisture levels was species dependent. At low moisture
levels, pea seeds were nearly unaffected by cooling rate (Table
I). Cooling soybean and sunflower seeds with moistures between 0.11 and 0.14 g/g and 0.08 and 0.09 g/g, respectively,
at 200'C/min resulted in poor germination. An increased
sensitivity to rapid cooling rates was observed as the seeds
were dried to even lower levels (Tables II and III).
DSC thermograms were used to determine the effect of
cooling rate on the thermal behavior of seed tissues. In pea
cotyledons with moistures of 0.10 g/g, there were no detectable thermal transitions whether cooled at a slow or rapid rate
(Fig. 2A). A previous study detected no thermal events be-
40
20
0
-20
-40
0
-60
D
-80
CL
I-
-100
-120
-140
-160
-180
-200
0
20
40
60
80
100
TIME (min)
Figure 1. Effect of various insulating materials on the rate at which whole seeds of soybean at 0.12 g/g were cooled to -1900C. The treatments
are as described in "Materials and Methods." Rates of cooling are 700, 200, 42, 8, 6, and 1 °C/min.
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1480
VERTUCCI
Table I. Effect of Cooling Rate on the Vigor of Pea Seeds Exposed
to -1900C at Various Moisture Levels
Vigor is expressed as the germination index, radicle length x
percent germination. Values represent the mean and SE (in parentheses) of 75 seeds.
Germination Index at Rate of Cooling (OC/min):
Moisture
0
1
6
8
42
200
700
g9g
0.41
0.39
0.36
0.31
0.28
Plant Physiol. Vol. 90, 1989
Table II. Effect of Cooling Rate on the Vigor of Soybean Seeds
Exposed to -1900C at Various Moisture Levels
Vigor is expressed as the germination index, radicle length x
percent germination. Values represent the mean and SE (in parentheses) of 75 seeds.
Germination Index at Rate of Cooling (OC/min):
Moisture
1
0
700
6
8
42
200
g/g
2.06
(0.32)
37.29
(8.85)
63.00
(7.53)
60.10 63.00 77.45 70.40 65.20
(4.44) (7.07) (8.27) (4.54) (8.07) (2.64) (7.22)
70.80 72.08 75.53 72.09 85.10 87.00 71.24
74.64
2.08
(3.91) (0.59)
79.00 6.50
(4.90) (5.58)
69.27 45.92
(5.24) (7.51)
79.36 62.91
3.06
(0.43)
23.85
(6.71)
43.70
(2.54)
1.83
(0.45)
20.92
(8.43)
44.00
(6.13)
8.09
(3.13)
29.67
(7.33)
64.50
(8.69)
15.18
(6.63)
32.64
(6.69)
66.11
(5.93)
0.39
0.22
66.23 73.00 72.64 82.25 85.00 81.38 69.18
(6.20) (6.03) (7.53) (5.10) (2.15) (6.05) (5.89)
77.69 73.73 74.67 86.69 85.17 83.08 84.27
0.37
0.16
82.46 75.64 83.36 82.43 90.23 72.36 71.06
(4.53) (5.94) (5.23) (6.44) (6.41) (3.75) (5.64)
80.85 75.71 92.83 84.38 92.40 78.54 86.00
0.34 116.83
(16.80)
0.29 89.33
(8.70)
0.26 83.08
0.10
74.15 80.43 81.92 83.64 82.62 73.86 80.23
(6.65) (2.74) (3.92) (6.15) (4.37) (3.65) (8.57)
80.87 81.00 76.00 86.38 83.87 79.93 87.07
0.06
73.08 77.23 86.92 74.29 80.00 72.92 86.50
(3.64) (3.37) (4.46) (4.92) (6.78) (3.19) (3.19)
63.79 64.93 63.31 70.46 77.64 77.92 73.00
0.04
56.85 72.07 65.54
(6.63) (5.82) (4.59)
57.86 59.07 68.42
(8.43) (5.71) (6.19)
66.79 72.86 80.45 80.79
(7.27) (7.07) (7.50) (5.24)
65.67 63.07 58.75 60.10
(8.20) (4.97) (5.15) (6.31)
tween 0.06 and 0.26 g/g (15). Heating runs of pea seeds at
higher moisture contents exhibited an endothermic peak at
-20°C, presumably from the melting of ice (Fig. 3). Heating
scans after rapid cooling at high moisture contents (< 0.26 g/
g) resulted in a series of small endo- and exothermic events
prior to the main endotherm as well as power shifts indicative
of second order transitions (Fig. 3). These events could be
eliminated by annealing the tissue at -25°C and recooling
rapidly (data not shown). The temperature at which the
thermal events occurred increased slightly as moisture content
decreased (Table IV).
When soybean cotyledons (0.08 g/g) were cooled at 5°C/
min or slower, there was, during warming, an endothermic
event at -40°C that had a large peak followed by a shoulder
(Fig. 2B). If the cotyledons were cooled at faster rates (50°C/
min is shown), the endotherm was present, although it was
broader. There was also a shift in the power at about -100°C
8.36
8.23 14.08 30.20 46.31
5.17
14.77
12.42 19.15 26.31
(7.96) (6.01) (6.26)
34.36 28.43 44.31
(10.05) (4.80) (9.78)
52.14 57.92 59.69
31.31
(7.65)
48.38
(8.12)
68.46
46.64
(6.59)
48.05
(9.28)
63.33
27.54
(4.14)
17.83
(4.13)
47.62
81.79 77.92 71.85 72.62 80.77 64.17 53.31
0.20
77.85 86.83 80.15 83.14 74.42 91.15 53.64
0.16
75.54 70.77 82.57 79.20 65.54 44.85 38.46
0.14
0.11
75.08 75.33 75.50 70.62 73.38 41.69 17.93
(8.65) (9.56) (10.76) (11.91) (10.00) (11.91) (6.98)
79.54 74.77 85.00 72.31 86.64 42.23 13.07
0.09
75.07 74.50 52.69 41.93 42.54 29.64 24.92
(6.83) (10.23) (7.26) (10.92) (12.90) (7.46) (11.94)
(4.86) (7.10) (10.34) (9.94) (9.10) (9.89) (11.06)
(7.15) (7.17) (10.93) (11.53) (7.53) (10.73) (6.10)
(13.56) (9.95) (11.38) (5.74) (9.35) (8.19) (6.91)
0.07
0.06
61.73 61.47 42.93 38.15 20.93 21.36 25.22
(9.01) (10.67) (10.91) (9.13) (6.24) (10.00) (6.41)
63.69 56.00 43.71 35.69 25.75 27.81 22.41
0.05
68.83 64.80 51.13 47.07 22.06 23.93 14.92
(7.86) (7.26) (4.97) (4.38) (5.26) (8.23) (5.06)
0.05
5.83
(11.20) (10.41) (7.57) (10.11) (9.24) (10.36) (9.96)
(6.17) (5.86) (3.75) (2.67) (7.56) (4.90) (4.28)
0.08
2.83
4.00
(2.35) (3.72) (2.21) (3.42)
(10.27) (8.69) (10.02) (9.26) (7.80) (10.19) (9.61)
0.22
(3.58) (5.84) (2.49) (2.71) (3.38) (3.64) (4.50)
0.13
101.50
0.00
(-)
(12.80) (0.22) (3.45) (7.43) (6.27) (7.88) (5.69)
(4.53) (6.59) (7.34) (5.80) (6.58) (3.89) (5.73)
0.18
0.00
(8.90) (-)a
(4.87) (5.20) (4.09) (6.29) (7.06) (5.85) (7.70)
0.24
73.92
(9.34) (9.77) (9.30) (8.60) (8.71) (7.93) (7.73)
(7.51) (6.80) (11.08) (10.46) (8.71) (8.59) (5.65)
65.36 58.88 57.86 53.25 36.64 28.88 19.73
(10.60) (9.74) (11.01) (9.08) (9.51) (8.26) (9.70)
a
Calculations not valid.
0.04
(Fig. 2B). This apparent shift in the base line could be eliminated if the sample was annealed at -35°C and then recooled
to -150°C at 200°C/min (data not shown). A similar effect
was observed in sunflower cotyledons, except that cooling
rates of 1°C/min or slower were necessary to eliminate the
power shifts at -92°C (Fig. 2C).
Moisture content affected the nature of the power shifts
observed in soybean cotyledons cooled at 200°C/min (Fig. 4).
As shown previously, a major endotherm is present at about
-40°C at all moistrue contents. When cotyldeons were cooled
at 200°C/min, discontinuities in the base line were also observed at about -1 00°C at all moisture levels. An exothermic
transition at -90°C was more pronounced as the moisture
content was increased from 0.02 to 0.21 g/g (Fig. 4). At 0.27
g/g moisture level, one large and two small exotherms were
observed prior to the main endotherm. These "pretransitions"
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1481
EFFECTS OF COOLING RATE ON SEED VIABILITY
Table Ill. Effect of Cooling Rate on the Vigor of Sunflower Seeds
Exposed to -190°C at Various Moisture Levels
Vigor is expressed as the germination index, radicle length x
percent germination. Values represent the mean and SE (in parentheses) of 50 seeds.
Germination Index at Rate of Cooling (OC/min):
Moisture
0
1
6
60.36
8
42
200
700
g/g
0.33
0.30
0.28
0.00
0.00
(6.95) (-)a
(-)
99.54
1.28
0.00
0.00 0.00
( (
) )
0.00 0.00
0.00
0.67
(-)
(0.15)
1.19
6.89
(4.85) (-) (0.38) (-)
(-)
(1.40) (1.26)
0.50
1.79
1.33
91.23
0.05
1.47
0.79
(6.12) (-) (0.51) (0.38) (0.94) (1.25) (0.91)
0.21
0.20
0.17
0.16
0.11
0.09
0.08
78.57
0.00
1.00
0.31
1.29
3.05
cc:
2.27
(6.37) (-)
(0.24) (-)
(0.92) (2.33) (1.86)
61.83
0.18
0.00
0.00
0.00
6.63
(6.19) (-)
(-)
(-)
(-)
(4.94) (0.88)
1.59
z
2
57.15 1.13 1.69 4.79 1.71 19.64 9.75
(5.41) (0.94) (2.13) (5.96) (0.92) (8.27) (3.87)
65.50 0.72 19.73 21.00 36.88 38.20 17.91
(5.94)
60.93
(7.04)
64.60
(6.22)
65.79
(0.62)
48.12
(7.70)
83.71
(6.03)
65.84
(7.78) (4.41) (7.13)
55.33 64.71 47.27
(6.57) (4.81) (7.22)
66.44 72.47 51.29
(5.45) (4.97) (5.12)
61.38 69.33 51.00
(5.73) (3.32)
47.93 48.24
(6.64) (5.61)
52.60 45.71
(7.01) (4.75)
34.92 39.50
(5.39) (6.06) (6.53) (4.97) (5.22) (6.72) (5.03)
0.06
0.04
63.85 62.00 61.59 74.08 44.07 37.46 42.00
(4.38) (3.90) (5.90) (6.75) (7.16) (5.98) (5.17)
70.83 79.89 60.47 57.85 54.00 42.00 43.33
(5.62) (6.99) (5.22) (5.21) (6.41) (5.94) (2.37)
68.10 74.07
(4.82) (5.25)
0.03 70.09 74.62
(4.71) (4.57)
0.02 65.77 69.79
(5.89) (7.11)
a Calculations not valid.
0.04
52.56
(4.79)
71.63
(6.14)
64.25
(6.13)
60.05
(4.97)
63.06
(6.63)
68.53
(5.63)
53.46
(4.98)
53.46
(5.07)
47.75
(6.41)
38.93
(6.33)
43.60
(7.89)
43.24
(5.98)
42.38
(4.64)
41.17 44.36
(6.11) (5.72)
TEMPERATURE (C)
were less obvious when seeds at 0.35 g/g were cooled at
200°C/min and eliminated if the seeds were cooled at 1°C/
min (Fig. 4). The intensities of the power shifts were diminished if cotyledons were cooled at slower rates (Table V).
As a demonstration that the apparent shifts in the baseline
observed in soybean and sunflower seeds at low moisture
contents (Fig. 2, B and C) may be due to glass transitions in
the lipid component of the tissues, DSC thermograms were
produced for the extracted lipid fractions (Fig. 5). For the
lipid fractions from both soybean and sunflower seeds, heating
thermograms after lipids were cooled at 200°C/min showed
discontinuities at about -90°C (Fig. 5, A and B). These
discontinuities could be reduced or diminished by cooling the
lipids at 1°C/min (Fig. 5, A and B) or by annealing rapidly
cooled tissue at -65°C (Fig. SC).
Figure 2. Effect of cooling rate on the thermal behavior of dry
(moisture contents 5 0.10 g/g) (A) pea, (B) soybean, and (C) sunflower
cotyledons. Samples were cooled to -1 500C at indicated rates then
warmed at 10°C/min. Heating thermograms were recorded using
DSC. Vertical arrows indicate a shift in power indicative of a glass
transition. The endothermic events at about -400C represent the
onset of the lipid transitions. Samples of about 20 mg were used.
DISCUSSION
This report establishes that the moisture content of the seed
is a critical variable when determining the effect of cooling
rate for cryopreservation (Tables I-III). Rapid cooling rates
enhance the germination of hydrated seeds, but lower the
germination of some (soybean and sunflower) dry seeds. The
sensitivity of dry seeds to rapid cooling rates was noticed in
the two species with high lipid contents. The lipid component
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1 482
VERTUCCI
Plant Physiol. Vol. 90, 1989
Figure 3. DSC thermograms of pea cotyledons heated
at 1 0°C/min after cooling to -1 500C at 200°C/min and
1 0°C/min. Rates of cooling are as indicated. The 25 mg
(dry weight) sample had a moisture content of 0.295 g/
g. The a, b, and c are indicative of the onset of the glass
transition, a devitrification event, and the onset of the
melting of water, respectively.
TEMPERATURE (C)
Table IV. Temperatures at Which Glass Formation Is Indicated by
DSC in Pea Cotyledons of Different Moisture Contents Cooled at
2000C/min
Data are taken from thermograms similar to those given in Figure
3.
Water Content
Temperature of Onset
0.28
0.30
0.31
0.38
0.42
Not detected
-75
-81.5
-84
Not detected
of Antemelting Peak
in these seeds underwent glass transitions when cooled rapidly
(Figs. 2 and 5). It is suggested that lipid vitrification, induced
by rapid cooling, may impart damage to the seed.
Cooling rate has been shown to affect survival in hydrated
biological systems (3, 5, 10, 11). Experiments with seed tissues
have previously demonstrated that rapid cooling rates resulted
in superior germination in lettuce seeds with moistures between 0.22 and 0.26 g/g, but were detrimental to sesame seeds
with moisture contents less than 0.06 g/g (12, 14).
As with other hydrated tissues and cells, there is a biphasic
response to cooling rates in soybean seed tissues with moistures high enough to contain freezable water. Soybean seeds
contain freezable water at moisture contents as low as 0.22 g/
g; however, zero germination was observed only at moisture
contents greater than 0.36 (15). Within the moisture range of
0.29 and 0.39 g/g, soybean seeds were damaged by either very
rapid cooling (700°C/min) or very slow cooling (1°C/min)
(Table II). Optimum rates of cooling for these tissues were
between 42 and 200°C/min (Table II). Hydrated sunflower
seeds showed a similar biphasic response to cooling rate (Table
III). However, high moisture pea seeds were mostly damaged
by slow cooling rates (Table I).
The biphasic response to cooling rate in fully hydrated cells
and tissues has been attributed to the plasmalemma permeability to water (3, 5, 10, 11). Supraoptimal cooling rates
encourage intracellular ice formation because there is insufficient time for cellular water to diffuse to the apoplast (3, 5,
10, 1 1). Suboptimal cooling rates result in 'solution effects'
injuries such as salt toxicity and desiccation damage (3, 1 1).
Evidence is accumulating which suggests that slow cooling
(which encourages extracellular ice growth) may also produce
mechanical forces which can deform cells or induce membrane structural changes (6-8, 1 1). Hypotheses regarding injuries incurred by suboptimal cooling rates generally pertain
to systems that are not desiccation tolerant. This latter type
of damage would probably not occur in seeds since they are
tolerant to severe dehydration (16). Thus, it seems unlikely
that the optimal rate of cooling in partially hydrated seeds
resulted from the diffusion of water to extracellular spaces.
Hence, even though partially hydrated seeds show a biphasic
response to cooling rates, explanations for damage which have
been derived from previously studied hydrated samples, may
not be pertinent.
A biphasic response to cooling rate for sunflower and
soybean seeds with moisture contents lower than 0.01 and
0.14 g/g, respectively, is not indicated by the data (Tables II
and III). In these tissues, cooling at about 1°C/min was the
most favorable of the rates tested. The damage incurred by
soybean and sunflower seeds at these low moisture contents
is probably not a result of intracellular ice formation, since
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1 483
EFFECTS OF COOLING RATE ON SEED VIABILITY
Table V. Power of the Apparent Shifts in Baseline in Soybean
Cotyledons with Different Moisture Contents Cooled at 1, 10, and
2000C/min
Data are taken from thermograms similar to those given in Figure
4. The changes in power are indicated by "p" in the thermograms.
To normalize data, values are corrected for by the dry weight of the
sample.
Power of Baseline Shifts
(mcal/s/g dry wt)
Water Content
g/g
0.015
0.115
0.161
0.208
0.275
0.355
with Cooling Rate at:
1 °C/min
1 0°C/min
200°C/min
0
0
0
0.0053
0
0.0062
0.0019
0.0077
0.0066
0.0053
0.0025
0.0085
0.0066
0.0129
0.0117
0.0090
0.0045
water is not freezable at these water contents (Figs. 2 and 4).
cooled ILICkni7
I
0
2
z
55
.U 150
7r
C
coled
.150
cftlCAmin
0-30
TEMPERATURE
Figure 4. DSC thermograms of soybean cotyledons with different
moisture contents heated at 10°C/min after cooling to -1500C at
200°C/min. The a represents the onset of the glass transition; b
represents a devitrification event in the lipid (b,) and aqueous (b2)
phases; c and d are the onset of the lipid and water melt, respectively;
p is the shift in power observed upon a glass transition. Sample size
ranged from 20 to 30 mg dw. Moisture contents are as indicated.
The full size of the melting endotherms for cotyledons with 0.275 and
0.355 g/g moisture are not given. The thermogram of the cotyledon
sample with 0.355 g/g moisture cooled at 1 °C/min is given in the
bottom curve.
In pea seeds with moisture contents less than 0.23 g/g, rate of
cooling had no effect on the viability of seed tissues cooled to
-196°C (Table I).
DSC thermograms of seed tissues with various moisture
contents cooled at various rates were used to compare viability
data with the thermal behavior of the seed tissue. In all cases
where cooling rate was important to seed survival, DSC data
indicated that vitrification events had occurred. Vitrification
is the solidification of a liquid by increases in viscosity, not
by crystallization (3). It is a second order phase transition that
is detectable as an apparent shift in the baseline in DSC (4, 5,
9). Vitrified solutions, or glasses, are formed by reducing the
concentration of a solvent relative to the solute or by cooling
rapidly enough to avoid nucleation and crystal growth (4, 5).
The temperature at which a glass occurs is strongly dependent
on the solvent concentration (1, 4, 13, 18).
Literature dealing with vitrification as a means for cryopreservation usually reports glass formation in aqueous solutions
(3, 5, 9, 13), and it has been suggested that water in partially
hydrated seeds exists as a glass (1, 18). Since moisture content
influences the temperature at which glasses are observed in
pea seeds between 0.31 and 0.38 g/g (Table IV), it is likely
that the vitrification events observed are due to aqueous
glasses. Williams and Leopold (18) reported similar trends of
aqueous glass formation in defatted corn embryos. Like the
corn embryos, glasses were not detectable in pea if moisture
content exceeded a critical value (0.42 g/g in pea, Table IV).
Glasses were observable in soybean tissues at all moisture
contents studied (Fig. 4, Table V). Unlike in peas, the temperature that the glass melted in soybeans did not change with
moisture content (Fig. 4). This is an indication that the glass
detected was not aqueous, and it is suggested that the apparent
shift in the base line observed at -100°C in soybean and at
-92°C in sunflower is due to glass formation in the lipid
component of the seeds. Lipids extracted from these seeds are
capable of forming glasses, and these occur within similar
temperature ranges (Fig. 5).
It is suggested that the effect of cooling rate on the viability
ofseeds is associated with the formation of glasses. In hydrated
samples where freezable water is present, but ice formation is
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
1484
VERTUCCI
Plant Physiol. Vol. 90, 1989
not lethal (15), glasses probably form in the aqueous component of the seed. Rapid cooling, which promotes glass formation, enhances seed survival. In pea, the size of the watermelting endotherm varies inversely with the cooling rate (Fig.
3), which may indicate that less ice was formed upon rapid
cooling. In soybeans, endotherms formed after cotyledons
had been cooled at various rates were of similar size (data not
shown); however, separation of the melting endotherms of
the water and lipid was better when samples were cooled
slowly (i.e. bottom 2 curves in Fig. 4). This suggests that there
is a greater lipid-water interaction when samples are cooled
rapidly.
In dry tissues, glass formation of the lipid component
corresponded with detrimental effects. Both dry soybean and
sunflower form glasses (Fig. 2) and are affected by rate of
cooling at -190°C (Tables II and III); pea does not exhibit
glass formation at low moisture contents (Fig. 2) and its
survival at - 190C is independent of cooling rate (Table I).
Why glasses in the lipid component may be damaging to
seeds, and why the damaging effect is only observed at low
moisture contents is not understood. It has been suggested
that glasses can crack if cooled rapidly below their transition
temperatures (17). Perhaps the rapid cooling treatments given
to seeds at various hydration levels produced cracks causing
mechanical damage to seed components.
This paper reports that the survival of seeds exposed to
liquid nitrogen temperatures is influenced by an interaction
between cooling rate and moisture content. Rapid cooling of
seeds with high moisture contents (where freezable water is
present) has beneficial effects, while rapid cooling ofdry seeds
with high lipid contents is detrimental. It is suggested that
glass transitions in the water and/or lipid components of the
seed are associated with the two effects.
I
cJ
w
ILL
ACKNOWLEDGMENTS
Appreciation is expressed to Jennifer Rochon and Wister Miller
for their technical assistance; Drs. R. J. Williams and A. G. Hirsh for
their enthusiasm and advice when the glass transition work was first
presented to them; Dr. A. C. Leopold for his helpful comments on
the manuscript; Sigco Research, Inc., for generously supplying sunflower seeds.
TEMPERATURE (C)
Figure 5. Effect of cooling rate on the thermal behavior of the
extracted oil from (A) soybean (8.6 mg) and (B) sunflower (5.2 mg)
seeds. Samples were cooled to -1500C at indicated rates, then
warmed at 10°C/min. Heating thermograms were recorded using
DSC. The p indicates a shift in power indicative of a glass transition
and the a represents the onset of a lipid transition. In (C), the same
soybean and sunflower lipid samples used in (A) and (B) were cooled
to -1500C at 200°C/min, heated to -650C, recooled at 200°C/min
to -1 500C and then finally warmed at 1 0°C/min. Thermograms were
taken of the final warming. The soybean oil sample was annealed to
-650C for 2 min, while the sunflower sample was annealed for
1 0 min.
LITERATURE CITED
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EFFECTS OF COOLING RATE ON SEED VIABILITY
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.

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