Nitrogen Temperatures - Effects of Cooling Rate on Seeds Exposed to Liquid
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Plant Physiol. (1989) 90, 1478-1485 Received for publication September 6, 1988
0032-0889/89/90/1 478/08/$01 .00/0 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 and sunflower (Helianthus annuus, cv No. 452, Sigco Re-
The effect of cooling rate on seeds was studied by hydrating search, Inc.) were used in germination and DSC' studies.
pea (Pisum sativum), soybean (Glycine max), and sunflower Moisture contents in seeds were controlled by either storing
(Hehlanthus annuus) seeds to different levels and then cooling seeds in various relative humidity chambers or adding known
them to -1900C at rates ranging from 10C/minute to 7000C/ quantities of water to weighed samples ( 15). Moisture contents
minute. When seeds were moist enough to have freezable water are expressed as g/g, dry weights being determined after seeds
(> 0.25 gram H20/gram dry weight), rapid cooling rates were had been heated at 95°C for 5 d. Moisture contents studied
optimal for maintaining seed vigor. If the seeds were cooled while ranged from about 0.02 to 0.4 g/g.
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 Whole Seed Experiments
gram dry weight), cooling rate had no effect on pea, but rapid To determine the effects of cooling rate on the viability of
cooling rates had a marked detrimental effect on soybean and seeds at 15 different moisture levels, seeds, equilibrated to
sunflower germination. Glass transitions, detected by differential given water contents, were sealed in plastic cryovials and
scanning calorimetry, were observed at all moisture contents in
sunflower and soybean cotyledons that were cooled rapidly. In cooled to liquid nitrogen temperatures at a variety of rates.
pea, glasses were detectable when cotyledons with high moisture Six cooling rates were achieved by embedding cryovials in a
levels were cooled rapidly. The nature of the glasses changed series of insulated materials similar, in principle, to those used
with moisture content. It is suggested that, at high moisture by Diaper (2): (a) seeds wrapped in Parafilm and immersed
contents, glasses were formed in the aqueous phase, as well as directly into liquid nitrogen, (b) cryovials immersed in liquid
the lipid phase if tissues had high oil contents, and this had nitrogen, (c) cryovials immersed in liquid nitrogen vapor, (d)
beneficial effects on the survival of seeds at low temperatures. cryovials in two padded envelopes and immersed in liquid
At low moisture contents, glasses were observed to form in the nitrogen vapor, (e) cryovials in five padded envelopes and
lipid phase, and this was associated with detrimental effects on immersed in liquid nitrogen vapor, and (f cryovials in an
seed viability. 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.
The rate at which hydrated biological samples are cooled After exposure for 16 h at -190'C, seeds were warmed on
to subfreezing temperatures has a great effect on their subse- the bench for 2 h. They were then rolled in germination paper,
quent viability (3, 10, 11). Most tissues exhibit a biphasic watered, and incubated for 96 h at 250C. Seed vigor is ex-
response to cooling rate in which they are severely damaged pressed as the germination index: radicle length after 96 h x
if cooled too slowly or too rapidly (3, 10, 1). The optimum percent germination. Each treatment consisted of 25 seeds.
rate is tissue dependent and is perhaps a function of the Experiments with soybean and pea seeds were repeated twice
permeability of the plasmalemma to water (10, 1). Optimum and experiments with sunflower were repeated once.
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 Differential Scanning Calorimetry
dramatic effects on tissue survival during exposure to low To determine how the rate of cooling affected the thermal
temperature. Rapid cooling of lettuce seeds, for example, can behavior of the seed tissue, 20 mg slices of the cotyledons
protect seeds from freezing injury (12), whereas rapid cooling were loaded into aluminum sample pans and cooled to
of sesame seeds can have detrimental effects ( 14). The purpose -150C in a Perkin Elmer DSC-4 at a variety of rates. The
of this paper is to explore further the nature of cooling effects effect of moisture content on the thermal behavior of seeds
on seeds in relation to the level of hydration. was studied using soybean and pea cotyledons, hydrated as
described above and cooled at 1, 10, and 200'C/min. All
MATERIALS AND METHODS samples were heated at 10C/min with the warming thermo-
Seeds from soybean (Glycine max, cv Williams'82, Dewine ' Abbreviations: DSC, differential scanning calorimetry; g/g, g
Seed Co.), pea (Pisum sativum, cv. Alaska, Burpee Seed Co.), H20/g dry weight.
Downloaded on March 11, 2021. - Published
1478 by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.EFFECTS OF COOLING RATE ON SEED VIABILITY 1 479
grams recorded. After the DSC measurements, the pans were 0.16 and 0.20 g/g (Table III). In most cases, however, rapid
punctured and dry weights were determined. cooling of high moisture seeds was not effective at maintaining
The thermal behavior of lipids extracted from soybean and seed vigor at control (uncooled) levels (Tables I-III). Freezing
sunflower seeds was determined by similar methods. The lipid sunflower seeds at moisture levels higher than 0.21 g/g killed
fraction was extracted with a chloroform:methanol (2:1) so- seeds regardless of cooling rates (Table III). Moisture levels
lution. The solvent was then evaporated off. Sample sizes for higher than 0.41 g/g in pea or 0.39 g/g in soybean were not
the lipid experiments ranged between 5 and 9 mg extract. studied.
At intermediate moisture levels, cooling rates between 1
RESULTS and 200C/min had little effect on the viability of pea, soy-
A series of cooling rates ranging from VC to 700'C/min bean, or sunflower seeds (Tables I-III). Germination of pea
were achieved for whole seed experiments as shown in Figure seeds at moistures of 0.31 g/g or less was not different than
1. These rates varied slightly with species and water content the untreated controls (Table I). When soybean seeds were
especially when cooling from 22 to -10C. Samples were cooled to - 190C at 0.26 g/g moisture, germination was not
warmed at about 12'C/min before running germination tests. affected by the cooling rate; however, it was lower than
Cooling rate had a variable effect on seed vigor dependent uncooled controls (Table II). There was no effect of cooling
upon seed moisture content and species. Fifteen moisture observed in soybean seeds at 0.20 and 0.22 g/g or in sunflower
treatments were studied in three different species (Tables I- seeds at 0.11 and 0.09 g/g (Tables II and III).
III). Cooling of sunflower and soybean seeds at any moisture The effect of cooling rate on the germination of seeds at
level at 700'C/min had detrimental effects on seed survival low moisture levels was species dependent. At low moisture
(Tables II and III). In contrast, pea seeds were notably resistant levels, pea seeds were nearly unaffected by cooling rate (Table
to damage due to rapid cooling (Table I). I). Cooling soybean and sunflower seeds with moistures be-
At high moisture contents, all three species were damaged tween 0.11 and 0.14 g/g and 0.08 and 0.09 g/g, respectively,
when exposed to - 190TC. When pea seeds with moisture at 200'C/min resulted in poor germination. An increased
contents between 0.36 and 0.41 g/g were cooled at rates of sensitivity to rapid cooling rates was observed as the seeds
40'C/min and faster, germination was improved over seeds were dried to even lower levels (Tables II and III).
that were cooled at slower rates (Table I). Similarly, rapidly DSC thermograms were used to determine the effect of
cooled (40-200C/min) soybean seeds with moistures be- cooling rate on the thermal behavior of seed tissues. In pea
tween 0.29 and 0.39 g/g germinated better than their slowly cotyledons with moistures of 0.10 g/g, there were no detect-
cooled counterparts (Table II). The same trend was also able thermal transitions whether cooled at a slow or rapid rate
observed for sunflower seeds with moisture contents between (Fig. 2A). A previous study detected no thermal events be-
40
20
0
-20
-40
0
-60
D
-80
CL -100
I-
-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.
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1480 VERTUCCI Plant Physiol. Vol. 90, 1989
Table I. Effect of Cooling Rate on the Vigor of Pea Seeds Exposed Table II. Effect of Cooling Rate on the Vigor of Soybean Seeds
to -1900C at Various Moisture Levels Exposed to -1900C at Various Moisture Levels
Vigor is expressed as the germination index, radicle length x Vigor is expressed as the germination index, radicle length x
percent germination. Values represent the mean and SE (in parenthe- percent germination. Values represent the mean and SE (in parenthe-
ses) of 75 seeds. ses) of 75 seeds.
Germination Index at Rate of Cooling (OC/min): Germination Index at Rate of Cooling (OC/min):
Moisture Moisture
0 1 6 8 42 200 700 0 1 6 8 42 200 700
g9g g/g
0.41 74.64 2.08 3.06 1.83 8.09 2.06
15.18 0.39 73.92 0.00 0.00 4.00 5.83 8.36 5.17
(3.91) (0.59) (0.43) (0.45) (3.13) (6.63)
(0.32) (8.90) (-)a (-) (2.35) (3.72) (2.21) (3.42)
0.39 79.00 6.50 23.85 20.92 29.67 32.64
37.29 0.37 101.50 2.83 8.23 14.08 30.20 46.31 14.77
(4.90) (5.58) (6.71) (8.43) (7.33) (6.69)
(8.85) (12.80) (0.22) (3.45) (7.43) (6.27) (7.88) (5.69)
0.36 69.27 45.92 43.70 44.00 64.50 63.00
66.11 0.34 116.83 12.42 19.15 26.31 31.31 46.64 27.54
(5.24) (7.51) (2.54) (6.13) (8.69) (5.93)
(7.53) (16.80) (7.96) (6.01) (6.26) (7.65) (6.59) (4.14)
0.31 79.36 62.91 60.10 63.00 77.45 70.40 65.20 0.29 89.33 34.36 28.43 44.31 48.38 48.05 17.83
(4.44) (7.07) (8.27) (4.54) (8.07) (2.64) (7.22) (8.70) (10.05) (4.80) (9.78) (8.12) (9.28) (4.13)
0.28 70.80 72.08 75.53 72.09 85.10 87.00 71.24 0.26 83.08 52.14 57.92 59.69 68.46 63.33 47.62
(4.87) (5.20) (4.09) (6.29) (7.06) (5.85) (7.70) (10.27) (8.69) (10.02) (9.26) (7.80) (10.19) (9.61)
0.24 66.23 73.00 72.64 82.25 85.00 81.38 69.18 0.22 81.79 77.92 71.85 72.62 80.77 64.17 53.31
(6.20) (6.03) (7.53) (5.10) (2.15) (6.05) (5.89) (11.20) (10.41) (7.57) (10.11) (9.24) (10.36) (9.96)
0.22 77.69 73.73 74.67 86.69 85.17 83.08 84.27 0.20 77.85 86.83 80.15 83.14 74.42 91.15 53.64
(4.53) (6.59) (7.34) (5.80) (6.58) (3.89) (5.73) (6.83) (10.23) (7.26) (10.92) (12.90) (7.46) (11.94)
0.18 82.46 75.64 83.36 82.43 90.23 72.36 71.06 0.16 75.54 70.77 82.57 79.20 65.54 44.85 38.46
(4.53) (5.94) (5.23) (6.44) (6.41) (3.75) (5.64) (4.86) (7.10) (10.34) (9.94) (9.10) (9.89) (11.06)
0.16 80.85 75.71 92.83 84.38 92.40 78.54 86.00 0.14 75.08 75.33 75.50 70.62 73.38 41.69 17.93
(3.58) (5.84) (2.49) (2.71) (3.38) (3.64) (4.50) (8.65) (9.56) (10.76) (11.91) (10.00) (11.91) (6.98)
0.13 74.15 80.43 81.92 83.64 82.62 73.86 80.23 0.11 79.54 74.77 85.00 72.31 86.64 42.23 13.07
(6.65) (2.74) (3.92) (6.15) (4.37) (3.65) (8.57) (7.15) (7.17) (10.93) (11.53) (7.53) (10.73) (6.10)
0.10 80.87 81.00 76.00 86.38 83.87 79.93 87.07 0.09 75.07 74.50 52.69 41.93 42.54 29.64 24.92
(6.17) (5.86) (3.75) (2.67) (7.56) (4.90) (4.28) (13.56) (9.95) (11.38) (5.74) (9.35) (8.19) (6.91)
0.08 73.08 77.23 86.92 74.29 80.00 72.92 86.50 0.07 61.73 61.47 42.93 38.15 20.93 21.36 25.22
(3.64) (3.37) (4.46) (4.92) (6.78) (3.19) (3.19) (9.01) (10.67) (10.91) (9.13) (6.24) (10.00) (6.41)
0.06 63.79 64.93 63.31 70.46 77.64 77.92 73.00 0.06 63.69 56.00 43.71 35.69 25.75 27.81 22.41
(7.86) (7.26) (4.97) (4.38) (5.26) (8.23) (5.06) (9.34) (9.77) (9.30) (8.60) (8.71) (7.93) (7.73)
0.05 56.85 72.07 65.54 66.79 72.86 80.45 80.79 0.05 68.83 64.80 51.13 47.07 22.06 23.93 14.92
(6.63) (5.82) (4.59) (7.27) (7.07) (7.50) (5.24) (7.51) (6.80) (11.08) (10.46) (8.71) (8.59) (5.65)
0.04 57.86 59.07 68.42 65.67 63.07 58.75 60.10 0.04 65.36 58.88 57.86 53.25 36.64 28.88 19.73
(8.43) (5.71) (6.19) (8.20) (4.97) (5.15) (6.31) (10.60) (9.74) (11.01) (9.08) (9.51) (8.26) (9.70)
a
Calculations not valid.
tween 0.06 and 0.26 g/g (15). Heating runs of pea seeds at
higher moisture contents exhibited an endothermic peak at (Fig. 2B). This apparent shift in the base line could be elimi-
-20°C, presumably from the melting of ice (Fig. 3). Heating nated if the sample was annealed at -35°C and then recooled
scans after rapid cooling at high moisture contents (< 0.26 g/ to -150°C at 200°C/min (data not shown). A similar effect
g) resulted in a series of small endo- and exothermic events was observed in sunflower cotyledons, except that cooling
prior to the main endotherm as well as power shifts indicative rates of 1°C/min or slower were necessary to eliminate the
of second order transitions (Fig. 3). These events could be power shifts at -92°C (Fig. 2C).
eliminated by annealing the tissue at -25°C and recooling Moisture content affected the nature of the power shifts
rapidly (data not shown). The temperature at which the observed in soybean cotyledons cooled at 200°C/min (Fig. 4).
thermal events occurred increased slightly as moisture content As shown previously, a major endotherm is present at about
decreased (Table IV). -40°C at all moistrue contents. When cotyldeons were cooled
When soybean cotyledons (0.08 g/g) were cooled at 5°C/ at 200°C/min, discontinuities in the base line were also ob-
min or slower, there was, during warming, an endothermic served at about -1 00°C at all moisture levels. An exothermic
event at -40°C that had a large peak followed by a shoulder transition at -90°C was more pronounced as the moisture
(Fig. 2B). If the cotyledons were cooled at faster rates (50°C/ content was increased from 0.02 to 0.21 g/g (Fig. 4). At 0.27
min is shown), the endotherm was present, although it was g/g moisture level, one large and two small exotherms were
broader. There was also a shift in the power at about -100°C observed prior to the main endotherm. These "pretransitions"
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.EFFECTS OF COOLING RATE ON SEED VIABILITY 1481
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 parenthe-
ses) of 50 seeds.
Germination Index at Rate of Cooling (OC/min):
Moisture
0 1 6 8 42 200 700
g/g
0.33 60.36 0.00 0.00 0.00 0.00 0.00 0.67
(6.95) (-)a (-) ( () ) (-) (0.15)
0.30 99.54 0.00 1.28 0.00 0.00 1.19 6.89
(4.85) (-) (0.38) (-) (-) (1.40) (1.26)
0.28 91.23 0.05 1.47 0.50 1.79 1.33 0.79
cc:
(6.12) (-) (0.51) (0.38) (0.94) (1.25) (0.91)
0.21 78.57 0.00 1.00 0.31 1.29 3.05 2.27
z
(6.37) (-) (0.24) (-) (0.92) (2.33) (1.86)
0.20 61.83 0.18 0.00 0.00 0.00 6.63 1.59
2
(6.19) (-) (-) (-) (-) (4.94) (0.88)
0.17 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)
0.16 65.50 0.72 19.73 21.00 36.88 38.20 17.91
(5.94) (0.62) (7.78) (4.41) (7.13) (5.73) (3.32)
0.11 60.93 48.12 55.33 64.71 47.27 47.93 48.24
(7.04) (7.70) (6.57) (4.81) (7.22) (6.64) (5.61)
0.09 64.60 83.71 66.44 72.47 51.29 52.60 45.71
(6.22) (6.03) (5.45) (4.97) (5.12) (7.01) (4.75)
0.08 65.79 65.84 61.38 69.33 51.00 34.92 39.50
(5.39) (6.06) (6.53) (4.97) (5.22) (6.72) (5.03)
0.06 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)
0.04 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)
0.04 68.10 74.07 52.56 60.05 53.46 38.93 43.24
(4.82) (5.25) (4.79) (4.97) (4.98) (6.33) (5.98)
0.03 70.09 74.62 71.63 63.06 53.46 43.60 42.38
(4.71) (4.57) (6.14) (6.63) (5.07) (7.89) (4.64)
0.02 65.77 69.79 64.25 68.53 47.75 41.17 44.36
(5.89) (7.11) (6.13) (5.63) (6.41) (6.11) (5.72)
a Calculations not valid.
TEMPERATURE (C)
were less obvious when seeds at 0.35 g/g were cooled at Figure 2. Effect of cooling rate on the thermal behavior of dry
200°C/min and eliminated if the seeds were cooled at 1°C/ (moisture contents 5 0.10 g/g) (A) pea, (B) soybean, and (C) sunflower
min (Fig. 4). The intensities of the power shifts were dimin- cotyledons. Samples were cooled to -1 500C at indicated rates then
warmed at 10°C/min. Heating thermograms were recorded using
ished if cotyledons were cooled at slower rates (Table V). DSC. Vertical arrows indicate a shift in power indicative of a glass
As a demonstration that the apparent shifts in the baseline transition. The endothermic events at about -400C represent the
observed in soybean and sunflower seeds at low moisture onset of the lipid transitions. Samples of about 20 mg were used.
contents (Fig. 2, B and C) may be due to glass transitions in
the lipid component of the tissues, DSC thermograms were DISCUSSION
produced for the extracted lipid fractions (Fig. 5). For the This report establishes that the moisture content of the seed
lipid fractions from both soybean and sunflower seeds, heating is a critical variable when determining the effect of cooling
thermograms after lipids were cooled at 200°C/min showed rate for cryopreservation (Tables I-III). Rapid cooling rates
discontinuities at about -90°C (Fig. 5, A and B). These enhance the germination of hydrated seeds, but lower the
discontinuities could be reduced or diminished by cooling the germination of some (soybean and sunflower) dry seeds. The
lipids at 1°C/min (Fig. 5, A and B) or by annealing rapidly sensitivity of dry seeds to rapid cooling rates was noticed in
cooled tissue at -65°C (Fig. SC). the two species with high lipid contents. The lipid component
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.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 between 42 and 200°C/min (Table II). Hydrated sunflower
DSC in Pea Cotyledons of Different Moisture Contents Cooled at seeds showed a similar biphasic response to cooling rate (Table
2000C/min III). However, high moisture pea seeds were mostly damaged
Data are taken from thermograms similar to those given in Figure by slow cooling rates (Table I).
3. The biphasic response to cooling rate in fully hydrated cells
Temperature of Onset and tissues has been attributed to the plasmalemma permea-
Water Content bility to water (3, 5, 10, 11). Supraoptimal cooling rates
of Antemelting Peak
encourage intracellular ice formation because there is insuf-
0.28 Not detected ficient time for cellular water to diffuse to the apoplast (3, 5,
0.30 -75 10, 1 1). Suboptimal cooling rates result in 'solution effects'
0.31 -81.5 injuries such as salt toxicity and desiccation damage (3, 1 1).
0.38 -84 Evidence is accumulating which suggests that slow cooling
0.42 Not detected (which encourages extracellular ice growth) may also produce
mechanical forces which can deform cells or induce mem-
in these seeds underwent glass transitions when cooled rapidly brane structural changes (6-8, 1 1). Hypotheses regarding in-
(Figs. 2 and 5). It is suggested that lipid vitrification, induced juries incurred by suboptimal cooling rates generally pertain
by rapid cooling, may impart damage to the seed. to systems that are not desiccation tolerant. This latter type
Cooling rate has been shown to affect survival in hydrated of damage would probably not occur in seeds since they are
biological systems (3, 5, 10, 11). Experiments with seed tissues tolerant to severe dehydration (16). Thus, it seems unlikely
have previously demonstrated that rapid cooling rates resulted that the optimal rate of cooling in partially hydrated seeds
in superior germination in lettuce seeds with moistures be- resulted from the diffusion of water to extracellular spaces.
tween 0.22 and 0.26 g/g, but were detrimental to sesame seeds Hence, even though partially hydrated seeds show a biphasic
with moisture contents less than 0.06 g/g (12, 14). response to cooling rates, explanations for damage which have
As with other hydrated tissues and cells, there is a biphasic been derived from previously studied hydrated samples, may
response to cooling rates in soybean seed tissues with mois- not be pertinent.
tures high enough to contain freezable water. Soybean seeds A biphasic response to cooling rate for sunflower and
contain freezable water at moisture contents as low as 0.22 g/ soybean seeds with moisture contents lower than 0.01 and
g; however, zero germination was observed only at moisture 0.14 g/g, respectively, is not indicated by the data (Tables II
contents greater than 0.36 (15). Within the moisture range of and III). In these tissues, cooling at about 1°C/min was the
0.29 and 0.39 g/g, soybean seeds were damaged by either very most favorable of the rates tested. The damage incurred by
rapid cooling (700°C/min) or very slow cooling (1°C/min) soybean and sunflower seeds at these low moisture contents
(Table II). Optimum rates of cooling for these tissues were is probably not a result of intracellular ice formation, since
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.EFFECTS OF COOLING RATE ON SEED VIABILITY 1 483
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 with Cooling Rate at:
1 °C/min 1 0°C/min 200°C/min
g/g
0.015 0 0.0062 0.0085
0.115 0 0.0019 0.0066
0.161 0 0.0077 0.0129
0.208 0.0053 0.0066 0.0117
0.275 0 0.0053 0.0090
0.355 0.0025 0.0045
water is not freezable at these water contents (Figs. 2 and 4).
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
cooled ILICkni7
I where cooling rate was important to seed survival, DSC data
2
0 indicated that vitrification events had occurred. Vitrification
z 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
55
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).
.U 150 C 7r
Literature dealing with vitrification as a means for cryopres-
ervation 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
coled cftlCAmin 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).
.150 0-30
Glasses were observable in soybean tissues at all moisture
contents studied (Fig. 4, Table V). Unlike in peas, the tem-
TEMPERATURE
perature that the glass melted in soybeans did not change with
Figure 4. DSC thermograms of soybean cotyledons with different moisture content (Fig. 4). This is an indication that the glass
moisture contents heated at 10°C/min after cooling to -1500C at detected was not aqueous, and it is suggested that the apparent
200°C/min. The a represents the onset of the glass transition; b
represents a devitrification event in the lipid (b,) and aqueous (b2) shift in the base line observed at -100°C in soybean and at
phases; c and d are the onset of the lipid and water melt, respectively; -92°C in sunflower is due to glass formation in the lipid
p is the shift in power observed upon a glass transition. Sample size component of the seeds. Lipids extracted from these seeds are
ranged from 20 to 30 mg dw. Moisture contents are as indicated. capable of forming glasses, and these occur within similar
The full size of the melting endotherms for cotyledons with 0.275 and temperature ranges (Fig. 5).
0.355 g/g moisture are not given. The thermogram of the cotyledon It is suggested that the effect of cooling rate on the viability
sample with 0.355 g/g moisture cooled at 1 °C/min is given in the ofseeds is associated with the formation of glasses. In hydrated
bottom curve. samples where freezable water is present, but ice formation is
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.1484 VERTUCCI Plant Physiol. Vol. 90, 1989
not lethal (15), glasses probably form in the aqueous compo-
nent of the seed. Rapid cooling, which promotes glass for-
mation, enhances seed survival. In pea, the size of the water-
melting 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).
I
cJ 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
w
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
I- mechanical damage to seed components.
LL
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.
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 sun-
flower seeds.
LITERATURE CITED
1. Burke MJ (1986) The glassy state and survival of anhydrous
biological systems. In AC Leopold, ed, Membranes, Metabo-
lism and Dry Organisms. Comstock, Ithaca, NY, pp 358-363
TEMPERATURE (C) 2. Diaper MP (1986) Practical techniques for cooling biological
samples at 0.3 - lOOC min-'. Cryo Lett 7: 279-290
Figure 5. Effect of cooling rate on the thermal behavior of the 3. Fahy GM, MacFarlane DR, Angeli CA, Meryman HT (1984)
extracted oil from (A) soybean (8.6 mg) and (B) sunflower (5.2 mg) Vitrification as an approach to cryopreservation. Cryobiology
seeds. Samples were cooled to -1500C at indicated rates, then 21: 407-426
warmed at 10°C/min. Heating thermograms were recorded using 4. Franks F (1982) Water. A Comprehensive Treatise, Vol 7. Water
DSC. The p indicates a shift in power indicative of a glass transition
and Aqueous Solutions at Subzero Temperatures. Plenum
Press, New York
and the a represents the onset of a lipid transition. In (C), the same 5. Franks F (1985) Biophysics and Biochemistry at Low Tempera-
soybean and sunflower lipid samples used in (A) and (B) were cooled tures. Cambridge University Press, New York
to -1500C at 200°C/min, heated to -650C, recooled at 200°C/min 6. Fujikawa S (1987) Mechanical force by growth of extracellular
to -1 500C and then finally warmed at 1 0°C/min. Thermograms were ice crystals is widespread cause for slow freezing injury in
taken of the final warming. The soybean oil sample was annealed to tertiary hyphae of mushrooms. Cryo Lett 8: 156-161
-650C for 2 min, while the sunflower sample was annealed for 7. Gordon-Kamm WJ, Steponkus PL (1984) Lameliar-to-hexagonal
1 0 min. II phase transitions in the plasma membrane of isolated pro-
toplasts after freeze-induced dehydration. Proc Natl Acad Sci
USA 81: 6373-6377
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.EFFECTS OF COOLING RATE ON SEED VIABILITY 1485
8. Gordon-Kamm WJ, Steponkus PL (1984) The behavior of the strawberries studied using differential scanning calorimetry. J
plasma membrane following osmotic contraction of isolated Food Sci 52: 146-149
protoplasts: implications in freezing injury. Protoplasma 123: 14. Stanwood PC (1987) Survival of sesame seeds at the temperature
83-94 (- 196'C) of liquid nitrogen. Crop Sci 27: 327-331
9. Hirsh AG, Williams RJ, Meryman HT (1985) A novel method 15. Vertucci CW (1989) Relationship between thermal transitions
of natural cryoprotection. Intracellular glass formation in and freezing injury in pea and soybean seeds. Plant Physiol 90:
deeply frozen Populus. Plant Physiol 79: 41-56 1121-1128
10. Liebo SP, Mazur P (1971) The role of cooling rates in low- 16. Vertucci CW, Leopold AC (1987) The relationship between water
temperature preservation. Cryobiology 8: 447-452 binding and desiccation tolerance in tissues. Plant Physiol 85:
11. Levitt J (1980) Responses of Plants to Environmental Stress Vol. 232-238
1. Chilling, Freezing and High Temperature Stresses. Academic 17. Williams RJ (1988) Association between ice nuclei in sucrose:
Press, New York H20 glasses and fracture interfaces. In Proceedings of the 17th
12. Roos EE, Stanwood PC (1981) Effects of low temperature, cool- North American Thermal Analysis Society Conference, Lake
ing rate and moisture content on seed germination of lettuce. Buena Vista, FL, pp 252-254
J Am Soc Hortic Sci 106: 30-34 18. Williams RJ, Leopold AC (1989) The glassy state in corn em-
13. Roos YH (1987) Effect of moisture on the thermal behavior of bryos. Plant Physiol 89: 977-981
Downloaded on March 11, 2021. - Published by https://plantphysiol.org
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.You can also read
