Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 311--318
© 1997 Heron Publishing----Victoria, Canada
Root cold tolerance of black spruce seedlings: viability tests in relation
to survival and regrowth
F. J. BIGRAS
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, Sainte-Foy, Québec G1V 4C7, Canada
Received March 25, 1996
Summary Root systems of 6-month-old, cold-hardened,
container-grown black spruce seedlings (Picea mariana (Mill.)
B.S.P.) were exposed to 0, −5, −10, −15, −20, or −22.5 °C.
Freezing-induced damage to fine roots, coarse roots and the
whole root system was assessed by various viability tests
including leakage of electrolytes, leakage of phenolic compounds, water loss, root and shoot water potentials, and live
root dry mass. To assess the long-term effects of freezing-induced root damage, seedling survival and regrowth were measured. Leakage of both electrolytes and phenolic compounds
differed among fine roots, coarse roots, and whole root systems. In coarse roots and the whole root system, but not in fine
roots, leakage of electrolytes, leakage of phenolic compounds,
water loss, and root and shoot water potentials were correlated
with percentage of live root dry mass which, in turn, was highly
correlated with seedling survival and regrowth. Compared with
live root dry mass, electrolyte and phenolic leakage, water loss,
and root and shoot water potentials were less well correlated
with seedling survival and regrowth. Among the viability tests,
electrolyte leakage of the whole root system correlated most
closely with seedling survival and regrowth. Under freezing
conditions that destroyed less than 50% of each seedling’s root
system, about 70% of the seedlings survived and subsequent
growth was little affected, whereas under freezing conditions
that destroyed 70% of each seedling’s root system, only about
30% of the seedlings survived and subsequent growth was
reduced compared with that of undamaged plants.
Keywords: electrolyte leakage, phenolic leakage, Picea mariana, water loss, water potential.
Introduction
Frost damage to root systems, which frequently occurs during
production of conifer seedlings in containers or during storage
of bare-root seedlings (Lindström 1986a, Simpson 1993), has
a negative impact on seedling survival and regrowth (Lindström 1986b, Bigras and Margolis 1996). Therefore, a cheap,
rapid and accurate procedure is needed to identify frost-damaged seedlings before they are shipped to plantation sites.
Various methods have been developed to estimate root damage after frost events. Rapid methods include measurements of
leakage of electrolytes, leakage of ninhydrin-reactive or phe-
nolic compounds, triphenyl tetrazolium chloride reduction,
sugar content, root respiration, shoot and root water potentials,
water loss and resistance to electric current. Tests that require
an incubation period are root growth capacity, visual examination of tissue browning, and measurement of fresh mass or dry
mass of undamaged roots. Although many methods have been
used to estimate root damage, few attempts have been made to
compare the results of the various tests with seedling survival
and growth (Bigras and Calmé 1994).
The objective of this research was to identify seedling viability tests that are highly correlated with seedling survival
and subsequent growth. Specifically, we subjected root systems of black spruce seedlings to a range of freezing temperatures to: (i) compare different root viability tests among
different root diameter classes; (ii) evaluate the impact of frost
damage on seedling survival and regrowth; and (iii) correlate
the results of the viability tests with seedling survival and
regrowth characteristics.
Materials and methods
Plant material
Black spruce seedlings (Picea mariana (Mill.) B.S.P.) (provenance 47°45′ N, 70°05′ W) were grown in unheated polyethylene-covered greenhouses at a commercial forest nursery
(Centre de production de plants forestiers de Québec Inc.,
Sainte-Anne-de-Beaupré, PQ) in a peat moss/vermiculite mixture (2/1, v/v). On May 20, 1992, three to four seeds were sown
per cavity (110 cm3) in container trays (45 cavities) and
thinned to one seedling per cavity 30 days later. Seedlings were
fertilized with liquid fertilizer from June 21 until September 13, and received a total of 6.5 mg N, 5.0 mg P, and 5.8 mg
K per cavity. Seedlings were placed outside on October 14
(mean height, 6.5 cm; stem diameter, 0.98 mm; shoot dry
mass, 117 mg; and root dry mass, 71 mg). On November 9,
seedlings were shipped to the Laurentian Forestry Centre,
Sainte-Foy, PQ (Canadian Forest Service) and placed in a
greenhouse at a day/night temperature of 10/6 °C in a natural
photoperiod. The seedlings were watered to saturation and no
fertilizer was added.
312
BIGRAS
Freezing tests
Before freezing, root systems were washed under running
water to completely remove substrate. The root system of each
seedling was inserted in a 16-mm test tube and the tubes were
immersed in a cooling bath (Model 2325, Forma Scientific
Inc., Mariatta, OH) with a programmable temperature controller (Models 512 and 519, Powers Process Control, Oakton, IL)
and exposed to temperatures of 0 (control), −5, −10, −15, −20,
and −22.5 °C. Temperature was lowered at a rate of 2.5 °C h−1
and then held constant for 30 min after each 5 °C drop in
temperature (2.5 °C drop, for sampling at −22.5 °C). Temperatures in the test tubes were recorded during the freezing tests
with thermistor probes (Type 107, Campbell Scientific, Logan,
UT). The cooling bath had a polystyrene cover under which the
shoots were exposed to air in the dark. Air temperature in the
box was about 22 °C. After freezing, tubes were kept at 2 °C
until the roots thawed.
Viability tests
After thawing, viability tests were performed and percent live
root dry mass was evaluated.
Experiment 1----electrolyte and phenolic leakage
The entire root systems of some seedlings were cut with a razor
blade into 10-mm segments; roots of other seedlings were first
divided into fine (< 0.3 mm) and coarse (≥ 0.3 mm) roots,
before being cut into 10-mm segments. Between 75 and
200 mg of roots was placed in a 10-mm test tube containing
10 ml of distilled water. Test tubes were agitated for 16 h
(Model 6010, Eberbach Corporation, Ann Arbor, MI) at room
temperature and electrical conductivity of the water measured
(Model CDM 83 Conductivity Meter, Radiometer, Copenhagen, Denmark). To estimate phenolic compounds, 1-ml
aliquots were taken from the tubes and absorbance was measured at 260 nm (Spectronic 1002, Bausch and Lomb, Milton
Roy Co., Rochester, NY). Tissues were killed by autoclaving
the tubes at 121 °C for 30 min, after which the tubes were
agitated for 1 h and then reanalyzed for electrolyte (EL) and
phenolic leakage (PL). Leakage was expressed as the ratio
(leakage of live tissues/leakage of dead tissues) × 100. These
methods slightly overestimate leakage for a given degree of
damage because solutes were dissolved in 10 ml of water
before autoclaving, but only 9 ml after autoclaving.
Experiment 2----water loss, shoot and root water potential
Seedlings were placed in water at room temperature for 1 h to
allow saturation (Ritchie 1990). Root systems were excised
1 cm above the uppermost lateral roots, dried with a paper
towel and inserted in a pressure chamber (PMS Instruments
Co., Corvallis, OR). Pressure was increased to 0.15 MPa and
the xylem sap expressed during the following 10 min was
collected on preweighed filter paper which was then
reweighed. For other seedlings, root and shoot water potentials
were measured with a pressure chamber.
Percent live root dry mass
After thawing, one group of seedlings from each experiment
was placed in a plastic bag in the dark at 4 °C for 15 days before
separating dead roots from live ones. Dead roots were identified by brown discoloration of the cambial zone and cortex.
Live and dead roots were dried separately for 24 h at 85 °C and
weighed; live root dry mass was evaluated as a percentage of
total root dry mass.
Regrowth characteristics
In both experiments, seven seedlings per treatment temperature were held at 2 °C in the dark for 60 days to break bud
dormancy. Seedlings were then repotted in individual containers (4 × 13.5 cm, 115 cm3) in a 4/1 (v/v) peat moss/vermiculite
mixture and placed in a greenhouse for 6 months at 20 °C
(minimum 16.5 °C; maximum 26.8 °C) in an 18-h photoperiod
extended with high-pressure sodium lamps providing a photon
flux of 300 µmol m −2 s −1 (PPFD) at seedling height. Seedlings
were fertilized with 20,20,20 N,P,K at a concentration of
2 g l −1. After 6 months, seedling survival was evaluated. A
seedling was considered dead and was discarded when all
needles were yellow. Stem diameter, total live-root dry mass,
and terminal- and lateral-shoot dry mass, and terminal shoot
growth were measured on the remaining seedlings. Stem diameter was measured about 1 cm above substrate level. Root
regrowth was evaluated as total live-root dry mass. Live roots
were identified, separated from dead roots, dried for 24 h at
85 °C and weighed. New terminal and lateral shoots were dried
and weighed. New terminal shoot length was measured to the
nearest millimeter.
Experimental design, sampling, and statistical analyses
The two experiments consisted of completely randomized
block designs with eight blocks in Experiment 1 (8 blocks × 6
temperatures × 10 seedlings) and six blocks in Experiment 2
(6 blocks × 6 temperatures × 12 seedlings). Each block consisted of one container (45 cavities) per temperature treatment
for a total of six containers per block. In Experiment 1, 10 seedlings were sampled per temperature treatment per block: the
severed root system of one seedling was divided into fine and
coarse roots, a whole root system was severed from another
seedling; one seedling was used to estimate percent live root
dry mass and seven seedlings were used to study regrowth
characteristics. In Experiment 2, 12 seedlings were sampled
per temperature treatment per block: two seedlings were used
to estimate water loss; two seedlings were used to measure root
and shoot water potentials; one seedling was used to estimate
percent live root dry mass and seven seedlings were used to
study regrowth characteristics. The MIXED procedure of SAS
was used to perform the analyses of variance (SAS Institute
Inc., Cary, NC, 1992). Main temperature effects were partitioned into polynomial contrasts. For Experiment 1, root parts
were partitioned into orthogonal contrasts and the interaction
between temperature and root parts was partitioned accordingly. Polynomial response surfaces were adjusted to the least
squares means. Transformations were required to stabilize the
residual error variance: logarithmic for phenolic leakage,
water loss and root water potential, square root for shoot water
potential, arc sine for percent live root dry mass, and an
empirical logistic transformation for survival (Cox 1970). Cor-
TREE PHYSIOLOGY VOLUME 17, 1997
ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
relations between transformed variables were established
based on data from dead and live seedlings for the variables
describing regrowth characteristics.
Results
Experiment 1----electrolyte and phenolic leakage
Electrolyte leakage (EL) and phenolic leakage (PL) increased
with decreasing temperatures (T) in a way that differed between root parts (R) (P = 0.0046 and 0.0179 for the interaction
between T and R for EL and PL, respectively) (Figure 1a for
EL, Figure 1b for PL and Table 1). Fine roots had the highest
amount of leakage and whole root systems had the lowest
amount of leakage; coarse roots showed intermediate values.
Percent live root dry mass decreased quadratically with decreasing temperatures (P = 0.0129 for T quadratic) (Figure 1c)
(Table 1) with about 50% of the root system being destroyed
at −20 °C.
After 6 months, more than 90% of the seedlings survived
exposure of their root systems to 0 to −15 °C, but percent
survival decreased to 70 and 45% for seedlings whose roots
313
had been exposed to temperatures of −20 and −25 °C, respectively (P ≤ 0.0001 for T quadratic) (Figure 1d) (Table 1). The
temperature treatments had no effect on stem diameter
(P = 0.3547) (Figure 1e), new terminal shoot length
(P = 0.7570) (Figure 1f), lateral (P = 0.5053) (Figure 1h),
terminal (P = 0.9790) (Figure 1h) or total (terminal + lateral)
shoot dry mass (P = 0.4920) (Figure 1h) of the surviving
seedlings (Table 1). Total live root dry mass decreased linearly
with temperature (P ≤ 0.0001) (Figure 1g) (Table 1).
Correlations between the viability tests and seedling survival and regrowth characteristics are presented in Table 2.
Both electrolyte and phenolic leakage of coarse roots and
whole root systems were negatively correlated with percent
live root dry mass (r = −0.49 to −0.74), whereas the corresponding correlations were weak for fine roots (r = −0.31,
−0.32). The viability tests were less strongly correlated with
root regrowth (total live root dry mass, TLIR) than with percent
live root dry mass (r = −0.34 to −0.66). Electrolyte leakage and
phenolic leakage of coarse roots and whole root systems and
percent live root dry mass were all correlated with seedling
survival (r = 0.55 to 0.78) and new terminal shoot length
(r = 0.53 to 0.78).
Figure 1. Relationship between freezing temperatures
and (a) electrolyte leakage of
fine roots (d), coarse roots
(m), and whole root system
(j), 2 SE = 7.22; (b) phenolic
leakage of fine roots (d),
coarse roots (m), and whole
root system (j), 2 SE = 2.22;
(c) %LIR, percent live root
dry mass, 2 SE = 0.13; (d)
seedling survival, 2 SE = 2.56;
(e) stem diameter, 2 SE = 0.16
to 1.36; (f ) new terminal shoot
length, 2 SE = 8.18 to 9.26;
(g) TLIR, total live root dry
mass, 2 SE = 0.048 to 0.052;
(h) dry mass of new lateral
(d), 2 SE = 0.048 to 0.56, terminal (m), 2 SE = 0.028 to
0.030, and total (j) shoots,
2 SE = 0.052 to 0.060. Each
symbol is the mean of eight
observations for graphs a, b,
and c, and of 56 observations
for graph d. For graphs e--h,
the number of observations
varied between 25 and 56.
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314
BIGRAS
Table 1. Observed significance (P > F) associated with the analysis of variance of variables of Experiment 1----electrolyte and phenolic leakage.
df1
Fixed effects (P > F)
Temperature (T)
T linear
T quadratic
Root parts (R)3
WRS versus FR
WRS versus CR
T×R
FR: T linear
CR: T linear
WRS: T linear
WRS: T quadratic
Random effects (P > |Z|)4
Block (B)
B×T
Seedlings (B T)
1
2
3
4
EL2
dfn
dfd
5
1
1
2
1
1
10
1
1
1
1
35
35
35
84
84
84
84
84
84
84
84
PL
%LIR
Survival
Stem
NTSL
diameter
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.0107
0.0129 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
0.0046
0.0179
≤ 0.0001
0.0002
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
0.0186
0.0003
0.3547
Dry mass
TLIR
0.7570 ≤ 0.0001
≤ 0.0001
Shoot
lat.
0.5053
Shoot
term.
0.9790
Shoot
lat. +
term.
0.4920
0.0854
0.0778
0.4159
0.2755
0.2421 0.2995
0.2226
0.1203
0.1429 0.0857
0.9179
0.0396 ≤ 0.0001 ≤ 0.0001
0.2018 0.0918
0.0023
0.0825
0.0374 0.3502
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
df, degrees of freedom; dfn, degrees of freedom of the numerator; dfd, degrees of freedom of the denominator.
EL, electrolyte leakage; PL, phenolic leakage; %LIR, percent live root dry mass; NTSL, new terminal shoot length; TLIR, total live root dry
mass; lat., lateral; term., terminal.
FR, fine roots; CR, coarse roots; WRS, whole root system.
The multiplication sign indicates an interaction; parentheses either enclose a symbol for an effect or indicate embedding of the source of variation
within the enclosed factors.
Experiment 2----water loss, shoot and root water potential
Water loss increased quadratically with decreasing temperatures (P = 0.0275 for T quadratic) (Figure 2a) (Table 3). More
than 0.002 g of water was lost when temperatures decreased
from −15 to −22.5 °C compared with 0.001 g or less in the 0
to −10 °C range. Although shoot and root water potentials at
0 °C were lower than at −5 °C and decreased rapidly from −5
to −10 °C, they remained quite stable at temperatures below
−10 °C (P = 0.0113 and 0.0117 for T quartic for shoots and
roots, respectively) (Figure 2b) (Table 3). Percent live root dry
mass decreased quadratically with decreasing temperatures
(P = 0.0155 for T quadratic) (Figure 2c) (Table 3), and about
70% of the root systems were destroyed by the −20 and
−22.5 °C treatments.
After 6 months, survival was about 90% for seedlings exposed to temperatures ranging between 0 and −10 °C; it decreased to 75% at −15 °C and reached 35 and 20% at −20 and
−22.5 °C, respectively (P = 0.0007 for T quadratic) (Figure 2d)
(Table 3). The temperature treatments had no effect on stem
diameter of surviving seedlings (P = 0.6309) (Figure 2e) (Table 3). New terminal shoot length (P = 0.0452 for T linear)
(Figure 2f), lateral (P = 0.0010 for T linear) (Figure 2h),
terminal (P = 0.0514 for T quadratic) (Figure 2h) and total
shoot dry mass (P = 0.0003 for T linear) (Figure 2h) decreased
with decreasing temperature (Table 3). Total live root dry mass
decreased linearly with decreasing temperature (P ≤ 0.0001 for
T linear) (Figure 2g) (Table 3).
Water loss and shoot and root water potentials were correlated with percent live root dry mass (r = 0.66 to 0.70), and, as
in Experiment 1, the correlations were lower for total root dry
mass (r = 0.52 to 0.65) (Table 4). Although water loss, shoot
and root water potentials and percent live root dry mass were
all correlated with survival (r = 0.48 to 0.80), they were not
correlated with regrowth characteristics such as new terminal
shoot length (r = 0.13 to 0.48).
Discussion
Electrolyte and phenolic leakage
Percent live root dry mass provides a direct estimate of root
damage, whereas both electrolyte and phenolic leakage provide indirect estimates. Correlations between the direct and
indirect viability tests showed that electrolyte leakage of
coarse roots and whole root systems as well as phenolic leakage of coarse roots were highly correlated with percent live
root dry mass. However, because of recovery of the root system
over time, the same tests were less closely correlated with total
live root dry mass measured after 6 months. Similarly, Bigras
and Calmé (1994) showed that both electrolyte and phenolic
leakage were strongly correlated with total live-root dry mass
measured 94 days after the freezing tests but less well correlated with total live-root dry mass at Day 204. Lindström and
Nyström (1987) found that triphenyl tetrazolium chloride reduction was closely correlated with root growth capacity of
TREE PHYSIOLOGY VOLUME 17, 1997
ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
315
Table 2. Correlation coefficients (r) and levels of probability (second line) among the viability tests, survival and morphological characteristics
measured during Experiment 1----electrolyte and phenolic leakage.
EL-FR
EL1
FR
EL
CR
EL
WRS
PL
FR
PL
CR
PL
WRS
1.00
0.67
0.0001
0.58
0.0001
0.49
0.0004
0.41
0.0037
0.44
--0.32
−0.31
−0.13
−0.31
−0.34
−0.08 −0.28
−0.17
0.0016 0.0260 0.0314 0.3614 0.0346 0.0176 0.5927 0.0504 0.2388
1.00
0.75
0.0001
0.42
0.0039
0.62
0.0001
0.47
−0.74
−0.55
−0.44
−0.61
−0.62
−0.20 −0.59
−0.36
0.0010 0.0001 0.0001 0.0024 0.0001 0.0001 0.1779 0.0001 0.0141
1.00
0.64
0.0001
0.75
0.0001
0.81
−0.71
−0.71
−0.52
−0.70
−0.66
−0.33 −0.63
−0.45
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0239 0.0001 0.0013
1.00
0.77
0.0001
0.69
0.0001
1.00
0.75
−0.64
−0.61
−0.49
−0.61
−0.60
−0.19 −0.48
−0.28
0.0001 0.0001 0.0001 0.0003 0.0001 0.0001 0.1897 0.0006 0.0516
EL-CR
EL-WRS
PL-FR
PL-CR
PL-WRS
%LIR
1.00
%LIR
Survival Stem
NTSL
diameter
TLIR
Shoot
lat.
DM
Shoot
term.
DM
0.31
−0.37
−0.22
−0.31
−0.38
−0.07 −0.21
−0.09
0.0311 0.0086 0.1419 0.0343 0.0085 0.6560 0.1570 0.5518
−0.49
−0.57
−0.38
−0.53
−0.47
−0.16 −0.44
−0.24
0.0005 0.0001 0.0075 0.0001 0.0008 0.2839 0.0017 0.0973
1.00
Survival
Stem diameter
NTSL
0.78
0.0001
0.71
0.0001
0.78
0.0001
0.80
0.0001
0.51
0.0002
0.76
0.0001
0.65
0.0001
1.00
0.89
0.0001
0.92
0.0001
0.87
0.0001
0.62
0.0001
0.90
0.0001
0.77
0.0001
1.00
0.84
0.0001
0.83
0.0001
0.64
0.0001
0.85
0.0001
0.77
0.0001
1.00
0.83
0.0001
0.53
0.0001
0.92
0.0001
0.73
0.0001
1.00
0.64
0.0001
0.80
0.0001
0.76
0.0001
1.00
0.52
0.0001
0.94
0.0001
1.00
0.75
0.0001
TLIR
Shoot
lat. DM
Shoot
term. DM
Shoot
lateral + terminal DM
1
Shoot
lat. +
term.
DM
1.00
DM, dry mass; EL, electrolyte leakage; FR, fine roots; lat., lateral; %LIR, percent live root dry mass; CR, coarse roots; NTSL, new terminal
shoot length; PL, phenolic leakage; term., terminal; TLIR, total live root dry mass; WRS, whole root system.
Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies
(L.) Karst), and lodgepole pine (Pinus contorta Dougl. ex.
Loud.). Lindström and Mattsson (1994) showed that reduced
root growth capacity of frost-damaged roots was correlated
with increased root electrolyte leakage.
Although both electrolyte and phenolic leakage of whole
root systems and coarse roots were correlated with survival,
percent live root dry mass showed the highest correlation with
survival. These results contrast with those of Palta et al. (1977)
who concluded that conductivity of effusate after thawing
(electrolyte leakage) can be used to evaluate final injury but not
to predict survival. Both electrolyte and phenolic leakage of
fine roots were less well correlated with seedling survival than
electrolyte and phenolic leakage from the whole root system
or coarse roots. In contrast, McKay and Mason (1991) reported
a highly negative correlation between rates of electrolyte leakage of fine roots and survival of Sitka spruce (Picea sitchensis
(Bong.) Carr.) and Douglas-fir (Pseudotsuga menziesii (Mirb.)
Franco) seedlings after storage at 1 °C, indicating that the
observed differences among root classes may be species specific.
Water loss, and shoot and root water potentials
Water loss and shoot and root water potentials, all of which
increased with decreasing temperatures and increasing root
damage, were correlated with percent live root dry mass (cf.
Bigras and Calmé 1994). Although water loss and root water
potential were also correlated with survival, percent live root
dry mass was more strongly correlated with this parameter.
Water loss values of 0.001 g or less were related to a percent
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316
BIGRAS
Figure 2. Relationship between
freezing temperatures and (a)
water loss, 2 SE = 0.14; (b)
water potential of shoots (m), 2
SE = 0.015, and roots (d), 2 SE
= 2.66; (c) %LIR, percent live
root dry mass, 2 SE = 0.17; (d)
survival, 2 SE = 1.21; (e) stem
diameter, 2 SE = 0.09 to 0.19;
(f) new terminal shoot length, 2
SE = 11.39 to 14.80; (g) TLIR,
total live root dry mass, 2 SE =
0.04 to 0.06; (h) dry mass of
new lateral (d), 2 SE = 0.017 to
0.024, terminal (m), 2 SE =
0.031 to 0.045, and total (j)
shoots, 2 SE = 0.045 to 0.062.
Each symbol is the mean of 12
observations for graphs a and b,
six observations for graph c,
and 42 observations for graph d.
For graphs e--h, number of observations varied between 6 and
41.
Table 3. Observed significance (P > F) associated with the analysis of variance of variables of Experiment 2----water loss, shoot and root water
potential.
df1
Water
loss
dfn dfd
Water
potential
Root
Live
Survival Stem
root DM
diam.2
New
Dry mass3
terminal
shoot
Shoot
length
Fixed effects (P > F)
Temperature (T)
5
T linear
1
T quadratic
1
T quartic
1
Random effects (P > |Z|)4
Block (B)
B×T
Seedlings (B T)
1
2
3
4
25
25
25
25
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.0275 0.0325
0.0155 0.0007
0.0117 0.0113
0.2043 0.9657 0.2014
0.3417 0.1927 0.0073
≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.5788
0.0004
0.3127
0.0004
0.6309
Total
Shoot
live root lat.
0.3117 ≤ 0.0001
0.0452 ≤ 0.0001
0.0331
0.0010
Shoot
term.
0.0300
0.0014
0.0514
Shoot
lat. +
term.
0.0122
0.0003
0.1601 0.2618 0.2401 0.2568 0.2176
0.1824 0.2433 0.2366 0.0362 0.3852 0.1530
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
df, degrees of freedom; dfn, degrees of freedom of the numerator; dfd, degrees of freedom of the denominator; DM, dry mass; diam., diameter.
15 degrees of freedom of the denominator because of missing values for blocks 2 and 4.
lat., lateral; term., terminal.
The multiplication sign indicates an interaction; parentheses either enclose a symbol for an effect or indicate embedding of the source of variation
within the enclosed factors.
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ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
317
Table 4. Correlation coefficients (r) and levels of probability (second line) among the viability tests, survival and morphological characteristics
measured during Experiment 2----water loss, shoot and root water potential.
WL
WL1
WPR
WPS
%LIR
Survival
Stem
diameter
NTSL
TLIR
Shoot
lat.
DM
Shoot
term.
DM
Shoot
lat. +
term.
DM
1.00
−0.70
0.0001
−0.71
0.0001
−0.66
0.0001
−0.64
0.0001
−0.55
0.0053
−0.13
0.4572
−0.65
0.0001
−0.63
0.0001
−0.61
0.0001
−0.63
0.0001
1.00
0.89
0.0001
0.70
0.0001
0.64
0.0001
0.49
0.0160
0.29
0.0830
0.65
0.0001
0.60
0.0001
0.62
0.0001
0.63
0.0001
1.00
0.66
0.0001
0.48
0.0028
0.32
0.1318
0.15
0.3811
0.52
0.0011
0.47
0.0041
0.46
0.0050
0.47
0.0035
1.00
0.80
0.0001
0.74
0.0001
0.48
0.0028
0.84
0.0001
0.74
0.0001
0.61
0.0001
0.68
0.0001
1.00
0.96
0.0001
0.49
0.0022
0.96
0.0001
0.95
0.0001
0.86
0.0001
0.92
0.0001
1.00
0.94
0.0001
0.95
0.0001
0.93
0.0001
0.86
0.0001
0.91
0.0001
1.00
0.61
0.0001
0.53
0.0009
0.39
0.0175
0.46
0.0048
1.00
0.94
0.0001
0.84
0.0001
0.91
0.0001
1.00
0.89
0.0001
0.96
0.0001
1.00
0.98
0.0001
WPR
WPS
%LIR
Survival
Stem diameter
NTSL
TLIR
Shoot
lat. DM
Shoot
term. DM
Shoot
lateral + terminal DM
1
1.00
DM, dry mass; lat., lateral; %LIR, percent live root dry mass; NTSL, new terminal shoot length; term., terminal; TLIR, total live root dry mass;
WL, water loss; WPR, root water potential; WPS, shoot water potential.
live root dry mass of 70% or more and to a survival rate of
around 90%. Comparable threshold values for water potential
could not be defined because the water potential values were
similar in the −10 to −22.5 °C temperature treatments.
McCreary (1984) and Brown et al. (1977) concluded that
xylem water potential was related to survival in Douglas-fir,
black locust (Robinia pseudoacacia L.) and western hemlock
(Tsuga heterophylla (Raf.) Sarg.) seedlings; however, Delisle
and D’Aoust (1994) could not find any significant relationship
between morphophysiological characteristics of Picea abies,
P. glauca (Moench) Voss., and P. mariana seedlings, including
xylem water potential and root growth capacity, and their
survival in the field.
Long-term effects of freezing-induced root damage
In Experiment 1, seedling survival was ≥ 90% when no more
than 30% of the root system was destroyed but survival
dropped to between 45 and 70% when about 50% of the root
system was destroyed by frost and decreased to 20--30% when
about 70% of the root system was destroyed. Destruction of
about 50% of a seedling’s root system had no measurable
effect on stem diameter, new terminal shoot length or shoot dry
mass after 6 months of regrowth in a greenhouse on a well-irrigated substrate. In contrast, Lindström (1986b, 1987) found
that exposing root systems of Norway spruce to −16 °C caused
a 50% reduction in root growth capacity and a 40% reduction
in shoot growth.
Compared with Experiment 1, which showed destruction of
50% of the root dry mass at −20 and −22.5 °C, 70% of the root
system was destroyed by the same temperature treatments in
Experiment 2, indicating that the seedlings were less cold
hardy. Of the surviving seedlings, destruction of 70% of the
root system caused significant decreases in new terminal shoot
length and total shoot dry mass (lateral plus terminal). Similarly, Bigras and Margolis (1997) showed that destruction of
about 50% of the root systems of Pinus banksiana Lamb.
seedlings resulted in decreased shoot growth and stem diameter. Langerud et al. (1991) destroyed half of the root systems
of Norway spruce by boiling and showed that shoot growth
was lower than in control plants after 20 days in a growth
chamber.
Of the tests examined, we conclude that percent live root dry
mass provides the most accurate measure of freezing-induced
root damage; unfortunately, this test is time consuming. Elec-
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
318
BIGRAS
trolyte leakage of the whole root system was ranked the second
most reliable test and may be useful for monitoring large
samples in the nursery; however, this test must be applied
immediately after a frost event, otherwise its reliability is
compromised.
Acknowledgments
The author expresses her gratitude to Eric Leclerc, technician, for his
collaboration during the project, Carole Hébert for statistical analyses
and Michèle Bernier-Cardou for statistical supervision. The author
also thanks Richard Gohier and Rosaire Tremblay from the Centre de
production de plants forestiers de Québec for their continuing collaboration. She also thanks Drs. André D’Aoust from the Canadian Forest
Service, Laurentian Forestry Centre, and Carole Coursolle from Université Laval for revision of the first draft.
References
Bigras, F.J. and S. Calmé. 1994. Viability tests for estimating root cold
tolerance of black spruce seedlings. Can. J. For. Res. 24:1039-1048.
Bigras, F.J. and H.A. Margolis. 1997. Shoot and root sensitivity of
containerized black spruce, white spruce and jack pine seedlings to
late fall freezing. New For. In press.
Brown, G.N., J.A. Bixby, P.K. Melcarek, T.M. Hinckley and R. Rogers. 1977. Xylem pressure potential and chlorophyll fluorescence
as indicators of freezing survival in black locust and western hemlock seedlings. Cryobiology 14:94--99.
Cox, D.R. 1970. The analysis of binary data. Methuen’s statistical
monographs. Methuen & Co. Ltd., London, 142 p.
Delisle, C. and A.L. D’Aoust. 1994. The relationship between the
morpho-physiological characteristics of containerized spruce seedlings and their field performance. In Making the Grade: An International Symposium on Planting Stock Performance and Quality
Assessment, Sault Ste. Marie, Ontario. Eds. D.S. Maki, T.M.
McDonough and T.L. Noland. Ontario Forest Research Institute.
72 p.
Langerud, B.R., P. Puttonen and E. Troeng. 1991. Viability of Picea
abies seedlings with damaged roots and shoots. Scand. J. For. Res.
6:59--72.
Lindström, A. 1986a. Outdoor winter storage of container stock on
raised pallets----effects on root zone temperature and seedling
growth. Scand. J. For. Res. 1:37--47.
Lindström, A. 1986b. Freezing temperature in the root zone----effects
on growth of containerized Pinus sylvestris and Picea abies seedlings. Scand. J. For. Res. 1:371--378.
Lindström, A. 1987. Winter storage and root hardiness of containerized conifer seedlings. Doctoral Thesis, Swedish Univ. of Agric.
Sci., Dept. Forest Yield Res., Garpenberg, Sweden.
Lindström, A. and C. Nyström. 1987. Seasonal variation in root
hardiness of container-grown Scots pine, Norway spruce, and
lodgepole pine seedlings. Can. J. For. Res. 17:787--793.
Lindström, A. and A. Mattsson. 1994. Cultivation of containerized
seedlings in Sweden----systems for frost protection and methods to
detect root injuries. Acta Hortic. (Wageningen) 361:429--440.
McCreary, D.D. 1984. Using a pressure chamber to detect damage to
seedlings accidentally frozen during cold storage. In The Challenge
of Producing Native Plants for the Intermountain Area. Proceedings, Intermountain Nurseryman’s Association Conference, Las
Vegas, NV. Ed. P.M. Murphy. USDA For. Serv. Gen. Tech. Rep.
INT-168, pp 58--60.
McKay, H.M. and W.L. Mason. 1991. Physiological indicators of
tolerance to cold storage in Sitka spruce and Douglas-fir seedlings.
Can. J. For. Res. 21:890--901.
Palta, J.P., J. Levitt and E.J. Stadelman. 1977. Freezing injury in onion
bud cells. II. Post thawing injury or recovery. Plant Physiol.
60:398--401.
Ritchie, G.A. 1990. A rapid method for detecting cold injury in conifer
seedling root systems. Can. J. For. Res. 20:26--30.
Simpson, D.G. 1993. Root cold hardiness of western Canadian conifers. In Proceedings, 12th Annual Meeting of the B.C. Forest Nursery Association, Penticton. B.C. Ministry of Forests, Victoria, pp
97--105.
TREE PHYSIOLOGY VOLUME 17, 1997
© 1997 Heron Publishing----Victoria, Canada
Root cold tolerance of black spruce seedlings: viability tests in relation
to survival and regrowth
F. J. BIGRAS
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, Sainte-Foy, Québec G1V 4C7, Canada
Received March 25, 1996
Summary Root systems of 6-month-old, cold-hardened,
container-grown black spruce seedlings (Picea mariana (Mill.)
B.S.P.) were exposed to 0, −5, −10, −15, −20, or −22.5 °C.
Freezing-induced damage to fine roots, coarse roots and the
whole root system was assessed by various viability tests
including leakage of electrolytes, leakage of phenolic compounds, water loss, root and shoot water potentials, and live
root dry mass. To assess the long-term effects of freezing-induced root damage, seedling survival and regrowth were measured. Leakage of both electrolytes and phenolic compounds
differed among fine roots, coarse roots, and whole root systems. In coarse roots and the whole root system, but not in fine
roots, leakage of electrolytes, leakage of phenolic compounds,
water loss, and root and shoot water potentials were correlated
with percentage of live root dry mass which, in turn, was highly
correlated with seedling survival and regrowth. Compared with
live root dry mass, electrolyte and phenolic leakage, water loss,
and root and shoot water potentials were less well correlated
with seedling survival and regrowth. Among the viability tests,
electrolyte leakage of the whole root system correlated most
closely with seedling survival and regrowth. Under freezing
conditions that destroyed less than 50% of each seedling’s root
system, about 70% of the seedlings survived and subsequent
growth was little affected, whereas under freezing conditions
that destroyed 70% of each seedling’s root system, only about
30% of the seedlings survived and subsequent growth was
reduced compared with that of undamaged plants.
Keywords: electrolyte leakage, phenolic leakage, Picea mariana, water loss, water potential.
Introduction
Frost damage to root systems, which frequently occurs during
production of conifer seedlings in containers or during storage
of bare-root seedlings (Lindström 1986a, Simpson 1993), has
a negative impact on seedling survival and regrowth (Lindström 1986b, Bigras and Margolis 1996). Therefore, a cheap,
rapid and accurate procedure is needed to identify frost-damaged seedlings before they are shipped to plantation sites.
Various methods have been developed to estimate root damage after frost events. Rapid methods include measurements of
leakage of electrolytes, leakage of ninhydrin-reactive or phe-
nolic compounds, triphenyl tetrazolium chloride reduction,
sugar content, root respiration, shoot and root water potentials,
water loss and resistance to electric current. Tests that require
an incubation period are root growth capacity, visual examination of tissue browning, and measurement of fresh mass or dry
mass of undamaged roots. Although many methods have been
used to estimate root damage, few attempts have been made to
compare the results of the various tests with seedling survival
and growth (Bigras and Calmé 1994).
The objective of this research was to identify seedling viability tests that are highly correlated with seedling survival
and subsequent growth. Specifically, we subjected root systems of black spruce seedlings to a range of freezing temperatures to: (i) compare different root viability tests among
different root diameter classes; (ii) evaluate the impact of frost
damage on seedling survival and regrowth; and (iii) correlate
the results of the viability tests with seedling survival and
regrowth characteristics.
Materials and methods
Plant material
Black spruce seedlings (Picea mariana (Mill.) B.S.P.) (provenance 47°45′ N, 70°05′ W) were grown in unheated polyethylene-covered greenhouses at a commercial forest nursery
(Centre de production de plants forestiers de Québec Inc.,
Sainte-Anne-de-Beaupré, PQ) in a peat moss/vermiculite mixture (2/1, v/v). On May 20, 1992, three to four seeds were sown
per cavity (110 cm3) in container trays (45 cavities) and
thinned to one seedling per cavity 30 days later. Seedlings were
fertilized with liquid fertilizer from June 21 until September 13, and received a total of 6.5 mg N, 5.0 mg P, and 5.8 mg
K per cavity. Seedlings were placed outside on October 14
(mean height, 6.5 cm; stem diameter, 0.98 mm; shoot dry
mass, 117 mg; and root dry mass, 71 mg). On November 9,
seedlings were shipped to the Laurentian Forestry Centre,
Sainte-Foy, PQ (Canadian Forest Service) and placed in a
greenhouse at a day/night temperature of 10/6 °C in a natural
photoperiod. The seedlings were watered to saturation and no
fertilizer was added.
312
BIGRAS
Freezing tests
Before freezing, root systems were washed under running
water to completely remove substrate. The root system of each
seedling was inserted in a 16-mm test tube and the tubes were
immersed in a cooling bath (Model 2325, Forma Scientific
Inc., Mariatta, OH) with a programmable temperature controller (Models 512 and 519, Powers Process Control, Oakton, IL)
and exposed to temperatures of 0 (control), −5, −10, −15, −20,
and −22.5 °C. Temperature was lowered at a rate of 2.5 °C h−1
and then held constant for 30 min after each 5 °C drop in
temperature (2.5 °C drop, for sampling at −22.5 °C). Temperatures in the test tubes were recorded during the freezing tests
with thermistor probes (Type 107, Campbell Scientific, Logan,
UT). The cooling bath had a polystyrene cover under which the
shoots were exposed to air in the dark. Air temperature in the
box was about 22 °C. After freezing, tubes were kept at 2 °C
until the roots thawed.
Viability tests
After thawing, viability tests were performed and percent live
root dry mass was evaluated.
Experiment 1----electrolyte and phenolic leakage
The entire root systems of some seedlings were cut with a razor
blade into 10-mm segments; roots of other seedlings were first
divided into fine (< 0.3 mm) and coarse (≥ 0.3 mm) roots,
before being cut into 10-mm segments. Between 75 and
200 mg of roots was placed in a 10-mm test tube containing
10 ml of distilled water. Test tubes were agitated for 16 h
(Model 6010, Eberbach Corporation, Ann Arbor, MI) at room
temperature and electrical conductivity of the water measured
(Model CDM 83 Conductivity Meter, Radiometer, Copenhagen, Denmark). To estimate phenolic compounds, 1-ml
aliquots were taken from the tubes and absorbance was measured at 260 nm (Spectronic 1002, Bausch and Lomb, Milton
Roy Co., Rochester, NY). Tissues were killed by autoclaving
the tubes at 121 °C for 30 min, after which the tubes were
agitated for 1 h and then reanalyzed for electrolyte (EL) and
phenolic leakage (PL). Leakage was expressed as the ratio
(leakage of live tissues/leakage of dead tissues) × 100. These
methods slightly overestimate leakage for a given degree of
damage because solutes were dissolved in 10 ml of water
before autoclaving, but only 9 ml after autoclaving.
Experiment 2----water loss, shoot and root water potential
Seedlings were placed in water at room temperature for 1 h to
allow saturation (Ritchie 1990). Root systems were excised
1 cm above the uppermost lateral roots, dried with a paper
towel and inserted in a pressure chamber (PMS Instruments
Co., Corvallis, OR). Pressure was increased to 0.15 MPa and
the xylem sap expressed during the following 10 min was
collected on preweighed filter paper which was then
reweighed. For other seedlings, root and shoot water potentials
were measured with a pressure chamber.
Percent live root dry mass
After thawing, one group of seedlings from each experiment
was placed in a plastic bag in the dark at 4 °C for 15 days before
separating dead roots from live ones. Dead roots were identified by brown discoloration of the cambial zone and cortex.
Live and dead roots were dried separately for 24 h at 85 °C and
weighed; live root dry mass was evaluated as a percentage of
total root dry mass.
Regrowth characteristics
In both experiments, seven seedlings per treatment temperature were held at 2 °C in the dark for 60 days to break bud
dormancy. Seedlings were then repotted in individual containers (4 × 13.5 cm, 115 cm3) in a 4/1 (v/v) peat moss/vermiculite
mixture and placed in a greenhouse for 6 months at 20 °C
(minimum 16.5 °C; maximum 26.8 °C) in an 18-h photoperiod
extended with high-pressure sodium lamps providing a photon
flux of 300 µmol m −2 s −1 (PPFD) at seedling height. Seedlings
were fertilized with 20,20,20 N,P,K at a concentration of
2 g l −1. After 6 months, seedling survival was evaluated. A
seedling was considered dead and was discarded when all
needles were yellow. Stem diameter, total live-root dry mass,
and terminal- and lateral-shoot dry mass, and terminal shoot
growth were measured on the remaining seedlings. Stem diameter was measured about 1 cm above substrate level. Root
regrowth was evaluated as total live-root dry mass. Live roots
were identified, separated from dead roots, dried for 24 h at
85 °C and weighed. New terminal and lateral shoots were dried
and weighed. New terminal shoot length was measured to the
nearest millimeter.
Experimental design, sampling, and statistical analyses
The two experiments consisted of completely randomized
block designs with eight blocks in Experiment 1 (8 blocks × 6
temperatures × 10 seedlings) and six blocks in Experiment 2
(6 blocks × 6 temperatures × 12 seedlings). Each block consisted of one container (45 cavities) per temperature treatment
for a total of six containers per block. In Experiment 1, 10 seedlings were sampled per temperature treatment per block: the
severed root system of one seedling was divided into fine and
coarse roots, a whole root system was severed from another
seedling; one seedling was used to estimate percent live root
dry mass and seven seedlings were used to study regrowth
characteristics. In Experiment 2, 12 seedlings were sampled
per temperature treatment per block: two seedlings were used
to estimate water loss; two seedlings were used to measure root
and shoot water potentials; one seedling was used to estimate
percent live root dry mass and seven seedlings were used to
study regrowth characteristics. The MIXED procedure of SAS
was used to perform the analyses of variance (SAS Institute
Inc., Cary, NC, 1992). Main temperature effects were partitioned into polynomial contrasts. For Experiment 1, root parts
were partitioned into orthogonal contrasts and the interaction
between temperature and root parts was partitioned accordingly. Polynomial response surfaces were adjusted to the least
squares means. Transformations were required to stabilize the
residual error variance: logarithmic for phenolic leakage,
water loss and root water potential, square root for shoot water
potential, arc sine for percent live root dry mass, and an
empirical logistic transformation for survival (Cox 1970). Cor-
TREE PHYSIOLOGY VOLUME 17, 1997
ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
relations between transformed variables were established
based on data from dead and live seedlings for the variables
describing regrowth characteristics.
Results
Experiment 1----electrolyte and phenolic leakage
Electrolyte leakage (EL) and phenolic leakage (PL) increased
with decreasing temperatures (T) in a way that differed between root parts (R) (P = 0.0046 and 0.0179 for the interaction
between T and R for EL and PL, respectively) (Figure 1a for
EL, Figure 1b for PL and Table 1). Fine roots had the highest
amount of leakage and whole root systems had the lowest
amount of leakage; coarse roots showed intermediate values.
Percent live root dry mass decreased quadratically with decreasing temperatures (P = 0.0129 for T quadratic) (Figure 1c)
(Table 1) with about 50% of the root system being destroyed
at −20 °C.
After 6 months, more than 90% of the seedlings survived
exposure of their root systems to 0 to −15 °C, but percent
survival decreased to 70 and 45% for seedlings whose roots
313
had been exposed to temperatures of −20 and −25 °C, respectively (P ≤ 0.0001 for T quadratic) (Figure 1d) (Table 1). The
temperature treatments had no effect on stem diameter
(P = 0.3547) (Figure 1e), new terminal shoot length
(P = 0.7570) (Figure 1f), lateral (P = 0.5053) (Figure 1h),
terminal (P = 0.9790) (Figure 1h) or total (terminal + lateral)
shoot dry mass (P = 0.4920) (Figure 1h) of the surviving
seedlings (Table 1). Total live root dry mass decreased linearly
with temperature (P ≤ 0.0001) (Figure 1g) (Table 1).
Correlations between the viability tests and seedling survival and regrowth characteristics are presented in Table 2.
Both electrolyte and phenolic leakage of coarse roots and
whole root systems were negatively correlated with percent
live root dry mass (r = −0.49 to −0.74), whereas the corresponding correlations were weak for fine roots (r = −0.31,
−0.32). The viability tests were less strongly correlated with
root regrowth (total live root dry mass, TLIR) than with percent
live root dry mass (r = −0.34 to −0.66). Electrolyte leakage and
phenolic leakage of coarse roots and whole root systems and
percent live root dry mass were all correlated with seedling
survival (r = 0.55 to 0.78) and new terminal shoot length
(r = 0.53 to 0.78).
Figure 1. Relationship between freezing temperatures
and (a) electrolyte leakage of
fine roots (d), coarse roots
(m), and whole root system
(j), 2 SE = 7.22; (b) phenolic
leakage of fine roots (d),
coarse roots (m), and whole
root system (j), 2 SE = 2.22;
(c) %LIR, percent live root
dry mass, 2 SE = 0.13; (d)
seedling survival, 2 SE = 2.56;
(e) stem diameter, 2 SE = 0.16
to 1.36; (f ) new terminal shoot
length, 2 SE = 8.18 to 9.26;
(g) TLIR, total live root dry
mass, 2 SE = 0.048 to 0.052;
(h) dry mass of new lateral
(d), 2 SE = 0.048 to 0.56, terminal (m), 2 SE = 0.028 to
0.030, and total (j) shoots,
2 SE = 0.052 to 0.060. Each
symbol is the mean of eight
observations for graphs a, b,
and c, and of 56 observations
for graph d. For graphs e--h,
the number of observations
varied between 25 and 56.
TREE PHYSIOLOGY ON-LINE at http://www.heronpublishing.com
314
BIGRAS
Table 1. Observed significance (P > F) associated with the analysis of variance of variables of Experiment 1----electrolyte and phenolic leakage.
df1
Fixed effects (P > F)
Temperature (T)
T linear
T quadratic
Root parts (R)3
WRS versus FR
WRS versus CR
T×R
FR: T linear
CR: T linear
WRS: T linear
WRS: T quadratic
Random effects (P > |Z|)4
Block (B)
B×T
Seedlings (B T)
1
2
3
4
EL2
dfn
dfd
5
1
1
2
1
1
10
1
1
1
1
35
35
35
84
84
84
84
84
84
84
84
PL
%LIR
Survival
Stem
NTSL
diameter
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.0107
0.0129 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
0.0046
0.0179
≤ 0.0001
0.0002
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001
0.0186
0.0003
0.3547
Dry mass
TLIR
0.7570 ≤ 0.0001
≤ 0.0001
Shoot
lat.
0.5053
Shoot
term.
0.9790
Shoot
lat. +
term.
0.4920
0.0854
0.0778
0.4159
0.2755
0.2421 0.2995
0.2226
0.1203
0.1429 0.0857
0.9179
0.0396 ≤ 0.0001 ≤ 0.0001
0.2018 0.0918
0.0023
0.0825
0.0374 0.3502
≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
df, degrees of freedom; dfn, degrees of freedom of the numerator; dfd, degrees of freedom of the denominator.
EL, electrolyte leakage; PL, phenolic leakage; %LIR, percent live root dry mass; NTSL, new terminal shoot length; TLIR, total live root dry
mass; lat., lateral; term., terminal.
FR, fine roots; CR, coarse roots; WRS, whole root system.
The multiplication sign indicates an interaction; parentheses either enclose a symbol for an effect or indicate embedding of the source of variation
within the enclosed factors.
Experiment 2----water loss, shoot and root water potential
Water loss increased quadratically with decreasing temperatures (P = 0.0275 for T quadratic) (Figure 2a) (Table 3). More
than 0.002 g of water was lost when temperatures decreased
from −15 to −22.5 °C compared with 0.001 g or less in the 0
to −10 °C range. Although shoot and root water potentials at
0 °C were lower than at −5 °C and decreased rapidly from −5
to −10 °C, they remained quite stable at temperatures below
−10 °C (P = 0.0113 and 0.0117 for T quartic for shoots and
roots, respectively) (Figure 2b) (Table 3). Percent live root dry
mass decreased quadratically with decreasing temperatures
(P = 0.0155 for T quadratic) (Figure 2c) (Table 3), and about
70% of the root systems were destroyed by the −20 and
−22.5 °C treatments.
After 6 months, survival was about 90% for seedlings exposed to temperatures ranging between 0 and −10 °C; it decreased to 75% at −15 °C and reached 35 and 20% at −20 and
−22.5 °C, respectively (P = 0.0007 for T quadratic) (Figure 2d)
(Table 3). The temperature treatments had no effect on stem
diameter of surviving seedlings (P = 0.6309) (Figure 2e) (Table 3). New terminal shoot length (P = 0.0452 for T linear)
(Figure 2f), lateral (P = 0.0010 for T linear) (Figure 2h),
terminal (P = 0.0514 for T quadratic) (Figure 2h) and total
shoot dry mass (P = 0.0003 for T linear) (Figure 2h) decreased
with decreasing temperature (Table 3). Total live root dry mass
decreased linearly with decreasing temperature (P ≤ 0.0001 for
T linear) (Figure 2g) (Table 3).
Water loss and shoot and root water potentials were correlated with percent live root dry mass (r = 0.66 to 0.70), and, as
in Experiment 1, the correlations were lower for total root dry
mass (r = 0.52 to 0.65) (Table 4). Although water loss, shoot
and root water potentials and percent live root dry mass were
all correlated with survival (r = 0.48 to 0.80), they were not
correlated with regrowth characteristics such as new terminal
shoot length (r = 0.13 to 0.48).
Discussion
Electrolyte and phenolic leakage
Percent live root dry mass provides a direct estimate of root
damage, whereas both electrolyte and phenolic leakage provide indirect estimates. Correlations between the direct and
indirect viability tests showed that electrolyte leakage of
coarse roots and whole root systems as well as phenolic leakage of coarse roots were highly correlated with percent live
root dry mass. However, because of recovery of the root system
over time, the same tests were less closely correlated with total
live root dry mass measured after 6 months. Similarly, Bigras
and Calmé (1994) showed that both electrolyte and phenolic
leakage were strongly correlated with total live-root dry mass
measured 94 days after the freezing tests but less well correlated with total live-root dry mass at Day 204. Lindström and
Nyström (1987) found that triphenyl tetrazolium chloride reduction was closely correlated with root growth capacity of
TREE PHYSIOLOGY VOLUME 17, 1997
ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
315
Table 2. Correlation coefficients (r) and levels of probability (second line) among the viability tests, survival and morphological characteristics
measured during Experiment 1----electrolyte and phenolic leakage.
EL-FR
EL1
FR
EL
CR
EL
WRS
PL
FR
PL
CR
PL
WRS
1.00
0.67
0.0001
0.58
0.0001
0.49
0.0004
0.41
0.0037
0.44
--0.32
−0.31
−0.13
−0.31
−0.34
−0.08 −0.28
−0.17
0.0016 0.0260 0.0314 0.3614 0.0346 0.0176 0.5927 0.0504 0.2388
1.00
0.75
0.0001
0.42
0.0039
0.62
0.0001
0.47
−0.74
−0.55
−0.44
−0.61
−0.62
−0.20 −0.59
−0.36
0.0010 0.0001 0.0001 0.0024 0.0001 0.0001 0.1779 0.0001 0.0141
1.00
0.64
0.0001
0.75
0.0001
0.81
−0.71
−0.71
−0.52
−0.70
−0.66
−0.33 −0.63
−0.45
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0239 0.0001 0.0013
1.00
0.77
0.0001
0.69
0.0001
1.00
0.75
−0.64
−0.61
−0.49
−0.61
−0.60
−0.19 −0.48
−0.28
0.0001 0.0001 0.0001 0.0003 0.0001 0.0001 0.1897 0.0006 0.0516
EL-CR
EL-WRS
PL-FR
PL-CR
PL-WRS
%LIR
1.00
%LIR
Survival Stem
NTSL
diameter
TLIR
Shoot
lat.
DM
Shoot
term.
DM
0.31
−0.37
−0.22
−0.31
−0.38
−0.07 −0.21
−0.09
0.0311 0.0086 0.1419 0.0343 0.0085 0.6560 0.1570 0.5518
−0.49
−0.57
−0.38
−0.53
−0.47
−0.16 −0.44
−0.24
0.0005 0.0001 0.0075 0.0001 0.0008 0.2839 0.0017 0.0973
1.00
Survival
Stem diameter
NTSL
0.78
0.0001
0.71
0.0001
0.78
0.0001
0.80
0.0001
0.51
0.0002
0.76
0.0001
0.65
0.0001
1.00
0.89
0.0001
0.92
0.0001
0.87
0.0001
0.62
0.0001
0.90
0.0001
0.77
0.0001
1.00
0.84
0.0001
0.83
0.0001
0.64
0.0001
0.85
0.0001
0.77
0.0001
1.00
0.83
0.0001
0.53
0.0001
0.92
0.0001
0.73
0.0001
1.00
0.64
0.0001
0.80
0.0001
0.76
0.0001
1.00
0.52
0.0001
0.94
0.0001
1.00
0.75
0.0001
TLIR
Shoot
lat. DM
Shoot
term. DM
Shoot
lateral + terminal DM
1
Shoot
lat. +
term.
DM
1.00
DM, dry mass; EL, electrolyte leakage; FR, fine roots; lat., lateral; %LIR, percent live root dry mass; CR, coarse roots; NTSL, new terminal
shoot length; PL, phenolic leakage; term., terminal; TLIR, total live root dry mass; WRS, whole root system.
Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies
(L.) Karst), and lodgepole pine (Pinus contorta Dougl. ex.
Loud.). Lindström and Mattsson (1994) showed that reduced
root growth capacity of frost-damaged roots was correlated
with increased root electrolyte leakage.
Although both electrolyte and phenolic leakage of whole
root systems and coarse roots were correlated with survival,
percent live root dry mass showed the highest correlation with
survival. These results contrast with those of Palta et al. (1977)
who concluded that conductivity of effusate after thawing
(electrolyte leakage) can be used to evaluate final injury but not
to predict survival. Both electrolyte and phenolic leakage of
fine roots were less well correlated with seedling survival than
electrolyte and phenolic leakage from the whole root system
or coarse roots. In contrast, McKay and Mason (1991) reported
a highly negative correlation between rates of electrolyte leakage of fine roots and survival of Sitka spruce (Picea sitchensis
(Bong.) Carr.) and Douglas-fir (Pseudotsuga menziesii (Mirb.)
Franco) seedlings after storage at 1 °C, indicating that the
observed differences among root classes may be species specific.
Water loss, and shoot and root water potentials
Water loss and shoot and root water potentials, all of which
increased with decreasing temperatures and increasing root
damage, were correlated with percent live root dry mass (cf.
Bigras and Calmé 1994). Although water loss and root water
potential were also correlated with survival, percent live root
dry mass was more strongly correlated with this parameter.
Water loss values of 0.001 g or less were related to a percent
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316
BIGRAS
Figure 2. Relationship between
freezing temperatures and (a)
water loss, 2 SE = 0.14; (b)
water potential of shoots (m), 2
SE = 0.015, and roots (d), 2 SE
= 2.66; (c) %LIR, percent live
root dry mass, 2 SE = 0.17; (d)
survival, 2 SE = 1.21; (e) stem
diameter, 2 SE = 0.09 to 0.19;
(f) new terminal shoot length, 2
SE = 11.39 to 14.80; (g) TLIR,
total live root dry mass, 2 SE =
0.04 to 0.06; (h) dry mass of
new lateral (d), 2 SE = 0.017 to
0.024, terminal (m), 2 SE =
0.031 to 0.045, and total (j)
shoots, 2 SE = 0.045 to 0.062.
Each symbol is the mean of 12
observations for graphs a and b,
six observations for graph c,
and 42 observations for graph d.
For graphs e--h, number of observations varied between 6 and
41.
Table 3. Observed significance (P > F) associated with the analysis of variance of variables of Experiment 2----water loss, shoot and root water
potential.
df1
Water
loss
dfn dfd
Water
potential
Root
Live
Survival Stem
root DM
diam.2
New
Dry mass3
terminal
shoot
Shoot
length
Fixed effects (P > F)
Temperature (T)
5
T linear
1
T quadratic
1
T quartic
1
Random effects (P > |Z|)4
Block (B)
B×T
Seedlings (B T)
1
2
3
4
25
25
25
25
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.0275 0.0325
0.0155 0.0007
0.0117 0.0113
0.2043 0.9657 0.2014
0.3417 0.1927 0.0073
≤ 0.0001 ≤ 0.0001 ≤ 0.0001
0.5788
0.0004
0.3127
0.0004
0.6309
Total
Shoot
live root lat.
0.3117 ≤ 0.0001
0.0452 ≤ 0.0001
0.0331
0.0010
Shoot
term.
0.0300
0.0014
0.0514
Shoot
lat. +
term.
0.0122
0.0003
0.1601 0.2618 0.2401 0.2568 0.2176
0.1824 0.2433 0.2366 0.0362 0.3852 0.1530
≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001 ≤ 0.0001
df, degrees of freedom; dfn, degrees of freedom of the numerator; dfd, degrees of freedom of the denominator; DM, dry mass; diam., diameter.
15 degrees of freedom of the denominator because of missing values for blocks 2 and 4.
lat., lateral; term., terminal.
The multiplication sign indicates an interaction; parentheses either enclose a symbol for an effect or indicate embedding of the source of variation
within the enclosed factors.
TREE PHYSIOLOGY VOLUME 17, 1997
ROOT COLD TOLERANCE OF BLACK SPRUCE SEEDLINGS
317
Table 4. Correlation coefficients (r) and levels of probability (second line) among the viability tests, survival and morphological characteristics
measured during Experiment 2----water loss, shoot and root water potential.
WL
WL1
WPR
WPS
%LIR
Survival
Stem
diameter
NTSL
TLIR
Shoot
lat.
DM
Shoot
term.
DM
Shoot
lat. +
term.
DM
1.00
−0.70
0.0001
−0.71
0.0001
−0.66
0.0001
−0.64
0.0001
−0.55
0.0053
−0.13
0.4572
−0.65
0.0001
−0.63
0.0001
−0.61
0.0001
−0.63
0.0001
1.00
0.89
0.0001
0.70
0.0001
0.64
0.0001
0.49
0.0160
0.29
0.0830
0.65
0.0001
0.60
0.0001
0.62
0.0001
0.63
0.0001
1.00
0.66
0.0001
0.48
0.0028
0.32
0.1318
0.15
0.3811
0.52
0.0011
0.47
0.0041
0.46
0.0050
0.47
0.0035
1.00
0.80
0.0001
0.74
0.0001
0.48
0.0028
0.84
0.0001
0.74
0.0001
0.61
0.0001
0.68
0.0001
1.00
0.96
0.0001
0.49
0.0022
0.96
0.0001
0.95
0.0001
0.86
0.0001
0.92
0.0001
1.00
0.94
0.0001
0.95
0.0001
0.93
0.0001
0.86
0.0001
0.91
0.0001
1.00
0.61
0.0001
0.53
0.0009
0.39
0.0175
0.46
0.0048
1.00
0.94
0.0001
0.84
0.0001
0.91
0.0001
1.00
0.89
0.0001
0.96
0.0001
1.00
0.98
0.0001
WPR
WPS
%LIR
Survival
Stem diameter
NTSL
TLIR
Shoot
lat. DM
Shoot
term. DM
Shoot
lateral + terminal DM
1
1.00
DM, dry mass; lat., lateral; %LIR, percent live root dry mass; NTSL, new terminal shoot length; term., terminal; TLIR, total live root dry mass;
WL, water loss; WPR, root water potential; WPS, shoot water potential.
live root dry mass of 70% or more and to a survival rate of
around 90%. Comparable threshold values for water potential
could not be defined because the water potential values were
similar in the −10 to −22.5 °C temperature treatments.
McCreary (1984) and Brown et al. (1977) concluded that
xylem water potential was related to survival in Douglas-fir,
black locust (Robinia pseudoacacia L.) and western hemlock
(Tsuga heterophylla (Raf.) Sarg.) seedlings; however, Delisle
and D’Aoust (1994) could not find any significant relationship
between morphophysiological characteristics of Picea abies,
P. glauca (Moench) Voss., and P. mariana seedlings, including
xylem water potential and root growth capacity, and their
survival in the field.
Long-term effects of freezing-induced root damage
In Experiment 1, seedling survival was ≥ 90% when no more
than 30% of the root system was destroyed but survival
dropped to between 45 and 70% when about 50% of the root
system was destroyed by frost and decreased to 20--30% when
about 70% of the root system was destroyed. Destruction of
about 50% of a seedling’s root system had no measurable
effect on stem diameter, new terminal shoot length or shoot dry
mass after 6 months of regrowth in a greenhouse on a well-irrigated substrate. In contrast, Lindström (1986b, 1987) found
that exposing root systems of Norway spruce to −16 °C caused
a 50% reduction in root growth capacity and a 40% reduction
in shoot growth.
Compared with Experiment 1, which showed destruction of
50% of the root dry mass at −20 and −22.5 °C, 70% of the root
system was destroyed by the same temperature treatments in
Experiment 2, indicating that the seedlings were less cold
hardy. Of the surviving seedlings, destruction of 70% of the
root system caused significant decreases in new terminal shoot
length and total shoot dry mass (lateral plus terminal). Similarly, Bigras and Margolis (1997) showed that destruction of
about 50% of the root systems of Pinus banksiana Lamb.
seedlings resulted in decreased shoot growth and stem diameter. Langerud et al. (1991) destroyed half of the root systems
of Norway spruce by boiling and showed that shoot growth
was lower than in control plants after 20 days in a growth
chamber.
Of the tests examined, we conclude that percent live root dry
mass provides the most accurate measure of freezing-induced
root damage; unfortunately, this test is time consuming. Elec-
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318
BIGRAS
trolyte leakage of the whole root system was ranked the second
most reliable test and may be useful for monitoring large
samples in the nursery; however, this test must be applied
immediately after a frost event, otherwise its reliability is
compromised.
Acknowledgments
The author expresses her gratitude to Eric Leclerc, technician, for his
collaboration during the project, Carole Hébert for statistical analyses
and Michèle Bernier-Cardou for statistical supervision. The author
also thanks Richard Gohier and Rosaire Tremblay from the Centre de
production de plants forestiers de Québec for their continuing collaboration. She also thanks Drs. André D’Aoust from the Canadian Forest
Service, Laurentian Forestry Centre, and Carole Coursolle from Université Laval for revision of the first draft.
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