Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol14.1994:

Tree Physiology 14, 1313--1325
© 1994 Heron Publishing----Victoria, Canada

Frost tolerance and bud dormancy of container-grown yellow
birch, red oak and sugar maple seedlings
SOPHIE CALMÉ,1 FRANCINE J. BIGRAS,2 HANK A. MARGOLIS1 and
CAROLE HÉBERT2
1

Centre de recherche en biologie forestière, Université Laval, Sainte-Foy, Quebec G1K 7P4, Canada

2

Natural Resources Canada, Canadian Forest Service--Quebec Region, P.O. Box 3800, Sainte-Foy,
Quebec G1V 4C7, Canada

Received December 14, 1993
Summary
Container-grown seedlings of red oak (Quercus rubra L.), sugar maple (Acer saccharum Marsh.) and
yellow birch (Betula alleghaniensis Britton) in their first year of growth were overwintered outdoors.
Tolerance of roots and stems to freezing were compared from late summer to the following spring.

Mitotic activity in the apical bud was related more closely to air temperature than to bud dormancy as
defined by days-to-bud-break. In all species, stem hardening was observed before days-to-bud-break
reached a maximum. Dormancy release (days-to-bud-break equal to zero) of yellow birch coincided with
loss of stem hardening in the spring. Roots hardened more slowly, had a lower frost tolerance than stems
in fall and winter, and dehardened earlier than stems in the spring. There were differences in stem and
root hardiness among the species, with yellow birch being the most tolerant, followed by sugar maple
and red oak. Primarily because of root sensitivity to frost, winter was a critical period for all three species,
but particularly for red oak.
Keywords: Quercus rubra, Betula alleghaniensis, Acer saccharum, root, shoot, bud break, mitotic frequency.

Introduction
In 1993, red oak (Quercus rubra L.), sugar maple (Acer saccharum Marsh.) and
yellow birch (Betula alleghaniensis Britton) represented 90% of the production of
hardwood seedlings in Quebec (G. Couture, personal communication). Because
these species are at the northern limit of their distribution in Quebec, their frost
tolerance is likely to be a key determinant in their ability to survive in both the nursery
and the plantation.
Attempts have been made to relate the bud dormancy status of nursery grown
seedlings to their field survival (Garber and Mexal 1980, Boyer and South 1985) and
to their resistance to environmental stresses such as freezing (Lavender 1991). A

knowledge of the precise nature of such relationships is of considerable practical
importance (Carlson et al. 1980, Carlson 1985). The two variables most frequently
used to determine the bud dormancy status of tree seedlings are (1) the heat sum
required for buds to break once they are placed in a growth-stimulating environment,
also referred to as days-to-bud-break, and (2) the mitotic activity of the apical bud,
i.e., the mitotic index or mitotic frequency (Owens and Molder 1973, 1976, Carlson

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

et al. 1980, Carlson 1985, Colombo et al. 1989, Fielder and Owens 1989, Cannell et
al. 1990, Kainer et al. 1991, Williams and South 1992). Several authors have found
a difference in the patterns of development of bud dormancy and mitotic activity
(Lavender 1985, Kainer et al. 1991), and some have even concluded that no direct
relationship exists between these two variables in the terminal bud (Cannell et al.
1990, Williams and South 1992).
The objectives of this study were to compare yellow birch, sugar maple and red
oak with respect to (i) frost tolerance of shoots and roots from late summer to spring,
(ii) timing of bud set, and (iii) changes in mitotic frequency and days-to-bud-break

that occur following apical bud formation.
Materials and methods
Seedling production
Yellow birch seeds (YB) (provenance: 46°30′ N, 78°50′ W) were sown on March 26,
1992, in Styroblock 20 containers (45 cavities per container, 340 cm3 per cavity;
Beaver Plastic Ltd., Edmonton, Alberta). Sugar maple (SM) (provenance: 46°08′ N,
70°38′ W) and red oak seeds (RO) (provenance: 45 °38′ N, 73 °15′ W) were sown in
Ventblock 28 containers (28 cavities per container, 340 cm3 per cavity; Beaver Plastic
Ltd.). The substrates were a 2/1 (v/v) mix of peat moss/vermiculite for yellow birch
and red oak, and a 4/1/1 (v/v) mix of peat moss/sand/vermiculite for sugar maple.
Seedlings were grown according to the cultural conditions used at the provincial
nursery in Berthier, Quebec (46°06′ N, 71°34′ W), in plastic-covered greenhouses
with irrigation and fertilization provided automatically. The plastic covers were
removed from the greenhouses in mid-July. On August 4, 1992, the seedlings were
transferred to the Laurentian Forestry Centre, Sainte-Foy, Quebec, repotted in Styroblock® 20 containers and kept outdoors. Seedlings were irrigated when necessary
and fertilized individually with soluble commercial preparations every second week,
following the schedule of the provincial nursery. Fertilization stopped on September
21, at which time it totaled 84.4 mg N, 23.6 mg P and 46.5 mg K per seedling for
yellow birch, 181.8 mg N, 36.2 mg P and 91.2 mg K for red oak, and 180.1 mg N,
33.0 mg P and 91.4 mg K for sugar maple. Morphological characteristics measured

at the end of August are presented in Table 1. During the course of the experiment at
the Laurentian Forestry Centre, substrate temperatures (in the center and at a depth
Table 1. Average (± standard error) shoot height and root collar diameter as measured at the beginning
of the experiment (August 26) by species (n = 60).
Species

Shoot height (cm)

Diameter at root collar (mm)

Yellow birch
Red oak
Sugar maple

55.2 ± 10.1
35.9 ± 4.3
47.8 ± 16.4

4.88 ± 0.71
4.46 ± 0.85

4.06 ± 0.70

FROST TOLERANCE AND BUD DORMANCY OF HARDWOOD SEEDLINGS

1315

of one-third of the cavity) and air temperatures (30 cm above the containers) were
recorded on an hourly basis with copper constantan thermocouples connected to a
data logger (model CR10, Campbell Scientific Inc., Logan, Utah).
Freezing tests and viability tests
In winter, the seedlings were thawed in a cold room at 4 °C until the substrate was
free of ice. The root systems of whole seedlings were gently washed under tap water
to remove the substrate. Once the roots had been washed, the seedlings were
individually put in black plastic bags and placed in modified programmable freezers.
Controls were placed in a cold room at 4 °C. Freezer temperature was maintained at
2 °C for about 6 h, and then decreased at a rate of 3 °C h −1, with a 1-h plateau
following each 3 °C drop in temperature. Temperatures were recorded in the bags
with copper constantan thermocouples connected to a data logger (model 21X,
Campbell Scientific Inc., Logan, Utah). The real temperatures obtained for each step
of the test, i.e., each drop--plateau sequence, were calculated as the mean of the

20 coldest minutes recorded, and thus differed from test to test.
Seedlings were sampled at the end of each step and immediately placed in a cold
room at 4 °C for 14 days. Freezing damage to roots was then evaluated. Excised roots,
cut just above the primary lateral roots, were examined under a stereo microscope,
and dead roots were removed with dissecting forceps and a razor blade. Primary dead
roots were identified by the brown color of the cambial zone; fine dead roots
exhibited a translucent and mushy cortex. Remaining roots were dried in a forced-air
oven at 75 °C for 24 h and then weighed. The basal end of each shoot was placed in
a 22 × 175 mm test tube filled with water and kept in a climate chamber at 24.4 ± 0.7
°C in a 16-h photoperiod with a photon flux density of 360 µmol m −2 s −1 (photosynthetically active radiation, PAR, 400--700 nm) for 6 days. Brown discolored phloem
and cambium were observed by cutting the shoots lengthwise with a razor blade. The
percentage of damage was determined by dividing the length of brown phloem and
cambium by the total shoot length.
Bud set
Apical bud formation was monitored during the fall. Buds were considered to be
formed when scales were entirely closed. Bud formation was considered complete
when about 80% of the seedlings had set terminal buds. Measurements of days-tobud-break (DBB) and mitotic frequency were begun on October 19.
Days-to-bud-break
On each sampling date, whole seedlings with their substrate were placed in styrofoam blocks in a climate chamber at 24.4 ± 0.7 °C in a 16-h photoperiod with a
photon flux density of 360 µmol m −2 s −1 (PAR). Seedlings were watered every

second day by soaking, but no fertilizer was added. Apical buds were checked for
their degree of flushing every day, until the green color of new leaves became visible.
Days-to-bud-break was determined when 100% of the seedlings had flushing buds.

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

Mitotic frequency
Excised apical buds were put in McClintock’s fixative (ethanol/acetic acid, 3/1, v/v)
(Johansen 1940) for at least 7 days. Longitudinal sections were then cut with a razor
blade while the bud was held in position in an elder core bit. Sections were placed in
Warmke’s solution (95% ethanol/5% HCl, v/v) (Johansen 1940) for 20 min to
remove pectins from the middle lamella, and then in Carnoy’s solution (ethanol/acetic acid/chloroform, 6/1/3, v/v) (Johansen 1940) for another 20 min to halt cell wall
softening. Sections were stained with 0.5% orcein in 45% acetic acid (w/v) for
20 min. Bud scales were then removed and sections were placed on a covered slide
and slightly warmed over a flame for about 3 min. Sections were observed under a
microscope at 400 × magnification. The mitotic frequency was the number of
dividing cells, as identified by the presence of chromosomes in prophase, metaphase,
anaphase and telophase, per unit of surface, i.e., per field of observation in the

microscope. Observations were made on three to seven sections. For sectioned
samples, mitotic frequency gives results that are similar to those obtained with
mitotic index (Owens and Molder 1973). However, unlike mitotic index, mitotic
frequency does not require a complete cell count and, for this logistical reason, it was
chosen as the measure of mitosis in this study.
Experimental design and statistical analyses
The experimental design was a split plot with 14 Styroblocks (whole plots) completely randomized within each of the four blocks. Each Styroblock corresponded to
one sampling date and contained the three species as subplots. Thus the experiment
consisted of a total of 2520 seedlings, i.e., 4 blocks × 13 sampling dates × 3 species
× 15 seedlings.
One seedling was sampled per block, date and species, for each variable except bud
set, for which all 15 seedlings were observed. There were 13 sampling dates, but
bud-related variables were observed only before or after bud set. The sampling dates
were August 26, September 17, October 5, October 19, November 2, November 16
and December 7 in 1992, and January 11, February 1, March 1, March 29, April 26
and May 10 in 1993.
The variances of mitotic frequency, bud set and DBB were analyzed according to
the experimental design. For freezing tolerance, data were analyzed with a more
general linear model where temperature and powers thereof were introduced into the
model as initial covariates rather than as factors. The model also involved interaction

between these covariates and species or sampling dates. Such interactions were later
removed from the model when the test that their coefficient was zero proved to be
nonsignificant. The random part of the models was reduced following a procedure
proposed by Milliken and Johnson (1984), and modified by Bernier-Cardou and
Genest (1992). Variance stabilizing transformations were needed. For DBB, the best
transformation found with the Box-Cox method (Fernandez 1992) was cubic root.
For stem damage and mitotic frequency, we used a logarithmic transformation, and
for bud set, a logistic transformation (Cox 1970).

FROST TOLERANCE AND BUD DORMANCY OF HARDWOOD SEEDLINGS

1317

Results
Freezing tolerance
Red oak, and to a lesser extent sugar maple, were damaged by severe frosts at the end
of December, because the seedlings, and particularly the root plugs, were not
protected by snow cover (Figure 1). Consequently, subsequent data for freezing
tolerance had to be discarded for these two species.
Shoot damage increased with decreasing temperature in a way that depended on

sampling date (P ≤ 0.0001 for both interactions between linear and quadratic
temperature effects and sampling date), but not on species (P > 0.05 for all interactions involving temperature and species, Table 2, Figure 2). In all three species, the
highest shoot frost tolerance was reached on or after November 2 (Figure 2) when
damage at − 33 °C was at most 2% for yellow birch and sugar maple, and between
25 and 60% for red oak. Yellow birch stems remained deeply hardened until March
29, but had started to deharden by April 26 (Figure 2).
Live root dry mass decreased with decreasing temperature, and the shape of the
relationship varied with both date and species (most interactions involving the linear
or the quadratic component of the temperature effect were significant at the 5% level,
Table 2, Figure 3). A 2.4- to 3.8-fold increase in root mass, depending on the species,

Figure 1. Minimum and maximum air temperatures recorded at a height of 30 cm above the containers,
and minimum and maximum soil temperatures recorded at a depth of one-third of the root plug during
the course of the experiment. Snow covered the containers when the snowpack exceeded 17 cm. Missing
data, indicated by the flat line at 0 °C, are due to mechanical problems with the data logger.

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

Table 2. Observed significance levels (P > F) associated with the analysis of covariance of freezing
tolerance.
Source of variation

Block (B)
Date (D)
B×D
Species (S)
D×S
Temperature (T)
T qua
T×D
T qua × D
T×S
T qua × S
T×D×S
T qua × D × S
1

Stem damage

Live root dry mass

df

P>F

df

P>F

3
12

0.0108
0.3535
--1
0.3171
0.8499
0.0001
0.0001
0.0001
0.0001
0.0696
0.1541
0.0622
0.0635

3
12
35
2
11
1
1
12
12
2
2
11
11

0.4878
0.3363
0.0046
0.0001
0.9154
0.0001
0.0001
0.0253
0.3711
0.0001
0.0020
0.0132
0.0408

2
12
1
1
12
12
2
2
12
12

A dash appears where a sum of squares was pooled to obtain a new error term.

Figure 2. Percentage of stem damage after exposure to different freezing temperatures for yellow birch
seedlings during hardening and dehardening, and red oak and sugar maple seedlings during hardening.

was observed from August 26 to November 2, indicating that roots continued to grow
in the fall as long as the temperature of the growing medium was above 0 °C. Root

FROST TOLERANCE AND BUD DORMANCY OF HARDWOOD SEEDLINGS

1319

Figure 3. Dry mass of remaining live roots following exposure to different freezing temperatures for
yellow birch seedlings during hardening and dehardening, and red oak and sugar maple seedlings during
hardening.

growth was visible as an increase in root volume and was probably associated with
an accumulation of carbohydrates. Afterward, seedling root mass tended to decrease
as freezing temperatures caused root mortality. Root hardening of the three species
began between October 5 and November 2, when minimum temperatures became
negative (Figure 3). Red oak roots reached a maximum tolerance at their last test on
December 7, with a lethal temperature of −23 °C, whereas on the same date, some
sugar maple root tissue remained alive at temperatures as low as −33 °C (Figure 3).
At least one third of the root tissue remained alive in yellow birch at the minimum
test temperature (about − 33 °C) between December 7 and February 1. Yellow birch
roots dehardened slowly from March 1 onward (Figure 3).
In all three species, shoots hardened more rapidly than roots. Hardening of the
aerial parts started on October 5 and reached a maximum on November 2, whereas
the root systems hardened on and after November 2, reaching their maximum on
December 7. Shoots also became more frost tolerant than roots, with no stem damage
at the lowest temperature tested (− 33 °C) for yellow birch and sugar maple, whereas
roots still suffered some damage at this temperature. For red oak, stem damage varied
between 25 and 60% at − 33 °C from November 2 until December 7, whereas all roots
were killed between −18 and − 23 °C at the same dates.

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

Bud set
Bud set varied with time according to species (P ≤ 0.0007 for the date × species
interaction, Table 3, Figure 4), but never reached 100%. In yellow birch, bud set
reached a maximum of around 90% on November 2, and sugar maple reached 98%
on November 16. Red oak buds were 95% formed when a warm period in October
caused another growth flush. Afterward, most of these seedlings did not set bud
again, probably because of a drop in temperature.
Days-to-bud-break
The time required for bud burst progressively decreased until the end of April, when
buds broke naturally outdoors (P ≤ 0.0001 for the main effect of date, Table 3,
Figure 5). The average time to bud break differed significantly among species (P ≤
0.0001 for the main effect of species, Table 3, Figure 5), and the difference between
any two species was constant over sampling dates (P = 0.1544 for the date × species
interaction, Table 3, Figure 5).
Table 3. Observed significance levels (P > F) associated with the analysis of variance of bud set,
days-to-bud-break and mitotic frequency.
Source of variation

Block (B)
Date (D)
B×D
Species
D×S
B×D×S
1

Bud set
df

P>F

3
4

0.6492
0.0001
--1
0.0003
0.0007
--

2
8

Days-to-bud-break

Mitotic frequency

df

P>F

df

P>F

0.1715
0.0001
-0.0001
0.1544
--

3
8
23
2
16
39

0.8358
0.0001
0.0483
0.0001
0.1032
0.0001

3
8
2
16

A dash appears where a sum of squares was pooled to obtain a new error term.

Figure 4. Percentage of apical bud set of red oak, sugar maple and yellow birch seedlings during the fall.
Each symbol is the mean of 60 seedlings. Standard errors on the logistic scale were 0.5354 at the most.

FROST TOLERANCE AND BUD DORMANCY OF HARDWOOD SEEDLINGS

1321

Figure 5. (A) Days-to-bud-break (DBB) for red oak, sugar maple and yellow birch seedlings in a growth
stimulating environment, and (B) mitotic frequency (number of dividing cells per field of observation in
the microscope) in apical buds of red oak, sugar maple and yellow birch seedlings for different sampling
dates. Each symbol is the mean of four seedlings. Symbols in parentheses are indicative because only
two blocks were included owing to previous frost damage to seedlings in the other two blocks (see
Results). Standard errors on the cubic root scale (DBB) and the logarithmic scale (mitotic frequency)
were at the most 0.3230 and 0.2553, respectively.

Mitotic frequency
Mitotic frequency varied over time (P ≤ 0.0001 for the main effect of sampling date,
Table 3, Figure 5) and among species (P ≤ 0.0001), but the three species exhibited
similar developmental patterns (P = 0.1032 for the date × species interaction). The
general trend was a decrease until January 11, with a minimum of zero dividing cells
for yellow birch, and about four for sugar maple and red oak. After March 1, mitotic
frequency began to increase until it could no longer be measured because buds had
broken. It should be noted that for red oak, values for February 1 and May 10 may
have been underestimated because buds in only two of the four blocks could be
observed; the others had been damaged by frost previously. Because it is unlikely that
cell mitotic activity continued once maximum temperatures had fallen below the
freezing point, or that it resumed during the short time spans when temperatures were
positive between January 11 and March 1 (Figure 1), results for all species are
discussed assuming that mitotic frequency was constant for the January 11, February 1 and March 1 tests.

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

Discussion
Freezing tolerance, as evaluated by artificial freezing tests, differed greatly between
red oak and the two other species. Natural freezing damage confirmed this difference.
Only a few (about 7%) red oak seedlings survived the winter outdoors. On the other
hand, artificial freezing tests do not reflect all aspects of freezing tolerance. Thus,
sugar maple and yellow birch, which appeared to be similar when tested in the
freezer, did not suffer the same damage outdoors, showing about 50 and 100%
survival, respectively, in the spring (data not shown). Because each artificial freezing
test temperature was maintained for only 1 h and estimated as the 20 coldest minutes
recorded, whereas outdoor frost can last many hours, freezing tolerance could be
overestimated (Lindström 1986a). Some researchers have found that prolonging the
exposure time to freezing conditions does increase damage (Levitt 1980, Zhurova
and Patlai 1981 in Lindström 1986a), but others have detected no effect of freezing
duration on tissue damage for up to 24 h (Levitt 1956, Lindström 1986b).
For both stems and roots, red oak was the least frost tolerant, followed by sugar
maple and yellow birch. Another experiment on red oak and yellow birch seedlings
of the same age and nursery of origin as the seedlings used in our experiment,
conducted at the same location using the same freezing test method, showed similar
results: stem frost tolerance (LT50) increased from −15 °C to − 30 °C for yellow birch
and from −15 °C to − 20 °C for red oak between October 18 and November 1
(H. Margolis, unpublished data).
Stems of yellow birch were hardy until March 29, but had started to deharden by
April 26. In contrast, roots began to deharden slowly as early as March 1, at which
time the minimum temperature they were able to survive was about −31 °C. Earlier
in February, the temperature in the containers had risen to a minimum of about − 2 °C
and a maximum of about − 0.75 °C. Roots may have been sensitive to this increase,
because the temperature of the substrate and frost tolerance paralleled each other for
about two months.
Maximum rest (i.e., when DBB is at its maximum) indicates that a chilling
requirement is needed for bud dormancy release. This occurred at least 49 to 67 days
before the minimum bud mitotic activity. The difference in timing between maximum rest and minimal apical cell division varies with species, provenance and year
(Carlson 1985, Cannell et al. 1990, Kainer et al. 1991, Williams and South 1992). On
the other hand, the onset of bud dormancy appeared to correspond to decreasing bud
cell activity (Figure 5). For red oak and sugar maple, mitotic frequency started to
decrease before maximum rest had occurred, although the dates were different for
each species. A similar pattern was observed in studies that measured both mitotic
index and DBB of slash pine (Pinus elliottii Engelm.) and loblolly pine (Kainer et al.
1991, Williams and South 1992).
Red oak reached maximal rest later than the other species; this may be related to
the late flush the seedlings exhibited in mid-October, which may have delayed
dormancy by maintaining a high cell activity late in the season. The reason why only
red oak resumed growth is not clear, but indicates that buds of red oak were quiescent

FROST TOLERANCE AND BUD DORMANCY OF HARDWOOD SEEDLINGS

1323

at that time, but not dormant. It is possible that red oak was more sensitive to the
warm temperatures recorded between October 5 and October 19, or to the heavy
precipitation around October 9, or both. Species that show a cyclic growth pattern
during the juvenile stage, such as many oaks, usually flush in response to favorable
environmental conditions (Dupré et al. 1985).
According to Cannell et al. (1990), mitotic activity is strongly dependent on
temperature and 5 °C represents the lower limit. In our study, cell activity virtually
ceased for all species between January 11 and March 1, when maximum air temperature was below 0 °C. Because cell mitotic activity roughly parallels temperature
(Carlson 1985, Williams and South 1992), it is surprising that both red oak and sugar
maple exhibited a mitotic frequency of four during the winter. However, the finding
that mitotic frequency was not zero may not necessarily mean cell activity was
maintained, because freezing temperatures could have stopped mitosis at the stages
the cells had reached at that time.
To our knowledge, no published data on mitotic activity of temperate hardwood
seedlings exist. Carlson (1985) and Williams and South (1992) found a stem apex
mitotic activity index slightly below 5 and 1%, respectively, for loblolly pine
seedlings, and Kainer et al. (1991) obtained a minimal mitotic index of about 1.8%
for slash pine seedlings. However, all of these studies were conducted in regions
where winters are relatively mild. Nevertheless, these results are contrary to other
reports on evergreen species where the mitotic index became nil in wintertime
(Owens and Molder 1973, Carlson et al. 1980, Cannell et al. 1990, Williams and
South 1992).
We observed that mitotic activity resumed in all three species in early March when
temperatures increased above the freezing point. Mitotic activity increased more
slowly in yellow birch than in sugar maple and red oak, although yellow birch had a
lower DBB requirement and bud break occurred earlier in May than in sugar maple
and red oak. It is doubtful, therefore, that a direct relationship exists between bud
dormancy and spring mitotic activity. This observation is supported by work on Sitka
spruce (Picea sitchensis (Bong.) Carr.), Douglas-fir (Cannell et al. 1990) and loblolly
pine (Williams and South 1992). A possible explanation proposed by Fielder and
Owens (1989) is that, because cell division in the rib meristem and pith precedes cell
elongation, it cannot be related to bud flushing. Moreover, mitotic frequency in the
overall bud could show a different pattern than mitotic frequency in the rib meristem
and subtending pith.
We conclude that overwintering outdoors results in damage to species that are not
well acclimated, especially because of root sensitivity to frost. Adequate winter
protection may improve survival. During the 1992 season of this study, snow arrived
late following severe frosts, and the snowpack was thin during the entire winter of
1992--1993.
Yellow birch was the most frost-tolerant species, followed by sugar maple and red
oak. Mitotic activity in the apical bud was more closely related to air temperature
than to bud dormancy as defined by the number of days to bud break (DBB). For the

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CALMÉ, BIGRAS, MARGOLIS AND HÉBERT

three species, stem hardening was observed before maximum DBB occurred. Dormancy release (DBB = 0) coincided with the loss of stem hardening in the spring for
yellow birch. Roots hardened more slowly and had less frost tolerance than stems in
fall and winter, but roots dehardened earlier than stems, perhaps in response to a rise
in minimum temperatures in the containers.
Acknowledgments
We thank Annie Grimard for the determination of mitotic frequency and Yves Dubuc for his technical
assistance throughout the project. We thank Michèle Bernier-Cardou for reviewing the first draft of the
manuscript and three anonymous reviewers. We are also grateful to the staff at the provincial nursery of
Berthier for supplying the seedlings. This research was funded by the ministère des Ressources naturelles
du Québec, the Canadian Forest Service--Quebec Region and the Natural Sciences and Engineering
Research Council of Canada.
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