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ORIGINAL PAPER
Short-day photoperiods affect expression of genes related
to dormancy and freezing tolerance in Norway spruce seedlings
Elisabeth Wallin
1,2
&Daniel Gräns
1,2
&Douglass F. Jacobs
3
&Anders Lindström
1,2
&
Nathalie Verhoef
4
Received: 20 February 2017 /Accepted: 4 July 2017
#The Author(s) 2017. This article is an open access publication
Abstract
&Key Message Gene expression analysis showed that
prolonged short day (SD) treatment deepened dormancy
and stimulated development of freezing tolerance of Picea
abies seedlings. Prolonged SD treatment also caused later
appearance of visible buds in autumn, reduced risks for
reflushing, and promoted earlier spring bud break.
&Context Short day (SD) treatment of seedlings is a common
practice in boreal forest tree nurseries to regulate shoot growth
and prepare t he seedlings for autumn p lanting or frozen storage .
&Aims The aim of this study was to examine responses of
Norway spruce (Picea abies (L.) Karst.) to a range of SD treat-
ments of different length and evaluate gene expression related
to dormancy induction and development of freezing tolerance.
&Methods The seedlings were SD treated for 11 h a day
during 7, 14, 21, or 28 days. Molecular tests were performed,
and the expression profiles of dormancy and freezing
tolerance-related genes were analyzed as well as determina-
tion of shoot growth, bud set, bud size, reflushing, dry matter
content, and timing of spring bud break.
&Results The 7-day SD treatment was as effective as longer
SD treatments in terminating apical shoot growth. However,
short (7 days) SD treatment resulted in later activation of
dormancy-related genes and of genes related to freezing toler-
ance compared to the longer treatments which had an impact
on seedling phenology.
&Conclusion Gene expression analysis indicated an effective
stimulus of dormancy-related genes when the SD treatment is
prolonged for at least 1–2 weeks after shoot elongation has
terminated and that seedlings thereafter are exposed to ambi-
ent outdoor climate conditions.
Keywords Picea abies .Molecular tests .Photoperiod .
Shootgrowthtermination .Bud formation .Storability
Handling Editor: Michael Tausz
Contribution of the co-authors Elisabeth Wallin performed all
experimental work, analyzed the data, and co-wrote the manuscript.
Daniel Gräns contributed in data interpretation and co-wrote the manu-
script.
Anders Lindström supervised the work and co-worked in paper writing.
Nathalie Verhoef was responsible for gene activity analyses.
D.F. Jacobs participated in data interpretation and paper writing.
*Elisabeth Wallin
ewa@du.se
Daniel Gräns
dga@du.se
Douglass F. Jacobs
djacobs@purdue.edu
Anders Lindström
ali@du.se
Nathalie Verhoef
nathalie.verhoef@nsure.nl
1
Dalarna University, 791 88 Falun, Sweden
2
School for Forest Management, Swedish University of Agricultural
Sciences, Box 43, 739 21 Skinnskatteberg, Sweden
3
Department of Forestry and Natural Resources, Purdue University,
West Lafayette, IN 47907-2061, USA
4
NSure, Binnenhaven 5, 6700 AA Wageningen, The Netherlands
Annals of Forest Science (2017) 74:59
DOI 10.1007/s13595-017-0655-9
1 Introduction
The quality of nursery seedling stock plays an important role
in determining reforestation success (Mattsson 1997). One
key attribute toward the end of nursery cultivation is dorman-
cy status, which affects seedling stress resistance during lifting
and handling, and freezing storability (Stattin et al. 2000).
Early development of adequate seedling freezing tolerance
may be important to reduce risks during autumn planting,
which may provide an effective means to extend the planting
season yet can be problematic for frost-sensitive species such
as Norway spruce (Picea abies (L.) Karst.) (Christersson
1978;KohmannandJohnsen2007).
The dormancy cycle of many boreal forest tree species is
strongly affected by seasonal changes in photoperiod (Ekberg
et al. 1979)and temperature (Heide1974). Short day (SD) treat-
ment (Rosvall-Åhnebrink 1982; Fløistad and Granhus 2010,
2013) of seedlings has shown to be an effective tool to regulate
height growth while also inducing dormancy. This treatment
involves reducing the photoperiod in late summer using black
curtains that completely cover the seedlings either inside the
greenhouses or outdoors in specially equippedareas. The inten-
sity and duration of SD treatments vary widely across regions
and nurseries, but typically fall within a range of 8–12 h of
photoperiod for 14–35 days (Rosvall-Åhnebrink 1982;
Konttinen et al. 2003;Jacobsetal.2008; Luoranen et al.
2009; MacDonald and Owens 2010; Fløistad and Granhus
2013). SD treatment promotes early growth cessation and in-
duces bud set (Dormling et al. 1968;Heide1974;Ekbergetal.
1979; Colombo et al. 1989,2001; Fløistad and Granhus 2013),
while also increasing seedling freezing tolerance (Dormling
1982; Colomboet al. 1989,2001;Jacobsetal.2008). Thus, this
cultural treatment is now regularly employed in container nurs-
eries across Canada and Scandinavia in production of boreal
conifers (Colombo et al. 2001).
SD treatments vary in their effects in seedling growth and
development depending on cultural conditions, species, and
provenance (Dormling et al. 1968). The dates for start and
termination of SD treatment are important because bud dor-
mancy is promoted if the natural critical night length (i.e.,
night lengthknown to promote bud set of a given provenance)
is reached by the end of treatment (Kohmann and Johnsen
2007) and this prevents reflushing (Fløistad and Granhus
2010,2013). The required natural critical night length varies
by provenance, whereby northern provenances and those from
high altitudes need shorter night lengths to induce dormancy
compared to southern provenances from lower altitudes
(Dormling 1973;Heide1974;Dormling1979;D
ormling
and Lundkvist 1983; Clapham et al. 1998). This suggests that
different provenances will respond variably to different timing
and durations of SD treatments. Fløistad and Granhus (2013)
showed that SD treatment (7, 10, 14, and 17 days) of Norway
spruce (origin 60°N, 10°E) significantly affected height
growth after just 7 days of treatment. Their results among
others also suggest that Norway spruce seedlings require a
longer SD treatment to avoid a second bud flush, especially
if the treatment starts early (Heide 1974; Fløistad and Granhus
2010,2013; Luoranen and Rikala 2015). Furthermore, Olsen
et al. (2014) reported that different day and night temperatures
during SD treatment variably affected bud development.
If SD treatments are too extensive (i.e., duration and/or
intensity), reduced photosynthesis may lead to a decline of
seedling growth and vigor. MacDonald and Owens (2010),
working with Douglas-fir (Pseudotsuga menziesii var.
menziesii (Mirb.) Franco), recommended SD treatments last-
ingfor3weeks(vs4–6 weeks) to avoid reductions in shoot
diameter and root mass. Similarly, Konttinen et al. (2003,
2007) found that prolonging daily light periods from 8 to
12–14 h during SD treatment resulted in better seedling per-
formance for provenances of northern Norway spruce (origin
60°40′N-64°40′N).
Determination of growth cessation and bud status is rela-
tively straightforward, while the estimation of dormancy level
and its relationship to subsequent freezing tolerance is more
complex. To our knowledge, even though SD treatment has
been used for several decades, there is still no practical way to
assess dormancy level and its relationship to the development
of subsequent freezing tolerance during ongoing SD treat-
ment. Enhanced knowledge of how patterns of gene expres-
sion correspond with phenological attributes, such as freezing
tolerance of seedlings, allows the possibility to use molecular
tests to postpone effects of environmental changes. Freeze-
induced electrolyte leakage provides a direct measure of seed-
ling freezing tolerance (Lindström et al. 2014); yet, develop-
ment of freezing tolerance lags behind dormancy development
(Weiser 1970;Fushigami and Nee 1987; Dormling 1993;
Clapham et al. 2001; Greer et al. 2001; Holliday et al.
2008), and so, its application is limited to several weeks after
completion of SD treatment. Previous studies have investigat-
ed gene expression connected to dormancy and freezing tol-
erance induced by SD treatment of species such as Norway
spruce (Asante et al. 2011), Sitka spruce (Holliday et al.
2008), and Douglas-fir (Balk et al. 2008). In 2006, NSure
(Wageningen, The Netherlands) marketed a testing procedure
for Norway spruce (ColdNSure™). This molecular test mea-
sures the activity of a set of genes involved in development of
freezing tolerance (Joosen et al. 2006; Stattin et al. 2012). The
ColdNSure™test, based on the relationship between the out-
come of freezing shoots to −25 °C and the expression of
freezing tolerance-related genes, has been shown to be as ef-
fective as freeze-induced electrolyte leakage in predicting
freezing tolerance and storability of Norway spruce (Stattin
et al. 2012). Furthermore, Stattin et al. (2011) identified
dormancy-related genes that respond upon SD treatment in
Norway spruce. These sets of genes could possibly be used
as a predictive tool to measure dormancy development during
59 Page 2 of 14 Annals of Forest Science (2017) 74:59
SD treatment and help nurseries to decide when it is appropri-
ate to terminate the treatment. The ColdNSure™test could be
used as an indicator of which SD program results in favorable
development of freezing tolerance and storability for a given
species and provenance.
Following planting, seedlings are susceptible to frost dam-
age after bud break (Dormling 1982; Christersson and von
Fircks 1988). The timing of bud break in spring is largely
affected by temperature accumulation (Hannerz 1994;
Sutinen et al. 2012), but other factors, such as photoperiod
and temperature during growth cessation and bud set, are also
important (Sögaard et al. 2007; Fløistad and Granhus 2010).
Konttinen et al. (2003) reported that SD treatment initiated in
the beginning of July with a duration of 3–4 weeks led to
earlier apical bud break in spring and thereby increased expo-
sure to frost damage compared to seedlings subjected to
shorter (2–3 weeks) SD treatment with starting dates in mid-
August. Sögaard et al. (2007) and Olsen et al. (2014)showed
that high temperature during SD treatment resulted in a later
bud break the following spring. It is possible that the activity
level of dormancy-related genes during growth cessation
could effectively forecast the timing of bud burst in spring.
The overall aim of this study was to investigate how the
activity of genes related to dormancy and freezing tolerance
influence the physiological responses of SD-treated seedlings.
Additionally, we sought to determine whether the gene ex-
pression profiles could be used to forecast seedling develop-
ment (i.e., termination of growth, bud set, freezing tolerance,
and timing of bud burst).
Our study hypotheses were that (i) the genes that are acti-
vated by SD treatment for developing dormancy can act as an
indicator for SD treatment termination, (ii) different durations
of SD treatment, and (iii) different conditions obtained indoors
and outdoors after termination of SD treatment will affect
gene- and physiological seedling responses.
2 Materials and methods
2.1 Seedling material and treatments
The experimental material consisted of container-grown 1.5-
year-old seedlings of Norway spruce cultivated at Nässja nurs-
ery (60° 15′N; 16° 50′E), Sweden. The seeds were collected
in the seed orchard Ålbrunna (59° 31′N, alt. 50 m), with mean
origin of the plus trees corresponding to lat. 60° 58′N, alt.
212 m. The seeds were sown on July 13, 2012 in 50-ml con-
tainers at a density of 820 cavities m
−2
(Plantek 121, Finland),
filled with peat (NMP Närkes AB, Sweden) and grown ac-
cording to standard routines at Nässja nursery. A total of 24
container units each consisting of 11 × 11 cavities were trans-
ferred from the Nässja nursery on July 9, 2013 and placed in
the open at the research station in Vassbo (60° 31′N; 15° 31′
E, alt. 130 m). Seedlings were fertilized weekly by adding a
complete mineral nutrient solution (Wallco, Sweden; N:P:K,
100:13:65) dissolved in the irrigation water at a rate of 3 g N
week
−1
m
−2
through the third week of September. On July 15,
2013, all seedlings were transferred indoors to a greenhouse
except for one batch (outdoor control) consisting of four con-
tainer units (replicates) that were placed outside. Another
batch (control indoor), also containing four units (replicates),
was placed indoors and exposed to natural day length. The
remaining 16 units were split in half, resulting in 32 smaller
units (each containing 55 seedlings), which were then ran-
domly placed in the blackout compartment indoors.
Seedlings were subjected to SD treatment of 11 h day and
13 h night, a commonly used blackout treatment for the se-
lected Norway spruce provenance. According to Dormling
and Lundkvist (1983), our Norway spruce provenance would
have a critical night length (when 50% of the population starts
to initiate bud set) of approximately 7 h, whichcorresponds to
a day length of 17 h. Seedlings were SD treated for 7 days (SD
7) after which four units were moved outdoors and four units
were kept indoors, with both environments providing natural
day length. This procedure was repeated after 14 (SD 14), 21
(SD 21), and 28 (SD 28) days (Fig. 1). The natural day length
that the SD 7 seedlings were exposed to after completion of
SD treatmentwas 17 h and 38 min, for SD 14; 17 h and 5 min,
SD 21; 16 h 32 min and SD 28; 15 h 55 min.
2.2 Measurements of height and bud development
In each container unit (replicate), the middle row of 11 seedlings
was marked for measurement of height and bud status.
Measurements of height were performed once a week during a
4-week period (July 15 through August 12, 2013). Thereafter,
measurements were made biweekly until September 23, and a
final measurement was completed on October 9. Different cate-
gories (based on Krutzsch (1973) with some alterations) were
used to classify apical bud set development, reflushing, and bud
break. Apical bud set development was measured at the same
time as height and classified as follows: no bud, initial indication
of bud, small bud, and large bud. Reflushing was monitored for
apicaland lateralbuds onlyon September9 using four categories:
no reflushing, bud needles visible, bud needles <10 mm, and bud
needles ≥10 mm. At the end of October 2013, outdoor-grown
seedlings were transferred into the greenhouse where all seed-
lings were kept until May 2014 (Fig. 2). On January 24, apical
bud size was measured using a caliper and classified into five
categories (1 = ≤1.0 mm, 2 = 1.1–2.0 mm, 3 = 2.1–3.0 mm,
4=3.1–4.0mm,5=4.1–5.0 mm). Apical bud break development
was measured weekly from March 12 to May 2 and classified as
follows: no bud break, needles visible, needles <10 mm, needles
≥10 mm. The previously described bud development categories
were combined into two major classes (1 and 2) to simplify data
analysis. This was done for bud set (no bud + initial indication of
Annals of Forest Science (2017) 74:59 Page 3 of 14 59
bud = S1, small bud + large bud = S2), reflushing (no
reflushing = R1, needles visible + needles <10 mm + needles
≥10 mm = R2), and bud break (no bud break = B1, needles
visible + needles <10 mm + needles ≥10 mm = B2).
2.3 Measurements of gene expression and dry matter
content
The gene expression profiles of the dormancy-related genes dur-
ing the period of SD treatment wasstudiedbytakingsamples
each week starting on July 15 (day 0), July 22 (day 7), July 29
(day 14), August 5 (day 21), and August 12 (day 28) followed by
a final sample on August 26 (day 42) (Fig. 1). Thereafter, samples
were collected at four different occasions (Sep 9, Sep 23, Oct 9,
and Oct 23) to determine the freezing tolerance and storability by
the molecular ColdNSure™test (Stattin et al. 2012). For each
treatment, the top 2 cm of eight randomly selected seedlings were
collected (two seedlings per replicate) and the needles were re-
moved with a sharp blade to uncover the apical bud; alternatively,
in case of early stages of bud development, the upper 3 mm of th e
shoot was used. The apical bud or the tip of the shoot was re-
moved and immediately frozen in liquid nitrogen and then stored
at −80 °C. General samples of the treatments (each containing
eight buds) were sent to the NSure laboratory where the molec-
ular measurements were performed. The bud tissue samples were
ground in liquid nitrogen, total RNA was isolated using the
Aurum Total RNA minikit (Bio-Rad), and thereafter, cDNA
was synthesized using the QScript cDNA supermix (Quanta
Fig. 2 Daily maximum and minimum greenhouse temperatures from
July 15, 2013 to Apr. 10, 2014. Temperature measured 1.6 m above
ground. Daily maximum and minimum outdoor temperatures from
July 15 to Oct. 28, 2013. Temperature measured 1.3 m above ground.
Seedlings grown outdoors were transferred indoors to the same
greenhouse as the other seedlings on Oct. 28 for winter storage at
minimum temperature of +7 °C. Natural day lengths are indicated in the
figure
Day 0 Day 7 Day 14 Day 21 Day 28 Day 42
Control ou
t
SD 7
SD 14
SD 21
SD 28
Control in
SD 7
SD 14
SD 21
SD 28
SD treatment followed by
outdoor growth
SD treatment followed by
indoor growth
July 15
July 22
July 29
Aug 5
Aug 26Aug 12
SD experiment ColdNSure, DMC
Samplin
g
occasions: 9/9 23/9 9/10 23/10
Fig. 1 Experimental design and sampling schedule for SD (short day)
experiment (11 h light; 13 h night). Untreated control seedlings (green)
were kept under natural light conditions indoors and outdoors during the
experiment. For the remaining seedlings, SD treatment started on July 15,
2013 in greenhouse for all SD treatments. Seedlings were divided into
four batches and subjected to four different SD treatment periods: 7
(orange), 14 (purple), 21 (blue),and28(red) days. Following
treatment, half of each batch was moved outdoors and the other
remained indoors, all exposed to natural day length. Samples for
molecular test of dormancy induction were taken each week during the
SD treatment period followed by a final sample on Aug. 26 (indicated by
open circles). Samples for freezing tolerance (ColdNSure™)were
collected at four different occasions: Sept. 9, Sept. 23, Oct. 9, and
Oct. 23 (indicated by solid circles). Samples for determination of DMC
were taken only on Oct. 23
59 Page 4 of 14 Annals of Forest Science (2017) 74:59
Biosciences). Gene expression was measured using PerfeCTa
SYBR Green Supermix (QuantoBio) and the CFX96 Real-
Time PCR detection system (Bio-Rad) under the following con-
ditions: denaturation at 95 °C for 3 min followed by 40 cycles of
amplification (95 °C for 10 s and 60 °C for 30 s). The level of gene
expression was given as delta delta threshold cycle (ddCt), which
is the relative difference between the gene of interest and internal
reference genes (i.e., genes that did not show any significant
difference in expression). The dormancy-related genes for
Norway spruce were previously identified by Stattin et al.
(2011) by using Single-Read Next Generation Illumina
Sequencing performed by ServiceXS (The Netherlands) on
SD-treated seedlings from the provenance Runesten. As a refer-
ence, a de novo assembly was used as described by Stattin et al.
(2012). Quantification of a gene was performed by counting the
number of reads per gene of each sample. For differential analy-
ses between the samples, DESeq (Anders and Huber 2010)was
used. For this project, four potential SD indicators (LN2,LN3,
LN4,andLN6) were selected, and specific primers weredesigned
using Primer3 (http://frodo.wi.mit.edu/primer3/).
Freezing tolerance was determined by using the freezing
tolerance-related genes according to the commercial molecular
test(ColdNSure™) usedby Swedish forest tree nurseries (Joosen
et al. 2006;Balketal.2007). The genes measured were CO1,
CO4,CO8,CO9,andCO10. The corresponding freezing toler-
ance status was calculated by NSure using a model for Norway
spruce similar to the model presented for Douglas-fir (Balk et al.
2008) and expressed as four different phases: ColdNSure™
phase 0 = cold sensitive and the indicator profiles match the
profiles of lots that are actively growing; no sign of freezing
tolerance can be recognized. Phase 1 = developing freezing tol-
erance; early signs of freezing tolerance development can be
recognized. Phase 2 = developing freezing tolerance; ap-
proaching full freezing tolerance. Phase 3 = freezing tolerant;
the indicator profiles match the profiles of lots that have ceased
growth and are fully tolerant, ready for lifting and storage.
On October 23, in addition to the ColdNSure™test, we
used a method described by Rosvall-Åhnebrink (1985)tode-
termine seedling storability by assessing dry matter content
(DMC) in the upper 2 cm of five shoots per replicate. As
reported by Rosvall-Åhnebrink (1985), the DMC should be
between 35 and 38% for 1-year-old spruce seedlings to be
classified as storable. The upper target level should be applied
for seedlings that are SD treated.
2.4 Environmental measurements
Temperatures during the experimental period were measured
using calibrated sensors attached to a data logger (Campbell
Scientific CR1000, UK) measuring every 5 min and producing
15-min averages. Temperature indoors was measured 1.6 m
above ground by a sensor placed in a ventilated radiation shield.
The outdoor sensor was placed 1.3 m above ground in a
ventilated radiation shield. Daily indoor and outdoor values are
presented in Fig. 2. During July, August, and the first days of
September 2013, the heating system was turned off. When
heating was turned on, the control system for regulation of tem-
perature in the greenhouse was not functioning satisfactorily dur-
ing a 3-week period in September. As a result, the minimum
temperatures indoors were above +20 °C (Sep 3–Sep 25). From
the end of September, to avoid freezing, the heating system of the
greenhouse was activated at temperatures below +7 °C.
2.5 Statistical analysis
Analysis of variance (ANOVA) was performed if the data
fulfilled the requirements of normal distribution and constant
variance. A general linear mixed hierarchical model with a
nested design was used (PROC MIXED) (SAS Institute Inc.
2002–2008) for shoot growth data obtained during the first
and the second week after starting the SD treatment:
Yijk ¼μþTiþRij þeijk ð1Þ
where Y
ijk
is the dependent variable (the shoot growth observa-
tion of the kth seedling of the jth replication in ith treatment) for
the first and second week, respectively, after starting the SD treat-
ment, μis the overall mean for shoot growth, T
i
is the fixed effect
of the ith treatment (i=1,2…,10) where the treatments used are
the 5 SD treatments for indoor and outdoor growth, respectively,
R
ij
is the random effect of the jth unit of replication (the jth con-
tainer unit, j = 1, 2…,4) within treatment i; from each replication,
11 seedlings were measured, ande
ijk
is the random error ~NID (0,
σ
2e
). Where ANOVA indicated significant treatment differences
(p< 0.05), Tukey’s studentized range test was used for pairwise
comparisons of treatment means (α= 0.05). This model was used
for data collected during the first and second week, respectively.
For the third and fourth weeks, only standard error of the treat-
ment means was calculated (Fig. 3), since the data was strongly
skewed and contained many observations of no growth.
Bud size was analyzed separately for indoor and outdoor
conditions using a general linear model (PROC GLM) (SAS
Institute Inc. 2002–2008).
Yij ¼μþSDiþeij ð2Þ
where Y
ij
is the dependent variable (the observation of the jth
seedling of the ith treatment level), μis the overall mean, SD
i
is the fixed effect of the ith SD treatment (i=1,2…,5) for
seedlings grown indoors and outdoors, respectively, and e
ij
is
the random error ~NID (0, σ
2e
). Where ANOVA indicated
significant treatment differences (p< 0.05), Tukey’s
studentized range test was used for pairwise comparisons of
treatment means (α=0.05).
DMC values were evaluated using a ttest (PROC TTEST)
(p< 0.05) (SAS Institute Inc. 2002–2008). Differences in bud
Annals of Forest Science (2017) 74:59 Page 5 of 14 59
set and bud break were analyzed by using the Chi-square test
(p< 0.05) in Microsoft Excel (2013).
3Results
3.1 Shoot growth
As expected, there were no significant differences in shoot elon-
gation between SD-treated seedlings during the first 7 days in the
blackout compartment. Seedlings moved from the blackout
compartment after 7, 14, 21, and 28 days to natural day length
conditions for growth either indoors or outdoors showed similar
elongation of shoots. For SD-treated seedlings, shoot growth
during the third and fourth week had almost declined to zero
(Fig. 3). Shoot growth of control seedlings was larger than for
SD-treated seedlings with the exception of the first 7 days. After
August 12 (4 weeks after SD treatment started), biweekly mea-
surements showed that most of the outdoor-grown control seed-
lings continued growing until mid-September (data not shown).
3.2 Bud set and bud size
During the first measurements (July 22, July 29, Aug 5, Aug 12),
no significant differences in bud set were found among treat-
ments (SD 7, 14, 21, 28) (data not shown). By August 26, almost
all seedlings (indoor and outdoor) that had received the shortest
SD treatment (SD 7) had set a visual bud (category S2) and the
frequency of bud set was significantly higher than for control
seedlings (Table 1). Also, on August 26, seedlings subjected to
the SD 7 treatment had a significantly higher frequency of bud set
compared to SD 14, 21, and 28 treated seedlings (Table 1). These
differences between SD-treated seedlings persisted until the mid-
dle of September, but seedlings in all treatments showed visible
apical buds toward the end of autumn. In January 2014, bud size
from the shorter SD treatments (SD 7 and SD 14) and the controls
remainedslightly largercompared with seedlings subjected to the
longer SD treatments (SD 21 and SD 28) (Fig. 4). There was a
weak negative correlation between length of SD treatment and
bud size. The trends were similar for both indoor (R
2
=0.17)and
outdoor conditions (R
2
=0.13).
3.3 Reflushing
On September 9, none of the seedlings subjected to the longer (14,
21, or 28 days) SD treatments showed any sign of reflushing,
regardless of the growth environment. The controls and SD 7 treat-
ed seedlings grown indoors showed reflushing among 2.3% of
apical buds and 2.3% of lateral buds (one seedling out of 44 of each
category). The corresponding values for apical and lateral buds for
the outdoor-grown seedlings were 11.4 and 0% for the controls and
0 and 9.1% for the SD 7 treated seedlings, respectively.
3.4 Molecular dormancy indicators
The three genes LN2,LN3,andLN4 (Fig. 5) used as indicators of
dormancy as well as LN6 (data not shown) all showed that longer
durations of SD treatment, i.e., SD 21 and SD 28, increased the
level of gene expression compared to the shorter treatments SD 7
and SD 14. The highest levels were obtained for the seedlings
grown outdoors following SD treatment (Fig. 5) indicating a
deeper dormancy. When the shorter SD treatments (SD 7 and
SD 14) were followed by natural day length, gene expression
transiently stabilized or decreased. The gene expression levels
Fig. 3 Mean weekly apical shoot elongation of seedlings kept indoors
and seedlings transferred outdoors after termination of SD (short day)
treatment, which was done in an indoor blackout compartment. SD
treatments (7, 14, 21, and 28 days) with a photoperiod of 11 h started
on July 15 (day 0). Seedlings were then measured on July 22 (day 7),
July 29 (day 14), Aug. 5 (day 21), and Aug. 12 (day 28). Control
seedlings were kept under natural day lengths, both indoors and
outdoors. Total number of seedlings in each treatment =44, n=4(each
replicate contains 11 seedlings). Data were analyzed independently for
the first and second week after starting the SD treatment and different
letters therein indicate significant differences among treatments at
p≤0.05 according to Tukey’stest.Vertical lines represent the standard
error. For the third and fourth week, ANOVA could not be performed due
to skewed distribution; only standard error of treatment means was
calculated
59 Page 6 of 14 Annals of Forest Science (2017) 74:59
measured at day 42 were similar for SD 21 and SD 28. At this final
sampling date, all selected dormancy-related genes showed higher
levels of gene expression in all SD treatments compared to the
untreated control.
3.5 Freezing tolerance determined by gene expression
According to the molecular ColdNSure™, test seedlings need
to reach phase 3 to be classified as freezing tolerant and stor-
able (Fig. 6). Seedlings left indoors only reached freezing
tolerance phase 1 except for treatment SD 28, which reached
phase 2 on October 23, the last date of sampling. All SD-
treated seedlings subjected to outdoor conditions had reached
freezing tolerance phase 1 on September 9, the first date of
sampling, while the control seedlings were still in phase 0
(Fig. 6). At the second date of sampling, September 23, all
SD-treated seedlings outdoors, except SD 7, reached freezing
tolerance phase 2. At the final two sampling occasions,
October 9 and October 23, all SD-treated seedlings outdoors
had reached phase 3 and were therefore classified as storable.
The outdoor control seedlings, however, did not reach phase 3
of the ColdNSure™test until the final sampling date.
Gene CO1,usedintheColdNSure™test for estimating freez-
ing tolerance, showed a different pattern of gene expression during
the autumn between seedlings grown indoors and outdoors (Fig.
7). A clear trend was observed outdoors indicating that longer SD
treatments resulted in higher levels of gene expression suggesting
improved freezing tolerance (Fig. 6). Gene expression was
Tabl e 1 The effect of different
SD treatments on bud set in
indoor and outdoor conditions
measured on August 26
Pairwise comparison
of treatment
Indoors Chi-square
test pvalue
Outdoors Chi-square
test pvalue
Bud No bud Bud No bud
Control 440 <0.001 539 <0.001
SD 7 41 3 40 4
Control 440 <0.001 539 0.156
SD 14 32 12 10 34
Control 4 40 0.080 539 0.006
SD 21 10 34 16 28
Control 4 40 0.398 5 39 0.725
SD 28 2 42 4 40
SD 7 41 3 0.011 40 4 <0.001
SD 14 32 12 10 34
SD 7 41 3 <0.001 40 4 <0.001
SD 21 10 34 16 28
SD 7 41 3 <0.001 40 4 <0.001
SD 28 242 440
Bud set had occurred if a small or large visible bud was detected (category S2, see “Materials and methods”).
Pairwise comparisons were performed using a χ
2
-test. Significant differences at the ≤0.05 level are marked in
italics. N=44seedlings
Fig. 4 Average apical bud size resulting from various lengths of SD
(short day) treatment (11 h light; 13 h night) for seedlings grown
indoors and outdoors. Measurements were performed on January 24,
2014 and classified into five different categories (1 = ≤1.0 mm,
2 = 1.1–2.0 mm, 3 = 2.1–3.0 mm, 4 = 3.1–4.0 mm, 5 = 4.1–5.0 mm).
Total number of seedlings in each treatment =44, n=4(eachreplicate
contains 11 seedlings). Data were analyzed independently for the indoor-
and outdoor-growthenvironments and different letters indicate significant
differences among treatments at p≤0.05, according to Tukey’s test.
Vertical lines represent the standard error
Annals of Forest Science (2017) 74:59 Page 7 of 14 59
generally very low among seedlings grown indoors at all sampling
dates. Similar trends were observed in data from other freezing
tolerance-related genes (CO4,CO8,CO9,CO10)usedinthe
ColdNSure™test (data not shown).
3.6 Dry matter content
On October 23, indoor-grown seedlings showed generally low
DMC values whereas seedlings grown outdoors displayed
considerably higher DMC values (Fig. 8). All seedlings grown
outdoors showed significantly (p< 0.001) higher DMC values
than the target value for storability for untreated seedlings
(DMC 35%). Only outdoor-grown SD-treated seedlings
reached the target value for SD-treated seedlings (DMC
38%). None of the treatments that were grown indoors
reached the target level for storability.
3.7 Bud break forthcoming spring
Bud break occurred earlier for a larger proportion of seedlings
grown outdoors compared to indoor-grown seedlings (Fig. 9).
A varying number of seedlings in all treatments except SD 7
indoors had initiated bud break on April 3. A specific analysis
of data from this date showed that a larger proportion of seed-
lings subjected to the longer (SD 21 and SD 28) treatments
broke bud earlier compared to shorter (SD 7 and SD 14) treat-
ments (p< 0.001 in both environments). A significantly
(p< 0.001) larger proportion of control seedlings from the
a) b)
c) d)
e) f)
Fig. 5 Gene expression profiles
of LN2 (a,b), LN3 (c,d), and LN4
(e,f) used for indicating
dormancy induction. The level of
gene expression is given as delta
delta threshold cycle (ddCt),
which is the relative difference
between the gene of interest and
reference genes (genes that did
not show any significant
difference in expression). SD
(short day) treatments (7, 14, 21,
and 28 days) with a photoperiod
of 11 h started on July 15.
Measurements were performed
on July 15 (day 0), July 22 (day
7),July29(day14),Aug.5(day
21), and Aug. 12 (day 28) and on
Aug. 26 (day 42). After
termination of SD treatment, the
Norway spruce seedlings were
either kept indoors (a,c,e)or
moved outdoors (b,d,f). Each
sampling day, eight apical buds
were collected from each SD
treatment and growing
environment and then analyzed
for gene expression as one general
sample
59 Page 8 of 14 Annals of Forest Science (2017) 74:59
outdoor growth environment had broken bud on April 3 com-
pared to control seedlings indoors.
4Discussion
Our results suggest that the duration of SD treatment can be
reduced compared to normal practice (4–5 weeks), but this
depends on whether the treatment objective is to stop growth
or to make seedlings freezing tolerant and storable at an early
date. In our study, the seedlings only required 1 week of SD
treatment for initiation of apical shoot growth cessation.
Similar results were obtained by Dormling et al. (1968),
Dormling (1973,1979,1993), Ekberg et al. (1979),
Konttinen et al. (2003,2007), Kohmann and Johnsen
(2007), as well as Fløistad and Granhus (2013).
Activity of dormancy-related genes as identified by Stattin
et al. (2011) indicated that the seedlings in our study need at
least 14 days of SD treatment, corresponding to a photoperiod
of 11 h for dormancy induction. Here, and in Stattin et al.
(2011), a longer duration (21–28 days) of SD treatment result-
ed in higher activity of the dormancy-related genes (Fig. 5). A
deeper state of dormancy reduces the risk of a second bud
flush during late autumn (Fløistad and Granhus 2010,2013).
Fig. 6 ColdNSure™freezing tolerance phases (adapted from Balk et al.
2007) at different dates of sampling (Sept. 9, Sept. 23, Oct. 9, Oct. 23) for
control and SD (short day) treated (11 h light; 13 h night) seedlings from
both indoor and outdoor growth environments. ColdNSure™phase
0 = cold sensitive; the indicator profiles match the profiles of lots that
areactivelygrowingandnosign of freezing tolerance could be
recognized. Phase 1 = developing freezing tolerance; early signs of
freezing tolerance development can be recognized. Phase
2 = developing freezing tolerance; tolerance level approaches full
freezing tolerance. Phase 3 = freezing tolerant; the indicator profiles
match the profiles of lots that have ceased growth and that are fully
tolerant, ready for lifting and storage. Each sampling date, eight apical
buds were collected from the controls and each SD treatment from both
growing environments and then analyzed for gene expression as one
general sample
Fig. 7 Expression profile of the gene CO1 used to indicate freezing
tolerance. The level of gene expression is expressed as delta delta
threshold cycle (ddCt), which is the relative difference between the
gene of interest and reference genes (genes that did not show any
significant difference in expression). Measurements were performed at
four different occasions, Sept. 9, Sept. 23, Oct. 9, and Oct. 23, 2013,
for Norway spruce seedlings previously subjected to SD (short day)
treatments with a photoperiod of 11 h of various lengths (0, 7, 14, 21,
and 28 days) starting on July 15. After SD treatment, seedlings were
either kept indoors or moved outdoors. Each sampling day, eight apical
buds were collected from each SD treatment and growing environment
and then analyzed for gene expression as one general sample
Annals of Forest Science (2017) 74:59 Page 9 of 14 59
In our study, the shorter SD treatments (SD 7 and SD 14)
resulted in a later activation not only of dormancy-related
genes, but also of genes associated with freezing tolerance
compared to the longer SD treatments (SD 21 and SD 28).
The results are supported by physiological tests showing that
longer SD treatments promote the development of freezing
tolerance (Rosvall-Åhnebrink 1982; Konttinen et al. 2003;
Kohmann and Johnsen 2007).
Based on the gene expression profiles, Norway spruce
seedlings subjected to warmer indoor conditions following
termination of SD treatment showed a slower development
of dormancy and obtained a lower level of freezing tolerance,
compared to seedlings subjected to outdoor conditions.
During September 3–25, the control system for regulation of
temperature in the greenhouse was not functioning satisfacto-
rily, which resulted in a minimum temperature of about 21 °C
(Fig. 2). This could be one of the reasons for the weak and
delayed development of freezing tolerance among seedlings
kept indoors (Fig. 6). As concluded by Weiser (1970), Heide
(1974), Christersson (1978), and Dormling and Lundkvist
(1983), both temperature and photoperiod are important fac-
tors affecting dormancy induction and development of freez-
ing tolerance as shown in our study. Our results suggest that
warmer autumns associated with climate change could cause
problems with the winter hardening processes of seedlings,
which can make autumn planting hazardous due to risk of
late-season frost. It may also delay the time point when seed-
lings are considered ready for long-term freezer storage
(Stattin et al. 2000).
4.1 Shoot growth
During the first week of SD treatment, there was no significant
difference in shoot growth between SD-treated seedlings and
untreated control seedlings indoors. Thus, shoot growth ter-
mination requires time to take effect following initial SD treat-
ment stimuli. Thereafter, all SD-treated seedlings had reduced
shoot growth compared to control seedlings in both indoor
and outdoor growth environments. By the third week, almost
no shoot growth was detected for any SD treatments as simi-
larly reported by Kohmann and Johnsen (2007).
Several studies suggest that less than 2 weeks of SD treat-
ment is enough to terminate apical shoot growth (Dormling
et al. 1968;Heide1974; Konttinen et al. 2003;Fløistadand
Granhus 2013). Our results indicate that 7 days of SD treat-
ment is as effective as longer SD treatments to stop apical
shoot growth. Further, other provenances of Norway spruce
than used in this study as well as different starting dates of SD
treatment could affect seedling shoot growth patterns (see,
e.g., Fløistad and Granhus 2013). Outdoor-grown control
seedlings of the local provenance of Norway spruce used for
this experiment still exhibited some shoot elongation as late as
the third week of September indicating that the seedlings
Fig. 9 Apical bud break during the spring of 2014 expressed as
percentages and measured weekly between March 20 and May 2.
Seedlings had been subjected to SD (short day) treatments (11 h light;
13 h night) of various lengths starting on July 15, 2013 and thereafter kept
indoors or outdoors. Control seedlings in both growing environments
were kept under natural day length. Seedlings previously grown and
stored in outdoor conditions were transferred indoors on Oct. 28, 2013
Fig. 8 Dry matter content (DMC %) for control seedlings and SD (short
day) treated (11 h light; 13 h night) seedlings grown indoors and outdoors
measured on Oct. 23, 2013. According to Rosvall-Åhnebrink (1985), the
target level for storability of SD-treated Norway spruce seedlings is ≥38%
(dashed line). DMC % values based on shoots from 20 seedlings, n=4.
Vertical lines represent the standard error
59 Page 10 of 14 Annals of Forest Science (2017) 74:59
would still be sensitive to frost exposure at this time. These
results were confirmed by our ColdNSure™test (Fig. 6)and
determination of DMC (Fig. 8). It has been shown that SD
treatment regulates growth (Fløistad and Granhus 2013)and
development of freezing tolerance (Konttinen et al. 2007;
Kohmann and Johnsen 2007). These physiological reactions
to SD treatment can be explained by the gene expression pat-
terns for dormancy and freezing tolerance, as exhibited in our
study.
4.2 Bud set and bud size
Bud set and bud development are well known tobe an energy-
consuming process, which is stimulated by, e.g., high temper-
ature (Dormling 1973; Olsen et al. 2014). Shorter SD treat-
ment (SD 7) resulted in earlier set of visible buds for a higher
frequency of seedlings compared to the longer (SD 14, SD 21
and SD 28) treatments both indoors and outdoors (Table 1). In
our case, a possible explanation for the later development of
visible buds and the smaller buds (Fig. 4) of seedlings endur-
ing longer periods of SD treatment is lack of photosynthetic
light rather than low temperatures. As it was hard to visually
detect the small buds from the longer SD treatments, it is
possible that there in fact were no differences in timing of
bud set among the different SD treatments in this study.
Despite the fact that control seedlings in our study showed
terminal shoot growth late in the season compared to SD-
treated seedlings, they still developed somewhat larger buds
than seedlings from the longer SD treatments (Fig. 4). These
results indicate that light intensity during the first stages of bud
development is important for further increasing bud size and
emphasize the importance of not exaggerating the length of
dark periods during SD treatment. It is well known that tem-
perature and light intensity are major factors driving bud de-
velopment for spruce species and that treatments such as SD
affect bud size and development of needle primordia, which
can affect apical shoot growth the forthcoming spring (sensu
Grossnickle 2000).
4.3 Autumn reflushing and bud break in spring
In our study, there were no signs of reflushing for the longer
durations (14, 21, or 28 days) of SD treatment, whereas
reflushing appeared among control and SD 7 treated seed-
lings. Our results confirm earlier studies by Konttinen et al.
(2007), Luoranen et al. (2009), as well as Fløistad and
Granhus (2013) showing that short or no periods of SD treat-
ment may increase the risk for reflushing. Fløistad and
Granhus (2013) as well as Kohmann and Johnsen (2007)re-
ported that an early starting date (mid or late June) of SD
treatment combined with a short (7–14 days) duration in-
creases the risk for reflushing. In our case, due to the relatively
late start (mid-July) of SD treatment, the seedlings from SD 7
and SD 14 met a natural night length that was only somewhat
shorter than their critical night length, and only a few of the
SD 7 treated seedlings showed reflushing. As SD treatment
ended, seedlings (especially longer durations of SD treat-
ments) were exposed to natural night lengths long enough to
further stimulate dormancy induction. Reflushing mostly oc-
curred among lateral buds of the SD 7 treated seedlings, as
similarly shown for silver birch (Betula pendula Roth)
(Junttila et al. 2003), which may be due to a lower level of
dormancy in lateral compared to apical buds (Fløistad and
Granhus, 2013).
Our study showed that all seedlings grown outdoors initi-
ated bud break the following spring about 1–2 weeks earlier
than seedlings grown indoors. This is in accordance with re-
sults from Olsen et al. (2014), who showed that bud break was
delayed in seedlings exposed to high (21 °C) day temperatures
compared to lower (15° or 18 °C) during SD treatment the
previous year. The results from our study also indicated that a
larger proportion of seedlings from the longer (SD 21 and SD
28) treatments broke bud somewhat earlier compared with the
shortest (SD 7) treatment. Konttinen et al. (2003)alsoshowed
that Norway spruce seedlings exposed to longer (3–4weeks)
SD treatments with a starting date in July had an earlier bud
break the following spring compared to shorter (1–2weeks)
SD treatments. In our study, all SD-treated seedlings had an
earlier bud break compared to untreated control seedlings, as
reported by others (e.g., Fløistad and Granhus 2010). In our
study, seedlings exposed to longer SD treatments and an out-
door climate showed a high activity of the dormancy-related
genes. The deeper dormancy induction for these seedlings
probably also results in earlier dormancy release, which leads
to an earlier bud burst in spring. Our results indicate that the
activity of the dormancy-related genes determines the timing
of dormancy induction, dormancy release, and forthcoming
bud burst. Thus, measurements of gene expression could be
used as a tool to predict bud burst in spring, which may help to
prevent the risk for spring frost damage (Fløistad and Granhus
2010) in field plantations. However, more research is needed
to find out absolute levels of gene expression that copes with
physiological reactions of seedlings.
4.4 Dormancy indicators
In previous studies, the physiological effects on seedlings from
SD treatments were evaluated by measuring different morpho-
logical parameters of seedlings (Dormling et al. 1968; Heide
1974;Ekbergetal.1979; Colombo et al. 1989,2001; Fløistad
and Granhus 2013). In our study, we evaluated relationships be-
tween gene expression and seedling physiology and growth. Our
results confirm several studies indicating that even short dura-
tions of SD treatment (SD 7) are effective to terminate growth.
However, the short duration does not stimulate the expression of
dormancy or freezing tolerance-related genes as much as the
Annals of Forest Science (2017) 74:59 Page 11 of 14 59
longer SD treatments. As outdoor-grown seedlings showed a
higher level of gene expression compared to indoor-grown seed-
lings, apparently cool temperatures are needed to promote rapid
dormancy development in addition to short days. Colombo et al.
(1989) also found that dormancy development is stimulated by
SD treatment combined with fluctuating ambient temperatures in
black spruce seedlings. As gene expression decreased or stabi-
lized after an early termination of SD treatment, our results em-
phasize the importance of continuing SD treatment to further
stimulate dormancy induction. If SD treatment starts early in
the season, e.g., at the end of June, the seedlings will be exposed
to long natural day lengths after the termination of SD treatment.
The recommendation from Kohmann and Johnsen (2007)isnot
to start SD treatment too early or else this may increase the risk for
reflushing and reduce development of freezing tolerance. The
startingdatewouldclearlyhaveaneffectongeneexpression,
and the extent to which this occurs warrants further research.
Measurements of the expression of dormancy-related genes
could possibly be used as a tool in forest tree nurseries to establish
when SD treatment can be terminated. Our results are promising,
but further studies are required to determine the appropriate
threshold values either to stop growth or to make seedlings stor-
able at an early date. A similar test to determine these values could
be developed as for the ColdNSure™test (Joosen et al. 2006;
Balk et al. 2008).
A system for continuous measurements of shoot growth dur-
ing blackout could also possibly be developed to predict when
SD treatment can be terminated. Further research is needed to
determine the time space between the termination of apical shoot
growth and initiation of dormancy. Our study indicates that to
obtain an effective stimulus of dormancy, SD treatment must
proceed for at least 1–2 weeks after termination of shoot growth
(see Figs. 3and 5). Among the genes tested, LN3 and LN6
generally showed a slower increase in time and lower expression
levels than LN2 and LN4. Therefore, LN2 and LN4 could better
serve as early indicators of dormancy.
4.5 Freezing tolerance
The ColdNSure™test used in our study for estimating freez-
ing tolerance is based on gene expression of several freezing
tolerance-related genes such as CO1 (Fig. 7). Longer SD treat-
ments resulted in higher levels of gene expression for CO1
and this trend was similar for CO4,CO8,CO9,andCO10,
demonstrating that seedlings can be cold stored earlier after
receiving a longer period of SD treatment (21–28 days). This
trend was most pronounced for outdoor compared to indoor-
grown seedlings showing that outdoor climate conditions
strongly impact the development of freezing tolerance, which
was also observed for the dormancy-related genes.
Comparison of only the control seedlings demonstrates the
role that climate conditions play in the development of autumn
freezing tolerance as the freezing tolerance-related genes
responded much more in outdoor- compared to indoor-
grown seedlings. From the October 23 DMC data, it is obvi-
ous that none of the seedlings kept indoors reached the target
level to be ready for cold storage (Fig. 8)while all seedlings
kept outdoors were considered storable (for target levels, see
Rosvall-Åhnebrink 1985). The ColdNSure™test (Fig. 6)sup-
ports the outcome of the DMC test as all indoor treatments had
only reached freezing tolerance phase 1 except for SD 28
where seedlings had reached phase 2. The results from the
ColdNSure™test (Fig. 6) also showed that outdoor-grown
seedlings from all SD treatments as well as the control were
considered storable on October 23, as they had reached freez-
ing tolerance phase 3. However, the test also indicated that all
SD treatments made outdoor-grown seedlings storable at an
earlier date compared to untreated control seedlings. We con-
sidered it important to include the DMC test in our study
because this test is commonly used in practical nursery oper-
ations. However, under certain circumstances, the DMC test
may be unreliable to forecast storability as shown by Colombo
(1990), Lindström (1996), and Lindström et al. (2014). These
observations led to the development of storability tests based
on the tolerance to freezing by determining electrolyte leakage
(Lindström and Håkansson 1996; Colombo 1997;Brönnum
2005) and eventually the ColdNSure™test (Joosen et al.
2006;Balketal.2007;Balketal.2008).
5 Conclusion
Compared to untreated seedlings, all SD treatments in this
study exhibited a rapid activation of genes related to dorman-
cy and freezing tolerance, thereby making SD-treated seed-
lings storable at an earlier date. The effect of SD treatment on
dormancy induction and development of freezing tolerance
determined by molecular tests were most obvious for the lon-
ger SD treatments (SD 21 and 28), but there were still clear
effects for the shorter SD treatments. Results from our obser-
vations of the expression of genes selected to measure dor-
mancy and freezing tolerance emphasize the importance of
controlling temperature and photoperiod to produce seedlings
that can withstand long-term frozen storage. Our DMC and
ColdNSure™tests indicate that seedlings kept indoors at high
night and day temperatures after termination of SD treatment
will not develop sufficient freezing tolerance for safe autumn
planting or frozen storage. To accomplish this goal within a
reasonable time, local provenances of Norway spruce need to
be SD treated in combination with normal outdoor climate
conditions. Decisions regarding when it is suitable to termi-
nate SD treatment in the nursery could be either based on the
expression of dormancy-related genes, or indirectly according
to when shoot growth stops. Our results indicate that an early
and continuous activation of dormancy-related genes results
in a reduced risk for reflushing, an early of development of
59 Page 12 of 14 Annals of Forest Science (2017) 74:59
tolerance tofreezing and storability and an early timing ofbud
break in spring.
Generally, our results suggest that a warmer climate during
autumn may adversely affect development of dormancy and
freezing tolerance of seedlings. Molecular tests open up pos-
sibilitiesto forecast plant reactions from environmental chang-
es. With further research, they may serve as tools for better
understanding factors influencing the phenology of plants and
provide decision support in nursery management.
Acknowledgements Thanks to Marianne Vemhäll for help with labo-
ratory work. Also thanks to Anders Lindgren, Bergvik Skog AB, Nässja
nursery for providing the seedlings used in the experiments. Thanks to
Claudia von Brömssen for her help with the statistics and to Claes
Hellqvist who helped creating the figures. We appreciate the constructive
comments given by the anonymous reviewers.
Compliance with ethical standards
Funding This study was supported by the Knowledge Foundation,
Bergvik Skog Plantor AB, SCA Skog AB Norrplant, Svenska
Skogsplantor AB, Södra Skogsplantor AB, Dalarna University, and the
Swedish University of Agricultural Sciences.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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