DSpace at VNU: Effect of storage time and pretreatment on seed germination of the threatened coniferous species Fokienia hodginsii

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Plant Species Biology (2014) ••, ••–••

doi: 10.1111/1442-1984.12062

NOTES AND COMMENTS

Effect of storage time and pretreatment on seed
germination of the threatened coniferous species
Fokienia hodginsii
DUC QUANG NGUYEN,* THI PHUONG HOA PHAN† and VAN TAN DAO‡
*University of Engineering and Technology, †Institute of Biotechnology, Vietnam National University Hanoi, and ‡Faculty of
Biology, Vietnam National University of Education Hanoi, Hanoi, Vietnam

Abstract
We report the effects of storage time and pretreatment on seed germination of Fokienia
hodginsii. Lower mean germination was observed in seeds stored for 2 years (6.41 ± 1.23
seeds/replicate) compared with those stored for 1 year (8.52 ± 1.06 seeds/replicate). Seeds
collected from a southern location had statistically higher mean germination (9.67 ± 1.28
seeds/replicate) than those collected from a northern location (7.99 ± 1.36 seeds/replicate).
Higher mean T50 was observed in seeds stored for 2 years (37.02 ± 4.43 days) compared
with those stored for 1 year (30.69 ± 5.06 days). Mean germination of untreated fresh seeds
was 9.97 ± 1.34 seeds/replicate and that of treated fresh seeds in 60°C water was
12.95 ± 1.24 seeds/replicate. Fresh seeds treated with 50°C and 70°C water had a significantly lower mean germination compared with untreated seeds and seeds treated in 60°C
water. Mean T50 was lowest in seeds treated with 60°C water.
Keywords: conservation, ex situ, Fokienia hodginsii, germination, seeds, Vietnam.
Received 28 February 2014; revision received 4 May 2014; accepted 28 July 2014

Introduction
Despite the existence of many other conservation

approaches used nowadays, ex situ conservation offers for
many threatened plants an important insurance policy for
the future. In ex situ conservation, species’ populations are
protected and managed outside their native or original
environment either as seed gene banks or field gene banks
(Kozlowski et al. 2012; Westengen et al. 2013). This
approach is preferred in situations where the populations
are in real danger of physical destruction or genetic deterioration due to excessive pressures in their natural
habitat. Ex situ conservation efforts are necessary to minimize the loss of genetic diversity (Fay 1992), reduce the
risk of extinction (Bowes 1999; Godefroid et al. 2010), and
produce propagation material for the expansion or reintroduction of populations (Brusa et al. 2007; Godefroid
et al. 2010; Ren et al. 2010). Understanding the seed germination requirements of the species to be reintroduced is
paramount to the success of reintroductions. Local adapCorrespondence: Duc Quang Nguyen
Email:
© 2014 The Society for the Study of Species Biology

tation to environmental conditions may result in strict
species-specific requirements for seed germination
(Rayburn et al. 2013). To efficiently and effectively produce
cultivated specimens for research and conservation purposes, requirements for seed germination of the species
must be known (Meyer & Monsen 1991; Cerabolini et al.
2004; Brusa et al. 2007). Differential germination responses
may be observed among seeds of the same species, if those
seeds are stored under different conditions and/or
treated differently before being sown (Cerabolini et al.
2004).
Fokenia hodginsii is a monotypic taxon of the
Cupressaceae family. The genus Fokienia comprises only one
living species and one fossil species (F. ravenscragensis).
This tree species is ancient and endemic to Laos, Vietnam,

and southern China, and it is currently listed as globally
threatened. Fokenia hodginsii is highly valued, both economically and culturally, throughout its distribution
range. The tree has become part of Vietnamese culture,
much like the Hinoki tree in Japan. The timber of this
species is durable and highly fragrant and is prized for
high-value furniture and craftwork as well as internal


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D. Q. NGUYEN ET AL.

paneling in houses. The demand for the timber is the
principal driver behind illegal logging, which is the most
serious threat to F. hodginsii despite it receiving legal protection in China and Vietnam. Even in high-profile
National Parks such as BiDoup Nui Ba and Chu Yang Sin,
mature trees are being felled despite constant monitoring.
The problem is even worse in more remote areas. In
Vietnam and Laos subpopulations have been heavily
exploited by legal and illegal logging over the last 50
years and are now highly fragmented and reduced in size.
In China, there has also been a significant decline; lowaltitude forests of F. hodginsii have been converted for
agriculture or forestry (Luu & Thomas 2000). Despite legal
protection, illegal logging continues to reduce the number
of mature trees of F. hodginsii throughout most of their
range.
Recently, the Vietnamese Government has introduced
many laws to protect F. hodginsii forests. Nevertheless,
trade in illegally harvested timber has caused local extinction or extreme scarcity of individuals in many localities.
At present, several plantation plans have been started in

some northern Vietnam areas, however, the planted areas
are not significant. Failure in producing seedlings was
frequently reported in many localities in northern
Vietnam. One of the difficulties in plantation programs is
the limited current knowledge regarding the biology, and
particularly ecology, of F. hodginsii. Research on germination ecology, storage conditions, and pretreatment of the
seeds of F. hodginsii is crucial in addressing questions of
conservation and sustainable harvest of this forest tree. To
the best of our knowledge, the present paper is the first to
address the questions regarding seed germination of
F. hodginsii under the effects of different storage and pretreatment conditions.

Materials and method

Seed collection
Fokienia hodginsii flowers twice a year. The first flowering
time occurs in spring from April to May and the cone is
ripe in October of the same year. The spring seeds are not
viable. The second flowering period is in autumn from
September to October, and the cone is ripe in October of
the next year. The autumn seeds are viable. All seeds used
in this study were collected during the period from early
November to early December 2010. Only seeds of the
mature fruits with a dark brown fruit coat were collected.
The seeds were dry-stored immediately after collection
for a period of either 1 or 2 years. Seeds of F. hodginsii
collected from a northernmost Vietnam location (Hagiang
Province at 22°02′N) were symbolized as “N-seeds” and
from a southernmost Vietnam location (Ninhthuan Province at 11°33′N) were symbolized as “S-seeds.” The
© 2014 The Society for the Study of Species Biology

N-seeds and S-seeds were classified into upper and lower
elevation groups according to the altitudes of the original
mother trees from which the seeds had been collected.
Seeds collected from higher altitudes (1990 m for S-seeds
and 1870 m for N-seeds) were called “upper seeds” and
seeds from lower altitudes (1610 m for S-seeds and
1500 m for N-seeds) were called “lower seeds”.

Effect of storage time on seed germination
Germination experiments were carried out in Hanoi
(Vietnam) during the period from 2011 to 2013 with a total
of 16 treatments that represented all combinations of differences in seed storage time (1 year and 2 years), latitudes of seed collection (N-seeds and S-seeds), and
altitudes of seed collection (upper and lower seeds for
northernmost and southernmost location). A total of 1600
seeds were equally distributed to across 16 treatments
with four replicate germination trials per treatment (25
seeds per replicate for a total of 100 seeds per treatment).

Effect of hot water pretreatment on seed germination
In 2009 when first plantation programs of F. hodginsii
were initiated, some households of northern Vietnam
started to try planting F. hodginsii in their own gardens
and they reported that the germination rates of fresh seeds
were low (approximately 20%). Some people tried pretreating the seeds with hot water (approximately 50°C),
and the germination rate was improved. They reported
that the germination rates were improved especially for
the seeds immersed in hot water from 2 to 4 h (pers.
comm.). In the present research, a second experiment was
performed in which seeds were pretreated by immersion

in three different temperatures of hot water (50°C, 60°C,
and 70°C) for either 3 or 4 h.
The seeds were then rinsed with KMnO4 0.05% to
remove the acidity, and were washed with cool water
before being sown. All seeds used in this experiment were
fresh seeds that had been left to dry naturally within 7 days
after collection from Hagiang Province, northern Vietnam.
A total of 600 seeds were equally distributed to six treatments (3 temperatures × 2 treatment times) with four replicate germination trials per treatment (25 seeds per
replicate for a total of 100 seeds per treatment). Untreated
seeds were sown at the same time to evaluate the effect of
pretreatment on seed germination, and four replicates (25
seeds per replicate) were also set up for the untreated lot.
The seeds were sown in standard germination boxes on
moist filter paper and continuously kept in a dark germination chamber. Germination was monitored every 5 days
with a green light in order to detect radicle emergence. A
seed was considered germinated when the radical pierced
the coats up to 2 mm.
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S E E D G E R M I N AT I O N O F F O K I E N I A H O D G I N S I I

Data analysis
All data in this study are reported as means ± one standard error. For the first experiment, a three-way anova for
a completely randomized design in program R (Version
2.12.0, R Development Core Team 2008) was used to test
for the main effects of storage time, latitudes, and altitudes on mean percentage germination and mean time to
50% germination (T50) as well as two-way and three-way
interactions between the predictor variables. For the
second experiment, a two-way anova was used to test for

the main effects of temperature and pretreatment time
on mean germination as well as two-way interactions
between the predictor variables. For both experiments, a
Turkey–Kramer adjustment for multiple comparisons
using the multcomp package in R (Hothorn et al. 2008) was
used to compare mean percentage germination and mean
T50 between treatments.

Results

Effect of storage time on seed germination
First germination was observed after 15 days, independent of treatment (Table 1). The approximate time to 50%
germination (T50) across treatments was 34 days. Out of
1600 seeds sown, a total of 502 seeds germinated (31.38%).
Mean germination was significantly affected by storage
time (P < 0.05), with lower mean germination observed in
seeds stored for 2 years (6.41 ± 1.23 seeds/replicate,
25.64%) compared with those stored for 1 year (8.52 ± 1.06
seeds/replicate, 34.08%) (Table 1). Independent of storage

3

time and altitudes, seeds collected from the southern location had statistically higher mean germination (9.67 ± 1.28
seeds/replicate, 38.68%) than those collected from the
northern location (7.99 ± 1.36 seeds/replicate, 31.96%;
P < 0.05; Table 1). Independent of storage time and latitudes, germination of seeds collected from upper and
lower altitudes did not differ significantly (7.78 ± 1.21
seeds/replicate, 31.12% and 7.84 ± 1.54, 31.36%, respectively, P > 0.05) (Table 1).
There were also significant differences in mean germination between certain treatment combinations (Table 1).
For example, the mean germination of 1-year stored seeds

collected from southern populations at lower sites
(9.78 ± 1.67 seeds/replicate) was significantly higher than
those collected from northern populations at both lower
and upper sites (8.02 ± 1.33 and 8.44 ± 1.98 seeds/
replicate, respectively) (P < 0.05) (Table 1). Independent of
latitudes and altitudes, the mean germination of 1-year
stored seeds was significantly higher than that of 2-year
stored seeds in all treatment combinations, except for the
2-year stored seeds collected from southern populations
at upper sites (7.53 ± 1.91 seeds/replicate) which was significantly higher than those of the other treatment combinations for 2-year stored seeds (P < 0.05) (Table 1).
On the other hand, T50 values were significantly different across all treatments (P < 0.05). Independent of latitudes and altitudes, mean T50 was significantly affected by
storage time (P < 0.05), with higher mean T50 was
observed in seeds stored for 2 years (37.02 ± 4.43 days)
compared with those stored for 1 year (30.69 ± 5.06 days)
(Table 1). There was no statistically significant effect of

Table 1 Mean germination and mean T50for the eight treatments representing pairwise combinations of seed storage time (1 or 2 years),
latitudes (northernmost or southernmost), and altitudes (upper or lower) of the original populations from which seeds were collected

Storage time (years)

Latitude

Altitude

Approximate
days to first
germination

1

1
1
1
2
2
2
2
Mean of 1-year stored seeds
Mean of 2-year stored seeds
Mean of northern seeds
Mean of southern seeds
Mean of upper seeds
Mean of lower seeds

Northern
Northern
Southern
Southern
Northern
Northern
Southern
Southern

Upper
Lower
Upper
Lower
Upper
Lower
Upper

Lower

15
43
15
22
34
33
19
30

Mean germination
(seeds/replicate)

Mean T50 (days)

8.44 ± 1.98a
8.02 ± 1.33a
9.45 ± 1.24ab
9.78 ± 1.67b
6.03 ± 1.77c
6.11 ± 1.55c
7.53 ± 1.91ab
6.07 ± 1.11c
8.52 ± 1.06
6.41 ± 1.23
7.99 ± 1.36
9.67 ± 1.28
7.78 ± 1.21
7.84 ± 1.54

33.76 ± 4.47a
31.54 ± 3.39a
36.88 ± 5.23b
29.43 ± 4.62a
37.09 ± 5.29b
35.23 ± 5.67ab
37.49 ± 5.55b
37.51 ± 3.69b
30.69 ± 5.06
37.02 ± 4.43
33.11 ± 3.29
33.16 ± 3.37
34.10 ± 4.11
33.95 ± 3.99

Values are means ± one standard error. Means in a column with the same superscript letter do not differ significantly from one another
according to Tukey’s test for multiple comparisons (α = 0.05).
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© 2014 The Society for the Study of Species Biology


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D. Q. NGUYEN ET AL.

Water temperature
(°C)
50

50
60
60
70
70
Untreated seeds

Treatment
time (h)
3
4
3
4
3
4

Approximate
days to first
germination

Mean germination
(seeds/replicate)

Mean T50 (days)

34
37
15
39
27

32
29

10.36 ± 1.68b
10.44 ± 1.56b
12.95 ± 1.24a
12.89 ± 1.33a
9.78 ± 1.65bc
8.90 ± 1.56c
9.97 ± 1.34bc

28.02 ± 4.89b
29.88 ± 5.44c
25.11 ± 5.13a
25.66 ± 4.22a
26.45 ± 3.14a
26.17 ± 4.01a
28.66 ± 4.55b

Table 2 Mean germination and mean
T50for the six treatments representing
pairwise combinations of seed pretreatment with water temperature (50, 60, and
70°C) and treatment time (3 and 4 h)

Values are means ± one standard error. Means in a column with the same superscript
letter do not differ significantly from one another according to Tukey’s test for multiple
comparisons (α = 0.05).

latitudes and altitudes on mean T50, but there were statistically significant differences in mean T50 values between
certain treatment combinations (Table 1). For instance, the

mean T50 value for seeds collected from southern population at lower site and stored for 1 year was significantly
lower than that for seeds collected from southern population at upper site (29 ± 4.62 and 36 ± 5.23, respectively,
P < 0.05) (Table 1).

Effect of hot water pretreatment on seed germination
Mean germination of fresh seeds of F. hodginsii was significantly affected by hot water pretreatment (Table 2).
Mean germination of untreated seeds was 9.97 ± 1.34
seeds/replicate (39.88%) (Table 2). Mean germination of
treated seeds in 60°C water for 3 h or 4 h was 12.95 ± 1.24
and 12.89 ± 1.33, respectively which were the highest
compared with that in 50°C and 70°C ((Table 2). Pretreatment with 70°C water caused a slight decrease in mean
germination compared with 50°C and untreated seeds,
however the difference was not statistically significant
(P > 0.05). It was noted that time of seed treatment in hot
water did not significantly affect mean germination as the
difference in the mean germination at 3 h and 4 h was not
statistically significant (P > 0.05) in all temperatures
(Table 2).
On the other hand, mean T50 was significantly lower
in seeds treated with 60°C water (25.11 ± 5.13 and
25.66 ± 4.22 seeds/replicate for 3 and 4 h, respectively)
and 70°C water (26.45 ± 3.14 seeds/replicate and
26.17 ± 4.01 seeds/replicate for 3 and 4 h, respectively)
compared with that of seeds treated with 50°C
(28.02 ± 4.89 seeds/replicate and 29.88 ± 5.44 seeds/
replicate for 3 h and 4 h, respectively) and untreated seeds
(28.66 ± 4.55 seeds/replicate) (P < 0.05) (Table 2). The twoand three-way interactions were not significant.
Germination was calculated at each time-step over the
360-day duration of the experiment for 60°C-water treated
© 2014 The Society for the Study of Species Biology

Fig. 1 Seed germination of Fokienia hodginsii over 360-day duration of the study. At each time step the bars represent the number
of newly germinated seeds observed for untreated seeds and for
seeds treated in 60°C water for 3 h. , 60°C treated; , untreated.

seeds (in 3 h) and untreated seeds (Fig. 1). Histograms of
the germination response and timing for treated seeds
compared with untreated seeds show that, in general,
treated seeds had more pronounced peaks in frequency
(approximately 25–30 days) than untreated seeds. Germination of treated seeds mostly occurred during the period
from 20 to 40 days, whereas germination of untreated
seeds occurred during the period from 25 to 40 days
(Fig. 1).

Discussion
Our past efforts to germinate cold-stored seeds of
F. hodginsii were unsuccessful, as seeds stored in 9°C for 9
months lost their germination ability (unpubl. data). In the
first experiment, we found that dry-stored seeds of
F. hodginsii were able to germinate after 2 years. There has
not been a thorough study on the germination ecology of
this threatened coniferous species. In the second experiPlant Species Biology ••, ••–••


S E E D G E R M I N AT I O N O F F O K I E N I A H O D G I N S I I
ment we found that mean germination of fresh seeds of
F. hodginsii was approximately 40% and that pretreatment
of seeds with 60°C water for 3–4 h raised mean germination to approximately 51%. Our data add critical preliminary insight into the germination biology of F. hodginsii
that adds to our understanding of the basic ecology of this
species and provides an initial guideline for germination

in ex situ conservation.
The present study revealed that, averaged over altitude
and latitude, mean germination of ≤ 2-year dry-stored
seeds of F. hodginsii was approximately 9% lower than that
of fresh seeds. Mean germination of 1-year stored seeds
was approximately 12% higher than that of 2-year stored
seeds. In another study, mean germination of 4-month
cold-stored seeds of F. hodginsii was approximately 20%
lower than that of fresh seeds (Canh et al., pers. comm.).
Consistent with the present study, Canh et al. also found
that cold-stored seeds of F. hodginsii were not able to germinate after 6-month storage. Some research has shown
that seed viability of coniferous species is significantly
reduced following periods of storage greater than 2–3
years (Berdeen et al. 2007). Seeds may deteriorate in dry
storage, losing vigor and becoming more sensitive to
stress during germination (Rajjou & Debeaujon 2008;
Probert et al. 2009). Temel et al. (2011) reported that mean
germination of seeds of black pine (Pinus nigra subsp.
pallasiana) stored for 10 years was approximately 38%
lower than that of fresh seeds. Liu et al. (2011) found that
mean germination of seeds of 489 grass species stored in
warm dry conditions had decreased by 16%–18%. Our
results suggest that ≤ 2 years dry storage of F. hodginsii
seeds for ex situ conservation purposes does not dramatically reduce seed viability. Although mean germination
was significantly different between some treatments, all
treatments had between 24% and 40% germination.
Future research on the seed storage of F. hodginsii should
include a more comprehensive germination trial in which
the length of dry storage time and temperature are
manipulated. Such research would help address some of

the limitations in our present study and provide useful
information for conservation strategy makers and practitioners. For example, mean germination may decrease
and T50 may be higher for ≥ 2-year dry stored seeds; T50
may be reduced if seeds of F. hodginsii are exposed to
higher temperatures as part of a germination trial.
Mean T50 of fresh seeds of F. hodginsii was approximately 29 days and that for 1-year stored and 2-year
stored were approximately 31 and 37 days, respectively. A
previous study on the effect of storage time on seed germination in white bark pine also found that seeds stored
for shorter periods of time germinated more quickly than
those stored for longer periods of time (Berdeen et al.
2007). However, Rayburn et al. (2013) found a reverse
trend in the threatened distylous primrose (Primula
Plant Species Biology ••, ••–••

5

cusickiana var. maguirei); that is, seeds stored for 2 years
had a higher germination percentage and lower mean T50
compared with seeds stored for 1 year.
Mean germination of seeds collected from southern
Vietnam populations was approximately 7% higher than
that of seeds collected from northern Vietnam populations. A previous study on black pine (Pinus nigra subsp.
pallasiana) (Temel et al. 2011) found that germination characteristics seemed to associate with population location,
especially with humidity of the region from which the
seeds were collected. While Vietnam lies entirely in the
tropics, there is quite a large difference in climate between
northern and southern regions. Northern Vietnam features a humid subtropical climate and has a full four
seasons, with much cooler temperatures than in the south,
as well as winters that can get quite cold. Southern
Vietnam, with its much hotter temperatures, has only two

main seasons: a dry season and a rainy season. The cold
experienced during northern winters is intensified by the
humidity. Snow can even be found to an extent up in the
mountains of the extreme northern regions in places,
especially on top of high mountains. In southern China,
Hou et al. (2005) reported that biological and phenological
characters of flower and cone of F. hodginsii populations
are closely linked to geography and climatic factors,
including location, altitude, and temperature of the
natural distribution area. Autumn flowering, fruit, maturity, and seed falling in mountainous areas occurred
earlier than in lower mountainous areas, and much earlier
than in hilly areas, and in high-altitude areas earlier than
in low-altitude areas. There are obvious geographic variations in cone diameter, cone height, seed number, 1000seed weight, and germination percentage among
populations of F. hodginsii in China (Hou et al. 2005).
Further research needs to be carried to study geographic
variation of the seed germination characteristics of
F. hodginsii populations in Vietnam. Our studies (Quang
et al. 2012; Nguyen & Nguyen 2012) on the genetic variation of F. hodginsii across its distribution range in Vietnam
found that southern populations had a relatively lower
amount of diversity than northern populations.
Our results revealed that pretreatment of seeds of
F. hodginsii with 60°C water in 3 h increased mean germination and decreased mean T50 and resulted in uniform
germination compared with untreated seeds. We
observed that after being left in a dry shaded place for 10
days, approximately 90% of the F. hodginsii fruit coats
opened naturally and seeds were split from the fruit coat.
However, the seed coat of F. hodginsii is relatively hard.
These seeds will usually germinate in 30–360 days, but
even under good conditions germination may be erratic.
The hard seed coat prevents imbibitions and gaseous

exchange that leads to physical dormancy and subsequently the foremost cause in poor and erratic germina© 2014 The Society for the Study of Species Biology


6

D. Q. NGUYEN ET AL.

tion. Overcoming hard-seededness is an important initial
step in the use of seeds of F. hodginsii in ex situ conservation and restoration programs. The present study showed
that pretreatment with 60°C water in 3 h made approximately 48% of the seeds of F. hodginsii germinate within
120 days, whereas approximately 40% of untreated seeds
germinated within 290 days.

Acknowledgment
Special thanks to The Vietnam National Foundation
for Science and Technology Development (NAFOSTED)
for the financial support, with project number 106.99–
2010.25. Special thanks to two reviewers with their
insightful and constructive comments and suggestions on
our manuscript.

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