Comparative study on seed germination characteristics of
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two species of Australia saltbush under salt stress
Deng Yunquan, Yuan Fang, Feng Zhongtao, Ding Tonglou, Song Jie, Wang Baoshan*
Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Jinan 250014,
China
Abstract: In order to examine the response characteristics and possible reasons of Atriplex
lentiformis and Atriplex undulata under salt stress at stage of seed germination, the seeds were
treated with different concentrations of NaCl (0, 50, 100, 200 and 300 mmol·L-1), 20 mmol·L-1
LiCl or mannitol whose iso-osmotic concentrations corresponding to 200 mmol·L-1 NaCl. The
results showed that the germination rate of two species of saltbush was depressed with the
increase of NaCl concentration, and A. lentiformis showed greater salt tolerance compared with A.
undulata. After removal of salt stress, the final germination ratio of A. lentiformis was over 93%,
while that of A. undulata was only 56%. Evans blue staining revealed that 200 mmol·L-1 NaCl did
not damage membrane permeability of A. lentiformis seed embryos, but significantly increased the
membrane permeability of A. undulata seed embryos and caused irreversible damage to them,
especially radicles. The results on water uptake indicated that the inhibition of NaCl on seed
germination was mainly due to osmotic stress instead of ionic toxicity, and A. lentiformis
exhibited higher salt tolerance due to its greater resistance to osmotic stress.
Key words: A. lentiformis; A. undulata; NaCl; seed germination; ionic effect; osmotic effect
1. Introduction
Atriplex is a typical secretohalophyte in arid and semi-arid regions of the whole world. Atriplex
species play an important role in vegetation reconstruction and soil conservancy for saline and arid
regions. There are 17 species and 2 varieties of Atriplex in China [1], among which A. tatarica and
A. centralasiatica had high salt tolerance [2-4]. Australian saltbush A. semibaccata R. Br. and A.
Canescens were introduced in Qinghai, Xinjiang in the late 1990s, which showed great potential
in enriching the biodiversity and improving the ecological environment in the saline and arid
regions [5]. A. lentiformis and A. undulata are saltbushes which were introduced from Australia.
The previous studies on A. lentiformis and A. undulata focused on the later stage of seedlings and
*Corresponding author: Tel: 0531-86180197, E-mail address: [email protected]
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little is known about response characteristics of the seeds under salt stress [6-10]. The adaptation
to the salinity during seed germination and seedling growth is very important for halophyte
population growth and construction in saline-alkaline condition [11]. In the present study, the
response characteristics and possible reasons of seed germination for A. lentiformis and A.
undulata under salt stress were determined to screen salt tolerant saltbushes being used in saline
soil.
2 Materials and methods
2.1 Materials
Two species of Australian saltbush seeds (Atriplex lentiformis and Atriplex undulata) collected
in 2010 were stored at 2C in refrigerator for further use.
2.2 Methods
2.2.1 Determination of salt tolerance of seeds
Uninformal and full seeds were washed with 0.1% HgCl2 for 5 minutes and rinsed with distilled
water. Four NaCl concentrations (50, 100, 200 and 300 mmol·L-1) were used to determine the salt
tolerance of seeds. NaCl was dissolved in distilled water and distilled water was as control (0).
Three replicates of 20 seeds each were germinated on two layers of filter paper in petri dishes
(9 cm in diameter) containing 5 mL each of the test solution respectively and the NaCl solutions
were changed completely every 2 days. The seeds were allowed to germinate at
25/15 °C day/night temperatures in the dark [12, 13]. A seed was considered to have germinated
when the emerging radicle elongated to 5 mm. The number of seeds germinated was counted daily
for 10 days, then some data were determined by the following formulas: Germination rate = the
total number of seeds germinated in saline solution / the total number of sown seeds ×100%
Relative germination rate=the germination rate of each salt treatments/ the germination rate of
control ×100% Germination energy=the total number of seeds when seed germination reached its
peak/ the total number of sown seeds ×100% Germination index (Gi)=Σ(Gt/Dt) and Vigor index
(Vi)=S×Gi, where Gt is the number of seeds germinated at t day, Dt is corresponding days of
germination, S is the mean weight of five seedlings after 5 days germination [14,15].
After 10 days, all ungerminated seeds were placed in distilled water and under the same
condition for 7 days to determine their ability to recover from salt stress. The final germination
rate was determined by the following formula: Final germination rate= the total number of seeds
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germinated after being transferred to distilled water / the total number of sown seeds ×100%
The membrane permeability of A. lentiformis and A. undulata seed embryos treated with 200
mmol·L-1 NaCl was detected with Evans blue staining [16].
2.2.2 Determination of ionic effect or osmotic effect of salt stress on seed germination
Salt-induced inhibition of seed germination could be attributed to osmotic stress or to specific
ion toxicity [17, 18]. The toxicity of Li+ is 10 times as that of Na+, and 20 mmol·L-1 LiCl has no
osmotic effect [19-23]. Three replicates of 20 seeds each were sown in Petri dishes (9 cm in
diameter) on two layers of filter paper moistened with 5 mL distilled water (control), 20 mmol·L-1
LiCl, 200 mmol·L-1 NaCl or isotonic mannitol, and each solution was changed completely every 2
days. Germinated seeds were recorded daily. Seeds were considered to have germinated when the
emerging radicle was at least 5 mm.
2.2.3 Determination of the water uptake
Water uptake was studied to validate whether the inhibition of salt stress on seed germination
was mainly caused by osmotic effect. Seeds sown in Petri dishes (9 cm in diameter) on two layers
of filter paper moistened with 10mL of distilled water (control), 20 mmol·L-1 LiCl, 200 mmol·L-1
NaCl or isotonic mannitol were weighed after incubation for 10 h at constant temperature (25°C)
in the dark. The relative increase in fresh weight (Wr) was calculated as Wr = (Wf –Wi)/Wi ×100%,
where Wi is the initial weight of seeds, and Wf is the weight after 10h [24, 25].
2.3 Data analysis
Data were subjected to one-way analysis of variance (ANOVA) using Duncan’s multiple range
test (P < 0.05).
3 Results
3.1 The effect of salt stress on seed germination
3.1.1 Germination rate and Germination energy
For two saltbush seeds, both germination rate and germination energy decreased progressively
as the level of salinity increased (Fig.1, 2). Low NaCl concentration did not significantly affect the
germination of A. lentiformis and A. undulata. However, at 200 mmol·L-1 NaCl, the germination
rate and germination energy of A. lentiformis were about 76.7% and 82.2% respectively while that
of A. undulata were 15% and 13.6% respectively. The trend of relative germination rate was
coincident with that of germination rate and germination energy (Fig. 3). These results suggested
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Germination rate/(%)
that A. lentiformis had higher salt tolerance than A. undulata.
A.lentiformis
120
a
100
A.undulata
ab
ab
c
80
cd
c
d
60
40
e
20
f
f
0
control
50
100
200
-1
NaCl concention/(mmol·L )
300
Fig.1 The effect of different concentrations of NaCl on the germination rate of A. lentiformis and A. undulata
Data are means ± SD, and each treatment has three replicates. Different letters (a, b, c, d, e, f) represent significant difference at P＜0.05 according to
Germination energy/(%)
Duncan’s multiple range test. The same notes are for the following figures.
A.lentiformis
100
80
a
a
ab ab
A.undulata
bc
60
c
d
40
20
e
e
e
0
control
50
100
200
-1
NaCl concention/(mmol·L )
300
Fig.2 The effect of different concentrations of NaCl on the germination energy of A. lentiformis and A. undulata
A.lentiformis
a a
Relative germination
rate/ (%)
120
100
A.undulata
a
ab
b
80
60
40
c
20
d
d
0
control
50
100
200
300
NaCl concention/ (mmol•L-1)
Fig.3 The effect of different concentrations of NaCl on the rel ative germination rate of A. lentiformis and A. undulata
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3.1.2 Germination index
Germination index can reflect the speed and uniformity of the germination. The germination
index of A. lentiformis and A. undulata decreased progressively as the level of salinity increased
and differed significantly under different NaCl concentrations (Fig. 4). However, at 200 mmol·L-1
NaCl, the germination index of A. lentiformis was 48.5% of control while A. undulata was only
Germination index
13.9%, which indicated A. lentiformis had higher salt tolerance than A. undulata.
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A.lentiformis
a
A.undulata
b
8
c
d
cd
6
e
e
4
2
f
g
g
0
control
50
100
200
NaCl concention/(mmol·L-1 )
300
Fig.4 The effect of different concentrations of NaCl on the germination index of A. lentiformis and A. undulata.
3.1.3 Vigor index
Vigor index represents the germination capacity and growing tendency of seedling. The vigor
index of A. lentiformis and A. undulata was significantly reduced as salinity increased (Fig. 5). At
100 mmol·L-1 NaCl, the vigor index of A. lentiformis decreased about 43.4% of control in
comparison to A. undulata decreasing 51.7%. At 200 mmol·L-1 NaCl, the vigor index of A.
lentiformis decreased 61.7% of control while A. undulata decreased 90.4%. These results
illustrated that A. undulata was more sensitive to NaCl than A. lentiformis.
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NaCl concention/(mmol·L-1 )
50
100
200
Decrease rate of vigor
index/(%)
0
20
40
60
80
100
A.lentiformis
A.undulata
Fig.5 The effect of different concentrations of NaCl on the vigor index of A. lentiformis and A. undulata
3.1.4 Final germination rate
When the ungerminated seeds were transferred to distilled water after ten-day exposure to
salinity, the differences in final germination rate between A. lentiformis and A. undulata were
obtained (Fig. 6). The final germination ratio of A. lentiformis was over 93% while that of A.
undulata was only 56%, which indicated that salt stress did not cause irreversible damage to seed
embryos and only temporarily inhibited seed germination of A. lentiformis. Did NaCl cause
irreversible damage to seed embryos of A. undulata?
Evans blue staining was used to detect the effect of 200 mmol·L-1 NaCl on membrane
permeability of A. lentiformis and A. undulata seed embryos. The results showed that 200
mmol·L-1 NaCl did not damage membrane permeability of A. lentiformis seed embryos, but
significantly increased the membrane permeability of A. undulata seed embryos and caused
irreversible damage to them, especially radicles (Fig. 7).
A.lentiformis
A.undulata
Final germination
rate/(%)
120
a
a
a
a
a
100
80
b
c
c
d
d
60
40
20
0
control
50
100
200
-1
NaCl concention/(mmol·L )
300
Fig.6 The effect of different concentrations of NaCl on the final germination rate of A. lentiformis and A. undulata
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Fig.7 Detection of membrane permeability of A. lentiformis and A. undulata seed embryos treated with 200 mmol·L-1 NaCl using Evans blue staining
3.2 Ionic effect or osmotic effect of salt stress on seeds germination
Germination rate of A. undulata and A. lentiformis at 20 mmol·L-1 LiCl was similar to control,
while germination was reduced in a considerable manner with 200 mmol·L-1 NaCl and isotonic
mannitol (Fig. 8). That’s to say the inhibitory effect of 200 mmol·L-1 NaCl on seed germination
was mainly due to osmotic stress. Moreover, the germination rate of A. undulata was more
remarkably reduced than that of A. lentiformis under 200 mmol·L-1 NaCl and isotonic mannitol, so
A. lentiformis had greater resistance to osmotic stress.
A.lentiformis
A.undulata
Germination rate/(%)
120
100
80
a
a
b
bc
bc
c
60
40
d
d
20
0
control
Fig.8
Na200
Treatments
Li20
Man200
Effect of different treatments on the germination rate of A.lentiformis and A. undulata
200 mmol·L-1 NaCl、20 mmol·L-1 LiCl and isosmotic treatments are indicated by Na200、Li20 and Man200. The same notes are for the following figure.
3.3 The effect of salt stress on water uptake
The relative increase in fresh weight of A. lentiformis seeds with each treatment was not
significantly changed (Fig. 9). However, the water uptake of A. undulata seeds was significantly
reduced under 200 mmol·L-1 NaCl and isotonic mannitol conditions compared with control and 20
mmol·L-1 LiCl, which further explained that the inhibition of NaCl on A. undulata seed
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germination was mainly due to osmotic stress and A. lentiformis had greater resistance to osmotic
stress.
A. lentiformis
Water uptake /(%)
70
60
a
A. undulata
a
a
a
a
50
b
b
40
a
30
20
10
0
control
Na200
Li20
Man200
Treatments
Fig.9 Effects of different treatments on the water uptake of A.lentiformis and A. undulata
4 Discussion and conclusion
The period of germination and establishment is the most critical stage in the life cycle, which is
a crucial factor in determining the species distribution and community components, and seeds of
most plant species have the highest resistance to extreme environmental stresses, whereas
seedlings are the most susceptible [26]. Saline habitat is one kind of the most stressful habitats
which is known to affect many physiological and ecological characteristics, such as plant growth,
development, reproduction and geographical distribution [27]. One of the important attributes of
halophyte seeds and possibly the principal characteristic distinguishing them from glycophytes
could be their seeds' ability to remain viable for long periods under extremely high salinity stress
and then germinate at a later time when soil water potentials were raised [11], and halophyte seeds
form multiple germination characteristics and survival mechanisms to adapt to the changeable
stress environment in prolonged evolution process [28].
Germination rate, germination index, germination energy and vigor index are the most common
indexes of germination which are used to estimate the germinating performance and seedling
growth [29]. Most of the studies demonstrated that distilled water was most suitable for the
germination of halophytic seeds, and that the germination rate gradually decreased with salinity
increasing [30, 31]. However, some experiments also showed that low salinity could improve seed
germination [32-34]. After assessment of germination rate and other indexes in our study, it was
found that the seed germination of A. lentiformis and A. undulata was inhibited by salt stress and
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the inhibition increased constantly with the increasing of NaCl concentration. On the one hand, the
results that a lot of ungerminated seeds could germinate quickly after rehydration suggested NaCl
only temporarily inhibited seed germination of A. lentiformis. The ability that seed germination
could rapidly recover when the seeds were transferred from stress environment to proper condition
had important significance on population stability and persistence, and that was also an important
mechanism for halophyte to adapt to salty environment. On the other hand, 200 mmol·L-1 NaCl
significantly increased the membrane permeability of A. undulata seed embryos and caused
irreversible damage to them, which was confirmed by Evans blue staining. So we could draw a
conclusion that A. lentiformis exhibited higher salt tolerance than A. undulata under different
NaCl concentrations especially at 200 mmol·L-1 NaCl.
The essence on salt tolerance of halophyte seeds was the synthetic suitability to ionic and
osmotic effect during germination, while ecological factors, such as solution types and ambient
temperature, affected salt tolerance of seeds through modifying ionic and osmotic effect of
solution [35]. The effect of NaCl on the seed germination of Suaeda physophora, Haloxylon
ammodendron and Haloxylon persicum was due to both osmotic effect and ion toxicity [36],
whereas ionic effect was the main reason for the inhibition of suaeda salsa seed germination at
low NaCl concentration [23]. Our study showed that the inhibition of NaCl on A. lentiformis and A.
undulata seed germination was mainly due to osmotic effect rather than ion toxicity, which
basically tallies with some conclusions gained by predecessors [37-40].
Abundant water was one of the indispensable factors for seed germination and the water
shortage of saline environment inhibits seed germination [41]. Our results on water uptake further
confirmed that the inhibition of NaCl on seed germination was mainly due to osmotic stress and A.
lentiformis had greater resistance to osmotic stress.
In conclusion, the inhibition of NaCl on the seed germination was mainly due to osmotic effect,
and A. lentiformis exhibited higher salt tolerance than A. undulata because of its greater resistance
to osmotic stress. However, it is desirable to further explore the mechanism by which A.
lentiformis seeds could alleviate the damage under osmotic stress.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Nos. 30870138 and
31070158), National science and technology project (No. 2009BADA7B05). We thank Prof.
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Gui-jun Yan from University of Western Australia for kindly providing saltbush seeds.
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