Annals of Botany 114: 991– 999, 2014
doi:10.1093/aob/mcu164, available online at www.aob.oxfordjournals.org
Intermediate complex morphophysiological dormancy in seeds of the cold desert
sand dune geophyte Eremurus anisopterus (Xanthorrhoeaceae; Liliaceae s.l.)
Jannathan Mamut1, Dun Yan Tan1,*, Carol C. Baskin1,2,3,* and Jerry M. Baskin1,2
1
Xinjiang Key Laboratory of Grassland Resources and Ecology and Ministry of Education Key Laboratory for Western Arid Region
Grassland Resources and Ecology, College of Grassland and Environment Sciences, Xinjiang Agricultural University,
Ürümqi 830052, China, 2Department of Biology and 3Department of Plant and Soil Sciences, University of Kentucky,
Lexington, KY 40546, USA
* For correspondence. E-mail: [email protected] or [email protected]
Received: 2 April 2014 Returned for revision: 19 May 2014 Accepted: 30 June 2014 Published electronically: 1 September 2014
† Background and Aims Little is known about morphological (MD) or morphophysiological (MPD) dormancy in
cold desert species and in particular those in Liliaceae sensu lato, an important floristic element in the cold deserts of
Central Asia with underdeveloped embyos. The primary aim of this study was to determine if seeds of the cold desert
liliaceous perennial ephemeral Eremurus anisopterus has MD or MPD, and, if it is MPD, then at what level.
† Methods Embryo growth and germination was monitored in seeds subjected to natural and simulated natural temperature regimes and the effects of after-ripening and GA3 on dormancy break were tested. In addition, the temperature requirements for embryo growth and dormancy break were investigated.
† Key Results At the time of seed dispersal in summer, the embryo length:seed length (E:S) ratio was 0.73, but it
increased to 0.87 before germination. Fresh seeds did not germinate during 1 month of incubation in either light
or darkness over a range of temperatures. Thus, seeds have MPD, and, after .12 weeks incubation at 5/2 8C, both
embryo growth and germination occurred, showing that they have a complex level of MPD. Since both after-ripening
and GA3 increase the germination percentage, seeds have intermediate complex MPD.
† Conclusions Embryos in after-ripened seeds of E. anisopterus can grow at low temperatures in late autumn, but if
the soil is dry in autumn then growth is delayed until snowmelt wets the soil in early spring. The ecological advantage
of embryo growth phenology is that seeds can germinate at a time (spring) when sand moisture conditions in the desert
are suitable for seedling establishment.
Key words: Cold desert perennial ephemeral, embryo growth, Eremurus anisopterus, germination ecology,
germination phenology, seed morphophysiological dormancy.
IN T RO DU C T IO N
Although species in various plant families including Apiaceae,
Araceae, Berberidaceae, Fumariaceae, Gentianaceae, Iridaceae,
Liliaceae sensu lato (s.l.) and Ranunculaceae occur in cold
deserts and their seeds have small underdeveloped embryos,
little is known about morphological (MD) and morphophysiological (MPD) dormancy in cold desert species (Baskin and
Baskin, 2014). Morphological dormancy means the embryo in
freshly matured seeds is small but differentiated into cotyledon(s) and hypocotyl/root, and grows before germination (root
emergence). Thus, after the embryo completes growth, MD is
broken, and the seed germinates. Morphophysiological dormancy is a combination of MD and physiological dormancy
(PD), the latter meaning that the embryo has a physiological
mechanism inhibiting germination that results in low growth
potential (Nikolaeva, 1969). In some species with MPD, the
embryo in freshly matured seeds is a mass of undifferentiated
cells, which subsequently differentiates into an underdeveloped
embryo that needs to grow before the seed germinates (Mondoni
et al., 2012). The PD part of MPD is broken by treatments such as
warm and cold stratification either before, during or after embryo
growth has occurred (Nikolaeva, 1969; Baskin and Baskin,
2014). Nine levels (kinds) of MPD have been described based
on timing of the breaking of PD and MD, temperature requirements for embryo growth and responses of seeds to gibberellins
(Baskin and Baskin, 2014).
One of the best represented families among Eurasian cold
desert geophytes is Liliaceae s.l. (Breckle, 1983; Walter and
Box, 1983a, b, c). However, little is known about the germination
ecophysiology of species of Liliaceae s.l. that occur in cold
deserts. Wu et al. (2005) reported that no seeds of Eremurus
inderiensis germinated during 40d of incubation in light or
darkness at 5, 10, 20 and 30 8C, a long ( presumably 8 months)
after-ripening period was required to break dormancy and a
freezing treatment ( –18 8C) was better than cold storage (0– 4
8C) for after-ripening. Mechanical scarification followed by
soaking in 0.01 M GA3 for 45 min promoted 53 % of the seeds
of E. spectabilis to germinate (Rahmanpour et al., 2005).
Ma et al. (2006) reported that the germination percentage of
selfed and crossed seeds of E. anisopterus and E. inderiensis
incubated at 10 8C was ≤40 %. However, they did not mention
whether the seeds were fresh or dry stored, or if they were incubated in light or darkness. Further, they did not identify the kind
of seed dormancy in either species. Freshly matured seeds of
Tulipa iliensis failed to germinate at 4, 10 or 10/4 8C, but after
# The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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992
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
2 months of dry storage they germinated to 70– 95 % at these
temperatures (Tang et al., 2009). It seems clear that seeds of
these three species were dormant. However, whether or not
embryos were underdeveloped and needed to grow inside the
seed before they could germinate was not determined. Thus,
we do not know if seeds of these three species had PD or MPD.
The purpose of our research initially was to investigate seed
dormancy of Eremurus anisopterus (Karelin & Kirilov) Regel.
However, our preliminary observations that seeds of this species
have small embryos relative to the amount of endosperm, along
with the results mentioned above that seeds of E. inderiensis
(Wu et al., 2005) and E. spectabilis (Rahmanpour et al., 2005)
have a physiological component of dormancy, suggested the
presence of MPD. In Liliaceae s.l., six levels of MPD (sensu
Baskin and Baskin, 2014) have been reported: deep simple epicotyl (Barton, 1936; Crocker and Barton, 1957; Liu et al., 1993;
Kondo et al., 2002, 2004), deep complex (Nikolaeva et al., 1985;
Baskin et al., 1995), non-deep complex epicotyl (Nikolaeva
et al., 1985), non-deep simple (Nikolaeva et al., 1985), deep
simple (Nikolaeva et al., 1985; Kondo et al., 2006) and non-deep
complex (Baskin and Baskin, 1985; Nikolaeva et al., 1985).
Thus, the aim of our research became more specific, i.e. to determine if seeds of E. anisopterus have MPD and, if so, the level of
MPD. To accomplish these objectives, we asked the following
questions. (1) Does the embryo grow prior to emergence of the
cotyledonary petiole that encloses the root–shoot axis (Tillich,
1995) (hereafter root)? (2) Are dormancy break and germination
promoted by warm and/or cold stratification? (3) Do GA3 and/or
dry storage (after-ripening) promote dormancy break and germination? (4) If the seeds are buried in soil under natural temperature
conditions, when do the embryos grow and seedlings emerge
above the soil surface?
MAT E RI ALS A ND METH O DS
Study organism and seed collection
Eremurus anisopterus [Liliaceae s.l. (Chen et al., 2000);
Xanthorrhoeaceae, Asparagales (APG, 2009)] is a perennial
ephemeral geophyte herb with a hermaphrodite-gynomonoecious
sexual system (Mamut et al., 2014). The species occurs in the
cold deserts of Kazakstan and north-west China (Chen et al.,
2000). In China, E. anisopterus grows on fixed and semi-fixed
sand dunes of the Gurbantunggut Desert of Xinjiang Uyghur
Autonomous Region. Flowering occurs from late April to early
May. Seeds mature in early June, after which they are dispersed
within a few days, and seedlings emerge in spring (Mamut et al.,
2014).
Freshly matured seeds were collected from a natural population of E. anisopterus growing on sand dunes in the Mosuowan
region of the Gurbantunggut Desert (85833′ 20.2′′ E, 44855′
19.4′′ N; 313 m a.s.l), on 17 June 2011 and 20 June 2012. After
collection, seeds were stored in paper bags under ambient conditions in the laboratory (16 – 30 8C, 10– 40 % relative humidity)
until used in experiments. This area is an inland cold desert
with typical temperate desert climate. Mean annual temperature
is 7 8C, the mean low temperature of the coldest month (January)
is – 23 8C and the mean high temperature of the hottest month
(July) is 33 8C. Annual precipitation (rain and snow) is
147 mm, 64 % of which occurs in spring and summer, and the
lowest (February) and highest (July) monthly amounts are
5 and 22 mm, respectively (data from Mosuowan weather station,
1991 – 2010). Mean annual potential evaporation is 1942mm
(Yuan and Tang, 2010).
Phenology of embryo growth and seedling emergence
To determine the initial length/size of the embryo, 25 seeds
collected in 2012 were placed on moist filter paper on 26 June
2012. After a 24 h period of imbibition at room temperature,
each seed was cut open with a razor blade and embryo length
and seed length were measured (E:S ratio), using a dissecting
microscope equipped with a micrometer.
To monitor phenology of embryo growth, seeds collected in
2012 were buried in the experimental garden on the campus of
Xinjiang Agricultural University in Ürümqi on 26 June 2012.
Thirty freshly matured seeds were placed in each of ten fine-mesh
nylon bags and buried to a depth of 3 cm in sand in clay pots
(30 cm diameter, 30 cm height) which were buried (the top of
the pot level with the soil surface) in soil in the experimental
garden. Sand in the pots was from the natural habitat, and it
was kept moist throughout the monitoring period. After burial
on 26 June 2012, 25 seeds were removed from one haphazardly
selected bag every 15 d until 7 November 2012 and the E:S ratio
determined as described above. The maximum (critical) embryo
length for germination was determined by measuring embryos in
seeds with the seed coat split open, indicating that root emergence was ready to occur.
To monitor the phenology of seedling emergence, five replicates of 50 freshly matured seeds collected in 2012 were sown
at a depth of 3 cm in sand in 30 cm diameter by 30 cm height
pots buried with the top level with the soil surface in the experimental garden on 26 June 2012. Sand in the pots was kept moist,
and seedling emergence was monitored at 1 week intervals, at
which time newly emerged seedlings were counted and removed
from the pots. Soil temperatures at a depth of 3 cm were recorded
at 2 h intervals throughout the burial period using Tiny Tag data
loggers (Model Micro Lite LITE5016, Fourier Technologies,
Beijing, China). Daily mean, maximum and minimum temperatures were calculated from these data.
Temperature requirements for embryo growth, dormancy break
and germination
To determine if warm stratification, cold stratification, warm
plus cold stratification or cold plus warm stratification is required
for dormancy break, a move-along experiment (Baskin and
Baskin, 2003) was conducted using seeds collected in June
2012. Fifty seeds were sown on wet filter paper in each of 24
Petri dishes of 12 cm diameter on 4 July 2012. Four dishes
each were placed in darkness at 5/2, 15/2, 20/10 and 30/15 8C
to serve as non-moved controls. Control seeds remained at
their respective temperature regime for the duration of the experiment. In the move-along portion of the experiment, one set
of seeds (four dishes of 50 seeds each) was placed in darkness
at 5/2 8C. After 12 weeks, they were moved from 5/2 8C to
15/2 8C (4 weeks)20/10 8C (4 weeks)30/15 8C (12 weeks)
20/10 8C (4 weeks). The second set of seeds in the move-along
portion of the experiment was started in darkness at 30/15 8C.
After 12 weeks, seeds were moved from 30/15 8C to 20/10 8C
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
(4 weeks)15/2 8C (4 weeks)5/2 8C (12 weeks)15/2 8C
(4 weeks) (Table 1). All seeds were checked for germination at
1 week intervals in green light, at which time seedlings were
removed and the substrate watered, if needed. The green light
source consisted of a 5W cool white incandescent tube wrapped
with an acrylic green sheet (Shandong Luke Electric Appliance
Co., Ltd, Shandong, China). The peak intensity of green light as
determined using a PR-650 spectrascan colorimeter (Photo
Research, Inc,, CA, USA) was at 530 nm. The average photon
flux density of green light at seed level, determined with a
TES-1332A digital llluminance meter (TES Electrical Electronic
Corp., Taipei, Taiwan), was approx. 1.8 mmol m – 2 s – 1. Six additional dishes of seeds were included in each of the two sequences to use
for determination of the E:S ratio. Prior to transferring seeds to the
next temperature in the sequence, the E:S ratio of 25 randomly
selected seeds in the extra dishes was determined as described
above. The initial E:S ratio was determined for embryos in 25
freshly matured seeds that had been incubated on moist filter
paper at room temperature for 24 h.
Effect of GA3 and dry storage (after-ripening)
on dormancy break
To determine the effects of GA3 on dormancy break, four
replicates of 25 seeds collected in June 2011 and stored dry at
room conditions (16– 30 8C, 10– 40 % relative humidity) for
0 (fresh, control), 2, 10 and 18 months were incubated in 0 (distilled water control), 0.1, 1 and 10 mmol L – 1 GA3 solutions at
5/2, 15/2, 20/10, 25/15 and 30/15 8C in both light/dark and darkness for 30 d. The daily photoperiod (cool white fluorescent
light, approx. 100 mmol m – 2 s – 1, 400–700 nm) in the light/dark
treatment was 12 h (hereafter light), and it corresponded to the
daily 12 h high-temperature period. Germination in light was monitored daily, at which time any germinated seeds were counted and
removed from the Petri dishes. Germination in darkness was determined only at the end of the 30 d experiment, and distilled water
was added every 7 d in green light.
Effect of burial at natural temperatures on dormancy break
Approximately 1000 seeds collected in June 2011 were placed
in each of 12 (10 × 12 cm) fine-mesh nylon bags on 30 June
2011. Each bag was buried to a depth of 3 cm in sand in 16 cm
diameter by 19.5 cm deep plastic pots with drainage holes (one
bag per pot), and then the pots were buried (the top of the pot
level with soil surface) in soil in the experimental garden.
These seeds were subjected to natural temperature and soil moisture conditions.
993
Germination tests were conducted on the day of burial and
after seeds had been buried for 1–12 months. Beginning on
30 July 2011, seeds were exhumed monthly for 1 year, except in
November and December 2011 and January and February 2012,
when the soil was frozen and seeds could not be exhumed. Four
replicates of 25 seeds each were incubated on wet filter paper in
9 cm diameter plastic Petri dishes incubated in light and in darkness at 5/2, 15/2, 20/10, 25/15 and 30/15 8C for 30 d.
Data analyses
Means and standard errors were calculated for germination
percentages and for E:S ratios. All statistical computations were
performed with the software SPSS 16.0 (SPSS Inc., Chicago,
IL, USA). Tukey’s HSD test was performed for multiple comparisons to determine significant (P , 0.05) differences (or
not) among levels in each treatment (Sokal and Rohlf, 1995).
Three-way analysis of variance (ANOVA) was used to test for
the significance of the main effects (temperature, light condition
and retrieval time) and their interaction on germination of seeds
in the ‘Effect of burial at natural temperatures on dormancy
break’ experiment. Four-way ANOVA was used to test for significance of main effects (GA3, temperature, light condition
and storage time) and their interaction on germination of seeds
in the ‘Effect of GA3 and dry storage on dormancy break’ experiment. All data were assessed for homogeneity of variance using
Levene’s test and log10 transformed if required, but untransformed data are presented in the figures.
R E S ULT S
Phenology of embryo growth and seedling emergence
The E:S ratio of freshly matured seeds of E. anisopterus was
0.73 + 0.01. Embryos grew only a little during summer 2012,
when mean daily maximum and minimum soil temperatures
were 34.6 and 18.8 8C, respectively. However, between 24
September and 7 November 2012, during which time mean
daily maximum and minimum soil temperatures were 15.9 and
3.2 8C, respectively, embryos grew rapidly (Fig. 1). Embryos
had reached the critical E:S ratio (0.87 + 0.01) required for
germination on 7 November 2012, at which time 4 % of the
seeds in the bag had germinated.
The first newly emerged seedlings of E. anisopterus were
observed on 5 April 2013 and the last ones on 18 April 2013;
during this time, mean daily maximum and minimum soil temperatures were 17.2 and 2.6 8C, respectively. During this
2 week period, 84.6 % of the seeds produced emergent seedlings;
no additional seedlings emerged thereafter (Fig. 1).
TA B L E 1. Outline for the move-along experiment
Weeks at treatment
temperature
12
4
4
12
4
Move along
5/2 8C winter 15/2 8C early spring 20/10 8C late spring 30/15 8C summer 20/10 8C early autumn
30/15 8C summer 20/10 8C early autumn 15/2 8C late autumn 5/2 8C winter 15/2 8C early spring
Controls
5/2 8C 5/2 8C 5/2 8C 5/2 8C 5/2 8C
15/2 8C 15/2 8C 15/2 8C 15/2 8C 15/2 8C
20/10 8C 20/10 8C 20/10 8C 20/10 8C 20/10 8C
30/15 8C 30/15 8C 30/15 8C 30/15 8C 30/15 8C
994
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
50
A
Max. temp
Mean temp.
Min. temp.
Temperature (C)
40
30
20
10
0
100
B
E:S ratio
Cumulative seedling emergence
0·92
80
E:S ratio
0·88
60
0·84
40
0·80
20
0·76
0
22-Apr
23-Mar
21-Feb
22-Jan
23-Dec
23-Nov
24-Oct
24-Sep
25-Aug
26-Jul
26-Jun
0·72
Cumulative seedling emergence (%)
–10
June 2012 to April 2013
F I G . 1. Daily maximum, daily minimum and daily mean soil temperatures at a
depth of 3 cm (A) and phenology of embryo growth and seedling emergence of
Eremurus anisopterus (B) in the experimental garden. Error bars in (B) are + s.e.
Temperature requirements for embryo growth, dormancy break
and germination
In the non-moved controls, the E:S ratio was 0.87 + 0.01 after
seeds were incubated at 5/2 and 15/2 8C for 16 and 4 weeks, respectively, and it was 0.83 + 0.01 and 0.78 + 0.02 after they
were incubated at 20/10 and 30/15 8C, respectively, for
36 weeks (Fig. 2A). Seeds incubated at 5/2 8C began to germinate after 8 weeks, and final germination (36 weeks) was 91 %.
Seeds incubated at 15/2 8C germinated to 37 % during the first
8 weeks, and final germination (36 weeks) was 42 %. Only 2 %
of the seeds incubated at 20/10 8C for 36 weeks germinated,
and no seeds germinated at 30/15 8C (Fig. 2B).
In the move-along treatment that began at 5/2 8C, the E:S ratio
after 12 weeks of incubation was 0.85 + 0.01 (Fig. 2C). Seeds
germinated to 7 % while they were at 5/2 8C, but germination
increased to 95 % when they were moved to 15/2 8C (Fig. 2D).
In the move-along treatment that began at 30/15 8C, the E:S
ratio was 0.84 + 0.01 after seeds were incubated at the sequence
of four temperatures for 32 weeks (Fig. 2C). After the seeds were
moved from 5/2 to 15/2 8C, they germinated to 99 % (Fig. 2D).
Effect of GA3 and dry storage (after-ripening) on dormancy break
A four-way ANOVA showed that the main factors and their
interactions had significant effects on germination percentages
(Table 2). Regardless of GA3 concentration or seed age,
germination was significantly higher in darkness than in light.
After 0 and 2 months of storage, the optimum germination temperature was 5/2 8C, but, after 10 and 18 months of storage
maximum, germination percentages at 15/2 and 5/2 8C were
equally high. Some seeds stored for 10 and 18 months and
treated with 10 mmol L – 1 GA3 germinated to 70 % at 20/10 8C
in darkness. In light, 15/2 and/or 5/2 8C were(was) optimal for
germination, and 10- and 18-month-old GA3-treated seeds germinated to ≤10 % in light at 20/10 8C. Regardless of seed age,
GA3 significantly increased germination in light and in darkness,
and the promotive effect of GA3 increased with seed age.
However, after 10 and 18 months of dry storage seeds incubated
in darkness at 15/2 8C germinated to about 75 % without GA3
treatment, but even at 10 and 18 months GA3 had a promotive
effect on germination, especially at 20/10 8C (Fig. 3).
Effect of burial at natural temperatures on dormancy break
The germination percentage of seeds was significantly affected
by temperature (P , 0.001), light condition (P , 0.001) and retrieval time (P , 0.001) (Table 3). Also, significant interactions
were observed in germination percentage between temperature
and light (P , 0.001), between temperature and retrieval time
(P , 0.001), between light and retrieval time (P , 0.001) and
between temperature, light and retrieval time (P , 0.001)
(Table 3). No fresh seeds germinated at any temperatures in
either light or constant darkness (Fig. 4). The optimum temperature regime for germination in both light and darkness was
15/2 8C. As burial time increased from 2 to 9 months, germination at 15/2 8C increased in darkness. However, seeds buried
for 9 months germinated to very low percentages in light and
only at 5/2 and 15/2 8C. Ninety per cent of the seeds exhumed
after 10 months (in late April 2012) had germinated in the bag,
and thus germination tests could not be conducted.
D IS C US S IO N
Freshly matured seeds of E. anisopterus did not germinate in
light or in darkness at 5/2, 15/2, 20/10, 25/15 and 30/15 8C
(Fig. 4), and seedlings of summer-sown seeds did not emerge
in the experimental garden until the following spring (Fig. 1).
Further, prior to root emergence, the E:S ratio increased from
0.73 to 0.87, a 19.2 % increase. Thus, seeds of E. anisopterus
have MPD, and both the breaking of PD and embryo growth
must occur before seeds can germinate. The presence of MPD
was expected, based on the wide occurrence of this class of dormancy in Liliaceae s.l. (see the Introduction). The next question
is what level of MPD do they have?
The nine levels of MPD can be sub-divided into two categories. (1) In simple levels of MPD, embryos grow at ≥15 8C, and
some (or all) of the PD is broken prior to embryo growth.
(2) In complex levels of MPD, embryos grow at temperatures favourable for cold stratification (approx. 0 – 10 8C), and PD and
MD can be broken at the same time (Nikolaeva, 1969; Baskin
and Baskin, 2014). When fresh seeds of E. anisopterus were
incubated at 5/2, 15/2, 20/10 and 30/15 8C for 36 weeks (i.e. controls for the move-along experiment), 91 % of them germinated
at 5/2 8C. That is, the breaking of PD and embryo growth occurred during the first 12 weeks of incubation at 5/2 8C, after
which seeds germinated at this temperature. In the case of the
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
0·92
5/2 C
15/2 C
20/10 C
30/15 C
A
E:S ratio
0·88
995
5/2 C(12wk)15/2 C(4wk)20/10 C(4wk)
30/15 C(12wk)20/10 C(4wk)
C
30/15 C(12wk)20/10 C(4wk)15/2 C(4wk)
5/2 C(12wk)15/2 C(4wk)
0·84
0·80
0·76
0·72
100
B
D
Germination (%)
80
15/2 C
15/2 C
60
40
20
0
0
4
8
12
16
20
24
28
32
36
0
4
8
12
Time (weeks)
16
20
24
28
32
36
Time (weeks)
F I G . 2. Embryo length to seed length (E:S) ratio (mean + s.e.) and germination (mean % + s.e.) of Eremurus anisopterus seeds in the move-along experiment.
(A, B) Controls; (C, D) move-along. Arrows indicate when seeds were moved to 15/2 8C.
TA B L E 2. Four-way ANOVA of effects of GA3, temperature, light condition, storage time and their interactions on seed germination
percentage of Eremurus anisopterus
GA3
Temperature (T)
Light (L)
Storage time (ST)
GA3 × T
GA3 × L
GA3 × ST
T×L
T × ST
L × ST
GA3 × T × L
GA3 × T × ST
GA3 × L × ST
T × L × ST
GA3 × T × L × ST
d.f.
SS
MS
F-value
P-value
3
4
1
3
12
3
9
4
12
3
12
36
9
12
36
16 802.900
52 180.400
35 283.600
31 041.900
15 759.600
7765.000
3477.500
31 159.400
34 157.600
17 171.600
10 947.000
7092.000
1399.000
24 839.400
5763.000
5600.967
13 046.100
35 283.600
10 347.300
2002.675
1313.300
2588.333
7789.850
2846.467
5723.867
912.250
197.000
155.444
2069.950
160.083
480.083
1.118
3.024
886.911
65.805
112.569
221.569
667.701
243.983
490.617
78.193
16.886
13.324
177.424
13.721
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
move-along treatment starting at 5/2 8C, both MD and PD were
broken at 5/2 8C, and seeds germinated immediately after
being moved to 15/2 8C (Fig. 2D). Thus, we can conclude that
the dormancy in E. anisopterus seeds fits into the complex category of MPD, but do seeds have non-deep, intermediate or
deep complex MPD?
An important consideration when trying to determine the level
of PD in seeds with MPD is the response of seeds to gibberellins.
GA3 promotes germination of seeds with non-deep PD, may
or may not promote germination of seeds with intermediate PD
and does not promote germination of seeds with deep PD
(Nikolaeva, 1969). GA3 was only moderately effective in promoting germination of fresh seeds of E. anisopterus, with a
maximum germination of 26 % for seeds incubated in
10 mmol L – 1 GA3. Thus, based on response of fresh seeds to
GA3, we conclude that they have intermediate complex MPD.
996
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
100
GA3 concentration (mmol L–1)
80
Germination (%)
Darkness: 0 months of storage
Light: 0 months of storage
0
0·1
1
10
60
40
c
b
20
0
100
a aa a
ab b
aa
aa
b
aa b
Darkness: 2 months of storage
Light: 2 months of storage
80
Germination (%)
b
a aa
c
60
40
b
c
20
0
100
a a ab
b
b
a a aa
a aa a
aa
a a
Light: 10 months of storage
c
abb
ab
a
c
60
40
b
20
0
100
b
a aa
a
aa
aa aa
aba
a a aa
Darkness: 18 months of storage
Light: 18 months of storage
c
80
Germination (%)
bb
Darkness: 10 months of storage
80
Germination (%)
aa
c
a
aa
a
a
60
b
40
0
bc
bb
20
a
c
b
ab
a
a
5/2
aa
15/2
a
b
20/10
a aa
25/15
Temperature (C)
a
a
30/15
5/2
a
15/2
a
20/10
aaa
a
25/15
30/15
Temperature (C)
F I G . 3. Effect of GA3 on germination (mean % + s.e.) of Eremurus anisopterus seeds in light and in darkness at different temperatures after 0 to 18 months of dry
storage under laboratory conditions. Bars with different letters within a burial time indicate significant differences (P , 0.001).
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
TA B L E 3. Three-way ANOVA of effects of temperature, light
condition, retrieval time and their interactions on germination of
Eremurus anisopterus seeds buried in soil in the experimental
garden
Source
d.f.
SS
MS
F-value
P-value
Temperature (T)
Light (L)
Retrieval time (R)
T×L
T×R
L×R
T×L×R
4
1
3
4
12
3
12
14 002.600
20 498.256
8641.369
13 208.900
5257.600
8114.769
4899.700
3500.650
20 498.256
2880.456
3302.225
438.133
2704.923
408.308
152.548
893.251
125.521
143.901
19.093
117.872
17.793
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
,0.001
100
A
5/2 °C
15/2 °C
20/10 °C
25/15 °C
30/15 °C
Germination (%)
80
60
40
20
aa
0
100
B
a
a
Germination (%)
80
a
60
b
40
b
b
b
bc
c
b
20
c
c
c
0
0
2
4
9
Burial time (months)
F I G . 4. Germination (mean % + s.e.) of Eremurus anisopterus seeds incubated
in light (A) and in constant darkness (B) at five temperature regimes following 0,
2, 4 and 9 months of burial in soil in the experimental garden. Bars with different
letters within a burial time indicate significant differences (P , 0.001).
Information on after-ripening in dry storage, i.e. breaking of
the PD, is also useful in determining the level of PD. Seeds
with non-deep PD can after-ripen completely, while those with
deep PD do not after-ripen. Seeds with intermediate PD can afterripen to some degree, which results in a decrease in the length of
the cold stratification period required to break PD (Nikolaeva,
1969). With an increase in the period of dry storage (afterripening), seeds of E. anisopterus became increasingly responsive to GA3, and 10- and 18-month-old seeds germinated
to about 75 % within 30d at 15/2 8C in darkness, without
any GA3. Thus, as seeds of E. anisopterus after-ripened in
997
dry storage, they required less and less time at both 5/2 and
15/2 8C, especially at 15/2 8C, to germinate (Fig. 3), which is
consistent for seeds with intermediate PD.
This is the first report of intermediate complex MPD in
Liliaceae s.l. Seeds of E. anisopterus do not have non-deep
complex MPD because a warm pre-treatment was not required
for them to respond to cold stratification, as evidence by 91 %
germination when they were incubated continuously at 5/2 8C.
Seeds did not have deep complex MPD because both afterripening and GA3 promoted dormancy break and germination
(Fig. 3). Seeds of E. inderiensis (Wu et al., 2005; Ma et al.,
2006) and E. spectabilis (Rahmanpour et al., 2005), which
were shown to be dormant, probably also have MPD, but not
enough information is available to assign them even to a dormancy class. Since the seeds were dormant, they would have
MPD if the embryo is underdeveloped and PD if the embryo is
fully developed (Baskin and Baskin, 2014).
In its cold desert sand dune habitat, seeds of E. anisopterus are
dispersed in early summer, and some after-ripening would occur
before the onset of cold weather in late autumn. Thus, when temperatures decrease in autumn and are within the range of those
required for cold stratification, any remaining PD could be
broken and embryos grow if the soil is moist. However, in the
cold deserts of north-west China, autumn and winter are frequently dry. There is snowfall during winter, and most of the
annual rainfall occurs in spring and summer. In fact, it often is
too dry in autumn for non-dormant seeds of annual species to germinate, and thus germination is delayed until soil is moistened by
snowmelt and/or rain in spring (e.g. Wang et al., 2010). In our
study, the soil was kept moist in the garden in autumn, and
embryos grew as seeds were exposed to cold-stratifying temperatures. However, seeds failed to germinate (emerge above the soil
surface) in the garden in autumn, probably due to temperatures
decreasing below those at which seeds can germinate by the
time embryos reached the critical E:S ratio for germination
(Fig. 1). If the soil is too dry for embryo growth in autumn, it is
expected that embryos would grow at cold-stratifying temperatures in early spring, when the soil is moistened by snowmelt
and/or rainfall. Regardless of when embryos grow (autumn or
early spring), seeds germinate in spring.
We cannot explain why only 42 % of the seeds incubated continuously at 15/2 8C germinated (Fig. 2B). After 4 weeks of incubation at 15/2 8C, the E:S ratio was 0.87; thus, lack of embryo
growth does not explain why 58 % of the seeds, which were
viable, failed to germinate. Further, 95– 100 % of the seeds germinated at 15/2 8C in the move-along experiment (Fig. 2D) and
.80 % in the GA3 (Fig. 3) and buried seed (Fig. 4) experiments,
showing that non-dormant seeds can germinate at this temperature regime. It seems that there are two possible reasons for the
low germination in the 15/2 8C control: the temperature regime
may have inhibited the breaking of PD or the seeds were
induced into secondary dormancy.
The germination percentage of E. anisopterus at 15/2 8C was
60– 80 % after 2 or 4 months of burial in soil (Fig. 4B). One
possible reason for the higher germination percentages after
2 –4 months of burial than in the move-along experiment is
that temperatures in the garden may have been more conducive
to after-ripening than those in the incubator. Whereas seeds in
the garden were frequently exposed to maximum temperatures
of 40 – 45 8C for about 1 month and a minimum temperature of
998
Mamut et al. — Complex morphophysiological dormancy in seeds of Eremurus
20 –15 8C during this time (Fig. 1A), seeds in the incubator were
exposed to 30/15 8C (12 weeks) and then to 20/10 8C (4 weeks).
Although some seeds that overwintered while buried in the
experimental garden germinated at 5/2 8C, the highest germination percentages were at 15/2 8C, which simulates field temperatures in early spring (Fig. 1). Regardless of seed age
(degree of after-ripening), test temperature or GA3 concentration, seeds germinated to a higher percentage in darkness than
in light. The promotive effect of darkness (vs. light) on germination is consistent with the germination requirements of seeds of
some psammophytes whose seeds germinate to higher percentages in darkness than in light (Baskin and Baskin, 2014). A
dark requirement for germination is adaptive in sandy habitats,
i.e. there is likely to be more water under the sand surface
(in darkness) than on the sand surface (in light). Thus, seedlings
from buried seeds would have a higher probability of survival
than those from seeds on the surface.
Much of the PD in seeds of E. anisopterus is broken by afterripening, and the remainder is broken rapidly during incubation
at low temperatures. In fact, seeds of E. anisopterus after-ripened
to a much greater degree than has been reported for any other
species whose seeds have the intermediate level of PD. That
not all the PD in seeds of E. anisopterus is broken by afterripening is shown by the promotive effect of GA3 on germination
of 10- and 18-month-old seeds incubated at 20/10 8C, especially
in darkness. The ecological advantage of PD being broken by a
combination of after-ripening and cold stratification and of
embryo growth occurring at low temperatures is that seeds can
germinate in early spring as soon as the soil becomes moist.
Thus, seedlings have the whole moist-cool season in which to
grow and become established. In contrast, if seeds had one of
the simple levels of MPD, for example non-deep simple MPD,
in which all the PD is broken by after-ripening (Baskin and
Baskin, 1990), a relatively high warm-stratifying temperature
would be required for embryo growth. The consequence of a
dry substrate in the habitat in autumn and of a high temperature
requirement for embryo growth would be a delay in germination
until part of the moist early spring period had passed.
After seeds of E. anisopterus are dispersed onto the surface of
their sand dune habitat, they will become buried to different
depths by sand movement. Depth of sand burial has been
shown to affect seed germination and seedling emergence and
survival in various species in different areas of the world (e.g.
Harty and McDonald, 1972; Pemadasa and Lovell, 1975;
Huang and Gutterman, 1978; Chen and Maun, 1999), including
arid/semi-arid regions of China (Zhu et al., 2009; Zheng et al.,
2012). Further study is needed to explain the ecology of the
seed and seedling stages of the life cycle of E. anisopterus and
how they are adapted to the harsh sand dune environment of
the Gurbantunggut Desert.
AC KN OW LED GEMEN T S
We thank Xuan Li for help with collection of data early in
the study. and Mihray Nur and Amanulla Eminniyaz for collection of seeds. This study was supported by the National
Science Foundation of China (U1130301, 31160063), the
Major National Scientific Research Program of China
(2014CB954200-2) and the International Science and Technology
Cooperation Program of China (ISTPC2011DFB30070).
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