The longevity of seeds during storage is influenced by
traits such as genetics and environment during maturation and harvest, seed
moisture content and storage conditions (Cromarty et al. 1982; Walters et
al. 2010). Low moisture content and low storage temperature slow down the
chemical reactions of seed aging by retaining cells structure and enzymes in a
stable state for long periods (Chang et al. 1996; Sun 1997; Bernal-Lugo
and Leopold 1998). Sugars are central to this process. As seed moisture is
reduced, soluble sugars form a grassy state which serves as a physical
stabilizer by suppressing deteriorative reactions (Franks 1994; Walters et
al. 2010).
Ultra-drying is a technique used to
reduce the seed moisture content to acceptable levels suitable for safe
long-term storage at ambient temperature (Li et al. 2007). Research on ultra-drying (Ellis et al.
1990; Huang et al. 2002) has focused mainly on its use as a low-cost
plant germplasm conservation method (Zheng et al. 1998) which is favored in developing countries because it eliminates the need to use refrigerated storage for seeds
(Ellis et al. 1989; Zheng 1994).
Potential
benefits of ultra-drying include improvement of seed
longevity and vigour (Koster 1991; Ellis et al. 1995; Li et al. 2007; Huo et al. 2011). Many
reports indicate that ultra-drying to a moisture content of 2 to 5% could
potentially increase seed life span by 4 to 39 times, depending on the species
(Harrington 1972; Ellis
et al. 1986). More
importantly, Ellis et al. (1996) and Demir and Ozcoban
(2007) showed that the loss in viability is more
rapid in dry (5.1–6.8% moisture content) than in ultra-dried (2.0–3.7% moisture
content) seeds of carrot, groundnut, lettuce, oilseed rape and onion stored
at 20°C for five years. In contrast, some studies have reported that ultra-drying
causes a significant reduction in the viability of stored seeds (Vertucci et al. 1994; Hu et al. 1998; and Engels 1998). This seems to occur when
seeds are dried below a critical water content which is species or even
cultivar specific (Woodstock et al. 1976;
Ellis et
al. 1988). However, Ellis (1992) has shown that real damage usually occurs when seeds
imbibed water during germination and not during the storage process. Based on
these research findings, it is now a standard recommendation that dry and ultra-dry
seeds should be
humidified prior to germination in order to raise their moisture contents
slowly in the initial stages of germination to minimise any loss to viability
(Powell and Matthews 1979).
The success of ultra-drying is
measured in terms of seed survival rate and vigor. The difference between
germination rates measured before and after drying seeds to different moisture
contents is indicative of deterioration caused primarily through oxidation
damage that affects seed survival (Walters et al. 2010). Germination
tests have been used extensively to show the positive results from ultra-drying
seeds (e.g. Ellis et al. 1993, 1995, 1996; Li et al. 2007;
Pérez-García et al. 2007; Demir and Ozcoban 2007).
However, very few previous reports (Li et
al. 2007; Pérez-García et
al. 2007, 2008) have examined the
effect of ultra-drying on seed vigour which is known
to influence significantly plant growth and yield (Ellis 1992). Seed vigour is a complex term which refers to the aspects of
seed performance which show variation such as the rate and uniformity of
germination, and seedling emergence and growth under variable environmental
conditions (Perry 1978). Enhanced tolerance to desiccation stress, resistance
to aging processes and improved germination and seedling growth have been noted
for seeds subjected to slow drying processes (Adams et al. 1983; Vertucci
and Farrant 1995) typical of ultra-drying techniques (Li et
al. 2007). Rosenberg and Rinne (1986) suggested that improved seedling growth
may be explained by the dependence of the initiation of cell division in the
root and shoot meristems on a drying treatment during seed development.
There is some
evidence that ultra-drying seeds of species and cultivar down to specific
critical moisture contents may also increase seedling tolerance to other
stresses caused by salinity and soil pH. For example,
Huo et al. (2015) reported preliminary results
showing that ultra-drying accelerated germination and seedling emergence of
alfalfa subjected to alkali stress. This suggests that ultra-drying may be a
useful process to improve establishment in soils considered marginal for
planting alfalfa, especially in western China where alfalfa is the most
important forage species currently sown to both improve soil quality and support ruminant production (Jiang et
al. 2007). However, before
these claims of stress tolerance can be substantiated, it is essential to gain
a better understanding of the effects of seed ultra-drying on the growth and
physiology of alfalfa in unstressed conditions. The aim of this study was to
investigate whether the ultra-drying of alfalfa seeds and then storing at room
temperature would provide better seedling growth and vigour,
and to identify possible mechanisms that explain differences in the growth of
alfalfa seedlings grown from normal and ultra-dried seed by monitoring key
physical and physiological indices in both seed
and seedlings.
Materials and Methods
Alfalfa seed source and ultra-drying treatment
Seeds
(~1 kg) of Medicago sativa L. cv.
Longdong were collected from 200 plants in October
2007 from the Lanzhou Forage Station (36°05′26” N, 103°41′57” E)
which forms part of Gansu Agricultural University, Gansu Province, China. The
initial seed purity at time of collection was 99 and 79% of initial germination
percentage (GP), and the initial moisture content (MC) of 9.05%. The seeds were stored at room temperature and humidity in
ventilated darkness for 1.5 years until
required for the experiments.
Approximately 160 g of stored seed was packaged in
porous nylon bags (20 g/bag), placed into a cooled hermetic desiccator and
buried in silica gel that diurnally dried at 120oC. The ratio
between seeds and silica gel was 1 to 10 (w/w) (Zeng et al. 2006). After
desiccation for 12, 24, 48, 72, 96, 120, 144 and 216 h, seed MC was reduced to
7.10, 6.94, 6.36, 5.73, 5.47, 5.19, 4.98 and 4.60%, respectively, from an
initial MC of 9.0% for the control (Table 1). Seed MC was determined by drying
at 105oC for 72 h (James and Don 2005). Ultra-dried and control
seeds were sealed in aluminium foil packages and stored in a desiccator
fulfilled with dried silica gel at room temperature (24.5oC ± 1.5oC).
Pre-germination
treatment of ultra-dried seeds
Prior
to use in the following series of experiments and tests, ultra-dried seeds were
rehydrated to avoid imbibitional damage (Ellis et
al. 1990). Seeds
were placed into sealed glass desiccator for 24 h (24.5oC ± 1.5oC)
containing saturated CaCl2 solution to produce a relative humidity
(RH) of 35%, and then hydrated for another 24 h using NH4Cl (RH =
75%) (Huang and Gao 2000).
Lipid peroxidation and total soluble sugar
in ultra-dried seeds
Lipid peroxidation of seeds was
measured in terms of malondialdehyde (MDA) content (µmol/g FW) (Loreto and Velikova 2001).
For each ultra-dried treatment four replicates each of 0.5 g seeds were
homogenized in 2 mL 20% (v/v) trichloroacetic acid containing 0.5% (v/v) thiobarbituric acid. The mixture was heated for 30 min at
100°C to release protein-bound MDA, centrifuged at 10000 × g for 10 min after
cooling, and the absorbance of the supernatant read at 532 nm and 600 nm. The
absorbance at 600 nm was subtracted from the 532 nm reading, and the
concentration of MDA was calculated by the means of extinction coefficient of
155 L mM–1 cm-1
(James and Don 2005).
Total
soluble sugars were determined using the Anthrone
reagent following the method of Stieger and Feller
(1994). Four replicates of 0.5 g seeds from each treatment were homogenized and
the centrifuged filtrate (12000 g 10 min) (Chandel et al. 1995)
mixed with anthrone reagent (20 mL ethanol, 100
mL 1 M H2SO4,
200 mg anthrone) and heated for 10 min in a
vigorously boiling water bath. Samples were cooled on ice for 10 min
to stop the color development. Absorbance was read at 623 nm and soluble sugar
determined using a glucose (0–100 µg) calibration (Roulin and Feller 2001).
Seed germination test
The experiment was set up to
investigate the effects of ultra-drying treatment on germination of alfalfa.
Seed surfaces were sterilized using 10% (v/v) Na-hypochlorite before the
germination test and seedling growth. Four replicates of 50 seeds each were
then arranged on two layers of filter paper moistened with 5 mL de-ionized
distilled water in 6 cm diameter petri dishes. Dishes were placed in an
incubator (SPX-100B-Z Biochemical Incubator; Shanghai Boxun
Medical Equipment Factory, Shanghai China) with constant temperature (24°C) and
complete darkness. Germination was defined when the length of the radicle was 2
mm (Song et al. 2005). Germinated
seeds were counted daily. Seed vigour and viability
were quantified by the germination index (GI) for each ultra-drying treatment
which was calculated as the product of the germination percentage and the
hypocotyl length after 4 days of germination (Vertucci
et al. 1994).
Seedling growth responses
Alfalfa seeds were grown in sand
cultures to test the effect of ultra-drying on seedlings growth, biochemical
and physiology responses. Ten replicates each of 25 germinated seeds for each
seed ultra-dried treatment were sown in individual polyvinyl plastics pots (12
cm in diameter and 15 cm high), filled with dry river sand (2 mm sieve mesh,
washed with de-ionized water for 3 times and autoclaved for 2h at 121°C). At
the sowing time seeds in each pot were irrigated with 25% Hoagland’s solution
(Hoagland and Arnon 1938). The same solution which
was placed in polyvinyl plastic trays to a depth of 1 cm,
was topped up to the marked level with distilled water daily and changed
weekly. Pots were arranged in the trays in a randomized block design.
The
experiment was performed in controlled environment growth chamber at
Gansu Agricultural University (36°05′18′′,
103°42′8′′E); altitude 1520 m). Typical conditions during the
22-day treatment period included were temperature of 24°C to 28°C, relative
humidity 45 to 93%, and maximum photon-flux density under 12 h of light 1430
µmol m–2 s–1.
Shoot
height of 10 seedlings chosen randomly in each treatment was recorded at 5, 10,
15 and 22 days after sowing. Leaf photosynthetic activity was characterized by
the number of active PS II reaction centers determined
by chlorophyll fluorescence, and was measured using a fluorimeter (FMS-2, Hansatech Instruments Ltd., U.K.) in randomly selected,
fully expanded leaves. The optimal quantum yield of PS II (Fv/Fm),
reflecting the functional activity of PS II
reaction centers, was measured on 10 seedling chosen randomly in each treatment
4 h before plants were harvested 22 days after sowing pre-germinated seeds.
Surviving seedlings in each
treatment were counted, harvested and the leaves, stems and roots of each plant
were separated. Leaf number, root length, and fresh biomass of shoots and roots
of each seedling were recorded. The leaf area of 10 fully expanded leaves
chosen randomly in each treatment was measured using a CI-203 Leaf Area Meter
(CI-203, CID, Inc., USA). Physiological
measurements were done as soon as possible after plants were harvested.
Root dehydrogenase activity related to respiration
capacity was measured by the triphenyltetrazolium
chloride (TTC) reduction technique (Huang and Gao
2000). Roots (0.5 g) of each treatment were placed into
test tubes with 60 mM Na2HPO4–KH2PO4
containing 0.6% (w/v) TTC and 0.05% (w/v) Tween 20, incubated the tubes
for 20 h at 30°C. All formazens formed from the
reduction of TTC by dehydrogenase enzymes in living roots was
extracted in 95% (v/v) ethanol for 4 h at 60°C (all temperature
control were in incubators). The absorbance of the extractants was
read at 480 nm.
The
chlorophyll (a + b) content of randomly sampled leaves (0.5 g) was determined
spectrophotometrically in dimethyl sulfoxide (DMSO) (Sigma Chem.
Co., USA) using the method of Fridgen and Varco (2004).
Leaves were placed in vials containing 10 mL of DMSO for approximately 45 min
until all the chlorophyll (a + b) was removed and kept on ice in
darkness until analysis. Absorption was measured using a Spectrophotometer
(S2000, WPA Co., U.K.). The concentrations of chlorophyll a, b, and total chlorophyll were calculated using equations described by Barnes et al. (1992).
Lipid
peroxidation in leaves was measured in terms of MDA content (Cakmak and Horst 1991). Four replicates of
previously frozen alfalfa leaves (0.5 g) from each ultra-drying treatment were
homogenized in 2 mL 20% (v/v) trichloroacetic acid containing 0.5% (v/v) thiobarbituric acid and measured as described above.
Buffer-soluble
carbohydrates were measured according to Stieger and Feller (1994). The centrifuged filtrate (10–20 µL)
was mixed with 1 mL anthrone reagent (20 mL
ethanol, 200 mg anthrone, 100 mL H2SO4)
and heated for 10 min in a vigorously boiling water bath. Colour development was stopped by incubating the sample
on ice for 10 min, and the A623 was measured. Glucose (0–100
µg) was used for calibration (Roulin and
Feller 2001).
Four
replicates of seedling leaves (0.5 g) derived from each treatment were
homogenized and analyzed for soluble sugars according to the method described
above.
Statistical analysis
For each treatment ten replicates were set up in a
completely randomized design. Four tissue sample replicates
%20377-384_files/image002.jpg)
Fig. 1: Root dehydrogenase activity
measured in 22-day old seedlings derived from seeds with different MC (%).
Columns identified with the same letter are not significantly different at P < 0.05, after applying Duncan test
were used for
physiology and biomass analyses. Data is presented as mean ± S.E. and
differences of variables between treatments were compared using 1-way ANOVA
followed by Duncan’s method where P <
0.05.
Results
Effect of ultra-drying seed biochemistry and germination
Seed biochemistry:
Ultra-dry seeds exhibited a higher vigor level than the air-dried control (MC =
9.1%) indicating that the biochemical processes of stored alfalfa seeds are
tolerant to dehydration down to about 5% MC. Soluble sugar concentration
increased exponentially (R2 = 0.794) to a maximum at 5% MC relative
to the control (P < 0.05) and then
declined back to the control concentration at 4.6% MC (Table 1). In contrast,
MDA concentration decreased (P < 0.05)
to a minimum at 5.2% and increased second only to the control MDA concentration
at 4.6% MC (Table 1). This suggested that MC of 5% is near the optimal for
alfalfa seed at which the integrity of the biochemical processes are
maintained. There was little decrease in seed MC when the drying period was
extended beyond 216 h.
Germination: The
germination percentage (GP) of ultra-dry alfalfa seeds with MC ranging from 5.7
to 5.0% were higher (P < 0.05)
than the air-dried control seed by 8 to 13% (Table 1). However, for the seeds
with a MC of 4.6%, GP was significantly (P
＜ 0.05) lower than control
which reflects the impact of changes in seed biochemistry at seed MC <5%,
particularly total soluble sugars and MDA (Table 1). A fitted curvilinear
relation indicated that total soluble sugar content of seed explained 93% of
the variation in germination with germination >92.5% at total sugar content >64
mg g-1 FW. In contrast, MDA concentration tended to reduce
germination with >95% germination when MDA was <8.98 nmol g-1
FW. There was no consistent effect of ultra-drying on hypocotyl length measured
4 days after the start of germination.
Effect
of ultra-drying seeds on seedling growth and biochemistry
Seedling
growth and root dehydrogenase activity: Seedling
derived from seeds dried to a MC of 5.5 to 5.2% had significantly higher (P < 0.05) total biomass than the
control (Table 2). This was due to a difference in root mass 33 to 64% higher (P < 0.05) than the control because
there was no significant difference in shoot biomass between seed treatments
(Table 2). The higher production of root mass was not due to a more extensive
root system as several other treatments produced significantly (P < 0.05) more root length than
control (Table 2), but rather to thicker roots produced in seedlings derived
from seeds with MC 5.5 to 5.2%. Root dehydrogenase activity measured
in 22-day-old seedlings showed a cyclic pattern with an initial significant (P < 0.05) decline to a minimum for
germinating seeds with 6.9% MC, increasing to a maximum at 5.2% and then
declined again to the same minimum for germinating seeds with 4.6% MC (Fig. 1).
Although, there was no difference in
shoot mass between treatments and significant differences in the dynamics of
the above ground biomass such as shoot height, leaf number and leaf area.
Differences in shoot height Table 1: Characteristics of M. sativa (cv. LongDong)
seeds dried to different moisture contents and stored for one year
|
Ultra- dry period (h) |
Moisture content (%) |
Germination percentage (%) |
Hypocotyl length (mm) |
MDA content (nmol g-1 FW) |
Soluble sugar content (mg g-1 FW) |
|
0 |
9.05 ± 0.07 a |
85.50 ± 4.43 c |
52.38 ± 2.49 cd |
11.62 ± 0.22 a |
56.99 ± 0.14 e |
|
12 |
7.10 ± 0.02 b |
78.50 ± 2.52 d |
69.00 ± 2.58 a |
9.49 ± 0.42 b |
62.31 ± 0.53 d |
|
24 |
6.93 ± 0.01 c |
72.50 ± 2.52 e |
63.13 ± 4.33 b |
9.27 ± 0.21 bc |
61.23 ± 1.20 d |
|
48 |
6.36 ± 0.01 d |
89.50 ± 4.12 bc |
43.25 ± 2.06 e |
9.17 ± 0.15 bc |
61.81 ± 1.04 d |
|
72 |
5.73 ± 0.03 e |
94.50 ± 3.0 ab |
52.13 ± 2.96 cd |
8.98 ± 0.37 c |
64.04 ± 0.91 c |
|
96 |
5.47 ± 0.02 f |
96.50 ± 3.42 a |
53.25 ± 4.94 cd |
7.11 ± 0.31 e |
66.25 ± 0.95 b |
|
120 |
5.19 ± 0.05 g |
96.00 ± 3.65 a |
49.75 ± 3.09 d |
6.64 ± 0.36 f |
67.73 ± 0.42 a |
|
144 |
4.98 ± 0.02 h |
92.50 ± 3.00 ab |
65.25 ± 4.92 ab |
8.38 ± 0.37 d |
66.44 ± 0.50 b |
|
216 |
4.60 ± 0.02 i |
79.00 ± 3.46 d |
57.25 ± 2.06 c |
9.50 ± 0.31 b |
63.74 ± 0.22 c |
Means ± S.E (n =4). Different letters indicate
significant difference at P < 0.05
(Duncan test)
Table 2: Biomass
of roots and shoots, root length for M.
sativa seedlings derived from seeds with different MC (%) for 22 d
|
Moisture content (%) |
Root length (cm) |
Shoot biomass (mg) FW |
Root biomass (mg)
FW |
Total biomass (mg) FW |
|
9.05 |
6.7 ± 0.3 e |
105.3 ± 26.7 bc |
84.0 ±14.2 c |
189.2 ± 20.6 c |
|
7.10 |
7.6 ± 0.3 bc |
103.0 ± 14.4 bc |
110.1 ± 14.4 b |
213.1 ± 21.2 bc |
|
6.94 |
7.1 ± 0.4 cde |
105.0 ± 20.0 bc |
103.5 ± 21.3 b |
208.5 ± 5.1 bc |
|
6.36 |
7.1 ± 0.3 de |
105.9 ± 9.0 bc |
111.0 ± 19.2 b |
216.9 ± 28.2 bc
|
|
5.73 |
7.0 ± 0.2 de |
111.9 ± 12.3 bc |
102.0 ± 15.2 b |
213.9 ± 16.7 bc |
|
5.47 |
7.8 ± 0.2 ab |
126.4 ± 9 .5 a |
112.0 ± 21.3 b |
238.4 ± 29.8 ab |
|
5.19 |
8.1 ± 0.3 a |
119.0 ± 15.9bc |
138.9 ± 19.5 a |
258.0 ± 24.1 a |
|
4.98 |
7.5 ± 0.4 bcd |
102.0 ± 11.6 bc |
109.1 ± 14.9 b |
211.0 ± 13.8 bc |
|
4.60 |
7.4 ± 0.5 bcd |
98.1 ± 21 .0 c |
105.6 ± 10.7 b |
203.7 ± 26.1 bc |
Values are means ± S.E (n =4). Different letters
indicate significant difference at P <
0.05 (Duncan test)
were most
pronounced at day 22 of growth with seedlings derived from seeds dried to 5.7%
reaching maximum height 25% higher (P <
0.05) than the control (Fig. 2A). Enhanced growth from ultra-dried seed
increased with seedling age since there were no differences in seedling height
observed at day 5 of growth and only a few differences at day 10 and 15 (Fig.
2A). Seedling height differences were correlated to differences in leaf number
and leaf area. For leaf number, only seedlings from seeds dried to 5.5% MC
produced significantly (P < 0.05)
more leaves than the control (Fig. 2B). However, the level of ultra-drying had
broader positive and negative effects on leaf area. Seedlings derived from
seeds dried within a range 5.7 to 5.2% had up to 15% more (P < 0.05) leaf area than the control, whereas seedlings from
seeds dried to 5% or less had 9% less leaf area than the control measured at
day 22 (Fig. 2C).
Chlorophyll
content of seedling was not significantly different as a result of
pre-germination seed treatment, except for seeds with a MC of 6.9 and 5.0%,
where a significant (P < 0.05)
reduction and increase in chlorophyll content, respectively was found (Fig.
3A). Irrespective of seed MC, seedlings in this study maintained nearly maximum
quantum efficiency of PS Ⅱ photochemistry (Fv/Fm) (Fig. 3B).
Leaf MDA
content is a physiological index which reflects the degree of plant’s ability
to withstand injury or stress. The results showed a pattern of significant (P < 0.05) decline in MDA content of
seedlings derived from seeds with MC ranging from 9.1 to 5.5%, and a
significant increase in MDA content for seedlings from seeds dried to a MC
<5.5% (Fig. 4A). This is consistent with changes of MDA measured in seeds
(Table 1). The soluble sugar content of seedling leaves decreased (P < 0.05) in control for seedling
derived from seeds within the MC range of 7.1 to 5.0% (Fig. 4B). However, the
soluble sugar content of seedlings increased (P < 0.05) by 28% for seedlings derived from seed with MC of 4.6%
relative to the control (Fig. 4B).
Discussion
Desiccation tolerance is
one of the most fundamental properties of seeds (Leprince
et al. 1993; Chandel et al. 1995;
Stéphane et al. 2018) and the ability of seeds to remain viable under
severe desiccation is dependent on the drying rate and final seed moisture
content (Hill et al. 2010). Studies indicate that a seed MC of around 5%
is optimal for seed storage (Harrington 1972), depending on storage temperature
(Ellis and Hong 2006). Our results confirm findings reported by Ellis and Hong
(2006) that the low moisture content limit for alfalfa seed is in the range 4.2
to 5.5% with the upper value applicable when seeds are stored at room
temperature (24℃) as in this experiment.
The higher (P < 0.01)
germination observed at seed MC of 5.0 to 5.7% (Table 1) is related to
concentrations
%20377-384_files/image004.png)
Fig. 2: Growth of Medicago sativa seedlings derived from seeds with different MC (%). Number of leaves (A) and Leaf area (mm2) (B) at 22 d and Shoot height (C) at 5, 10, 15 and 22 d DAT, respectively. Values represent means ± S.E., n = 4. Values sharing same letters differ non-significantly (P > 0.05)
of total sugars and MDA. The
accumulation of sugars appears central to a seed’s ability to retain viability
under desiccation by protecting membranes and proteins, develop a glassy state
for intracellular water and provide a carbon reserve for germination (Hoekstra et
al. 2001). Although total sugar concentration was increased by ultra-dying
to 5% seed MC and explained a high proportion of the variability in germination
of alfalfa seed dried to different moisture contents, this correlation most
likely reflects an altered carbohydrate composition rather than simply the
total sugar concentration per se. Tetteroo et
al. (1994) reported that during slow dehydration of alfalfa seed the
carbohydrate content changed with oligosaccharides concentrations increased and
both glucose and fructose content decreased. This study measured total sugar
content, but the implication of increased total sugar
\%20377-384_files/image006.png)
Fig. 3: Chlorophyll
content (A) and optimal quantum
yield of PS II
photochemistry (Fv/Fm) (B) of seedlings derived from seeds with
different MC (%) for 22 d. Optimal quantum yield of PS
II
photochemistry (Fv/Fm) (B)
at midday in randomly selected, fully expanded leaves. Values represent means ±
S.E., n=4. Values sharing same letters differ non-significantly (P > 0.05)
%20377-384_files/image008.png)
Fig. 4: MDA content (A) and soluble sugar content (B) of leaves of seedling derived from
seeds with different MC (%) at 22 d. Values represent means ± S.E., n=4. Values
sharing same letters differ non-significantly (P > 0.05)
content observed at 5% seed MC
together with Tetteroo
et al. (1994) finding that a glassy state was formed during dehydration
with oligosaccharides as the possible stabilizer resulting in less
deterioration of ultra-dried seed than the control.
Another positive effect of
ultra-drying was a reduction in lipid peroxidation which has been identified as
the major cause of reduced viability, vigour and
germination percentage of stored seeds (McDonald 1999; Cakmak et al. 2010). Determination of
MDA provides a convenient method to quantify the extent of lipid peroxidation
(Goel et al. 2003). The lower level (P < 0.05) of MDA measured for alfalfa
seed with an MC of 5.5% in present experiment suggests that these seeds had an
efficient antioxidant defensive system that limited the degree of lipid
peroxidation relative to seeds dried to other moisture contents (Li et al.
2007) for Liminium aureum
(L.) Hill. Zhu and Chen (2007) reported that for
moderate ultra-drying of peanut (MC 2.0%) lipid peroxidation and radical
emergence where inhibited relative to peanut dried to MC 0.9%, especially after
accelerated aging. Seed aging does seem to exert an influence on lipid
peroxidation (Huo et al. 2011) and ultra-drying
treatment of accelerated aged alfalfa seeds to 5.72% seed MC helped to maintain
seed vigor due to lower lipid peroxidation.
Changes in seed biochemical composition caused by
ultra-drying were also linked to improved seedling performance other than
germination. While it is well documented that early seedling growth is
correlated directly with the quantity and utilization of seed reserves (Westoby et al.
1992), most often expressed as differences in seed size (Turnbull et al. 2008), there are few reports
describing the effects of changes in seed biochemical composition on seedling
growth. The study results showed a higher root mass and thicker roots produced
in seedlings derived from seeds with MC 5.5 to 5.2% suggesting that relative
nutrient allocation to these seedlings was higher than controls. Fits with Lloret et
al. (1999)
observation linking higher relative growth rate with nutrient allocation
(especially sugars) to root development.
Conclusion
The results provided an important insight into the
possible importance of ultra-drying as a practical means to improve alfalfa
establishment. And seedling derived from seeds dried to MC of 5.2% had higher
biomass, and higher root dehydrogenase activity. Enhanced growth from ultra-dried
seed increased with age with seedlings derived from seeds dried within a range
5.7 to 5.2%. Perhaps more important is the effect of the ultra-drying process
on seed biochemistry that promotes tolerance in alfalfa to stress in both
saline and alkaline conditions warrant for further studies.
Acknowledgements
This work was financially supported
by the National Natural Science Foundation of China (NSFC) (41561072) and the
National Natural Science Foundation of China (NSFC) (31660691) and Natural
Science Incentive project of Guizhou Province ([2018]5778-05) and Guizhou
Education University Academic Discipline Project (Biology). We thank Professor Shangli Shi and Professor Liming Hua (College of Grassland
Science, Gansu Agricultural University, China) for critical reading and correction
of the manuscript. We also thank anonymous reviewers for their substantial
contributions during the development of the manuscript.
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