2.2 Measurements and Data CompilationThe germination of the three seeds was observed every day, and the
number of the seeds that had germinated were counted. The germination
potential was measured after 7 days of germination, and the percentage
of germination was calculated after 10 days. After counting the
germination number on the tenth day, 10 germinated seeds were randomly
selected with tweezers. The surface water was dried with filter paper,
and the length of buds and roots were measured with a vernier caliper,
and the germination rate, germination potential and germination index
were determined. The number of seeds that germinated was recorded until
no new seeds had germinated for three consecutive days. The seed
germination vigor (GV), final germination rate (FGR) and
germination index (GI) were calculated as described below:GV = (nt / N) × 100% 1
where nt is the cumulative number of germinated
seeds in 7 days, and N is the number of seeds used for the
treatment.FGR= (n / N) ×100% 2
where n is the number of seeds germinated in the treatment.GI = ∑ (Gt / Dt) 3
where Gt is the number of germinated seeds at t days, andDt is the number of the corresponding germination days.2.3 Statistical AnalysisStatistical analyses were conducted using SPSS 16.0 (SPSS, Inc.,
Chicago, IL, USA). It primarily includes a one-way analysis of variance
(ANOVA), two-way ANOVA, multiple comparative analysis and regression
simulation among others. The graphics were generated using SigmaPlot
10.0 (SYSTAT, Chicago, IL, USA).
3.1 Effects of pH on herbage seed germinationFigure 1. Germination parameters of D. glomerata,L. perenne, and M. sativa seeds under different pH values.
Capital letters indicate differences between species. Lowercase letters
indicate the difference between different treatments.
With the increase in pH value, the germination rate, germination
potential and germination index of the seeds of D. glomerata,L. perenne, and M. sativa first increased and then
decreased. The germination rate, germination potential and germination
index of M. sativa reached their maximum values when the pH value
was 6.61, 5.35, and 6.61, which was 83.33%, 76.0%, and 4.17,
respectively. The germination rate, germination potential and
germination index of D. glomerata and L. perenne were the
highest when pH was 6.61. Among them, the germination rate of D.
glomerata and L. perenne were 26.67% and 52.67%; the
germination potential was 12.67% and 25.33%, and the germination index
was 1.33 and 2.63, respectively (Figure 1).Figure 2. The lengths of seed buds and roots of D.
glomerata, L. perenne, and M. sativa seeds treated with
different pH values. Capital letters indicate differences between
species. Lowercase letters indicate the difference between different
treatments.
The bud and root lengths of D. glomerata, L. perenne, andM. sativa seeds all presented an “M” trend with increasing pH
values. The bud length and root length of the three types of seeds
reached their maximum value when the pH was 5.35 (Figure 2). The bud
lengths of D. glomerata, L. perenne, and M. sativawere 3.32 cm, 6.10 cm, and 1.42 cm, respectively, while the root lengths
were 4.22 cm, 5.03 cm, and 4.55 cm, respectively.3.2 Effects of CaCl2 on the germination of herbage
seedsThe germination rate, germination potential, and germination index ofD. glomerata, L. perenne, and M. sativa seeds all
increased first and then decreased as the concentration of
CaCl2 increased (Figure 3). The germination rate,
germination potential, and germination index of D. glomerata were
the highest when the CaCl2 concentration was 25 mmol/L,
which resulted in values of 38.0%, 20.0%, and 1.90, respectively. The
germination rate, germination potential, and germination index ofL. perenne were the highest when the CaCl2concentration was 100 mmol/L, 50 mmol/L, and 100 mmol/L, which were
68.67%, 52.67%, and 3.43, respectively. The germination rate,
germination potential, and germination index of M. sativa were
their highest when the CaCl2 concentration was 25
mmol/L, 0 mmol/L, and 25 mmol/L, which were 90.67%, 83.33%, and 4.53,
respectively.Figure 3. Germination parameters of D. glomerata,L. perenne, and M. sativa seeds under different
CaCl2 concentrations (mmol/L). Capital letters indicate
differences between species. Lowercase letters indicate the difference
between different treatments.
Similar to the differential responses for pH values, the bud lengths of
the M. sativa and L. perenne seeds presented an “M”
trend with increasing concentrations of CaCl2, while the
seeds of D. glomerata presented an “W” trend. The bud lengths
of M. sativa and L. perenne seeds were the highest when
the CaCl2 was 25 mmol/L, while the bud lengths ofD. glomerata seeds reached their maximum when there was no
CaCl2 in the treatment. The root lengths of the M.
sativa and D. glomerata seeds presented an “W” trend with
increasing CaCl2 concentrations similar to the bud
length of D. glomerata, while the L. perenne seeds
presented an “M” trend. The bud lengths of D. glomerata,L. perenne, and M. sativa seeds were the highest when the
concentration of CaCl2 was 50 mmol/L, 0 mmol/L, and 25
mmol/L, respectively (Figure 4).Figure 4. Seed bud and root lengths of D. glomerata,L. perenne, and M. sativa seeds under different
concentrations of CaCl2 (mmol/L). Capital letters
indicate differences between species. Lowercase letters indicate the
difference between different treatments.3.3 Interactive effects of pH and CaCl2 on the
germination of herbage seedsThe germination rates all showed M. sativa >L. perenne > D. glomerata. The seed
germination rate of the three herbage species all increased first and
then decreased with the increase in pH values at the same concentration
of CaCl2. The maximum seed germination rate was mostly
at pH 6.61. In contrast, except for D. glomerata at pH 5.3, at
the same pH value, the rate of germination of the three herbage species
increased first and then decreased with the increase in
CaCl2 concentration, and the seed germination rate was
mostly the highest when the CaCl2 concentration was 50
mmol/L or 20 mmol/L. The maximum value of the germination rate ofD. glomerata, L. perenne. and M. sativa was 40.67%, 58.0
%, and 88.0%, respectively, when the concentration of
CaCl2 was 25 mmol/L, 50 mmol/L, and 25 mmol/L,
respectively, and the pH values were all 6.61, respectively. When the pH
was 4.55, and the concentration of CaCl2 was 150 mmol/L
and 200 mmol/L, the seeds of D. glomerata did not germinate
(Figure 5).Figure 5. Germination rate of D. glomerata, L.
perenne, and M. sativa seeds under the interaction of pH and
CaCl2. Bar represents the mean ± standard deviation (n =
3). Letters represent the level of significance based on a one-way
analysis of variance (ANOVA) with a post hoc Duncan’s test (P ≤
0.05).
The seed germination potential experiments all also showed that M.
sativa > L. perenne > D.
glomerata. The germination rate of the D. glomerata, L. perenneseeds both increased first and then decreased with the increase in pH
value at the same CaCl2 concentration. The seeds
generally were the most likely to germinate at pH 6.61. In contrast, the
germination of D. glomerata and L. perenne seeds increased
first and then decreased with the increase in CaCl2concentration at the same pH value. The seeds of D. glomerata andL. perenne germinated at their highest rates when the
CaCl2 concentration was 25 mmol/L and 50 mmol/L,
respectively. The maximum value of the germination potential of D.
glomerata, L. perenne and M. sativa seeds was 15.33%, 35.33%,
and 84.0% when the CaCl2 concentration was 25 mmol/L,
50 mmol/L, and 25 mmol/L, respectively, and the pH values were all 6.61,
5.35, and 5.35, respectively. The seeds of D. glomerata did not
germinate when the pH was 4.55 or 5.35 and the CaCl2concentration was 150 mmol/L and 200 mmol/L or when the pH was 8.0 or
9.18 and the CaCl2 concentration was 200 mmol/L (Figure
6).Figure 6. Germination potential of D. glomerata,L. perenne, and M. sativa seeds under the interaction of
pH and CaCl2. The bar represents the mean ± standard
deviation (n=3). Letters represents the level of significance according
to a one-way analysis of variance (ANOVA) with a post hoc Duncan’s test
(P ≤ 0.05).
The germination index all also showed M. sativa >L. perenne > D. glomerata. The seed
germination index of the three herbage species all increased first and
then decreased with the increase in pH value at the same
CaCl2 concentration. The seeds germinated at the highest
rate when the pH was 6.61. In contrast, except for D. glomerataat pH 5.35, the seed germination index of the three herbage species
increased first and then decreased with the increase in
CaCl2 concentration at the same pH value. The seeds
primarily germinated at their highest rate when the
CaCl2 concentration was 50 mmol/L or 25 mmol/L. The
maximum value of the germination index of D. glomerata, L. perenne
and M. sativa was 2.03, 2.90, and 4.40 when the CaCl2concentration was 25, 50, and 25 mmol/L, respectively, and pH value was
5.35, 6.61, and 6.61, respectively. The seeds of D. glomerata did
not germinate when the CaCl2 concentration was 150
mmol/L and 200 mmol/L at pH 4.55 or when the CaCl2concentration was 200 mmol/L at pH 5.35 (Figure 7).Figure 7. Germination index of D. glomerata, L.
perenne, and M. sativa seeds under the interaction of pH and
CaCl2. The bar represents the mean ± standard deviation
(n = 3). Letters represent the level of significance based on a one-way
analysis of variance (ANOVA) with a post hoc Duncan’s test (P ≤
0.05).Table 2. Results (F-values) of an ANOVA of the pH and
CaCl2 and their interactions on the germination rate,
germination potential, and germination index of D. glomerata,L. perenne, and M. sativa