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).
Result
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