The aims of this work were to investigate the effect of individual factors and their levels on germination and dormancy and to determine the advisable conditions for germination, as well as to recommend a germination test method to evaluate the full germination capacity of
Seeds of two different origins were used for the germination experiments. Lot A was harvested in September 2012 in Vienna, Austria (48°15′23″N, 16°29′5″E). Lot B was obtained from a harvest in September 2011 in Rheinstetten-Forchheim, Germany (48°58′1″N, 8°20′3″E). The viability and the theoretical germination capacity of both seed lots (rep = 4 × 50) were determined by a tetrazolium test, which also counts seeds with a physiological dormancy as viable (Baskin and Baskin, 2014). The germination potential was 97.5 ± 2.2% for lot A and 98.5 ± 0.9% for lot B, providing an excellent basis for the subsequent tests.
The experimental design was based on several factors and factor levels. These were chosen based on literature values for germination requirements of achenes, other members of the Asteraceae, the results of preliminary experiments, and the climate conditions of
Medium: pleated paper (PP); top of paper (TP) Pretreatment: water; KNO3 – 200 mg/L; GA3 (Merck KGaA) – 500 mg/L Light (PAR between 400 and 700 nm): 24-h light; 12-h light Temperature: 20°C; 30°C; 20/30°C Prechilling: no prechilling; 7 days / 10°C; 7 days / 5°C; 7 days / 0°C
Each combination corresponded to a germination test method and was tested with seed lots A and B in a growth chamber (from the Kühlanlagenbau Fritz Lachmayr GesmbH, Kremsmünster, Austria). Based on sample size calculation (power 90%,
The evaluation was carried out after 7, 14, and 21 days. The seedlings were classified as normal seedlings, abnormal seedlings, or un-germinated seeds according to the International Rules for Seed Testing (ISTA, 2015). However, for evaluating dormancy, normal and abnormal seeds were both counted as germinated seeds.
The data set was analyzed with the statistics software SPSS V22.0. The requirements of the statistical tests were checked before and were fulfilled. Residuals were checked for normal distribution and homoscedasticity by using residual plots and statistical tests. An analysis of variance (ANOVA) was carried out with a mixed model approach to determine if the selected factors and their two-way interactions had a significant effect on germination capacity. The factors “medium, pretreatment, light, temperature and prechilling” were defined as fixed effects and the seed lot was defined as random effect. The significance level was chosen as 95%. Additionally, post-hoc analysis (Tukey-HSD) was applied for significant factors to separate the most effective factor levels.
Finally, the most promising factor levels were combined in a recommended germination test method and validated with the predicted expected values of the linear model with main effects and interactions.
The results of the germination capacity experiments ranged between 0 and 96% germinated seeds. With an unfavorable combination of factors, no seeds germinated, and with favorable combinations, up to 96% germinated. This effectively corresponds to the full germination potential of around 98%, which was determined in advance by a tetrazolium test. In addition, these tests showed that the embryos were fully developed and that water could enter through the seed coat to the embryo. Thus, a morphological and physical dormancy could be excluded.
All main factors and also almost all two-way interactions had a highly significant effect on germination (Table 1). The interaction of the individual factors caused a large degree of fluctuation. The standard deviation was between 20% and 30% for all factors.
Results of the mixed model analysis (ANOVA) Tabelle 1. Ergebnisse der gemischten Modellanalyse (ANOVA) The seeds from lot A had a germination capacity (overall factors and factor levels) of 34.0a ± 26.1%. Lot B, with 56.2b ± 27.9%, had significantly higher germination rates than lot A.Medium 14.6 <0.001 Light 221.3 <0.001 Temperature 6063.3 <0.001 Pretreatment 528.0 <0.001 Prechilling 540.7 <0.001 Lot 3213.7 <0.001 Medium × light 14.5 <0.001 Light × prechilling 18.0 <0.001 Light × pretreatment 2.7 0.065 Light × temperature 13.9 <0.001 Medium × prechilling 4.3 0.005 Medium × pretreatment 22.1 <0.001 Medium × temperature 75.3 <0.001 Pretreatment × prechilling 11.8 <0.001 Temperature × prechilling 39.8 <0.001 Temperature × pretreatment 11.5 <0.001
The mean value of germinated seeds with PP was about 1.7% higher than with TP (Table 2). Seeds in TP showed fungal infections. In contrast, the folds in the pleated paper (PP) served as a barrier, preventing fungi from growing from one seed to the next. PP seems better suited for the germination of
Mean value and standard deviation of germinated seeds for selected factor levels Tabelle 2. Mittelwert und Standardabweichung gekeimter Samen für ausgewählte Faktorstufen The factor levels marked with different letters (a–d) are significantly different (α= 5%) by Tukey-HSD test. See additional data in Table 4.Factors Factor levels Medium Pleated paper (PP) Top of paper (TP) 48.3b ± 30.7% 46.6a ± 30.4% Pretreatment Water KNO3 (200 mg/L) GA3 (500 mg/L) 39.3a ± 29.8% 47.0b ± 30.6% 55.9c ± 28.9% Light 24-hours light 12-hours light 44.3a ± 29.4% 50.6b ± 31.4% Temperature 20°C 30°C 20/30°C 18.6a ± 18.7% 48.5b ± 21.0% 75.2c ± 20.2% Prechilling No prechilling 7 days / 10°C 7 days / 5°C 7 days / 0°C 33.4a ± 26.6% 48.8b ± 31.1% 51.8c ± 29.9% 55.7d ± 29.6%
Resulting germination test method for Silphium perfoliatum L. to exploit the germination potential Tabelle 3. Resultierende Keimfähigkeitstestmethode für Silphium perfoliatum L. zur Ausschöpfung des Keimfähigkeitspotentials Alternating temperature and light regime: first temperature 12 hours with light, second temperature 12 hours without light. Alternating temperature and light regime: first temperature 12 hours with light, second temperature 12 hours without light.Species Substrate Temperature (°C) First count (days) Final count (days) Recommendations for breaking dormancy Additional directions Additional advice PP 20/30 7 21 GA3; prechill - prechill 7 days / 0°C; L/D
Multiple Comparisons of the selected factors temperature, pretreatment and prechilling Tabelle 4. Mehrfachvergleiche der ausgewählten Faktoren Temperatur, Vorbehandlung und Vorkühlung The mean difference is significant at the 0.05 level. Based on observed means. The error term is Mean Square(Error) = 101.407.95% Confidence Interval (I) temperature (J) temperature Mean Diff. (I-J) Std. Error Sig. Lower Bound Upper Bound 20°C 30°C -29.87 0.514 <0.001 -31.08 -28.67 20<=>30°C -56.56 0.514 <0.001 -57.77 -55.35 30°C 20°C 29.87 0.514 <0.001 28.67 31.08 20<=>30°C -26.69 0.514 <0.001 -27.89 -25.48 20<=>30°C 20°C 56.56 0.514 <0.001 55.35 57.77 30°C 26.69 0.514 <0.001 25.48 27.89 (I) pretreatment (J) pretreatment H2O KNO3 -7.76 0.514 <0.001 -8.96 -6.55 GA3 -16.68 0.514 <0.001 -17.89 -15.48 KNO3 H2O 7.76 0.514 <0.001 6.55 8.96 GA3 -8.93 0.514 <0.001 -10.13 -7.72 GA3 H2O 16.68 0.514 <0.001 15.48 17.89 KNO3 8.93 0.514 <0.001 7.72 10.13 (I) prechilling (J) prechilling no prechilling 7d / 0°C -22.28 0.593 <0.001 -23.81 -20.76 7d / 5°C -18.39 0.593 <0.001 -19.91 -16.86 7d / 10°C -15.31 0.593 <0.001 -16.84 -13.79 7d / 0°C no prechilling 22.28 0.593 <0.001 20.76 23.81 7d / 5°C 3.90 0.593 <0.001 2.37 5.42 7d / 10°C 6.97 0.593 <0.001 5.45 8.50 7d / 5°C no prechilling 18.39 0.593 <0.001 16.86 19.91 7d / 0°C -3.90 0.593 <0.001 -5.42 -2.37 7d / 10°C 3.07 0.593 <0.001 1.55 4.60 7d / 10°C no prechilling 15.31 0.593 <0.001 13.79 16.84 7d / 0°C -6.97 0.593 <0.001 -8.50 -5.45 7d / 5°C -3.07 0.593 <0.001 -4.60 -1.55
GA3 and KNO3 solutions can affect the metabolic activity of seeds (Baskin and Baskin, 2004). Our experiments show that the treatment with GA3 and KNO3 solutions led to significantly higher germination than the untreated control with water (Table 2 and Figure 1). The greatest effect on germination capacity was achieved with GA3. GA3 has the opposite effect to abscisic acid (ABA) and the exact ratio of GA3 to ABA determines, on a plant-hormone level, whether a seed will start to germinate or remain dormant. ABA accumulates in the embryo during seed ripening and is responsible for primary dormancy (Kucera et al., 2005). In contrast, GA3 induces growth of the embryo and increases the availability of nutrients (Koornneef et al., 2002; Hilhorst, 1995). The ratio of GA3 and ABA at
Light and its intensity can have a substantial effect on dormancy and germination (Oh et al., 2006). Light had a positive effect on the germination of
Favorable environmental conditions, like specific temperature ranges, are important for germination; otherwise the seeds cannot germinate (Baskin and Baskin, 2004) or fall into secondary dormancy (Hilhorst, 1998; Finch-Savage and Leubner-Metzger, 2006). If seeds have physiological dormancy, alternating temperatures can be used to break their dormancy (Long et al., 2014).
Temperature had the greatest effect on germination capacity over all the five factors in our experiment. The differences between the temperature variants were highly significant. As Table 2 and Figure 1 show, the temperature cycles of 20/30°C every 12 hours showed distinctly more germination than constant temperatures at 20°C or 30°C. In combination with the GA3 solution, an average germination capacity of over 85% was achieved.
Trölenberg et al. (2012) extensively studied the effect of various constant and changing temperatures on the germination of
Several decades ago, Sokolov and Gritsak (1972) and Troxler and Daccord (1982) recommended sowing
The results confirmed that prechilling has a positive effect on the germination capacity. The germination capacity of the variants with prechilling (7 days / 10°C; 7 days / 5°C; 7 days / 0°C) was highly significantly higher than the untreated control (no prechilling). The differences between the individual prechilling temperatures were not pronounced, but still significant. Therefore, a prechilling phase at 0°C over 7 days is advisable to achieve a high germination capacity and to break the physiological dormancy.
The results are consistent with the conclusions of Vetter et al. (2010) and Trölenberg et al. (2012). They recommend a prechilling phase at 5°C over 5 and 7 days respectively. Franzaring et al. (2014) used a four-week prechilling period in climate chambers for their growth experiments with
The most promising factor levels were combined in a germination test method, which allows the assessment of the germination capacity of
The results of the germination characteristic of
In future experiments, for example, the influence of seed moisture content, harvest date and seed age on dormancy and different seed preparation should be tested and the laboratory-scale results need to be confirmed at field scale. Therefore applying additional pretreatments, like other biologically active compounds, priming and pelleting, are of great interest. For this, the results of this work can be incorporated.