Seed germination percentage
Dormancy is a mechanism for seed self-protection, which may give annual plants the possibility of longevity in harsh environments was reported by
Al-Namazi et al., (2020). Seed dormancy is influenced by a variety of factors such as mother genetics, growth environment and maturation
(Gresta et al., 2007). Jaganathan (2016) has found that seeds matured under dry, warm conditions are more likely to form impermeable seed coats.
Chen et al., (2019) have been proven fresh seed testa form impermeability after two or three years of dry storage.
Hudson et al., (2015) found that 25% of the seeds of plants are physically dormant because of their impermeable seed coat.
The study clearly measured the germination percentage of seeds from groups A and B. The hardness rate of hard seeds was as high as 99.33%, but when the seed coat was scratched the hardness rate decreased to 1.33% (Table 2). The final germination percentage of the seeds that had been scarified was much higher than that of the non-treated seeds. These results are in accordance with most other studies
(Al-Namazi et al., 2020; Baskin and Baskin 2004;
Naser et al., 2013).
Determination of water absorption
Hudson et al., (2015) showed that innate dormancy and impermeable seed coat are important factors affecting delayed seed germination. After seed maturation and drying, the seed coat provides coverage and protection for the embryo, forms a barrier between the embryo and its immediate environment and may trigger seed dormancy by controlling water uptake
(Qutob et al., 2008).
In the germination trials (Fig 1), the seeds treated with H
2SO
4 had a significantly (p<0.05) higher germination rates than the control group, showing that seed coat impermeability was an important reason preventing the seeds from absorbing moisture. Most research has indicated that a hard and dense seed coat structure causes a mechanical barrier to water absorption and the germination of seeds by
Zhu et al., (2005). In this study, the water uptake curves over 48 h varied significantly among treatments (Fig 1a). The initial weight of seeds in group A was 0.042 g and the final seed weight was 0.050 g. The weight of seeds in group B was 0.121 g at 48 h, which was 2.42 times higher than that of group A (Fig 1b). This indicates that the water absorption of seeds increased significantly after the seed coat had been scarified.
Inhibitory effects on Medicago falcata L. seed
Germination inhibitors have been demonstrated to delay or inhibit germination of seeds of different plants. Inhibitors are not only present in seeds and fruits, but are present in individual plant parts depending on the species
(Evenari, 1949). Some secondary metabolites present in plant seeds may prevent germination. For example, 58 endogenous compounds were identified in the endocarp of
Cinnamomum migao seeds, including a phenolic compound, aldehydes, ethers and lipids, among others
(Chen et al., 2022).
In this study, the seed growth of the three materials showed an overall decreasing trend with increasing concentration of the extract, indicating that a high concentration of the extract had a certain inhibition effect on seed germination and growth (Fig 2). When treated with 80% and 100% extract concentration, the GR of B1 was reduced by 10.07% and 9.48%, respectively, compared to the control, but there was no significant (
p>0.05) difference in seedling GP, seedling growth potential or seedling length at 80% and 100% concentrations compared to the control treatment (Fig 2B1). This indicates that the high concentration of seed inhibitor hinders the germination of alfalfa seeds, the finding that is in agreement with
Zhao et al., (2019) and
Chen et al., 2022). GR of M2 seeds after 20%, 40% and 60% concentration treatments were unchanged compared to the control and all treatments had a GR of approximately 24%, while GR after 80% and 100% treatments were 7.02% and 7.86% lower than the control, respectively. In addition, there was no significant (
p>0.05) difference in seedling growth potential and seedling length following each treatment (Fig 2M2). The GR under the 100% treatment was significantly (p<0.05) lower than the 20%, 40%, 60% and control treatments and the GR of M3 decreased with increasing inhibitor concentration. This indicates that the dormancy of
M. falcata L. is not only due to the impermeability of the seed coat, but may also be due to other reasons. The results of this study are similar with
Bolingue et al. (2010), which found that the model legume
Medicago truncatula exhibits both PY and PD. But further confirmation is needed regarding the existence of other types of
M. falcata L.dormancy.
Initial point of water entry into seeds
The hilum, lens and micropyle are all considered to be the initial points where water enters the seed. The hilum is the mark of the seed and funiculus when the seed is shedding, with a depression in the middle of the hilum. It is symmetrical on both sides and separated by an obvious slit that points to a set of tracheid bars inside the hilum and extends to the micropyle in one section of the hilum. In legumes, seeds that are released from dormancy absorb water mainly through the micropyle and lens and these structures are associated with the swelling of seeds after breaking dormancy
(Baskin, 2003;
Gama et al., 2013; Baskin et al., 2008; de
Paula et al., 2012). Lersten (1982) showed that seeds with a thicker seed coat require a shorter swelling time for water to diffuse through the micropyle to the hilum and transport water directly to the radicle via the tracheid bar.
In our study, the untreated seeds did not swell within 48 h. Fig 3 shows the staining of the seeds after the release of physical dormancy (Fig 3). At 2 h, some of the seeds absorbed the staining solution and began to swell and the hilum and the micropyle were the first to turn blue and the color deepened at 4 h, indicating that the hilum and the micropyle were the initial and main water absorption sites of the seeds. This also indicated that water uptake gradually increased with a longer seed soaking time. At 6 hours, the coloring solution permeated from the micropyle to the radicle and embryo root, followed by its diffusion to the upper end of the seed coat at 12 hours, ultimately resulting in complete coverage of the entire seed coat. After 24 hours, the rupture of the inner epidermis of the seed could be observed under a stereomicroscope. At this point, the dye solution was absorbed by the inner part of the seed and water uptake began in the embryo, with movement patterns similar to those of the seed coat. Similar results were found
Erika et al., (2016).
The initial water uptake stage of seeds occurs at other parts of the seed ridge, but this site may vary depending on the species. There are also differences in the initial water uptake sites of legume seeds after release of dormancy and it is believed that the lens, hilum and micropyle are all closely related to the water uptake of seeds under test
Smýkal et al., (2014) and
Harrison et al., (2021). In this experiment, sulfuric acid was used to treat alfalfa seeds and the initial water absorption point was determined to be the hilum without destroying the seed coat structure, but the initial water absorption site of seeds treated with scarification was the seed coat break, so this experiment only analyzed the water absorption of seeds after concentrated sulfuric acid treatment.
Break dormancy
Reports on the breaking of PY in legume seeds indicate that the main principle of breaking dormancy using physical or chemical methods is to weaken the water absorption barrier of the seed coat and enhance the water absorption capacity of seeds. In this study, different treatments were used to release alfalfa seeds from hardness and the effects of different methods to reduce seed dormancy were dissimilar. There were highly significant differences in GR and GP after sandpaper + ultrasonic treatment (Fig 4) compared with the control (
P<0.01), with increases of 95.33% and 90.95%, respectively. The difference in germination rate between the two treatment groups was also significant (
P<0.05), indicating that the combined effect of sandpaper and ultrasound to release dormancy was stronger than that of sandpaper alone. As a rapid and effective non-destructive method, ultrasound can not only improve the GR of seeds and the growth state of seedlings, but also delay the decline in seed GR during storage, improve the vitality of aging seeds and enhance the stress resistance of seeds. Similar results were reported by
Babaei et al., (2020); Abbaspour et al., (2019); Ding et al., (2018) and
Shekari et al., (2015) in dormancy release studies.
Acid corrosion treatment can corrode the local seed coat, break the barrier of fenestrated tissue and increase the permeability of the seed coat. In this study, 98% concentrated sulfuric acid was used to treat seeds (for 5, 10, 20, 40, 60 and 120 min) and the GI of seeds varied greatly with different treatment durations (Table 3). Germination ability showed a trend of first increasing and then decreasing with increasing soaking time. Germination capacity decreased sharply and the GR and GI were significantly lower than other treatments except with 5 min treatment (
P<0.05), indicating that the inappropriate treatment duration did not achieve the expected effect or harmed the seeds themselves. In this study, 98% sulfuric acid treatment for 40 min was more effective at relieving the PY of alfalfa seeds and the operation was simple and not laborious compared with the sandpaper treatment.
Temperature treatment is also a common method to relieve seed dormancy. However, there are differences in the treatment time and temperature required for different varieties.
Patane et al., (2006) have found that hot water treatment for 20 minutes can significantly improve the GR of
Astragalus hamosus seeds, but there was no significant effect on the germination rate of
Medicago orbicularis seeds. The seed GR of alfalfa was 3%~23% after hot water treatment at different temperatures (Fig 5). The GR of alfalfa treated at 70ºC for 10 min was higher than that treated at 50ºC (
P<0.05). At the same temperature, when the treatment time was increased to 20 min or 30 min, the GR of seeds was significantly lower than that at 10 min, but there was no significant difference in GP among different treatments (
P>0.05). The germination index of seeds treated at 90ºC was lower than that treated at 70
oC (
P<0.05). The VI of 50ºC, 60ºC and 90ºC treatments were significantly lower than the other two treatments (
P<0.05). Therefore, the appropriate temperature could effectively improve the germination rate of seeds and enhance the vigor of seedlings. Hot water treatment also has a positive impact on the release of dormancy in other leguminous plants, such as
Ceratonia siliqua L. and
Acacia mearnsii seeds under treatments
Cavallaro et al. (2021) and
Sao Jose et al., (2019).
