Effects of Effects of Effects of Effects of eeeexogenous GA xogenous GA xogenous GA xogenous GA 3333 and warm water on dormancy breaking and warm water on dormancy breaking and warm water on dormancy breaking and warm water on dormancy breaking germination characteristics of germination characteristics of germination characteristics of germination characteristics of Eucommia ulmoides Eucommia ulmoides Eucommia ulmoides Eucommia ulmoides

AbstractAbstractAbstract Seeds of the plant Eucommia ulmoides Oliver, have prominent dormant characteristics. In the present study, seed dormancy and germination of E. ulmoides were investigated by treating them with exogenous GA 3 and different water temperatures. The results revealed that exogenous GA 3 and warm water soaking treatments were beneficial to the dormancy breaking and germination of seeds. The germination rate of seeds treated with 50 ℃ water was 84.67%. In comparison, the seeds treated with 300 mg·L -1 GA 3 had only a germination rate of 45%. This low germination rate resulted probably due to the low temperature of the GA 3 solution. Another important reason is the presence of eucommia gum in the seed coat of E. ulmoides which hinders the germination process. During germination, the SOD activity in the seeds treated with GA 3 (300 mg·L -1 ) and warm water (50 °C) increased significantly. Increased SOD activity reduced the degree of oxidative damage of the plasma membrane and resulted a continuous decrease in TBARS content. Thereby, the seeds were prompted to develop in a direction conducive to germination. In addition, GA 3 (300 mg·L -1 ) and warm water (50 ℃ ) treatments increased the content of endogenous GA 3 , IAA, and ZR in the seeds, and decreased the content of endogenous ABA. This had resulted significantly higher ratios of GA 3 /ABA, IAA/ABA and ZR/ABA than those ratios of distilled water (room temperature) treated plants. In short, 50 °C warm water treatment has a more marked germination promoting effect on E. ulmoides seeds.


Introduction Introduction Introduction Introduction
Eucommia ulmoides Oliver, is belonging to the Order Garryales and Family Eucommiaceae. It is a rare and valuable medicinal plant in the People's Republic of China. In China, the plant has been designated as a national second-class protected species, and is widely distributed in the subtropical to temperate regions. The provinces where the plant grows are Hunan, Guizhou, Sichuan, Henan and Shanxi (Wu et al., 2019). China has a long history of knowledge and use of E. ulmoides for at least 2,000 years. The famous Chinese herbology 'Compendium of Materia Medica', published in the Ming Dynasty, has systematically preserved the documents on comprehensive knowledge and recorded the experiences on E. ulmoides from previous dynasties (Feng and Liang, 1996). Equipped with modern science and technologies, people now have re-discovered the medicinal 2 value of E. ulmoides. In particular, the value of some secondary metabolites produced by E. ulmoides e.g., aucubin, pinoresinal diglycoside and chlorogenic acid, and these metabolites are the main components of antihypertensive drugs. Another secondary metabolite produced by this plant, syringin diglucoside, has the functions of enhancing memory, calming sedation, and lowering cholesterol (Li and Yan, 1986;Tang et al., 1998). In addition to its exceptional medicinal value, E. ulmoides is also an important rubber-producing plant worldwide. Eucommia, as an isomer of natural rubber (Hamann et al., 2002), has become the best natural polymer material alternative to natural rubber (Du et al., 2003;Chen et al., 2014). Furthermore, E. ulmoides is also a good timber plant species, and has been widely planted in urban gardening as an excellent ornamental plant as well.
Seedling cultivation by sowing, is one of the main means for producing E. ulmoides seedlings. But so far, the researchers have mainly focused on the dormancy and germination characteristics of seeds. However, there is no research on the physiological characteristics during the germination of seed. Shen and He (2005) revealed two reasons regarding the germination of E. ulmoides seeds, and these are the sensitivity to germination temperature and the mechanical restraint from the peel gum which restrict and hinder the germination of seeds and cause dormancy. Wu and Huang (1989) reported that the most suitable conditions for the germination of seeds are: mixed sand, wet storage and a temperature of 20-25 °C. The research of Zhou (2016) showed that soaking seeds with warm water or solutions of plant growth regulators (i.e., gibberellin) can significantly increase the germination rate. The seed germination process is accompanied by a series of complex and orderly physiological changes such as respiratory metabolism, metabolism and transformation of storage substance. Exploring the changes in enzymes and endogenous hormones involved in these physiological activities can provide insights into the germination and emergence mechanisms of E. ulmoides seeds.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
In October 2018, seeds were collected from the E. ulmoides Planting Centre in Yichuan County, Henan Province. Healthy seeds were screened via soaking with tap water in a pot. The seeds were stirred thoroughly in the pot for 8-10 min. The floating seeds were discharged as abortive. The seeds remained in the bottom of the pot were rinsed with water, dried and used as the experimental material. Seeds having full shape and uniform size were selected and sterilised by soaking with 5% sodium hypochlorite solution for 10 min. The sterilised seeds were then rinsed with distilled water for three times and dried.

Seed germination assay
The dried seeds were soaked separately in two groups. Group 1: at room temperature (18 ℃ ± 2 ℃), the seeds were separately soaked in solutions containing 0 (CK), 100, 150, 200, 250, 300 mg·L -1 GA3 for 24 h. In this treatment CK is a distilled water control. Group 2: the seeds were put into beakers in a water bath setting temperatures at 0 (CK), 40, 50, 60 and 70 ℃. In this treatment seeds of CK were incubated at room temperature and considered as control of the experiment. The germination assay was carried out by using the 'Petri dish/filter' paper method. The treated seeds were uniformly arranged in a 9 cm Petri dish (10 seeds · dish -1 ) covered with double-layer filter paper. For each replicate, 5 Petri dishes were used. Each treatment had three replicates and placed in the growth chambers with a setting of 12/12 h and 25/20℃ day/night cycle. The germination rate assay was performed for a total of 15 days. Germination of the experimental seeds were considered when the radicle length reached 1/2 of the seed length. The number of seed germinations was counted every day and distilled water was added regularly to keep the filter paper moist. The seed germination rate was calculated at the end of the experiment.

Statistical analysis
One-way analysis of variance (ANOVA) was used to compare the differences in the same index after with exogenous GA3 or different water temperatures treatments. Statistical values are expressed as mean (± SE). All statistical analysis were carried out using SPSS 18.0.

Results Results Results
With the increase of GA3 concentration, the germination rate of seeds increased rapidly ( Figure 1). For the 300 mg·L -1 treatment, the germination rate reached 45%, and it was significantly different from the CK (P < 0.05) ( Figure 1). In addition, soaking seeds in warm water also significantly promoted the germination. But the germination rate gradually decreased with the increase of water temperature (Figure 1 A). Among them, the germination rate of the 50 ℃ warm water treatment reached its maximum value (84.67%) and was significantly higher than the CK (P<0.05) (Figure 1 B). The SOD activity of seeds with different soaking treatments continued increased during the germination process ( Figure 2). Among them, the SOD activity of GA3 (300 mg·L -1 ) and warm water (50 ℃) treatment showed significant differences compared to the CK on the 10th and 5th day respectively (P < 0.05) ( Figure 2A). In addition, the SOD activity values of GA3 (300 mg·L -1 ) and warm water (50 ℃) treatments were significantly higher than that of distilled water (room temperature) treatment from the 5th day. The TBARS content of seeds with different treatments continued to decreased during the germination process ( Figure 2B). Among them, the TBARS content of GA3 (300 mg·L -1 ) and warm water (50 ℃) treatment showed significant differences compared to the CK on the 10th and 5th day respectively (P < 0.05). However, the difference between TBARS content in seeds treated by distilled water (room temperature) compared to CK did not reach a significant level on the 15th day.   The contents of endogenous GA3, IAA and ZR in the seeds of different soaking treatments increased continuously, while the content of ABA decreased continuously during the germination process ( Figure 3). Among them, the ABA content decreased to a significant level (P < 0.05) compared to CK on the 15th day ( Figure 3C), but the endogenous GA3, IAA and ZR contents were not different from CK in distilled water treatment (room temperature). In addition, the endogenous GA3 and IAA contents were significantly different from the control group on the 10th day (P<0.05) ( Figure 3A, B), but the ABA content was significantly different from the control group on the 5th day (P<0.05) in GA3 (300 mg·L -1 ) and warm water (50 ℃) treatments. Finally, the ZR content increased significantly compared to CK on the 15th day in the GA3 (300 mg·L -1 ) treatment (P<0.05), while the warm water (50 ℃) treatment showed a significant difference from the CK on the 5th day (P<0.05) ( Figure 3D).
The ratios of endogenous GA3/ABA, IAA/ABA and ZR/ABA of the seeds under different soaking treatments continued to increase during the germination process ( Figure 4). The endogenous GA3/ABA and IAA/ABA of the distilled water (room temperature) treatment increased significantly compared to CK on the 15th day (P<0.05) ( Figure 4A, B), while the ZR/ABA values was not significant difference from the CK on 15th day ( Figure 4C). In the treatment of GA3 solution (300 mg·L -1 ), endogenous GA3/ABA and IAA/ABA increased significantly compared to CK on the 5th day (P<0.05), while ZR/ABA values increased significantly compared to CK on the 15th day (P<0.05). However, in warm water (50 ℃) treatment, endogenous GA3/ABA, IAA/ABA, and ZR/ABA values all increased significantly compared to CK on the 5th day (P<0.05).       The values are shown as mean±SE. Different letters above the bars denote significant differences (P<0.05) in endogenous hormone ratio with Duncan's test with different treat time.

Discussion Discussion Discussion
Effects of different soaking treatments on the germination of seeds Gibberellin, as a signaling substance, plays an important role in promoting seed germination (Hamdaoui et al., 2021). A large number of studies have shown that exogenous GA3 treatment is helpful for seed dormancy and germination (Lai et al., 2017;Ma et al., 2018;Shang et al., 2019;Agostini et al., 2022). In addition, soaking seeds in warm water has the effect of softening the seed coat and increasing the permeability of the seed coat. Therefore, warm water soaking at a suitable temperature is beneficial for seed germination (Chen et al., 2017;Seng and Cheong, 2020;Yousif et al., 2020). This study confirmed that exogenous GA3 and warm water soaking treatments are beneficial to the dormancy breaking and germination of E. ulmoides seeds. However, when the concentration of GA3 solution reached 300 mg·L -1 , the seed germination rate was only 45%, which was far from the result obtained by Zhou (2016). According to Zhou (2016), the germination rate of seed over 80% occurred at 150 mg·L -1 GA3 solution. The main reason for the low germination rate in this study was the selection of experimental time period (November -December) when a relatively low temperature (experimental room temperature 18 ℃ ± 2 ℃) and short time prevailed. So, the soaking treatment with GA3 solution, though, can soften the seed coat with the effect of replacing its physiological post-ripening to some extent, the optimum was not fulfilled in the present study. In this experiment, the seed soaking with warm water promoted the germination of seeds to a great extent. Among them, the germination rate of warm water treatment at 40 °C and 50 ℃ both reached more than 80%. Afterwards, as the water temperature continues to 7 rise, the germination rate of the seeds gradually decreases. This may be related to the fact that seeds have already entered the stage of water swelling are still in a high-temperature water environment, which damages the embryo of the seeds and causes some seeds to die. This result is consistent with the observations obtained from Astragalus membranaceus Bge. var. (Chen et al., 2017) and Swainsonia salsula (Pall.) DC (Wang et al., 2011).

Changes in physiological and biochemical characteristics of seed germination
During the germination process, various metabolic activities are initiated inside the seeds. These active metabolic processes will produce a large number of reactive oxygen species, which will cause certain oxidative damage to cells and tissues (Yang et al., 2015;Farooq et al., 2021;Zhang et al., 2022). Plant antioxidant enzyme systems play an important role in removing and mitigating the damage caused by active oxygen to seed germination (Manjavachi et al., 2022). SOD, as the primary substance for scavenging free radicals in plants, converts the more toxic O 2-. into less toxic H2O2 through a disproportionation reaction. In this study, during the germination process, the SOD activities in seeds treated with GA3 (300 mg·L -1 ) and warm water (50℃) were significantly higher than that of the distilled water (room temperature) treatment. This indicates that different soaking treatments can significantly increase SOD activity, thereby reducing the damage of reactive oxygen species to the cell membrane system.
TBARS is an important indicator of the damage of plant plasma membranes. The changes in the membrane system will have a certain impact on seed viability (Xu et al., 2022). In the present study, during the germination process, the TBARS content in the seeds of different treatments continued to decrease. The TBARS contents in seeds treated with GA3 (300 mg·L -1 ) and warm water (50 ℃) were significantly lower than that of the distilled water (room temperature) treatment. These results indicate that different soaking treatments can reduce the degree of oxidative damage to the plasma membrane, which is beneficial for breaking seed dormancy.
Seed germination is closely related to changes in the content of endogenous hormones. Various hormones in seed can induce the decomposition and synthesis of storage substances and eventually cause seed germination (Ge et al., 2023). Endogenous GA3 accelerates the decomposition and synthesis of internal materials by increasing various enzyme activities, thereby promoting seed germination . ABA has the effect of antagonizing GA3. By inhibiting the normal metabolism of nucleic acids and interfering with the synthesis of ribonucleic acids, the seeds cannot conduct normal metabolic activities, which eventually leads to dormancy of the seeds Gao et al., 2021). In this study, the endogenous GA3 contents of seeds with GA3 (300 mg·L -1 ) and warm water (50 ℃) treatments were significantly higher than that in the distilled water (room temperature) treatment. It may be due to the continuous infiltration of external GA3 or because soaking seeds in warm water caused more bound GA3 in the seeds to gradually become free, and eventually led to an increase in endogenous GA3 content. In addition, the endogenous ABA contents of seeds in different treatments were significantly lower than that in distilled water (room temperature) treatment, and they continued to decline during the germination process. This shows that exogenous GA3 and warm water soaking treatment promote the increase of the endogenous GA3 content of the seeds. The treatment also reduces the endogenous ABA content, thereby promoting the seed to develop in a direction favourable to germination.
There are different research conclusions about the relation between IAA and seed dormancy. Some studies have suggested that IAA can promote the termination of seed dormancy (Zhao et al., 2020;Wu et al., 2022). While others suggested that the content of IAA has little relation with the termination of seed dormancy (Lu et al., 2014). ZR has an antagonistic effect on seed germination inhibitors, and to a certain extent it is negatively correlated with seed dormancy . In this study, the contents of endogenous IAA and ZR in seeds treated with GA3 (300 mg·L -1 ) and warm water (50 ℃) treatment were significantly higher than those treated with distilled water (room temperature). It again showed that exogenous GA3 and 8 warm water-soaked seeds enhanced the content of endogenous hormones that promote germination and eventually leaded the germination of E. ulmoides seeds.
In contrast to a single endogenous hormone, changes in the balance between different hormones are more important for seed dormancy and germination. Especially, the ratio between promoting and inhibiting growth hormones (Su et al., 2018;Wang et al., 2020). In this study, the ratio of GA3/ABA, IAA/ABA, and ZR/ABA in seeds treated with GA3 (300 mg·L -1 ) and warm water (50 ℃) were significantly higher than those treated with distilled water (room temperature). GA3 and warm water soaking treatments not only increased the growth hormone content in the seeds, but also reduced the content of endogenous hormones that inhibit growth. Thereby creating internal conditions that were beneficial to the germination of seeds. In addition, starting from the 5th day, the endogenous GA3, IAA and ZR contents, and the values of GA3/ABA, IAA/ABA, and ZR/ABA were significantly higher than the GA3 (300 mg·L -1 ) treatment. On the other hand, while the ABA content, which is not conducive to seed germination, was lower than that of GA3 (300 mg·L -1 ) treatment.

Conclusions Conclusions Conclusions Conclusions
Exogenous GA3 and warm water soaking treatments were beneficial to the dormancy breaking and germination of seeds. But compared to 300 mg·L -1 GA3, 50℃ warm water treatment has a more marked germination promoting effect on E. ulmoides seeds. This result is also consistent with the results of seed germination rate.

Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions
NW wrote the manuscript and designed experiments; SSX performed out experiments; NW and SSX analysed the experimental results. Both authors read and approved the final manuscript.
Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.

11
 Material disclaimer: The authors are fully responsible for their work and they hold sole responsibility for the articles published in the journal.  Maps and affiliations: The publisher stay neutral with regard to jurisdictional claims in published maps and institutional affiliations.  Responsibilities: The editors, editorial board and publisher do not assume any responsibility for the article's contents and for the authors' views expressed in their contributions. The statements and opinions published represent the views of the authors or persons to whom they are credited. Publication of research information does not constitute a recommendation or endorsement of products involved.