Background

Sophora davidii, also known as Tiemashao, Langyaci, Kuci, (Names of Sophora davidii in Chinese) Leguminosae papillionoideae sophora, and bush, widely distributes in the provinces of Hubei, Gansu, Shaanxi, Guizhou, Yunnan, Sichuan and Jiangsu of China. It could be a pioneer tree species for protecting water and soil excellently, recovering vegetation, and improving soil (The Institute of Botany, Chinese Academy of Sciences, 1981; Xu, 1997; Chen, 2002; Li et al., 2010).

The seed dormancy usually refers to the phenomenon that a viable seed can’t germinate normally under suitable conditions. It is a crucial characteristic of seed plants to adapt to environment changes and ensure seed germination at the most suitable time. It would be great significance to maintain the continuity of species. Current research showed several reasons of seeds dormancy: (1) the limitation of seed coat, which was airtight and impervious, or the seed coat was too thick; (2) the seeds that unfinished after-ripening needed to be treated with low temperature; (3) embryo was not fully developed; (4) the existence of inhibiting substances (Bewley, 1997; Yang et al., 2003; Tang et al., 2004; Pan et al., 2006; Pan and Xu, 2010). The treatments of hard seeds include three methods: physical method, chemical method and biological method. The physical methods include scraping seed coat, high temperature treatment, low temperature treatment, etc.; the chemical methods include acid etching, lye, hormone, etc.; the biological methods include the slow function of microorganism in nature, etc. (Li et al., 2010). At present, the research on seed coat treatment and its effect on seed germination rate had been carried out extensively. Among them, the authors of the passage: Study on the Properties of Hard and Germination of SophoraViciifolia Seed (Guo et al., 2010) dealt with the seeds of Sophora davidii by different methods. The research showed that the best method was breaking in seed coat by machinery, and the worst method was treated by hot water.

There are many kinds of GA, such as GA1, GA3, GA7, etc., it could effectively eliminate the dormancy of seeds, roots and buds, increase the content of IAA, promote cell division, induce synthesis α-amylase, speed up the starch hydrolysis in endosperm, and then, break seed dormancy. In this way, it could promote the germination of seed and the elongation of stem, and also facilitate the forming of male flower, plant blossoms, meanwhile promote the germination of seed (Wang et al., 2017). GA1, GA3, GA4, contained 3β-OH groups, so it had high bioactivity. And GA3 had the highest bioactivity among them, and it was the most widely used kind (Li D.L., 2013), which could improve the growth of seed and fruit, inhibit maturation, inhibit the dormancy and the senescence of lateral buds, as well as the formation of tuber (Pan et al., 2006). The research, which studied the germination effects of plant hormone (eg: IAA, 6-BA, GA3, etc.) and plant growth regulators on wheat seeds showed that GA3 might have optimal effects on improving the germination of seeds, but the germination rate was low and without the growth conditions of the plant after germination.

The application of GA was an ideal way to break seed dormancy in view of the wide spread and practicability of agriculture. But now, there was little research had been done to study the seeds of Sophora davidii with GA. In this study, the Sophora davidii seeds were treated by GA with different concentrations for different times. Then we measured physiological and biochemical indexes of the Sophora davidii seeds, as well as the sites of water absorption. This study aimed to clarify the effect on the growth conditions of roots and the seed germination by GA3 with different concentrations.

1 Results and Analyses

1.1 Morphological characteristics of Sophora davidii seeds

By observing the whole and anatomical map of the Sophora davidii seeds under anatomic microscope and recording the data, we can see that the seed of Sophora davidii is smaller and the seed coat is thicker (Table 1). The volume and quality of Sophora davidii seeds were relatively small which indicated that the injected-water volume was smaller and the seed coat was thicker, meanwhile, it would be the barrier to the absorption of water and gas exchange, and had bound effect on germination of seeds.

1.2 The effect on germination rates of Sophora davidii seeds treated by GA with different concentrations

The polishing group and unfinished group treated by GA3 with the same concentration gradient under the same condition were observed, from which we found the significant difference of germination rates. Took the seeds treated by GA3 with 200 mg/L and 500 mg/L concentration as an example. The germination rate of polishing group treated by GA3 up to 51% on the 3^th day, and the lowest rate was 32%; meanwhile, the group treated by GA3 without polishing started to sprout on the 5^th day. Compared the state of seeds treated by GA3 in two groups with the control group, we found the seeds treated by GA3 which sprouted on the1^st day and 5^th day was earlier than the seeds in control group which sprouted on the2^nd day and 8^th day. In the polishing group treated by GA3, GA3 with 200 mg/L concentration showed the biggest accelerating effect for seed germination. In the group without polishing treated by GA3, 100 mg/L concentration showed the biggest accelerating effect for seed germination (Figure 1; Figure 2).

Figure 1 Germination rates of Sophora davidii seeds treated by GA with different concentrations for different times after polishing

Figure 2 Germination rates of Sophora davidii seeds treated by GA with different concentrations for different times without polishing

Compared with the control group treated with vaseline seal, the germination rate of unsealed group was 38.46% from the beginning of germination to the 4^th day. However, from 5^th day to 13^th day, the maximum germination rate of every group except sealing raphe and hilum was 53.33%, which was higher than no sealing control group. The lowest germination rates of sealing raphe and hilum groups showed that raphe and hilum couldplay a big part in the water absorption and the germination of seeds. But there was a higher germination rate after sealing raphe and a lower germination rate after sealing hilum, which showed that the main water absorption site was raphe. In addition, the seeds could still germinate after sealing raphe and hilum, from which we found that other parts also could absorb water except raphe and hilum (Figure 3).

1.3 Effect of GA3 with different concentrations on root length of Sophora davidii

In the group treated by GA3 after polishing, compared with the control group treated with distilled water, the root growth rate of the seeds treated with GA3 was faster. And GA3 with 100 mg/L concentration had the biggest impact on the growth of roots, the average roots length appeared to 40.67 cm on the 10^th day (Figure 4).

Figure 4 Root length of Sophora davidii after the treatment of GA with different concentrations for different times after polishing

In the group treated by GA3 with no polishing, compared with the group treated by distilled water, GA3 with different concentration gradients all could make a more positive effect on roots, that is, in the same times, the roots treated by GA3 could grow better. For example, when the concentration of GA3 was 100 mg/L, the average root length was 42 cm on the 10^th day, but only 20 cm in the group treated by distilled water (Figure 5).

In the group with sealing treatment, the root of Sophora davidii seeds treated by distilled water grew gently, the length of root was shorter at the same times (Figure 6).

1.4 Effect of GA3 with different concentrations on the activity of a-amylase in Sophora davidii seeds

With the increase of GA concentrations, the activity of a-amylase increased first and decreased subsequently, and the optimal concentration was 200 mg/L, as well as the activity of a-amylase could up to 2.135 mg/(g·min). Treated by the GA with different concentrations, the activity of a-amylase also increased firstly and decreased subsequently with the treating time was prolonged. And the activity of a-amylase was highest on the 6^th day (Table 2).

2 Discussion

The hard seed of Sophora davidii belongs to the seed coat restriction, and the seed coat is very tough, which hindered the release of CO₂ and the entry of O₂, thus inhibited the seed respiration, It could not guarantee the enough energy supplied for the germination of seeds, and constrained the embryo. The results of this research confirmed the theoretical characteristics of Sophora davidii: smaller volume and quality, smaller water-absorption area, but it had thicker seed coats, during the process of germination and imbibition, embryo absorption and gas exchange would be impeded and limited, thus decreased the germination rate of seeds.

Without any treatment, the germination rate of Sophora davidii seeds is extremely low. And it could not accurately reflect the actual germination ability of the seed, and could not obtain greater economic benefit in actual production. This research found that the mechanically abrading promoted the germination of seeds, the germination rate could reach 94% on 10^th day, but the activity of a-amylase was weak and the germination rate was slow. From the 5^th day to the 10^th day, the optimal GA3 concentration of the seeds germination was 100 mg/L, but treated by optimal concentration of GA3, the germination rate of seeds on the 10^th was still 1.90%. It was an abnormal condition, and the reason for it was that the seeds were unfinished, the culture medium was not absorbed in time, the concentration of culture medium increased and the optimal concentration moved forward over time. The seeds treated by GA3 up to the maximum of germination on the 6^th day, which showed that the GA3 had a positive effect on the seed germination, could improve the activity of amylase in seeds, promote seed germination in advance, and it also could shorten the germination cycle.

Under the conditions of polishing treatment, the optimal concentration of GA3 for seeds was 200 mg/L, in case of treatment without polishing, the optimal concentration of GA3 for seeds was 100 mg/L. When the seeds treated by GA3 with 200 mg/L concentration after polishing, it had the strongest activity of amylase, the fast germination and the higher germination rate. GA3 improved the growth of roots after the germination of Sophora davidii seeds. And the optimal concentration of GA3 for seeds was 100 mg/L, because GA3 had induction for amylase, which could be stimulants of cell division. And the required concentration of the roots growth was different from the germination. The whole seed of Sophora davidii could absorb water, but the main part was hilum, the raphe was weak. This research provided a theoretical basis for the germination mechanism of Sophora davidii seeds, and it also could help the cultivation of Sophora davidii.

3 Materials and Methods

3.1 Experimental materials

The seeds of Sophora davidii picked from College of Agriculture, Guizhou University on October 20^th 2015 (Landmark: 26°25'39.62"N, 106°40'5.81"E).

3.2 Experimental reagents

GA3, starch, maltose, citric acid, sodium citrate, 3, 5-dinitrosalicylic acid, sodium hydroxide and seignette salt were taken from the laboratory of College of Agriculture, Guizhou University.

3.3 The preparation of reagents

Prepared the following solutions: (1) 1000 ml mother solution of GA3 with the concentration of 1000 mg/l. (2) 500 ml citric acid buffer solution with PH5.6 and the concentration of 0.1 mol/l. (3) 1000 ml 3, 5-dinitrosalicylic acid preserved in a dark place, sealed and refrigerated. (4) 100 ml maltose standard solution with the concentration of 1 mg/ml. (5) 100 ml NaOH with the concentration of 0.4 mol/l. (6) 500 ml starch solution with the concentration of 1%.

3.4 The treatment of seeds

This research took 300 seeds from provided seeds randomly, polished them by gauze until the scratches on them. And put them into constant temperature oven (25°C) with warm water of 25°C. After 36 h, the seeds should be cleaned and divided into 6 groups randomly, 50 seeds in each groups(Li, 2004); We took another 300 seeds without polishing, and put them into constant temperature oven (25°C) with warm water of 25°C. After 36 h, the seeds would be cleaned and divided into 6 groups randomly, 50 seeds in each groups.

3.5 The treatment methods of GA3

This research grouped the soaked seeds according to Table 3: (1) the group treated with GA3 after polishing; (2) the group treated with GA3 without polishing; (3) The group treated with sealing after polishing. Using solutions from 5 kinds of concentration gradients as culture solution for (1) and (2) respectively under the same conditions, cultivated them in 5 culture dishes, which had multi-layer gauze, set control group, and put them in constant temperature oven (25°C); (3) treated by sealing, and then cultivated it in culture dish with gauze and took distill water as culture solution. Then set a control group without sealing, and put it in constant temperature oven (25°C).

3.6 The physiological parameters monitoring of Sophora davidii seeds

In order to monitoring the physiological parameters of Sophora davidii seeds, this research used methods of weighing average quality of seeds, measuring the length, width and thickness, shooting the morphology of dry seed, measuring the thickness of seed coats, etc. by sensitive balance, vernier caliper, 01ympus SZX12 anatomical lens and the App of Image-Proplus.

3.7 The measurement of a-amylase activity

By 3,5-dinitrosalicylic acid method, this research measured the a-amylase activity of Sophora davidii seeds, which treated by GA with polishing on the 1^st day, 3^rd day, 6^th day and 9^th day respectively, measured the activity of seeds indirectly (Wang, 2006).

3.8 The measurement of the seeds germination rate

The seeds germination rate of (1), (2), (3) were measured respectively:

3.9 The measurement of the root growth of seeds

The each concentration and the growth of roots treated in (1), (2), (3) were measured respectively, the results were showed by scatter diagram.

3.10 The treatment of experimental data

This experiment used Image-Proplus, Excel and SPASS20.0 to handle and analyze statistical data, each experiment repeated at least 3 times.

Authors’ contributions

LJ was the executor of the experimental design and research. XR completed the data analysis and LC completed the first draft of this thesis. ZYF took part in the experiments, and YY was the designer and principal of this project. TXX directed the design of experiment, and SW was in charge of the writing and revising this thesis. All the authors read and approved the final manuscript.

Acknowledgments

This study was funded by the Science and technology projects in Guizhou (No. LH [2016] 7210), the key laboratory construction projects of the regulation of plant physiology and development in Guizhou, the key laboratory construction projects in Guizhou (No. z [2011] 4005), the innovation team in education ministries of biodiversity protection and sustainable utilization in karst mountain, the program for Changjiang Scholars and Innovative Research Team (IRT1227) and the program of doctor research start-up in Guizhou Normal University (11904-0514012).

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