A seed priming agent for elytrigia repens, its preparation method and application

Abstract

This invention discloses a seed initiator for *Elymus chinensis* (Leymus chinensis) under salt stress, its preparation method, and its application. The seed initiator regulates the endogenous hormone levels in *Elymus chinensis* seeds and significantly enhances the activity of antioxidant enzymes, thereby effectively alleviating salt stress-induced oxidative stress and antioxidant enzyme activity. It significantly improves the seed emergence rate, germination index, and vigor index of *Elymus chinensis* seeds, and shortens the average germination time, with better results than water-based initiation treatment. Comparative experiments confirm that this invention is not a universal seed treatment technology, but rather exhibits a specific promoting effect on the germination of *Elymus chinensis* seeds under salt stress. Furthermore, the preparation process is simple and applicable to pre-sowing seed treatment of *Elymus chinensis* and ecological restoration applications in saline-alkali soil areas. It is suitable for widespread application in the field of forage crop cultivation.

Description

Technical Field

[0001] This invention relates to the field of forage planting technology, specifically to a seed initiator for *Leymus chinensis*, its preparation method, and its application. Background Technology

[0002] Elymus nutans, a perennial high-quality forage grass belonging to the genus Elymus in the Poaceae family, is characterized by its strong adaptability, high nutritional value, and outstanding ecological restoration value. It has significant potential for widespread application in high-salinity areas such as Northwest my country and the Qinghai-Tibet Plateau. The seed germination period is one of the most sensitive stages to salt stress during the establishment of Elymus nutans. Salt stress reduces water absorption rate, inhibits radicle breakthrough, delays germination, and induces the accumulation of reactive oxygen species and membrane lipid peroxidation, resulting in a significant decrease in germination rate, germination index, and seedling vigor.

[0003] Seed initiation is a technique that improves seed germination uniformity and stress adaptability through pre-sowing water or chemical regulation. In recent years, nanomaterials, due to their size effect, high specific surface area, and strong interfacial activity, have been used in agricultural scenarios such as seed initiation, nutrient delivery, and stress regulation. Existing nano-seed treatment technologies are mostly concentrated on cash crops such as rice, cucumber, tomato, and soybean. The material systems often involve precious metal nanoparticles, composite nanomaterials, or carbon-based quantum dots. Some processes are complex and costly, and there is insufficient application foundation for the salt stress germination of forage seeds, especially drooping wheatgrass, in high-altitude / saline-alkali areas.

[0004] Currently, some studies have attempted to use nanomaterials to treat seeds in order to improve their stress resistance. For example, Chinese patent application CN119908367A discloses a method for promoting seed germination using nanomaterials. However, the inventors of this patent have found the following shortcomings in the prior art after in-depth research: (1) Its mechanism of action may mainly rely on the "water-induced" effect caused by the physical adsorption and water absorption of nanomaterials. This is a non-specific physical stimulus that may produce similar but weak effects on all seeds; (2) It lacks efficient and specific solutions for salt stress, a specific stress, and for the specific species *Leymus chinensis*; (3) Its effect is limited and does not reflect the differential regulation of germination behavior in different species. Therefore, the creativity and progress of its technology are limited. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a seed initiator for *Leymus chinensis* that can solve the problems of low germination rate, inhibited germination process, decreased vigor and oxidative damage of *Leymus chinensis* seeds under salt stress.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the seed initiator of *Leymus chinensis* is composed of a ZnO-NPs suspension, wherein the concentration of the ZnO-NPs suspension is 25-200 mg / L, the particle size of the ZnO-NPs is 30-80 nm, and the purity of the ZnO-NPs is ≥99.9%.

[0007] Furthermore, the concentration of the ZnO-NPs suspension is 25 mg / L.

[0008] This invention also provides a method for preparing a seed initiator for *Leymus chinensis*, wherein the method for preparing the ZnO-NPs suspension includes the following steps:

[0009] A. Weigh a certain amount of ZnO-NPs particles, then pour the ZnO-NPs into a beaker and add a certain volume of distilled water to obtain a ZnO-NPs suspension with a concentration of 25-200 mg / L.

[0010] B. The ZnO-NPs suspension was subjected to magnetic stirring.

[0011] C. The ZnO-NPs suspension treated in step B is subjected to ultrasonic treatment at room temperature to obtain a ZnO-NPs suspension, which is the seed initiator for Leymus chinensis.

[0012] Furthermore, in step B, a magnetic stirrer is used for magnetic stirring, with the magnetic stirrer rotating at 800 rpm, the temperature at 25°C, and the processing time at 10 min.

[0013] Furthermore, in step C, the ultrasonic frequency of the ultrasonic treatment is 30 kHz, and the treatment time is 30 min.

[0014] This invention also provides the application of a seed initiator for *Leymus chinensis* in promoting salt-tolerant germination of *Leymus chinensis* seeds.

[0015] The beneficial effects of this invention are as follows: The *Leymus chinensis* seed initiator described in this invention can regulate the endogenous hormone levels of *Leymus chinensis* seeds, significantly increase the activity of antioxidant enzymes such as SOD, POD, CAT, APX, GR, and DHAR, and increase the content of AsA and GSH. This effectively alleviates oxidative stress and antioxidant enzyme activity induced by salt stress, significantly improves the whitening rate, germination index, and vigor index of *Leymus chinensis* seeds, and shortens the average germination time. The effect is superior to water initiation treatment. Comparative experiments have confirmed that the same treatment has no significant germination-promoting effect on oat seeds of the same family, indicating that this invention has species specificity. Its mechanism of action surpasses the non-specific water initiation effect. This invention is not a universal seed treatment technology, but rather exhibits a specific promoting effect on the germination of *Leymus chinensis* seeds under salt stress. Furthermore, the preparation process of this invention is simple and applicable to pre-sowing seed treatment of *Leymus chinensis* and ecological restoration applications in saline-alkali soil areas. Attached Figure Description

[0016] Figure 1 The germination of *Leymus chinensis* seeds under different treatments in different embodiments on day 12;

[0017] Figure 2 The whitening rate of *Leymus chinensis* seeds under different treatments on day 6 and day 12 in different embodiments;

[0018] Figure 3 Germination index of *Leymus chinensis* seeds under different treatments in different embodiments;

[0019] Figure 4 Vigor index of *Leymus chinensis* seeds under different treatments in different embodiments;

[0020] Figure 5 Root and leaf lengths of *Leymus chinensis* seeds under different treatments in different embodiments;

[0021] Figure 6 Antioxidant enzyme activity of *Leymus chinensis* seeds under different treatments in different embodiments;

[0022] Figure 7 The antioxidant content of *Leymus chinensis* seeds under different treatments in different embodiments is shown. Detailed Implementation

[0023] To address the problems of low germination rate, inhibited germination process, decreased vigor, and oxidative damage in *Leymus chinensis* seeds under salt stress, this invention provides a *Leymus chinensis* seed initiator. This initiator regulates the endogenous hormone levels in *Leymus chinensis* seeds, significantly increases the activity of antioxidant enzymes such as SOD, POD, CAT, APX, GR, and DHAR, and enhances AsA and GSH content. This effectively alleviates salt stress-induced oxidative stress and antioxidant enzyme activity, significantly improving the whitening rate, germination index, vigor index, and shortening the average germination time of *Leymus chinensis* seeds. The effect is superior to water initiation treatment. Comparative experiments confirm that the same treatment has no significant germination-promoting effect on oat seeds of the same family, indicating that this invention is species-specific. Its mechanism of action surpasses the non-specific water initiation effect. This invention is not a universal seed treatment technology, but rather exhibits a specific promoting effect on the germination of *Leymus chinensis* seeds under salt stress. The seed initiator for *Leymus chinensis* consists of a ZnO-NPs suspension with a concentration of 25-200 mg / L, a particle size of 30-80 nm, and a purity of ≥99.9%. Under moderate salt stress conditions simulated by NaCl, 25 mg / L of ZnO-NPs significantly improved seed emergence rate, germination index, and vigor index, with better results than water initiation treatment.

[0024] This invention also provides a method for preparing a seed initiator for *Leymus chinensis*, wherein the method for preparing the ZnO-NPs suspension includes the following steps:

[0025] A. Weigh a certain amount of ZnO-NPs particles, then pour the ZnO-NPs into a beaker and add a certain volume of distilled water to obtain a ZnO-NPs suspension with a concentration of 25-200 mg / L.

[0026] B. The ZnO-NPs suspension was subjected to magnetic stirring. A magnetic stirrer was used for the magnetic stirring process, with a rotation speed of 800 rpm, a temperature of 25°C, and a processing time of 10 min.

[0027] C. The ZnO-NPs suspension treated in step B is subjected to ultrasonic treatment at room temperature to obtain ZnO-NPs suspension, which is the seed initiator of Leymus chinensis. The ultrasonic frequency of the ultrasonic treatment is 30 kHz and the treatment time is 30 min.

[0028] The above preparation process is simple and applicable to pre-sowing seed treatment of *Leymus chinensis* and ecological restoration applications in saline-alkali soil areas.

[0029] This invention also provides the application of *Leymus chinensis* seed initiator in promoting salt-tolerant germination of *Leymus chinensis* seeds. The verification method for this application includes: selecting plump, uniformly sized, and disease-free *Leymus chinensis* seeds; soaking the seeds in a ZnO-NPs suspension and initiating them at 25°C in the dark for 24 hours, shaking once every 3 hours during this period; after initiation, removing the seeds, draining or wiping off surface moisture, and air-drying them at room temperature to their original weight; placing the treated seeds in a germination medium containing 200 mmol / L NaCl and germinating them at 25°C under 8 hours of light / 16 hours of darkness.

[0030] Example 1 - Effects of Zn200: 200 mg / L ZnO-NPs suspension on seeds of Leymus chinensis and oats.

[0031] Step 1: Weigh 50 mg of ZnO-NPs particles, then pour the ZnO-NPs into a beaker and add distilled water to make up to 250 mL to obtain a ZnO-NPs suspension with a concentration of 200 mg / L.

[0032] Step 2: The ZnO-NPs suspension is magnetically stirred using a magnetic stirrer at a speed of 800 rpm, a temperature of 25°C, and a processing time of 10 min.

[0033] Step 3: The ZnO-NPs suspension treated in step B is subjected to ultrasonic treatment at room temperature to obtain ZnO-NPs suspension, which is the seed initiator of Leymus chinensis. The ultrasonic frequency of the ultrasonic treatment is 30 kHz and the treatment time is 30 min.

[0034] Step 4: Select plump and uniformly sized seeds of *Leymus chinensis* and oats. Soak 200 seeds in each of two separate petri dishes containing 25 mL of ZnO-NPs suspension. Initiate the seeds at 25°C in the dark for 24 h (*Leymus chinensis*) or 12 h (oats), shaking once every 3 h. After initiation, remove the seeds, remove surface moisture, and air-dry to their original weight. The resulting treatment is recorded as Zn200.

[0035] Step 5: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0036] Step 6: Arrange the seeds treated in steps 1 to 4 evenly (spaced ≥ 1 cm), 50 seeds per dish, with 4 replicates;

[0037] Step 7: Place the petri dish in a light incubator for germination. Add an equal amount of NaCl solution every 3-4 days to maintain humidity. The photoperiod is 8h / 16h, and the temperature is 25℃.

[0038] Step 8: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association (ISTA), and record the number of seeds that show white sprouts every 24 hours;

[0039] Step 9: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0040] Step 10: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0041] Example 2 - Effects of Zn100:100 mg / L nano zinc oxide suspension on seeds of Leymus chinensis and oats

[0042] Step 1: Using a 100ml graduated cylinder, accurately measure 100 mL of 200 mg / L ZnO-NPs suspension and add distilled water to 200 mL to dilute it to 100 mg / L ZnO-NPs suspension;

[0043] Step 2: Select plump and uniformly sized seeds of *Leymus chinensis* and oats. Soak 200 seeds in each of two petri dishes containing 25 mL of ZnO-NPs suspension. Initiate the seeds at 25°C in the dark for 24 h (*Leymus chinensis*) or 12 h (oats), shaking once every 3 h. After initiation, remove the seeds, remove surface moisture, and air-dry to their original weight. The resulting treatment is recorded as Zn200.

[0044] Step 3: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0045] Step 4: Arrange the seeds treated in steps 1 to 4 evenly (spaced ≥ 1 cm), 50 seeds per dish, with 4 replicates;

[0046] Step 5: Place the petri dish in a light incubator for germination. Replenish with an equal amount of NaCl solution every 3-4 days to maintain humidity. The photoperiod is 8h / 16h, and the temperature is 25℃.

[0047] Step 6: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association (ISTA), and record the number of seeds showing white sprouts every 24 hours;

[0048] Step 7: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0049] Step 8: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0050] Example 3 - Effects of Zn50: 50 mg / L nano zinc oxide suspension on seeds of Leymus chinensis and oats

[0051] Step 1: Using a 100ml graduated cylinder, accurately measure 100 mL of 100 mg / L ZnO-NPs suspension and add distilled water to 200 mL to dilute it to 50 mg / L ZnO-NPs suspension.

[0052] Step 2: Select plump and uniformly sized seeds of *Leymus chinensis* and oats. Soak 200 seeds in each of two petri dishes containing 25 mL of ZnO-NPs suspension. Initiate the seeds at 25°C in the dark for 24 h (*Leymus chinensis*) or 12 h (oats), shaking once every 3 h. After initiation, remove the seeds, remove surface moisture, and air-dry to their original weight. The resulting treatment is recorded as Zn200.

[0053] Step 3: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0054] Step 4: Arrange the seeds treated in steps 1 to 4 evenly (spaced ≥ 1 cm), 50 seeds per dish, with 4 replicates;

[0055] Step 5: Place the petri dish in a light incubator for germination. Replenish with an equal amount of NaCl solution every 3-4 days to maintain humidity. The photoperiod is 8h / 16h, and the temperature is 25℃.

[0056] Step 6: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association (ISTA), and record the number of seeds showing white sprouts every 24 hours;

[0057] Step 7: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0058] Step 8: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0059] Example 4 - Effects of Zn25: 25 mg / L nano zinc oxide suspension on seeds of Leymus chinensis and oats

[0060] Step 1: Using a 100ml graduated cylinder, accurately measure 100 mL of 50 mg / L ZnO-NPs suspension and add distilled water to 200 mL to dilute it to 25 mg / L ZnO-NPs suspension.

[0061] Step 2: Select plump and uniformly sized seeds of *Leymus chinensis* and oats. Soak 200 seeds in each of two petri dishes containing 25 mL of ZnO-NPs suspension. Initiate the seeds at 25°C in the dark for 24 h (*Leymus chinensis*) or 12 h (oats), shaking once every 3 h. After initiation, remove the seeds, remove surface moisture, and air-dry to their original weight. The resulting treatment is recorded as Zn200.

[0062] Step 3: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0063] Step 4: Arrange the seeds treated in steps 1 to 4 evenly (spaced ≥ 1 cm), 50 seeds per dish, with 4 replicates;

[0064] Step 5: Place the petri dish in a light incubator for germination. Replenish with an equal amount of NaCl solution every 3-4 days to maintain humidity. The photoperiod is 8h / 16h, and the temperature is 25℃.

[0065] Step 6: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association (ISTA), and record the number of seeds showing white sprouts every 24 hours;

[0066] Step 7: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0067] Step 8: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0068] Comparative Example 1-CK0: did not induce stress in seeds of *Leymus chinensis* and oats.

[0069] Step 1: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of distilled water;

[0070] Step 2: Select plump and uniformly sized seeds of Leymus chinensis and oat seeds and arrange them evenly (spaced ≥ 1 cm), with 50 seeds per dish and 4 replicates.

[0071] Step 3: Place the petri dish in a constant temperature incubator for germination, and add an equal amount of distilled water every 3-4 days to maintain humidity.

[0072] Step 4: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association, and record the number of seeds that show white sprouts every 24 hours.

[0073] Step 5: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0074] Step 6: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0075] Comparative Example 2-CK1: Stress alone did not induce seed rot in *Leymus chinensis* and oat seeds.

[0076] Step 1: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0077] Step 2: Select plump and uniformly sized seeds of Leymus chinensis and oat seeds and arrange them evenly (spaced ≥ 1 cm), with 50 seeds per dish and 4 replicates.

[0078] Step 3: Place the petri dish in a constant temperature incubator for germination, and add an equal amount of NaCl solution every 3-4 days to maintain humidity.

[0079] Step 4: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association, and record the number of seeds that show white sprouts every 24 hours.

[0080] Step 5: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings. Ten seedlings were measured for each replicate.

[0081] Step 6: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0082] Comparative Example 3-WT: Water-induced elymus seeds and oat seeds

[0083] Step 1: Select plump and uniformly sized seeds of Leymus chinensis and oat seeds. Soak 200 seeds in two petri dishes containing 25 mL of distilled water. Place them in a light incubator in the dark for 24 h and 12 h respectively, shaking once every 3 hours.

[0084] Step 2: After 12 hours and 24 hours, take out the seeds, wash them, wipe them dry, spread them flat on filter paper, and let them dry at room temperature until the moisture content returns to the original level.

[0085] Step 3: Line a 11.5×11.5cm petri dish with 3 layers of filter paper and add 10mL of 200mmol / L NaCl solution;

[0086] Step 4: Arrange the treated seeds evenly (spaced ≥ 1 cm), 50 seeds per dish, with 4 replicates;

[0087] Step 5: Place the petri dish in a constant temperature incubator for germination, and add an equal amount of NaCl solution every 3-4 days to maintain humidity;

[0088] Step 6: Perform germination tests on *Leymus chinensis* and oat seeds according to the standards of the International Seed Testing Association, and record the number of seeds that show white sprouts every 24 hours.

[0089] Step 7: On day 12, measure the leaf length (LL), root length (RL), and fresh weight (FW) of oat and crested wheatgrass seedlings, with 10 seedlings measured in each replicate.

[0090] Step 8: After sampling, the antioxidant enzyme activity and antioxidant content of *Leymus chinensis* seeds were determined using kits purchased from Beijing Solarbio Technology Co., Ltd. and Suzhou Keming Biotechnology Co., Ltd.

[0091] In the above examples, ZnO-NPs were purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., with a purity ≥99.9%, a particle size of 30-80 nm, and a specific surface area of ​​21.5 m² / g. The seeds of *Leymus chinensis* were selected as 'Sichuan West Leymus chinensis', with plump, uniformly sized seeds free from pests and diseases. The oat seed variety was 'Tyson', used for species-specific comparison experiments.

[0092] Seed emergence rate (%) = (Number of all seeds that showed signs of emergence in the experiment / Number of seeds supplied for the experiment) × 100%

[0093] Germination index = Germination index GI = ∑Gt / Dt; where: Gt is the number of germinated germinations on day t; Dt is the corresponding number of germination days.

[0094] Vitality index = ∑(Gt / Dt) × FW; where: Gt is the number of germinations on day t, Dt is the number of germination days (d), and FW is the fresh weight of seedlings.

[0095] The average germination time MGT = ∑(Gt×Dt) / ∑Gt; where: Gt is the number of germinations on day t; Dt is the corresponding number of germination days.

[0096] All data were analyzed using SPSS 26 one-way ANOVA, and multiple comparisons were performed using Duncan's new multiple range method. The significance level was P<0.05.

[0097] Comparative Example 1 (CK0) was the control group without initiation and without stress; Comparative Example 2 (CK1) was the control group without initiation but under stress; Comparative Example 3 (WT) was the control group initiated with distilled water and then under salt stress. Table 1 shows the effects of ZnO-NPs on the whitening rate, germination index, and vigor index of *Leymus chinensis* seeds.

[0098] Table 1. Effects of ZnO-NPs on the seed emergence rate, germination index, and vigor index of *Leymus chinensis*.

[0099]

[0100] Note: Different lowercase letters indicate that the differences between the means in the same column are significant at the P<0.05 level.

[0101] Figure 1 The germination of *Leymus chinensis* seeds under different treatments in different embodiments on day 12; Figure 2 The whitening rate of *Leymus chinensis* seeds under different treatments on day 6 and day 12 in different embodiments; Figure 3 Germination index of *Leymus chinensis* seeds under different treatments in different embodiments; Figure 4 The vigor index of *Leymus chinensis* seeds under different treatments in different embodiments; as shown in Table 1 and... Figure 1-4It was found that 200 mmol / L NaCl significantly inhibited the germination of *Leymus chinensis* seeds, with the emergence rate, germination index, and vigor index of CK1 decreasing to 37.5%, 13.14, and 1.27, respectively. Compared with CK1, ZnO-NPs initiation treatment generally improved the germination ability under salt stress, with Zn25 showing the most significant effect, increasing the emergence rate, germination index, and vigor index by 109.3%, 175.0%, and 235.0%, respectively; compared with water-initiated WT, Zn25 increased the emergence rate, germination index, and vigor index by approximately 34.1%, 55.4%, and 52.3%, respectively. Zn50 and Zn100 also showed some promoting effect, but their overall improvement was not as good as Zn25; the germination index of Zn200 treatment was not significantly better than WT. Therefore, 25 mg / L is the preferred concentration of this invention.

[0102] Table 2 shows the effects of ZnO-NPs on the fresh weight of seeds, root length and leaf length of Leymus chinensis.

[0103] Table 2 Effects of ZnO-NPs on seed fresh weight, root length and leaf length of *Leymus chinensis*

[0104]

[0105] Note: Different lowercase letters indicate that the differences between the means in the same column are significant at the P<0.05 level.

[0106] Figure 5 Root and leaf lengths of *Leymus chinensis* seeds under different treatments in different embodiments; from Table 2 and... Figure 5 Salt stress significantly reduced the fresh weight, root length, and leaf length of *Leymus chinensis* seedlings. Compared with CK1, the fresh weight and leaf length of the Zn25, Zn50, and Zn100 treatment groups were significantly increased, with Zn25 increasing leaf length by 472.7%, indicating that this treatment can effectively promote early seedling growth.

[0107] Table 3 Effects of ZnO-NPs on antioxidant enzyme activity in Leymus chinensis seeds (1)

[0108]

[0109] Table 4 Effects of ZnO-NPs on antioxidant enzyme activity in Leymus chinensis seeds (2)

[0110]

[0111] Note: Different lowercase letters indicate that the differences between the means in the same column are significant at the P<0.05 level.

[0112] Figure 6 The antioxidant enzyme activities of *Leymus chinensis* seeds under different treatments in different embodiments; as shown in Tables 3-4 and... Figure 6It was found that, compared with the normal control CK0, the salt stress control CK1 showed abnormal changes in the activities of SOD, POD, and CAT, and significantly reduced the activities of APX, GR, DHAR, and MDHAR, indicating that the seeds were under severe oxidative stress and the antioxidant cycle was inhibited. After treatment with ZnO-NPs, the activities of antioxidant enzymes increased, thereby scavenging excess reactive oxygen species. Among them, the Zn25 treatment group showed the most outstanding effect: compared with CK1, the activities of SOD, POD, and CAT increased significantly by 125.5%, 125.4%, and 127.6%, respectively; compared with WT, they increased significantly by 86.5%, 84.4%, and 95.4%, respectively; indicating that Zn25 effectively activated the antioxidant enzyme system of the seeds; at the same time, the activities of APX, GR, DHAR, and MDHAR were significantly higher than those of CK1 and WT. The Zn50 and Zn100 treatment groups showed some improvement in enzyme activity, but overall it was not as good as Zn25. The Zn200 treatment group had relatively low enzyme activity, and excessively high concentrations may inhibit the enzyme system response, so it is not suitable as the preferred concentration.

[0113] Table 5. Effects of ZnO-NPs on antioxidant content in Leymus chinensis seeds.

[0114]

[0115] Figure 7 The antioxidant content of *Leymus chinensis* seeds under different treatments in different embodiments; as shown in Table 5. Figure 7 It was found that salt stress caused an imbalance in the non-enzymatic antioxidant system of *Leymus chinensis* seeds. Compared with CK0, the contents of AsA, DHA, GSH, and GSSG in the CK1 and WT treatment groups were all imbalanced to varying degrees, with AsA and GSH contents being lower. After ZnO-NPs initiation treatment, the AsA content in the Zn25 treatment group increased by 50.8% compared to CK1 and by 64.5% compared to WT, and was the highest among all treatments; the DHA content was significantly higher than that in CK1 and WT; the GSH content increased by approximately 67.5% compared to CK1 and by approximately 57.0% compared to WT; the GSSG content increased accordingly, indicating that the glutathione redox cycle was effectively activated. The Zn50 and Zn100 treatment groups showed some antioxidant recovery effect, but the AsA and GSH contents were lower than those in Zn25; the recovery of antioxidant content in the Zn200 treatment group was limited.

[0116] Table 6 Effects of ZnO-NPs on oat seed whitening rate, germination index, and vigor index

[0117]

[0118] Note: Different lowercase letters indicate that the differences between the means in the same column are significant at the P<0.05 level.

[0119] As shown in Table 6, compared with CK1 and WT, the various concentrations of nano-zinc oxide treatment did not significantly promote the germination rate and germination index of oat seeds. This indicates that the nanomaterials of the present invention have no positive effect on the germination of oat seeds.

[0120] Table 7 Effects of ZnO-NPs on fresh weight, root length, and leaf length of oat seeds.

[0121]

[0122] Note: Different lowercase letters indicate that the differences between the means in the same column are significant at the P<0.05 level.

[0123] Table 7 shows that, compared with the salt stress group, the various concentrations of nano-zinc oxide treatment did not significantly promote the fresh weight and leaf length of oat seeds; 100 mg / L nano-zinc oxide significantly promoted root growth, but this does not mean it had a promoting effect on the oat seeds as a whole. This indicates that the nanomaterials of this invention have no positive effect on the germination of oat seeds.

[0124] Based on the germination phenotype, germination index, and physiological indicators, the preferred technical solution of this invention is a 25 mg / L ZnO-NPs initiation treatment; it significantly improves the whitening rate, germination index, and vigor index under salt stress; and by significantly increasing the activity of antioxidant enzymes such as SOD, POD, CAT, APX, GR, and DHAR, it increases the content of AsA and GSH, thereby reducing oxidative damage during seed germination.

[0125] Low concentrations of Zn25 showed the best performance. While Zn50 and Zn100 improved some germination or antioxidant indicators, their overall improvement was not as good as Zn25. High concentrations of Zn200 showed inhibitory effects on some indicators or had no significant promoting effect. Furthermore, the same treatment had no significant effect on promoting germination in oat seeds, further demonstrating that this invention is not a universal seed treatment technology, but rather a highly efficient solution specifically designed for the specific species *Leymus chinensis* under the specific stress of salt stress.

Claims

1. A seed initiator for *Leymus chinensis*, characterized in that: It consists of a ZnO-NPs suspension with a concentration of 25-200 mg / L, a ZnO-NPs particle size of 30-80 nm, and a ZnO-NPs purity of ≥99.9%.

2. The *Leymus chinensis* seed initiator as described in claim 1, characterized in that: The concentration of the ZnO-NPs suspension was 25 mg / L.

3. The method for preparing the *Leymus chinensis* seed initiator as described in claim 1, characterized in that: Includes the following steps: A. Weigh a certain amount of ZnO-NPs particles, then pour the ZnO-NPs into a beaker and add a certain volume of distilled water to obtain a ZnO-NPs suspension with a concentration of 25-200 mg / L. B. The ZnO-NPs suspension was subjected to magnetic stirring. C. The ZnO-NPs suspension treated in step B is subjected to ultrasonic treatment at room temperature to obtain a ZnO-NPs suspension, which is the seed initiator for Leymus chinensis.

4. The method for preparing the *Leymus chinensis* seed initiator as described in claim 3, characterized in that: In step B, a magnetic stirrer is used for magnetic stirring. The magnetic stirrer rotates at 800 rpm, the temperature is 25°C, and the processing time is 10 minutes.

5. The method for preparing the seed initiator of *Leymus chinensis* as described in claim 3, characterized in that: In step C, the ultrasonic frequency of the ultrasonic treatment is 30 kHz, and the treatment time is 30 min.

6. The application of the *Leymus chinensis* seed initiator as described in claim 1 or 2 in promoting salt-tolerant germination of *Leymus chinensis* seeds.

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