Method for improving seedling emergence of Astragalus adsurgens seeds under high-temperature drought stress in Coeruin sandy land

Abstract

The invention belongs to the technical field of seed processing, and provides a method for improving seedling emergence of Astragalus adsurgens seeds under high temperature and drought stress in Coeruin sandy land, the Astragalus adsurgens seeds are subjected to three-layer coating by seed initiation and biochar coating technologies and then are subjected to sand bed germination cultivation, and the germination effect of the method is evaluated; the result shows that the optimal use ratio of the coating is as follows: the powder of the first layer of coating is bentonite and talcum powder, and the mass ratio of the bentonite to the talcum powder to the seeds is 1: 1: 2; the second layer of coating powder is prepared from magnesium stearate, bentonite, talcum powder and biochar, and the mass ratio of the magnesium stearate to the bentonite to the talcum powder to the biochar to the seeds is 10: 5: 5: 4: 10; the third layer of coating powder is talcum powder, and the mass ratio of the talcum powder to the seeds is 2: 5; the three-layer coating treatment can significantly improve the seed vigor and the SOD activity, improve the emergence rate of the Astragalus adsurgens seedlings, promote the root growth of the Astragalus adsurgens seedlings, and solve the seedling emergence problem of the Astragalus adsurgens seeds under the high-temperature drought stress of the Colexin sandy land.

Description

Technical Field

[0001] This invention belongs to the field of seed processing technology, specifically relating to a method for improving the germination of *Salvia splendens* seeds under high temperature and drought stress in the Horqin Sandy Land. Background Technology

[0002] The Horqin Sandy Land is the largest of my country's four major sandy lands, characterized by a temperate semi-arid monsoon climate. The average annual temperature is 5.2–6.4℃, annual precipitation is 350–500 mm, and annual evaporation is 1600–2400 mm. Precipitation is unevenly distributed in time and space, gradually decreasing from southeast to northwest. 70% of the precipitation is concentrated in summer, while winter rainfall is scarce. The Horqin Sandy Land is dominated by fixed and semi-fixed sand dunes, with soil types primarily consisting of chestnut soil, black soil, and aeolian sand. These soils are coarse, arid, and infertile, unfavorable for plant growth, and the vegetation is mainly composed of psammophytic plants. With global warming and excessive human exploitation of land, soil desertification and degradation have intensified, and frequent high-temperature and drought stress events pose a serious threat to the ecological environment.

[0003] Astragalus adsurgens, a perennial herbaceous plant belonging to the genus Astragalus in the legume family, is named for its strong ability to resist wind and sand. It possesses biological characteristics such as cold resistance, drought resistance, wind and sand resistance, tolerance to poor soil, and salt tolerance. With its well-developed root system, Astragalus adsurgens effectively stabilizes the soil and prevents soil erosion, playing a vital role in the restoration of degraded grassland ecosystems and holding significant value for improving the ecological environment of the Horqin Sandy Land.

[0004] PEG initiation is a seed pretreatment method that involves placing seeds in a PEG solution to allow them to slowly absorb and swell, thus preparing them physiologically for germination. It regulates the physiological and metabolic activities within the seed, promotes the decomposition and transformation of stored substances, and provides more energy and nutrients for seed germination. At the same time, it can also significantly improve the seed's water absorption capacity, enzyme activity, and antioxidant capacity, thereby increasing the germination rate and seedling resistance, making them more adaptable to adverse environmental conditions such as high temperature and drought.

[0005] Biochar is a carbon-rich and stable organic material produced by the pyrolysis of biomass under anaerobic conditions. It possesses a large specific surface area, a porous internal structure, and abundant surface functional groups, thus exhibiting excellent adsorption and water retention capabilities. Using biochar as an active ingredient in seed coatings can effectively promote seed germination and seedling growth, enhance seedling resilience, and improve their survival ability under drought and other stress conditions, providing a new approach to solving vegetation restoration problems in arid regions.

[0006] Seed hybrid treatment technology combines different seed treatment methods to leverage their respective advantages and achieve better results. For example, combining initiation technology and coating technology can enhance the physiological activity of seeds during the pretreatment stage by initiation, and then provide protection and nutrition through coating, thereby enhancing the overall performance of the seeds and making them more competitive under complex environmental conditions, thus improving the success rate and stability of vegetation restoration.

[0007] In summary, this invention proposes a method to improve the seed germination of *Amaranthus praecox* seeds under high temperature and drought stress in the Horqin Sandy Land, thereby enhancing seed vigor and increasing the germination rate of *Amaranthus praecox* seedlings. Summary of the Invention

[0008] The purpose of this invention is to provide a method for improving the germination of alfalfa seeds under high temperature and drought stress in the Horqin Sandy Land. The method involves coating alfalfa seeds and applying the coating process to seedling cultivation, providing a new approach and means for improving the germination of alfalfa seeds under high temperature and drought stress.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] This invention provides a method for improving the germination of *Saussurea involucrata* seeds under high temperature and drought stress in the Horqin Sandy Land, specifically including the following steps:

[0011] (1) Seed initiation: Wet the filter paper with a PEG-6000 solution with a mass fraction of 5%~30%, sprinkle the seeds between two layers of filter paper for seed initiation, and dry at room temperature after initiation until the seed quality is restored to its original quality.

[0012] (2) Seed coating: Pour the seeds into the coating machine and coat them in three layers in sequence. In the second coating process, use corn straw biochar for biochar coating.

[0013] (3) Germination in sand bed: River sand is used as the germination medium. The coated seeds are placed in a light incubator for cultivation.

[0014] Preferably, the seed initiation conditions are: initiation with a 10% PEG solution for 6 hours.

[0015] Preferably, the three-layer coating specifically includes:

[0016] (1) First coating: Pour the seeds into the coating machine and adjust the speed until the seeds rotate along the inner wall and form obvious elliptical circles. Then, add 1% CMC aqueous solution to the atomizing plate to moisten the seeds but not stick them together. Then, add coating powder to it in small amounts and multiple times using a spatula until all the coating powder has been added.

[0017] (2) Second coating: After the first coating is completed, the seeds are idling in the coating machine for 1 minute. 1.5% CMC aqueous solution is added to the atomizing plate. At the same time, the second coating powder is added in small amounts and multiple times. The operation is repeated until the powder is added.

[0018] (3) Third coating: After the first two coatings are completed, the seeds are idled in the coating machine for 2 minutes. A 1.5% CMC aqueous solution is added to the atomizing plate, and the third coating powder is added in small amounts several times until all the powder is added.

[0019] Preferably, the powder for the first coating layer is bentonite, talc, and the mass ratio of bentonite, talc, and seeds is 1:1:2; the powder for the second coating layer is magnesium stearate, bentonite, talc, and biochar, and the mass ratio of magnesium stearate, bentonite, talc, biochar, and seeds is 10:5:5:4:10; and the powder for the third coating layer is talc, and the mass ratio of talc to seeds is 2:5.

[0020] Preferably, the germination of the sand bed specifically includes: in a 12×12×5 cm... 3 Dry sand and distilled water were added to a transparent petri dish, mixed and spread evenly. 100 seeds were placed in each dish, the lid was closed and the dish was weighed. The petri dishes were then placed in a light incubator and cultured for 14 days. The mass ratio of dry sand to distilled water was 50:1.

[0021] Preferably, the incubator conditions are: 35℃ light for 8 hours and 20℃ darkness for 16 hours, with water replenished evenly to the original weight daily using a small spray bottle during the incubation period.

[0022] The present invention also provides alfalfa seedlings obtained by the above method.

[0023] The present invention also provides the application of the above-described method in the cultivation of alfalfa seedlings.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention organically combines PEG initiation with corn stalk biochar coating technology to form a unique seed treatment method. PEG initiation can optimize the seed's imbibition characteristics and enhance its antioxidant capacity; biochar coating provides a water-retention and nutrient supply environment. The two work synergistically to significantly improve seed stress resistance and effectively solve the problem of seedling emergence of *Alternanthera philoxeroides* in the high-temperature and drought environment of the Horqin Sandy Land.

[0026] 2. This invention utilizes PEG and corn stalk biochar, both of which are environmentally friendly materials. The preparation of corn stalk biochar realizes the resource utilization of agricultural waste, while also having soil improvement functions. It can promote the optimization of sandy soil structure and the enhancement of fertility, and drive the sustainable restoration of sandy ecosystems, which is in line with the concepts of ecological protection and sustainable development. Attached Figure Description

[0027] Figure 1 These are the seed coating steps in this invention;

[0028] Figure 2 This invention relates to the effect of imbibition time on the water absorption rate of alfalfa seeds.

[0029] Figure 3 This invention describes the effect of PEG induction on the germination characteristics of *Saussurea involucrata* seeds under high temperature and drought stress (A represents germination rate; B represents average germination time; C represents germination rate index; P < 0.05, ns indicates no significant difference within groups; different letters indicate significant differences, the same letter indicates no significant difference between groups).

[0030] Figure 4 This invention describes the effect of PEG on the growth of *Saussurea involucrata* seedlings under high temperature and drought stress (A represents root length; B represents seedling length; C represents seedling fresh weight; D represents vigor index; P < 0.05, ns indicates no significant difference, within-group comparison; different letters indicate significant differences, the same letter indicates no significant difference, between-group comparison).

[0031] Figure 5 This invention relates to the effect of corn straw biochar coating on the germination characteristics of *Saussurea involucrata* seeds under high temperature and drought stress (A represents germination rate; B represents average germination time; C represents germination rate index; different letters indicate significant differences, the same letter indicates no significant differences, intergroup comparison).

[0032] Figure 6 This invention describes the effect of corn straw biochar coating on the growth of *Saussurea involucrata* seedlings under high temperature and drought stress (A represents root length; B represents seedling length; C represents seedling fresh weight; D represents vigor index; different letters indicate significant differences, the same letter indicates no significant differences, intergroup comparison).

[0033] Figure 7 This invention describes the effects of different treatments on the germination characteristics of *Saussurea involucrata* seeds under high temperature and drought stress (A represents germination rate; B represents average germination time; C represents germination rate index; different letters indicate significant differences, the same letter indicates no significant differences, and comparisons are made between groups).

[0034] Figure 8This invention describes the effects of different treatments on the growth of *Saussurea involucrata* seedlings under high temperature and drought stress (A represents root length; B represents seedling length; C represents seedling fresh weight; D represents vigor index; different letters indicate significant differences, the same letter indicates no significant differences, comparison between groups).

[0035] Figure 9 This invention utilizes multispectral imaging technology to evaluate *Alternanthera philoxeroides* seedlings under different treatments.

[0036] Figure 10 This invention describes the effects of different treatments on saprolegniaxin activity under high temperature and drought stress (A represents CAT activity; B represents POD activity; C represents SOD activity; D represents MDHAR activity; E represents GR activity; F represents APX activity; different letters indicate significant differences, the same letter indicates no significant differences, intergroup comparison). Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example Seed Initiation Process

[0041] 1.1 Seed-induced

[0042] Satamarisk seeds: Harvested in 2022, purchased from Jiuquan Daye Seed Industry Co., Ltd., Jiuquan City, Gansu Province. Seeds were stored in a low-temperature, low-humidity storage cabinet (CZ-1000FC) at the Forage Seed Laboratory of China Agricultural University, at a temperature of 4℃ and a humidity of 60%. Purity: ≥99%; Germination rate: ≥90%; Initial moisture content: 10%.

[0043] Initiation solution: Weigh different weights of PEG-6000 powder, dissolve in distilled water and mix thoroughly to prepare a PEG solution of a certain mass fraction as a seed initiator for alfalfa.

[0044] Seed priming procedure: Place three layers of filter paper in a petri dish, add 20 mL of PEG-6000 solution, and then sprinkle the seeds on top. To ensure sufficient imbibition, place three layers of filter paper moistened with the same concentration of PEG-6000 solution on top of the seeds. Primate at 20°C for a certain period of time. After priming, pour out the seeds, wrap them in gauze, rinse them three times in distilled water, then blot the surface moisture with filter paper, and finally allow them to dry at room temperature to their original weight, thus obtaining primed seeds.

[0045] 1.2 Seed coating technology

[0046] Coating powder: The inert substances include magnesium stearate, bentonite, and talc; the active substance is corn stalk biochar, purchased from Zhengzhou Lvhang Water Purification Materials Co., Ltd., with a particle size of 200 mesh. The inert and active substances are mixed in different proportions to serve as the raw material for preparing the coating.

[0047] Coating binder: 1.0% and 1.5% sodium carboxymethyl cellulose (CMC) aqueous solution is used as an adjuvant to allow the powder to adhere to the seed surface to form a shell.

[0048] Seed coating equipment: 5XW-110A seed coating machine.

[0049] Seed coating process steps:

[0050] First layer (isolation layer): Pour the seeds into the coating machine and adjust the speed until the seeds rotate along the inner wall and form obvious elliptical rings. Figure 1 A) Ensure the seeds touch the baffle. Next, add a 1% CMC aqueous solution to the atomizing tray to moisten the seeds without causing them to stick together. Then, using a spatula, add the powder in small amounts multiple times until all the powder has been added.

[0051] Second layer (active substance addition layer): After the first layer of coating is completed, the seeds are idled in the coating machine for 1 minute. 1.5% CMC aqueous solution is added dropwise to the atomizing plate. At the same time, the second layer of powder is added in small amounts and multiple times. The operation is repeated until the powder is completely added.

[0052] The third layer (protective layer): After the seeds have completed the first two coating layers, run them idle in the coating machine for 2 minutes. Add 1.5% CMC aqueous solution to the atomizing plate, and add the third layer powder in small amounts several times until all the powder has been added. This can improve the hardness and smoothness of the coated seeds.

[0053] The seed condition needs to be constantly monitored during the coating process. Figure 1 B) The speed of the coating machine can be adjusted appropriately according to the seed condition to reduce seed adhesion and improve powder flying, thus ensuring coating effect.

[0054] 1.3 Hybrid Processing Technology

[0055] Seeds treated with a suitable mass fraction of PEG solution were then coated with corn straw biochar to study the effect of the combined treatment on improving seed stress resistance.

[0056] Application Example 1: PEG-initiated treatment to improve the heat and drought resistance of alfalfa seeds

[0057] 1. Experimental Procedure

[0058] Randomly selected, uniform alfalfa seeds with a moisture content of 10% were cultured according to the germination test procedure for grass seeds (GB / T2930.4—2017). From these, 400 seeds with consistent imbibition were selected. Seeds were collected every 2 hours in the early stage and every 6 hours in the later stage. After collection, the surface moisture was blotted dry with filter paper, and the dried seeds were weighed. Each operation was repeated 4 times. The water absorption rate at each time point was calculated based on the weighing results, and an imbibition curve for alfalfa seeds was plotted.

[0059] Water absorption rate (%) = [(Weight of seeds after water absorption - Weight of dry seeds) / Weight of dry seeds] × 100%

[0060] Based on the drawn absorption curve, select one initiation time in the first and second stages of absorption.

[0061] After initiating *Alternanthera philoxeroides* seeds with PEG-6000 solutions at concentrations of 5%, 10%, 15%, 20%, 25%, and 30% (w / w), germination tests were conducted on the initiated seeds in a sand bed, following the germination test procedure for grass seeds (GB / T 2930.4-2017). River sand was used as the germination medium. Before use, the sand was sieved through a 0.8 cm square sieve and then washed 3-4 times with distilled water to achieve a pH of 6.0-7.5. The moist sand was then dried at 130°C. The germination medium was 12×12×5 cm. 3 Add 400g of dry sand and 8g of distilled water to a transparent petri dish, mix well, and spread evenly. Place 100 seeds in each dish, cover, and weigh. Place the petri dishes in a light incubator (GXZ-380B-LED) for 14 days. Set the incubator conditions as follows: 35℃ for 8 hours of light and 20℃ for 16 hours of darkness. During the incubation period, add water evenly to the original weight daily using a small spray bottle, and measure the following indicators:

[0062] (1) Emergence rate (%) = number of seedlings in the last count / total number of seeds tested × 100%.

[0063] (2) Average emergence time = ∑nt / ∑n, where n is the number of seeds whose embryos break through the surface of the sand at time t, and t is the statistical time.

[0064] (3) Emergence rate index = ∑(Gt / Dt), where Gt is the number of embryos breaking through the sand on day t, and Dt is the number of days (d) for embryos to break through the sand.

[0065] (4) Vitality index = ∑(Gt / Dt) × FW, where FW is fresh weight (g).

[0066] (5) Root length, seedling length, and seedling fresh weight: After the germination test, 10 seedlings were randomly selected from each replicate of each treatment to measure the root length (cm), seedling length (cm), and fresh weight (g).

[0067] 2. Experimental Results

[0068] Based on the measurement results, an absorption curve was plotted. Figure 2 The results showed that the water absorption of alfalfa seeds exhibited a rapid-slow-rapid change over time, with three stages: 0-8 h, 8-16 h, and after 16 h. Therefore, 6 h and 14 h were selected as initiation times for PEG initiation treatment.

[0069] like Figure 3 As shown in Figure A, at 6 h of initiation, the germination rate of *Saussurea involucrata* seeds treated with all PEG was significantly higher than that of CKb1 (P<0.05), with the 10% PEG initiation treatment showing the highest germination rate, reaching 91%. The germination rates of low-concentration PEG initiation treatments (5%–20%) showed no significant difference compared to CKb2, while the germination rates of high-concentration treatments (25% and 30%) were significantly lower than those of CKb2 (P<0.05). At 30 h of initiation, the germination rates of all initiation treatments were significantly higher than those of CKb1 but lower than those of CKb2. The germination rates of most treatments initiated at 14 h were significantly lower than those initiated at 6 h, indicating that prolonged initiation time significantly inhibited germination.

[0070] like Figure 3 As shown in Figure B, at 6 h of initiation, the average emergence time after all PEG initiation treatments was significantly shorter than that after CKb1 (P<0.05), but the differences between treatments were not significant. At 14 h of initiation, the average emergence time after all PEG initiation treatments was significantly shorter than that after CKb1 and CKb2 (P<0.05). Except for 30% PEG, the average emergence time after 14 h of initiation for other concentrations was significantly shorter than that after 6 h of initiation (P<0.05).

[0071] like Figure 3 As shown in Figure C, the emergence rate index of all PEG-initiated treatments was significantly higher than that of CKb1 (P<0.05). The emergence rate index decreased with increasing PEG concentration. At higher PEG concentrations, there was a difference between 6 h and 14 h of initiation treatment, with significant differences observed at PEG concentrations of 20% and 30% (P<0.05).

[0072] like Figure 4 As shown in Figure A, at 6 h of initiation, the root length of *Alternanthera philoxeroides* seedlings was significantly higher than that of CKb1, with lower concentrations of PEG showing a more significant effect. The 5% and 10% PEG initiation treatments increased root length by 24.9% and 23.2% respectively compared to CKb1 (P<0.05). At 14 h of initiation, the root length of the seedlings was significantly higher than that of CKb2 (P<0.05). Overall, 6 h of initiation had a better promoting effect on root length than 14 h, and this difference was significant at the 5% PEG treatment level (P<0.05).

[0073] like Figure 4 As shown in Figure B, the effect of PEG initiation on seedling growth is similar to that on root length, with different initiation treatments showing varying degrees of improvement in seedling length. At 6 h of initiation, lower concentrations of PEG were more effective in promoting seedling growth, with the 10% PEG initiation treatment showing a 24.9% increase compared to CKb1. At 14 h of initiation, the seedling lengths of the 10%–25% PEG treatments were all significantly higher than those of CKb1, although there were no significant differences between them. Overall, 6 h of initiation had a better promoting effect on seedling growth than 14 h.

[0074] like Figure 4 As shown in C, all PEG initiation methods increased the fresh weight of seedlings. At 6 h of initiation, low to medium concentrations of PEG had a more significant effect on increasing the fresh weight of seedlings, with the 10% PEG treatment increasing the fresh weight by 40.8% and 16.5% compared to CKb1 and CKb2, respectively. The changes in seedling fresh weight at 14 h of initiation were similar to those at 6 h, but overall, initiation at 6 h was more effective than initiation at 14 h.

[0075] like Figure 4 As shown in Figure D, the activity index of all PEG-initiated treatments was significantly higher than that of CKb1 (P<0.05). However, the higher the PEG concentration, the smaller the effect on improving the activity index. After 6 h of initiation, the activity index of the 10% PEG treatment was 159.1% higher than that of CKb1, while that of the 30% PEG treatment was only 77.1% higher. After 14 h of initiation, the activity index of all PEG-initiated treatments was significantly higher than that of CKb1 (P<0.05). There was no significant difference between the PEG treatments at different concentrations after 6 h and 14 h of initiation, indicating that extending the initiation time had no significant effect on improving the activity index.

[0076] Application Example 2

[0077] Corn stalk biochar coating treatment to improve the heat and drought resistance of alfalfa seeds

[0078] The seeds were subjected to a three-layer coating treatment, with corn stalk biochar added to the second layer. The biochar-to-seed mass ratios were 1:15, 1:10, 1:5, and 1:3, denoted as Bio15, Bio10, Bio5, and Bio3, respectively. Two control groups were set up: CKa1: naked seeds without coating treatment, and CKa2: seeds with only basic coating treatment (i.e., no biochar added to the second layer).

[0079] Table 1 Coating Powder Formulation Table

[0080]

[0081] Note: Powder required for corn stalk biochar coating per 5g of net seeds.

[0082] After coating 5g of *Alternanthera philoxeroides* seeds according to Table 1, a sand bed germination test was conducted on the coated seeds according to the germination test procedure for grass seeds (GB / T2930.10-2017). River sand was used as the germination medium. Before use, the sand was passed through a 0.8 cm square sieve and then washed 3-4 times with distilled water to adjust its pH to 6.0-7.5. The moist sand was then dried at 130°C. The germination medium was 12×12×5 cm. 3 Add 400g of dry sand and 8g of distilled water to a transparent petri dish, mix well, and spread evenly. Place 100 seeds in each dish, cover, and weigh. Place the petri dishes in a light incubator (GXZ-380B-LED) for 14 days. Set the incubator conditions as follows: 35℃ for 8 hours of light and 20℃ for 16 hours of darkness. During the incubation period, add water evenly to the original weight daily using a small spray bottle, and measure the following indicators:

[0083] (1) Emergence rate (%) = number of seedlings in the last count / total number of seeds tested × 100%.

[0084] (2) Average emergence time = ∑nt / ∑n, where n is the number of seeds whose embryos break through the surface of the sand at time t, and t is the statistical time.

[0085] (3) Emergence rate index = ∑(Gt / Dt), where Gt is the number of embryos breaking through the sand on day t, and Dt is the number of days (d) for embryos to break through the sand.

[0086] (4) Vitality index = ∑(Gt / Dt) × FW, where FW is fresh weight (g).

[0087] (5) Root length, seedling length, and seedling fresh weight: After the germination test, 10 seedlings were randomly selected from each replicate of each treatment to measure the root length (cm) and fresh weight (g).

[0088] The results showed that all coating treatments improved the germination rate of *Alternanthera philoxeroides* seeds under high temperature and drought conditions (P<0.05). Compared with Cka1, Bio15, Bio10, Bio5, and Bio3 showed increases of 35.7%, 28.95%, 45.1%, and 35.2%, respectively, but there was no significant difference between any of the Bio treatments and Cka2. Regarding average germination time, there was no significant difference between Bio15 and Bio10 and Cka1, while Bio5 and Bio3 showed shorter germination times compared to Cka1. As for the germination rate index, all coating treatments showed a significant increase compared to Cka1 (P<0.05), with Bio5 and Bio3 showing increases of 69.3% and 62.9%, respectively.

[0089] The promotion of *Alternanthera philoxeroides* seedlings by biochar coating treatment was mainly reflected in seedling length, fresh weight, and vigor index, while root length showed no significant change (P > 0.05). Regarding seedling length, Bio15, Bio10, and Bio5 increased by 19.2%, 13.9%, and 16.3% compared to Cka1, respectively, but these three treatments showed no significant difference from Cka2 (P > 0.05). Regarding fresh weight, Bio15, Bio10, Bio5, and Bio3 increased by 24.4%, 28.7%, 36.9%, and 28.2% compared to Cka1, respectively, with Bio5 showing a significant increase of 13.7% compared to Cka2. Regarding vigor index, Bio5 and Bio3 showed substantial improvements, increasing by 132.2% and 109.5% compared to Cka1, respectively.

[0090] Considering all indicators, the Bio5 treatment was the most effective in improving the emergence and seedling growth of *Saussurea involucrata* under combined high temperature and drought stress.

[0091] Application Example 3

[0092] A mixed treatment method for germination followed by coating of saxaul seeds.

[0093] First, the alfalfa seeds were treated with the optimal initiation conditions selected in Application Example 2 (initiation with 10% PEG solution for 6 h). After the alfalfa seeds were dried back to their original weight, they were then treated with the optimal coating conditions selected in Example 2 (corn straw biochar:seed mass ratio of 1:5). This completed the mixed treatment of the alfalfa seeds (denoted as Combine).

[0094] Germination tests were conducted on mixed-treated *Saussurea involucrata* seeds in a sand bed, following the germination test procedures for grass seeds (GB / T 2930.10-2017). River sand was used as the germination medium. Before use, the sand was passed through a 0.8 cm square sieve and then washed 3-4 times with distilled water to achieve a pH of 6.0-7.5. The moist sand was then dried at 130°C. Germination was carried out in a 12×12×5 cm bed. 3Add 400g of dry sand and 8g of distilled water to a transparent petri dish, mix well, and spread evenly. Place 100 seeds in each dish, cover, and weigh. Place the petri dishes in a light incubator (GXZ-380B-LED) for 14 days. Set the incubator conditions as follows: 35℃ for 8 hours of light and 20℃ for 16 hours of darkness. During the incubation period, add water evenly to the original weight daily using a small spray bottle, and measure the following indicators:

[0095] (1) Emergence rate (%) = number of seedlings in the last count / total number of seeds tested × 100%.

[0096] (2) Average emergence time = ∑nt / ∑n, where n is the number of seeds whose embryos break through the surface of the sand at time t, and t is the statistical time.

[0097] (3) Emergence rate index = ∑(Gt / Dt), where Gt is the number of embryos breaking through the sand on day t, and Dt is the number of days (d) for embryos to break through the sand.

[0098] (4) Vitality index = ∑(Gt / Dt) × FW, where FW is fresh weight (g).

[0099] (5) Root length, seedling length, and seedling fresh weight: After the germination test, 10 seedlings were randomly selected from each replicate of each treatment to measure the root length (cm) and fresh weight (g).

[0100] The results showed that the germination rate of *Alpinia serratifolia* seeds after mixed treatment was significantly higher than that after untreated treatment (P<0.05), but lower than that after priming treatment (P<0.05), and there was no significant difference compared with the coating treatment. Mixed treatment significantly shortened the germination time (P<0.05), with the average germination time under mixed treatment being approximately 28% shorter than that after untreated treatment. The germination rate index after mixed treatment was significantly higher than that after untreated and coating treatments (P<0.05).

[0101] Compared with untreated, the initiation treatment, coating treatment, and mixed treatment all significantly (P<0.05) increased the seedling length of *Saussurea involucrata* seedlings under high temperature and drought stress, but the differences among them were not significant. Except for CKa2, all other treatments significantly promoted the root length of *Saussurea involucrata*. The initiation treatment, coating treatment, and mixed treatment all significantly (P<0.05) increased the fresh weight, but the differences among them were not significant. The fresh weight of the PEG treatment was significantly greater than that of CKb2, and the fresh weight of the Bio treatment was significantly greater (P<0.05) than that of CKa2, indicating that corn straw biochar and PEG played a role in the process. The trend of seedling vigor index was similar to that of fresh weight; the vigor index of the PEG treatment was significantly greater (P<0.05) than that of CKb2, and the vigor index of the Bio treatment was significantly greater (P<0.05) than that of CKa2.

[0102] Application Example 4

[0103] After the experiment in Application Example 3, multispectral images of alfalfa seedlings under different treatments were collected, and the images were converted to nCDA to obtain RGB images and nCDA images of alfalfa seedlings under different treatments.

[0104] The results showed that: the multispectral evaluation images indicated ( Figure 9 Under high temperature and drought stress, untreated *Alternanthera philoxeroides* seedlings showed low vigor and appeared red, while the vigor of seedlings treated with different methods all showed a trend towards turning blue, indicating an improvement in their vigor. The seedlings treated with PEG were bluer than those treated with CKb2, and the seedlings treated with Bio were bluer than those treated with CKa2, suggesting that PEG initiation and corn straw biochar coating have certain effects.

[0105] Application Example 5

[0106] After the experiment in Example 3, a certain number of *Arundinaria heterophylla* seedlings were randomly selected from the petri dishes for enzyme activity assays, including: catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), and ascorbate peroxidase (APX).

[0107] The results showed that, compared with the control (CK), the CAT activity of *Alternanthera philoxeroides* seedlings treated with priming was significantly increased (P<0.05), while the CAT activity of the coating treatment and the mixed treatment showed no significant change. Figure 10 A). The SOD activity of the Bio-treatment and Combine-treatment was significantly higher than that of other treatments (P<0.05). Figure 10 C). Compared with CK, the POD activity of all treatments showed varying degrees of increase ( Figure 10 B), where the POD activity of PEG-treated biochar was significantly higher than that of CKb2 (P<0.05), and the POD activity of Bio-treated biochar was significantly higher than that of CKa2 (P<0.05), indicating that corn straw biochar and PEG played certain roles. Compared with CK, the activity of MDHAR was increased the most under PEG treatment, by about 180%, followed by Combine treatment, which increased by about 107%. Figure 10 D). The changes in GR were similar to those in APX; PEG treatment, Bio treatment, and Combine treatment were all significantly higher than CK, while there were no significant differences among the three. Figure 10 (EF in the text).

[0108] In summary, the optimal coating conditions for cultivating *Alternanthera philoxeroides* seeds after three-layer coating are as follows: the first layer consists of bentonite and talc powder, with a weight ratio of bentonite, talc powder, and seeds of 1:1:2; the second layer consists of magnesium stearate, bentonite, talc powder, and biochar, with a weight ratio of magnesium stearate, bentonite, talc powder, biochar, and seeds of 10:5:5:4:10; and the third layer consists of talc powder, with a weight ratio of talc powder and seeds of 2:5. This three-layer coating treatment significantly enhances seed vigor and SOD activity, increases the emergence rate of *Alternanthera philoxeroides* seedlings, and promotes root growth. The method of this invention can be applied to the emergence of *Alternanthera philoxeroides* seeds under high temperature and drought stress in the Horqin Sandy Land.

[0109] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for improving seed germination of *Salvia splendens* seeds under high temperature and drought stress in the Horqin Sandy Land, characterized in that, Specifically, the following steps are included: (1) Seed initiation: Wet the filter paper with a PEG-6000 solution with a mass fraction of 5%~30%, sprinkle the seeds between two layers of filter paper for seed initiation, and dry at room temperature after initiation until the seed quality is restored to its original quality. (2) Seed coating: Pour the seeds into the coating machine and coat them in three layers in sequence. In the second coating process, use corn straw biochar for biochar coating. (3) Germination in sand bed: River sand is used as the germination medium. The coated seeds are placed in a light incubator for cultivation.

2. The method according to claim 1, characterized in that, The seed initiation conditions were as follows: initiation with a 10% PEG solution for 6 hours.

3. The method according to claim 1, characterized in that, The three-layer coating specifically includes: (1) First coating: Pour the seeds into the coating machine and adjust the speed until the seeds rotate along the inner wall and form obvious elliptical circles. Then, add 1% CMC aqueous solution to the atomizing plate to moisten the seeds but not stick them together. Then, add coating powder to it in small amounts and multiple times using a spatula until all the coating powder has been added. (2) Second coating: After the first coating is completed, the seeds are idling in the coating machine for 1 minute. 1.5% CMC aqueous solution is added to the atomizing plate. At the same time, the second coating powder is added in small amounts and multiple times. The operation is repeated until the powder is added. (3) Third coating: After the first two coatings are completed, the seeds are idled in the coating machine for 2 minutes. A 1.5% CMC aqueous solution is added to the atomizing plate, and the third coating powder is added in small amounts several times until all the powder is added.

4. The method according to claim 3, characterized in that, The first coating powder consists of bentonite, talc, and seeds in a mass ratio of 1:1:2; the second coating powder consists of magnesium stearate, bentonite, talc, and biochar in a mass ratio of 10:5:5:4:10; and the third coating powder consists of talc in a mass ratio of 2:

5.

5. The method according to claim 1, characterized in that, The specific germination process in the sand bed includes: in a 12×12×5 cm... 3 Dry sand and distilled water were added to a transparent petri dish, mixed and spread evenly. 100 seeds were placed in each dish, the lid was closed and the dish was weighed. The petri dishes were then placed in a light incubator and cultured for 14 days. The mass ratio of dry sand to distilled water was 50:

1.

6. The method according to claim 5, characterized in that, The incubator conditions are: 35℃ light for 8 hours and 20℃ darkness for 16 hours. During the incubation period, water is added evenly to the original weight daily using a small spray bottle.

7. The alfalfa seedlings obtained by the method as described in claims 1-6.

8. The application of the method as described in claims 1-7 in the cultivation of alfalfa seedlings.

Images