A seed initiator based on sporopollenin nanoparticles and its application

Abstract

This invention provides a seed initiator based on sporopollenin nanoparticles and its application. The seed initiator is prepared by dispersing sporopollenin nanoparticles in deionized water. This invention utilizes an oxidation method to decompose sporopollenin into nanoparticles, simultaneously altering its surface chemical composition, and applying it to plant seed initiation, thus expanding the application range of sporopollenin materials. Using the sporopollenin nanoparticle seed initiator for seed initiation in pakchoi can improve the seed germination rate and the antioxidant capacity of seedling leaves under salt stress.

Description

Technical Field

[0001] This invention belongs to the field of plant stress response and pesticide formulation processing, specifically relating to a seed initiator based on sporophytin nanoparticles and its application. Background Technology

[0002] In recent years, soil salinization has become increasingly serious due to extreme weather, improper irrigation methods, fertilizer application, and industrial pollution, posing a significant challenge to global food security. Soil salinization affects more than 1.381 billion hectares of land. 20% of irrigated land is directly affected by soil salinization, and the accumulation of salt in the soil severely restricts agricultural production.

[0003] Excessive salt in the soil inhibits seed germination and can also lead to ionic and osmotic stress, thereby affecting plant morphology, metabolism, physiological activities, and biomass, ultimately resulting in plant damage and reduced yield.

[0004] Chinese cabbage (Brassica chinensis L.) is a plant with low salt tolerance. When Chinese cabbage seeds are sown directly in the field, salt can not only delay seed germination and seedling emergence, but also lead to uneven seedling emergence. Increased salt concentration in the soil will adversely affect the photosynthetic efficiency, ion absorption and transport of Chinese cabbage, seriously affecting its growth and yield. Sporopyrin is widely found in the outer wall of pollen / spores. Sporopyrin microcapsules (SECs) obtained through extraction are used as carriers for drugs or proteins due to their abundant source, renewability, and high toughness. However, the application of sporopyrin nanoparticles as plant seed initiators to resist salt stress has not yet been reported. Summary of the Invention

[0005] One of the objectives of this invention is to provide a seed initiator based on sporophytin nanoparticles.

[0006] The seed initiator based on sporophytin nanoparticles provided by this invention is prepared by dispersing sporophytin nanoparticles in deionized water.

[0007] The average particle size of the sporophytin nanoparticles is 300nm-500nm; In the seed initiator based on sporophytin nanoparticles, the mass percentage of sporophytin nanoparticles can be 0.005%-1%, specifically 0.01%.

[0008] The sporophytin nanoparticles were prepared by a method comprising the following steps: 1) The pollen is degreased to obtain degreased pollen; 2) Degrading the sporophyte components in defatted pollen to obtain sporophytin microcapsules; 3) The sporopyram microcapsules are degraded into sporopyram nanoparticles by oxidation.

[0009] In step 1) of the above method, the pollen is rapeseed pollen; In step 1), the defatting process is performed as follows: a) the pollen is placed in hot acetone and stirred; b) the acetone is poured out, collected by vacuum filtration, and dried to obtain defatted pollen. In a), the temperature of the hot acetone is 50°C, and the ratio of the hot acetone to pollen is 50-80 mL: 100 g. The stirring speed is 220 rpm; The stirring time is 0.5 to 3 hours; Degreased pollen is collected by vacuum filtration.

[0010] The operation in step 2) above is as follows: add defatted pollen to a potassium hydroxide aqueous solution and reflux under heating and stirring; The potassium hydroxide aqueous solution is a 5-10 w / v potassium hydroxide aqueous solution; The ratio of potassium hydroxide aqueous solution to defatted pollen is 50-80 mL: 10 g; The reflux temperature is 60-80°C, specifically 70°C, and the time is 2 hours.

[0011] Step 2) also includes collecting sporophytin microcapsules by vacuum filtration after reflux and washing the sporophytin microcapsules with deionized water until pH=7.

[0012] Step 3) involves adding the obtained sporophytin microcapsules to a sodium hypochlorite solution and refluxing with stirring to degrade the sporophytin into nanoparticles. The mass concentration of the sodium hypochlorite solution can be 1%-30%. The ratio of sodium hypochlorite solution to sporophytin microcapsules can be 100–500 mL: 10 g; The reflux temperature can be 50–90°C, and the time can be 0.5–4 hours, more specifically 2 hours; Step 3) of the above method may further include centrifuging to collect the sporophytin nanoparticles, followed by washing with water to remove residual sodium hypochlorite. The centrifugation conditions can be 8,000 rpm for 3-5 min; The washing process can be repeated 5 to 7 times.

[0013] The second objective of this invention is to provide the application of sporopyrrin nanoparticles or the above-mentioned seed initiators based on sporopyrrin nanoparticles in enhancing the salt stress resistance of seeds.

[0014] Specifically, the application involves using a seed initiator based on sporophytin nanoparticles to initiate the germination of pakchoi seeds, thereby improving the germination rate of pakchoi seeds under salt stress and the antioxidant capacity of seedling leaves.

[0015] This invention also provides a seed initiation method for improving the salt tolerance of Chinese cabbage.

[0016] The seed initiation method for improving the salt tolerance of Chinese cabbage provided by the present invention includes immersing Chinese cabbage seeds in the above-mentioned seed initiator based on sporophytin nanoparticles, shaking well to ensure that the initiation solution is in full contact with the seeds for 3-4 hours, pouring out the suspension, and absorbing the moisture from the seeds.

[0017] The above method includes a step of disinfecting the bok choy seeds before immersing them in a seed initiator.

[0018] Specifically, the seeds can be disinfected by soaking them in a 0.5% (by mass) sodium hypochlorite solution for 5 minutes, and then rinsed with deionized water 4-5 times.

[0019] This invention utilizes an oxidation method to decompose sporopollenin into nanoparticles, simultaneously altering its surface chemical composition. This allows for its application in plant seed initiation, expanding the application range of sporopollenin materials. Using sporopollenin nano-seed initiators for seed initiation in pakchoi can enhance the seed germination rate and antioxidant capacity of seedling leaves under salt stress.

[0020] This invention provides a seed initiation method to improve the salt tolerance of Chinese cabbage. The method uses sporophytin nano-seed initiator to initiate the seed development of Chinese cabbage and applies salt stress treatment during the seed germination stage to explore its response to physiological and biochemical changes such as seed germination characteristics and antioxidant enzyme system of Chinese cabbage. This provides a theoretical basis for the high-quality cultivation and production of Chinese cabbage in saline-alkali land. Attached Figure Description

[0021] Figure 1 The morphological changes of natural rapeseed pollen after different chemical treatments.

[0022] Figure 2 The particle size distribution of sporophytin nanoparticles formed after alkali treatment and oxidation treatment.

[0023] Figure 3 The effects of different treatments on the germination rate of Chinese cabbage seeds at different time points were investigated (CK: no sporophytin treatment + no salt stress treatment; CK-BFS: sporophytin treatment only; S: salt stress treatment only; S-BFS: sporophytin treatment + salt stress treatment).

[0024] Figure 4Phenotypic results of Chinese cabbage seedlings induced by sporophytin nanoparticles (CK: no sporophytin treatment + no salt stress treatment; CK-BFS: sporophytin treatment only; S: salt stress treatment only; S-BFS: sporophytin treatment + salt stress treatment).

[0025] Figure 5 The effects of sporophytin nanoparticles on the growth and root length of pak choi seedlings under salt stress were investigated (CK: no sporophytin treatment + no salt stress treatment; CK-BFS: sporophytin treatment only; S: salt stress treatment only; S-BFS: sporophytin treatment + salt stress treatment).

[0026] Figure 6 The effects of sporophytin nanoparticles on the fresh and dry weight of pak choi seeds under salt stress were investigated (CK: sporophytin-free treatment + salt stress-free treatment; CK-BFS: sporophytin-only treatment; S: salt stress-only treatment; S-BFS: sporophytin-only treatment + salt stress treatment).

[0027] Figure 7 The effects of sporophytin nanoparticles on the content of antioxidant enzymes in Chinese cabbage seedlings under salt stress were investigated (CK: no sporophytin treatment + no salt stress treatment; CK-BFS: sporophytin treatment only; S: salt stress treatment only; S-BFS: sporophytin treatment + salt stress treatment). Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1: Preparation of seed initiator based on sporopollenin nanoparticles Step 1: Place rapeseed pollen (100 g, Henan Zhuoyu Bee Industry Co., Ltd.) in hot acetone (80 mL, 50°C, Sinopharm Holdings Co., Ltd.) and stir (220 rpm) for 2 hours. Then pour out the acetone, remove the water by vacuum filtration, and dry to obtain defatted pollen.

[0031] Step 2: Add defatted pollen (10g) to 50mL of potassium hydroxide aqueous solution (10%, w / v, Sinopharm Holdings Co., Ltd.), reflux for 2h with heating and stirring (70°C, 220 rpm), collect sporophytin microcapsules by vacuum filtration, wash with water 3 times until the pH of the sporophytin aqueous solution is 7, filter the water from the sporophytin microcapsules and dry them; Step 3: Add 10 g of sporopyram microcapsules to a 10% sodium hypochlorite solution (200 mL, Sinopharm Holdings Co., Ltd.) and keep stirring under reflux (80°C) for 2 hours to degrade sporopyram into nanoparticles. Collect the sporopyram nanoparticles by centrifugation (8,000 rpm) for 5 minutes, and then wash thoroughly with water (100 mL) 6 times to remove residual sodium hypochlorite.

[0032] Figure 1 These represent the morphological changes of natural rapeseed pollen after different chemical treatments. Figure 1 It can be seen that after alkali treatment, the cytoplasm inside the pollen has been removed, and the germination pore of the pollen has been completely split open, but the microcapsule structure of sporophytin is still preserved, with a particle size range of 15-30 micrometers; after alkali treatment and oxidation treatment, the microcapsule structure of sporophytin is completely destroyed, and the sporophytin shell is degraded into a large number of nanoparticles with a particle size of less than 1 micrometer (average particle size of 338 nanometers).

[0033] Step four: Disperse the obtained sporophytin nanoparticles in deionized water to prepare sporophytin seed initiators of different concentrations, with particle size distributions as shown in the figure. Figure 2 As shown. Figure 2 This is the particle size distribution of a 0.01% (w / w) concentration of sporophytin seed initiator.

[0034] Example 2: Preparation of Seed Initiator The sporophytin nanoparticles prepared in Example 1 were dispersed in deionized water to prepare a 0.01% (w / w) sporophytin nanoparticle seed initiator.

[0035] Example 3: Effect of seed initiator on seed germination of Chinese cabbage under salt stress Test methods Seed treatment: Select 100 uniformly sized, plump Chinese cabbage seeds. Rinse with clean water and sift out the sunken seeds. Disinfect the seeds by soaking them in a 0.5% (w / v) sodium hypochlorite solution for 5 minutes. Rinse the seeds 5 times with deionized water and air dry at room temperature. Seed initiation process: Place the seeds in a 50mL centrifuge tube and add 20mL of 0.01% (w / v) of spore-pollen nanoparticle seed initiator to submerge the seeds. Place the centrifuge tube containing the seeds and initiator on a mechanical shaker (150 rpm) in the dark to ensure full contact between the initiation solution and the seeds. After culturing for 4 hours, pour out the suspension, blot dry with absorbent paper, spread evenly on clean absorbent paper, and allow the seeds to dry again at room temperature for 2 hours for subsequent germination tests. 3. Salt stress treatment process: 3.1 Experimental Design Several experimental groups were set up, including a water control group, an initiation treatment group, a salt stress group, and a salt stress + initiation treatment group. 3.2 Seed germination The paper germination method was used to culture the induced seeds. The seeds were placed in petri dishes lined with two layers of filter paper. During germination culture, 5 mL of 200 mM NaCl solution was added to each dish. The water control group was treated with 5 mL of distilled water. The dishes were placed in a dark incubator for further culture. Liquid was added regularly every day. Germination was recorded as the criterion for sprouting. The number of germinated seeds was counted every 6 hours for a period of 36 hours.

[0036] 3.3 Methods for determining seed germination indices Several seedlings were selected from each petri dish, washed with distilled water, and dried with filter paper. The hypocotyl length and root length were measured after different priming treatments. The germination standard was that the hypocotyl length was 1 / 2 of the seed length.

[0037] 3.4 Measurement of seedling length and root length of Chinese cabbage seedlings Seven days after growth, five Chinese cabbage seedlings were selected from each petri dish, washed with distilled water, and dried with filter paper. The seedling length and root length after different initiation treatments were then measured.

[0038] 3.5 Methods for determining physiological and biochemical indicators Physiological parameters were measured on 7-day-old Chinese cabbage seedlings. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined by visible spectrophotometry.

[0039] 4. Experimental Results 4.1 Effects of sporophytin nanoparticle initiators on seed germination Depend on Figure 3It can be seen that after initiation with 0.01wt% sporopollen nanoparticle initiator, the germination rate of pakchoi seeds was significantly improved. The control group seeds started germinating after 8 hours, while after treatment with sporopollen, the germination rate increased from 2% in the control group to 4% after 8 hours. Salt stress can inhibit the germination rate of seeds. Under salt stress conditions, untreated seeds started germinating after 16 hours, while after initiation with sporopollen, the germination rate increased from 3% in the control group to 36.5% after 16 hours. Sporopollen initiation can significantly improve the germination rate of pakchoi seeds.

[0040] 4.2 Effects of sporophytin nanoparticle initiators on the biomass of pakchoi Depend on Figure 4-6 It can be seen that, after initiation with 0.01wt% sporophytin nanoparticle initiator, compared with the water control group, the root length increased from 2.67cm to 3.67cm, the seedling length increased from 5.47cm to 6.03cm, the fresh weight increased from 208.93mg to 267.17mg, and the dry weight increased from 66mg to 87.6mg. Compared with the salt stress treatment group, after initiation with 0.01wt% sporophytin nanoparticle initiator, the root length increased from 1.37cm to 2.37cm, the seedling length increased from 3.37cm to 4.83cm, the fresh weight increased from 126.4mg to 221mg, and the dry weight increased from 41.4mg to 80.63mg.

[0041] 4.3 Effect of sporophytin nanoparticle initiator on the content of antioxidant enzymes in pakchoi Depend on Figure 7 It can be seen that under salt stress, after initiation with 0.01wt% sporopollenin nanoparticle initiator, CAT activity increased from 149.91 U / g to 191.5 U / g, SOD activity increased from 27.80 U / g to 54.5 U / g, and POD activity increased from 165.0 U / g to 588.0 U / g. This shows that initiation with 0.01wt% sporopollenin nanoparticle initiator can enhance the antioxidant enzyme activity of pakchoi seedlings and alleviate the impact of salt damage on pakchoi seed germination and seedling growth.

[0042] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of a seed initiator based on sporopollenin nanoparticles in enhancing seed salt stress resistance, characterized in that, The application involves using a seed initiator based on sporophytin nanoparticles to initiate the germination of pakchoi seeds, thereby improving the germination rate of pakchoi seeds under salt stress and the antioxidant capacity of seedling leaves. The seed initiator based on sporopollenin nanoparticles is prepared by dispersing sporopollenin nanoparticles in deionized water. The average particle size of the sporophytin nanoparticles is 300nm-500nm. In the seed initiator based on sporophytin nanoparticles, the mass percentage content of sporophytin nanoparticles is 0.005%-1%; The sporophytin nanoparticles were prepared by a method comprising the following steps: 1) The pollen is degreased to obtain degreased pollen; 2) Degrading the sporophyte components in defatted pollen to obtain sporophytin microcapsules; 3) The sporopyramin microcapsules are degraded into sporopyramin nanoparticles using an oxidation method, thus obtaining the product; In step 1), the defatting process is performed as follows: a) the pollen is placed in hot acetone and stirred; b) the acetone is poured out, collected by vacuum filtration, and dried to obtain defatted pollen. Step 2) involves adding defatted pollen to a potassium hydroxide aqueous solution and refluxing it while heating and stirring. The potassium hydroxide aqueous solution is a 5-10 w / v potassium hydroxide aqueous solution; Step 3) involves adding the obtained sporophytin microcapsules to a sodium hypochlorite solution and refluxing with stirring to degrade the sporophytin into nanoparticles. The sodium hypochlorite solution has a mass concentration of 1%-30%.

2. The application according to claim 1, characterized in that, In step 1), the pollen is rapeseed pollen; In step 1)a), the temperature of the hot acetone is 50°C, and the ratio of the hot acetone to pollen is 50-80 mL: 100 g. The stirring speed is 220 rpm; The stirring time is 0.5 to 3 hours.

3. The application according to claim 1, characterized in that, In step 2), the ratio of potassium hydroxide aqueous solution to defatted pollen is 50-80 mL: 10 g; The reflux temperature is 60-80°C, and the time is 2 hours; Step 2) also includes collecting sporophytin microcapsules by vacuum filtration after reflux and washing the sporophytin microcapsules with deionized water until pH=7.

4. The application according to claim 1, characterized in that, In step 3), the ratio of sodium hypochlorite solution to sporophytin microcapsules is 100-500 mL: 10 g; The reflux temperature is 50–90°C, and the time is 0.5–4 hours.

5. A seed initiation method for improving the salt tolerance of Chinese cabbage, characterized in that, The method includes immersing Chinese cabbage seeds in a seed initiator based on sporophytin nanoparticles and shaking it to ensure that the initiation solution is in full contact with the seeds for 3-4 hours. The seed initiator based on sporophytin nanoparticles is prepared by dispersing sporophytin nanoparticles in deionized water. The average particle size of the sporophytin nanoparticles is 300nm-500nm. In the seed initiator based on sporophytin nanoparticles, the mass percentage content of sporophytin nanoparticles is 0.005%-1%; The sporophytin nanoparticles were prepared by a method comprising the following steps: 1) The pollen is degreased to obtain degreased pollen; 2) Degrading the sporophyte components in defatted pollen to obtain sporophytin microcapsules; 3) The sporopyramin microcapsules are degraded into sporopyramin nanoparticles using an oxidation method, thus obtaining the product; In step 1), the defatting process is performed as follows: a) the pollen is placed in hot acetone and stirred; b) the acetone is poured out, collected by vacuum filtration, and dried to obtain defatted pollen. Step 2) involves adding defatted pollen to a potassium hydroxide aqueous solution and refluxing it while heating and stirring. The potassium hydroxide aqueous solution is a 5-10 w / v potassium hydroxide aqueous solution; Step 3) involves adding the obtained sporophytin microcapsules to a sodium hypochlorite solution and refluxing with stirring to degrade the sporophytin into nanoparticles. The sodium hypochlorite solution has a mass concentration of 1%-30%.

6. The method according to claim 5, characterized in that, In step 1), the pollen is rapeseed pollen; In step 1)a), the temperature of the hot acetone is 50°C, and the ratio of the hot acetone to pollen is 50-80 mL: 100 g. The stirring speed is 220 rpm; The stirring time is 0.5 to 3 hours.

7. The method according to claim 5, characterized in that, In step 2), the ratio of potassium hydroxide aqueous solution to defatted pollen is 50-80 mL: 10 g; The reflux temperature is 60-80°C, and the time is 2 hours; Step 2) also includes collecting sporophytin microcapsules by vacuum filtration after reflux and washing the sporophytin microcapsules with deionized water until pH=7.

8. The method according to claim 5, characterized in that, In step 3), the ratio of sodium hypochlorite solution to sporophytin microcapsules is 100-500 mL: 10 g; The reflux temperature is 50–90°C, and the time is 0.5–4 hours.

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