A gibberellin-loaded mesoporous silica rice leaf surface cadmium-reducing agent, a preparation method and a spraying method

Abstract

The present application provides a kind of mesoporous silica supported gibberellin rice leaf surface cadmium reducing agent, preparation method and spraying method, belong to the safe production technology field of medium-light pollution farmland.The preparation method provided by the present application is first prepared nano mesoporous silica particles using sol-gel method, then gibberellin is loaded on nano mesoporous silica particles, finally PVP is encapsulated, and a surfactant is added to obtain it.The rice leaf surface cadmium reducing agent prepared by the preparation method of the present application reduces the translocation of node 1 to grain and reduces the transpiration rate of rice leaf surface, thereby reducing the direct absorption and translocation of Cd, and by encapsulation, the oxidation and degradation of gibberellin can be effectively avoided, with the characteristics of high efficiency, low cost, simple operation, simple raw materials and the like.

Description

Technical Field

[0001] This invention relates to the field of safe production technology in farmland with moderate to light pollution, and in particular to a cadmium-reducing agent for rice leaves loaded with gibberellin from mesoporous silica, its preparation method, and its application method. Background Technology

[0002] The main pathways for Cd entry into rice include root absorption (lateral transport), xylem migration and translocation to node 1 (vertical transport), and redistribution of Cd through the leaf phloem into the grain. Cd is primarily concentrated in the ectoderm of the rice grain. Existing research suggests that rice nodes, especially node 1, significantly intercept Cd transport to the grain. The cross-sectional area of ​​the dispersed vascular bundles in node 1 is considered a crucial factor in Cd accumulation in rice. Long-distance Cd transport is an energy-intensive process; theoretically, increasing the length of the first internode (panicle neck) can increase Cd transport distance and reduce transport capacity. However, there are no reports on whether the first internode (panicle neck) plays a role in Cd redistribution. Internode elongation in rice is mainly regulated by gibberellin signaling molecules within the plant. The length of the panicle neck is primarily determined by gibberellin (GA4) in node 1 during the heading stage. Spraying gibberellin to increase panicle neck length has been reported in rice production; however, multiple applications are generally required.

[0003] Mesoporous nano-silica has been widely used in drug delivery and pesticide loading, exhibiting significant sustained-release effects. Foliar inhibition, due to its ease of operation and low input cost, is one of the important methods for safe production in Cd-contaminated paddy fields. Currently, the main foliar inhibition materials are primarily inorganic elements, especially trace elements. The main principle is to reduce cadmium absorption in rice through the antagonistic effect between elements. However, its Cd-reducing effect varies greatly due to multiple factors, including soil environment (pH and redox conditions), the abundance or deficiency of trace elements in the soil, and rice varieties. Therefore, there is an urgent need to develop a low-cost, green, and highly efficient foliar Cd-reducing agent. Among these, plant hormones, due to their small dosage and significant effect on adjusting rice growth and yield formation, have been widely used in paddy fields and other crop production. However, they are easily decomposed in the environment and have a significant concentration effect on plant growth (low dosage promotes growth, high dosage inhibits growth).

[0004] Based on this, the present invention proposes for the first time to assemble plant hormones using mesoporous materials to reduce the accumulation of Cd in rice, and further verifies its Cd-reducing effect on rice with moderate to mild Cd pollution through pot experiments and field experiments. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a rice foliar cadmium-reducing agent supported on mesoporous silica and gibberellin, a preparation method and a spraying method. The rice foliar cadmium-reducing agent obtained by the preparation method provided by this invention can effectively reduce the concentration of Cd in rice and significantly improve the compliance rate of rice in rice fields with excessive Cd.

[0006] The technical solution adopted by this invention to solve its technical problem is as follows: By loading gibberellin (GA4) onto mesoporous silica particles and then encapsulating it with PVP, a nano-foliar cadmium-reducing agent is prepared. This nanomaterial can be well absorbed by plants and, through encapsulation and slow release, can be applied to the leaves of rice planted with excessive Cd in the soil during the critical growth period of brown rice (heading-grain filling). This continuously produces gibberellin signaling molecules, effectively increasing the length of the panicle neck (distance from the first node to the panicle node), reducing transpiration in the panicle, and increasing the concentration of pectin in the cell walls of the stems and leaves, thereby reducing the accumulation of Cd during the grain filling process of rice. This can effectively reduce the concentration of Cd in rice and significantly improve the compliance rate of rice in rice fields with excessive Cd.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a rice foliar cadmium-reducing agent supported on mesoporous silica and gibberellin, comprising the following steps:

[0008] (1) Using tetraethyl orthosilicate as the silicon source and a template agent, nano-mesoporous silica particles were prepared by sol-gel method. After the reaction was completed, the precipitate was centrifuged, dried and calcined at high temperature to remove the template agent to obtain PHSN powder.

[0009] (2) The obtained PHSN powder was dispersed in gibberellin ethanol-water solution, stirred at low temperature to allow it to react fully, and then PHSN@GA4 powder was obtained by centrifugation and freeze drying.

[0010] (3) Add the PHSN@GA4 obtained above to a PVP solution with pH=5, stir at low temperature for 36h, centrifuge-filter and freeze dry to obtain PVP-encapsulated PHSN@GA4;

[0011] (4) The PHSN@GA4 powder encapsulated in PVP was uniformly dispersed in the aqueous phase and a surfactant was added to obtain a cadmium-reducing agent for rice leaves.

[0012] Preferably, step (1) is as follows: the template agent is dissolved in ammonia-ethanol mixed solution, and tetraethyl orthosilicate is added dropwise. When the solution turns milky white, the reaction is stirred at 85°C. After the reaction is completed, the precipitate is collected. The precipitate is washed, centrifuged, dried and calcined to obtain PHSN powder.

[0013] More preferably, the template agent is a hexadecyltrimethylammonium bromide and a triblock copolymer.

[0014] Preferably, step (2) is as follows: the obtained PHSN powder is dispersed in an ethanol solution of gibberellin, stirred at low temperature for 72 hours, and after the reaction is completed, the obtained solution is centrifuged, washed, and freeze-dried to obtain PHSN@GA4 powder.

[0015] More preferably, the stirring temperature is less than or equal to 25°C.

[0016] More preferably, the mass ratio of PHSN powder to gibberellin is 1:10.

[0017] Preferably, the surfactant is Tween-80; the concentration of Tween-80 is 0.5‰.

[0018] Preferably, the concentration of the PVP solution in step (3) is 5 μM.

[0019] Preferably, in step (3), the amount of PHSN@GA4 added is 0.1g of PHSN@GA4 per 10mL of 5μM pH=5 PVP solution.

[0020] The present invention also provides a rice foliar cadmium-reducing agent prepared by the preparation method described in the above technical solution, which is supported on mesoporous silica.

[0021] The present invention also provides a method for spraying a rice foliar cadmium-reducing agent loaded with mesoporous silica and gibberellin, wherein the rice foliar cadmium-reducing agent is sprayed once during the rice booting-heading stage, and the spraying concentration is 5-10 mg / L.

[0022] Beneficial technical effects:

[0023] This invention utilizes mesoporous silica nanoparticles loaded with gibberellin and encapsulated using PVP to prepare a foliar inhibitor that can significantly reduce Cd content in rice. The foliar inhibitor prepared in this invention primarily works by increasing the length of the rice panicle neck, reducing the translocation of node 1 to the grain, decreasing the transpiration rate of rice leaves, and thus reducing the direct absorption and translocation of Cd. Encapsulation effectively prevents the oxidative degradation of gibberellin, reducing environmental losses. This method is characterized by high efficiency, low cost, simple operation, and readily available raw materials. Attached Figure Description

[0024] Figure 1 The images shown are transmission electron microscope (TEM) images of PHSN@GA4 obtained in Example 1, where the left image is PHSN@GA4 and the right image is PHSN.

[0025] Figure 2 The image shows the BJH pore size distribution of PHSN@GA4 obtained in Example 1.

[0026] Figure 3The nitrogen (N2) adsorption-desorption isotherm of PHSN@GA4 obtained in Example 1;

[0027] Figure 4 The infrared spectrum of PHSN@GA4 obtained in Example 1;

[0028] Figure 5 The effect of PHSN@GA4 foliar spraying on gas exchange parameters of rice (differences between mean photosynthetic parameters are represented by different letters (p<0.05);

[0029] Figure 6 Figure showing the effect of PHSN@GA4 foliar spraying on internode length in rice;

[0030] Figure 7 The effect of foliar spraying of PHSN@GA4 on the relative expression of Cd transporter protein in node 1 of rice (differences between mean parameters are represented by different letters (p<0.05));

[0031] Figure 8 The effects of foliar spraying of PHSN@GA4 and GA4 on Cd accumulation in rice of different rice varieties (different letters indicate the differences in mean Cd parameters of brown rice after different foliar spraying treatments for the same variety (p<0.05)). Detailed Implementation

[0032] The present invention will be further illustrated below with reference to the embodiments, but the present invention is not limited to the embodiments described below.

[0033] Example 1

[0034] (1) Dissolve hexadecyltrimethylammonium bromide (CTAB) and triblock copolymer (Pluronic P123) in an ammonia-ethanol mixture, and add tetraethyl orthosilicate (TEOS) dropwise. When the solution turns milky white, stir overnight at 85°C. The white precipitate is washed, centrifuged, dried and calcined to obtain mesoporous silica.

[0035] (2) Weigh 0.5g of the mesoporous silica powder obtained in step (1) and disperse it in 100mL of gibberellin (GA4) aqueous solution with a concentration of 50mg / mL. Stir magnetically for 72h at 25℃. After the reaction is completed, centrifuge the solution and save the supernatant (for determining the gibberellin concentration). Wash the precipitate with deionized water several times and freeze dry to obtain PHSN@GA4 powder.

[0036] (3) Add 0.1 g of the obtained PHSN@GA4 to 10 mL of PVP (5 μM) solution with pH = 5, stir at low temperature for 36 h, centrifuge-filter and freeze dry to obtain PVP-encapsulated PHSN@GA4; determine the gibberellin (GA4) content in the supernatant of the PVP-encapsulated mesoporous silica nanoparticles (PHSN@GA4) powder by liquid chromatography, and calculate that the GA4 loading rate in PHSN@GA4 is 75.4%.

[0037] (4) Weigh 150 mg of the PHSN@GA4 prepared above, add it to 1 L of pure water, and add Tween 80 at a concentration of 0.5‰. Under 25℃ conditions, the cadmium-reducing agent for rice leaves is obtained by ultrasonic treatment for 30 min.

[0038] Based on the product obtained in this embodiment, the following experimental examples were conducted, and the specific results are as follows:

[0039] Experimental Example 1: Characterization of cadmium-reducing agents on rice foliar surface (TEM, BET, FTIR)

[0040] Experimental methods: (1) Scanning electron microscopy (SEM) analysis: A small amount of PHSN and PHSN@GA4 powders were ultrasonically dispersed in anhydrous ethanol, and the dispersed samples were dropped onto a copper grid. After freeze-drying, the samples were observed under a transmission electron microscope (HITACHIHT7800), and the particle size was calculated using image software; (2) Fully automated specific surface area and porosity (BET) analysis: The specific surface area was measured by nitrogen adsorption-desorption test using a TriStarII 3020 (Micromeritics Instruments Corp, Norcross, GA, USA). The specific surface area was calculated by the Brunauer-Emmett-Teller (BET) method. Before analysis, PHSN and PHSN@GA4 were degassed at 80℃ for 12 hours. When P / P0 is in the range of 0.05-0.25, calculate the BET specific surface area and pore size distribution of the particles; (3) Infrared spectroscopy (FTIR) analysis: Place some samples in a mortar and grind them thoroughly, add a certain amount of dry KBr powder, mix evenly and compress into tablets, and use infrared spectroscopy (Thermo Scientific Nicolet iS20) for detection, wherein the spectral scanning range is 400-4000 cm⁻¹. -1 The test results are as follows Figure 1 .

[0041] Experimental results: Figure 1 It can be seen that mesoporous silica has a significant encapsulation effect; from Figure 2 It can be seen that the pore sizes of the prepared PHSN and PHSN@GA4 are 8.83 nm and 4.38 nm, respectively. Figure 3 It can be seen that the specific surface area of ​​the prepared mesoporous material is 159.3973 m². 2 / g and 159.39m 2 / g(BET), Figure 4 It can be seen that GA4 was successfully loaded into mesoporous silica.

[0042] Experimental Example 2: Effects of foliar cadmium-reducing agents on cadmium absorption and translocation in rice

[0043] Test method:

[0044] 1. Test location: Greenhouse room, Soil Research Institute, Xuanwu District, Nanjing City, Jiangsu Province.

[0045] 2. Soil type: Yellow-brown paddy soil with a pH of 5.86 and a total cadmium content of 0.85 mg / kg. 8 kg of soil is used per pot, and the soil is fertilized with 200-120-150 mg / kg (N-P2O5-K2O).

[0046] 3. Rice variety selection and planting method: Fengliangyouxiang No. 1 was raised on March 15, 2022, and transplanted into 9L plastic buckets on April 15, 2022. Water management characteristics: flooding in the early stage and drainage during the grain filling stage (22 days before harvest). The rice entered the grain filling stage on August 5, 2022, and was harvested on August 27.

[0047] 4. Foliar spraying treatment: Foliar spraying will be carried out during the grouting period (August 5, 2022) from 16:00 to 17:00, with the following four treatments:

[0048] Control: Spray with clean water;

[0049] Treatment 1: Spray mesoporous silica (PHSN);

[0050] Treatment 2: Apply GA4 separately;

[0051] Treatment 3: Spray mesoporous silica@gibberellin (PHSN@GA4);

[0052] The spraying concentration was 100 μmol / L, and each treatment was repeated 4 times.

[0053] 5. Indicator Observation

[0054] Within 14 days of foliar spraying, the photosynthetic gas exchange parameters of rice were measured using a Li-6800 portable photosynthesis system (see attached figure). Figure 5(Photosynthetic rate, transpiration rate, stomatal conductance, intercellular carbon dioxide) Rice was collected at maturity, and 10 dominant tillers were selected. Leaf area index, internode length, yield and its components (thousand-grain weight, seed setting rate, number of grains per panicle) were measured. Rice was divided into 5 parts, including root, stem, leaf, glumes, and brown rice. After drying, grinding and sieving, the rice was digested with HNO3-HClO4 (4:1). The digestion solution was diluted, brought to volume and filtered. The Cd concentration of each organ was determined using ICP-MS. The effects of foliar spraying of mesoporous silica@gibberellin (PHSN@GA4) on rice yield and its components are shown in Table 1. The effects of foliar spraying of mesoporous silica@gibberellin (PHSN@GA4) on the Cd content of each organ of rice are shown in Table 2.

[0055] Table 1. Effects of foliar spraying of mesoporous silica@gibberellin (PHSN@GA4) on rice yield and its components.

[0056]

[0057] Table 2. Effects of foliar spraying of mesoporous silica@gibberellin (PHSN@GA4) on Cd content in various organs of rice.

[0058]

[0059] As shown in Table 1, based on the results of yield and its components, both foliar spraying with gibberellin and treatment with gibberellin loaded on mesoporous silica can increase rice yield, with yields increasing by 11.8% and 24.7% respectively compared to the control. It can be clearly seen that spraying with gibberellin can increase the number of grains per panicle in rice, while mesoporous silica alone has no significant effect on rice yield. In terms of rice photosynthetic parameters, spraying with gibberellin can improve its photosynthetic efficiency.

[0060] Table 2 shows that PHSN alone did not significantly reduce Cd in rice, and GA4 alone had no effect on reducing Cd in rice leaves and roots, but mainly affected stems, glumes, and brown rice. However, PHSN@GA4 significantly reduced the Cd content in stems, glumes, and brown rice. This invention also tested the effects of foliar spraying of mesoporous silica@gibberellin (PHSN@GA4) on internode length and the relative expression level of Cd transporter protein in rice node 1. Figure 6 and Figure 7 It can be seen that gibberellin spraying significantly increased the length of internode 1 in rice and upregulated the relative expression levels of OsHMA2 and OsHMA3 in node 1, promoting the accumulation of Cd in the first node and internode of rice, further reducing the translocation of Cd into the grain, thereby reducing the Cd content in rice.

[0061] Experiment Example 2: Effects of spraying GA4 and PHSN@GA4 on the yield and Cd reduction of different rice varieties

[0062] Test method:

[0063] 1. Test location: Weizhuang Village, Qichun County, Huanggang City, Hubei Province.

[0064] 2. Test soil: Yellow-brown paddy soil with a pH of 5.86 and a total cadmium content of 0.85 mg / kg. Each plot was 20 m². 2 The randomized block design was repeated 4 times, and the fertilizer was 15-8-12 kg of N-P2O5-K2O per acre.

[0065] 3. Experimental Design and Treatments: Four varieties of single-season mid-season rice—Fengliangyouxiang No. 1, Kenliangyou 801, Huangkexiang No. 1, Fudao 99, and Liangyou 537—were selected. Seedlings were raised on March 15, 2022, and transplanted to plots on April 15, 2022. Water management was characterized by early-stage flooding followed by drainage during the grain-filling stage (22 days before harvest). The rice entered the grain-filling stage on August 2, 2022, and matured and was harvested on August 31, 2022. The same control methods were used for all diseases, pests, and weeds.

[0066] 4. Foliar spraying treatment: Foliar spraying will be carried out during the grouting period (August 10, 2022) from 15:00 to 16:00, with the following four treatments:

[0067] Control: Spray with clean water;

[0068] Treatment 1: Spray mesoporous silica (PHSN);

[0069] Treatment 2: Apply GA4 separately;

[0070] Treatment 3: Spray mesoporous silica@gibberellin (PHSN@GA4);

[0071] The spraying concentration was 100 μmol / L, and each treatment was repeated 4 times.

[0072] 5. Indicator Observation:

[0073] Rice grains were collected at maturity after different spraying processes. Five rice plants were randomly selected from each plot. The rice grains underwent a process of drying, hulling, baking, grinding, and sieving. The digestion was performed using HNO3-HClO4, and the analysis was performed using ICP-MS.

[0074] The results of the above comparative experiments demonstrate that the application of PHSN@GA4 showed a significant Cd-reducing effect in all five main cultivated varieties, while the application of GA4 alone did not show a Cd-reducing effect in each variety. This may be because the application of GA4 alone is not the same as the application of GA4 alone. Therefore, the preferred method of the present invention has significant technical advantages over the conventional application of GA4 alone, and the effect is outstanding.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a rice foliar cadmium-reducing agent supported on mesoporous silica and gibberellin, characterized in that, Includes the following steps: (1) Using tetraethyl orthosilicate as the silicon source and a template agent, nano-mesoporous silica particles were prepared by sol-gel method. After the reaction was completed, the resulting precipitate was centrifuged, dried, and calcined at high temperature to remove the template agent to obtain PHSN powder. (2) The obtained PHSN powder was dispersed in gibberellin ethanol-water solution, stirred to allow it to react fully, and then PHSN@GA4 powder was obtained by centrifugation and freeze drying. (3) Add the PHSN@GA4 obtained in step (2) to a PVP solution with pH=5, stir at low temperature for 36 h, centrifuge-filter and freeze dry to obtain PVP-encapsulated PHSN@GA4; (4) The PHSN@GA4 powder encapsulated in PVP was uniformly dispersed in the aqueous phase and a surfactant was added to obtain a cadmium-reducing agent for rice leaves. The template agent is a hexadecyltrimethylammonium bromide and a triblock copolymer; The mass ratio of PHSN powder to gibberellin is 1:

10.

2. The preparation method according to claim 1, characterized in that, The specific steps (1) are as follows: the template agent is dissolved in a mixture of ammonia and ethanol, and tetraethyl orthosilicate is added dropwise. When the solution turns milky white, the reaction is stirred at 85°C. After the reaction is completed, the precipitate is collected. The precipitate is washed, centrifuged, dried and calcined to obtain PHSN powder.

3. The preparation method according to claim 1, characterized in that, The specific steps (2) are as follows: the obtained PHSN powder is dispersed in the ethanol solution of gibberellin and stirred for 72 hours. After the reaction is completed, the obtained solution is centrifuged, washed, and freeze-dried to obtain PHSN@GA4 powder.

4. The preparation method according to claim 1 or 3, characterized in that, The stirring temperature is less than or equal to 25°C.

5. The preparation method according to claim 1, characterized in that, The surfactant is Tween-80; the concentration of Tween-80 is 0.5‰.

6. The rice foliar cadmium-reducing agent prepared by the preparation method according to any one of claims 1-5, which is supported on mesoporous silica.

7. A method for spraying a rice foliar cadmium-reducing agent supported on mesoporous silica and gibberellin, characterized in that, The cadmium-reducing agent for rice foliar application as described in claim 6 is applied once during the booting-heading stage of rice, at a concentration of 5-10 mg / L.

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