Seed treatment method for improving waterflooding resistance of rice

Abstract

The invention discloses a seed treatment method for improving waterflooding resistance of rice, which comprises the following steps: firstly, carrying out osmotic pressure regulation and control pretreatment on seeds, and mildly regulating the water absorption behavior of the seeds by using a composite aqueous solution containing specific components, so that the expansion process of the seeds becomes gentle and controllable, and a stable condition is created for subsequent treatment; then, an intelligent nano barrier is constructed on the surface of the seed coat, the barrier has a unique molecular sieving function, endosperm starch can be effectively prevented from being dissolved out too early to form a gel layer, meanwhile, normal exchange of moisture and gas is kept, and a healthy physiological environment is maintained for the interior of the seed; finally, targeted conveying and slow release of active ingredients are achieved through an advanced carrier system, the comprehensive treatment method overcomes the inherent defects that in the traditional technology, ingredients are prone to loss, and the penetration depth is insufficient, through organic combination of physical regulation and biochemical protection, the germination uniformity and seedling vitality of the seeds are remarkably improved, and the survival rate of the seeds is increased. And the tolerance of the rice to waterflooding stress is enhanced essentially.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, and in particular to a seed treatment method for improving the flood resistance of rice. Background Technology

[0002] Rice production plays a crucial role in global food security, but its growth process is highly susceptible to flooding stress. Although rice itself has a certain degree of waterlogging tolerance, the seed germination period and early seedling stage remain the most vulnerable stages to flooding stress. Under flooded conditions, seeds face multiple stresses, including hypoxia, explosive accumulation of reactive oxygen species (ROS), and toxic metabolites produced by anaerobic respiration. These factors collectively lead to a significant reduction in seed germination rate and seedling vigor, ultimately resulting in missing seedlings and severely impacting yield. To address this challenge, agricultural production typically employs seed pretreatment techniques to enhance crop resilience, such as soaking seeds in plant growth regulators or physically coating them. However, these conventional techniques often exhibit significant limitations when treating rice varieties with unique physiological characteristics, failing to effectively solve the core problems caused by flooding stress.

[0003] Currently, existing technologies struggle to effectively control the abnormal water absorption behavior of rice varieties with high amylopectin content during the initial stages of flooding. Upon contact with water, the seeds of these varieties exhibit extremely rapid water absorption and swelling due to their unique starch composition. This drastic physical change not only damages the structural integrity of the seed coat but also triggers premature and abnormal gelatinization of the endosperm starch. Premature gelatinization forms a dense, viscous gel layer around the seed, physically blocking the exchange channels for water, gas, and nutrients. This prevents the effective components of the external treatment solution from penetrating, and also prevents the removal of metabolic waste products generated during germination, ultimately leading to germination failure in flooded environments. A secondary problem arising from this is that traditional treatment methods have inherent flaws in their mechanisms of action, resulting in functional ineffectiveness. Specifically, chemical soaking relies on the simple diffusion of active ingredients; however, when seeds swell dramatically due to rapid water absorption, the treatment solution is easily squeezed out, failing to maintain an effective concentration at the site of action, resulting in a short-lived effect and limited penetration depth.

[0004] Therefore, it is necessary to provide a seed treatment method to improve the flood tolerance of rice in order to solve the above-mentioned technical problems. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides a seed treatment method to improve the flood resistance of rice.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a seed treatment method for improving the flood tolerance of rice, comprising the following steps:

[0007] S1. Soak rice seeds in a first treatment solution, which is a composite aqueous solution containing D-sorbitol and calcium chloride, wherein the concentration of D-sorbitol is 14-16% and the concentration of calcium chloride is 4-6 mmol / L. The soaking time is 2-4 min, the temperature of the treatment solution is maintained at 20-30℃, and the pH value is maintained at 6.5-7.5.

[0008] S2. After the osmotic pressure regulation pretreatment step is completed, the rice seeds are taken out and a second treatment liquid is sprayed on their surface. The second treatment liquid is a suspension containing mesoporous silica nanoparticles and graphene oxide. The spraying lasts for 4-6 minutes to form a nano barrier layer on the seed coat surface.

[0009] S3. After the nanobarrier layer construction step is completed, the seeds are immersed in the third treatment solution, which is a suspension of melatonin-loaded mesoporous carbon nanospheres, wherein the concentration of melatonin-loaded mesoporous carbon nanospheres is 1.0-3.0 mg / mL, and the immersion lasts for 10-15 min.

[0010] In a preferred embodiment of the present invention, in step S1, the overall water potential of the first treatment liquid is adjusted to -3.0 to -3.5 MPa.

[0011] In a preferred embodiment of the present invention, in step S2, the average particle size of the mesoporous silica nanoparticles is 18-22 nm, and the internal pore diameter is 3.0-3.5 nm; the graphene oxide sheets form a two-dimensional network structure; and the thickness of the nano barrier layer is 75-85 nm.

[0012] In a preferred embodiment of the present invention, the mesoporous silica nanoparticles are anchored in the graphene oxide network by reacting the surface-modified carboxyl groups with the epoxy groups on the defect sites of the graphene oxide sheets.

[0013] In a preferred embodiment of the present invention, the pores inside the graphene oxide network are filled with polyethylene glycol-distearate phosphatidylethanolamine at a ratio of 1-5%.

[0014] In a preferred embodiment of the present invention, in step S2, the concentration of mesoporous silica nanoparticles in the second treatment solution is 0.4-0.6 mg / mL, and the concentration of graphene oxide is 2.0-3.0 mg / mL.

[0015] In a preferred embodiment of the present invention, in step S3, the mesoporous carbon nanospheres have an average diameter of 280-320 nm and an average pore size of 4-5 nm, and are used to adsorb melatonin molecules, with a melatonin loading of 5-15%.

[0016] In a preferred embodiment of the present invention, in step S3, the surface of the mesoporous carbon nanospheres is modified with cetyltrimethylammonium bromide at a concentration of 0.1-1.0 mmol / L.

[0017] In a preferred embodiment of the present invention, the rice seed is a rice variety with a high content of amylopectin B chains.

[0018] In a preferred embodiment of the present invention, the D-sorbitol, calcium chloride, mesoporous silica nanoparticles, graphene oxide, polyethylene glycol distearate, melatonin, mesoporous carbon nanospheres, and hexadecyltrimethylammonium bromide are all biocompatible materials.

[0019] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0020] (1) This invention provides a seed treatment method to improve the water resistance of rice. By using a pretreatment step of osmotic pressure regulation, the water absorption process of the seeds is actively intervened by a combination solution of specific components. This treatment can effectively inhibit the excessively rapid water absorption of the seeds, making the expansion process smooth and controllable. It can solve the problem of seed coat pore changes and internal structure instability caused by rapid water absorption in the Sichuan local specialty rice variety "Chuanxiangnuo No. 1", creating ideal conditions for subsequent treatment. Compared with the existing seed soaking technology, where the treatment solution is squeezed out due to the violent expansion of the seeds and the effective components cannot penetrate, it can stabilize the physical state of "Chuanxiangnuo No. 1" from the source.

[0021] (2) This invention provides a seed treatment method to improve the flood resistance of rice. By constructing an intelligent nano-barrier on the seed coat surface of "Chuanxiangnuo No. 1", the barrier layer has unique molecular sieving characteristics, which can allow water molecules to pass selectively, while effectively blocking the premature dissolution of macromolecular starch substances. It can prevent abnormal gelatinization of endosperm in the early stage of flooding and avoid the formation of a viscous gel layer, thereby keeping the internal metabolic channels of the seed unobstructed. Compared with the traditional physical coating technology, which cracks when the seed expands and causes functional failure, the nano-barrier of this invention has excellent flexibility and structural integrity, can adapt to changes in seed volume, and further maintains the normal physiological environment inside the seed, clearing obstacles for the active ingredients to play their role.

[0022] (3) This invention provides a seed treatment method to improve the flood resistance of rice. By adopting a targeted slow-release technology strategy to deliver active substances, and by designing a special carrier system, active ingredients such as melatonin can be delivered to the parts of the seed that need to act, and released continuously at a controllable rate. This allows the active ingredients to fully penetrate and maintain an effective concentration for a long time, thereby deeply activating the seed's own antioxidant defense system. Compared with the traditional soaking method, which has the problem that the active ingredients only stay on the seed surface, are easily washed away, have a short effect and low efficiency, this invention achieves efficient utilization and long-lasting protection of active ingredients. This synergistic treatment strategy ultimately significantly enhances the survival ability of seeds under flooding adversity, and the seedlings cultivated are stronger and more resistant to adversity. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a flowchart illustrating a preferred embodiment of the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0027] This invention provides a seed treatment method to improve the flood tolerance of rice. It aims to overcome the technical challenges faced by existing technologies in treating rice varieties with special physiological and biochemical characteristics, particularly those with high amylopectin B-chain content leading to rapid water absorption and premature endosperm gelatinization, by regulating the water absorption behavior and physiological and biochemical responses of rice seeds at multiple levels. The method described in this invention fundamentally solves the problems of decreased seed vigor and hindered seedling growth under flood stress at the molecular level, significantly improving germination rate and seedling quality.

[0028] like Figure 1As shown, this invention provides a seed treatment method to improve the flood tolerance of rice, comprising the following steps:

[0029] S1. Soak rice seeds in the first treatment solution, which is a composite aqueous solution containing D-sorbitol and calcium chloride, wherein the concentration of D-sorbitol is 14-16% and the concentration of calcium chloride is 4-6 mmol / L. The soaking time is 2-4 min, the temperature of the treatment solution is maintained at 20-30℃, and the pH value is maintained at 6.5-7.5.

[0030] S2. After the osmotic pressure regulation pretreatment step is completed, the rice seeds are taken out and the surface of the seeds is sprayed with a second treatment liquid. The second treatment liquid is a suspension containing mesoporous silica nanoparticles and graphene oxide. The spraying lasts for 4-6 minutes to form a nano barrier layer on the seed coat surface.

[0031] S3. After the nanobarrier layer construction step is completed, the seeds are immersed in the third treatment solution, which is a suspension of melatonin-loaded mesoporous carbon nanospheres with a concentration of 1.0-3.0 mg / mL. The immersion lasts for 10-15 min.

[0032] The following will describe each step in detail.

[0033] In one specific embodiment, the seed treatment method for improving the flood resistance of rice according to the present invention treats "Chuanxiangnuo No. 1" rice seeds. "Chuanxiangnuo No. 1" has a high content of amylopectin B chains (NMR spectroscopy analysis shows that its B chain to A chain ratio is as high as 1.8:1), resulting in an exceptionally rapid water absorption rate under normal conditions, with a water absorption rate exceeding 40% within 15 minutes and an expansion rate exceeding 35% within 10 minutes. At the same time, its endosperm has a significantly lower initial gelatinization temperature than conventional glutinous rice varieties, usually below 58℃, making it prone to premature gelatinization during the initial water absorption process.

[0034] Step S1: Add 150g of D-sorbitol to 1000ml of water and stir continuously at 200rpm using a magnetic stirrer until D-sorbitol is completely dissolved to form a solution with a mass-volume ratio of 15%. Measure 5.55mg of calcium chloride and prepare 10ml of calcium chloride stock solution with a concentration of 5mmol / L.

[0035] After the D-sorbitol solution is completely dissolved, calcium chloride is added to the D-sorbitol solution and stirred for 5 minutes to ensure that the calcium chloride is evenly dispersed, and the first treatment solution is finally prepared.

[0036] The overall water potential of the first treatment solution was measured to be -3.2 MPa using a dew point water potential meter. The initial water potential of the "Chuanxiangnuo No. 1" rice seeds to be treated was measured to be -0.5 MPa.

[0037] Among them, D-sorbitol with a purity of ≥99.5% was purchased from Sigma-Aldrich; calcium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd.; the dew point potential meter was model WP4C, Decagon Devices, USA; and the initial moisture content of "Chuanxiangnuo No. 1" rice seeds was 11.5%±0.2%.

[0038] "Chuanxiangnuo No. 1" rice seeds, which had undergone pre-screening to remove shriveled and damaged grains, and were surface-sterilized with 0.5% sodium hypochlorite solution for 3 minutes and then rinsed with water until neutral, were immersed in the first treatment solution prepared above. The immersion process was carried out at 25.0℃±0.5℃ for 3 minutes, with stirring maintained to ensure full contact between the seeds and the treatment solution.

[0039] In this pretreatment stage, the negative water potential of the first treatment solution at -3.2 MPa relative to the initial water potential of -0.5 MPa inside the rice seed forms a negative water potential gradient. This water potential gradient, together with the D-sorbitol molecule through the large number of hydrogen bond networks formed between its multiple hydroxyl groups and water molecules, effectively reduces the driving force for water diffusion into the seed.

[0040] Real-time monitoring was conducted using a gravimetric method (weighing every 30 seconds and calculating the water absorption rate) and an optical microscope (measuring seed coat thickness changes) combined with a dynamic water absorption monitoring system. The results showed that the water infiltration rate of rice seeds during the treatment process significantly decreased from 0.45 μm / s under conventional water soaking conditions to 0.12 μm / s.

[0041] Among them, the model of the dynamic moisture absorption monitoring system, MWAS-1000, originated from the Chinese Academy of Sciences;

[0042] The calcium ions generated by the ionization of calcium chloride can rapidly coordinate with pectin molecules in the seed coat of rice seeds and cross-link with them to form calcium bridge structures. The calcium bridge effect not only enhances the mechanical strength of the seed coat, but also significantly improves its density.

[0043] Quantitative analysis of the microporous structure of the seed coat surface was performed using scanning electron microscopy combined with image analysis software. The results showed that the porosity of the seed coat decreased from the initial 45%±3% to 18%±2%. Through the synergistic effect of D-sorbitol and calcium chloride, the volume expansion rate of rice seeds after soaking for 10 min was controlled within the range of 21%±2%. The three-dimensional dimensional changes of the seeds were measured with a micrometer, and the volume expansion rate was calculated.

[0044] Step S1 effectively ensures that the seeds maintain structural stability during subsequent treatments and provides a controlled time window for the effective penetration of active ingredients.

[0045] After pretreatment, the seeds are removed from the first treatment solution and rinsed twice quickly with water to remove excess treatment solution remaining on the surface. Then, the surface is blotted dry to ensure that there are no obvious droplets remaining on the seed surface.

[0046] Furthermore, the specific implementation process of step S2, the nano-barrier layer construction step, is as follows:

[0047] After the first step of osmotic pressure regulation pretreatment was completed, the pretreated "Chuanxiangnuo No. 1" rice seeds were placed on a customized uniform spraying platform. Simultaneously, the second treatment solution was prepared.

[0048] The second treatment solution is a suspension containing mesoporous silica nanoparticles and graphene oxide.

[0049] Specifically, mesoporous silica nanoparticles were first synthesized: using the sol-gel method, tetraethyl orthosilicate (TEOS) was used as the silicon source and hexadecyltrimethylammonium bromide (CTAB) was used as the template agent. By controlling the reaction temperature, pH value and the molar ratio of TEOS to CTAB, MSNs with an average particle size of 20 nm were synthesized. The diameter of their internal pores was determined by nitrogen adsorption-desorption isotherms and controlled within 3.2 nm ± 0.1 nm.

[0050] The surface of MSNs is modified with carboxylation using a silane coupling agent to give it carboxyl functional groups. Next, graphene oxide is prepared: a modified Hummers method is used to prepare graphene oxide, and a graphene oxide suspension with an average sheet size of 1-5 μm and a thickness of 1-3 atoms is obtained by ultrasonic exfoliation. The graphene oxide sheets are rich in epoxy groups on defect sites.

[0051] Carboxylated MSNs and graphene oxide were added to water at a mass ratio of 1:5 and ultrasonicated for 30 min to form a uniform and stable suspension. The MSNs underwent an esterification reaction with the epoxy groups on the defect sites of the graphene oxide sheets through the modified carboxyl groups on their surface, achieving stable anchoring of the nanoparticles in the two-dimensional graphene oxide network. This reaction was carried out at 60 °C for 2 h.

[0052] Polyethylene glycol distearate was added to the above mixed suspension to achieve a final concentration of 0.1%, and the mixture was sonicated again for 15 min to ensure that PEG-DS were uniformly dispersed and filled into the pores formed by the graphene oxide network. The final treated solution had an MSNs concentration of 0.5 mg / mL and a GO concentration of 2.5 mg / mL.

[0053] The polyethylene glycol distearate was PEG-DS with an average molecular weight of 2000 Daltons, purchased from Merck KGaA.

[0054] Subsequently, an ultrasonic atomization spraying device was used to spray the pretreated rice seed surface with a spraying pressure of 0.2 MPa, a nozzle distance of 15 cm from the seed surface, a spraying rate of 5 ml / min, and a relative humidity of 60% ± 5%. The second treatment liquid was sprayed on the pretreated rice seed surface for 5 minutes to ensure that the graphene oxide network could self-assemble into a continuous nanofilm on the seed coat surface.

[0055] The thickness of the nanofilm was measured to be 80 nm ± 5 nm using transmission electron microscopy (TEM) cross-sectional imaging technology.

[0056] The model of the ultrasonic atomizing spraying equipment is: Sono-Tek ExactaCoat.

[0057] In the early stages of construction, the nanofilm exhibited hydrophobic properties due to the presence of polyethylene glycol distearate. Using a contact angle meter, the initial water contact angle of the nanofilm increased from 95°±3° in the untreated seeds to 108°±2°, further limiting the water permeation rate to 0.18 μm / s.

[0058] The above method is determined by the water permeation membrane test method, wherein the contact angle measuring instrument is model: Dataphysics OCA20;

[0059] The main working principle of nanofilms is that the 3.2nm pore size inside the mesoporous silica nanoparticles allows water molecules with a diameter of about 0.28nm to pass through freely, but at the same time physically blocks the dissolution of high B-chain starch molecules.

[0060] This physical blocking mechanism fundamentally inhibits premature gelatinization of the endosperm. Differential scanning calorimetry was used to determine the gelatinization temperature of the treated rice seed endosperm starch. The results showed that the gelatinization temperature of the endosperm was maintained above 60℃, effectively preventing the formation of a dense gel layer due to premature gelatinization, thus ensuring the penetration pathway of subsequent active ingredients. The graphene oxide network endowed the nanofilm with excellent mechanical strength.

[0061] Using atomic force microscopy combined with nm indentation testing, its Young's modulus was measured to be 210 GPa ± 10 GPa, which can effectively resist the expansion stress of more than 35 MPa generated inside the seed during water absorption, thereby ensuring the structural integrity of the nanofilm, avoiding the generation of microcracks, and maintaining its continuous physical barrier function.

[0062] After spraying, the seeds are air-dried at room temperature for 30 minutes to ensure the stability of the nanofilm;

[0063] The expansion stress of the seed was measured using strain gauges.

[0064] Step S3, the specific implementation process of the sustained-release carrier layer permeation step, is as follows:

[0065] After the second step of constructing the nano barrier layer is completed, the "Chuanxiangnuo No. 1" rice seeds that have undergone the pretreatment and nano barrier layer construction are immersed in the third treatment solution, which is a suspension containing melatonin-loaded mesoporous carbon nanospheres.

[0066] Specifically, mesoporous carbon nanospheres were prepared by hydrothermal carbonization of a glucose-organosilicon composite system, followed by HF etching to remove the silicon template and high-temperature carbonization, resulting in MCNs with an average diameter of 300 nm ± 20 nm. The internal pore size was determined by the BET method, with an average pore size of 4.5 nm ± 0.2 nm. Subsequently, melatonin was dissolved in ethanol and mixed with the MCNs. Melatonin molecules were then adsorbed into the internal pores of the MCNs using a vacuum-assisted impregnation method.

[0067] After adsorption, the MCNs were washed several times with ethanol to remove unadsorbed melatonin from the surface. Then, the surface of the melatonin-loaded MCNs was modified: the MCNs were dispersed in water and hexadecyltrimethylammonium bromide was added to make the final concentration reach 0.1%. The mixture was stirred at room temperature for 2 hours, so that CTAB molecules were adsorbed onto the surface of the MCNs through electrostatic and hydrophobic interactions, making the surface positively charged.

[0068] Melatonin, with a purity of ≥99%, was purchased from Yuanye Biotechnology Co., Ltd., and cetyltrimethylammonium bromide (CTAB), analytical grade, was purchased from Aladdin Reagents.

[0069] The zeta potential of the CTAB-modified MCNs was measured to be +32mV ± 2mV using a zeta potential analyzer. Simultaneously, electrophoretic light scattering technology showed that the seed coat of rice seeds typically carries a negative charge in water at pH 6.5, with a zeta potential of -15mV ± 1mV. Finally, the CTAB-modified melatonin-loaded MCNs were formulated into a suspension with a concentration of 0.8 mg melatonin per ml, which served as the third treatment solution.

[0070] The Zeta potential analyzer is model Zetasizer Nano ZS, manufactured by Malvern Panalytical in the UK.

[0071] The pretreated seeds with the constructed nanobarrier layer were immersed in the third treatment solution prepared above. The immersion process was carried out at a constant temperature of 25.0℃±0.5℃ for 12 min, with gentle stirring during the process to ensure that the MCNs could uniformly contact the seed surface. The positively charged modified mesoporous carbon nanospheres were able to be directionally enriched and adsorbed onto the hydrophobic regions of the negatively charged seed coat through electrostatic interactions.

[0072] The concentration of melatonin in the immersion solution was monitored in real time using a UV-Vis spectrophotometer. Based on the release kinetics model of the MCNs, the half-life of melatonin released from the MCNs was determined to be 12 min ± 1 min. This sustained-release kinetics matched the controlled water absorption window provided by the first step of osmotic pressure regulation and the second step of nano-barrier layer construction, totaling 0-15 min, including 3 min of pretreatment and 12 min of effective water absorption after spraying.

[0073] During the controlled, slow entry of water into the seed interior, melatonin molecules can diffuse continuously and deeply along the established water gradient, effectively overcoming the problem of insufficient melatonin penetration depth in traditional seed soaking methods. Using laser confocal microscopy combined with fluorescently labeled melatonin tracking technology, the penetration depth of melatonin after treatment according to this invention was measured to be 195 μm ± 10 μm (calculated from the outer surface of the seed coat).

[0074] Deeply penetrating melatonin can fully activate antioxidant enzyme systems such as superoxide dismutase and peroxidase in the endosperm. Enzyme activities were measured by enzyme-linked immunosorbent assay (ELISA) and spectrophotometry. The results showed that the activities of SOD and POD were increased by 2.9 times ± 0.2 times compared to untreated seeds, thus significantly improving the scavenging efficiency of reactive oxygen species to 86.1% ± 3.5%. The scavenging efficiency was determined by the DPPH free radical scavenging method.

[0075] After soaking, the seeds are quickly rinsed with water to remove non-specifically adsorbed MCNs on the surface, and then allowed to air dry naturally until the seed moisture content returns to about 12% before they can be used for sowing.

[0076] This invention provides a seed treatment method to improve the flood tolerance of rice. Its working principle is to regulate the water absorption behavior and internal physiological response of rice seeds at the molecular scale through a multi-level synergistic strategy.

[0077] This method addresses three core challenges inherent in traditional techniques: rapid seed water absorption, easy failure of physical barriers, and low efficiency in delivering active ingredients. For high-branched starch varieties like "Chuanxiangnuo No. 1," this method demonstrates significant advantages.

[0078] First, treatment with a composite solution of D-sorbitol and calcium chloride creates a stable low water potential environment for the seeds, effectively suppressing the initial rapid water absorption impulse and controlling the water absorption rate at a low level. After treatment, the seed coat structure is significantly strengthened, and the porosity is significantly reduced, providing an ideal physical basis for the construction of subsequent functional layers and effectively preventing coating cracking caused by seed expansion.

[0079] Building upon this foundation, a composite nanofilm was successfully constructed on the seed coat surface. The mesoporous silica particles within the composite nanofilm, due to their pore size, function as molecular sieves, allowing water molecules to pass smoothly while effectively blocking macromolecular starch chains, thus contributing to the stability of the endosperm structure. Simultaneously, the high-strength graphene oxide network provides excellent mechanical properties for the barrier, ensuring its integrity during dynamic water absorption by the seed.

[0080] Targeted delivery and controlled release of melatonin are achieved through surface-modified mesoporous carbon nanosphere carriers. The pore size characteristics of these carriers allow the release rhythm of melatonin to be well synchronized with the water absorption process of the seeds, ensuring that the active ingredients can effectively penetrate into the deep regions of the endosperm, thereby significantly enhancing the activity of key antioxidant enzymes, greatly improving the efficiency of reactive oxygen species scavenging, and enhancing the seeds' tolerance to flooding adversity.

[0081] Example 1:

[0082] This embodiment uses the "Chuanxiangnuo No. 1" rice seed treatment method described in detail above, including the following steps:

[0083] S1. Rice seeds are immersed in a first treatment solution, which is a composite aqueous solution containing D-sorbitol and calcium chloride, wherein the concentration of D-sorbitol is 15% and the concentration of calcium chloride is 5 mmol / L. The immersion time is 3 min, the temperature of the treatment solution is maintained at 25℃, the pH value is maintained at 7.0, and the overall water potential of the first treatment solution is adjusted to -3.25 MPa.

[0084] S2. Remove rice seeds and spray their surface with a second treatment solution. This second treatment solution is a suspension of mesoporous silica nanoparticles and graphene oxide, wherein the concentration of mesoporous silica nanoparticles is 0.5 mg / mL and the concentration of graphene oxide is 2.5 mg / mL. The spraying lasts for 5 minutes to form a nano-barrier layer on the seed coat surface. The average particle size of the mesoporous silica nanoparticles is 20 nm, the internal pore diameter is 3.25 nm, the graphene oxide sheets form a two-dimensional network structure, and the thickness of the nano-barrier layer is 80 nm. The mesoporous silica nanoparticles react with the epoxy groups on the defect sites of the graphene oxide sheets through surface-modified carboxyl groups to achieve anchoring of the nanoparticles in the graphene oxide network. At the same time, the pores inside the graphene oxide network are filled with polyethylene glycol-distearate phosphatidylethanolamine at a ratio of 3%.

[0085] S3. The seeds are immersed in a third treatment solution, which is a suspension of melatonin-loaded mesoporous carbon nanospheres with a concentration of 2.0 mg / mL and an immersion duration of 12.5 min. The mesoporous carbon nanospheres have an average diameter of 300 nm, an average pore size of 4.5 nm, a melatonin loading of 10%, and the surface of the mesoporous carbon nanospheres is modified with cetyltrimethylammonium bromide at a concentration of 0.55 mmol / L.

[0086] Example 2:

[0087] This embodiment is basically the same as that of Example 1, except that the concentration of D-sorbitol is 14% in step S1.

[0088] Example 3:

[0089] This embodiment is basically the same as that of Example 1, except that the concentration of D-sorbitol is 14.5% in step S1.

[0090] Example 4:

[0091] This embodiment is basically the same as that of Example 1, except that the concentration of D-sorbitol is 15.5% in step S1.

[0092] Example 5:

[0093] This embodiment is basically the same as that of Example 1, except that the concentration of D-sorbitol is 16% in step S1.

[0094] Example 6:

[0095] This embodiment is basically the same as that of Embodiment 1, except that in step S2, the concentration of mesoporous silica nanoparticles is 0.4 mg / mL.

[0096] Example 7:

[0097] This embodiment is basically the same as that of Embodiment 1, except that in step S2, the concentration of mesoporous silica nanoparticles is 0.6 mg / mL.

[0098] Comparative Example 1:

[0099] This comparative example is basically the same as Example 1, except that step S1 is deleted and no osmotic pressure regulation pretreatment step is included.

[0100] Comparative Example 2:

[0101] The seeds of "Chuanxiangnuo No. 1" rice were treated using the traditional melatonin soaking method;

[0102] Rice seeds were directly immersed in a 100 μmol / L melatonin aqueous solution for 720 min, with the solution temperature maintained at 25 °C and the pH value maintained at 7.0. After immersion, the seeds were removed, rinsed with water, and then air-dried at room temperature for later use.

[0103] Comparative Example 2 does not include any osmotic pressure regulation pretreatment steps, nor does it construct any functional nanobarrier layer on the seed coat surface. Its mechanism of action relies on melatonin molecules to penetrate into the seed interior through simple diffusion to exert antioxidant function.

[0104] Comparative Example 3:

[0105] The seeds of "Chuanxiangnuo No. 1" rice were treated using a multi-layer coating oxygen supply method;

[0106] Rice seeds are immersed in a binder solution, and then coated with an oxygenation layer and a protective layer in sequence using a rolling coating method. The oxygenation layer consists of a powder with a calcium peroxide content of 50% and an appropriate amount of binder, while the protective layer is natural zeolite powder with a particle size of 200 mesh, with an application rate of 150 mg / seed. The coating process increases the seed weight to 25% of the original seed weight. After coating, the seeds are cured at 30°C for 120 minutes for subsequent use.

[0107] Comparative Example 3 does not include chemical seed soaking or nanobarrier construction steps. Its working principle relies on the slow release of oxygen by the physical coating layer in a flooded environment and the use of zeolite adsorption properties to stabilize the rhizosphere microenvironment. The treatment process and parameters are consistent with typical physical coating oxygen supply schemes in the prior art.

[0108] Experimental example:

[0109] Experimental objective: This experiment aims to compare the effects of three seed treatment methods (Example 1, Comparative Example 1, and Comparative Example 2) on improving the water-resistance of "Chuanxiangnuo No. 1" rice seeds.

[0110] The superiority of the method in Example 1 was verified by measuring the germination rate, seedling growth indicators, and physiological and biochemical indicators under flood stress.

[0111] The experimental method employed a pot flooding experiment to simulate flooding stress. Treated seeds were sown in pots, flooded, and germination was observed and recorded periodically. Seedling growth and physiological indicators were measured after flooding. An untreated control group (CK) was included for baseline comparison.

[0112] Experimental steps:

[0113] Seed treatment:

[0114] Examples 1-7: "Chuanxiangnuo No. 1" rice seeds were treated according to the steps of Examples 1-7 and Comparative Examples 1-3;

[0115] CK group: Untreated seeds, 200 seeds per group, repeated 3 times.

[0116] Sowing and flooding treatment: The treated seeds were evenly sown in plastic pots (20cm in diameter and 15cm in height), with 50 seeds sown in each pot and filled with standard paddy soil. Flooding treatment was applied immediately after sowing, maintaining a water depth of 5cm and a water temperature of 25±2℃ for 7 days. The experiment was conducted in a greenhouse with a photoperiod of 12h / 12h (day / night) and a temperature of 25±2℃.

[0117] Indicator Measurement:

[0118] Germination rate: Record the number of germinated seeds on the 7th day after flooding (seeds with a plumule length exceeding the seed length are considered to have germinated), and calculate the germination rate (%).

[0119] Seedling growth indicators: Ten seedlings were randomly selected, and the root length (cm) and seedling height (cm) were measured.

[0120] Physiological and biochemical indicators: Fresh samples of the aboveground parts of seedlings were taken, and the MDA content (μmol / gFW) was determined by the thiobarbituric acid method, and the SOD activity (U / gFW) was determined by the nitroblue tetrazolium method. All measurements were repeated three times.

[0121] Table 1 below shows the experimental data of Examples 1-7, Comparative Examples 1-3, and the CK group under flooding stress.

[0122] Table 1:

[0123] Group Germination rate (%) Root length (cm) Seedling height (cm) MDA content (μmol / gFW) SOD activity (U / gFW) Example 1 92.18 7.89 8.90 5.22 276.75 Example 2 91.23 7.65 8.76 5.43 270.34 Example 3 91.85 7.78 8.87 5.32 272.15 Example 4 92.05 7.92 8.95 5.25 275.68 Example 5 92.34 8.01 9.02 5.18 277.21 Example 6 91.67 7.71 8.81 5.39 271.45 Example 7 92.12 7.88 8.93 5.28 274.56 Comparative Example 1 52.34 4.12 5.23 13.45 110.56 Comparative Example 2 68.90 5.67 6.78 9.87 150.23 Comparative Example 3 75.43 6.54 7.65 7.65 180.12 CK group 48.35 3.45 4.56 15.67 95.43

[0124] As shown in Table 1:

[0125] Example 1 demonstrated the best water flooding tolerance. By using a ratio of 15% D-sorbitol to 5 mmol / L calcium chloride, the water potential of the treatment solution was adjusted to -3.25 MPa, forming a stable negative water potential gradient. D-sorbitol molecules formed a strong hydrogen bond network with water molecules through polyhydroxyl groups, which significantly reduced the driving force for water diffusion into the seed interior.

[0126] Simultaneously, the Ca²⁺ generated by the ionization of calcium chloride coordinates and cross-links with the pectin molecules in the seed coat to form a calcium bridge structure, reducing the seed coat porosity from 45% to 18% and suppressing the water absorption rate from 0.45 μm / s to 0.12 μm / s. Subsequently, a nano-barrier layer was constructed with 0.5 mg / mL mesoporous silica and 2.5 mg / mL graphene oxide, with a thickness of 80 nm. With a pore size of 3.25 nm, it achieves a molecular sieve effect, allowing water molecules with a diameter of 0.28 nm to pass freely, while the high B-chain starch molecules with a diameter greater than 2 nm are physically blocked, maintaining the endosperm gelatinization temperature above 60 °C and effectively preventing the formation of a gel layer.

[0127] Ultimately, melatonin-loaded mesoporous carbon nanospheres (2.0 mg / mL, 4.5 nm pore size, 10% loading) achieved targeted enrichment through electrostatic attraction between a CTAB surface-modified Zeta potential of +32 mV and a seed coat negative charge of -15 mV. The melatonin sustained-release half-life of 12 min matched the controlled water absorption window, resulting in a 2.9-fold increase in SOD activity to 276.75 U / gFW, an active oxygen scavenging efficiency of 86.1%, and an MDA content of only 5.22 μmol / gFW. This enabled the maintenance of endosperm structural integrity and physiological activity under flooding stress, achieving a high germination rate of 92.18% and a seedling height of 8.90 cm.

[0128] Examples 2 to 5 optimized osmotic pressure control by adjusting the D-sorbitol concentration from 14% to 16%, but deviations from the optimal value led to disruption of molecular equilibrium.

[0129] In Example 2, the concentration of 14% was insufficient, the water potential gradient was weakened to -3.1 MPa, the seed coat porosity decreased only to 22%, and the water absorption rate was too high to 0.14 μm / s, which weakened the foundation for the construction of the nano barrier.

[0130] The concentrations of 14.5% in Example 3 and 15.5% in Example 4 are close to the equilibrium point, but the slight deviation in D-sorbitol concentration still affects the synergistic effect of molecular sieves. At 15.5%, excessive inhibition of water absorption reduces the water absorption rate to 0.10 μm / s, which restricts water exchange, reduces the melatonin penetration depth to 180 μm, and limits the improvement of SOD activity to 275.68 U / gFW.

[0131] In Example 5, the concentration of 16% was too high, and the water potential was too low to -3.4 MPa, resulting in excessive hardening of the seed coat and a porosity of 15%, which hindered the dynamic exchange of water and active ingredients. Although the germination rate was slightly higher to 92.34%, the slight advantage of MDA content (5.18 μmol / g FW) and SOD activity (277.21 U / g FW) failed to translate into seedling growth advantage. The seedling height was 9.02 cm, indicating that excessive osmotic pressure regulation interfered with the internal physiological rhythm of the seed and disrupted the molecular-level balance.

[0132] Examples 6 and 7 optimized the nanobarrier structure by adjusting the mesoporous silica concentration from 0.4 mg / mL to 0.6 mg / mL, but deviations from the optimal value of 0.5 mg / mL weakened the molecular sieve function.

[0133] In Example 6, the concentration of 0.4 mg / mL was insufficient, the barrier layer thickness was less than 70 nm, and the porosity increased to 25%, resulting in the partial dissolution of high B-chain starch molecules with a diameter of 3 to 5 nm. The endosperm gelatinization temperature dropped to 58.5 °C, forming a local gel layer that blocked the water channels, causing the MDA content to rise to 5.39 μmol / g FW.

[0134] In Example 7, the concentration of 0.6 mg / mL was too high, the barrier layer was too thick (90 nm), the pores were blocked, the water molecule passage rate dropped to 0.15 μm / s, hindering the melatonin sustained-release kinetics, the half-life was extended to 15 min, resulting in uneven distribution of melatonin inside the seed, a penetration depth of 170 μm, and SOD activity of only 271.45 U / g FW.

[0135] Examples 6 and 7 all suffered from an imbalance in the pore size and thickness of the barrier layer, which disrupted the selectivity of the molecular sieve and caused the physical blocking of the nano-barrier and the permeability of water molecules to lose their synergy. As a result, they lagged behind Example 1 in terms of seedling root length (7.71cm to 7.88cm) and seedling height (8.81cm to 8.93cm).

[0136] Comparative examples 1 to 3 failed to address the core molecular mechanisms of flooding stress;

[0137] In Comparative Example 1, the deletion of S1 caused the seed to rapidly absorb water at a rate of 0.45 μm / s due to the lack of osmotic pressure regulation. The seed coat porosity remained above 45%, and the seed coat structure ruptured during water absorption and swelling, failing to form a continuous nano-barrier. This resulted in abnormal gelation of the endosperm starch below 58°C, forming a dense gel layer with a thickness greater than 5 μm. This physically blocked the exchange of water and gas, causing a sharp drop in the O2 concentration inside the seed, explosive accumulation of ROS, MDA of 13.45 μmol / gFW, and SOD activity of only 110.56 U / gFW.

[0138] Comparative Example 2: Traditional melatonin-soaked seeds, while containing melatonin, did not regulate the water absorption rate. Rapid seed expansion caused the treatment solution to be squeezed out, resulting in melatonin only remaining on the surface with a penetration depth of less than 50 μm. This failed to activate the deep endosperm antioxidant enzyme system, leading to a limited increase in SOD activity (150.23 U / gFW), a low reactive oxygen species scavenging efficiency of 65.2%, an MDA content of 9.87 μmol / gFW, and a germination rate of 68.90%. Both methods failed because they lacked a molecular-level regulatory chain, preventing the active ingredients from effectively targeting key sites.

[0139] Comparative Example 3, with the deletion of S2, showed that due to the lack of a nano-barrier, the high B-chain starch molecules (3-5 nm in diameter) could freely enter the endosperm, causing the endosperm gelatinization temperature to drop to 57.5℃. Abnormal gelatinization resulted in the formation of a continuous gel layer with a thickness greater than 6 μm, blocking water exchange. The O2 concentration was 1.5%, the ROS accumulation rate was 1.0 μmol / gFW / min, the MDA content was 12.34 μmol / gFW, the SOD activity was 125.34 U / gFW, the reactive oxygen species scavenging efficiency was 72.5%, the germination rate was 55.67%, and the seedling root length was 3.85 cm and the seedling height was 4.82 cm. This indicates that the absence of molecular-level regulatory chains prevented the active ingredients from effectively acting on key targets.

[0140] The CK group, as the untreated control, completely lost its waterlogging tolerance. In the early stage of waterlogging, the seeds absorbed water at a conventional rate of 0.45 μm / s. The seed coat porosity was 45% uncontrolled. The high-branched starch B chain B / A ratio of 1.8:1 underwent abnormal gelatinization below 58°C, forming a continuous gel layer with a thickness greater than 8 μm. This completely blocked the exchange channels for water, O2, and metabolic waste, resulting in oxygen deficiency inside the seeds. The O2 concentration was less than 2%, the ROS accumulation rate reached 1.2 μmol / gFW / min, the irreversible membrane lipid peroxidation MDA was 15.67 μmol / gFW, and the SOD activity was inhibited to 95.43 U / gFW, which was only 34.5% of that in Example 1. The antioxidant defense system was completely inactivated.

[0141] Meanwhile, the premature dissolution of endosperm starch forms a physical barrier, preventing the external treatment solution from penetrating and the internal metabolic waste from being discharged. This ultimately results in a germination rate of only 48.35%, a seedling root length of 3.45cm, and a seedling height of 4.56cm, fully demonstrating the core value of this invention in solving flooding stress at the molecular level.

[0142] Based on the preferred embodiments of the present invention described above, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A seed treatment method for improving submergence tolerance in rice, characterized by, Includes the following steps: S1. Soak rice seeds in a first treatment solution, which is a composite aqueous solution containing D-sorbitol and calcium chloride, wherein the concentration of D-sorbitol is 14-16% and the concentration of calcium chloride is 4-6 mmol / L. The soaking time is 2-4 min, the temperature of the treatment solution is maintained at 20-30℃, and the pH value is maintained at 6.5-7.

5. S2. After the osmotic pressure regulation pretreatment step is completed, the rice seeds are taken out and a second treatment liquid is sprayed on their surface. The second treatment liquid is a suspension containing mesoporous silica nanoparticles and graphene oxide. The spraying lasts for 4-6 minutes to form a nano barrier layer on the seed coat surface. S3. After the nanobarrier layer construction step is completed, the seeds are immersed in the third treatment solution, which is a suspension of melatonin-loaded mesoporous carbon nanospheres, wherein the concentration of melatonin-loaded mesoporous carbon nanospheres is 1.0-3.0 mg / mL, and the immersion lasts for 10-15 min.

2. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 1, characterized by: In S1, the overall water potential of the first treatment liquid is adjusted to -3.0 to -3.5 MPa.

3. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 1, characterized by: In step S2, the average particle size of the mesoporous silica nanoparticles is 18-22 nm, and the internal pore diameter is 3.0-3.5 nm; the graphene oxide sheets form a two-dimensional network structure; and the thickness of the nano barrier layer is 75-85 nm.

4. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 3, characterized by: The mesoporous silica nanoparticles are anchored in the graphene oxide network by reacting the surface-modified carboxyl groups with the epoxy groups on the defect sites of the graphene oxide sheets.

5. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 1, characterized by: The pores inside the graphene oxide network are filled with polyethylene glycol-distearate phosphatidylethanolamine at a ratio of 1-5%.

6. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 1, characterized by: In S2, the concentration of mesoporous silica nanoparticles in the second treatment solution is 0.4-0.6 mg / mL, and the concentration of graphene oxide is 2.0-3.0 mg / mL.

7. The seed treatment method for improving the ability of rice to tolerate flooded water according to claim 1, characterized by: In step S3, the mesoporous carbon nanospheres have an average diameter of 280-320 nm and an average pore size of 4-5 nm, and are used to adsorb melatonin molecules, with a melatonin loading of 5-15%.

8. The seed treatment method for improving the flood tolerance of rice according to claim 1, characterized in that: In step S3, the surface of the mesoporous carbon nanospheres is modified with cetyltrimethylammonium bromide at a concentration of 0.1-1.0 mmol / L.

9. A seed treatment method for improving the flood tolerance of rice according to claim 1, characterized in that: The rice seeds are from a rice variety with a high content of B-chain amylopectin.

10. A seed treatment method for improving the flood tolerance of rice according to claim 1, characterized in that: The D-sorbitol, calcium chloride, mesoporous silica nanoparticles, graphene oxide, polyethylene glycol distearate, melatonin, mesoporous carbon nanospheres, and hexadecyltrimethylammonium bromide are all biocompatible materials.

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