Method for promoting low-temperature germination of rapeseeds, nano initiator and preparation method

Abstract

The invention belongs to the field of agriculture, particularly relates to the field of seed germination regulation and control, and more particularly relates to a method for promoting rape seed germination under low-temperature stress, an initiator and a preparation method, the initiator composed of a Mn-doped mesoporous cerium oxide (Mn-CeO2) nano material and ultrapure water is adopted for seed initiation, and the seed germination is promoted under low-temperature stress. The germination rate and germination potential of seeds can be remarkably improved in a low-temperature stress environment, the stress resistance of crops can be improved, and generated stress memory for enhancing low-temperature stress can be maintained to the seedling stage.

Description

Technical Field

[0001] The invention belongs to the field of agriculture, in particular to the field of seed germination regulation, and more specifically to a method for promoting low-temperature germination of rapeseed, a nano initiator and a preparation method. Background Art

[0002] Rapeseed itself is a cool-loving and cold-resistant crop. It can safely overwinter at temperatures between 0°C and -2°C. It can generally withstand temperatures between -3-5°C, and some varieties with strong cold resistance can even withstand temperatures between -5-8°C. In extreme low temperatures, such as -7°C to -8°C, rapeseed will suffer severe frost damage.

[0003] Rapeseed's growth will be significantly inhibited at low temperatures, with yellowing leaves and softening stems. In severe cold (below 0°C), rapeseed roots may be damaged by frost, with the leaves still green but the entire plant frozen and dead. At the same time, rapeseed buds are at risk of frost damage at temperatures below 0°C. In mild cases, the cortex will rupture but growth can resume, while in severe cases, the stalks will shrink and droop, break, and the buds will turn yellow and dry due to freezing, wither and die. After the frozen parts of the buds resume growth, they often show symptoms of longitudinal cracking of the stems, which is not conducive to the plant's resistance to lodging and disease, and is more susceptible to disease and lodging than normal rapeseed; frozen buds can easily cause the frozen buds to fall off, seriously affecting yields.

[0004] In response to the problem of low temperature stress, the technical solutions commonly used in the existing technology include screening frost-resistant varieties, inter-cultivation and soil cultivation, application of phosphorus and potassium fertilizers, stalk removal, stalk picking and timely irrigation, etc. The technical solution for rapeseed seeds is mainly to cultivate new low-temperature and cold-resistant rapeseed varieties through breeding methods. Summary of the invention

[0005] Aiming at the problem that rapeseed seeds have low germination rate and weak germination potential under low temperature environment, the present invention provides a method for promoting low temperature germination of rapeseeds, comprising the following steps: (1) Select rapeseed seeds with full grains and perform pretreatment; (2) Soaking the treated rapeseed seeds in an initiator consisting of Mn-CeO2 nanomaterials and ultrapure water at 22°C in the dark for 24 hours; (3) The soaked seeds are taken out and germinated under artificial controlled conditions; The Mn-CeO2 nanomaterial is a mesoporous cerium oxide nanomaterial doped with 10% metal Mn.

[0006] Preferably, the concentration of Mn-CeO2 nanomaterials is 20 mg / L; and the ratio of seed weight to solution volume is 1:5 (g / mL).

[0007] Furthermore, the Mn-CeO2 nanomaterial is in the form of irregular particles with an average size of 8.4±2.7 nm, and the average hydrodynamic diameter of the nanomaterial in ultrapure water is 195.8±2.2 nm, and the average zeta potential is -24.7±0.3 mV, indicating that the Mn-CeO2 nanomaterial has the characteristics of small size and negative charge.

[0008] Preferably, the germination conditions are 14 h day / 10 h night, light intensity of 12,000 Lux, relative humidity of 60%, normal temperature setting of 22° C., and low temperature setting of 8° C.

[0009] Further preferably, ultrapure water is appropriately added during the germination process to compensate for evaporated water and ensure that the seeds are always moist.

[0010] At the same time, the invention also discloses a nano-initiator suitable for the germination method, wherein the initiator consists of Mn-CeO2 nanoparticles (NPs) and ultrapure water, and the Mn-CeO2 nanomaterial is a mesoporous cerium oxide nanomaterial doped with 10% metal Mn.

[0011] Further preferably, the preparation method of the Mn-CeO2 nanomaterial in the initiator comprises: (1) Preparation of MCM-48 template, (2) preparing Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials, specifically, adding the template MCM-48 into an ethanol solution of cerium (III) nitrate hexahydrate and manganese (II) nitrate hexahydrate, and stirring at 60°C; The product was filtered and dried, and then calcined in air at 400 °C for 2 h to obtain Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials.

[0012] Preferably, the addition ratio of the cerium (III) nitrate hexahydrate, manganese (II) nitrate hexahydrate and the ethanol solution is 3.0 g of cerium (III) nitrate hexahydrate and 0.2 g of manganese (II) nitrate hexahydrate are added to 50 mL of ethanol solution.

[0013] Preferably, the addition ratio of the template MCM-48 to cerium (III) nitrate hexahydrate and manganese (II) nitrate hexahydrate is 3.0 g of template MCM-48, 3.0 g of cerium (III) nitrate hexahydrate and 0.2 g of manganese (II) nitrate hexahydrate.

[0014] In a preferred technical solution, the dried product is heated to 400° C. at a rate of 2° C. / min in air and calcined for 2 h.

[0015] Further preferably, the prepared Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterial is placed in a 2 M NaOH aqueous solution and dissolved at 60° C. for 6 hours, and then the product is collected by centrifugation and washed with distilled water and ethanol in sequence to obtain a high-purity Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterial.

[0016] At the same time, the present invention further discloses a method for preparing the MCM-48 template, which is as follows: dissolving hexadecyltrimethylammonium bromide (5.0 g) and Pluronic F-127 (20.5 g) in a mixed solvent containing distilled water (960 mL) and ethanol (340 g); after the surfactant is completely dissolved, adding 28% ammonium hydroxide solution (100 g) at room temperature while vigorously stirring; then adding tetraethyl orthosilicate (18 g), and continuing to stir at 1000 rpm for 1 minute; then standing at room temperature for 24 hours, ultra-high-speed centrifugation to obtain a white product; washing the product with water and ethanol until the pH value of the solution becomes neutral, and then calcining at 550° C. for 5 hours to remove the pore-forming template to obtain the product MCM-48 template.

[0017] The present invention uses an initiator composed of Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials and ultrapure water to perform seed priming, which can not only significantly improve the germination rate and germination potential of rapeseed seeds under a low temperature stress environment, but also improve the low temperature resistance of rapeseed in the seedling stage. The enhanced stress memory of low temperature stress can be maintained until the seedling stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 TEM image of Mn-CeO2 nanomaterials.

[0019] Figure 2 The hydrodynamic diameter and zeta potential diagram of Mn-CeO2 nanomaterials.

[0020] Figure 3 Schematic diagram of the effect of Mn-CeO2 nanomaterials on the germination rate of rapeseed seeds under normal temperature and low temperature conditions. The data are mean ± standard deviation (n=3).

[0021] Figure 4 Schematic diagram of the effect of different concentrations of Mn-CeO2 nanomaterials on the germination rate of rapeseed seeds under low temperature conditions. The data are mean ± standard deviation (n=3).

[0022] Figure 5 This is an analysis chart of the germination potential of rapeseed at normal and low temperatures after seeds were soaked in Mn-CeO2 nanoparticles. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the present invention is further described below in conjunction with specific embodiments.

[0024] The reagents and instruments not specially described or indicated in the examples are all conventional commercial products and can be purchased. The conditions not specially described or indicated can be carried out according to the conventional conditions in the art or the instructions of the sellers of the relevant products.

[0025] Example 1 Preparation of Mn-CeO2 Nanomaterials (1) Preparation of MCM-48 template: Hexadecyltrimethylammonium bromide (5.0 g) and Pluronic F-127 (20.5 g) were dissolved in a mixed solvent containing distilled water (960 mL) and ethanol (340 g). After the surfactant was completely dissolved, 28% ammonium hydroxide solution (100 g) was added while vigorously stirring at room temperature. Then, ethyl orthosilicate (18 g) was added and stirred at 1000 rpm for 1 minute. Then, the mixture was allowed to stand at room temperature for 24 h and ultracentrifuged to obtain a white product. The product was washed with water and ethanol until the pH value of the solution became neutral, and then calcined at 550 °C for 5 h to remove the porous template.

[0026] (2) Preparation of Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials: The impregnation-calcination method was used for synthesis. The specific method is as follows: the template MCM-48 (3.0 g) was added to a 50 mL ethanol solution containing cerium (III) nitrate hexahydrate (3.0) and manganese (II) nitrate hexahydrate (0.2 g) and stirred at 60°C. After drying, the impregnated sample was heated to 400°C in air at a rate of 2°C / min and calcined for 2 h. The template was then dissolved in a 2 M NaOH aqueous solution at 60°C for 6 h, and the product was collected by centrifugation and washed with distilled water and ethanol, respectively. Example 2

[0027] The particle size and potential of the nanomaterials obtained in Example 1 were measured using a dynamic light scattering instrument (Malvern Zetasizer, Nano). Specifically, 5 μL of the suspension of Mn-CeO2 nanomaterials (200 mg / L) was added to a carbon film-supported copper mesh (Beijing Zhongxing Bairui Technology Co., Ltd., Beijing, China) and freeze-dried. The microstructure and morphology of the nanoparticles were characterized using a transmission electron microscope (JEM-200CX, JEOL, Tokyo, Japan) equipped with a CCD digital imaging system (GATAN782, USA) with a point resolution of 0.3 nm and an accelerating voltage of 80-200 kV. The results are shown in Figure 2. Figure 1TEM images show that Mn-CeO2 NPs are irregular particles with an average size of 8.4±2.7 nm.

[0028] The hydrodynamic diameter and zeta potential of Mn-CeO2 nanoparticles in ultrapure water were measured by dynamic light scattering. Figure 2 As shown, the average hydrodynamic diameter of Mn-CeO2 nanoparticles in ultrapure water is 195.8±2.2 nm and the average zeta potential is -24.7±0.3 mV. Example 3

[0029] Select rapeseed seeds with full grains, first disinfect the seed surface with 1% sodium hypochlorite for 10 minutes, and then rinse with ultrapure water for 5 times to complete the seed pretreatment. Subsequently, the disinfected seeds were soaked in 40 mg / L Mn-CeO2 NPs and ultrapure water at 22°C in the dark for 24 h, and the ratio of seed weight to solution volume was 1:5 (g / mL). After the end, the rapeseed seeds were placed in a culture dish containing 3 mL of solution. Each culture dish contained 50 seeds, and each treatment contained 3 replicates. The experiment used an artificial climate chamber (Ningbo Ledian Instrument Manufacturing Co., Ltd., model: RLD-1000D-4) for seed germination and seedling growth. The culture parameters were: 14 h day / 10 h night, light intensity 12 000 Lux, relative humidity 60%, normal temperature setting 22°C, low temperature setting 8°C, and 1-2 mL of ultrapure water was added to each culture dish during the culture period to compensate for the evaporated water.

[0030] The germination rate was evaluated by recording the number of seeds germinated every 24 h, with the radicle length not less than the seed diameter as the germination standard, and the record was made for 7 days. On the 4th day, 1-2 mL of ultrapure water was added to each culture dish to compensate for the evaporated water. The germination potential and germination rate were counted on the 5th and 7th days respectively. After the germination, 10 seedlings were randomly selected to measure the fresh weight of the seedlings. The dry weight of the seedlings was measured after drying at 60℃ for 48 h, and the germination index and vitality index were calculated: Germination rate = N 1 / N ×100%, Germination potential = N 2 / N ×100% in, N Represents the number of seeds tested, N 1 is the normal number of germinated grains at the end of the seventh day, N 2 represents the normal number of germinated grains on the 3rd day.

[0031] The experimental results are shown in Figure 3 .

[0032] It can be seen that the germination rate of rapeseed seeds treated with Mn-CeO2 nanomaterials at room temperature was higher than that of the water control group, indicating that Mn-CeO2 seed priming can significantly promote the germination rate of rapeseed seeds at room temperature. Compared with the room temperature water treatment group, the water treatment group under low temperature stress (8°C) showed delayed germination, and the germination rate stabilized after 5 days. On the 7th day, the germination rate reached 98% and 94.7% and ( Figure 3 ), indicating that low temperature has a significant negative effect on rapeseed seed germination. Under low temperature stress conditions, Mn-CeO2 NPs seed soaking significantly increased the germination rate of rapeseed compared with the water control. Example 4

[0033] According to the method in Example 3, the concentration of Mn-CeO2 nanomaterials was set to 20, 40, and 80 mg / L respectively, and the clean water treatment was used as the control. The germination experiment was carried out at low temperature to compare the effects of different concentrations of Mn-CeO2 nanomaterials on germination. The experimental results are shown in Figure 4 and Figure 5 .

[0034] The results showed that 20 mg / L Mn-CeO2 nanomaterials had the best effect on promoting rapeseed germination at low temperature. After soaking with Mn-CeO2 nanoparticles, the germination potential of rapeseed in the normal temperature group increased from 69.3 to 72.7, and the germination potential of rapeseed in the low temperature group increased from 56.7 to 69.0. Example 5

[0035] According to the method in Example 3, 20 mg / L Mn-CeO2 nanomaterials were used to prime the seeds of rapeseed varieties Ningza 1818 and Ningza 158. Clear water treatment was used as a control. After the priming, the seeds were sown in the soil, 2 seeds were sown in each pot, and each treatment was repeated three times. The seeds were placed in a light incubator for cultivation, with an air humidity of 60% and a light intensity of 150 μmol m -2 s -1 , the photoperiod was 14h / 10h, and the day and night temperature was 23°C / 18°C. When the rapeseed reached the four-leaf stage, six seedlings were subjected to low temperature (2°C / 0°C) stress for 48 h. After the stress, the frost damage index was calculated to explore whether the "stress memory" at the seed stage can be carried to the later development stage and enhance the low temperature resistance of rapeseed seedlings.

[0036] The results of the effect of nanoparticle seed soaking on the low temperature resistance of rapeseed seedlings at the four-leaf stage are shown in Table 1; Table 1.

[0037] After the stress ended, the frost damage index (0.75) of the Ningza 1818 and Ningza 158 seedlings in the Mn-CeO2NPs seed soaking treatment group was lower than that of the clear water control group (1), indicating that the "stress memory" induced by Mn-CeO2NPs at the very beginning (24 hours) of the plant life cycle can be carried to the later developmental stage, enhancing the low temperature resistance of rapeseed seedlings.

[0038] The above is a specific implementation 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 are also considered to be within the protection scope of the present invention.

Claims

1. A method for promoting low temperature germination of rapeseed, characterized in that: The following steps are involved: (1) Select rapeseed seeds with full grains and perform pretreatment; (2) Soaking the treated rapeseed seeds in an initiator consisting of Mn-CeO2 nanomaterials and ultrapure water at 22°C in the dark for 24 hours; (3) The soaked seeds are taken out and germinated under artificial controlled conditions; The Mn-CeO2 nanomaterial is a mesoporous cerium oxide nanomaterial doped with 10% metal Mn.

2. The method for promoting low temperature germination of rapeseed according to claim 1, characterized in that: The concentration of Mn-CeO2 nanomaterials was 20 mg / L; the ratio of seed weight to solution volume was 1:5 (g / mL).

3. The method for promoting low temperature germination of rapeseed according to claim 1, characterized in that: The Mn-CeO2 nanomaterial is in the form of irregular particles with an average size of 8.4±2.7 nm, an average hydrodynamic diameter of the nanomaterial in ultrapure water of 195.8±2.2 nm, and an average zeta potential of -24.7±0.3 mV.

4. The method for promoting low temperature germination of rapeseed according to claim 1, characterized in that: The germination conditions are 14 h / day and 10 h / night, light intensity of 12,000 Lux, relative humidity of 60%, normal temperature set at 22°C, and low temperature set at 8°C.

5. A nano-initiator suitable for the method for promoting low-temperature germination of rapeseed according to any one of claims 1 to 4, characterized in that: The nano-initiator consists of Mn-CeO2 nanoparticles (NPs) and ultrapure water, and the Mn-CeO2 nanomaterial is a mesoporous cerium oxide nanomaterial doped with 10% metal Mn.

6. A method for preparing the Mn-CeO2 nanomaterial in the nano-initiator described in claim 5, characterized in that it comprises: (1) Preparation of MCM-48 template, (2) preparing Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials, specifically, adding the template MCM-48 into an ethanol solution of cerium (III) nitrate hexahydrate and manganese (II) nitrate hexahydrate, and stirring at 60°C; The product was filtered and dried, and then calcined in air at 400 °C for 2 h to obtain Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterials.

7. The method according to claim 6, characterized in that: The addition ratio of the cerium (III) nitrate hexahydrate, manganese (II) nitrate hexahydrate and the ethanol solution is 3.0 g of cerium (III) nitrate hexahydrate and 0.2 g of manganese (II) nitrate hexahydrate are added to 50 mL of the ethanol solution.

8. The method according to claim 6, characterized in that: The addition ratio of the template MCM-48, cerium (III) nitrate hexahydrate, and manganese (II) nitrate hexahydrate is 3.0 g of template MCM-48, 3.0 g of cerium (III) nitrate hexahydrate, and 0.2 g of manganese (II) nitrate hexahydrate.

9. The method according to claim 6, characterized in that: The dried product was calcined in air at a temperature of 2 °C / min to 400 °C for 2 h.

10. The method according to claim 6, characterized in that: The prepared Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterial was placed in a 2 M NaOH aqueous solution and dissolved at 60°C for 6 hours. The product was then collected by centrifugation and washed with distilled water and ethanol in sequence to obtain a high-purity Mn-doped mesoporous cerium oxide (Mn-CeO2) nanomaterial.

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