Application of copper-doped carbon quantum dots in improving salt tolerance of crop seeds and seedlings

Abstract

The application belongs to the technical field of agriculture, and particularly relates to application of copper-doped carbon quantum dots in improving salt tolerance of crop seeds and seedlings. The application finds that, after the crop seeds are treated by the copper-doped carbon quantum dots, the germination rate, germination index and germination potential of the seeds under salt stress can be obviously improved; after the crop seedlings are treated by the copper-doped carbon quantum dots, the fresh weight of the aboveground part and the fresh weight of the underground part of the crop under salt stress can be obviously improved. In addition, after the crop seedlings are treated by the copper-doped carbon quantum dots, the chlorophyll content and the antioxidant performance of the crop can be obviously improved.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural technology and more specifically relates to the application of copper-doped carbon quantum dots in improving the salt tolerance of crop seeds and seedlings. Background Art

[0002] Soil salinization affects arable land in many countries and is a major factor limiting crop yields. Salt stress first inhibits seed germination, reducing seed emergence rates in the field, leading to uneven emergence and reduced yields later in life. Second, salt stress continues after seed emergence, exposing plants to osmotic, ionic, and oxidative stresses during their growth stages. Therefore, improving salt tolerance in crops during the seed and seedling stages to maintain healthy vegetative growth is crucial.

[0003] Conventional approaches to addressing soil salt stress currently include farmland water management, soil improvement, optimized agronomic practices, and the breeding and cultivation of salt-tolerant crops. While these measures are somewhat effective, they are resource-intensive and time-consuming. For example, desalting farmland irrigation water can dilute and remove salt from the soil, but saline-alkali lands are predominantly found in water-scarce regions of my country, draining already scarce irrigation water resources. Conventional agronomic practices, such as crop rotation or intercropping with salt-tolerant crops, can mitigate the effects of salt on crops, but not all crops are suitable for these practices. Breeding salt-tolerant crops requires breeding or genetic engineering techniques, which are difficult to accomplish quickly. Therefore, new, easily accessible technologies are urgently needed to alleviate crop salt stress.

[0004] Copper (Cu) is an essential trace element for plant growth. Although plants require relatively small amounts of copper, it plays an important role in various physiological and biochemical processes, particularly enzyme activation, antioxidant activity, disease resistance, and plant growth and development. First, copper is a key component of many enzymes, particularly peroxidase, polyphenol oxidase, cytochrome, and superoxide dismutase, which play a key role in antioxidant activity under salt stress. Second, copper participates in key plant growth processes such as lignin synthesis, auxin metabolism, photosynthesis, electron transport, and flowering and fruiting, playing a vital role in plant growth and development.

[0005] As an emerging field in agriculture, nano-agriculture has great application prospects in improving agricultural production efficiency, reducing environmental pollution, improving crop quality and promoting sustainable agricultural development. For example, Lovely et al. found that nano-copper oxide can promote the growth of radish under salt stress (Lovely Mahawar C; Marek Maria Barboricova;etal.Effect of copper oxide and zinc oxide nanoparticles on photosynthesis and physiology of Raphanus sativus L.under salinity stress[J].Plant Physiologyand Biochemistry,2023,Vol.206:108281). However, high concentrations of metal oxides, such as copper oxide nanoparticles, can harm plants, leading to reduced crop growth and yield (Feigl,G.(2023).The impact of copper oxide nanoparticles on plant growth:a comprehensive review.Journal ofPlant Interactions,18(1).https: / / doi.org / 10.1080 / 17429145.2023.2243098). Therefore, a more biocompatible alternative is needed to reduce the toxicity of nanomaterials while improving the salt tolerance of crops. Carbon quantum dots (CQDs) are a new type of bioluminescent carbon nanomaterial with good biocompatibility, water solubility, and low toxicity. They have great application potential in bioimaging, fluorescence sensing, drug delivery, plant growth, etc. However, research on copper-doped carbon quantum dots in improving plant growth is relatively limited. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing technology and provide an application of copper-doped carbon quantum dots in improving the salt tolerance of crop seeds and seedlings.

[0007] The present invention aims to provide an application of copper-doped carbon quantum dots in scavenging hydroxyl radicals, superoxide anions and hydrogen peroxide in vitro.

[0008] The purpose of the present invention is to provide a method for improving the salt tolerance of crop seeds.

[0009] Another object of the present invention is to provide a method for improving the salt tolerance of crops at the seedling stage.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides application of copper-doped carbon quantum dots in improving salt tolerance of crop seeds and seedlings.

[0012] Through extensive research, the inventors found that, at equivalent concentrations, applying either copper ions or carbon quantum dots alone had no significant effect on the fresh weight of aboveground or belowground parts under salt stress, indicating that applying either alone does not significantly promote the growth and development of crops under salt stress. However, the inventors discovered that combining copper and carbon quantum dots to create copper-doped carbon quantum dots exhibits a synergistic effect, significantly promoting the growth and development of crop seeds and seedlings under salt stress.

[0013] Preferably, the improving of the salt tolerance of crop seeds and seedlings is: improving at least one of the germination rate, fresh weight, germination index, and germination potential of crop seeds under salt stress, and improving at least one of the aboveground fresh weight, underground fresh weight, chlorophyll content, and antioxidant activity of crop seedlings under salt stress.

[0014] Preferably, the crop is cotton.

[0015] The present invention also provides the use of copper-doped carbon quantum dots in scavenging hydroxyl radicals, superoxide anions and hydrogen peroxide in vitro.

[0016] The inventors have found through extensive research that the copper-doped carbon quantum dots of the present invention have the ability to scavenge free radicals, superoxide anions and hydrogen peroxide in vitro, and can function as a nano-enzyme.

[0017] Preferably, the copper-doped carbon quantum dots include the following preparation method: mixing copper nitrate, urea, citric acid, and N,N-dimethylformamide, performing a solvent thermal reaction, and post-treating to obtain copper-doped carbon quantum dots.

[0018] Furthermore, the temperature of the solvent thermal reaction is 160-200°C.

[0019] Furthermore, the solvent thermal reaction time is 4 to 6 hours.

[0020] Furthermore, the copper nitrate, urea, citric acid, and N,N-dimethylformamide are mixed in a ratio of 0.2-0.4 g: 1-2 g: 0.5-1 g: 10 mL.

[0021] Furthermore, the post-treatment is to centrifuge and take the supernatant, mix it with ethanol, and finally take the precipitate and dry it.

[0022] Preferably, the volume ratio of the supernatant to ethanol is 1:1.5-2.5.

[0023] Furthermore, the present invention provides a method for improving the salt tolerance of crop seeds, wherein the crop seeds are soaked in a copper-doped carbon quantum dot solution; wherein the concentration of the copper-doped carbon quantum dot solution is 20 to 50 mg / L.

[0024] Preferably, the mass ratio of the crop seeds to the copper-doped carbon quantum dot solution is 1:4-6.

[0025] Preferably, the concentration of the copper-doped carbon quantum dot solution is 20-26 mg / L.

[0026] Preferably, the soaking time is 7 to 9 hours.

[0027] Additionally, the present invention provides a method for improving the salt tolerance of crops at the seedling stage, comprising applying a solution containing copper-doped carbon quantum dots to the surface of the crops; wherein the concentration of the copper-doped carbon quantum dots in the solution is 250 to 2000 mg / L.

[0028] Preferably, the concentration of copper-doped carbon quantum dots in the solution is 500-1000 mg / L.

[0029] Preferably, the concentration of copper-doped carbon quantum dots in the solution is 450-550 mg / L.

[0030] Preferably, the solution further contains an organosilicon surfactant.

[0031] Furthermore, the organosilicon surfactant is a polysiloxane polyether surfactant.

[0032] More preferably, the volume concentration of the organosilicon surfactant in the solution is 0.5‰.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides the use of copper-doped carbon quantum dots for improving salt tolerance in crop seeds and seedlings. Research has found that treatment of crop seeds with copper-doped carbon quantum dots significantly improves the germination rate, germination index, and germination potential under salt stress. Treatment of crop seedlings with copper-doped carbon quantum dots also significantly increases the aboveground and belowground fresh weights of the crops under salt stress. Furthermore, treatment of crop seedlings with copper-doped carbon quantum dots significantly increases chlorophyll content and antioxidant properties.

[0035] In addition, copper-doped carbon quantum dots can be used as seed soaking agents or foliar fertilizers on crops, which can quickly improve the salt tolerance of crops in a short period of time (5 to 7 days). It has low cost and high efficiency. Copper-doped carbon quantum dots are harmless to the environment and will not cause potential biosafety issues such as heavy metal accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The UV-visible absorption spectra and fluorescence spectra of the copper-doped carbon quantum dots prepared in Examples 1 to 4 and the carbon quantum dots prepared in Comparative Example 1 are shown. Figure 1 Figure A is the UV-visible absorption spectrum. Figure 1 Figure B is the fluorescence spectrum.

[0037] Figure 2 This is a structural characterization diagram of the copper-doped carbon quantum dots prepared in Example 1. Figure 2 Figure A is the transmission electron microscope image and lattice diagram of copper-doped carbon quantum dots. Figure 2 Figure B is the particle size distribution and average particle size diagram. Figure 2 The C diagram in the figure is the potential diagram. Figure 2 Figure D is the UV-visible absorption spectrum and the optimal excitation emission fluorescence spectrum. Figure 2 Figure E is the X-ray diffraction pattern.

[0038] Figure 3 The following is a phenotypic diagram and germination rate statistical diagram of cotton seeds grown under salt stress after being treated with copper-doped carbon quantum dots. Figure 3 Figure A is the cotton seed germination phenotype diagram. Figure 3 Figure B shows the germination rate of cotton seeds. Figure 3 Figure C shows the fresh weight of cotton seeds. Figure 3 Figure D in the figure is the germination index of cotton seeds. Figure 3 Figure E shows the germination potential of cotton seeds.

[0039] Figure 4 The following are the phenotypic diagrams and statistical data of the aboveground and underground fresh weight of cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots. Figure 4 Figure A is the growth phenotype of cotton seedlings. Figure 4 Figure B is a statistical chart of the fresh weight of the aboveground part of cotton after spraying copper-doped carbon quantum dots at the seedling stage. Figure 4 Figure C is a statistical chart of the fresh weight of the underground part of cotton after spraying copper-doped carbon quantum dots at the seedling stage.

[0040] Figure 5 The phenotypic diagram and statistical diagram of the fresh weight of aboveground and underground parts of cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots, carbon quantum dots and copper nitrate. Figure 5 Figure A is the growth phenotype of cotton seedlings. Figure 5 Figure B is a statistical chart of fresh weight of aboveground part of cotton seedlings. Figure 5 Figure D is a statistical chart of fresh weight of underground parts of cotton seedlings.

[0041] Figure 6 The following is a statistical chart of chlorophyll content in cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots. Figure 6 Figure A is a statistical graph of chlorophyll a content. Figure 6 Figure B is a statistical diagram of chlorophyll b content. Figure 6Figure C is a statistical graph of total chlorophyll content. Figure 6 Figure D is a statistical chart of carotenoid content data.

[0042] Figure 7 This is a diagram of chlorophyll fluorescence parameters of cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots.

[0043] Figure 8 This is a statistical chart of the content of hydrogen peroxide and superoxide anion radicals in cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots. Figure 8 Figure A is a statistical diagram of hydrogen peroxide content. Figure 8 Figure B is a statistical diagram of superoxide anion radical content data.

[0044] Figure 9 The following is a statistical chart of antioxidant enzyme activity data in cotton seedlings under salt stress after treatment with copper-doped carbon quantum dots. Figure 9 Figure A is a statistical diagram of SOD enzyme activity data. Figure 9 Figure B is the statistical diagram of POD enzyme activity data. Figure 9 Figure C is a statistical graph of CAT enzyme activity data. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0047] Example 1

[0048] The preparation method of copper-doped carbon quantum dots (CuCDs) is as follows:

[0049] 0.2 g of copper nitrate and 2 g of urea were dissolved in 10 mL of N,N-dimethylformamide (DMF). 1 g of citric acid was then added and allowed to fully dissolve. The mixture was then reacted at 160°C for 6 hours. After cooling to room temperature, the solution was centrifuged at 12,000 rpm for 20 minutes to remove the precipitate. The supernatant was mixed with ethanol at a volume ratio of 1:2 and centrifuged at 12,000 rpm for 10 minutes. The precipitate was then freeze-dried to obtain CuCDs powder.

[0050] Example 2

[0051] 0.2 g of copper nitrate and 1 g of urea were dissolved in 10 mL of N,N-dimethylformamide (DMF). 0.5 g of citric acid was then added and allowed to fully dissolve. The mixture was then reacted at 160°C for 6 hours. After cooling to room temperature, the solution was centrifuged at 12,000 rpm for 20 minutes to remove the precipitate. The supernatant was mixed with ethanol and centrifuged at 12,000 rpm for 10 minutes. The precipitate was then freeze-dried to obtain CuCDs powder.

[0052] Example 3

[0053] 0.4 g of copper nitrate and 2 g of urea were dissolved in 10 mL of N,N-dimethylformamide (DMF). 1 g of citric acid was then added and allowed to fully dissolve. The mixture was then reacted at 160°C for 6 hours. After cooling to room temperature, the solution was centrifuged at 12,000 rpm for 20 minutes to remove the precipitate. The supernatant was mixed with ethanol at a volume ratio of 1:2 and centrifuged at 12,000 rpm for 10 minutes. The precipitate was then freeze-dried to obtain CuCDs powder.

[0054] Example 4

[0055] 0.2 g of copper nitrate and 1 g of urea were dissolved in 10 mL of N,N-dimethylformamide (DMF). 0.5 g of citric acid was then added and allowed to fully dissolve. The mixture was then reacted at 200°C for 4 hours. After cooling to room temperature, the solution was centrifuged at 12,000 rpm for 20 minutes to remove the precipitate. The supernatant was mixed with ethanol at a volume ratio of 1:2 and centrifuged at 12,000 rpm for 10 minutes. The precipitate was then freeze-dried to obtain CuCDs powder.

[0056] Comparative Example 1

[0057] Compared with Example 1, copper nitrate was not added in this comparative example, and other preparation methods and conditions were the same, and carbon quantum dots CDs were finally prepared.

[0058] Experimental Example 1 Material Characterization

[0059] Experimental materials: copper-doped carbon quantum dots (CuCDs) prepared in Examples 1 to 4, and CDs prepared in Comparative Example 1.

[0060] UV-visible absorption spectra of CuCDs and CDs were obtained using an AOE UV-1800PC spectrophotometer. Fluorescence spectra were obtained using a Shimadzu RF-6000 fluorescence spectrophotometer in a quartz cuvette (10 mm × 10 mm). The potential of CuCDs was measured using a Malvern Zetasizer nano ZS. Transmission electron microscopy (TEM) was performed using a FEI Talos F200c TEM at 200 kV. X-ray diffraction (XRD) was performed on a Bruker D8 ADVANCE X-ray diffractometer.

[0061] Figure 1 The UV-visible absorption spectra of the CuCDs prepared in Examples 1 to 4 and the CDs prepared in Comparative Example 1 ( Figure 1 Figure A in the figure) and fluorescence spectra ( Figure 1 Figure B in the figure), Figure 1 CuCDs-1 represents the CuCDs prepared in Example 1, CuCDs-2 represents the CuCDs prepared in Example 2, CuCDs-3 represents the CuCDs prepared in Example 3, and CuCDs-4 represents the CuCDs prepared in Example 4. A comprehensive comparison shows that CuCDs-1 (CuCDs prepared in Example 1) has the highest fluorescence intensity, indicating that this copper-doped carbon dot has the highest fluorescence efficiency. Therefore, the CuCDs prepared using this synthesis scheme were selected for subsequent experiments.

[0062] Figure 2 This is a related characterization diagram of CuCDs prepared in Example 1, wherein: Figure 2 Figure A shows the transmission microscope imaging of CuCDs and carbon dot lattice. The results show that CuCDs are uniformly distributed nanomaterials with a lattice of 0.24nm. Figure 2 Figure B shows the particle size distribution and average particle size of CuCDs. The particle size of CuCDs is 2.04±0.04nm. Figure 2 Figure C shows that the CuCDs potential was measured at -27.83±0.33mV. Figure 2 Figure D shows the UV-visible absorption spectrum and optimal fluorescence spectrum of CuCDs. Figure 2 Figure E in the figure is the X-ray diffraction analysis (XRD) of CuCDs. The above results show that Example 1 successfully synthesized copper-doped carbon quantum dots with a negative surface potential and a particle size of 2.04 nm.

[0063] Experimental Example 2

[0064] Experimental materials: copper-doped carbon quantum dots (CuCDs) prepared in Example 1.

[0065] The ability of copper-doped carbon nanodots to scavenge reactive oxygen species in vitro was evaluated using the following methods:

[0066] The hydrogen peroxide (H2O2) content was determined using the "Hydrogen Peroxide Content Determination Kit" (A064-1-1, Nanjing Jiancheng Biological Co., Ltd.). Since hydrogen peroxide can react with molybdic acid to form a complex with a characteristic absorption peak at 405 nm, the efficiency of the material in removing hydrogen peroxide can be calculated. The sample was added according to the sample addition process in the kit instructions, and CuCDs (final concentration of 10 mg L -1 and 50mg·L -1 ) solution, mix well, and measure the absorbance at a wavelength of 405 nm and a light path of 1 cm.

[0067] Superoxide anion (O2 - The in vitro clearance efficiency of ) was determined using a SOD activity assay kit (WST-1, Shanghai Beyotime Biotechnology Co., Ltd.). First, the O2· - It reacts with WST-1 to generate a water-soluble dye that can be detected (maximum absorption at 450nm), and the absorbance value is linearly related to the concentration. -1 and 50mg·L -1 ) solution was added to the mixture of xanthine and xanthine oxidase, incubated at 37°C for 30 min, and the absorbance at 450 nm was measured using a microplate reader.

[0068] · OH scavenging ability determination, specific preparation method: take 3mL containing Tris-HCl (0.1mol·L -1 , pH = 4.7), methyl violet (24 μmol·L -1 ), FeSO4(1.08mmol·L -1 ), H2O2(0.1mol·L -1 ) of CuCDs (final concentration was 10 mg·L -1 or 50 mg·L -1 ) solution, the above solution without CuCDs was used as standard control, and 3 mL of methyl violet (24 μmol·L -1 ) solution served as a blank control. Each test tube was placed in a 37°C constant temperature water bath for 30 min. After the reaction, the absorbance of each tube was measured at 582 nm. The measured data were recorded and the hydroxyl radical scavenging rate of CuCDs was calculated according to the following formula:

[0069] CuCDs scavenging rate of hydroxyl radicals (%) = (1-△α582 / △A582)*100

[0070] Where: △α582 is A blank control-A determination

[0071] △A582 is A blank control-A standard control

[0072] The test results are shown in Table 1. It can be seen from Table 1 that the concentration is 10 mg·L -1 and 50mg·L -1 The scavenging rates of CuCDs for hydrogen peroxide, hydroxyl radicals, and superoxide anions were 16.99% and 47.42%, 0.74% and 17.27%, and 4.49% and 93.77%, respectively, indicating that CuCDs can function as a nanoenzyme mimic.

[0073] Table 1 Cu-doped carbon nanodots scavenging H2O2, ·OH, and ·O2 in vitro - Ability

[0074] <![CDATA[H2O2]]> OH <![CDATA[O2· - ]]> <![CDATA[10mg·L -1 CuCDs]]> 16.99％ 0.74％ 4.49％ <![CDATA[50mg·L -1 CuCDs]]> 47.42％ 17.27％ 93.77％

[0075] Experimental Example 3

[0076] Experimental materials: copper-doped carbon quantum dots (CuCDs) prepared in Example 1.

[0077] Copper-doped carbon nanodots improve cotton seed germination rate and growth under salt stress

[0078] Cotton (variety: Huamian 3097) seed disinfection, sowing and introduction treatment: The seeds were disinfected with 5% sodium hypochlorite and then rinsed three times with purified water. After drying, the CuCDs prepared in Example 1 were mixed with deionized water to prepare seed soaking agents with mass concentrations of 25 mg / L and 50 mg / L. Deionized water was used for seed soaking as a control (Control). The seeds were immersed in the above-mentioned soaking agent at room temperature, with a mass ratio of seed mass to seed soaking agent of 1:5, and the soaking time was 8 hours. Two layers of germination paper were laid in the germination box, and 10 mL of 200 mM NaCl solution was added to each box. The soaked cotton seeds were evenly placed on the germination paper, with 30 seeds (5×6) placed in each culture dish, with four replicates set up. The culture dishes were placed in a culture room with a light intensity of 16 hours and a dark intensity of 8 hours for cultivation. The number of seeds that germinated was counted daily, and the fresh weight was weighed after 7 days. The germination rate, germination potential, and germination index were calculated according to the following formula.

[0079]

[0080] Where n represents the statistical date of germination, and its value is 1-7.

[0081] Figure 3 Figure A shows the germination phenotype of cotton 7 days after 8 hours of CuCDs introduction. It can be seen that the cotton seeds grew better after the introduction of CuCDs, and the cotton under the two concentrations was significantly better than the control treatment. Figure 3The BE graph in the figure shows the germination rate, fresh weight, germination index, and germination potential of the introduced cotton seeds at day 7. The results show that the germination rate, germination index, and germination potential of the seeds introduced with CuCDs were significantly higher than those of the control treatment.

[0082] Experimental Example 4

[0083] Experimental materials: copper-doped carbon quantum dots (CuCDs) prepared in Example 1, CDs prepared in Comparative Example 1, and copper nitrate.

[0084] Experimental method: Cotton (variety: Huamian 3097) seeds were sterilized. After soaking in pure water at 25°C for 3 hours, the seeds were sown in 10 cm × 10 cm plastic nutrient pots filled with nutrient soil (peat: vermiculite: perlite = 1:1:1). When the cotyledons of the seedlings were fully expanded, the seedlings were transferred to a 5 L black plastic square pot filled with 1 / 2 Hoagland nutrient solution (refer to the existing conventional formula, self-prepared). The seedlings were kept at a temperature of 28°C / 18°C (day / night), a 14 h photoperiod, and 200 μmol m -2 ·s -1 of light intensity and 70% to 75% humidity. When the seedlings grow to the point where the second true leaf is fully expanded, the experimental treatment is carried out. First, the optimal application concentration is screened, and deionized water is used to prepare CuCDs solutions with concentrations of 250 mg / L, 500 mg / L, 1000 mg / L, and 2000 mg / L. The control group (Control) is deionized water, and the organosilicon surfactant Silwet L-77 is added with a final volume concentration of 0.5‰. After mixing, the solution is sprayed on the cotton leaves. Dark adaption is carried out in low light for 3 hours, and then the cotton seedlings are placed in 1 / 2 Hoagland nutrient solution with a concentration of 200 mM NaCl (prepared by referring to the existing conventional formula) and cultured in a culture room. After 5 days, the phenotype of the cotton seedlings is observed and the fresh weight of the aboveground and underground parts of the cotton is calculated.

[0085] Figure 4 The results show the phenotypes and aboveground and belowground fresh weights of cotton plants under salt stress, including a control and treatments with different concentrations of CuCDs solution. The results show that spraying with CuCDs solution improved the phenotypes and aboveground and belowground fresh weights of cotton plants under salt stress. In particular, application of 500mg / L and 1000mg / L of CuCDs significantly improved the phenotypes and biomass of cotton plants under salt stress, indicating that CuCDs can enhance salt tolerance in cotton seedlings.

[0086] Figure 5 For cotton, the concentration of 500 mg / L CuCDs, CDs and Cu 2+After the solution (copper ion concentration in copper nitrate solution was 500 mg / L) was treated as described above, the phenotype, fresh weight of aboveground and underground parts of cotton under salt stress were measured. The results showed that compared with the control group, CDs treatment improved the salt tolerance of cotton but there was no significant difference, while the biomass of cotton treated with CuCDs under salt stress increased significantly, and was better than that of CDs and Cu alone at the same concentration. 2+ This indicates that copper-doped carbon dots can improve the salt tolerance of cotton seedlings, which is comparable to the effects of CDs and Cu alone. 2+ Compared with the above, it has a synergistic effect.

[0087] Experimental Example 5

[0088] Experimental materials: cotton leaves treated with 500 mg / L CuCDs solution and deionized water after the experimental method in Experimental Example 4.

[0089] Chlorophyll content detection

[0090] 0.1 g of fresh cotton leaves treated with a 500 mg / L CuCDs solution and deionized water and exposed to salt stress for 5 days (as in Example 4) were weighed, cut into small pieces, and placed in a 15 mL centrifuge tube. 10 mL of a 1:1 (v:v) mixture of anhydrous ethanol and acetone was added to the centrifuge tube and incubated in the dark for 24 hours to fully extract the chlorophyll. The absorbance of the extract at 440 nm, 644 nm, and 662 nm was measured using a microplate reader, and the absorbance was calculated according to the following formula:

[0091] Chlorophyll a concentration (mg / L, Ca) = 9.784*A662-0.99*A644

[0092] Chlorophyll b concentration (mg / L, Cb) = 21.426*A644-4.65*A662

[0093] Total chlorophyll concentration (mg / L) = chlorophyll a concentration + chlorophyll b concentration

[0094] Carotenoid concentration (mg / L) = 4.695*A440-0.268*(Ca+Cb)

[0095] Pigment content (mg / g) = [pigment concentration (mg / L) * total amount of extract (mL)] / [leaf weight (g) * 1,000], where the pigment is chlorophyll a, chlorophyll b, or carotenoids.

[0096] Measurement results Figure 6 As shown in the results, application of CuCDs to cotton under salt stress can effectively maintain the chlorophyll content in leaves, and the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were significantly increased compared with the control group.

[0097] Experimental Example 6

[0098] Experimental materials: cotton leaves treated with 500 mg / L CuCDs solution and deionized water after the experimental method in Experimental Example 4.

[0099] Using a chlorophyll fluorescence imaging system (IMAGING-PAM, WALZ, Germany), chlorophyll fluorescence parameters were analyzed in cotton seedlings from each treatment group (500 mg / L CuCDs solution and deionized water) after five days of salt stress as described in Experiment 4. Plants were kept in darkness for at least 30 minutes before measurement. The pulsed light intensity settings were adjusted based on the real-time fluorescence value (Ft) of each region of interest, ensuring that the Ft value was within the range of 0.01 to 0.02, which indicates optimal measurement performance. Parameters measured included maximum photochemical efficiency (Fv / Fm), non-actinic fluorescence quenching (NPQ), actinic fluorescence quenching (qL), and actual photosynthetic quantum yield (Y(II)) under PSII light.

[0100] Figure 7 The results showed that under salt stress, the Fv / Fm and Y(II) of cotton in the CuCDs group were significantly improved, while NPQ and qL increased but without significant differences. This indicates that the application of CuCDs to cotton under salt stress significantly improves the photosynthetic efficiency of chlorophyll.

[0101] Experimental Example 7

[0102] Experimental materials: cotton leaves and roots treated with 500 mg / L CuCDs solution and deionized water after treatment according to the experimental method in Experimental Example 4.

[0103] H2O2 content was determined using the "Hydrogen Peroxide Content Assay Kit" (A064-1-1, Nanjing Jiancheng Biological Co., Ltd.) according to the manufacturer's instructions. Accurately weigh 0.1 g of cotton leaf and root samples (treated with 500 mg / L CuCDs solution and deionized water and exposed to salt stress for 5 days, as described in Example 4) and add 1 mL of 0.05 mol / L sodium phosphate buffer (pH 7.5). Grind the mixture for 120 seconds (65 Hz) using a grinder, then centrifuge at 12,000 g at 4°C for 20 minutes. The supernatant was collected for later use. Samples were added according to the manufacturer's instructions, mixed, and then the absorbance was measured at a wavelength of 405 nm, a 1 cm optical path, and zeroed with deionized water.

[0104] O2 ·-The content of superoxide anion was determined using a superoxide anion content detection kit (Solarbio, China) according to the manufacturer's instructions. 0.1 g of cotton leaf and root samples treated with 500 mg / L CuCDs solution and deionized water and exposed to salt stress for 5 days (as in Example 4) were weighed and added to 1 mL of the extract in the kit. The samples were ground in an ice bath using a grinder at 65 Hz for 180 sec and 12,000 rpm. -1 Centrifuge at 4°C for 20 min and collect the supernatant for later use. Add the sample according to the sample addition process in the instruction manual, adjust the zero value with distilled water, and measure the absorbance of the sample at 530 nm using a spectrophotometer.

[0105] Figure 8 The results showed that the application of CuCDs could significantly reduce H2O2 and O2 in cotton leaves and roots. ·- The results show that the CuCDs prepared in the embodiment of the present invention have good antioxidant properties and can remove excessive H2O2 and superoxide anion radicals produced by cotton under salt stress, further promoting the growth of cotton under salt stress, maintaining the homeostasis of active oxygen in cotton, and thus alleviating the salt stress of cotton.

[0106] Experimental Example 8

[0107] 0.1 g of fresh cotton leaf and root samples treated with 500 mg / L CuCDs solution and deionized water and subjected to salt stress for 5 days were weighed, 1 mL of 0.05 mol / L sodium phosphate buffer (pH = 7.5) was added, and the leaves and roots were ground in an ice bath using a grinder (65 Hz) for 120 sec and then centrifuged at 12000 r / min. -1 , centrifuge at 4°C for 20 min, and the supernatant is the enzyme extract.

[0108] Superoxide dismutase (SOD) assay: The superoxide dismutase (SOD) assay was performed as described by Wang Xuekui (2006). To a 5 mL test tube, 1.5 mL of 0.05 mol / L PBS, 0.3 mL of 0.13 mol / L methionine solution, 0.3 mL of 0.75 mmol / L NBT solution, 0.3 mL of EDTA-Na2 solution, and 0.3 mL of 0.02 mmol / L riboflavin solution were added. 0.05 mL of enzyme extract was added (sodium phosphate buffer was used instead of enzyme solution in the light and dark control tubes). Finally, 0.25 mL of distilled water was added to bring the total volume to 3 mL. After mixing, the dark control tube was placed in the dark, and the other tubes were reacted under 4000 lx sunlight for 20 min. The absorbance of each tube was measured at 560 nm. SOD activity was calculated according to the following formula:

[0109] Total SOD activity (U / g) = (ALC-AE)*V / (0.5*(ALC-ADC*W*VT)

[0110] Where: ALC is the absorbance of the light control tube; ADC is the absorbance of the dark control tube; AE is the absorbance of the sample tube; V is the total volume of the sample solution (mL); VT is the amount of sample used during the measurement (mL); W is the fresh weight of the sample (g).

[0111] Peroxidase (POD) assay:

[0112] Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (PBS, 0.2 M, pH 6), add 0.076 mL of stock guaiacol (2-methoxyphenol), heat and stir to dissolve, and after cooling, add 0.112 mL of 30% H2O2. Enzyme activity determination: Take 3 mL of the above reaction mixture, add 50 μL of the enzyme extract, mix thoroughly, and zero the mixture using PBS as a control. Measure the absorbance of the mixture at 470 nm for 180 seconds, reading every 30 seconds. An increase of 0.01 in absorbance per minute is defined as 1 unit (U). Calculate peroxidase activity according to the following formula:

[0113] POD activity (U / g·min)=(ΔA470*V) / (W*VT*T*0.01)

[0114] Where: △A470 is the change in absorbance during the reaction time; W is the fresh weight of the sample (g); T is the reaction time; V is the volume of enzyme solution used during the determination (mL); VT is the total volume of the extracted enzyme solution (mL).

[0115] Catalase (CAT) assay: Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (0.15 M, pH = 7), add 0.3092 ml of 30% H2O2 stock solution, and shake well. Enzyme activity assay: Take 3 mL of the above reaction mixture, add 20 μL of enzyme extract, adjust to zero using the buffer as a control, and measure the absorbance of the mixture at 240 nm. Measure for a total of 180 seconds, reading once every 30 seconds. A decrease in absorbance of 0.01 per minute is defined as 1 enzyme activity unit (U). Calculate CAT activity according to the following formula:

[0116] CAT activity (U / g·min) = (ΔA240*V) / (W*VT*T*0.01)

[0117] Where: △A240 is the change in absorbance during the reaction time; W is the fresh weight of the sample (g); T is the reaction time; V is the volume of enzyme solution used during the determination (mL); VT is the total volume of the extracted enzyme solution (mL).

[0118] Figure 9The results of SOD, POD, and CAT enzyme activity measurements in the aboveground and underground parts of cotton after 5 days of salt stress showed that the application of CuCDs prepared in the examples of the present invention significantly increased the POD and CAT activities in the aboveground part of cotton under salt stress, while there was no significant difference in the underground part. However, the application of CuCDs did not increase the SOD activity of cotton under salt stress, which may be due to the strong O2· - It has the ability to clear bacteria and replaces the SOD enzyme in cotton.

[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of copper-doped carbon quantum dots in improving salt tolerance of crop seeds and seedlings, characterized in that: The crop is cotton.

2. The application according to claim 1, characterized in that The copper-doped carbon quantum dots include the following preparation method: copper nitrate, urea, citric acid, and N,N-dimethylformamide are mixed, subjected to solvent thermal reaction, and post-treated to obtain the copper-doped carbon quantum dots.

3. The application according to claim 2, characterized in that: The temperature of the solvent thermal reaction is 160-200°C.

4. The application according to claim 2, characterized in that: The solvent thermal reaction time is 4 to 6 hours.

5. The application according to claim 2, characterized in that: The copper nitrate, urea, citric acid, and N,N-dimethylformamide are mixed in a ratio of 0.2-0.4 g: 1-2 g: 0.5-1 g: 10 mL.

6. A method for improving the salt tolerance of crop seeds, characterized in that: The crop seeds are soaked in a copper-doped carbon quantum dot solution; wherein the concentration of the copper-doped carbon quantum dot solution is 20-50 mg / L; and the crop is cotton.

7. The method according to claim 6, characterized in that The mass ratio of the crop seeds to the copper-doped carbon quantum dot solution is 1:4-6.

8. A method for improving salt tolerance of crops at the seedling stage, characterized in that: A solution containing copper-doped carbon quantum dots is applied to the surface of a crop; wherein the concentration of the copper-doped carbon quantum dots in the solution is 250-2000 mg / L; and the crop is cotton.

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