Method for promoting germination of brassica napus seeds under low-temperature stress by using allantoin

Abstract

The invention relates to the technical field of crop planting, and provides a method for promoting germination of brassica napus seeds under low-temperature stress by using allantoin. The method comprises the following steps: firstly, disinfecting cabbage type rape seeds, and then soaking the cabbage type rape seeds in an allantoin solution with a specific concentration under room-temperature and weak-light conditions; the treated seeds do not need to be washed and are directly placed in a culture dish paved with double-layer filter paper, a proper amount of distilled water is added, and then the seeds are cultured in an incubator simulating a low-temperature environment. Experimental results show that the germination rate, the germination index and the vitality index of the brassica napus seeds are remarkably improved through allantoin treatment, and the average germination time is shortened. Mechanism research finds that allantoin improves the low-temperature resistance of the brassica napus by various ways of increasing the content of free water of seedlings, increasing the content of soluble sugar and proline, enhancing the activity of antioxidant enzyme, reducing active oxygen and malonaldehyde accumulation and the like. The method disclosed by the invention is low in cost and environment-friendly, and provides powerful guarantee for field production such as late direct seeding of the brassica napus.

Description

Technical Field

[0001] The invention relates to the technical field of crop planting, and more specifically to a method for promoting the germination of Brassica napus seeds under low temperature stress by utilizing allantoin. Background Art

[0002] Brassica napus L. is one of the main oil crops. Its seeds can be used for edible oil and biodiesel production. It also plays an important role in food and drug development, feed and fertilizer production, and horticultural landscape design. The annual planting area of ​​Brassica napus in my country exceeds 7 million hectares, accounting for more than 40% of the total area of ​​oil crops in the country. It is the core crop of the winter fallow field rotation system in the Yangtze River Basin. However, its production is often affected by low temperature stress, especially low temperature adversity during the seedling and flowering stages, which can cause yield losses of 20%-30%. Therefore, improving the low temperature resistance of Brassica napus is the key to ensuring its stable and high yield. Seed germination is a stage in the life cycle of plants with extremely weak stress resistance. Low temperature will reduce the water absorption rate of Brassica napus seeds and make it difficult for the radicle to break through the seed coat; at the micro level, it will inhibit the activity of key hydrolases such as α-amylase and protease, destroy mitochondrial function, reduce adenosine triphosphate (ATP) synthesis, and trigger reactive oxygen species (ROS) bursts, leading to damage such as cell membrane lipid peroxidation. In field production, uneven emergence and weak seedlings caused by low temperatures will further aggravate the risk of diseases and pests, forming a vicious circle.

[0003] Plant growth regulators (PGRs) have been widely used to enhance the low temperature tolerance of crops. For example, gibberellin (GA3) can promote seed germination by inducing the expression of α-amylase genes; salicylic acid (SA) and polyamines can reduce ROS damage by enhancing the activity of antioxidant enzymes. In recent years, allantoin, as a natural nitrogen-containing compound, has attracted attention due to its multiple physiological functions. Studies have found that allantoin can promote cytokinin synthesis and remove ROS by activating superoxide dismutase (SOD) and peroxidase (POD). At present, exogenous allantoin treatment has been shown to significantly improve the germination rate and seedling survival rate of rice and wheat seeds under low temperature, but its application in Brassica napus has not been systematically reported.

[0004] In response to the problem of limited germination of Brassica napus seeds at low temperatures, the development of efficient and environmentally friendly regulation technologies is the focus of current research. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by utilizing allantoin. The method comprises the following steps: soaking the Brassica napus seeds in an allantoin solution, treating the seeds under room temperature and weak light conditions, and then culturing the seeds. The problems of slow germination, poor uniformity, and weak growth potential of the Brassica napus seeds under low temperature conditions are effectively alleviated. The method is particularly suitable for field production such as late direct seeding. In addition, allantoin has the advantages of being environmentally friendly and having a low cost, thereby providing a strong guarantee for the production of Brassica napus.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin comprises the following steps:

[0008] Step S1, sterilizing the Brassica napus seeds with a 70% ethanol solution for 2 minutes, and then washing with distilled water for 3-5 times;

[0009] Step S2, placing the sterilized Brassica napus seeds into a centrifuge tube, injecting 2-6 mmol / L of allantoin solution into the centrifuge tube according to a concentration gradient design, and soaking the seeds for 7 hours at room temperature and in a weak light environment;

[0010] Step S3, taking out the soaked rapeseed seeds, placing them directly in a culture dish containing two layers of filter paper without rinsing, and injecting 3 mL of distilled water;

[0011] Step S4, placing the culture dish in an artificial incubator, setting the temperature to 7°C under a light intensity of 20000Lx for 8 hours; setting the temperature to 1°C under a dark condition with a light intensity of 0Lx for 16 hours; and controlling the relative humidity at 60%-70% for low temperature environment cultivation.

[0012] As a further embodiment of the present invention, the concentration of the allantoin solution is preferably 4 mmol / L.

[0013] As a further solution of the present invention, the soaked Brassica napus seeds can germinate more quickly in a low temperature environment, and the growth potential and resistance of the seedlings are stronger.

[0014] As a further embodiment of the present invention, the Brassica napus seeds are low temperature sensitive variety ZD622 or low temperature tolerant variety PS1.

[0015] As a further embodiment of the present invention, the allantoin improves the resistance to low temperature by promoting the germination rate, growth potential, biomass and average germination time of Brassica napus seeds.

[0016] As a further scheme of the present invention, compared with a control group of rapeseed seeds that have not been treated with allantoin except for disinfection, the rapeseed seeds treated by the method can increase the germination rate of the low-temperature sensitive variety ZD622 by 25%-60%, the germination index by 100%-170%, shorten the average germination time by 20%-40%, increase the fresh weight by 25%-60%, increase the dry weight by 5%-12%, increase the vitality index by 85%-190%, and increase the root length by 60%-100%; and can increase the germination rate of the low-temperature tolerant variety PS1 by 15%-30%, the germination index by 40%-90%, shorten the average germination time by 20%-30%, increase the fresh weight by 15%-25%, increase the dry weight by 8%-13%, increase the vitality index by 45%-100%, and increase the root length by 40%-55%.

[0017] As a further embodiment of the present invention, the allantoin increases the metabolic rate of Brassica napus seedlings by increasing the free water content of the seedlings, thereby improving the resistance to low temperature.

[0018] As a further scheme of the present invention, compared with a control group of rapeseed seeds that have not been treated with allantoin except for disinfection, the Brassica napus seeds treated by the method can increase the free water content of the low-temperature sensitive variety ZD622 by 19%-52%, the bound water content changes between -5% and +7%, and the total water content changes between -2% and +6%; and can increase the free water content of the low-temperature tolerant variety PS1 by 18%-26%, reduce the bound water content by 2%-11%, and increase the total water content by 8%-9%.

[0019] As a further solution of the present invention, the allantoin increases the soluble sugar and proline content of Brassica napus seedlings, thereby increasing the content of soluble substances and permeability substances to protect the cell membrane lipid system, etc., thereby enhancing resistance to low temperature.

[0020] As a further scheme of the present invention, compared with a control group of rapeseed seeds that have not been treated with allantoin except for disinfection, the Brassica napus seeds treated by the method can increase the soluble sugar content of the low-temperature sensitive variety ZD622 by 10%-15% and the free amino acid content by 3%-5% after treatment with 4mmol / L allantoin; increase the proline content of the low-temperature sensitive variety ZD622 by 1%-3% after treatment with 6mmol / L allantoin; and increase the soluble sugar content of the low-temperature tolerant variety PS1 by 25%-45% after treatment with 2-6mmol / L allantoin, and increase the proline content by 5%-10% after treatment with 4mmol / L allantoin.

[0021] As a further embodiment of the present invention, the allantoin enhances the resistance to low temperature by increasing the antioxidant enzyme activity of Brassica napus seedlings.

[0022] As a further scheme of the present invention, compared with a control group of rapeseed seeds that were not treated with allantoin except for disinfection, the rapeseed seeds treated by the method can increase the superoxide dismutase activity of the low-temperature sensitive variety ZD622 by 45%-55% and the peroxidase activity by 20%-30% after being treated with 4mmol / L allantoin; the catalase activity of the low-temperature sensitive variety ZD622 by 65%-80% after being treated with 6mmol / L allantoin; for the low-temperature tolerant variety PS1, the superoxide dismutase activity can be increased by 10%-20% after being treated with 6mmol / L allantoin, the peroxidase activity can be increased by 25%-35% after being treated with 2mmol / L allantoin, and the catalase activity can be increased by 200%-250% after being treated with 4mmol / L allantoin.

[0023] As a further embodiment of the present invention, the allantoin enhances the resistance to low temperature by reducing the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) in Brassica napus seedlings.

[0024] As a further scheme of the present invention, the rapeseed seeds treated by the method can control the accumulation of active oxygen and malondialdehyde in the rapeseed seedlings compared with the rapeseed seed control group that is not treated with allantoin except for disinfection; for the low-temperature sensitive variety ZD622, the allantoin treatment can reduce the accumulation of superoxide anions to 85%-92% of the control group, the accumulation of hydrogen peroxide to 80%-85% of the control group, and the accumulation of malondialdehyde to 65%-75% of the control group; for the low-temperature tolerant variety PS1, the allantoin treatment can reduce the accumulation of superoxide anions to 70%-75% of the control group, the accumulation of hydrogen peroxide to 65%-70% of the control group, and the accumulation of malondialdehyde to 70%-80% of the control group.

[0025] Compared with the prior art, the method of the present invention for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin has the following beneficial effects:

[0026] The present invention proposes for the first time the application of allantoin in improving the germination performance of Brassica napus seeds under low temperature environment, and further finds that allantoin shortens the average germination time by improving the germination rate and growth potential of rapeseed seeds; at the same time, allantoin can increase the free water content of seedlings, promote the accumulation of osmotic substances in seedlings, increase the activity of antioxidant enzymes, and reduce the content of reactive oxygen species (ROS) and malondialdehyde (MDA), thereby synergistically enhancing the resistance of seedlings to low temperature stress, and effectively alleviating the problems of slow germination of Brassica napus seeds, necrosis of young embryos and slow growth of seedlings under low temperature environment; in addition, as a natural compound, allantoin has the two major advantages of low cost and environmental friendliness, and has huge production and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a comparison of the germination status of Brassica napus ZD622 and PS1 seeds on the 14th day under different treatments of the present invention.

[0028] Figure 2 This is a statistical diagram of changes in antioxidant enzyme activities in Brassica napus ZD622 and PS1 seedlings under different treatments of the present invention.

[0029] Figure 3 This is a comparison of the accumulation of ROS and MDA in Brassica napus ZD622 and PS1 seedlings under different treatments of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] The embodiment of the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin. The seeds of the Brassica napus seeds (ZD622 and PS1) were provided by the Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, wherein ZD622 is a cold stress sensitive variety and PS1 is a cold stress tolerant variety. Allantoin was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd.

[0033] First, weigh the gradient mass of allantoin and prepare allantoin solutions of 0, 2, 4, and 6 mmol / L with distilled water. Among them, the recommended concentration determined in this embodiment is 4 mmol / L, that is, the optimal allantoin content added per 1 L of water. The concentration gradient of allantoin is shown in Table 1.

[0034] Table 1 Concentration gradient of allantoin

[0035]

[0036] The present invention uses different concentrations to conduct comparative experiments, and a total of five groups of treatments are set up, including:

[0037] The CK group, i.e., the control group, means that rapeseed seeds had no extra pretreatment except disinfection;

[0038] Water group, indicating that rapeseed seeds were disinfected and then soaked in distilled water for 7 hours;

[0039] Group N2, indicating that rapeseed seeds were disinfected and then immersed in 2mmol / L allantoin solution for 7 hours;

[0040] Group N4, indicating that rapeseed seeds were disinfected and then immersed in 4mmol / L allantoin solution for 7 hours;

[0041] Group N6, indicating that rapeseed seeds were disinfected and then immersed in 6mmol / L allantoin solution for 7 hours;

[0042] The experiment was conducted on two varieties, ZD622 and PS1, and the design is shown in Table 2:

[0043] Table 2 Treatment with different concentrations

[0044]

[0045] At room temperature, select an appropriate amount of plump, healthy, and morphologically similar Brassica napus ZD622 and PS1 seeds, quickly disinfect them with 70% ethanol solution for 2 minutes, and then rinse them with distilled water several times. The seeds were divided into 2mL centrifuge tubes with different numbers, 30 seeds per tube, and injected with equal amounts of allantoin solution according to the concentration gradient design, and soaked for 7 hours. The seeds of each treatment group were taken out from the soaking solution (distilled water and gradient allantoin solution) without rinsing, and were placed in a culture dish containing two layers of filter paper, and 3mL of distilled water was injected to facilitate the seeds to fully absorb water and germinate without causing waterlogging.

[0046] Each culture dish was placed in an artificial incubator, and the environmental parameters were set as follows: the temperature was 7°C under light (20000Lx) for 8 hours; the temperature was 1°C under dark (0Lx) for 16 hours; the relative humidity was maintained at 60%-70%. Since the rapeseed seeds need to be protected from light for germination, the culture dishes were completely wrapped with black material to block light. The number of rapeseed seeds that germinated was counted daily, and biomass statistics, photos, and physiological measurements were performed on the 14th day.

[0047] On the 14th day, ZD622 and PS1 rapeseed seedlings were photographed and recorded. Figure 1 It can be seen that after allantoin pretreatment, the number of rapeseed seeds that germinated was significantly higher than that in the CK group and the Water group, and more seedlings completed cotyledon hulling, and the seedling morphology was more complete.

[0048] In the embodiment of the present invention, the biomass index of rapeseed seedlings was measured, including the biomass indexes of rapeseed seedlings, including germination rate, germination index, average germination time, fresh weight, dry weight, vitality index and root length. The dry weight was measured after the fresh sample was placed in an oven at 75°C for 3 hours. The values ​​in the table are mean values ​​± standard deviation. According to Tukey's test, different letters after the mean in the same column in the table indicate significant differences at the P<0.05 level. The measurement results are shown in Table 3:

[0049] Table 3 Biomass of ZD622 and PS1 seeds germinated under low temperature stress

[0050]

[0051] The test results showed that allantoin pretreatment significantly promoted the rapid germination of Brassica napus seeds under low temperature stress, showing characteristics such as higher germination rate, germination index, vitality index and shorter germination time. This indicates that allantoin can quickly mobilize rapeseed seeds to break shells and germinate under low temperature stress, and complete initial nutritional growth in a shorter time.

[0052] Example 2

[0053] The embodiment of the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin. The seeds of the Brassica napus seeds (ZD622 and PS1) were provided by the Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, wherein ZD622 is a cold stress sensitive variety and PS1 is a cold stress tolerant variety. Allantoin was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd.

[0054] First, allantoin in gradient mass was weighed and prepared with distilled water into 0, 2, 4, and 6 mmol / L allantoin solutions.

[0055] The present invention uses different concentrations to conduct comparative experiments, and a total of five groups of treatments are set up, including:

[0056] The CK group, i.e., the control group, means that rapeseed seeds had no extra pretreatment except disinfection;

[0057] Water group, indicating that rapeseed seeds were disinfected and then soaked in distilled water for 7 hours;

[0058] Group N2, indicating that rapeseed seeds were disinfected and then immersed in 2mmol / L allantoin solution for 7 hours;

[0059] Group N4, indicating that rapeseed seeds were disinfected and then immersed in 4mmol / L allantoin solution for 7 hours;

[0060] Group N6, indicating that rapeseed seeds were disinfected and then immersed in 6mmol / L allantoin solution for 7 hours;

[0061] The experiment was conducted simultaneously on two varieties, ZD622 and PS1.

[0062] At room temperature, select an appropriate amount of plump, healthy, and morphologically similar Brassica napus ZD622 and PS1 seeds, quickly disinfect them with 70% ethanol solution for 2 minutes, and then rinse them with distilled water several times. The seeds were divided into 2mL centrifuge tubes with different numbers, 30 seeds per tube, and injected with equal amounts of allantoin solution according to the concentration gradient design, and soaked for 7 hours. The seeds of each treatment group were taken out from the soaking solution (distilled water and gradient allantoin solution) without rinsing, and were placed in a culture dish containing two layers of filter paper, and 3mL of distilled water was injected to facilitate the seeds to fully absorb water and germinate without causing waterlogging.

[0063] Each culture dish was placed in an artificial incubator, and the environmental parameters were set as follows: the temperature was 7°C under light (20000Lx) for 8 hours; the temperature was 1°C under dark (0Lx) for 16 hours; the relative humidity was maintained at 60%-70%. Since the rapeseed seeds need to be protected from light for germination, the culture dishes were completely wrapped with black material to block light. Samples were taken on the 14th day of germination for moisture status determination.

[0064] The free water, bound water and total water content of rapeseed seedlings in the embodiment of the present invention are determined by weighing method (XU Liangzheng, LIU Huina. Course of plant physiological experiment [M]. Beijing: Science Press, 2022. XU LZ, LIU HN. Course of plant physiological experiment [M]. Beijing: Science Press, 2022). The values ​​in the table are mean ± standard deviation. According to Tukey's test, different letters after the mean in the same column in the table indicate significant differences at the P < 0.05 level. The measurement results are shown in Table 4:

[0065] Table 4 Water content of ZD622 and PS1 seedlings under low temperature stress

[0066]

[0067] The results showed that allantoin pretreatment can effectively improve the water metabolism capacity of rapeseed seedlings under low temperature stress, especially increasing the proportion of free water in plant cells, which is conducive to maintaining cell turgor pressure, promoting cell elongation and division, and thus accelerating the establishment of seedling morphology. Improving water metabolism capacity is one of the important physiological mechanisms by which allantoin enhances the low temperature resistance of Brassica napus.

[0068] Example 3

[0069] The embodiment of the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin. The seeds of the Brassica napus seeds (ZD622 and PS1) were provided by the Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, wherein ZD622 is a cold stress sensitive variety and PS1 is a cold stress tolerant variety. Allantoin was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd.

[0070] First, allantoin in gradient mass was weighed and prepared with distilled water into 0, 2, 4, and 6 mmol / L allantoin solutions.

[0071] The present invention uses different concentrations to conduct comparative experiments, and a total of five groups of treatments are set up, including:

[0072] The CK group, i.e., the control group, means that rapeseed seeds had no extra pretreatment except disinfection;

[0073] Water group, indicating that rapeseed seeds were disinfected and then soaked in distilled water for 7 hours;

[0074] Group N2, indicating that rapeseed seeds were disinfected and then immersed in 2mmol / L allantoin solution for 7 hours;

[0075] Group N4, indicating that rapeseed seeds were disinfected and then immersed in 4mmol / L allantoin solution for 7 hours;

[0076] Group N6, indicating that rapeseed seeds were disinfected and then immersed in 6mmol / L allantoin solution for 7 hours;

[0077] The experiment was conducted simultaneously on two varieties, ZD622 and PS1.

[0078] At room temperature, select an appropriate amount of plump, healthy, and morphologically similar Brassica napus ZD622 and PS1 seeds, quickly disinfect them with 70% ethanol solution for 2 minutes, and then rinse them with distilled water several times. The seeds were divided into 2mL centrifuge tubes with different numbers, 30 seeds per tube, and injected with equal amounts of allantoin solution according to the concentration gradient design, and soaked for 7 hours. The seeds of each treatment group were taken out from the soaking solution (distilled water and gradient allantoin solution) without rinsing, and were placed in a culture dish containing two layers of filter paper, and 3mL of distilled water was injected to facilitate the seeds to fully absorb water and germinate without causing waterlogging.

[0079] Each culture dish was placed in an artificial incubator, and the environmental parameters were set as follows: the temperature was 7°C under light (20000Lx) for 8 hours; the temperature was 1°C under dark (0Lx) for 16 hours; the relative humidity was maintained at 60%-70%. Since the rapeseed seeds need to be protected from light for germination, the culture dishes were completely wrapped with black material to block light. Sampling was performed on the 14th day of germination to determine the content of soluble sugar, free amino acids and proline in the seedlings.

[0080] The embodiment of the present invention uses a multifunctional microplate reader (H1MD Take3 Trio, BioTek, America) to measure various indicators. The determination of soluble sugar adopts the anthrone colorimetric method (Li Hesheng. Principles and techniques of plant physiological and biochemical experiments [M]. Higher Education Press, 2000.); the determination of free amino acids adopts the ninhydrin colorimetric method (Wang Xuekui, Huang Jianliang. Principles and techniques of plant physiological and biochemical experiments [M]. Higher Education Press, 2015.); the determination of proline adopts the colorimetric method (Wang Xuekui, Huang Jianliang. Principles and techniques of plant physiological and biochemical experiments [M]. Higher Education Press, 2015.). The values ​​in the table are mean ± standard deviation. According to Tukey's test, different letters after the mean in the same column in the table indicate significant differences at the P<0.05 level. The measurement results are shown in Table 5:

[0081] Table 5 Soluble sugar, free amino acids and proline contents in ZD622 and PS1 seedlings under low temperature stress

[0082]

[0083]

[0084] The results showed that allantoin pretreatment could increase the content of soluble substances in rapeseed seedling tissues under low temperature stress to a certain extent, among which 4mM allantoin treatment showed a better effect, significantly increasing the soluble sugar content and proline content of PS1. However, the content of free amino acids did not change significantly, so these results may indicate that allantoin pretreatment does not directly promote the production of soluble and osmotic substances, but an indirect promotion effect.

[0085] Example 4

[0086] The embodiment of the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin. The seeds of the Brassica napus seeds (ZD622 and PS1) were provided by the Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, wherein ZD622 is a cold stress sensitive variety and PS1 is a cold stress tolerant variety. Allantoin was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd.

[0087] First, allantoin in gradient mass was weighed and prepared with distilled water into 0, 2, 4, and 6 mmol / L allantoin solutions.

[0088] The present invention uses different concentrations to conduct comparative experiments, and a total of five groups of treatments are set up, including:

[0089] The CK group, i.e., the control group, means that rapeseed seeds had no extra pretreatment except disinfection;

[0090] Water group, indicating that rapeseed seeds were disinfected and then soaked in distilled water for 7 hours;

[0091] Group N2, indicating that rapeseed seeds were disinfected and then immersed in 2mmol / L allantoin solution for 7 hours;

[0092] Group N4, indicating that rapeseed seeds were disinfected and then immersed in 4mmol / L allantoin solution for 7 hours;

[0093] Group N6, indicating that rapeseed seeds were disinfected and then immersed in 6mmol / L allantoin solution for 7 hours;

[0094] The experiment was conducted simultaneously on two varieties, ZD622 and PS1.

[0095] At room temperature, select an appropriate amount of plump, healthy, and morphologically similar Brassica napus ZD622 and PS1 seeds, quickly disinfect them with 70% ethanol solution for 2 minutes, and then rinse them with distilled water several times. The seeds were divided into 2mL centrifuge tubes with different numbers, 30 seeds per tube, and injected with equal amounts of allantoin solution according to the concentration gradient design, and soaked for 7 hours. The seeds of each treatment group were taken out from the soaking solution (distilled water and gradient allantoin solution) without rinsing, and were placed in a culture dish containing two layers of filter paper, and 3mL of distilled water was injected to facilitate the seeds to fully absorb water and germinate without causing waterlogging.

[0096] Each culture dish was placed in an artificial incubator, and the environmental parameters were set as follows: the temperature was 7°C under light (20000Lx) for 8 hours; the temperature was 1°C under dark (0Lx) for 16 hours; the relative humidity was maintained at 60%-70%. Since the rapeseed seeds need to be protected from light for germination, the culture dishes were completely wrapped with black material to block light. Samples were taken on the 14th day of germination to measure the activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in the seedlings.

[0097] In the examples of the present invention, a multifunctional microplate reader (H1MD Take3 Trio, BioTek, America) was used to measure various indicators.

[0098] The activity determination of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the embodiment of the present invention refers to Xu et al. (Xu L, Li J, Najeeb U, Li X, Pan J, Huang Q, Zhou W, Liang Z. Synergistic effects of EDDS and ALA on phytoextraction of cadmium as revealed by biochemical and ultrastructural changes in sunflower (Helianthus annuus L.) tissues. J Hazard Mater. 2021 Apr 5; 407: 124764. doi: 10.1016 / j.jhazmat.2020.124764. Epub 2020 Dec 8. PMID: 33348204.) The method comprises the following steps: 0.1 g of fresh seedling sample is extracted with 50 mM potassium phosphate buffer (pH 7.8), centrifuged at 12000 g, 4°C for 12 min, and the supernatant is used for the determination of SOD, POD and CAT activities.

[0099] The CAT activity in the embodiment of the present invention is detected by H 2 O 2 To decompose the enzyme, 10 μL of enzyme solution was added to 190 μL of reaction solution (containing 2 mM EDTA-2Na and 10 mM H2O2) and the absorbance decrease rate (ΔA·min -1 ).

[0100] The POD activity detection in the present embodiment is based on the guaiacol oxidation method. 10 μL of enzyme solution is added to 190 μL of reaction solution (0.3% guaiacol and 0.4% hydrogen peroxide), and then the absorbance change rate (ΔA·min) of the reaction system at 470 nm is immediately measured. -1 ).

[0101] The SOD activity in the present invention was determined by inhibiting the NBT photoreduction method, wherein 0.1 mL of enzyme solution and 2.9 mL of NBT reaction solution (containing 13 mM methionine, 75 μM NBT, 0.1 mM EDTA and 2 μM riboflavin) were added to the reaction tube, and 0.1 mL of ultrapure water was used instead of the enzyme solution in the control tube. The reaction system was treated under 4000 lx light and dark conditions for 20 minutes, and the absorbance was measured at 560 nm.

[0102] The experimental results are as follows Figure 2As shown in the figure, (a), (b), and (c) show the changes in antioxidant enzyme activities in seedlings of Brassica napus ZD622 and PS1 under different treatments, where the values ​​are the mean ± standard deviation of three biological replicates (n = 9), and different lowercase letters indicate statistically significant differences at the P ≤ 0.05 level by Tukey's multiple comparison test; (a) is SOD activity; (b) is POD activity; (c) is CAT activity. Compared with the control group (CK) and the water treatment group (Water), the SOD activity of the sensitive variety ZD622 seedlings treated with 4mmol / L allantoin increased most significantly, increasing by 48.6% and 10.0%, respectively; similarly, the 4mmol / L concentration treatment had the best promoting effect on POD activity, increasing by 26.2% and 113.3%, respectively; and the 6mmol / L concentration treatment had the best promoting effect on CAT activity, increasing by 72.4% and 47.2%, respectively.

[0103] For the seedlings of the tolerant variety PS1, the 6mmol / L allantoin treatment had the best promoting effect on SOD activity, which increased by 13.9% and 46.1% respectively; the 2mmol / L treatment had the best promoting effect on POD activity, which increased by 32.1% and 33.3% respectively; the 4mmol / L treatment had the best promoting effect on CAT activity, which increased by 239.9% and 483.3% respectively.

[0104] These results show that allantoin pretreatment can significantly increase the antioxidant enzyme activity of Brassica napus seeds during germination, thereby enhancing its tolerance to low temperature environments. Different varieties have different responses to different concentrations of allantoin, but overall they all show an improvement in antioxidant capacity.

[0105] Example 5

[0106] The embodiment of the present invention provides a method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin. The seeds of the Brassica napus seeds (ZD622 and PS1) were provided by the Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, wherein ZD622 is a cold stress sensitive variety and PS1 is a cold stress tolerant variety. Allantoin was purchased from Hangzhou Chentong Biochemical Technology Co., Ltd.

[0107] First, allantoin in gradient mass was weighed and prepared with distilled water into 0, 2, 4, and 6 mmol / L allantoin solutions.

[0108] The present invention uses different concentrations to conduct comparative experiments, and a total of five groups of treatments are set up, including:

[0109] The CK group, i.e., the control group, means that rapeseed seeds had no extra pretreatment except disinfection;

[0110] Water group, indicating that rapeseed seeds were disinfected and then soaked in distilled water for 7 hours;

[0111] Group N2, indicating that rapeseed seeds were disinfected and then immersed in 2mmol / L allantoin solution for 7 hours;

[0112] Group N4, indicating that rapeseed seeds were disinfected and then immersed in 4mmol / L allantoin solution for 7 hours;

[0113] Group N6, indicating that rapeseed seeds were disinfected and then immersed in 6mmol / L allantoin solution for 7 hours;

[0114] The experiment was conducted simultaneously on two varieties, ZD622 and PS1.

[0115] At room temperature, select an appropriate amount of plump, healthy, and morphologically similar Brassica napus ZD622 and PS1 seeds, quickly disinfect them with 70% ethanol solution for 2 minutes, and then rinse them with distilled water several times. The seeds were divided into 2mL centrifuge tubes with different numbers, 30 seeds per tube, and injected with equal amounts of allantoin solution according to the concentration gradient design, and soaked for 7 hours. The seeds of each treatment group were taken out from the soaking solution (distilled water and gradient allantoin solution) without rinsing, and were placed in a culture dish containing two layers of filter paper, and 3mL of distilled water was injected to facilitate the seeds to fully absorb water and germinate without causing waterlogging.

[0116] Each culture dish was placed in an artificial incubator, and the environmental parameters were set as follows: the temperature was 7°C under light (20000Lx) for 8 hours; the temperature was 1°C under dark (0Lx) for 16 hours; the relative humidity was maintained at 60%-70%. Since rapeseed seeds need to be protected from light for germination, the culture dishes were completely wrapped with black materials to block light. Samples were taken on the 14th day of germination to determine the effects of allantoin treatment on the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) in rapeseed seedlings under low temperature stress.

[0117] The determination of MDA and ROS in the embodiments of the present invention was performed using a multifunctional microplate reader (H1MD Take3Trio, BioTek, America). The malondialdehyde (MDA) content was determined according to the method of Li et al. (Li J, Yang C, Liu H, et al. 5-aminolevolinic acid enhances sunflower resistance to Orobanche cumana (Broomrape) [J]. Industrial Crops and Products, 2019, 140: 111467. DOI: 10.1016 / j.indcrop.2019.111467.), using the TBA colorimetric method, 0.1 g of fresh seedling samples were extracted with 50 mM potassium phosphate buffer (pH 7.8), 5 mL of 0.5% TBA (10% trichloroacetic acid configuration) was added to 1 mL of supernatant, the reaction system was immediately ice-bathed after boiling water bath for 30 min, and then the supernatant was taken and the absorbance at 532 nm and 600 nm was measured.

[0118] The determination of reactive oxygen species (ROS) in the embodiments of the present invention includes hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2- ) two parts.

[0119] The hydrogen peroxide (H 2 O 2 ) content was detected using a titanium tetrachloride colorimetric kit (Solarbio, China), and the extraction and determination were performed according to the method provided by the kit, and the absorbance of the final solution was measured at 415 nm.

[0120] The superoxide anion O in the embodiment of the present invention 2- The determination was based on the method of Li et al. (Li J, Yang C, Liu H, et al. 5-aminolevolinic acid enhances sunflower resistance to Orobanche cumana (Broomrape) [J]. Industrial Crops and Products, 2019, 140: 111467. DOI: 10.1016 / j.indcrop.2019.111467.), which included extracting 0.1 g of fresh seedling sample with 65 mM potassium phosphate buffer (pH 7.8), then adding 0.1 mL of 10 mM hydroxylamine hydrochloride to 1 mL of supernatant, incubating for 1 h, then adding 1 mL of 17 mM p-aminobenzenesulfonic acid and 1 mL of 7 mM α-naphthylamine, and measuring the absorbance of the final solution at 530 nm.

[0121] The experimental results are as follows Figure 3 As shown, (a), (b), and (c) show the accumulation of ROS and MDA in Brassica napus ZD622 and PS1 seedlings under different treatments, where the values ​​are the mean ± SD of three biological replicates (n = 9), and different lowercase letters indicate statistically significant differences at the P ≤ 0.05 level by Tukey's multiple comparison test; (a) is O 2- level; (b) H 2 O 2 (c) MDA level. Excessive accumulation of ROS and MDA can lead to adverse consequences such as damage to the cell membrane lipid system, protein damage and nucleic acid damage. The experimental results showed that after allantoin pretreatment, ZD622 and PS1 seedlings grown to 14 days under low temperature stress had a significant difference in O 2- , H 2 O 2 The accumulation of the three indicators of MDA was alleviated.

[0122] Compared with the control group (CK) and the water treatment group (Water), the best treatment group among the three allantoin treatment groups of ZD622 increased O 2- , H 2 O 2 The accumulation of 1,2-dimethylaminopropene and 1,6-dihydropyridine was 89.6% and 76.0%, 82.6% and 87.5%, and 68.7% and 80.2% of the first two groups, respectively. For PS1, the best allantoin treatment group made the accumulation of these three indicators 71.5% and 78.9%, 66.6% and 86.4%, and 75.2% and 71.9% of the first two groups, respectively.

[0123] These results collectively indicate that allantoin pretreatment can promote the germination of ZD622 and PS1 seeds in low temperature environments, and its mechanism includes reducing the accumulation of ROS and MDA and increasing the activity of antioxidant enzymes, thereby improving the low temperature adaptability of Brassica napus seedlings.

[0124] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0125] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting the germination of Brassica napus seeds under low temperature stress by using allantoin, characterized in that: The following steps are involved: Step S1, disinfecting the Brassica napus seeds with an ethanol solution, and then washing them with distilled water for 3-5 times; Step S2, placing the sterilized Brassica napus seeds into a centrifuge tube, injecting an equal amount of allantoin solution into the centrifuge tube according to a concentration gradient design, and soaking the seeds at room temperature and in a weak light environment; Step S3, taking out the soaked rapeseed seeds, placing them directly in a culture dish containing filter paper without rinsing, and injecting distilled water; Step S4, placing the culture dish in an artificial incubator, setting the light cycle and temperature cycle to change alternately, and controlling the relative humidity, and performing low-temperature environment cultivation, wherein the temperature during the light period is higher than the temperature during the dark period, simulating the day and night temperature difference of a natural low-temperature environment.

2. The method of claim 1, wherein the method comprises: The concentration of the ethanol solution in step S1 is 70%, and the disinfection treatment time is 2 minutes; the concentration of the allantoin solution in step S2 is 2-6mmol / L, and the seed soaking treatment time is 7 hours; the culture dish in step S3 contains double-layer filter paper, and the amount of distilled water injected is 3mL; the illumination intensity of the illumination condition in step S4 is 20000Lx, the temperature is 7°C, and the duration is 8 hours; the illumination intensity of the dark condition is 0Lx, the temperature is 1°C, and the duration is 16 hours; the relative humidity is controlled at 60%-70%.

3. The method of claim 1, wherein the method comprises: The culture dish was completely wrapped with black material to block out light.

4. The method of claim 1, wherein the method comprises: The Brassica napus seeds are low temperature sensitive variety ZD622 or low temperature tolerant variety PS1.

5. The method of claim 1, wherein the method comprises: Compared with a control group of rapeseed seeds which are not treated with allantoin except for disinfection, the rapeseed seeds treated by the method can increase the germination rate of the low-temperature sensitive variety ZD622 by 25%-60%, increase the germination index by 100%-170%, shorten the average germination time by 20%-40%, increase the fresh weight by 25%-60%, increase the dry weight by 5%-12%, increase the vitality index by 85%-190%, and increase the root length by 60%-100%; and can increase the germination rate of the low-temperature tolerant variety PS1 by 15%-30%, increase the germination index by 40%-90%, shorten the average germination time by 20%-30%, increase the fresh weight by 15%-25%, increase the dry weight by 8%-13%, increase the vitality index by 45%-100%, and increase the root length by 40%-55%.

6. The method of claim 1, wherein the method comprises: Compared with a control group of rapeseed seeds that are not treated with allantoin except for disinfection, the cabbage rapeseed seeds treated by the method can increase the free water content of the low-temperature sensitive variety ZD622 by 19%-52%, the bound water content changes between -5% and +7%, and the total water content changes between -2% and +6%; and can increase the free water content of the low-temperature tolerant variety PS1 by 18%-26%, reduce the bound water content by 2%-11%, and increase the total water content by 8%-9%.

7. The method of claim 1, wherein the method comprises: Compared with a control group of rapeseed seeds that have not been treated with allantoin except for disinfection, the rapeseed seeds treated with the method show that 4 mmol / L allantoin treatment can increase the soluble sugar content of a low-temperature sensitive variety ZD622 by 10%-15% and the free amino acid content by 3%-5%; 6 mmol / L allantoin treatment can increase the proline content of the low-temperature sensitive variety ZD622 by 1%-3%; and for a low-temperature tolerant variety PS1, 2-6 mmol / L allantoin treatment can increase the soluble sugar content by 25%-45%, and 4 mmol / L allantoin treatment can increase the proline content by 5%-10%.

8. The method of claim 1, wherein the method comprises: Compared with a control group of rapeseed seeds which are not treated with allantoin except for disinfection, the rapeseed seeds treated with the method can increase the superoxide dismutase activity of a low-temperature sensitive variety ZD622 by 45%-55% and the peroxidase activity by 20%-30% after being treated with 4mmol / L allantoin; the catalase activity of the low-temperature sensitive variety ZD622 by 6mmol / L allantoin can be increased by 65%-80%; for a low-temperature tolerant variety PS1, the superoxide dismutase activity can be increased by 10%-20% after being treated with 6mmol / L allantoin, the peroxidase activity can be increased by 25%-35% after being treated with 2mmol / L allantoin, and the catalase activity can be increased by 200%-250% after being treated with 4mmol / L allantoin.

9. The method of claim 1, wherein the method comprises: Compared with a control group of rapeseed seeds that are not treated with allantoin except for disinfection, the rapeseed seeds treated by the method can control the accumulation of active oxygen and malondialdehyde in the rapeseed seedlings; for the low-temperature sensitive variety ZD622, the allantoin treatment can reduce the accumulation of superoxide anions, hydrogen peroxide and malondialdehyde to 85%-85% and 65%-75% of the control group; for the low-temperature tolerant variety PS1, the allantoin treatment can reduce the accumulation of superoxide anions, hydrogen peroxide and malondialdehyde to 70%-75% and 65%-70% of the control group.

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