A method for improving seed germination under salt stress and its application

Abstract

The application discloses a method for improving seed germination under salt stress and application thereof, and belongs to the technical field of crop production. The method provided by the application can significantly improve the germination ability of seeds under salt stress. Seed soaking treatment with 10 muM of coffee acid methyl ester can significantly improve the germination rate, germination potential and germination index of watermelon seeds and melon seeds under 250 mM NaCl simulated salt stress, and has no adverse effect on the germination of watermelon and melon seeds under normal conditions. The seed treated by the method provided by the application grows into seedlings with healthy growth, and no obvious abnormality is observed compared with the seedlings grown under normal conditions, indicating that the method provided by the application is safe and has no toxic effect on plant growth. The method is simple, effective and low in cost, and can be used to improve the seed germination ability under salt stress, so as to improve the utilization ability of saline-alkali land and improve the yield and quality of crops in saline-alkali land.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop production, in particular to a method for improving seed germination under salt stress and application thereof. BACKGROUND

[0002] Soil salinization refers to the process of gradually accumulating soluble salts in soil due to natural factors or human activities, which adversely affects plant growth and production. This type of soil causes growth inhibition of plants, which is called salt stress. Salt stress affects soil structure, reduces soil fertility, and makes it difficult for plants to absorb water. Most physiological processes of plants are affected to varying degrees, such as inhibited seed germination, slowed growth rate, smaller leaves, weaker photosynthesis, shorter growth period, and early senescence. The specific hazards include the following aspects:

[0003] 1) Osmotic pressure effect: the increase of soil salt increases the osmotic pressure of soil solution, making it difficult for plant roots to absorb water. In order to maintain the osmotic balance between the inside and outside of cells, crops need to consume more energy to regulate osmotic pressure, which will inhibit the normal growth and development of crops.

[0004] 2) Ion toxicity: Excessive accumulation of soluble salts such as sodium ions (Na+) and chloride ions (Cl-) to a certain concentration can cause direct toxicity to crops, interfere with normal physiological metabolic processes such as photosynthesis, enzyme activity, nutrient absorption and transport, affect the growth point and tender tissue of crops, and cause symptoms such as leaf yellowing and wilting.

[0005] 3) Nutrient imbalance: Excessive salt will reduce the proportion of available nutrients in the soil, especially the availability of important nutrients such as potassium, calcium, and magnesium, leading to nutrient absorption disorders in crops, further affecting crop yield and quality.

[0006] 4) Root damage: In a high-salt environment, crop root growth is hindered, root hairs are reduced, and the ability of roots to absorb water and nutrients is affected, which also reduces the ability of crops to resist pathogen attacks.

[0007] 5) Oxidative stress: Increased salt can also cause a decrease in oxygen content in the soil, causing the root system to be in a state of hypoxia, affecting root respiration, and thus exacerbating the physiological stress of crops.

[0008] Seed germination is the prerequisite for normal growth and development of plants, and is a very important period in the life process of plants, which is related to the establishment of seedlings and subsequent growth, and has important biological significance. However, the seed germination process is particularly susceptible to external mechanical damage, diseases and pests, and stress, so seed germination is also considered to be the most vulnerable stage of the plant life cycle. Many studies have shown that high water potential in salt stress soil leads to slow seed water absorption speed, and even cannot complete the swelling; for the seeds that successfully swell, salt stress can inhibit the function of mitochondria in cells, damage the structure of cell membranes, interfere with hormone balance, inhibit the mobilization of seed reserves, cause osmotic stress, change the reaction conditions of enzymes, affect enzyme activity, and cause active oxygen metabolism system disorder, etc., which has a serious impact on seed germination rate, germination potential, radicle length, plumule length, seed vigor, etc. under the action of various factors.

[0009] Therefore, how to alleviate the influence of salt stress on seed germination is a problem to be solved in the field of crop planting. Although the traditional method for alleviating the influence of salt stress on seed germination has certain effect, it has problems such as high cost and complex operation. Therefore, it is of great significance to find a simple, effective and economical method for alleviating salt stress. SUMMARY

[0010] The purpose of the present application is to provide a method for improving seed germination under salt stress and its application, so as to provide a simple, effective, safe and low-cost method for alleviating the adverse effects of salt stress on seed germination.

[0011] To achieve the above-mentioned purpose, the present application provides a method for improving seed germination under salt stress, which uses a methyl caffeate solution as a seed introduction agent. The seed to be treated is soaked in the seed introduction agent, and then normal germination is performed.

[0012] Preferably, the methyl caffeate solution is prepared by dissolving methyl caffeate in DMSO and then diluting the solution with sterile ultrapure water to the working concentration.

[0013] Preferably, the concentration of the methyl caffeate solution is 0-1000 μM.

[0014] Preferably, the optimal concentration of the methyl caffeate solution is 10 μM.

[0015] Preferably, the seed to be treated is soaked in the seed introduction agent at 25-28℃ in the dark for 12h, and then the excess seed introduction agent solution is absorbed and germination is performed.

[0016] Preferably, the seed to be treated is watermelon seed or melon seed.

[0017] The application of a method for improving seed germination under salt stress as described above in the preparation of seed germination performance improvement.

[0018] A product for improving seed germination performance, the product has the above-mentioned methyl caffeate as a single component or an effective component.

[0019] Therefore, the application provides a method for improving seed germination under salt stress and application thereof, and the specific technical effects are as follows:

[0020] (1) The method provided by the application can significantly improve the germination ability of seeds under salt stress. Seed soaking treatment with 10 μM methyl caffeate can significantly improve the germination rate, germination potential and germination index of watermelon seeds and melon seeds under 250 mM NaCl simulated salt stress, and has no adverse effect on the germination of watermelon and melon seeds under normal conditions.

[0021] (2) The seed treated by the method provided by the application grows into seedlings with healthy growth, and no obvious abnormalities are observed compared with seedlings grown under normal conditions, indicating that the method provided by the application is safe and has no toxic effect on plant growth.

[0022] (3) The method provided by the application is simple, effective and low in cost, and can be used to improve the seed germination ability under salt stress, so as to improve the utilization ability of saline-alkali land and improve the yield and quality of crops in saline-alkali land.

[0023] The technical solutions of the application will be further described in detail below with the aid of the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the description of the embodiments of the application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0025] Figure 1 is a photo of part of the seeds germinated in the first embodiment of the application for 60h;

[0026] Figure 2 is the statistical results of the germination rate (A), germination potential (B) and germination index (C) in the first embodiment of the application;

[0027] Figure 3 is a photo of seeds treated by different methods in the second embodiment of the application and cultured in darkness at 25-28℃ for 60h;

[0028] Figure 4 is the statistical results of the germination rate (A), germination potential (B) and germination index (C) of different treatments in the second embodiment of the application;

[0029] Figure 5Figure 1 is a side view (A) and a top view (B) of the seedlings generated from different treated seeds in Example 2 of the present application;

[0030] Figure 6 Figure 3 is a photograph of the seeds treated differently in Example 3 of the present application and cultured in darkness at 25-28°C for 60h;

[0031] Figure 7 Figure 4 is the statistical results of the germination rate (A), germination potential (B) and germination index (C) of the seeds treated differently in Example 3 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0033] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below by means of the accompanying drawings and examples. The following detailed description is the description of examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meanings as generally understood by those skilled in the art to which the present application belongs.

[0034] The instruments and reagents used in the examples are obtained through commercial channels.

[0035] Example 1

[0036] The optimal concentration of the working solution of the seed priming agent was investigated, and the specific steps were as follows:

[0037] S1, preparation of salt stress solution: 1.17g of sodium chloride was dissolved in 100mL of sterilized ultrapure water to prepare a 200mM salt stress solution.

[0038] S2, preparation of seed priming agent working solution: 23.30mg of methyl caffeate (MC) was added to 50μL of dimethyl sulfoxide (DMSO), and after stirring and dissolving, 119.950mL of sterilized ultrapure water was added and stirred to obtain the seed priming agent mother liquor. 100mL of the seed priming agent working solution with a concentration of 1000μM was taken, and the remaining mother liquor was respectively taken 0μL, 10μL, 100μL, 1mL and 10mL of the seed priming agent, and dissolved in 100mL, 99.99mL, 99.9mL, 99mL and 90mL of sterilized ultrapure water to prepare the seed priming agent working solutions with concentrations of 0, 0.1, 1, 10 and 100μM, respectively.

[0039] S3, select full, no disease and pest of 'Xinong No. 8' watermelon seeds, in the clean bench, with 3% sodium hypochlorite solution for 30s, then rinse with sterile ultrapure water for 3 times, with sterile filter paper to remove excess water, then the seeds were randomly divided into 7 groups, three replicates in each group, 21 seeds in each replicate, respectively soaked in different concentrations of introduction agent prepared in step S2 working solution, at room temperature, light, for 12h, recorded as experimental group, named MC0 group, MC0.1 group, MC1 group, MC10 group, MC100 group and MC1000 group respectively. The same method and conditions with sterile ultrapure water soaked seeds as control group (recorded as Control).

[0040] S4, to the sterile culture dish in turn laid a layer of sterile paper, a layer of filter paper, with step S1 prepared salt stress solution wet sterile filter paper, the experimental group seeds after soaking with sterile filter paper to remove excess liquid, interval placed on the wet filter paper, cover a layer of sterile filter paper, then cover the culture dish cover. The control group was sown by the same method, but the filter paper and sterile paper were wetted with sterile water. The experimental group and the control group were cultured at 25-28℃ in the dark for 60h, and the germination rate (Germination rate, GR), germination potential (Germination potential, GP) and germination index (Germination index, GI) were calculated. The photos of part of seeds in the 60th hour of germination are shown in Figure 1

[0041] Germination rate (germination percentage, GP) = n / N x 100 (%), wherein n and N are the number of germinated seeds and the total number of seeds tested, respectively.

[0042] Germination energy (germinating energy, GE) = nx / N x 100 (%), wherein nx and N are the number of germinated seeds in a specified day (48h) and the total number of seeds tested, respectively.

[0043] Germination index (germination index, GI) = ∑(Gt / Dt), wherein Gt is the number of germination in the tth day, and Dt is the corresponding germination day.

[0044] The results are shown in part A of Figure 2 As shown in part A of the results, with the increase of the concentration of introduction agent, the germination rate showed a trend of gradually increasing first and then decreasing, low concentration of introduction agent (0-10μM) promoted the germination of watermelon seeds under salt stress, while high concentration (100 and 1000μM) of introduction agent inhibited the germination of seeds. The effect of 10μM introduction agent was the best, and the germination rate of watermelon seeds was the highest in the 60th hour of germination, which was significantly different from the control group.

[0045] ​The results of germination potential and germination index are similar to the trend of the results of germination rate, as shown in parts B and C of Figure 2 As shown in parts B and C of

[0046] Example Two

[0047] To investigate the effect of 10 μM methyl caffeate on the germination of watermelon seeds under normal conditions and 250 mM NaCl simulation conditions, the specific steps are as follows:

[0048] S1, prepare a salt stress solution, dissolve 1.4625 g of sodium chloride in 100 mL of sterilized ultrapure water to prepare a 250 mM salt stress solution.

[0049] S2, prepare the seed primer working solution, prepare the seed primer mother liquor by the method of step S2 of example one, take 1 mL of the seed primer mother liquor and dissolve it in 100 mL of sterilized ultrapure water to prepare a seed primer working solution with a concentration of 10 μM, and use the addition of 4.17 μL / L DMSO in sterile ultrapure water as the control working solution.

[0050] S3, select 180 full and disease-free "Xinong No. 8" watermelon seeds, soak them in 3% sodium hypochlorite solution for 30 s in a clean bench, then rinse them with sterilized ultrapure water for 3 times, absorb the excess water with sterile filter paper, and then randomly divide the seeds into 4 groups, each with three repeats, and each repeat has 15 seeds. The germination method is the same as step S3 of example one, and the four groups are treated as follows:

[0051] Control group (control): soak the seeds in the control working solution, and wet the filter paper and sterile toilet paper used for laying and covering with sterile ultrapure water;

[0052] MC group: soak the seeds in the seed primer working solution prepared in S2, and wet the filter paper and sterile toilet paper used for laying and covering with sterile ultrapure water;

[0053] NaCl group: soak the seeds in the control working solution, and wet the filter paper and sterile toilet paper used for laying and covering with the salt stress solution prepared in step S1;

[0054] MC+NaCl group: soak the seeds in the seed primer working solution prepared in S2, and wet the filter paper and sterile toilet paper used for laying and covering with the salt stress solution prepared in step S1.

[0055] S4, cultivate the seeds in each group under dark conditions at 25-28°C, count the number of germinated seeds at 24h, 36h, 48h, and 60h of cultivation, respectively, and the photos of part of the seeds after 60h of cultivation are as shown in Figure 3The seed germination rate was calculated using the formula in Example 1, and the results are shown in Figure 4 Part A of Table 1.

[0056] The seed germination potential and germination index were calculated using the formula in Example 1, and the results are shown in Figure 4 Parts B and C of Table 1. The seed germination rate, germination potential, and germination index of the MC group were basically the same as those of the control group within 0-60 h; compared with the control group, the seed germination rate, germination potential, and germination index of the NaCl group were significantly reduced; compared with the NaCl group, the seed germination rate of the MC+NaCl group was significantly improved within 36-60 h, and the seed germination potential and germination index were also significantly higher than those of the NaCl group. The above results show that seed soaking with 10 μM methyl caffeate has no adverse effect on the germination of watermelon seeds under normal conditions, but can significantly enhance the germination ability of watermelon seeds under 250 mM salt stress. The seedling photos of different groups are shown in Figure 5 Table 2. The seedlings generated from the seeds treated with the working solution of the seed priming agent grow healthily, and no obvious abnormalities are observed compared with the seedlings grown under normal conditions, indicating that the method provided by the present application is safe and has no toxic effect on plant growth.

[0057] Example Three

[0058] The effect of 10 μM methyl caffeate on the germination of watermelon seeds under normal conditions and 250 mM NaCl simulation conditions was investigated, and the specific steps are as follows:

[0059] S1, prepare a salt stress solution, dissolve 1.4625 g of sodium chloride in 100 mL of sterilized ultrapure water to prepare a 250 mM salt stress solution.

[0060] S2, prepare the seed priming agent working solution, prepare the seed priming agent mother liquor by the method of step S2 in Example 1, and dissolve 1 mL of the seed priming agent mother liquor in 100 mL of sterilized ultrapure water to prepare a seed priming agent working solution with a concentration of 10 μM. The sterile ultrapure water containing 4.17 μL / L DMSO was used as a control working solution.

[0061] S3, select 180 full and disease-free 'Yangjiaomeng' melon seeds, soak them in 3% sodium hypochlorite solution for 30 s on the clean bench, then rinse them with sterilized ultrapure water for 3 times, and then use sterile filter paper to absorb the excess water. After that, the seeds are randomly divided into 4 groups, each with three repeats, and each repeat contains 15 seeds. The germination method is the same as that in step S3 of Example 1, and the four treatment groups are as follows:

[0062] Control group (control): soak the seeds in the control working solution, and wet the filter paper and sterile toilet paper used for laying and covering with sterile ultrapure water;

[0063] MC group: the seeds were soaked with the priming agent working solution prepared in step S2, and the filter paper and sterile toilet paper were wetted with sterile ultrapure water;

[0064] NaCl group: the seeds were soaked with the control working solution, and the filter paper and sterile toilet paper were wetted with the salt stress solution prepared in step S1;

[0065] MC+NaCl group: the seeds were soaked with the priming agent working solution prepared in S2, and the filter paper and sterile toilet paper were wetted with the salt stress solution prepared in step S1.

[0066] S4, the seeds in each group were cultured at 25-28°C in the dark, and the number of germinated seeds was counted at 24h, 36h, 48h and 60h of culture, respectively. The photos of part of the seeds cultured for 60h are shown in Figure 6 The seed germination rate was calculated by the formula in Example 1, and the results are shown in A of Figure 7

[0067] The seed germination potential and germination index were calculated by the formula in Example 1. The results are shown in B and C of Figure 7 The seed germination rate, germination potential and germination index of the MC group were basically the same as those of the control group. Compared with the control group, the seed germination rate, germination potential and germination index of the NaCl group were significantly reduced. Compared with the NaCl group, the seed germination rate of the MC+NaCl (i.e. the seed was soaked with methyl caffeate and then germinated under salt stress) was significantly improved at 36-60h, and the seed germination potential and germination index were also significantly higher than or higher than those of the NaCl group. It was shown that the soaking of 10 μM methyl caffeate could promote the germination of the melon seeds under 250 mM salt stress and had no adverse effect on the germination of the melon seeds under normal conditions.

[0068] Therefore, the method provided by the present application can significantly improve the germination ability of the seeds under salt stress. The soaking of 10 μM methyl caffeate can significantly improve the seed germination rate, germination potential and germination index of the watermelon and melon seeds under the salt stress simulated by 250 mM NaCl, and has no adverse effect on the germination of the watermelon and melon seeds under normal conditions. The seedlings generated from the seeds treated by the method provided by the present application grow vigorously, and no obvious abnormalities are observed compared with the seedlings grown under normal conditions, indicating that the method provided by the present application is safe and has no toxic effect on the plant growth. The method is simple, effective and low in cost, and can be used to improve the seed germination ability under salt stress, so as to improve the utilization ability of the saline-alkali land and the yield and quality of the crops grown in the saline-alkali land.

[0069] ​It should be pointed out finally that the above examples are only used to illustrate the technical solutions of the present application but not to limit it, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can still be modified or replaced equivalently, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method of improving seed germination under salt stress conditions, characterized in that: The seed to be treated is soaked in the methyl caffeate solution as the priming agent, and then normally sowed; The methyl caffeate solution is prepared by dissolving methyl caffeate in DMSO and then diluting the solution with sterilized ultrapure water to a working concentration; The seed to be treated is soaked in the priming agent at 25-28 DEG C in the dark for 12 hours, and then the excess priming agent solution is absorbed and the seed is germinated; The seed to be treated is watermelon seed or melon seed; The working concentration of the methyl caffeate solution is 10 micromole.

Images