Method for increasing the germination rate of wheat seeds under salt stress

Abstract

The application discloses a method for improving the germination rate of wheat seeds under salt stress, and comprises the following steps: after the wheat seeds are sterilized, the seeds are soaked in a mixed bacterial solution of B. velezensis, B. thuringiensis and B. frigoritolerans for a set time, and then the seeds are dried at low temperature and planted and germinated. The method can improve the germination rate of the wheat seeds under salt stress.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of wheat seed treatment, and particularly relates to a method for improving the germination rate of wheat seeds under salt stress. BACKGROUND

[0002] The statements herein are provided only to complement the background of the present application and are not necessarily prior art.

[0003] High salt stress causes changes in the internal structure and external morphology of plants to a certain extent by inhibiting the physiological activities of plants. The adverse effects of salt stress can seriously affect the growth and development of plants, and even cause the death of plants. Wheat has strong environmental adaptability, and has growth characteristics such as cold tolerance, drought tolerance, and salt and alkali tolerance, so it can grow in different climates and soil conditions. However, when encountering high salt stress conditions, salt invades the endosperm and germ of wheat seeds, resulting in low germination rate or direct death. At the same time, the wheat germinated under high salt stress has the disadvantages of low growth rate, reduced leaf extension rate, and reduced leaf area of the plant. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide a method for improving the germination rate of wheat seeds under salt stress.

[0005] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme.

[0006] The present application provides a method for improving the germination rate of wheat seeds under salt stress, comprising the following steps:

[0007] After sterilizing the wheat seeds, immerse them in a mixed bacterial solution of B . Bacillus velezensis 、 B . Bacillus thuringiensis and B . Brevibacterium frigoritolerans for a set time, dry at low temperature, and then plant and germinate.

[0008] In some embodiments, the sterilization method of the wheat seeds is as follows: after washing with disinfectant ethanol for a set time, wash with sterile distilled water, then wash with 8%-10% hydrogen peroxide, and finally wash with Hoagland nutrient solution.

[0009] Preferably, the volume percentage of the disinfectant ethanol is 75%.

[0010] Preferably, the washing time with disinfectant ethanol is 3-6 min.

[0011] In some embodiments, B . Bacillus velezensis 、 B . Bacillus thuringiensisand B . Brevibacterium frigoritolerans The OD of the mixed bacteria solution of 600 nm 0.3-2.0, and the CFU ratio of the three bacteria is 0.8-1.2:0.8-1.2:0.8-1.2.

[0012] Preferably, B . Bacillus velezensis , B . Bacillus thuringiensis and B . Brevibacterium frigoritolerans In the mixed bacteria solution of

[0013] Preferably, the soaking time of the seed in the mixed bacteria solution is 1-5h after the seed is sterilized.

[0014] In some embodiments, after the wheat seeds soaked in the mixed bacteria solution are planted in the soil and germinated, the method further comprises the step of applying the mixed bacteria solution to the soil of the wheat seedling roots.

[0015] Preferably, the application is once every 4-7 days.

[0016] The beneficial effects achieved by the one or more embodiments of the application are as follows:

[0017] (1) The method of the application can improve the germination rate of wheat seeds under salt stress conditions;

[0018] (2) The method can minimize the damage to the germination of salt-stressed wheat seeds, and provide a new idea and method for alleviating salt stress. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of the application form a part of the application and serve to provide further understanding of the application, the exemplary embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application.

[0020] Figure 1 is a control diagram of the germination rate of wheat seeds treated with different strains in the embodiments of the application, and the order of the germination rate corresponding to each salt concentration is the same as that of the legend;

[0021] Figure 2 is a control diagram of the germination rate of wheat seeds treated with mixed strains in the embodiments of the application, and the order of the germination rate corresponding to each salt concentration is the same as that of the legend;

[0022] Figure 3 is a pot experiment of wheat in the embodiments of the application, wherein (A) is blank; (B) is joint; (C) Bacillus velezensis; (D) Bacillus thuringiensis and (E) Brevibacterium frigoritolerans ;

[0023] Figure 4 Effects of the salt-tolerant microorganism of the embodiments of the present application on wheat growth parameters, wherein (A) plant height; (B) root length; (C) fresh weight; (D) dry weight. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0025] A method for improving the germination rate of wheat seeds under salt stress using microorganisms, comprising the following steps:

[0026] (1) Seed sterilization

[0027] The wheat seeds were sterilized using the following protocol: 75% ethanol wash for 4 min; wash with sterile distilled water three times; 10% H2O2 soak for 20 min; wash with 50 mL Hoagland's nutrient solution.

[0028] (2) Preliminary experiment

[0029] The sterilized wheat seeds were soaked in different salt concentration gradients (0 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM) of the bacterial solution (the bacterial solution was a mixture of the three bacterial strains, OD 600 nm 1.5, and the CFU ratio of the three strains was 1:1:1) for 4 h, 20 seeds per treatment, and the wheat seeds soaked in sterile culture solution were used as a control. After drying at room temperature for 2 h, the treated seeds were evenly placed in sterile moist Petri dishes for culture, and the growth conditions were observed and the germination rate was calculated. B Bacillus velezensis , B . Bacillus thuringiensis and B . Brevibacterium frigoritolerans

[0030] In the preliminary experiment, there was a significant difference (P < 0.01) in the germination rate of wheat seeds under salt stress between the untreated and strain-treated seeds. As shown in Table 1, the germination rate of the control seeds decreased with increasing salt concentration, and the germination rate of the strain-treated seeds was higher than that of the control. Compared with the wheat seeds treated with a single strain, the germination rate of the wheat seeds treated with the three strains together was the best. Figure 1

[0031] As shown in Table 2, the germination rate of the control seeds decreased with increasing salt concentration, and the germination rate of the strain-treated seeds was higher than that of the control. Compared with the wheat seeds treated with a single strain, the germination rate of the wheat seeds treated with the three strains together was the best. Figure 2 ​​​As shown, the germination rate of wheat seeds soaked in the mixed bacterial solution was higher than that of untreated seeds, and the germination rate of seeds treated with all three strains of bacteria was higher than that of seeds treated with two strains of bacteria. This is sufficient to demonstrate that wheat seeds treated with all three strains of bacteria are the most effective in alleviating salt stress and improving germination rate in the mixed bacterial solution.

[0032] Therefore, in subsequent pot experiments, three strains of bacteria were used together to alleviate salt stress in plants, providing a clear contrast between the promoting effects of single strains and mixed strains on plant growth under salt stress.

[0033] Subsequent wheat pot experiments included the following steps:

[0034] (1) Seed pre-culture

[0035] Sterilized wheat seeds were soaked in the strain B . Bacillus velezensis , B . Bacillus thuringiensis , B . Brevibacterium frigoritolerans The seeds were soaked in a combined bacterial solution of three strains (1:1:1) for 4 hours, then dried at room temperature for 2 hours. The treated plant seeds were then placed in a petri dish (containing moist sterile filter paper) and germinated at 25°C.

[0036] (2) Amplification of bacterial strains

[0037] Bacillus belye B . Bacillus velezensis The accession number is CICC 21082, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0038] Bacillus thuringiensis B . Bacillus thuringiensis The accession number is CICC 20553, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0039] Brevibacterium thermotolerans B . Brevibacterium frigoritolerans The accession number is CGMCC 1.10488, and it was purchased from the China General Microbiological Culture Collection Center.

[0040] Each bacterium was inoculated into its corresponding culture medium and incubated statically for 12 hours to serve as the "seed" culture medium. A sterile culture medium was used as a control.

[0041] Using a sterile pipette, transfer 100 mL of culture medium into each 250 mL Erlenmeyer flask. Inoculate each flask with 10 mL of "seed" culture medium and incubate at a constant temperature with shaking (28±2℃, 120 rpm) for 72 h to allow OD to develop. 600 nm It reaches 0.3~2.0.

[0042] (3) Inoculation of the bacterial strains

[0043] The seedlings of similar size and vigor were transplanted into the flower pots with soil prepared in advance, three seedlings were transplanted into each flower pot, and the seedlings treated with sterile culture solution were used as controls.

[0044] The cultivated bacterial strains were washed 3 times to remove the culture medium, and then resuspended in a sterile liquid medium without NaCl (to maintain the growth conditions of the bacterial strains) to an OD 600 nm of 1.5, and applied alone or in combination to the roots of the plants, and the control group was added with an equal amount of sterile liquid medium. Then, every 5 days, the plants were inoculated with a bacterial suspension (suspended in sterile water) with the same concentration, and the control group was added with an equal amount of sterile water.

[0045] The same dose of bacterial strains was applied to each flower pot, as shown in Table 1.

[0046] Table 1 Inoculation of bacterial strains

[0047]

[0048] In Table 1, 10% refers to the mass percentage of bacterial solution in soil.

[0049] In Table 1, combination refers to a CFU ratio of 1:1:1 for the three bacterial strains.

[0050] After 28 days of constant temperature cultivation of wheat, the whole seedlings were removed from the pots, washed clean, and the morphological parameters were measured. The plant height, bud (root) length were measured using a ruler, and the fresh weight, dry weight (60°C drying for 24 h) and other growth parameters were measured using an electronic balance.

[0051] (4) Results of the pot experiment

[0052] The results of the wheat pot experiment are shown in Figure 3 , and in each figure, the NaCl stress values of wheat from left to right are 0, 50, 100, 200, 300, and 400 mM, respectively. Figure 3 Aand Figure 4 It can be seen that the increase of salt stress inhibits the growth of wheat. When exposed to salt stress, B . Bacillus velezensis, B . Bacillus thuringiensis and B . Brevibacterium frigoritolerans Both single and combined action can promote the growth of plants to different extents. Figure 4 It is shown that the plant parameters of wheat without the action of bacterial strains decrease with the increase of salt stress. The use of a single bacterial strain on wheat under salt stress can increase the plant height, root length, and fresh weight and dry weight. Under the action of a single bacterial strain, the growth parameters of wheat under salt stress, B .Brevibacterium frigoritolerans In addition, the study on the combined action of the strains on wheat was carried out through a pot experiment. The results show that the parameters of the wheat treated by the combined action of the strains are better than those treated by the single action, whether or not there is salt stress.

[0053] It is worth noting that the effect of the strains is combined action>single action>blank control. Under high salt stress (400 mM), compared with the blank, the plant height, root length, fresh weight and dry weight of the wheat treated by the combined action of the three strains are increased by 1.78, 2.00, 2.13 and 2.81 times respectively. Compared with the single action of the strains, the wheat can obtain better growth conditions through the combined action of the strains.

[0054] The above merely describes preferred embodiments of the present application but is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of increasing the germination rate of wheat seeds under salt stress, characterized in that: comprising the following steps: After sterilizing the wheat seeds, the seeds were soaked in mixed bacterial solution of Bacillus velezensis ( Bacillus velezensis ), Bacillus thuringiensis ( Bacillus thuringiensis ) and Brevibacillus chagii ( Brevibacterium frigoritolerans ) for a certain time, dried at low temperature, and then planted and germinated; The preservation number of Bacillus velezensis is CICC 21082, which is purchased from China Industrial Microbial Culture Collection Center; The preservation number of Bacillus thuringiensis is CICC 20553, which is purchased from China Industrial Microbial Culture Collection Center; The preservation number of Brevibacterium frigoritolerans is CGMCC 1.10488, which is purchased from China General Microbiological Culture Collection Center; The CFU ratio of the three bacteria in the mixed bacteria solution of Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans is 0.8-1.2:0.8-1.2:0.8-1.2; After the seed is sterilized, the soaking time in the mixed bacteria solution is 1-5h.

2. The method of increasing the seed germination rate of wheat under salt stress according to claim 1, characterized in that: The sterilization method of the wheat seed is as follows: after washing with disinfectant ethanol for a certain time, washing with sterile distilled water, then washing with 8%-10% hydrogen peroxide, and finally washing with Hoagland nutrient solution.

3. The method of increasing the seed germination rate of wheat under salt stress according to claim 2, characterized in that: The volume percentage of disinfectant ethanol is 75%.

4. The method of increasing the seed germination rate of wheat under salt stress according to claim 2, characterized in that: The washing time with disinfectant ethanol is 3-6min.

5. The method of increasing the seed germination rate of wheat under salt stress according to claim 1, characterized in that: The CFU ratio of the three bacteria in the mixed bacteria solution of Bacillus velezensis, Bacillus thuringiensis and Brevibacterium frigoritolerans is 1:1:

1.

6. The method of increasing the seed germination rate of wheat under salt stress according to claim 1, characterized by: After the seed is sterilized, the soaking time in the mixed bacteria solution is 2-4h.

7. The method of increasing the seed germination rate of wheat under salt stress according to claim 1, characterized by: After the wheat seeds soaked in the mixed bacteria solution are planted in the soil and germinated, the step of applying the mixed bacteria solution to the soil of the wheat seedling roots is further included.

8. The method of increasing the seed germination rate of wheat under salt stress according to claim 7, characterized in that: The application is carried out every 4-7 days. The application is carried out every 4-7 days.

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