Initiating liquid and method for promoting germination and growth of stylosanthes guianensis seeds under saline-alkali stress

Abstract

The invention provides an initiation solution for promoting germination and growth of stylosanthes guianensis seeds under saline-alkali stress. The initiation solution comprises nano-zinc oxide particles, compared with the prior art, the method has the advantages that the Zinc oxide nanoparticle solution is used for treating the stylosanthes guianensis seeds, and then the seeds are dried, so that the germination rate and germination potential of the stylosanthes guianensis seeds under saline-alkali stress are remarkably improved, the salt tolerance of the stylosanthes guianensis is remarkably enhanced, and the stylosanthes guianensis shows efficient and rapid saline-alkali relieving capacity. And a seed priming technology is adopted, so that the process of cultivating stylosanthes guianensis in a saline-alkali environment is greatly shortened.

Description

Technical Field

[0001] The present application relates to the technical field of forage seed production, and in particular to a priming solution for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress, and a method for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress. Background Art

[0002] Stylosanthes guianensis is a perennial herbaceous plant belonging to the genus Stylosanthes of the Leguminosae family. It likes humid tropical climates and is widely distributed in tropical and subtropical areas. It has high yield and is rich in protein. It is widely used as grass feed for livestock in tropical regions of the world. It can also be used as a leguminous green manure plant and forest and fruit grass ecological engineering construction. At the same time, because of its drought resistance, acid and barren soil resistance, and anthrax resistance, it is often used for slope restoration, slope greening, and rocky desertification control, and has a wide range of applications in agricultural production. Stylosanthes has certain limitations on its tolerance to salt and alkali concentrations. Stylosanthes generally shows good acid resistance and grows well in acidic to slightly alkaline soil environments, but when the soil pH exceeds 7.5, its growth may be inhibited. Therefore, Stylosanthes is suitable for lower salt and alkali concentrations and is not suitable for growing in soils with high salt and alkali concentrations, and its physiological activities will be significantly affected. Specifically, salt and alkali stress will cause the relative water content of Stylosanthes to decrease, photosynthesis to be inhibited, and electrolyte permeability to increase. Stylosanthes has poor tolerance to salt stress, which limits its promotion and planting in saline-alkali areas. Therefore, it is an urgent problem to study the effects of salt-alkali stress on the physiological activities of Stylosanthes, improve the tolerance of Stylosanthes in saline-alkali environments, and increase the breadth of its planting areas.

[0003] Due to the above problems, the current development technology of Stylosanthes is slow, the breeding cycle is long, and the germination rate is low. Based on this, it is urgent for those skilled in the art to provide a method for promoting the germination of Stylosanthes seeds and the growth of seedlings under saline-alkali stress. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an initiating solution for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress, wherein the initiating solution comprises nano zinc oxide particles.

[0005] Optionally, the initiating liquid is a nano zinc oxide aqueous dispersion, and the nano zinc oxide aqueous dispersion includes the nano silver oxide particles; the nano silver oxide particles have a purity of 99.8% and a particle size of 90 nm.

[0006] Optionally, the preparation is prepared according to the following method:

[0007] An appropriate amount of the nano zinc oxide particles is weighed, and the nano zinc oxide particles are dispersed in deionized water by ultrasonic vibration.

[0008] Optionally, the concentration of the nano zinc oxide particles in the configured initiating solution is 100 mg / L.

[0009] Optionally, include the following steps:

[0010] The steps of breaking dormancy, disinfecting, washing and drying are sequentially performed on the stylosanthes seeds;

[0011] The stylosanthes seeds are placed in a centrifuge tube, and a triggering solution is added, wherein the triggering solution is the triggering solution according to any one of claims 1 to 3; and the centrifuge tube is placed in a shaking incubator for cultivation;

[0012] After the incubation, pour out the suspension, use absorbent paper to absorb the water, spread it evenly on clean absorbent paper, and dry the Stylosanthes seeds at room temperature for subsequent germination;

[0013] The initiated Stylosanthes seeds are germinated and cultured by using a paper germination method, a sodium chloride solution is added during the germination culture, and the seeds are placed in an artificial climate incubator for continued culture until germination.

[0014] The present invention also provides a method for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress, wherein the Stylosanthes seeds are sequentially subjected to dormancy breaking, disinfection, rinsing, and air-drying, comprising the following steps:

[0015] The dormancy of the Stylosanthes seeds is broken by bathing them in a constant temperature water bath at 80° C. for 3 minutes;

[0016] The stylosanthes seeds are disinfected with 75% ethanol for 1 minute;

[0017] The stylosanthes seeds were rinsed with sterile water for 3 to 5 times, and then disinfected with 10% sodium hypochlorite for 5 minutes, and then rinsed with sterile water for 3 to 5 times and dried.

[0018] Optionally, the “placing the centrifuge tube in a shaking incubator for culturing” comprises the following steps:

[0019] The centrifuge tube was placed in the shaker in a dark environment, the shaker was set to 50 revolutions per minute, the culture temperature was 28° C., the culture time was 8 h, and the drying time was 48 h.

[0020] Optionally, the germination culture of the primed Stylosanthes seeds using the paper germination method comprises the following steps:

[0021] The primed Stylosanthes seeds were placed in a culture dish covered with filter paper; the filter paper was 2 layers.

[0022] Optionally, the concentration of the sodium chloride solution is 100 mmol / L or 200 mmol / L.

[0023] Optionally, the incubation time of the artificial climate incubator is 7 days; the light time is 12 hours and the dark time is 12 hours per day; and the temperature of the incubator is 25°C.

[0024] Compared with the prior art, the present invention significantly improves the germination rate and germination potential of Stylosanthes seeds under saline-alkali stress by treating them with zinc oxide nanoparticle solution and then drying the seeds, significantly enhances the salt tolerance of Stylosanthes, and shows efficient and rapid salt-alkali mitigation ability. In addition, by adopting seed priming technology, the cultivation process of Stylosanthes in saline-alkali environment is greatly shortened. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0026] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." are not excluded from the package.

[0027] The present invention provides an initiation liquid for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress, wherein the initiation liquid comprises nano zinc oxide particles. In a preferred embodiment, the initiation liquid is a nano zinc oxide aqueous dispersion, wherein the nano zinc oxide aqueous dispersion comprises the nano silver oxide particles; the purity of the nano silver oxide particles is 99.8%, and the particle size is 90 nm.

[0028] In an optional embodiment, the preparation is prepared according to the following method: weigh an appropriate amount of the nano zinc oxide particles, and disperse the nano zinc oxide particles in deionized water by ultrasonic vibration. In this embodiment, preferably, the concentration of the nano zinc oxide particles in the prepared initiating solution is 100 mg / L.

[0029] The present invention also provides a method for promoting germination of Stylosanthes seeds and growth of seedlings under saline-alkali stress, comprising the following steps:

[0030] The steps of breaking dormancy, disinfecting, washing and drying are sequentially performed on the stylosanthes seeds;

[0031] The stylosanthes seeds are placed in a centrifuge tube, and a triggering solution is added, wherein the triggering solution is the triggering solution according to any one of claims 1 to 3; and the centrifuge tube is placed in a shaking incubator for cultivation;

[0032] After the incubation, pour out the suspension, use absorbent paper to absorb the water, spread it evenly on clean absorbent paper, and dry the Stylosanthes seeds at room temperature for subsequent germination;

[0033] The initiated Stylosanthes seeds are germinated and cultured by using a paper germination method, a sodium chloride solution is added during the germination culture, and the seeds are placed in an artificial climate incubator for continued culture until germination.

[0034] In an optional embodiment, the steps of breaking dormancy, disinfecting, rinsing and drying the Stylosanthes seeds in sequence include the following steps:

[0035] The dormancy of the Stylosanthes seeds is broken by bathing them in a constant temperature water bath at 80° C. for 3 minutes;

[0036] The stylosanthes seeds are disinfected with 75% ethanol for 1 minute;

[0037] The stylosanthes seeds were rinsed with sterile water for 3 to 5 times, and then disinfected with 10% sodium hypochlorite for 5 minutes, and then rinsed with sterile water for 3 to 5 times and dried.

[0038] In an optional embodiment, the “placing the centrifuge tube in a shaking incubator for culturing” comprises the following steps:

[0039] The centrifuge tube was placed in the shaker in a dark environment, the shaker was set to 50 revolutions per minute, the culture temperature was 28° C., the culture time was 8 h, and the drying time was 48 h.

[0040] In an optional embodiment, the germination culture of the primed Stylosanthes seeds using the paper germination method comprises the following steps:

[0041] The primed Stylosanthes seeds were placed in a culture dish covered with filter paper; the filter paper was 2 layers.

[0042] In an optional embodiment, the concentration of the sodium chloride solution is 100 mmol / L or 200 mmol / L.

[0043] In an optional embodiment, the artificial climate incubator has an incubation time of 7 days, a light time of 12 hours and a dark time of 12 hours per day, and a temperature of 25°C in the incubator.

[0044] The following are specific embodiments provided by the present invention:

[0045] Example 1: Comparison of treatment with different concentrations of initiating liquid

[0046] The specific steps include:

[0047] S1: Prepare an initiator solution, weigh an appropriate amount of nano zinc oxide particles (ZnO NPs, 99.8% high purity, particle size of 90nm, purchased from MacLean Company) to prepare a nano zinc oxide solution; the nano zinc oxide solution is prepared by dispersing the nano zinc oxide particles in deionized water through ultrasonic vibration (130W, 20kHz, 30°C), wherein the ultrasonic vibration is not more than 30min; in this step, initiator solution samples with different nano zinc oxide concentrations are prepared, and the concentrations are 10mg / L, 20mg / L, 50mg / L, 100mg / L, 200mg / L and 500mg / L, respectively.

[0048] S2: Seed collection and processing. Remove impurities and shrunken grains from the Stylosanthes seeds and set them aside. Pick the Stylosanthes seeds with uniform size, full grains and uniform color. Rinse the seeds with clean water and select the full seeds that sink. Place the Stylosanthes seeds in a constant temperature water bath at 80°C for 3 minutes to break the dormancy of the Stylosanthes seeds. Then disinfect the Stylosanthes seeds with 75% ethanol for 1 minute. Then rinse the Stylosanthes seeds with sterile water for 3 to 5 times, and then disinfect the Stylosanthes seeds with 10% sodium hypochlorite for 5 minutes. Finally, rinse the Stylosanthes seeds with sterile water for 3 to 5 times, dry them and set them aside.

[0049] S3: In the initiation process, the seeds were placed in 6 centrifuge tubes of 50 ml, and 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 500 mg / L of initiation solution were added to immerse the seeds, respectively, with a solid-liquid ratio of 1:5, and placed in a 28°C shaker for 8 hours, and the shaker was set to 50 revolutions per minute. After the incubation, the suspension was poured out, the water was absorbed with absorbent paper, and the seeds were evenly spread on clean absorbent paper. The seeds were dried at room temperature for subsequent germination;

[0050] S4: Seed germination. The seeds after initiation in step S3 were germinated and cultured using the paper germination method. The seeds treated with 10mg / L, 20mg / L, 50mg / L, 100mg / L, 200mg / L, and 500mg / L initiation solutions were placed in a culture dish covered with two layers of filter paper. 40 stylosanthes seeds were placed in each culture dish. Five replicates were made for each treatment, and 10mL of sterile water was added to each dish. The seeds were placed in an artificial climate incubator and cultured for 7 days. The light time in the incubator was 12h per day, the dark time was 12h per day, and the temperature was 25°C. An appropriate amount of sterile water was added every other day. The number of seeds germinating each day was recorded using the whitening as the germination standard. Example 2: Comparison of optimal concentrations of nano-zinc oxide initiation under different saline-alkali stresses

[0051] The specific steps include:

[0052] S1: Prepare an initiator solution, weigh an appropriate amount of nano zinc oxide particles (ZnO NPs, 99.8% high purity, particle size of 90nm, purchased from MacLean Company) to prepare a nano zinc oxide solution, wherein the nano zinc oxide solution is prepared by dispersing the nano zinc oxide particles in deionized water through ultrasonic vibration (130W, 20kHz, 30°C), wherein the ultrasonic vibration is not more than 30min; prepare an initiator solution with a nano zinc oxide concentration of 100mg / L.

[0053] S2: Seed collection and processing, remove impurities and shrunken grains from the stylosanthes seeds and set aside, select the stylosanthes seeds with uniform size, full grains and uniform color, rinse the seeds with clean water and select the full seeds that sink, place the stylosanthes seeds in a water bath at 80°C for 3 minutes to break the dormancy of the stylosanthes seeds; then disinfect the stylosanthes seeds with 75% ethanol for 1 minute; then rinse the stylosanthes seeds with sterile water for 3 to 5 times, and then disinfect the stylosanthes seeds with 10% sodium hypochlorite for 5 minutes; finally, rinse the stylosanthes seeds with sterile water for 3 to 5 times, dry them and set aside;

[0054] S3: In the initiation process, the seeds were placed in a 50 ml centrifuge tube, 100 mg / L of initiation solution was added to immerse the seeds, the solid-liquid ratio was 1:5, and the seeds were placed in a 28°C shaker for 8 hours, and the shaker was set to 50 revolutions per minute. After the incubation, the suspension was poured out, the water was absorbed with absorbent paper, and the seeds were evenly spread on clean absorbent paper. The seeds were dried at room temperature for subsequent germination;

[0055] S4: Seed germination, the seeds after initiation in step S3 were germinated and cultured by the germination method on paper, and the seeds treated with 100 mg / L initiation solution were placed in a culture dish covered with 2 layers of filter paper, and 40 stylosanthes seeds were placed in each culture dish, and 10 mL of sterile water, 100 mmol / L and 200 mmol / L sodium chloride solutions were added respectively, and the culture conditions of no stress and two saline-alkali concentration stress were set, and five replicates were performed for each culture condition. It was placed in an artificial climate incubator and continued to be cultured for 7 days. The light time in the artificial incubator was 12 hours per day, the dark time was 12 hours, the temperature was 25 ° C, the filter paper was changed every 2 days, and 10 mL of sterile water and two different concentrations of sodium chloride solutions were supplemented after the filter paper was changed. The number of seeds germinated per day was recorded with the whitening as the germination standard.

[0056] Example 3: Determination of physiological indicators

[0057] After the plant samples obtained from the above experiment were rinsed with sterile water, the surface moisture was absorbed by filter paper and placed in a mortar, and liquid nitrogen was added for grinding. After grinding, 0.1 g of plant sample was weighed and homogenized in 1 mL of extract solution in an ice bath, and then the tissue homogenate was poured into a 2 mL centrifuge tube and centrifuged at 8000 g for 10 min at 4 ° C. The samples were tested for plant catalase, ascorbate peroxidase, glutathione peroxidase, and peroxidase activities using BC0205 CAT, BC0225 APX, BC1195 GSH, and BC0095 POD kits (Solarbio, Beijing, China) according to the kit instructions.

[0058] Comparative Example 1

[0059] This comparative example is used as a control group. The germination of salt-tolerant seeds of Stylosanthes in this comparative example specifically includes the following steps:

[0060] S1: Seed collection and processing, remove impurities and shrunken seeds from the stylosanthes seeds and set them aside, select the stylosanthes seeds with uniform size, full grains and uniform color, rinse the seeds with clean water and select the full seeds that sink, and use a constant temperature water bath at 80°C for 3 minutes to break the dormancy of the stylosanthes seeds; disinfect the seeds with 75% ethanol for 1 minute, rinse them with sterile water for 3 to 5 times, and then disinfect the seeds with 10% sodium hypochlorite for 5 minutes, rinse them with sterile water for 3 to 5 times, and dry them for later use;

[0061] S2: Seed germination, the seeds after initiation in step S1 were germinated and cultured by the paper germination method, the unprimed seeds were placed in a culture dish covered with 2 layers of filter paper, 40 stylosanthes seeds were placed in each culture dish, 10 mL of sterile water, 10 mL of 100 mmol / L and 200 mmol / L sodium chloride solutions were added respectively, and the culture conditions of no stress and two saline-alkali concentration stress were set, and five replicates were repeated for each culture condition. It was placed in an artificial climate incubator and continued to be cultured for 7 days. The light time in the artificial incubator was 12 hours per day, the dark time was 12 hours, the temperature was 25°C, the filter paper was changed every 2 days, and 10 mL of sterile water and two different concentrations of sodium chloride solutions were supplemented after the filter paper was changed. The number of seeds germinated per day was recorded with the whitening as the germination standard.

[0062] The germination rates and germination potentials of Examples 1 to 3 and Comparative Example 1 were calculated respectively, and the root length, seedling length, fresh weight, dry weight, and physiological indices of the uninduced treatment and the induced treatment with 100 mg / L nano zinc oxide solution were measured. The measurement results are shown in Tables 1 to 3.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067]

[0068] Table 3

[0069]

[0070] The experimental results show that, as shown in Table 1-3, the germination rate of the stylosanthes seeds after being triggered by the 100mg / L nano zinc oxide triggering solution for 8 hours was increased from 53.75% of the untriggered treatment to 82.50%, the germination potential of the stylosanthes seeds was increased from 45.00% of the untriggered treatment to 78.12%, and the root length was increased from 24.54mm of the untriggered treatment to 65.17mm. Under the stress of 200mmol / L sodium chloride solution, the germination rate of the stylosanthes seeds was increased from 18% of the untriggered treatment to 31%, the germination potential of the stylosanthes seeds was increased from 18.12% of the untriggered treatment to 31.25%, and the root length was increased from 4.43mm of the untriggered treatment to 10.55mm, which significantly improved the germination speed of the stylosanthes seeds under saline-alkali stress and alleviated the effect of salt damage on seedling growth.

[0071] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A priming solution for promoting the germination and growth of Stylosanthes seeds under saline-alkali stress, characterized in that: The initiating solution includes nano zinc oxide particles.

2. The initiating solution according to claim 1, characterized in that: The initiating liquid is a nano zinc oxide water dispersion, and the nano zinc oxide water dispersion includes the nano silver oxide particles; the purity of the nano silver oxide particles is 99.8%, and the particle size is 90nm.

3. The initiating solution according to claim 2, characterized in that: The preparation is configured according to the following method: An appropriate amount of the nano zinc oxide particles is weighed, and the nano zinc oxide particles are dispersed in deionized water by ultrasonic vibration.

4. The initiating solution according to claim 3, characterized in that: The concentration of the nano zinc oxide particles in the configured initiating solution is 100 mg / L.

5. A method for promoting seed germination and seedling growth of Stylosanthes japonica under saline-alkali stress, characterized in that: The following steps are involved: The steps of breaking dormancy, disinfecting, washing and drying are sequentially performed on the stylosanthes seeds; The stylosanthes seeds are placed in a centrifuge tube, and a triggering solution is added, wherein the triggering solution is the triggering solution according to any one of claims 1 to 3; and the centrifuge tube is placed in a shaking incubator for cultivation; After the incubation, pour out the suspension, use absorbent paper to absorb the water, spread it evenly on clean absorbent paper, and dry the Stylosanthes seeds at room temperature for subsequent germination; The initiated Stylosanthes seeds are germinated and cultured by using a paper germination method, a sodium chloride solution is added during the germination culture, and the seeds are placed in an artificial climate incubator for continued culture until germination.

6. The method for promoting germination and growth of Stylosanthes seeds under saline-alkali stress according to claim 5, characterized in that: The method of sequentially breaking dormancy, disinfecting, rinsing and drying the Stylosanthes seeds comprises the following steps: The seeds were bathed in a constant temperature water bath for 3 minutes to break the dormancy of the Stylosanthes seeds, and the water bath temperature was 80° C.; The stylosanthes seeds are disinfected with 75% ethanol for 1 minute; The stylosanthes seeds were rinsed with sterile water for 3 to 5 times, and then disinfected with 10% sodium hypochlorite for 5 minutes, and then rinsed with sterile water for 3 to 5 times and dried.

7. The method according to claim 5, characterized in that The "placing the centrifuge tube in a shaking table for culturing" comprises the following steps: The centrifuge tube was placed in the shaker in a dark environment, the shaker was set to 50 revolutions per minute, the culture temperature was 28° C., the culture time was 8 h, and the drying time was 48 h.

8. The method according to claim 5, characterized in that The method of germinating and culturing the primed Stylosanthes seeds by using the paper germination method comprises the following steps: The primed Stylosanthes seeds were placed in a culture dish covered with filter paper; the filter paper was 2 layers.

9. The method according to claim 5, characterized in that The concentration of the sodium chloride solution is 100 mmol / L or 200 mmol / L.

10. The method according to claim 5, characterized in that The artificial climate incubator has a culture time of 7 days, a light time of 12 hours and a dark time of 12 hours per day, and a temperature of 25°C.

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