Asparagus seed viability determination method

Abstract

The invention provides a method for determining the viability of asparagus seeds, and belongs to the technical field of seed determination. The method comprises the steps of seed pretreatment and dyeing, the seed pretreatment comprises the following steps: randomly selecting asparagus seeds, removing surface impurities, soaking the asparagus seeds in an acetic acid solution with the mass being 5-7 times that of the asparagus seeds for 28-32 minutes, taking out the asparagus seeds after soaking is finished, putting the asparagus seeds into a soaking solution for soaking for 2.3-2.6 hours, taking out the asparagus seeds after soaking is finished, putting the asparagus seeds into a penetrating fluid for continuous soaking for 1.0-1.3 hours, taking out the asparagus seeds after soaking is finished, and taking out the asparagus seeds after soaking is finished. The umbilicus faces downwards, 1 / 3-1 / 2 of endosperm is transversely cut from bottom to top through a scalpel, the embryos are longitudinally cut, and pretreated seeds are obtained; the soaking solution is a mixture of deionized water and nonylphenol polyoxyethylene ether; the penetrating fluid is a mixture of deionized water, sodium dioctyl sulfosuccinate and laurocapram; the determination method provided by the invention has the advantages of short dyeing time and high accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of seed determination, and particularly relates to a method for determining the vitality of asparagus seeds. Background Art

[0002] Asparagus is a perennial vegetable that can be used as both food and medicine, belonging to the genus Asparagus of the Asparagaceae family. The edible tender stems have extremely high nutritional and medicinal value, and it enjoys the reputation of "the king of vegetables". It can be divided into white asparagus, green asparagus and purple asparagus according to its color. It is rich in vitamins, amino acids, proteins and mineral elements, and the content of these substances is much higher than that of other vegetables and fruits. "Compendium of Materia Medica" records that asparagus can be used as a traditional Chinese medicine, and "Dictionary of Chinese Medicine" records that the medicinal parts are tender stems and tubers, but the two have different effects. The active ingredients such as steroidal saponins, flavonoids, phenolic acids, polysaccharides contained in asparagus give it anti-tumor, blood pressure lowering, blood sugar lowering, blood lipid lowering, and immune regulation effects. In recent years, with the increase in international market demand, asparagus has been in short supply. Currently, my country's asparagus production and export volumes are both ranked first in the world.

[0003] The cultivation and reproduction of asparagus mainly relies on seeds. High-quality seeds are a prerequisite for producing high-quality and high-yield asparagus, which directly affects the standardized planting and cultivation of asparagus. Asparagus seeds themselves germinate slowly and unevenly, and are easily affected by storage time, storage conditions, and internal and external environmental stresses. They deteriorate, age, and their vitality decreases, failing to meet seed standards, causing great losses to production.

[0004] Seed viability refers to the potential ability of seeds to germinate or the vitality of the embryo. Seeds with high vigor can germinate quickly and uniformly, and can grow normally under harsh environmental conditions, thereby increasing the yield per unit area; therefore, it is crucial to screen seeds with high vigor before sowing.

[0005] At present, the detection methods of seed vitality include artificial accelerated aging method, 2,3,5-triphenyltetrazolium chloride (TTC) staining method, conductivity measurement, etc. In addition, with the development of spectral technology, spectral imaging technology has gradually become a rapid and non-destructive method for detecting seed vitality. However, the corresponding detection standards for spectral imaging technology have not yet been issued, and the cost of detection instruments and equipment is relatively high, which is not conducive to promotion; the artificial accelerated aging method has high reliability and small application limitations, but the test time is long and is affected by many factors. The moisture content of seeds, aging treatment time and the development of mold will affect the accuracy of the results; the conductivity measurement method can measure a large number of samples at the same time, but it requires specific equipment and operating skills, and the measurement results are greatly affected by the internal composition of the seeds and the external environment; the TTC detection method has become an important basis for judging seed vitality due to its advantages such as convenience, rapidity and sensitivity. Therefore, TTC staining is of great significance in the rapid detection of seed vitality.

[0006] The International Seed Testing Association and China's Crop Inspection Procedures recommend that TTC staining be included as one of the methods for determining seed viability. TTC staining is easily restricted by seed type and staining conditions, and there are great differences in the conditions for TTC staining of seeds of different crops. Therefore, it is necessary to establish a corresponding set of TTC staining techniques for different crops.

[0007] At present, there is no report on the research of asparagus seed viability determination method; If you want to understand the vitality of asparagus seeds, you can only do it through a germination test; however, in actual production, asparagus seeds have problems such as slow germination, long germination cycle, and uneven seedlings. Some seeds with low vitality take up to 25 days to germinate, which greatly extends the detection period. In addition, the germination test is also affected by the germination bed, germination temperature, moisture, light and other environmental factors. If the environmental conditions are not suitable, the test results will also be biased and cannot accurately reflect the vitality of the seeds.

[0008] Therefore, providing a method for determining the viability of asparagus seeds by TTC staining technology, which can effectively shorten the determination period and improve the accuracy of the determination results, is a technical problem that needs to be solved urgently in the prior art. Summary of the invention

[0009] In order to solve the technical problems existing in the prior art, the present invention provides a method for determining the vitality of asparagus seeds, which is determined by TTC staining technology, has a short determination cycle and high accuracy.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for determining the vitality of asparagus seeds, comprising seed pretreatment and dyeing steps, specifically as follows: 1. Seed pretreatment After randomly selecting asparagus seeds and removing surface impurities, the seeds are placed in a 5-7 times mass acetic acid solution for soaking for 28-32 minutes, taken out after the soaking, and immersed in a soaking liquid for soaking for 2.3-2.6 hours, taken out after the soaking, and immersed in a permeate for further soaking for 1.0-1.3 hours, and taken out after the soaking, with the hilum facing downward, and 1 / 3-1 / 2 of the endosperm is cut horizontally from bottom to top with a scalpel, and the embryo is cut longitudinally to obtain pretreated seeds; The mass concentration of the acetic acid solution is 0.3-0.5%; The soaking liquid is a mixture of deionized water and nonylphenol polyoxyethylene ether, and the mass ratio of the deionized water to nonylphenol polyoxyethylene ether is 100:3.0-3.5; The permeate is a mixture of deionized water, dioctyl sodium sulfosuccinate and laurocapram, and the mass ratio of the deionized water, dioctyl sodium sulfosuccinate and laurocapram is 100:3.8-4.2:2.5-3.0.

[0011] 2. Staining The pretreated seeds are placed in a light-proof container, 4-6 times the mass of dyeing solution is added, the temperature is controlled to be 35-40°C for dyeing, and the dyeing time is 0.5-1.0h. After the dyeing is completed, the seeds are taken out and rinsed with clean water until the rinse solution is colorless to obtain dyed seeds. After the dyeing, the seed embryos with a dyeing area of ​​more than 2 / 3 and no tissue damage are observed to be active dyeing seeds, otherwise they are inactive seeds; The dyeing solution is composed of a tetrazolium solution and a dyeing auxiliary, and the mass ratio of the tetrazolium solution to the dyeing auxiliary is 10.3-10.7:1.1-1.4; The preparation method of the tetrazolium solution comprises the following steps: adding potassium dihydrogen phosphate to deionized water, stirring evenly, and obtaining a mother solution 1; adding disodium hydrogen phosphate to deionized water, stirring evenly, and obtaining a mother solution 2; mixing the mother solution 1 with the mother solution 2, stirring evenly, and obtaining a phosphate buffer solution; placing 2,3,5-triphenyl tetrazolium chloride in the phosphate buffer solution, stirring evenly, and obtaining a tetrazolium solution; In the mother solution 1, the mass ratio of the deionized water to potassium dihydrogen phosphate is 1000:8.8-9.2; In the second mother solution, the mass ratio of the deionized water to disodium hydrogen phosphate is 1000:9.2-9.6; The volume ratio of the mother liquor 1 to the mother liquor 2 is 1:1.2-1.8; The mass concentration of the tetrazolium solution is 0.5-1.0%; The dyeing auxiliary agent is composed of deionized water, zinc citrate and Tween 80, and the mass ratio of the deionized water, zinc citrate and Tween 80 is 110:0.25-0.30:0.7-0.8.

[0012] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention adopts TCC dyeing to measure the vitality of asparagus seeds. First, the asparagus seeds are pretreated. Specifically, the asparagus seeds are first placed in an acetic acid solution. On the one hand, the acetic acid can remove impurities and attachments on the surface of the seeds and improve the cleanliness of the seeds; on the other hand, the seed coat can be softened, which is helpful for the penetration of the dyeing liquid during the dyeing process, thereby improving the accuracy of the dyeing result; then, the asparagus seeds are soaked in a soaking liquid. The nonylphenol polyoxyethylene ether in the soaking liquid dissolves and disperses the wax components on the surface of the seeds, accelerates the water absorption performance of the seeds, and then combined with the permeation liquid, it can reduce the water surface The surface tension makes it easier for water to penetrate into the seeds, improving the penetration capacity of the dyeing solution. Dioctyl sodium sulfosuccinate has good dispersibility and wetting properties, which helps the dyeing solution to be evenly distributed on the surface and inside of the seeds, improves the uniformity and accuracy of dyeing, and shortens the dyeing time. In the dyeing step, the dyeing auxiliary can effectively improve the compatibility with the tetrazolium solution, help to fully contact and react with living cells, accelerate the respiration of the seeds, further shorten the dyeing time, and ensure the accuracy of the measurement results. The measured seed vitality is close to the germination rate, and the measurement results are accurate and effective. 2. The method of the present invention is used to determine the vitality of asparagus seeds. The dyeing time is short, only 0.5-1.0h, the germination rate of asparagus seeds is 90.7%, and the vitality is 94.3-96.7%. DETAILED DESCRIPTION

[0013] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0014] Example 1 1. Seed pretreatment After randomly selecting asparagus seeds to remove surface impurities, the seeds were placed in a 6-fold mass 0.4wt% acetic acid solution for soaking for 30 min, taken out after the soaking, and immersed in the soaking liquid for soaking for 2.5 h. After the soaking, the seeds were taken out and immersed in the permeate for further soaking for 1.2 h. After the soaking, the seeds were taken out with the hilum facing downwards, and 2 / 5 of the endosperm was cut horizontally from bottom to top with a scalpel, and the embryo was cut longitudinally to obtain pretreated seeds; The soaking liquid is a mixture of deionized water and nonylphenol polyoxyethylene ether, and the mass ratio of the deionized water to nonylphenol polyoxyethylene ether is 100:3.2; The permeate is a mixture of deionized water, dioctyl sodium sulfosuccinate and laurocapram, and the mass ratio of the deionized water, dioctyl sodium sulfosuccinate and laurocapram is 100:4.0:2.7.

[0015] 2. Staining The pretreated seeds were placed in a light-proof container, 5 times the mass of dyeing solution was added, the temperature was controlled to be 40°C for dyeing, and the dyeing time was 0.5h. After the dyeing was completed, the seeds were taken out and rinsed with clean water until the rinse solution was colorless to obtain the dyed seeds. After the dyeing, the seed embryos with a dyeing area of ​​more than 2 / 3 and no tissue damage were observed to be active dyeing seeds, otherwise they were inactive seeds; The dyeing solution is composed of a tetrazolium solution and a dyeing auxiliary, and the mass ratio of the tetrazolium solution to the dyeing auxiliary is 10.5:1.3; The preparation method of the tetrazole solution is as follows: add 9.078g of potassium dihydrogen phosphate to 1000g of deionized water, stir evenly, and obtain a mother solution 1; add 9.472g of disodium hydrogen phosphate to 1000g of deionized water, stir evenly, and obtain a mother solution 2; mix the mother solution 1 and the mother solution 2 at a volume ratio of 1:1.5, stir evenly, and obtain a phosphate buffer solution; place 2,3,5-triphenyl tetrazolium chloride in the phosphate buffer solution, stir evenly, and obtain a tetrazole solution with a mass concentration of 0.5%; The dyeing auxiliary agent is composed of deionized water, zinc citrate and Tween 80, and the mass ratio of the deionized water, zinc citrate and Tween 80 is 110:0.27:0.8.

[0016] Example 2 1. Seed pretreatment After randomly selecting asparagus seeds to remove surface impurities, the seeds were placed in a 0.3wt% acetic acid solution of 5 times their mass for immersion for 28 minutes. After the immersion, the seeds were taken out and immersed in the immersion solution for 2.3 hours. After the immersion, the seeds were taken out and immersed in the permeate for 1.0 hours. After the immersion, the seeds were taken out with the hilum facing downwards, and 1 / 3 of the endosperm was cut horizontally from bottom to top with a scalpel, and the embryo was cut vertically to obtain pretreated seeds. The soaking liquid is a mixture of deionized water and nonylphenol polyoxyethylene ether, and the mass ratio of the deionized water to nonylphenol polyoxyethylene ether is 100:3.0; The permeate is a mixture of deionized water, dioctyl sodium sulfosuccinate and laurocapram, and the mass ratio of the deionized water, dioctyl sodium sulfosuccinate and laurocapram is 100:3.8:3.0.

[0017] 2. Staining The pretreated seeds were placed in a light-proof container, 4 times the mass of dyeing solution was added, the temperature was controlled to be 35°C for dyeing, and the dyeing time was 1.0h. After the dyeing was completed, the seeds were taken out and rinsed with clean water until the rinse solution was colorless to obtain the dyed seeds. After the dyeing, the seed embryos with a dyeing area of ​​more than 2 / 3 and no tissue damage were observed to be active dyeing seeds, otherwise they were inactive seeds; The dyeing solution is composed of a tetrazolium solution and a dyeing auxiliary, and the mass ratio of the tetrazolium solution to the dyeing auxiliary is 10.3:1.1; The preparation method of the tetrazole solution is as follows: add 8.8g of potassium dihydrogen phosphate to 1000g of deionized water, stir evenly, and obtain mother solution 1; add 9.2g of disodium hydrogen phosphate to 1000g of deionized water, stir evenly, and obtain mother solution 2; mix the mother solution 1 and the mother solution 2 at a volume ratio of 1:1.2, stir evenly, and obtain a phosphate buffer solution; place 2,3,5-triphenyl tetrazolium chloride in the phosphate buffer solution, stir evenly, and obtain a tetrazole solution with a mass concentration of 1.0%; The dyeing auxiliary agent is composed of deionized water, zinc citrate and Tween 80, and the mass ratio of the deionized water, zinc citrate and Tween 80 is 110:0.25:0.7.

[0018] Example 3 1. Seed pretreatment After removing surface impurities, randomly selected asparagus seeds were placed in a 7-fold mass 0.5wt% acetic acid solution for soaking for 32 minutes. After soaking, the seeds were taken out and immersed in the soaking solution for soaking for 2.6 hours. After soaking, the seeds were taken out and immersed in the permeate for soaking for 1.3 hours. After soaking, the seeds were taken out with the hilum facing downwards, and 1 / 2 of the endosperm was cut horizontally from bottom to top with a scalpel, and the embryo was cut longitudinally to obtain pretreated seeds. The soaking liquid is a mixture of deionized water and nonylphenol polyoxyethylene ether, and the mass ratio of the deionized water to nonylphenol polyoxyethylene ether is 100:3.5; The permeate is a mixture of deionized water, dioctyl sodium sulfosuccinate and laurocapram, and the mass ratio of the deionized water, dioctyl sodium sulfosuccinate and laurocapram is 100:4.2:2.5.

[0019] 2. Staining The pretreated seeds were placed in a light-proof container, 6 times the mass of dyeing solution was added, the temperature was controlled to be 38°C for dyeing, and the dyeing time was 1.0h. After the dyeing was completed, the seeds were taken out and rinsed with clean water until the rinse solution was colorless to obtain the dyed seeds. After the dyeing, the seed embryos with a dyeing area of ​​more than 2 / 3 and no tissue damage were observed to be active dyeing seeds, otherwise they were inactive seeds; The dyeing solution is composed of a tetrazolium solution and a dyeing auxiliary, and the mass ratio of the tetrazolium solution to the dyeing auxiliary is 10.7:1.4; The preparation method of the tetrazole solution is as follows: add 9.2g of potassium dihydrogen phosphate to 1000g of deionized water, stir evenly, and obtain mother solution 1; add 9.6g of disodium hydrogen phosphate to 1000g of deionized water, stir evenly, and obtain mother solution 2; mix the mother solution 1 and the mother solution 2 at a volume ratio of 1:1.8, stir evenly, and obtain a phosphate buffer solution; place 2,3,5-triphenyl tetrazolium chloride in the phosphate buffer solution, stir evenly, and obtain a tetrazole solution with a mass concentration of 0.8%; The dyeing auxiliary agent is composed of deionized water, zinc citrate and Tween 80, and the mass ratio of the deionized water, zinc citrate and Tween 80 is 110:0.30:0.75.

[0020] Comparative Example 1 Based on Example 1, the changes are as follows: In the pretreatment step, the soaking solution and the permeate were replaced with deionized water in equal amounts; The rest of the operations are the same.

[0021] Comparative Example 2 Based on Example 1, the changes are as follows: In the staining step, the dyeing auxiliary agent in the staining solution is replaced with an equal amount of tetrazolium solution; and the staining temperature is changed to 37°C; The rest of the operations are the same.

[0022] Determination experiment 1. Germination rate test 100 seeds of the asparagus variety "Champion" were selected and placed in a culture dish with 2 layers of moist filter paper. The experiment was repeated three times. The daily germination light time was controlled to be 12.0 h, the temperature during light was 25°C, the dark time was 12.0 h, the temperature during dark was 25°C, the experiment lasted for 20 days, and the germination rate was 90.7%.

[0023] 2. Seed Viability Test 500 seeds of the asparagus variety "Champion" were selected and divided into 5 groups, each with 100 seeds, denoted as AE. Then, the seed viability was measured according to the methods of Examples 1-3 and Comparative Examples 1-2, and 3 repeated tests were set to calculate the seed viability; wherein the viability = number of seeds with vitality staining / 100×100%; The test results are as follows:

[0024] The invention adopts TCC dyeing to measure the vitality of asparagus seeds. The asparagus seeds are pretreated first, specifically, the asparagus seeds are first placed in an acetic acid solution. On the one hand, the acetic acid can remove impurities and attachments on the surface of the seeds, thereby improving the cleanliness of the seeds; on the other hand, the seed coat can be softened, which is helpful for the penetration of the dyeing liquid during the dyeing process, thereby improving the accuracy of the dyeing result; then, the asparagus seeds are soaked in a soaking liquid, and the nonylphenol polyoxyethylene ether in the soaking liquid dissolves and disperses the wax components on the surface of the seeds, thereby accelerating the water absorption performance of the seeds, and then combined with the permeation liquid, the water surface can be reduced. Tension makes it easier for water to penetrate into the seeds, improving the penetration capacity of the dyeing solution, and dioctyl sodium sulfosuccinate has good dispersibility and wetting properties, which helps to evenly distribute the dyeing solution on the surface and inside of the seeds, improves the uniformity and accuracy of dyeing, and shortens the dyeing time; in the dyeing step, the dyeing auxiliary can effectively improve the compatibility with the tetrazolium solution, helps to fully contact and react with living cells, accelerates the respiration of the seeds, further shortens the dyeing time, ensures the accuracy of the measurement results, and the measured seed vitality is close to the germination rate, and the measurement results are accurate and effective.

[0025] In the pretreatment step of Comparative Example 1, the soaking liquid and the permeate were replaced with deionized water in equal amounts. The surface tension of water was high, and the surface of the seeds contained more wax components, so it was difficult for water to penetrate into the seeds. The seeds had poor water absorption capacity, which also reduced the permeability of the dyeing liquid to the seeds, making it difficult for the dyeing liquid to dye the seeds, resulting in poor uniformity of dyeing and inaccurate measurement results. In comparative example 2, in the dyeing step, the dyeing auxiliary agent is replaced with tetrazolium solution, which has poor contact with the living cells inside the seeds and low reactivity, and the respiration of the seeds is slow, which affects the determination of the vitality of the seeds. In addition, comparative example 2 also reduces the dyeing temperature, which affects the reaction between the dehydrogenase of the active seeds and the tetrazolium solution during the dyeing process. The final measured seed vitality and germination rate results are quite different, and the accuracy of the measurement results is low.

[0026] Unless otherwise specified, all ratios and percentages described in the present invention are by mass ratios and percentages are by mass percentages.

[0027] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the vitality of asparagus seeds, characterized in that: It includes seed pretreatment and dyeing steps; The seed pretreatment step comprises soaking the asparagus seeds in an acetic acid solution for 28-32 minutes, then soaking them in a soaking solution for 2.3-2.6 hours, and then soaking them in a permeation solution for 1.0-1.3 hours, taking them out, with the hilum facing downward, and cutting 1 / 3-1 / 2 of the endosperm from bottom to top with a scalpel, and cutting the embryo longitudinally to obtain pretreated seeds; The soaking liquid is a mixture of deionized water and nonylphenol polyoxyethylene ether; the permeation liquid is a mixture of deionized water, dioctyl sodium sulfosuccinate and laurocapram; The dyeing step comprises placing the pretreated seeds in a light-proof container, adding a quality dyeing solution, controlling the temperature to be 35-40° C. for dyeing, and dyeing for 0.5-1.0 h. After the dyeing is completed, observing that the seed embryo dyeing area after dyeing is more than 2 / 3 and there is no tissue damage, which is a vitality-dyed seed, otherwise it is a non-vitality seed; The dyeing solution is composed of a tetrazolium solution and a dyeing auxiliary agent; The mass concentration of the tetrazolium solution is 0.5-1.0%; The dyeing auxiliary agent consists of deionized water, zinc citrate and Tween 80.

2. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: In the seed pretreatment step, the mass concentration of the acetic acid solution is 0.3-0.5%; The mass ratio of the asparagus seeds to the acetic acid solution is 1:5-7.

3. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: In the seed pretreatment step, in the soaking solution, the mass ratio of the deionized water to nonylphenol polyoxyethylene ether is 100:3.0-3.

5.

4. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: In the seed pretreatment step, in the permeate, the mass ratio of the deionized water, dioctyl sodium sulfosuccinate and laurocapram is 100:3.8-4.2:2.5-3.

0.

5. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: In the dyeing step, the mass ratio of the pretreated seeds to the dyeing solution is 1:4-6; In the dyeing solution, the mass ratio of the tetrazolium solution to the dyeing auxiliary agent is 10.3-10.7:1.1-1.

4.

6. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: The preparation method of the tetrazole solution comprises the following steps: adding potassium dihydrogen phosphate to deionized water, stirring evenly, and obtaining a mother solution one; adding disodium hydrogen phosphate to deionized water, stirring evenly, and obtaining a mother solution two; mixing the mother solution one with the mother solution two, stirring evenly, and obtaining a phosphate buffer solution; placing 2,3,5-triphenyl tetrazolium chloride in the phosphate buffer solution, stirring evenly, and obtaining a tetrazole solution.

7. A method for determining the vitality of asparagus seeds according to claim 6, characterized in that: In the mother solution 1, the mass ratio of the deionized water to potassium dihydrogen phosphate is 1000:8.8-9.2; In the second mother solution, the mass ratio of the deionized water to disodium hydrogen phosphate is 1000:9.2-9.6; The volume ratio of the mother liquor one to the mother liquor two is 1:1.2-1.

8.

8. The method for determining the vitality of asparagus seeds according to claim 1, characterized in that: In the dyeing step, in the dyeing auxiliary agent, the mass ratio of the deionized water, zinc citrate and Tween 80 is 110:0.25-0.30:0.7-0.8.

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