CN110082248B - Method for determining use concentration of leaching liquor in biological assay of seed germination inhibitor and application of method - Google Patents
Method for determining use concentration of leaching liquor in biological assay of seed germination inhibitor and application of method Download PDFInfo
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- CN110082248B CN110082248B CN201910371707.6A CN201910371707A CN110082248B CN 110082248 B CN110082248 B CN 110082248B CN 201910371707 A CN201910371707 A CN 201910371707A CN 110082248 B CN110082248 B CN 110082248B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N5/02—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by absorbing or adsorbing components of a material and determining change of weight of the adsorbent, e.g. determining moisture content
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Abstract
The invention provides a method for determining the use concentration of a leaching solution in biological measurement of a seed germination inhibitor and application thereof, belonging to the technical field of seed biology. The invention relates to a method for determining the use concentration of a leaching solution in biological assay of a seed germination inhibitor, which comprises the following steps: measuring the dry weight value of the first seed, measuring the fresh weight value of the seed when the second seed absorbs water until the second seed absorbs water and is saturated, subtracting the dry weight value from the fresh weight value of the seed to obtain a difference value, and dividing the dry weight value by the difference value to obtain a ratio, namely the concentration of the leaching solution stock solution during the biological measurement of the seed germination inhibitor; the concentration of the leaching liquor stock solution is less than or equal to the concentration of the leaching liquor stock solution, namely the use concentration of the leaching liquor in the biological determination of the seed germination inhibitor; the second seed is the same kind, quality and quantity as the first seed. The utilization concentration of the leaching liquor determined by the method provided by the invention is utilized to carry out biological assay, and the state of inhibiting substance activity in seeds can be well reflected.
Description
Technical Field
The invention relates to the technical field of seed biology, in particular to a method for determining the use concentration of a leaching solution in biological assay of a seed germination inhibitor and application thereof.
Background
Seed dormancy refers to the phenomenon that viable seeds cannot germinate under appropriate environmental conditions (light, temperature, moisture, oxygen). Different species have different endogenous dormancy mechanisms. At present, three main reasons of seed dormancy are considered to be water absorption obstacle of seed coat (pericarp), embryo dormancy and germination inhibiting substances in seeds.
Plant seed germination inhibitors are one of the important causes of seed dormancy. The inhibiting substance is a substance capable of delaying or inhibiting germination of a seed of the same or different plant. Many plant seeds are proved to contain germination inhibiting substances which can inhibit the germination of plant seeds such as Chinese cabbage, lettuce and the like and even self embryo.
In the prior art, the concentration of seed leaching liquor adopted during inhibitor bioassay is different and has no uniform and proper standard, which influences the accuracy of judging the activity of the germination inhibiting substances in the seeds by using test results to a certain extent, thereby influencing the judgment of the relationship between the activity of the germination inhibiting substances of the seeds and the dormancy of the seeds.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for determining the concentration of leachate used in the bioassay of a seed germination inhibitor and applications thereof. The utilization concentration of the leaching liquor determined by the method provided by the invention is utilized to carry out biological assay, and the state of inhibiting substance activity in seeds can be well reflected.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for determining the use concentration of a leaching solution in biological assay of a seed germination inhibitor, which comprises the following steps:
(1) measuring the dry weight value of the first seed and the fresh weight value of the second seed when the second seed absorbs water until the water absorption is saturated;
(2) subtracting the dry weight value from the fresh weight value to obtain a difference value;
(3) dividing the dry weight value by the difference to obtain a ratio;
the ratio is the concentration of the leaching liquor stock solution in the biological determination of the seed germination inhibitor;
the using concentration of a leaching solution is less than or equal to the concentration of a raw solution of the leaching solution during the biological measurement of the seed germination inhibitor;
the second seed is the same in kind, weight and quantity as the first seed;
the concentration unit of the leaching liquor stock solution is g/ml.
Preferably, the number of the first seeds is more than or equal to 25.
Preferably, the number of times of the measurement is 3 or more.
Preferably, the fresh weight value of the second seed is measured once every 1.5-2.5 h when the second seed is saturated after water absorption.
Preferably, the fresh weight value is measured every 2 h.
Preferably, the concentration of the leaching solution stock solution diluted by 10 times is less than or equal to the using concentration of the leaching solution in the biological assay of the seed germination inhibitor.
Preferably, the concentration of the leaching solution stock solution diluted by 5 times is less than or equal to the using concentration of the leaching solution in the biological assay of the seed germination inhibitor.
Preferably, the concentration of the leaching solution stock solution diluted by 2 times is less than or equal to the using concentration of the leaching solution in the biological assay of the seed germination inhibitor.
The invention also provides application of the leaching liquor use concentration determined by the method in the technical scheme in reflecting the activity state of the substances in the seeds during the biological assay of the seed germination inhibitors.
Preferably, the seeds comprise fraxinus rhynchophylla seeds, pinus tabulaeformis seeds or syringa amurensis seeds.
The invention provides a method for determining the use concentration of a leaching solution in biological assay of a seed germination inhibitor. The method calculates the maximum concentration value of the seed leaching liquor when the seeds are subjected to inhibitor bioassay by measuring the dry weight of the first seeds and the fresh weight of the second seeds when the water absorption reaches saturation, and further determines the use concentration of the leaching liquor when the seed germination inhibitor bioassay is carried out. The use concentration of the leaching liquor determined by the method of the invention is used for biological measurement, and the state of inhibiting substance activity in seeds can be well reflected.
Detailed Description
The invention provides a method for determining the use concentration of a leaching solution in biological assay of a seed germination inhibitor, which comprises the following steps:
(1) measuring the dry weight value of the first seed and the fresh weight value of the second seed when the second seed absorbs water until the water absorption is saturated;
(2) subtracting the dry weight value from the fresh weight value to obtain a difference value;
(3) dividing the dry weight value by the difference to obtain a ratio;
the ratio is the concentration of the leaching liquor stock solution in the biological determination of the seed germination inhibitor;
the using concentration of a leaching solution is less than or equal to the concentration of a raw solution of the leaching solution during the biological measurement of the seed germination inhibitor;
the second seed is the same in kind, weight and quantity as the first seed;
the concentration unit of the leaching liquor stock solution is g/ml.
The method for determining the dry weight value of the first seed is not particularly limited in the present invention, and is preferably determined by a drying method, and the drying temperature is preferably 105 ℃. The number of the seeds to be measured is not particularly limited in the present invention, and is preferably 25 or more. The number of times of measuring the seed is not particularly limited in the present invention, and is preferably at least 3 times.
The method for determining the fresh weight value of the second seeds when the water absorption of the second seeds reaches saturation is not particularly limited, and after seeds are soaked in preferably clear water, the fresh weight value of the second seeds is measured every 1.5-2.5 hours; more preferably, the fresh weight of the second seed is measured every 2 h. In the present invention, the weight and amount of the first seed are determined to be the same as the second seed.
In the present invention, the concentration of the leaching solution used in the bioassay of the seed germination inhibitor is preferably equal to or more than the concentration of the leaching solution stock solution diluted by 10 times, more preferably the concentration of the leaching solution stock solution diluted by 5 times, and most preferably the concentration of the leaching solution stock solution diluted by 2 times.
In the invention, the leaching liquor is obtained by extracting different parts of seeds with water and a hydrophilic solvent to extract hydrophilic inhibiting substances; or extracting the water-insoluble inhibitor with organic reagent. And then treating the selected Chinese cabbage seeds with the leaching liquor for biological determination, and analyzing the germination rate data of the obtained seeds to determine whether the leaching liquor has inhibitory activity.
The determination of the use concentration of the seed germination inhibitor leaching liquor is obtained by calculating the fresh weight of the seeds when the water absorption of the seeds reaches saturation by utilizing the dry weight of the seeds.
The invention also provides application of the leaching liquor use concentration determined by the method in the technical scheme in reflecting the activity state of the substances in the seeds during the biological assay of the seed germination inhibitors. The present invention is not particularly limited in the kind of the seed, and preferably includes a Fraxinus rhynchophylla seed, an Pinus tabulaeformis seed or a Syringa amurensis seed.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Weighing 100 pieces of fraxinus rhynchophylla seeds, drying at 105 ℃, and measuring the dry weight to be 2.67 g; and weighing 100 pieces of the same mass of the fraxinus rhynchophylla seeds, soaking the seeds in clear water, and measuring the fresh weight of 7.50g when the seeds are saturated by water. Subtracting the dry weight value from the fresh weight value of the fraxinus rhynchophylla seeds to obtain a difference value of 4.83 g; the dry weight value was divided by the difference to give a ratio of 0.55.
According to the calculation result, the concentration of the fraxinus rhynchophylla seed leach liquor stock solution is determined to be 0.55g/ml, the concentration of the doubled solution obtained by concentrating the stock solution is 1.11g/ml, and the concentrations of the diluted solutions obtained by diluting the stock solution by 2.5 times and 5 times are respectively 0.22g/ml and 0.11 g/ml. Adding 3ml of each of the above 4 solutions into a culture dish filled with a layer of filter paper, adding 3ml of distilled water as control, placing 50 Chinese cabbage seeds in each culture dish, and repeating for 4 times. Placing the culture dishes with different treatments in a constant-temperature (25 ℃) illumination incubator for germination test, and measuring the germination rates of the seeds of the Chinese cabbages with different treatments after 24 hours, wherein the results of the germination test are shown in table 1.
TABLE 1 Effect of Fraxinus rhynchophylla seed leach solutions of different concentrations on cabbage seed germination
As can be seen from Table 1, when the concentration range of the fraxinus rhynchophylla seed leaching liquor is 0.11-0.55 g/ml, compared with the 92% germination rate of the Chinese cabbage seeds in the distilled water control group, the germination rate of the Chinese cabbage seeds is reduced, but partial seeds can germinate, and the germination rate reaches 18% -88%; but when the concentration of the leaching liquor exceeds 0.55g/ml of the leaching stock solution, namely the concentration reaches 1.11g/ml, the germination rate of the Chinese cabbage seeds is 0. Therefore, when the using concentration of the leaching liquor in the bioassay of the seed germination inhibitor is less than or equal to 0.55g/ml, the activity state of the germination inhibitor in the seeds can be well reflected; when the concentration of the leaching solution exceeds 0.55g/ml of the leaching stock solution, the activity state of the germination inhibiting substances in the seeds cannot be accurately reflected.
Example 2
Weighing 100 Chinese pine seeds, drying at 105 ℃, and measuring the dry weight to be 3.44 g; and weighing 100 Chinese pine seeds with the same mass, soaking the seeds in clear water, and measuring the fresh weight of 15.54g when the seeds are saturated by water. Subtracting the dry weight value from the fresh weight value of the Chinese pine seeds to obtain a difference value of 12.10 g; the dry weight value was divided by the difference to give a ratio of 0.28.
According to the calculation results, the concentration of the stock solution of the Chinese pine seed leaching liquor is determined to be 0.28g/ml, the concentration of the doubled solution obtained by concentrating the stock solution is 0.57g/ml, and the concentrations of the diluted solutions obtained by diluting the stock solution by 2.5 times and 5 times are respectively 0.11g/ml and 0.06 g/ml. Adding 3ml of each of the above 4 solutions into a culture dish filled with a layer of filter paper, adding 3ml of distilled water as control, placing 50 Chinese cabbage seeds in each culture dish, and repeating for 4 times. Placing the culture dishes with different treatments in a constant-temperature (25 ℃) illumination incubator for germination test, and measuring the germination rates of the seeds of the Chinese cabbages with different treatments after 24 hours, wherein the results of the germination test are shown in table 2.
TABLE 2 influence of extract of Chinese pine seeds of different concentrations on the germination percentage of Chinese cabbage seeds
As can be seen from Table 2, when the concentration of the Chinese pine seed leaching liquor is in the range of 0.06-0.28 g/ml, compared with the Chinese cabbage seed germination rate of 92% in the distilled water control group, the Chinese cabbage seed germination rate is reduced, but partial seeds can germinate, and the germination rate reaches 78% -97%; but when the concentration of the leaching liquor exceeds 0.28g/ml of the leaching stock solution, namely the concentration reaches 0.57g/ml, the germination rate of the Chinese cabbage seeds is 0. Therefore, when the using concentration of the leaching liquor in the bioassay of the seed germination inhibitor is less than or equal to 0.28g/ml, the activity state of the germination inhibitor in the seeds can be well reflected; when the concentration of the leaching solution exceeds 0.28g/ml of the leaching stock solution, the activity state of the germination inhibiting substances in the seeds cannot be accurately reflected.
Example 3
Weighing 100 Syringa amurensis seeds, drying at 105 ℃, and measuring the dry weight to be 3.14 g; 100 Syringa amurensis seeds with the same mass are weighed and soaked in clear water, and the fresh weight is measured to be 6.89g when the water absorption reaches saturation. Subtracting the dry weight value from the fresh weight value of the Syringa amurensis seeds to obtain a difference value of 3.75 g; the dry weight value was divided by the difference to give a ratio of 0.84.
According to the calculation results, the concentration of the stock solution of the Syringa amurensis seed leaching liquor is determined to be 0.84g/ml, the concentration of the doubled solution obtained by concentrating the stock solution is 1.67g/ml, and the concentrations of the diluted solutions obtained by diluting the stock solution by 2.5 times and 5 times are respectively 0.33g/ml and 0.17 g/ml. Adding 3ml of each of the above 4 solutions into a culture dish filled with a layer of filter paper, adding 3ml of distilled water as control, placing 50 Chinese cabbage seeds in each culture dish, and repeating for 4 times. Placing the culture dishes with different treatments in a constant-temperature (25 ℃) illumination incubator for germination test, and measuring the germination rates of the seeds of the Chinese cabbages with different treatments after 24 hours, wherein the results of the germination test are shown in a table 3.
TABLE 3 influence of Syringa amurensis seed leach liquors at different concentrations on the germination rate of cabbage seeds
As can be seen from Table 3, when the concentration range of the Syringa amurensis seed leaching liquor is 0.17-0.84 g/ml, compared with the 92% germination rate of the Chinese cabbage seeds in the distilled water control group, the germination rate of the Chinese cabbage seeds is reduced, but partial seeds can germinate, and the germination rate reaches 60% -88%; however, when the concentration of the leaching liquor exceeds 0.84g/ml of the leaching stock solution, namely the concentration reaches 1.67g/ml, the germination rate of the Chinese cabbage seeds is only 11 percent. Therefore, when the using concentration of the leaching liquor in the bioassay of the seed germination inhibitor is less than or equal to 0.84g/ml, the activity state of the germination inhibitor in the seeds can be well reflected; when the concentration of the leaching solution exceeds 0.84g/ml of the leaching stock solution, the activity state of the germination inhibiting substances in the seeds cannot be accurately reflected.
From the results of examples 1 to 3, it is understood that the state of the inhibitory substance activity in the seeds can be reflected well when the concentration of the leachate determined by the method of the present invention is used for bioassay.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for determining the use concentration of a leaching solution in bioassay of a seed germination inhibitor is characterized by comprising the following steps of: (1) measuring the dry weight value of the first seed and the fresh weight value of the second seed when the second seed absorbs water until the water absorption is saturated; (2) subtracting the dry weight value from the fresh weight value to obtain a difference value; (3) dividing the dry weight value by the difference to obtain a ratio; the ratio is the concentration of the leaching liquor stock solution in the biological determination of the seed germination inhibitor; the using concentration of a leaching solution is less than or equal to the concentration of a raw solution of the leaching solution during the biological measurement of the seed germination inhibitor; the second seed is the same in kind, weight and quantity as the first seed; the concentration unit of the leaching liquor stock solution is g/ml.
2. The method of claim 1, wherein the number of the first seeds is 25 or more.
3. The method according to claim 1, wherein the number of times of measurement is 3 or more.
4. The method according to claim 1, wherein the fresh weight value of the second seed is measured every 1.5-2.5 h during the period when the water absorption reaches saturation.
5. The method according to claim 4, wherein the fresh weight value is measured every 2 h.
6. The method of claim 1, wherein the concentration of said leachate stock solution diluted 10-fold is less than or equal to the concentration of leachate used in said bioassay of seed germination inhibitors.
7. The method of claim 1, wherein said leachate stock concentration is diluted 5-fold to a concentration that is less than or equal to the concentration of leachate used in said bioassay of seed germination inhibitors.
8. The method of claim 1, wherein the concentration of the leachate stock solution diluted 2-fold is less than or equal to the concentration of leachate used in the bioassay of the seed germination inhibitor.
9. Use of the extract in a bioassay of seed germination inhibitors determined according to any one of claims 1 to 8, in a concentration reflecting the activity status of an inhibitor in the seed.
10. The use of claim 9, wherein the seeds comprise fraxinus rhynchophylla seeds, pinus tabulaeformis seeds, or syringa amurensis seeds.
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