KR20090081107A - Agent for detecting seed viability and a detection method of seed viability using the agent - Google Patents

Abstract

An agent for measuring seed vitality and measuring method using the same are provided to exactly examine the vitality of seed using a computer. A method for measuring the vitality of seed comprises: a step of mixing 50 to 150mug/mL of resazurin with 1 to 4mg/mL of yeast in distilled water; a step of dipping a seed in an agent for measuring seed vitality; and a step of determining the color change of the agent from blue to red; a step of measuring optical density and scanning with a multiplate reader; and a step of analyzing the color intensity of scan image.

Description

Seed vitality formulation and agent for measuring seed vitality {Agent for detecting seed viability and a detection method of seed viability using the agent}

The present invention relates to an agent for measuring seed vitality and a method for measuring seed vitality using the same. More specifically, the content of seed cells in a germinated seed when a seed is immersed in a solution of resazurin- yeast for measuring seed vitality and the solution ( Carbohydrates, amino acids, etc.) leak out, and yeast is activated, and the vitality of the seed is measured in detail by judging the degree of reduction of lesazurin by the color change of lesazurin by respiration of the activated yeast.

All seeds cannot maintain 100% germination rate because their physiological quality is not 100% uniform and also degrades during seed storage. If the seed's physiological quality can be accurately predicted by germinating and ungerminated seeds and abnormal seedlings producing abnormal seedlings, the seed industry does not evaluate seed products or do so by seeding in agriculture. This can be very useful because the condition of the seed can be known in advance.

In the conventional seed vitality test, the tetrazolium test is widely used in the world, which measures the enzyme activity of seed cells to test whether the cells are alive or dead and destroys the seed to tetrazolium. How to color. This method has a problem of destroying the seed and having a lot of experience and low accuracy to visually judge the color of the seed. In addition, there is an electric conductivity test method to test the vitality of the seed by testing the charge material leaking from the seed, which measures the electric conductivity of the leaked charge material by immersing many seeds at once without measuring the seed by one unit. It is a method used to compare the relative quality of seeds. In addition, by absorbing the water of the seeds using the sinapine (amino acid) or amino acid leak from the cruciferous vegetable seeds, coated with cellulose (dry) to dry the fluorescent color or amino acid of cinnapine in the cellulose layer ninhydrin (ninhydrin) There is a method to visually test the development of undeveloped seeds by color development. This is a non-destructive method that can be evaluated on a single seed basis, but the process is complex and difficult to accurately mechanically test.

Resazurin is an indicator of the redox state and when oxygen is lost and reduced, it changes from blue to red oxazin-resorufin. Lesazurin is also used as a pH indicator, blue when the pH is above 6.8 and red when below 5.3. Resazurin is used to test the quality of milk. If milk contains bacteria when lesazurin is added to the milk, the resazurin is reduced by the breathing of the bacteria and the color turns blue.

Seeds leak out through the cell membranes where carbohydrates, amino acids, proteins and minerals within the seeds are destroyed as the cell membranes break down and absorb moisture as the degeneration progresses. The degree of destruction of the cell membrane is proportional to the degree of degeneration of the seed, and therefore the amount of nutrients leaked from the seed is also proportional. However, normal seeds are less likely to break cell membranes, so they do not leak out of the seeds when they absorb moisture.

As a result of repeated studies focusing on these facts, the present inventors found that the nutrients leaked from the seeds activate the yeast, and as the yeast becomes vigorous, the respiration products reduce the lesazurin to induce a change in color. The present invention was able to confirm the state.

Accordingly, an object of the present invention is to provide a method for visually or mechanically measuring the vitality of seeds by a simple method in a short time using a solution in which yeast is mixed with lesazurin.

The object of the present invention is to prepare a resazurin-yeast solution, which is a formulation for measuring seed vitality, soaking the seeds in the solution to confirm that the color changes according to the vitality of the seed, and observed by visual observation or mechanical methods Achieved by establishing a way to do it.

The present invention is characterized in that it provides a formulation for measuring seed vitality which is composed of 50 to 150 μg / mL of resazurin and 1 to 4 mg / mL of yeast in distilled water. The seed vitality measurement agent is preferably used by mixing the resazurin concentration 50μg / mL, yeast concentration 4mg / mL.

The present invention is characterized by providing a seed vitality measuring method characterized in that by measuring the immersion of the seed in the formulation for measuring the seed vitality to determine whether the change of the color of the formulation from the blue to red over time.

In another aspect, the present invention is to put the formulation for measuring seed activity in a 96-well plate by immersing the seeds one by one to visually observe whether the color of the formulation changes from blue to red over time, using a multiplate reader It provides a method for measuring seed vitality, characterized in that by measuring the absorbance or after scanning the color intensity of the scanned image.

It is preferable to induce a color change in the 96 well plate containing the seed vitality measuring agent and the seed in a thermostat of 20 to 40 ℃, most preferably maintaining the temperature of the thermostat at 35 ℃ suitable for activation of yeast . The color change time depends on the size of the seeds or the amount of leakage, usually 2-4 hours is appropriate, especially 4 hours for cabbage seeds, 2 hours for seedless seeds.

Among the seed vitality measurement methods, the absorbance may be measured using a multi-plate reader, or the color intensity of a scanned image may be measured, and then analyzed more precisely using a mechanical device. The term “mechanical device” refers to a device such as a computer in which an analysis program is embedded.

The present invention relates to a method for measuring the vitality of a seed using a resazurin-yeast solution, by immersing the seed in the resazurin-yeast solution and observing the color of the resazurin solution from blue to red to visually increase the vitality of the seed. In addition to measuring the absorbance of lesazurin or scanning discolored containers, the images can be analyzed by computer to provide an accurate test of seed vitality. In addition, it is possible to measure the vitality of the seed in a short time without destroying the seed, which is a very useful invention for proving the quality of the seed in advance in the field of agriculture and agriculture, such as distribution and storage.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example  1: for seed vitality measurement Lesazurin Preparation of Yeast Solution

Lesazurin  And preparation of yeast

To prepare a resazurin-yeast solution for seed vitality, resazurin was purchased from Sigma, and the yeast was obtained from Sigcharomyces cerevisiae as a Sigma product (YSC2). , Bakers yeast, Type II) was purchased and used. The yeast can achieve the same results using any species.

Lesazurin Preparation of Mixed Solution of Yeast

Resazurin and yeast were mixed in distilled water to prepare a standard solution for seed vitality measurement. The concentration of resazurin in the solution was 50 μg / mL and the concentration of yeast was 4 mg / mL.

The concentration of lesazurin may be performed to be between 50 and 150 μg / mL, but the optimal concentration was 50 μg / mL. If the concentration of resazurin is higher than the optimal concentration, the reaction rate is slow, and if the concentration is too low, there is a disadvantage that it is difficult to discern the change in color. In addition, the concentration of yeast can be adjusted to be between 1 and 4 mg / mL, but the optimal concentration was 4 mg / mL. When the concentration of yeast is lower than the optimum concentration, the reaction is slow, and when the concentration is high, it is difficult to distinguish colors.

Example  2 : Lesazurin -Vitality Measurement of Cabbage Seed Using Yeast Standard Solution

The vitality of the seed was measured using the resazurin-yeast solution prepared in Example 1, as shown in FIG. 1.

Preparation of normal and artificial degenerative cabbage seeds

Normal Chinese cabbage seeds (Rice cabbage) was purchased from Nongwoo Bio Co., Ltd. as a test material. The normal cabbage seed having a germination rate of 100% was adjusted to 20% of the seed moisture content and placed at 45 ° C. for 3 days to prepare a degenerated cabbage seed by artificially degenerating so that the germination rate was 0%.

Visual inspection using Lesazurin-Yeast Standard Solution

Seeds leak contents from seeds depending on the degree of degeneration. Yeast is a microorganism that uses carbohydrates as a nutrient source. Therefore, as carbohydrates leak from seeds, yeast is activated and respiration is active. Later it turns from blue to red of various intensities, making it a suitable condition for visual or mechanical measurement of color. The vitality of the seed was measured as follows.

200-500 μL of the resazurin-yeast solution prepared in Example 1 was put in each well of a 96 well plate (96 well clear polyethylene microplate), and then 48 normal seeds and 48 degenerative seeds were mixed and placed into a well plate 35 The temperature of the solution was changed from blue to red or colorless as time went into the incubator was measured.

The amount of lesazurin-yeast to enter per well of a 96 well plate of the method was sufficient to observe the color change at 200-500 μL per seed. In addition, the temperature of the incubator for observation of the color change can be in the range of 20-40 ℃, but if the temperature is low, the reaction is very slow, even if too high, the reaction is too fast is not preferable. The color change time was different from seed to seed, but cabbage seeds were clearly visible by 4 hours.

The results of the experiment are shown in FIG. 2, and as can be seen from FIG. 2, normal seeds appear blue and degenerated seeds turn red, so that the seedlings of the resazurin-yeast solution may be discolored to accurately determine which seeds degenerate. there was.

Lesazurin -Using yeast standard solution Absorbance difference  According to

Seeds were taken from the 96 well plates and seeded into the well plate positions, and the 96 well plates were read by absorbance (570 nm) in a multiplate reader (Opsys MR, DYNEX Technology USA)). Absorbance was measured. The absorbances of the 96 well plates were read at a time using the corresponding software (Revelation Quick Link Software) on a computer and classified according to the absorbance (see FIG. 3). The germination test of the seed germination, fire germination and After investigating the abnormal germination seeds, the absorbance of each seed was compared and the relationship between the change of color of the resazurin solution and the germination of seeds was investigated.

Absorbance measurements of the discolored 96 well plate using a multiplate reader are shown in FIG. 3 and Table 1. FIG.

Difference in Absorbance Between Normal and Degenerated Seeds  Normal seed  Degenerative seeds  Discolored seeds (dog)   0   48  Colorless seeds (dog)   48   0  Absorbance (570nm) 2.379 ± 0.02 ^*  0.636 ± 0.205

*: Mean ± standard error

As shown in the above results, it was possible to test exactly which seed degenerated due to discoloration of the resazurin-yeast solution for normal seed and degenerated seed, and the absorbance of each seed was measured. A big difference was shown. Therefore, the absorbance measurement was able to confirm the vitality state of the seed accurately.

Example  3: Black, normal and abnormal germination of cabbage and radish seeds

Assay of Absorbance Using Resazurin-Yeast Standard Solution

Three varieties of cabbage ( Brassica oleracea L.) (dynamic, tara, red) and radish ( Raphanus) sativus L.) 2 varieties (Changbaek, R-347) were purchased from Nongshim Biotech Co., Ltd. and used as test materials. For each cultivar, induce discoloration using lesazurin-yeast solution, and sowing each seed into normal seed (normal germination seed), abnormal seed (seed that produces abnormal seedlings), and fire germination seed. Table 2 shows the absorbance of the corresponding lesazurin-yeast solution.

Seed vitality resazurin absorbance of three varieties of cabbage and two varieties of radish  Seed varieties  Normal seed  Abnormal seeds  Ungerminated seeds Dynamic Cabbage 1.960 ± 0.03 ^* 1.491 ± 0.06 0.657 ± 0.03 Tara cabbage 2.045 ± 0.02 1.571 ± 0.05 0.725 ± 0.02 Flushing Cabbage 2.060 ± 0.02 1.608 ± 0.04 1.036 ± 0.03 Albino 1.274 ± 0.03 0.997 ± 0.03 0.654 ± 0.03 R-347 1.566 ± 0.02 1.302 ± 0.05 0.869 ± 0.04

As shown in Table 2, there is a difference in absorbance according to the types of seeds, but the absorbance decreases in the order of normal seeds, abnormal seeds, and unspoiled seeds. It is related to the amount of water.

Based on the results, the results of Table 2 were inputted using an analytical computer program, and used as standard test values. The results of reanalysis based on the standard test values were able to quickly measure seed quality. In this way, one measure of absorbance in the same cultivar can then be used as a standard test for testing other seeds, which can be used to accurately and quickly determine the quality of the seed by a computer program.

Color Difference Using Lesazurin-Yeast Standard Solution

The images were also analyzed by scanning the colored plates, which are shown in FIG. 4. The image was exactly the same as the absorbance measurement result. Image analysis can also be performed using digital photographs as shown in FIG. 2 without necessarily scanning the colored 96 well plates. However, scanning from a scanner has the advantage of being simpler. Image analysis is performed by importing a scanned picture or photo onto a program from a computer using image analysis software (SigmaScan Pro 5.0, Systat Software, Inc. USA) and then depending on the color discrimination or color intensity of the image. You could calculate numbers or percentages right away.

Figure 1 is a simplified diagram of the process of measuring and analyzing the vitality of the seed using the solution of the resazurin- yeast mixture.

Figure 2 shows a photograph of a 96 well plate in which the resazurin-yeast solution is discolored according to the seed activity.

Figure 3 shows the results obtained by measuring the absorbance of the resazurin-yeast solution using a multi-plate reader 96 color plate discolored according to the seed activity.

4 is a scan image of the resazurin-yeast solution discolored according to seed vitality.

Claims (3)

Formulation for measuring seed vitality, characterized in that it is composed by mixing 50 ~ 150μg / mL resazurin (yeast) 1 ~ 4mg / mL in distilled water. Seed vitality measurement method characterized in that the immersion of the seed in the seed vitality measurement agent according to claim 1 to determine whether the color of the preparation changes from blue to red color over time. The method according to claim 2, wherein the seed vitality measuring agent is placed in a 96-well plate and the seeds are immersed one by one to visually observe whether the color of the agent changes from blue to red over time, and is a multiplate reader. Seed vitality measurement method characterized in that by measuring the absorbance using a measuring device or after scanning the color intensity of the scanned image.

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