KR101754830B1 - A method for predicting germination of seeds by color development of phosphorus from seed leachate - Google Patents

Abstract

The present invention relates to a method for predicting seed germination rate using phosphorus (P) coloring of a seed leaching solution, and provides a method of accurately predicting the seed germination power in a short time without destroying the seed by visual observation or by using a mechanical absorbance measurement There is an excellent effect.

Description

[0001] The present invention relates to a method for predicting seed germination using seedling leaching solution,

The present invention relates to a novel method for predicting seed germination rate by confirming a phosphorus (P) color reaction using a seed leaching solution.

Seeds are not 100% homogeneous in their physiological quality and can not maintain germination rate at 100% because they degrade during seed storage. As the seeds are degraded and cell membranes are destroyed and water is absorbed, the carbohydrates, amino acids, proteins and minerals in the seeds leaks out through the destroyed cell membranes. The degree of destruction of the cell membrane is also proportional to the degree of degeneration of the seed, and the amount of the cellular component that leaks from the seed is proportional to the amount. Normal seeds, on the other hand, are either sound or less destructive to the cell membrane, and even if they absorb water, they do not emit any leaks from the seeds or leak very finely. Therefore, if we can accurately predict the physiological quality of seeds, it would be very useful because we can know the status of seeds without evaluating seed products in seed industry or conducting germination tests in agriculture.

The tetrazolium test is commonly used worldwide for seed activation testing, which measures the enzymatic activity of seed cells to test whether the cells are alive or dead. In this method, seeds are destroyed and developed with tetrazolium. Thus, there is a problem that seeds are destroyed and the color of the seeds is visually inspected, requiring a lot of experience and low accuracy. An electrical conductivity test is a method of testing the potential of seeds by examining the charge substances that leak from the seeds. The electrical conductivity of the leaked charge substances is measured by immersing the seeds in large amounts at one time. Thus, this method is the method used to compare the relative quality of seeds. Alternatively, seeds may be coated with cellulose by absorbing water using a fluorescent substance, such as sinapine or amino acid, exposed to cruciferous vegetables and seeds, and then dried. Thus, the fluorescence of cinnamin in the cellulose layer, There is a method of visually checking that coloring of the adsorbed amino acid causes coloring of noxious sub-species. This method is nondestructive and can be evaluated on a single seed basis, but the process is complicated and difficult to accurately test mechanically.

As a method for measuring seed viability by immersing a seed in a resazurin-yeast solution, the present inventors have disclosed a method for predicting seed germination treated with a ninhydrin reagent disclosed in Korean Patent Laid-Open No. 10-2009-0081107 Have been disclosed by the present inventor in Korean Patent Application No. 10-2012-0071915.

Accordingly, an object of the present invention is to provide a method for quickly and easily measuring and predicting accurate seed germination by coloring phosphorus (P) components contained in a seed leach solution.

The above object of the present invention is achieved by a method for producing a seed culture, comprising the steps of: preparing a seed leaching solution by soaking the seed; Treating the seed leaching solution with phosphorus (P) coloring reagent; Determining the seed germination rate of the leach solution visually using phosphorus (P) color development; Seed germination rate was achieved by measuring absorbance or color intensity of phosphorus (P) color.

The present invention not only visually predicts the vitality of the seed using the phosphorus (P) color of the seed leach solution, but also statistically analyzes the probability of seed germination ability by measuring the degree of phosphorus (P) There is an excellent effect of predicting the germination of seeds.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing phosphorus (P) color photographs (A) and absorbance (B) and germination rate (C) of degradation time of Chinese cabbage seed extract according to the present invention.
FIG. 2 is a graph showing the seed germination behavior of the Chinese cabbage seed extract according to the absorbance interval after phosphor (P) development.
FIG. 3 is a graph showing the seed germination pattern of the seedless leach solution according to the absorbance interval after phosphor (P) coloring according to the present invention.
4 is a photographic view showing phosphor (P) color development of a Chinese cabbage seed leaching solution according to the present invention.

The present invention provides a method of predicting seed germinability by coloring a phosphorus (P) component contained in a seed leach solution.

A preferred seed type of the present invention is all the degraded seeds that leach phosphorus and other species of Brassicaceae vegetable such as radish and cabbage.

According to the present invention, one cabbage and one seedless seed are placed in a 96-well plate, and 100 μL of distilled water and 150 μL of distilled water are immersed in the cabbage seeds. Depending on the size of the seeds, it is necessary to add distilled water to the seed so that the seeds are sufficiently locked. It is important that the immersion time is the time for the seed to fully absorb the moisture, and the immersion time for the Chinese cabbage seed or the seedless is suitable for 3 to 4 hours.

The phosphorus (P) coloring reagent is ① sulfuric acid (Merck & Co., Germany), ② 25% ammonium molybdenate (Samseon Pure Chemical Industries, Korea), ③ 0.1M ascorbic acid , ④ reagent of tartaric antimonyl kalium (1 mg antimony (Sb) / mL, Sigma-Aldrich, USA), and the volume ratio of ①: ②: ③: ④ = 10: 3: 6: 1 .

In the case of Chinese cabbage and seedless seeds, 40 μL of phosphorus (P) coloring reagent is required per seed, so the amount of reagent should be adjusted according to the number of seeds. 50 μL of the leached solution of the seeds is immersed in a new 96-well plate and 40 μL of phosphorus (P) coloring reagent is added to produce a blue color according to the concentration of phosphorus (P) component .

A specific temperature for color development is not required, and the color is developed at room temperature, and coloring is completed within 5 minutes after addition of the coloring reagent. When seeds other than Chinese cabbage and non-seeds are used, it is important to find the best coloring conditions such as immersion time, amount of immersion distilled water, amount of seed leaching solution and amount of phosphorus (P) coloring agent depending on seed size.

Among the seed vitality measurement methods, the absorbance can be measured using a multiplate reader or the color intensity of the developed image can be measured and then analyzed more precisely using a mechanical device. The term 'mechanical device' means a device such as a computer having an analysis program embedded therein.

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

Example : Phosphorus (P) component of cabbage seed leaching solution Color

Chinese cabbage seeds were subjected to artificial degeneration treatment at 0 hours, 24 hours, 48 hours, and 72 hours by Deamamplus (Dongbu Hanon, 2010) to artificially deteriorate seed quality. Degradation treatment conditions were as follows: Seed moisture content was adjusted to 20%, vacuum packed, and then treated in a constant temperature water bath at 45 ° C for several hours. One hundred and sixty (40 × 4 repeats) of each treated seeds were placed in 96-well plates and immersed in 100 μL of distilled water for 3 hours. After immersion, seed leachate (50 uL) was added to each seed and mixed with 40 uL of phosphorus (P) color reagent.

As a result of the experiment, the color was immediately developed as shown in Fig. 1A according to the degree of seed degeneration. As shown in FIG. 1B, the average of the absorbance of the leached liquid of each seed was higher with longer seed degradation time, and the linear regression coefficient was highly correlated with r ² = 0.84. The longer the seed regeneration period, the lower the germination rate, and the linear regression coefficient was highly correlated with r ² = 0.96 (FIG. 1C).

Therefore, it was found that the higher the phosphorus (P) color of the seed leach (the higher the absorbance), the lower the germination rate. Based on the above results, it was possible to visually observe the degree of color development of the image (FIG. A) in which the 96 well plate in which the leachate was developed was scanned to predict the seed quality. The absorbance was measured at 630 nm using a multiplate reader (Opsys MR, Dynex Technology, Chantilly, VA, USA), and germination was performed by seeding the seeds corresponding to each absorbance.

Experimental Example  1: phosphorus (P) content of cabbage seed leaching solution Color  Relationship between absorbance and germination

Cabbage ( B. rapa ssp. 5 varieties were cultivated in the spring (1995, Heungnong), and spring (1995, Heungin) in the winter. It was used as a degenerate state. Each seed of 160 seeds (40 × 4 repeats per repetition) was placed in a 96-well plate and immersed in 100 μL of distilled water for 3 hours. 50 μL of each seed leach was taken and mixed with 40 μL of phosphorus (P) color reagent . After measuring the absorbance with a multiplate reader, each seed was sown and germination test was performed. That is, a 630 nm absorbance filter was attached to a multiplate reader, and a 96-well plate was inserted to examine the absorbance. Eight holes in the 96-well plate were blanks, but seeds were not added and only the phosphorus (P) coloring reagent was added. The 80 samples of the seeds immersed in the seeds were analyzed by the corresponding software (Revelation Quick Link Software) , And classified according to the absorbance.

In order to clarify the relationship between the germination and absorbance values of seeds, the absorbance was divided into five stages of 0.000-0.100, 0.101-0.200, 0.201-0.300, 0.301-0.400, and 0.401 at 0.100 intervals, and the seed germination morphology Respectively. The germination of the seeds belonging to each absorbance range was classified into normal grains, abnormal graves and non-grains, and the results are shown in FIG.

As a result, the ratio of normal seedlings was highest at lower absorbance of 0.100 or less, and the ratio of normal seedlings was lower as the absorbance value was higher. Thus, the absorbance value and the normal seedling ratio were statistically highly correlated (r ² = 0.98). In addition, the seeds of unburnt seeds increased proportionally with higher absorbance values, and almost all seeds were found to be unfertilized seeds at> 0.401.

Experimental Example  2: phosphorus (P) of seedless leach solution Color  Relationship between absorbance and germination

Raphanus (1995, Heungnong), sputum radish (2011, rice paddy ), seeds (Kangwon, Korea) One seed of 160 seeds (40 replicates per replicate) per each cultivar was placed in a 96-well plate and immersed in 150 μL of distilled water for 3 hours, and then 50 μL of each seed leach was taken. (P) were mixed with 40 uL of chromogenic reagent. After measuring absorbance with a multiplate reader, each seed was sown and germination test was performed.

In order to investigate the relationship between the germination and absorbance values of seeds, the absorbance was divided into five stages as 0.000-0.100, 0.101-0.200, 0.201-0.300, 0.301-0.400, and 0.401 at intervals of 0.100 as in the case of the cabbage seeds, The seed germination morphology was examined. The germination of the seeds belonging to each absorbance range was classified into normal grains, abnormal graves and non-grains, and the results are shown in FIG.

As a result, the ratio of normal seedlings was highest at lower absorbance of 0.100 or less, and the ratio of normal seedlings was lower as the absorbance value became higher. As a result, the absorbance value and the normal seedling ratio were statistically highly correlated (r ² = 0.98 ). In addition, the unburned seeds increased proportionally with higher absorbance values, and almost all of them were found to be unfertilized seeds at> 0.401.

Experimental Example  3: Phosphorus (P) of Chinese cabbage and seedless leaching solution Color development  Method of predicting germination

The absorbance of the seed leaching solution was ranked in descending order from the lowest value to the highest value. Then, the range was divided into 0.100 intervals, and the germination probability was statistically determined for seeds within each range. The logistic regression model was used. The logistic regression coefficient for seed germination in each absorbance range was obtained and the seed germination probability was obtained by the factor.

Table 2 shows the germination probabilities of the Chinese cabbage seeds and the seedless seeds in Table 2. The logistic regression coefficients of each coloring range of Chinese cabbage and non-seed were statistically significant.

Seed germination probability of cabbage seed leaching solution by absorbance interval Absorbance interval Germination probability 0.000-0.100 0.982 0.101-0.200 0.921 0.201-0.300 0.709 0.301-0.400 0.338 > 0.401 0.096

Seed germination probability by absorbance range of seedless leach solution Absorbance interval Germination probability 0.000-0.100 0.973 0.101-0.200 0.885 0.201-0.300 0.620 0.301-0.400 0.257 > 0.401 0.068

In other words, the higher the degree of color development, the lower the probability of germination, and the germination rate can be estimated by the following equation (1) according to the germination probabilities of unknown cabbage and seedless seedlings.

(Where S is the number of seeds in the corresponding absorbance interval, P is the probability of germination, TS is the total number of seeds, and n is the absorbance).

That is, if the absorbance interval is 1, 2, 3, ..., n, multiply the number of seeds in each section by the germination probability, divide by the total number of seeds, and multiply by 100 to obtain the estimated germination rate. Therefore, without germinating the unknown Chinese cabbage seeds, the germination rate of the seeds can be easily calculated according to each coloring degree by the formula (1).

Experimental Example  4: phosphorus (P) content of cabbage seed leaching solution Colored  Image analysis

The 96 well plate colored with phosphorus (P) coloring reagent was scanned with a scanner (Fig. 4) using Chinese cabbage seed (Bulam plus) leach solution as shown in Fig. 1 to analyze images. The image was analyzed with a computerized image analyzer to analyze the type of color and its intensity, and the relationship with seed germination was analyzed through a computer program. As a result, the same results as in Experimental Example 1 were obtained. The image analysis is performed by the image analysis software (SigmaScan Pro 5.0, Systat Software, which is a software for image analysis), which can measure the vitality of the seed in a short time without destroying the seed It is a very useful invention in seed industry and agriculture life industry because it has excellent effect of providing method.

Claims (1)

A seed soaking step in which each seed is soaked in distilled water to soak in water for 3 to 4 hours to absorb moisture;
Mixing 50 μl of the seed leach solution for each of the seeds immersed in the leach solution with 40 μl of phosphorus (P) coloring reagent;
A visual prediction step of visually predicting the germination rate of each seed by coloring the sediment sample within 5 minutes;
An absorbance measurement step of irradiating the absorbent sample with a multiplate reader equipped with a 630 nm absorbance filter;
A seed classification step of classifying each seed into a predetermined absorbance interval according to the absorbance;
And a germination rate estimating step of estimating the seed germination rate of the classified absorbance interval by the following equation,
In the seed dipping step, the seeds are put into each well plate, and the phosphorus (P) coloring reagent is mixed with 5N sulfuric acid, 25% ammonium molybdate, 0.1M ascorbic acid and antimonyl potassium tartrate in a volume ratio of 10: 3: 1 < / RTI >
Method of predicting seed germination rate by using color of seed leaching solution

(Where S is the number of seeds in the corresponding absorbance interval, P is the probability of germination, TS is the total number of seeds, and n is the absorbance).

Images