Method for rapidly detecting seed activity based on fluorescein diacetate and application

Abstract

The invention discloses a method for rapidly detecting seed activity and application, FDA has no fluorescence and is hydrolyzed by seed esterase to generate fluorescein, so that a fluorescence signal is specifically generated. The seed vigor is detected through the FDA probe, the detection time is remarkably shortened, the detection sensitivity and accuracy are improved, high-throughput detection is achieved, and a set of efficient, accurate and large-scale technical scheme is provided for large-scale seed vigor screening in the agricultural field.

Description

Technical Field

[0001] This invention relates to a method for detecting seed vigor based on fluorescein diacetate, belonging to the field of seed quality testing technology. Background Technology

[0002] Seed vigor is a key indicator for evaluating seed quality. It reflects the overall potential of a seed or seed batch to germinate quickly and uniformly and grow into a normal seedling under various field conditions.

[0003] High-viability seeds are crucial for improving seedling emergence rates, reducing sowing costs, increasing crop yields, and promoting mechanized production. Therefore, it is necessary to conduct regular viability testing on stockpiled germplasm resources to ensure their viability is maintained long-term.

[0004] Currently, the gold standard method for seed vigor testing is the standard germination test (GB / T3543.4-1995). The test procedure includes: randomly selecting samples from clean seeds, placing them in a suitable germination bed, and culturing them under specified temperature, light, and water conditions; periodically checking and recording the number of normal seedlings, identifying seedlings according to the standard; and finally calculating the germination rate and reporting the results. However, this testing method takes several days to several weeks, has poor timeliness, and is subject to stringent environmental conditions such as temperature and humidity (e.g., temperature fluctuations need to be controlled within ±1℃), making it impossible to achieve real-time and rapid detection of seed vigor.

[0005] Rapid on-site seed vigor testing technology can quickly identify high-vigor seeds, directly guide sowing decisions, reduce ineffective costs, and is crucial for ensuring field emergence rates, saving costs, and increasing yields and income.

[0006] Currently, the main methods for rapid on-site detection of seed vigor include tetrazolium staining (TTC method), electrical conductivity method, and infrared spectroscopy.

[0007] The TTC colorimetric reaction relies on dehydrogenase activity to distinguish between live and dead seeds, and the staining time varies from 5 to 20 hours depending on the seed size. This method can only qualitatively determine whether a seed is alive or dead, and cannot assess the vigor and uniformity of seedlings (i.e., the level of vitality) like a germination test. Furthermore, it is highly subjective, with low sensitivity and accuracy.

[0008] The conductivity method assesses seed vigor by measuring the conductivity of the seed soaking solution, thus providing a rapid and non-destructive assessment. This method requires approximately 24 hours of soaking, and its results have a relatively low correlation with seed vigor. While simple to operate, it is a rapid detection method with relatively low accuracy and reliability.

[0009] Infrared spectroscopy indirectly and non-destructively assesses the vigor and quality of seeds by scanning the characteristic absorption peaks of organic compounds. Its advantages are speed and high throughput, but it requires the establishment of accurate models and has high equipment costs, making it difficult to promote.

[0010] Therefore, developing a seed vigor testing technology that is fast, accurate, low-cost, and suitable for field application is of great practical significance and urgency for precision agriculture, seed conservation, and ensuring food security.

[0011] Fluorescein diacetate (FDA) is an important fluorescent probe widely used in cell viability and enzyme activity detection. As a fluorescein derivative, FDA itself does not possess fluorescent properties; however, the acetate group in its molecular structure can be specifically hydrolyzed by intracellular esterases, releasing fluorescein molecules with green fluorescence (excitation / emission wavelengths of approximately 490 / 520 nm). This unique biochemical property makes FDA an effective tool for distinguishing between living and dead cells: living cells, with their intact cell membranes and normal metabolic activity, retain the fluorescein produced by hydrolysis; while dead cells, due to membrane damage and cessation of metabolic activity, cannot accumulate fluorescent signals.

[0012] In terms of applications, the FDA is widely used in medical research (such as drug screening and cytotoxicity assessment), microbiology (microbial activity testing), and environmental science (pollutant biotoxicity testing). In the medical field, the FDA is commonly used for anticancer drug screening, stem cell viability testing, pathogen activity analysis, and cytotoxicity experiments. In biological research, the FDA can be used for monitoring microbial metabolic activity, parasite drug sensitivity testing, and sperm quality assessment.

[0013] In summary, esterase activity is one of the core indicators of cell viability, and the FDA often uses it as an effective detection tool.

[0014] Seeds are composed of living cells, and esterase activity is expected to serve as an important indicator of seed viability.

[0015] Therefore, this patent uses FDA as a fluorescent probe to detect the esterase activity of seeds, and then evaluates seed vigor, aiming to establish a rapid, efficient, accurate and low-cost method for detecting seed vigor. Summary of the Invention

[0016] The purpose of this invention is to provide a method for rapid detection of seed vigor, in order to solve the problems of long time consumption, low sensitivity and accuracy, and high cost of existing rapid detection methods. It aims to provide an efficient, accurate and scalable technical solution for large-scale seed vigor screening in the agricultural field.

[0017] To achieve the above objectives, the present invention adopts the following technical solution:

[0018] A rapid method for detecting seed viability involves co-incubating seed extract and FDA in a buffer solution and then measuring the fluorescence value using a fluorescence microplate reader at an excitation wavelength of 480-495 nm and an emission wavelength of 510-530 nm.

[0019] Specifically, the excitation wavelength of the fluorescence microplate reader can be set to any value within the range of 480-495nm, such as 480nm, 485nm, 490nm, and 495nm, while the emission wavelength can be set to any value within the range of 510-530nm, such as 510nm, 515nm, 520nm, 525nm, and 530nm.

[0020] Preferably, it includes the following steps:

[0021] Step a. Grind the seeds to be tested into powder using a grinder;

[0022] Step b. Weigh the seed powder, add PBS buffer, and vortex.

[0023] Step c. Centrifuge to remove the precipitate and obtain the seed extract;

[0024] Step d. Prepare FDA preservation solution;

[0025] Step e. Prepare the FDA working solution;

[0026] Step f. Start the fluorescent staining reaction: Mix the FDA working solution with the seed extract and incubate. Measure the fluorescence value using a fluorescent microplate reader.

[0027] In any of the above schemes, the preferred option is that in step b, 0.2 g of seed powder is weighed, 800 μl of phosphate buffer (PBS, pH 7.2-7.4) is added, and the mixture is vortexed for 5 min.

[0028] In any of the above schemes, the preferred method is to centrifuge at 10,000 rpm (~11,180×g) for 10 min in step c, discard the precipitate, and obtain the seed extract.

[0029] In any of the above schemes, the preferred option is that, in step d, the FDA preservation solution is prepared by weighing 1 mg of FDA powder, dissolving it in 1 ml of dimethyl sulfoxide, shaking and mixing to prepare a 1 mg / ml FDA preservation solution, and storing it at 4°C in the dark.

[0030] In any of the above schemes, the preferred option is that, in step e, the FDA working solution is prepared (prepared fresh for use): the FDA preservation solution is diluted 3 / 1000 with PBS buffer (final concentration 3 μg / ml) and stored away from light.

[0031] In any of the above schemes, the preferred method is that, in step f, 45 μl of FDA working solution is mixed with 5 μl of esterase extract and incubated at 37°C in the dark for 20 min.

[0032] In any of the above schemes, it is preferred that, in step g, the excitation wavelength of the fluorescence microplate reader is 480-495nm and the emission wavelength is 510-530nm.

[0033] This invention also provides the application of the above-mentioned detection method in the detection of wheat seed vigor.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Significantly improve detection efficiency: The detection cycle is shortened to less than 1 hour, and the fluorescence microplate reader is used to replace manual subjective interpretation, realizing high-throughput and high-precision analysis.

[0036] (2) Overcoming the limitations of seed structure differences: By extracting esterases for in vitro reactions, the interference of seed coat barrier and membrane permeability differences on substrate permeation is overcome, which significantly enhances the universality and reproducibility of the method in different seed types.

[0037] (3) Balancing accuracy and convenience: While maintaining laboratory-level precise quantification, it reduces reliance on complex instruments and provides a feasible method for rapid detection in field or storage environments.

[0038] (4) Low detection cost. FDA is a commonly used fluorescent dye, which is inexpensive, stable, and easy to store. The matching single-channel fluorescent microplate reader is technically mature, easy to operate, and easy to deploy and promote on a large scale. Attached Figure Description

[0039] Figure 1 : Basic flowchart of FDA seed viability testing.

[0040] Figure 2 Standard germination test to detect changes in seed vigor at different aging times.

[0041] Figure 3 TTC method for detecting changes in seed vigor at different aging times.

[0042] Figure 4 : Detection of seed vigor changes at different aging times using conductivity method.

[0043] Figure 5 FDA method for detecting changes in seed vigor at different aging times. Detailed Implementation

[0044] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. It should be noted that the described embodiments represent only some implementations of the present invention, and not all implementations. Other implementations obtained by those skilled in the art without creative work based on the embodiments of the present invention also fall within the protection scope of the present invention. The dosages mentioned in the present invention are determined according to national technical specifications and actual production conditions. Unless otherwise specified, all raw materials used in the present invention are commercially purchased.

[0045] Example 1

[0046] wheat seed aging test.

[0047] Spread the wheat seeds evenly on a metal mesh frame, place the metal mesh frame in a sealed box, ensuring that the seeds are in a single layer and not stacked, so that all the seeds can be evenly exposed to the high temperature and high humidity environment.

[0048] Pour an appropriate amount of water (1-2cm) into the bottom of the box. Do not add too much water, as this may submerge the seeds.

[0049] The container is sealed to ensure good airtightness and placed in a 42℃ constant temperature incubator for aging treatment.

[0050] Once the predetermined time has elapsed, immediately remove the sealed box and move it to a well-ventilated environment to dry, allowing the moisture content of the seeds to return to a level similar to that of unaged seeds.

[0051] This experiment set up two aging times: 72 hours and 120 hours.

[0052] Example 2

[0053] Standard germination test for wheat seeds.

[0054] According to the standard germination test method specified in the "Regulations for the Inspection of Crop Seeds" (GB / T3543-1995), the seeds were first disinfected in a 0.5% sodium hypochlorite solution for 2 minutes, followed by rinsing twice with distilled water to thoroughly remove any residual disinfectant. After disinfection, the seeds were placed in a germination box and cultured in a constant temperature incubator at 20℃. Each replicate used 100 seeds, with a total of three replicates. Germination was judged when the sprout length of the wheat seed exceeded half its own length. The number of germinated seeds was recorded daily starting from the second day of germination.

[0055] On the 7th day of cultivation, the wheat seedlings were removed, the seed coats were removed, and they were placed in an aluminum box and baked at 80℃ for 48 hours. The dry weight of the seedlings was then measured, and the dry weight of a normal single seedling was calculated accordingly.

[0056] Test seeds: Wheat seeds to be tested (at least 100 seeds, repeated 3 times).

[0057] Incubator: temperature control (20±1℃), humidity control (relative humidity 90%~95%).

[0058] Experimental steps:

[0059] (1) Seed disinfection and pretreatment: Soak seeds in 70% ethanol for 3 minutes, rinse with distilled water 3 times to remove surface microorganisms; soak (25℃, 6 h) to break dormancy.

[0060] (2) Seed culture: 100 seeds are evenly distributed in the seed germination box and cultured in the incubator at 20°C. Water is added regularly every 12 hours to ensure that the germination box is moist but not waterlogged.

[0061] (3) Observation record: Germination standard: radicle ≥ seed length, plumule ≥ 1 / 2 seed length (ISTA standard).

[0062] (4) Calculation of results:

[0063] Germination rate (%) = (Number of germinated seeds on day X / Number of seeds in the experiment) × 100;

[0064] Germination potential (%) = (Number of germinated seeds on day 3 / Number of seeds in the experiment) × 100;

[0065] Germination index (GI) = ∑(Gt / Dt);

[0066] Vitality Index (VI) = GI × S0;

[0067] Gt represents the number of germinations per day, Dt represents the number of days corresponding to Gt, and S0 represents the dry weight of the seedlings at the end of germination.

[0068] (4) Germination index results ( Figure 2 ): Table 1. Changes in germination index and vigor index of wheat seeds at different aging times. wheat seeds Germination Index (GI) Vitality Index VI Normal seeds 316.7 1029.3 Seeds aged for 72 hours 265.3 750.8 Seeds aged for 120 hours 222.1 573.0

[0069] Example 3.

[0070] TTC staining method for detecting wheat seed vigor.

[0071] Preparation of TTC solution (prepare fresh before use): Weigh 0.1g of TTC powder and add it to 100ml of PBS solution to prepare a 0.1% TTC aqueous solution (pH 7.2-7.4). Store in a brown bottle away from light.

[0072] Sample preparation and pretreatment: Take 100 wheat seeds and soak them for 6 hours to allow them to fully swell, making them easier to dissect and stain.

[0073] Dissecting the wheat embryo: Carefully cut the wheat seed longitudinally with tweezers and a knife to expose the embryo.

[0074] TTC staining reaction: The treated seeds were completely immersed in TTC solution and placed in a dark environment for staining at room temperature for 6 h.

[0075] Termination of reaction and observation: After the predetermined time has been reached, discard the TTC solution and gently rinse the seeds 2-3 times with pure water to terminate the reaction. Observe the staining results immediately.

[0076] like Figure 3 As shown, both aged and non-aged seeds can be stained with TTC. In terms of staining depth, non-aged seeds generally show deeper and more uniform staining.

[0077] Therefore, the TTC staining method is not sensitive enough to changes in seed vigor, and its result interpretation relies heavily on subjective experience.

[0078] Example 4

[0079] Electrical conductivity method for detecting wheat seed vigor.

[0080] Weigh 5 g of wheat seeds treated for different aging times and place them in 100 mL beakers.

[0081] Remove the seeds, but retain the soaking solution;

[0082] The conductivity values ​​of each soaking solution were measured using a conductivity meter, and the results are shown in Table 3.

[0083] Although the conductivity method can quantitatively demonstrate seed vigor and indicate vigor decline caused by aging (Table 2, ...), Figure 5 However, the results showed significant deviations from the standard germination experiment in terms of variation range and trend, indicating that the sensitivity and accuracy of this method need further improvement.

[0084] Table 2. Changes in electrical conductivity of wheat seeds at different aging times.

[0085] wheat seeds Normal seeds Seeds aged for 72 hours Seeds aged for 120 hours Electrical conductivity (μS / cm) 39.2 32.5 34.3

[0086] Example 5

[0087] FDA method for testing wheat seed viability.

[0088] (1) Take 50 seeds to be tested and grind them into powder using a grinder.

[0089] (2) Weigh 0.2g of seed powder, add 800μl of PBS buffer (pH 7.2-7.4), and vortex for 5min.

[0090] (3) Centrifuge at 10,000 rpm (~11,180×g) for 10 min, discard the precipitate, and obtain the seed extract.

[0091] (4) Preparation of FDA preservation solution: Weigh 1 mg of FDA powder, dissolve it in 1 ml of dimethyl sulfoxide, shake and mix well to prepare 1 mg / ml FDA preservation solution, and store at 4°C protected from light.

[0092] (5) Preparation of FDA storage solution (prepare fresh for immediate use): Dilute the FDA storage solution 3 / 1000 with PBS buffer (final concentration 3 μg / ml) and store away from light.

[0093] (6) Mix 45 μl of FDA working solution with 5 μl of esterase extract, incubate at 37°C in the dark for 20 min, and measure the fluorescence value using a fluorescence microplate reader. Read the value at 37°C for 20 sec.

[0094] As shown in Table 3 and Figure 5 As shown, the FDA method can accurately reflect the changes in seed vigor after aging, and its trend and magnitude are highly consistent with the standard germination experiment, confirming the reliability of the method.

[0095] Table 3. FDA fluorescence values ​​of wheat seeds at different aging times wheat seeds Normal seeds Seeds aged for 72 hours Seeds aged for 120 hours FDA Fluorescence Intensity (RFU) 2710040 1987539 1632362

Claims

1. A method for rapidly detecting seed vigor, characterized in that, Seed extract and fluorescein diacetate (FDA) were co-incubated in phosphate-buffered saline (PBS), and the fluorescence value was measured using a fluorescence microplate reader at an excitation wavelength of 480-495 nm and an emission wavelength of 510-530 nm.

2. The method for rapid detection of seed vigor according to claim 1, characterized in that, Includes the following steps: Step a. Grind the seeds to be tested into powder using a grinder; Step b. Weigh the seed powder, add PBS buffer, and vortex. Step c. Centrifuge to discard the precipitate, obtaining the seed extract. Step d. Prepare FDA preservation solution; Step e. Prepare the FDA working solution; Step f. Start the fluorescent staining reaction: Mix the FDA working solution with the seed extract and incubate. Measure the fluorescence value using a fluorescent microplate reader.

3. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step b, weigh 0.2g of seed powder, add 800μl of PBS, and vortex for 5min.

4. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step c, centrifuge at 10,000 rpm (~11,180×g) for 10 min, discard the precipitate, and obtain the seed esterase extract.

5. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step d, the FDA preservation solution is prepared as follows: Weigh 1 mg of FDA powder, dissolve it in 1 ml of dimethyl sulfoxide, shake to mix, and prepare a 1 mg / ml FDA preservation solution. Store at 4°C protected from light.

6. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step e, the FDA working solution is prepared (prepare fresh for immediate use): dilute the FDA preservation solution 3 / 1000 with PBS buffer (final concentration 3 μg / ml) and store in the dark.

7. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step f, mix 45 μl of FDA working solution with 5 μl of esterase extract and incubate at 37°C in the dark for 20 min.

8. The method for rapid detection of seed vigor as described in claim 2, characterized in that, In step g, the excitation wavelength of the fluorescence microplate reader is 480-495nm and the emission wavelength is 510-530nm.

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