A method for quickly detecting the germination rate of Gastrodia elata seeds

Abstract

The present invention belongs to the field of detecting the germination rate of Gastrodia elata seeds, and discloses a method for quickly detecting the germination rate of Gastrodia elata seeds. First, the Gastrodia elata seeds are stained by the tetrazolium chloride method. Randomly absorb the stained Gastrodia elata seeds and place them under a stereomicroscope to take pictures of the stained Gastrodia elata seeds. Process the stained pictures through Image J software to calculate the average redness degree value of the Gastrodia elata seeds. At the same time, conduct a symbiotic germination experiment on the same batch of Gastrodia elata seeds and germination fungi. After the seeds germinate, count the number of germinated Gastrodia elata seeds and the number of ungerminated seeds respectively, and calculate the germination rate. Obtain multiple groups of average redness degree values and germination rates of Gastrodia elata seeds through simultaneous staining experiments and germination experiments on multiple batches of different Gastrodia elata seeds. Then, conduct a linear regression analysis between the redness degree value and the germination rate to obtain a regression equation. Substitute the redness degree value obtained by TTC staining of the newly obtained Gastrodia elata seeds into the regression equation, and the germination rate of this batch of Gastrodia elata seeds can be calculated, indicating the germination ability of Gastrodia elata seeds.

Description

Technical Field

[0001] The present invention belongs to the field of detecting the germination rate of Gastrodia elata seeds, and relates to a method for rapidly detecting the germination rate of Gastrodia elata seeds. Background Art

[0002] Gastrodia elata is a commonly used precious traditional Chinese medicine, which is the tuber of the orchid plant Gastrodia elata, and has the effects of calming endogenous wind and stopping convulsions, suppressing hyperactive liver yang, dispelling wind and dredging collaterals; it is used for infantile convulsions, epilepsy convulsions, tetanus, headache and dizziness, paralysis of hands and feet, limb numbness, rheumatic arthralgia, etc. Gastrodia elata has been a nourishing substance that is both food and medicine since ancient times. In November 2023, China included Gastrodia elata in the "List of Substances that are Both Food and Traditional Chinese Medicines in Accordance with Tradition", that is, substances that are both food and medicine. The demand for Gastrodia elata has increased sharply.

[0003] The natural distribution of Gastrodia elata is very wide. Due to the large demand for Gastrodia elata, the wild resources have decreased, and it mainly relies on artificial cultivation to meet people's needs. Gastrodia elata is cultivated in more than 20 provinces such as Sichuan, Yunnan, Guizhou, Shaanxi, Hubei, Hunan, and Anhui. There are two ways of reproduction of Gastrodia elata: one is sexual reproduction, and the other is asexual reproduction. Among them, sexual reproduction with seeds is the main one. The viability / germination rate of Gastrodia elata seeds directly affects the yield, quality and economic benefits of Gastrodia elata.

[0004] The structure of Gastrodia elata seeds is simple and they are easy to inactivate.

[0005] Since Gastrodia elata seeds are as fine as powder, with a simple structure and no endosperm. The Gastrodia elata seeds stored under natural conditions quickly inactivate, and the storage period is only a few days. Therefore, in Gastrodia elata cultivation, usually after the fruits mature in March - May, the slightly cracked fruits are picked, dried, and then the seeds are shaken out for sowing. With the rapid expansion of the modern Gastrodia elata cultivation area, the cultivation area is wide, the storage time is long, and the seeds purchased and transported across regions are very easy to inactivate, with a low germination rate, or even all inactivate and do not germinate, causing serious losses in production. Therefore, it is necessary to evaluate the germination rate and / or viability of Gastrodia elata seeds before sowing, determine the appropriate seeding rate, and even whether the seeds are sown.

[0006] At present, the determination results of seed viability are highly subjective and have large errors.

[0007] The viability and germination rate of seeds are the main indicators for evaluating the quality of seeds. The viability of seeds refers to the potential ability of seeds to germinate or the vitality of the embryo, which is usually determined by the red tetrazolium chloride (TTC) staining method. The principle of the TTC staining method is that the embryo of viable seeds is stained red, while the embryo of non-viable seeds is not stained. The seed viability is expressed as the percentage of the number of viable seeds divided by the total number of seeds. Due to the different strengths of seed viability, the degree of red staining is different, and the criteria for inspectors to observe and judge the red color by the naked eye are also different, making it difficult to accurately count the number of stained seeds and obtain an accurate viability value. Moreover, seeds with weak viability and light staining cannot germinate, affecting the yield and quality of cultivated Gastrodia elata. Only seeds with a germination rate can germinate. Therefore, the viability is also directly expressed by the germination rate of the seeds.

[0008] The operation of measuring the germination rate of Gastrodia elata seeds through symbiotic germination experiments with germination fungi is complex and time-consuming.

[0009] For ordinary crop seeds such as corn and rice, it only takes 5 to 7 days to directly measure the germination rate through germination experiments. However, Gastrodia elata seeds need to symbiotically germinate with fungi such as Mycena osmundicola or Mycena dendrobii in the genus Mycena (collectively referred to as germination fungi), and the operation is complex. Moreover, the germination process of Gastrodia elata seeds includes the invasion of germination fungi into embryo cells → the embryo swelling and breaking through the epidermis → the formation of protocorms, and the whole process takes more than 30 days. If the Gastrodia elata seeds stored under natural conditions wait for such a long time to know the germination rate, the seeds have all lost their viability and cannot be used for sowing and cultivation.

[0010] The prior art "A method for the preservation and viability detection of Gastrodia elata seeds" (Patent No.: CN116584252A; Inventors: Huang Xiaorun, Huang Wanbing, Lu Yingying, etc. Publication date: August 15, 2023) discloses two methods for detecting the viability of Gastrodia elata seeds. One is the germination method by culturing germination fungi: using Mycena dendrobii as the germination fungus, with water agar as the culture medium, dark-culturing at 20°C to 25°C, and observing the germination of Gastrodia elata seeds with a stereomicroscope every 5 days until the Gastrodia elata seeds grow into protocorms visible to the naked eye, counting the number of protocorms, and then calculating the germination rate of Gastrodia elata seeds as the viability of Gastrodia elata seeds. This method takes a long time. Another method is the TTC staining method: using a 1% TTC solution, dark-staining statically at 30°C for 48 hours, then observing the seed staining under a stereomicroscope, and regarding the seeds with the embryo stained orange or pink as viable seeds, and the seed viability = (number of viable seeds / total number of seeds) × 100%. This method has subjectivity in judging the seed color, resulting in uncertain viability results.

[0011] The prior art "A method for germinating Gastrodia seeds of the genus Gastrodia" (Patent No.: CN118489330A; Inventors: Xu Qian, Hu Xiaojian, Lin Liang, etc. Publication date: August 16, 2024) discloses a method for measuring the viability and germination rate of Gastrodia seeds. The method for measuring the viability of Gastrodia seeds by the TTC staining method is as follows: Gastrodia seeds are stained in 1% TTC staining solution in the dark at 30°C for 48 h, and then the number of seeds with embryos stained red is counted under a stereomicroscope to calculate the viability of the seeds. The method for measuring the germination rate is as follows: Mycena osmundicola is inoculated on a potato dextrose agar medium first, and then Gastrodia seeds are inoculated 3 weeks later, and cultured under dark conditions at 23°C for 6 - 8 weeks to calculate the germination rate of the seeds. This method still has problems such as large errors in viability measurement and a germination rate measurement time of more than 9 weeks.

[0012] The prior art "A method for evaluating the germination effect of Gastrodia germination fungi on Gastrodia seeds of the genus Gastrodia" (Patent No.: CN118483016A; Inventors: Lin Liang, Hu Xiaojian, Xu Qian, etc. Publication date: August 13, 2024) discloses different preservation methods of Gastrodia seeds and the germination effects of different germination fungi, and involves methods for measuring the viability and germination rate of Gastrodia seeds. This method is the same as the above patent (Patent No.: CN118489330A) and also has problems such as large errors in viability measurement and a long time for germination rate measurement.

[0013] It can be seen that the prior art has problems such as strong subjectivity in detecting the viability of Gastrodia seeds by the TTC staining method, large errors in the measurement results of different technicians, a long time and complex operation in the symbiotic germination experiment for measuring the germination rate of Gastrodia seeds. Therefore, in order to solve the above technical problems, the technical solution of the present invention is designed. Summary of the Invention

[0014] The purpose of the present invention is to provide a method for quickly detecting the germination rate of Gastrodia seeds, which solves the above technical problems.

[0015] The technical solution adopted by the present invention is as follows:

[0016] A method for quickly detecting the germination rate of Gastrodia seeds includes TTC staining of Gastrodia seeds, quantification of the redness degree, determination of the germination rate of Gastrodia seeds, correlation analysis between the redness degree value and the germination rate, determination of the redness degree value of new seeds, and calculation of the germination rate;

[0017] Staining treatment of Gastrodia seeds

[0018] Gastrodia seeds are stained by the 2,3,5 - triphenyltetrazolium chloride (TTC) method to obtain stained Gastrodia seeds;

[0019] Quantification of the redness degree

[0020] Take pictures of the stained Gastrodia elata seeds with a stereomicroscope, and use Image J software to calculate the average red degree value of the Gastrodia elata seeds in the picture (total red degree value of Gastrodia elata seeds / total number of Gastrodia elata seeds) to obtain a quantitative seed red degree value;

[0021] Determination of the germination rate of Gastrodia elata seeds (determination of the actual germination rate)

[0022] Carry out symbiotic germination culture of Gastrodia elata seeds and germination fungi until the germination state of Gastrodia elata seeds is observed; count the number of germinated Gastrodia elata seeds and ungerminated seeds under an optical microscope, and calculate the actual germination rate of the Gastrodia elata seeds (percentage of the number of germinated Gastrodia elata seeds divided by the total number of Gastrodia elata seeds);

[0023] Correlation analysis between the red degree value and the germination rate

[0024] The red degree value after staining of the same batch of Gastrodia elata seeds and the germination rate obtained from the germination experiment are a set of data; conduct correlation analysis between the red degree values and germination rates of multiple groups of different Gastrodia elata seeds, and establish a regression equation;

[0025] Determination of the red degree value of new Gastrodia elata seeds and calculation of the germination rate

[0026] Stain the newly obtained Gastrodia elata seeds by the 2,3,5 - triphenyltetrazolium chloride (TTC) method, take pictures with a stereomicroscope, process the pictures with Image J software, and calculate the average red degree value; substitute the average red degree value into the above - established regression equation to calculate the germination rate of the seeds (indicating the germination ability of this batch of Gastrodia elata seeds).

[0027] The working principle of the present invention is as follows: First, stain Gastrodia elata seeds by the TTC method; place the stained seeds under a stereomicroscope for taking pictures to obtain pictures of the stained Gastrodia elata seeds; use Image J software to process the pictures, visually display the different red degrees of different seeds with different red areas, and then calculate the average red degree value of all Gastrodia elata seeds in the picture. At the same time, conduct a symbiotic germination experiment on the same batch of Gastrodia elata seeds and germination fungi until the seeds have germinated (protocorms) are observed, count the number of germinated Gastrodia elata seeds and the number of ungerminated seeds, and calculate the actual germination rate (percentage of the number of germinated Gastrodia elata seeds divided by the total number of Gastrodia elata seeds).

[0028] Conduct staining experiments on multiple batches of different Gastrodia elata seeds to obtain red degree values, and at the same time conduct germination experiments to obtain the corresponding germination rates. Conduct linear regression analysis between the red degree values and the actual germination rates of multiple groups of different Gastrodia elata seeds to obtain a regression equation. Substitute the average red degree value obtained by TTC staining, taking pictures, and picture processing of the newly obtained Gastrodia elata seeds into the regression equation, and the germination rate of the Gastrodia elata seeds can be calculated.

[0029] Therefore, in the present invention, after the Gastrodia seeds are stained with TTC, they are photographed by a stereomicroscope. The stained images are processed by Image J software. At the same time, a threshold is set, and the pixels with a red degree higher than the threshold are marked as red areas, while the pixels with a red degree lower than the threshold are marked as non-red areas. Finally, the total area of the red areas (standardization of image area recognition) is calculated to obtain the total red degree value of the Gastrodia seeds. Then, the average red degree value of the Gastrodia seeds (total red degree value of Gastrodia seeds / total number of Gastrodia seeds) is calculated, so as to quantitatively detect the red degree of the Gastrodia seeds, obtain the red degree value objectively reflecting the stained Gastrodia seeds, avoid the errors of different testers' visual judgments, and make the measured results more objective.

[0030] At the same time, through the germination experiment on this batch of Gastrodia seeds, the actual germination rate is obtained. Then, through the correlation analysis between the average red degree values and the germination rates of multiple batches of different Gastrodia seeds, a regression equation is established. For the newly obtained Gastrodia seeds, only the average red degree value after staining needs to be measured and substituted into the regression equation to obtain the germination rate, which shortens the time for measuring the germination rate of Gastrodia seeds by more than 30 days.

[0031] Furthermore: The parameters for photographing the stained Gastrodia seeds with a stereomicroscope are as follows: LDE top light Light = 2, bottom light Light = 0, objective lens 0.63X, eyepiece 1X, magnification 42X; select the RGB mode, white balance select red 30%, green 64%, blue 15%, exposure time 2600.00 ms, saturation set to maximum; photograph after adjusting the field of view clearly; randomly select more than 3 fields of view for the same batch of stained Gastrodia seeds to take pictures respectively, and obtain more than 3 pictures.

[0032] Furthermore: After the cultivation of Gastrodia seeds in the actual germination experiment is completed, randomly select 1 cm 2 of the culture medium with Gastrodia seeds, and count the germinated seeds and ungerminated seeds respectively under an optical microscope. The number of Gastrodia seeds on each culture medium should be greater than 50 grains, and multiple portions should be taken in parallel, and the average value of the obtained germination rates is calculated.

[0033] Furthermore: After mixing the newly obtained Gastrodia seeds, take a part of the Gastrodia seeds at random and obtain the average red degree value of this batch of Gastrodia seeds according to the staining method and image processing method, and substitute it into the regression equation to calculate the germination rate of this batch of Gastrodia seeds.

[0034] Furthermore: After the Gastrodia elata seeds are stained by the TTC method, the redness degrees of Gastrodia elata seeds with different viability are different. They are photographed under the stereomicroscope under the same conditions, and then the images are processed by Image J software. At the same time, a threshold is set. The pixels with a redness degree value higher than the threshold are marked as the red area, while the pixels with a redness degree value lower than the threshold are marked as the non-red area. Calculate the total area of the red area (standardization of image area recognition) to obtain the total redness degree value of the Gastrodia elata seeds, and then calculate the average redness degree value of the Gastrodia elata seeds (total redness degree value of Gastrodia elata seeds / total number of Gastrodia elata seeds), so as to quantitatively detect the redness degree of Gastrodia elata seeds.

[0035] Furthermore: The threshold is set to setThreshold(200, 255, "raw").

[0036] Furthermore: During the process of processing the image by Image J software, the total number of Gastrodia elata seeds in the selected image is automatically counted.

[0037] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0038] 1. A method for quickly detecting the germination rate of Gastrodia elata seeds quantifies the results of staining Gastrodia elata seeds, thereby making the judgment of the viability of Gastrodia elata seeds more objective; at the same time, measures the germination rate of this batch of Gastrodia elata seeds; analyzes the relationship between the quantified redness degree value and the actual germination rate and establishes a regression equation; stains and quantifies the newly obtained Gastrodia elata seeds to obtain the average redness degree value and substitutes it into the regression equation, then the germination rate of the Gastrodia elata seeds can be calculated.

[0039] 2. In the present invention, by standardizing the staining conditions of Gastrodia elata seeds and the photography, and at the same time quantitatively processing the redness degree of the stained image of Gastrodia elata seeds by Image J software, objective numerical values of the viability of Gastrodia elata seeds are obtained, avoiding the visual differences of different testers observing Gastrodia elata seeds with the naked eye under the microscope and the errors in the viability measurement results caused by different judgment criteria for stained seeds, which directly affect the production of Gastrodia elata.

[0040] 3. In the present invention, substituting the average redness degree value obtained from the staining experiment into the regression equation can predict the germination rate of the Gastrodia elata seeds; compared with the actual germination rate of the same batch of Gastrodia elata seeds, the predicted germination rate is close to the actual germination rate, verifying that the method for detecting the germination rate of Gastrodia elata seeds in the present invention is fast and accurate.

[0041] 4. In the present invention, only by measuring the average redness degree value (vitality) of Gastrodia elata seeds and substituting it into the regression equation, the germination rate of the Gastrodia elata seeds can be calculated, shortening the time required to obtain the germination rate through the symbiotic germination experiment of Gastrodia elata seeds from more than 30 days to less than 2 days, greatly shortening the time and operating cost of the germination experiment, providing the germination rate data in a timely manner, and providing a basis for the sowing of Gastrodia elata. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings, where:

[0043] Figure 1 is a flowchart of the present invention;

[0044] Figure 2 is a linear relationship diagram between the average redness degree value of Gastrodia elata seeds and the germination rate in the present invention;

[0045] Figure 3 is a schematic diagram of Gastrodia elata seeds taken under a stereomicroscope after being stained by the TTC method in the present invention;

[0046] Figure 4 is in the present invention, through Image J software Figure 3 The red schematic diagram of the schematic diagram in is quantitatively processed;

[0047] Figure 5 is a comparison diagram of germinated and ungerminated Gastrodia elata seeds in the present invention (where the letter a represents the seeds germinated into protocorms, and the letter b represents the ungerminated seeds). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention, that is, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0049] Accordingly, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0050] It should be noted that relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0051] The features and performance of the present invention will be further described in detail below in conjunction with embodiments.

[0052] Embodiment 1

[0053] A method for quickly detecting the germination rate of Gastrodia elata seeds according to the present invention, as Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 shown, the specific implementation manner of this embodiment is as follows:

[0054] Select 20 batches of different Gastrodia elata seeds as model samples. After mixing the Gastrodia elata seeds of the same batch, randomly take several portions of Gastrodia elata seeds. Half of each portion of Gastrodia elata seeds is used for measuring the average redness value by the TTC staining method; the other half is used for the germination experiment to measure the germination rate.

[0055] Perform TTC staining on Gastrodia elata seeds: Stain 20 batches of Gastrodia elata seeds separately. The TTC staining conditions are a TTC solution concentration of 1.5%, a temperature of 30 °C, and a light-shielded staining time of 40 h;

[0056] Photographing the stained seeds: After the staining is completed, randomly pick up the stained seeds and place them under a stereomicroscope to take pictures of the stained Gastrodia seeds. The photography parameters are as follows: For the LDE top light, Light = 2; for the bottom light, Light = 0; objective lens 0.63X, eyepiece 1X, magnification 42X; select the RGB mode, and for white balance, select red 30%, green 64%, blue 15%, exposure time 2600.00 ms, saturation set to maximum; after adjusting the field of view to be clear, take pictures; randomly select more than 3 fields of view from the same batch of stained Gastrodia seeds to take pictures respectively, and obtain more than 3 pictures.

[0057] Counting the Gastrodia seeds in the pictures: Use Image J software to analyze the pictures. Write a program through macros, use the Subtract Background, Gaussian Blur, and Image Calculator plugins to remove the influence of the background, and then use the Analyze Particles plugin to limit the size of the particles recognized as background particles to calculate the total number of Gastrodia seeds.

[0058] Calculating the average redness value of Gastrodia seeds in the pictures: In Image J software, write a program through macros, use the Subtract Background, Gaussian Blur, and Image Calculator plugins to remove the influence of the background, convert the pictures to the LAB color space. After selecting the A channel, set setThreshold(200, 255, "raw"), mark the pixels with redness higher than the threshold as the red area, and mark the pixels with redness lower than the threshold as the non - red area, and calculate the total area of the red area in the pictures; divide the total red area value by the total number of Gastrodia seeds to obtain the average redness value of Gastrodia seeds in the pictures. The average redness values of 20 batches of different red Gastrodia seeds are shown in Table 1.

[0059]

[0060] Determining the germination rate of Gastrodia seeds by the germination experiment method:

[0061] Weigh 30 g of sucrose, 2.37 g of MS medium solid powder, and 6.5 g of agar powder and place them in a conical flask. Add 1 L of ultrapure water to prepare 1 / 2MS medium; wrap the petri dishes with kraft paper and place them in a high - pressure steam sterilizer together with 1 bottle of ultrapure water, and sterilize at 121 °C for 30 min; prepare 100 mL each of 70% alcohol and 0.1% sodium hypochlorite solution for disinfecting Gastrodia seeds; prepare filter paper, metal spatulas, glass funnels, conical flasks, glass droppers, and cut sealing films; and spray 10% sodium hypochlorite solution on the sealing films.

[0062] Place the above items, culture medium and pure water together in a biosafety cabinet and irradiate under ultraviolet light for 1 h; mix the Gastrodia elata seeds with a metal spoon, take about 10 mg of red Gastrodia elata seeds and place them on the filter paper in the funnel; add 70% alcohol to the Gastrodia elata seeds with a dropper, disinfect for 30 s, and repeat the disinfection 3 times; then use a dropper to add 0.1% sodium hypochlorite solution to soak the Gastrodia elata seeds for 20 minutes, and then rinse the Gastrodia elata seeds with 0.1% sodium hypochlorite solution 5 times; place the sterilized Gastrodia elata seeds together with the filter paper in the ventilation port of the biosafety cabinet, and dry the Gastrodia elata seeds.

[0063] Take out the germinated bacteria from the refrigerator and inoculate 1 cm 3 Germination bacteria; 5 mg of Gastrodia elata seeds are evenly sprinkled around the germination bacteria; the culture dish is sealed with the above-mentioned sterilized sealing film, and placed in a sealed bag sprayed with 10% sodium hypochlorite solution, and then placed in a constant temperature incubator, and cultured in the dark at 25°C for 30 days until the Gastrodia elata seeds germinate into protocorms.

[0064] After the culture was completed, 1 cm 2 The culture medium with Gastrodia elata seeds was used to count the number of Gastrodia elata seeds that had germinated and those that had not germinated under an optical microscope. The number of Gastrodia elata seeds on each culture medium should be greater than 50, and multiple samples were taken for observation in parallel to calculate the average value of the actual germination rate. The actual germination rates of 20 batches of different red Gastrodia elata seeds are shown in Table 1.

[0065] Correlation analysis between redness value and germination rate: Linear correlation analysis was performed between the average redness value of the above 20 batches of different red Gastrodia elata seeds and the actual germination rate, and the normalization equation was y = 5×10 -5 x–23.605, R 2 =0.8803 (where y is the germination rate, x is the redness value, and R is the correlation coefficient). R is used to measure the correlation between the redness value of Gastrodia elata seeds and the germination rate; the closer the R value is to 1, the better the correlation between the two. In this method, the R value reached 0.938 (R 2 is 0.8803), indicating that there is a good correlation between the redness value of red Gastrodia elata seeds and the germination rate.

[0066] Reliability verification of predicted germination rate:

[0067] Five different batches of red Gastrodia elata seeds were taken. Half of each batch of red Gastrodia elata seeds were measured for the average redness value by TTC staining method, and the other half were measured for the actual germination rate of Gastrodia elata seeds by germination experiment. The average redness value of Gastrodia elata seeds was substituted into the above regression equation to calculate the predicted germination rate of Gastrodia elata seeds. The difference between the predicted germination rate and the actual germination rate of these five batches of red Gastrodia elata seeds was less than 5% (Table 2), indicating that the prediction results of this method are accurate and reliable.

[0068]

[0069] The present invention enables obtaining the average redness degree value after TTC staining each batch of Gastrodia elata seeds, and substituting this average redness degree value into the regression equation in the present invention can calculate and predict the germination rate, greatly shortening the time of the germination experiment.

[0070] After the existing TTC method stains Gastrodia elata seeds, the Gastrodia elata seeds are observed to show different degrees of red under a microscope. However, different testers have different judgment criteria for whether the Gastrodia elata seeds are stained red, resulting in different statistical quantities of viable seeds and different calculated viability values. Due to the small size of Gastrodia elata seeds, there are also errors in manually counting the number of viable seeds and non-viable seeds under the microscope, ultimately leading to large errors in the judgment results of the viability of Gastrodia elata seeds.

[0071] Therefore, the present invention performs standardized photography on the seeds after TTC staining, processes the images of the stained Gastrodia elata seeds using Image J software, and automatically identifies and calculates the total number of Gastrodia elata seeds. At the same time, a threshold is set in Image J software. Pixels with a redness degree value higher than the threshold are marked as red regions, while pixels with a redness degree value lower than the threshold are marked as non-red regions. Gastrodia elata seeds with different redness degrees present red regions of different sizes. Summing up all the red regions gives the total area of the red regions (standardization of image area recognition), that is, the total redness degree value of the Gastrodia elata seeds in this image. Then, the average redness degree value of the Gastrodia elata seeds (total redness degree value of Gastrodia elata seeds / total number of Gastrodia elata seeds) is calculated, enabling quantification of the redness degree of Gastrodia elata seeds and obtaining viability data objectively reflecting the stained Gastrodia elata seeds.

[0072] This enables the present invention not to require testers to observe and count the number of seeds with the naked eye under a microscope and calculate the viability, avoiding the error in the viability of seeds caused by the visual error and different judgment criteria of testers, thereby affecting the production of Gastrodia elata.

[0073] Example Two

[0074] A method for quickly detecting the germination rate of Gastrodia elata seeds according to the present invention, as Figure 1 shown, the specific implementation manner of this embodiment is:

[0075] Staining temperature test: To initially select the staining temperature of red Gastrodia elata seeds, when the concentration of the TTC staining solution was set to 1.5% and the staining time was 36 h, the average redness degree values of Gastrodia elata seeds at staining temperatures of 25, 30, 35, and 40 °C were compared.

[0076] Dyeing time test: To select the dyeing time for Gastrodia elata seeds, with the concentration of TTC staining solution set at 1.5% and the staining temperature at 30°C, the average redness degree values of Gastrodia elata seeds with dyeing times of 12, 24, 36, 48, and 60 h were compared.

[0077] Selection of TTC staining solution concentration: To select the concentration of TTC staining solution for Gastrodia elata seeds, with the dyeing time set at 36 h and the staining temperature at 30°C, the average redness degree values of Gastrodia elata seeds with staining solution concentrations of 0.1, 0.5, 1, 1.5, and 2% were compared.

[0078] Based on the results of the above single-factor experiments, a three-factor and three-level response surface experiment (Box-Behnken) was designed with the staining temperature A, dyeing time B, and staining solution concentration C to screen the optimal conditions for staining Gastrodia elata seeds with the TTC method: the temperature is 30°C, the dyeing time is 40 h, and the staining solution concentration is 1.5%.

[0079] The above is only an example of the Gastrodia elata seeds of the present invention. For seeds of different variants of Gastrodia elata (Gastrodia elata Bl. f. elata, Gastrodia elata Bl. f. glauca S. Chow, Gastrodia elata Bl. f. viridis Makino, Gastrodia elata Bl. f. flavida S. Chow, and their hybrid Gastrodia elata), except for adjusting the TTC staining conditions, other technical routes and parameters are still applicable and are not intended to limit the protection scope of the present invention. Any modifications, equivalent replacements, and improvements made by those skilled in the art within the principles and technical routes of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for quickly detecting the germination rate of Gastrodia elata seeds, characterized in that: Including TTC staining of Gastrodia elata seeds, quantification of red degree, determination of the germination rate of Gastrodia elata seeds, correlation analysis between the red degree value and the germination rate, determination of the red degree value of new seeds and calculation of the germination rate; Staining treatment of Gastrodia elata seeds The Gastrodia elata seeds are stained by the red tetrazolium method to obtain the stained Gastrodia elata seeds; Quantification of red degree Take pictures of the stained Gastrodia elata seeds with a stereomicroscope, and use Image J software to calculate the average red degree value of the Gastrodia elata seeds in the picture to obtain the quantified red degree value of the seeds; Determination of the germination rate of Gastrodia elata seeds The Gastrodia elata seeds are co-cultured with the germination fungus until the germination state of the Gastrodia elata seeds is observed; count the number of germinated Gastrodia elata seeds and non-germinated seeds under the microscope, and calculate the actual germination rate of the Gastrodia elata seeds; Correlation analysis between the red degree value and the germination rate The red degree value after staining of the same batch of Gastrodia elata seeds and the germination rate obtained from the germination experiment are called a set of data; Perform linear correlation analysis on the red degree values and germination rates of multiple groups of different Gastrodia elata seeds, and establish a regression equation; Determination of the red degree value of new Gastrodia elata seeds and calculation of the germination rate Stain the newly obtained Gastrodia elata seeds by the red tetrazolium method, take pictures with a stereomicroscope, process the pictures with Image J software, and calculate the red degree value; substitute the red degree value into the above regression equation to calculate the germination rate of the seeds.

2. The method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: Take standardized pictures of the stained Gastrodia elata seeds with a stereomicroscope. The photography parameters are: LDE top light Light = 2, bottom light Light = 0, objective lens 0.63X, eyepiece 1X, magnification 42X; select the RGB mode, white balance select red 30%, green 64%, blue 15%, exposure time is 2600.00ms, saturation is set to maximum; take pictures after adjusting the Gastrodia elata seeds in the field of view clearly; randomly select more than 3 fields of view of the same batch of stained Gastrodia elata seeds to take pictures respectively to obtain more than 3 pictures.

3. A method for quickly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: After the completion of the gastrodia elata seed germination experiment culture, randomly select 1 cm 2 For the culture medium with gastrodia elata seeds, count the germinated and ungerminated gastrodia elata seeds respectively under an optical microscope. The number of gastrodia elata seeds on each culture medium should be greater than 50, and multiple parallel samples are taken. Calculate the average value of the germination rate obtained.

4. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: After mixing the newly obtained Gastrodia elata seeds, take any part of the Gastrodia elata seeds according to the staining method and the processing method of the stained seed pictures to obtain the average red degree value of this batch of Gastrodia elata seeds, and substitute it into the regression equation to calculate the germination rate of the Gastrodia elata seeds.

5. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: Stain the Gastrodia elata seeds by the TTC method. Different batches of Gastrodia elata seeds show different red degrees due to different viability. Then process the stained seed pictures with Image J software, and set the threshold at the same time. Mark the pixels with a red degree higher than the threshold as the red area, and mark the pixels with a red degree lower than the threshold as the non-red area. Finally, calculate the total area of the red area to obtain the total red degree value of the Gastrodia elata seeds, and then calculate the average red degree value of the Gastrodia elata seeds to quantitatively detect the red degree of the Gastrodia elata seeds.

6. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 5, characterized in that: The threshold is set to setThreshold, 200, 255, "raw".

7. A method for quickly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: Automatically identify and count the total number of Gastrodia elata seeds in the selected pictures during the process of processing the pictures with Image J software.

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