Method for rapidly detecting germination rate of gastrodia elata seeds

Abstract

The invention belongs to the field of gastrodia elata seed germination rate detection, and discloses a method for rapidly detecting gastrodia elata seed germination rate, which comprises the following steps: dyeing gastrodia elata seeds by a red tetrazole method, randomly sucking the dyed gastrodia elata seeds, placing the gastrodia elata seeds under a stereoscopic microscope, shooting a dyed gastrodia elata seed graph, processing the dyed graph by Image J software, and determining the gastrodia elata seed germination rate. Calculating the average red degree value of the gastrodia elata seeds; meanwhile, performing a symbiotic germination experiment on the same batch of gastrodia elata seeds and germination bacteria, respectively counting the number of germinated seeds and the number of non-germinated seeds of the gastrodia elata after the seeds are germinated, and calculating the germination rate; dyeing experiments and germination experiments are carried out on multiple batches of different gastrodia elata seeds at the same time to obtain the average red degree value and germination rate of multiple groups of gastrodia elata seeds; performing linear regression analysis between the red degree value and the germination rate to obtain a regression equation; and substituting the red degree value obtained through TTC dyeing of the newly obtained gastrodia elata seeds into the regression equation to calculate the germination rate of the gastrodia elata seeds in the batch, thereby indicating the germination capability of the gastrodia elata seeds.

Description

Technical Field

[0001] The invention belongs to the field of detecting the germination rate of gastrodia elata seeds and relates to a method for quickly detecting the germination rate of gastrodia elata seeds. Background Art

[0002] Gastrodia elata is a commonly used precious Chinese medicine. It is the tuber of the orchid plant Gastrodia elata. It has the effects of calming wind and relieving spasms, calming liver yang, and dispelling wind and unblocking meridians. It is used for infantile convulsions, epilepsy, tetanus, headache, dizziness, limb paralysis, limb numbness, rheumatism, etc. Gastrodia elata has been a tonic that is both a food and a medicine since ancient times. In November 2023, my country included Gastrodia elata in the "List of Substances that are Traditionally Both Food and Chinese Medicinal Materials", that is, food and medicine substances. The demand for Gastrodia elata has increased sharply.

[0003] Gastrodia elata is widely distributed in nature. Due to the large demand for Gastrodia elata and the reduction of wild resources, it mainly relies on artificial cultivation to meet people's needs. More than 20 provinces, including Sichuan, Yunnan, Guizhou, Shaanxi, Hubei, Hunan, and Anhui, cultivate Gastrodia elata. There are two ways of reproduction of Gastrodia elata: one is sexual reproduction and the other is asexual reproduction. Among them, sexual reproduction by seeds is the main method. The vitality / germination rate of Gastrodia elata seeds directly affects the yield, quality and economic benefits of Gastrodia elata.

[0004] Gastrodia elata seeds have a simple structure and are easily inactivated.

[0005] Since the seeds of Gastrodia elata are as small as powder, have a simple structure and no endosperm. The seeds of Gastrodia elata stored under natural conditions quickly become inactive and have a shelf life of only a few days. Therefore, in the cultivation of Gastrodia elata, the slightly cracked fruits of Gastrodia elata are usually picked after the fruits mature in March-May, and after drying, the seeds are shaken out for sowing. With the rapid expansion of modern Gastrodia elata cultivation area, the cultivation area is wide, the storage time is long, and the seeds purchased and transported across regions are easily inactivated, with a low germination rate, or even completely inactivated and not germinating, causing serious losses in production. Therefore, it is necessary to evaluate the germination rate and / or vitality of Gastrodia elata seeds before sowing, determine the appropriate sowing amount, and even whether the seeds should be sown.

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

[0007] Seed vitality and germination rate are the main indicators for evaluating seed quality. Seed vitality refers to the potential ability of seeds to germinate or the vitality of seed embryos, which is usually determined by red tetrazolium (TTC) staining. The principle of TTC staining is to dye the embryos of viable seeds red, while the embryos of inactive seeds are not stained. Seed vitality 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 vitality, the degree of red dyeing is different, and the inspection personnel observe and judge the red standard by naked eye. It is difficult to accurately count the number of dyed seeds and obtain an accurate vitality value. In addition, seeds with weak vitality and light dyeing cannot germinate, which affects the yield and quality of cultivated Gastrodia elata. Only seeds with germination rate can germinate. Therefore, the germination rate of seeds is also used to directly indicate vitality.

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

[0009] It only takes 5 to 7 days to directly determine the germination rate of seeds of common crops such as corn and rice through germination experiments. However, Gastrodia seeds need to coexist with fungi such as Agaricus oxyphylla or Dendrobium candidum to germinate (collectively referred to as germination fungi), which is complicated to operate. Moreover, the germination process of Gastrodia seeds includes the invasion of germination fungi into embryo cells → the embryo expands and breaks through the epidermis → the formation of protocorms, and the whole process takes more than 30 days. If the germination rate of Gastrodia seeds stored under natural conditions is known for such a long time, the seeds are completely inactivated at this time and cannot be used for sowing and cultivation.

[0010] The prior art "A method for preserving and detecting the vitality of Gastrodia elata seeds" (patent number: CN116584252A; inventors: Huang Xiaorun, Huang Wanbing, Lu Yingying, etc. Application publication date: 2023.08.15) discloses two methods for detecting the vitality of Gastrodia elata seeds. One is the germination method of germination bacteria culture: using Dendrobium officinale as the germination bacteria, water agar as the culture medium, dark culture at 20℃~25℃, and observing the germination of Gastrodia elata seeds with a stereo microscope 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 vitality of Gastrodia elata seeds. This method takes a long time. Another method is TTC staining: use 1% TTC solution, stand in the dark at 30℃ for 48h to stain, then observe the seeds under a stereo microscope, and the seeds with orange or pink embryos are considered viable seeds, and seed viability = (number of viable seeds / total number of seeds) × 100%. This method is subjective in judging seed color, resulting in uncertain results on viability.

[0011] Prior art "A method for germinating Gastrodia elata seeds of the genus Gastrodia elata" (patent number: CN118489330A; inventors: Xu Qian, Hu Xiaojian, Lin Liang, etc. Application publication date 2024.08.16), published in the method for determining the vitality and germination rate of Gastrodia elata seeds. The method for determining the vitality of Gastrodia elata seeds by TTC staining is: Gastrodia elata seeds are stained in 1% TTC staining solution at 30°C in the dark for 48 hours, and then the number of seeds with embryos stained red is counted under a stereomicroscope to calculate the vitality of the seeds. The method for determining the germination rate is: first inoculate Osmanthus fragrans on potato glucose agar medium, then inoculate Gastrodia elata seeds 3 weeks later, and culture them in the dark 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 vitality determination and the time for germination rate determination is as long as more than 9 weeks.

[0012] The current technology "A method for evaluating the germination effect of germination fungi of Gastrodia elata seeds" (patent number: CN118483016A; inventors: Lin Liang, Hu Xiaojian, Xu Qian, etc. Application publication date: 2024.08.13) discloses different storage methods for Gastrodia elata seeds and the germination effects of different germination fungi, and involves a method for determining the vitality and germination rate of Gastrodia elata seeds. This method is consistent with the above-mentioned patent (patent number: CN118489330A), and also has problems such as large errors in vitality determination and long time for germination rate determination.

[0013] It can be seen that the existing technology has the problems of strong subjectivity in the TTC staining method for detecting the vitality of Gastrodia elata seeds, large errors in the measurement results of different technicians, long time and complex operation in the symbiotic germination experiment for determining the germination rate of Gastrodia elata seeds, etc. 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 elata seeds, thereby solving the above-mentioned technical problems.

[0015] The technical solution adopted by the present invention is as follows: A method for rapidly detecting the germination rate of gastrodia elata seeds, comprising TTC staining of gastrodia elata seeds, quantification of redness, determination of the germination rate of gastrodia elata seeds, correlation analysis between the redness value and the germination rate, determination of the redness value of new seeds and calculation of the germination rate; Dyeing of Gastrodia elata seeds The Gastrodia elata seeds were dyed by red tetrazolium (TTC) method to obtain dyed Gastrodia elata seeds; Quantification of redness Use a stereo microscope to take pictures of stained Gastrodia elata seeds, and use Image J software to calculate the average redness value of Gastrodia elata seeds in the picture (total redness value of Gastrodia elata seeds / total number of Gastrodia elata seeds) to obtain a quantified seed redness value; Determination of germination rate of Gastrodia elata seeds (determination of actual germination rate) The Gastrodia elata seeds are subjected to symbiotic germination culture with germination fungi until the germination state of the Gastrodia elata seeds is observed; the number of Gastrodia elata germinated seeds and the number of ungerminated seeds are counted under an optical microscope, and the actual germination rate of the Gastrodia elata seeds (the percentage of the number of Gastrodia elata germinated seeds divided by the total number of Gastrodia elata seeds) is calculated; Correlation analysis between redness value and germination rate The red degree value of the same batch of Gastrodia elata seeds after dyeing and the germination rate obtained by the germination experiment are a set of data; the correlation between the red degree value and the germination rate of multiple groups of different Gastrodia elata seeds is analyzed to establish a regression equation; Determination of the redness value of new Gastrodia elata seeds and calculation of germination rate The newly obtained Gastrodia elata seeds were stained with red tetrazolium (TTC) method, photographed under a stereomicroscope, and processed with Image J software to calculate the average red degree value; the average red degree value was substituted into the regression equation established above to calculate the seed germination rate (indicating the germination ability of the batch of Gastrodia elata seeds).

[0016] The working principle of the present invention is as follows: firstly dye the Gastrodia elata seeds by the TTC method; place the dyed seeds under a stereo microscope for photography to obtain a dyed Gastrodia elata seed image; process the image by using Image J software, visually display the redness of different seeds with different red areas of different sizes, and then calculate the average redness value of all Gastrodia elata seeds in the image. At the same time, a symbiotic germination experiment is performed on the same batch of Gastrodia elata seeds and germination bacteria until the seeds are observed to have germinated (protocorms), the number of Gastrodia elata germinated seeds and the number of ungerminated seeds are counted, and the actual germination rate (the percentage of the number of Gastrodia elata germinated seeds divided by the total number of Gastrodia elata seeds) is calculated.

[0017] A dyeing experiment was performed on multiple batches of different Gastrodia elata seeds to obtain redness values, and a germination experiment was performed at the same time to obtain the corresponding germination rate. A linear regression analysis was performed between the redness values ​​of multiple groups of different Gastrodia elata seeds and the actual germination rate to obtain a regression equation. The average redness value obtained by TTC dyeing, photography, and image processing of the newly obtained Gastrodia elata seeds was substituted into the regression equation to calculate the germination rate of the Gastrodia elata seeds.

[0018] Therefore, the present invention photographs the gastrodia elata seeds after TTC staining through a stereo microscope, processes the stained image through Image J software, sets a threshold value at the same time, marks pixels with a red degree higher than the threshold value as a red area, and marks pixels with a red degree lower than the threshold value as a non-red area, finally calculates the total area of ​​the red area (standardization of image area recognition), obtains the total red degree value of the gastrodia elata seeds, and then calculates the average red degree value of the gastrodia elata seeds (total red degree value of the gastrodia elata seeds / total number of gastrodia elata seeds), so that the red degree of the gastrodia elata seeds is quantitatively detected, and the red degree value objectively reflecting the gastrodia elata seeds after staining is obtained, thereby avoiding errors in judgment by different detection personnel based on naked eyes and making the measurement result more objective.

[0019] At the same time, the actual germination rate was obtained by conducting a germination experiment on the batch of Gastrodia elata seeds. Then, a regression equation was established by analyzing the correlation between the average red degree values ​​of multiple batches of different Gastrodia elata seeds and the germination rate. The newly obtained Gastrodia elata seeds only need to measure their average red degree values ​​after dyeing and substitute them into the regression equation to obtain the germination rate, which shortens the time for measuring the germination rate of Gastrodia elata seeds by more than 30 days.

[0020] Further: the parameters for photographing the Gastrodia elata seeds with a stereo microscope after staining are: LDE top light Light = 2, bottom light Light = 0, objective lens 0.63X, eyepiece 1X, and magnification 42X; select RGB mode, select red 30%, green 64%, and blue 15% for white balance, the exposure time is 2600.00ms, and the saturation is set to the maximum; adjust the field of view to be clear and then take pictures; randomly select more than 3 fields of view for the same batch of Gastrodia elata stained seeds and take pictures separately to obtain more than 3 pictures.

[0021] Further: After the cultivation of Gastrodia elata seeds in the actual germination experiment, 1 cm 2 The germinated seeds and ungerminated seeds on the culture medium with Gastrodia elata seeds are counted under an optical microscope. The number of Gastrodia elata seeds on each culture medium should be greater than 50, and multiple samples are taken in parallel to calculate the average germination rate.

[0022] Furthermore: after the newly obtained Gastrodia elata seeds are mixed, a portion of the Gastrodia elata seeds are randomly selected and subjected to the dyeing method and the image processing method to obtain the average red degree value of the batch of Gastrodia elata seeds, and the average red degree value is substituted into the regression equation to calculate the germination rate of the Gastrodia elata seeds.

[0023] Further: the Gastrodia elata seeds stained by the TTC method have different degrees of redness. They are photographed under the same conditions under a stereomicroscope, and then the images are processed by Image J software. At the same time, a threshold is set, and pixels with redness values ​​higher than the threshold are marked as red areas, while pixels with redness values ​​lower than the threshold are marked as non-red areas. The total area of ​​the red area is calculated (standardization of image area recognition) to obtain the total redness value of the Gastrodia elata seeds, and then the average redness value of the Gastrodia elata seeds is calculated (total redness value of the Gastrodia elata seeds / total number of Gastrodia elata seeds), so that the redness of the Gastrodia elata seeds can be quantitatively detected.

[0024] Further: The threshold is set to setThreshold(200, 255, "raw").

[0025] Furthermore: the total number of Gastrodia elata seeds in the selected image is automatically counted during the image processing by Image J software.

[0026] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. A method for quickly detecting the germination rate of Gastrodia elata seeds, quantifying the results of Gastrodia elata seed dyeing, thereby making the judgment of the vitality of Gastrodia elata seeds more objective; at the same time, determining the germination rate of the batch of Gastrodia elata seeds; analyzing the quantified red degree value and the actual germination rate and establishing a regression equation; dyeing and quantifying the newly obtained Gastrodia elata seeds to obtain an average red degree value, substituting it into the regression equation, and then calculating the germination rate of the Gastrodia elata seeds.

[0027] 2. In the present invention, the dyeing conditions of Gastrodia elata seeds are standardized, the photography is standardized, and the red degree of the Gastrodia elata seed dyeing image is quantified by Image J software to obtain the objective value of the vitality of Gastrodia elata seeds, thereby avoiding the visual differences of different inspectors observing Gastrodia elata seeds under a microscope and the errors between the vitality measurement results caused by different judgment standards for dyed seeds, which directly affect the production of Gastrodia elata.

[0028] 3. In the present invention, the average red color value obtained from the staining experiment is substituted into the regression equation to 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 similar to the actual germination rate, which verifies that the present invention is fast and accurate in detecting the germination rate of Gastrodia elata seeds.

[0029] 4. In the present invention, it is only necessary to determine the average red color degree value (vitality) of the Gastrodia elata seeds and substitute it into the regression equation to calculate the germination rate of the Gastrodia elata seeds. The time for obtaining the germination rate through the Gastrodia elata seed symbiotic germination experiment is shortened from more than 30 days to less than 2 days, which greatly shortens the time and operation cost of the germination experiment, provides germination rate data in a timely manner, and provides a basis for Gastrodia elata sowing. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative work, among which: Figure 1 It is a schematic diagram of the process of the present invention; Figure 2 is a linear relationship diagram between the average red degree value of Gastrodia elata seeds and the germination rate in the present invention; Figure 3 is a schematic diagram of Gastrodia elata seeds photographed under a stereo microscope after being stained by the TTC method in the present invention; Figure 4 In the present invention, Image J software is used to Figure 3 The red schematic diagram is quantified by the schematic diagram in the figure; Figure 5 It is a comparison diagram of the Gastrodia elata seeds that have germinated and those that have not germinated in the present invention (wherein the letter a represents seeds that have germinated into protocorms, and the letter b represents seeds that have not germinated). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying 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 embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0032] Therefore, 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 invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0033] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0034] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0035] Embodiment 1

[0036] The present invention provides a method for rapidly detecting the germination rate of Gastrodia elata seeds. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown, the specific implementation of this embodiment is: Twenty different batches of red Gastrodia elata seeds were selected as model samples. After mixing the same batch of red Gastrodia elata seeds, several portions of red Gastrodia elata seeds were randomly selected. Half of each portion of red Gastrodia elata seeds was used for TTC staining to determine the average red color value; the other half was used for germination experiment to determine the germination rate.

[0037] TTC staining of red Gastrodia elata seeds: 20 batches of red Gastrodia elata seeds were stained separately, and the TTC staining conditions were TTC solution concentration of 1.5%, temperature of 30℃, and light-proof staining time of 40h; Photographing the stained seeds: After the staining is completed, randomly absorb the stained seeds and place them under a stereo microscope to photograph the stained Gastrodia elata seeds. The photographic parameters are: LDE top light Light = 2, bottom light Light = 0, objective lens 0.63X, eyepiece 1X, magnification 42X; select RGB mode, white balance selects red 30%, green 64%, blue 15%, exposure time 2600.00ms, saturation is set to maximum; adjust the field of view to be clear before photographing; randomly select more than 3 fields of view of the same portion of Gastrodia elata stained seeds and photograph them separately to obtain more than 3 pictures; Counting the Gastrodia elata seeds in the image: Analyze the image using Image J software, write a program through macros, use Subtract Background, Gaussian Blur, and Image Calculator plug-ins to remove the influence of the background, and then use the Analyze Particles plug-in to limit the size of particles identified as background to calculate the total number of Gastrodia elata seeds; Calculate the average red value of the Gastrodia elata seeds in the image: In Image J software, use the macro program to remove the background effect using the Subtract Background, Gaussian Blur, and Image Calculator plug-ins, convert the image to LAB space, select the A channel, set setThreshold (200, 255, "raw"), mark the pixels with redness higher than the threshold as red areas, and mark the pixels with redness lower than the threshold as non-red areas, calculate the total area of ​​the red area in the image; divide the total red area value by the total number of Gastrodia elata seeds to obtain the average red value of the Gastrodia elata seeds in the image. The average red values ​​of 20 batches of different red Gastrodia elata seeds are shown in Table 1.

[0038]

[0039] Germination test method to determine the germination rate of Gastrodia elata seeds: Weigh 30 g of sucrose, 2.37 g of MS medium solid powder, and 6.5 g of agar powder into a conical flask, add 1 L of ultrapure water to prepare 1 / 2 MS medium; wrap the culture dish with kraft paper, put it in a high-pressure steam sterilizer together with a bottle of ultrapure water, and sterilize it at 121°C for 30 min; prepare 100 mL each of 70% alcohol and 0.1% sodium hypochlorite solution for disinfection of Gastrodia elata seeds; prepare filter paper, metal medicine spoon, glass funnel, conical flask, glass dropper, and cut the sealing film; and spray 10% sodium hypochlorite solution on the sealing film.

[0040] 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.

[0041] Take out the germinated bacteria from the refrigerator and inoculate 1 cm 3Germination 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.

[0042] 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.

[0043] 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.

[0044] Reliability verification of predicted germination rate: 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.

[0045]

[0046] The invention allows each batch of gastrodia elata seeds to be subjected to TTC dyeing to obtain an average red degree value, and the average red degree value is brought into the regression equation of the invention to calculate and predict the germination rate, thereby greatly shortening the time of the germination experiment.

[0047] After the existing TTC method is used to dye the Gastrodia elata seeds, it is observed under a microscope that the Gastrodia elata seeds show different degrees of red. Different testers have different judgment standards on whether the Gastrodia elata seeds are dyed red, and the statistical number of viable seeds is different, so the calculated vitality values ​​are different. Since the Gastrodia elata seeds are small, there are errors in manually counting the number of viable seeds and the number of non-viable seeds under a microscope, which ultimately leads to large errors in the judgment results of the vitality of the Gastrodia elata seeds.

[0048] Therefore, the present invention is to carry out standardized photography to the seeds after dyeing by TTC method, process the Gastrodia elata seed image after dyeing with Image J software, automatically identify and calculate the total number of Gastrodia elata seeds. Simultaneously, a threshold is set in the Image J software, pixels with red degree values ​​higher than the threshold are marked as red areas, and pixels with red degree values ​​lower than the threshold are marked as non-red areas, Gastrodia elata seeds with different red degrees present red areas of different sizes, all red areas are added up, the total area of ​​the red areas (standardization of image area recognition) is obtained, i.e., the total red degree value of Gastrodia elata seeds in the figure, and then the average red degree value of Gastrodia elata seeds (total red degree value of Gastrodia elata seeds / total number of Gastrodia elata seeds) is calculated, so that the red degree of Gastrodia elata seeds is quantified, and the vitality data after the Gastrodia elata seeds are objectively reflected are obtained.

[0049] The present invention does not require inspectors to observe and count the number of seeds and calculate the viability with naked eyes under a microscope, thus avoiding errors in seed viability caused by visual errors and different judgment standards of inspectors, which in turn affect the production of Gastrodia elata.

[0050] Embodiment 2

[0051] The present invention provides a method for rapidly detecting the germination rate of Gastrodia elata seeds. Figure 1 As shown, the specific implementation of this embodiment is: Dyeing temperature test: In order to preliminarily select the dyeing temperature of red Gastrodia elata seeds, the concentration of TTC dyeing solution was set to 1.5%, and the dyeing time was 36 h. The average red color values ​​of Gastrodia elata seeds at dyeing temperatures of 25, 30, 35, and 40°C were compared.

[0052] Dyeing time test: In order to select the dyeing time of Gastrodia elata seeds, the concentration of TTC dyeing solution was set to 1.5% and the dyeing temperature was set to 30℃. The average red color values ​​of Gastrodia elata seeds with dyeing time of 12, 24, 36, 48 and 60 h were compared.

[0053] Selection of TTC staining solution concentration: In order to select the concentration of TTC staining solution for Gastrodia elata seeds, the staining time was set to 36 h and the staining temperature was set to 30 °C. The average red color values ​​of Gastrodia elata seeds with staining solution concentrations of 0.1, 0.5, 1, 1.5, and 2% were compared.

[0054] According to the above single factor experimental results, a three-factor three-level response surface experiment (Box-Behnken) was designed with dyeing temperature A, dyeing time B, and dyeing solution concentration C to screen out the optimal conditions for dyeing red Gastrodia elata seeds by TTC method: temperature of 30℃, dyeing time of 40h, and dyeing solution concentration of 1.5%.

[0055] The above description is only an embodiment of the red Gastrodia elata seeds of the present invention. Except for adjusting the TTC staining conditions, other technical routes and parameters are still applicable to the seeds of different variants of Gastrodia elata (red Gastrodia elata, black Gastrodia elata, green Gastrodia elata, yellow Gastrodia elata and hybrid Gastrodia elata). It is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions and improvements made by any technician familiar with the field within the principles and technical routes of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for rapidly detecting the germination rate of Gastrodia elata seeds, characterized in that: It includes TTC staining of Gastrodia elata seeds, quantification of redness, determination of germination rate of Gastrodia elata seeds, correlation analysis between redness value and germination rate, determination of redness value of new seeds and calculation of germination rate; Dyeing of Gastrodia elata seeds The Gastrodia elata seeds are dyed by red tetrazolium method to obtain dyed Gastrodia elata seeds; Quantification of redness The dyed Gastrodia elata seeds were photographed with a stereo microscope, and the average redness value of the Gastrodia elata seeds in the image was calculated with Image J software to obtain a quantified redness value of the seeds; Determination of germination rate of Gastrodia elata seeds The Gastrodia elata seeds are subjected to symbiotic germination culture with germination bacteria until the germination state of the Gastrodia elata seeds is observed; the number of Gastrodia elata germinated seeds and the number of ungerminated seeds are counted under a microscope to calculate the actual germination rate of the Gastrodia elata seeds; Correlation analysis between redness value and germination rate The redness value of the same batch of Gastrodia elata seeds after dyeing and the germination rate obtained from the germination experiment are called a set of data; The linear correlation analysis between the redness values ​​and germination rates of different groups of Gastrodia elata seeds was conducted to establish a regression equation. Determination of the redness value of new Gastrodia elata seeds and calculation of germination rate The newly obtained Gastrodia elata seeds were stained with red tetrazolium method, photographed with stereo microscope, processed with Image J software, and the red degree value was calculated; the red degree value was substituted into the above regression equation to calculate the seed germination rate.

2. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: The stained Gastrodia elata seeds were photographed in a standardized manner using a stereo microscope, and the photographic parameters were: LDE top light Light = 2, bottom light Light = 0, objective lens 0.63X, eyepiece 1X, and magnification 42X; RGB mode was selected, white balance selected red 30%, green 64%, and blue 15%, the exposure time was 2600.00ms, and the saturation was set to the maximum; the Gastrodia elata seeds in the field of view were adjusted to be clear and then photographed; more than 3 fields of view were randomly selected from the same batch of stained Gastrodia elata seeds and photographed separately to obtain more than 3 images.

3. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: After the Gastrodia elata seed germination experiment was completed, 1 cm 2 The number of germinated and ungerminated Gastrodia elata seeds on the culture medium with Gastrodia elata seeds is counted under an optical microscope. The number of Gastrodia elata seeds on each culture medium should be greater than 50. Multiple samples are taken in parallel, and the average germination rate is calculated.

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, randomly select a portion of the Gastrodia elata seeds and use the dyeing method and the processing method of the dyed seed diagram to obtain the average red degree value of the batch of Gastrodia elata seeds. Substituting it into the regression equation, the germination rate of the Gastrodia elata seeds can be calculated.

5. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: Gastrodia elata seeds were stained by the TTC method. Different batches of Gastrodia elata seeds showed different degrees of red due to different vitality. The stained seed images were then processed by Image J software, and a threshold was set. Pixels with a red degree higher than the threshold were marked as red areas, while pixels with a red degree lower than the threshold were marked as non-red areas. Finally, the total area of ​​the red area was calculated to obtain the total red degree value of the Gastrodia elata seeds, and then the average red degree value of the Gastrodia elata seeds was calculated, so that the red degree of the Gastrodia elata seeds could be quantitatively detected.

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

7. A method for rapidly detecting the germination rate of Gastrodia elata seeds according to claim 1, characterized in that: The total number of Gastrodia elata seeds in the selected image was automatically identified and counted during image processing using Image J software.

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