Method for rapidly detecting the fullness rate of orchid seeds

Abstract

This invention discloses a rapid method for detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; and seed recovery. During microscopic examination, a cold light source is used, transmitted upwards from below the seed. This method solves the problems of difficulty and long processing times associated with germination detection or TZ staining for plumpness detection. The detection method provided by this invention can rapidly, simply, efficiently, and accurately detect the plumpness of orchid seeds. Furthermore, the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for long-term use. The effect is significant, opening up a completely new technical route for the rapid detection of orchid seed plumpness.

Description

Technical Field

[0001] This invention relates to the field of germplasm resource preservation technology, and in particular to a method for rapidly detecting the plumpness of orchid seeds. Background Technology

[0002] According to *Flora of China* and related literature, there are over 27,500 species in the Orchidaceae family, with 1,723 found in my country. Many species in this family possess significant ornamental and medicinal value, impacting various aspects of art, culture, economy, medicine, and daily life. For example, *Cymbidium goeringii*, a renowned traditional Chinese orchid, is deeply loved by the Chinese people; *Paphiopedilum* species have long flowering periods and are excellent floral germplasm resources; *Dendrobium* species all possess high medicinal value, with *Dendrobium nobile* being an extremely rare and precious traditional Chinese medicine recorded in the *Pharmacopoeia of the People's Republic of China*. It is not only an essential ingredient in *Mailuoning* injection but also an effective component of several traditional Chinese medicines and health products; *Dendrobium officinale* is an important rare and endangered medicinal plant resource in China, its stems used medicinally for their effects of clearing heat and nourishing yin, moistening the lungs and promoting fluid production, tonifying the kidneys and stomach, and improving eyesight.

[0003] However, due to the close relationship between orchids and symbiotic fungi in their life cycle, orchid reproduction is extremely demanding in terms of habitat requirements. Simultaneously, orchid reproduction is highly dependent on other organisms (such as pollinators), making them highly sensitive to human disturbance and environmental factors. Furthermore, due to their valuable ornamental and medicinal properties, severe over-harvesting has led to a continuous decline in wild resources. These complex reasons have ultimately resulted in many orchid species currently facing extinction. The most clearly identified major factors contributing to their endangerment are the overexploitation and utilization of their habitats. Among existing orchid conservation methods, ex-situ conservation is considered one of the most effective, and seed banks are one of the most economical and effective ex-situ conservation methods. The most crucial prerequisite for ex-situ conservation is obtaining healthy, plump seeds; therefore, finding a rapid, efficient, and accurate method to detect the plumpness and quality of orchid seeds is particularly important.

[0004] Seeds are important materials for the conservation of orchid species diversity and the long-term preservation of germplasm. It is particularly important to conduct systematic research on the storage characteristics and long-term preservation methods of orchid seeds. Whether it is germplasm resource collection and preservation, seed biology research, in-situ conservation, ex-situ conservation and research utilization, it is necessary to collect and preserve a large number of orchid seeds. It is very important to preserve as many high-quality seeds as possible in a limited space.

[0005] However, traditional methods of seed quality testing (germination testing or TZ staining) are difficult and time-consuming. Germination is extremely difficult, and the TZ staining rate is hard to determine accurately. Therefore, there is an urgent need to find a fast and accurate method to detect the plumpness of orchid seeds and solve the problem of seed quality during the collection, cleaning, germination, and preservation of orchid seeds. At the same time, it is impractical to directly observe the plumpness of orchid seeds with the naked eye to detect seed quality, because orchid seeds are the smallest known seeds in the world, mostly as fine as dust. For example, 50,000 seeds of *Cymbidium goeringii* weigh only 0.025g, and 100 million seeds of *Cymbidium goeringii* weigh only 50g. Its seeds are about 110μm long and weigh about 0.5μg. For such tiny seeds, even under a microscope, it is often difficult to clearly see the internal state of the seed, making it difficult to detect whether the seed is plump.

[0006] Therefore, exploring a rapid method for detecting the plumpness of orchid seeds is of great significance for the long-term preservation of seeds, the establishment of orchid germplasm resource banks, and the improvement of the orchid germplasm resource preservation system. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for rapidly detecting the plumpness of orchid seeds.

[0008] The objective of this invention is achieved through the following technical solution: a method for rapidly detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; and seed recovery; wherein, during microscopic examination, a cold light source is used to transmit the cold light source upward from below the seed; Furthermore, the sampling step involves placing the seeds in a sampling tray and shaking them thoroughly, then randomly selecting 2 to 4 seed samples, with each seed sample containing 80 to 100 seeds. Furthermore, the pretreatment step is to place the seed sample on the microscope base or slide, add physiological saline to the microscope base or slide, and let it stand for 2-3 minutes until the seeds are moistened to obtain the sample to be tested. Furthermore, the microscopic examination step is as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, and adjust the focus until the seed morphology is clear. Furthermore, the cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx; Furthermore, the plumpness detection step is to count the number of seeds, plump seeds, and empty seeds in a sample, and calculate the seed plumpness according to the following formula (Ⅰ); (I); Furthermore, the sampling step is preceded by a fruit collection step; A further technical solution is to harvest the fruit when the capsule is about to split open or when the seeds have just begun to disperse.

[0009] The present invention has the following advantages: 1. This invention employs an improved microscopic examination method, solving the problems of high difficulty and long cycle in germination detection or TZ staining detection of seed plumpness. The detection method provided by this invention can quickly, easily, efficiently, and accurately detect the plumpness of orchid seeds. Moreover, the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for a long time, with significant effects. It opens up a completely new technical route for the rapid detection of orchid seed plumpness. 2. Preferably, this invention uses microscopic examination to observe whether orchid seeds are plump or shriveled. During the microscopic examination, a cold light source is transmitted from below the seed upwards, penetrating the seed, while the observer is above the seed. The cold light source can avoid overheating and damaging the seed. When the cold light source penetrates from below, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the seed morphology to be clearly presented in the field of vision. From the test results, the boundary between the seed coat and the embryo of plump orchid seeds is clear and distinct, and the morphology of the embryo is clear, while the embryo structure cannot be observed inside the seed coat of shriveled orchid seeds. 3. Preferably, during sampling, the seeds are placed in a sampling tray and shaken well before random sampling is performed to ensure the uniformity of the samples and the randomness of the sampling; taking 3 samples, each with 80 to 100 seeds, is beneficial to the accuracy of the results. 4. Preferably, the seeds are pretreated by placing the seed sample on the microscope base or slide and adding physiological saline. Physiological saline helps maintain the seed cell morphology and avoids changes in seed morphology during observation. At the same time, physiological saline helps make the seed texture more uniform and increases light transmittance, which helps avoid unclear images due to agglomeration when cold light is transmitted. 5. Preferably, in this invention, the cold light source located below the stage is turned on, allowing the cold light source to penetrate the seeds. The magnification is selected and the focus is adjusted until the seeds are clear. The power of the cold light source is 30-50W, the color temperature is 3200-6500K, and the illuminance is 50000-120000Lx. The cold light source can avoid overheating and damaging the seeds. Too high an illuminance will cause the seeds to turn white and make it difficult to identify plump seeds. Too low an illuminance will result in insufficient light transmission and make it difficult to identify empty or shriveled seeds. 6. Preferably, this invention provides a method for calculating seed plumpness. Through the calculation formula, the seed plumpness in a sample can be calculated, which is beneficial for providing data support for subsequent seed preservation and germination, and opens up a seed detection technology route that does not require germination or TZ staining. 7. Preferably, the present invention does not cause any damage to the seeds during the seed plumpness detection process. The seeds after the detection is completed can also be used for germination, long-term preservation, and morphological photography. Attached Figure Description

[0010] Figure 1 The results were observed using the method described in Example 5 for Cymbidium goeringii.

[0011] Figure 2 The results were observed using the method of Comparative Example 1 for Cymbidium sclerophyllum.

[0012] Figure 3 The results were observed using the method in Comparative Example 2 for Cymbidium sclerophyllum.

[0013] Figure 4 The results were observed using the method described in Example 6 for the Ryukyu Forked Orchid.

[0014] Figure 5 The results were observed using the method of Comparative Example 3 for the Ryukyu Forked Orchid.

[0015] Figure 6 The results were observed using the method in Comparative Example 4 for the Ryukyu Forked Orchid.

[0016] Figure 7 The results are for Example 4.

[0017] Figure 8 This is a schematic diagram of the morphology of an empty seed. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0021] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0022] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship commonly used when the product of this invention is in use, or the orientation or positional relationship commonly understood by those skilled in the art. They are only used for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. In addition, the terms "first," "second," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0023] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0024] Example 1: A method for rapidly detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; seed recovery; wherein, during microscopic examination, a cold light source is transmitted from the bottom of the seed upwards; this invention adopts an improved microscopic examination method, solving the problems of difficulty and long cycle in germination detection or TZ staining detection of plumpness. The detection method provided by this invention can quickly, easily, efficiently, and accurately detect the plumpness of orchid seeds, and the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for long-term use; the effect is significant; it opens up a completely new technical route for the rapid detection of orchid seed plumpness; using microscopic examination, the plumpness or shriveledness of orchid seeds is observed. During the microscopic examination, a cold light source is transmitted from the bottom of the seed upwards, penetrating the seed, while the observer is above the seed. The cold light source can avoid overheating and damaging the seed. When the cold light source penetrates from bottom to top, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the morphology of the seed to be clearly presented in the field of view. Figure 1 , 4 Based on the test results of 7, the boundary between the seed coat and the embryo of plump orchid seeds is clear and distinct, and the embryo morphology is clear, while the embryo structure cannot be observed in the seed coat of shriveled orchid seeds.

[0025] The sampling steps are as follows: after placing the seeds in a sampling tray and shaking them thoroughly, 2 to 4 seed samples are randomly selected, with each seed sample containing 80 to 100 seeds; during sampling, the seeds are placed in the sampling tray and shaken thoroughly before randomly selecting samples to ensure the uniformity of the samples and the randomness of the sampling; taking 3 samples, with each sample containing 80 to 100 seeds, is beneficial to the accuracy of the results.

[0026] The pretreatment step is as follows: place the seeds on the microscope base or slide, add physiological saline solution, and let stand for 2-3 minutes until the seeds are moistened to obtain the sample to be tested; pretreatment of the seeds involves placing the seed sample on the microscope base or slide and adding physiological saline solution. Physiological saline solution helps maintain the seed cell morphology and avoids changes in seed morphology during observation. At the same time, physiological saline solution helps make the seed texture more uniform, increases light transmittance, and helps avoid unclear images due to aggregation when cold light source is transmitted.

[0027] The microscopic examination steps are as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, and adjust the focus until the seed morphology is clear. The cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx. After turning on the cold light source located below the stage, allowing it to penetrate the seeds, select the magnification and adjust the focus until the image is clear. The cold light source can prevent overheating from concentrated light, which can damage the seeds. Excessive illuminance causes the seeds to appear whitish, making it difficult to identify plump seeds. Insufficient illuminance results in insufficient light transmission, making it difficult to identify empty or shriveled seeds.

[0028] The seed plumpness detection step is to count the total number of seeds, the number of plump seeds and the number of empty seeds in a sample, and calculate the seed plumpness according to the following formula (Ⅰ): Through the calculation formula, the seed plumpness in a sample can be calculated, which is beneficial to provide data support for the subsequent preservation and germination of seeds, and opens up a seed detection technology route that does not require germination or TZ staining. (I); Before the sampling step, there is also a fruit collection step; before sampling, the capsules of Salicaceae seeds are collected, and the seeds can be obtained after opening the capsules.

[0029] The optimal time for fruit collection is when the capsule is about to split open or when the seeds have just begun to disperse. For some species in the Salicaceae family, the fruit turns yellow, and fruits collected just before splitting open have high seed viability, which is beneficial for subsequent observation. For other species, the fruit does not turn yellow at maturity, so fruits are collected when the seeds have just begun to disperse. Collecting too early results in immature seeds, a high rate of empty shriveles, and seeds that are not resistant to dehydration. Collecting too late leads to the large fruits already dispersed, and seed viability rapidly declines, which is detrimental to later processing and long-term preservation.

[0030] Example 2: A method for rapidly detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; seed recovery; wherein, during microscopic examination, a cold light source is transmitted from the bottom of the seed upwards; this invention adopts an improved microscopic examination method, solving the problems of difficulty and long cycle in germination detection or TZ staining detection of plumpness. The detection method provided by this invention can quickly, easily, efficiently, and accurately detect the plumpness of orchid seeds, and the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for long-term use; the effect is significant; it opens up a completely new technical route for the rapid detection of orchid seed plumpness; using microscopic examination, the plumpness or shriveledness of orchid seeds is observed. During the microscopic examination, a cold light source is transmitted from the bottom of the seed upwards, penetrating the seed, while the observer is above the seed. The cold light source can avoid overheating and damaging the seed. When the cold light source penetrates from bottom to top, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the morphology of the seed to be clearly presented in the field of view. Figure 1 , 4 Based on the test results of 7, the boundary between the seed coat and the embryo of plump orchid seeds is clear and distinct, and the embryo morphology is clear, while the embryo structure cannot be observed in the seed coat of shriveled orchid seeds.

[0031] The sampling steps are as follows: after placing the seeds in a sampling tray and shaking them thoroughly, two seed samples are randomly selected, with 80 seeds in each sample. The sampling process involves placing the seeds in the sampling tray, shaking them thoroughly, and then randomly selecting samples to ensure the uniformity of the samples and the randomness of the sampling. Taking two samples, with 80 seeds in each sample, helps to improve the accuracy of the results.

[0032] The pretreatment step is as follows: place the seeds on the microscope base or slide, add physiological saline to the microscope base or slide, and let stand for 2 minutes until the seeds are moistened to obtain the sample to be tested; the seeds are pretreated by placing the seed sample on the microscope base or slide and adding physiological saline. Physiological saline helps maintain the seed cell morphology and avoids changes in seed morphology during observation. At the same time, physiological saline helps make the seed texture more uniform, increases light transmittance, and helps avoid unclear images due to agglomeration when cold light is transmitted.

[0033] The microscopic examination steps are as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, orchid seeds are generally magnified to 4x-30x, and adjust the focus until the seed morphology is clear. The cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx. After turning on the cold light source located below the stage, allowing it to penetrate the seeds, select the magnification and adjust the focus until the image is clear. The cold light source can prevent overheating from concentrated light, which can damage the seeds. Excessive illuminance causes the seeds to appear whitish, making it difficult to identify plump seeds. Insufficient illuminance results in insufficient light transmission, making it difficult to identify empty or shriveled seeds.

[0034] The seed plumpness detection step is to count the total number of seeds, the number of plump seeds and the number of empty seeds in a sample, and calculate the seed plumpness according to the following formula (Ⅰ): Through the calculation formula, the seed plumpness in a sample can be calculated, which is beneficial to provide data support for the subsequent preservation and germination of seeds, and opens up a seed detection technology route that does not require germination or TZ staining. (I); Before the sampling step, there is also a fruit collection step; before sampling, the capsules of Salicaceae seeds are collected, and the seeds can be obtained after opening the capsules.

[0035] The optimal time for fruit collection is when the capsule is about to split open or when the seeds have just begun to disperse. For some species in the Salicaceae family, the fruit turns yellow, and fruits collected just before splitting open have high seed viability, which is beneficial for subsequent observation. For other species, the fruit does not turn yellow at maturity, so fruits are collected when the seeds have just begun to disperse. Collecting too early results in immature seeds, a high rate of empty shriveles, and seeds that are not resistant to dehydration. Collecting too late leads to the large fruits already dispersed, and seed viability rapidly declines, which is detrimental to later processing and long-term preservation.

[0036] Example 3: A method for rapidly detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; seed recovery; wherein, during microscopic examination, a cold light source is transmitted from the bottom of the seed upwards; this invention adopts an improved microscopic examination method, solving the problems of difficulty and long cycle in germination detection or TZ staining detection of plumpness. The detection method provided by this invention can quickly, easily, efficiently, and accurately detect the plumpness of orchid seeds, and the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for long-term use; the effect is significant; it opens up a completely new technical route for the rapid detection of orchid seed plumpness; using microscopic examination, the plumpness or shriveledness of orchid seeds is observed. During the microscopic examination, a cold light source is transmitted from the bottom of the seed upwards, penetrating the seed, while the observer is above the seed. The cold light source can avoid overheating and damaging the seed. When the cold light source penetrates from bottom to top, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the morphology of the seed to be clearly presented in the field of view. Figure 1 , 4 Based on the test results of 7, the boundary between the seed coat and the embryo of plump orchid seeds is clear and distinct, and the morphological structure of the embryo is clearly visible, while the embryo structure cannot be observed in the seed coat of shriveled orchid seeds.

[0037] The sampling steps are as follows: after placing the seeds in the sampling tray and shaking them thoroughly, four seed samples are randomly selected, with 100 seeds in each sample. The sampling process involves placing the seeds in the sampling tray, shaking them thoroughly, and then randomly selecting samples to ensure the uniformity of the samples and the randomness of the sampling. Taking four samples, with 100 seeds in each sample, helps to improve the accuracy of the results.

[0038] The pretreatment step is as follows: place the seeds on the microscope base or slide, add physiological saline to the microscope base or slide, and let stand for 3 minutes until the seeds are moistened to obtain the sample to be tested; the seeds are pretreated by placing the seed sample on the microscope base or slide and adding physiological saline. Physiological saline helps maintain the seed cell morphology and avoids changes in seed morphology during observation. At the same time, physiological saline helps make the seed texture more uniform, increases light transmittance, and helps avoid unclear images due to agglomeration when cold light is transmitted.

[0039] The microscopic examination steps are as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, and adjust the focus until the seed morphology is clear. The cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx. After turning on the cold light source located below the stage, allowing it to penetrate the seeds, select the magnification and adjust the focus until the image is clear. The cold light source can prevent overheating from concentrated light, which can damage the seeds. Excessive illuminance causes the seeds to appear whitish, making it difficult to identify plump seeds. Insufficient illuminance results in insufficient light transmission, making it difficult to identify empty or shriveled seeds.

[0040] The seed plumpness detection step is to count the total number of seeds, the number of plump seeds and the number of empty seeds in a sample, and calculate the seed plumpness according to the following formula (Ⅰ): Through the calculation formula, the seed plumpness in a sample can be calculated, which is beneficial to provide data support for the subsequent preservation and germination of seeds, and opens up a seed detection technology route that does not require germination or TZ staining. (I); Before the sampling step, there is also a fruit collection step; before sampling, the capsules of Salicaceae seeds are collected, and the seeds can be obtained after opening the capsules.

[0041] The optimal time for fruit collection is when the capsule is about to split open or when the seeds have just begun to disperse. For some species in the Salicaceae family, the fruit turns yellow, and fruits collected just before splitting open have high seed viability, which is beneficial for subsequent observation. For other species, the fruit does not turn yellow at maturity, so fruits are collected when the seeds have just begun to disperse. Collecting too early results in immature seeds, a high rate of empty shriveles, and seeds that are not resistant to dehydration. Collecting too late leads to the large fruits already dispersed, and seed viability rapidly declines, which is detrimental to later processing and long-term preservation.

[0042] Example 4: A method for rapidly detecting the plumpness of orchid seeds, comprising the following steps: sampling; pretreatment; microscopic examination; plumpness detection; seed recovery; wherein, during microscopic examination, a cold light source is transmitted from the bottom of the seed upwards; this invention adopts an improved microscopic examination method, solving the problems of difficulty and long cycle in germination detection or TZ staining detection of plumpness. The detection method provided by this invention can quickly, simply, efficiently, and accurately detect the plumpness of orchid seeds, and the orchid seeds detected by this method are undamaged and can continue to germinate or be stored for long-term use; the effect is significant; it opens up a completely new technical route for the rapid detection of orchid seed plumpness; using microscopic examination, the plumpness or shriveledness of orchid seeds is observed. During the microscopic examination, a cold light source is transmitted from the bottom of the seed upwards, penetrating the seed, while the observer is above the seed. The cold light source can avoid overheating and damaging the seed. When the cold light source penetrates from bottom to top, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the morphology of the seed to be clearly presented in the field of view. Figure 1 , 4 Based on the test results of 7, the boundary between the seed coat and the embryo of plump orchid seeds is clear and distinct, and the embryo morphology is clear, while the embryo structure cannot be observed in the seed coat of shriveled orchid seeds.

[0043] The sampling steps are as follows: after placing the seeds in the sampling tray and shaking them thoroughly, three seed samples are randomly selected, with 90 seeds in each sample. The sampling process involves placing the seeds in the sampling tray, shaking them thoroughly, and then randomly selecting samples to ensure the uniformity of the samples and the randomness of the sampling. Taking three samples, with 90 seeds in each sample, helps to improve the accuracy of the results.

[0044] The pretreatment step is as follows: place the seeds on the microscope base or slide, add physiological saline to the microscope base or slide, and let stand for 2 minutes until the seeds are moistened to obtain the sample to be tested; the seeds are pretreated by placing the seed sample on the microscope base or slide and adding physiological saline. Physiological saline helps maintain the seed cell morphology and avoids changes in seed morphology during observation. At the same time, physiological saline helps make the seed texture more uniform, increases light transmittance, and helps avoid unclear images due to agglomeration when cold light is transmitted.

[0045] The microscopic examination steps are as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, and adjust the focus until the seed morphology is clear. The cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx. After turning on the cold light source located below the stage, allowing it to penetrate the seeds, select the magnification and adjust the focus until the image is clear. The cold light source can prevent overheating from concentrated light, which can damage the seeds. Excessive illuminance causes the seeds to appear whitish, making it difficult to identify plump seeds. Insufficient illuminance results in insufficient light transmission, making it difficult to identify empty or shriveled seeds.

[0046] The seed plumpness detection step is to count the total number of seeds, the number of plump seeds and the number of empty seeds in a sample, and calculate the seed plumpness according to the following formula (Ⅰ): Through the calculation formula, the seed plumpness in a sample can be calculated, which is beneficial to provide data support for the subsequent preservation and germination of seeds, and opens up a seed detection technology route that does not require germination or TZ staining. (I); Before the sampling step, there is also a fruit collection step; before sampling, the capsules of Salicaceae seeds are collected, and the seeds can be obtained after opening the capsules.

[0047] The optimal time for fruit collection is when the capsule is about to split open or when the seeds have just begun to disperse. For some species in the Salicaceae family, the fruit turns yellow, and fruits collected just before splitting open have high seed viability, which is beneficial for subsequent observation. For other species, the fruit does not turn yellow at maturity, so fruits are collected when the seeds have just begun to disperse. Collecting too early results in immature seeds, a high rate of empty shriveles, and seeds that are not resistant to dehydration. Collecting too late leads to the large fruits already dispersed, and seed viability rapidly declines, which is detrimental to later processing and long-term preservation.

[0048] Example 5: The seeds of *Cymbidium goeringii* were tested using the same method as in Example 4, and the results are as follows: Figure 1 As shown; from Figure 1 As can be seen above: the seeds of the hard-leaved orchid do not appear whitish, the shape of the seeds is clearly presented in the field of vision, the boundary between the seed coat and the embryo of the plump hard-leaved orchid seeds is clear and distinct, and the morphology and structure of the embryo are clear, which can be identified as plump seeds.

[0049] Comparative Example 1: The seeds of *Cymbidium goeringii* were tested using the same method as in Example 4, except that top lighting was used. That is, during microscopic examination, the cold light source and the observer's eye were always positioned above the sample. The results are as follows: Figure 2 As shown; from Figure 2As can be seen above, the seeds of the hard-leaved orchid appear whitish and reflective, and the morphology and structure of the embryo cannot be clearly observed in the seed coat. It is impossible to determine whether there is an embryo inside the seed coat or whether the seed is plump.

[0050] Comparative Example 2: The seeds of *Cymbidium goeringii* were tested using the same method as in Example 4, except that no pretreatment was performed on the samples; that is, physiological saline was not added to the microscope tray or slide. The results are as follows: Figure 3 As shown, from Figure 3 As can be seen above, the seeds of the hard-leaved orchid are grayish, and the boundary between the seed coat and the embryo is blurred. It is impossible to clearly observe the morphology and structure of the embryo in the seed coat, and it is impossible to determine whether it is an embryo or other aggregate, or whether it is an unripe seed.

[0051] Example 6: The seeds of *Cymbidium lycopersicum* were tested using the same method as in Example 4, and the results are as follows: Figure 4 As shown; from Figure 4 As can be seen above, the seeds of Ryukyu Forked Columnar Orchid do not appear whitish, and the shape of the seeds is clearly presented in the field of vision. The boundary between the seed coat and the embryo of the plump Ryukyu Forked Columnar Orchid seeds is clear and distinct, and the morphological structure of the embryo is clear, which can be identified as plump seeds.

[0052] Comparative Example 3: Seeds of *Cymbidium lycopersicum* were tested using the same method as in Example 4, except that top lighting was used. That is, during microscopic examination, the cold light source and the observer's eye were always positioned above the sample. The results are as follows: Figure 5 As shown; from Figure 5 As can be seen above, the seeds of Ryukyu Forked Columnar Orchid have a whitish reflective appearance. The morphology and structure of the embryo cannot be clearly observed in the seed coat, making it impossible to determine whether there is an embryo inside the seed coat or whether the seed is plump.

[0053] Comparative Example 4: Seeds of *Cymbidium lycopersicum* were tested using the same method as in Example 4, except that no pretreatment was performed on the samples; that is, physiological saline was not added to the microscope tray or slide. The results are as follows: Figure 6 As shown, from Figure 6 As can be seen above, the seeds of Ryukyu Forked Columnar Orchid are grayish, and the boundary between the seed coat and the embryo is blurred. It is impossible to clearly observe the morphology and structure of the embryo in the seed coat, and it is impossible to determine whether it is an embryo or other aggregate, or whether it is an immature seed.

[0054] The detection rates of orchid seeds under different treatment conditions are shown in Table 1 below.

[0055] Table 1. Detection rate of Orchid seeds under different treatment conditions As shown in Table 1, the detection methods for orchid seeds listed in Examples 5 and 6 have excellent detection effects, good light transmittance, uniform texture, and a detection rate of 100%, indicating that the orchid seed preservation method provided by this invention has a very good treatment effect. In contrast, the orchid seed detection methods used in Comparative Examples 1-4 have poor detection effects, cannot accurately determine the fullness of orchid seeds, and have low detection rates.

[0056] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rapidly detecting the seed filling rate of a species of the family Orchidaceae, characterized in that, The process includes the following steps: sampling; pretreatment; microscopic examination; plumpness detection; and seed recovery. During microscopic examination, a cold light source is used to transmit the cold light upward from below the seed. The pretreatment step is as follows: place the seed sample on the microscope base or slide, add physiological saline to the microscope base or slide, and let it stand for 2-3 minutes until the seeds are moistened to obtain the sample to be tested; physiological saline helps to make the seed texture more uniform, increases light transmittance, and helps to avoid unclear images due to agglomeration when cold light is transmitted. The microscopic examination steps are as follows: place the sample to be tested on the microscope stage, turn on the cold light source below, select the magnification, and adjust the focus until the seed morphology is clear. The cold light source has a power of 30-50W, a color temperature of 3200-6500K, and an illuminance of 50000-120000Lx; During microscopic examination, a cold light source is transmitted upwards from below the seed, penetrating it, while the person observes the seed from above. The cold light source avoids overheating and damaging the seed. When the cold light source is transmitted upwards, the propagation path of the reflected light is downwards, thus no longer causing light interference, allowing the morphology of the seed to be clearly presented in the field of vision. The testing process will not cause any damage to the seeds. After the testing is completed, the seeds can be used for germination, long-term preservation, and morphological photography.

2. The method for rapidly detecting the seed plumpness rate of orchid seeds according to claim 1, characterized in that: The sampling steps are as follows: after placing the seeds in a sampling tray and shaking them thoroughly, randomly select 2 to 4 seed samples, with each seed sample containing 80 to 100 seeds.

3. The method for rapidly detecting the seed fullness rate of ferns according to claim 1, characterized in that: The seed plumpness detection step is as follows: count the number of seeds, plump seeds, and empty seeds in a sample, and calculate the seed plumpness rate according to the following formula (1): Seed plumpness rate = (number of plump seeds / number of seeds tested) × 100% (1).

4. The method for rapidly detecting the seed fullness rate of ferns according to claim 1, characterized in that: The sampling step is preceded by a fruit collection step.

5. The method for rapidly detecting the seed fullness rate of fern according to claim 4, characterized in that: The fruits are harvested when the capsules are about to split open or when the seeds have just begun to disperse.

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