Seed treatment method for globeflower aseptic seedling cultivation and application of seed treatment method
By repeatedly rinsing and disinfecting with alcohol, hot water, and sodium hypochlorite solution, combined with soaking in gibberellin solution, the problem of difficult disinfection and contamination of nasturtium seeds was solved, and sterile seedlings were cultivated.
Patent Information
- Application Number
- CN202511633167.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-10
- Publication Date
- 2025-12-19
AI Technical Summary
Golden lotus seeds are small and light, and their hydrophobic nature makes it difficult for them to immerse in liquids. Furthermore, traditional disinfection methods cannot completely sterilize them, leading to seed contamination and making them unsuitable for aseptic seedling cultivation.
After pretreatment with 75% alcohol and hot water at 56-57℃, the seeds were rinsed and disinfected multiple times with 75% alcohol and 2% sodium hypochlorite solution to break dormancy. They were then soaked in gibberellin solution to ensure complete immersion, and finally cultured on sterile culture medium.
This method achieves pollution-free cultivation of nasturtium seeds during aseptic culture, minimizes damage, and successfully cultivates aseptic seedlings, solving the problems of seed contamination and damage in traditional methods.
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Abstract
Description
Technical Field
[0002] This invention belongs to the field of biotechnology, and in particular relates to a seed treatment method for the cultivation of sterile seedlings of golden lotus and its application. Background Technology
[0003] *Trollius chinensis* is a perennial herb belonging to the genus *Trollius* in the family Ranunculaceae. Its seeds are tiny, measuring 1.62-1.91 mm in length and 0.95-1.15 mm in width, with a thousand-seed weight of 0.66-1.50 g. The seeds are small and lightweight, with a hydrophobic seed coat. When soaked in liquid, the seeds float on the surface, even after absorbing sufficient water. Furthermore, due to their small size and light weight, the seeds are difficult to separate completely from the seed coat and other impurities during harvesting, resulting in low seed purity. In tissue culture experiments, it was found that living materials such as leaves, stems, petals, and sepals are severely intolerant to traditional disinfectants such as alcohol, mercuric chloride, and sodium hypochlorite. After disinfection, these materials turn brown and black within minutes, rendering them unusable. Therefore, living *Trollius chinensis* materials cannot be used as explants for tissue culture research. Generally, plant seeds are protected by a seed coat, making them more resistant to damage from disinfectants compared to living materials. Therefore, by sterilizing and disinfecting *Trollius chinensis* seeds before sowing them in a sterile culture medium, and obtaining sterile seedlings as explants after germination, the problem of sterilizing living materials can be solved. However, sterilizing *Trollius chinensis* seeds is also difficult. Traditional sterilization methods, regardless of the duration, cannot completely sterilize the seeds. Even extreme methods such as soaking the seeds in 75% alcohol for 2 hours fail to achieve complete sterilization, resulting in complete contamination even after sowing in sterile MS medium, primarily due to fungal contamination. Therefore, providing a seed treatment method for cultivating sterile *Trollius chinensis* seedlings, ensuring that treated seeds are not contaminated after sowing on a sterile culture medium, has become a pressing technical challenge in this field. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a seed treatment method for the cultivation of sterile seedlings of golden lotus, wherein the seeds obtained can effectively avoid contamination problems during the cultivation process and minimize damage to the seeds.
[0005] This invention provides a seed treatment method for the cultivation of sterile seedlings of golden lotus, comprising the following steps: After breaking dormancy, soak the golden lotus seeds in 75% alcohol for 2-3 minutes. After the first rinse, soak them in sterile water at 56℃-57℃ for 5-10 minutes. Then soak them in 75% alcohol for 3-7 minutes. After the second rinse, soak them in 2% sodium hypochlorite solution for 8-12 minutes for a third rinse.
[0006] Preferably, the dormancy breaking is achieved by soaking the nasturtium seeds in a gibberellin solution of 400-600 mg / L for 8-10 days.
[0007] Preferably, when soaking the golden lotus seeds in the gibberellin solution, the gibberellin solution is changed every 3 to 5 days.
[0008] Preferably, rinsing is performed using sterile water at 20~28℃.
[0009] Preferably, the first rinse is performed until the solution is clear; the second rinse is performed 3 to 5 times; and the third rinse is performed 3 to 5 times.
[0010] Preferably, after the first rinse, the golden lotus seeds are placed in a cloth bag for subsequent soaking and rinsing.
[0011] This invention provides the application of the seed treatment method in the cultivation of sterile seedlings of Natalius.
[0012] The present invention also provides a method for cultivating sterile seedlings of golden lotus, comprising the following steps: placing golden lotus seeds obtained by the seed treatment method on a sterile culture medium for sterile culture.
[0013] Preferably, the sterile culture medium is MS culture medium without hormones.
[0014] Preferably, the culture temperature is 12~18℃ and the culture time is 40~50 days.
[0015] The beneficial effects of this invention are: This invention involves breaking the dormancy of *Trollius chinensis* seeds, pretreating them with 75% alcohol and hot water at 56-57°C, and then sterilizing them with a solution of 75% alcohol and 2% sodium hypochlorite. The resulting *Trollius chinensis* seeds can be used to cultivate sterile seedlings, ensuring the seeds remain uncontaminated during cultivation and minimizing damage, thus guaranteeing successful germination and the production of sterile seedlings. This seed treatment method solves the problem of obtaining *Trollius chinensis* explants due to the insensitivity of living *Trollius chinensis* material to disinfectants and the difficulty of thoroughly sterilizing *Trollius chinensis* seeds using traditional methods. This provides strong support for the collection of explants in *Trollius chinensis* tissue culture technology. Detailed Implementation
[0016] This invention provides a seed treatment method for the cultivation of sterile seedlings of golden lotus, comprising the following steps: After breaking dormancy, soak the golden lotus seeds in 75% alcohol for 2-3 minutes. After the first rinse, soak them in sterile water at 56℃-57℃ for 5-10 minutes. Then soak them in 75% alcohol for 3-7 minutes. After the second rinse, soak them in 2% sodium hypochlorite solution for 8-12 minutes for a third rinse.
[0017] This invention does not specifically limit the source of the golden lotus seeds; commercially available products or self-collected seeds can be used. The preferred self-collection steps are as follows: During the golden lotus seed maturity season (e.g., mid-to-late August), select wild or cultivated golden lotus plants free from pests and diseases, with plump, mature seeds. Collect the follicles and bring them indoors to air dry naturally. Gently tap the follicles to release the seeds, being careful not to break the follicle shell too much, making separation difficult and reducing seed purity. Repeatedly sift the seeds using a sieve to separate them from impurities such as the seed coat. Store the seeds in kraft paper bags at -20°C for later use.
[0018] In this invention, the dormancy of collected or purchased *Trollius chinensis* seeds is broken. Preferably, the dormancy-breaking process involves placing the *Trollius chinensis* seeds in a container and soaking them in a gibberellin solution of 400-600 mg / L for 8-10 days. More preferably, the concentration of the gibberellin solution is 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, or 600 mg / L, and the soaking time is 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. The soaking is preferably carried out at room temperature, preferably 20℃-28℃, more preferably 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, or 28℃. During the soaking process, it is preferable to place a layer of absorbent paper on the liquid surface or place the seeds in a cloth bag to prevent the seeds from floating on the liquid surface due to their light weight and hydrophobicity, ensuring that the seeds are completely submerged in the liquid. The absorbent paper is preferably filter paper, and the cloth bag is preferably a non-woven fabric bag or a gauze bag. When soaking golden lotus seeds in gibberellin solution, it is preferable to change the gibberellin solution every 3 to 5 days, more preferably every 3, 3.5, 4, 4.5 or 5 days, to ensure that the gibberellin solution is clear and to prevent the solution from spoiling.
[0019] After breaking dormancy, the seeds of the golden lotus are soaked in 75% alcohol for 2-3 minutes, preferably 2 minutes, 2.5 minutes, or 3 minutes. After soaking, a first rinse is performed, preferably until the solution is clear. During soaking, impurities in the seeds absorb water and sink. Utilizing the light and hydrophobic properties of the golden lotus seeds, repeated rinsing until clear during the first rinse separates the seeds from most impurities, reducing the chance of contamination during cultivation. After the first rinse, the golden lotus seeds are preferably placed in a cloth bag for subsequent soaking and rinsing to prevent them from floating due to their light weight, ensuring complete immersion in the solution and preventing seed loss during rinsing. The cloth bag is preferably a non-woven fabric bag or a gauze bag.
[0020] In this invention, after the first rinse, the seeds are soaked in sterile water at 56-57°C for 5-10 minutes; the temperature of the sterile water is more preferably 56°C, 56.5°C, or 57°C, and the soaking time is more preferably 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. This invention does not have a specific limitation on the source of the sterile water; commercially available sterile water or water obtained by boiling and cooling cold water can be used. This invention pre-treats the seeds with hot sterile water, effectively solving the problem that traditional disinfection and sterilization methods cannot completely sterilize the seeds, ensuring that the seeds are not contaminated during sterile cultivation and successfully obtaining sterile seedlings. Furthermore, the temperature of the sterile water selected in this invention not only ensures that the seeds are not contaminated during cultivation but also ensures successful germination, significantly superior to treatment at other temperatures.
[0021] After soaking in sterile water at 56-57℃, the sterile water is poured out to disinfect and sterilize the nasturtium seeds. This disinfection and sterilization is preferably performed in a clean bench. The disinfection and sterilization process begins with soaking in 75% alcohol for 3-7 minutes, more preferably 3, 4, 5, 6, or 7 minutes. After the alcohol soak, a second rinse is performed, 3-5 times, more preferably 3, 4, or 5 times, to remove the alcohol from the seed surface. Following the second rinse, the seeds are soaked in a 2% sodium hypochlorite solution for 8-12 minutes, more preferably 8, 9, 10, 11, or 12 minutes, for further disinfection and sterilization. Finally, a third rinse is performed, 3-5 times, more preferably 3, 4, or 5 times, to remove the sodium hypochlorite from the surface of the nasturtium seeds. In this invention, during the disinfection and sterilization process using alcohol immersion and sodium hypochlorite solution immersion, stirring is preferably performed to allow the disinfectant to come into full contact with the golden lotus seeds, thereby achieving a better disinfection and sterilization effect. This invention does not have any special limitations on the stirring method; conventional stirring methods in the art can be used. In some embodiments, the immersion container can be shaken or stirred using sterile tweezers.
[0022] In this invention, the first, second, and third rinses all use sterile water at room temperature. The room temperature is preferably 20-28°C, more preferably 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, or 28°C. In embodiments of this invention, since the room temperature is not constant throughout the experiment, it is sufficient as long as the sterile water used for rinsing is within the range of 20-28°C.
[0023] This invention utilizes alcohol and sodium hypochlorite solution to disinfect and sterilize nasturtium seeds, further enhancing the effect of hot sterile water treatment. While ensuring sterilization effectiveness, it shortens the hot sterile water treatment time, simplifies the disinfectant treatment steps, and minimizes damage to the nasturtium seeds. It is more effective than traditional disinfection and sterilization methods.
[0024] This invention provides the application of the seed treatment method in the cultivation of sterile seedlings of Natalius.
[0025] This invention also provides a method for cultivating sterile seedlings of *Trollius chinensis*, comprising the following steps: placing the *Trollius chinensis* seeds obtained by the seed treatment method on a sterile culture medium for aseptic cultivation. The *Trollius chinensis* seeds treated using the seed treatment method of this invention can achieve a pollution-free cultivation process, successfully cultivating sterile seedlings of *Trollius chinensis*.
[0026] The sterile culture medium is preferably hormone-free MS medium. This invention does not have a specific limitation on the source of the culture medium; commercially available products commonly used in the field are acceptable. The culture temperature is preferably 12-18℃, more preferably 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, or 18℃. The culture time is preferably 40-50 days, more preferably 40 days, 42 days, 44 days, 45 days, 46 days, 48 days, or 50 days. This invention does not have specific limitations on the specific steps of sterile culture, such as inoculation methods; conventional sterile seedling culture methods used in the field are acceptable.
[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Unless otherwise specified, the following embodiments are all conventional methods.
[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0030] Example 1 1. A seed treatment method for the cultivation of sterile seedlings of golden lotus, comprising the following steps: (1) Collection, seed production and storage of golden lotus seeds: In mid-to-late August, during the seed ripening season of nasturtiums, select disease-free plants with plump, mature seeds, collect the follicles, and bring them indoors to air dry naturally. Gently tap the follicles to release the seeds, repeatedly sift them to separate the seeds from impurities, and store the seeds in kraft paper bags in a -20°C refrigerator for later use.
[0031] (2) Breaking dormancy of golden lotus seeds Place the golden lotus seeds in a container and soak them in a 400 mg / L gibberellin solution. Place a layer of filter paper on the surface of the liquid to ensure the seeds are completely submerged. Soak the seeds at 25°C for 9 days, changing the gibberellin solution every 3 days to keep the solution clear and prevent it from spoiling.
[0032] (3) Pretreatment of golden lotus seeds 75% alcohol treatment: After soaking the golden lotus seeds in gibberellin solution, drain them, soak them in 75% alcohol for 2 minutes, and then rinse them repeatedly with room temperature sterile water until the solution is clear.
[0033] Hot water treatment: After rinsing, put the seeds into a non-woven fabric bag and place the non-woven fabric bag in a sterile container. Pour 56℃ hot water (from boiling water that has cooled naturally to 56℃) into the container containing the non-woven fabric bag and soak for 5 minutes. Then pour out the hot water and immediately add room temperature sterile water to transfer the bag to a clean bench.
[0034] (4) Sterilization of golden lotus seeds In a clean bench, the nasturtium seeds treated with 56°C hot water, along with the non-woven fabric bag, were transferred to another sterile container and soaked in 75% alcohol for 5 minutes. During the soaking process, the solution was stirred continuously with sterile tweezers. After completion, the solution was rinsed three times with room temperature sterile water. The seeds, along with the non-woven fabric bag, were then transferred to another sterile container and soaked in 2% sodium hypochlorite solution for 10 minutes. During the soaking process, the solution was stirred continuously. After completion, the solution was rinsed three times with room temperature sterile water.
[0035] 2. A method for cultivating sterile seedlings of golden lotus, comprising the following steps: Take a sterile inoculation tray and pour in an appropriate amount of room temperature sterile water. Transfer the golden lotus seeds treated in step 1 of this embodiment, along with the non-woven fabric bag, to the inoculation tray. Use sterile scissors to cut open the non-woven fabric bag and pour the seeds into the inoculation tray. Rinse the seeds adhering to the non-woven fabric bag repeatedly with room temperature sterile water until clean, then discard the non-woven fabric bag. After draining the room temperature sterile water from the inoculation tray, use sterile tweezers to pick up an appropriate amount of seeds (about 100 seeds) and place them in a tissue culture bottle. The tissue culture bottle contains pre-prepared, high-temperature and high-pressure sterilized MS medium (Qingdao High-tech Park Haibo Biotechnology Co., Ltd., product number HBZ0609) that does not contain any hormones. Then, add a small amount of room temperature sterile water to the tissue culture bottle and gently shake the bottle to evenly disperse the seeds on the surface of the medium with the water flow. Seal the bottle with the cap. After all inoculation is completed, transfer the culture bottle (tissue culture bottle) to a 15°C incubator for 45 days.
[0036] Example 2 1. A seed treatment method for the cultivation of sterile seedlings of golden lotus, comprising the following steps: (1) Collection, seed production and storage of golden lotus seeds: In mid-to-late August, during the seed ripening season of nasturtiums, select disease-free plants with plump, mature seeds, collect the follicles, and bring them indoors to air dry naturally. Gently tap the follicles to release the seeds, repeatedly sift them to separate the seeds from impurities, and store the seeds in kraft paper bags in a -20°C refrigerator for later use.
[0037] (2) Breaking dormancy of golden lotus seeds Place the golden lotus seeds in a container and soak them in a 500 mg / L gibberellin solution. Place a layer of filter paper on the surface of the liquid to ensure the seeds are completely submerged. Soak the seeds at 20°C for 8 days, changing the gibberellin solution every 4 days to keep the solution clear and prevent it from spoiling.
[0038] (3) Pretreatment of golden lotus seeds 75% alcohol treatment: After soaking the golden lotus seeds in gibberellin solution, drain them, soak them in 75% alcohol for 3 minutes, and then rinse them repeatedly with room temperature sterile water until the solution is clear.
[0039] Hot water treatment: After rinsing, put the seeds into a non-woven fabric bag, place the non-woven fabric bag in a sterile container, pour sterile water at 57°C into the container containing the non-woven fabric bag, soak for 8 minutes, then pour out the sterile water at 57°C, immediately add room temperature sterile water and transfer to a clean bench.
[0040] (4) Sterilization of golden lotus seeds In a clean bench, the nasturtium seeds treated with 57°C sterile water, along with the non-woven fabric bag, were transferred to another sterile container and soaked in 75% alcohol for 3 minutes. During the soaking process, the solution was stirred continuously with sterile tweezers. After completion, the solution was rinsed 4 times with room temperature sterile water. The seeds, along with the non-woven fabric bag, were then transferred to another sterile container and soaked in 2% sodium hypochlorite solution for 8 minutes. During the soaking process, the solution was shaken continuously. After completion, the solution was rinsed 4 times with room temperature sterile water.
[0041] 2. A method for cultivating sterile seedlings of golden lotus, comprising the following steps: Take a sterile inoculation tray and pour in an appropriate amount of room temperature sterile water. Transfer the golden lotus seeds treated in step 1 of this embodiment, along with the non-woven fabric bag, to the inoculation tray. Use sterile scissors to cut open the non-woven fabric bag and pour the seeds into the inoculation tray. Rinse the seeds adhering to the non-woven fabric bag repeatedly with room temperature sterile water until clean, then discard the non-woven fabric bag. After draining the room temperature sterile water from the inoculation tray, use sterile tweezers to pick up an appropriate amount of seeds (about 100 seeds) and place them in a tissue culture bottle containing pre-prepared, high-temperature and high-pressure sterilized MS medium (Qingdao High-tech Park Haibo Biotechnology Co., Ltd., product number HBZ0609). Then, add a small amount of room temperature sterile water to the tissue culture bottle and gently shake the bottle to evenly disperse the seeds on the surface of the medium with the water flow. Seal the bottle with the cap. After all inoculation is completed, transfer the culture bottle to a 12°C incubator for 40 days.
[0042] Example 3 1. A seed treatment method for the cultivation of sterile seedlings of golden lotus, comprising the following steps: (1) Collection, seed production and storage of golden lotus seeds: In mid-to-late August, during the seed ripening season of nasturtiums, select disease-free plants with plump, mature seeds, collect the follicles, and bring them indoors to air dry naturally. Gently tap the follicles to release the seeds, repeatedly sift them to separate the seeds from impurities, and store the seeds in kraft paper bags in a -20°C refrigerator for later use.
[0043] (2) Breaking dormancy of golden lotus seeds Place the golden lotus seeds in a container and soak them in a 600 mg / L gibberellin solution. Place a layer of filter paper on the surface of the liquid to ensure the seeds are completely submerged. Soak the seeds at 28°C for 10 days, changing the gibberellin solution every 5 days to keep the solution clear and prevent it from spoiling.
[0044] (3) Pretreatment of golden lotus seeds 75% alcohol treatment: After soaking the golden lotus seeds in gibberellin solution, drain them and soak them in 75% alcohol for 2.5 minutes. Then rinse them repeatedly with room temperature sterile water until the solution is clear.
[0045] Hot water treatment: After rinsing, put the seeds into a non-woven fabric bag and place the non-woven fabric bag in a sterile container. Pour sterile water at 56.5°C into the container containing the non-woven fabric bag and soak for 10 minutes. Then pour out the sterile water at 56.5°C and immediately add room temperature sterile water to transfer the solution to a clean bench.
[0046] (4) Sterilization of golden lotus seeds In a clean bench, the nasturtium seeds, treated with sterile water at 56.5℃, were transferred along with a non-woven fabric bag to another sterile container. The solution was then soaked in 75% alcohol for 7 minutes, with constant stirring using sterile tweezers during the soaking process. After soaking, the solution was rinsed five times with sterile water at room temperature. The seeds were then transferred again along with the non-woven fabric bag to another sterile container and soaked in a 2% sodium hypochlorite solution for 12 minutes, with constant stirring during the soaking process. Finally, the solution was rinsed five times with sterile water at room temperature.
[0047] 2. A method for cultivating sterile seedlings of golden lotus, comprising the following steps: Take a sterile inoculation tray and pour in an appropriate amount of room temperature sterile water. Transfer the golden lotus seeds treated in step 1 of this embodiment, along with the non-woven fabric bag, to the inoculation tray. Use sterile scissors to cut open the non-woven fabric bag and pour the seeds into the inoculation tray. Rinse the seeds adhering to the non-woven fabric bag repeatedly with room temperature sterile water until clean, then discard the non-woven fabric bag. After draining the room temperature sterile water from the inoculation tray, use sterile tweezers to pick up an appropriate amount of seeds (about 100 seeds) and place them in a tissue culture bottle containing pre-prepared, high-temperature and high-pressure sterilized MS medium (Qingdao High-tech Park Haibo Biotechnology Co., Ltd., product number HBZ0609). Then, add a small amount of room temperature sterile water to the tissue culture bottle and gently shake the bottle to evenly disperse the seeds on the surface of the medium with the water flow. Seal the bottle with the cap. After all inoculation is completed, transfer the culture bottle to an 18°C incubator for 50 days.
[0048] Comparative Examples 1-20 The only difference between Comparative Examples 1-20 and Example 1 is that: ①The temperature of the hot water in step 1 (3) is different, and the number of times and the time of hot water soaking are different. See Table 1 for details; ② In step 1, (4) is as follows: In the clean bench, transfer the hot water-treated golden lotus seeds along with the non-woven bag to another sterile container, soak them in 2% sodium hypochlorite solution for 15 minutes, and stir the solution continuously with sterile tweezers during the soaking process. After completion, rinse them 3 times with room temperature sterile water. Then transfer the seeds along with the non-woven bag to another sterile container, soak them in 75% alcohol for 5 minutes, and stir continuously during the soaking process. After completion, rinse them 3 times with room temperature sterile water. Then transfer the seeds along with the non-woven bag to another sterile container, soak them in 0.2% mercuric chloride solution for 10 minutes, and stir continuously during the soaking process. After completion, rinse them 3 times with room temperature sterile water.
[0049] The rest are the same as in Example 1.
[0050] Table 1. Hot water temperature and soaking time for Comparative Examples 1 to 20
[0051] Note: "Natural cooling" refers to soaking the nasturtium seeds in hot water of the corresponding temperature until the water cools naturally to the same temperature as room temperature (20~28℃). For example, if the hot water temperature is 100℃, the treatment is repeated twice, and the time is for natural cooling. If the room temperature is 25℃, the nasturtium seeds are soaked in hot water at 100℃ until the water temperature cools naturally to 25℃, and then the treatment is repeated once with hot water at 100℃.
[0052] Comparative Example 21 The only difference from Example 1 is that the hot water soaking in steps 1 and (3) is done once, and the soaking time is 10 minutes. Steps 1 and (4) are as follows: In a clean bench, transfer the hot water-treated golden lotus seeds along with the non-woven bag to another sterile container, soak them in 75% alcohol for 5 minutes, and stir the solution continuously with sterile tweezers during the soaking process. After completion, rinse three times with room temperature sterile water. Then transfer the seeds along with the non-woven bag to another sterile container, soak them in 0.2% mercuric chloride for 10 minutes, and stir continuously during the soaking process. After completion, rinse three times with room temperature sterile water. The rest is the same as in Example 1.
[0053] Comparative Example 22 The only difference from Example 1 is that the hot water soaking in steps 1 and (3) is once, and the soaking time is 10 minutes. The rest are the same as in Example 1.
[0054] Comparative Example 23 The only difference from Example 1 is that steps (3) and (4) in step 1 are different; the rest are the same as in Example 1. Steps (3) and (4) are as follows: (3) Pretreatment of golden lotus seeds After soaking the golden lotus seeds in gibberellin solution, drain them, soak them in 75% alcohol for 3 minutes, and then rinse them repeatedly with room temperature sterile water until the solution is clear. Place the rinsed seeds in non-woven fabric.
[0055] (4) Sterilization of golden lotus seeds In a clean bench, the rinsed golden lotus seeds, along with the non-woven fabric bag, are transferred to a sterile container and soaked in a 2% sodium hypochlorite solution for 15 minutes. During the soaking process, the solution is stirred continuously with sterile tweezers. After completion, the solution is rinsed three times with room temperature sterile water. The seeds, along with the non-woven fabric bag, are then transferred to another sterile container and soaked in 75% alcohol for 5 minutes, stirring continuously. After completion, the solution is rinsed three times with room temperature sterile water. Finally, the seeds, along with the non-woven fabric bag, are transferred to another sterile container and soaked in 0.2% mercuric chloride solution for 10 minutes, stirring continuously. After completion, the solution is rinsed three times with room temperature sterile water.
[0056] Comparative Example 24 The only difference between this example and Comparative Example 23 is step (4) in step 1. The rest is the same as Comparative Example 22. Step (4) is as follows: (4) Sterilization of golden lotus seeds In a clean bench, transfer the rinsed golden lotus seeds along with the non-woven bag to a sterile container and soak them in 75% alcohol for 2 hours, stirring constantly during the soaking process. After completion, rinse them three times with room temperature sterile water.
[0057] Test case During the cultivation process, the culture flasks of Examples 1-3 and Comparative Examples 1-24 were visually observed, and the presence of contamination was recorded (mucus indicated bacterial contamination, and mold on the culture medium indicated fungal contamination; both fungal and bacterial contamination were counted as contamination). The percentage of contaminated culture flasks out of the total number of culture flasks was counted and calculated to obtain the contamination rate (contamination rate = number of contaminated culture flasks / total number of culture flasks * 100%). The germination of seeds in the culture flasks was also observed. The results are shown in Table 2.
[0058] Table 2 Seed germination status and contamination rate of each group
[0059] As can be seen from Comparative Examples 23-24, when the seeds of Nastas davidii were treated with traditional disinfection and sterilization methods, all culture bottles were contaminated after sowing, and sterile Nastas davidii seedlings could not be obtained.
[0060] The results of Comparative Examples 1-13 show that while a hot water treatment temperature of 100℃ can ensure that the culture medium for cultivating sterile seedlings from *Trollius chinensis* seeds is not contaminated, temperatures exceeding 70℃ cause irreversible damage to the seeds, resulting in the failure of germination in all culture bottles and making sterile seedling cultivation impossible. However, when the hot water treatment temperature is between 56 and 60℃, all seeds in the culture bottles germinate with low contamination rates, below 10%. As the hot water temperature decreases further, the contamination rate of the culture bottles increases significantly, exceeding 25%, and even reaching over 80% at 54℃, rendering them unsuitable for cultivating sterile seedlings.
[0061] Meanwhile, the results of Comparative Examples 14-20 show that when using three traditional disinfectants—"15 min 2% sodium hypochlorite solution + 5 min 75% alcohol + 10 min 0.2% mercuric chloride"—a 15-minute hot water treatment at 56-57°C can achieve a contamination rate of 0.0%. Comparative Example 21, by reducing the use of sodium hypochlorite solution and using 75% alcohol and 0.2% mercuric chloride, showed a 6.5% increase in contamination rate compared to Comparative Example 19. Examples 1-3 and Comparative Example 22, by reducing the use of mercuric chloride in traditional disinfection methods and using 2% sodium hypochlorite solution and 75% alcohol, only require 5-10 minutes of hot water treatment to achieve a contamination rate of 0.0%, successfully obtaining sterile seedlings suitable as explant donors. This indicates that the seed treatment method of the present invention can minimize seed damage while ensuring the sterile cultivation of *Trollius chinensis* seedlings.
[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A seed treatment method for aseptic seedling cultivation of golden lotus, characterized in that, Includes the following steps: After breaking dormancy, soak the golden lotus seeds in 75% alcohol for 2-3 minutes. After the first rinse, soak them in sterile water at 56-57℃ for 5-10 minutes. Then soak them in 75% alcohol for 3-7 minutes. After the second rinse, soak them in 2% sodium hypochlorite solution for 8-12 minutes for a third rinse.
2. The seed treatment method according to claim 1, characterized in that, The process of breaking dormancy involves soaking the seeds of *Trollius chinensis* in a gibberellin solution of 400-600 mg / L for 8-10 days.
3. The seed treatment method according to claim 2, characterized in that, When soaking the golden lotus seeds in the gibberellin solution, the gibberellin solution should be changed every 3 to 5 days.
4. The seed treatment method according to claim 1, characterized in that, Rinsing is performed using sterile water at 20-28℃.
5. The seed treatment method according to claim 1, characterized in that, The first rinse is performed until the solution is clear; the second rinse is performed 3 to 5 times; the third rinse is performed 3 to 5 times.
6. The seed treatment method according to claim 1, characterized in that, After the first rinse, the golden lotus seeds are placed in a cloth bag for subsequent soaking and rinsing.
7. The application of the seed treatment method according to any one of claims 1 to 6 in the cultivation of sterile seedlings of Napier lotus.
8. A method for cultivating sterile seedlings of golden lotus, characterized in that, The process includes the following steps: placing the golden lotus seeds obtained by the seed treatment method according to any one of claims 1 to 6 on a sterile culture medium for aseptic culture.
9. The cultivation method according to claim 8, characterized in that, The sterile culture medium is MS medium without hormones.
10. The cultivation method according to claim 8, characterized in that, The culture temperature is 12~18℃, and the culture time is 40~50 days.