Method for cultivating young morchella mycelium based on fungal liquid

Abstract

This invention discloses a method for cultivating *Cymbidium goeringii* seedlings using a fungal liquid culture, belonging to the field of medicinal plant cultivation / traditional Chinese medicine cultivation. The method includes the following steps: S1, preparing a culture medium containing 70% broadleaf hardwood sawdust (particle size less than 10mm), 16% wheat bran, 10% corn cob (particle size 6mm), 6% soil (particle size less than 3mm), 1% gypsum, and 0.1% potassium dihydrogen phosphate; adding spring water and stirring until the water content is 50%-55%, adjusting the pH to 6.5-7.0, and then sterilizing at high temperature and cooling for later use; S2, selecting mature, unopened *Cymbidium goeringii* fruit pods and disinfecting them by soaking them in 75% medical alcohol for 5 seconds in a Class 100 cleanroom environment. This invention is based on the forefront of modern plant microbiology and biotechnology. By carefully selecting various high-quality raw materials and strictly controlling the particle size, while precisely adjusting the proportions of each raw material to achieve the optimal nutrient balance of the culture medium and accurately controlling the water content and pH value, and then performing scientific high-temperature sterilization, a suitable nutritional, pH-appropriate, and sterile growth environment is provided for *Cymbidium goeringii* seedlings.

Description

Technical Field

[0001] The present invention relates to the field of medicinal plant cultivation / herbal medicine cultivation, and particularly to a method for cultivating Cremastra appendiculata seedlings based on fungal liquid culture. Background Art

[0002] Cremastra appendiculata, as a plant with important medicinal value and certain edible value, occupies a unique position in the field of traditional Chinese medicine and the specialty agricultural product market. It contains various bioactive components and has significant effects in clearing heat and detoxifying, dissipating carbuncles and nodules, etc., and is widely used in various traditional Chinese medicine prescriptions. The market demand continues to grow steadily. At the same time, with people's pursuit of healthy diet and special food materials, Cremastra appendiculata has gradually attracted attention in the edible field, further promoting the expansion of its market demand. However, the current output of Cremastra appendiculata is difficult to meet the increasing market demand, as follows: In the cultivation of Cremastra appendiculata seedlings, the culture medium is the basis for providing nutrients and the growth environment. However, the existing methods for preparing the culture medium are relatively crude, the selection of raw materials lacks strict standards, often using some broad-leaved miscellaneous tree leaves, humus soil, wood blocks, sheep manure, etc. with uneven quality, and the control of the particle size of raw materials is not accurate, resulting in uneven texture of the culture medium. In terms of the raw material ratio, there is no scientific basis and it is mostly prepared by experience, making the nutrient components of the culture medium unbalanced and unable to provide comprehensive and stable nutrient support for the seedlings. At the same time, the control of the water content and pH value of the culture medium is not accurate, and it is difficult to create a microenvironment suitable for the growth of seedlings. In addition, the sterilization treatment is not thorough enough, and harmful microorganisms are easily left behind, increasing the risk of seedlings being infected by pests and diseases, seriously affecting the survival rate and growth quality of seedlings.

[0003] Seed treatment is the key starting link for cultivating high-quality seedlings. In the existing technology, when obtaining seeds, the screening of fruit capsules is not strict enough, and some immature or damaged fruit capsules are often mixed in, resulting in uneven seed quality. In the disinfection process, the operation is not standardized and the disinfection is not thorough, which easily makes the seeds carry germs and lay hidden dangers for subsequent cultivation. In the seed soaking and mixing process, the lack of a seed soaking link leads to insufficient water absorption of seeds, reducing the germination rate, and the control of parameters such as the stirring method is not accurate, and the fusion of seeds and germination fungi is not sufficient, affecting the germination rate and germination speed of seeds. During the cultivation process, the regulation of environmental parameters such as temperature, humidity, light, and carbon dioxide concentration is not fine enough, and a relatively crude management method is mostly adopted, and it is impossible to provide suitable environmental conditions according to the needs of different growth stages of seedlings, resulting in slow growth, poor development of seedlings, extended cultivation cycles, and low production efficiency.

[0004] Hardening-off and transplanting are crucial processes for seedlings transitioning from artificial cultivation to the natural environment. Current techniques lack scientifically sound operational guidelines for hardening-off seedlings. The hardening-off environment differs significantly from the natural environment, and improper timing and methods prevent seedlings from effectively adapting to these changes, leading to stunted growth or even death after transplanting. Furthermore, the selection of transplanting sites often lacks scientific evaluation, with inadequate consideration of soil fertility and drainage. Inappropriate row and plant spacing results in insufficient growing space or wasted land resources. Insufficient root protection during transplanting easily damages the roots, and the lack of effective post-transplant care, such as inadequate watering and proper mulching to retain moisture, further reduces seedling survival rates and impacts the final yield of *Sagittaria sagittaria*. Therefore, we provide a method for cultivating *Sagittaria sagittaria* seedlings using liquid fungal culture. Summary of the Invention

[0005] The purpose of this invention is to solve the problems in the prior art by proposing a method for cultivating Maocigu seedlings based on fungal liquid culture.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for cultivating *Sagittaria sagittifolia* seedlings using liquid fungal culture includes the following steps: S1. Prepare a culture medium containing 70% broadleaf hardwood sawdust with a particle size of less than 10mm, 16% wheat bran, 10% corn cob with a particle size of 6mm, 6% soil with a particle size of less than 3mm, 1% gypsum, and 0.1% potassium dihydrogen phosphate. Add spring water and stir until the water content is 50%-55%, adjust the pH value to 6.5-7.0, and sterilize at high temperature and cool for later use. S2. Select mature, unopened fruit pods of Sagittaria sagittifolia and disinfect them by soaking them in 75% medical alcohol for 5 seconds in a Class 100 cleanroom environment. S3. Soak the disinfected Sagittaria sagittifolia seeds in the prepared liquid germination inoculum for 1.5 to 2 hours; S4. After thoroughly mixing the soaked seeds with the germination bacteria liquid inoculum and the prepared culture medium, pack them into boxes. S5. Cultivate *Gnaphalium affine* seedlings under controlled temperature conditions of 21℃-25℃, controlled humidity of 60%-90%, controlled light of 600lx-800lx, and controlled gas (carbon dioxide concentration) of 900ppm-1200ppm. S6. When the seedlings grow to 3cm-10cm, move them to a plastic greenhouse at natural temperature for 7-10 days to harden them off, and then transplant them.

[0007] Preferably, during the preparation of the culture medium, high-temperature sterilization can be performed in two ways: one is to keep the medium at 100°C for 10 hours to ensure that the medium is fully heated and effectively kill various bacteria and insect eggs; the other is to keep the medium at 121°C for 5 hours to quickly and thoroughly sterilize it using a high-temperature and high-pressure environment. The cooling process should be carried out in a clean environment and allowed to cool naturally to 22°C-26°C for later use to prevent re-contamination during the cooling process.

[0008] Preferably, in the seed soaking step, the aseptic operating procedures are strictly followed. 1000ml of cultured germination bacteria liquid inoculum is extracted in a Class 100 clean bench. 100 sterilized and mature, unopened fruit segments of *Sagittaria sagittifolia* are carefully selected, the fruit segments are carefully cut open, and the seeds inside are completely removed and soaked in the extracted germination bacteria liquid inoculum to ensure full contact between the seeds and the inoculum, creating favorable conditions for subsequent germination.

[0009] Preferably, in the seed mixing step, 5000ml of the cultured germination bacteria liquid inoculum is first placed in a clean mixing container, and then the soaked seeds are slowly poured in. The mixture is then thoroughly stirred using a sterilized stirring tool to ensure that the germination bacteria are evenly attached to the seed surface. Subsequently, 50kg of the prepared culture medium is added to the mixing container in multiple portions, and each addition is thoroughly stirred to ensure that the seeds, germination bacteria liquid inoculum, and culture medium are evenly mixed without any local accumulation or clumping.

[0010] Preferably, in the boxing step, a culture box with filter ventilation holes that has undergone strict sterilization is selected. The size of the filter ventilation holes must be large enough to meet the gas exchange requirements while effectively preventing the invasion of miscellaneous bacteria. The mixture after seed dressing is slowly poured into the culture box while gently shaking the culture box to ensure that the mixture is evenly distributed. The filling thickness is precisely controlled at 5cm-7cm to ensure that the growth space of the hairy arrowhead seedlings is suitable. After filling, the lid is quickly closed and food-grade tape is used to seal the lid firmly to the culture box to prevent external contamination.

[0011] Preferably, the cultivation process is divided into two stages: In the initial stage, the sealed cultivation box is placed in a temperature-controlled device, precisely controlling the temperature at 21℃-25℃, maintaining humidity at 60% using a humidifier, and using a gas control device to maintain the carbon dioxide concentration at 900ppm-1200ppm. This stage lasts for 13 days, providing a stable and suitable environment for seed germination. In the subsequent stage, the cultivation box lid is carefully removed, and the box is transferred to a cultivation room with light, temperature, humidity, and gas control functions. The temperature is again precisely controlled at 21℃-25℃, the humidity is increased to 85%-95% using a humidifier, and a light intensity of 600lx-800lx is provided using a light control device, with 8 hours of light per day. Simultaneously, the carbon dioxide concentration is controlled at 900ppm-1200ppm using a gas control device. This process continues for 80-100 days until the seedlings grow to 3cm-10cm.

[0012] Preferably, in the seedling hardening step, the cultivated *Sagittaria sagittifolia* seedlings, along with their culture boxes, are carefully transferred to a plastic greenhouse at a natural temperature. A humidity control device is installed in the greenhouse to precisely control the moisture content of the culture medium at 55%-65%, providing a buffer transition for the seedlings to adapt to the external environment. During the seedling hardening period, the growth status of the seedlings and the changes in the humidity of the culture medium are observed regularly every day, and the humidity is adjusted in a timely manner according to the actual situation to ensure that the seedlings grow healthily during the 7-10 day hardening period and gradually adapt to the natural environment.

[0013] Preferably, the Maocigu seedlings cultivated using this method have achieved a transplant success rate of over 95% after extensive practical transplanting experiments, which is significantly higher than that of traditional seedling cultivation methods. At the same time, the cost of Maocigu seedlings with a size of 3cm-10cm is only 0.2 yuan per seedling, which is significantly lower than that of traditional seedling cultivation methods, effectively improving the economic benefits of Maocigu cultivation and providing reliable technical support for the large-scale cultivation of Maocigu.

[0014] Preferably, this method has wide seasonal adaptability and is not limited by the traditional seedling season, allowing seedling cultivation to be carried out in winter, spring, and summer. During the seedling cultivation process, precise environmental control can provide stable growth conditions for the *Cistanche deserticola* seedlings, ensuring that the seedlings can grow and develop normally in different seasons. After the seedlings are cultivated, they can be transplanted in spring, summer, and autumn according to the actual situation. Moreover, the seedling cultivation time is significantly shortened to 90-120 days compared to the traditional method, effectively improving the efficiency of *Cistanche deserticola* cultivation and shortening the cultivation cycle.

[0015] Preferably, this method has significant ecological and environmental advantages in the seedling cultivation process. In the traditional seedling cultivation process, in order to obtain enough leaves and humus, it is often necessary to collect forests on a large scale, which not only easily leads to soil erosion, but also causes serious damage to forest biodiversity. In contrast, this method uses a specific culture medium formula, which does not require the large-scale collection of leaves and humus, thereby reducing the dependence on forest resources from the source, effectively protecting the forest ecological environment, conforming to the concept of sustainable development, and providing a useful reference for the development of eco-friendly agriculture.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By carefully selecting a variety of high-quality raw materials and strictly controlling particle size, while precisely adjusting the proportions of each material and accurately controlling moisture content and pH value, and then undergoing scientific high-temperature sterilization, a suitable, pH-balanced, and sterile growth environment is provided for *Cistanche deserticola* seedlings. This method of preparing the culture medium achieves the effect of providing seedlings with comprehensive and balanced nutrients, reducing the probability of pests and diseases, helping seedlings to grow healthily and robustly in the initial stage, and improving seedling survival rate and overall quality.

[0017] 2. Starting with rigorous selection of mature, unopened fruit pods, and meticulous disinfection and seed extraction processes, the seeds are ensured to remain uncontaminated. During seed soaking and mixing, soaking time and stirring methods are precisely controlled to ensure complete integration of the seeds with the germination bacteria. Then, through appropriate packaging and phased, precise control of environmental parameters, including temperature, humidity, light, and carbon dioxide concentration, this series of meticulous procedures aims to improve seed germination rate, accelerate seedling growth, shorten the cultivation cycle, and increase production efficiency.

[0018] 3. After the seedlings reach a certain stage of growth, they are transplanted to a plastic greenhouse at a natural temperature for hardening-off, with precise control of the culture medium's moisture content to allow them to gradually adapt to changes in the natural environment. After hardening-off, suitable plots are selected for scientific transplanting, with reasonable row and plant spacing. Care is taken to protect the roots during transplanting and watering thoroughly. The seedlings are then covered with crushed rice straw or stalks (10mm-20mm in length). This hardening-off and transplanting method enhances the seedlings' adaptability to the natural environment and improves transplant survival rates, laying a solid foundation for the subsequent growth, development, and yield increase of *Sagittaria sagittifolia*. Attached Figure Description

[0019] Figure 1 This is a schematic flowchart of the method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture proposed in this invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] Example, refer to Figure 1 This study describes a method for cultivating *Sagittaria sagittifolia* seedlings using liquid culture. The culture medium is fundamental to the seedling growth, and its quality directly impacts the cultivation outcome. In the raw material preparation stage, for broadleaf hardwood sawdust, we not only require a particle size below 10mm but also rigorously screen it to remove any impurities or moldy parts. Sawdust from broadleaf trees such as oak and birch is a high-quality choice, and 70kg was collected after careful sieving. Fresh, odorless, and insect-free wheat bran was selected, and 16kg was weighed to ensure sufficient nutrients. Corn cobs were controlled to a particle size of 6mm, and 10kg was collected after sieving to ensure uniform texture. Soil was collected from humus-rich topsoil in mountain forests, sieved through a 3mm sieve, and 6kg was collected to provide abundant minerals and beneficial microorganisms. Simultaneously, 1kg of gypsum and 0.1kg of potassium dihydrogen phosphate were prepared to ensure the purity of the chemicals and prevent clumping due to moisture.

[0022] Further, during mixing, pour all raw materials into a large mixer sequentially, slowly adding spring water while observing the moisture content by touch until the mixture reaches a moisture content of 50%-55%, achieving a consistency where it can be formed into a clump when squeezed but crumbles easily when dropped. Next, adjust the pH value using pH test paper or a pH meter. If it's outside the 6.0-6.5 range, add citric acid solution if too high, and lime solution if too low. Stir thoroughly and test again after each addition until it stabilizes within the appropriate range. Finally, perform high-temperature sterilization. Two methods can be chosen: one is to maintain the temperature at 100℃ for 10 hours, with a slow and steady increase; the other is to maintain the temperature at 121℃ for 5 hours, again paying attention to the rate of increase. After sterilization, place the sterilization bags in a clean, well-ventilated cooling room to cool naturally to 22℃-26℃, avoiding contamination during this period.

[0023] Furthermore, the quality of *Sagittaria sagittifolia* seeds is crucial to the success of seedling cultivation. During the ripening season, mature, unopened fruit pods are selected from deep within the forest. These pods are brightly colored, firm, and free from obvious damage or signs of pests or diseases. One hundred pods are selected and placed in a clean plastic basket for later use. The baskets containing the fruit pods are then disinfected. The baskets are moved to a Class 100 cleanroom environment, such as a Class 100 clean bench, and sprayed with 75% medical alcohol until each pod is moistened. After soaking for 5 seconds, the pods are removed and placed on sterile filter paper to drain. Once the alcohol has evaporated, the pods are carefully opened with sterilized tweezers or a scalpel, and the seeds are extracted intact and collected in clean, sterile petri dishes. The entire process must strictly adhere to aseptic techniques to prevent seed contamination and ensure the smooth progress of subsequent cultivation.

[0024] Further, in the seed soaking process, within a Class 100 clean bench, 1000ml of the cultured liquid inoculum of germinating bacteria is accurately drawn from the container using a sterilized pipette and transferred to a clean, sterile container. The seeds from the culture dish are then slowly poured in, and gently stirred with a sterilized glass rod to ensure full contact between the seeds and the inoculum. Soaking is continued for 1.5 to 2 hours, with stirring every 30 minutes to ensure even soaking.

[0025] For seed dressing, first perform an initial mixing: pour 5000ml of the prepared germination bacteria liquid inoculum into a large, clean mixing container, then add the soaked seeds. Use a sterilized mixing tool to stir for 10-15 minutes to ensure the germination bacteria are evenly coated on the seed surface. Next, perform a second mixing: add 50kg of the prepared culture medium in several batches, about 10kg each time, stirring thoroughly after each addition to prevent local accumulation or clumping. The entire process takes about 30-40 minutes until the mixture is homogeneous.

[0026] Furthermore, before filling the boxes, select culture boxes that have undergone strict sterilization and have filter ventilation holes (approximately 0.2mm in diameter) to check the airtightness and the unobstructedness of the ventilation holes. Slowly pour the mixed seed solution into the culture box, gently shaking it as you pour to ensure even distribution. Use a special ruler to measure and ensure that the filling thickness is precisely controlled between 5cm and 7cm. After filling, quickly close the lid and seal it securely with food-grade tape.

[0027] The cultivation process is divided into two stages. In the initial stage, the sealed culture boxes are placed in a temperature-controlled environment, such as a constant-temperature incubator, with precise temperature control between 21℃ and 25℃. Humidity is maintained at 60% using a humidifier, such as an ultrasonic humidifier, and the carbon dioxide concentration is maintained at 900ppm-1200ppm using a gas control device, such as a carbon dioxide concentration regulator. Cultivation continues for 13 days, during which the equipment is checked daily to ensure stable environmental parameters. In the subsequent stage, after 13 days, the culture box lids are carefully removed, and the boxes are transferred to a cultivation room with controlled light, temperature, humidity, and gas parameters. Temperature is again precisely controlled at 21℃-25℃, humidity is increased to 85%-95%, and LED plant growth lights provide 600lx-800lx light intensity for 8 hours daily. Simultaneously, the carbon dioxide concentration is maintained at 900ppm-1200ppm. Cultivation continues for 80-100 days, until the seedlings reach 3cm-10cm in height. Observe and record the seedling growth in detail every week, including height, number of leaves, color, etc., and adjust the cultivation environment parameters in a timely manner.

[0028] Further, when the seedlings grow to 3-10cm, hardening-off is carried out. Carefully transfer the well-cultivated seedlings, along with their culture boxes, to a plastic greenhouse at ambient temperature. Avoid shaking the culture boxes during the transfer process to prevent damage to the seedlings. Install humidity control devices, such as a misting system, in the greenhouse to precisely control the moisture content of the culture medium at 55%-65%. Observe the seedling growth and changes in culture medium humidity daily using a hygrometer, and adjust the humidity according to the actual situation to ensure healthy growth of the seedlings during the 7-10 day hardening-off period, gradually adapting to the natural environment.

[0029] After the seedling hardening-off period, transplant the seedlings. Select a fertile, well-drained, and sunny plot of land. Deeply plow and harrow the land, level it, and make raised beds approximately 1.2m wide and 20cm high. Carefully remove the seedlings from the culture boxes, preserving as much root culture medium as possible during separation. Transplant them into the raised beds at a row spacing of 20cm and a plant spacing of 15cm, ensuring the planting depth covers the root culture medium by 1-2cm. After transplanting, water thoroughly to help the seedlings establish. Cover the raised beds with a thin layer of straw or stalks to maintain soil moisture and temperature, promoting seedling survival and laying a good foundation for the subsequent growth of *Sagittaria sagittifolia*.

[0030] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for cultivating *Sagittaria sagittifolia* seedlings using liquid fungal culture, characterized in that, Includes the following steps: S1. Prepare a culture medium containing 70% broadleaf hardwood sawdust with a particle size of less than 10 mm, 16% wheat bran, 10% corn cob with a particle size of 6 mm, 6% soil with a particle size of less than 3 mm, 1% gypsum, and 0.1% potassium dihydrogen phosphate. Add spring water and stir until the water content is 50%-55%, adjust the pH value to 6.5-7.0, and sterilize at high temperature and cool for later use. S2. Select mature, unopened fruit pods of *Sagittaria sagittifolia* and disinfect them by soaking them in 75% medical alcohol for 5 seconds in a Class 100 cleanroom environment. S3. Soak the disinfected Sagittaria sagittifolia seeds in the prepared liquid germination inoculum for 1.5 to 2 hours; S4. After thoroughly mixing the soaked seeds with the germination bacteria liquid inoculum and the prepared culture medium, pack them into boxes. S5. Cultivate *Sagittaria sagittifolia* seedlings under controlled temperature of 21℃-25℃, controlled humidity of 60%-90%, controlled light of 600lx-800lx, and controlled gas (carbon dioxide concentration) of 900ppm-1200ppm. S6. When the seedlings grow to 3cm-10cm, move them to a plastic greenhouse at natural temperature for 7-10 days to harden them off, and then transplant them.

2. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, In the preparation of the culture medium, high-temperature sterilization can be performed in two ways: one is to keep it at 100°C for 10 hours to ensure that the inside of the culture medium is fully heated and effectively kill various bacteria and insect eggs; the other is to keep it at 121°C for 5 hours to quickly and thoroughly sterilize it using a high-temperature and high-pressure environment. The cooling process needs to be carried out in a clean environment to cool it to 22°C-26°C for later use to prevent re-contamination during the cooling process.

3. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, In the seed soaking step, strictly follow the aseptic operating procedures. In a Class 100 clean bench, extract 1000ml of cultured germination liquid inoculum. Precisely select 100 sterilized and mature, unopened fruit segments of *Sagittaria sagittifolia*, carefully cut open the fruit segments, remove the seeds inside intact, and soak them all in the extracted germination liquid inoculum to ensure full contact between the seeds and the inoculum, creating favorable conditions for subsequent germination.

4. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, In the seed mixing step, first, 5000ml of the cultured germination bacteria liquid inoculum is placed in a clean mixing container. Then, the soaked seeds are slowly poured in and thoroughly mixed using a sterilized mixing tool to ensure that the germination bacteria are evenly attached to the seed surface. Subsequently, 50kg of the prepared culture medium is added to the mixing container in multiple batches, and thoroughly mixed after each addition to ensure that the seeds, germination bacteria liquid inoculum, and culture medium are evenly mixed without local accumulation or clumping.

5. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, In the boxing step, a culture box with filter ventilation holes that has undergone strict sterilization is selected. The size of the filter ventilation holes must be large enough to meet the gas exchange requirements while effectively preventing the invasion of miscellaneous bacteria. The mixture after seed dressing is slowly poured into the culture box while gently shaking the box to ensure even distribution of the mixture. The filling thickness is precisely controlled at 5cm-7cm to ensure suitable growth space for the hairy arrowhead seedlings. After filling, the lid is quickly closed and sealed firmly to the culture box using food-grade tape to prevent contamination by external bacteria.

6. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, The cultivation process is divided into two stages: In the initial stage, the sealed cultivation box is placed in a temperature-controlled environment, precisely maintaining the temperature at 21℃-25℃. Humidity is controlled at 60% using a humidity control device, and the carbon dioxide concentration is maintained at 900ppm-1200ppm using a gas control device. This stage lasts for 13 days, providing a stable and suitable environment for seed germination and mycelial growth. In the subsequent stage, the cultivation box lid is carefully removed, and the box is transferred to a cultivation room with light, temperature, humidity, and gas control functions. Alternatively, the cultivation environment is precisely temperature-controlled at 21℃-25℃, and humidity is increased to 85%-95% using a humidifier. A light intensity of 600lx-800lx is provided using a light control device, with 8 hours of light per day. Simultaneously, the carbon dioxide concentration is controlled at 900ppm-1200ppm using a gas control device. Cultivation continues for 80-100 days until the seedlings reach 3cm-10cm in height.

7. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to claim 1, characterized in that, In the seedling hardening process, the cultivated *Sagittaria sagittifolia* seedlings, along with their culture boxes, are carefully transferred to a plastic greenhouse at a natural temperature. A humidity control device is installed in the greenhouse to precisely control the moisture content of the culture medium at 55%-65%, providing a buffer transition for the seedlings to adapt to the external environment. During the hardening period, the growth status of the seedlings and the changes in the humidity of the culture medium are observed regularly every day, and the humidity is adjusted in a timely manner according to the actual situation to ensure that the seedlings grow healthily during the 7-10 day hardening period and gradually adapt to the natural environment.

8. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to any one of claims 1-7, characterized in that, The seedlings of *Cistanche deserticola* cultivated using this method have achieved a transplant success rate of over 95% after extensive practical transplanting experiments, significantly higher than that of traditional seedling cultivation methods. Furthermore, the cost of *Cistanche deserticola* seedlings with a diameter of 3cm-10cm is only 0.2 yuan per seedling, a substantial reduction compared to traditional seedling cultivation methods. This effectively improves the economic benefits of *Cistanche deserticola* cultivation and provides reliable technical support for large-scale cultivation.

9. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to any one of claims 1-7, characterized in that, This method has wide seasonal adaptability and is not limited by the traditional seedling season. Seedling operations can be carried out in winter, spring and summer. During the seedling process, precise environmental control can provide stable growth conditions for the germination of hairy arrowhead seeds and the growth of seedlings, ensuring that seedlings can grow and develop normally in different seasons. After the seedlings are cultivated, they can be transplanted in spring, summer and autumn according to the actual situation. The seedling time is significantly shortened to 90-120 days compared with the traditional method, which effectively improves the efficiency of hairy arrowhead cultivation and shortens the cultivation cycle.

10. The method for cultivating *Sagittaria sagittifolia* seedlings based on fungal liquid culture according to any one of claims 1-7, characterized in that, This method has significant ecological and environmental advantages in the seedling cultivation process. In the traditional seedling cultivation process, in order to obtain enough leaves and humus, it is often necessary to collect forests on a large scale, which not only easily leads to soil erosion, but also causes serious damage to forest biodiversity. In contrast, this method uses a specific culture medium formula, which does not require the large-scale collection of leaves and humus, thereby reducing the dependence on forest resources from the source, effectively protecting the forest ecological environment, conforming to the concept of sustainable development, and providing a useful reference for the development of eco-friendly agriculture.

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