An indoor seedling raising method of morchella

Abstract

The application discloses an indoor seedling raising method of Tricholoma mongolicum, and relates to the technical field of indoor seedling raising, which comprises the following steps: preparing a low-nutrient symbiotic germination culture medium, taking water and agar as the basis, containing 0-5 g / L sugar substances and humus soil extract, and forming a sterile environment beneficial to the symbiosis of fungi; selecting associated fungi to prepare mycelium fragment suspension as symbiotic inoculants through liquid fermentation; performing surface disinfection and sterile ultrasonic treatment on Tricholoma mongolicum seeds; sowing the seeds on the surface of the culture medium and inoculating the symbiotic inoculants under sterile conditions, and culturing under artificial climate control of temperature and light, so as to promote the symbiotic relationship between the fungi and the seeds; and obtaining symbiotic seedlings after the seeds swell and differentiate into protocorms or young leaves. The symbiotic germination mechanism of associated fungi is introduced into the indoor sterile culture system, the emergence rate can reach more than 90%, the emergence uniformity is high, the seedlings are not limited by seasons and climate, the seedlings are self-brought mycorrhizal roots, the transplanting survival rate is high, and the reliable seedling guarantee is provided for the large-scale artificial planting of Tricholoma mongolicum.

Description

Technical Field

[0001] This invention relates to the field of indoor seedling technology, and in particular to an indoor seedling cultivation method for *Cymbidium goeringii*. Background Technology

[0002] Mao Cigu, also known as Shan Cigu or Du Suan Lan, is a rare perennial medicinal plant belonging to the genus Rhododendron of the Orchidaceae family. Its dried pseudobulb is a traditional and precious Chinese medicinal material with effects such as clearing heat and detoxifying, resolving phlegm and dissipating nodules. Modern pharmacological studies have also confirmed that it has significant activity in anti-tumor and anti-angiogenesis. The market demand is huge. Due to long-term over-harvesting, the wild resources of Mao Cigu have been drastically depleted and it has been listed as a national second-class key protected wild plant. Large-scale artificial cultivation has become the only way to meet market demand and protect wild resources.

[0003] The large-scale propagation of *Cistanche deserticola* seedlings is a core bottleneck restricting the development of the artificial cultivation industry. Its seeds are as fine as dust and lack endosperm, requiring specific symbiotic fungi for germination under natural conditions. While existing field direct seeding methods using symbiotic fungi can achieve seedling emergence, they are severely constrained by season and climate, resulting in unstable germination rates, susceptibility to contamination, long cycles, and uneven seedling quality, making standardized production difficult. Tissue culture methods, while enabling factory-scale production, rely entirely on exogenous hormones and are separated from the symbiotic fungal environment, leading to easily degraded seedling quality and low transplant survival rates. Furthermore, in vitro aseptic sowing methods often use high-sugar culture media, disrupting the natural nutritional interaction between seeds and symbiotic fungi, resulting in slow seedling growth and a long recovery period. Summary of the Invention

[0004] The purpose of this invention is to provide an indoor seedling cultivation method for *Cymbidium goeringii* to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an indoor seedling cultivation method for *Cymbidium goeringii*, comprising S1, preparation of a symbiotic culture medium: preparing a low-nutrient symbiotic germination culture medium, wherein the culture medium is based on water and agar, and contains a reduced concentration of sugars (content of 0-5 g / L) and humus extract, and is sterilized at high temperature and then dispensed for use, so as to form a sterile environment conducive to fungal symbiosis rather than simply providing nutrients; S2. Preparation of symbiotic inoculant: Select symbiotic fungi that can promote the germination of *Cymbidium goeringii* seeds, and after liquid fermentation culture, filter the obtained mycelium and prepare a mycelial fragment suspension with controllable concentration as a symbiotic inoculant. S3. Aseptic treatment of seeds: The seeds of *Cynanchum paniculatum* are subjected to surface disinfection and aseptic ultrasonic treatment in sequence. The aseptic ultrasonic treatment is intended to increase seed coat permeability and break dormancy to obtain pretreated seeds. S4. Symbiotic inoculation and culture: Under aseptic conditions, the pretreated seeds are sown on the surface of the symbiotic germination medium, and then inoculated with the symbiotic agent. Afterwards, the seeds are placed under artificial climate conditions for temperature and light control culture to promote the establishment of a symbiotic relationship between the fungi and the seeds and initiate germination. S5. Seedling formation: After the seeds swell and differentiate into protocorms or young leaves, the indoor sterile symbiotic seedling process is completed, and symbiotic seedlings are obtained.

[0006] Preferably, in S1, the components and concentrations of the symbiotic germination culture medium are: 150-250 mL / L of humus extract, 0-3 g / L of sucrose, 6-8 g / L of agar, with the remainder being water, and the pH being natural.

[0007] Preferably, in S2, the associated fungus is one of Coprinus radiata and Coprinus alba.

[0008] Preferably, in step S2, the method for preparing the mycelial fragment suspension is as follows: the associated fungus is inoculated into a liquid fermentation medium and cultured with shaking at a temperature of 22-26°C and a rotation speed of 120-160 r / min for 5-7 days. The mycelial balls are collected by filtration with sterile gauze, repeatedly rinsed with sterile water, resuspended with sterile water, and broken up by a stirrer to prepare a suspension with mycelial fragments of 0.5-2 mm in length. The concentration is adjusted to contain 50-200 mycelial fragments per milliliter.

[0009] Preferably, in step S3, the conditions for the aseptic ultrasonic treatment are: frequency 30-50 kHz, treatment time 20-60 seconds, and treatment temperature 20-28°C.

[0010] Preferably, in step S4, the inoculation method of the symbiotic agent is either spray inoculation or drip inoculation, and the inoculation amount is 0.05 to 0.2 mL of the mycelial fragment suspension per square centimeter of culture medium surface.

[0011] Preferably, in step S4, the temperature and light control conditions for cultivation are: cultivation temperature 20–26°C, light intensity 1500–3000 Lux, and light cycle 12–16 h / d.

[0012] Preferably, the preparation method of the humus extract is as follows: take humus from the native habitat of *Sagittaria sagittifolia* and distilled water at a mass-volume ratio of 1:5 to 1:10, boil for 20 to 30 minutes, let stand and cool, and then filter the supernatant to obtain the humus extract.

[0013] Preferably, after S5, an acclimatization and transplanting step is included: the obtained symbiotic seedlings are gradually hardened off in a culture container by opening the lid for 2 to 4 days, and then transplanted into a sterilized mixed substrate of peat moss and perlite in a volume ratio of 1 to 3:1. The seedlings are then cultured for another 10 to 20 days under conditions of 20 to 25°C, 75 to 85% humidity, and diffused light, and then transplanted to the field.

[0014] Preferably, in S4, the initial stage of the temperature- and light-controlled culture involves dark culture for 3 to 8 days, and then the culture is switched to light culture after the mycelium colonizes the seed surface, in order to promote the preferential colonization of fungi and the establishment of a symbiotic relationship.

[0015] The technical effects and advantages of this invention are as follows: (1) This invention innovatively introduces the symbiotic germination mechanism of associated fungi into the indoor sterile culture system. By using ultrasonic pretreatment to break seed dormancy and increase seed coat permeability, and combined with low sugar symbiotic culture medium to form a nutrient stress environment, the seeds are directed to establish a symbiotic relationship with specific associated fungi, so that the germination rate can reach more than 90%, the uniformity of germination is significantly improved, and the whole process is carried out under artificial climate conditions, which completely gets rid of the limitations of traditional direct seeding in the field that are restricted by season and climate, and can realize standardized and batch production all year round and all weather.

[0016] (2) This invention does not add any exogenous plant hormones during the entire germination and seedling process. Instead, it uses hormone analogs and signaling molecules naturally produced by symbiotic fungi during metabolism to promote seed germination and protocorm differentiation. This fundamentally avoids the problems of somatic cell variation and seedling quality degradation caused by long-term hormone induction in tissue culture. The obtained symbiotic seedlings have their own mycorrhizal symbiotic system, well-developed root system, strong resistance, and a transplant survival rate of over 90%, which is significantly better than traditional tissue culture virus-free seedlings.

[0017] (3) The entire process of this invention is carried out under strict aseptic conditions. Aseptic barriers are established in all stages from culture medium sterilization, seed disinfection, preparation of microbial agents to inoculation and culture. The contamination rate of miscellaneous bacteria is extremely low, the production success rate is high, and the risk is controllable. Moreover, the overall technical solution is easy to operate and the equipment requirements are moderate. It is easy to promote quickly in seedling enterprises and agricultural cooperatives, providing a stable and reliable seedling guarantee for the large-scale artificial cultivation of Maocigu. It has significant economic and social benefits. Attached Figure Description

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic flowchart of the indoor seedling cultivation method for *Cymbidium goeringii* according to the present invention. Detailed Implementation

[0019] 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. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] This invention provides, for example Figure 1 The method for indoor seedling cultivation of *Sagittaria sagittifolia* includes S1, preparation of symbiotic culture medium: preparing a low-nutrient symbiotic germination culture medium, which is based on water and agar, and contains sugars at a reduced concentration of 0-5 g / L and humus extract. After high-temperature sterilization, it is dispensed for use to form a sterile environment conducive to fungal symbiosis rather than simply providing nutrients. The components and concentrations of the symbiotic germination culture medium are: 150-250 mL / L humus extract, 0-3 g / L sucrose, 6-8 g / L agar, with the remainder being water, and the pH being natural. The preparation method of humus extract is as follows: Collect surface humus from the native habitat of *Sagittaria sagittifolia*, remove impurities, weigh the humus and mix it with distilled water at a mass-to-volume ratio of 1:8, place in a heating container and boil for 25 minutes. After naturally cooling, use double-layer gauze for coarse filtration to remove large particles of impurities, and then filter through qualitative filter paper. Collect the filtrate, which is the humus extract. Store at 4℃ for later use. Weigh each component according to the above formula. First, add agar to an appropriate amount of distilled water and heat until completely dissolved. Then, add the humus extract and sucrose in sequence, stir thoroughly to dissolve, and then dilute to the target volume with distilled water. While hot, dispense into tissue culture bottles or petri dishes, each bottle containing approximately 30-50 mL. After dispensing, place in an autoclave and sterilize at 121℃ and 0.1 MPa for 20 minutes. After removal, allow to cool and solidify naturally to obtain the symbiotic germination medium, which is then ready for use.

[0021] S2. Preparation of symbiotic inoculant: Associated fungi with the ability to promote the germination of *Cymbidium goeringii* seeds were selected. After liquid fermentation culture, the obtained mycelium was filtered and prepared into a mycelial fragment suspension with controllable concentration as a symbiotic inoculant. The associated fungi were either *Coprinus radiata* or *Coprinus spp.*. The preparation method of the mycelial fragment suspension was as follows: The associated fungi were inoculated into the liquid fermentation medium and cultured with shaking at a temperature of 22-26℃ and a rotation speed of 120-160 r / min for 5-7 days. The mycelial balls were collected by filtering with sterile gauze, rinsed repeatedly with sterile water, resuspended with sterile water, and broken up with a stirrer to prepare a suspension with mycelial fragment length of 0.5-2 mm. The concentration was adjusted to contain 50-200 mycelial fragments per milliliter. The selected symbiotic fungus was *Coprinus radiata*, which was isolated from the rhizosphere soil of *C. radiata*'s native habitat. This fungus is currently the only symbiotic fungus that significantly promotes seed germination in *C. radiata*. The preparation method for the mycelial fragment suspension was as follows: the activated *Coprinus radiata* strain was inoculated into a liquid fermentation medium. The medium formula was: 200 g / L potato broth, 10 g / L glucose, 2 g / L peptone, 1 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate, with a natural pH. The liquid volume was 40% of the Erlenmeyer flask's capacity. The medium was cultured for 6 days in the dark on a constant-temperature shaker at 24℃ and 140 r / min. After the culture, a large number of uniformly dispersed mycelial balls appeared in the culture medium. The mycelial balls were collected by filtration through sterile double-layer gauze and rinsed repeatedly with sterile water three times to thoroughly remove residual culture medium components. The washed mycelial balls were resuspended in an equal volume of sterile water and transferred to a sterile tissue homogenizer. They were then stirred at a medium-low speed for 10-15 seconds to prepare a suspension with mycelial fragments of 0.5-2 mm in length. The mycelial fragments were counted under a microscope using a hemocytometer. The concentration of the suspension was adjusted with sterile water to approximately 100 mycelial fragments per milliliter to obtain the symbiotic agent, which should be prepared and used immediately.

[0022] S3. Aseptic treatment of seeds: The seeds of *Cynanchum paniculatum* were subjected to surface disinfection and aseptic ultrasonic treatment in sequence. The aseptic ultrasonic treatment was intended to increase the permeability of the seed coat and break dormancy to obtain pretreated seeds. The conditions for aseptic ultrasonic treatment were: frequency 30-50KHz, treatment time 20-60 seconds, and treatment temperature 20-28℃. For surface disinfection and aseptic ultrasonic treatment, select mature, plump *Gnaphalium affine* capsules harvested that year. First, disinfect the surface by soaking in a 75% ethanol solution for 30 seconds, then rinse once with sterile water. Next, soak in a 0.1% mercuric chloride solution for 8 minutes, agitating continuously during this time. After removal, rinse repeatedly with sterile water 4-5 times to thoroughly remove residual mercuric chloride. Cut open the surface-disinfected capsules with a sterile scalpel in a laminar flow hood, remove the internal powdery seeds, place them in sterile centrifuge tubes, and add an appropriate amount of sterile water to prepare a seed suspension. Place the centrifuge tubes containing the seed suspension in an ultrasonic cleaner for aseptic ultrasonic treatment under the following conditions: frequency 40 kHz, treatment time 40 seconds, and treatment temperature 25℃. Remove immediately after ultrasonic treatment for later use. This treatment effectively increases the micro-cracks in the seed coat, improves permeability, breaks dormancy, and facilitates the subsequent invasion of fungal hyphae and the establishment of a symbiotic relationship.

[0023] S4. Symbiotic Inoculation and Cultivation: Under aseptic conditions, pretreated seeds are sown on the surface of the symbiotic germination medium, followed by inoculation with a symbiotic agent. The medium is then placed under controlled temperature and light conditions to promote the establishment of a symbiotic relationship between the fungus and the seeds, initiating germination. The symbiotic agent can be inoculated using either spraying or drip inoculation, with an inoculation volume of 0.05–0.2 mL of mycelial fragment suspension per square centimeter of medium surface. The controlled temperature and light conditions are as follows: cultivation temperature... At 20–26℃, with a light intensity of 1500–3000 Lux and a photoperiod of 12–16 h / d, humus from the native habitat of *Sagittaria sagittifolia* and distilled water were mixed at a mass-to-volume ratio of 1:5–1:10, boiled for 20–30 minutes, and after cooling, the supernatant was filtered to obtain the humus extract. The initial stage of the temperature- and light-controlled culture was carried out in the dark for 3–8 days until the mycelium colonized the seed surface, and then the culture was transferred to light to promote the preferential colonization of fungi and the establishment of a symbiotic relationship.

[0024] For both spray inoculation and drop inoculation, in a sterile environment under a laminar flow hood, use a pipette or sterile dropper to pick up the seed suspension treated with sterile ultrasound in step S3 and evenly spread it on the surface of the symbiotic germination medium prepared in step S1, ensuring the seeds are evenly distributed but not overlapping. Then, inoculate with the symbiotic agent. For drop inoculation, use a sterile pipette to pick up the mycelial fragment suspension prepared in step S2 and, according to the standard of 0.1 mL per square centimeter of medium surface, evenly drop the mycelial fragment suspension onto the surface of the medium after sowing, ensuring full contact between the mycelial fragments and the seeds. After inoculation, seal the culture bottle or petri dish and place it in an artificial climate incubator for temperature and light controlled cultivation. The cultivation conditions were: temperature 23℃, light intensity 2000 Lux, and a daily light cycle of 14 hours of light and 10 hours of darkness. This stage aims to promote the rapid colonization of *Coprinus radiata* mycelium on the seed surface and its invasion into the seed interior, establishing a functional symbiotic relationship. The fungus absorbs water and humic nutrients from the culture medium to supply the seeds and initiate the seed germination process. Regular observations were conducted during the cultivation process. Usually, 7–10 days after inoculation, the mycelium began to wrap around and coat the seeds; 15–20 days later, the seeds showed obvious water absorption and swelling, and their color changed from pale yellow to light green, indicating that the symbiotic relationship had been successfully established and germination had begun.

[0025] S5. Seedling Development: After the seeds swell and differentiate into protocorms or young leaves, the indoor sterile symbiotic seedling process is completed, and symbiotic seedlings are obtained. After S5, there is also an acclimatization and transplanting step: the obtained symbiotic seedlings are gradually opened in the culture container for 2-4 days to harden off, and then transplanted into a sterilized mixed substrate of peat moss and perlite in a volume ratio of 1-3:1. They are then cultured for 10-20 days under the conditions of temperature 20-25℃, humidity 75-85%, and diffused light, and then transplanted to the field. Continue culturing under the above conditions. After about 40 to 50 days, the symbiotic system will further develop, and the seeds will swell to form protocorms that are visible to the naked eye. After about 60 to 80 days, the protocorms will begin to differentiate into the first young leaves. Once the vast majority of individuals have completed leaf differentiation and grown into symbiotic seedlings about 2 to 3 cm tall, the indoor aseptic symbiotic seedling production process is complete, and symbiotic seedlings are obtained.

[0026] The working principle of this invention is as follows: First, rhizosphere soil from the native habitat of *Cymbidium goeringii* is collected in nature. Associated fungi (such as *Coprinus radiata* or *Coprinus spp.*) that promote seed germination are isolated and screened. After purification and identification, the original strain is obtained. Then, the original strain is inoculated into a liquid fermentation medium and cultured on a temperature-controlled shaker to obtain mycelium. After filtration and washing, a mycelial fragment suspension with controllable concentration is prepared, which is the symbiotic agent. Simultaneously, a low-nutrient symbiotic culture medium is prepared with humus extract and low-concentration sucrose as its core. After autoclaving, an aseptic culture medium is prepared. Culture substrate plates: After surface disinfection and aseptic ultrasonic treatment, *Cistanche deserticola* seeds are sown on the surface of the culture medium; then inoculated with symbiotic agents, and cultured under controlled temperature and light conditions in an artificial climate. Ultrasonic treatment increases seed coat permeability, and the low-sugar culture medium creates nutrient stress, driving the seeds to establish a symbiotic relationship with the associated fungi. The fungi provide nutrients and germination signals to the seeds, causing them to swell and form protocorms and differentiate into leaves; when the seedlings grow to a height of about 2-3 cm, the indoor aseptic symbiotic seedling process is completed, and *Cistanche deserticola* seedlings carrying active symbiotic mycorrhizae are obtained.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for indoor seedling cultivation of *Sagittaria sagittifolia*, characterized in that, include: S1. Preparation of symbiotic culture medium: Prepare a low-nutrient symbiotic germination culture medium, which is based on water and agar, contains a reduced concentration of sugars (content of 0-5 g / L) and humus extract, and is sterilized at high temperature and then dispensed for use to form a sterile environment conducive to fungal symbiosis rather than simply providing nutrients. S2. Preparation of symbiotic inoculant: Select symbiotic fungi that can promote the germination of *Cymbidium goeringii* seeds, and after liquid fermentation culture, filter the obtained mycelium and prepare a mycelial fragment suspension with controllable concentration as a symbiotic inoculant. S3. Aseptic treatment of seeds: The seeds of *Cynanchum paniculatum* are subjected to surface disinfection and aseptic ultrasonic treatment in sequence. The aseptic ultrasonic treatment is intended to increase seed coat permeability and break dormancy to obtain pretreated seeds. S4. Symbiotic inoculation and culture: Under aseptic conditions, the pretreated seeds are sown on the surface of the symbiotic germination medium, and then inoculated with the symbiotic agent. Afterwards, the seeds are placed under artificial climate conditions for temperature and light control culture to promote the establishment of a symbiotic relationship between the fungi and the seeds and initiate germination. S5. Seedling formation: After the seeds swell and differentiate into protocorms or young leaves, the indoor sterile symbiotic seedling process is completed, and symbiotic seedlings are obtained.

2. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S1, the components and concentrations of the symbiotic germination culture medium are: 150-250 mL / L of humus extract, 0-3 g / L of sucrose, 6-8 g / L of agar, with the remainder being water, and the pH being natural.

3. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S2, the associated fungus is one of Coprinus radiata and Coprinus alba.

4. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S2, the preparation method of the mycelial fragment suspension is as follows: the associated fungus is inoculated into a liquid fermentation medium and cultured with shaking at a temperature of 22-26℃ and a rotation speed of 120-160 r / min for 5-7 days. The mycelial balls are collected by filtration with sterile gauze, rinsed repeatedly with sterile water, resuspended with sterile water, and broken up by a stirrer to prepare a suspension with mycelial fragments of 0.5-2 mm in length. The concentration is adjusted to contain 50-200 mycelial fragments per milliliter.

5. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S3, the conditions for the aseptic ultrasonic treatment are: frequency 30-50 kHz, treatment time 20-60 seconds, and treatment temperature 20-28°C.

6. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In step S4, the symbiotic agent is inoculated using either spraying or dripping inoculation, with an inoculation amount of 0.05–0.2 mL of mycelial fragment suspension per square centimeter of culture medium surface.

7. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S4, the temperature and light control conditions for cultivation are: cultivation temperature 20-26℃, light intensity 1500-3000 Lux, and light cycle 12-16 h / d.

8. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 2, characterized in that, The method for preparing the humus extract is as follows: take humus from the native habitat of *Sagittaria sagittifolia* and distilled water at a mass-volume ratio of 1:5 to 1:10, boil for 20 to 30 minutes, let stand and cool, and then filter the supernatant to obtain the humus extract.

9. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, Following S5, an acclimatization and transplanting step is also included: the obtained symbiotic seedlings are gradually hardened off in a culture container by opening the lid for 2 to 4 days, and then transplanted into a sterilized mixed substrate of peat moss and perlite in a volume ratio of 1 to 3:

1. The seedlings are then cultured for another 10 to 20 days under conditions of 20 to 25°C, 75 to 85% humidity, and diffused light, before being transplanted to the field.

10. The indoor seedling cultivation method for *Sagittaria sagittifolia* according to claim 1, characterized in that, In S4, the initial stage of the temperature- and light-controlled culture involves dark culture for 3 to 8 days. After the mycelium colonizes the seed surface, the culture is then switched to light culture to promote preferential colonization of fungi and the establishment of a symbiotic relationship.

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