A method for direct seeding of fern spores

Abstract

This invention belongs to the field of medicinal fern spore propagation technology. To address the technical problems of complex operation, high cost, and insufficient seedling effect in existing fern spore seedling cultivation methods, this invention discloses a direct seeding method for fern spores, comprising the following steps: collecting fern spores and passing them through a No. 6 pharmacopoeia sieve for later use; selecting a cultivation box with a transparent lid and an opaque bottom; filling the cultivation box with the culture medium; sterilizing the box; then directly seeding the spores; and finally, sealing the transparent lid tightly to continue fern spore seedling cultivation. This invention employs a closed, enclosed cultivation method, which reduces the frequency of watering during seedling cultivation, reduces the risk of microbial contamination, and protects the survival environment of the prothallus and sporophyte. Simultaneously, optimizations are made in spore maturity selection, culture medium selection, culture medium depth and sterilization method, spore seeding density, and fertilization from prothallus to sporophyte, comprehensively improving spore germination rate and sporophyte formation rate, thus enhancing the effectiveness of direct seeding of fern spores.

Description

Technical Field

[0001] This invention belongs to the field of medicinal fern spore propagation technology, specifically relating to a method for direct seeding of *Adiantum capillus-veneris* spores. Background Technology

[0002] The Chinese medicinal herb *Drynaria fortunei* is *Drynaria fortunei*, a plant belonging to the genus *Drynaria* of the family Pteridaceae. Drynaria roosii The dried rhizome of Nakaike.

[0003] Oak fern has high requirements for its natural growth environment. It usually grows epiphytically on tree trunks and rocks, and rarely grows directly in soil. It has high requirements for air temperature, humidity, and light, and has a long growth cycle. In addition, the current market use of the traditional Chinese medicine Drynaria fortunei has led to a shortage of Drynaria fortunei resources. Artificial propagation can reduce the consumption of wild oak fern resources and increase the yield of Drynaria fortunei. Among them, oak fern spore seedling cultivation is a simple and efficient seedling cultivation method.

[0004] However, existing methods for propagating fern spores have the following shortcomings: (1) Traditional fern spore cultivation techniques are generally carried out in an open-air environment or require daily watering to keep the substrate moist. The planting and management are complicated, increasing labor costs. At the same time, it increases the frequency of air exchange with external sources, making it easy for microorganisms such as mold in the air to contaminate the culture medium, resulting in mold growth and algae bloom.

[0005] (2) Research on the selection of mature spores is relatively superficial: At present, all direct seeding methods for fern spores mention the selection of mature spores, but the methods for selecting mature spore powder have not been explored in depth, resulting in unclear selection criteria for mature spores and unstable seedling results.

[0006] (3) Insufficient exploration of sterilization methods for culture media: The safety of existing substrate sterilization methods is not adequately considered. For example, sterilization by autoclaving has a low safety factor and is difficult to operate; or sterilization by adding exogenous agents such as cyromazine soluble powder can easily threaten health if not operated properly.

[0007] (4) Insufficient consideration of economic benefits and promotion: The existing fern spore seedling technology has problems such as high planting technology requirements, complex operation, high labor management costs, and high cost and difficulty in obtaining seedling auxiliary materials. It has failed to solve the problem of efficient synergy between economic benefits and planting management in artificial fern seedling cultivation.

[0008] (5) Using exogenous substances (such as sucrose, vitamins, natural plant extracts, etc.) is an effective means to improve and accelerate the germination of spores and the growth of prothalliums. However, most existing technologies use exogenous hormones such as growth regulators, which pose safety risks. There are also reports of using other nutrients (such as sucrose, vitamins, natural plant extracts, etc.) to promote spore development. However, existing technologies generally add nutrients directly to the substrate without considering the dispersion of nutrients, and different nutrients have different seedling promotion effects.

[0009] (6) Insufficient research on how to improve the success rate of prothallium to sporophyte: After fertilization, the prothallium grows into a sporophyte. As the young sporophyte gradually grows and becomes independent, the prothallium on which it is attached gradually withers and dies. Existing technologies do not mention how to improve the fertilization probability of the prothallium. If the prothallium is not fertilized, it cannot grow into a sporophyte, that is, spore seedling cultivation cannot be successful.

[0010] (7) Insufficient compatibility of culture substrate components: When using different materials to prepare mixed culture substrates, the existing technology only focuses on the influence of substrate type on spore seedling cultivation, but ignores the compatibility between different substrates, resulting in many substrate materials not being used properly.

[0011] In summary, the current method of harvesting wild oak fern faces safety and sustainability issues, impacting the sustainable supply of the traditional Chinese medicine *Drynaria fortunei*. Furthermore, existing artificial propagation techniques for oak fern have significant shortcomings and room for improvement in areas such as spore maturity selection, culture medium selection and sterilization methods, planting management, success rate from prothallium to sporophyte, elucidation of the yield-increasing mechanism, and considerations of economic benefits and scalability. Therefore, there is an urgent need to develop a simple, low-cost, and effective direct-sowing method for oak fern spore propagation. Summary of the Invention

[0012] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a method for direct seeding of *Adiantum capillus-veneris* spores. This method employs a closed-environment culture method using a cultivation box with a transparent lid and an opaque bottom, which reduces the frequency of watering during seedling cultivation (resulting in more stable humidity), reduces the risk of microbial contamination, and ensures a suitable environment for the prothallium and sporophyte. Furthermore, optimizations are made in spore maturity selection, culture medium selection, culture medium depth and sterilization methods, spore direct seeding density, and fertilization from prothallium to sporophyte, comprehensively improving spore germination rate and sporophyte formation rate, thus enhancing the effectiveness of direct seeding of *Adiantum capillus-veneris* spores.

[0013] The first objective of this invention is to select a closed-environment culture method for direct seeding of fern spores, and to optimize aspects such as spore maturity selection, culture medium selection, culture medium depth and sterilization method, spore direct seeding density, and fertilization from prothallium to sporophyte, so as to improve spore germination rate and sporophyte formation rate.

[0014] To achieve the first objective, the present invention adopts the following technical solution: A method for direct seeding of *Adiantum capillus-veneris* spores includes the following steps: (1) Spore collection: Collect fresh leaves with mature sporangia on the back of the fern, brush off the sporangia with a sterilized brush, and collect the spore powder for later use. (2) Substrate selection and packaging: Use any one or two of peat moss and vermiculite as the culture medium. Select a culture box with a transparent lid and an opaque bottom, put the culture medium into the culture box, and sterilize it. (3) Direct spore release: The spore suspension prepared in step (1) is mixed with water and sprayed evenly onto the culture medium. (4) After direct sowing of spores, cover the transparent cap tightly and manage the period from spores to prothallus and the period from prothallus to sporophyte.

[0015] (5) When the sporophyte grows into a fern seedling with more than 2 leaves, transplant the seedlings, manage fertilizer and water, and control pests and diseases in accordance with conventional methods.

[0016] Preferably, in step (1), the collected spore powder is screened through a No. 6 pharmacopoeia sieve after sterilization to obtain spore powder with better maturity.

[0017] Preferably, in step (2), the culture medium includes any one or two of peat moss and vermiculite. More preferably, the culture medium includes peat moss or peat moss and vermiculite in a volume ratio of (1-10):1. Even more preferably, the culture medium is peat moss.

[0018] Preferably, in step (2), the depth of the incubation box is 4-6 cm, the depth of the culture medium after being packed in the box is 2-3 cm, and the humidity of the culture medium is 70-90%.

[0019] Preferably, in step (2), the sterilization method after the culture medium is placed into the incubation box is steam sterilization, and the sterilization time is not less than 35 minutes. Sufficient sterilization time is necessary to ensure that the mold in the substrate and incubation box is eliminated.

[0020] Preferably, in step (3), the density of direct spore seeding is 1-8 mg / m³. 2The concentration of the spore suspension is 0.04-0.2 g / L. This invention requires selecting a suitable direct spore density to ensure that the resulting sporophytes are neither too dense nor too sparse. Overly dense sporophytes result in small, thin sporophytes, which are detrimental to transplant survival.

[0021] Preferably, in step (4), during the management from spore to prothallus and from prothallus to sporophyte, the transparent lid of the cultivation box is opened for 10-120 seconds every 7-15 days to exchange air. Because the humidity of the culture medium changes little due to the closed cultivation, the frequency of watering during the seedling stage can be reduced (the traditional method requires watering every day). The seedling status of the fern can be observed while opening the transparent lid of the cultivation box every 7-15 days. If the humidity is too low, distilled water is added to the appropriate humidity (70-90%). If the humidity is too high, the excess water needs to be drained.

[0022] Preferably, in step (4), the prothallus to sporophyte stage is treated by soaking in water. Distilled water is added to the culture box and the prothallus is soaked (submerging the prothallus) for 15-30 minutes. During this period, the culture box is shaken every 3-5 minutes. After soaking, excess water is removed to restore the original humidity (70-90%), and the remaining management is carried out. This method uses a flowing liquid environment to accelerate the fertilization of the fern prothallus.

[0023] Except for the air exchange every 7-15 days and the need to open the lid of the incubator during the soaking method, the lid does not need to be opened at other times, which reduces the contact time between the outside and the incubator and ensures a sterile environment inside the incubator.

[0024] Preferably, in step (4), the management during the period from spore to prothallus includes: light intensity of 1000-2000 lx, daytime temperature of 22-27℃, nighttime temperature of 14-20℃, and light duration of 8-14 h.

[0025] Management during the period from prothallus formation to sporophyte formation includes: light intensity of 1500-2500 lx, daytime temperature of 22-27℃, nighttime temperature of 14-20℃, and light duration of 8-14 h.

[0026] In practical production applications, peat moss resources are relatively scarce and expensive compared to vermiculite (especially high-quality imported Pinscher peat moss). Therefore, adding vermiculite to peat moss is a relatively economical method. However, vermiculite is lightweight and tends to float on the surface after absorbing water, resulting in uneven dispersion of the composite substrate. The second objective of this invention is to provide a culture substrate for direct seeding of *Adiantum capillus-veneris* spores. This involves treating vermiculite and then combining it with peat moss as a substrate, thereby improving the promoting effect of this composite substrate on *Adiantum capillus-veneris* seedling cultivation, and particularly compensating for the shortcomings of combining peat moss and vermiculite.

[0027] To achieve the second objective, the present invention adopts the following technical solution: A culture medium for direct seeding of *Adiantum capillus-veneris* spores comprises peat moss and vermiculite in a volume ratio of (1-3):1, wherein the vermiculite is treated with plant colloids before use. The specific method includes: Plant colloids are added to hot water and stirred until evenly mixed. Then vermiculite is added and mixed evenly. The water is evaporated under reduced pressure while stirring to obtain the processed vermiculite.

[0028] Preferably, the plant gum is seaweed gum, agar, carrageenan, guar gum, or alginate. More preferably, the plant gum is seaweed gum.

[0029] Preferably, the temperature of the hot water is 40-60℃, the amount of hot water added is 50-500 times the mass of the plant gum, and the mass ratio of the plant gum to vermiculite is 1:(50-200).

[0030] Technical effects of the present invention: 1. This invention utilizes a closed-loop cultivation method for seedling raising, employing a cultivation box with a transparent lid and an opaque bottom. This reduces the frequency of watering during the seedling stage (resulting in more stable humidity), minimizes the risk of microbial contamination, and ensures a suitable environment for the prothallus and sporophyte. Furthermore, the cultivation box can be opened for 10-120 seconds every 7-15 days to exchange air and ensure adequate oxygen supply. Additionally, the opaque bottom of the cultivation box promotes sporophyte formation. Overall, this method improves the sporophyte formation rate and seedling emergence rate, thereby increasing the success rate of direct seeding of *Adiantum capillus-veneris*.

[0031] 2. In this invention, during the process from prothallus to sporophyte, the prothallus is soaked in water. The fluid liquid environment increases the contact opportunities between sperm and egg, promotes fertilization of the prothallus and the growth of sporophyte, and further improves the success rate of prothallus to sporophyte transformation.

[0032] 3. This invention optimizes aspects such as spore maturity, culture substrate type, culture substrate depth and sterilization method, spore direct seeding density, and fertilization from prothallium to sporophyte, thereby simultaneously improving spore germination rate and sporophyte formation rate, and comprehensively improving seedling cultivation effect.

[0033] 4. The seedling cultivation method of this invention is simple to operate, the seedling materials are widely available and easily obtained, and the seedling time is short (prothallus forms in about 25 days, sporophyte seedlings form in 50-75 days, and seedlings can be transplanted once they have grown up); moreover, it does not use exogenous hormones, reducing the health controversies that may arise from the introduction of exogenous hormones into the fern seedlings; the sealed cultivation reduces the growth of mold between cultivation boxes and reduces the risk of mold entering the respiratory tract of growers. In summary, the seedling cultivation method of this invention is a simple, economical, widely applicable, and safe solution.

[0034] 5. In this invention, vermiculite is treated with plant colloids and then combined with peat moss as a culture medium. The plant colloids are rich in nutrients, which can not only effectively promote the development of prothallium into sporophyte and increase the sporulation rate through the introduction of nutrients from plant colloids, but also, compared with directly adding plant colloids to the culture medium, the plant colloids adsorbed by vermiculite can be slowly released, exerting their effects in a long-lasting and stable manner.

[0035] The preferred plant gum is seaweed gum. After treatment with seaweed gum, the mixing and dispersion of vermiculite and peat moss are better, which can promote the uniformity of spore germination and make up for the shortcomings of peat moss and vermiculite compound. Attached Figure Description

[0036] Figure 1 Image of a fern prothallus obtained from direct seeding of fern spores according to the present invention; Figure 2 Image of a fern sporophyte obtained from direct seeding of fern spores according to the present invention; Figure 3 The development of *Adiantum capillus-veneris* sporophytes obtained from direct seeding of *Adiantum capillus-veneris* spores in Examples 1-4 of this invention is shown. Figure 4 Germination of ferns from different groups in a preliminary screening experiment for culture substrate; Figure 5 From top to bottom, the development of the prothallus of *Adiantum capillus-veneris* is shown in Example 1, Comparative Example 3, and Comparative Example 2. Figure 6 The development of *Quercus acutissima* seedlings in Comparative Example 4; Figure 7 This is a comparison of the sporophyte development of *Adiantum quercetin* when the bottom of the culture box in Comparative Example 5 was transparent. Figure 8 The sporophyte formation of fern in batch 4 of Examples 1 and 5; Figure 9 The development of the prothallus of *Adiantum querceae* in Comparative Example 10; Figure 10 The image shows an electron microscope scan (left) and a photograph (right) of the fern spores obtained by passing them through a No. 6 pharmacopoeia sieve in Example 1. Figure 11 To compare the growth of *Adiantum capillus-veneris* seedlings obtained by passing through the No. 4 pharmacopoeia sieve in Example 12; Figure 12 The image shows a scanning electron microscope (SEM) image (left) and a photograph (right) of the fern spores obtained by passing through the No. 5 pharmacopoeia sieve in Comparative Example 13. Figure 13 To show the growth of *Quercus acutissima* seedlings obtained by passing through the No. 5 pharmacopoeia sieve in Comparative Example 13; Figure 14 The spore germination status of Examples 4, 6, 9, and 10 are shown. Detailed Implementation

[0037] The above-mentioned solution will be further described below with reference to specific embodiments; it should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments; the implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0038] Unless otherwise specified in the following examples, all raw materials are commercially available or prepared by conventional methods in the art.

[0039] In the following examples, the peat soil is pure Danish imported 0.01-10 mm Pinscher peat soil (pH 5.5-6.0, EC value 0-300 μS / cm); the vermiculite has a particle size of 5-15 mm; and the desalinated coconut brick has a particle size of 1-10 mm and an EC value of over 1000 μS / cm. Example 1

[0040] This embodiment provides a method for direct seeding of fern spores, including the following steps: (1) Spore collection: Collect fresh leaves with mature sporangia on the back of the fern. After brushing off the sporangia with a sterilized brush, sieve out the fully mature spore powder with a sterilized No. 6 pharmacopoeia sieve and store it in a sterile dry seed storage tank.

[0041] (2) Substrate selection and packaging: Peat moss was used as the culture medium. A culture box (5.2 cm × 160 mm × 110 mm) with a transparent lid and a black bottom was selected. The culture medium was filled into the culture box to a depth of 2.5 cm. Water was added to make the humidity of the culture medium 70-90%. Then the culture box was covered and placed in a steam sterilizer (one corner of the lid of the culture box can be slightly loosened during the sterilization process). After the water at the bottom of the steam sterilizer boiled for 40 minutes, it was cooled.

[0042] (3) Direct spore release: The spore powder prepared in step (1) is mixed with water to make a spore suspension of 0.1 g / L, and the direct spore release density is 2.3 mg / m³. 2 Spray the spore suspension evenly into the culture medium, and then cover it tightly with a transparent cap.

[0043] (4) Management during the period from spore to prothallus: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0044] Management during the period from prothallus formation to sporophyte formation: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0045] During the spore-to-prothallus stage and the prothallus-to-spore stage, open the transparent lid of the cultivation box for 90 seconds every 10 days to allow air exchange. Because the sealed cultivation results in minimal changes in the humidity of the culture medium, the frequency of watering during seedling cultivation can be reduced (traditional methods require daily watering). The seedlings can be observed by opening the transparent lid of the cultivation box every 10 days. If the humidity is too low, add distilled water to bring it to a suitable level (70-90%); if the humidity is too high, drain off the excess water.

[0046] (5) When the sporophyte grows into a fern seedling with more than 2 leaves, transplant the seedlings, manage fertilizer and water, and control pests and diseases in accordance with conventional methods. Example 2

[0047] The difference between this embodiment and Embodiment 1 is that the culture medium is peat moss and vermiculite in a volume ratio of 3:1. Example 3

[0048] The difference between this embodiment and Embodiment 1 is that the culture medium is peat moss and vermiculite in a volume ratio of 2:1. Example 4

[0049] The difference between this embodiment and Embodiment 1 is that the culture medium is peat moss and vermiculite in a volume ratio of 1:1. Example 5

[0050] Based on Example 1, this example provides a method for direct seeding of fern spores. In order to improve the success rate of the transition from prothallus to sporophyte, a soaking method is used during the transition from prothallus to sporophyte. Distilled water is added to the culture box to soak the prothallus (submerging the prothallus) for 20 minutes. During this period, the culture box is shaken every 4 minutes. After soaking, excess water is removed to restore the original humidity (70-90%), and other management is carried out.

[0051] The rest is the same as in Example 1. Example 6

[0052] In this embodiment, to further improve the effect of peat moss and vermiculite composite as a culture medium, vermiculite was treated with seaweed gum before use.

[0053] This embodiment provides a method for direct seeding of fern spores, including the following steps: (1) Spore collection: Collect fresh leaves with mature sporangia on the back of the fern. After brushing off the sporangia with a sterilized brush, sieve out the fully mature spore powder with a sterilized No. 6 pharmacopoeia sieve and store it in a sterile dry seed storage tank.

[0054] (2) Substrate selection and packaging: A 1:1 volume ratio of peat moss and vermiculite treated with seaweed gum was used as the culture substrate. A cultivation box (5.2 cm × 160 mm × 110 mm) with a transparent lid and a black bottom was selected. The culture substrate was filled into the cultivation box to a depth of 2.5 cm, and water was added to maintain the humidity of the culture substrate at 70-90%. The cultivation box was then covered and placed in a steam sterilizer. After the water at the bottom of the steam sterilizer boiled for 40 minutes, it was cooled.

[0055] The method for treating vermiculite with seaweed gum is as follows: Add seaweed gum to 100 times its weight of hot water (50℃) and stir until homogeneous. Then add vermiculite and mix thoroughly. Under stirring conditions, evaporate the water under reduced pressure to obtain the treated vermiculite. The mass ratio of seaweed gum to vermiculite is 1:100.

[0056] (3) Direct spore release: The spore powder prepared in step (1) is mixed with water to make a spore suspension of 0.1 g / L, and the direct spore release density is 2.3 mg / m³. 2 Spray the spore suspension evenly into the culture medium, and then cover it tightly with a transparent cap.

[0057] (4) Management during the period from spore to prothallus: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0058] Management during the period from prothallus formation to sporophyte formation: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0059] During the spore-to-prothallus stage and the prothallus-to-spore stage, open the transparent lid of the cultivation box for 90 seconds every 10 days to allow air exchange. While opening the transparent lid every 10 days, observe the fern seedling growth. If the humidity is too low, add distilled water to bring it to a suitable level; if the humidity is too high, drain off excess water.

[0060] (5) When the sporophyte grows into a fern seedling with more than 2 leaves, transplant the seedlings, manage fertilizer and water, and control pests and diseases in accordance with conventional methods. Example 7

[0061] In this embodiment, to further improve the effect of peat moss and vermiculite composite as a culture medium, vermiculite was treated with seaweed gum before use.

[0062] This embodiment provides a method for direct seeding of fern spores, including the following steps: (1) Spore collection: Collect fresh leaves with mature sporangia on the back of the fern. After brushing off the sporangia with a sterilized brush, sieve out the fully mature spore powder with a sterilized No. 6 pharmacopoeia sieve and store it in a sterile dry seed storage tank.

[0063] (2) Substrate selection and packaging: Peat moss and vermiculite treated with seaweed gum in a volume ratio of 2:1 were used as the culture substrate. A culture box (5.2 cm × 160 mm × 110 mm) with a transparent lid and a black bottom was selected. The culture substrate was filled into the culture box to a depth of 2 cm, and water was added to make the humidity of the culture substrate 70-90%. Then the culture box was covered and placed in a steam sterilizer for steam sterilization (one corner of the lid of the culture box can be slightly loosened during the sterilization process). After the water at the bottom of the steamer boiled for 35 minutes, it was cooled.

[0064] The method for treating vermiculite with seaweed gum is as follows: Add seaweed gum to 50 times its weight of hot water (40℃) and stir until homogeneous. Then add vermiculite and mix thoroughly. Under stirring conditions, evaporate the water under reduced pressure to obtain the treated vermiculite. The mass ratio of seaweed gum to vermiculite is 1:50.

[0065] (3) Direct spore release: The spore powder prepared in step (1) is mixed with water to make a spore suspension of 0.04 g / L, and the direct spore release density is 1.7 mg / m³. 2 Spray the spore suspension evenly into the culture medium, and then cover it tightly with a transparent cap.

[0066] (4) Management during the period from spore to prothallus: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0067] Management during the period from prothallus formation to sporophyte formation: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0068] During the spore-to-prothallus stage and the prothallus-to-spore stage, open the transparent lid of the cultivation box for 60 seconds every 7 days to allow air exchange. While opening the transparent lid every 7 days, observe the fern seedling growth. If the humidity is too low, add distilled water to bring it to a suitable level; if the humidity is too high, drain off the excess water.

[0069] (5) When the sporophyte grows into a fern seedling with more than 2 leaves, transplant the seedlings, manage fertilizer and water, and control pests and diseases in accordance with conventional methods. Example 8

[0070] In this embodiment, to further improve the effect of peat moss and vermiculite composite as a culture medium, vermiculite was treated with seaweed gum before use.

[0071] This embodiment provides a method for direct seeding of fern spores, including the following steps: (1) Spore collection: Collect fresh leaves with mature sporangia on the back of the fern. After brushing off the sporangia with a sterilized brush, sieve out the fully mature spore powder with a sterilized No. 6 pharmacopoeia sieve and store it in a sterile dry seed storage tank.

[0072] (2) Substrate selection and packaging: Peat moss and vermiculite treated with seaweed gum in a volume ratio of 3:1 were used as the culture substrate. A culture box (5.2 cm × 160 mm × 110 mm) with a transparent lid and a black bottom was selected. The culture substrate was filled into the culture box to a depth of 3 cm, and water was added to make the humidity of the culture substrate 70-90%. Then the culture box was covered and placed in a steam sterilizer (one corner of the lid of the culture box can be slightly loosened during the sterilization process). After the water at the bottom of the steamer boiled for 50 minutes, it was cooled.

[0073] The method for treating vermiculite with seaweed gum is as follows: Add seaweed gum to 500 times its weight of hot water (60℃) and stir until homogeneous. Then add vermiculite and mix thoroughly. Under stirring conditions, evaporate the water under reduced pressure to obtain the treated vermiculite. The mass ratio of seaweed gum to vermiculite is 1:200.

[0074] (3) Direct spore release: The spore powder prepared in step (1) is mixed with water to make a spore suspension of 0.2 g / L, and the direct spore release density is 8 mg / m³. 2 Spray the spore suspension evenly into the culture medium, and then cover it tightly with a transparent cap.

[0075] (4) Management during the period from spore to prothallus: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0076] Management during the period from prothallus formation to sporophyte formation: light intensity 2000 lx, daytime temperature 26℃, nighttime temperature 16℃, light duration 12 h.

[0077] During the spore-to-prothallus stage and the prothallus-to-spore stage, open the transparent lid of the cultivation box for 120 seconds every 14 days to allow for air exchange. While opening the transparent lid every 14 days, observe the fern seedling growth. If the humidity is too low, add distilled water to bring it to a suitable level; if the humidity is too high, drain off the excess water.

[0078] (5) When the sporophyte grows into a fern seedling with more than 2 leaves, transplant the seedlings, manage fertilizer and water, and control pests and diseases in accordance with conventional methods. Example 9

[0079] The difference between this embodiment and Embodiment 6 is that seaweed gum is replaced with agar. Example 10

[0080] The difference between this embodiment and Embodiment 6 is that carrageenan is replaced with seaweed gum.

[0081] I. Basic Seedling Raising Procedures of the Invention Observations on the growth and development of *Adiantum capillus-veneris* in Examples 1-4 showed that *Adiantum capillus-veneris* spores germinated into prothalli after 23-24 days (see...). Figure 1 ), 52-53 days later the prothallus develops into the sporophyte (see Figure 2 The development of the sporophyte in Examples 1-4 is shown in [the original text]. Figure 3 The spore germination rate, sporophyte formation rate, and seedling emergence rate of each group were calculated and statistically analyzed, and the results are shown in Table 1.

[0082] Among them, the spore germination rate (%) = 100% × (prothallium area / upper surface area of ​​culture substrate).

[0083] Sporophyte formation rate (%) = 100% × (sporophyte area / prothallus area).

[0084] The number of *Acer buergerianum* seedlings was counted using the S-shaped five-point sampling method, and the emergence rate was calculated per unit area (dm²). 2 The number of oak fern seedlings counted.

[0085] Table 1

[0086] As shown in Table 1, the *Quercus* fern of Examples 1-4 of this invention exhibited good growth and development, with spore germination rates ranging from 90.0% to 95.0%, sporophyte formation rates all exceeding 50%, and seedling emergence rates of 200-300 plants / dm². 2 In Example 1, when peat moss was used alone as the culture medium, the oak fern developed well, with a spore germination rate as high as 95.0%, a sporophyte formation rate of 85%, and a seedling emergence rate of 300 plants / dm². 2 .

[0087] II. The Influence of Culture Substrate Type on Spore Germination Rate 1. Preliminary screening test of culture medium In the early stages of this invention research, a cultivation box with a transparent lid and a transparent bottom was selected for the cultivation of *Adiantum capillus-veneris* spores, and the effects of different culture media on the cultivation of *Adiantum capillus-veneris* spores were studied. The grouping numbers for the different culture media are as follows: ① Desalinated coconut brick: pine bark: green zeolite = 1:1:1 (volume ratio).

[0088] ② Humus: Pine bark: Green zeolite = 1:1:1 (volume ratio).

[0089] ③ Humus: Pine bark = 1:1 (volume ratio).

[0090] ④ Pure pine bark.

[0091] ⑤ Pure humus soil.

[0092] ⑥ Humus: Green zeolite = 1:1 (volume ratio).

[0093] ⑦ Desalinated coconut bricks: pine bark = 1:1 (volume ratio).

[0094] ⑧ Pure desalinated coconut bricks.

[0095] ⑨ Desalinated coconut brick: green zeolite = 1:1 (volume ratio).

[0096] See results Figure 4 The seedling growth effects of the different culture media were not ideal. In particular, almost no prothallium growth was observed in the substrate containing desalinated coconut bricks and the pure pine bark substrate, indicating that pine bark is not suitable as a substrate for the cultivation of fern spores alone. Desalinated coconut bricks, due to their high electrical conductivity of over 1000 μS / cm, are not suitable for the growth and development of fern spores under high salt conditions.

[0097] 2. Second screening test of culture medium Following the preliminary screening experiments of the aforementioned culture media, it was observed that in some groups, green vesicle-like substances grew at the bottom and around the substrate during prothallus growth. This was considered to be due to the influence of light transmission at the bottom of the culture box. Therefore, in the second screening experiment, culture boxes with transparent lids and black bottoms were selected for the cultivation of fern spores. The substrates were then replaced with peat moss, vermiculite, and yellow soil for further screening.

[0098] To investigate the effect of culture substrate type on spore germination rate, the following comparative examples are provided based on Example 1 of this invention: Comparative Example 1 The difference between this comparative example and Example 1 is that the culture medium is peat moss and vermiculite in a volume ratio of 1:2.

[0099] Comparative Example 2 The difference between this comparative example and Example 1 is that the culture medium is yellow soil.

[0100] Comparative Example 3 The difference between this comparative example and Example 1 is that the culture medium is peat soil and yellow soil in a volume ratio of 1:1.

[0101] *Adiantum capillus-veneris* spores were cultivated according to the methods of Example 1 and Comparative Examples 1-3, respectively, and their spore germination was compared. The prothallus development of Example 1, Comparative Examples 2 and 3 is shown in [reference needed]. Figure 5 Therefore, it can be concluded that the yellow soil commonly found in nature is not suitable as a culture medium for the cultivation of fern spores.

[0102] The spore germination rates for each group are shown in Table 2.

[0103] Table 2

[0104] As shown in Tables 1 and 2, compared with Comparative Examples 2 to 3, the present invention uses peat moss or peat moss + vermiculite as a culture medium for the cultivation of fern spores, and the spore germination rate is higher, reaching over 90%.

[0105] Comparing Examples 1-4 and Comparative Example 1, it can be seen that as the proportion of peat in the peat and vermiculite gradually decreases, the spore germination rate also gradually decreases. In particular, when the volume ratio of peat to vermiculite in Comparative Example 1 is less than 1:1, the spore germination rate drops to below 90%, indicating that when peat and vermiculite are used as a culture medium, the proportion of vermiculite should not be too high.

[0106] III. The Effect of Cultivation Boxes on the Sporozoite Seedling Development of *Adiantum capillus-veneris* To investigate the effects of whether the cultivation box has a transparent lid and whether the bottom of the box is transparent on the seedling cultivation of *Adiantum capillus-veneris* spores, the following comparative examples are provided based on Example 1: Comparative Example 4 The difference between this comparative example and Example 1 is that the culture box does not have a transparent lid, and the culture box is kept open during the seedling stage.

[0107] Comparative Example 5 The difference between this comparative example and Example 1 is that a culture box with a transparent bottom is used.

[0108] 1. The impact of enclosed cultivation on mold and algae bloom. The spores of *Adiantum capillus-veneris* were cultivated according to the methods of Example 1 and Comparative Example 4, respectively. The mold and algae bloom conditions (the area ratio of mold and algae bloom in the cultivation box) and the time for the formation of prothallus and sporophyte were compared. The results are shown in Table 3.

[0109] Table 3

[0110] Table 3 shows that in Example 1, the use of a sealed cultivation box with a transparent lid resulted in virtually no mold or algae blooms, and the time from spore to prothallus and from prothallus to sporophyte was relatively short. In contrast, the conventional seedling cultivation method in Comparative Example 4 without a transparent lid resulted in 20-40% mold and algae blooms, and the time from spore to prothallus and from prothallus to sporophyte was much longer. This demonstrates that sealed cultivation is crucial for reducing the risk of microbial contamination and ensuring a suitable environment for the survival of prothallus and sporophyte. The development of the *Quercus acutissima* seedlings in Comparative Example 4 is shown in [Table 3]. Figure 6 The algae bloom was obvious, and the algae development was poor.

[0111] 2. Effect of bottom transparency of the cultivation box on spore germination rate and sporophyte formation rate The spores of *Adiantum capillus-veneris* were cultivated according to the methods of Example 1 and Comparative Example 5, respectively. The spore germination rate and sporophyte formation rate were compared. The results are shown in Table 4.

[0112] Table 4

[0113] Table 4 shows that the transparency of the bottom of the culture box has little effect on the spore germination rate. However, in Comparative Example 5, due to the light penetration at the bottom, green vesicle-like substances grew on the bottom and surrounding area (see Table 4). Figure 7 This affects the growth of its prothallus, and the sporophyte formation rate is significantly lower than in Example 1, indicating that the transparency of the bottom of the cultivation box is detrimental to the development of oak fern.

[0114] IV. The effect of the soaking method during the prothallus to sporophyte stage on the sporophyte formation rate of multiple batches of *Fernonia querceta* seedlings. Five batches (three replicates per batch) of *Adiantum capillus-veneris* spores were cultivated according to the methods of Examples 1 and 5, respectively. The sporophyte formation rate of different batches in each group was statistically analyzed, and the success rate from prothallus to sporophyte was calculated. The results are shown in Table 5. A comparison of sporophyte formation in batch 4 of Examples 1 and 5 is shown in Table 5. Figure 8 .

[0115] Success rate of prothallus to sporophyte (%) = 100% × (number of batches in the test group with a sporophyte formation rate of 50% or more / total number of batches 5).

[0116] Table 5

[0117] From Table 5 and Figure 8 It is known that using the soaking method during the prothallus to sporophyte stage can not only improve the sporophyte formation rate, but also increase the success rate of prothallus to sporophyte development in different batches of seedlings. This is because the fluid environment increases the contact opportunities between sperm and egg, promoting fertilization of the prothallus and the emergence of the sporophyte.

[0118] V. The impact of steam sterilization of culture medium on mold and algae bloom. To investigate the effect of steam sterilization of the culture medium on spore germination rate, the following comparative examples are provided based on Example 1: Comparative Example 6 The difference between this comparative example and Example 1 is that the culture box containing the culture medium is not steam sterilized.

[0119] Comparative Example 7 The difference between this comparative example and Example 1 is that the culture box containing the culture medium was steam sterilized for 25 minutes.

[0120] The spores of *Adiantum capillus-veneris* were cultivated according to the methods of Example 1, Comparative Example 6, and Comparative Example 7, respectively. The mold and algae bloom conditions (the area ratio of mold and algae bloom in the cultivation box) and the time for the formation of prothallus and sporophyte were compared. The results are shown in Table 6.

[0121] Table 6

[0122] As shown in Table 6, compared with Comparative Examples 6 and 7, the steam sterilization of the culture medium in Example 1 of the present invention for a sufficient time can effectively avoid the occurrence of mold and algae bloom, and the time for the formation of prothallium and sporophyte will not be affected.

[0123] VI. The effect of direct spore density on sporophyte development To investigate the effect of direct spore density on sporophyte development, the following comparative examples are provided based on Example 1: Comparative Example 8 The difference between this comparative example and Example 1 is that the direct seeding density of spores is 0.5 mg / m³. 2 .

[0124] Comparative Example 9 The difference between this comparative example and Example 1 is that the direct seeding density of spores was 6.0 mg / m³. 2 .

[0125] The spores of *Adiantum capillus-veneris* were cultivated according to the methods of Example 1, Comparative Example 8, and Comparative Example 9, respectively. The spore germination rate, the time to form sporophytes, and the size of the sporophytes were compared. The results are shown in Table 7.

[0126] Table 7

[0127] As shown in Table 7, the spore direct seeding density of this invention (1-8 mg / m³) is [data missing]. 2 This method is more suitable for propagating fern spores. When the density of direct spore seeding is too low, the spore germination rate is low. When the density of direct spore seeding is too high, the time from prothallium to sporophyte is prolonged, and the sporophyte is relatively small.

[0128] VII. Effect of substrate depth on spore germination rate To investigate the effect of different culture substrate depths on spore germination rate, the following comparative examples are provided based on Example 1: Comparative Example 10 The difference between this comparative example and Example 1 is that the depth of the culture medium is 1.5 cm.

[0129] Comparative Example 11 The difference between this comparative example and Example 1 is that the depth of the culture medium is 3.5 cm.

[0130] The methods for cultivating *Adiantum capillus-veneris* spores were carried out according to Example 1, Comparative Example 10, and Comparative Example 11, respectively. The spore germination rate and prothallium growth were compared. The results are shown in Table 8. The prothallium development in Comparative Example 10 is shown in... Figure 9 Browning occurred.

[0131] Table 8

[0132] From Table 8 and Figure 9 It is known that the substrate depth (2-3 cm) of the present invention is more suitable for the cultivation of *Adiantum capillus-veneris* spores. When the substrate depth is too low, although there is no significant difference in spore germination rate, humidity management is difficult, and air humidity fluctuates greatly. When the humidity is high, browning is likely to occur during the subsequent prothallus growth, while when the humidity is low, prothallus growth is slow. When the substrate depth is too high, the spore germination rate decreases.

[0133] 8. Maturity screening of *Adiantum capillus-veneris* spores using different pharmacopoeia sieves. When collecting fresh fern spores, some immature spores also emerge from the sporangia, along with unemerged sporangia of lower maturity. If the sieve mesh is large, a small amount of larger, grayish-white powder may remain in the spore powder, easily clogging small spray bottles (used for direct spore release). Furthermore, coarser particles in the spore suspension tend to adhere to the spray bottle walls. Conversely, if the sieve mesh is small, the spore powder is difficult to pass through. Only by selecting a suitable sieve to screen the fern spores can relatively pure, mature spore powder be obtained, ensuring the resulting spore suspension does not clog the spray bottle.

[0134] To investigate the effects of different pharmacopoeia sieves on direct spore release and spore germination, the following comparative examples are provided based on Example 1: Comparative Example 12 The difference between this comparative example and Example 1 is that the No. 6 pharmacopoeia sieve is replaced with a No. 4 pharmacopoeia sieve.

[0135] Comparative Example 13 The difference between this comparative example and Example 1 is that the No. 6 pharmacopoeia sieve is replaced with the No. 5 pharmacopoeia sieve.

[0136] Comparative Example 14 The difference between this comparative example and Example 1 is that the No. 6 pharmacopoeia sieve is replaced with the No. 7 pharmacopoeia sieve.

[0137] The spores of *Adiantum capillus-veneris* were screened using different pharmacopoeia sieves according to the methods of Example 1 and Comparative Examples 12-14, respectively. The spore germination effect was: Example 1 > Comparative Example 13 > Comparative Example 12.

[0138] in: See the electron micrograph and photograph of *Adiantum capillus-veneris* spores obtained by passing through a No. 6 pharmacopoeia sieve in Example 1. Figure 10 The spore powder appears to be relatively pure, with no obvious impurities. The prepared spore suspension was successfully sprayed directly into the soil using a spray bottle, and spore germination (prothallus growth is observed) was good. Figure 5 ) and sporophyte growth (see Figure 3 They are all very uniform.

[0139] Comparative Example 12, which passed through a No. 4 pharmacopoeia sieve, showed that its spore suspension easily clogged the sprayer during direct seeding, resulting in uneven spore germination and uneven growth of the resulting *Quercus acutissima* seedlings, which grew in clumps and clusters (see...). Figure 11 ).

[0140] The electron micrographs and photographs of *Adiantum capillus-veneris* spores obtained by passing through the No. 5 pharmacopoeia sieve in Comparative Example 13 are shown below. Figure 12 The image shows some white particulate impurities. The spore suspension prepared from this solution easily clogs the spray bottle during direct seeding, resulting in uneven spore germination and uneven growth of the oak fern seedlings (see...). Figure 13 ).

[0141] When comparing Example 14, the filtration process was very difficult, and the yield of fern spores was low, making it unsuitable.

[0142] IX. The Influence of Plant Gum on the Sporulation of *Adiantum capillus-veneris* Table 1 shows that using peat moss + vermiculite as the culture medium for *Adiantum capillus-veneris* spore cultivation is less effective than using peat moss alone. To improve the economic efficiency of this cultivation method and make better use of different substrate materials, it is necessary to improve the compatibility between peat moss and vermiculite by treating the vermiculite with nutrient-rich plant colloids to further promote the growth and development of *Adiantum capillus-veneris* spores.

[0143] To investigate the effects of different plant gums and the methods of adding plant gums on spore propagation, Examples 6 to 10 are provided, and the following comparative examples are provided based on Example 6: Comparative Example 15 The difference between this comparative example and Example 6 is that the seaweed gum is directly mixed with the culture medium (peat soil and vermiculite in a volume ratio of 1:1) and used uniformly.

[0144] 1. The effect of plant gums on the growth of fern spores The methods of Examples 1, 4, 6-10 and Comparative Example 15 were used to cultivate fern spores. The spore germination rate, sporophyte formation rate and seedling emergence rate were compared. The results are shown in Table 9.

[0145] Table 9

[0146] As shown in Table 9, compared with Example 4, Examples 6 to 10 of the present invention used different plant gums (seaweed gum, agar, carrageenan) to treat vermiculite, which all promoted the growth of fern spores and increased the spore germination rate, sporophyte formation rate and seedling emergence rate.

[0147] Compared with Example 7, Comparative Example 15 changed the way seaweed gum was added, and the growth of fern spores was also affected.

[0148] 2. Effects of plant colloid treatment on vermiculite dispersion, humidity uniformity, and spore germination uniformity in composite culture media. Depend on Figure 3 The sporophyte development of *Adiantum buergerianum* in Examples 1-4 shows that when peat moss and vermiculite were used as a composite substrate in Examples 2-4, some sporophytes grew unevenly. However, when peat moss was used alone as the substrate in Example 1, the sporophyte growth was more uniform. This indicates that there are compatibility issues when peat moss and vermiculite are used as a culture medium.

[0149] The methods of Examples 4, 6, 9 and 10 were used to cultivate fern spores. During the cultivation, the dispersion of the culture medium was observed. Every 10 days, when the transparent lid of the cultivation box was opened, the humidity of the culture medium was measured by a five-point sampling method. The standard deviation of the humidity at different sampling points was calculated. The results are shown in Table 10.

[0150] Table 10

[0151] As shown in Table 10, compared with other plant gums (agar, carrageenan), the treatment of vermiculite with seaweed gum has a better effect on improving the dispersibility of the composite substrate and further improves the moisture uniformity of the substrate (standard deviation as low as 1.258). The seedlings of oak fern have very strict requirements for humidity, and maintaining uniform humidity is conducive to the growth of oak fern.

[0152] See below for spore germination data from different groups Figure 14 As can be seen, the uniformity of spore germination is as follows: Example 6 > Example 4 ≈ Example 9 ≈ Example 10.

[0153] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for direct seeding of *Adiantum capillus-veneris* spores, characterized in that: Collect fern spores and pass them through a No. 6 pharmacopoeia sieve for later use. Select a culture box with a transparent lid and an opaque bottom, fill the culture medium into the culture box, sterilize it, and then administer the culture at a dose of 1-8 mg / m³. 2 Direct sowing of spores is carried out, and after direct sowing, a transparent cap is tightly covered to cultivate fern spores. The culture medium comprises peat moss and vermiculite in a volume ratio of (1-3):

1. The vermiculite is treated with plant colloids before use. The specific method includes: Add plant colloids to hot water and stir until evenly mixed. Then add vermiculite and mix evenly. Under stirring conditions, evaporate the water under reduced pressure to obtain the processed vermiculite. The plant gum is seaweed gum.

2. The method according to claim 1, characterized in that: The culture medium is placed at a depth of 2-3 cm in the incubation box, and the humidity of the culture medium is 70-90%.

3. The method according to claim 1, characterized in that: The culture medium is sterilized by steam sterilization after being placed in the incubation box for no less than 35 minutes.

4. The method according to claim 1, characterized in that: After direct spore seeding, management was carried out during the period from spore to prothallus and then during the period from prothallus to sporophyte. During these periods, the transparent lid of the culture box was opened for 10-120 seconds every 7-15 days to exchange air.

5. The method according to claim 1, characterized in that: The temperature of the hot water is 40-60℃, and the amount of hot water added is 50-500 times the mass of the plant gum. The mass ratio of the plant gum to vermiculite is 1:(50-200).

6. The method according to claim 4, characterized in that: During the period from prothallus to sporophyte, the soaking method is used. Distilled water is added to the culture box and the prothallus is soaked for 15-30 minutes. After soaking, excess water is removed to restore the original humidity.

7. The method according to claim 4, characterized in that: Management during the period from spore to prothallus includes: light intensity of 1000-2000 lx, daytime temperature of 22-27℃, nighttime temperature of 14-20℃, and light duration of 8-14h. Management during the period from prothallus formation to sporophyte formation includes: light intensity of 1500-2500 lx, daytime temperature of 22-27℃, nighttime temperature of 14-20℃, and light duration of 8-14h.

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