A set of cyatheasporae culture medium and segmented seedling raising method

Abstract

The present application belongs to the technical field of medicinal plant spore breeding, and relates to a group of culture media for breeding of Cyatheas spores and a segmented breeding method. The present application provides a group of culture media for breeding of Cyatheas spores; and based on the culture media and the segmented breeding, Cyatheas sporophyte seedlings are obtained. The present application has two stages for breeding, the first stage is to induce Cyatheas spore germination and prothallus proliferation by tissue culture, and the second stage is to plant the proliferated prothallus into a mixed nutrient medium and spray nutrient solution to obtain sporophyte seedlings. The present application has the advantages that the breeding operation is simple and easy to operate, the problems that it is difficult for the prothallus of Cyatheas to transform into sporophyte and it is extremely difficult to produce sporophyte due to the yellow-brown fluff produced by the prothallus in the process of tissue culture can be solved, the hardening-off transition period after single tissue culture breeding can be omitted, the harm to the original plant of Cyatheas caused by the use of division propagation can be reduced, the dependence on tissue culture breeding of Cyatheas explants can be reduced, and the damage to the ecology caused by the collection of wild Cyatheas plants can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant spore propagation technology, specifically involving a set of culture media for fern spore seedling cultivation and a segmented seedling cultivation method. Background Technology

[0002] Drynaria, also known as Quercus variabilis (Drynaria roosii Nakaike), is a plant belonging to the genus Drynaria in the family Drynariaceae. The Drynariaceae family comprises 8 genera and approximately 100 species. In my country, about 30 species from 5 genera are cultivated, distributed from tropical to tropical Oceania. In my country, they are mainly produced in Zhejiang, Hubei, Guangdong, Guangxi, and Sichuan provinces.

[0003] The rhizome of *Drynaria fortunei* is used medicinally, rich in flavonoids, alkaloids, phenols, and other active ingredients. It possesses properties such as dispersing blood stasis and relieving pain, promoting bone healing, and treating toothache, back pain, and chronic diarrhea. In recent years, due to indiscriminate harvesting, natural resources have been gradually depleted, and wild resources have been severely damaged. In 2002, *Drynaria fortunei* was recommended for inclusion in the National Endangered Species List. Plants in the *Drynaria* family are mostly epiphytic on rocks or tall tree trunks, requiring specific environmental conditions. Their suitable temperature, humidity, and soil pH ranges are relatively narrow, making them difficult to cultivate and posing significant challenges to germplasm preservation and utilization.

[0004] In recent years, there have been some reports on the artificial propagation technology of *Acer buergerianum*. The patent "A method for propagating *Acer buergerianum*" (CN1788549A) established the in vitro culture system of *Acer buergerianum* for the first time. Subsequently, the patents "A method for rapid propagation of *Acer buergerianum*" (CN103430853A) and "A method for in vitro preservation of *Acer buergerianum*" (CN105660415A) reported methods for rapid in vitro propagation and preservation of *Acer buergerianum* based on tissue culture technology. The patent "A method for rapid propagation of *Acer buergerianum* seedlings by tissue culture using the Green Globular Body (GGB) method" (CN111406652B) reported a method for rapid propagation of *Acer buergerianum* seedlings by tissue culture using the Green Globular Body (GGB) method. In addition, patents such as "A Method for Year-Round Propagation of Oak Fern Seedlings" (CN106613809A), "A Method for Artificial Propagation of Oak Fern" (CN109691372A), and "A Method for Improving the Propagation Rate of Oak Fern Rhizomes" (CN110337926A) have all reported on the artificial cultivation technology of Oak Fern. However, there is currently no large-scale artificial cultivation of Oak Fern. Therefore, it is urgent to accelerate the use of Oak Fern spores to propagate seedlings for artificial cultivation to solve the problem of the shortage of raw materials for the traditional Chinese medicine Drynaria fortunei.

[0005] Although it is possible to induce the germination of prothallus and sporophyte from fern spores using tissue culture, the entire process is very lengthy, and the transformation from prothallus to sporophyte is extremely difficult during tissue culture. This often results in the prothallus producing a large amount of yellowish-brown hairs, making it very difficult for sporophyte to develop. Summary of the Invention

[0006] The purpose of this invention is to provide a set of culture media for the cultivation of *Adiantum capillus-veneris* spores and a segmented cultivation method. Using the culture media of this invention for the cultivation of *Adiantum capillus-veneris* seedlings can rapidly produce a large number of sporophyte seedlings, eliminating the hardening-off period after cultivating a single set of seedlings.

[0007] This invention provides a set of culture media for the cultivation of *Adiantum capillus-veneris* spores. The culture media include a spore germination medium, a prothallium proliferation medium, a mixed nutrient substrate, and a nutrient solution. The spore germination medium is based on 1 / 2 MS medium and further includes 0.01–1.0 mg / L GA3 and 6.0–7.5 g / L agar. The prothallium proliferation medium is based on MS medium and further includes 0.01–1.0 mg / L 6-BA, 0.0001–0.003 mg / L NAA, 0.01–1.0 mg / L GA3, 0–30 g / L sucrose, and 6.0–7.5 g / L agar. The mixed nutrient substrate includes peat moss, potting soil, and fine sand. The nutrient solution is a 1 / 2 MS basic medium and a 1 / 1000 amino acid water-soluble fertilizer.

[0008] Preferably, the mass ratio of peat soil, nutrient soil and fine sand in the mixed nutrient substrate is (0.1-4):(0.1-2):(0.1-2).

[0009] The present invention also provides the application of the culture medium described in the above technical solution in the seedling cultivation of fern spores.

[0010] This invention also provides a method for segmented seedling cultivation of *Adiantum capillus-veneris* spores based on the culture medium described above, comprising the following steps:

[0011] The sterilized fern spores were inoculated into the spore germination medium for spore germination culture, and the spores germinated into clumps of green prothallia.

[0012] The prothallus was transferred to the prothallus proliferation medium and subcultured to obtain the subcultured prothallus.

[0013] After subculture, the prothallus was spread evenly on a mixed culture medium and cultured in nutrient solution to obtain sporophytes.

[0014] Preferably, the disinfection method for fern spores includes: first disinfecting with an ethanol aqueous solution containing 75% ethanol by volume for 30 seconds, rinsing with sterile water more than 5 times; then disinfecting with an HgCl2 aqueous solution containing 0.1% HgCl2 by mass for 10 minutes, rinsing with sterile water more than 10 times.

[0015] Preferably, the temperatures for spore germination culture and subculture are 18–25°C.

[0016] Preferably, the spore germination culture and subculture are carried out in an alternating light and dark environment; the light culture time in each light-dark alternation cycle is 12-14 h / d; and the light intensity of the light culture is 1500-2500 lx.

[0017] Preferably, the spore germination culture time is 15-25 days; the subculture time is 30-150 days.

[0018] Preferably, the prothallus after subculture is laid flat on the mixed culture medium at intervals of 0.1 to 1 cm; the nutrient solution is used by watering thoroughly and spraying to keep it moist.

[0019] Preferably, the sporophyte culture time is 15 to 30 days.

[0020] This invention provides a set of culture media for the cultivation of *Adiantum capillus-veneris* spores. Cultivation using this culture medium is simple and easy to perform, overcoming the difficulties in the transformation from prothallus to sporophyte during tissue culture, and the problem of extremely difficult sporophyte formation due to the production of yellowish-brown pubescence on the prothallus. It also reduces the harm to the original *Adiantum capillus-veneris* plant from division propagation and the dependence on *Adiantum capillus-veneris* explants, thus reducing the ecological damage caused by the whole-plant harvesting of wild *Adiantum capillus-veneris* plants. Experimental results show that, using this culture medium, *Adiantum capillus-veneris* spores germinate in 15 days. After the prothallus grows to 30 days, it is subcultured. After 4-5 subcultures, approximately 120 days later, the spores can be implanted into a mixed nutrient substrate for sporophyte culture. During this period, nutrient solution is sprayed to maintain moisture and provide further nutrition, resulting in sporophyte seedlings in 15-30 days. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Images of the wild oak fern's habitat provided for this invention;

[0023] Figure 2 Images of wild oak fern sporophytes and their sori provided for this invention;

[0024] Figure 3 Microscopic images of spore germination provided by this invention;

[0025] Figure 4 This is an image of a prothallus after spore germination and growth for a period of time, provided by the present invention.

[0026] Figure 5 This is an image of a prothallus formed after spore germination, provided by the present invention.

[0027] Figure 6 The images show the growth of the prothallus provided by this invention after it has been inoculated with a mixed nutrient substrate. Detailed Implementation

[0028] This invention provides a set of culture media for the cultivation of *Adiantum capillus-veneris* spores. The culture media include a spore germination medium, a prothallium proliferation medium, a mixed nutrient substrate, and a nutrient solution. The spore germination medium is based on 1 / 2 MS medium and further includes 0.01–1.0 mg / L GA3 and 6.0–7.5 g / L agar. The prothallium proliferation medium is based on MS medium and further includes 0.01–1.0 mg / L 6-BA, 0.0001–0.003 mg / L NAA, 0.01–1.0 mg / L GA3, 0–30 g / L sucrose, and 6.0–7.5 g / L agar. The mixed nutrient substrate includes peat moss, potting soil, and fine sand. The nutrient solution is a 1 / 2 MS basic medium and a 1 / 1000 amino acid water-soluble fertilizer. In this invention, the spore germination medium preferably includes 0.03–0.07 mg / L GA3, more preferably 0.05 mg / L. In this invention, the prothallus proliferation medium preferably includes 0.01–0.1 mg / L of 6-BA, more preferably 0.05 mg / L of 6-BA. In this invention, the prothallus proliferation medium preferably includes 0.001 mg / L of NAA. In this invention, the prothallus proliferation medium preferably includes 0.01–0.1 mg / L of GA3, more preferably 0.05 mg / L of GA3. In this invention, the prothallus proliferation medium preferably includes 10–20 g / L of sucrose, more preferably 10 g / L of sucrose. In this invention, the mass ratio of peat moss, nutrient soil, and fine sand in the mixed nutrient substrate is preferably (0.1–4):(0.1–2):(0.1–2), more preferably 2:1:1.

[0029] This invention also provides the application of the culture medium described in the above-mentioned technical solution in the cultivation of *Adiantum capillus-veneris* spores. This invention utilizes the culture medium described in the above-mentioned technical solution, preferably through tissue culture and substrate-based segmented cultivation. *Adiantum capillus-veneris* spores are first induced to germinate into prothalli through tissue culture, and after subculturing the prothalli to produce more prothalli, they are then placed in a mixed culture medium (combined with nutrient solution) for cultivation. This rapidly forms a large number of sporophyte seedlings and eliminates the need for a hardening-off transition period after cultivating a single group of seedlings.

[0030] This invention also provides a method for segmented seedling cultivation of *Adiantum capillus-veneris* spores based on the culture medium described above, comprising the following steps:

[0031] The sterilized fern spores were inoculated into the spore germination medium for spore germination culture, and the spores germinated into clumps of green prothallia.

[0032] The prothallus was transferred to the prothallus proliferation medium and subcultured to obtain the subcultured prothallus.

[0033] After subculture, the prothallus was spread evenly on a mixed culture medium and cultured in nutrient solution to obtain sporophytes.

[0034] This invention involves inoculating sterilized *Adiantum capillus-veneris* spores into a spore germination medium for spore germination culture, yielding clods of green prothallus from the germinated spores. In this invention, the preferred sterilization method for *Adiantum capillus-veneris* spores includes: first, sterilization with 75% ethanol for 30 seconds, followed by rinsing with sterile water at least 5 times; then, sterilization with an HgCl2 aqueous solution containing 0.1% HgCl2 for 10 minutes, followed by rinsing with sterile water at least 10 times. In this invention, the preferred temperature for spore germination culture is 18–25°C, more preferably 22°C. In this invention, the spore germination culture is preferably conducted under alternating light and dark conditions; the preferred light culture time in each light-dark cycle is 12–14 h / d, more preferably 12 h / d, and the preferred dark culture time is 8–12 h / d, more preferably 12 h / d; the preferred light intensity for light culture is 1500–2500 lx, more preferably 2000 lx. In this invention, the spore germination culture time is preferably 15-25 days, more preferably 15 days. After spore germination, they further grow into prothallia. In this invention, the time for Quercus acutissima spores to germinate and form prothallia is preferably 35 days.

[0035] After obtaining the prothallus, which germinates into a clump of green spores, the prothallus is transferred to a prothallus proliferation medium for subculture to obtain subcultured prothallus. In this invention, the subculture temperature is preferably 18–25°C, more preferably 22°C. In this invention, the subculture is preferably carried out under alternating light and dark conditions; the light culture time in each light-dark cycle is preferably 12–14 h / d, more preferably 12 h / d, and the dark culture time is preferably 8–12 h / d, more preferably 12 h / d; the light intensity for light culture is preferably 1500–2500 lx, more preferably 2000 lx. In this invention, the subculture time is preferably 30–150 days, more preferably 150 days. In this invention, during the subculture period, the prothallus is preferably transferred 4–5 times, more preferably 5 times.

[0036] After obtaining the subcultured prothallus, this invention lays the subcultured prothallus flat on a mixed culture medium and uses nutrient solution for sporophyte culture to obtain sporophytes. In this invention, the subcultured prothallus are preferably laid flat on the mixed culture medium at intervals of 0.1–1 cm, more preferably 0.5 cm. In this invention, the nutrient solution is preferably applied by thoroughly watering and then spraying to maintain moisture. Specifically, this invention preferably involves thoroughly watering the subcultured prothallus on the mixed culture medium, and then spraying with nutrient solution every 2 days to keep the surface of the prothallus moist. After laying flat, it is preferable to gently press the prothallus with tweezers. In this invention, the sporophyte culture time is preferably 15–30 days, more preferably 15–20 days, to produce sporophyte seedlings.

[0037] The segmented seedling cultivation method described in this invention preferably refers to the cultivation of sporophyte seedlings in two stages. The first stage involves inducing germination of sporophytes through tissue culture and promoting the proliferation of prothalliums; the second stage involves the growth of prothalliums into sporophytes in a mixed culture medium (containing nutrient solution). Using this segmented seedling cultivation method is simple and easy to implement, overcoming the difficulties in the transformation of *Adiantum capillus-veneris* prothalliums into sporophytes during tissue culture, as well as the difficulty in sporophyte formation due to the production of yellowish-brown pubescence on prothalliums. It also eliminates the need for a hardening-off period after single-group seedling cultivation, further reducing the harm to the original *Adiantum capillus-veneris* plant from division propagation and the reliance on *Adiantum capillus-veneris* explants for tissue culture seedling cultivation, and minimizing the ecological damage caused by harvesting wild *Adiantum capillus-veneris* plants.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a set of culture media for fern spore cultivation and a segmented cultivation method provided by the present invention, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] A method for inducing spore germination from *Adiantum capillus-veneris* spores, comprising the following steps:

[0041] (1) Collect fresh wild oak fern leaves ( Figure 1 The leaves have sori ( Figure 2 The collected oak fern leaves were placed in kraft paper bags and brought back. The mature, yellowish-brown sori on the leaves were carefully scraped off with a scalpel.

[0042] (2) Soak the 100μm cell filter sieve and cell culture plate in 75% alcohol for 5 minutes, then remove them and place them on a clean bench for 30 minutes of ultraviolet light sterilization.

[0043] (3) Three wells in a 6-well cell culture plate are labeled 1, 2, and 3. The sporangia of *Adiantum capillus-veneris* scraped in step (1) are placed in the sterilized cell filter sieve from step (2) and then placed in well 1 of the cell culture plate. 75% alcohol is then poured into well 1 for 10 seconds for disinfection, followed by rinsing the spores 5 times with sterile water. The cell filter sieve containing the sporangia is then placed in well 2 of the cell culture plate, followed by 0.1% HgCl2 solution for disinfection for 15–20 minutes, and then rinsed 10 times with sterile water. The cell filter sieve containing the sporangia is then placed in well 3 of the cell culture plate.

[0044] (4) Using a sterile inoculation loop, inoculate the sporangia from well 3 in step (3) into the spore germination medium. The spore germination medium consists of 1 / 2 MS + 0.05 mg / L GA3 + 7.0 g / L agar. Then, place the medium inoculated with sporangia in an artificial climate chamber and incubate at a temperature of 22°C, a light intensity of 2000 lx, and a photoperiod of 12 h until spore germination (see...). Figure 3 ) and further grow into a prothallus ( Figure 4 The process takes 35 days.

[0045] (5) Transfer the prothallus to a proliferation subculture medium after about 40 days. Figure 5 The subculture medium consisted of MS medium containing 0.05 mg / L 6-BA, 0.001 mg / L NAA, 0.05 mg / L GA3, 10 g / L sucrose, and 7.0 g / L agar. The culture was subcultured five times for a total of 150 days.

[0046] (6) The subcultured prothallus were transferred into a mixed nutrient substrate, laid flat on the substrate at 0.5 cm intervals, and thoroughly watered with nutrient solution. Nutrient solution was sprayed every 2 days to keep the surface of the prothallus moist and provide nutrients. After 15 days of culture, sporophytes gradually formed (see...). Figure 6The mixed substrate is a mixture of peat soil, nutrient soil and fine sand (mass ratio of 2:1:1), and the sprayed nutrient solution is 1 / 2MS + 1 / 1000 Caifat amino acid water-soluble fertilizer.

[0047] Comparative Example 1

[0048] The effects of different basic culture media on the germination of *Adiantum capillus-veneris* spores are shown in Table 1.

[0049] Table 1 Effects of different basic culture media on the germination of *Adiantum capillus-veneris* spores.

[0050]

[0051]

[0052] As shown in Table 1, the germination time of *Adiantum capillus-veneris* spores varied considerably with changes in the basic culture medium. The combination of 1 / 2 MS medium + 0.05 mg / L GA3 + 7 g / L agar resulted in the shortest germination time. Other MS basic media with different proportions could induce germination, but the time was longer. B5, White, and WPM basic media failed to induce germination.

[0053] Comparative Example 2

[0054] The effects of adding different plant growth regulators on the germination of fern spores and the formation of prothallus are shown in Table 2.

[0055] Table 2 Effects of different plant growth regulators on the germination of *Adiantum capillus-veneris* spores and the formation of prothallus.

[0056] <![CDATA[1 / 2 MS + 0.01 mg / L GA₃ + 7 g / L agar]]> 47 <![CDATA[1 / 2MS + 0.03 mg / L GA₃ + 7 g / L agar]]> 41 <![CDATA[1 / 2 MS + 0.05 mg / L GA₃ + 7 g / L agar]]> 35 <![CDATA[1 / 2MS + 0.07 mg / L GA₃ + 7 g / L agar]]> 39 <![CDATA[1 / 2MS + 0.1 mg / L GA₃ + 7 g / L agar]]> 53

[0057] As shown in Table 2, under the same basic culture medium, the addition of different concentrations of GA3 resulted in different germination induction times. The formulation of 1 / 2 MS + 0.05 mg / L GA3 + 7 g / L agar induced the shortest germination time for *Adiantum capillus-veneris* spores. Other concentrations of GA3 could also induce germination of *Adiantum capillus-veneris* spores, but the time was longer. The purpose of this invention is to shorten the time from germination to prothallus development of *Adiantum capillus-veneris* spores as much as possible.

[0058] Comparative Example 3

[0059] Using MS as the basic culture medium, a four-factor, three-level study was conducted to investigate the effects of different combinations of plant growth regulators and sugar concentrations on the subculturing of fern sporophytes. The results are shown in Table 3.

[0060] Table 3. Effects of different plant growth regulators and sugar concentrations on the germination of *Adiantum capillus-veneris* spores and the formation of prothallus.

[0061]

[0062]

[0063] As shown in Table 3, under the same basic culture medium, the addition of different concentrations of 6-BA, NAA, GA3, and sucrose had a significant impact on the proliferation and subculturing of *Adiantum capillus-veneris* prothallus. When the sugar concentration was high, yellowish-brown pubescence was produced, which was not conducive to subculturing and the formation of sporophyte seedlings. In the absence of sugar, the prothallus died after 150 days of subculturing. The formula used in this invention, MS + 0.05 mg / L 6-BA + 0.001 mg / L NAA + 0.05 mg / L GA3 + 10 g / L sucrose + 7.0 g / L agar, could achieve prothallus proliferation well without producing yellowish-brown pubescence.

[0064] Comparative Example 4

[0065] The effects of different mixed substrates on sporophyte formation of *Adiantum capillus-veneris* are shown in Table 4.

[0066] Table 4. Effects of different mixed matrices on sporophyte formation of *Adiantum capillus-veneris*.

[0067] A mixed substrate of peat moss, potting soil, and fine sand (mass ratio 2:1:1). 20 A mixed substrate of peat moss, potting soil, and fine sand (mass ratio 1:2:1). 27 A mixed substrate of peat moss, potting soil, and fine sand (mass ratio 1:1:2). 36

[0068] As shown in Table 4, different proportions of mixed substrates have a significant impact on the sporophyte formation time of *Adiantum capillus-veneris*. Among them, the mixed substrate of peat moss, nutrient soil, and fine sand (mass ratio of 2:1:1) has the shortest sporophyte formation time.

[0069] Comparative Example 5

[0070] The effects of different nutrient solutions on the growth of *Adiantum capillus-veneris* sporophytes were shown in Table 5 when a mixed substrate of peat moss, nutrient soil, and fine sand (mass ratio of 2:1:1) was used.

[0071] Table 5 Effects of different nutrient solutions on the growth of *Adiantum capillus-veneris* sporophytes.

[0072]

[0073]

[0074] As shown in Table 5, different nutrient solutions have a significant impact on the formation time and growth of *Adiantum capillus-veneris* sporophytes. Among them, the nutrient solution of 1 / 2MS + 1 / 1000 Caifat amino acid water-soluble fertilizer (company name: Fuwoshi; standard: NY1429-2010; registration certificate number: Nongfei (2019) Zhunzi 14486; formulation: aqueous solution; net content: 500g) showed the best performance, which can shorten the sporophyte formation time and promote better sporophyte growth.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for raising fern spores, characterized in that, The culture medium includes a spore germination medium, a prothallus proliferation medium, a mixed nutrient substrate, and a nutrient solution. The spore germination medium is based on 1 / 2 MS medium, with the addition of 0.05 mg / L GA3 and 6.0–7.5 g / L agar. The prothallus proliferation medium is based on MS medium, with the addition of 0.01–0.1 mg / L 6-BA, 0.001–0.003 mg / L NAA, 0.05 mg / L GA3, 10 g / L sucrose, and 6.0–7.5 g / L agar. The mixed nutrient substrate consists of peat moss, nutrient soil, and fine sand. The mass ratio of peat moss, nutrient soil, and fine sand in the mixed nutrient substrate is 2:1:

1. The nutrient solution is 1 / 2 MS medium and 1 / 1000 amino acid water-soluble fertilizer.

2. The application of the culture medium according to claim 1 in the cultivation of fern spores.

3. A method for segmented seedling cultivation of *Adiantum capillus-veneris* spores based on the culture medium described in claim 1, characterized in that, Includes the following steps: The sterilized fern spores were inoculated into the spore germination medium for spore germination culture, and the spores germinated into clumps of green prothallia. The prothallus was transferred to the prothallus proliferation medium and subcultured to obtain the subcultured prothallus. After subculture, the prothallus was spread evenly on a mixed culture medium and cultured in nutrient solution to obtain sporophytes.

4. The seedling raising method according to claim 3, characterized in that, The disinfection methods for fern spores include: first disinfecting with a 75% ethanol solution for 30 seconds, then rinsing with sterile water at least 5 times; then disinfecting with a 0.1% HgCl2 solution for 10 minutes, and rinsing with sterile water at least 10 times.

5. The seedling raising method according to claim 3, characterized in that, The temperatures for spore germination culture and subculture were 18~25℃, respectively.

6. The seedling raising method according to claim 3 or 5, characterized in that, The spore germination culture and subculture were carried out in an alternating light and dark environment; the light culture time in each light-dark cycle was 12-14 h / d; the light intensity of the light culture was 1500-2500 lx.

7. The seedling raising method according to claim 3, characterized in that, The spore germination culture time is 15-25 days; the subculture time is 30-150 days.

8. The seedling raising method according to claim 3, characterized in that, The prothallus cells after subculture are laid flat on the mixed culture medium at intervals of 0.1-1 cm; the nutrient solution is used by watering thoroughly and spraying to keep it moist.

9. The seedling raising method according to claim 3, characterized in that, The sporophyte culture time is 15-30 days.

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