Method for improving prothallus and sporophyte inductivity of cibotium barometz

Abstract

The invention discloses a method for improving the induction rate of prothalli and sporophytes of cibotium barometz, which comprises the following steps: S1, sterilizing a substrate by using a bactericide which comprises thiophanate-methyl and tebuconazole, and after sterilization, reducing the water content of the substrate to 40-50% to obtain the sterilized substrate; s2, contacting the bottom of the substrate with a hormone aqueous solution to obtain a substrate absorbing the hormone aqueous solution; s3, sowing the cibotium barometz spores on the surface of the substrate for culture. The method shortens the spore germination time of the cibotium barometz, improves the prothallus and sporophyte induction rate, and is simple and easy to implement. By adopting the method to cultivate the golden dog seedlings, prothalli are induced in 22 days on average, and sporophytes are induced in 66 days on average. The method can be widely applied to sowing and seedling raising of cibotium barometz, and seedling raising time and labor cost are saved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of seedling propagation, and particularly relates to a method for improving the induction rate of prothallus and sporophyte of Cibotium barometz (L.) J. Sm. BACKGROUND

[0002] Cibotium barometz (L.) J. Sm. is a perennial tree fern plant of Cibotium, a national second-class protected plant, and an important raw material of traditional Chinese medicine (included in Chinese Pharmacopoeia as “Cibotium barometz”). Cibotium barometz is not only a traditional medicinal plant, but also has unique ornamental effects. However, the market demand for artificially propagated spores of Cibotium barometz cannot be met, which leads to the serious destruction of the habitat of wild Cibotium barometz and the imminent extinction of the wild population. In addition, the natural propagation rate of Cibotium barometz spores is low, and the number of wild Cibotium barometz is rapidly decreasing. Some scholars have initially carried out research on the tissue culture and seedling raising technology of Cibotium barometz. This method requires high equipment and technical personnel, and increases the cost of seedling raising. Therefore, it is of great significance to develop a high-efficiency soil seeding and seedling raising technology of Cibotium barometz to realize its protection and subsequent development and utilization.

[0003] The propagation of Cibotium barometz spores has high requirements for environmental conditions, and the soil, temperature and humidity, and light conditions will all affect the spore propagation. If the conditions are not suitable, the spore germination rate is low, and the formation rate of prothallus and sporophyte is low and the development is slow. Currently, some scholars have carried out research on the soil seeding and seedling raising technology of Cibotium barometz. For example, the Chinese patent application with the publication number CN119522821A “A soil seeding and seedling raising method of Cibotium barometz” uses peat soil as the substrate, and seeds the spores of Cibotium barometz in the substrate to obtain the sporophyte seedlings. The specific method is to supplement water to the substrate after seeding to keep the water content of the substrate at 85-95%. After 45 days of seeding, the gametophyte is sprayed with nutrient solution every 5 days for 6 times. This method is complicated and labor-intensive. The Chinese patent application with the publication number CN116569796A “A spore seedling raising method of Cibotium barometz” requires the substrate to be fermented and crushed, and the shortest seedling raising time is 152 days (measured when the seedlings are 6-10 cm high). The Chinese patent application with the publication number CN117694188A “A method for raising spore seedlings of Cibotium barometz” sterilizes the seedling raising substrate and adds an inducer to the suspension, and sprays the suspension on the substrate for culture. However, this scheme does not mention the sterilization method and whether the water content of the substrate needs to be maintained. These schemes have too many processes, and some of them lack complete data on the influence of external conditions on spore propagation. SUMMARY

[0004] The application aims to provide a method for improving the induction rate of protoplast and sporont of golden dog, which can promote the rapid germination of spores of golden dog, improve the induction rate of protoplast and sporont, and has the advantages of simple operation, time and labor saving, and high induction rate.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is:

[0006] A method for improving the induction rate of protoplast and sporont of golden dog, comprising the following steps:

[0007] S1, sterilizing the substrate by using a fungicide, wherein the fungicide comprises thiophanate-methyl and dazomet•tebuconazole, and after sterilization, the water content of the substrate is reduced to 40-50%, thereby obtaining the sterilized substrate;

[0008] S2, contacting the bottom of the substrate sterilized in step S1 with a hormone aqueous solution, thereby obtaining the substrate absorbing the hormone aqueous solution;

[0009] S3, sowing the spores of golden dog on the surface of the substrate absorbing the hormone aqueous solution in step S2 for cultivation.

[0010] The corresponding treatments 19-27 of the screening experiment are that the substrate is put into a container and flattened, the mixture of "thiophanate-methyl + dazomet•tebuconazole" is used to soak through the substrate, and then sowing and cultivation are performed. The results show that the nine treatments not only have high contamination rate, but also the induction time of uncontaminated protoplast is prolonged to 40-63 days, and as the mold reproduction invades the protoplast, the protoplast is prone to death.

[0011] The application makes creative adjustment on this, and the following three key technical points interact with each other: (1) the seedling substrate is fully soaked by using a fungicide, so as to ensure that the seedling process is not contaminated (thiophanate-methyl and dazomet•tebuconazole in the fungicide can synergistically sterilize, so as to ensure that the substrate is not moldy); (2) the water content of the sterilized substrate is reduced to 40-50%, at this time, the substrate is relatively fluffy, the substrate is flattened, and the spores obtain sufficient light (only the control group of sterilization without shade drying will lead to high contamination rate, prolonged induction time of protoplast, and protoplast susceptible to mold); (3) the bottom of the substrate continuously absorbs the hormone aqueous solution, so as to provide nutrients and water for the rapid germination of spores and the induction of protoplast. The above three key technical points interact with each other, thereby improving the induction rate of protoplast and sporont.

[0012] According to the embodiments of the application, the application can be further optimized, and the following is the technical scheme formed after optimization:

[0013] In step S1, the fungicide is a mixture of 800-1000 times of liquid of thiophanate-methyl with an effective component mass content of 60-80% and 1500-2000 times of liquid of dazomet•tebuconazole with an effective component mass content of 20-40%, and 3-5 parts of volume of the fungicide is mixed with 1 part of volume of the substrate.

[0014] The effective component of thiophanate-methyl includes thiophanate-methyl (chemical name: 1,2-bis(3-methoxy carbonyl-2-thiourea) benzene). The effective component of azoxystrobin • tebuconazole includes pyraclostrobin and tebuconazole.

[0015] In one preferred embodiment, in step S2, the hormone aqueous solution includes 6-benzyl amino adenine and indole acetic acid.

[0016] Preferably, 6-benzyl amino adenine 0.08-0.10 mg and indole acetic acid 0.10-0.15 mg are included in 1000 ml of the hormone aqueous solution.

[0017] In one preferred embodiment, in step S2, the substrate sterilized in step S1 is placed in a seedling container, the seedling container has a slit at the bottom, and the length of the slit is 2 / 3-3 / 4 of the length of the seedling container.

[0018] In one preferred embodiment, in step S2, the seedling container is a square box, the height of the box is 4.0-5.0 cm, the height of the substrate is 1 / 4-1 / 3 of the height of the box, and the surface of the substrate is flat.

[0019] In one preferred embodiment, in step S2, the bottom of the seedling container is soaked in the hormone aqueous solution, the depth of the hormone aqueous solution is 2.0-3.0 cm, and the soaking time is 10-12 h.

[0020] In one preferred embodiment, in step S3, after the chrysosporium spores are sown on the surface of the substrate soaked with the hormone aqueous solution in step S2, the lid is covered and sealed, and the culture is carried out for 35-40 d.

[0021] Preferably, the lid is sealed with a plastic wrap. The bottom of the plastic box is soaked in the aqueous solution and the plastic wrap is sealed, which provides nutrients and water for the rapid germination of spores and the induction of protoplasts.

[0022] In one preferred embodiment, in step S3, after the culture is carried out for 35-40 d, the seedling container is not sealed, the lid does not need to be opened, the box is soaked in sterile water, the water depth is 2.0-3.0 cm, the soaking time is 20-24 h, and then the seedling container is sealed again after soaking.

[0023] After a certain period of culture, the bottom of the container is soaked in sterile water and the plastic wrap is sealed, which provides nutrients and water for the induction of protoplasts.

[0024] In one preferred embodiment, in step S3, the culture room temperature is 25-30 DEG C, the light intensity is 2500-4500 lux, and the light time is 12-14 h / d. Too high or too low temperature is not conducive to spore germination and later growth and development. The protocorm and thallus induction time is shorter when the light intensity is 3500-4500 lux.

[0025] Preferably, in step S3, the light intensity is 3500-4500 lux.

[0026] In one preferred embodiment, in step S3, the seeding amount is (0.03-0.04) g / m 2 .

[0027] In step S1, after sterilization, the substrate is naturally dried to a water content of 40-50%.

[0028] In step S1, the substrate comprises peat soil and grass carbon soil. Preferably, the substrate comprises 1-4 parts by volume of peat soil and 1-2 parts by volume of grass carbon soil.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The present application screens a low-cost but effective sterilization method. By improving the seeding conditions and method, the spores are sown on the substrate with a flat surface, and water is supplemented in time from the bottom of the substrate. The combination of several key factors finally improves the protocorm and thallus induction rate. Using the method to cultivate the Chrysemelmba chryseroides seedlings, the protocorm is induced in an average of 22 d, and the thallus is induced in 66 d. The method can be widely used in large-scale Chrysemelmba chryseroides seeding and seedling raising, saving the seedling raising time and labor cost.

[0031] 1. The present application uses suitable mixed fungicides to fully kill harmful bacteria in the substrate by long-time soaking of the substrate, and no miscellaneous bacteria are produced during the culture process. Compared with high-temperature and high-pressure sterilization, the present application reduces the production cost and improves the market competitiveness, and is suitable for factory production.

[0032] 2. After the substrate is sterilized, it is naturally dried to a water content of 40-50%, at which time the substrate is fluffy, and the substrate surface is flat when sown in the container. By soaking the water solution containing hormones from the bottom, the substrate absorbs water, and the spores are distributed on the flat substrate surface, ensuring that the protocorm false roots can fully absorb nutrients and water, and can maximize the light source, which is conducive to spore germination and protocorm induction.

[0033] 3. After 35-40 d of culture, a large number of protocorms are formed, at which time the water is supplemented by soaking sterile water, and the container is sealed, which provides conditions for the development and fertilization of the protocorm eggs, thereby improving the thallus induction rate.

[0034] Using this method, prothallus (visible to the naked eye) is induced after an average of 22 days of culture, and microsporophyte is induced after 66 days. Both are bright green in color, and the seedlings emerge uniformly. The culture cycle is significantly shortened. This invention can be applied on a large scale to the sowing and seedling cultivation of Golden Retriever. Attached Figure Description

[0035] Figure 1 The protophylls (40d) induced by treatment 6 in this embodiment of the invention.

[0036] Figure 2 The sporophyte (90d) induced by treatment 6 in this embodiment of the invention. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0038] The embodiments described herein are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0039] Example 1:

[0040] This invention uses a mixture of: ① a substrate consisting of 2 parts by volume of Northeast peat soil (purchased from Lvwo Nursery in Hengdaohe Town, with moderate decomposition, loose and breathable, pH=5.5-6.0; organic matter content >60%, total humic acid content >35%, free humic acid >30%, total nitrogen content >1.5%, total phosphorus content >0.1%; cellulose content accounting for more than 50% of organic matter) and 1 part by volume of imported peat soil (purchased from JIFFY in the Netherlands, particle size ≤20mm); ② a square transparent plastic box, 5.0cm high, with a slit cut at the bottom, the length of which is 3 / 4 of the box length; ③ an aqueous solution containing 0.10mg of 6-benzylaminopurine and 0.15mg of indoleacetic acid per 1000ml, with the remainder being water; ④ spores are evenly sown on the substrate surface (0.04g / m²). 2 Cover the plastic box with the lid, seal the plastic box with plastic wrap, and incubate under specific temperature and light conditions. ⑤ After 40 days of incubation, remove the plastic wrap, without opening the plastic box lid, immerse the box in sterile water, seal the container with new plastic wrap, and incubate under specific temperature and light conditions.

[0041] Different sterilization methods are used for treatment:

[0042] (A) Soak the substrate in ① above in advance with a fungicide mixture of "thiophanate-methyl + azoxystrobin·tebuconazole". The fungicide is a mixture of 800 times dilution of thiophanate-methyl (70% active ingredient by mass) and 1500 times dilution of azoxystrobin·tebuconazole (30% active ingredient by mass). Mix 5 parts by volume of the fungicide mixture with 1 part by volume of the substrate. Allow it to air dry naturally until the moisture content is 50%. Spread the substrate in the container in ② above, ensuring the surface is flat. Before sowing, soak the container in the aqueous solution in ③ above for 10 hours. Sow according to the spore quantity and method in ④ above to induce prothallium. Induce sporophytes according to the method in ⑤ above.

[0043] (B) Using 1.1 kg / cm 2 The above-mentioned seedling substrate was sterilized at 121℃ under high pressure for 25 minutes, and then the substrate was placed into the container described in step ②. Before sowing, the container was soaked in the aqueous solution described in step ③ for 10 hours. Sowing was carried out according to the spore quantity and method described in step ④ to induce prothallus. Sporophyte induction was carried out according to the method described in step ⑤.

[0044] (C) Place the above-mentioned substrate ① into a container and spread it evenly. Thoroughly drench the substrate with a mixture of "thiophanate-methyl + azoxystrobin·tebuconazole". The fungicide is a mixture of 800 times dilution of thiophanate-methyl (70% active ingredient by mass) and 1500 times dilution of azoxystrobin·tebuconazole (30% active ingredient by mass). Mix 5 parts by volume of the fungicide mixture with 1 part by volume of the substrate. Sow according to the spore quantity and method described in ④ above to induce prothallus. Induce sporophytes according to the method described in ⑤ above.

[0045] Test temperatures: (α) 20~24℃; (β) 25~30℃; (γ) 31~35℃.

[0046] Experimental illumination: (I) 0; (II) 2500~3500 lux; (III) 3500~4500 lux, illumination duration 12h / d.

[0047] A randomized block design was used, with each treatment replicated three times, and 10 seedling boxes sown per replicate. Prothallus germination time: visible green dots. Sporophyte germination time: visible sporophytes. Contamination rate: calculated based on visible mold coverage. Prothallus induction rate: calculated at 80 days after sowing, based on prothallus coverage in the seedling container, regardless of prothallus maturity; Sporophyte induction rate: calculated at 120 days after sowing, based on sporophyte coverage in the seedling container, regardless of sporophyte size.

[0048]

[0049]

[0050] As shown in Table 1-2, the thoroughness of substrate sterilization and moisture content are crucial during the sowing and seedling raising process of Golden Retrievers.

[0051] Treatments 1-9 involved pre-soaking the substrate in a mixture of thiophanate-methyl, azoxystrobin, and tebuconazole, followed by natural air drying to a moisture content of 40-50%. Before sowing, the substrate was soaked in the solution at the bottom of the container. After sowing, the container was sealed with plastic wrap, and later, the plastic wrap was opened and the substrate was soaked in sterile water. No mold grew in any of these treatments. Treatments 10-18 showed mold growth in the substrate starting on the second day after sowing. Within five days, the mold covered the entire container, ultimately preventing spore germination. Treatments 19-27 showed a small amount of mold growth in the substrate 20 days after sowing, with a contamination rate as high as 70.0-86.7% after 100 days. As the mold increased, the induced prothallus and sporophyte were also largely infected and died.

[0052] A smooth seedling substrate is also beneficial for spore germination and growth. Because the substrates for treatments 1-9 were naturally air-dried, they were loose in the containers and did not lose their smoothness after soaking in water. This allowed spores to obtain sufficient nutrients, water, and light, resulting in rapid spore germination. The prothallus induction time was 22-53 days, with a maximum induction rate of 93.2%; the sporophyte induction time was 66-93 days, with a maximum induction rate of 89.9%. Treatments 19-27 involved filling containers with the substrate and leveling it, then drenching the substrate with a mixture of thiophanate-methyl, azoxystrobin, and tebuconazole before sowing and cultivation. The result was that these nine treatments not only had a high contamination rate, but the induction time for uncontaminated prothallus was prolonged to 40-63 days. With the proliferation and infection of mold, the prothallus were highly susceptible to death.

[0053] Light and temperature are also crucial for spore germination and development. Results from treatments 1, 4, 7, 19, 22, and 25 showed that *Cibotium barometz* spores would not germinate without light. Although the induction rates were similar for treatments 5 (2500–3500 lux) and 6 (3500–4500 lux), the induction times for prothalliums and sporophytes in treatment 5 were on average 10 and 14 days later than in treatment 6, respectively. Temperatures that were too high or too low were detrimental to spore germination and later growth. Treatments 3, 6, and 9 were at temperatures of 20–24℃, 25–30℃, and 31–35℃, respectively, with prothallium germination times of 30 days, 22 days, and 53 days, and induction rates of 88.7%, 93.2%, and 45.0%, respectively. The sporophyte germination times were 75 days, 66 days, and 93 days, with induction rates of 83.8%, 89.9%, and 43.2%, respectively. Therefore, the optimal conditions for culturing *Cyprinus pubescens* are 3500–4500 lux of light and 25–30℃, under which the induction rates of prothallium and sporophyte are high.

[0054] Soil-seeding of *Cephalotaxus fortunei* is an open-type seedling cultivation method. The seedling environment has high humidity (generally above 95%), and the substrate is highly susceptible to mold infection under high temperature and humidity conditions; even a small amount of mold can multiply rapidly. Therefore, the thoroughness of substrate sterilization, spore exposure to light, and sufficient moisture content during cultivation are all crucial for spore germination, prothallus and sporophyte induction. Using the fungicide and treatment method of this invention, no mold was generated during the seedling cultivation process. Simultaneously, the substrate surface treated by this invention is smooth, ensuring sufficient light, nutrients, and moisture for spore germination and growth. After a large number of prothallus are formed, the water required for growth and development is replenished by immersing sterile water at the bottom of the container. With suitable temperature and light, the prothallus induction rate for treatments 5 and 6 is ≥91.5%, and the sporophyte induction rate is ≥85.2%.

[0055] Comparative Example 1

[0056] The difference between this comparative example and treatment 6 in Example 1 is that it does not include tebuconazole and thiophanate-methyl, but instead uses thiophanate-methyl to soak the above-mentioned substrate in advance, air-dry it naturally to a moisture content of 40-50%, and then sow it according to the container and method described in this invention, and cultivate it under the conditions of temperature 25-30°C and light intensity 3500-4500 lux.

[0057] As the spore culture time increased, the amount of mold gradually increased. The average contamination rate was 20.3% after 120 days of sowing. Prothallium was induced after 25 days, with an induction rate of 83.3%; sporophyte induction time was 70 days, with an induction rate of 75.5%. The experimental results show that the contamination rate of this comparative example is higher than that of the treatment with the mixture of "thiophanate-methyl + azoxystrobin·tebuconazole", and the induction rates of both prothallium and sporophyte are lower than those of treatment 6 in Example 1.

[0058] Comparative Example 2

[0059] The difference between this comparative example and treatment 6 in Example 1 is that it does not include thiophanate-methyl, but instead uses tebuconazole and azoxystrobin to soak the above-mentioned substrate in advance, and then air-dry it naturally until the moisture content is 40-50%. It is then sown according to the container and method described in this invention and cultured under the conditions of temperature 25-30°C and light intensity 3500-4500 lux.

[0060] As spore germination time increased, mold gradually increased. The average contamination rate was 16.5% after 120 days of sowing. Prothallium was induced at 25 days, with an induction rate of 75.7%; sporophyte induction time was 73 days, with an induction rate of 68.9%. The experimental results show that the contamination rate of this comparative example is higher than that of soaking in the mixture of "thiophanate-methyl + azoxystrobin·tebuconazole", and the induction rates of both prothallium and sporophyte are lower than those of treatment 6 in Example 1.

[0061] Comparative Example 3

[0062] The difference between this comparative example and treatment 6 in Example 1 is that the substrate was soaked in a mixture of "thiophanate-methyl + tebuconazole" and placed in a container before it was naturally air-dried (the substrate was squeezed tightly and there was water between the fingers). It was not soaked in the above-mentioned aqueous solution (each 1000 ml of the hormone aqueous solution contains 0.08-0.10 mg of 6-benzylaminopurine and 0.10-0.15 mg of indoleacetic acid). After direct sowing, the box was sealed with plastic wrap and cultured under the conditions of 25-30°C and 3500-4500 lux of light.

[0063] No mold developed in the golden retriever cultured under these conditions. The prothallus induction time was 28 days, with an induction rate of 70.2%; the sporophyte induction time was 70 days, with an induction rate of 65.2%. Compared with treatment 6 in Example 1, the induction time for prothallus and sporophyte in this comparative example was basically the same, but the induction rate was lower.

[0064] Comparative Example 4

[0065] The difference between this comparative example and treatment 6 in Example 1 is that the substrate was not soaked in the above-mentioned aqueous solution (each 1000 ml of the hormone aqueous solution contains 0.08-0.10 mg of 6-benzylaminopurine and 0.10-0.15 mg of indoleacetic acid) before sowing. Instead, the spores were directly sown on the substrate with a moisture content of 40-50%. After sowing, the box was sealed with plastic wrap and cultured under the conditions of 25-30°C and 3500-4500 lux of light.

[0066] After 40 days of sowing, no prothalliums were induced. The plastic wrap was removed, but the lid was not opened. The container was then immersed in sterile water, and after soaking, the plastic container was sealed with plastic wrap and cultured for another 60 days. Prothalliums were induced after 60 days, and the prothallium induction rate was 71.4% after 100 days. Both the induction time and the induction rate were lower than those of treatment 6 in Example 1.

[0067] Comparative Example 5

[0068] The difference between this comparative example and treatment 6 in Example 1 is that the above aqueous solution does not contain 0.08-0.10 mg of 6-benzylaminopurine or 0.10-0.15 mg of indoleacetic acid (i.e., it is pure water without any added hormones). Other treatments are the same, and the samples are cultured at a temperature of 25-30°C and a light intensity of 3500-4500 lux.

[0069] The induction time for prothallium and sporophyte was basically the same as that of treatment 6 in Example 1, but the induction rates at 80 days after sowing were 88.8% and 73.2%, respectively, which were lower than those of treatment 6.

Claims

1. A method for improving the induction rate of prothallus and sporophyte in *Cibotium barometz*, comprising the following steps: S1. The substrate is sterilized with a bactericide, including thiophanate-methyl and tebuconazole. After sterilization, the moisture content of the substrate is reduced to 40-50%, and the sterilized substrate is obtained. S2. Bring the bottom of the substrate after sterilization in step S1 into contact with the hormone aqueous solution to obtain a substrate that has absorbed the hormone aqueous solution. S3. Spread the golden bream spores on the surface of the substrate containing the hormone solution in step S2 and culture them.

2. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 1, characterized in that, In step S1, the bactericide is a mixture of 800-1000 times dilution of thiophanate-methyl with an effective ingredient content of 60-80% and 1500-2000 times dilution of tebuconazole with an effective ingredient content of 20-40%. 3-5 parts by volume of the bactericide are mixed with 1 part by volume of the matrix.

3. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 1, characterized in that, In step S2, the hormone aqueous solution includes 6-benzylaminopurine and indoleacetic acid.

4. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 1, characterized in that, In step S2, the substrate sterilized in step S1 is placed in a seedling container. The bottom of the seedling container has a slit, and the length of the slit is 2 / 3 to 3 / 4 of the length of the seedling container.

5. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 4, characterized in that, In step S2, the seedling container is a square box with a height of 4.0 to 5.0 cm, the substrate height is 1 / 4 to 1 / 3 of the box height, and the substrate surface is flat.

6. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 4, characterized in that, In step S2, the bottom of the seedling container is immersed in a hormone solution, the depth of which is 2.0-3.0 cm, and the immersion time is 10-12 hours.

7. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 4, characterized in that, In step S3, after the golden dog spores are sown on the surface of the substrate containing the hormone solution in step S2, the lid is covered and sealed, and the substrate is cultured for 35-40 days.

8. The method for improving the induction rate of prothallus and sporophyte of Cibotium barometz according to claim 4, characterized in that, In step S3, after culturing for 35-40 days, keep the seedling container unsealed and do not open the lid. Soak the container in sterile water to a depth of 2.0-3.0 cm for 20-24 hours. After soaking, seal the seedling container.

9. The method for improving the induction rate of prothallus and sporophyte of *Cibotium barometz* according to any one of claims 1-8, characterized in that, In step S3, the culture room temperature is 25-30℃, the light intensity is 2500-4500 lux, and the light duration is 12-14 h / d.

10. The method for improving the induction rate of prothallus and sporophyte of *Cibotium barometz* according to any one of claims 1-8, characterized in that, The seeding rate in step S3 is (0.03~0.04) g / m². 2 .

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