Direct sowing seedling raising method for cibotium barometz spores

Abstract

The invention relates to a direct seeding and seedling raising method for cibotium barometz spores, and belongs to the technical field of plant planting. The invention provides a direct seeding seedling raising method for cibotium barometz spores. The method comprises the following steps: directly seeding the spores to obtain sporophyte bud seedlings; transplanting the sporophyte bud seedlings to obtain sporophyte seedlings; seedling hardening is carried out on the sporophyte seedlings; the sporophyte seedlings are subjected to seedling hardening when the height is 10-15 cm and the number of leaves is 3-4. By means of the direct seeding seedling raising method, the seedling yield is remarkably increased, the seedling yield of the obtained sporophyte seedlings is 85% or above, large-scale industrial popularization and planting can be conducted, and the market supply amount is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of plant cultivation technology, specifically relating to a method for direct seeding of Cibotium barometz spores. Background Technology

[0002] The medicinal herb *Cibotium barometz* is the dried rhizome of *Cibotium barometz* (L.) J.Sm., a plant belonging to the family Cibotaceae. The genus *Cibotium* comprises about 10 species, mainly distributed in tropical and subtropical regions of Central America, the Hawaiian Islands, and Asia. In China, three species with significant genetic differences are found: *Cibotium barometz* (L.) J.Sm., *Cibotium cumingii* De Vol., and a newly discovered species, *Sino-Burmaense* sp.nov., found in northwestern Yunnan, China, and northeastern Myanmar. Its dried rhizome is the medicinal part, primarily used for strengthening bones and muscles, invigorating the lower back and knees, and tonifying the liver and kidneys. It is widely used in various health products and traditional Chinese medicine preparations, with an annual demand exceeding 4,000 tons.

[0003] Due to limited and protected wild resources, there is currently little legally available stock on the market, making it difficult for pharmaceutical companies to obtain it. To ensure the supply of Cibotium barometz medicinal resources, large-scale industrial-scale cultivation is necessary to guarantee market supply. Currently, few entities possess mature Cibotium barometz seedling propagation technology, resulting in a limited supply of seedlings.

[0004] Patent document CN 107646657 A discloses a method for propagating the tillers of *Cibotium barometz*, which involves digging up the rhizomes of *Cibotium barometz*, propagating them through tillering, and then cultivating and managing them under certain conditions. This invention can produce qualified seedlings with a high survival rate, but wild *Cibotium barometz* cannot be harvested in large quantities, and its tillering coefficient is relatively low, making it difficult to propagate large quantities of seedlings on the market. Patent document CN 109699495B discloses a method for improving the germination rate of *Cibotium barometz* spores, which improves the germination rate of *Cibotium barometz* spores by adjusting the culture medium ratio and adding a certain concentration of growth hormone. However, this invention only addresses the process of spore germination into prothalliums, and does not cover the subsequent transformation of prothalliums into sporophytes and the later transplanting for outdoor planting. Patent document CN 119144541 A discloses a prothallus proliferation culture medium and rapid propagation method for the rare and endangered plant *Cibotium barometz*. This method shortens the maturation time from prothallus to gametophyte by preparing a prothallus proliferation culture medium, accelerating the development of prothallus into sporophytes. However, this method only allows for cultivation and propagation in laboratory tissue culture bottles, resulting in limited propagation coefficients and quantities. Furthermore, this method does not describe or research the outdoor cultivation techniques for *Cibotium barometz* sporophytes, such as greenhouse or field cultivation. Moreover, a significant technological gap exists between the laboratory and actual production, making it difficult to directly achieve large-scale industrial seedling production. Currently, due to the limitations of wild *Cibotium barometz* and the lack of improvements to the entire seedling cultivation process, cultivating qualified seedlings suitable for industrial-scale promotion and planting is extremely difficult. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defect of the existing technology that makes it difficult to industrialize and cultivate qualified seedlings of Cibotium barometz on a large scale, thereby providing a method for direct seeding of Cibotium barometz spores.

[0006] Therefore, the present invention provides the following technical solution:

[0007] This invention provides a method for direct seeding of Cibotium barometz spores, the method comprising: direct seeding of spores to obtain sporophyte seedlings; transplanting of sporophyte seedlings to obtain sporophyte seedlings; hardening off the sporophyte seedlings; and hardening off the sporophyte seedlings when they are 10-15 cm tall and have 3-4 leaves.

[0008] In one embodiment of the present invention, the substrate for seedling hardening is a mixture of yellow clay and peat moss.

[0009] In one embodiment of the present invention, the volume ratio of yellow clay to peat soil in the mixture is 3:7 to 6:4.

[0010] In one embodiment of the present invention, the hardening process of the sporophyte seedlings is carried out in an outdoor hardening greenhouse;

[0011] Preferably, the shading rate of the seedling hardening greenhouse is 65% to 85%.

[0012] In one embodiment of the present invention, the step of obtaining sporophyte seedlings by direct spore seeding includes the following steps: spore collection and purification, spore treatment, and direct spore seeding.

[0013] In one embodiment of the present invention, the spore treatment involves soaking the spores in 75% alcohol for 5 minutes for disinfection, centrifuging them for 5 minutes, discarding the supernatant, and repeating the above process 3 times.

[0014] In one embodiment of the present invention, the direct sowing of spores is carried out under tissue culture conditions, with a sowing density of 60–100 mg / m². 2 The tissue culture temperature is 20–28℃, and the light intensity is 3000–5000 lux.

[0015] In one embodiment of the present invention, during the transformation from prothallus to sporophyte (60-120 days after sowing), a seedling growth promoter (containing humic acid water-soluble fertilizer) is sprayed every 5 days at a concentration of 800-1000 times; from sporophyte seedling to seedling transplanting (120 days after sowing), a rooting agent (containing amino acid water-soluble fertilizer) is sprayed every 5 days at a concentration of 800-1000 times.

[0016] In one embodiment of the present invention, in the sporophyte seedling transplanting step, when the sporophyte seedlings grow to 1.0-3.0 cm, they are transplanted into new peat moss trays or preservation boxes and continued to be managed in the same tissue culture room under the same conditions.

[0017] In one embodiment of the present invention, the seedling qualification standard is: plant height 20-40cm, number of leaves not less than 6, and golden hairs at the base of the petiole.

[0018] The technical solution of this invention has the following advantages:

[0019] 1. This invention provides a method for direct seeding of Cibotium barometz spores, comprising: direct seeding of spores to obtain sporophyte seedlings; transplanting of sporophyte seedlings to obtain sporophyte seedlings; hardening off the sporophyte seedlings; and hardening off the sporophyte seedlings when they are 10-15 cm tall and have 3-4 leaves. This invention has found that during the hardening off process of sporophyte seedlings, the smaller or taller the sporophyte seedlings are transplanted, the lower the survival rate in the later stages of transplanting. The main reason for this is likely that smaller or taller seedlings are more prone to leaf collapse during post-transplant management, adhering to the substrate soil surface, eventually leading to leaf rot and death, ultimately hindering large-scale industrial cultivation of qualified seedlings. Further research by this invention has found that selecting sporophyte seedlings at a height of 10-15 cm and with 3-4 leaves for hardening off results in a high rate of qualified seedlings and shortens the prolonged recovery period caused by leaf collapse or rot.

[0020] 2. This invention provides a method for direct seeding of Cibotium barometz spores. The substrate for hardening off the seedlings comprises a mixture of yellow clay and peat moss in a volume ratio of 3:7 to 6:4. Research in this invention has found that the substrate used for hardening off significantly impacts the survival rate in the later stages of transplanting. The reason for this may be that substrate mixtures in this volume ratio range (3:7 to 6:4) have a slightly acidic pH and a relatively loose composition, suitable for the growth and development of Cibotium barometz rhizomes. Pure yellow clay, on the other hand, tends to be compacted, resulting in slow rhizome growth; pure peat moss, with its acidic conditions and insufficient nutrients, also leads to slow rhizome growth. Further research in this invention has shown that a mixture of yellow clay and peat moss in a volume ratio of 3:7 to 6:4 can obtain seedlings with a high qualification rate and shorten the seedling establishment period.

[0021] 3. This invention provides a method for direct seeding of Cibotium barometz spores. The hardening-off process of the sporophyte seedlings is carried out in an outdoor hardening-off greenhouse with a shading rate of 65%–85%. Research in this invention has found that the shading rate during hardening-off significantly affects the survival rate in the later stages of transplanting. The reason for this may be that transplanting seedlings outdoors and managing them in a double-layered shade net greenhouse provides ideal conditions for the growth of Cibotium barometz seedlings, effectively improving the qualification rate and survival rate. With a single layer of shade netting, the leaves of Cibotium barometz seedlings are easily scorched, resulting in a lower seedling survival rate. With more than two layers of shade netting, the growth of Cibotium barometz seedlings is slow, and the qualified seedling rate is low. Further research in this invention has found that a shading rate of 65%–85% can obtain seedlings with a high qualification rate and shorten the seedling establishment period.

[0022] 4. This invention provides a method for direct seeding of Cibotium barometz spores. Soaking the spores in 75%–90% alcohol and centrifuging, repeated 2–4 times, effectively disinfects the spore powder. Centrifugation removes other fern spores, moss spores, and molds from the spore powder, reducing contamination during spore germination. Furthermore, the alcohol is volatile and does not affect subsequent experiments.

[0023] 5. This invention provides a method for direct seeding of Cibotium barometz spores, with the sowing density controlled at 60–100 mg / m². 2 This can effectively control the appropriate growth density of prothallium; if the density is lower or higher than this range, the space and number of prothalliums that germinate will be limited, which will inhibit the growth of prothalliums.

[0024] Furthermore, maintaining a temperature of 20–28°C in the culture room can effectively promote spore germination and growth. If the temperature exceeds 28°C, the prothallus is prone to mold and death; if the temperature is below 20°C, the prothallus grows extremely slowly, and the time for germination into sporophyte seedlings is delayed by more than 45 days compared to the recommended temperature.

[0025] Furthermore, a light intensity of 3000–5000 lux can effectively promote spore germination, prothallium growth, and sporophyte growth; light intensities below this range will slow down the processes of spore germination, prothallium growth, and sporophyte growth; light intensities above this range will scorch the leaves of the prothallium and sporophyte, eventually leading to their death.

[0026] Furthermore, during the transformation from prothallus to sporophyte (60-120 days after sowing), spray with a seedling growth promoter every 5 days; from sporophyte seedling to transplanting (120 days after sowing until transplanting), spray with a rooting hormone solution every 5 days. Spraying these two agents at two different stages after spore germination can effectively promote their growth. The first seedling growth promoter can effectively promote the growth and development of prothallus leaves, effectively carry out photosynthesis, promote their faster growth and maturation into gametophytes, and promote the subsequent fusion of sperm and archegonia; the second rooting hormone solution can effectively promote the rooting and strengthening of sporophyte seedlings, absorb more nutrients, and promote the growth process of sporophyte seedlings.

[0027] 6. Using the method of this invention for direct seeding of *Cibotium barometz* spores, the initial transformation into prothallus occurs 15 days after sowing, and the initial transformation into sporophyte occurs 60 days after sowing (complete transformation into sporophyte occurs 120 days after sowing). After hardening off, the time to reach qualified seedling size (20-40cm tall, 6-8 pinnate compound leaves, with golden down at the base of the petiole) is 270-400 days, with a seedling qualification rate of over 85%. This represents a significant improvement compared to the 20% qualification rate obtained by using prepared culture media in tissue culture bottles in existing technologies. This invention is the first to propose a complete seedling cultivation system for direct seeding of *Cibotium barometz* spores, significantly improving the seedling qualification rate and cultivating qualified seedlings suitable for industrial-scale cultivation. Attached Figure Description

[0028] Figure 1 The germination of *Cibotium barometz* spores is the entire growth process of a sporophyte seedling. (A) *Cibotium barometz* spore powder (yellow powder with an irregular tetrahedral structure); (B) First stage of spore germination (a green substance appears on the surface of white vermiculite, i.e., the prothallus); (C) Second stage of spore germination (the prothallus grows further, forming adventitious roots, and gradually differentiates into antheridium and archegonia); (D) Third stage of spore germination (after fertilization by sperm in the archegonia of the female gametophyte, the sporophyte gradually forms); (E) Fourth stage of spore germination (the sporophyte absorbs nutrients from the prothallus and gradually grows to form a complete root system).

[0029] Figure 2 Effects of different sowing densities of Cibotium barometz on prothallus germination and growth. (A) Sowing density 100 mg / m² 2 (B) Sowing density 20 mg / m² 2 (C) Sowing density 40 mg / m² 2 (D) Sowing density 200.0 mg / m² 2

[0030] Figure 3 The effects of different substrate ratios in nutrient pots on the growth and development of sporophytes of Cibotium barometz. (A) From left to right: pure yellow clay, yellow clay: peat moss = 5:5, pure peat moss, pure coconut coir; (B) pure yellow clay; (C) yellow clay: peat moss = 5:5; (D) pure peat moss; (E) pure coconut coir. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0033] Example 1: Direct seeding method for Cibotium barometz spores

[0034] The specific process is as follows: the entire growth process of *Cibotium barometz* spores germinating into sporophyte seedlings is as follows. Figure 1 As shown:

[0035] 1. Spores grow into sporophyte seedlings in the tissue culture room:

[0036] (1) Spore collection and purification: Spores must be collected from pinnate leaves of Cibotium barometz with spores within the approved collection location and quantity range. Mature spores are those whose sporangia have turned brown and a small portion has dehisced. Place the collected leaves in a clean, dry room with a clean tarpaulin or plastic film on the floor. Spread the collected leaves loosely and let them air dry naturally. Turn them over several times during this period. Most of the sporangia will dehisce and the spores will scatter. After shaking the leaves, collect the sporangia and spores. First, use a 20-mesh coarse sieve to remove branches and leaves, then use a 200-mesh fine sieve. The spores that pass through the sieve are purified spores. Place them in a resealable bag and label it with the collection location and date. Store them in a 4°C constant temperature refrigerator.

[0037] (2) Spore treatment: Soak in 75% alcohol for 5 minutes for disinfection, centrifuge for 5 minutes, and discard the supernatant. Repeat the above process 3 times.

[0038] (3) Direct sowing of spores: Dilute the sterilized spore powder with a certain amount of purified water and determine the optimal sowing density as 100 mg / m³. 2 The tissue culture room was set at a temperature of 28℃ and a light intensity of 5000 lux. Between 60 and 120 days after sowing, a seedling growth promoter (containing humic acid water-soluble fertilizer, produced by Henan Shuanghui Agricultural Technology Development Co., Ltd.) was sprayed every 5 days at a concentration of 800 times. After 120 days after sowing, a root-promoting solution (containing amino acid water-soluble fertilizer, produced by Shandong Hainengda Agricultural Technology Development Co., Ltd.) was sprayed every 5 days at a concentration of 800 times.

[0039] 2. Transplanting sporophyte seedlings: When the sporophyte seedlings are about 3.0cm tall, transplant them into new peat moss trays or preservation boxes, and continue to manage them in the same conditions in the tissue culture room.

[0040] 3. Hardening off sporophyte seedlings in nutrient pots: When the sporophyte seedlings are 10cm tall and have 3 leaves, they are hardened off by being placed in nutrient pots with a volume ratio of yellow clay to peat moss of 5:5. They are then transplanted outdoors and placed in a greenhouse with a double-layer shade net (80% shading rate) for management.

[0041] The experimental results are shown in Tables 1-3. Following this embodiment, the time required for all spores to germinate and transform into prothallia is approximately 58 days, and the time required for all prothallia to transform into sporophytes is approximately 62 days (total time: 120 days). The total number of prothallia per unit area is approximately 400 plants / dm². 2 Approximately 200 sporophytes per unit area / dm² 2 Approximately 270 days are required for the sporophytes to reach the standard for qualified seedlings, given the slight contamination. The sporophyte growth rate should be based on the rate at which the seedlings reach the qualified standard.

[0042] Example 2: Effects of different breeding conditions on the growth and development of Cibotium barometz

[0043] The specific process is as follows:

[0044] Based on Example 1, the following conditions were adjusted: spore disinfection and sterilization method, spore sowing density, tissue culture room temperature, light intensity, dilution of seedling growth promoter and rooting hormone, transplanting height of sporophyte seedlings, hardening height of sporophyte seedlings, substrate ratio in nutrient pots, and shading rate of hardening greenhouse. Other conditions remained the same as in Example 1. The specific experimental conditions are as follows:

[0045] The spores were sterilized by soaking in 75% alcohol and centrifuging, repeated three times; the spore sowing density was 80 mg / m³. 2 The tissue culture room was set at a temperature of 25℃ and a light intensity of 4000 lux; the seedling growth promoter and rooting agent were diluted 900 times; the sporophyte seedlings were transplanted to a height of 2.0 cm; the sporophyte seedlings were hardened off when they reached a height of 12 cm and had 4 leaves; the volume ratio of yellow clay to peat moss in the nutrient pots was 3:7; the seedlings were transplanted outdoors and managed in a greenhouse with a double-layer shade net (65% shading rate).

[0046] The experimental results are shown in Tables 1-3. Following this embodiment, the time required for all spores to germinate and transform into prothallia is approximately 60 days, and the time required for all prothallia to transform into sporophytes is approximately 70 days (total time: 130 days). The total number of prothallia per unit area is approximately 352 plants / dm². 2 The total number of sporophytes per unit area is approximately 176 per dm². 2 Approximately 280 days are required for the sporophytes to reach the standard for qualified seedlings, with the contamination level being slight. The sporophyte growth rate should be based on the rate at which the seedlings reach the qualified standard.

[0047] Example 3: Effects of different breeding conditions on the growth and development of Cibotium barometz

[0048] The specific process is as follows:

[0049] The following conditions were adjusted: spore disinfection and sterilization method, spore sowing density, tissue culture room temperature, light intensity, dilution of seedling growth promoter and rooting hormone, transplanting height of sporophyte seedlings, hardening height of sporophyte seedlings, substrate ratio in nutrient pots, and shading rate of hardening greenhouse. Other conditions remained consistent with those in Example 1. The specific experimental conditions are as follows:

[0050] The spores were sterilized by soaking in 75% alcohol and centrifuging, repeated three times; the spore sowing density was 60 mg / m³. 2 The tissue culture room was set at a temperature of 22℃ and a light intensity of 3000 lux; the seedling growth promoter and rooting agent were diluted 1000 times; the sporophyte seedlings were transplanted to a height of 1.0 cm; when the sporophyte seedlings reached a height of 15 cm and had 4 leaves, they were placed in cups for hardening off; the volume ratio of yellow clay to peat moss in the nutrient pots was 6:4; the seedlings were transplanted outdoors and placed in a double-layered shade net greenhouse (85% shading rate) for management;

[0051] The experimental results are shown in Tables 1-3. Following this embodiment, the time required for all spores to germinate and transform into prothallia is approximately 65 days, and the time required for all prothallia to transform into sporophytes is approximately 70 days (total time: 135 days). The total number of prothallia per unit area is approximately 360 plants / dm². 2 Approximately 180 sporophytes per unit area (dm²) 2 Approximately 300 days is required for the sporophytes to reach the standard for qualified seedlings, with the contamination being minor. The growth rate of the sporophytes should be based on the rate at which they reach the standard for qualified seedlings.

[0052] Table 1. Growth of prothallus after sowing of Cibotium barometz spores under different cultivation conditions

[0053]

[0054] Note the prothallus contamination status: Slightly contaminated means the contamination rate is less than 5% of the total; moderately contaminated means the contamination rate is 10-20% of the total; moderately contaminated means the contamination rate is less than 30-50% of the total; severely contaminated means the contamination rate is greater than 50% of the total. The contamination rate is calculated as (moldy area of ​​the substrate seedbed / total area of ​​the substrate seedbed) * 100%. Normally, the seedbed surface is green after spore germination, but after contamination, white or black spots will appear on the surface.

[0055] Table 2. Growth of Cibotium barometz from prothallus to sporophyte stage under different cultivation conditions.

[0056]

[0057] Note the level of sporophyte contamination: Slight contamination rate is less than 5% of the total; moderate contamination rate is 10-20%; relatively severe contamination rate is less than 30-50%; and severe contamination rate is greater than 50%. The time required for prothallium to sporophyte transformation includes the time required for spore germination to develop into prothallium. The contamination rate is calculated as (moldy area of ​​the substrate seedbed / total area of ​​the substrate seedbed) * 100%. Normally, the seedbed surface is green after spore germination; contamination will result in white or black spots on the surface. The time required for prothallium to sporophyte transformation includes the time required for spore germination to develop into prothallium and the time required for all prothalliums to transform into sporophytes.

[0058] Table 3. Growth of Cibotium sporophytes during the hardening-off stage under different cultivation conditions.

[0059]

[0060] Note: Sporophyte seedling qualification rate: The proportion of qualified *Cibotium barometz* seedlings to the total number of seedlings per unit area. The time required for the sporophyte to reach the qualified seedling standard includes the time required for spore germination to the prothallus, the time required for all prothallus to transform into sporophytes, and the time required for all sporophytes to reach the qualified seedling standard. The qualified sporophyte seedling (seedling) standard is: plant height 20-40cm, number of leaves not less than 6, and golden hairs at the base of the petiole.

[0061] The results showed that the contamination level of prothallium varied depending on the disinfection method used, with the lowest contamination rate observed after three cycles of alcohol immersion disinfection and centrifugation. Different sowing densities resulted in varying total prothallium counts per unit area, with the optimal sowing density being 60–100 mg / m². 2 In the tissue culture room, the temperature is set at 20-28℃ and the light intensity at 3000-5000 lux, which are the optimal conditions for spore germination and prothallium growth. At 60-120 days and after 120 days, a seedling growth promoter and a rooting agent are used respectively, with the concentration controlled between 800-1000 times, which can effectively promote the growth of prothalliums and sporophytes. During the hardening-off stage, sporophyte seedlings with a height of 10-15cm and 3-4 leaves are transplanted into nutrient pots with a yellow clay:peat soil volume ratio of 3:7-6:4, and managed under two layers of shade netting (shading rate of 65%-85%). This results in the fastest growth rate and the highest rate of reaching the standard for mature *Cibotium barometz* seedlings.

[0062] Comparative Examples 1-3: Effects of different sowing densities on the total number of prothalliums per unit area

[0063] The specific process is as follows:

[0064] Based on Example 1, the sowing density was adjusted to 20 mg / m². 2 40mg / m2 and 200mg / m 2 Under the same conditions as in Example 1, the effects of different sowing densities on the germination and growth of protophylls were compared. The experimental results are attached. Figure 2 As shown.

[0065] The results showed that different sowing densities had a significant impact on the total number of prothallus per unit area. Figure 2 A. Sowing density is 100 mg / m² 2 A relatively large number of them germinate into prothallus; Figure 2 B is 20 mg / m³ 2 The optimal sowing density results in the fewest prothallus germinations. Figure 2 C is 40 mg / m 2 The seeding density is the second highest, followed by the number of prothalliums that germinate. Figure 2 D is 200 mg / m 2 At a certain sowing concentration, a large number of prothallia germinate, and the prothallia grow well overall. However, the high density of prothallia leads to the death of a small number of prothallia. This indicates that the sowing density of spores should not be too low or too high; a density of 60–100 mg / m³ is recommended. 2 between.

[0066] Comparative Examples 4–6: Effects of different spore disinfection methods on spore contamination rate and germination rate

[0067] The specific process is as follows:

[0068] Based on Example 1, the sterilization methods were adjusted to pure alcohol, sodium hypochlorite, and potassium permanganate, respectively, while other conditions remained the same as in Example 1. The effects of different spore disinfection methods on spore contamination rate and germination rate were compared, and the experimental results are shown in Table 4 below.

[0069] Table 4. Effects of different spore disinfection methods on spore contamination rate and germination rate.

[0070]

[0071]

[0072] The results showed that the contamination level of prothallus varied depending on the disinfection method used. The lowest contamination rate was observed after three cycles of alcohol immersion and centrifugation. Other disinfectants, such as pure alcohol, caused bacterial surface proteins to coagulate into a protective layer, and spores were easily dehydrated and inactivated. Sodium hypochlorite sterilization was difficult to control in terms of time; the longer the time, the lower the spore germination rate. Potassium permanganate sterilization had strong oxidizing properties, and the sterilization time was also difficult to control; the longer the sterilization time, the lower the spore activity. Furthermore, multiple spore washings were required after sterilization, making the process complex, and its use was not recommended.

[0073] Comparative Examples 7-9: The effects of transplanting and hardening off sporophyte seedlings of different heights on the seedling qualification rate of Cibotium barometz are detailed below:

[0074] Based on Example 1, the transplanting height of sporophyte seedlings was adjusted to 3cm, 7cm and 20cm respectively, while other conditions remained the same as in Example 1. The effects of transplanting sporophyte seedlings of different heights on the seedling qualification rate of Cibotium barometz were compared. The experimental results are shown in Table 5 below.

[0075] Table 5. Effects of transplanting and hardening of sporophyte seedlings of different heights on the seedling qualification rate of Cibotium barometz.

[0076]

[0077] The results showed that the smaller the sporophyte seedlings (less than 10 cm) or the taller they were transplanted (greater than 15 cm), the lower their survival rate in the later stages of transplanting. This was mainly because smaller or taller seedlings were more prone to having their leaves fall over and stick to the surface of the substrate soil after watering during post-transplanting management, eventually leading to leaf rot and death.

[0078] Comparative Examples 10-12: The effects of different substrate ratios in nutrient pots on the growth and development of sporophytes of Cibotia repens are detailed below:

[0079] Based on Example 1, the substrate ratios of the nutrient pots were adjusted to pure yellow clay, pure peat moss, and pure coconut coir, respectively, while keeping other conditions consistent with Example 1. The effects of different substrate ratios on the growth and development of Cibotium barometz sporophytes were compared. The experimental results are shown in Table 6 below. Figure 3 As shown.

[0080] Table 6. Effects of different substrate ratios on the growth and development of sporophytes of Cibotium simonii.

[0081]

[0082] The results showed that the seedlings of *Cibotium barometz* grew fastest and best in nutrient pots with a 5:5 volume ratio of yellow clay to peat moss; followed by pure yellow clay, pure peat moss, and pure coconut coir. This 5:5 ratio of substrate to soil mixture provides a slightly acidic pH and a relatively loose substrate composition, which is suitable for the growth and development of *Cibotium barometz* rhizomes. In contrast, pure yellow clay resulted in a compacted substrate, leading to slow rhizome growth; and pure peat moss, with its insufficient acidity and nutrients, also resulted in slow rhizome growth.

[0083] Comparative Examples 13–15: Effects of different shading rates in breeding sheds on the growth and development of Cibotium barometz.

[0084] The specific process is as follows:

[0085] Based on Example 1, the shading rate of the breeding shed was adjusted to 30%, 50%, and 95%, respectively, while other conditions remained the same as in Example 1. The effects of different shading rates on the growth and development of Cibotium barometz were compared, and the experimental results are shown in Table 7 below.

[0086] Table 7. Effects of breeding sheds with different shading rates on the growth and development of Cibotium barometz.

[0087]

[0088] The results showed that after transplanting, the sporophyte seedlings of Cibotium barometz, when placed in a greenhouse with 80% shading, reached the qualified seedling standard in 270 days, with a sporophyte qualification rate of 95%. When placed in a greenhouse with 30% shading, the time required was 300 days, with a sporophyte qualification rate of 40%. Under 50% shading, the time was 280 days, with a sporophyte qualification rate of 50%. And under 95% shading, the time was 340 days, with a sporophyte qualification rate of 80%. This indicates that light conditions play a crucial role in the later growth and development of sporophyte seedlings after transplanting, and that controlled light conditions within a certain range promote faster growth of sporophyte seedlings.

[0089] Comparative Example 16: Effects of different breeding conditions on the growth and development of Cibotium barometz

[0090] The specific process is as follows:

[0091] Based on Example 1, the following conditions were adjusted: spore disinfection and sterilization method, spore sowing density, tissue culture room temperature, light intensity, dilution of seedling growth promoter and rooting hormone, transplanting height of sporophyte seedlings, hardening height of sporophyte seedlings, substrate ratio in nutrient pots, and shading rate of hardening greenhouse. Other conditions remained the same as in Example 1. The specific experimental conditions are as follows:

[0092] The spores were sterilized by soaking in 75% alcohol and centrifuging, repeated twice; the spore sowing density was 200 mg / m³. 2 The tissue culture room was set at a temperature of 30℃ and a light intensity of 5000 lux; the seedling growth promoter and the rooting agent were diluted 600 times; the sporophyte seedlings were transplanted to a height of 1.0 cm; the sporophyte seedlings were placed in cups to harden off when they reached a height of 15 cm and had 5 leaves; the nutrient pots contained pure yellow mud; the seedlings were transplanted outdoors and placed in a single-layer shade net greenhouse (with a shading rate of 30%) for management.

[0093] Comparative Example 17: Effects of different breeding conditions on the growth and development of Cibotium barometz

[0094] The specific process is as follows:

[0095] Based on Example 1, the following conditions were adjusted: spore disinfection and sterilization method, spore sowing density, tissue culture room temperature, light intensity, dilution of seedling growth promoter and rooting hormone, transplanting height of sporophyte seedlings, hardening height of sporophyte seedlings, substrate ratio in nutrient pots, and shading rate of hardening greenhouse. Other conditions remained the same as in Example 1. The specific experimental conditions are as follows:

[0096] The spores were sterilized by soaking in 75% alcohol and centrifuging, repeated twice; the spore sowing density was 40 mg / m³. 2 The tissue culture room was set at a temperature of 18℃ and a light intensity of 2000 lux; the seedling growth promoter and rooting agent were diluted 400 times; the sporophyte seedlings were transplanted to a height of 3.0 cm; the sporophyte seedlings were placed in cups to harden off when they reached a height of 5 cm and had 2 leaves; the nutrient pots contained pure yellow mud; the seedlings were transplanted outdoors and placed in a single-layer shade net greenhouse (with a shading rate of 30%) for management.

[0097] Comparative Example 18: Effects of Different Breeding Conditions on the Growth and Development of Cibotium barometz

[0098] The specific process is as follows:

[0099] Based on Example 1, the following conditions were adjusted: spore disinfection and sterilization method, spore sowing density, tissue culture room temperature, light intensity, dilution of seedling growth promoter and rooting hormone, transplanting height of sporophyte seedlings, hardening height of sporophyte seedlings, substrate ratio in nutrient pots, and shading rate of hardening greenhouse. Other conditions remained the same as in Example 1. The specific experimental conditions are as follows:

[0100] The spores were sterilized by soaking in 75% alcohol and centrifuging, with no repetition; the spore sowing density was 20 mg / m³. 2 The tissue culture room was set at a temperature of 15℃ and a light intensity of 1000 lux; the seedling growth promoter and rooting hormone were diluted 1500 times; the sporophyte seedlings were transplanted to a height of 3.0 cm; sporophyte seedlings were hardened off when they reached a height of 5 cm and had 2 leaves; the volume ratio of yellow clay to peat moss in the nutrient pots was 3:7; the seedlings were transplanted outdoors and placed in a multi-layered shade net greenhouse (90% shading rate) for management. The experimental results are shown in Tables 8-10.

[0101] Table 8. Growth of prothallus after sowing of Cibotium barometz spores under different cultivation conditions

[0102]

[0103] Note the prothallus contamination status: Slightly contaminated means the contamination rate is less than 5% of the total; moderately contaminated means the contamination rate is 10-20% of the total; moderately contaminated means the contamination rate is less than 30-50% of the total; severely contaminated means the contamination rate is greater than 50% of the total. The contamination rate is calculated as (moldy area of ​​the substrate seedbed / total area of ​​the substrate seedbed) * 100%. Normally, the seedbed surface is green after spore germination, but after contamination, white or black spots will appear on the surface.

[0104] Table 9. Growth of Cibotium barometz from prothallus to sporophyte stage under different cultivation conditions.

[0105]

[0106] Note the sporophyte contamination status: Slight contamination rate is less than 5% of the total; moderate contamination rate is 10-20%; relatively severe contamination rate is less than 30-50%; and severe contamination rate is greater than 50%. The contamination rate is calculated as (moldy area of ​​the substrate seedbed / total area of ​​the substrate seedbed) * 100%. Normally, the seedbed surface is green after spore germination; after contamination, white or black spots will appear on the surface. The time required for prothallium to sporophyte transformation includes the time required for spore germination to reach the prothallium and the time required for all prothalliums to transform into sporophytes.

[0107] Table 10 Growth of Cibotium genus sporophytes during hardening-off stage under different cultivation conditions.

[0108]

[0109]

[0110] Note: Sporophyte seedling qualification rate: The proportion of qualified *Cibotium barometz* seedlings to the total number of seedlings per unit area. The time required for the sporophyte to reach the qualified seedling standard includes the time required for spore germination to the prothallus, the time required for all prothallus to transform into sporophytes, and the time required for all sporophytes to reach the qualified seedling standard. The qualified sporophyte seedling (seedling) standard is: plant height 20-40cm, number of leaves not less than 6, and golden hairs at the base of the petiole.

[0111] The experimental results are shown in Tables 8-10. Significant differences were observed in the growth of prothalliums, the growth from prothallium to sporophyte stage, and the hardening-off stage of *Cibotium barometz* spores under different cultivation conditions. This indicates that adjusting factors such as spore sterilization methods, spore sowing density, tissue culture room temperature, light intensity, dilution of growth promoters and rooting hormone, sporophyte seedling transplanting height, sporophyte seedling hardening-off height, substrate ratio in nutrient pots, and shading rate in the hardening-off greenhouse all significantly affect the final qualified rate of *Cibotium barometz* sporophyte seedlings. To improve the final qualified rate of *Cibotium barometz* sporophyte seedlings, strict control must be exercised over each stage of the early cultivation process.

[0112] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for direct seeding of Cibotium barometz spores, characterized in that, The seedling cultivation method includes: Sporophyte seedlings are obtained by direct spore seeding; Transplanting sporophyte seedlings yields sporophyte seedlings; Hardening off sporophyte seedlings: The sporophyte seedlings are hardened off when they are 10-15cm tall and have 3-4 leaves.

2. The method for direct seeding of Cibotium barometz spores as described in claim 1, characterized in that, The substrate for seedling hardening is a mixture of yellow clay and peat moss.

3. The method for direct seeding of Cibotium barometz spores as described in claim 2, characterized in that, The volume ratio of yellow clay to peat soil in the mixture is 3:7 to 6:

4.

4. A method for direct seeding of Cibotium barometz spores as described in any one of claims 1 to 3, characterized in that, The hardening-off process for the sporophyte seedlings was carried out in an outdoor hardening-off greenhouse; Preferably, the shading rate of the seedling hardening greenhouse is 65% to 85%.

5. A method for direct seeding of Cibotium barometz spores as described in any one of claims 1 to 4, characterized in that, The steps for obtaining sporophyte seedlings by direct spore seedling production include the following steps: spore collection and purification, spore treatment, and direct spore seedling production.

6. The method for direct seeding of Cibotium barometz spores as described in claim 5, characterized in that, The spores were treated by soaking in 75%–90% alcohol for disinfection and centrifugation and washing 2–4 times.

7. A method for direct seeding of Cibotium barometz spores as described in claim 5 or 6, characterized in that, The direct seeding of spores was carried out under tissue culture conditions, with a seeding density of 60–100 mg / m². 2 The tissue culture temperature is 20-28℃, and the light intensity is 3000-5000 lux. During the transformation from prothallus to sporophyte, a seedling growth promoter is applied at a concentration of 800-1000 times. Before the sporophyte seedlings are transplanted, a rooting agent is applied at a concentration of 800-1000 times.

8. A method for direct seeding of Cibotium barometz spores as described in any one of claims 1 to 7, characterized in that, In the sporophyte seedling transplantation step, when the sporophyte seedlings grow to 1.0–3.0 cm, they are transplanted and continued to be managed under the same tissue culture conditions.

9. A method for direct seeding of Cibotium barometz spores as described in any one of claims 1 to 8, characterized in that, The substrate for transplanting is peat moss.

10. A method for direct seeding of Cibotium barometz spores as described in any one of claims 1 to 9, characterized in that, The seedling qualification standards are as follows: plant height 20-40cm, number of leaves not less than 6, and golden hairs at the base of the petiole.

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