Spore propagation method of cibotium barometz

Abstract

The invention provides a spore propagation method of cibotium barometz, and relates to the technical field of plant propagation. The method provided by the invention comprises the following steps: sowing Cibotium barometz spores in a composite culture medium, culturing under the conditions of natural illumination and normal temperature, promoting gametophyte transformation by adopting atomized water spraying instead of traditional oscillation insemination, and culturing seedlings in combination with a non-woven fabric seedling culture container. By means of environmental conditions such as natural light and the like, the bionic technology is adopted for breeding, daily management and protection are simple and economical, low-cost, rough-management and efficient batch seedling raising are achieved, the rate of converting gametophytes into sporophytes is greatly increased, the period from spores to transplantable seedlings of cibotium barometz is shortened to 70-80 days, the transplanting survival rate can be increased to 100%, and the survival rate of cibotium barometz is increased to 50%. And a seedling raising guarantee is provided for batch planting of cibotium barometz.

Description

Technical Field

[0001] This invention relates to the field of plant propagation technology, and in particular to a method for spore propagation of *Cibotium barometz*. Background Technology

[0002] *Cibotium barometz* (L.) J. Sm., belonging to the genus *Cibotium* in the family Ceratophyllaceae, order Polypodiales, is distributed in tropical and subtropical Asia. In China, it is mainly found in the southern and southwestern regions. It gets its name from its rhizome, which is prostrate, thick, and has an upward-curving tip covered with dense, long, soft, golden-yellow hairs, resembling a golden-haired dog lying on the ground. Its unique morphology makes it valuable for ornamental purposes. The rhizome, also known as "dog's spine," is included in the *Pharmacopoeia of the People's Republic of China* (2020 edition) and is believed to have effects such as tonifying the liver and kidneys, strengthening the waist and knees, and dispelling rheumatism. It is an ideal medicinal material for treating rheumatic bone pain, weakness in the legs and knees, and urinary incontinence. The long, soft, golden hairs on the rhizome can also be applied externally to treat external bleeding, and have long been used in folk medicine in China and other parts of Asia for stopping bleeding from external injuries.

[0003] In recent years, people have gained a deeper understanding of the medicinal, health, culinary, and nutritional value of golden retrievers, leading to a surge in exploitation. Wild golden retriever resources have been overexploited, while artificial breeding and cultivation have lagged behind, resulting in a sharp decline in my country's golden retriever resources year after year, with some areas already depleted.

[0004] Compared with division and natural reproduction, spore reproduction has made significant breakthroughs in efficiency and adaptability, and is the core means of large-scale production and species protection. At present, the spore reproduction technology of golden retriever dogs has the following defects: (1) The environmental control requirements are strict, the energy consumption is large and the daily management methods are complicated; (2) The method of soaking in water and using a shaker to accelerate fertilization is not only complicated to operate, but also the gametophytes are easily contaminated when soaked in water, resulting in a low gametophyte conversion rate; (3) The use of special shakers and constant temperature incubators makes it highly dependent on equipment and difficult to apply on a large scale; (4) It takes 180-280 days from spores to transplantable seedlings, the reproduction cycle is too long, the transplant survival rate is low, and it cannot meet the needs of commercialization.

[0005] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a method for spore propagation of Cibotium barometz.

[0007] In a first aspect, the present invention provides a method for spore propagation of *Cibotium barometz*, comprising the following steps: (1) Spore sowing: Sterilized golden dog spores were sown on a culture medium made of a mixture of Northeast black soil, vermiculite and coconut coir; (2) Spore germination: Under natural light, normal temperature, ambient humidity of 65%-85%, and water content of culture medium of 60%-70%, the spores are cultured until they develop into mature gametophytes; (3) Gametophyte fertilization: When the gametophyte matures, fertilization is promoted by atomizing water spray; (4) Sporophyte formation: Apply nutrient solution regularly to promote sporophyte development when the first young sporophylle appears after fertilization; (5) Seedling transplanting: After the seedlings have developed to a suitable stage, transplant them into a breathable container.

[0008] Optionally, the volume ratio of Northeast black soil, vermiculite and coconut coir in the culture medium in step (1) is (1-3):1:1.

[0009] Optionally, the culture medium described in step (1) is sterilized by high-pressure steam at 120°C for 30 minutes.

[0010] Optionally, the particle sizes of the Northeast black soil, vermiculite and coconut coir mentioned in step (1) are 0.1mm-2mm, 0.1mm-1mm and 0.1mm-2mm, respectively.

[0011] Optionally, the spore germination in step (2) includes cultivation under natural light with a shading rate of 40%-60%, ambient temperature of not less than 10℃, ambient humidity of 70%-85%, and substrate moisture content of 60%-70% until it develops into a mature gametophyte.

[0012] Optionally, the droplet size of the atomized water spray in step (3) is controlled to be 50μm-100μm.

[0013] Optionally, the frequency of atomized water spraying in step (3) is once a day, with a water volume of 200 mL / 2000 cm² each time, and the spraying continues for 7-10 days.

[0014] Optionally, the nutrient solution in step (4) includes water-soluble fertilizers containing macro-elements.

[0015] Optionally, the nutrient solution has a mass percentage concentration of 0.001%-0.005%.

[0016] Optionally, the nutrient solution is applied once every 7 days, with each application consisting of 200 mL / 2000 cm².

[0017] Optionally, the seedling transplantation in step (5) includes transplanting the seedling into a breathable container when the first true leaf of the seedling grows to 1cm-1.5cm.

[0018] Optionally, the breathable container in step (5) includes a nonwoven container.

[0019] Optionally, the transplanting substrate comprises fermented rice husks, fully burned rice husk ash, perlite, and garden soil mixed evenly in a volume ratio of 1:1:0.5:1.

[0020] Optionally, the method achieves a cycle from spore to transplantable seedling in 70-80 days.

[0021] Optionally, the transplant survival rate obtained by the method can reach 100%. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the spore reproduction process of *Cibotium barometz* in Embodiment 3 of the present invention; wherein, A: mature spores; B: sowing device and substrate; C: germinating gametophyte; D: developing gametophyte (gametophyte diameter is 2mm-3mm); E: mature gametophyte; F: spore seedlings differentiated from gametophyte; G: spore seedlings to be transplanted; H: 90 days after transplanting spore seedlings; I: 180 days after transplanting spore seedlings; Figure 2 This is a schematic diagram of transplanting golden dog fern seedlings in non-woven fabric bags according to Embodiment 4 of the present invention; Figure 3 This is a schematic diagram of transplanting golden dog fern seedlings in plastic pots according to Embodiment 5 of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0024] This invention provides a method for spore propagation of *Cibotium barometz*, comprising sowing *Cibotium barometz* spores in a composite culture medium, culturing under natural light and room temperature conditions, using atomized water spraying instead of traditional shaking for fertilization to promote gametophyte transformation, and cultivating seedlings in non-woven fabric seedling containers. This invention utilizes environmental conditions such as natural light and employs biomimetic technology for propagation, simplifying daily management and saving costs. It achieves low-cost, low-management, and highly efficient mass seedling production, significantly increasing the rate of gametophyte transformation into sporophytes. This shortens the cycle from spore to transplantable seedlings to 70-80 days, and increases the transplant survival rate to 100%, providing a seedling guarantee for the mass cultivation of *Cibotium barometz*.

[0025] This invention provides a method for spore propagation of Cibotium barometz, comprising the following steps: (1) Spore sowing: Sterilized golden dog spores were sown on a culture medium made of a mixture of Northeast black soil, vermiculite and coconut coir; (2) Spore germination: Under natural light, normal temperature, ambient humidity of 65%-85%, and water content of culture medium of 60%-70%, the spores are cultured until they develop into mature gametophytes; (3) Gametophyte fertilization: When the gametophyte matures, fertilization is promoted by atomizing water spray; (4) Sporophyte formation: Apply nutrient solution regularly to promote sporophyte development when the first young sporophylle appears after fertilization; (5) Seedling transplanting: After the seedlings have developed to a suitable stage, transplant them into a breathable container.

[0026] In some embodiments, the volume ratio of Northeast black soil, vermiculite, and coconut coir in the culture medium used in step (1) is (1-3):1:1. In fact, Northeast black soil has a high organic matter content and is rich in various trace elements, providing slowly released natural nutrients for spore germination and early gametophyte growth. It also has a high cation exchange capacity, allowing it to adsorb nutrients and water, preventing loss. Vermiculite's structure creates numerous pores, preventing water accumulation in the substrate, ensuring sufficient oxygen for the roots, preventing root rot, and maintaining the substrate's moisture content. Coconut coir's structure absorbs and retains moisture, working synergistically with vermiculite to achieve a moist but not wet state. Furthermore, it prevents the black soil from becoming too heavy and compacted, making the substrate structure looser and facilitating the extension and attachment of gametophyte rhizoids. Through the compounding of Northeast black soil, vermiculite, and coconut coir, the culture medium can simultaneously meet the three core requirements of moisture retention, aeration, and nutrient supply.

[0027] In some embodiments, the culture medium used in step (1) is autoclaved at 120°C for 30 minutes.

[0028] In some embodiments, the particle sizes of the Northeast black soil, vermiculite and coconut coir used in step (1) are 0.1mm-2mm, 0.1mm-1mm and 0.1mm-2mm, respectively.

[0029] In some embodiments, spore germination in step (2) includes cultivation under natural light with a shading rate of 40%-60%, ambient temperature of not less than 10°C, ambient humidity of 70%-85%, and substrate moisture content of 60%-70% until the spores develop into mature gametophytes.

[0030] In some embodiments, the droplet size of the atomized water spray in step (3) is controlled to be 50μm-100μm. In fact, the fertilization of gametophytes in golden retrievers must rely on water as a medium. Although the soaking and shaking in traditional techniques also provide a water environment, the methods are crude and have great side effects. The present invention can control the droplet size of the atomized water spray to create the optimal water film thickness, promote the fertilization of mature gametophytes. The water mist in the range of 50μm-100μm can be evenly attached to the surface of the gametophytes to form an extremely thin and continuous water film, which can both allow sperm to swim and promote fertilization and not completely isolate the air to ensure respiration and avoid suffocation and decay caused by long-term soaking.

[0031] In some embodiments, the frequency of atomized water spraying in step (3) is once a day, with a spray volume of 200 mL / 2000 cm² each time, and the spraying is continued for 7-10 days. In fact, the control of the spray volume and spray frequency is to ensure that all gametophytes on the culture surface are evenly covered with a water film, maintaining the continuity of the water film, while preventing water accumulation at the bottom of the seedling box. In a highly controllable, biomimetic, and low-damage manner, a necessary aquatic medium environment is created and maintained for the fertilization process of Cibotium barometz gametophytes.

[0032] In some embodiments, the nutrient solution used in step (4) includes a macro-element water-soluble fertilizer.

[0033] In some embodiments, the mass percentage concentration of the nutrient solution used in step (4) is 0.001%-0.005%.

[0034] In some embodiments, the frequency of applying nutrient solution in step (4) is once every 7 days, and the amount sprayed each time is 200mL / 2000cm².

[0035] In some embodiments, the seedling transplantation in step (5) includes transplanting the seedling into a breathable container when the first true leaf of the seedling grows to 1cm-1.5cm.

[0036] In some embodiments, the breathable container used in step (5) includes a non-woven fabric container. In fact, compared to the closed system of plastic pots, the non-woven fabric container is an open system, and its material is made of interwoven fibers with countless micropores. These micropores allow air to circulate freely, while also allowing excess water to evaporate or permeate from all directions, ensuring that the substrate is neither too dry nor too wet, and always maintaining an ideal moisture content to avoid waterlogging and root rot.

[0037] In some embodiments, the transplanting substrate used comprises fermented rice husks, fully combusted rice husk ash, perlite, and garden soil mixed evenly in a volume ratio of 1:1:0.5:1.

[0038] In some embodiments, the spore propagation method using *Cibotium barometz* can achieve a cycle of 70-80 days from spores to transplantable seedlings.

[0039] In some embodiments, the transplant survival rate obtained by using the spore propagation method of Cibotium barometz can reach 100%.

[0040] Example 1

[0041] This embodiment 1 provides a method for spore propagation of *Cibotium barometz*, including the following steps: S1, Golden Retriever Spore Collection

[0042] On August 20th (in the southern Jiangxi region), sporangia on healthy, disease-free *Cibotium barometz* plants, showing yellowish-green or yellowish-brown spores that had not yet split open, were collected. During collection, while minimizing damage to the plant, the small pinnae bearing the sporangia were cut off with scissors, placed in a kraft paper bag, sealed, and left to air dry in a cool, well-ventilated place for 7 days. Before sowing, the dried spores were poured out and passed through a 60-mesh sieve to thoroughly separate impurities from the spores. The cleaned spores were then collected in a brown wide-mouthed bottle and stored in a refrigerator at 4°C.

[0043] S2, Golden Retriever Spore Sowing

[0044] The culture medium used for sowing is mainly a mixture of Northeast black soil (0.1mm-2mm particle size), vermiculite (0.1mm-1mm particle size), and coconut coir, provided by Ma Honghe Chunqing Substrate Container Factory in Xinyu High-tech Zone, in a volume ratio of 1:1:1. The above substrate is packed into high-temperature resistant sterilization bags (Labshark high-temperature resistant garbage bags), sterilized by high-pressure steam at 120℃ for 30 minutes, cooled, and then spread evenly in white seedling frames (37cm×30cm×7.5cm). The soil layer is compacted to a thickness of 10cm, and then sprayed with purified water (which can effectively reduce fungal infection) until the soil moisture content reaches 60%-70%.

[0045] When sowing, place 1g of spores on filter paper, then place the filter paper in the center of a 60-mesh sieve. While tapping the filter paper, slowly move the paper to spread the spores, ensuring that the spores are evenly distributed in the seedling frame.

[0046] After sowing, cover the seedling frame with a transparent cover with a breathable valve and turn the valve to the closed position to maintain substrate moisture. After sowing, water as needed with a sprayer depending on the substrate moisture.

[0047] S3, Germination of sporophyte of *Cibotium barometz*

[0048] After sowing, place the seedling frames directly in a greenhouse (25℃-30℃) with 70%-80% humidity and 50% shade, without any special treatment. Spray water every other day, stopping when only a small amount of water remains on the surface of the substrate. Maintain these conditions; the sporophytes will begin to germinate 30 days after sowing. Once the sporophytes have germinated, turn on the ventilation valve. After the gametophytes develop to a diameter of 2mm-3mm, the water used for spraying can be replaced with tap water. Spray with a low-concentration plant nutrient solution (500mL tap water mixed with 5mL undiluted plant nutrient solution) every 7 days to effectively promote gametophyte development and maturity.

[0049] S4, Golden Retriever gametophyte fertilization

[0050] Sixty days after spore sowing, the gametophyte sexual organs mature, which can be identified by the distinct wavy morphology of the outer edge of the gametophyte. After the sexual organs mature, it is necessary to open the transparent plastic cover daily and spray water evenly, with about 50 mL of water sprayed per tray. After 15 days, the mature gametes begin to differentiate into young sporophytes.

[0051] S5, Sporophyte formation of *Cibotium barometz*

[0052] Once young spores begin to differentiate at the edge of the gametophyte, open the lid and spray water evenly every other day, applying approximately 30 mL of water per tray. After 15 days of young sporophyte differentiation and seedling emergence, maintain air and substrate humidity at 60%-70%, keep the temperature at room temperature (10℃-30℃), and provide 50% shade. Spraying with a low-concentration plant nutrient solution (10 mL of undiluted plant nutrient solution in 500 mL of tap water) every 7 days can effectively promote seedling growth.

[0053] S6, Transplanting of Golden Retriever Seedlings

[0054] After the seedlings emerge, wait 15 days until the young sporophytes develop 1-2 young leaves. Then, transplant the seedlings using tweezers into non-woven fabric containers (5.5cm*8cm). After 33 days of growth, when the seedling leaves completely cover the surface of the substrate bag, transplant them again into non-woven fabric containers (8cm*10cm). Repeat this transplanting process three times during the seedling growth period to promote growth until the seedlings begin to differentiate underground runners. During transplanting, use a uniform substrate consisting of fermented rice husks, fully burned rice husk ash, perlite, and garden soil mixed in a volume ratio of 1:1:0.5:1, with a substrate moisture content of 60%.

[0055] Example 2

[0056] This embodiment 2 provides a method for spore propagation of *Cibotium barometz*, including the following steps: S1, Golden Retriever Spore Collection

[0057] On August 20th (in southern Jiangxi), sporangia on healthy, disease-free *Cibotium barometz* plants, showing yellowish-green or yellowish-brown spores that had not yet split open, were collected. During collection, the small pinnae bearing the sporangia were cut off with scissors, placed in kraft paper bags, sealed, and left to air dry in a cool, well-ventilated place for 7 days, while minimizing damage to the plant. Before sowing, the dried spores were poured out and passed through a 60-mesh sieve to thoroughly separate impurities from the spores. The cleaned spores were then collected in brown wide-mouthed bottles and stored in a refrigerator at 4°C.

[0058] S2, Golden Retriever Spore Sowing

[0059] The growing medium used for sowing is mainly a mixture of Northeast black soil (0.1mm-2mm particle size), vermiculite (0.1mm-1mm particle size), and coconut coir, provided by Ma Honghe Chunqing Substrate Container Factory in Xinyu High-tech Zone, in a volume ratio of 2:1:1. The above substrate is packed into high-temperature resistant sterilization bags (Labshark high-temperature resistant garbage bags), sterilized by high-pressure steam at 120℃ for 30 minutes, cooled, and then spread evenly in white seedling frames (37cm×30cm×7.5cm). The soil is compacted to a thickness of 10cm, and then sprayed with purified water (which can effectively reduce fungal infection) until the soil moisture content reaches 60%-70%.

[0060] When sowing, place 1g of spores on filter paper, then place the filter paper in the center of a 60-mesh sieve. While tapping the filter paper, slowly move the paper to spread the spores, ensuring that the spores are evenly distributed in the seedling frame.

[0061] After sowing, cover the seedling frame with a transparent cover with a breathable valve and turn the valve to the closed position to maintain substrate moisture. After sowing, water as needed with a sprayer depending on the substrate moisture.

[0062] S3, Germination of sporophyte of *Cibotium barometz*

[0063] After sowing, place the seedling frames directly in a greenhouse (25℃-30℃) with 70%-80% humidity and 50% shade, without any special treatment. Spray water every other day, stopping when only a small amount of water remains on the surface of the substrate. Maintain these conditions; the sporophytes will begin to germinate 25 days after sowing. Once the sporophytes have germinated, turn on the ventilation valve. After the gametophytes develop to a diameter of 2mm-3mm, the water used for spraying can be replaced with tap water. Spray with a low-concentration plant nutrient solution (500mL tap water mixed with 5mL undiluted plant nutrient solution) every 7 days to effectively promote gametophyte development and maturity.

[0064] S4, Golden Retriever gametophyte fertilization

[0065] Forty-five days after spore sowing, the gametophyte sexual organs mature, which can be identified by the distinct wavy morphology of the outer edge of the gametophyte. After the sexual organs mature, it is necessary to open the transparent plastic cover daily and spray water evenly, with about 50 mL of water sprayed per tray. After 15 days, the mature gametes begin to differentiate into young sporophytes.

[0066] S5, Sporophyte formation of *Cibotium barometz*

[0067] Once young spores begin to differentiate at the edge of the gametophyte, open the lid and spray water evenly every other day, applying approximately 30 mL of water per tray. After 15 days of young sporophyte differentiation and seedling emergence, maintain air and substrate humidity at 60%-70%, keep the temperature at room temperature (10℃-30℃), and provide 50% shade. Spraying with a low-concentration plant nutrient solution (10 mL of undiluted plant nutrient solution in 500 mL of tap water) every 7 days can effectively promote seedling growth.

[0068] S6, Transplanting of Golden Retriever Seedlings

[0069] After the seedlings emerge, wait 12 days until the young sporophytes develop 1-2 young leaves. Then, transplant the seedlings using tweezers into non-woven fabric containers (5.5cm*8cm). After 25 days of growth, when the seedling leaves completely cover the surface of the substrate bag, they can be transplanted again into non-woven fabric containers (8cm*10cm). Repeat this transplanting process three times during the seedling growth period to promote growth until the seedlings begin to differentiate underground runners. The cultivation substrate should be a uniform mixture of fermented rice husks, fully burned rice husk ash, perlite, and garden soil in a volume ratio of 1:1:0.5:1, with a moisture content of 60%.

[0070] Example 3

[0071] This embodiment 3 provides a method for spore propagation of *Cibotium barometz*, including the following steps: S1, Golden Retriever Spore Collection

[0072] On August 20th (in the southern Jiangxi region), sporangia on healthy, disease-free *Cibotium barometz* plants, showing yellowish-green or yellowish-brown spores that had not yet split open, were collected. During collection, while minimizing damage to the plant, the small pinnae bearing the sporangia were cut off with scissors, placed in a kraft paper bag, sealed, and left to air dry in a cool, well-ventilated place for 7 days. Before sowing, the dried spores were poured out and passed through a 60-mesh sieve to thoroughly separate impurities from the spores. The cleaned spores (… Figure 1 A) Collect the contents into a brown wide-mouthed bottle and store in a refrigerator at 4°C.

[0073] S2, Golden Retriever Spore Sowing

[0074] The growing medium used for sowing is mainly a mixture of Northeast black soil (0.1mm-2mm particle size), vermiculite (0.1mm-1mm particle size), and coconut coir, provided by Ma Honghe Chunqing Substrate Container Factory in Xinyu High-tech Zone, in a volume ratio of 3:1:1. The above substrate is packed into high-temperature resistant sterilization bags (Labshark high-temperature resistant garbage bags), sterilized by high-pressure steam at 120℃ for 30 minutes, cooled, and then spread evenly in white seedling frames (37cm×30cm×7.5cm). The soil is compacted to a thickness of 10cm, and then sprayed with purified water (which can effectively reduce fungal infection) until the soil moisture content reaches 60%-70%.

[0075] When sowing, place 1g of spores on filter paper, then place the filter paper in the center of a 60-mesh sieve. While tapping the filter paper, slowly move the paper to spread the spores, ensuring that the spores are evenly distributed in the seedling frame.

[0076] After sowing, cover the seedling frame with a transparent cover that has a breathable valve, and turn the valve to the closed position to maintain substrate moisture. After sowing, water as needed with a spray bottle depending on the substrate moisture. Figure 1 (B in the middle).

[0077] S3, Germination of sporophyte of *Cibotium barometz*

[0078] After sowing, place the seedling trays directly in a greenhouse (25℃-30℃) with 70%-80% humidity and 50% shade; no special treatment is required. Water by misting every other day, stopping when only a small amount of water remains on the surface of the substrate. Maintaining these conditions, gametophytes will begin to germinate after 18 days. Figure 1 (C) The aeration valve is opened by rotating the sporophyte only after spore germination. This occurs after the gametophyte has developed to a diameter of 2mm-3mm (C). Figure 1 (D) The water used for spraying can be replaced with tap water. A low concentration of plant nutrient solution (500mL tap water to 5mL undiluted plant nutrient solution) can be sprayed once every 7 days, which can effectively promote the development and maturation of gametophytes. Figure 1 (E in the text).

[0079] S4, Golden Retriever gametophyte fertilization

[0080] Forty days after spore sowing, the gametophyte sexual organs mature, which can be identified by the distinct wavy morphology of the gametophyte's outer edge. After sexual organ maturity, the transparent plastic cover should be opened daily for even misting and watering, with approximately 50 mL of water sprayed per tray. Mature gametes begin to differentiate into young sporophytes after 12 days. Figure 1 (F in the middle).

[0081] S5, Sporophyte formation of *Cibotium barometz*

[0082] Once young spores begin to differentiate at the edge of the gametophyte, open the lid and spray water evenly every other day, applying approximately 30 mL of water per tray. After 15 days of young spore differentiation and seedling emergence, maintain air and substrate humidity at 60%-70%, keep the temperature at room temperature (10℃-30℃), and provide 50% shade. Spraying with a low-concentration plant nutrient solution (10 mL of undiluted plant nutrient solution in 500 mL of tap water) every 7 days can effectively promote seedling growth.

[0083] S6, Transplanting of Golden Retriever Seedlings

[0084] After the seedlings emerge, wait 10 days for the young sporophyte to develop 1-2 young leaves. Figure 1 After step G), use tweezers to transplant the seedlings into non-woven fabric container bags (5.5cm*8cm). After 30 days of growth, when the seedling leaves completely cover the surface of the substrate bag, they can be transplanted again into non-woven fabric container bags (8cm*10cm). Figure 1 H in the text). During the seedling growth period, transplant three times to promote seedling growth until the seedlings begin to differentiate underground stolons (H). Figure 1 (I) The cultivation substrate is uniformly selected from fermented rice husks, fully burned rice husk ash, perlite and garden soil mixed evenly in a volume ratio of 1:1:0.5:1, and the moisture content of the cultivation substrate is 60%.

[0085] Comparative Example 1

[0086] Comparative Example 1 provides a method for spore propagation of *Ceratophyllum demersum*, including the following steps: S1, Golden Retriever Spore Collection

[0087] On August 20th (in the southern Jiangxi region), sporangia on healthy, disease-free *Cibotium barometz* plants, showing yellowish-green or yellowish-brown spores that had not yet split open, were collected. During collection, while minimizing damage to the plant, the small pinnae bearing the sporangia were cut off with scissors, placed in a kraft paper bag, sealed, and left to air dry in a cool, well-ventilated place for 7 days. Before sowing, the dried spores were poured out and passed through a 60-mesh sieve to thoroughly separate impurities from the spores. The cleaned spores were then collected in a brown wide-mouthed bottle and stored in a refrigerator at 4°C.

[0088] S2, Golden Retriever Spore Sowing

[0089] The growing medium used for sowing is mainly habitat soil (0.1mm-2mm particle size) from the surrounding wild populations of the Golden Retriever in Quannan County. The above substrate is packed into high-temperature sterilization bags (Labshark high-temperature garbage bags), sterilized by high-pressure steam at 120℃ for 30 minutes, then cooled and spread evenly in white seedling frames (37cm×30cm×7.5cm). The soil is compacted to a thickness of 10cm, and then sprayed with purified water (which can effectively reduce fungal infection) until the soil moisture content reaches 60%-70%.

[0090] When sowing, place 1g of spores on filter paper, then place the filter paper in the center of a 60-mesh sieve. While tapping the filter paper, slowly move the paper to spread the spores, ensuring that the spores are evenly distributed in the seedling frame.

[0091] After sowing, cover the seedling frame with a transparent cover with a breathable valve and turn the valve to the closed position to maintain substrate moisture. After sowing, water as needed with a sprayer depending on the substrate moisture.

[0092] S3, Germination of sporophyte of *Cibotium barometz*

[0093] After sowing, place the seedling trays directly in a greenhouse (25℃-30℃) with 70%-80% humidity and 50% shade, without any special treatment. Spray water every other day, stopping when only a small amount of water remains on the surface of the substrate. Maintain these conditions; the sporophytes will begin to germinate 22 days after sowing. Once the sporophytes have germinated, turn on the ventilation valve. After the gametophytes develop to a diameter of 2mm-3mm, the water used for spraying can be replaced with tap water. A low-concentration plant nutrient solution (500mL tap water mixed with 5mL undiluted plant nutrient solution) can be sprayed every 7 days to effectively promote gametophyte development and maturity.

[0094] S4, Golden Retriever gametophyte fertilization

[0095] Forty-two days after spore sowing, the gametophyte sexual organs mature, which can be identified by the distinct wavy morphology of the outer edge of the gametophyte. After the sexual organs mature, it is necessary to open the transparent plastic cover daily and spray water evenly, with about 50 mL of water sprayed per tray. After 15 days, the mature gametes begin to differentiate into young sporophytes.

[0096] S5, Sporophyte formation of *Cibotium barometz*

[0097] Once young spores begin to differentiate at the edge of the gametophyte, open the lid and spray water evenly every other day, applying approximately 30 mL of water per tray. After 15 days of young sporophyte differentiation and seedling emergence, maintain air and substrate humidity at 60%-70%, keep the temperature at room temperature (10℃-30℃), and provide 50% shade. Spraying with a low-concentration plant nutrient solution (10 mL of undiluted plant nutrient solution in 500 mL of tap water) every 7 days can effectively promote seedling growth.

[0098] S6, Transplanting of Golden Retriever Seedlings

[0099] After the seedlings emerge, wait 15 days until the young sporophytes develop 1-2 young leaves. Then, transplant the seedlings using tweezers into non-woven fabric containers (5.5cm*8cm). After 36 days of growth, when the seedling leaves completely cover the surface of the substrate bag, transplant them again into non-woven fabric containers (8cm*10cm). Repeat this transplanting process three times during the seedling growth period to promote growth until the seedlings begin to differentiate underground runners. During transplanting, use a uniform substrate consisting of fermented rice husks, fully burned rice husk ash, perlite, and garden soil mixed in a volume ratio of 1:1:0.5:1, with a substrate moisture content of 60%.

[0100] Example 4

[0101] This embodiment 4 provides a method for spore propagation of *Cibotium barometz*, which differs from embodiment 3 in that, in step S6, the seedlings are transplanted using tweezers. Non-woven fabric seedling bags (12cm × 14cm) are used as cultivation containers. The cultivation substrate consists of fermented rice husks, fully burned rice husk ash, perlite, and garden soil mixed in a volume ratio of 1:1:0.5:1. The moisture content of the cultivation substrate is 60%. 30 bags of seedlings are transplanted. Figure 2 As shown.

[0102] Example 5

[0103] This embodiment 5 provides a method for spore propagation of *Cibotium barometz*, which differs from embodiment 3 in that, in step S6, the seedlings are transplanted using tweezers. Plastic seedling pots (15cm in diameter) are used as cultivation containers. The cultivation substrate consists of fermented rice husks, fully burned rice husk ash, perlite, and garden soil mixed in a volume ratio of 1:1:0.5:1. The moisture content of the cultivation substrate is 60%. 30 seedlings are transplanted. Figure 3 As shown.

[0104] The culture medium is closely related to the seedling growth. For Examples 1 to 3 and the comparative example, vigorous and robust seedlings with one or two young leaves were selected. Ten seedlings with consistent growth were selected from each replicate. The total plant length, taproot length, plant height, leaf area, root-to-shoot ratio, and rejuvenation index were measured (Table 1). The total plant length was measured using a ruler from the root tip to the shoot tip meristem; the taproot length was measured using a ruler from the base of the rootstock to the meristem; the plant height was measured using a ruler from the base of the crown to the meristem; the leaf area was calculated by scanning the unfolded leaves of the plant using an EPSONScan scanner and then using ImageJ software; the root-to-shoot ratio was the ratio of the fresh weight of the underground part (root system) to the aboveground part (crown) of the seedling. The test results are shown in Table 1.

[0105] Table 1: Effects of different substrate formulations on the growth of *Cibotium barometz* larvae seedlings

[0106]

[0107] Analyzing the data in Table 1 above, we can find that: (1) Effects of different substrate formulations on root growth of Golden Retriever seedlings

[0108] Root growth is related to the rhizosphere environment. The larger the pores in the substrate formulation, the better it is for the roots to anchor the seedlings and absorb nutrients. Comparing the treatments, it can be seen that the formulation of Example 1 has the largest proportion of vermiculite, and its taproot length and root tip number are significantly higher than those of Example 2, Example 3 and Comparative Example 1. Conversely, the large proportion of Northeast black soil will restrict the growth of seedling roots.

[0109] (2) Effects of different substrate formulations on the plant height of Golden Retriever seedlings

[0110] There were significant differences in the plant height of the Golden Retriever seedlings among the different formulations: a comparison of the treatments showed that the Northeast Black Soil had the largest proportion in the formulation of Example 3, and its plant height was significantly higher than that of Example 1, Example 2 and Comparative Example 1.

[0111] (3) Effects of different substrate formulations on leaf area of ​​Golden Retriever seedlings

[0112] There are certain differences in the leaf area of ​​Golden Retriever seedlings among the different formulations: a comparison of the treatments shows that as the proportion of Northeast black soil in the formulation increases, the leaf area also gradually increases, with the highest leaf area observed in Example 3.

[0113] (4) Effects of different substrate formulations on the seedling vigor index of Golden Retriever seedlings

[0114] The seedling vigor index is an indicator reflecting the overall quality of seedlings, while the root-to-shoot ratio reflects the correlation between the aboveground and underground parts of the seedling. The seedling vigor index and root-to-shoot ratio were compared and measured using different substrate formulations (Table 1). Significant differences in the seedling vigor index of *C. truncatum* seedlings after transplanting were observed among the different formulations. The highest seedling vigor index was found in the treatment of Example 3 (0.092 and 0.098), while the seedling vigor index of the treatment of Example 1 was significantly lower than that of the other treatments (0.065 and 0.068). The root-to-shoot ratios varied significantly among the treatments, with the highest being in the treatment of Example 1 (0.561 and 0.612) and the lowest being in the treatment of Example 3 (0.373 and 0.331).

[0115] By comparing different substrate formulations, the seedlings of *Cibotium barometz* treated with the substrate formulation in Example 3 showed significantly higher plant height, leaf area, and seedling vigor index compared to those in Examples 1, 2, and the comparative example. This indicates that the higher the aboveground biomass of *Cibotium barometz* seedlings, the higher their recovery index, resulting in a shorter recovery time and higher survival rate after transplanting.

[0116] The container used for transplanting seedlings has a significant impact on the survival rate and rejuvenation effect of seedlings after transplanting. In order to screen the optimal cultivation container, experiments were conducted on Examples 4 and 5, recording the survival rate (survival rate = number of surviving plants / 100 × 100%) and growth (plant height, crown width, and number of leaves) of the seedlings on the 15th, 30th, 60th, and 90th days after transplanting. The experimental results are detailed in Table 2.

[0117] Table 2: Effects of different transplanting containers on seedling growth

[0118]

[0119] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for spore propagation of *Cibotium barometz*, characterized in that, Includes the following steps: (1) Spore sowing: Sterilized golden dog spores were sown on a culture medium made of a mixture of Northeast black soil, vermiculite and coconut coir; (2) Spore germination: Under natural light, normal temperature, ambient humidity of 65%-85%, and water content of culture medium of 60%-70%, the spores are cultured until they develop into mature gametophytes; (3) Gametophyte fertilization: When the gametophyte matures, fertilization is promoted by atomizing water spray; (4) Sporophyte formation: Apply nutrient solution regularly to promote sporophyte development when the first young sporophylle appears after fertilization; (5) Seedling transplanting: After the seedlings have developed to a suitable stage, transplant them into a breathable container.

2. The breeding method according to claim 1, characterized in that, In step (1), the volume ratio of Northeast black soil, vermiculite, and coconut coir in the culture medium is (1-3):1:1; and / or, the culture medium is sterilized by high-pressure steam at 120℃ for 30 minutes; and / or, the particle sizes of the Northeast black soil, vermiculite, and coconut coir are 0.1mm-2mm, 0.1mm-1mm, and 0.1mm-2mm, respectively.

3. The breeding method according to claim 1, characterized in that, The spore germination described in step (2) includes cultivation under natural light with a shading rate of 40%-60%, ambient temperature of not less than 10℃, ambient humidity of 70%-85%, and substrate moisture content of 60%-70% until the spores develop into mature gametophytes.

4. The breeding method according to claim 1, characterized in that, The droplet size of the atomized water spray in step (3) is controlled to be 50μm-100μm.

5. The breeding method according to claim 1, characterized in that, The frequency of atomized water spraying in step (3) is once a day, with a water volume of 200 mL / 2000 cm² each time, and the spraying continues for 7-10 days.

6. The breeding method according to claim 1, characterized in that, The nutrient solution in step (4) includes water-soluble fertilizers containing macro-elements; and / or, the mass percentage concentration of the nutrient solution is 0.001%-0.005%; and / or, the frequency of application of the nutrient solution is once every 7 days, and the amount sprayed each time is 200mL / 2000cm².

7. The breeding method according to claim 1, characterized in that, The seedling transplantation in step (5) includes transplanting the seedlings into a breathable container when the first true leaf of the seedling grows to 1cm-1.5cm.

8. The breeding method according to claim 1, characterized in that, The breathable container mentioned in step (5) includes a non-woven fabric container; and / or, the transplanting substrate includes fermented rice husks, fully burned rice husk ash, perlite and garden soil mixed evenly in a volume ratio of 1:1:0.5:

1.

9. The breeding method according to claim 1, characterized in that, The method achieves a cycle of 70-80 days from spores to transplantable seedlings.

10. The breeding method according to claim 1, characterized in that, The transplant survival rate obtained by the method is 100%.

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