A method for germination of cibotium barometz spores and a method for breeding cibotium barometz

Abstract

This invention relates to a method for the germination of Cibotium barometz spores and a method for the propagation of Cibotium barometz, belonging to the field of plant culture technology. The propagation method of this invention includes the following steps: disinfection: disinfecting Cibotium barometz spores with a 10% sodium hypochlorite solution for 5-10 minutes; spore germination: the germination medium used includes 1 / 4 MS medium, 4.5-7 g·L⁻¹. ‑1 Agar and 0-30 g·L ‑1 Sucrose; Sporophyte differentiation: The sporophyte differentiation media used included 1 / 2 MS medium and 0-1 mg / L... ‑1 6-BA, 0.5-1 mg·L ‑1 2,4-D, 4.5-7 g·L ‑1 Agar and 25-30 g·L ‑1 Sucrose; Sporophyte strengthening: The sporophyte strengthening medium used included 1 / 4 MS medium, 4.5–7 g·L⁻¹. ‑1 Agar, 30 g·L ‑1 Sucrose and 1-2 g·L ‑1 Activated carbon; hardening and transplanting: After the tissue culture seedlings are sealed, the seal is gradually lifted, and they are then transferred to a specific mixed substrate for cultivation to obtain commercial seedlings. This method can improve the spore germination rate by using a specific sterilization procedure, and achieves the effect of producing more and better seedlings by using specific culture media and hardening preparations at each stage.

Description

Technical Field

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

[0002] *Cibotium barometz* (L.) J.Sm., commonly known as the golden-haired dog's spine, is a plant belonging to the genus *Cibotium* in the family Ceratophyllaceae. Traditional Chinese medicine considers *Cibotium barometz* to be bitter, sweet, and warm in nature, possessing the effects of dispelling wind and dampness, tonifying the liver and kidneys, and strengthening the lower back and knees. It is commonly used to treat lower back pain, wind-cold-dampness syndrome, and numbness in the hands and feet. The wild resources of *Cibotium barometz* are widely distributed, mainly in subtropical regions such as Yunnan, Guizhou, Guangdong, and Guangxi. Due to factors such as industrial development and ecological degradation, the wild reserves of *Cibotium barometz* are continuously decreasing, and it has been listed as a second-class protected plant by the state. To ensure the sustainable utilization of *Cibotium barometz* resources, artificial cultivation and propagation have become important means.

[0003] Golden-haired Cibotium barometz can be propagated through three methods: sexual reproduction, division, and tissue culture. Sexual reproduction is time-consuming and easily limited by season and climate. While division is highly efficient and produces consistent genetic traits, it has a low propagation coefficient and causes significant damage to wild resources. Tissue culture technology utilizes the regenerative capacity of cells to cultivate complete plants, featuring a short cycle, high yield, and controllability, providing high-quality materials to meet the needs of industrial development.

[0004] In the tissue culture methods of Cibotium barometz, there are many conflicting opinions regarding spore disinfection procedures and culture systems. Currently, 70% alcohol combined with 0.1% mercuric chloride or 5% sodium hypochlorite is commonly used for spore disinfection. However, some disinfection procedures are cumbersome, mercuric chloride is highly toxic, and the germination effect after disinfection is unclear. In addition, previous studies have mostly established rapid tissue propagation systems through steps such as sporophyte germination, prothallium proliferation, sporophyte induction, sporophyte proliferation, sporophyte rooting, and hardening-off. This process is time-consuming, requires changing multiple culture media, increases the complexity of the experiment, and results in fewer seedlings. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a method for the germination of Cibotium barometz spores and a method for the propagation of Cibotium barometz, by adopting a specific and simple disinfection procedure with the highest spore germination rate, and by optimizing the culture medium formula and seedling preparation at each stage to achieve the effect of producing more and better seedlings.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first objective of this invention is to provide a method for the germination of Cibotium barometz spores, comprising the following steps:

[0008] Disinfection: Disinfect Cibotium barometz spores with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores.

[0009] Spore germination: The suspension containing Cibotium barometz spores is inoculated into a germination medium and cultured to allow the Cibotium barometz spores to germinate, transform into immature prothallus, and then into mature prothallus; wherein, the germination medium comprises 1 / 4 MS medium, 4.5-7 g·L⁻¹ agar and 0-30 g·L⁻¹ sucrose.

[0010] The germination method of *Cibotium barometzii* spores of the present invention involves sterilizing the spores with a 10% sodium hypochlorite solution for 5-10 minutes during the sterilization step, and using a medium containing 1 / 4 MS at 4.5-7 g·L⁻¹ during the spore germination step. -1 Agar and 0-30 g·L -1 Cultivating sucrose in a germination medium significantly improves the germination rate of *Cibotium barometzii*, enabling effective germination. Furthermore, the 1 / 4 MS medium used in this germination medium contains suitable amounts and proportions of inorganic nutrients, sufficient to meet the nutritional and physiological needs of *Cibotium barometzii* germination. The lower inorganic salt concentration in the 1 / 4 MS medium is also beneficial for germination. Additionally, this germination medium facilitates the effective transformation of germinated *Cibotium barometzii* spores into juvenile and mature prothallia.

[0011] As a preferred embodiment of the present invention, the method for germinating Cibotium barometz spores includes a material screening step before the disinfection step: collecting leaves containing sporangia of Cibotium barometz plants; collecting spores after the sporangia dehisce; and screening them successively using 60-mesh and 150-mesh sieves to obtain pure Cibotium barometz spores. The Cibotium barometz spores are then stored at 4°C for later use. This method removes sporangia and impurities, yielding pure, golden-yellow Cibotium barometz spores, which are then used for subsequent steps such as disinfection.

[0012] As a preferred embodiment of the present invention, in the method for germinating Cibotium barometz spores, during the disinfection step, the Cibotium barometz spores are first soaked in sterile water overnight, and then disinfected with a 10% sodium hypochlorite solution. Soaking the Cibotium barometz spores in sterile water overnight allows them to absorb water, promotes metabolism, and increases the germination rate.

[0013] In a preferred embodiment of the present invention, the germination method for Cibotium barometz spores includes a disinfection time of 5-7 minutes during the disinfection step. By disinfecting the Cibotium barometz spores with a 10% sodium hypochlorite solution for 5-7 minutes, the germination rate can reach 100%, ensuring effective germination of the spores.

[0014] As a preferred embodiment of the present invention, in the germination method of Cibotium barometz spores of the present invention, the concentration of the suspension containing Cibotium barometz spores is 0.3-0.7 mg / mL, more preferably 0.4-0.6 mg / mL, and most preferably 0.5 mg / mL.

[0015] A second objective of this invention is to provide a method for propagating Cibotium barometz, comprising the disinfection and spore germination steps of any of the above-described methods for Cibotium barometz spore germination; and further comprising the following steps:

[0016] Sporophyte differentiation: Mature prothallus masses formed after spore germination are inoculated into sporophyte differentiation medium and cultured to transform the mature prothallus masses into sporophyte masses; wherein, the sporophyte differentiation medium comprises 1 / 2 MS medium and 0-1 mg·L⁻¹... -1 6-BA, 0.5-1 mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g·L -1 sucrose;

[0017] Sporophyte seedling strengthening: Sporophytes from the sporophyte cluster are inoculated into a sporophyte seedling strengthening medium for culture to strengthen the sporophytes and obtain tissue culture seedlings; wherein, the sporophyte seedling strengthening medium includes 1 / 4 MS medium, 4.5-7 g·L⁻¹ -1 Agar, 30 g·L -1 Sucrose, 1-2 g·L -1 Activated carbon;

[0018] Hardening and transplanting: After the tissue culture seedlings obtained from the sporophyte strengthening step are sealed, the seal is gradually lifted. Then, the tissue culture seedlings are transferred to a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1 for cultivation to obtain commercial seedlings.

[0019] The method for propagating Cibotium barometz of the present invention improves the germination rate of Cibotium barometz spores by disinfecting them with a 10% sodium hypochlorite solution for 5-10 minutes during the disinfection step, thus enabling effective germination. Furthermore, by specifically designing the formulations of the germination medium used in the spore germination step, the sporophyte differentiation medium used in the sporophyte differentiation step, and the sporophyte seedling strengthening medium used in the sporophyte seedling strengthening step, as well as by specifically designing the method and mixed substrate for the seedling hardening and transplanting step, the propagation coefficient of Cibotium barometz of the present invention can be high, achieving the effect of producing more seedlings.

[0020] In the method for propagating Cibotium barometz of the present invention, the spores of Cibotium barometz are disinfected with a 10% sodium hypochlorite solution for 5-10 minutes in the disinfection step, and in the spore germination step, a medium containing 1 / 4 MS and 4.5-7 g·L⁻¹ is used. -1 Agar and 0-30 g·L -1 Cultivation in a sucrose germination medium improved the germination rate of *Cibotium barometzii*, ensuring effective germination. Furthermore, the use of 1 / 4 MS medium in the germination medium provided an appropriate quantity and ratio of inorganic nutrients, sufficient to meet the nutritional and physiological needs of *Cibotium barometzii* germination. The lower inorganic salt concentration in 1 / 4 MS medium also favored germination. Simultaneously, this germination medium facilitated the effective transformation of germinating *Cibotium barometzii* spores into immature and mature prothallophyll, with a high proliferation coefficient from immature to mature prothallophyll. By using 1 / 2 MS medium containing 0-1 mg·L⁻¹ of sucrose in the sporophyte differentiation step… -1 6-BA, 0.5-1 mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g·L -1 Cultured in sucrose-based sporophyte differentiation medium, mature prothalliums can be induced to transform into sporophytes, promoting plant growth and root elongation in sporophytes, resulting in a high number of differentiated sporophytes and a high proliferation coefficient from mature prothalliums to sporophytes. Furthermore, the addition of 6-BA and 2,4-D (plant growth regulators) to the sporophyte differentiation medium stimulates sporophyte plant growth, enhancing the physiological functions of the stimulated parts. This was achieved by using 1 / 4 MS medium at 4.5-7 g / L... -1 Agar, 30 g·L -1 Sucrose and 1-2 g·L -1 Cultivating tissue-cultured seedlings in a sporophyte-strengthening medium with activated carbon can promote plant growth and root development. By first sealing the vigorous tissue-cultured seedlings during the hardening-off and transplanting process, and then gradually releasing the seal before transferring them to a mixed substrate with a peat moss:perlite:coconut coir ratio of 3:1:1, the survival rate of the tissue-cultured seedlings can be improved, enabling them to be converted into qualified commercial seedlings. Through the synergistic effect of the above steps, the propagation method of *Cibotium barometz* of this invention achieves the effect of producing more and better seedlings.

[0021] As a preferred embodiment of the present invention, in the method for propagating Cibotium barometz, the germination medium in the spore germination step comprises 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar and 30 g·L -1 Sucrose; in the sporophyte differentiation step, the sporophyte differentiation medium comprises 1 / 2 MS medium, 0.5 mg·L⁻¹ -16-BA, 1 mg·L -1 2,4-D, 7 g·L -1 Agar and 30 g·L -1 Sucrose; In the sporophyte strengthening step, the sporophyte strengthening culture medium comprises 1 / 4 MS medium, 7 g·L⁻¹ -1 Agar, 30 g·L -1 Sucrose, 1 g·L -1 Activated carbon. This invention employs a specific sterilization procedure in the sterilization step and a specific method including 1 / 4 MS medium and 7 g·L⁻¹ in the germination culture step. -1 Agar and 30 g·L -1 The germination medium for sucrose, used in sporophyte differentiation, includes specific media such as 1 / 2 MS medium and 0.5 mg·L⁻¹. -1 6-BA, 1 mg·L -1 2,4-D, 7 g·L -1 Agar and 30 g·L -1 The sucrose sporophyte differentiation medium, used in the sporophyte vigorization step, employs specific media including 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar, 30 g·L -1 Sucrose, 1 g·L -1 The activated carbon sporophyte culture medium, combined with specific treatment methods and a specific mixed substrate in the seedling hardening and transplanting steps, enables the propagation method of Cibotium barometz of the present invention to achieve the effect of producing more seedlings.

[0022] In a preferred embodiment of the present invention, in the spore germination step, the suspension containing *Cibotium barometz* spores is inoculated into the germination medium and cultured for 21-30 days to allow the *Cibotium barometz* spores to germinate; culturing continues for 60 days to allow the germinated *Cibotium barometz* spores to transform into immature prothalli; the immature prothalli are then transferred to a new germination medium and cultured for another 30 days to allow the immature prothalli to transform into mature prothalli; in the sporophyte differentiation step, the mature prothalli aggregates formed after spore germination are inoculated into the sporophyte differentiation culture and cultured for 6... The process begins with 0 days to transform mature prothallus clusters into sporophyte clusters. In the sporophyte strengthening step, sporophytes from the sporophyte clusters are inoculated into a sporophyte strengthening culture medium and cultured for 30-60 days to obtain tissue culture seedlings. In the hardening-off and transplanting step, the tissue culture seedlings obtained after the sporophyte strengthening step are sealed in tissue culture bottles for 3 days. From day 4 to 6, the degree of opening the bottle caps is gradually increased, and the seedlings are then transferred to a mixed substrate with a peat moss:perlite:coconut coir ratio of 3:1:1 for further cultivation to obtain commercial seedlings. This method for propagating Cibotium barometz produces a high number of seedlings.

[0023] In a preferred embodiment of the present invention, during the seedling hardening and transplanting step, the tissue culture seedlings obtained after the sporophyte strengthening step are sealed in tissue culture bottles under hardening conditions for 3 days. On the 4th day, the bottle cap is unscrewed 1 / 4; on the 5th day, the cap is unscrewed 1 / 2; on the 6th day, the cap is fully opened; and on the 7th day, the tissue culture seedlings are removed from the tissue culture bottles and transplanted into a mixed substrate with a mass ratio of peat moss:perlite:coconut coir = 3:1:1. By employing the above method to first seal the strengthened tissue culture seedlings and then gradually unseal them before transplanting them into a mixed substrate with a mass ratio of peat moss:perlite:coconut coir = 3:1:1, the survival rate of the tissue culture seedlings can be improved.

[0024] As a preferred embodiment of the present invention, in the seedling hardening and transplanting step, after the tissue culture seedlings are taken out of the tissue culture bottle, the culture medium at the roots of the tissue culture seedlings is rinsed clean, 40%-50% of the leaves above the base of the tissue culture seedlings are removed, and then the seedlings are transplanted into a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1.

[0025] As a preferred embodiment of the present invention, the method for propagating Cibotium barometz comprises the following steps: in the spore germination step, the spores are cultured under an ambient temperature of 25±3℃ and a daily light exposure of 16 hours under fluorescent lamps; in the sporophyte differentiation step, the spores are cultured under an ambient temperature of 25±3℃ and a daily light exposure of 16 hours under fluorescent lamps; in the sporophyte seedling strengthening step, the spores are cultured under an ambient temperature of 25±3℃ and a daily light exposure of 16 hours under fluorescent lamps; in the seedling hardening and transplanting step, the tissue culture seedlings are sealed under natural diffused light conditions; and after transplanting, the tissue culture seedlings are cultured under natural diffused light conditions. Attached Figure Description

[0026] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings.

[0027] Figure 1 This is a flowchart of the breeding method for Cibotium barometz according to the present invention;

[0028] Figure 2 The diagram shows the germination of Cibotium barometz spores in Examples 3, 1, and 2 (from left to right);

[0029] Figure 3 The diagram shows the germination of Cibotium barometz spores in Comparative Examples 1, 2, and 3 (from left to right).

[0030] Figure 4 This is a diagram showing the sporophyte differentiation in Example 4;

[0031] Figure 5 This is a diagram showing the vigorous growth of sporophytes in Example 4;

[0032] Figure 6This is a diagram showing the seedling hardening and transplanting process in Example 4. Detailed Implementation

[0033] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from the conventional market. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0034] 1. Materials

[0035] Select healthy, disease-free Cibotium barometz plants and harvest leaves with plump, unopened, brown sporangia on the underside. Spread the harvested leaves flat on newspaper and place them in a cool, well-ventilated area to allow the sporangia to open. Collect the spores scattered on the newspaper and sift them through a 60-mesh sieve and then a 150-mesh sieve to remove sporangium shells and impurities, leaving only pure, golden-yellow spore powder. Store the spore powder at 4°C for later use.

[0036] 2. Reagents and Instruments

[0037] Reagents: Sodium hypochlorite, agar, sucrose, anhydrous ethanol (Tianjin Damao Chemical Reagent Factory), 1 / 4 MS medium, 1 / 2 MS medium, potato broth (Haibo Biotechnology Co., Ltd.), 6-BA, 2,4-D, HCl, NaOH, activated carbon (Guangzhou Chemical Reagent Factory). Please refer to Tables 1 and 2 for the specific names and formulations of some reagents.

[0038] Table 1. Reagent English-Chinese Comparison Table

[0039]

[0040] Table 2 MS medium formulation

[0041]

[0042]

[0043] Note: In 1 / 2MS and 1 / 4MS media, the amounts of macroelements potassium nitrate, ammonium nitrate, potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride are halved in that order, while the others remain unchanged.

[0044] Instruments and equipment: Autoclave (Shanghai Boneng Instrument Co., Ltd., CT-90A), Clean bench (Suzhou Jingjing Group Suzhou Jingantai Air Technology Co., Ltd., VS-1300L-U), Microscope (Olympus Biosciences, CX43), Ultrapure water system (Xiamen Kexi Instrument Co., Ltd., UPF-40L-RO), pH meter (Shanghai Sanxin Instrument Factory, PHB-3), Electronic balance (Sartorius Group, Germany, BS110S), etc.

[0045] 3. Method of the present invention

[0046] 3.1 Germination method of Cibotium barometz spores according to the present invention

[0047] This invention provides a method for the germination of Cibotium barometz spores, comprising the following steps:

[0048] Disinfection: Disinfect Cibotium barometz spores with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores.

[0049] Spore germination: The suspension containing Cibotium barometz spores was inoculated into a germination medium and cultured to allow the Cibotium barometz spores to germinate, transform into immature prothallus, and then into mature prothallus; wherein, the germination medium comprises 1 / 4 MS medium, 4.5-7 g·L⁻¹ -1 Agar and 0-30 g·L -1 sucrose.

[0050] Example 1

[0051] This embodiment provides a method for the germination of Cibotium barometz spores, including the following steps:

[0052] (1) Disinfection: Disinfect the Cibotium barometz spores with 10% sodium hypochlorite solution for 5 min; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores.

[0053] Specifically: Weigh 10 mg of Cibotium barometz spores and place them in a 2 mL centrifuge tube. Add 1 mL of sterile water and soak overnight. Aspirate the supernatant and add 1.5 mL of 10% sodium hypochlorite solution for sterilization for 5 minutes. During the 5-minute sterilization, shake the centrifuge tube occasionally to ensure even contact between the Cibotium barometz spores and the sodium hypochlorite solution. Finally, allow the centrifuge tube to stand naturally for 1.5-2 minutes before the 5-minute sterilization time to allow the Cibotium barometz spores to precipitate naturally. After sterilization (i.e., after the Cibotium barometz spores have precipitated), aspirate the sterilization solution and wash the tube 4 times with sterile water to obtain the Cibotium barometz spore precipitate. Add 1 mL of sterile water to the Cibotium barometz spore precipitate and transfer it to a tissue culture flask. Continue to add 19 mL of sterile water to dilute it to a concentration of 0.5 mg / mL of Cibotium barometz spore suspension for later use.

[0054] (2) Spore germination: The suspension containing Cibotium barometz spores was inoculated into a tissue culture flask containing germination medium to induce germination of Cibotium barometz spores; wherein, the germination medium included 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar and 30 g·L -1 sucrose.

[0055] Specifically: Prepare germination medium (1 / 4 MS medium + 7 g·L⁻¹). -1 Agar + 30g·L -1 Sucrose (pH 5.8) was used to prepare the germination medium, which was then transferred to tissue culture flasks and sterilized at 121°C for 20 minutes. The flasks were then cooled to solidify. 0.5 mL of a 0.5 mg / mL suspension of Cibotium barometz spores (prepared as described above) was pipette-added to the germination medium in each flask (approximately 0.25 mg of spores per flask). The mixture was shaken well to ensure even distribution of the spores. Eight flasks were inoculated, and the process was repeated three times. The flasks were then placed in a culture room and cultured under fluorescent light at 25±3°C with 16 hours of light per day for 30 days to allow spore germination. (Growth was observed and recorded daily after inoculation. After 30 days, the contamination rate, germination rate, and earliest germination time were recorded.)

[0056] After culturing for 30 days, continue culturing for another 60 days to allow the germinating spores to transform into young prothallus.

[0057] Then, the young prothallus in each culture bottle was divided into 8 bottles, and 5 clusters of young prothallus with a diameter of 1 cm were inoculated in each bottle. This was repeated 3 times. At this time, the weight of prospores contained in each cluster of young prothallus was 5-6.25 μg. After culturing for another 30 days, the diameter of the prothallus was about 2.5 cm, that is, the young prothallus was transformed into a mature prothallus.

[0058] Example 2

[0059] This embodiment provides a method for the germination of Cibotium barometz spores, which is basically the same as that in Embodiment 1, except that the disinfection time in this embodiment is 7 minutes.

[0060] Example 3

[0061] This embodiment provides a method for the germination of Cibotium barometz spores, which is basically the same as that in Embodiment 1, except that the disinfection time in this embodiment is 10 minutes.

[0062] Comparative Example 1

[0063] This comparative example provides a method for the germination of Cibotium barometz spores, which is basically the same as that in Example 1, except that this comparative example uses 4% sodium hypochlorite solution for disinfection for 3 minutes.

[0064] Comparative Example 2

[0065] This comparative example provides a method for the germination of Cibotium barometz spores, which is basically the same as that in Example 1, except that this comparative example uses 4% sodium hypochlorite solution for disinfection for 5 minutes.

[0066] Comparative Example 3

[0067] This comparative example provides a method for the germination of Cibotium barometz spores, which is basically the same as that in Example 1, except that this comparative example uses 4% sodium hypochlorite solution for disinfection for 7 minutes.

[0068] 3.2 Breeding method of Cibotium barometz according to the present invention

[0069] This invention provides a method for breeding Golden Retrievers. Please refer to [link / reference]. Figure 1 This includes the following steps:

[0070] Disinfection: Disinfect Cibotium barometz spores with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores.

[0071] Spore germination: The suspension containing Cibotium barometz spores was inoculated into a germination medium and cultured to allow the Cibotium barometz spores to germinate, transform into immature prothallus, and then into mature prothallus; wherein, the germination medium comprises 1 / 4 MS medium, 4.5-7 g·L⁻¹ -1 Agar and 0-30 g·L -1 sucrose;

[0072] Sporophyte differentiation: Mature prothallus masses formed after spore germination are inoculated into sporophyte differentiation medium and cultured to transform the mature prothallus masses into sporophyte masses; wherein, the sporophyte differentiation medium comprises 1 / 2 MS medium and 0-1 mg·L⁻¹... -1 6-BA, 0.5-1 mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g·L -1 sucrose;

[0073] Sporophyte seedling strengthening: Sporophytes from the sporophyte cluster are inoculated into a sporophyte seedling strengthening medium for culture to strengthen the sporophytes and obtain tissue culture seedlings; wherein, the sporophyte seedling strengthening medium includes 1 / 4 MS medium, 4.5-7 g·L⁻¹ -1 Agar, 30 g·L -1 Sucrose, 1-2 g·L -1 Activated carbon;

[0074] Hardening and transplanting: After the sporophyte seedling strengthening step, the tissue culture seedlings are sealed and then gradually unsealed. The tissue culture seedlings are then transferred to a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1 for cultivation to obtain commercial seedlings.

[0075] Example 4

[0076] This embodiment provides a breeding method for Golden Retrievers. Please refer to [link / reference needed]. Figure 1 The process includes the following steps (wherein the disinfection and germination steps are the same as in Example 1):

[0077] (1) Disinfection: Disinfect the Cibotium barometz spores with 10% sodium hypochlorite solution for 5 min; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores.

[0078] Specifically: Weigh 10 mg of Cibotium barometz spores and place them in a 2 mL centrifuge tube. Add 1 mL of sterile water and soak overnight. Aspirate the supernatant and add 1.5 mL of 10% sodium hypochlorite solution for sterilization for 5 minutes. During the 5-minute sterilization, shake the centrifuge tube occasionally to ensure even contact between the Cibotium barometz spores and the sodium hypochlorite solution. Finally, allow the centrifuge tube to stand naturally for 1.5-2 minutes before the 5-minute sterilization time to allow the Cibotium barometz spores to precipitate naturally. After sterilization (i.e., after the Cibotium barometz spores have precipitated), aspirate the sterilization solution and wash the tube 4 times with sterile water to obtain the Cibotium barometz spore precipitate. Add 1 mL of sterile water to the Cibotium barometz spore precipitate and transfer it to a tissue culture flask. Continue to add 19 mL of sterile water to dilute it to a concentration of 0.5 mg / mL of Cibotium barometz spore suspension for later use.

[0079] (2) Spore germination: The suspension containing Cibotium barometz spores was inoculated into a tissue culture flask containing germination medium and cultured to allow the Cibotium barometz spores to germinate, transform into immature prothallus, and then into mature prothallus; wherein, the germination medium includes 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar and 30 g·L -1 sucrose.

[0080] Specifically: Prepare germination medium (1 / 4 MS medium + 7 g·L⁻¹). -1 Agar + 30g·L -1 Sucrose (pH 5.8) was used to prepare the germination medium, which was then transferred to tissue culture flasks and sterilized at 121°C for 20 minutes. The flasks were then cooled to solidify. 0.5 mL of a 0.5 mg / mL suspension of Cibotium barometz spores (prepared as described above) was pipetted into the germination medium in each flask (approximately 0.25 mg of spores per flask). The mixture was shaken well to ensure even distribution of the spores. Eight flasks were inoculated, and the process was repeated three times. The flasks were then placed in a culture room and cultured under fluorescent light at 25±3°C with 16 hours of light per day for 30 days to allow spore germination.

[0081] After culturing for 30 days, continue culturing for another 60 days to allow the germinating spores to transform into young prothallus.

[0082] Then, the young prothallus in each culture bottle was divided into 8 bottles, and 5 clusters of young prothallus with a diameter of 1 cm were inoculated in each bottle. This was repeated 3 times. At this time, the weight of prospores contained in each cluster of young prothallus was 5-6.25 μg. After culturing for another 30 days, the diameter of the prothallus was about 2.5 cm, that is, the young prothallus was transformed into a mature prothallus.

[0083] (3) Sporophyte differentiation: The mature prothallus formed after spore germination is inoculated into a tissue culture flask containing sporophyte differentiation medium and cultured to convert the mature prothallus into a sporophyte; wherein, the sporophyte differentiation medium includes 1 / 2 MS medium and 0.5 mg·L⁻¹ medium. -1 6-BA, 1 mg·L -1 2,4-D, 7 g·L -1 Agar, 30 g·L -1 sucrose.

[0084] Specifically: Prepare sporophyte differentiation medium (1 / 2 MS medium + 0.5 mg·L⁻¹). -1 6-BA + 1 mg·L -1 2,4-D + 7 g·L -1 Agar + 30g·L -1 Sucrose (pH 5.8) was used to prepare the sporophyte differentiation medium. The medium was then placed into tissue culture bottles and sterilized at 121°C for 20 minutes. After sterilization, the medium was cooled and allowed to solidify. The mature prothallium masses formed in step (2) were cut into prothallium masses with a diameter of about 1 cm (each mass of prothallium contained about 1-1.25 μg of protospores) and inoculated into the sporophyte differentiation medium in the tissue culture bottles. Five prothallium masses were inoculated into each bottle, and eight bottles were inoculated each time. The inoculation was repeated three times, with each bottle constituting one replicate. The tissue culture bottles were placed in a culture room and cultured for 60 days under the same conditions as for spore germination.

[0085] (4) Sporophyte Enrichment: The sporophytes of the sporophyte clusters after the sporophyte differentiation step are inoculated into tissue culture flasks containing sporophyte enrichment medium for cultivation, thereby enriching the sporophytes and obtaining tissue culture seedlings; the sporophyte enrichment medium includes 1 / 4 MS medium and 7 g·L⁻¹ medium. -1 Agar, 30 g·L -1 Sucrose, 1 g·L -1 Activated carbon.

[0086] Specifically: Prepare a sporophyte seedling strengthening medium (1 / 4 MS medium + 7 g·L⁻¹). -1 Agar + 30g·L -1 Sucrose + 1g·L -1Activated carbon (pH 5.8) was used to prepare the sporophyte culture medium, which was then placed in tissue culture bottles and sterilized at 121°C for 20 minutes. The culture was then cooled to solidify and set aside. Individual sporophytes were separated from the sporophyte clusters, and the prothallus at the base was removed before inoculation onto the sporophyte culture medium in tissue culture bottles. The culture was then inoculated for 30 days. Eight bottles were inoculated at a time, with each bottle constituting one replicate, for a total of three replicates. Each bottle contained five sporophytes.

[0087] (5) Hardening and transplanting: After the sporophyte seedling strengthening step, the tissue culture seedlings obtained are sealed in the tissue culture bottle for 3 days. On the 4th to 6th day, the degree of opening of the bottle cap is gradually increased. The tissue culture seedlings are then transferred from the tissue culture bottle to a mixed substrate of peat moss: perlite: coconut coir = 3:1:1 for cultivation to obtain commercial seedlings.

[0088] Specifically: Tissue culture seedlings were used as transplanting material. The seedlings were sealed in their bottles for 3 days. On the 4th day, the bottle cap was loosened by 1 / 4 and the seedlings were sprayed with tap water. On the 5th day, the bottle cap was opened by 1 / 2 and the seedlings were sprayed with tap water. On the 7th day, the bottle cap was fully opened and the seedlings were sprayed with tap water. On the 7th day, the seedlings were removed from the bottles with tweezers, and the culture medium was washed off the roots under running water. The cleaned seedlings were temporarily placed in a water basin, and 40%-50% of the leaves above the base were removed. They were then transplanted one by one into a seedling tray containing a mixed substrate (charcoal:perlite:coconut coir = 3:1:1 by weight). The trays were thoroughly watered with tap water and then the lid was closed to maintain humidity. The seedlings were then placed under natural diffused light for 30 days to obtain marketable seedlings.

[0089] Example 5

[0090] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that in embodiment 4, except that the disinfection time in this embodiment is 7 minutes.

[0091] Example 6

[0092] This embodiment provides a breeding method for Cibotium barometz, which is basically the same as that in embodiment 4, except that the disinfection time in this embodiment is 10 minutes.

[0093] Example 7

[0094] This embodiment provides a method for breeding Cibotium barometz, which is basically the same as that in Example 4, except that the germination medium in this embodiment includes 1 / 4 MS medium and 4.5 g·L⁻¹. -1 Agar and 0 g·L -1 Sucrose (i.e., sucrose-free).

[0095] Example 8

[0096] This embodiment provides a method for breeding Cibotium barometz, which is basically the same as that in Example 4, except that the germination medium in this embodiment includes 1 / 4 MS medium and 4.5 g·L⁻¹.-1 Agar and 25 g·L -1 sucrose.

[0097] Example 9

[0098] This embodiment provides a method for propagating Cibotium barometz, which is basically the same as that in Example 4, except that the sporophyte differentiation medium in this embodiment includes 1 / 2 MS medium and 1 mg·L⁻¹. -1 6-BA, 0.5 mg·L -1 2,4-D, 4.5 g·L -1 Agar and 30 g·L -1 sucrose.

[0099] Example 10

[0100] This embodiment provides a method for breeding Cibotium barometz, which is basically the same as that in Example 4, except that the sporophyte differentiation medium in this embodiment includes 1 / 2 MS medium and 0 mg·L⁻¹. -1 6-BA (i.e., without 6-BA), 1 mg·L -1 2,4-D, 7 g·L -1 Agar and 25 g·L -1 sucrose.

[0101] Example 11

[0102] This embodiment provides a method for propagating Cibotium barometz, which is basically the same as that in Example 4, except that the sporophyte seedling culture medium in this embodiment includes 1 / 4 MS medium and 4.5 g·L⁻¹. -1 Agar, 30 g·L -1 Sucrose, 2g·L -1 Activated carbon.

[0103] Example 12

[0104] This embodiment provides a method for breeding Cibotium barometz, which is basically the same as that in embodiment 4, except that in the sporophyte seedling strengthening step, the sporophyte is cultured in the sporophyte seedling strengthening culture medium for 60 days to obtain tissue culture seedlings.

[0105] 4 Results

[0106] 4.1 Germination results of Cibotium barometz spores

[0107] The contamination rate, germination rate, and earliest germination time of Examples 1-3 and Comparative Examples 1-3 were statistically analyzed respectively. The results are shown in Table 3.

[0108] Any abnormal tissue culture phenomena observed in the culture medium are considered contamination.

[0109] Germination rate: Number of germination bottles / Number of inoculated bottles × 100%;

[0110] Earliest germination time: Germination is determined by the appearance of small green dots on the culture medium when observed with the naked eye.

[0111] Table 3 Germination results of Cibotium barometz spores

[0112] Disinfection methods Pollution rate (%) Germination rate (%) Earliest germination time (d) Example 1 10% sodium hypochlorite for 5 minutes 0 100 21 Example 2 10% sodium hypochlorite for 7 minutes 0 100 23 Example 3 10% sodium hypochlorite for 10 minutes 0 86.66 26 Comparative Example 1 4% sodium hypochlorite for 3 minutes 0 72.22 29 Comparative Example 2 4% sodium hypochlorite for 5 minutes 0 83.33 29 Comparative Example 3 4% sodium hypochlorite for 7 minutes 0 94.44 28

[0113] As shown in Table 3, the present invention, by disinfecting the *Cibotium barometz* spores with 10% sodium hypochlorite for 5-10 minutes, can significantly improve the germination rate of *Cibotium barometz* spores, enabling effective germination and earlier germination time. Preferably, referring to Examples 1 and 2, the present invention, by disinfecting the *Cibotium barometz* spores with 10% sodium hypochlorite for 5-7 minutes, can achieve a 100% germination rate, and the earliest germination time is significantly earlier than that of Comparative Examples 1-3.

[0114] 4.2 Breeding results of Golden Retrievers

[0115] Germination rate, earliest germination time, prothallium proliferation coefficient, differentiation and reproduction coefficient, total proliferation coefficient, and variance of total proliferation coefficient for Examples 4-12 were statistically analyzed. The results are shown in Table 4.

[0116] Germination rate: Number of germination bottles / Number of inoculated bottles × 100%;

[0117] Earliest germination time: Germination is determined by the appearance of small green dots on the culture medium as observed by the naked eye;

[0118] Prothallus proliferation coefficient: refers to the proliferation coefficient of the spore germination process from immature prothallus to mature prothallus;

[0119] Differentiation and reproduction coefficient: refers to the proliferation coefficient of sporophyte differentiation from mature prothallus to sporophyte in the sporophyte differentiation process;

[0120] Total proliferation coefficient: refers to the product of the prothallus proliferation coefficient and the differentiation and reproduction coefficient;

[0121] Tissue culture seedling survival rate: refers to the number of surviving tissue culture seedlings / the total number of transplanted tissue culture seedlings during the hardening-off and transplanting process.

[0122] Table 4 Results of proliferation coefficient

[0123]

[0124] In the propagation methods of Cibotium barometz in Examples 4-12 of this invention, the contamination rate of Cibotium barometz spores was 0% in both the disinfection and spore germination steps. The germination rate of Cibotium barometz spores in Examples 4-5 and 7-12 was 100%, while the germination rate of Cibotium barometz spores in Example 6 was 86.66%, which, although lower than that in Examples 4-5 and 7-12, still showed a high germination effect. In the propagation methods of Cibotium barometz in Examples 4-12, green germination material could be observed with the naked eye in the disinfection and spore germination steps during the 21st-26th day of cultivation. Furthermore, during the process of culturing the germinating Cibotium barometz spores into young prothalliums and then into mature prothalliums, the prothallium proliferation coefficient from young prothalliums to mature prothalliums was 5.13-5.73. In the sporophyte differentiation step, during the 60-day culture on the differentiation medium to allow mature prothalliums to differentiate into sporophytes, the differentiation and proliferation coefficient of mature prothalliums into sporophytes is 7.13-8.00. During this process, the sporophyte plants grow taller and have longer roots. In the sporophyte seedling strengthening step, the tissue culture seedlings grow taller and have more roots; the proliferation coefficient for the entire culture cycle is 39.43-44.03. In the seedling hardening and transplanting step, by first sealing the tissue culture seedlings and then gradually unscrewing the cap before transferring them to a mixed substrate with a mass ratio of peat moss:perlite:coconut coir = 3:1:1, the survival rate of the tissue culture seedlings can reach 92%, which is significantly higher than the survival rate of tissue culture seedlings transplanted without sealing treatment (83%) and the survival rate of tissue culture seedlings transplanted immediately after sealing without gradually unscrewing the cap (83%). Therefore, the method for propagating Cibotium barometz of this invention produces a high number of seedlings.

[0125] Considering all performance parameters, the propagation method of *Cibotium barometz* in Example 4 yielded the best results and the highest number of seedlings. The propagation method of *Cibotium barometz* in Example 4 involved disinfecting the spores with a 10% sodium hypochlorite solution for 5 minutes during the disinfection step, and using a mixture of 1 / 4 MS medium and 7 g / L... -1 Agar and 30 g·L -1 Cultivation in a sucrose-based germination medium resulted in a 100% germination rate for Cibotium barometz spores, with germination beginning as early as day 21 of cultivation. Please refer to [link / reference needed]. Figure 2 The germination effect of Example 4 (i.e., the corresponding Figure 2 Example 1) is good. Germinating *Cibotium barometz* spores were further cultured in the above germination medium to transform into immature prothallophytes, which then transformed into mature prothallophytes. Example 4 showed the highest prothallophyte proliferation coefficient from immature to mature prothallophytes. The *Cibotium barometz* propagation method in Example 4 used a mixture of 1 / 2 MS medium and 0.5 mg·L⁻¹... -1 6-BA, 1 mg·L -1 2,4-D, 7 g·L -1Agar and 30 g·L -1 The plants were cultured on a sucrose-based sporophyte differentiation medium to convert mature prothallus masses into sporophyte masses. Example 1 showed the highest differentiation and proliferation coefficient from mature prothallus to sporophyte. Furthermore, this process also promoted the growth of sporophyte plants and the elongation of roots. For details on sporophyte differentiation, please refer to [link to relevant documentation]. Figure 4 Example 4 describes the propagation method of Cibotium barometz, which uses a mixture of 1 / 4 MS medium and 7 g·L⁻¹ in the sporophyte seedling strengthening step. -1 Agar, 30 g·L -1 Sucrose, 1 g·L -1 Cultivating tissue culture seedlings in a sporophyte-enhancing medium using activated carbon can increase the height and root number of the seedlings. For details on sporophyte enhancement, please refer to [link to relevant documentation]. Figure 5 Example 4 describes a method for propagating Cibotium barometz. In the hardening-off and transplanting step, the tissue culture seedlings are first sealed, then the bottle caps are gradually opened before being transferred to a mixed substrate with a peat moss:perlite:coconut coir ratio of 3:1:1. This method achieves a 92% survival rate for the tissue culture seedlings. For details on hardening-off and transplanting, please refer to [link to relevant documentation]. Figure 6 Therefore, the propagation method of Cibotium barometz in Example 4 produced the most seedlings. This method achieved the effect of producing more and better seedlings by using a sterilization procedure with the highest spore germination rate and by optimizing the culture medium formula and hardening-off configuration at each stage.

[0126] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and the present invention also intends to include these modifications and variations.

Claims

1. A method for breeding Golden Retrievers, characterized in that, Includes the following steps: Disinfection: Disinfect Cibotium barometz spores with 10% sodium hypochlorite solution for 5-10 minutes; collect the disinfected Cibotium barometz spores and add sterile water to prepare a suspension containing Cibotium barometz spores; Spore germination: The suspension containing Cibotium barometz spores was inoculated into a germination medium and cultured to allow the Cibotium barometz spores to germinate, transform into immature prothallus, and then into mature prothallus; wherein, the germination medium consisted of 1 / 4 MS medium and 4.5-7 g·L⁻¹. -1 Agar and 0-30 g·L -1 Composition of sucrose; Sporophyte differentiation: Mature prothallus masses formed after spore germination are inoculated into sporophyte differentiation medium and cultured to transform the mature prothallus masses into sporophyte masses; wherein, the sporophyte differentiation medium consists of 1 / 2 MS medium and 0-1 mg·L⁻¹... -1 6-BA, 0.5-1 mg·L -1 2,4-D, 4.5-7 g·L -1 Agar and 25-30 g·L -1 Composition of sucrose; Sporophyte strengthening: Sporophytes from the sporophyte cluster are inoculated into a sporophyte strengthening medium for cultivation to obtain tissue culture seedlings; wherein, the sporophyte strengthening medium consists of 1 / 4 MS medium and 4.5-7 g·L⁻¹. -1 Agar, 30 g·L -1 Sucrose and 1-2 g·L -1 Composition of activated carbon; Hardening and transplanting: After the sporophyte seedling strengthening step, the tissue culture seedlings obtained are sealed and then gradually unsealed. The tissue culture seedlings are then transferred to a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1 for cultivation.

2. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: Before the disinfection step, there is also a material screening step: collect the leaves of the sporangia of Cibotium barometz, collect the spores after the sporangia split open, and screen them with 60-mesh and 150-mesh sieves to obtain pure Cibotium barometz spores.

3. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: In the disinfection process, the Cibotium barometz spores were first soaked in sterile water overnight, and then disinfected with a 10% sodium hypochlorite solution.

4. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: The disinfection process takes 5-7 minutes.

5. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium barometz spores is 0.3-0.7 mg / mL.

6. The breeding method of Cibotium barometz according to claim 5, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium barometz is 0.4-0.6 mg / mL.

7. The breeding method of the Golden Retriever's spine according to claim 6, characterized in that: In the disinfection step, the concentration of the suspension containing Cibotium barometz spores is 0.5 mg / mL.

8. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: In the spore germination step, the germination medium consists of 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar and 30 g·L -1 Composition of sucrose; In the sporophyte differentiation step, the sporophyte differentiation medium consists of 1 / 2 MS medium and 0.5 mg·L⁻¹. -1 6-BA, 1 mg·L -1 2,4-D, 7 g·L -1 Agar and 30 g·L -1 Composition of sucrose; In the sporophyte strengthening step, the sporophyte strengthening culture medium consists of 1 / 4 MS medium and 7 g·L⁻¹. -1 Agar, 30 g·L -1 Sucrose and 1 g·L -1 Composition of activated carbon.

9. The breeding method of the Golden Retriever's spine according to claim 1, characterized in that: In the spore germination step, the suspension containing Cibotium barometz spores is inoculated into the germination medium and cultured for 21-30 days to allow the Cibotium barometz spores to germinate; culture is continued for 60 days to allow the germinated Cibotium barometz spores to transform into immature prothallus; the immature prothallus is transferred to a new germination medium and cultured for another 30 days to allow the immature prothallus to transform into mature prothallus; In the sporophyte differentiation step, the mature prothallus formed after spore germination is inoculated into the sporophyte differentiation culture and cultured for 60 days to transform the mature prothallus into a sporophyte. In the sporophyte seedling strengthening step, the sporophytes in the sporophyte cluster are inoculated into the seedling strengthening culture medium and cultured for 30-60 days to strengthen the sporophytes and obtain tissue culture seedlings; In the seedling hardening and transplanting step, the tissue culture seedlings obtained after the sporophyte strengthening step are sealed in tissue culture bottles for 3 days. On the 4th to 6th day, the degree of opening of the bottle cap is gradually increased. Then, the tissue culture seedlings are transferred to a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1 for cultivation.

10. The breeding method of the Golden Retriever's spine according to claim 9, characterized in that: In the hardening and transplanting step, the tissue culture seedlings obtained after the sporophyte strengthening step are sealed in tissue culture bottles under hardening conditions for 3 days. On the 4th day, the bottle cap is unscrewed 1 / 4, on the 5th day, the bottle cap is unscrewed 1 / 2, on the 6th day, the bottle cap is fully opened, and on the 7th day, the tissue culture seedlings are taken out of the tissue culture bottle and transplanted into a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:1 for cultivation.

11. The breeding method of the Golden Retriever's spine according to claim 10, characterized in that: In the seedling hardening and transplanting step, after the tissue culture seedlings are taken out of the tissue culture bottle, the culture medium at the roots of the tissue culture seedlings is rinsed clean. After removing 40%-50% of the leaves above the base of the tissue culture seedlings, they are transplanted into a mixed substrate with a mass ratio of peat moss: perlite: coconut coir = 3:1:

1.

12. The breeding method of the Golden Retriever's spine according to claim 10, characterized in that: During the spore germination, sporophyte differentiation, and sporophyte seedling strengthening stages, the culture was carried out at an ambient temperature of 25±3℃ and under fluorescent lamp illumination for 16 hours per day. In the seedling hardening and transplanting stage, the hardening conditions were natural diffused light, and the seedlings were then cultured under natural diffused light after transplanting.

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