A method for the propagation of golden spores and the rejuvenation of seedlings

Abstract

This invention provides a method for the artificial cultivation of Cibotium barometz spores and the rejuvenation of young spore seedlings. The method for artificially cultivating Cibotium barometz includes the following steps: transplanting the young Cibotium barometz sporophytes to be rejuvenated into a substrate for rejuvenation culture; wherein the substrate is a mixture of leaf mold and peat moss in a volume ratio of (1-1.5):1 or 1:1, or a mixture of leaf mold, vermiculite, and garden soil in a volume ratio of 4:1:3. This method is low-cost, technically simple, and yields high output. The seedlings grow rapidly, making it suitable for large-scale mass production. It effectively meets the needs for rapid propagation of Cibotium barometz and can be widely applied in landscaping, medicinal production, and scientific research.

Description

Technical Field

[0001] This invention relates to the field of forest tree seedling technology, specifically to a method for the propagation of golden spores and the rejuvenation of seedling larvae. Background Technology

[0002] *Cibotium barometz* (Linn.) J.Sm., belonging to the family Cibotiaceae and the genus *Cibotium*, is a perennial, tall, tree-like fern used for medicinal purposes. It is mainly distributed in tropical and subtropical Asia, particularly in southern my country. The fern has a thick, prostrate rhizome, with a cluster of large leaves emerging from the top. The petioles are brownish-red, and the base is covered with a cushion of golden-yellow hairs. It prefers diffused light and often grows in shady, warm, and moist forest environments. It is an internationally renowned ornamental plant and an important traditional Chinese medicine. *Cibotium barometz* is famous for its unique appearance and medicinal value. Its upright form, large, spreading leaves, and beautiful appearance give it high ornamental value, making it important in horticulture. Its rhizome (known as "Gouji" in Chinese medicine) is listed in the Chinese Pharmacopoeia (2020 edition) and has the effects of dispelling wind and dampness, tonifying the liver and kidneys, and strengthening the waist and knees. It is often used to treat rheumatic pain, soreness and weakness of the waist and knees, and weakness of the lower limbs. The scales on the rhizome can also be used externally to stop bleeding. In addition, in recent years, various active ingredients (such as flavonoids and polysaccharides) have been extracted from the golden retriever using modern technology, providing new directions for pharmaceutical technology development, and its health and nutritional value is gradually gaining attention.

[0003] Despite its immense ornamental, medicinal, and health-promoting value, the wild population of *Fernonia simonii* is declining year by year due to unregulated exploitation. In recent years, over-harvesting of wild *Fernonia simonii* resources, neglecting its growth patterns and distribution characteristics, has led to a sharp decline in its numbers. Currently, *Fernonia simonii* is listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (Appendix II, CITES 2016) and is also listed as a Class II protected plant in the National Key Protected Wild Plants List (First Batch). Its endangered status is mainly attributed to difficulties in propagation, slow growth, and the extensive traditional cultivation methods lacking specialized management techniques for ferns. Current cultivation methods are unsatisfactory in terms of seedling survival rate, seedling growth rate, and plant quality, leading to a heavy reliance on wild resources for the development of *Fernonia simonii*, further exacerbating the resource scarcity problem. With the increasing demand for *Fernonia simonii* resources in horticulture, medicinal use, and scientific research, protecting wild *Fernonia simonii* resources and developing effective propagation methods has become an urgent priority.

[0004] The propagation of Cibotium barometz mainly includes three methods: sexual reproduction (spore propagation), asexual reproduction (division propagation), and in vitro propagation (tissue culture). Among these, spore propagation often suffers from problems such as low germination rate, uneven emergence, slow seedling growth, and a high proportion of weak seedlings, resulting in long seedling cycles, low survival rates, poor seedling adaptability, and even large-scale seedling death. These problems seriously restrict the promotion and application of large-scale artificial propagation of Cibotium barometz and have become a key technical bottleneck that urgently needs to be solved. Summary of the Invention

[0005] To address the shortage of Golden Retriever dog resources, the inventors of this invention, through extensive experimentation, have developed a method for the artificial cultivation and propagation of Golden Retriever dogs. This method boasts a high survival rate and short production time. This invention addresses seedling problems such as low spore germination rate, uneven emergence, and yellowing of gametophytes and sporophytes, offering a solution characterized by uniform, rapid, and robust seedling emergence.

[0006] The method for artificially cultivating Cibotium barometz provided by the present invention includes the following steps: transplanting the Cibotium barometz sporophyte seedlings to be rejuvenated into a substrate for rejuvenation culture; wherein the substrate is a mixture of leaf mold and peat moss in a volume ratio of (1-1.5):1 or 1:1, or a mixture of leaf mold, vermiculite and garden soil in a volume ratio of 4:1:3.

[0007] Leaf mold, also known as humus, is a nutrient-rich soil formed from the decomposition and fermentation of plant leaves and branches by microorganisms in the soil. As a specific example, leaf mold contains 45.7% organic matter and has a bulk density of 0.36 g / cm³. 3 The pH (water immersion) is 6.0, and the soil is black with organic matter particles.

[0008] Peat soil refers to soils found in certain river and lake sedimentary plains and mountain valleys. Due to long-term water accumulation and dense aquatic vegetation, under anaerobic conditions, a large amount of incompletely decomposed plant remains accumulate and form a peat layer. As a specific example, peat soil has a combined organic matter and humic acid content of 50% or more, a porosity of 70-80, and a pH (water immersion) of 5.6-6.5.

[0009] Preferably, in the above method, when the first true leaf of the sporophyte seedling grows to 0.3-0.5 cm, it is transplanted and rejuvenated.

[0010] Preferably, the conditions for the rejuvenation culture include: a temperature of 23–25°C, a light intensity of 2500–3500 Lux, a light duration of 12–14 hours or 14 hours per day, and a relative humidity of 65%–85% in the space where the sporophyte seedlings are located. The space where the sporophyte seedlings are located is the space formed by transplanting the sporophyte seedlings into seedling containers and covering the seedling containers with a transparent lid.

[0011] Preferably, the water content of the substrate in the rejuvenation culture is 60% to 65% or 60%.

[0012] Preferably, the rejuvenation cultivation also includes the step of applying nutrient solution; specifically, the nutrient solution is a water-soluble fertilizer containing macro-elements; more specifically, the nutrient solution is applied starting on the 90th day after transplanting, the water-soluble fertilizer containing macro-elements is diluted to a concentration of 0.2% (mass percentage concentration) aqueous solution, and sprayed once every 7 days, with 200ml sprayed per 1760 square centimeters each time.

[0013] Among them, water-soluble fertilizers containing macronutrients are a commonly used type of fertilizer for plant growth. They are mainly composed of three essential macronutrients for plants: nitrogen (N), phosphorus (P), and potassium (K). These elements play a crucial role in plant growth and development, and because they are completely soluble in water and easily absorbed by plants, they are widely used in agricultural production, such as drip irrigation, spraying, and foliar fertilization. The formula we use is a balanced type, containing 20% ​​nitrogen (N), 20% phosphorus (P2O5), and 20% potassium (K2O) (model: 20-20-20+TE, standard NY / T 1107-2020).

[0014] Preferably, the method further includes a seedling hardening step, in which the sporophyte seedlings to be hardened are planted separately, and the substrate is a mixture of leaf mold and peat moss in a volume ratio of (1-1.5):1 or 1:1.

[0015] Preferably, the sporophyte seedlings to be hardened are sporophyte seedlings with a height of 8.5-10.0 cm or sporophyte seedlings with 5 to 8 leaves per plant.

[0016] Preferably, the conditions for hardening off the seedlings include: a temperature of 25-30℃, an air humidity of 60%-65% in the greenhouse, a light intensity of 3000-3700 lux, and a light duration of 12-14 hours.

[0017] Preferably, the method includes the following steps: sowing Cibotium barometz spores in a substrate, subjecting them to dark treatment, and then cultivating them until sporophyte seedlings are obtained for rejuvenation.

[0018] Preferably, the conditions for the dark treatment include: culturing in darkness for 44–48 hours or 48 hours; specifically, the conditions for the dark treatment include: culturing in darkness at a temperature of 23–25°C for 44–48 hours or 48 hours; more specifically, the conditions for the dark treatment include: culturing in darkness, with an initial substrate humidity of 60% or 60%–65%, a temperature of 23–25°C, and in a sealed environment for 44–48 hours or 48 hours; even more specifically, the conditions for the dark treatment include: culturing in darkness, with an initial substrate humidity of 60% or 60%–65%, a temperature of 23–25°C, and in a sealed environment for 44–48 hours or 48 hours, without adding water during this period. The "sealed" condition refers to covering the seedling container with a transparent lid and then sealing the gap between the container and the lid to form a sealed environment where the spores or sporophyte seedlings are all within the seedling container.

[0019] Preferably, the step of reculturing to obtain sporophyte seedlings to be rejuvenated includes culturing to gametophyte sexual organs under the following conditions: ambient temperature of 23–25°C, light intensity of 2500–3500 Lux, 12–14 hours or 14 hours of light per day, and relative humidity of the air in the gametophyte environment of 65–85%. Specifically, culturing to gametophyte sexual organs to maturity is carried out under the following conditions: ambient temperature of 23–25°C, light intensity of 2500–3500 Lux, 12–14 hours or 14 hours of light per day, relative humidity of the air in the gametophyte environment of 65–85%, and a sealed environment. The relative humidity of the air in the gametophyte environment refers to the relative humidity of the air inside the seedling container with a transparent lid. A sealed environment refers to the space formed by covering the seedling container with a transparent lid and sealing the gap between the container and the lid. If the relative humidity of the air is not within the range of 65-85% during this period, add water to bring the relative humidity of the air to 65-85%, and immediately restore the airtight state after adding water.

[0020] Preferably, after the gametophyte sex organs mature, the following steps are included:

[0021] (1) Adjust the relative humidity of the air in the gametophyte environment to 65-85%, and then culture it for 10 days under sealed conditions and constant temperature of 25℃ without adding water. Specifically, adjust the relative humidity of the air in the gametophyte environment to 65-85%, and then culture it for 10 days under sealed conditions, constant temperature of 25℃, light intensity of 2500-3500 Lux, and 12-14 hours or 14 hours of light per day without adding water. The relative humidity of the air in the gametophyte environment refers to the relative humidity of the air inside the seedling container with a transparent lid. Sealed means that the seedling container is covered with a transparent lid, and the gap between the container and the lid is sealed to form a spatial environment.

[0022] (2) Then, remove the sealed conditions and place the gametophytes in a greenhouse environment at 23-25°C, with a light intensity of 2500-3500 Lux, 12-14 hours of light per day, and a relative humidity of 22-25% for 22-24 hours. The relative humidity of the gametophytes in this environment is equivalent to that in a greenhouse. After removing the sealed environment, the gametophytes are exposed to the greenhouse, where the relative humidity is 22-25%. Removing the sealed conditions means removing the transparent cover.

[0023] (3) Promote fertilization by spraying water mist.

[0024] Preferably, the method includes the following steps: after fertilization, culturing until sporophyte seedlings germinate; more specifically, after fertilization, culturing until sporophyte seedlings germinate under the following conditions: ambient temperature of 23–25°C, light intensity of 2500–3500 Lux, 12–14 hours of light per day, and relative humidity of 65–85%; even more specifically, the culturing is carried out under sealed conditions. Sealed means that the seedling container is covered with a transparent lid, and the gap between the container and the lid is sealed to create a closed spatial environment.

[0025] Preferably, after the sporophyte seedlings germinate, they are cultured under the following conditions until vigorous sporophyte seedlings are obtained: light intensity of 3000-4000 Lux, 12-14 hours or 14 hours of light per day, temperature of 26-27°C, and relative humidity of air maintained at 55%-70%; wherein the relative humidity of air refers to the relative humidity of air inside the seedling container covered with a transparent lid.

[0026] Preferably, the substrate used for spore inoculation is peat moss, specifically Pinscher peat moss, with a particle size of 0–6 mm and a pH value of 5.5–6.0.

[0027] Preferably, the initial moisture content of the substrate used for spore inoculation is 60%.

[0028] In this article, sporophyte seedling and larval seedling have the same meaning and can be used interchangeably.

[0029] The method for cultivating *Cibotium barometz* seedlings provided by this invention enables rapid, large-scale seedling production from spores, significantly improving the spore germination rate (to 3000 seedlings / tray, i.e., 3000 seedlings / 0.176 square meters). Seedling emergence is highly uniform, and the gametophytes and sporophytes are robust. This method also shortens the seedling growth cycle to 120 days. In summary, this method is low-cost, technically simple, and yields high output. The seedlings grow rapidly and are suitable for large-scale mass production, effectively meeting the rapid propagation needs of *Cibotium barometz*. It can be widely applied in landscaping, medicinal production, and scientific research. Attached Figure Description

[0030] Figure 1 It is the spore leaf of the golden-haired dog.

[0031] Figure 2 It is a mature gamete of a golden retriever.

[0032] Figure 3 It is a seedling of the golden retriever dog.

[0033] Figure 4 Different treatments were used to rejuvenate the substrate.

[0034] Figure 5 This shows the seedling recovery status of group T1 on day 15.

[0035] Figure 6 The growth and development of seedlings in different groups on day 45.

[0036] Figure 7 This shows the growth and development of seedlings in group T1 on day 90.

[0037] Figure 8 This shows the growth and development of seedlings in group T1 on day 120.

[0038] Figure 9 This is a picture of the growth and development of a Golden Retriever puppy on day 150. Detailed Implementation

[0039] I. Collection of Golden Retriever Spores

[0040] Collect cotyledons from mature, healthy plants with a sporangium dehiscence rate of 20%–30%. Place the cotyledons in a rectangular paper bag made of 2–3 layers of newspaper, seal it, and let it air dry in a cool, well-ventilated place for 10–15 days. Before sowing, gently lift the dried paper bag, cut off a corner, and tap the spores onto the white paper. If the spores are mixed with impurities, spread them evenly, tilt the white paper, and gently tap to separate the spores from the impurities. Repeat this process until all impurities are removed. Collect the purified spores in centrifuge tubes and store them at 4°C to ensure spore viability and quality.

[0041] II. Inoculation with golden dog spores

[0042] The soil used for sowing was imported Danish Pinscher peat moss (particle size 0-6mm, pH 5.5-6.0) provided by Beijing Zhonghong Lianke Biotechnology Co., Ltd. The peat moss was wrapped in newspaper, sterilized by high-pressure steam at 125℃ for 25 minutes, cooled, and then spread evenly in a 55cm×32cm×8cm culture tray, compacting to a soil layer thickness of 5cm. Tap water was then sprayed until the soil moisture content was approximately 60%. For sowing, 0.01ml of spores was placed at the fold line of a folded A4 sheet of paper, and the paper was gently tapped to evenly distribute the spores along the fold line into the culture tray. After sowing, the seedling tray was covered with a transparent lid, and the gaps between the lid and tray were sealed with black electrical tape to keep the substrate moist. No further watering was required after sowing. The seedling trays were then covered with an opaque black cloth and placed in a culture room at an ambient temperature of 23-25℃ for 48 hours to promote synchronous spore germination and more uniform seedling growth. After the above-mentioned dark treatment, the temperature of the cultivation environment is controlled at 23-25℃, the light intensity is 2500-3500 Lux, and the light is 14 hours a day; and the relative humidity of the air (the relative humidity of the air in the seedling tray) is maintained at 65%-85% by timely watering, that is, spraying water into the substrate in the seedling tray to make the humidity in the seedling tray reach 65%-85%. Immediately cover the seedling tray with a transparent lid, and seal the gap between the tray and the lid with electrical tape. Maintain this sealed environment until the following germination begins before opening the lid.

[0043] III. Artificial Assisted Fertilization and Sporulation Transformation

[0044] Ten to fifteen days after sowing, the spores of *Cibotium barometz* begin to germinate. Approximately 60 to 80 days later, the gametophyte's reproductive organs (antheridia and archegonia) mature and can be observed under a microscope. After maturation, the humidity is checked by opening the lid and spraying water mist to maintain 65-85%. The culture tray is then sealed at a constant temperature of 25°C for 10 days without watering, under a light intensity of 2500-3500 Lux and 14 hours of light per day. Subsequently, the transparent plastic lid is opened, but instead of spraying to maintain humidity, the tray is left uncovered and allowed to air dry naturally in a greenhouse for 24 hours (during which time the temperature is 23-25°C, the light intensity is 2500-3500 Lux, 14 hours of light per day, and the relative humidity in the culture room is 22-25%) to promote full development of the antheridia and increase sperm concentration. After drying, the reproductive organs are thoroughly saturated with water through frequent, light spraying (once in the morning, noon, and evening) to facilitate fertilization. After fertilization, seal the environment again for 7 days. Cover the culture tray with a transparent lid and seal it with electrical tape. Do not move or spray water during this period (the relative humidity inside the tray should be 65-85%) until the sporophyte seedlings germinate. If no seedlings are observed after 7 days, open the transparent lid for ventilation for 3 days, then repeat the spraying and sealing steps. Throughout the fertilization process, maintain a temperature of 23-25℃, a light intensity of 2500-3500 Lux, and 14 hours of light per day. After the seedlings emerge, increase the light intensity to 3000-4000 Lux, 14 hours of light per day, maintain a room temperature of 26-27℃, and keep the relative humidity (inside the tray) at 55%-70%. With this treatment, the germination rate of *Cibotium barometz* spores can reach 3000 plants / tray (3000 plants / 0.176 square meters).

[0045] IV. Seedling rejuvenation

[0046] After the sporophyte seedlings of *Cibotium barometz* emerged, their growth was observed every 3 days. When the first true leaf reached 0.3–0.5 cm in length, the seedlings were transplanted into culture trays of the same size as those used for sowing, allowing for division and rejuvenation. The study found that the transplanting substrate had a significant rejuvenation effect on *Cibotium barometz* seedlings. To screen for the optimal substrate, the experiment used leaf mold and peat moss as the main materials, and coconut coir, vermiculite (5–8 mm), pond mud, and garden soil as auxiliary materials to prepare different substrate combinations (specific ratios are shown in Table 1). The substrate moisture content was 60%. The experiment included 4 treatments (T1–T4), with 3 trays of seedlings transplanted from each treatment substrate, 100 seedlings per tray, 1 tray as one replicate, and a total of 3 replicates.

[0047] Coconut bricks, vermiculite, pond mud, and garden soil were all purchased from Huihuang Gardening Supplies Store in Longgang District, Shenzhen.

[0048] Leaf mold, also known as humus, is a nutrient-rich soil formed from the decomposition and fermentation of plant leaves and branches in the soil by microorganisms. In this example, the leaf mold was purchased from Shenzhen Lvguocheng Landscape Co., Ltd. Its organic matter content was 45.7%, and its bulk density was 0.36 g / cm³. 3 The pH (water immersion) is 6.0, and the soil is black with organic matter particles.

[0049] Peat soil: Peat soil refers to soil formed in certain river and lake sedimentary plains and mountain valleys where, due to long-term water accumulation and dense aquatic vegetation, a large amount of incompletely decomposed plant remains accumulate and form a peat layer under anaerobic conditions. In this example, the peat soil was purchased from Changchun Zhuangmiao Peat Technology Co., Ltd., and the product name is "New Seedling Substrate - Jintian Brand". This peat soil has a total organic matter and humic acid content of ≥50%, a porosity of 70-80, and a pH (water immersion) of 5.6-6.5.

[0050] Table 1. Composite seedling substrate formula (volume ratio)

[0051]

[0052] The substrate used for division and rejuvenation does not require sterilization. The division containers are the same seedling trays and transparent covers used for sowing, but no longer sealed with tape. After division, maintain the cultivation temperature at 23–25℃, the light intensity at 2500–3500 Lux, and the light duration at 14 hours per day. Water as needed to maintain a relative humidity of 65%–85% in the seedling trays; spray water mist to increase humidity when it is below 65%, and provide appropriate ventilation when it is above 85%. Observe the seedling establishment and the severity of pests and diseases, and record the survival rate (%) of sporophyte seedlings on days 15, 30, 45, 60, 75, and 90 after division. The survival rate is calculated as: number of surviving seedlings / 100 × 100%. Plant height is measured on days 30, 60, and 90. See Table 2 for detailed experimental results.

[0053] Table 2. Growth and development of *Cibotium barometz* larvae under different treatments.

[0054]

[0055] Note: Different lowercase letters after the data in the same column indicate significant differences (p < 0.05).

[0056] Table 2 shows that different seedling substrates significantly affected the growth and development of *Cibotium barometz* larvae. The standard for successful seedling establishment was the emergence of the second true leaf. Only the T1 group substrate (leaf mold 1: peat moss 1) reached this standard within 20 days, with plant height reaching 4.5–5.5 cm within 90 days, demonstrating good growth and a final survival rate of 79.17%. In contrast, the other three groups failed to achieve effective seedling establishment and exhibited poor growth: the survival rates of the T2 and T3 groups dropped to 4.00% and 4.33% respectively by day 60, with all seedlings dying within 75 days; the survival rate of the T4 group was only 9.67% by day 75, with all seedlings dying within 90 days. Furthermore, all four substrate treatments showed varying degrees of algal contamination, with the T1 group showing the least contamination, while the other three groups showed more severe contamination, potentially adversely affecting seedling establishment and growth. This experiment shows that the substrate of leaf mold (1): peat moss (1) is most suitable for the transplanting and rejuvenation of Cibotium barometz seedlings. In this substrate, the seedlings recovered within 20 days and reached a height of 4.5-5.5 cm within 90 days, with a survival rate as high as 79.17%, which is significantly better than other substrate treatment groups.

[0057] V. Water and Fertilizer Management

[0058] Ninety days after transplanting and rejuvenation, nutrient solution was sprayed on the seedlings for continued cultivation. The nutrient solution used was a water-soluble fertilizer containing macro-elements (standard NY / T 1107-2020), diluted to a concentration of 0.2% (mass percentage). 200ml was sprayed per tray every 7 days. Under these cultivation conditions, the light intensity was maintained at 2500–3500 Lux, the indoor temperature at 23–25℃, and the relative humidity (inside the seedling trays) at 65%–85%. Approximately 120 days after nutrient solution spraying, the seedlings of *Cibotium barometz* reached a height of 8.5–10.0 cm, with each seedling producing 5–8 leaves. At this stage, the seedlings were growing well and could be transplanted into individual pots to harden them off, promoting independent growth and improving their adaptability. When hardening off seedlings by dividing them into pots, seedlings with a height of 8.5–10.0 cm are individually planted into flowerpots. The hardening substrate is the same as that used in group T1, consisting of leaf mold (1) and peat moss (1), which is the best substrate selected in previous experiments and helps promote healthy seedling growth. After planting, water them promptly and place them in a hardening greenhouse, maintaining the temperature between 25–30℃. Spray water mist 2–3 times a day for 10 minutes each time to ensure that the relative humidity of the culture air is maintained at 60%–65%. The light intensity for hardening off seedlings is 3000–3700 lux, and the light duration is 12–14 hours. After about 30 days of hardening off (approximately 150 days since the spore seedlings were divided), the plant height can reach 12–15 cm, and the number of leaves increases to 6–9, showing good growth and ready for further cultivation.

Claims

1. A method for artificially breeding Golden Retrievers, comprising the following steps: The method includes the following steps: sowing golden retriever spores in a substrate, first subjecting the plants to darkness, and then cultivating them until sporophyte seedlings are obtained for rejuvenation; the substrate used for spore inoculation is peat moss; The conditions for the dark processing include: Incubate in darkness, with an initial substrate humidity of 60%–65%, a temperature of 23–25°C, and in a sealed environment for 44–48 hours without adding water. The steps of reculturing to obtain sporophyte seedlings to be rejuvenated include: culturing to gametophyte sexual organs maturity under the following conditions: ambient temperature of 23~25℃, light intensity of 2500~3500Lux, 12~14 hours of light per day, relative humidity of the air in the gametophyte environment of 65~85%, and sealing. If the relative humidity of the air is not within the range of 65~85% during the period, water is added to make the relative humidity of the air 65~85%, and after adding water, the sealed state is immediately restored. After the gametophyte sex organs mature, the following steps are included: (1) Adjust the relative humidity of the air in the gametophyte environment to 65-85%, and then culture it for 10 days under closed, constant temperature of 25℃, light intensity of 2500-3500 Lux and light intensity of 12-14 hours per day, without adding water during the period; (2) Then remove the sealed conditions and place the gametophytes at 23~25℃, with a light intensity of 2500~3500 Lux, 12~14 hours of light per day, and a relative humidity of 22~25% for 22~24 hours. (3) Promote fertilization by spraying water mist; After fertilization, the seedlings are cultured under the following conditions until they germinate: ambient temperature of 23-25℃, light intensity of 2500-3500 Lux, 12-14 hours of light per day, and relative humidity of 65-85%; the culture is carried out under sealed conditions. After the sporophyte seedlings germinate, cultivate them under the following conditions until rejuvenated sporophyte seedlings are obtained: light intensity of 3000~4000 Lux, 12~14 hours of light per day, temperature of 26~27℃, and relative humidity of air maintained at 55%~70%; When the first true leaf of the sporophyte seedling grows to 0.3-0.5cm, it should be transplanted and rejuvenated. The sporophyte seedlings of the golden-haired dog that are to be rejuvenated are transplanted into a substrate for rejuvenation culture; the substrate is a mixture of leaf mold and peat moss in a volume ratio of (1~1.5):

1.

2. The method according to claim 1, characterized in that: The substrate is a mixture of leaf mold and peat moss in a 1:1 volume ratio.

3. The method according to claim 2, characterized in that: The conditions for rejuvenation culture include: a temperature of 23-25℃, a light intensity of 2500-3500 Lux, a light duration of 12-14 hours per day, and a relative humidity of 65%-85% in the space where the sporophyte seedlings are located.

4. The method according to claim 3, characterized in that: The water content of the substrate in the rejuvenation culture is 60%~65%.

5. The method according to claim 4, characterized in that: The rejuvenation cultivation also includes the step of applying nutrient solution; the nutrient solution is a water-soluble fertilizer containing macro-elements; the nutrient solution is applied starting on the 90th day after transplanting, the water-soluble fertilizer containing macro-elements is diluted to a concentration of 0.2% (mass percentage concentration) aqueous solution, and sprayed once every 7 days, each time spraying 200ml per 1760 square centimeters.

6. The method according to claim 5, characterized in that: The method also includes a seedling hardening step, in which the sporophyte seedlings to be hardened are planted separately, and the substrate is a mixture of leaf mold and peat moss in a volume ratio of (1~1.5):

1.

7. The method according to claim 6, characterized in that: The sporophyte seedlings to be hardened are sporophyte seedlings with a height of 8.5-10.0cm or sporophyte seedlings with 5 to 8 leaves per plant.

8. The method according to claim 7, characterized in that: The conditions for hardening off the seedlings include: a temperature of 25-30℃, a relative humidity of 60%-65% in the greenhouse, a light intensity of 3000-3700 lux, and a light duration of 12-14 hours.

9. The method according to claim 8, characterized in that: The substrate is a mixture of leaf mold and peat moss in a 1:1 volume ratio.

10. The method according to any one of claims 1-9, characterized in that: The peat soil is Pins peat soil, with a particle size of 0~6mm and a pH value of 5.5~6.0.

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