Method for artificial breeding of golden jackals using spores

Abstract

The present application belongs to the technical field of golden retriever breeding, and particularly relates to a method for artificially breeding golden retriever by using spores, which comprises the following steps: a substrate is prepared according to the following formula by weight: 100 parts of farmland soil, 20-25 parts of sawdust, 10-15 parts of perlite, 10-12 parts of wood ash, 0.05-0.15 parts of manganese sulfate and 0.3-0.5 parts of sodium selenite; spores are germinated at an ambient temperature of 24-26 DEG C; and the germinated seedlings are transplanted into a greenhouse for cultivation. The present application ensures high spore germination rate and seedling survival rate, and the transplanted seedlings can grow smoothly.

Description

Technical Field

[0001] This invention belongs to the field of golden retriever breeding technology, specifically relating to a method for artificially breeding golden retrievers using spores. Background Technology

[0002] Cibotium barometz (L.) J.Sm. is a tree-shaped fern belonging to the Cibotaceae family and the Cibotium genus. It occupies an important position in the ecosystem. Currently, existing technologies for the study of Cibotium barometz present a multi-dimensional situation, mainly including: (1) morphological diversity research, which aims to reveal the pattern of morphological variation of Cibotium barometz in different geographical regions and ecological environments, and to provide a solid morphological basis for species classification, identification and evolutionary research; (2) community classification research, which focuses on the distribution of Cibotium barometz in different ecological communities and its relationship with surrounding plants, aiming to clarify the suitable ecological niche of Cibotium barometz and provide scientific guidance for the protection and restoration of its habitat; (3) genetic diversity Diversity studies, from a molecular biology perspective, utilize molecular marker technology to analyze genetic differences within and between populations of *Cephalotaxus fortunei*, understand the genetic structure and evolutionary history of the population, and provide a genetic basis for formulating reasonable conservation strategies; (4) Allelopathic studies focus on the impact of chemical substances produced by *Cephalotaxus fortunei* plants on the growth and development of other surrounding organisms; (5) Reproduction studies, such as gametophyte development stage studies, track the various stages of *Cephalotaxus fortunei* from spore germination to gametophyte development and fertilization, study the needs and responses of gametophytes at different developmental stages to environmental factors, and provide a theoretical basis for optimizing gametophyte development conditions during artificial breeding. All of these studies require *Cephalotaxus fortunei* plants, such as roots, stems, and leaves, as research materials, so the demand for a large number of *Cephalotaxus fortunei* plants is relatively high.

[0003] However, the breeding process of Golden Retrievers is significantly affected by season and temperature. Seasonal changes and temperature fluctuations make it difficult to predict the breeding season, resulting in a consistently low breeding coefficient. This situation makes it difficult to obtain large numbers of breeding stock in the wild, severely limiting the expansion of the Golden Retriever population and its distribution range.

[0004] Although existing technologies include research on obtaining Golden Retriever seedlings using spore propagation, Golden Retriever spore propagation is extremely sensitive to environmental conditions. Factors such as soil pH, fertility, and temperature can all adversely affect spore implantation and germination, resulting in low spore germination rates and restricting the breeding of Golden Retrievers. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for artificially breeding golden retrievers using spores.

[0006] The purpose of this invention is to provide a method for artificially breeding Golden Retrievers using spores, comprising:

[0007] Substrate preparation steps: The substrate formula, by weight, is as follows: 100 parts garden soil, 20-25 parts sawdust, 10-15 parts perlite, 10-12 parts wood ash, 0.05-0.15 parts manganese sulfate, and 0.3-0.5 parts sodium selenite. Garden soil, sawdust, and wood ash have good air permeability, drainage, and moderate water retention, making them ideal substrate materials for the germination of *Cibotium barometz* spores. Garden soil, sawdust, and wood ash also provide carbon sources and other nutrients. Perlite provides air permeability. Sodium selenite provides selenium nutrition and, in combination with manganese sulfate, stimulates the germination of *Cibotium barometz* spores.

[0008] Spore germination occurs at an optimal temperature of 24℃ to 26℃; this temperature range is the optimal temperature for germination of Cibotium barometz spores, ensuring the efficient execution of various physiological and biochemical reactions within the spores.

[0009] Seedling cultivation: Germinating seedlings are transplanted to greenhouses for further cultivation. Greenhouses provide seedlings with a relatively stable temperature, humidity, and light environment, reducing the impact of external environmental fluctuations on seedling growth.

[0010] Preferably, the substrate formula for the above-mentioned method of artificially breeding golden retrievers using spores is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite.

[0011] Preferably, in the above-mentioned method for artificially breeding golden retrievers using spores, the sawdust is poplar sawdust powder.

[0012] Preferably, in the above-mentioned method for artificially propagating Cibotium barometz using spores, spore germination takes place indoors, with light conditions set at 2500-3000 lux for 10-12 hours. This avoids scorching the spores from excessive light while meeting their light requirements for photosynthesis, simulating the light rhythm in the natural environment, and promoting normal spore germination and early growth.

[0013] Preferably, in the above-mentioned method for artificially breeding golden retrievers using spores, the indoor humidity is controlled at 70% to 80%.

[0014] Preferably, in the above-mentioned method of artificially breeding golden retrievers using spores, the temperature of the greenhouse is 24℃~26℃ and the humidity is 70%~75%.

[0015] Preferably, in the above-mentioned method of artificially breeding golden retriever dogs using spores, the soil moisture content is maintained at 60% to 65%.

[0016] This invention provides an application of a substrate in improving the efficiency of artificial breeding of Golden Retrievers, wherein the substrate is the aforementioned substrate.

[0017] Preferably, improving the efficiency of artificial breeding of golden retriever dogs refers to increasing the spore germination rate, seedling acquisition rate, seedling survival rate after transplanting, and promoting seedling development.

[0018] Preferably, promoting seedling development refers to accelerating the production of new leaves and increasing leaf length and width.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention significantly improves spore germination rates through precise control of the substrate formula (100 parts garden soil, 20-25 parts sawdust, 10-15 parts perlite, 10-12 parts wood ash, 0.05-0.15 parts manganese sulfate, and 0.3-0.5 parts sodium selenite), the germination environment, and the seedling cultivation process. The addition of manganese sulfate and sodium selenite to the substrate formula, along with controlling the ambient temperature at 24℃-26℃ during the spore germination stage, allows spores to break dormancy and initiate the germination process in a shorter time. During the seedling cultivation stage, cultivation in a greenhouse ensures high spore germination and seedling survival rates, and the transplanted seedlings grow successfully, effectively improving the survival rate (up to 95%) of later understory planting.

[0021] This invention enables the production of a large number of seedlings of uniform physiological age and growth, ensuring seedling consistency in the initial stage. In subsequent seedling cultivation and transplanting, standardized environmental settings and management measures further promote uniform seedling growth. This characteristic makes the technology highly suitable for large-scale seedling production and cultivation, meeting market demand for a large quantity of high-quality Golden Retriever seedlings and driving the development of the Golden Retriever artificial breeding industry. Detailed Implementation

[0022] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0024] The sources of the raw materials used in the following examples and comparisons are as follows:

[0025] (1) Golden Retriever spores were collected and preserved using the following method: The collection site was Lushan City, Jiujiang City, Jiangxi Province. Mature and healthy Golden Retriever plants were selected. Maturity was indicated by the sori being dark brown and plump, at which point the spores were fully developed and had good germination ability. Using sterilized scissors, leaflets with mature spores were collected and placed in clean paper bags. The paper bags had good air permeability, which prevented spore loss and ensured air circulation to avoid spore deterioration. The paper bags containing spore samples were brought back to the laboratory and allowed to air dry in the laboratory environment. After the spores fell naturally from the leaves, they were collected and then stored at 4℃ for later use.

[0026] (2) Field soil, collected from Guling Town, Lushan City, Jiujiang City, Jiangxi Province.

[0027] (3) Sawdust, purchased from Caoxian Shenhang Wood Industry Co., Ltd., is premium pure poplar sawdust powder.

[0028] (4) Perlite, purchased from Lingshou County Chenyang Mineral Products Co., Ltd., with a particle size of 1mm to 3mm.

[0029] (5) Wood ash, purchased from Hebei Xiongan Xinying Technology Co., Ltd., pH 8.5-9, water absorption 40%, dark brown.

[0030] To investigate the effect of different amounts of manganese sulfate on the artificial breeding efficiency of golden retrievers, we set up Examples 1 to 3, a blank control, and a single-factor control 1.

[0031] Example 1

[0032] A method for artificially breeding golden retrievers using spores includes:

[0033] Step 1, substrate preparation

[0034] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.05 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0035] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0036] Step 2, spore germination

[0037] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0038] On day 30, the number of germinating spores was observed and counted under a microscope. The germination of spores is marked by the prothallus breaking through the spore wall and the emergence of green filamentous and plate-like structures. The seedling survival rate was calculated.

[0039] Spore germination rate = (number of spores that have germinated into sporophytes / 300) × 100%.

[0040] On day 90, observe and record the number of seedlings at the two-leaf-one-heart stage, and calculate the seedling survival rate:

[0041] Seedling acquisition rate = (number of seedlings at the two-leaf-one-heart stage / 300) × 100%.

[0042] Step 3, Seedling Cultivation

[0043] When the seedlings have been cultivated for a period of time and have grown to 3 leaves and 2 cm in height, they have developed a certain degree of independent living ability and can be transplanted to a greenhouse. After transplanting, spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0044] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0045] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0046] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0047] Example 2

[0048] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.3 parts sodium selenite.

[0049] Specifically, the method in Example 2 includes:

[0050] Step 1, substrate preparation

[0051] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0052] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0053] Step 2, spore germination

[0054] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0055] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0056] Step 3, Seedling Cultivation

[0057] When the seedlings have been cultivated for a period of time and have grown to 3 leaves and 2 cm in height, they have developed a certain degree of independent living ability and are ready for transplanting. Transplanting should then be done in a greenhouse. After transplanting, water regularly each week to maintain soil moisture at 60%, but avoid waterlogging to prevent root rot.

[0058] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0059] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0060] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0061] Example 3

[0062] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.15 parts manganese sulfate, and 0.3 parts sodium selenite.

[0063] Specifically, the method in Example 3 includes:

[0064] Step 1, substrate preparation

[0065] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.15 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0066] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0067] Step 2, spore germination

[0068] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated and cultured indoors, with each treatment repeated three times. The spore germination environment was set at 25℃, with a light condition of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0069] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0070] Step 3, Seedling Cultivation

[0071] When the seedlings have been cultivated for a period of time and have grown to 3 leaves and 2 cm in height, they have developed a certain degree of independent living ability and are then transplanted into a greenhouse. After transplanting, the seedlings are placed inside the greenhouse for further cultivation. Water is sprayed regularly each week to maintain soil moisture at 60%, but waterlogging should be avoided to prevent root rot.

[0072] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0073] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0074] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0075] Blank control

[0076] A method for artificially breeding golden retrievers using spores is basically the same as that in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, and 10 parts wood ash.

[0077] Specifically, methods for blank control include:

[0078] Step 1, substrate preparation

[0079] The substrate formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, and 10 parts wood ash. The substrate is obtained by thoroughly mixing all the raw materials.

[0080] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0081] Step 2, spore germination

[0082] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0083] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0084] Step 3, Seedling Cultivation

[0085] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0086] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0087] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0088] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0089] Single-factor control 1

[0090] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, and 0.05 parts manganese sulfate.

[0091] Specifically, the methods for single-factor control group 1 include:

[0092] Step 1, substrate preparation

[0093] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, and 0.05 parts manganese sulfate. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0094] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0095] Step 2, spore germination

[0096] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0097] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0098] Step 3, Seedling Cultivation

[0099] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0100] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0101] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0102] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0103] To investigate the effects of different sodium selenite dosages on the artificial breeding efficiency of Golden Retrievers, we set up Example 2, Examples 4-5, a blank control, and a single-factor control 2. Examples 4-5 and the single-factor control 2 are as follows.

[0104] Example 4

[0105] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite.

[0106] Specifically, the method in Example 4 includes:

[0107] Step 1, substrate preparation

[0108] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0109] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0110] Step 2, spore germination

[0111] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0112] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0113] Step 3, Seedling Cultivation

[0114] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0115] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0116] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0117] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0118] Example 5

[0119] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.5 parts sodium selenite.

[0120] Specifically, the method in Example 5 includes:

[0121] Step 1, substrate preparation

[0122] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.5 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0123] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0124] Step 2, spore germination

[0125] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0126] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0127] Step 3, Seedling Cultivation

[0128] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0129] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0130] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0131] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0132] Single-factor control 2

[0133] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, and 0.3 parts sodium selenite.

[0134] Specifically, the methods for single-factor control group 2 include:

[0135] Step 1, substrate preparation

[0136] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0137] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0138] Step 2, spore germination

[0139] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0140] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0141] Step 3, Seedling Cultivation

[0142] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0143] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0144] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0145] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0146] To investigate the effects of other matrix formulation ingredients on the artificial breeding efficiency of Golden Retrievers, we set up Example 4 and Example 6.

[0147] Example 6

[0148] A method for artificially breeding golden retrievers using spores is basically the same as the operation in Example 1, except that the substrate formula is as follows: 100 parts garden soil, 25 parts sawdust, 15 parts perlite, 12 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite.

[0149] Specifically, the method in Example 6 includes:

[0150] Step 1, substrate preparation

[0151] The matrix formula, by weight, is as follows: 100 parts garden soil, 25 parts sawdust, 15 parts perlite, 12 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0152] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0153] Step 2, spore germination

[0154] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 25℃, with a light exposure of 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0155] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0156] Step 3, Seedling Cultivation

[0157] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0158] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0159] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0160] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0161] To investigate the effects of other breeding conditions on the efficiency of artificial breeding of Golden Retrievers, we set up Example 1 and Examples 7 to 9.

[0162] Example 7

[0163] A method for artificially breeding golden retrievers using spores includes:

[0164] Step 1, substrate preparation

[0165] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.05 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0166] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0167] Step 2, spore germination

[0168] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The spore germination environment was set at 24℃, with a light condition of 2700 lux for 11 hours followed by 13 hours of darkness. The indoor humidity was controlled at 75%.

[0169] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0170] Step 3, Seedling Cultivation

[0171] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0172] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0173] The temperature in the greenhouse is set at 24℃ and the humidity at 80%.

[0174] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0175] Example 8

[0176] A method for artificially breeding golden retrievers using spores includes:

[0177] Step 1, substrate preparation

[0178] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.05 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0179] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0180] Step 2, spore germination

[0181] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. The cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The ambient temperature for spore germination was set at 26℃, and the light conditions were set at 3000 lux for 12 hours followed by 12 hours of darkness. The indoor humidity was controlled at 75%.

[0182] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0183] Step 3, Seedling Cultivation

[0184] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 65%, but avoid waterlogging to prevent root rot.

[0185] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0186] The temperature in the greenhouse is controlled at 26℃ and the humidity at 75%.

[0187] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0188] Example 9

[0189] In addition to Example 1, understory cultivation is also included.

[0190] The method in this embodiment is as follows:

[0191] A method for artificially breeding golden retrievers using spores includes:

[0192] Step 1, substrate preparation

[0193] The matrix formula, by weight, is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.05 parts manganese sulfate, and 0.3 parts sodium selenite. The matrix is ​​obtained by thoroughly mixing all raw materials.

[0194] Before use, the substrate should be sterilized, such as by sterilizing it under high temperature and high pressure at 121℃ for 20 minutes to thoroughly kill pathogens, insect eggs, and other harmful microorganisms in the soil, creating a sterile and excellent environment for spore germination. After sterilization, fill the substrate into nutrient cups, water thoroughly, and set aside.

[0195] Step 2, spore germination

[0196] Golden Retriever spores were rinsed three times with sterile water to remove dust and pathogens. The rinsed spores were then evenly sown onto the substrate surface in nutrient cups, with 50 spores in each cup (22.3 cm long, 15.3 cm wide, and 7.9 cm high). A total of 300 spores were treated. During spore germination, the cups were covered with transparent plastic lids to maintain humidity and incubated indoors. Each treatment was repeated three times. The ambient temperature was set at 25℃, and the light conditions were set at 2500 lux for 10 hours followed by 14 hours of darkness. The indoor humidity was controlled at 70%.

[0197] On day 30, the number of germinating spores was observed and counted under a microscope. On day 90, the number of seedlings at the two-leaf-one-heart stage was observed and recorded. The spore germination rate and seedling acquisition rate were calculated according to the formula in Example 1.

[0198] Step 3, Seedling Cultivation

[0199] When the seedlings have grown to 3 leaves and 2 cm in height after a period of cultivation, they have developed a certain degree of independent living ability and are ready for transplanting. After transplanting, place the seedlings in a greenhouse for further cultivation. Spray water regularly every week to maintain the soil moisture content at 60%, but avoid waterlogging to prevent root rot.

[0200] The substrate formula used for transplanting was the same as that in step 1. Each treatment group was replicated with 100 seedlings. The growth status of the seedlings was recorded within one month, and the average value of each indicator was taken.

[0201] The temperature in the greenhouse is controlled at 25℃ and the humidity at 70%.

[0202] The light transmittance of the greenhouse is controlled at 50%, and the greenhouse provides a diffused light environment.

[0203] Step 4: After the seedling cultivation is completed, carry out understory cultivation.

[0204] The selection of understory cultivation sites is crucial for the subsequent growth of Golden Retrievers. Through research and screening, subtropical Chinese fir forests with an average annual temperature above 5℃ have become ideal understory cultivation sites.

[0205] Covering the soil surface under the trees with mulch film increases soil temperature, retains soil moisture, and promotes rapid root growth. Then, transplant the seedlings and build a shade structure over them. The shade structure provides suitable diffused light. After two months of growth, the seedlings gradually adapt to the understory environment. At this point, remove the mulch film and shade structure, allowing the seedlings to continue growing and developing in the natural understory ecosystem, making full use of the abundant natural resources to promote their robust growth.

[0206] To more clearly illustrate the differences between the above embodiments and the comparisons, Table 1 and Table 2 are provided in this invention.

[0207] Table 1. Differences in matrix formulations (based on dry weight parts)

[0208] Example 1 100 20 10 10 0.05 0.3 Example 2 100 20 10 10 0.1 0.3 Example 3 100 20 10 10 0.15 0.3 Example 4 100 20 10 10 0.1 0.4 Example 5 100 20 10 10 0.1 0.5 Example 6 100 25 15 12 0.1 0.4 Blank control 100 20 10 10 0 0 Single-factor control 1 100 20 10 10 0.05 0 Single-factor control 2 100 20 10 10 0 0.3

[0209] For other differences in breeding conditions, please refer to Table 2.

[0210] Table 2 Other Breeding Conditions

[0211]

[0212] The experimental results are shown in Table 3.

[0213] Table 3 Experimental Results

[0214]

[0215]

[0216] The results in Table 3 are analyzed as follows:

[0217] (1) Spore germination rate

[0218] Using the method of this invention, the spore germination rate and seedling acquisition rate are significantly improved. The spore germination rate of the blank control is only 33%, and the seedling acquisition rate is only 10%. The spore germination rate and seedling acquisition rate of single-factor control 1 and single-factor control 2 are also significantly lower than those of the other example groups.

[0219] Spore germination rate directly reflects the natural regeneration capacity and potential development trend of Cibotium barometz. A higher spore germination rate means that more seeds can successfully break dormancy. From an ecosystem perspective, a high spore germination rate ensures the population proportion of Cibotium barometz in the community, maintaining the biodiversity and structural stability of the ecosystem. For artificial breeding of Cibotium barometz, spore germination rate is even more crucial. Artificial breeding of Cibotium barometz aims to expand the population for ecological restoration or conservation research. By increasing the spore germination rate, breeding costs can be reduced and breeding efficiency increased. A high spore germination rate not only rapidly increases the population of Cibotium barometz but also provides abundant material for subsequent genetic diversity research, helping to cultivate varieties more adaptable to environmental changes. Therefore, the effect of this invention in improving spore germination rate has significant ecological implications.

[0220] (II) Seedling acquisition rate and seedling survival rate after transplanting

[0221] The method of this invention can maintain a seedling survival rate of over 70%, while the control group only has 10%. The survival rate of seedlings after transplanting in the blank control group is 22%, while the method of this invention can stably maintain a seedling survival rate of 83% to 85% after transplanting, greatly improving the conversion rate of breeding results.

[0222] The seedling acquisition rate is highly correlated with material utilization and work efficiency. The method of this invention can improve the seedling acquisition rate, which means that a lot of cost and time costs can be saved, and material utilization and work efficiency can be improved.

[0223] The survival rate of transplanted seedlings refers to the proportion of seedlings that successfully survive and grow to a certain stage after germination. It is a crucial link in the growth process of *Cibotium barometz*, directly affecting the transition of the population from the seedling stage to the mature stage. From an ecological balance perspective, the survival rate of transplanted seedlings affects the energy flow and material cycle of the entire ecosystem. As producers in the ecosystem, the survival of *Cibotium barometz* seedlings means that more photosynthetic products are fixed, providing food and habitat for other organisms. In artificial cultivation, improving the survival rate of transplanted seedlings is the core to ensuring cultivation success. A high survival rate reduces resource waste during cultivation, improves the economic and ecological benefits of artificial propagation, and accelerates the rejuvenation and population expansion of *Cibotium barometz*.

[0224] (III) Uniformity of growth

[0225] This method yields seedlings of uniform physiological age and growth. The coefficients of variation for all seedling growth indicators are controlled within 10%. This indicates that this breeding technique effectively ensures the uniformity of seedling growth, providing a solid foundation for large-scale seedling production and cultivation, facilitating unified management and maintenance, and improving production efficiency.

[0226] Uniformity of growth refers to the degree of consistency in the growth of individuals within a population of *Cibotium barometz*. In the natural environment, populations of *Cibotium barometz* with high uniformity of growth are more competitive. Uniform growth allows for full utilization of environmental resources and reduces competition among individuals. For artificial cultivation and management, uniformity of growth facilitates standardized cultivation management and harvesting, saving human and material resources and improving harvesting efficiency and product quality.

[0227] (iv) Seedling development

[0228] This invention evaluates seedling development by measuring the number of new leaves produced, leaf length, and leaf width. The results show that, compared with the blank control, single-factor control 1, and single-factor control 2, the method of this invention significantly improves the number of new leaves produced, leaf length, and leaf width.

[0229] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0230] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A method for artificially breeding golden retrievers using spores, characterized in that, include: The substrate formula is as follows by weight: 100 parts garden soil, 20-25 parts sawdust, 10-15 parts perlite, 10-12 parts wood ash, 0.05-0.15 parts manganese sulfate, and 0.3-0.5 parts sodium selenite. Spores germinate, and the optimal temperature for spore germination is 24℃~26℃; The germinating seedlings are transplanted to greenhouses for further cultivation; The method is used to improve the spore germination rate, seedling acquisition rate, seedling survival rate after transplanting, and promote seedling development of *Cibotium barometz*. Promoting seedling development refers to accelerating the production of new leaves and increasing leaf length and width.

2. The method for artificially breeding golden retrievers using spores according to claim 1, characterized in that, The substrate formula is as follows: 100 parts garden soil, 20 parts sawdust, 10 parts perlite, 10 parts wood ash, 0.1 parts manganese sulfate, and 0.4 parts sodium selenite.

3. The method for artificially breeding Golden Retrievers using spores according to claim 1, characterized in that, The sawdust is poplar sawdust powder.

4. The method for artificially breeding Golden Retrievers using spores according to claim 3, characterized in that, Spore germination was carried out indoors, with light conditions set at 2500 lux to 3000 lux for 10 to 12 hours.

5. The method for artificially breeding Golden Retrievers using spores according to claim 4, characterized in that, Set the indoor humidity to 70%~80%.

6. The method for artificially breeding golden retrievers using spores according to claim 1, characterized in that, The temperature in the greenhouse is 24℃~26℃, and the humidity is 70%~75%.

7. The method for artificially breeding Golden Retrievers using spores according to claim 6, characterized in that, Maintain soil moisture content at 60%~65%.

Images