Tea fungus strain cxg003 with early fruiting, cultivation method and application thereof

Abstract

The application belongs to the technical field of edible fungi, and discloses a fruiting early Agrocybe aegerita strain CXG003 and a culture and cultivation method and application. The fruiting early Agrocybe aegerita strain is CXG003, which is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, has a preservation number of CCTCC NO:M 20251057, a preservation date of May 14, 2025, and a classification name of Agrocybe aegerita. Cyclocybe chaxingu The culture method comprises the following steps: culture medium preparation, including preparation of PDA culture medium, PDB culture medium and regeneration medium; preparation of protoplasts, picking of Agrocybe aegerita monokaryon strains; and hybridization of Agrocybe aegerita monokaryon to obtain Agrocybe aegerita strains. The protoplast concentration can reach 1.07x10 8 6 / mL, and the fruiting time is early.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi technology, and particularly relates to the early-bearing mushroom strain CXG003 of *Pleurotus ostreatus*, its cultivation method, and its application. Background Technology

[0002] Tea wood mushroom Cyclocybe chaxingu The commercial name is tea tree mushroom, which is one of the main cultivated edible fungi. It belongs to the phylum Basidiomycota, order Agaricales, family Tubariaceae, and genus Amanita. Cyclocybe Tea wood mushrooms are rich in nutrients, have a delicious taste, a rich aroma, a crisp and tender stem, a thick cap, and a unique flavor. They are a type of edible fungus that is high in protein, low in fat, rich in fungal polysaccharides, and relatively high in minerals.

[0003] Tea wood mushroom cultivation has a long period, with multiple fruiting cycles, typically lasting from around April to October. This extended fruiting cycle makes it susceptible to pests and diseases, hindering its industrial-scale production. This is a major bottleneck for the development of the tea wood mushroom industry. Therefore, cultivating tea wood mushroom varieties with shorter fruiting cycles is an urgent problem to be solved.

[0004] Mushrooms sphagnum are edible fungi; however, the long production cycle of existing varieties leads to severe pests and diseases, and they cannot be mass-produced, thus limiting the development of the mushroom industry. For many years, people have longed to cultivate mushroom varieties with a short fruiting cycle to reduce pests and diseases and achieve mass production. However, due to high technical difficulty and long research and development cycles, a variety with early fruiting has not yet been cultivated. The strain of this invention has early fruiting and a short cycle; it solves this long-standing technical problem in the mushroom industry.

[0005] Tea wood mushroom is a traditional edible mushroom industry product. Most practitioners and researchers in related fields generally believe that the biological characteristic of tea wood mushroom is its long fruiting cycle, which is difficult to change. This invention, using advanced protoplast technology, and through experimental research and extensive testing, overcomes the aforementioned technical biases and cultivates a high-quality tea wood mushroom strain with early fruiting and a short cycle. This invention transforms the traditional, outdated, and scattered greenhouse cultivation of tea wood mushroom into large-scale, factory-style cultivation. Summary of the Invention

[0006] To overcome the problems existing in the related technologies, the present invention discloses embodiments that provide an early-fruiting strain of Mushroom CXG003, as well as its cultivation method and application, specifically involving a strain CXG003 with a short fruiting cycle.

[0007] The technical solution is as follows: An early-fruiting strain of *Pleurotus ostreatus*, specifically strain CXG003, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20251057, deposited on May 14, 2025, and classified as follows: Cyclocybe chaxingu CXG003.

[0008] Another object of the present invention is to provide a method for cultivating an early-bearing *Pleurotus ostreatus* strain, the method comprising:

[0009] S1, Culture medium preparation, including the preparation of PDA medium, PDB medium and regeneration medium;

[0010] S2, Protoplast preparation, selection of monokaryotic strains of *Pleurotus ostreatus*;

[0011] S3, a hybrid strain of *Pleurotus ostreatus* was obtained by hybridization of *Pleurotus ostreatus* monokaryotic cells.

[0012] In step S1, the preparation of PDA culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a boiled potato aqueous solution; weighing glucose and agar, adding them to the filtered potato water, stirring evenly with a glass rod, then pouring them into beakers and dispensing them, sterilizing them; cooling, pouring the liquid culture medium into sterile petri dishes in a laminar flow hood, letting the solution in the petri dishes cool and solidify, and then sealing them with sealing film for later use;

[0013] The preparation of PDB culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a cooked potato solution; weighing glucose, adding it to the filtered potato water while stirring with a glass rod, pouring it into a beaker, making up to volume, dispensing the prepared solution into Erlenmeyer flasks, sterilizing, and cooling for later use;

[0014] The preparation of the regeneration culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water and boiling them in an induction cooker. After the potato water is boiled, pour it into a beaker and filter it with gauze to obtain a boiled potato solution. Weigh glucose, agar, and mannitol, add them to the filtered potato water, stir well with a glass rod, pour it into a beaker, make up to volume, dispense it into Erlenmeyer flasks, sterilize, cool, and pour the liquid culture medium into sterile petri dishes in a laminar flow hood until the solution in the petri dishes cools and solidifies. Seal the petri dishes with sealing film for later use.

[0015] In step S2, protoplast preparation involves selecting monokaryotic strains of *Pleurotus ostreatus*, including:

[0016] Protoplast preparation includes the following steps: First, prepare protoplasts for *Pleurotus ostreatus* ND5 and LJH001. Specifically, inoculate *Pleurotus ostreatus* ND5 and LJH001 spawn into the center of a disposable petri dish containing PDA medium. When the mycelium has covered two-thirds of the dish, use a sterilized punch to create a solid mycelial block at the edge, inoculate it into PDA medium, and then culture it. Filter the resulting liquid mycelium through gauze and collect it in centrifuge tubes for later use. Then, enzymatically hydrolyze the obtained mycelium with a lysozyme in a water bath to obtain protoplasts.

[0017] The selection of monokaryotic strains of *Pleurotus ostreatus* involved: diluting the number of protoplasts with mannitol-stabilized solution, spreading them onto regeneration medium using a spreader, and then incubating them in the dark in a constant temperature incubator.

[0018] When regenerated colonies grow on the regeneration plate, each regenerated colony is picked out with a sterile needle and transferred to PDA medium, and placed in an incubator for separate culture. After culture, regenerated hyphae are picked onto a glass slide for preparation and placed under an inverted microscope to observe clamp connections. Strains without clamp connections are transferred back to a new PDA medium, placed in an incubator for separate culture, and observed again for clamp connections for secondary verification. Monokaryotic hyphae without clamp connections are transferred to a new PDA medium for culture.

[0019] The obtained mycelia were enzymatically hydrolyzed using a 2% concentration of lysozyme in a water bath at 29-31℃ for 3.5-4.5 hours, resulting in a protoplast concentration of 1.07 × 10⁻⁶. 8 Protoplasts / mL were diluted with 0.6 mol / L mannitol osmotic stabilizer to a protoplast count of 1 × 10⁻⁶. 2 per mL.

[0020] In step S3, the *Phyllostachys edulis* monokaryotic hybrid strains obtained by hybridization include:

[0021] Step 1: Using *Pleurotus ostreatus* ND5 and LJH001 as parents, monokaryotic strains of ND5 and LJH001 inoculated on PDA medium were cultured at a constant temperature. After culture, multiple mycelial blocks were punched out and placed on PDA medium for confrontation culture, and then placed in an incubator for dark culture. As the mycelia on the two mycelial blocks continued to grow, the mycelia at both ends intertwined to form a groove-shaped or raised antagonistic line. Multiple mycelial blocks were picked from the antagonistic line and transferred to a new PDA medium, and then cultured in a constant temperature incubator in the dark.

[0022] Step 2, initial screening of hybrid strains of *Phyllostachys edulis*; after hybridization, the mycelium of the *Phyllostachys edulis* monokaryotic strain is obtained, which is the *Phyllostachys edulis* hybrid strain.

[0023] Step 3: SRAP molecular marker analysis of hybrid strains, including SRAP primer design, SRAP-PCR amplification, and horizontal agarose gel electrophoresis.

[0024] Another object of the present invention is to provide the application of the early-fruiting *Tetracentron sinense* strain in the breeding of early-fruiting hybrid *Tetracentron sinense*.

[0025] Another objective of this invention is to provide a cultivation method for an early-bearing *Tetracentron sinense* strain, obtained by cultivating the aforementioned early-bearing *Tetracentron sinense* hybrid strain.

[0026] The cultivation method for the early-bearing *Tetracentron sinense* strain includes:

[0027] Take out the hybrid strain of *Pleurotus ostreatus* and the parent strain, transfer them to PDA medium in a clean bench, seal them and place them in a constant temperature incubator for static cultivation in the dark until the mycelium covers the plate, then take them out for use. The culture medium formula includes: corn cob 51-52%, cottonseed hull 21-22%, wheat bran 16.5-17.5%, corn flour 6-7%, gypsum 0.5-1.5%, and lime 0.5-1.5%.

[0028] Weigh the raw materials according to the cultivation formula ratio, mix the raw materials evenly, add water and mix well, then mix the mixed culture medium evenly and pack it into medium-sized polypropylene plastic bags. Put the bagged spawn bags into a high-temperature and high-pressure sterilizer for sterilization at 121℃ for 2 hours. After sterilization, wait until the temperature drops below 50℃ before removing the spawn bags from the high-temperature and high-pressure sterilizer. Place the sterilized spawn bags in a pre-sterilized incubator to cool, and then place the cooled spawn bags into a sterile ultra-clean workbench. Inoculate the prepared spawn with an inoculation spatula, and inoculate each spawn bag with spawn blocks.

[0029] After inoculation, place the bags in a constant temperature incubator for light-proof cultivation until the mycelium completely covers the bags. Move the bags into the fruiting room, open the bags and harvest the fruiting bodies. Cut off the plastic film from the upper part of the bag. Harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

[0030] The conditions for mushroom growth are: temperature set at 24-26℃ and relative humidity at 90-95%.

[0031] Combining all the above technical solutions, the beneficial effects of this invention are as follows:

[0032] This invention significantly improves the biological efficiency of the first fruiting cycle compared to existing technologies. The protoplast concentration of this invention can reach 1.07 × 10⁻⁶. 8The strain's fruiting rate (per 1000 cells / mL) improves the breeding method of *Pleurotus ostreatus* and increases breeding efficiency. This strain also produces fruiting earlier. The aroma and flavor of the *Pleurotus ostreatus* CXG003 strain of this invention are significantly superior to existing mushrooms on the market, further demonstrating that this invention has the effect of increasing consumer acceptance.

[0033] The strain of this invention produces fruit early, shortens the cycle, saves labor and cultivation costs, is suitable for factory production, and can be used for large-scale and automated production.

[0034] Existing varieties of *Tetracentron sinense* suffer from long fruiting cycles, hindering industrial-scale production and leading to issues such as excessive pesticide residues. The strain of this invention fruits early and has a short cycle, reducing pests and diseases and making it suitable for industrial-scale production. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure;

[0036] Figure 1 This is a flowchart of the cultivation method for the early-bearing *Tea Leaf Fungus* strain provided in this embodiment of the invention;

[0037] Figure 2 This is an effect diagram of the hyphae of the ND5 monokaryotic strain without clamping linkage provided in the embodiments of the present invention;

[0038] Figure 3 This is an effect diagram of the hyphae of the LJH001 monokaryotic strain without clamping linkage provided in the embodiments of the present invention;

[0039] Figure 4 This is a diagram showing the clamping effect of the successfully hybridized strains provided in this embodiment of the invention;

[0040] Figure 5 This is an electrophoretic pattern obtained by running horizontal agarose gel electrophoresis after PCR amplification, as provided in this embodiment of the invention. Detailed Implementation

[0041] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] Example 1: This embodiment of the invention provides an early-fruiting *Pleurotus ostreatus* strain, CXG003, deposited at the China Center for Type Culture Collection (Wuhan, China), accession number CCTCC NO: M20251057, deposit date: May 14, 2025, and test result: viable; classified and named as follows: Cyclocybe chaxingu CXG003.

[0044] Example 2, as Figure 1 As shown, a method for cultivating an early-fruiting *Pleurotus ostreatus* strain is applied to the cultivation of early-fruiting *Pleurotus ostreatus* strains. This cultivation method includes:

[0045] S1, Culture medium preparation, including the preparation of PDA medium, PDB medium and regeneration medium;

[0046] S2, Protoplast preparation, selection of monokaryotic strains of *Pleurotus ostreatus*;

[0047] S3, a hybrid strain of *Pleurotus ostreatus* was obtained by hybridization of *Pleurotus ostreatus* monokaryotic cells.

[0048] For example, in step S1, the preparation of PDA culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a boiled potato aqueous solution; weighing glucose and agar, adding them to the filtered potato water, stirring evenly with a glass rod, then pouring them into beakers and dispensing them, sterilizing them; cooling, pouring the liquid culture medium into sterile petri dishes in a laminar flow hood, letting the solution in the petri dishes cool and solidify, and then sealing them with sealing film for later use;

[0049] The preparation of PDB culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water, boiling them in an induction cooker, pouring the cooked potatoes into a beaker, filtering them with gauze to obtain a cooked potato solution; weighing glucose, adding it to the filtered potato water while stirring with a glass rod, pouring it into a beaker, making up to volume, dispensing the prepared solution into Erlenmeyer flasks, sterilizing, and cooling for later use;

[0050] The preparation of the regeneration culture medium includes: weighing fresh potatoes, cutting them into small pieces, adding distilled water and boiling them in an induction cooker. After the potato water is boiled, pour it into a beaker and filter it with gauze to obtain a boiled potato solution. Weigh glucose, agar, and mannitol, add them to the filtered potato water, stir well with a glass rod, pour it into a beaker, make up to volume, dispense it into Erlenmeyer flasks, sterilize, cool, and pour the liquid culture medium into sterile petri dishes in a laminar flow hood until the solution in the petri dishes cools and solidifies. Seal the petri dishes with sealing film for later use.

[0051] In step S2, protoplast preparation involves selecting monokaryotic strains of *Pleurotus ostreatus*, including:

[0052] Protoplast preparation includes the following steps: First, prepare protoplasts for *Pleurotus ostreatus* ND5 and LJH001. Specifically, inoculate *Pleurotus ostreatus* ND5 and LJH001 spawn into the center of a disposable petri dish containing PDA medium. When the mycelium has covered two-thirds of the dish, use a sterilized punch to create a solid mycelial block at the edge, inoculate it into PDA medium, and then culture it. Filter the resulting liquid mycelium through gauze and collect it in centrifuge tubes for later use. Then, enzymatically hydrolyze the obtained mycelium with a lysozyme in a water bath to obtain protoplasts.

[0053] The selection of monokaryotic strains of *Pleurotus ostreatus* involved: diluting the number of protoplasts with mannitol-stabilized solution, spreading them onto regeneration medium using a spreader, and then incubating them in the dark in a constant temperature incubator.

[0054] When regenerated colonies grow on the regeneration plate, each regenerated colony is picked out with a sterile needle and transferred to PDA medium, and placed in an incubator for separate culture. After culture, regenerated hyphae are picked onto a glass slide for preparation and placed under an inverted microscope to observe clamp connections. Strains without clamp connections are transferred back to a new PDA medium, placed in an incubator for separate culture, and observed again for clamp connections for secondary verification. Monokaryotic hyphae without clamp connections are transferred to a new PDA medium for culture.

[0055] The obtained mycelia were enzymatically hydrolyzed using a 2% concentration of lysozyme in a water bath at 29-31℃ for 3.5-4.5 hours, resulting in a protoplast concentration of 1.07 × 10⁻⁶. 8 Protoplasts / mL were diluted with 0.6 mol / L mannitol osmotic stabilizer to a protoplast count of 1 × 10⁻⁶. 2 per mL.

[0056] In step S3, the *Phyllostachys edulis* monokaryotic hybrid strains obtained by hybridization include:

[0057] Step 1: Using *Pleurotus ostreatus* ND5 and LJH001 as parents, monokaryotic strains of ND5 and LJH001 inoculated on PDA medium were cultured at a constant temperature. After culture, multiple mycelial blocks were punched out and placed on PDA medium for confrontation culture, and then placed in an incubator for dark culture. As the mycelia on the two mycelial blocks continued to grow, the mycelia at both ends intertwined to form a groove-shaped or raised antagonistic line. Multiple mycelial blocks were picked from the antagonistic line and transferred to a new PDA medium, and then cultured in a constant temperature incubator in the dark.

[0058] Step 2, initial screening of hybrid strains of *Phyllostachys edulis*; after hybridization, the mycelium of the *Phyllostachys edulis* monokaryotic strain is obtained, which is the *Phyllostachys edulis* hybrid strain.

[0059] Step 3: SRAP molecular marker analysis of hybrid strains, including SRAP primer design, SRAP-PCR amplification, and horizontal agarose gel electrophoresis.

[0060] Example 3: Application of early-fruiting *Tetracentron sinense* strains in the breeding of early-fruiting hybrid *Tetracentron sinense*.

[0061] Example 4: A cultivation method for an early-fruiting *Pleurotus ostreatus* strain, obtained by cultivating an early-fruiting *Pleurotus ostreatus* strain.

[0062] The cultivation method for the early-bearing *Tetracentron sinense* strain includes:

[0063] Take out the hybrid strain of *Pleurotus ostreatus* and its parent strain, transfer them to PDA medium in a clean bench, seal them and place them in a constant temperature incubator for static cultivation in the dark until the mycelium covers the plate, then remove them for use. The culture medium formula includes: 51-52% corn cob, 21-22% cottonseed hull, 16.5-17.5% wheat bran, 6-7% corn flour, 0.5-1.5% gypsum, and 0.5-1.5% lime.

[0064] Weigh the raw materials according to the cultivation formula ratio, mix the raw materials evenly, add water and mix well, then mix the mixed culture medium evenly and pack it into medium-sized polypropylene plastic bags. Put the bagged spawn bags into a high-temperature and high-pressure sterilizer for sterilization at 121 ℃ for 2 hours. After sterilization, wait until the temperature drops below 50 ℃ before removing the spawn bags from the high-temperature and high-pressure sterilizer. Place the sterilized spawn bags in a pre-sterilized incubator to cool, and then place the cooled spawn bags into a sterile ultra-clean workbench. Inoculate the prepared spawn bags with an inoculation spatula, and inoculate each spawn bag with spawn blocks.

[0065] After inoculation, place the bags in a constant temperature incubator for light-proof cultivation until the mycelium completely covers the bags. Move the bags into the fruiting room, open the bags and harvest the fruiting bodies. Cut off the plastic film from the upper part of the bag. Harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

[0066] The conditions for fruiting are: temperature set at 24-26 ℃ and relative humidity at 90-95%.

[0067] For example, the above-described technical solutions of the present invention can produce protoplasts and / or spores and / or mycelia and / or fruiting bodies and / or fungal clubs.

[0068] The application of this invention will be further described below with reference to specific data and experimental data.

[0069] Application Example 1: Method for preparing culture medium.

[0070] PDA medium: Weigh 200 g of fresh potatoes, cut them into 1 cm × 1 cm pieces, add distilled water, and boil in an induction cooker for 10-15 minutes, until the potato pieces can be easily mashed with tweezers. After boiling, pour the potatoes into a beaker and filter through four layers of gauze to obtain 1 L of boiled potato water. Weigh 20 g of glucose and 20 g of agar, add them to the filtered potato water, stir well with a glass rod, and then pour into a beaker, bringing the volume to 1000 mL. Dispense into Erlenmeyer flasks, allow the pH to set naturally, and sterilize at 121 ℃ for 20 minutes. Cool to approximately 50 ℃, and in a laminar flow hood, pour the liquid medium into sterile petri dishes until the solution in the petri dishes cools and solidifies. Seal the dishes with plastic wrap for later use.

[0071] PDB medium: Weigh 200 g of fresh potatoes, cut them into 1 cm × 1 cm pieces, add distilled water, and boil in an induction cooker for 10-15 minutes, or until the potato pieces can be easily mashed with tweezers. After boiling, pour the potatoes into a beaker and filter through four layers of gauze to obtain 1 L of boiled potato solution. Weigh 20 g of glucose, add it to the filtered potato water, and stir well with a glass rod while adding. Pour the solution into a beaker and bring the volume to 1000 mL. Dispense the prepared solution into Erlenmeyer flasks, allow the pH to set naturally, sterilize at 121 ℃ for 20 minutes, and cool before use.

[0072] Regeneration medium: Weigh 200 g of fresh potatoes, cut them into 1 cm × 1 cm pieces, add distilled water, and boil in an induction cooker for 10-15 minutes, until the potato pieces can be easily mashed with tweezers. After boiling, pour the potato water into a beaker and filter it through four layers of gauze to obtain 1 L of boiled potato solution. Weigh 20 g of glucose, 20 g of agar, and 109.3 g of mannitol, add them to the filtered potato water, stir well with a glass rod, and pour into a beaker, bringing the volume to 1000 mL. Dispense into Erlenmeyer flasks, allow the pH to set naturally, and sterilize at 121 ℃ for 20 minutes. Cool to approximately 50 ℃, and in a laminar flow hood, pour the liquid culture medium into sterile petri dishes until the solution in the petri dishes cools and solidifies. Seal the dishes with plastic wrap for later use.

[0073] Application Example 2: Protoplast preparation, selecting monokaryotic strains of *Pleurotus ostreatus*.

[0074] The parental strains ND5 and LJH001 were purchased from Jiangxi Licai Edible Fungus Co., Ltd.

[0075] First, protoplasts of *Pleurotus ostreatus* ND5 and LJH001 were prepared. The steps were as follows: *Pleurotus ostreatus* ND5 and LJH001 spawn were inoculated into the center of a disposable Petri dish containing PDA medium. When the mycelium had covered two-thirds of the dish, three solid mycelial blocks (6-10 mm holes) were punched at the edge using a sterile punch. These blocks were then inoculated into PDA medium and cultured for 11 days. The resulting liquid mycelium was filtered through double-layered gauze and collected in 10 mL centrifuge tubes for later use. The obtained mycelium was then enzymatically hydrolyzed using a 2% concentration of lysozyme (purchased from the Guangdong Institute of Microbiology). The water bath hydrolysis temperature was controlled at 29℃-31℃, preferably 29.5℃, and the hydrolysis time was 3.5-4.5 h, preferably 4 h. The protoplast concentration reached 1.07 × 10⁻⁶. 8 The number of protoplasts was 1 × 10⁶ / mL, diluted with 0.6 mol / L mannitol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) osmotic stabilizer solution to a total protoplast count of 1 × 10⁶. 2 The sample was collected at a concentration of 100 cells / mL and spread onto the regeneration medium using a spreader. The medium was then placed in a constant temperature incubator at 25 °C for incubation in the dark, and observed 1-2 times daily.

[0076] When regenerated colonies appear on the regeneration plate, each colony is picked up individually with a syringe and transferred to PDA medium, then incubated separately at 25°C. After 3-5 days of incubation, regenerated hyphae are picked and prepared on glass slides for observation of clamp connections under an inverted microscope. Strains without clamp connections are then transferred back to fresh PDA medium and incubated separately at 25°C for 3-5 days, followed by a second observation to verify the presence of clamp connections. Monokaryotic hyphae without clamp connections are then transferred to fresh PDA medium for further culture. Figure 2 The image shows the hyphae of the ND5 monokaryotic strain without clamp connections. Figure 3 The image shows the hyphae of the LJH001 monokaryotic strain without clamp connections.

[0077] This invention, through protoplast preparation of *Agaricus bisporus* ND5 and LJH001, achieves a protoplast yield of 2.50 × 10⁻⁶ compared to existing methods. 7 The concentration of protoplasts in this invention can reach 1.07 × 10⁻⁶ per mL. 8 A concentration of more than 100 cells / mL demonstrates the advantages of this invention in protoplast preparation.

[0078] Application Example 3: Mushroom strain of Mushroom were obtained by hybridization of Mushroom monokaryotes.

[0079] Step 1: Using *Pleurotus ostreatus* ND5 and LJH001 as parents, monokaryotic strains of ND5 and LJH001 inoculated onto PDA medium were cultured at 25 ℃ for 5-10 days. After 5-10 days, 0.6 cm diameter mycelial blocks were punched out and placed on 60 mm PDA medium for confrontation culture, with the mycelial blocks 3 cm apart. They were then placed in a 25 ℃ incubator in the dark for 10-15 days. As the mycelia on the two mycelial blocks continue to grow, the mycelia at both ends will intertwine to form a groove-like or raised antagonistic line. Three 0.3 cm × 0.3 cm mycelial blocks were picked from the antagonistic line and transferred to a new PDA medium. They were then cultured in a 25 ℃ incubator in the dark for 3-7 days.

[0080] Step 2: Initial screening of hybrid strains of *Pleurotus ostreatus*.

[0081] Because monokaryotic strains exhibit isolated mycelial morphology without clamp connections when observed under a microscope, while hybrid strains show clamp connections in their mycelia upon microscopic examination. Figure 4 The image shows the effect of clamping linkage in the successfully hybridized strain.

[0082] Step 3: SRAP molecular marker analysis of hybrid strains.

[0083] Step 3.1, SRAP primer design.

[0084] me2 (upstream primer sequence 5'-3'): TGAGTCCAAACCGGAGC (SEQ ID NO: 1);

[0085] em3 (downstream primer sequence 5'-3'): GACTGCGTACGAATTGAC (SEQ ID NO: 2);

[0086] Step 3.2, then perform subsequent SRAP-PCR amplification;

[0087] Amplification reaction system: template DNA 1 μL, upstream primer 1 μL, downstream primer 1 μL; 2 × Tap PCRMaster Mix 12.5 μL; ddH2O 9.5 μL;

[0088] Amplification procedure:

[0089] Pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1 min, annealing at 35 ℃ for 1 min, refolding at 72 ℃ for 1 min, 5 cycles; denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min, extension at 72 ℃ for 1 min, 35 cycles; extension at 72 ℃ for 10 min, storage at 12 ℃.

[0090] The primer sequences mentioned above are from the literature: LIU JH, DING FH, SONG HY, et al. Analysis of genetic diversity among Chinese Cyclocybe chaxingu strains using ISSR and SRAP markers [J]. PeerJ, 2022, 10:e14037.

[0091] Step 3.3, horizontal agarose gel electrophoresis;

[0092] After PCR amplification, the electrophoretic pattern was obtained by horizontal agarose gel electrophoresis and photographed. For example... Figure 5 Wherein, P1 is ND5, P2 is LJH001, and 1 represents the strain CXG003 with a short fruiting cycle of the present invention.

[0093] To demonstrate the effectiveness of this invention, a control experiment is needed to verify its advantages: Experiment 1, cultivation experiment of hybrid strain of Mushroom arborescens.

[0094] The spawn from the hybrid strain of *Pleurotus ostreatus* and its parent strain was transferred to PDA medium in a clean bench, sealed, and placed in a dark incubator at 25°C for static cultivation. Once the mycelium had fully colonized the plate, it was removed and ready for use. The substrate formula is as follows:

[0095] The ingredients are: corn cob 51-52%, cottonseed hull 21-22%, wheat bran 16.5-17.5%, corn flour 6-7%, gypsum 0.5-1.5%, and lime 0.5-1.5%. The preferred composition is: corn cob 51.8%, cottonseed hull 21.7%, wheat bran 17.4%, corn flour 6.7%, gypsum 1.2%, and lime 1.2%. The total dry weight is 200 g.

[0096] Weigh the raw materials according to the cultivation formula ratio, mix them evenly, then add water and mix thoroughly. The moisture content of the substrate bags should be controlled at around 60%. The moisture content is ready when, when squeezed tightly by hand, water droplets should remain suspended without dripping. Mix the prepared substrate evenly and fill it into medium-sized polypropylene plastic bags (14 × 28 cm), pressing firmly with your fist as you fill until the substrate is slightly springy. Seal the bags with a ring seal. The weight of each bag is 0.5 kg. Place the filled bags into a high-temperature, high-pressure autoclave for sterilization. Before sterilization, check the bags for any damage. Sterilize at 121 ℃ for 2 hours. After sterilization, remove the bags from the autoclave when the temperature drops below 50 ℃. Place the sterilized bags in a pre-sterilized incubator to cool to below 10 ℃, ready for inoculation. Place the cooled inoculum bags into a sterile laminar flow hood and inoculate the prepared inoculum using an inoculation spatula. For example, inoculate each inoculum bag with 5-10 pieces of inoculum measuring 0.5 cm × 0.5 cm. Maintain aseptic operation during the inoculation process.

[0097] After inoculation, place the bags in a constant temperature incubator for cultivation in the dark. The incubator temperature is set to 25 ℃ and the relative humidity to 70%. In the early stages of cultivation, the mycelium begins to germinate slowly in the bags. After about a week of mycelial germination, turn the bags over to check for contamination. Dispose of any contaminated bags promptly to prevent further contamination. Cultivation should be carried out in the dark until the mycelium completely covers the bags. Transfer the bags to the fruiting room and wait for primordia differentiation before opening the bags to harvest the fruiting bodies. Fruiting conditions are: temperature set at 24-26 ℃, relative humidity controlled at 90-95%, and diffused light. When opening the bags to harvest, cut off the plastic film from the upper part of the bag to prevent water accumulation and ensure the survival rate of the small mushroom buds. Harvest when the fruiting bodies have grown to a point where the edge of the cap is lighter in color and the membrane under the cap is about to break.

[0098] Experimental Example 2: Determination of agronomic traits of hybrid strains of *Pleurotus ostreatus*.

[0099] Determination of fruiting time of Mushroom arborescens: The fruiting time of the first batch of mushrooms is the interval between the inoculation of the strain into the spawn bag and the first harvest of Mushroom arborescens fruiting bodies.

[0100] Yield determination of *Pleurotus ostreatus*: Weigh and record the fresh weight of the fruiting bodies grown on the first batch of *Pleurotus ostreatus* spawn bags. See Table 1; calculate the biological efficiency of *Pleurotus ostreatus* according to the formula. Biological efficiency = (Fresh weight of one batch of mushrooms / Dry weight of substrate) × 100%.

[0101] Table 1 Yield of Mushroom Monokaryotic Hybrids

[0102]

[0103] Experiments show that the existing technology for mushroom cultivation (Tea Leaf Mushroom) has a fruiting period of about 50-60 days. This invention achieves a fruiting period earlier than the existing technology, and the biological efficiency of the first batch of mushrooms produced by this invention is significantly improved compared to the existing technology. Furthermore, the culture medium formula used in the existing technology is: cottonseed hulls 63.8%, wheat bran 21%, peanut cake 8%, corn flour 5%, gypsum 1.4%, and lime 0.8%. Through screening and improvements to different culture media and cultivation processes, this invention achieves a protoplast concentration of 1.07 × 10⁻⁶ during the breeding process. 8 The number of cells / mL improved the breeding method of Mushroom arborescens and increased breeding efficiency.

[0104] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0105] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention and within the spirit and principles of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An early-bearing strain of *Tetracentron sinense*, characterized in that, The early-bearing *Tea spore* strain is CXG003, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251057, deposited on May 14, 2025, and classified as follows: Cyclocybe chaxingu .

2. The application of the early-fruiting *Tea fern* strain of claim 1 in the breeding of early-fruiting hybrid *Tea fern*.

3. A cultivation method for an early-bearing *Pleurotus ostreatus* strain, characterized in that, The early-bearing *Tea spore* strain of claim 1 was cultivated.

4. The cultivation method of the early-bearing *Tetracentron sinense* strain according to claim 3, characterized in that, The cultivation method for the early-bearing *Tetracentron sinense* strain includes: The *Pleurotus ostreatus* strain described in claim 1 was taken out and transferred to PDA medium in a clean bench. It was then sealed and placed in a constant temperature incubator for static cultivation in the dark until the mycelium covered the plate. The culture medium formula includes: 51-52% corn cob, 21-22% cottonseed hulls, 16.5-17.5% wheat bran, 6-7% corn flour, 0.5-1.5% gypsum, and 0.5-1.5% lime. Weigh the raw materials according to the cultivation material formula ratio, mix the raw materials evenly, add water and mix well, then mix the mixed culture material evenly and put it into medium-sized polypropylene plastic bags. Put the bagged mushroom bags into a high-temperature and high-pressure sterilizer for sterilization at 121℃ for 2 hours. After sterilization, wait until the temperature drops below 50℃ and then take the mushroom bags out of the high-temperature and high-pressure sterilizer. Place the sterilized mushroom bags in a pre-sterilized incubator to cool, and then put the cooled mushroom bags into a sterile ultra-clean workbench. Inoculate the prepared tea tree mushroom spawn with an inoculation spatula, and inoculate each mushroom bag with a block of mycelium. After inoculation, place the bags in a constant temperature incubator for light-proof cultivation until the mycelium completely covers the bags. Move the bags into the fruiting room, open the bags and harvest the fruiting bodies. Cut off the plastic film from the upper part of the bag. Harvest when the fruiting bodies grow to the edge of the cap and the film under the cap is about to break.

5. The cultivation method of the early-bearing *Tetracentron sinense* strain according to claim 4, characterized in that, The conditions for mushroom growth are: temperature set at 24-26℃ and relative humidity at 90-95%.

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