Chrysanthemum breeding method

Abstract

The application provides a chrysosporium inosporum breeding method and relates to the technical field of edible mushroom cultivation. The breeding method is as follows: the chrysosporium fruiting body is hung and cultured, and after the spores in the fruiting body are separated out, the spores are moved to a flat plate culture medium to culture the chrysosporium single colony, two chrysosporium single colonies are randomly picked and cross lines are drawn on the same clean flat plate culture medium to carry out hybridization culture, and the liquid strain of phanerochaete chrysosporium is simultaneously introduced as a mycelium growth auxiliary, is evenly smeared on the culture medium, and the ear is induced. The breeding method solves the technical problems of the existing cultivation method, such as complex operation, tedious steps, high requirement for equipment and long cultivation time, and realizes the technical effects of fast spore culture speed, simple operation, low equipment requirement and meeting the needs of factory production.

Description

Technical Field

[0001] This invention relates to the field of edible fungi cultivation technology, specifically to a method for breeding Auricularia auricula-judae spores. Background Technology

[0002] *Auricularia auricula-judae* is a fungus belonging to the family Auriculariaceae and the genus *Auricularia*. The basidiosperm is large, hemispherical to irregularly shaped, and massive, ranging from brain-like to split into several deeply grooved and thick lobes, 3-12 cm in diameter and 2-8 cm high. The hymenium is widespread on the exposed surface, forming a broad, loose area containing the basidia; the probasidium is initially elliptical to ovoid; the mature hypobasidium is spherical, nearly spherical, rarely pear-shaped to have a short stalk-like base, 13-25 μm long and 11.5-19.5 μm wide. *Auricularia auricula-judae* is distributed in high-altitude forests of Fujian, Sichuan, Yunnan, Shaanxi, Shanxi, Guizhou, and Tibet in China, with the highest production in northwestern Yunnan and southeastern Tibet. It grows mostly in summer and autumn, at altitudes of 1800-3200 meters, often solitary or in groups on decaying wood of broad-leaved trees such as those in the Fagaceae and Betulaceae families in broad-leaved forests and mixed coniferous and broad-leaved forests. Golden ear fungus is rich in fat, protein, and trace elements such as phosphorus, sulfur, manganese, iron, magnesium, calcium, and potassium. It is a nutritious tonic and can also be used medicinally. It is warm in nature with a slightly cooling effect, and has a sweet taste. It can resolve phlegm, stop coughing, relieve asthma, regulate qi, and soothe the liver and intestines. It is mainly used to treat lung heat, excessive phlegm, colds with cough, asthma, and high blood pressure.

[0003] Because golden ear fungus has high edible and medicinal value, and wild golden ear fungus is expensive and scarce, artificial cultivation is carried out. Existing golden ear fungus breeding processes typically employ single-spore isolation, where golden ear fungus spores are isolated under a microscope. This method requires high operational skill, involves cumbersome cultivation steps, and is time-consuming, failing to meet practical production needs. Therefore, this paper proposes a simpler golden ear fungus spore breeding method with a shorter cultivation time. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a method for breeding Auricularia auricula-judae spores, which solves the technical problems of high operational requirements, cumbersome cultivation steps, and long cultivation time in existing cultivation methods.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for breeding *Auricularia auricula-judae* spores is described as follows: *Auricularia auricula-judae* fruiting bodies are cultured using a suspension method. After spores are isolated from the fruiting bodies, they are transferred to sterilized screening plates to cultivate single colonies. Two single colonies are randomly selected and intersected on the same sterilized fruiting plate for hybridization. Simultaneously, a liquid inoculum of *Trichoderma harzianum* is inoculated as a mycelial growth aid. This inoculum is evenly spread on a plate or test tube to induce fruiting. The principle of mycelial growth aid is that the mycelium of *Trichoderma harzianum* secretes products that induce *Auricularia auricula-judae* spores to germinate and produce mycelium. The mycelium of *Trichoderma harzianum* absorbs nutrients, providing nutrition for the production of *Auricularia auricula-judae* fruiting bodies.

[0009] The specific steps are as follows:

[0010] S1. Processing of golden ear fruit bodies:

[0011] Select high-quality golden ear fruiting bodies, cut them into 4-5cm cubes, and spray alcohol evenly on the surface of the golden ear fruiting bodies for disinfection.

[0012] S2. Sterilization:

[0013] The culture flask containing distilled water was suspended and sterilized at 121℃ under high pressure and humidity. The distilled water content was 20mL and the culture flask capacity was 500mL.

[0014] S3. Collect spores:

[0015] Disinfect the clean bench by irradiating it with ultraviolet light for 15 minutes. Place the culture bottle and the fruiting body cube of Auricularia auricula-judae on the clean bench. Disinfect the tweezers, pick up the fruiting body cube of Auricularia auricula-judae, hang it on the hook, and suspend it in the culture bottle. Incubate at 20°C in the dark for 2 days and collect the Auricularia auricula-judae spores.

[0016] S4. Spore treatment:

[0017] After collection, remove the cap of the culture bottle and the cube of golden ear fruit in the ultra-clean workbench, and then use a pipette to draw the distilled water containing spores from the bottom onto the sterile screening plate culture medium, and shake it to distribute it evenly.

[0018] S5. Spore culture:

[0019] Place the screening plate culture medium in the dark and incubate at 20°C for 4-5 days; multiple single colonies visible to the naked eye will appear.

[0020] S6. Hybridization-induced ear formation:

[0021] Randomly select a single colony of Auricularia auricula-judae and streak it on a sterile agar plate. Then randomly select another single colony of Auricularia auricula-judae and streak it on the same agar plate, intersecting the previous line. At the same time, inoculate 0.5 ml of Auricularia auricula-judae liquid inoculum, shake well on the agar plate, and induce auricular growth.

[0022] S7. Hybrid Culture:

[0023] Culture at 18-19℃ in the dark for 25-30 days; the appearance of immature ears indicates successful hybridization.

[0024] Preferably, the screening plate culture medium formula in step S4 is: 200g potato, 20g glucose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, 1L water, and 30mg ampicillin.

[0025] Preferably, the formula for the agar plate culture medium in step S6 is: 200g potato, 20g sucrose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, and 1L water.

[0026] Preferably, the preparation method of the liquid culture of *Trichoderma harzianum* in step S6 is as follows: 1) Prepare and sterilize the liquid culture medium, the formula of which is: 20g glucose, 3g yeast powder, 3g peptone, 1g potassium dihydrogen phosphate, and 1g magnesium sulfate; 2) Place the *Trichoderma harzianum* mother culture, the sterilized liquid culture medium, inoculation tools, etc., into a clean bench for ultraviolet sterilization for 15 minutes; 3) Light an alcohol lamp, open the mother culture plate in front of the flame, and use a scalpel to cut 1cm of the plate. 2 4) Stir the mother culture of different sizes and put the chopped mother culture into the liquid culture medium; 5) Stir the liquid culture with magnetic stirring and incubate at 23°C in the dark for 5 days.

[0027] (III) Beneficial Effects

[0028] This invention provides a method for breeding Auricularia auricula-judae spores. Compared with existing technologies, it has the following advantages: Existing methods use single-spore isolation under a microscope, which requires high operational skills, involves cumbersome cultivation steps, and is time-consuming, failing to meet practical production needs. The Auricularia auricula-judae spore breeding method of this invention first directly suspends and cultivates the fruiting bodies of Auricularia auricula-judae. After spore emergence, the spores are collected and then cultured separately on screening plates. New strains are then cultivated through hybridization of different colonies, and fruiting bodies are induced. This significantly shortens the cultivation time, simplifies the operation, and reduces equipment requirements, meeting the needs of practical industrial production. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1This is an operational diagram illustrating the collection of spores in Embodiment 1 of the present invention;

[0031] Figure 2 This is a diagram illustrating the process of picking a single colony for hybridization in Example 1 of the present invention;

[0032] Figure 3 This is a photograph of the actual product of the mixed culture of the liquid bacterial strain for wool and leather in Example 1 of the present invention;

[0033] Figure 4 This is a picture of the primordia that grew after the hybrid strain was inoculated into the original culture bottle following the completion of the cultivation in Example 1 of the present invention.

[0034] Figure 5 This is a photograph of the fruit waiting to ripen according to Embodiment 1 of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This application provides a method for breeding Auricularia auricula-judae spores, which solves the technical problems of existing cultivation methods, such as complex operation, cumbersome steps, high equipment requirements, and long cultivation time. It achieves the technical effects of fast spore cultivation, simple operation, low equipment requirements, and the ability to meet the needs of industrial production.

[0037] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0038] Golden ear fungus: A complex organism containing two species, Auricularia auricula-judae and Auricularia velutipes, such as the fruiting body of Auricularia auricula-judae.

[0039] Auricularia auricula colony: An organism containing only one species of auricularia auricula, existing in the form of hyphae, basidiospores, or yeast-like spores.

[0040] Leatherwort liquid culture: A culture containing only one species, Leatherwort.

[0041] Hybrid strains: Cultures containing two species, Auricularia auricula-judae and Leuciscus spp., in which a parasitic relationship has been established between the two species.

[0042] Example 1:

[0043] A method for breeding Auricularia auricula-judae spores, comprising: suspending and culturing the fruiting bodies of Auricularia auricula-judae; after the spores are isolated from the fruiting bodies, transferring the spores to a sterile screening plate medium to cultivate single colonies of Auricularia auricula-judae; randomly selecting two single colonies of Auricularia auricula-judae and drawing intersecting lines on the same sterile fruiting plate medium for hybridization culture; simultaneously inoculating with liquid culture of Trichoderma harzianum as an aid for mycelial growth; spreading evenly on a plate medium or test tube to induce fruiting.

[0044] The specific steps are as follows:

[0045] S1. Processing of golden ear fruit bodies:

[0046] Select high-quality golden ear fruiting bodies, cut them into 4-5cm cubes, and spray alcohol evenly on the surface of the golden ear fruiting bodies for disinfection.

[0047] S2. Sterilization:

[0048] The culture flask containing distilled water was suspended and sterilized at 121℃ under high pressure and humidity. The distilled water content was 20mL and the culture flask capacity was 500mL.

[0049] S3. Collect spores:

[0050] The laminar flow hood was sterilized by irradiating it with UV light for 15 minutes. The culture flask and *Auricularia auricula-judae* fruiting body cubes were placed on the laminar flow hood. The tweezers were sterilized, and the *Auricularia auricula-judae* fruiting body cubes were picked up, hung on hooks, and lowered into the culture flask. The flasks were incubated at 20°C in the dark for 2 days. *Auricularia auricula-judae* spores were then collected. Figure 1 As shown;

[0051] S4. Spore treatment:

[0052] After collection, remove the cap of the culture bottle and the cube of golden ear fruit in the ultra-clean workbench, and then use a pipette to draw the distilled water containing spores from the bottom onto the sterile screening plate culture medium, and shake it to distribute it evenly.

[0053] The screening plate culture medium formula is as follows: 200g potato, 20g glucose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, 1L water, and 30mg ampicillin.

[0054] S5. Spore culture:

[0055] When the screening plate culture medium is placed in the dark and incubated at 20°C for 4 days, multiple single colonies visible to the naked eye will appear.

[0056] S6. Hybridization-induced ear formation:

[0057] like Figure 2As shown, a single colony of *Auricularia auricula-judae* was randomly selected and streaked onto a sterile agar plate. Then, another single colony of *Auricularia auricula-judae* was randomly selected and streaked onto the same agar plate, intersecting the previous streak. Simultaneously, 0.5 ml of *Trichoderma harzianum* liquid inoculum was inoculated, and the mixture was shaken well on the agar plate to induce ear emergence. Figure 3 As shown;

[0058] The formula for the agar plate culture medium is as follows: 200g potato, 20g sucrose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, and 1L water.

[0059] The preparation method of the liquid culture of *Trichoderma harzianum* is as follows: 1) Prepare and sterilize the liquid culture medium. The liquid culture medium formula is: 20g glucose, 3g yeast powder, 3g peptone, 1g potassium dihydrogen phosphate, and 1g magnesium sulfate; 2) Place the *Trichoderma harzianum* mother culture, the sterilized liquid culture medium, inoculation tools, etc. into a clean bench for ultraviolet sterilization for 15 minutes; 3) Light an alcohol lamp, open the mother culture plate in front of the flame, and use a scalpel to cut 1cm of the plate. 2 4) Place the chopped mother culture into the liquid culture medium; 5) Stir the liquid culture with magnetic stirring and incubate at 23°C in the dark for 5 days.

[0060] S7. Hybrid Culture:

[0061] like Figure 4 , Figure 5 As shown, after culturing at 18°C ​​in the dark for 30 days, the appearance of young ears indicates successful hybridization.

[0062] Example 2:

[0063] A method for breeding Auricularia auricula-judae spores, comprising: suspending and culturing the fruiting bodies of Auricularia auricula-judae; after the spores are isolated from the fruiting bodies, transferring the spores to a sterile screening plate medium to cultivate single colonies of Auricularia auricula-judae; randomly selecting two single colonies of Auricularia auricula-judae and drawing intersecting lines on the same sterile fruiting plate medium for hybridization culture; simultaneously inoculating with liquid culture of Trichoderma harzianum as an aid for mycelial growth; spreading evenly on a plate medium or test tube to induce fruiting.

[0064] The specific steps are as follows:

[0065] S1. Processing of golden ear fruit bodies:

[0066] Select high-quality golden ear fruiting bodies, cut them into 4-5cm cubes, and spray alcohol evenly on the surface of the golden ear fruiting bodies for disinfection.

[0067] S2. Sterilization:

[0068] The culture flask containing distilled water was suspended and sterilized at 121℃ under high pressure and humidity. The distilled water content was 20mL and the culture flask capacity was 500mL.

[0069] S3. Collect spores:

[0070] Disinfect the clean bench by irradiating it with ultraviolet light for 15 minutes. Place the culture bottle and the fruiting body cube of Auricularia auricula-judae on the clean bench. Disinfect the tweezers, pick up the fruiting body cube of Auricularia auricula-judae, hang it on the hook, and suspend it in the culture bottle. Incubate at 20°C in the dark for 2 days and collect the Auricularia auricula-judae spores.

[0071] S4. Spore treatment:

[0072] After collection, remove the cap of the culture bottle and the cube of golden ear fruit in the ultra-clean workbench, and then use a pipette to draw the distilled water containing spores from the bottom onto the sterile screening plate culture medium, and shake it to distribute it evenly.

[0073] The screening plate culture medium formula is as follows: 200g potato, 20g glucose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, 1L water, and 30mg ampicillin.

[0074] S5. Spore culture:

[0075] When the screening plate culture medium is placed in the dark and incubated at 20°C for 5 days, multiple single colonies visible to the naked eye will appear.

[0076] S6. Hybridization-induced ear formation:

[0077] Randomly select a single colony of *Auricularia auricula-judae* and streak it onto a sterile agar plate. Then, randomly select another single colony of *Auricularia auricula-judae* and streak it onto the same agar plate, intersecting the previous streak. Simultaneously, inoculate 0.5 ml of *Trichoderma harzianum* liquid inoculum, shake well on the agar plate, and induce ear growth.

[0078] The formula for the agar plate culture medium is as follows: 200g potato, 20g sucrose, 1g potassium dihydrogen phosphate, 1g magnesium sulfate, 1g peptone, 1g yeast powder, 20g agar, and 1L water.

[0079] The preparation method of the liquid culture of *Trichoderma harzianum* is as follows: 1) Prepare and sterilize the liquid culture medium. The liquid culture medium formula is: 20g glucose, 3g yeast powder, 3g peptone, 1g potassium dihydrogen phosphate, and 1g magnesium sulfate; 2) Place the *Trichoderma harzianum* mother culture, the sterilized liquid culture medium, inoculation tools, etc. into a clean bench for ultraviolet sterilization for 15 minutes; 3) Light an alcohol lamp, open the mother culture plate in front of the flame, and use a scalpel to cut 1cm of the plate. 2 4) Place the chopped mother culture into the liquid culture medium; 5) Stir the liquid culture with magnetic stirring and incubate at 23°C in the dark for 5 days.

[0080] S7. Hybrid Culture:

[0081] Cultured at 19℃ in the dark for 25 days, the appearance of immature ears indicates successful hybridization.

[0082] Experimental data

[0083] Table 1 below shows the relevant data on cultivation time and maturation time, as well as the description of the quality of golden ear fungus in the preparation method of Example 1.

[0084] Table 1 - Relevant experimental data and quality of the preparation method in Example 1

[0085] Test data Example 1 Average incubation time (d) 40 Time to full maturity (d) 40 Mature quality The tablet contains small, white or transparent, young golden ear fruiting bodies that are visible to the naked eye.

[0086] 1. Existing methods for isolating spores under a microscope using single spores are complex, cumbersome, and require sophisticated equipment, resulting in long cultivation times that cannot meet practical production needs. The present invention's *Auricularia auricula-judae* spore breeding method first directly suspends and cultivates *Auricularia auricula-judae* fruiting bodies. After spore emergence, the spores are collected and then cultured separately on agar plates. New strains are then cultivated through hybridization of different colonies, and fruiting bodies are induced. This significantly shortens the cultivation time, simplifies the operation, and reduces equipment requirements, thus meeting the needs of actual industrial production.

[0087] 2. The present invention further improves the formulation of the culture medium, which is more conducive to the growth of colonies and strains.

[0088] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0089] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for breeding Auricularia auricula-judae spores, characterized in that, The specific steps are as follows: S1. Processing of golden ear fruit bodies: Select high-quality golden ear fruiting bodies, cut them into 4-5cm cubes, and disinfect the surface. S2. Sterilization: The culture flasks containing distilled water were suspended and sterilized under high pressure and humidity at 121°C. S3. Collect spores: Disinfect the clean bench, place the culture bottle and the Auricularia auricula fruiting body cube on the clean bench, disinfect the tweezers, pick up the Auricularia auricula fruiting body cube, hang it on the hook, and suspend it in the culture bottle. Incubate at 20℃ in the dark for 2 days, and collect Auricularia auricula spores. S4. Spore treatment: After collection, remove the cap of the culture bottle and the cube of golden ear fruit in the ultra-clean workbench, and then use a pipette to draw the distilled water containing spores from the bottom onto the sterile screening plate culture medium, and shake it to distribute it evenly. S5. Spore culture: Place the screening plate culture medium in the dark and incubate at 20°C for 4-5 days; multiple single colonies visible to the naked eye will appear. S6. Hybridization-induced ear formation: Randomly select a single colony of Auricularia auricula-judae and streak it on a sterile agar plate. Then randomly select another single colony of Auricularia auricula-judae and streak it on the same agar plate, intersecting the previous line. At the same time, inoculate 0.5 ml of Auricularia auricula-judae liquid inoculum, shake well on the agar plate, and induce auricular growth. S7. Hybrid Culture: Culture at 18-19℃ in the dark for 25-30 days; the appearance of young ears indicates successful hybridization.

2. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The screening plate culture medium formula in step S4 is as follows: 200 g potato, 20 g glucose, 1 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 1 g peptone, 1 g yeast powder, 20 g agar, 1 L water, and 30 mg ampicillin.

3. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The formula for the agar plate culture medium in step S6 is as follows: 200 g potato, 20 g sucrose, 1 g potassium dihydrogen phosphate, 1 g magnesium sulfate, 1 g peptone, 1 g yeast powder, 20 g agar, and 1 L water.

4. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The preparation method of the liquid culture of *Trichoderma harzianum* in step S6 is as follows: 1) Prepare and sterilize the liquid culture medium; 2) Place the *Trichoderma harzianum* mother culture, the sterilized liquid culture medium, and the inoculation tools into a clean bench for ultraviolet sterilization for 15 minutes; 3) Light an alcohol lamp, open the mother culture plate in front of the flame, and use a scalpel to cut 1 cm of the plate. 2 4) Stir the mother culture of different sizes and put the chopped mother culture into the liquid culture medium; 5) Stir the liquid culture with magnetic stirring and incubate at 23°C in the dark for 5 days.

5. The method for breeding Auricularia auricula-judae spores as described in claim 4, characterized in that, The liquid culture medium formula is as follows: 20 g glucose, 3 g yeast powder, 3 g peptone, 1 g potassium dihydrogen phosphate, and 1 g magnesium sulfate.

6. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The disinfection method in step S1 is as follows: uniformly spray alcohol onto the surface of the fruiting body of Auricularia auricula-judae for disinfection.

7. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The distilled water content in step S2 is 20 mL, and the culture flask capacity is 500 mL.

8. The method for breeding Auricularia auricula-judae spores as described in claim 1, characterized in that, The disinfection method for the clean bench in step S3 is as follows: disinfection is performed by irradiating with ultraviolet light for 15 minutes.

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