Tremella aurantialba spore cultivation method based on single colony streak cross breeding
The single-colony streak hybridization breeding method simplifies the steps of Auricularia auricula-judae spore cultivation, shortens the cultivation time, and solves the problems of complex operation and long time in the existing technology, achieving the effect of simplified operation and rapid cultivation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DONGZHI HESHUISHAN TECH CO LTD
- Filing Date
- 2024-01-30
- Publication Date
- 2026-05-12
AI Technical Summary
Existing methods for breeding and cultivating golden ear fungus have high operational requirements, complicated steps, and long cultivation time, which cannot meet the actual production needs.
A single-colony streak hybridization breeding method was adopted. The fruiting bodies of Auricularia auricula were cultured in suspension, and the spores were collected and cultured into single colonies on screening plates. These colonies were then hybridized with liquid strains of Mycorrhizal trichomoniasis to induce fruiting. This method simplifies the operation steps and shortens the cultivation time.
It has achieved a method for cultivating Auricularia auricula-judae spores that is simple to operate, requires low equipment, has a short cultivation time, and meets the needs of industrialized production.
Smart Images
Figure CN122004087A_ABST
Abstract
Description
[0001] This application is a divisional application of the invention application filed on January 30, 2024, with Chinese application number 2024101332830 and entitled "Method for Breeding Golden Ear Spores". Technical Field
[0002] This invention relates to the field of edible fungi cultivation technology, specifically to a method for cultivating Auricularia auricula spores based on single-colony streak hybridization breeding. Background Technology
[0003] *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.
[0004] 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 and cultivation 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 and faster method for golden ear fungus spore cultivation based on single-colony streak hybridization breeding. Summary of the Invention
[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for cultivating Auricularia auricula spores based on single-colony streak hybridization breeding, which solves the technical problems of high operational requirements, cumbersome cultivation steps, and long cultivation time in existing cultivation methods.
[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: The method for cultivating Auricularia auricula-judae spores based on single-colony streak hybridization breeding involves the following steps: S1. Obtain surface-sterilized golden ear fruiting body blocks; S2. Place the culture flask containing distilled water in a high-temperature autoclave at 121℃; S3. After picking up the fruiting body of Auricularia auricula-judae and hanging it on the hook, put it into a culture bottle and culture it at 20°C in the dark for 2 days to collect the Auricularia auricula-judae spores; S4. Pour the distilled water containing spores from the bottom of the culture flask onto a sterile screening plate medium and shake to distribute it evenly. S5. Place the screening plate culture medium in the dark at 20°C for 4-5 days to obtain multiple single colonies that are visible to the naked eye; S6. Randomly select two single colonies of Auricularia auricula-judae and draw intersecting lines on the same sterile agar plate for hybridization culture. At the same time, inoculate with liquid culture of Auricularia auricula-judae and shake well on the agar plate to induce ear growth. Culture S7 at 18~19℃ in the dark for 25~30 days; the appearance of young ears indicates successful hybridization.
[0007] Preferably, the screening plate culture medium formula in step S4 is: 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.
[0008] Preferably, the formula for the agar plate culture medium in step S6 is: 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.
[0009] 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: 20 g glucose, 3 g yeast powder, 3 g peptone, 1 g potassium dihydrogen phosphate, and 1 g 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 1 cm 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.
[0010] (III) Beneficial Effects This invention provides a method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding. 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 cultivation method of this invention first directly suspends and cultivates *Auricularia auricula-judae* fruiting bodies. After spore emergence, the spores are collected and then cultured separately on screening plates. New strains are then cultivated through different colony hybridization breeding methods, 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
[0011] 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.
[0012] Figure 1 This is an operational diagram illustrating the collection of spores in Embodiment 1 of the present invention; Figure 2 This is a diagram illustrating the process of picking a single colony for hybridization in Example 1 of the present invention; 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; 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. Figure 5 This is a photograph of the fruit waiting to ripen according to Embodiment 1 of the present invention. Detailed Implementation
[0013] 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.
[0014] This application provides a method for cultivating Auricularia auricula spores based on single-colony streak hybridization breeding, 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 can meet the needs of industrial production.
[0015] 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.
[0016] Golden ear fungus: A complex organism containing two species, Auricularia auricula-judae and Leuciscus fusiforme, such as the fruiting body of Auricularia auricula-judae.
[0017] Auricularia auricula colony: An organism containing only one species of auricularia auricula, existing in the form of hyphae, basidiospores, or yeast-like spores.
[0018] Leatherwort liquid culture: A culture containing only one species, Leatherwort.
[0019] Hybrid strains: Cultures containing two species, Auricularia auricula-judae and Leuciscus spp., in which a parasitic relationship has been established between the two species.
[0020] Example 1: A method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding is as follows: *Auricularia auricula-judae* fruiting bodies are cultured by suspension. After spores are isolated from the fruiting bodies, the spores are transferred to sterilized screening plates to cultivate single colonies. Two single colonies are randomly selected and streaked on the same sterilized fruiting plate for hybridization. Simultaneously, liquid inoculum of *Trichoderma harzianum* is introduced as a mycelial growth aid. The inoculum is evenly spread on a plate or test tube to induce fruiting.
[0021] 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 evenly spray alcohol on the surface of the golden ear fruiting bodies for disinfection. S2. Sterilization: The culture flask containing distilled water was suspended and sterilized at 121 °C under high pressure and humidity. The distilled water content was 20 mL and the culture flask capacity was 500 mL. S3. Collect spores: 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; 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. The screening plate culture medium formula 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. S5. Spore culture: 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. S6. Hybridization-induced ear formation: like Figure 2 As 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; The formula for the agar plate culture medium 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. 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: 20 g glucose, 3 g yeast powder, 3 g peptone, 1 g potassium dihydrogen phosphate, and 1 g 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 1 cm 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. S7. Hybrid Culture: like Figure 4 , Figure 5 As shown, after culturing at 18 ℃ in the dark for 30 days, the appearance of immature ears indicates successful hybridization.
[0022] Example 2: A method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding is as follows: *Auricularia auricula-judae* fruiting bodies are cultured by suspension. After spores are isolated from the fruiting bodies, the spores are transferred to sterilized screening plates to cultivate single colonies. Two single colonies are randomly selected and streaked on the same sterilized fruiting plate for hybridization. Simultaneously, liquid inoculum of *Trichoderma harzianum* is introduced as a mycelial growth aid. The inoculum is evenly spread on a plate or test tube to induce fruiting.
[0023] 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 evenly spray alcohol on the surface of the golden ear fruiting bodies for disinfection. S2. Sterilization: The culture flask containing distilled water was suspended and sterilized at 121 °C under high pressure and humidity. The distilled water content was 20 mL and the culture flask capacity was 500 mL. S3. Collect spores: Disinfect the clean bench by irradiating it with a UV lamp 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 ℃ in the dark for 2 days and collect the Auricularia auricula-judae 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. The screening plate culture medium formula 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. S5. Spore culture: 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. S6. Hybridization-induced ear formation: 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 line. Simultaneously, inoculate 0.5 ml of *Trichoderma harzianum* liquid inoculum, shake well on the agar plate, and induce ear growth. The formula for the agar plate culture medium 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. 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: 20 g glucose, 3 g yeast powder, 3 g peptone, 1 g potassium dihydrogen phosphate, and 1 g 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 1 cm 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. S7. Hybrid Culture: Cultured at 19 ℃ in the dark for 25 days, the appearance of immature ears indicates successful hybridization.
[0024] Experimental data 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.
[0025] Table 1 - Relevant experimental data and quality of the preparation method in Example 1 1. Existing methods for isolating spores under a microscope using single-spore isolation are complex, cumbersome, and require sophisticated equipment, resulting in long cultivation times that cannot meet practical production needs. The present invention provides a method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding. First, *Auricularia auricula-judae* fruiting bodies are directly cultured in suspension. After spore emergence, the spores are collected and then cultured separately on agar plates. New strains are then cultivated through hybridization breeding of different colonies, and fruiting bodies are induced. This significantly shortens the cultivation time, simplifies operation, and reduces equipment requirements, thus meeting the needs of practical industrial production. 2. The present invention further improves the formulation of the culture medium, which is more conducive to the growth of colonies and strains.
[0026] 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.
[0027] 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 cultivating Auricularia auricula-judae spores based on single-colony streak hybridization breeding, characterized in that, The specific steps are as follows: S1. Obtain surface-sterilized golden ear fruiting body blocks; S2. Place the culture flask containing distilled water in a high-temperature autoclave at 121℃; S3. After picking up the fruiting body of Auricularia auricula-judae and hanging it on the hook, put it into a culture bottle and culture it at 20°C in the dark for 2 days to collect the Auricularia auricula-judae spores; S4. Pour the distilled water containing spores from the bottom of the culture flask onto a sterile screening plate medium and shake to distribute it evenly. S5. Place the screening plate culture medium in the dark at 20°C for 4-5 days to obtain multiple single colonies that are visible to the naked eye; S6. Randomly select two single colonies of Auricularia auricula-judae and draw intersecting lines on the same sterile agar plate for hybridization culture. At the same time, inoculate with liquid culture of Auricularia auricula-judae and shake well on the agar plate to induce ear growth. Culture S7 at 18~19℃ in the dark for 25~30 days; the appearance of young ears indicates successful hybridization.
2. The method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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 cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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 cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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, 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 1 cm 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.
5. The method for cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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 cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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 cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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 cultivating *Auricularia auricula-judae* spores based on single-colony streak hybridization breeding 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.