Ganoderma leukotrichum basidiospore germination culture solution and application thereof
Through the combination of malt extract, yeast extract, glucose and acetic acid with specific ratios, the problem of difficult germination of ganoderma lucidum spores is solved, and efficient germination of ganoderma lucidum spores of ganoderma lucidum spores is achieved, which improves breeding potential.
Patent Information
- Application Number
- CN202510154654.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-12
- Publication Date
- 2025-08-12
AI Technical Summary
The prior art is difficult to effectively germinate the spores of white-meat Ganoderma lucidum, which limits the genetic diversity and breeding efficiency of white-meat Ganoderma lucidum breeding.
A germination culture medium for ganoderma lucidum spores is provided, consisting of malt extract, yeast extract, glucose and acetic acid. The germination of ganoderma lucidum spores is promoted through specific proportions and treatment methods.
The germination rate of ganoderma lucidum spores has been significantly improved to 5.20%, simplified the germination process and reduced costs.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of edible fungus breeding, and in particular relates to a basidiospore germination culture solution of white-fleshed Ganoderma lucidum and application thereof. Background Art
[0002] White Ganoderma belongs to the Basidiomycota, Hymenomycetes, Polyporaces, Ganodermaceae, and Ganoderma genus. It has a mild and warm nature, is non-toxic, slightly bitter, and has no side effects. It is primarily used to tonify qi and calm the mind, relieve cough and asthma, combat hypoxia, and fight aging.
[0003] As a species of the genus Ganoderma, white-fleshed Ganoderma also exhibits tetrapolar heterothallic assimilation, meaning that two different nuclei coexist within the hyphae of a single cell in order to undergo reproductive growth and form a fruiting body. Common breeding methods for white-fleshed Ganoderma include hybridization, but the genetic diversity among currently used white-fleshed Ganoderma strains is low. The probability of superparental hybridization in hybrids, mutagenized strains, or fusions derived from these parental materials through hybridization, mutagenesis, or protoplast fusion is very low, severely impacting breeding efficiency and the development of high-yield, high-quality white-fleshed Ganoderma varieties. Basidiospores of white-fleshed Ganoderma are mononuclear reproductive cells produced through meiosis in the late stages of growth and development. As such, they possess rich genetic diversity, providing excellent material for the breeding of new white-fleshed Ganoderma varieties. However, ungerminated white-fleshed Ganoderma basidiospores cannot generate primary hyphae, making hybridization, protoplast fusion, or mutagenesis ineffective for breeding white-fleshed Ganoderma varieties. White Ganoderma spores have a double wall, making them very tough and difficult to break with conventional chemical and physical methods. They are also extremely difficult to germinate in nature, and the germination rate is extremely low under laboratory conditions, which limits the selection and breeding of new varieties of white Ganoderma.
[0004] Although the use of protoplast uninucleation technology can solve the problem of the extremely low germination rate of basidiospores of white-fleshed Ganoderma lucidum and the difficulty in obtaining uninucleated strains, the uninucleated strains obtained through protoplast uninucleation technology do not undergo meiosis, resulting in a low genetic diversity of the obtained uninucleated strains. Therefore, it is difficult to obtain new strains with extensive variation through protoplast uninucleation technology.
[0005] In order to solve the problem of white-fleshed Ganoderma lucidum breeding, especially laboratory breeding, it is necessary to develop a new white-fleshed Ganoderma lucidum basidiospore germination culture medium and a white-fleshed Ganoderma lucidum basidiospore germination method. Summary of the Invention
[0006] The present invention first provides a basidiospore germination culture solution of white-fleshed Ganoderma lucidum, which is composed of the following components:
[0007] Each 100 mL of the germination culture solution includes: 0.2-0.5 g of malt extract, 0.02-0.08 g of yeast extract, 0.02-0.06 g of glucose, 100-200 μL of acetic acid, and distilled water to 100 mL;
[0008] Wherein, per 100 mL of the germination culture medium, malt extract is 0.2-0.5 g, for example, 0.2 g, 0.3 g, 0.4 g or 0.5 g, preferably 0.3-0.4 g; yeast extract is 0.02-0.08 g, for example, 0.02 g, 0.03 g, 0.04 g, 0.05 g, 0.06 g, 0.07 g or 0.08 g, preferably 0.04-0.06 g; glucose is 0.02 -0.06g, for example, it can be 0.02g, 0.03g, 0.04g, 0.05g or 0.06g, preferably 0.04-0.05g; 100-200μL of acetic acid, for example, it can be 100μL, 110μL, 120μL, 130μL, 140μL, 150μL, 160μL, 170μL, 180μL, 190μL or 200μL, preferably 140-160μL.
[0009] The preferred culture medium for germination of basidiospores of white-fleshed Ganoderma lucidum of the present invention is composed of the following components:
[0010] Each 100 mL of the germination culture solution includes: 0.3-0.4 g of malt extract, 0.04-0.06 g of yeast extract, 0.04-0.05 g of glucose, 140-160 μL of acetic acid, and distilled water to 100 mL.
[0011] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture medium of the present invention comprises the following steps:
[0012] Malt extract, yeast extract, glucose and distilled water were mixed according to the formula amount and sterilized at 121° C. for 15 minutes, and then the formula amount of acetic acid was added.
[0013] The present invention also provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum, the method comprising the following steps:
[0014] After the white-fleshed Ganoderma lucidum basidiospore suspension is shaken and cultured in the germination culture medium for 2-4 days, the culture is aspirated for dilution and spread on a PDA plate to ensure that the spore concentration on each PDA plate is 500-1000. The plate is then cultured in the dark at 20-26° C. until the white-fleshed Ganoderma lucidum basidiospores germinate.
[0015] Preferably, the temperature of the dark culture is 20-26°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or 26°C, preferably 23-24°C.
[0016] The present invention aims to provide a basidiospore germination culture solution of white-fleshed Ganoderma lucidum and a germination method of the basidiospore of white-fleshed Ganoderma lucidum. The basidiospore germination culture solution of white-fleshed Ganoderma lucidum can significantly improve the germination rate of the basidiospore of white-fleshed Ganoderma lucidum, and the composition of the basidiospore germination culture solution of white-fleshed Ganoderma lucidum is simple and the cost is low. The germination method using the basidiospore germination culture solution of white-fleshed Ganoderma lucidum is simple and easy.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] (1) The present invention provides a method for increasing the germination rate of the basidiospores of the white-fleshed Ganoderma lucidum to 5.20% by adding an appropriate amount of acetic acid to the germination culture medium of the white-fleshed Ganoderma lucidum basidiospores, so that the acetic acid, malt extract, glucose, and yeast extract cooperate with each other;
[0019] (2) The composition of the basidiospore germination culture medium of white-fleshed Ganoderma lucidum provided by the present invention is simple, and the method of germinating white-fleshed Ganoderma lucidum basidiospores using the basidiospore germination culture medium is simple and easy, and can be widely used.
[0020] The malt extract, yeast extract, glucose and the like in the basidiospore germination culture solution of white-fleshed Ganoderma lucidum provided by the present invention provide necessary nutrients such as carbon and nitrogen sources for the germination of the white-fleshed Ganoderma lucidum basidiospores. The white-fleshed Ganoderma lucidum basidiospores have tough two-layer walls, which are difficult to break by conventional physical and chemical methods. However, the addition of acetic acid at a specific content in the present invention can destroy the walls of the white-fleshed Ganoderma lucidum basidiospores and promote the absorption of nutrients by the white-fleshed Ganoderma lucidum basidiospores. A lower content of acetic acid cannot activate the cell membranes of the white-fleshed Ganoderma lucidum basidiospores, while a higher content of acetic acid is likely to damage the white-fleshed Ganoderma lucidum basidiospores, thereby reducing the germination rate of the white-fleshed Ganoderma lucidum basidiospores. The malt extract, yeast extract, glucose and acetic acid of the specific composition of the present invention synergize with each other to jointly improve the germination rate of the white-fleshed Ganoderma lucidum basidiospores. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a diagram showing the formation of a monokaryotic strain from the basidiospores of Ganoderma lucidum described in Application Example 1 after germination in the germination culture medium. DETAILED DESCRIPTION
[0022] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.
[0023] Malt extract (product number LP0039B, purchased from Oxiod);
[0024] Yeast extract (product number LP0021B, purchased from Oxiod);
[0025] White-fleshed Ganoderma leucocontextum was collected from Milin County, Tibet.
[0026] Example 1
[0027] This embodiment provides a germination culture medium for basidiospores of white-fleshed Ganoderma lucidum, wherein each 100 mL of the germination culture medium comprises the following components: 0.35 g of malt extract, 0.05 g of yeast extract, 0.045 g of glucose, distilled water to 100 mL, and 150 μL of acetic acid;
[0028] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture liquid is as follows: malt extract, yeast extract, glucose and distilled water are mixed according to the formula amount, sterilized at 121° C. for 15 minutes, and then glacial acetic acid is added in the formula amount to obtain the white-fleshed Ganoderma lucidum basidiospore germination culture liquid with a volume of 100 mL.
[0029] Example 2
[0030] This embodiment provides a germination culture medium for basidiospores of white-fleshed Ganoderma lucidum, wherein each 100 mL of the germination culture medium comprises the following components: 0.3 g of malt extract, 0.04 g of yeast extract, 0.04 g of glucose, distilled water to 100 mL, and 140 μL of acetic acid;
[0031] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture liquid is as follows: malt extract, yeast extract, glucose and distilled water are mixed according to the formula amount, sterilized at 121° C. for 15 minutes, and then glacial acetic acid is added in the formula amount to obtain the white-fleshed Ganoderma lucidum basidiospore germination culture liquid with a volume of 100 mL.
[0032] Example 3
[0033] This embodiment provides a germination culture medium for basidiospores of white-fleshed Ganoderma lucidum, wherein each 100 mL of the germination culture medium comprises the following components: 0.4 g of malt extract, 0.06 g of yeast extract, 0.05 g of glucose, distilled water to 100 mL, and 160 μL of acetic acid;
[0034] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture liquid is as follows: malt extract, yeast extract, glucose and distilled water are mixed according to the formula amount, sterilized at 121° C. for 15 minutes, and then glacial acetic acid is added in the formula amount to obtain the white-fleshed Ganoderma lucidum basidiospore germination culture liquid with a volume of 100 mL.
[0035] Example 4
[0036] This embodiment provides a germination culture solution for basidiospores of white-fleshed Ganoderma lucidum, wherein each 100 mL of the germination culture solution comprises the following components: 0.2 g of malt extract, 0.02 g of yeast extract, 0.02 g of glucose, distilled water to 100 mL, and 100 μL of acetic acid;
[0037] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture liquid is as follows: malt extract, yeast extract, glucose and distilled water are mixed according to the formula amount, sterilized at 121° C. for 15 minutes, and then glacial acetic acid is added in the formula amount to obtain the white-fleshed Ganoderma lucidum basidiospore germination culture liquid with a volume of 100 mL.
[0038] Example 5
[0039] This embodiment provides a germination culture solution for basidiospores of white-fleshed Ganoderma lucidum, wherein each 100 mL of the germination culture solution comprises the following components: 0.5 g of malt extract, 0.08 g of yeast extract, 0.06 g of glucose, distilled water to 100 mL, and 200 μL of acetic acid;
[0040] The preparation method of the white-fleshed Ganoderma lucidum basidiospore germination culture liquid is as follows: malt extract, yeast extract, glucose and distilled water are mixed according to the formula amount, sterilized at 121° C. for 15 minutes, and then glacial acetic acid is added in the formula amount to obtain the white-fleshed Ganoderma lucidum basidiospore germination culture liquid with a volume of 100 mL.
[0041] Comparative Example 1
[0042] This comparative example provides a basidiospore germination culture solution of Ganoderma lucidum, which is the same as Example 1 except that acetic acid is not added to every 100 mL of the basidiospore germination culture solution of Ganoderma lucidum.
[0043] Comparative Example 2
[0044] This comparative example provides a basidiospore germination culture solution of Ganoderma lucidum, which is the same as Example 1 except that the content of acetic acid in every 100 mL of the basidiospore germination culture solution is 50 μL.
[0045] Comparative Example 3
[0046] This comparative example provides a culture medium for germinating basidiospores of Ganoderma lucidum, which is the same as that of Example 1 except that the content of acetic acid in every 100 mL of the culture medium is 250 μL.
[0047] Application Example 1
[0048] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. The steps of the method are as follows: adding the basidiospores of white-fleshed Ganoderma lucidum to the basidiospore germination culture medium of white-fleshed Ganoderma lucidum provided in Example 1, and shaking culturing for 2-4 days. Then, 100 μL of the basidiospore suspension of white-fleshed Ganoderma lucidum is aspirated and diluted to ensure that the number of spores coated on each PDA plate is 500-1000, and culturing at 23° C. in the dark.
[0049] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0050] Application Example 2
[0051] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. The steps of the method are as follows: adding the basidiospores of white-fleshed Ganoderma lucidum to the basidiospore germination culture medium of white-fleshed Ganoderma lucidum provided in Example 1, and shaking culturing for 2-4 days. Then, 100 μL of the basidiospore suspension of white-fleshed Ganoderma lucidum is aspirated and diluted to ensure that the number of spores coated on each PDA plate is 500-1000, and culturing at 24° C. in the dark.
[0052] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0053] Application Example 3
[0054] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. The steps of the method are as follows: adding the basidiospores of white-fleshed Ganoderma lucidum to the basidiospore germination culture medium of white-fleshed Ganoderma lucidum provided in Example 1, and shaking culturing for 2-4 days. Then, 100 μL of the basidiospore suspension of white-fleshed Ganoderma lucidum is aspirated and diluted to ensure that the number of spores coated on each PDA plate is 500-1000, and culturing at 20° C. in the dark.
[0055] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0056] Application Example 4
[0057] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. The steps of the method are as follows: adding the basidiospores of white-fleshed Ganoderma lucidum to the basidiospore germination culture medium of white-fleshed Ganoderma lucidum provided in Example 1, and shaking culturing for 2-4 days. Then, 100 μL of the basidiospore suspension of white-fleshed Ganoderma lucidum is aspirated and diluted to ensure that the number of spores coated on each PDA plate is 500-1000, and culturing at 25° C. in the dark.
[0058] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0059] Application Example 5
[0060] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Example 2, the rest is the same as that of Application Example 1.
[0061] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0062] Application Example 6
[0063] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Example 3, the rest is the same as that of Application Example 1.
[0064] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0065] Application Example 7
[0066] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Example 4, the rest is the same as that of Application Example 1.
[0067] The number of monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum in the germination culture medium was observed under a microscope, and the germination rate was calculated.
[0068] Application Example 8
[0069] This application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Example 5, the rest is the same as that of Application Example 1.
[0070] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0071] Application Example 9
[0072] This application example provides a method for germinating basidiospores of Ganoderma lucidum. Except for not performing shaking culture, the rest is the same as that of Application Example 1.
[0073] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0074] Comparative Application Example 1
[0075] This comparative application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Comparative Example 1, the rest is the same as that of Application Example 1.
[0076] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0077] Comparative Application Example 2
[0078] This comparative application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Comparative Example 2, the rest is the same as that of Application Example 1.
[0079] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0080] Comparative Application Example 3
[0081] This comparative application example provides a method for germinating basidiospores of white-fleshed Ganoderma lucidum. Except that the germination culture medium used is the germination culture medium provided in Comparative Example 3, the rest is the same as that of Application Example 1.
[0082] The monokaryotic strains formed by germination of basidiospores of Ganoderma lucidum on PDA plates were observed under a microscope, the colony numbers were counted, and the germination rate was calculated.
[0083] The germination rate of the basidiospores of white meat Ganoderma lucidum is the ratio of the number of monokaryotic strains germinated on the PDA plate to the total number of basidiospores of white meat Ganoderma lucidum uniformly spread on the plate. Taking Application Example 1 as an example, the monokaryotic strains formed by the basidiospores of white meat Ganoderma lucidum spread on the PDA plate after shaking culture are as follows: Figure 1 As shown, the number of monokaryotic strains obtained by germination was 52, the total number of basidiospores of Ganoderma lucidum evenly spread on the DPA plate was 1000, and the germination rate of basidiospores of Ganoderma lucidum was (52 / 1000) × 100% = 5.20%. The germination rates of basidiospores of Ganoderma lucidum in Application Examples 1-9 and Comparative Application Examples 1-3 are shown in Table 1.
[0084] Table 1 Comparison of germination rates of basidiospores of white-fleshed Ganoderma lucidum
[0085]
[0086]
[0087] As shown in Table 1, acetic acid was added to the germination culture solutions provided in Examples 1-5. In Application Examples 1-8, germination of basidiospores of Ganoderma lucidum using the germination culture solutions provided in Examples 1-5 resulted in germination rates of 3.90% to 5.20%. Compared to Example 1, Comparative Example 1, which did not add acetic acid and used the germination culture solution provided in Comparative Application Example 1, achieved a germination rate of 2.44%, significantly lower than the 5.20% achieved in Example 1. Although acetic acid was added to the germination culture solution provided in Comparative Example 2, the acetic acid content was too low compared to Example 1, failing to effectively promote germination of basidiospores of Ganoderma lucidum. When germinating basidiospores of Ganoderma lucidum using the germination culture solution provided in Comparative Application Example 2, the germination rate was 2.89%, significantly lower than the 5.20% achieved in Example 1. Compared to Example 1, the acetic acid content in the germination culture solution provided in Comparative Example 3 was too high, adversely affecting germination of basidiospores of Ganoderma lucidum. When the germination culture medium provided in Comparative Application Example 3 was used to germinate basidiospores of white-fleshed Ganoderma lucidum, the germination rate was 2.00%, which was significantly lower than the 2.44% when the germination culture medium provided in Comparative Application Example 1 was used to germinate basidiospores. This indicates that the amount of acetic acid added and its synergistic effect with other ingredients have a significant effect on the germination rate of basidiospores of white-fleshed Ganoderma lucidum.
[0088] Meanwhile, Application Example 9 was identical to Application Example 1 except that the shaking culture was not performed. However, the germination rate of the basidiospores of Ganoderma lucidum in Application Example 9 was only 3.14%, far lower than the 5.20% of Application Example 1. This indicates that in the present invention, not only is it important to provide a basidiospore germination culture solution for basidiospore germination, but shaking the basidiospores of Ganoderma lucidum is also very important for improving the germination rate of the basidiospores of Ganoderma lucidum.
[0089] In summary, the present invention provides a simple composition for the germination culture medium for basidiospore germination of Ganoderma lucidum. By adding an appropriate amount of acetic acid and subjecting the basidiospores of Ganoderma lucidum to shaking culture, the germination of the basidiospores of Ganoderma lucidum is promoted, and the germination rate of the basidiospores of Ganoderma lucidum is increased to 5.20%. The use of the germination culture medium for germinating the basidiospores of Ganoderma lucidum is simple to operate and is easy to promote and apply.
[0090] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.
Claims
1. A basidiospore germination culture medium of white flesh Ganoderma lucidum, characterized in that It consists of the following components: Each 100 mL of the germination culture solution includes: 0.2-0.5 g of malt extract, 0.02-0.08 g of yeast extract, 0.02-0.06 g of glucose, 100-200 μL of acetic acid, and distilled water to 100 mL.
2. The basidiospore germination culture medium of Ganoderma lucidum according to claim 1, which is composed of the following components: Each 100 mL of the germination culture solution includes: 0.3-0.4 g of malt extract, 0.04-0.06 g of yeast extract, 0.04-0.05 g of glucose, 140-160 μL of acetic acid, and distilled water to 100 mL.
3. A method for germinating basidiospores of white-fleshed Ganoderma lucidum, characterized in that The steps include: After the white-fleshed Ganoderma lucidum basidiospore suspension is shaken and cultured in the germination culture medium for 2-4 days, the culture is aspirated for dilution and spread on a PDA plate to ensure that the spore concentration on each PDA plate is 500-1000. The plate is then cultured in the dark at 20-26° C. until the white-fleshed Ganoderma lucidum basidiospores germinate.
4. The method for germinating basidiospores of Ganoderma lucidum according to claim 3, wherein the temperature of the dark-proof culture is 23-24°C.