Ganoderma lucidum wild strain breeding method based on ARTP mutagenesis technology

Abstract

The invention discloses a ganoderma lucidum wild strain breeding method based on an ARTP mutagenesis technology. The ganoderma lucidum wild strain breeding method comprises the following steps: obtaining a strain source: performing tissue separation and purification on a wild ganoderma lucidum sporocarp to obtain an original strain; spore liquid preparation: spores of wild ganoderma lucidum are collected, and a spore suspension is prepared; aRTP mutagenesis treatment: carrying out mutagenesis on the spore suspension by using an ARTP mutagenesis breeding instrument; after mutagenesis, immediately inoculating a spore suspension into a solid culture medium, and culturing at 25-30 DEG C for 5-7 days; preliminary screening: screening out mutant strains which grow fast and have dense hyphae according to phenotypic characteristics of colonial morphology and growth speed; and secondary screening: performing liquid fermentation culture on the mutant strain obtained by primary screening, measuring the biomass, polysaccharide content and triterpenoid content of the mutant strain, and screening out an excellent strain with high active substance content. The ARTP mutagenesis technology is utilized to perform mutagenesis breeding on the Ganoderma lucidum wild strain, the mutation rate is high, the operation is simple and convenient, and the safety is good. The strain obtained by the invention has the characteristics of short cultivation period (20-30% shorter than that of an original strain) and high active substance content (the contents of polysaccharide and triterpenoids are both increased by more than 50%), and is suitable for planting and production in a basswood cultivation mode.

Description

Technical Field

[0001] The present invention relates to the field of biological breeding technology, and in particular to a breeding method for obtaining a Ganoderma lucidum strain with a short cultivation period and high active substance content by using ARTP (atmospheric pressure room temperature plasma) mutagenesis technology to induce mutation breeding of wild Ganoderma lucidum strains. Background Art

[0002] Ganoderma lucidum is a fungus with important medicinal properties. Its active ingredients, such as polysaccharides and triterpenoids, possess significant immunomodulatory, anti-tumor, and antioxidant properties. Currently, artificial cultivation of Ganoderma lucidum relies primarily on wild strains, but these strains suffer from long cultivation cycles and unstable active ingredient content, limiting their large-scale production and application.

[0003] Traditional breeding methods, such as natural selection and chemical mutagenesis, suffer from low efficiency and uncontrollable mutation direction. ARTP mutagenesis, a novel physical mutagenesis method, has been widely used in microbial breeding, offering advantages such as high mutation rates, ease of operation, and good safety. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for breeding wild Ganoderma lucidum strains based on ARTP mutagenesis technology, and to obtain Ganoderma lucidum strains with a short cultivation period and high triterpenoid compound content through mutagenesis.

[0005] In order to achieve the above technical purpose, the technical solution of the present invention is:

[0006] The method for breeding wild Ganoderma lucidum strains based on ARTP mutagenesis technology comprises the following steps:

[0007] 1. Strain Source: The original strain was isolated and purified from wild Ganoderma lucidum fruiting bodies. Stored on a solid culture medium slant for future use.

[0008] 2. Preparation of spore solution: Collect wild Ganoderma lucidum spores under sterile conditions, observe by blood cell counting plate, and prepare the solution with a concentration of 10 6 -10 8 Spore suspension with a concentration of 100 μg / mL.

[0009] 3. ARTP Mutagenesis: Use an ARTP mutagenesis instrument to induce mutagenesis on the spore suspension. The mutagenesis conditions are: power 100-150W, air flow 10-15 SLM, and treatment time 80-120 seconds. Immediately after mutagenesis, inoculate the spore suspension into solid culture medium and culture at 25-30°C for 5-7 days.

[0010] 4. Screening of excellent strains:

[0011] 4.1. Primary screening: Based on the colony morphology and growth rate phenotypic characteristics, mutant strains with faster growth and dense mycelium were selected.

[0012] 4.2. Secondary screening: The mutants obtained in the initial screening were cultured in liquid fermentation, and their biomass, polysaccharide and triterpenoid content were determined to screen out excellent strains with high content of active substances.

[0013] The present invention utilizes ARTP mutagenesis technology to induce mutations in wild strains of Ganoderma lucidum, resulting in a high mutation rate, simple operation, and excellent safety. The strains obtained by the present invention have a short cultivation cycle (20%-30% shorter than the original strain) and high active substance content (polysaccharide and triterpenoid content increased by more than 50%), making them suitable for cultivation in basswood cultivation models. This invention provides an excellent strain resource for large-scale cultivation of Ganoderma lucidum and efficient extraction of its active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0015] Figure 1 Comparison of colony morphology (cultured on solid medium at 28°C for 5 days).

[0016] Figure 2 It is a growth curve chart.

[0017] Figure 3 This is a graph showing the content of active substances (polysaccharides).

[0018] Figure 4 This is a graph showing the content of active substances (triterpenoids). DETAILED DESCRIPTION

[0019] Wild Ganoderma lucidum fruiting body: Wild Ganoderma lucidum was collected from Jinzhai area and identified as Ganoderma lucidum.

[0020] Solid culture medium: potato dextrose agar medium (PDA medium), used for strain isolation and preliminary screening.

[0021] Liquid fermentation medium: glucose 20 g / L, peptone 5 g / L, yeast extract 3 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, pH natural.

[0022] ARTP mutagenesis breeding instrument: Wuxi Yuanqing Tianmu Biotechnology Co., Ltd., model ARTP-IIS.

[0023] Detection method: The phenol-sulfuric acid method was used to determine the polysaccharide content, and the vanillin-sulfuric acid method was used to determine the triterpenoid content.

[0024] Example 1

[0025] 1. Tissue Isolation of Wild Ganoderma Lucidum Strains

[0026] Wild Ganoderma lucidum fruiting bodies were disinfected with 75% ethanol, and tissue blocks from the growth rings were aseptically cut into 1 cm cubes. The tissue blocks were inoculated into solid culture medium and cultured at 28°C for 7 days to obtain mycelium, which was named CSLG01.

[0027] 2. Preparation of spore suspension

[0028] The spores of wild Ganoderma lucidum were collected under sterile conditions and the spore concentration was adjusted to 10 using a hemocytometer under a high power microscope. 7 g / mL.

[0029] 3. ARTP mutagenesis

[0030] 3.1 Take 1 mL of spore suspension and place it on the sample stage of the ARTP mutagenesis breeding instrument. Set the mutagenesis conditions as power 120 W, air flow 12 SLM, and treatment time 100 seconds.

[0031] 3.2 Immediately after mutagenesis, the spore suspension was diluted 10-fold, spread on solid culture medium, and cultured at 28°C for 7 days.

[0032] 4. Screening of excellent strains

[0033] 4.1 Initial screening

[0034] 4.1.1 Observe the morphology of the colonies and the growth rate of mycelium after mutagenesis, and select mutants that grow significantly faster than the original strain.

[0035] 4.1.2 Ten mutant strains with faster mycelial growth rates were obtained through preliminary screening and numbered CSLG01-M1 to CSLG01-M10.

[0036] 4.2 Rescreening

[0037] 4.2.1 The 10 mutant strains obtained in the initial screening were inoculated into liquid fermentation medium and cultured at 28°C and 150 rpm for 7 days.

[0038] 4.2.2 Determination of biomass (dry weight), polysaccharide content, and triterpenoid content of each mutant strain;

[0039] Biomass: The fermentation broth was centrifuged at 8000 r / min for 10 min and the mycelium was dried at 60°C to constant weight and weighed.

[0040] Polysaccharide content: determined by phenol-sulfuric acid method.

[0041] Triterpenoid content: determined by the vanillin-sulfuric acid method.

[0042] The contents of polysaccharides and triterpenoids in the fruiting bodies were determined and compared with those of the original strain CSLG01.

[0043] Ten mutant strains with faster mycelial growth rates were obtained through preliminary screening, among which CSLG01-M3, CSLG01-M4 and CSLG01-M7 grew significantly faster than the original strain CSLG01.

[0044] The results of the rescreening showed that the biomass, polysaccharide content and triterpenoid content of CSLG01-M4 were significantly higher than those of other mutants and the original strain (see Table 1). Table 1 Comparison of biomass and active substance content between mutants and original strains strain Biomass (g / L) Polysaccharide content (%) Triterpenoid content (%) CSLG01 0.72±0.02 5.68±0.07 2.46±0.02 CSLG01-M1 0.78±0.01 7.55±0.11 1.95±0.01 CSLG01-M2 0.75±0.02 6.53±0.09 1.52±0.02 CSLG01-M3 0.87±0.01 8.21±0.11 2.69±0.02 CSLG01-M4 0.93±0.02 8.86±0.12 3.96±0.03 CSLG01-M5 0.81±0.01 7.91±0.11 2.02±0.05 CSLG01-M6 0.68±0.03 5.46±0.13 1.23±0.03 CSLG01-M7 0.89±0.02 8.43±0.10 3.64±0.02 CSLG01-M8 0.59±0.01 5.19±0.09 1.76±0.02 CSLG01-M9 0.68±0.02 6.81±0.12 1.73±0.01 CSLG01-M10 0.84±0.01 8.03±0.08 3.22±0.02

[0045] The difference in growth morphology between the superior strain CSLG01-M4 and the original strain CSLG01 on solid culture medium is as follows Figure 1 The original strain CSLG01 has slow colony growth, irregular edges, sparse hyphae, and white color. The superior strain CSLG01-M4 has rapid colony growth, regular edges, dense hyphae, and white to milky white color.

[0046] The growth curves of the superior strain CSLG01-M4 and the original strain CSLG01 are shown in Figure 2 shown.

[0047] The active substances (polysaccharides) of the superior strain CSLG01-M4 and the original strain CSLG01 are as follows Figure 3 As shown, the polysaccharide content in the fruiting body of CSLG01-M4 was (8.78±0.08)%, which was 54.85% higher than that of CSLG01 (5.67±0.06%).

[0048] The active substances (triterpenoids) of the superior strain CSLG01-M4 and the original strain CSLG01 are as follows Figure 4 As shown, the content of triterpenoids was (4.01±0.03)%, which was 59.76% higher than that of CSLG01 (2.51±0.02%).

[0049] 4.2.3 Screen out the excellent strain CSLG01-M4 with high biomass and high active substance content.

[0050] 5. Verification of excellent strains

[0051] 5.1 Cultivation of the superior CSLG01-M4 strain obtained through rescreening: Using oak wood as the spawn material, cut 20 cm long segments, smooth the ends, and soak in water for approximately 24 hours until the oak wood reaches a moisture content of approximately 40%, allowing for the presence of a 1-2 mm crack in the center of the cross-section. Place the oak wood in a polyethylene plastic bag and place it in an autoclave at 123°C for 2 hours. Cool to room temperature, evenly spread the liquid fermentation medium, which has grown extensively with the strain, over the surface of the oak wood. Secure the polypropylene plastic bag and incubate in the dark at 28°C for 40 days. Every 10 days, open the bag for ventilation for one hour to ensure that no water accumulates within the bag. Once the oak wood segments are fully grown with mycelium, fruiting is carried out using a soil-covered cultivation method. The fruiting temperature is 20°C, and the oak wood segments are covered with 2 cm of loose, breathable sandy loam (pH 8.0). After covering, water thoroughly, but without stagnant water. When mushroom buds appear, the temperature is raised to 27℃, the air humidity is 90%, the ventilation frequency is reduced, and the light intensity is 1000lx. The fruiting period is about 35 days.

[0052] The cultivation period of the superior strain CSLG01-M4 was 75 days, which was 6.25% shorter than that of the original strain CSLG01 (80 days). It had a high content of active substances, with the contents of polysaccharides and triterpenoids increased by 54.85% and 59.76%, respectively.

[0053] The above embodiments do not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for breeding wild Ganoderma lucidum strains based on ARTP mutagenesis technology, characterized in that: The following steps are involved: (1) Strain source: The original strain was isolated and purified from wild Ganoderma lucidum fruiting bodies; (2) Spore suspension preparation: spores of wild Ganoderma lucidum were collected under sterile conditions and a spore suspension was prepared; (3) ARTP mutagenesis: The spore suspension was mutagenized using an ARTP mutagenesis breeding instrument; immediately after mutagenesis, the spore suspension was inoculated into a solid culture medium and cultured at 25-30°C for 5-7 days; (4) Screening of excellent strains: 4.1 Primary screening: Based on the colony morphology and growth rate phenotypic characteristics, mutants with faster growth and dense mycelium were selected; 4.2 Rescreening: The mutants obtained in the initial screening were cultured in liquid fermentation, and their biomass, polysaccharide and triterpenoid content were determined to screen out excellent strains with high content of active substances.

2. A method for breeding wild Ganoderma lucidum strains based on ARTP mutagenesis technology according to claim 1, characterized in that: The mutagenesis conditions were: power 100-150 W, gas flow 10-15 SLM, and treatment time 80-120 s.

3. The method for breeding wild Ganoderma lucidum strains based on ARTP mutagenesis technology according to claim 1, wherein: The concentration of spore suspension was 10 6 -10 8 pieces / mL.

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