Nutrient solution, propagation solution and culture method for culturing colored bean puffball strains

Abstract

The invention relates to the technical field of strain culture, and particularly discloses a nutrient solution, an expanding propagation solution and a culture method for culturing colored bean puffball strains. The nutrient solution provided by the invention comprises 18-21g / L of glucose, peptone, yeast powder, monopotassium phosphate, citric acid, calcium chloride, diammonium hydrogen phosphate, vitamin B1, wort, magnesium sulfate heptahydrate, ferric chloride, boric acid, manganese chloride tetrahydrate, zinc sulfate heptahydrate, copper sulfate pentahydrate and sodium molybdate dehydrate. The nutrient solution, the solid culture medium and the propagation liquid provided by the invention are rich and balanced in nutrition, and are suitable for culture and propagation of colored bean puffball, so that the production cycle of a stock culture stage and a production seed propagation stage are obviously shortened, and the experiment cost is greatly reduced; and the components are clear and stable, the components are accurately proportioned, the quality is stable, the difference between batches is small, and the stability and repetition of experimental results are facilitated.

Description

Technical Field

[0001] The present application relates to the technical field of bacterial strain cultivation, and in particular to a nutrient solution, a propagation solution and a cultivation method thereof for cultivating colored bean puffball bacterial strains. Background Art

[0002] Colored bean puffball (Pisolithus tinctorius), also known as bean bag fungus, belongs to the genus Colored bean puffball of the family Pisolithaceae of the order Puffballales. It is an ectomycorrhizal fungus that often forms mycorrhizal symbiosis with pine plants. The presence of mycorrhiza can significantly improve the host plant's transformation, absorption and transportation of various mineral elements and trace elements such as phosphorus, zinc and calcium in the soil, promote the production of hormones and antibiotics, and the absorption of water, thereby enhancing the synthesis of chlorophyll in seedlings, promoting the photosynthesis of host plants, and helping to improve ecological adaptability, plasticity, disease resistance and stress resistance. In the process of seedling cultivation, the use of mycorrhizal biotechnology to cultivate mycorrhized seedlings can not only improve the quality of seedlings, enable seedlings to be planted earlier, enhance the adaptability and resistance of seedlings to adverse environments, but also improve the survival rate of afforestation and accelerate the early growth of young forests. The survival rate of afforestation using mycorrhizal seedlings can be increased by 2 to 3 times, and the growth indicators of seedlings can be increased by 3 to 4 times. Therefore, the cultivation and propagation of ectomycorrhizae will help improve the survival rate of seedlings. However, although there are studies on the cultivation and propagation of ectomycorrhizal fungi, the research is mainly in the laboratory stage. There is still a lack of in-depth and systematic research on how to mass-produce the fungus and how to develop mycorrhizal agent production technology for pine plants. Summary of the invention

[0003] In order to solve the above technical problems, the present application provides a nutrient solution, a propagation solution and a culture method thereof for culturing colored bean puffball strains.

[0004] In the first aspect, the present application provides a nutrient solution for rapid cultivation of colored bean puffball strains, specifically comprising the following components in concentrations: glucose 18-21 g / L, peptone 1.5-2 g / L, yeast powder 5-6 g / L, potassium dihydrogen phosphate 0.45-0.5 g / L, citric acid 0.08-0.12 g / L, calcium chloride 0.04-0.06 g / L, diammonium hydrogen phosphate 0.20-0.30 g / L, vitamins B11.5-2.5mg / L, malt juice 4-6g / L, magnesium sulfate heptahydrate 0.13-0.17g / L, ferric chloride 0.01-0.03g / L, boric acid 2.75-2.85mg / L, manganese chloride tetrahydrate 3.65-3.7mg / L, zinc sulfate heptahydrate 2.3-2.4mg / L, copper sulfate pentahydrate 0.92-0.93mg / L, sodium molybdate dihydrate 0.26-0.28mg / L.

[0005] Preferably, the nutrient solution for rapid cultivation of colored bean puffball strains specifically includes the following components in concentrations: glucose 19-20 g / L, peptone 1.6-1.8 g / L, yeast powder 5.2-5.8 g / L, potassium dihydrogen phosphate 0.47-049 g / L, citric acid 0.09-0.11 g / L, calcium chloride 0.045-0.055 g / L, diammonium hydrogen phosphate 0.22-0.28 g / L, vitamin B1 1.7-2.2mg / L, malt juice 4.5-5.5g / L, magnesium sulfate heptahydrate 0.14-0.16g / L, ferric chloride 0.015-0.025g / L, boric acid 2.77-2.83mg / L, manganese chloride tetrahydrate 3.66-3.69mg / L, zinc sulfate heptahydrate 2.32-2.38mg / L, copper sulfate pentahydrate 0.923-0.928mg / L, sodium molybdate dihydrate 0.265-0.275mg / L.

[0006] Preferably, the nutrient solution for rapid cultivation of colored bean puffball strains specifically includes components of the following concentrations: glucose 19.5g / L, peptone 1.7g / L, yeast 5.5g / L, potassium dihydrogen phosphate 0.48g / L, citric acid 0.1g / L, calcium chloride 0.05g / L, diammonium hydrogen phosphate 0.25g / L, vitamin B1 2mg / L, malt extract 5g / L, magnesium sulfate heptahydrate 0.14-0.16g / L, ferric chloride 0.015-0.025g / L, boric acid 2.77-2.83mg / L, manganese chloride tetrahydrate 3.66-3.69mg / L, zinc sulfate heptahydrate 2.32-2.38mg / L, copper sulfate pentahydrate 0.923-0.928mg / L, sodium molybdate dihydrate 0.265-0.275mg / L.

[0007] It also includes components at the following concentrations: unsaturated fatty acids 0.2-0.6 mg / L; The unsaturated fatty acid is composed of a mixture of linoleic acid and arachidonic acid in a weight ratio of 10:0.5-1.5.

[0008] In a specific embodiment, the dosage of the unsaturated fatty acid can be 0.2 mg / L, 0.4 mg / L, or 0.6 mg / L.

[0009] In a specific embodiment, the weight ratio of linoleic acid to arachidonic acid may be 10:0.5, 10:1, or 10:1.5.

[0010] During the experiment, the applicant found that the unsaturated fatty acids composed of linoleic acid and arachidonic acid as the nutrient solution for culturing colored bean puffball fungi can provide the necessary nutrients for growth, which is essential for maintaining the normal physiological functions and metabolic activities of the fungi. And the appropriate concentration of linoleic acid and arachidonic acid can promote the growth of colored bean puffball fungi; these fatty acids can serve as carbon sources and energy sources, providing the fungi with the energy and material basis required for growth, thereby accelerating the reproduction and growth of the fungi.

[0011] In a second aspect, the present application provides a solid culture medium for rapid cultivation of colored bean puffball strains, comprising the above-mentioned nutrient solution; and agar powder with a final concentration of 16-18 g / L; the solid culture medium uses KOH to adjust the pH value to 5.5-6.0.

[0012] In a third aspect, the present application provides a propagation solution for rapidly culturing colored bean puffball strains, comprising a matrix and the above-mentioned nutrient solution; the volume ratio of the nutrient solution to the matrix is ​​100:50-60.

[0013] Preferably, the matrix is ​​composed of a mixture of vermiculite, peat, wheat bran and corn flour in a volume ratio of 8-12:1-3:0.5-1.5:0.5-1.5.

[0014] Preferably, the matrix is ​​composed of a mixture of vermiculite, peat, wood chips, wheat bran, crushed garden waste and corn flour in a volume ratio of 8-10:1-3:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5.

[0015] In a specific embodiment, the volume ratio of the vermiculite, peat, sawdust, wheat bran, crushed garden waste, and corn flour can be 8:1:0.5:0.5:0.5:0.5, 8:2:1:1:1:1, 8:3:1.5:1.5:1.5:1.5, 9:1:0.5:0.5:0.5:0.5, 9:2:1:1:1:1, 9:3:1.5:1.5:1.5:1.5:1.5, 10:1:0.5:0.5:0.5:0.5, 10:2:1:1:1:1, 10:3:1.5:1.5:1.5:1.5.

[0016] Preferably, in the matrix, the performance parameters of the vermiculite are: particle size of 40-80 mesh, silicon dioxide content of 50-60%, density of 1.2-1.25 g / cm 3 ; Purchased from Xinyang Houpu Mining Co., Ltd.; The particle size of the peat is ≤40 mesh; The performance parameters of the sawdust are: porosity is 2.35, linear expansion rate is 1%; The particle size of the wheat bran is ≤40 mesh; The particle size of the crushed garden waste is ≤40 mesh; The particle size of the corn flour is ≤40 mesh.

[0017] In a fourth aspect, the present application provides a method for rapid cultivation and propagation of colored bean puffball fungi, characterized in that the method comprises using the nutrient solution described in any one of claims 1 to 3 for rapid cultivation and propagation; or using the solid culture medium described in claim 4 for rapid cultivation and propagation; or using the propagation solution described in claim 5 for rapid cultivation and propagation; specifically comprising the following steps in sequence: Stock culture: inoculate the healthy plate bacteria into the solid culture medium, place it in a constant temperature incubator at 27-29°C and culture it for 24-35 days to complete the stock culture; Propagation: The strain obtained by culturing the original strain is inoculated into the propagation solution, placed in an incubator at 26-28°C and cultured for 55-75 days to obtain the production strain.

[0018] Preferably, the method for rapid culture and propagation of the colored bean puffball strains further comprises the following steps: Use a sterile cork puncher to take multiple mycelium discs with a diameter of 4-6mm and a thickness of 1.3-1.7mm from the edge of the mother colony after the original seed culture, inoculate them into sterile nutrient solution, and place them on a shaker at 28℃ for reciprocating shaking culture to obtain a large amount of original seeds.

[0019] In summary, the technical solution of this application has the following effects: The nutrient solution, solid culture medium, and enrichment solution provided in this application are matched and combined by specific components in specific dosages, and are rich in nutrition and balanced, containing carbon sources such as glucose and nitrogen sources such as peptone, as well as various minerals such as potassium dihydrogen phosphate and magnesium sulfate heptahydrate, and nutrients such as vitamin B1, which can meet the nutritional needs of the growth of ectomycorrhizal fungi of colored bean puffballs. In addition, the above formula is easy to prepare, and each component can be weighed according to the formula, and high-pressure sterilization can be performed after packaging, without the need for complex preparation processes and special equipment.

[0020] The nutrient solution, solid culture medium and propagation solution provided in the present application are rich in nutrition and balanced, and are particularly suitable for the cultivation and propagation of colored bean puffballs. The production cycle of the original seed cultivation stage and the production seed propagation stage are significantly shortened, which greatly reduces the experimental cost.

[0021] The nutrient solution, solid culture medium, and propagation solution provided in the present application can be used together to promote the growth of specific fungi, and can provide suitable nutrition for the ectomycorrhizal fungi of colored bean puffball, which is beneficial to the extension of its mycelium and the accumulation of biomass; and the ingredients are clear and stable, the ingredients are precisely proportioned, the quality is stable, and the batch-to-batch differences are small, which is beneficial to the stability and repetition of experimental results, and the user can accurately control the culture conditions.

[0022] This application studies the propagation and culture conditions of colored bean puffball to solve the problems of complex culture and propagation in existing technologies and slow growth of ectomycorrhizae, and provides technical support for the large-scale standardized production, application and promotion of ectomycorrhizal agents for pine plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 These are growth status pictures taken on the 2nd, 4th, 6th, 8th, 10th, 12th, 14th and 28th days during the original culture stage of the colored bean puffball mycelium in Example 3.

[0024] Figure 2 This is a growth status diagram of the colorful bean puffball mycelium expansion stage in Example 3, which was recorded every three days (0-60 days). DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application.

[0026] The sources of the experimental materials used in the experiment of this application are as follows: malt extract (model Y017FC) was purchased from Hangzhou Baisi Biotechnology Co., Ltd. When used, 130.1g of this product was added to 1000mL of distilled water, stirred and heated to boil until completely dissolved, sterilized at 121℃ for 15min, and set aside; yeast powder (model Y017FC) was purchased from Beijing Hongrun Baoshun Technology Co., Ltd.; the performance parameters of vermiculite A are: particle size of 40-80 mesh, silicon dioxide content of 50-60%, density of 1.2-1.25g / cm 3 ; Purchased from Xinyang Houpu Mining Co., Ltd.; The performance parameters of vermiculite B are: particle size 1-3mm, density 2.4-2.7g / cm 3 , purchased from Lingshou County Xubang Mineral Products Processing Plant; the performance parameters of peat are: particle size ≤40 mesh, purchased from Xiangniu Fertilizer Co., Ltd. The performance parameters of sawdust are: porosity 2.35, linear expansion rate 1%, purchased from Cao County Shenhang Wood Industry Co., Ltd.; the performance parameters of wheat bran are: particle size ≤40 mesh, purchased from Jinan Shuangshi Environmental Technology Co., Ltd.; the performance parameters of garden waste crushed material are: particle size ≤40 mesh, homemade; the performance parameters of corn flour are: particle size ≤80 mesh, purchased from Jiyang District Jide Family Farm. Example

[0027] Examples 1-7 Examples 1-7 respectively provide a nutrient solution, a propagation solution and a culture method thereof for culturing colored bean puffball strains.

[0028] The difference between the above embodiments is that the concentrations of the components in the nutrient solution are different, as shown in Table 1.

[0029] (1) The solid culture medium is prepared as follows: according to Table 1, based on 1000 mL of the culture medium, weigh the corresponding weight of each component raw material, weigh 17 g of agar powder, dissolve each component in deionized water, adjust the pH value to 5.5-6.0 (25° C.) with KOH, make up the volume, sterilize at 121° C. and autoclave for 1 h, and obtain the solid culture medium after cooling.

[0030] (2) The method for preparing the enrichment solution is as follows: according to Table 1, based on 1000 mL of nutrient solution, weigh the corresponding weight of each component raw material, dissolve each component in deionized water, and obtain a nutrient solution; Vermiculite A, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 9:2:1:1:1:1 to obtain a matrix; Mix the nutrient solution and matrix evenly in a volume ratio of 100:54 to obtain the propagation solution.

[0031] (3) The rapid culture and propagation method of colored bean puffball strains is: Stock culture: inoculate the robust plate bacteria into the solid culture medium, place it in a constant temperature incubator at 28°C, observe the growth and maturation cycle, and complete the stock culture; Propagation: The strain obtained from the original seed culture is inoculated into the propagation solution, placed in a 27°C incubator for culture, and the period in which the mycelium almost completely infects the substrate is recorded to obtain the production strain.

[0032] Table 1 Concentration of each component in the nutrient solution in Examples 1-7 and Comparative Examples 1-2 Embodiment 8-11 Examples 8-11 respectively provide a nutrient solution, a propagation solution and a culture method for culturing colored bean puffball strains.

[0033] The differences between the above embodiment and embodiment 3 are specifically as follows.

[0034] In Example 8: the unsaturated fatty acid is composed of a mixture of linoleic acid and linolenic acid in a weight ratio of 10:1.

[0035] In Example 9, the unsaturated fatty acid is composed of a mixture of linoleic acid and arachidonic acid in a weight ratio of 1:10. In Example 10, the unsaturated fatty acid is composed of a mixture of linoleic acid and arachidonic acid in a weight ratio of 10:0.5. In Example 11, the unsaturated fatty acid is composed of a mixture of linoleic acid and arachidonic acid in a weight ratio of 10:1.5. The other process parameters in the above embodiment are the same as those in embodiment 3.

[0036] Examples 12-18 Examples 12-18 respectively provide a nutrient solution, a propagation solution and a culture method for culturing colored bean puffball strains.

[0037] The differences between the above embodiment and embodiment 3 are specifically as follows.

[0038] In Example 12: vermiculite B, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 9:2:1:1:1:1 to obtain a matrix.

[0039] In Example 13: vermiculite A, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 7:4:0.2:1.8:0.2:1.8 to obtain a matrix.

[0040] In Example 14: vermiculite A, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 15:0.5:1.8:0.2:1.8:0.2 to obtain a matrix.

[0041] In Example 15: vermiculite A, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 8:3:0.5:1.5:0.5:1.5 to obtain a matrix.

[0042] In Example 16: vermiculite A, peat, wood chips, wheat bran, crushed garden waste, and corn flour were mixed in a volume ratio of 12:1:1.5:0.5:1.5:0.5 to obtain a matrix.

[0043] In Example 17: the nutrient solution and the matrix are evenly mixed in a volume ratio of 100:50 to obtain the propagation solution.

[0044] In Example 18: the nutrient solution and the matrix are evenly mixed in a volume ratio of 100:60 to obtain the propagation solution.

[0045] The other process parameters in the above embodiment are the same as those in embodiment 3.

[0046] Comparative Example Comparative Example 1-2 Comparative Examples 1-2 respectively provide a nutrient solution, a propagation solution and a culture method thereof for culturing colored bean puffball strains.

[0047] The difference between the comparative example and Example 3 is that the dosage of each component in the nutrient solution is different, as shown in Table 1.

[0048] The other process parameters in the above comparative example are the same as those in Example 3.

[0049] Comparative Examples 3-5 Comparative Examples 3-5 respectively provide a nutrient solution, a propagation solution and a culture method thereof for culturing colored bean puffball strains.

[0050] The differences between the above comparative example and Example 3 are specifically as follows.

[0051] In Comparative Example 3: an equal amount of ammonium sulfate was used instead of manganese chloride tetrahydrate.

[0052] In Comparative Example 4: the pH value of the solid culture medium was adjusted to 6.5 using KOH.

[0053] In comparative example 5: the nutrient solution and the matrix were evenly mixed in a volume ratio of 100:68 to obtain the propagation solution.

[0054] The other process parameters in the above comparative example are the same as those in Example 3.

[0055] Performance testing Record the growth status of the colored bean puffball mycelium cultured in the culture medium, record the growth and maturity cycle during the stock culture stage, and record the cycle of the mycelium completely infecting the substrate during the expansion stage. Figure 1 The growth status diagrams shown are taken at the 2nd, 4th, 6th, 8th, 10th, 12th, 14th and 28th days during the original culture stage of the colored bean puffball mycelium in the parallel experiment A group of Example 3; Figure 2 The figure shows the growth status of the colored bean puffball mycelium in Group A of the parallel experiment of Example 3, which was recorded every three days (0-60 days) during the propagation stage.

[0056] Test results: as shown in Table 1.

[0057] Table 1 Cultivation and propagation performance of colored bean puffball strains in the examples and comparative examples Combination Figure 1 and Figure 2 Using the technical solution provided in Example 3, it can be seen that the growth and maturation period of the colored bean puffball mycelium cultured on solid culture medium during the original seed culture stage is about 28 days, and the period in which the mycelium almost completely infects the substrate during the expansion stage is about 60 days.

[0058] Combining the test results of the embodiments and comparative examples in Table 1, the growth and maturation cycle of the colored bean puffball mycelium and the cycle of the production and expansion stage are significantly shortened; and the quality of the results of each group of parallel tests is stable, and the difference between batches is small, which is conducive to the stability and repetition of the experimental results. The user can accurately control the culture conditions, which greatly reduces the experimental cost.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A nutrient solution for rapidly culturing colored bean puffball strains, characterized in that: Specifically, it includes the following components in the following concentrations: glucose 18-21g / L, peptone 1.5-2g / L, yeast powder 5-6g / L, potassium dihydrogen phosphate 0.45-0.5g / L, citric acid 0.08-0.12g / L, calcium chloride 0.04-0.06g / L, diammonium hydrogen phosphate 0.20-0.30g / L, vitamin B1 1.5-2.5mg / L, malt juice 4-6g / L, magnesium sulfate heptahydrate 0.13-0.17g / L, ferric chloride 0.01-0.03g / L, boric acid 2.75-2.85mg / L, manganese chloride tetrahydrate 3.65-3.7mg / L, zinc sulfate heptahydrate 2.3-2.4mg / L, copper sulfate pentahydrate 0.92-0.93mg / L, sodium molybdate dihydrate 0.26-0.28mg / L.

2. The nutrient solution for rapidly culturing colored bean puffball strains according to claim 1, characterized in that: Specifically, the components include the following concentrations: glucose 19-20g / L, peptone 1.6-1.8g / L, yeast powder 5.2-5.8g / L, potassium dihydrogen phosphate 0.47-049g / L, citric acid 0.09-0.11g / L, calcium chloride 0.045-0.055g / L, diammonium hydrogen phosphate 0.22-0.28g / L, vitamin B1 1.7-2.2mg / L, malt juice 4.5-5.5g / L, magnesium sulfate heptahydrate 0.14-0.16g / L, ferric chloride 0.015-0.025g / L, boric acid 2.77-2.83mg / L, manganese chloride tetrahydrate 3.66-3.69mg / L, zinc sulfate heptahydrate 2.32-2.38mg / L, copper sulfate pentahydrate 0.923-0.928mg / L, sodium molybdate dihydrate 0.265-0.275mg / L.

3. The nutrient solution for rapidly culturing colored bean puffball strains according to claim 1, characterized in that: The composition also includes the following concentrations: unsaturated fatty acids 0.2-0.6 mg / L; the unsaturated fatty acids are in a weight ratio of 10: It is composed of a mixture of 0.5-1.5 linoleic acid and arachidonic acid.

4. A solid culture medium for rapidly culturing colored bean puffball strains, characterized in that: The invention comprises the nutrient solution according to any one of claims 1 to 3; and agar powder with a final concentration of 16 to 18 g / L; the solid culture medium is adjusted to a pH value of 5.5 to 6.0 by using KOH.

5. A propagation solution for rapid cultivation of colored bean puffball strains, characterized in that: It comprises a matrix and the nutrient solution according to any one of claims 1 to 3; the volume ratio of the nutrient solution to the matrix is ​​100:50-60.

6. The enrichment solution according to claim 5, characterized in that The matrix is ​​composed of a mixture of vermiculite, peat, wheat bran and corn flour in a volume ratio of 8-12:1-3:0.5-1.5:0.5-1.

5.

7. The enrichment solution according to claim 5, characterized in that The matrix is ​​composed of a mixture of vermiculite, peat, wood chips, wheat bran, crushed garden waste and corn flour in a volume ratio of 8-10:1-3:0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.

5.

8. The enrichment solution according to any one of claims 6 to 7, characterized in that The performance parameters of the vermiculite in the matrix are: particle size of 40-80 mesh, silicon dioxide content of 50-60%, density of 1.2-1.25g / cm 3 ; Purchased from Xinyang Houpu Mining Co., Ltd.; The particle size of the peat is ≤40 mesh; The performance parameters of the sawdust are: porosity is 2.35, linear expansion rate is 1%; The particle size of the wheat bran is ≤40 mesh; The particle size of the crushed garden waste is ≤40 mesh; The particle size of the corn flour is ≤40 mesh.

9. A method for rapid cultivation and propagation of colored bean puffball strains, characterized in that: Rapidly culturing and propagating using the nutrient solution described in any one of claims 1 to 3; or rapidly culturing and propagating using the solid culture medium described in claim 4; or rapidly culturing and propagating using the propagation solution described in claim 5; specifically comprising the following steps in sequence: Stock culture: inoculate the healthy plate bacteria into the solid culture medium, place it in a constant temperature incubator at 27-29°C and culture it for 24-35 days to complete the stock culture; Propagation: The strain obtained by culturing the original strain is inoculated into the propagation solution, placed in an incubator at 26-28°C and cultured for 55-75 days to obtain the production strain.

10. The method for rapid cultivation and propagation of the colored bean puffball strain according to claim 1, characterized in that: The following steps are also included: Use a sterile cork puncher to take multiple mycelium discs with a diameter of 4-6mm and a thickness of 1.3-1.7mm from the edge of the mother colony after the original seed culture, inoculate them into sterile nutrient solution, and place them on a shaker at 28℃ for reciprocating shaking culture to obtain a large amount of original seeds.

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