Sleet collection and preservation method for sterile spores of lentinus edodes

Abstract

The invention belongs to the technical field of shiitake mushroom breeding, and particularly relates to a shiitake mushroom sterile spore gill collection and preservation method which comprises the steps of shiitake mushroom harvesting, gill collection, dry film acquisition, germination promotion identification and preservation, process management and scientific preparation of a culture medium. The method has the advantages of simple spore collection operation method, high purity, simple physiological identification, long preservation time and the like.

Description

Technical Field

[0001] This invention belongs to the field of shiitake mushroom breeding technology, specifically relating to a method for collecting and preserving the gills of sterile shiitake mushroom spores. Background Technology

[0002] Hybrid breeding includes single-spore hybridization, multi-spore hybridization, and single-double hybridization, which mainly achieves gene transfer and excellent gene aggregation between the two parents through hybridization, creating new edible fungi varieties with excellent commercial traits and quality. Hybrid breeding is one of the most widely used and effective methods in the selection and breeding of new shiitake mushroom varieties. Single spore acquisition is a key step in hybrid breeding and directly affects the success or failure of the selection and breeding. Aseptic single spores of shiitake mushrooms are usually collected by natural ejection from fresh fruiting bodies that are 7-8 parts mature, and then the spores are cultured. Currently, the existing methods for collecting edible fungi spores include: (1) Instrument ejection method: using a special spore collector (a bell jar, support, gauze, etc. to form a sterile environment), the fruiting body naturally ejects spores into the culture dish under controlled conditions, and then collects them with sterile water and transfers them to the culture medium for culture. However, this method depends on specific equipment, the operation steps are complicated, the sterile maintenance requirements are high, and it is easy to introduce contamination due to improper operation. (2) Petri dish fixation method: The fruiting body is fixed on the culture medium in the petri dish, and sterile paper is placed below to directly receive the ejected spores. However, the fruiting body will release tissue fluid or debris, which can easily contaminate the spores. Large fruiting bodies are difficult to fix, and the applicability is limited. (3) Physical fixation method: The fruiting body is fixed with rubber bands, and sulfuric acid paper is placed below in the petri dish to receive the spores. However, the fixation stability of this method is insufficient, the spore collection surface is limited, and it is difficult to maintain sterile conditions continuously, resulting in a high risk. (4) Hanging centrifugation preservation method: The fruiting body is made into a hanging structure and placed in a sterile bag. After the spores are ejected naturally, the robust spores are separated by centrifugation and then preserved for a long time with preservation solution. However, this method requires centrifugation equipment, has requirements on the initial concentration of spores, and the hanging method needs to be adjusted and adapted according to the morphology of the fruiting body, which increases the difficulty of operation.

[0003] Patent CN109601247A discloses a high-temperature resistant shiitake mushroom culture medium formula and its cultivation method, including step 3: spore isolation; cutting off the base of the stipe of each wild shiitake mushroom specimen from step 1, disinfecting the surface of the fruiting body of the wild shiitake mushroom specimen with 0.1% to 0.2% mercuric chloride solution or 75% alcohol for 1 to 2 minutes; then rinsing in sterile water, and wiping the surface of the fruiting body with sterile gauze to remove moisture and disinfectant; fixing the fruiting body, and placing a culture dish b under each fruiting body, and letting it stand for 1 to 2 days; when a large number of spores on the gills fall into the culture dish b, forming a layer of powdery spores; injecting 3 to 5 ml of sterile water into each culture dish b, and gently stirring to suspend the spores evenly in the water.

[0004] The patent with publication number CN108094043A discloses a method of soaking mushroom spawn in a 0.1% mercuric chloride solution for 1-2 minutes, rinsing it 3-5 times with sterile water, blotting the surface moisture with filter paper, placing the mushroom spawn with its gills facing down on a petri dish supported by wire, placing sterilized filter paper inside the petri dish, and after 12-20 hours, a layer of white spore imprints will be scattered on the filter paper inside the petri dish, thus obtaining shiitake mushroom spores.

[0005] The aforementioned patented technologies all disclose methods for cultivating spores using gills, but their purpose is to expand cultivation or select superior varieties. They cannot achieve long-term preservation of spores or simple rejuvenation. In addition, the aforementioned patented technologies all involve spore cultivation of the entire fruiting body, which is inconvenient to collect and has a high risk of contamination, resulting in an unsatisfactory spore printing rate. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention proposes a method for collecting and preserving the gills of sterile spores of shiitake mushrooms.

[0007] Specifically, this is achieved through the following technical solutions: A method for collecting and preserving the gills of aseptic spores from shiitake mushrooms, comprising the following steps: Step 1: Shiitake mushroom harvesting: When the second flush of shiitake mushrooms is produced, select fruiting bodies with round and intact shapes, excellent agronomic traits, and whose caps have just ruptured. Place them in a sterile collection bag and bring them back to the laboratory. Once the caps have completely ruptured and the gills have unfolded, they can be used for spore collection. Step 2: Gill Collection: Wipe the surface of the shiitake mushrooms with 75% alcohol, cut off the stems, and place them upside down in a laminar flow hood to dry the surface moisture; use sterile dissecting scissors to cut off the rolled edges along the edge of the fruiting body cap; use sterile forceps to pick up a single gill and place it on an agar medium, with 3-5 gills per petri dish, the gills being 8-20mm in size; use the grafting membrane opening and place the mushrooms upside down in a laminar flow hood for incubation. The agar medium is prepared by dissolving 15-20g of agar powder in 1.0L of deionized water and sterilizing at 121℃ for 20min. After the agar medium is prepared, it is cooled to 55-65°C and poured into disposable culture dishes at a ratio of 15-20 mL / dish in a clean bench. After cooling, the grafting membrane is used for later use.

[0008] The disposable culture dish is 90cm in size.

[0009] Step 3 Dry film acquisition: Observe the ejection of spores from the gills at intervals of 12-48 hours. When there are obvious white spore marks visible to the naked eye on the lid of the culture dish, replace the bottom of the spore collection dish with a new sterile disposable culture dish; mark the position of the spore marks with a marker, invert the dish in the laminar flow hood to dry the moisture at the spore marks, and seal it for later use. Step 4 Germination Identification: Using aseptic technique, repeatedly rinse the spore prints with sterile water using a pipette to prepare a spore suspension. Collect the spore prints from 3-5 culture dishes into 2 mL sterile centrifuge tubes. Dilute the spore prints to 10-20 spores / mL using a serial dilution method. Inoculate the diluted spore solution onto enriched PDA medium using a pipette, inoculating 0.1-0.3 mL onto each culture dish. Spread evenly with a spreader and seal the dish. Incubate at 20-25°C for 10-15 days. After 7 days of incubation, observe the spore germination. Once spores have germinated, pick single spore mycelium and transfer it to enriched PDA medium. Incubate at 20-25°C for 8-12 days. Examine the single spores under a microscope using coverslips to check for clamp connections. If no clamp connections are found in any of the 30 fields of view for each single spore strain, it can be confirmed as a single spore.

[0010] The PDA-enriched culture medium is prepared by first boiling 20g of wheat bran and 200g of potato in deionized water for 20 minutes, then filtering the mixture through 8 layers of gauze. The collected filtrate is then mixed with 15-25g of glucose, 1.5-2.5g of yeast extract, 1.5-2.5g of fermented corn steep liquor, 0.8-1.2g of potassium dihydrogen phosphate, 0.3-0.7g of magnesium sulfate, and 5-15mg of gibberellin. The mixture is then replenished with deionized water to a final volume of 1000mL and sterilized at 121℃ for 30 minutes.

[0011] Using the PDA-enriched medium of the present invention, the growth rate of spores can be significantly increased by more than 10%.

[0012] Step 5 Preservation: When the single spores have germinated to 2 / 3, use a 7mm punch to punch holes along the edge of the colony and inoculate them into the middle of the centrifuge tube of the single spore preservation medium. Inoculate 2-3 agar blocks into each centrifuge tube and prepare the grafting membrane opening. Place the tube vertically with the opening facing up and culture at 23-25℃ until the mycelium has fully grown. After full growth, store in a refrigerator at 4℃.

[0013] The single-spore preservation medium consists of 73-82% hardwood sawdust, 8-12% wheat bran powder, 3-7% rice bran powder, 1-3% soybean meal powder, and 0.5-1.5% light calcium carbonate powder, with a moisture content of 55-60%. The mixture is then transferred to a 50mL centrifuge tube, with the filling volume being 1 / 2 to 2 / 3 of the centrifuge tube, and sterilized at 121℃ for 2 hours.

[0014] The mycelium obtained by the five steps of this invention can be preserved for 5-8 years. If it is needed during the preservation period, it can be taken out and rejuvenated with PDA-enriched medium.

[0015] Beneficial effects: This invention provides a simple method for collecting, physiologically identifying, and preserving sterile spores of shiitake mushrooms. By designing and managing the steps of shiitake mushroom harvesting, gill collection, dry film acquisition, germination induction identification, and preservation, and combining them with the scientific preparation of culture medium, this method has the advantages of simple spore collection operation, high purity, simple physiological identification, and long preservation time.

[0016] This invention utilizes gill culture to cultivate spores, avoiding the complex operations of fixing and ejecting the entire or part of the fruiting body. Instead, it involves aseptically picking and placing individual gills directly onto agar medium. This method minimizes sources of contamination (such as stipes and caps) at the source and significantly simplifies the operational process.

[0017] This invention transforms the spore print ejected onto the inner wall of the petri dish lid into a dry, pure "spore membrane" by replacing the bottom of the petri dish after the gills eject spores. The resulting spore print is dry and filled, avoiding secondary pollution and the impact of spore germination during the preparation of spore suspension, thus obtaining standardized spore material that can be directly stored or used.

[0018] This invention utilizes a scientifically formulated PDA-enriched culture medium: by adding ingredients such as wheat bran filtrate, yeast extract, fermented corn steep liquor, and gibberellin, it significantly enhances spore germination speed and mycelial growth vitality (increasing growth rate by more than 10%), thus shortening the breeding cycle.

[0019] This invention achieves stable preservation of single-spore mycelium for 5-8 years at 4℃ by scientifically preparing a single-spore preservation culture medium and combining it with a miniaturized centrifuge tube container. The revival process is also simple, which greatly facilitates the long-term management and use of germplasm resources. Attached Figure Description

[0020] Figure 1 The high-quality shiitake mushroom spawn used in Example 1; Figure 2 Image of the cap of a shiitake mushroom at 7-8 minutes of maturity used in Example 1; Figure 3 : Diagram of the gills of a 7-8 minute mature fruiting body of shiitake mushroom used in Example 1; Figure 4 : Diagram of the gills of a 7-8 minute mature fruiting body of shiitake mushroom used in Example 1; Figure 5 In Example 1, spores were collected from the gills in a petri dish; Figure 6 : Shiitake mushroom spore prints collected in the petri dish in Example 1; Figure 7 : The 5-year-old monokaryotic mycelium of shiitake mushrooms preserved in Example 1; Figure 8 : Growth rate of shiitake mushroom single spores in Example 1. Detailed Implementation

[0021] To enable those skilled in the field of edible mushroom breeding technology to better understand the technical solution of this invention and further clarify its purpose, technical solution, and key technical points, the invention is further described in detail below with reference to embodiments. However, the embodiments of this invention are not limited to the following. Those skilled in the art can make appropriate adjustments or modifications to the following embodiments without departing from the concept of this invention, and all such modifications should be considered to fall within the protection scope of this invention.

[0022] Rapid collection of aseptic spores from shiitake mushrooms is not only a key technological link connecting basic research and industrial applications, but also a crucial cornerstone for promoting the transformation of the edible fungi industry towards high efficiency, standardization, and intelligence. The method for rapid collection and preservation of aseptic spores from shiitake mushrooms described in this invention is not only an innovation in preservation technology, but also an important supporting tool for the modern shiitake mushroom breeding system. By providing a standardized, highly efficient, and long-lasting spore material treatment solution, it fundamentally improves the scientific rigor, precision, and sustainability of breeding work, and has profound significance for promoting the innovative development of the shiitake mushroom seed industry.

[0023] Example 1 Step S1: Prepare agar medium using 20.0g agar powder and 1.0L deionized water, sterilized at 121℃ for 20min. Cool to 55℃, pour 20mL into disposable 90cm petri dishes in a clean bench, and use the grafting membrane for later use.

[0024] Step S2: When the shiitake mushrooms produce their second flush, select fruiting bodies that are round and well-formed, have excellent agronomical traits, and whose veil has just ruptured. Place them in a sterile collection bag and bring them back to the laboratory. Once the veil has completely ruptured and the gills have unfolded, they can be used for spore collection.

[0025] Step S3: Wipe the surface of the shiitake mushrooms with 75% alcohol, cut off the stems, and place them upside down in a laminar flow hood to dry the surface moisture. Use sterile dissecting scissors to trim the rolled edges along the edge of the fruiting body's cap; use sterile forceps to pick up a single gill and place it on an agar plate, with 4 gills per plate, each gill being 10-15 mm in size. Use the grafting membrane opening and place the plate upside down in the laminar flow hood for incubation.

[0026] Step S4: Observe the ejection of spores from the gills at 12-48 hours intervals. When there are visible white spore marks on the lid of the culture dish, replace the bottom of the spore collection dish with a new sterile disposable culture dish. Mark the location of the spore marks with a marker, invert the dish in a laminar flow hood, dry the spore marks, and seal it for later use.

[0027] Step S5: Prepare PDA-enriched culture medium. Filter 20g of wheat bran and 200g of potato through 8 layers of gauze after boiling water for 20 minutes. Add 20g of glucose, 2g of yeast extract, 2g of fermented corn steep liquor, 1.0g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 10mg of gibberellin, and deionized water to a final volume of 1000mL. Sterilize at 121℃ for 30 minutes.

[0028] Step S6: Using aseptic technique, repeatedly rinse the spore prints with sterile water using a pipette to prepare a spore suspension. Collect the spore prints from 3-5 culture dishes into 2 mL sterile centrifuge tubes. Dilute the spore prints to 10-20 spores / mL using a serial dilution method. Inoculate the spore dilution onto enriched PDA medium using a pipette, 0.2 mL per dish. Spread evenly with a spreader and seal the dish. Incubate at 20-25°C. Observe spore germination after 7 days of incubation. Once spores have germinated, pick single spore mycelium and transfer it to enriched PDA medium. Incubate at 20-25°C for 8-12 days. Examine single spores under a microscope using coverslips for clamp connections. For each single spore strain, three replicates are required. If no clamp connections are observed in 30 fields of view for each strain, it can be confirmed as a single spore. See details. Figure 8 It can be seen that using the PDA-enriched medium in this embodiment can significantly promote the growth rate of spores by more than 10%; while using the PDA medium as a control group. Step S7: Prepare the single-spore preservation medium: 78% hardwood sawdust, 10% wheat bran powder, 5% rice bran powder, 2% soybean meal powder, 1% light calcium carbonate powder, with a moisture content of 55-60%. Transfer to 50mL centrifuge tubes, filling the tubes to 1 / 2-2 / 3 capacity. Sterilize at 121℃ for 2 hours.

[0029] Step S8: When the single spores have germinated to 2 / 3 of their full size, use a 7mm punch to inoculate the colony along the edge into the center of a centrifuge tube containing the single spore preservation medium. Inoculate 2-3 agar blocks into each centrifuge tube, reserving the grafting membrane opening. Place the tubes vertically with the opening facing upwards and incubate at 23-25℃ until the mycelium has fully grown. After full growth, store at 4℃; it can be preserved for 5-8 years. If experimental use is required during this period, it can be removed and rejuvenated using PDA-enriched medium.

Claims

1. A method for collecting and preserving the gills of sterile spores from shiitake mushrooms, characterized in that, Includes the following steps: Step 1: Shiitake mushroom harvesting: When the second flush of shiitake mushrooms is produced, select fruiting bodies with round and intact shapes, excellent agronomic traits, and whose caps have just ruptured. Place them in a sterile collection bag and bring them back to the laboratory. Once the caps have completely ruptured and the gills have unfolded, they can be used for spore collection. Step 2: Gill Collection: Wipe the surface of the shiitake mushrooms with 75% alcohol, cut off the stems, and place them upside down in a laminar flow hood to dry the surface moisture; use sterile dissecting scissors to cut off the rolled edges along the edge of the fruiting body cap; use sterile forceps to pick up a single gill and place it on an agar medium, with 3-5 gills per petri dish, the gills being 8-20mm in size; use the grafting membrane opening and place the mushrooms upside down in a laminar flow hood for incubation. Step 3: Dry film acquisition: Observe the ejection of spores from the gills at intervals of 12-48 hours. If there are obvious white spore marks visible to the naked eye on the lid of the culture dish, replace the bottom of the spore collection dish with a new sterile disposable culture dish; mark the position of the spore marks with a marker, invert the dish in a laminar flow hood to dry the moisture at the spore marks, and seal it for later use. Step 4 Germination Identification: Using aseptic technique, repeatedly rinse the spore prints with sterile water using a pipette to prepare a spore suspension. Collect spore prints from 3-5 culture dishes into 2 mL sterile centrifuge tubes. Dilute the spore prints to 10-20 spores / mL using a serial dilution method. Inoculate the diluted spore solution onto PDA-enriched medium using a pipette, inoculating 0.1-0.3 mL onto each culture dish. Spread evenly with a spreader and seal the dish. Incubate at 20-25°C for 10-15 days. After 7 days of incubation, observe the spore germination. Once spores have germinated, pick single spore mycelium and transfer it to PDA-enriched medium. Incubate at 20-25°C for 8-12 days. Examine the single spores under a microscope using coverslips for clamp connections. If no clamp connections are observed in 30 fields of view for each single spore strain, it can be confirmed as a single spore. Step 5 Preservation: When the single spores have germinated to 2 / 3, use a 7mm punch to punch holes along the edge of the colony and inoculate them into the middle of the centrifuge tube of the single spore preservation medium. Inoculate 2-3 agar blocks into each centrifuge tube and prepare the grafting membrane opening. Place the tube vertically with the opening facing up and culture at 23-25℃ until the mycelium has fully grown. After full growth, store in a refrigerator at 4℃.

2. The method for collecting and preserving the gills of aseptic spores of shiitake mushrooms as described in claim 1, characterized in that, The agar medium is prepared by dissolving 15-20g of agar powder in 1.0L of deionized water and sterilizing at 121℃ for 20min.

3. A method for collecting and preserving the gills of aseptic spores of shiitake mushrooms as described in claim 1 or 2, characterized in that, After the agar medium is prepared, it is cooled to 55-65°C and poured into disposable culture dishes at a ratio of 15-20 mL / dish in a clean bench. After cooling, the grafting membrane is used for later use.

4. The method for collecting and preserving the gills of aseptic spores of shiitake mushrooms as described in claim 3, characterized in that, The disposable culture dish is 90cm in size.

5. The method for collecting and preserving the gills of aseptic spores of shiitake mushrooms as described in claim 1, characterized in that, The PDA-enriched culture medium is prepared by first boiling 20g of wheat bran and 200g of potato in deionized water for 20 minutes, then filtering the mixture through 8 layers of gauze. The collected filtrate is then mixed with 15-25g of glucose, 1.5-2.5g of yeast extract, 1.5-2.5g of fermented corn steep liquor, 0.8-1.2g of potassium dihydrogen phosphate, 0.3-0.7g of magnesium sulfate, and 5-15mg of gibberellin. The mixture is then replenished with deionized water to a final volume of 1000mL and sterilized at 121℃ for 30 minutes.

6. The method for collecting and preserving the gills of aseptic spores of shiitake mushrooms as described in claim 1, characterized in that, The single-spore preservation medium consists of 73-82% hardwood sawdust, 8-12% wheat bran powder, 3-7% rice bran powder, 1-3% soybean meal powder, and 0.5-1.5% light calcium carbonate powder, with a moisture content of 55-60%. The mixture is then transferred to a 50mL centrifuge tube, with the filling volume being 1 / 2 to 2 / 3 of the centrifuge tube, and sterilized at 121℃ for 2 hours.

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