CN113455287A - Method for directly separating morchella tissue by PDA (personal digital Assistant) plate to obtain pure strain - Google Patents
Method for directly separating morchella tissue by PDA (personal digital Assistant) plate to obtain pure strain Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses a method for directly separating morel tissues by a prefabricated PDA (personal digital assistant) flat plate to obtain pure strains, which comprises the following steps: preparing a PDA agar plate, preparing a penicillin sucrose bacteriostatic solution, separating and pretreating sporocarp, primarily separating and culturing hyphae, purifying and culturing a second crossing bacteriostatic solution, and observing and transferring seeds. According to the technical scheme, toadstool tissue hyphae possibly polluted by mixed bacteria in the first flat plate separation process are transferred to another flat plate by a stainless steel puncher to be cultivated by spanning antibiotic liquid, the purpose of hypha purification is achieved, and the hyphae are separated and purified through two flat plate operations and then transferred to a test tube for storage and use. The strain separating and purifying technology of the invention has the advantages that the sporocarp is not disinfected, the complicated disinfection and sterilization material preparation work before strain separation is reduced, a large amount of time and manpower and material resources are saved, the operation is convenient, the observation is easy, the cost is low, and the method can also be applied to the purification, propagation and storage of test tube stock seeds with longer time in a refrigerator.
Description
Technical Field
The invention relates to the technical field of strain isolation culture of morchella, in particular to a method for obtaining pure strains by directly separating morchella tissues by a PDA (personal digital Assistant) flat plate and crossing a strain inhibiting culture solution, which is also suitable for secondary purification and propagation of strains stored in a refrigerator for a long time.
Background
At present, domestic morchella strains (test tube stock strains) are obtained by 2 methods, namely a spore ejection method and a tissue separation method. In the two methods for obtaining the strains, after morchella esculenta is collected, sporocarp is subjected to comparatively complicated and strict operation procedures such as alcohol disinfection, sterile water cleaning and the like and then inoculated to a test tube for separating and culturing hyphae, and the defects of long seed taking preparation process, universal pollution problem and high operation technical requirement exist in the actual operation.
Disclosure of Invention
In order to solve the problems, the invention uses a simple and quick technical method to solve the problems of high requirement on the strain separation environmental condition, large difficulty in tissue selection slicing operation, complicated preparation work in the prior art and the like, i.e. morchella sporophores are directly picked to carry out primary plate primary hypha separation on inner wall tissues of morchella sporophores within 18-24 hours (the time that spores naturally eject in the air is most abundant) of the collected morchella sporophores without disinfection treatment, and if mixed bacteria pollution occurs in the plate separation process, a technical method spanning antibiotic bacteriostatic solution is used, i.e. the characteristic that the morchella can climb to the space on a sterile and smooth object (glass and plate) is skillfully utilized, and the possibly polluted mixed bacteria can inhibit the growth by dripping the antibiotic-containing solution, and purifying morchella spores or tissue hyphae on a PDA spanned agar plate for the second time, observing and selecting agar blocks with thick hyphae and rich sclerotia through a microscope, transferring the agar blocks to a test tube, and cultivating and using or storing strains.
The invention adopts the following technical scheme:
the invention discloses a method for directly separating morchella tissues by a PDA (personal digital Assistant) flat plate to obtain pure strains, which comprises the following steps of:
s1, preparing a PDA-enriched agar fungus culture medium, which comprises the following steps:
s1a, preparing a tree juice mixed solution: boiling cortex Eucommiae, folium Cinnamomi Japonici, radix Ulmi Pumilae, stigma Maydis, folium indocalami tessellati, and testa Tritici in water, adding water, boiling, adjusting pH with quicklime powder, and collecting supernatant;
s1b, preparing basic PDA agar: cooling potato filtered juice, adding glucose, potassium dihydrogen phosphate, magnesium sulfate, agar powder, peptone and radix Panacis Quinquefolii powder, heating, melting and boiling together for later use, wherein:
the preparation method of the potato filtered juice comprises the following steps: cutting fresh potatoes into small particles of about 20 × 30 mm, adding water, heating, boiling, collecting supernatant, filtering with 3 layers of gauze, and collecting filtrate to obtain potato filtrate.
S1c, adding the melted basic PDA agar culture medium prepared in the step S1b into the tree juice mixed solution prepared in the step S1a, adding water, heating and boiling for 30 minutes, respectively subpackaging test tubes and triangular flasks, placing the test tubes into an autoclave for sterilization, placing the test tubes on inclined planes, and pouring the triangular flask agar hectare to a 9 cm flat plate for use;
s2, preparing a penicillin sucrose bacteriostatic solution;
s3, preparing a PDA selective culture medium plate by using a plate punching method or a plastic water pipe prefabricating method;
s4, collecting sporocarp;
s5, transporting fruiting bodies;
s6, pretreatment for fruit body separation:
drying the sporophore in the dark at the temperature of 20 +/-1 ℃ in a laboratory, storing for 18-24 hours to activate morchella tissue, and starting seed taking operation at the optimal window period of spontaneous ejection of sporophore spores;
s7, cutting and placing the fruit body tissue:
the inoculation personnel disinfect both hands and inoculation equipment, cut a 30 x 40 mm incision at the joint of the sporocarp pileus of morchella esculenta, cut 2 x 3 mm tissue blocks on the white vaporific protuberant tissue visible to naked eyes by adopting aseptic operation, place the tissue blocks on a PDA agar plate, and place 5 points on one plate according to the mode of one particle at the center and one particle at the opposite angles at four sides for primary hypha primary separation culture.
S8, after the first tissue isolation culture for 72 hours, selecting front end agar blocks with thick and sparse hyphae and moderate growth speed (about 1 cm per day) seen by naked eyes, observing that the front end of the hyphae has typical morchella hyphae bifurcation and the rear end hyphae angle gradually changes from an acute angle to an obtuse angle by a microscope, cutting the front end agar blocks, transferring the front end agar blocks to the central part of a PDA selection flat plate which is manufactured in advance, keeping the distance between the edge and the outer edge PDA flat plate to be about 1.5 mm, and then dripping 0.1 milliliter (about 20 drops) of 100 million units of penicillin sucrose into the circular agar gaps by using a sterile syringe to inhibit the diffusion of mixed bacteria.
And S9, observing and transferring, observing the growth condition of the morchella mycelium plate when the mycelium just grows on the PDA plate, cutting the front end of the PDA agar with sclerotia to transfer to the PDA test tube inclined plane obtained in the step S1c after confirming the morchella mycelium according to the method S8, and preserving the strain after culturing for 5-7 days or performing transfer culture.
Further, in the step S1a, the required components are 180-220 parts by weight of cortex eucommiae; 80-120 parts of sweet osmanthus leaves; indocalamus leaf 80-120 weight portions. 80-120 parts of elm root; 45-55 parts of corn stigma; 95-105 g of bran, 22000 parts of first water adding 5-00 parts, 2300 parts of second water adding 1500-00 parts, boiling for 1.8-2.2 hours, and adjusting the target PH to 7.8-8.2;
further, in the step S1b, the components required for preparing the potato filtered juice are 850 parts by weight of fresh potatoes and 4050 parts by weight of water, and the boiling time is 25-35 minutes; 3000 ml of potato filtered juice required by preparing basic PDA agar, cooling at 54-58 ℃, adding 75-85g of glucose, 3-5g of monopotassium phosphate, 1.5-2.5g of magnesium sulfate, 75-85g of agar powder, 15-18g of peptone and 8-12g of American ginseng powder, heating, melting and boiling for 3 minutes;
further, in the step S1c, the required components are that the volume part of the mixed liquid is 380-720, the basic PDA agar culture medium prepared in the step S1b is added with water to 3950-4050, the boiling time is 1 minute, the sterilization temperature is 115 ℃, and the time is 45 minutes.
In a preferred embodiment of the present invention, the preparation of the PDA enriched agar fungal culture medium required by the present invention comprises: the method comprises the following steps:
preparing a tree juice mixed solution: taking 200g of eucommia bark; 100g of sweet osmanthus leaves; indocalamus leaf 100g elm root 100 g; 50g of corn stigma; 100g of bran is added with 20 liters of water and boiled, then 2 liters of water is added and boiled for 2 hours, the PH value is adjusted to about 8.0 by quicklime powder, and the supernatant is taken for standby;
preparing a basic PDA agar plate: cooling 3000 ml of potato filtered juice to about 56 ℃, adding 80g of glucose, 4g of monopotassium phosphate, 2g of magnesium sulfate, 80g of agar powder, 16g of peptone and 10g of American ginseng powder, heating, melting and boiling for 3 minutes for later use, wherein the preparation of the potato filtered juice comprises the following steps: cutting 800g of fresh potatoes into small particles of about 20 x 30 mm, adding 4000 ml of tap water, heating and boiling for 30 minutes, taking supernatant, filtering by using 3 layers of gauze, and taking filtrate to obtain potato filtered juice.
Taking 400 ml of the prepared tree juice mixed solution, adding 3000 ml of melted prepared basic PDA agar culture medium, adding water to 4000 ml, heating and boiling for 1 minute, respectively subpackaging test tubes and triangular flasks, placing in an autoclave for sterilizing at 115 ℃ for 45 minutes, placing the test tubes on an inclined plane, and pouring the triangular flask agar hectare into a 9 cm flat plate for use.
Further, in step S2, the process of preparing the penicillin sucrose antimicrobial solution includes: taking 1 bottle of a commercially available 400-ten-thousand-unit penicillin potassium ampoule, and taking 4 milliliters of bacteria trace sucrose biochemical fermentation identification tube liquid by using a sterile syringe to dilute the bacteria trace sucrose biochemical fermentation identification tube liquid to 100-thousand-unit penicillin sucrose antibacterial liquid per milliliter for later use.
Further, in step S3, preparing a PDA selection medium plate using a plate punching method includes: selecting a stainless steel puncher with the diameter of 10-15 mm, punching a hole in the center of a PDA basic flat plate under the aseptic operation environment, and removing PDA agar in the central groove part for later use by aseptic operation.
Alternatively, in the step S3, in the embodiment of the present invention, a plastic water pipe prefabrication method may also be used to prepare a PDA selective medium plate, including:
selecting a commercial high-temperature resistant plastic PP-R water pipe with the diameter of 20 mm, cutting the pipe into short pipes with the length of 15 mm, and sterilizing the short pipes and PDA flat agar together for 45 minutes at 115 ℃; pouring agar to 90 mm flat plate, putting sterilized prefabricated 15 x 20 mm short pipe at the center of 90 mm flat plate, waiting for the flat plate agar to cool, taking out the central prefabricated heat-resistant plastic PP-R short pipe by aseptic operation, culturing at 28 deg.C for 3 days, and using after confirming the sterility.
Further, in the step S4, the morchella sporophores are collected together with morchella sporophores soil when the morchella sporophores are collected, and the collected morchella sporophores are morchella sporophores with the earliest fruiting, standard mushroom type and incompletely opened mushroom cap folds.
Further, in step S5, the collected fruiting body is put into a 500 ml sterile plastic bottle without a cover, so as to avoid shaking during transportation, avoid contacting other objects and keep the air smooth.
Further, in step S7, the tissue blade No. 11 is held to cut the tissue block.
Further, in the step S8, the agar block is cut out using a circular stainless steel rubber punch. In step S9, the mycelia of morchella were confirmed at a magnification of 40 times using a microscope.
Compared with the prior art, the invention has the advantages that:
according to the technical scheme, toadstool hyphae possibly polluted by mixed bacteria in the first flat plate separation process are transferred to another pre-prepared PDA selective culture medium flat plate with a groove in the center through a special simple method to grow across antibiotic liquid, and the purpose of toadstool purification is achieved.
The strain isolation culture technology of the invention has the advantages that the sporocarp can not be disinfected, the complicated disinfection and sterilization material preparation work before strain isolation is reduced, a large amount of time, manpower and material resources are saved, and the method is simple and easy to master.
The plate crossing culture method is easy to obtain in the market or on the network of material and equipment such as heat-resistant prefabricated plastic pipes, agar punches, antibiotics, trace sucrose fermentation pipes and the like, and is convenient and quick to implement.
Compared with a test tube separation and purification method, the method for selecting and purifying the strains on the flat plate has the advantages of more convenient operation, easier observation and lower cost.
Actual cultivation data of an inventor in 2019-2020 two years show that the toadstool strain cultivated by using the secret PDA plate purification method is superior to the toadstool strain cultivated by using the conventional test tube purification method in purity, and the cultivation yield is more satisfactory.
Drawings
FIG. 1 is a flow chart of medium preparation.
FIG. 2 is a flow chart of plate-punching method for preparing PAD plate and transferring culture.
Fig. 3 is a schematic view of a portion of the process flow in fig. 2.
FIG. 4 is a flow chart of the PAD plate prepared by the plastic water pipe prefabrication method and the transfer culture.
Fig. 5 is a schematic view of a part of the flow chart in fig. 4.
FIG. 6 shows the growth of Morchella strains after 12 hours of culture in crossover experiment F-2143.
FIG. 7 shows the microscopic morphology of morchella mycelium successfully cultivated in 1938-2 land dike.
FIG. 8 shows the microscopic morphology of 1938-2 Morchella mycelium at day 3 of the crossover experiment.
FIG. 9 is a statistical chart comparing the yields of strains purified using conventional, secret PDA agar plates.
FIG. 10 is a photograph of the recording of the dripping of penicillin sucrose solution in the agar groove of the second PDA selective medium plate after the first hypha cutting and transferring operation
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated to the manufacturer, such as the punch used for punching, and are commercially available.
As shown in FIG. 1, this example requires the use of a PDA-enriched agar fungal culture medium, which is prepared by the following steps:
1. preparing a tree juice mixed solution: taking 200g of eucommia bark; 100g of sweet osmanthus leaves; 100g of elm root; indocalamus leaf 100 g; 50g of corn stigma; 100g of bran is added with 20 liters of water and boiled, then 2 liters of water is added and boiled for 2 hours, the PH value is adjusted to about 8.0 by quicklime powder, and the supernatant is taken for standby.
2. Preparing basic PDA agar: 3000 ml of potato filtered juice (800g of fresh potatoes, which are cut into small particles of about 20 x 30 mm, added with 4000 ml of tap water, heated and boiled for 30 minutes, then supernatant is filtered by 3 layers of gauze, and filtered to obtain filtrate) is cooled to about 56 ℃, 80g of glucose, 4g of monopotassium phosphate, 2g of magnesium sulfate, 80g of agar powder, 16g of peptone and 10g of American ginseng powder are added, and the mixture is heated, melted and boiled for 3 minutes for later use.
3. And (3) taking 400 ml of the tree juice mixed solution prepared in the step (1), adding 3000 ml of the melted basic PDA agar culture medium prepared in the step (2), adding water to 4000 ml, uniformly mixing, heating and boiling for 1 minute, respectively subpackaging test tubes and triangular flasks (used for manufacturing spanning plates), and sterilizing for 45 minutes at 115 ℃ in a pressure cooker. The test tube was placed on a slant and the flask was inverted to a 9 cm plate.
In this embodiment, the preparation work required for the method of obtaining pure strains by directly separating morchella tissues across the bacteriostatic culture solution through the PDA plate is as follows:
preparing a penicillin sucrose antibacterial solution: a pharmacy purchases one ampoule of 400 ten thousand units of penicillin potassium, 4 milliliters of commercially available bacterial micro-sucrose biochemical fermentation identification tube liquid is taken by aseptic injection and diluted to 100 ten thousand unit concentration penicillin sucrose bacteriostatic solution per milliliter for use, wherein the micro-sucrose fermentation tube liquid mainly has the function of diluting antibiotics and also has the function of providing nutrition for morchella mycelium.
As shown in fig. 2-5, the present invention shows two methods for manufacturing a cross-over PDA flat panel, i.e. a flat panel punching method and a plastic water pipe prefabrication method, in this embodiment, a plastic water pipe prefabrication method is used to manufacture a central groove PDA flat panel.
Firstly, preparation work: according to the step S3 of the PDA agar culture medium of the embodiment, a common PDA plate is prepared for cutting and placing the morchella tissue for the first time.
Secondly, preparing a central groove PDA selective flat plate:
the plastic water pipe prefabricating method comprises the following steps: selecting a commercial high-temperature-resistant PP-R water pipe with the diameter of 20 mm, cutting the pipe into short pipes with the length of 15 mm, and placing the short pipes and PDA flat agar together in a pressure cooker to sterilize for 45 minutes at 115 ℃. Pouring agar to 90 mm flat plate, putting sterilized prefabricated 15 × 20 mm short pipe in the center of 90 mm flat plate, waiting for the flat plate agar to cool, taking out the central prefabricated heat-resistant plastic short pipe by aseptic means, culturing at 28 deg.C for 3 days, and keeping under aseptic condition.
In this embodiment, the hole puncher of punching operation is commercial stainless steel hole puncher, purchases on the net.
In this embodiment, the method for directly separating morchella tissue across the bacteriostatic culture solution by using the PDA plate to obtain pure strains includes the following steps:
collecting sporocarps:
the collected morchella sporocarp is required to be the morchella sporocarp which has the earliest fruiting and standard mushroom type and has not completely opened mushroom cap folds. Collecting morchella sporophore soil together.
And (3) fruiting body transportation:
carefully put into a 500 ml sterile plastic bottle without a cover, avoid shaking during transportation to the greatest extent, avoid contacting other articles and keep the air smooth.
Pretreatment for fruiting body separation:
the sporophore is dried and stored in dark for 18-24 hours under the environment condition of 20 +/-1 ℃ in a laboratory, the morchella tissue is activated at the optimal temperature, and the seed taking operation is started at the optimal window period of the sporophore spore natural ejection.
Cutting and placing the fruit body tissues:
an inoculating person sterilizes double hands and inoculating equipment (tissue scissors, a tissue clamp, a tissue knife and a PDA flat plate with the diameter of 9 cm and rich), firstly, a 30 x 40 mm cut is cut at the joint of a morchella sporocarp pileus handle, the morchella sporocarp is tightly held by the left hand, and a 11 # tissue blade is held by the right hand, 2 x 3 mm tissue blocks are cut on white vaporific protuberant tissues visible to naked eyes by adopting sterile operation through a knife tip and are quickly placed on a common PDA agar flat plate, and 5 points are placed on one flat plate according to the mode of one central particle and one square diagonal particle to carry out primary hypha separation culture.
After the first tissue isolation culture for 72 hours, through microscope observation, selecting front end agar blocks with sparse and thick hyphae and moderate growth speed (about 1 cm per day) visible to naked eyes, observing that typical morchella hyphae bifurcate at the front end of the hyphae and the rear end hyphae gradually change from an acute angle to an obtuse angle by using a microscope, cutting the appropriate agar blocks (with the diameter of 12 mm) by using a circular stainless steel rubber puncher through aseptic operation, transferring the appropriate agar blocks to a PDA (personal digital assistant) crossing the center of a plate which is manufactured in advance, keeping the distance between the edge and the PDA plate at the outer edge to be about 1.5 mm, then dripping 20 drops (about 0.1 ml) of 100 ten thousand units of penicillin sucrose into the circular agar gap by using an aseptic syringe to inhibit the diffusion of mixed bacteria, and recording the operation of dripping penicillin sucrose in a graph 10.
Observing and transferring, observing the growth condition of the morchella mycelium plate when the mycelium just grows on the PDA plate, determining that the mycelium is morchella mycelium by combining microscope magnification of 40 times, cutting the front end of the PDA agar with sclerotia, transferring the front end of the PDA agar to a test tube inclined plane, and preserving the strain after culturing for 5-7 days or performing transferring culture.
Observing test tubes, stock seeds and cultivated seeds: in 2019 and 2020, the toadstool test tube mother strains 1938-2 and P2-B1 nucleuses separated and cultured by the method have good experimental cultivation effect in 2020; FIG. 7 shows the microscopic hyphal morphology of 1938-2 Morchella esculenta. FIG. 8 shows the microscopic morphology of 1938-2 Morchella hyphae across the 3 rd day of the experiment. The strains M-2142 and F-2143 obtained by the same seed production method are duplicated in 2021 years, and the mycelia are pure and strong, rich in sclerotia and slow in pigment formation when observed under a microscope, have all the characteristics of excellent morchella mycelia, and grow fast and vigorously after being transformed and cultivated, and the pigment formation is slow. FIG. 6 shows the growth of the Morchella strains after 12 hours of culture across the experiment F-2143.
The following table records the strain expansion and cultivation records of Morchella esculenta in 2019-.
TABLE 2019 + 2020 Morchella culture strain expansion/culture record
In addition, the following table two shows the records of cultivation experiments in the flat dam village of black township in the city of unitary yang soil family, the municipality of the Chongqing city.
Statistics of relevant data of cultivation experiment of Morchella esculenta in flat dam (2021.1.5)
The third lower table is the morchella strain preserved, transferred and observed record 5 and 28 months before 2021.
Table III morchella species observation record
Example two:
comparative experiments with different media compared to example one, this example uses a commercially available medium and a self-made medium with a commercially available medium formulation, the experimental procedure is identical to the examples, and the results are as follows:
table four: table for recording growth conditions of morchella mycelium by using PDA agar plates with different formulas
Table four illustrates:
the hyphae grow, plus means about 0.5 cm per day, plus means about 1 cm, plus + means about 1.5 cm;
sclerotia formed, + indicates that the plate has 3-5 sclerotia, + indicates 5-10, + + + indicates 10-20, and, + +++ indicates a full plate sclerotia.
For the PDA test tube provided by the technical scheme of the invention and the conventional commercial PDA fungus agar separation and expansion cultivation, about 2 mu average yield per mu of about 55.8 kg/mu of 7 strains (7 greenhouses) are cultivated in 2020 by using the conventional PDA test tube expansion method, and about 1.6 mu average yield per mu of 2 strains (6 greenhouses) is 154 kg by using the PDA flat plate purification method provided by the invention, and the specific yield is shown in figure 9 and the following table.
The following table shows the data related to the cultivation experiment of morel in flat dam village of black township in unitary Yangxian prefecture, Chongqing city, and fig. 9 shows the comparison of the yield of the purified strain using conventional test tubes with the PDA agar plate of the present invention, which is evident that the cultivation of the purified strain using the PDA agar plate of the present invention has a higher yield.
TABLE V comparison of the yields of strains purified using conventional tubes and PDA agar plates of the invention
Note: number 01 mixed as mixed strain.
In the first flat plate separation process, morchella mycelium possibly polluted by mixed bacteria is transferred to another flat plate through a special, rapid and simple method to grow across antibiotic liquid, the purpose of morchella purification is realized, the mycelium is separated and purified through two simple operations of flat plate mycelium and is transferred to a test tube for storage, wherein 2 strains in 2019 and 2020: 1928-2(3 strain) and P2-B1 nucleus (11 strain) are verified by experimental cultivation, and the fruiting yield effect is satisfactory.
The strain separation culture technology of the invention has the advantages that the sporocarp is not disinfected, the complicated disinfection and sterilization material preparation work before strain separation is reduced, and a large amount of time, manpower and material resources are saved; the cross-plate culture method is easy to buy in the market or on the network by using heat-resistant prefabricated plastic pipes, agar punches, antibiotics and micro-sucrose fermentation pipes; compared with a test tube separation and purification method, the method for selecting and purifying the strains on the flat plate has the advantages of more convenient operation, easier observation and lower cost.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical scope of the present invention by equivalent or modified solutions and modifications within the technical scope of the present invention.
Claims (10)
1. A method for directly separating morchella tissues by a PDA (personal digital Assistant) flat plate to obtain pure strains is characterized by comprising the following steps of:
s1, preparing a PDA-enriched agar fungus culture medium, which comprises the following steps:
s1a, preparing a tree juice mixed solution: decocting cortex Eucommiae, folium Cinnamomi Japonici, folium indocalami tessellati, radix Ulmi Pumilae, stigma Maydis, testa Tritici, and radix Panacis Quinquefolii powder in water, adding water, decocting, adjusting pH with calx powder, and collecting supernatant;
s1b, preparing basic PDA agar: cooling the potato filtered juice, adding glucose, potassium dihydrogen phosphate, magnesium sulfate, agar powder and peptone, heating, melting and boiling for later use, wherein:
the preparation method of the potato filtered juice comprises the following steps: cutting fresh potatoes into small particles of 20 × 30 mm, adding water, heating and boiling for 30 minutes, taking supernatant, filtering by using 3 layers of gauze, and taking filtrate to obtain potato filtered juice;
s1c, adding the melted basic PDA agar culture medium prepared in the step S1b into the tree juice mixed solution prepared in the step S1a, adding water, heating and boiling, respectively subpackaging test tubes and triangular flasks, placing the test tubes into an autoclave for sterilization, placing the test tubes on an inclined plane, and pouring the triangular flask agar to a 9 cm flat plate for use;
s2, preparing a penicillin sucrose bacteriostatic solution;
s3, preparing a PDA selective culture medium plate by using a plate punching method or a plastic water pipe prefabricating method;
s4, collecting sporocarp;
s5, transporting fruiting bodies;
s6, pretreatment for fruit body separation:
drying the sporophore in the dark at the temperature of 20 +/-1 ℃ in a laboratory, storing for 18-24 hours to activate morchella tissue, and starting seed taking operation at the optimal window period of spontaneous ejection of sporophore spores;
s7, cutting and placing the fruit body tissue:
the inoculation personnel disinfect both hands and inoculation equipment, cut a 30 x 40 mm incision at the joint of the sporocarp pileus of morchella esculenta, cut 2 x 3 mm tissue blocks on the white vaporific protuberant tissue visible to naked eyes by adopting aseptic operation, place the tissue blocks on a PDA agar plate, and place 5 points on one plate according to the mode of one particle at the center and one particle at the opposite angles at four sides for primary hypha primary separation culture.
S8, after carrying out first tissue isolation culture for 72 hours, selecting a front agar block with thick and sparse hyphae and moderate growth speed seen by naked eyes, observing through a microscope to confirm that typical morchella hyphae exists, namely the front end of the hyphae is forked, the rear hyphae angle gradually changes from an acute angle to an obtuse angle, cutting a proper agar block to transfer to the central part of a PDA (personal digital assistant) plate manufactured in advance, keeping the distance between the edge and the outer edge PDA plate at 1.5 mm, and then dripping 0.1 ml of 100 ten thousand units of penicillin sucrose into a circular agar gap groove by using a sterile injector to inhibit the diffusion of mixed bacteria;
and S9, observing and transferring, observing the growth condition of the morchella mycelium plate when the mycelium just grows on the PDA plate, cutting the agar at the front end of the PDA to transfer to the PDA test tube inclined plane obtained in the step S1c after confirming the morchella mycelium according to the method S8, and preserving the strain after culturing for 5-7 days or performing transfer culture.
2. The method as claimed in claim 1, wherein the step S1a comprises the steps of separating Morchella esculenta tissue directly from the PDA plate to obtain pure strain, wherein the required components comprise 180-220 parts by weight of eucommia ulmoides bark; 80-120 parts of sweet osmanthus leaves; 80-120 parts of indocalamus leaves; 80-120 parts of elm root; 45-55 parts of corn stigma; 95-105 g of bran, 22000 parts of first water adding 5-00 parts, 2300 parts of second water adding 1500-00 parts, boiling for 1.8-2.2 hours, and adjusting the target PH to 7.8-8.2;
in the step S1b, the components required for preparing the potato filtered juice comprise, by weight, 850 parts of fresh potatoes and 4050 parts of water, wherein the boiling time is 25-35 minutes; 3000 ml of potato filtered juice required by preparing basic PDA agar, 54-58 ℃ of cooling temperature, 75-85g of glucose, 3-5g of monopotassium phosphate, 1.5-2.5g of magnesium sulfate, 75-85g of agar powder, 15-18g of peptone and 8-12g of American ginseng powder, and the heating, melting and boiling time is 3 minutes;
in the step S1c, the required components are 380-720 parts by volume of the mixed liquid, and the basic PDA agar culture medium prepared in the step S1b is added with 3950-4050 parts by volume of water, the boiling time is 1 minute, the sterilization temperature is 115 ℃ and the time is 45 minutes.
3. The method as claimed in claim 2, wherein the step S2 of preparing the penicillin sucrose solution comprises: taking 1 bottle of 400-ten-thousand-unit penicillin potassium ampoule, taking 4 ml of bacteria micro-sucrose biochemical fermentation identification tube liquid by using a sterile syringe, and diluting to 100-thousand-unit penicillin sucrose antibacterial liquid per ml for later use.
4. The method of claim 3, wherein the step S3 of preparing the PDA basal medium plate by plate punching comprises:
selecting a stainless steel puncher with the diameter of 10-15 mm, punching a hole in the center of a PDA basic flat plate under the aseptic operation environment, and removing PDA agar in the central groove part for later use by aseptic operation.
5. The method of claim 3, wherein the step S3 of preparing the PDA selective medium plate by using a plastic hose prefabrication method comprises:
selecting a 20 mm diameter high temperature resistant plastic PP-R water pipe, cutting into short pipes with the length of 15 mm, and sterilizing the short pipes and PDA flat agar together for 45 minutes at 115 ℃; pouring agar to 90 mm flat plate, putting sterilized prefabricated 15 × 20 mm short pipe in the center of 90 mm flat plate, waiting for the flat plate agar to cool, taking out the central prefabricated heat-resistant plastic PP-R short pipe by aseptic means, culturing at 28 deg.C for 3 days, and using after confirming the sterility.
6. The method of claim 4 or 5, wherein the morchella sporocarp is collected together with morchella sporocarp soil in the step S4, and the collected morchella sporocarp is the morchella sporocarp with earliest fruiting, standard mushroom type and incompletely opened cap fold.
7. The method of claim 6, wherein in step S5, the collected fruit body is placed in a 500 ml sterile plastic bottle without a cover, and the collected fruit body is transported without shaking, contacting other objects and keeping the air smooth.
8. The method as claimed in claim 7, wherein in step S7, 11 tissue blades are held to cut out tissue blocks.
9. The method for directly separating toadstool tissues from the PDA plate to obtain pure strains as claimed in claim 8, wherein in the step S8, agar blocks are cut out by using a round stainless steel rubber punch.
10. The method for directly separating toadstool tissues to obtain pure strains according to claim 9, wherein in the step S9, toadstool hyphae are confirmed by using a microscope at a magnification of 40 times.
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