First, the spore separation method
Mushroom spore separation is the use of the characteristics of mature fruiting bodies of sexually-derived spores that can be automatically ejected from the fruit body layer. The spores are germinated into hyphae under sterile conditions and suitable culture medium to obtain purebred species. a way. Spore isolation can be divided into two kinds of single spore separation and polysporum separation method, because it is isolated from the natural spores (it refers to the nucleus has been nuclear matching process) to separate the pure mycelium, so the production of polysporum separation method to use To maintain the original characteristics of the strain, and the single spore separation method is mainly used for strain screening and cross breeding. Spore separation is mainly divided into two steps, the first is to collect spores, followed by isolation of single or multiple spores.
(a) collecting spores
1, the selection of mushroom species collection of spores First, we must choose good mushrooms that are separated fruiting bodies. The mushroom mushroom requirements are generally the first tidal mushroom, the mushroom is earlier, solitary, the individual is robust, and the typical fruit body is characteristic. After careful observation and screening, a mark is made on the mushroom bed to allow the mushroom to fully grow until it is about to be broken. Membrane picking mushrooms for spore ejection.
2. Spore Ejection Collection Mushrooms can be immersed in a 0.1% Hg solution for about one minute after being harvested, removed with scorpions and rinsed several times with sterile water, and then the surface water is blotted dry with sterile filter paper, or used directly. % The alcohol swab gently controls the sputum cap and stipe for surface disinfection. Then use a sterile knife to cut off the excess stipe (about 1.5-2cm can be left), the mushroom upright, the mushroom shank down into the support made of aluminum wire, into the first prepared with sterile filter paper Fill the plate with a bell jar (as shown). This spore collection device needs to be autoclaved beforehand. The spore ejecting collector of the fruiting body was placed in a room at a temperature of 15 to 20°C. After about 24 to 48 hours, a brown mushroom spore printed on the sterile filter paper was observed. Under aseptic conditions, filter paper strips collected from mushroom spores are placed in a sterile empty test tube, vacuum-dried at a temperature, and placed in a refrigerator for long-term storage.
(b) Spore separation
1, polysporum separation method that is a plurality of spores inoculated on the same medium, so that they germinate grow together and grow together, so as to obtain a method of pure breeding, due to the complementary species of multiple spores, can basically maintain The stability of the parent, this method is relatively simple, in the past more common in mushroom seed production.
(1) Bevel line method: According to the aseptic procedure, use a sterile inoculating loop to remove a small amount of spores from the spore-receiving filter paper strip and mark it on the PDA test tube slant medium from bottom to top. Do not use force so as not to scratch the surface of the culture medium. After the inoculation is completed, inoculate the kind of burning test tube, plug the tampon and place it in a constant temperature incubator at 24°C. After the spores have germinated (generally about 15-20 days), select the germination. Fast and growing colonies were transferred to new test tube cultures for further culture.
(2) Coating separation method: According to the method of aseptic processing, take a small piece of filter paper with spores, place it in a small flask with sterile water, and shake well to make a spore suspension, and then use it. Sterilize the test tube, take the spore suspension, drop 1-2 drops into the PDA test tube or culture medium, rotate the test tube so that the spore suspension is evenly distributed on the slope, or coat the suspension on the plate with a glass coating rod. Evenly. After the spores were germinated at a constant temperature of 24°C on the medium, several well-proportioned and fast-growing colonies were selected and transferred to another test tube slant medium. The thermostatic culture was the parent species.
2, single spore separation is to separate the collected spore groups individually, let it germinate into mycelium alone to obtain pure breeding methods, single spore separation method, according to the separation means can be divided into the following three.
(1) Dilution and separation method: This is achieved by continuously diluting the spore suspension so that the final spore concentration of the spores is controlled at 300-500 spores/ml, and 0.1 ml of spore solution is injected into the plate to uniformly coat the dispersed spores. Spore colonies, the steps are as follows:
1 Preparation of sterile water test tubes: Take 10 test tubes, one of which contains 100 ml of distilled water, and the other 9 bottles of 9 ml of distilled water, and autoclave the sterile water.
2 Sporulation suspension: Take a small piece of filter paper with spores collected, immerse in 10 ml sterile water test tube, shake it to disperse the spore into a suspension, and use a sterile 1 ml pipette to suck 1 ml of spore suspension In the second tube, shake it to make it staging, and then take 1 ml from the second tube and inject it into the third tube. Repeat this dilution until the spore concentration reaches 300-500 spores/ml.
3 medium culture plate: Prepare PDA medium, place it in a flask, autoclave, prepare the plate to be plated and autoclave again. When the sterilized PDA is cooled to 50-60°C, no Under sterile conditions, pour 15-20 ml of PDA into the Petri dish. Place the Petri dish flat and the medium to a smooth surface with uniform thickness.
4 Spore coating: Aseptically, draw 0.1 ml of spore suspension droplets onto the surface of the plate medium, spread the spores evenly on the plate using a T-Bar, and cover with a stimulated hyphae Petri dish. -25°Cincubator.
5 Select single spore germinating colonies: general spores begin to germinate and grow hyphae after 5 days of culture. Petri dishes are microscopically examined to confirm that the spore germinating colonies are formed by the growth of a single spore, and can be punched with a punch. The wells were transferred to fresh PDA bevel tubes for further culture. The outer diameter of the puncher should be smaller than the field of view of the microscope. Before the punching, it is necessary to check whether there are spores or other colonies around the colony, so that the punching will not touch the surrounding spores or colonies to ensure pure breeding.
(2) Capillary method: After the spore suspension is diluted, the spores are suspended in droplets using a capillary pipette so that each droplet contains only one spore, thereby achieving single spore separation. The method is as follows:
1 The spore suspension was prepared by the same dilution method and the spore concentration was controlled at 200-300 spores/ml.
2 Preparation of capillary dropper: Take a clean glass tube and pull it into a thin tube with an outer diameter of 1 mm on an alcohol torch. Cool it slightly, then heat it and quickly extend it so that the inner diameter of the tube tip is about 200 μm. After plugging the cotton at the rear end of the pipette, it is sterilized and used after being placed in the disinfection box.
3 spot microscopy: Use a capillary pipette to pipette approximately 0.1 ml of the diluted spore suspension (with 30 drops drip). Under aseptic conditions, quickly point to the center of the marking circle on the inner wall of the lid. The drop should be less than the low value. The field of view of the microscope. After the sample is placed, the petri dish is still covered with the bottom of the dish with water agar. After the adhesive tape is fixed, the drop of the inner wall of the lid of the dish is checked with a low sharpness to confirm that it is a single spore, that is, on the lid of the dish. Mark the mark.
4 Push-up and stimulation of culture medium: Use a small piece of PDA medium (approximately 22 mm squares) with an inoculation needle to push onto the droplets labeled as single spores so that the spores in the droplets can be absorbed to the edge of the medium. The plate petri dish cover with previously cultivated mushroom aerial mycelium is removed, set on the bottom of the mycelium plate, and then the dish of the separated and affixed culture medium is covered on the glass dish covered with the mycelium plate. , So that the two lids are docked and sealed with adhesive tape (0.5 cm for ventilation), so as to form a culturing chamber that stimulates the germination of single spores. Incubate at 24 °C in the incubator until single spores germinate and tear in time. Under the tape, the inoculated shovel was used to pick up the germinated single spore mycelium patch, transferred to a fresh PDA slant tube, set in a constant temperature box at 24 ° C, you can get single spores purebred.
(3) Apparatus separation method: Using a micromanipulator, single spores were directly selected and transferred to a PDA plate for spore stimulation and germination to obtain pure spores of single spores.
1 spore suspension preparation: the same dilution method, the spore concentration is controlled at 1000 / ml;
2 plate coating: the diluted spore suspension droplets 0.1 ml on the plate, with a uniform T rod coated evenly, each plate contains about 100 spores;
3 Microscopy separation: After the uniform agar plate surface to be coated is dried with moisture, it can be observed under a microscope. When spores are seen in the center of the field of vision, and there are no other spores around the field of vision, a micromanipulator can be used to manipulate the glass needle to spores. The surface of the culture medium is picked, and the spores are then transferred to the surface of the PDA plate for spore germination. The spore germination culture must be stimulated with the aerial mycelium of the mushroom to increase the germination rate. The specific operation method and the coating separation method the same.
(4) Single spore colony reception: When the spores are stimulated for 10 days, the mycelium can be seen to grow into small colonies with the naked eye. The spores should be used to separate the colonies. The spore germination should be observed every day. If not, When the colonies are transferred, the colonies will grow rapidly and affect the isolation of later germinating single spores.
Second, the organizational separation method
Tissue separation is a method using the fruit body tissue to separate pure mycelium. Tissue separation is a kind of asexual propagation method. The chromosomes of both parents have not been reconstituted, so the tissue separation can basically keep the biological characteristics of the parents. The rods are easy to operate and widely used in the village. In the past, the traditional stable strains often used this method for bacterial species. The separation is not obvious, but with the application of mushroom hybrid strains, due to the instability of the hybrid itself, the tissue separation often causes the degradation of the strain, which is rarely used in production at present, but only when the wild strains are isolated. Commonly used. The method is as follows:
1 Picking mushrooms: The standard and spore collection requirements are the same;
2 Sterilization of the mushroom body: The base of the fruit stipe was removed from the fruiting body, placed in the inoculation box, and the surface of the mushroom body was sterilized with 75% alcohol;
3 Tissue separation: The scalpel is sterilized by flame, and a knife is cut longitudinally in the middle of the mushroom shank. The bacterium is opened by hand and then inoculated with an inoculation shovel to cut five small squares at the junction of the mushroom cap and mushroom stalk, and then a small piece of tissue is picked. Rapidly transfer to PDA slant culture medium, set the culture at 24 °C, until the mycelium of the tissue block grew without contamination is pure.
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