How to operate the shiitake mushroom mother seed and breeding specifically
The tissues, spores or hypha of mushroom wood are separated, purified and cultured by tube, and then used to propagate the original species or preserve the bacterial species, which is called the mother species. But how to breed specifically? There are several key points.
1. Strain isolation
1. Organization separation
To cultivate good strains, you must have a good mother species, which is derived from robust individual mushrooms. Therefore, you must first choose fresh mushroom buds with normal development, robust individuals and no insect bites as the material for separation. . After the mushroom buds are collected, they do not need to be washed with water or disinfected with syrup. Put the mushrooms into a clean petri dish, use a sterilized hand or a scalpel to cut the mushrooms apart, cut the soybean-sized mushrooms from the part where the lid and the handle are in contact, place them on the prepared medium, and stuff them with cotton. Can.
2. Spore separation
Choose strong and eighth mature shiitake mushrooms, remove the stalks, hang them on a special wire hook on a glass funnel, and cover the petri dish (the petri dish is placed in an enamel basin with gauze) After leaving it for 12-20 hours, most of the spores (seeds) on the gills will fall into the petri dish, forming a layer of white powder, which can be used as a separate culture.
When spores are inoculated, a small amount of spores are taken out with an inoculation needle, and a spore suspension is prepared with sterile water. Then use the inoculation needle to take out a small amount of suspension and inoculate it on the culture medium in the petri dish by streaking. After the spores germinate and the bacteria are formed, the colony with the spores germinating and growing best is selected for purification by tube propagation.
3. Separation of mushroom wood hyphae
Select a small piece of mushroom wood that has not grown or has just grown but has resumed mycelial growth (invisible to the naked eye). Take it back to the room without surface disinfection. Use a belt to wash the part to be separated. Take off the bark, then sterilize the belt with alcohol, and sterilize the punch on the flame of an alcohol lamp. After sterilization, immediately punch in the part of the mushroom that has been peeled, quickly take out the belt, and use the sterilized small scissors. Cut out a piece the size of a soybean grain in the middle of the red mushroom block, and connect it to the culture medium.
For the above three separation methods, the entire process should be carried out in a sterile room, and all utensils should be strictly disinfected to prevent contamination by bacteria. The sterile room should be sprayed with 5% carbolic acid beforehand, and then irradiated by ultraviolet light for 20-30 minutes if possible. It is best to spread a piece of wet gauze disinfected with 2% Lysur on the inoculation table.
After the strains are separated and inoculated, they are cultured at about 25°C. In two or three days, white cotton-like hyphae can be seen growing from the mushroom flesh or wood block, and the white spores can be seen germinating from the spores in three or four days. Hyphae, the whole test tube is full of hyphae for more than ten days. During the cultivation process, if there is blue, green, yellow or paste on the culture medium in the test tube, it means that it has been contaminated by bacteria and cannot be used. Therefore, it is usually necessary to divide the strains into several tubes to select the best-growing ones for purification or transfer to tube propagation as the mother species.
Among the above three separation methods, the tissue separation method is commonly used. Because of the small variation of asexual reproduction, it is easy to operate, and it is not easy to bring in miscellaneous bacteria. In the absence of shiitake mushrooms, mushroom wood hyphae separation can be used. If you want to use the hybrid method to breed, you must use the spore separation method, and you must pick individual spores for hybridization.
Tissue separation is not easy to bring in miscellaneous bacteria, but spores often bring in miscellaneous bacteria during separation, so it must be purified. That is, pick the mushroom hyphae on the culture medium and put them on a new culture medium, and then obtain pure mushroom hyphae seeds for several consecutive times. This process is called purification.
Fourth, the transfer and breeding of female breeds
Splitting the female seed of one test tube into many test tubes for propagation is called transfer tube breeding. One tube of female species can turn around 30 tubes. If the inoculum is large, the hyphae grow quickly; if the inoculum is small, the hyphae grow slowly. Method: Use a sterilized inoculation hook to hook the mother seed and the culture medium into a small piece, about 0.5 square centimeters, into the middle of the mother seed culture medium to facilitate the rapid propagation of hyphae.
The entire process should also be aseptic. The culture temperature is about 25°C. One day after inoculation, white hyphae can be seen growing from the inoculum. After more than ten days, the hyphae grow up in the whole tube and can be used to access the original seed culture medium or preserve it as the mother seed.
V. Mother species preservation
It is best to use potato, dextrose or white sugar medium for preservation of strains, but not wood juice agar medium, because the hyphae of the latter grow fast and are easy to age. The mother species can be stored for 2-3 months at room temperature (for example, if the time is too long, the water in the culture gene will evaporate and shrink, and the strain will age), if it continues to be needed, it must be transferred to the tube for storage in time. During the rainy season and the hot and humid summer, special attention should be paid to the contamination of bacteria.
At low temperatures, the medium water evaporates slowly, and the mycelial metabolism is slow or stopped, and the bacteria can be stored for a longer time. In the refrigerator at 1-4°C, the bacteria can be stored for about half a year, but care must be taken not to make the cotton plug damp and grow bacteria. The storage temperature should not be too low, otherwise the sloping medium will freeze and dehydrate, and the bacterial characteristics will easily decline or die.
The mother species stored at low temperature for a long time must be placed at a suitable temperature before use, so that it can be used immediately after it is awakened (long-term storage is easy to decline), otherwise it will not be easy to survive the transfer. In order to prevent the bacteria from degenerating or mutating due to multiple tube transfers, they should be separated and stored again within 2-3 years.