Detailed Explanation of Mother Culture Production Techniques for Gastrodia elata Armillaria and Germination Fungi

For artificial cultivation of Gastrodia elata, during the sexual reproduction process, two symbiotic fungi are needed: germination fungi and Armillaria. In production practice, the author has worked out a culture medium and production technique on which both symbiotic fungi can grow. The specific method is as follows.

1 Culture medium preparation

1.1 Liquid culture medium Potato (remove buds, peel, slice) 100g, hardwood sawdust, wheat bran, cornmeal 50g each, tap water or natural water 1300mL, boil in a double boiler for 30 minutes, then filter with double-layer gauze. Take the filtrate and add water to make up to 1000mL, add glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 0.5%, 10mL VB1. After all are dissolved and shaken evenly, dispense into 500mL Erlenmeyer flasks or jar bottles, about 150mL per bottle, or into test tubes, with the amount being 1/4 of the test tube volume. Plug Erlenmeyer flasks and test tubes with cotton, and for jar bottles, cover with 2 layers of kraft paper or 4 layers of newspaper plus one layer of plastic film, then tie the mouth with a rubber band. Characteristics: high technical content, fast propagation, ready to use once grown well, but cannot be stored for a long time.

1.2 Solid slant culture medium This is a commonly used culture medium in edible mushroom production. Prepare the filtrate according to the procedure in "1.1", add agar 18g to the filtrate, heat to dissolve and add water to make up to 1000mL, then while hot add glucose 20g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, peptone 5g, 10mg VB1, after all are dissolved and shaken evenly, dispense into test tubes, with the amount being 1/4 of the test tube volume, then plug the mouth with cotton.

1.3 Sawdust culture medium Hardwood sawdust 50%, corncob (crushed into small particles) 25%, wheat bran 15%, cornmeal 10%, plus glucose 0.5%, sucrose 0.5%, gypsum 1%, lime 0.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1%, 2% VB1. Mix the raw materials with dry materials such as gypsum and lime evenly, then dissolve all the above soluble auxiliary materials in water and mix into the raw materials, so that the water content is 70%, after fully mixing evenly, put into test tubes and press properly. The filling amount is 1/2 of the test tube volume, then plug the mouth with cotton. Characteristics: storage-resistant, convenient for mailing.

2 Sterilization

Use a portable high-pressure sterilization pot, or a household pressure cooker can be used instead.

2.1 When sterilizing liquid and slant solid culture media, keep the steam pressure stable at 1.4kg/sq cm for 30 minutes, and take them out when the temperature in the pot drops to 50-70°C. After the liquid culture medium is taken out, put it directly into the inoculation box, and inoculate when the temperature drops below 25°C; the slant solid culture medium needs to be tilted into a slant while still in liquid state (the slant length accounts for about 3/5 of the test tube length), and after the culture medium naturally solidifies, move it into the inoculation box.

2.2 When sterilizing sawdust culture medium, keep the steam pressure stable at 1.4kg/sq cm for 45-60 minutes, and after natural cooling, take it out and put it directly into the inoculation box.

Note: The sterilization pot should be filled with enough water in advance. After it starts boiling, be sure to wait until the internal cold air is completely exhausted before pressing the valve. After reaching the pressure, reduce the heat source and maintain the pressure. After sterilization, the pressure should not be reduced too quickly, to prevent the cotton plug from being blown off.

3 Inoculation

It must be carried out under aseptic conditions and in strict accordance with aseptic operation requirements.

3.1 Inoculating liquid culture medium It is only necessary to pick 1-2 pieces of mother culture the size of a peanut (culture pieces with mycelium) from the mother culture test tube and inoculate them into the Erlenmeyer flask. When inoculating into test tube liquid culture medium, the culture pieces can be smaller.

3.2 Inoculating slant culture medium Armillaria needs to be inoculated when there is condensed water on the slant; the condensed water is beneficial to the germination of Armillaria. Inoculate 1-3 pieces of mother culture onto each test tube slant; when inoculating germination fungi, it is necessary to wait until there is no condensed water on the surface of the culture medium before inoculating. Inoculate 1-2 pieces of mother culture onto each test tube slant.

3.3 Sawdust culture medium After sterilization, the culture medium cannot be stored and needs to be inoculated as soon as possible. When inoculating, use an inoculation hook to properly push and press the culture pieces so that they are in close contact with the culture medium, which is beneficial to the germination and growth of the spawn. After inoculation, they need to be placed upright.

4 Cultivation

After inoculation, put them into a constant-temperature incubator or cultivation room as soon as possible, and culture at a constant temperature of 22-24°C.

4.1 During the cultivation of liquid spawn, start shaking culture on a shaker from the 2nd day; when operating manually, it needs to be shaken by hand 6-8 times a day. If it is in test tubes, after shaking it needs to be placed at a slant, and cultivation can be terminated in about 7 days (the cultivation time for germination fungi is 2-3 days longer than for Armillaria), and it should be used for production as soon as possible. Mature culture liquid should be clear and transparent, light tea-colored or earthy yellow. If it is turbid, has a film on the liquid surface, or appears as a large amount of foam, it is contamination by unwanted microorganisms and should be discarded.

4.2 During cultivation of slant spawn, it needs to be placed flat, and in about 48 hours the mycelium can be seen germinating and gradually extending radially around. When the mycelium runs through the slant culture medium in 10-15 days (the cultivation time for germination fungi needs to be longer), it can be used for production. Placed in a refrigerator at 0-4°C, Armillaria can be preserved for 40 days, and germination fungi can be preserved for 60 days.

4.3 After inoculation, sawdust spawn needs to be placed upright for cultivation to prevent the culture pieces from vibrating. After 15-20 days of cultivation, when Armillaria rhizomorphs or germination fungus mycelium run through to the bottom of the bottle, it can be used for production. Preserved in a refrigerator at 0-4°C, the quality remains unchanged for 6 months. When sawdust spawn that has been stored in the refrigerator for a long time is used for production, in order to prevent contamination by unwanted microorganisms during inoculation, the spawn can be taken by breaking the bottom of the test tube for inoculation.