Community-based mushroom production and processing, part 2: Growing mushroom through the tissue culture method

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By Patricia Bianca S. Taculao

Mushrooms have become a favorite ingredient in many healthy meals and snacks. Because of this numerous farmers have decided to try out mushroom production. 

Through growing these edible fungi, they have created an opportunity for them to not only meet the demands of the market when it comes to healthy food but also produce value-added products made from mushrooms. 

In the previous article, Anecita M. Troza, a Science Research Assistant of the Department of Agriculture’s Regional Field Office 13 in Caraga (DA-RFO 13) gave a brief background on mushrooms, its health contents, and some challenges that mushroom farmers might face in today’s market. But in this article, Troza will share how to grow mushrooms through various culture methods. 

Ways to grow mushroom 

During the webinar of AgriTalk 2020’s Caraga leg, held in partnership with the Department of Agriculture’s Agricultural Training Institute and Manila Bulletin, Troza said that some ways to grow mushrooms is through the tissue culture method, rapid multiplication or subculture, and spawn preparation. 

The tissue culture method uses a fragment of tissue from an animal or plant which is then transferred to an artificial environment where they can continue to develop. In mushroom production, this method has for stages: the pure culture of fresh mushroom, rapid multiplication or subcultures, spawn preparation, and planting fruit for production. 

But before anyone should begin following this method, the first thing they should do is create the culture media where the mycelia, or the vegetative part of fungi, will grow. 

Luckily, this process doesn’t necessarily require high-end equipment. Low-cost supplies can work well if utilized properly. 

A common medium used is potato dextrose agar, which is made from potatoes, sugar, gulaman bars, and distilled water. The potato can be substituted with sweet potato, rice wash, monggo extract, and corn extract to name a few. 

For the other materials or equipment, a stove, casserole, flat bottles, and an autoclave which is a machine that uses steam under pressure to kill harmful bacteria, viruses, fungi, and spores. 

In the absences of an autoclave, an ordinary pressure cooker can take its place. 

To begin, collect rice wash water or the water that’s been used to wash rice before cooking. Then work on the potato dextrose agar by taking 200g of clean, peeled, and chopped potatoes and placing it in a pot or casserole along with the collected rice water on top of an open stove. Next, add one liter of clean water and let it boil for five to 10 minutes. Strain the solution and add clean water to complete the one liter measurement. 

Potato dextrose agar (PDA) contains a carbohydrate source which serves as a growth stimulant while the potato serves as a nutrient base for most fungi. The agar-agar acts as the solidifying agent. 

Add 20 grams of powdered agar-agar and two bars of white gulaman in the solution. Keep stirring until it reaches a boil then add 20 grams of white sugar and stir until completely dissolved. 

“White sugar is added instead of brown to keep the media from becoming brown which makes it difficult to determine if the culture is contaminated,” Troza said.

Once everything is well incorporated, transfer the media into clean bottles, Make sure that each bottle has 35 to 45 mL per bottle. While transferring the media, keep stirring to prevent thickening or coagulation. 

Let the media cool before sealing the bottle with a cotton, scratch paper or cellophane. This is done to prevent water vapors from being trapped inside the bottles and adding excess water into the mix which could be a source of contamination. 

The bottles will then be sterilized in the autoclave. If without an autoclave, a pressure cooker works well as an alternative. To do this, arrange the bottles inside a pressure cooker and set the pressure at 15 PSI (pounds per square inch). Once the gauge has reached the set pressure,  allow the sterilization process to take place for 30 to 45 minutes.

After the sterilization, arrange the bottles in a flat and wide position to promote good growth of mycelia. 

Pure culture or tissue culture method 

For the first step, of the tissue culture method, collect a young and healthy mushroom fruit. Sterilize the specimen by soaking it in a three percent chlorine solution, cotton swabbing, and soaking in denatured alcohol. 

Cut the mushroom fruit into half and collect the innermost tissue. Inject the tissue into a bottle that’s previously prepared with media and sterilized. 

Lastly, incubate the bottle with the tissue for 14 to 21 days in a dark, clean, and cold room. Frequently check on the bottles for mycelial growth and monitor for contamination.  

Rapid multiplication or subculture method 

In this process, the mycelia or specimen is taken from the bottles done with the tissue culture method and then transferred into new bottles. Inject the mycelia into the center of the new media. 

Cover the bottles with foil or cotton before allowing them to be incubated in a dark, clean, and cold room to promote mycelial growth. 

Spawn preparation 

This method uses grains such as rice and monggo beans. Wash the grains and boil in a pot of water. The level of water and the grains should be the same. Stir the media until the water has been either evaporated or absorbed by the grains. 

Stop the process of cooking by aerating the grains or allowing them to air out. Next, fill the bottles with grains and cover with cotton, cellophane, foil, or paper. 

Next, sterilize the bottles at 15 PSI for 30 to 45 minutes. After sterilization, let the bottles cool for at least three hours before transferring the mycelia into other bottles to be incubated for 14 to 21 days. 

Substrate preparation

In mushroom production, substrates are known as bulk materials which mushroom mycelia can use as a good source of energy and nutrition. Common materials include rice straw, corn cobs, and saw dust. 

To create mushroom substrates, first make sure that the materials are chopped or crushed. 

For rice straws, soak it in a drum full of water for one night before draining. As for corn cobs or saw dust, thoroughly mix 80 percent cobs or saw dust, 10 percent rice bran and 10 percent lime. Add water before covering it to let it ferment up to seven days. Mix it every day.

Bag the substrate after the process. 60 percent moisture content should be achieved.

After bagging, put a PVC pipe to help with the shape and ease the inoculation process later. Insert a cotton ball as well before covering the bag with paper or cellophane. 

In sterilizing the bags, arrange the bags in a metal drum with water. Place something between the bags and the water, preferably something with holes, to keep the bags from being soaked in the water and just take steam. 

The sterilization will take about eight hours after the water in the drum boils. 

A day after the sterilization, transfer the bags to a clean room before inoculation or injecting the mycelia inside.

Next, arrange the fruiting bags for incubation. This process usually takes one month depending on the substrate. After a month, the bags will be ready to produce mushrooms. 

Once the bags have begun to bear fruit, the next concern of the mushroom farmer should be proper care and maintenance. 

The proper care and maintenance of mushrooms will be tackled in part 3 of the article.

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Patricia Bianca S. Taculao
Patricia Taculao, or Patty as she likes to be called, is a content producer for Manila Bulletin Digital Lifestyle. She graduated from University of Santo Tomas with a bachelor’s degree in Journalism. She loves to spend her free time, reading, painting, and watching old movies.

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