Mushroom Tissue Culture: Growing Fungi From Cells

Mushroom tissue culture is a method of growing mushrooms by transferring a fragment of tissue from a mushroom fruit to an artificial environment, where it can develop and grow. This process is also known as cloning, as it preserves the exact genetic character of the contributing mushroom. Tissue culture can be taken from spores or from the living mushroom. The first step in mushroom tissue culture is to collect a young, healthy mushroom fruit and sterilise it. The mushroom fruit is then cut in half, and the innermost tissue is placed into a bottle that has been prepared with media and sterilised. The bottle is then incubated for 14 to 21 days in a dark, clean, and cold room. The process of tissue culture is used to facilitate mushroom research and artificial cultivation.

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The process of mushroom tissue culture

The growth medium can be prepared using potato-dextrose-agar (PDA). This involves shredding and boiling raw potatoes, straining the mixture, and adding dextrose, agar, and other optional ingredients like peptone or yeast extract. The growth medium can also be gelatin-based, which involves dissolving sugar and gelatin powder in water and boiling the mixture.

Once a pure strain has been developed, the next step is to increase the mycelial mass by growing the mycelium on enriched agar media in a Petri dish and then on grain or sawdust/bran. The mycelium is then transferred to grain-filled jars (G1 Masters), which can be used to create another generation of spawn (G2) or inoculate bulk substrates.

Mushroom tissue culture is a highly individualised process, and it is important to maintain the sterility of the laboratory environment to prevent contamination. Overall, the tissue culture method offers a simple and effective way to grow mushrooms by transferring a fragment of tissue to an artificial environment for development.

Preparing the growth medium

PDA is one of the easiest and most effective growth media to make. Here's one way to make it:

• Shred 100 g of raw potato and boil in 600 mL of water for at least 30 minutes.
• Let the mixture cool, then strain it through a fine sieve to remove solids.
• Add the potato extract to a graduated cylinder and make up the volume to 500 mL.
• Transfer the mixture to a 1000 mL flask.
• Add 5 g of dextrose (glucose), 9 g of agar (a gelling agent), and 0.1-0.5 g of peptone or yeast extract (or both) to the flask.
• Stir the mixture well and adjust the pH to 5.8.
• Heat the mixture on a hot plate or in a microwave oven until the agar and other ingredients are dissolved. Do not let it boil over.

Another option for a growth medium is gelatin. Here's how to make it:

• Dissolve 2 g of sugar and 18 g of gelatin powder in 125 mL of water.
• Boil the mixture for 5-10 minutes, stirring constantly to avoid burning it. If you have a pressure cooker, using it for this step can reduce the contamination rate.
• Let the mixture cool down to about 50°C (122°F).
• Pour the mixture into plates or containers, such as Petri plates or small glass jars.

Once you have prepared your growth medium, you can move on to the next step of mushroom tissue culture, which is collecting and sterilizing the mushroom tissue. This process involves cutting a small piece of tissue from the center of a young, healthy mushroom fruit and sterilizing it with a chlorine solution and alcohol. With the growth medium and tissue ready, you can then transfer the tissue to the medium and begin the incubation process.

Collecting and sterilising the mushroom tissue

Collecting and sterilising mushroom tissue

Mushroom tissue culture is a highly individualised art, but it is the simplest way to grow mushrooms once mastered. Tissue culture can be taken from spores or from a living mushroom. The latter approach preserves the exact genetic character of the mushroom.

To collect mushroom tissue, begin with a young, fresh mushroom with no signs of decay. Wipe down all work surfaces and disinfect tools with 70% isopropyl alcohol. Pull apart the mushroom cap to expose the inside, uncontaminated tissues. Using sterile fine-tipped tweezers, pluck tiny pieces of the inside tissue and transfer them onto an agar or gelatin growth medium. This should ideally be done inside a still-air transfer chamber or a laminar flow hood to avoid airborne contamination.

Once you have a pure strain, the next step is to increase the mycelial mass. This is done by growing the mycelium on enriched agar media in a Petri dish and then on grain or sawdust/bran. The Petri dish allows cultivators to easily identify and avoid contaminants such as moulds and bacteria. The mycelium is then transferred to sterilised grain or sawdust-filled jars (G1 Masters). These can be used to inoculate more jars (G2) or bulk substrates such as straw, wood, or compost.

To sterilise mushroom substrates, or bulk materials, the materials must first be chopped or crushed. For rice straws, soak the material in water overnight and then drain. For corn cobs or sawdust, mix 80% cobs or sawdust, 10% rice bran, and 10% lime, before adding water and letting the mixture ferment for up to seven days. Bag the substrate after mixing. The mixture should achieve 60% moisture content. Sterilise the bags at 15 PSI for 30 to 45 minutes. After sterilisation, let the bags cool for at least three hours.

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Incubating the tissue

Preparation of Growth Media

Before incubating the tissue, it is essential to prepare the growth media that will support the development of the mushroom mycelium. One of the most commonly used growth media is Potato-Dextrose-Agar (PDA). To prepare PDA, follow these steps:

• Wash and cut about 100-200 grams of raw potatoes into small cubes. Removing the skin is optional.
• Boil the potato cubes in 500-600 mL of water for at least 30 minutes to an hour.
• Allow the potato broth to cool, then strain it through a fine sieve or cheesecloth to remove solids and obtain a clear broth.
• Add water to the potato broth to make a total volume of 500 mL.
• Transfer the potato broth to a 1000 mL flask or cylinder.
• Add 5 grams of dextrose (glucose) and 9 grams of agar (a gelling agent) to the potato broth. You can also add 0.1-0.5 grams of peptone or yeast extract for additional nutrients.
• Stir the mixture well and adjust the pH to 5.8.
• Heat the mixture on a hot plate or in a microwave oven until the agar and other ingredients are completely dissolved. Do not let it boil over.
• Pour the liquid PDA into your chosen containers, such as Petri dishes, small glass jars, or small bottles. Leave about 5-10 mm of space from the top.

Tissue Transfer

Once your growth media is ready, it's time to transfer the mushroom tissue:

• Select a young, fresh, and healthy mushroom for tissue culture.
• Pull apart the mushroom cap to expose the inside, uncontaminated tissues.
• Using sterile fine-tip tweezers or a sterilized knife/scalpel, carefully pluck or cut a small piece of tissue from the centre of the mushroom cap, avoiding the outer surface.
• Keep the tissue sample on the cutting tool and quickly transfer it to the centre of the agar surface in your chosen container. A larger tissue sample will have a better chance of success.
• Repeat the process several times with different containers to increase the likelihood of obtaining at least one contaminant-free mycelium.

Incubation Conditions

After transferring the tissue, it's time to create optimal conditions for incubation:

• Incubate the cultures in a dark environment, away from direct light.
• Maintain the temperature at room temperature (around 23°C) during incubation.
• Ensure that the relative humidity is suitable for mushroom growth, generally between 60-80%.
• Allow sufficient airflow to the cultures to prevent excess moisture buildup, which can promote bacterial or fungal contamination.

Monitoring and Sub-Culturing

During the incubation period, it is crucial to monitor the cultures regularly:

• Inspect the cultures daily for signs of bacterial or mould contamination. Moulds tend to grow faster and produce coloured spores, while pure mushroom mycelium will appear as a solid, fuzzy white colony.
• If contamination occurs, discard the affected cultures and maintain sterile conditions to prevent further issues.
• If the mushroom mycelium grows but is also contaminated, you can attempt to sub-culture it onto a fresh, uncontaminated growth medium under aseptic conditions.
• Depending on the mushroom species, the incubation period from tissue transfer to full growth of mycelium can take about 10-15 days.
• Once the mycelium covers the entire growth medium, store the mature mycelium in a cool place or the refrigerator to maintain its vitality.

Incubating mushroom tissue requires careful attention to detail, a sterile environment, and regular monitoring. By following these steps, you can successfully develop pure mushroom cultures for further cultivation and spawning.

Maintaining the culture

Preparation

Before attempting any transfer or culture work, it is essential to have prepared media plates for pure culturing. You can use Petri plates or small glass jars as your containers. Two common growth media used for culturing the mycelium of oyster mushrooms are agar-based and gelatin-based.

Sterilization

Sterility is of paramount importance to prevent contamination. Ensure that all equipment, including knives, scalpel, and tweezers, are sterilized. Collect a young and healthy mushroom fruit and sterilize it by soaking it in a three percent chlorine solution, cotton swabbing, and soaking it in denatured alcohol.

Tissue Collection

Pull apart the mushroom cap to expose the inside uncontaminated tissues. Using sterile fine-tip tweezers, pluck tiny pieces of the inside tissue and transfer them onto the prepared agar or gelatin media. This step is ideally done inside a still-air transfer chamber or a laminar flow hood with micron filters to further minimize the risk of airborne contamination.

Incubation

Incubate the cultures in a dark, clean, and cold room. Maintain a temperature of 4°C (39°F) for non-tropical Pleurotus strains. Inspect the cultures daily and discard any that show signs of bacterial or mold contamination.

Sub-culturing

Observe for the growth of new hyphae. When it becomes evident that the new hyphae are growing out of the transferred mushroom pieces without any contamination, it's time to sub-culture onto a new growth medium under aseptic conditions. To do this, cut a small piece of tissue from the uncontaminated area and transfer it to a new agar plate or gelatin medium.

Storage

Assign unique codes or numbers to each new culture and store them as mother cultures (pure cultures). Sub-culture every 6-12 months to maintain the viability of the strain. For long-term storage, agar media is recommended over gelatin media.

Spawn Preparation

Once you have successfully maintained a pure strain, you can increase the mycelial mass by transferring it to grain or sawdust/bran that has been sterilized in jars. These grain-filled jars are known as G1 Masters. From G1 Masters, you can create G2 spawn and even G3 spawn if desired.

Remember, mushroom tissue culture is an art, and with practice, you'll refine your techniques to ensure the successful maintenance of your cultures.

Frequently asked questions