Mushroom cloning, also known as tissue culturing, is a method used by cultivators to reproduce the exact genetic characteristics of a specific mushroom specimen. Unlike starting from spores, which can lead to unpredictable genetic variations, cloning ensures you capture desirable traits, such as increased size, higher yield, or resistance to disease. This technique involves taking a small piece of tissue from a mushroom and transferring it to a nutrient-rich medium, allowing the vegetative part of the fungus, called mycelium, to grow in isolation.
Preparation and Necessary Supplies
The most common reason for failure in mushroom cloning is contamination, making a sterile working environment necessary for success. This requires setting up a workspace that minimizes the movement of airborne contaminants like mold spores and bacteria. A Still Air Box (SAB), a simple, clear plastic container with armholes, provides an affordable and effective solution by preventing air currents from disturbing your work.
Before beginning, all surfaces inside the SAB must be thoroughly sanitized with a 70% isopropyl alcohol solution. This alcohol concentration is effective at disinfecting while still allowing for a quick evaporation time. You will need a sterile scalpel or a sharp razor blade, a lighter or torch for flame sterilization, and gloves and a mask to minimize contamination from your hands and breath.
The tissue sample needs a nutrient-rich base on which to grow, provided by nutrient agar poured into sterile petri dishes. Two common types are Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA), both containing carbohydrates and nutrients that support mycelial growth. MEA is slightly more acidic, which discourages bacterial contaminants, while PDA works well for a wide range of fungi.
Have your agar plates prepared and sealed before the procedure begins. The agar, a gelatinous substance derived from algae, provides the surface for the mycelium to colonize. Having all tools, the agar plates, and the mushroom specimen inside the sanitized SAB before starting reduces the need to open the box later, maintaining the still air environment.
The Tissue Transfer Technique
The cloning process begins with selecting a mushroom specimen that is young and healthy. The goal is to obtain tissue from the interior of the mushroom, which is naturally protected from airborne contaminants. The best area for a sample is usually the stem, or stipe, as the outer surface of the cap and gills are more exposed to the environment.
Instead of cutting the mushroom with a knife, which can drag contaminants inward, gently tear the mushroom open lengthwise to expose the clean inner tissue. Next, the scalpel must be heat sterilized by holding it in a flame until the metal glows red hot.
Allow the scalpel to cool completely before using it to excise a small piece of tissue, roughly the size of a grain of rice, from the center of the mushroom stem. A hot scalpel would instantly kill the delicate living cells. Working quickly and deliberately inside the SAB, lift the lid of the sterile petri dish just enough to insert the scalpel tip.
The tissue sample is then gently placed onto the surface of the agar medium, aiming for the center of the dish. Pressing the tissue deeply is unnecessary, as contact is sufficient for the mycelium to begin growing. The petri dish lid must be immediately replaced to prevent airborne microbes from settling on the exposed nutrient surface. After closing, seal the dish with a material like Parafilm or electrical tape to prevent the agar from drying out and maintain sterility during incubation.
Culturing the Clone and Next Steps
Once the tissue transfer is complete, the sealed petri dish needs to be placed in an incubation environment to encourage mycelial growth. Ideal conditions for most cultivated species involve a dark area with a stable temperature, typically ranging from 70 to 75 degrees Fahrenheit. This temperature range supports the vegetative growth of the fungus.
Within three to ten days, you should observe the growth of fine, white, thread-like structures radiating outward from the tissue sample. This growth is the mycelium. Inspect the plates for any signs of contamination, which often appear as fuzzy green or black spots of mold, or wet, slimy bacterial films.
If contamination is present, you may still be able to save the clone by performing a sub-culture. This involves cutting a small piece of clean mycelium furthest away from the contaminant and transferring it to a fresh agar plate. This isolation process is repeated until a pure culture is achieved. Once the mycelium has fully colonized the agar plate, it is ready for the next stage.
A fully colonized agar culture can be used to inoculate a larger volume of substrate. This is most often done by transferring small wedges of the agar culture into sterilized grain spawn, such as rye or millet. Alternatively, the agar can be used to inoculate a liquid culture, a nutrient-rich water solution that allows the mycelium to grow rapidly in suspension. Both methods effectively expand the clone, preparing it for eventual transfer to a bulk substrate for mushroom production.