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Cardboard Cloning Tek

A simple method for cloning without agar.

Here is a simple method for cloning without agar. It is also particularly useful when cloning a single specimen, and when contamination is a factor (you can use cardboard for isolation from contaminants in a similar fashion). Cardboard is semi-selective towards mycelium growth, and not favorable for the growth of most contaminants. This method can be used on many species, and works especially well for wood-lovers. Ps. Cubensis is illustrated here:

Cut a 1x1” piece of ordinary corrugated cardboard. Peel off one of the smooth sides, exposing the internal ribbed surface. Discard the flat piece. Try to avoid cardboard with large amounts of ink or glue. Clean off all remnants of cardboard fibers that were left over from removing the other piece. You want all the small fibers clear to prevent them from sticking up and drying out. You should also remove any glue residue if present as best you can before cooking.

Take a canning jar and fill ¼ full with water. Screw a plastic lid prepared with a poly-fill wad on the top to filter incoming air drawn in while cooling. Place the trimmed cardboard wedge in the microwave and cook at boiling for 10-15 minutes. I achieve this by microwaving at full power until it boils, then turn the heat setting down. I find a low enough setting to allow cycles of boiling, then cooling without boiling over. You may need to experiment to find the ideal. This cooking is done to not only hydrate and sterilize the cardboard, but to cook the glue out as well. Upon cooling the jar is capped with an alcohol soaked coffee filter for transport back to the lab. Pressure cooking is not necessary with cardboard in my experience.

Now find your mushroom to clone. Prime sites for tissue extraction are the thick tissue at the base of the stem, and the fleshy tissue inside the cap above the gills. Here the cap and stem are torn in half (not cutting, the blade can be a vector for contamination) and a small piece of inner tissue is carefully extracted (with a flame sterilized blade). Make sure you touch and extract nothing but clean tissue inside the mushroom, and do not get any gill tissue (or spores). If you use the base of a stem, be sure to extract tissue at least a half inch above the base to avoid contaminated tissue that is in contact with the substrate.

The excised tissue is then dunked in a 10% peroxide dilution (10ml 3% diluted with 90ml sterile water), and transferred to a petri with the cardboard. You can use any container you like provided it can seal in moisture and not dry the cardboard out. It is also important to note that the cardboard should be moist, not saturated. Too wet will cause slow, weak growth and will encourage contaminants. Place the dunked tissue deep into one of the ribs in the cardboard piece, to maximize contact area. Cover and incubate at the temperature required for your species.

After incubation for 1-2 weeks, the cardboard will be colonized inside and out (and on the back). You can then transfer pieces of this to Agar or strait to grain for spawn production. This method works very well, and should not be overlooked. It is especially useful when you don’t want to cook up an entire batch of agar to clone one mushroom.


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