Emma Wilson
Cross-breeding mushrooms, or hybridizing mushrooms, is a process that involves combining two or more strains of mushrooms to create a new strain. This process aims to retain the best characteristics of the parent strains, such as potency and ease of growth. While it is possible to crossbreed mushrooms, it can be tedious, time-consuming, and challenging. The success rate of crossbreeding varies, and it may take multiple attempts to achieve a successful cross. Additionally, the qualities passed down from the parent strains to the offspring can be unpredictable. Several techniques are employed to crossbreed mushrooms, including mycelial mating, protoplast fusion, and molecular genetic transformation, each requiring precise methods and specialized equipment.
| Characteristics | Values |
|---|---|
| Mushroom hybridization technique | Isolation and germination of single spores of both parents, followed by mycelium fusion in a petri dish |
| Requirements | Single spores, petri dishes, agar, swabs, and grain |
| Challenges | Tedious and time-consuming, spores may not mate and form hybrids |
| Success Rate | Varies, typically low (e.g., 1 in 4 to 16 crosses succeed for P. cubensis) |
| Commercial Applications | Development of new strains for the mushroom industry, such as H. marmoreus |
| Advanced Techniques | Mycelial mating, protoplast fusion, molecular genetic transformation, somatic cell fusion, gene editing |
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What You'll Learn
Isolating single spores from both parents
To cross-breed mushrooms, you need to isolate single spores from both parents. This process is precise and controlled but tedious and time-consuming. It requires the use of many petri dishes to isolate single spores and achieve a few viable crosses.
Firstly, you need to extract the spores from the mushroom. To do this, take a fresh, healthy mushroom with a firm cap and moist gills. Remove the stem from the mushroom, leaving the cap with the gills intact. Place the cap gill-side down onto a piece of paper or sterilized tin foil. Alternatively, you can use a heat-sterilized knife to carefully remove the skirt of the cap, avoiding the gills. Place a drop of water on the cap and cover it with a bowl. Leave it for 2-4 hours.
After this, you should be able to see the spores. To preserve them, put a single drop of distilled water on the print and place a slide cover on top. You can also observe the spores under a microscope. To do this, sterilize your water, syringes, and tools in a pressure cooker. Place 10 mL of distilled water in a glass or shot glass, cover it with aluminium foil, and wrap your syringes and tools in aluminium as well. Place everything in a pressure cooker. Then, place a small amount of spores on a slide and observe under the microscope.
Once you have extracted the spores from both parent mushrooms, you can proceed to the next step of the mushroom hybrid technique. This involves creating a petri dish of monokaryotic mycelium from a single spore. Streak an agar petri dish with a small amount of spores, and at the end of the streak, there should be just a few spores on the agar. As soon as you notice germination, transfer a small bit of mycelium to a fresh plate, selecting isolated germination points away from any clusters. The mycelium from a single spore grows slowly and cannot fruit. You can confirm that the mycelium is monokaryotic by observing it under a microscope and looking for the lack of clamp connections.
Once you have your petri dish of monokaryotic mycelium, you can maintain it by periodically transferring it to new plates. You can then add the spores of the second parent to the mix. Open the spawn jar lid and scrape in some spores of the second parent, or make a fresh syringe and inject the spores. Shake the jar and let it incubate for at least a week or two.
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Germination of spores in a petri dish
To crossbreed mushrooms, you will need to begin with single-spore isolates from two different strains, placed on one petri dish. This process will need to be repeated several times as fungi have a complex mating incompatibility system, and only one in several (4 to 16) crosses will be successful.
To begin the germination process, you will need to prepare your work surface and petri dishes. Sanitize the surface with a solution of 1 tablespoon of unscented bleach and 1 gallon of cold water. Wipe the surface with a lint-free cloth and allow it to dry. Prepare a bowl of isopropyl rubbing alcohol and place a metal paper clip in it for each mushroom you will be working with. Allow the paper clips to soak for 10 minutes and then transfer them to a lint-free cloth to air dry.
Next, prepare your petri dishes. You will need pre-sterilized plastic petri dishes, which you can place on your sanitized work surface. Prepare an agar mixture by boiling 1 cup of water and then removing it from the heat. Add 1 tablespoon of agar and stir until dissolved. Add the agar mixture to each petri dish, filling each about three-quarters of the way or 1/4 inch from the top.
Now you are ready to prepare your mushrooms. Position a cardboard or soft-plastic box about 1/2 inch from the side of the petri dishes. Take the paper clips and bend them to create a right angle. Insert one end of each paper clip into the side of the box so that it resembles an upside-down L. The height of insertion will depend on the size of your mushrooms. Insert the other end of the paper clip into the center of the bottom of a mushroom stem, so each paper clip is attached to its own mushroom. The mushrooms should be suspended over the petri dishes without touching them. Allow the mushrooms to hang over the dishes for 24 hours. You may need to gently squeeze the mushrooms to facilitate the release of their spores into the dishes. Seal the dishes with their covers and place them in a dark room with an ambient temperature between 55 and 80 degrees Fahrenheit.
Within two to three days, you should see mycelia strands growing in a web formation in the petri dishes, indicating successful germination. Once the dishes are completely colonized with mycelia strands, you can proceed to the next step of crossbreeding.
At this point, you should have a petri dish of monokaryotic mycelium, which can be used to make crosses with another strain. You can confirm that the mycelium is monokaryotic by examining it under a microscope; monokaryotic mycelium lacks clamp connections. To make a cross, inoculate some spawn with this mycelium and let it grow to near completion. Once the spawn is fully run with mycelium but not completely white, you can add the second parent by scraping in some spores or injecting them with a fresh syringe. Shake the jar and let it incubate for at least a week or two.
Note that this process is tedious and time-consuming, and the results of crossbreeding mushrooms can be unpredictable. It may take several attempts to get a successful cross, and the offspring may not always resemble the parent strains.
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Mating of monokaryotic mycelia
The mycelium from a single spore grows slowly and cannot fruit. It is important to confirm that the mycelium is monokaryotic, which can be done by examining it under a microscope. Monokaryotic mycelium lacks clamp connections and is visually distinct. This step can be skipped if a microscope is unavailable. Once a petri dish of monokaryotic mycelium is obtained, it can be used to make multiple crosses.
For the next step, some spawn is inoculated with the monokaryotic mycelium and allowed to grow until the spawn is fully run with mycelium but not completely white. This monokaryotic jar can then be introduced to the second parent by adding spores of the second parent to the jar or by injecting spores with a fresh syringe. The jar is shaken and allowed to incubate for at least a week or two.
The process of mating monokaryotic mycelia involves the fusion of two compatible monokaryotic mycelia to form a dikaryotic mycelium. This dikaryotic mycelium has the potential to develop fruiting bodies, such as mushrooms. The two nuclei from the fused monokaryotic mycelia remain unfused in the dikaryotic mycelium and eventually fuse in the reproductive cells of the mature fruiting body. This process is essential for the formation of fruiting bodies, which is a critical stage in the mushroom life cycle, especially for edible mushrooms with high economic value.
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Inoculating spawn with mycelium
Inoculation is the process of introducing mushroom spores or spawn to a suitable growth medium. Mushroom farmers typically use mushroom spawn and liquid culture to inoculate substrates, as they usually contain mycelium cloned from a proven strain with desirable characteristics. The type of mushroom you are growing will affect the inoculation rate, and you may need to use more spawn when growing fussier species that are slow colonizers.
Before inoculating grain spawn, it is important to wipe down your grain jar and agar dish with alcohol and set them up in front of a laminar flow hood. If you don't have a laminar flow hood, try to make a "still air box" to minimise airflow. Flame-sterilise your scalpel or blade until it is red-hot, then quickly cool it off by dipping it into the agar dish. You should hear an audible sizzle. With your cooled blade, cut a piece of mycelium from the agar dish and transfer it to the jar.
If you are using a syringe, it is important to note that no syringe is 100% clean. Use a minimal amount of spore solution and injection ports or polyfill holes to prevent contamination. You can also use micro-pore tape to cover the holes in your jar, which will help with gas exchange and keep out airborne contaminants.
When inoculating spawn with mycelium, it is important to allow it to grow to near completion. Once the spawn is fully run with mycelium but not completely white, you can proceed to the next step. You can also speed up the process by breaking up the grain and mixing the contents of the jar once there has been about 30% colonisation. This spreads the mycelium more evenly and gets the jar colonised quicker.
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Cross-breeding with somatic cell fusion or gene editing
Cross-breeding mushrooms is a precise and time-consuming process. It involves the isolation of single spores and the use of multiple Petri dishes to create viable crosses. This method requires the isolation of monokaryotic mycelium from a single spore, which can be done by streaking an agar Petri dish with a small amount of spores.
While the traditional method of cross-breeding mushrooms is tedious, modern techniques such as gene editing and somatic cell fusion offer new possibilities. Gene editing, for example, uses the CRISPR/Cas9 system to modify the genome of mushrooms. This technique allows for the development of better markers for gene-editing breeding and can be combined with metabolism engineering and in-silico tools analysis for more precise results.
Somatic cell fusion, on the other hand, involves stripping the cell wall with enzymes and then forcing the cells to merge using chemicals. This method is not commonly performed outside specialized labs due to the use of dangerous agents like venom. Results from somatic cell fusion are often unstable and rarely produce fruitful outcomes.
Another technique that has been used to create mushroom hybrids is protoplast fusion. This method has been applied interspecifically, intraspecifically, and interhetero generically to modify phenotypic traits and improve bioefficiency and nutritional properties. Protoplast fusion has successfully realized genetic manipulation, especially in fungi lacking sexual reproduction capacity.
Overall, while traditional cross-breeding methods for mushrooms are precise but time-consuming, modern techniques like gene editing and somatic cell fusion offer new avenues for creating mushroom hybrids. These techniques can improve efficiency, nutritional value, and yield, as well as overcome sexual incompatibilities between different species.
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Frequently asked questions
The first step is to isolate and germinate single spores of both parents.
You should then let the mycelium from both parents cross in a petri dish.
Once the spawn is fully run with mycelium but not completely white, you can add the second parent in the cross.
