Mushrooms have very different physiological properties between different varieties. However, there are also significant variation in the property of a mushroom culture depending on its unique genetic makeup. Within the mycology and mushroom cultivation world, we refer to these differences in mushroom cultures as strains.

Cultivators have poured great resources into isolating out superior-performing strains of mushrooms. They select for desirable properties over multiple generations of the fungus lifecycle. They select for characteristics such as fast colonization time (often referred to as mycelium running) to improve cultivation turnaround time. They select for strong immunity towards competing organisms so that their colony is resistant to contamination. They select for sturdy fruiting bodies to make mushrooms less perishable and easier to transport to stores without becoming damaged. They select for productivity so that they can increase yield from each mushroom fruiting block. They select for high nutritional content to offer a more superior product to their customers.

This selection process can continue endlessly, but when one finds a "winning strain," they would want to "clone" and "save" an exact copy of it, so that they can continue to grow that strain once again in the future.

In this blog post, I will cover the general biology of mushroom genetics and how us mycologists go about preserving desirable genetics in the form of mushroom clones.

Mushroom Taxonomy

Mushrooms belong to the Fungi Kingdom, which includes a vast array of organisms. Fungi are distinct from plants and animals in that they do not have chlorophyll and cannot photosynthesize. Instead, they obtain nutrients by breaking down organic matter in their environment.

When you obtain spores from a mushroom such as an oyster mushroom, or lion's mane mushroom, you are obtaining a particular species of mushroom. In the example of Hericium coralloides, the first word Hericium refers to the Genus, whereas the coralloides refers to the species. You can have other species of Hericium, such as Hericium coralloides, Hericium americanum, Hericium erinaceus, and many more.

What is a mushroom strain?

To better understand the concept of a strain, we should first go over what a Species exactly means. A Species is defined as a group of organisms that are unable to interbreed with other Species and produce healthy, fertile offsprings. Members of a Species can only breed and reproduce within that same Species.

In biology, a strain refers to a genetic variant, or a subtype within a species. This means members of the same Species, but different strains can still breed and produce viable offsprings. This is an important concept to understand when cloning mushrooms, because you can define which mushroom cultures can share genetic information between each other. For instance, Hericium coralloides cannot reproduce with Hericium americanum because they belong to different Species.

How do Fungi reproduce?

Mushrooms reproduce both sexually and asexually. Sexual reproduction involves the fusion of two haploid nuclei, one from each parent, to form a diploid zygote. Asexual reproduction, on the other hand, involves the cloning of an individual organism.

Asexual reproduction occurs through either fragmentation, budding, or spore generation.  Fragmentation refers to the segmentation of the fungal hyphae that can develop into separate mycelium. Budding is where the fungal cell divides mitotically, and the new nucleus "buds" off from the mother cell in asymmetric division, but this is only commonly seen in budding yeast.

Finally, we have spores, which is the most predominant mode of asexual reproduction. Spores are produced by one parent through mitosis, which means that the spores are identical to the parent. Spores are a method for Fungi to disperse their progeny among the environment, as well as enter a dormant phase when the environment might become inhospitable for growth.

Mushrooms have a complex life cycle that includes both a haploid and diploid phase. The haploid phase is represented by the spores and the mycelium, which is the vegetative part of the fungus. The diploid phase is represented by the mushroom itself, which produces spores through meiosis. We will cover this more in-depth in the next section.

Sexual Reproduction

Sexual reproduction introduces genetic variation to your mushroom Fungi species. Before we begin, it is important to note that Fungi exist as haploids for most of their lives, and only merge to form diploids at the zygote state briefly, then undergo meiosis to reestablish the haploid stage once again. During sexual reproduction, the first step involves plasmogamy, where two compatible haploid cells fuse to form the dikaryotic stage (two haploid nuclei stage). The second step is karyogamy, where the haploid nuclei fuse to form a diploid zygote (single nucleus), which is also referred to as the zygote. It's at this fusing of haploid nuclei stage that we get swapping of genetic information, and introduction to variability in our mushroom strains. In the third and last step, meiosis occurs where the gametes of different mating types are formed. Spores are generated and released into the environment. Therefore, the spores that are generated from this sexual reproduction are the genetic combination of the two parent mycelia. It's through this method that we can generate biological diversity in our mushroom strains.

Note that we covered spore generation during both sexual and asexual reproduction, and this is true. Mycelia can generate spores in both forms of reproduction. The spores that we collect from mushroom fruiting bodies are the result of sexual reproduction, meaning that each of the spores contain some genetic variation.

Crossing Spores

Now that we understand a bit more about the mushroom lifecycle, we can move onto how we can cross different strains of mushrooms to select for properties that we want. From spores, new mycelia will grow, and this new mycelia once again has the opportunity to "fuse" with other mycelia and merge their haploid cells to generate a diploid zygote, which can then undergo meiosis again to produce spores that have scrambled genetic information; Therefore, we can control the fusing process by doing serial dilutions of our mushroom spores, and "streaking" them onto an agar plate to grow. The goal here is to dilute out the mushroom spores so much that when they start growing on an agar plate, they are a single colony of haploid mycelia sharing the same genetic information or DNA. Then you can combine the mycelia of two isolated homogenous colonies and let their haploids fuse, mix, and produce a new genetic strain of mushrooms.

Another simpler way to cross and mix spores, is to mix them within a test tube or syringe, and inoculate an agar petri dish with a high concentration of spores. Once they start developing into mycelia, they will be in close proximity and cross their haploids naturally. These mycelia will then grow outwards in a single colony. But because there are many crossed and mated mycelia strains competing, they will generate this "sectoring" effect where you will see wedges like pizza slices in your agar plate. Each of those wedges or slides will be its own distinct strain of mycelia. You can then isolate each wedge out and let it grow separately on a new agar plate. This second passage will produce a mycelial ring that grows evenly (same mycelium running speed), and similar morphology which confirms that the mycelium colony you've isolated is a pure strain.

Mycelium culture started from concentrated spore solution produces sectoring in the first passage (P1).

A single colony of mycelia growing on MEA petri dish after second passage (P2).

You will notice that as you continue to cross mushroom strains, you will naturally select for properties that are best suited for your growing environment. For instance, if you live in a warmer, drier climate, the mushroom culture you purchased from a colder, more humid region might not be doing so well. However, if you manage to repeat this crossing and selection process multiple times, you would naturally be selecting for strains that are naturally more adapted to your growing environment and conditions. Further, you will also see that certain wedges of mycelia will grow faster than others. This is also another form of selection that mushroom cultivators can select for. You can pick sections of mycelia that grow faster than the others to expand out. Later during fruiting, you can also keep track of which strains are more productive. That's why its super important to keep detailed notes of your crosses and mushroom strains.

Cloning

Once you've repeated the crossing and selection, and arrived at a mushroom mycelia strain you think has all the desirable characteristics, you can clone that strain. This means that you can keep taking small sections of that mycelia and passaging it onto new agar petri dishes to let it grow out. This is the fragmentation part of asexual reproduction that we covered earlier.

Saving or storing your strains long term

You can continue to "clone" your desired strain this way and even put it in the refrigerator for long term storage. If you want to have a backup of this strain, you can let the mycelium develop into fruits, and collect the spores in a spore print. The spore print will have mixing of the genetics, but it will be mixed within the same mycelial colony, instead of with an outside colony. This way, you can preserve the genetics in a more stable spore form that although is not an exact copy, but will share similar properties as the parent clone.

Step by step how to clone mushrooms

Cloning a mushroom strain involves taking a small piece of mycelium and growing it on an agar petri dish. This small piece of mycelium can be taken from another colonized agar petri dish or from a mushroom fruiting body as well. Agar is a gelatinous substance that is derived from algae and is commonly used in microbiology to grow microorganisms. The mycelium is placed on the agar, and over time, it will grow and form a new colony.

To clone a mushroom strain, you will need to start with a mushroom mycelia that is healthy and free from contamination. Once you have selected your mushroom mycelia, you will need to clean the surface with alcohol to sterilize it.

Using a sterile scalpel or razor blade, carefully remove a small piece of tissue from either your petri dish or from the stem or cap of the mushroom. The tissue should be no larger than a grain of rice. Place the tissue on a sterile agar petri dish and incubate it at the appropriate temperature and humidity for the species of mushroom you are working with.

Over time, the mycelium will grow and form a new colony on the agar. This new colony is genetically identical to the parent mushroom and is referred to as a clone. The clone can be used to start a new culture or to inoculate a substrate for mushroom cultivation.

Conclusion

Mushrooms are a fascinating group of organisms with a wide range of applications. Understanding the genetics of mushrooms is an important step towards improving their cultivation and harnessing their potential. Cloning a mushroom strain is a simple and effective way to propagate a specific genotype for further study or cultivation. With the right tools and techniques, anyone can learn how to clone mushroom strains using agar petri dishes.

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