In November of 1999, the Chinese spacecraft "Shenzhou I" orbited our planet 14 times in its 21-hour journey through space. Inside, the spacecraft was loaded with state-of-the-art equipment and research materials. While no human was on board this flight, there were living organisms, amongst them numerous cultures of the highly prized Reishi mushroom (Ganoderma lucidum).
Shenzhou-1 return capsule displayed at China Science and Technology Museum. Within it’s contents were included various living cultures of Reishi mushroom. Photo by Shujianyang.
The intent was not to take the cultures on an extraterrestrial joy ride. It was actually quite the opposite. They exposed them to cosmic radiation, microgravity, and the harsh conditions of space. Nothing to do with a joy ride.
The purpose of this was to see how it affected the fungal cultures and if, by chance, beneficial mutations could occur in these extreme conditions. This process, known as induced mutagenesis, is the topic of study for many researchers and has been successfully used to produce novel strains of mushrooms.
Thankfully, you don’t have to send fungi to space to perform induced mutagenesis. Since at least the late ‘60s (Garibova, 1969), researchers have been experimenting with induced mutagenesis by exposing cultures to UV light and different chemical mutagens. While these sorts of studies essentially “roll the genetic dice” by causing mutations at random, researchers undergo a strategic process to select strains with a high potential to have beneficial traits.
While there is some area of debate around these sorts of breeding techniques, I will clarify that the resulting strains are not considered GMO organisms. They do not use methods to introduce foreign DNA from organisms but simply utilize a process that significantly speeds up the rate of mutations, which does occur in nature.
After all, anyone who has worked with mushrooms in a laboratory knows traditional breeding techniques take a lot of time and patience. By successively growing out spores or isolating cultures, growers can develop breeds that have better yields or are more suited to their environmental conditions. While these approaches definitely work, true breakthroughs are rare, often coming only a handful of times in a dedicated grower’s career.
Improving Temperature Tolerance Of Fungal Strains
A 2013 study titled “Breeding of new high-temperature-tolerant strains of Flammulina velutipes“ explored the potential of utilizing mutagenesis to produce a strain of Enoki (Flammulina velutipes) more tolerant to warmer temperatures. This mushroom, highly prized in Asia, traditionally requires cold temperatures of 60°F (15°C) or below during cultivation. This makes it challenging to grow in many climates, requiring strict climate control with high energy costs for a successful crop.
Classic shape of cultivated Enoki. Long stems and pale color are created by fruiting conditions with high CO2 and low light. Photo by frankenstoen.
In attempts to develop a novel strain, researchers exposed the mycelium of Enoki to two chemical mutagens and UV radiation. The chemical mutagens included Ethyl methane sulfonate (EMS), which is very common in these studies, and Lithium chloride (LiCl). Some samples received combinations of multiple treatments, including these chemical mutagens and UV radiation. Mycelium was given doses of these mutagens that resulted in 80–85% lethality.
After these treatments, the remaining mycelia were incubated at 33 °C for 15 days. This temperature was shown to be fatal to the parental strain, suggesting that any surviving cultures must have some sort of heat tolerance. Indeed, some of the mycelial fragments grew just fine at these high temperatures, and those that displayed typical morphologies in culture were selected for further study. Researchers repeated this procedure six times and subjected the mycelia that maintained stable high-temperature tolerance to antagonistic testing to identify true mutants.
Afterward, these cultures were grown on a bulk substrate to test their fruiting capabilities. Fruiting temperatures of 20 °C were selected, as it was determined to be the highest temperature at which fruiting body formation could occur. At fruiting temperatures of 20 °C, the non-mutated parental strains had a very slight fruiting tendency, very few primordia, and a biological efficiency of only 4.87%. In contrast, three mutant strains grew successfully at these temperatures with biological efficiencies of 31.64%, 27.74%, and 37.83%.
Ultimately, these researchers successfully developed heat-tolerant strains of Enoki through mutagenesis. Currently, I know there are strains of heat-tolerant Enoki on the market, but I am unsure if they are derived from this specific study. A similar study was conducted on King Oyster (Pleurotus eryngii), where strains capable of growing at 32°C and fruiting conditions at 18 °C were developed.
Improving Low-Temperature Tolerance Of Paddy Straw Mushroom
Likewise, researchers have also experimented with improving some species tolerance to lower temperatures. This was the case for this 2016 study that aimed to enhance the tolerance of the tropical Paddy Straw Mushroom (Volvariella volvacea) to low temperatures.
Typically Paddy Straw Mushrooms. Photo by Dorami Chan.
The goal of this study wasn’t only to produce a strain that grows well at low temperatures but also to overcome the fact that fruiting bodies of Paddy Straw Mushroom don’t traditionally handle the low temperatures used in refrigeration.
Due to its low-temperature intolerance, the fruiting bodies of Paddy Straw Mushrooms quickly soften, liquefy, and rot at refrigeration temperatures. This results in a short shelf-life, making it a significant challenge for distribution and commercialization.
In this study, researchers used induced mutagenesis, as discussed above, as well as a novel technique known as “Genome Shuffling.” This technique entails taking multiple parental strains of the fungus and creating hybrids via a method called protoplast fusion.
Below, I will describe this technique in simple terms. If it’s over your head, all you need to know is that Genome Shuffling allows researchers to make hybrids of various parental strains simultaneously. Through repeated hybridizations, researchers select strains that display the desired traits.
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Numerous parental strains are chosen to provide a high genetic diversity. In the case of this Paddy Straw Mushroom study, 16 parental strains were used, representing a majority of cultivated strains in Southeast Asia at the time.
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These parental strains are then exposed to enzymes that degrade their cell walls. This leaves you with cells that do not have a cell wall, known as protoplasts.
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At this point, the scientists expose the protoplasts to mutagens, like UV light, to further increase the genetic diversity of the samples.
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Afterward, protoplasts of different strains are mixed in a solution. Without their cell walls, genetic material from parent strains can fuse and mix.
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The fusion process results in new cells that contain the genetic material from parent cells, creating hybrids with potentially new characteristics.
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This process is repeated multiple times. At each stage, the varieties with desired traits are selected. This entire process is known as genome shuffling.
By the end of their study, the researchers had successfully developed strains with significantly higher low-temperature tolerance. These strains not only grew well with a high yield, but they had an increased shelf-life during refrigeration. Two of their newly developed strains had 75% longer shelf life than the best-performing parental strain.
Improving Microbial Resistance, Mushroom Color, and Spore Production
These breeding techniques aren’t just used to improve mushroom temperature tolerance. No, they are utilized for tons of other applications.
One team of researchers used induced mutagenesis to reduce the spore production of Oyster Mushrooms (Pleurotus ostreatus). The reason was to minimize potential health hazards associated with spore inhalation. Their results were published in this 2014 study titled “Strain Improvement in Pleurotus Ostreatus using UV light and ethyl methyl sulfonate as mutagens.”
Instead of exposing mycelium to mutagens, as discussed in previous studies, these researchers exposed the spores of three parental strains to Ethyl methane sulfonate (EMS) and UV light. Not only did they successfully breed a strain with reduced spore production, but they also developed a mutant with unique coloration.
Another group of researchers attempted the opposite. In their study titled “A Novel Strain Breeding of Ganoderma lucidum UV119 (Agaricomycetes) with High Spores Yield and Strong Resistant Ability to Other Microbes' Invasions,” they discuss their process of breeding a novel strain of Reishi (Ganoderma lucidum) with increased spore production.
This is because the spores of Reishi have become a highly valued medicinal product in Chinese medicine, but few strains have been developed for this specific purpose. On top of this, researchers hoped to create a strain with high microbial resistance since many spore-producing strains were significantly vulnerable to contamination.
Growth of mutagenesis on PDA plate dominated by Trichoderma. LZ-1: Longzhi No.1. (A): UV119 grew on PDA plate with Trichoderma; (B): LZ-1 grew on PDA plate with Trichoderma. (Chuanhong Tang 2023; CC BY 4.0)
Using the genome shuffling techniques discussed earlier, researchers developed a strain with these desired characteristics. Of the 165 mutated strains they produced, one was particularly effective.
This strain had a fruiting body yield of approximately 9% higher than the parental yield, with spore production that was almost 20% greater. When cultivated in field conditions, the novel strain had a 99.15% success rate compared to the 69.20% rate of the parental strain, suggesting improved tolerance to microorganisms. Microorganism resistance was also visible in co-culture experiments.
Production of spores during the strain cultivation. (A): Spore collection; (B): LZ-1; (C): mutagenic strain UV119. (Chuanhong Tang 2023; CC BY 4.0)
Beyond the Lab
While induced mutagenesis may seem like a complex process confined to professional research, some techniques, like UV irradiation, could be easily adapted by hobbyist growers or small farms. While I won’t go into detail about how to do it since I don’t have personal experience with it, you can check out the articles in this text to get an idea of their procedure. There are dozens, if not hundreds, of other studies on this subject that can be found online.
Just do your research carefully and adhere to all safety protocols! Even UV light can be dangerous if misused; take it seriously. Do not utilize chemical mutagens mentioned here unless you have extensive experience handling hazardous chemicals.
We hope you enjoyed this article and that it brought you unique insights! Happy growing!
