Welcome to The Spore Works

Part Three

Initially, I wasn’t even thinking about mushroom breeding. While working with the PF Albino and growing a few pure white mushrooms, I decided to check out the gills under the microscope. I wanted to see if these mushrooms were spore-less, like everyone assumed. Surprisingly, spores were observed, and like the rest of the fruit body, the spores were colorless. With some effort, a light partial spore print, that was barely visible on foil, was produced from a very mature cap. The spores appeared white in deposit but there weren’t many and they were difficult to see. To check for spore viability, I placed some of the white spores on malt agar in petri dishes.

The spores germinated in only a few days. I noticed that there were very few colonies growing and they were spaced some distance apart. I assume this was due to the low number of spores and maybe low viability. The isolated germinations made me think that each colony might be from a single spore (monokaryotic). If this was true, I might be able to use these monokaryotic mycelia for breeding experiments. Maybe, even make an Albino Penis Envy! To prevent fusing with other mycelia from nearby spores and generating a dikaryotic mycelium, I quickly transferred the tiniest samples of tissue to new agar plates.

At this time, I should go over the very basics of mushroom sex for those that aren’t familiar with mushroom genetics. At its simplest, two compatible spores are needed to produce a mushroom producing dikaryotic mycelium. A mycelium generated from a single spore (monokaryotic) won’t produce mushrooms (usually) but is very useful for breeding new mushroom varieties. Crossing different monokaryotic mycelia is by simple mixing or allowing them grow together across a medium such as an agar plate.

To confirm that the mycelium was monokaryotic, a small sample was placed under the microscope to look for clamp connections. A clamp connection looks like a little bump or bulge between hyphal cells and indicates nuclear movement within a dikaryotic mycelium. A mycelium with no clamp connections should be monokaryotic, which is what I wanted to see. I want to keep this simple, so that is all I’m going to cover on clamp connections. More information and pictures can be easily found online.

At this same time, I was reading my newly acquired copy of “Genetics and Breeding of Edible Mushrooms” by A.C. Chang. I noted this passage on nuclear migration on page 47.

In many Basidiomycetes a phenomenon occurs following hyphal fusion which permits the establishment of a heterokaryotic condition in growing cells. This phenomenon is nuclear migration by which is meant the migration of nuclei through the established mycelium of a confronting strain. Nuclear migration was first described by Buller from observations of the formation of clamp connections, indicative of dikaryotic hyphae, at a distance from the site of fusion of compatible strains greater than could be explained by hyphal growth. Nuclear migration rates of different species may vary widely from 0.5mm/h in Coprinus lagopus (Buller, 1931) to 40mm/h in Coprinus congregatus (Ross, 1976). The advantage to the organism of nuclear migration is that the heterokaryotic condition is not limited to those heterokaryotic cells that have been formed by plasmogamy, but can be established rapidly in large numbers of cells and be perpetuated in the growing cells as is the case in the clamp forming dikaryotic hyphae of Basidiomycetes

I am pretty lazy and I didn't want to spend time isolating more single spores from other cubes and then do several controlled crosses and hope for the best. I was now aware of a mechanism where a monokaryotic mycelium can become a dikaryotic mycelium by nuclear migration, and not just at the new growth at the point of mycelial contact. Essentially the entire monokaryotic culture becomes dikaryotic by replicating and moving nuclei in a sort of a chain reaction through the already existing mycelial network.

Sooooooo.......... here is what I did.

I put the microscopically confirmed monokaryoitic PF Albino culture into a jar of sterile grain (grass seed in this case) and let it colonize almost completely. Every grain was covered with mycelium, but it would need a few more days to get really dense. Then I made a fresh solution (adding just dry spores also worked) of normally pigmented Penis Envy (PE) cubensis spores and injected that into the nearly colonized grain. I shook the jar to mix. The idea is to deny any uncolonized substrate to the injected PE spores for colonization if they combined (mated) with other PE spores. In theory, the PE spores will germinate and some will combine with other nearby germinated PE spores, but some will combine with the PF Albino mycelium. Nuclear migration should then transform the entire jar of monokaryotic PF Albino mycelium into several strains of dikaryotic PF Albino X Penis Envy mycelium. There shouldn't be any substrate left for any significant amount of pure Penis Envy dikaryotic mycelium to grow and later form mushrooms.

The colonized grain needs to be fruited directly and not spawned to a bulk substrate. Surprisingly, this method worked on the first attempt and produced some exceptionally normal looking Psilocybe cubensis mushrooms, with no hints of mutations or abnormalities. Since both varieties are supposedly descendants of early collections from the Amazon, this may be a glimpse into what the original mushrooms looked like when first collected.

PF Albino x Penis Envy (F1) mushrooms grown from cased grass seed. (above)

Now that we have varietal hybrid mushrooms, the hard part is over. I can just take spore prints of this generation and use them for new multi spore cultures. The first generation is genetically 50% of each parent and all the mushrooms look the same. But the later generations are where the action is, and we should expect to see all sorts of crazy combinations of traits. And that is what I observed.

A few examples of the F2 generation showing various forms. (above)

After selecting spores from promising specimens from the F2 generation, I finally got an early version of what I was looking for. An albino mushroom with a penis shaped cap in the F3 generation and another interesting form that I called the PE Uncut.

The very first Albino Penis Envy (APE) 2006

After a few more generations for stabilization and we have the APE we have today. As with the original Penis Envy, the reduced caps don’t produce or release many spores. But spores are present and can be swabbed from the gills in adequate amounts for propagation. I also developed an in-house method of separating and concentrating spores with a centrifuge for syringes.

I created the APE purely with aesthetics in mind and didn’t intend to produce a super potent variety of Psilocybe cubensis. Anecdotal reports always rated the APE highly potent but real results finally came to light in 2022 with the advent of laboratory testing in the Psilocybin Cup by Hyphae Labs, where APE ranked at or near the top in potency.

https://www.oaklandhyphae510.com/post/oakland-hyphae-hyphae-labs-present-the-spring-psilocybin-cup-2022-final-results

And again in 2023 with Colorado’s first Psychedelic Cup.

https://www.5280.com/what-we-learned-at-colorados-first-psychedelic-cup/

John Workman