8 replies to this topic

[ElrikEriksson]

Breeding fungi is not as hard as many believe. Internet legends would have you convinced that fungal husbandry requires very expensive lab equipment, a university degree, exotic reagents such as rattlesnake venom, and a fanatic level of patience and precision to allow for manipulation of individual spores. This is simply not the case.
At the most simple one can intimately mix spores from two varieties, use that mixture to do several small grows, and continue the best of the resulting mushrooms to the next generation. This can lead you to improved mushrooms, and they may indeed be hybrids, but unless there are very easily identified traits from both parent varieties you will not know for certain if what you got is a hybrid or simply a good selection of one of the parents. By all means if your goal is simply to have better mushrooms and there are two varieties you love, Costa Rican and Mazatapec for instance, please do mix the spores and breed them on for several generations. Your result may be awesome. But dont call them 'Costa Rican X Mazatapec', as the parentage by that method can not be certain. Just name them after your girlfriend or something.
The purpose of this thread is to inspire people to go to the next level and correctly create specific crosses with specific, defined goals.
This will require some specific equipment many growers do not have, but not much.
You will need a still air box, which growers likely will have. You will also need petri dishes, telephone brand agar [the gold standard agar for amateur microbiology], a nutrient such as malt extract or instant potato + sugar or powdered dry dog food, a dye such as methylene blue, and lastly you will need a microscope that gives a clear image at 400X. If you have those materials, we can proceed.

When an individual mushroom spore sprouts it forms a single monokaryotic strain. This monokaryon only has half of the genetic material a fruiting [dikaryotic] mushroom strain has. The monokaryon grows and spreads  like normal mushroom mycelium except it isn't rhizomorphic and it will never fruit. When two cubensis monokaryons meet they have a minimum of a 25% chance of being sexually compatible, the odds improve if they are not closely related and can theoretically get as high as 100%. To hybridize like a pro you first need to isolate, stabilize, and verify at least one monokaryotic strain.
There are various tactics to catch a monokaryon. I go for arguably the simplest method. I prepare malt extract agar plates, get a small but visible speck of spores on a loop, streak it back and forth into a stripe down the middle of the petri dish, turn 90°, and then cross-swipe all over the dish. With luck this will spread the spores thinly enough for you to catch free monokaryons before they mate. You must transfer them soon after they grow to a size that is just visible to the eye if you want good success. When I first start seeing them I will sometimes wait 12-18 hours so more spores sprout and I can figure out which of those newer sprouts are in areas with a lower spore density.
To make the transfers I use hobby knife blades. I do not flame these, I just put them in test tubes in groups of 10, cover the tubes with foil, and put them in the foil covered bread pans I have other tools in for oven sterilization. I use these blades to cut a square about 1,5mm by 1,5mm around a lone, isolated monokaryon candidate. Then I cut just under the surface of the agar so the wedge is as small as a grain of millet and quite thin.
Here you can see a plate 24 hours after I removed 12 transfers. At this point it would have been too late, but most of the transfers I made just a day before proved to be monokaryotic. I try to streak plates more thinly than this, these were just awesome spores.

I generally put three to four transfers onto each new agar plate. When the new colonies grow to 1,5 to 2 cm they can be tested. Test them and take transfers well before they touch! Be sure to label the bottom of the petri and number your slides so you know which slide came from which colony. I like to number the plates and then number the colonies left to right, top to bottom when viewed from the top. I then name the isolates plate.colony. Like colony 3 from plate 4 when working with Cuban would be Cuban 4.3.

Now comes the microscope. The easiest way to get mycelium from an agar plate to a microscope slide that I've found is to set up a slide with a coverslip on top, put a small drop of dye off center on the slip, take a piece of clear plastic tape from an alcohol sanitized roll and just tap the sticky side against the mycelium so the mycelium leaves a 'thumb print' on the tape. Then stick the tape to the coverslip so the dye spreads out under it as you lay it down. Flip the slip+tape over and firmly press out any bubbles and excess dye. Don't let the tape slide across the slip or it'll damage the cells. Methylene blue in a concentration of 0.25 to 0.5% is a wonderful dye for this application.
You can use the lowest magnification to get a view of a good group of the cells, then switch to 400X and if your microscope is a good one you should be very nearly in focus, just needing to go a little bit one way or the other. What you are looking for are clamp connections.

A fungal cell has two nuclei. When a cell divides the daughter cell only gets one, the second is then given to the cell by this little loop that forms at the end of the cell where the cell also has the ability of branching. Don't be fooled by baby branch spurs, it'll be a loop connecting two adjacent cells that you're looking for. If a colony has any clamp connections it is dikaryotic and not what your looking for.
Monokaryotic mycelium, the stuff you want, has cell-cell junctions that all look like hot dogs lined up end to end, like this:

But don't be fooled, if you handled the cells roughly they can make splits that look like cell-cell junctions. Also, if a colony has just bred it might only have a few clamp connections and if a clamp connection is pointing up or down it may not be visible. Hunt around, look at other sections, be suspicious.
The ones that look healthy with no clamps are the colonies you want to transfer from the group testing plates to new individual plates to grow out. You'll also want to watch out for chaotic or distorted cell growth, that's bad, you want nice healthy orderly cells.
You'll want to test them with the microscope one last time when the now isolated colony has grown to 3 cm or larger on its new plate. This is to make sure it wasnt a colony that has just bred or was carying a still-viable spore. If its still monokaryotic, then your ready to move on. You may be thinking this will involve making two monokaryons breed on agar, but for general breeding work there is an easier way that also generally gives better results. You now want to colonize jars of grain with your healthiest monokaryon.

To colonize a jar of grain I grow the monokaryon out on agar as above, and then I dump some sterile water into the dish and scrub the mycelia off into the water with a wire loop I made out of thick steel wire and heated to bright red hot and dropped in water to give it a rough texture. This mycelial suspension is injected into sterilized jars of grain with a wide bore needle. Once the jar is 100% colonized with white you then shake the jar, inject some spores of the prospective parent #2, shake again, and give it about 10 days for the spores to germinate and breed to the monokaryon.
At this point the spawn is an F1 hybrid between the single isolated 'Parent #1' monokaryotic strain and the dozens or hundreds of strains that came out of the 'Parent #2' spores. You can spawn this to a minimal amount of minimally nutritious bulk substrates and fruit it. A 1:1 or 1:2 spawn to sub ratio using coir is fine. There may be some unbred monokaryotic mycelium but it'll acquire a second nucleus as it grows out into the substrate. There will be specks of dikaryotic mycelium that formed from two Parent #2 spores, but if the grain was fully colonized with the monokaryon and the bulk substrate isn't highly nutritious the non-hybrid mycelium should not gain enough mass to form mushrooms. Here is my first hybrid that was actually better than the average cube, PE6 4.1 X PESH F1, it's got good visual intensity while being exuberantly euphoric and fun, but not speedy:

Now, there's a fine point hidden in the last paragraph. You formed the F1 from live mycelium and got mushrooms from this mycelium. At no time do mushrooms have F1 hybrid spores the way tomatoes can have F1 hybrid seeds. Spore prints taken from the above grown mushrooms should be labeled as the F2 generation because that's what they will grow, if grown. The spores collected from that F2 generation will make the F3 and so on.
At this point we're still dealing with hybrids, but the title of this thread says varieties. The difference is selection and stabilization. If a hybrid is grown out through 8 or more generations with strict selection criteria its traits can be stabilized enough for it to constitute a new variety. When breeding for a new variety the selection criteria should be simple and firm. Unless you plan to grow out hundreds of lines from every generation to select from then your list of traits you wish to stabilize should be short. This greatly improves your chances of getting individuals from each generation that have all your desires traits, preventing you from making harmful compromises. Keeping the list short also increases your chances of getting multiple individuals from each generation that fit, you can mix these spores together to grow the next generation with less inbreeding.
If you are very dedicated to isolating and stabilizing the desired traits you can grow numerous mini-tubs and then from each tub try to find one mushroom with the desired traits which can be cloned and sectored out so you can grow out pure isolated strains with your desired traits from which to harvest spores for the next generation. If you have gotten this far you already have everything needed to clone and isolate pure strains.

I hope those of you with an interest in creating new custom tailored bloodlines of psychoactive mushrooms see that the process is neither mysterious, nor restricted to people working in microbiology labs. The entire process from parent spores to a new stabilized variety can be performed in your kitchen, if you like, and the most expensive item is the microscope. And it need not be particularly expensive, a good high school level microscope can suffice.

[RutgerHauer]

Great write-up Elrik! This should go straight into the vaults somewhere with a pin on it, for safe keeping and easy access. I will at some point try out this method for sure. Thanks a lot for sharing this, it is great to see the progress you have made.

Edited by RutgerHauer, 23 January 2020 - 03:27 AM.

[ChocolateStarfish]

Oh WOW, im going to give this a shot in the next month. Doesn't sound too crazy hard.

[Boebs]

Amazing write up!!

[ElrikEriksson]

I'm glad this topic interests people
I feel I should mention some main points of the genetics of selection.
When two mushrooms are crossed and the F1 generation is grown it will be a combination of the dominant traits from both parents. Often times this can make some more distinctive traits vanish, producing mushrooms that look more like regular cubensis. This phenomena is primarily caused by the unique traits of one parent being genetically recessive. So, for instance, leucistic pigmentation or albinism may seem to vanish. If that happens, dont worry, the F1 still has all the genetics from both parents. Though it should be remembered that a monokaryon only carries half of the genetic blueprint of its variety. It'll generally be better to use the more domesticated variety to derive the monokaryon from, unless you want to play the lottery and make multiple hybrids with multiple monokaryons, looking for the one that holds on to an unstable trait.
Due to the mechanics of mendelian genetics, the F2 generation is an orgy of diversity. A full explanation of mendelian genetics is probably beyond the scope of this post, but its not very complicated and I suggest people read up on it. For now I think it will be sufficient to say that in the F1 generation traits can be hidden or mixed and it is the F2 generation where traits start separating out into clearly different combinations. If, in general, you want to keep the number of simultaneous grows limited it should be the F2 generation where you make an exception and grow as many little tubs as is practical. If you can only do four or six little tubs at a time, perhaps do several successive grows of the F2 generation to really look for the most promising individuals. That's where the biggest burst of diversity is. Another trick is to use several liners in a tub so you can make one tub act like a bunch of tubs. This 6 quart dubtub is growing four semi-isolates in separate liners.

Several liners in one tub takes much less space than as many smaller tubs. I'll soon be working with an unstable mutant and I'll be doing six, possibly even 8 liners in each of those six quart tubs so I can hunt the best mutants to isolate and breed.
Also, when I said to keep your list of target traits short that doesn't mean you will be limited to just those traits. Once your most important goals are stabilized your bloodline may still contain unstable or partially stabilized traits that you could then breed to stabilize. For instance, I have a Transkei bloodline that is stabilized as a highly rhizomorphic and very aggressive colonizer and I have an Ecuador bloodline that tends to be poorly or at least inconsistently rhizomorphic and is an average colonizer. I have my most aggressive Transkei monokaryon colonizing grain jars now and I will soon inject Ecuador spores. My primary selection criteria for the F2 generation will be vigorous and highly rhizomorphic mycelium. Once I have selected out those strains I will test them for effect and potency as a secondary criteria, the average and above average strains will be the parents of the F3 generation. This will help keep the genes for good effect and good potency in the mix, and possibly increase them, while I stabilize aggressive mycelial characteristics. Then, once the bloodline is stable as a great colonizer I may choose to put some exclusive focus on potency and effect. If I don't get busy with another bloodline first.
I like to assay non-mutant varieties at 7 grams because, for me, it makes both potency and individual character stand out quite well. One thing I haven't resolved is how to bioassay loads of strains in a reasonable time frame when I only dose once a week to keep tolerance stable and near zero. Testing for effect and potency is a major bottleneck in breeding, it seems.
I know, "oh poor guy, more nearly-free drugs than he can use".
Life is hard

[macgyver]

Out of likes, thank you for taking the time to write this out. I agree that this should be pinned somewhere, maybe mad scientists. I will definitely be starting something soon with this information.

 

MVP Elrik

[ElrikEriksson]

Here is a project I just started.

People often complain about the apparent degradation of Penis Envy genetics. I had a 9 year old PE print that I accidentally stored improperly, I tried and tried to revive it but its toast. But, heck, just reviving the old would be no challenge anyway. Time for some fun! I specifically sought out and obtained a print from a clearly degraded PE lineage. I thinly streaked some plates and caught six promising prospective monokaryons.

I still need to test and retest them, but from their initial presentation on the plates I'm quite hopeful. While I'm doing that the MS plates will be used to do several PE grows. I may use the same parent print to do more groups of grows after that.

When the monos are proven and ready I'll hybridize each one to spores of PE6, which is a reasonably stabilized but largely unimproved and average cube potency cross of old school PE and Texas. This constitutes a backcross. Each little tub will have the corresponding degraded PE monokaryon as one parent and numerous PE6 spore germinations contributing as the second parent. The result will be 75% PE genetics in each tub. I'll select the best group based on printability, potency, and most of all resemblance to classic PE and I will take that bloodline to the F2 and apply the same selection criteria. I'll start each F2 group with just a speck of spores and I'll do 8 or 16 mini-grows at a time, as many times as needed to find something very promising.

While all of that is going on I'll do more grows of the degraded PE, selecting for printability and most of all resemblance to classic PE.

At this point I can combine the two, deriving monokaryons from the more genetically homogeneous PE and crossing them to the still quite genetically diverse spores from the best PE X PE6 F2 generation.

I will then have a hybrid that is 87,5% PE DNA but, hopefully, with improved genetics bred in. From that, further selection can continue for isolation and stabilization of desired traits.

 

This is the kind of thing you can do with monokaryons.

 

If reality isn't as you would like it, make a new reality!

[BrotherDekatessera]

At this point the spawn is an F1 hybrid between the single isolated 'Parent #1' monokaryotic strain and the dozens or hundreds of strains that came out of the 'Parent #2' spores. &&0){for(var>)throw>

 

 

Is it for sure?

 

 

unless the monokaryons were mated in a dish, and the result spawned, how do we have any idea whats in the grain?

 

if we dump spores in to a colonized monokaryon, we may not get any crosses at all? does the possibility not exist for some of the spores to germinate unto themselves, or for several of them to mate the mono- from parent A? causing a hodhepodge competing with each other and no real knowledge to us of whats what? 

 

or does waiting for the mono to fully colonize make that a non-issue? and if thats the claim, how sure are we that its a factual claim?

 

not trying to poke holes in your post, just very interested in breeding and Im trying to get the full scope that I can on the subject.

 

other than that, thanks for this post. like everything from watering a weed plant to extracting DMT all the cant-handle-its and armchair experts on the internet make it seem like it requires two trips to the moon and a list of doctorates. what I find most alarming about these online psychadelic forums is that it tends to be alot of  the well known and "respected" "oldheads" that are virulently against anything beyond their own reach. makes for a rather shitty research environment for anyone looking to go beyond the monkey-could-do-it monotub. 

Edited by BrotherDekatessera, 28 September 2020 - 01:44 PM.

[ElrikEriksson]

It's a method that has been used by professional mycologists. It's important to let the spawn fully colonize with monokaryon because, as you say, if given a chance spore-spore matings could take hold in uncolonized grain.

With the grain fully colonized with monokaryon and the spores only having days or a week to work on the mass there may be specks of non-hybrid dikaryons that formed but they wont be of sufficient mass to form mushrooms.

And, obviously, you wouldn't want to spawn to a highly nutritious bulk substrate. I just use straight coir.

 

Mon-mon crossings on agar are also possible, of course. But it is much more work to get a good grow out of such a method, and produces an instant genetic bottleneck.