Muad.Dweeb said:

...
Back to RustyWhyte, Pasty put spores from two varieties with distinct traits into the same agar plate, and mixed them. This is arguably the simplest way currently known to cross tetrad ...

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lothar48 said:

Great thread! So many questions! For the moment, here are a few:

You use F* a few times. Are you referring to fruit generations?

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Makes sense to me and is probably an easy start. That said, what approach of crossing have you've been using? I've been considering working a successive dilution method to isolate monokaryons from prints. From there I'd go to plates, select isolates, transfer to clean plates for compatibility (mating) testing, then moving on to normal spawn->fruiting.

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Lastly (for now), where do you see this going as a community effort?

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What I don't like about using monokaryons is that you are kind of adding a layer of blindness to the process, in that you don't know what traits you're going to get from that mon until you've fruited it as a paired dikaryon...

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... I think that these discussions are needed. 

1) there are a ton of people with the baseline skill needed that want to do this already, and finding the information succinctly in the OMC can be a huge chore.

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2) a lot of people doing this already aren't accomplishing their goals. You can spend 5 minutes in mycogroups on any social media and see it first hand. Some of these people just need guidance, others are actually being predatory and putting out new "crosses" and "isolates" seemingly by the hour for gullible noobs. The answer to both is education, IMO.

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Most of the means to create a cross are incredibly easy and accessible to anyone who can produce clean spawn and pull off fruiting decently. Every viable method has it's own pros and cons, but they generally are super easy with no need for special chems or equipment. The real work all takes place AFTER the cross to achieve, first, the desired combination of traits, then producing consistency of the trait (s). It takes a lot of work on the other side of the actual pairing. and depending on the choices you make, it can be as successful as APE or RW, or turn out like PE6 and stabilized to the wrong traits.

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I guess where I *hope* this goes, is that for those who wish to undertake breeding, there is a comprehensive, digestible knowledge of the biology, the theory, and the practice of fungal sexuality and selective breeding (I'm focusing strictly on cubes, but most of it is broadly applicable).

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Muad.Dweeb said:

Here are some of the streaking methods I've used.

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lothar48 said:

One thing I've been mulling over is a rapid, small scale fruiting process such that a) you're not breeding to waste (product you can't use), and b) keeps things reasonably space constrained. How do you approach the many F iterations?

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Fried_Sushi said:

lothar48 said:

One thing I've been mulling over is a rapid, small scale fruiting process such that a) you're not breeding to waste (product you can't use), and b) keeps things reasonably space constrained. How do you approach the many F iterations?

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You might want to check this out
Pf cakes in a sandwich bag Using Ziplocs (Ziplocks) as Fruiting Chambers

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I have the same thinking. Last year I started using what I think is a killer combo, though I'm still working on getting the FAE just right. I use the Betty Crocker Easy Seal pint cups and Surefresh 1L cups that I find at a local dollar store. The 1L fits right over the threaded lip of the pint cup. I'm using a modified PF Tek substrate, which is basically just BRF and CV sub mixed 1:3. I cut crockpot liners down and use them as liners, otherwise it's terribly difficult to avoid side pinning. I modified the lids with filter patches and omit the dry verm layer, since I'm working in front of an FFU. It is a huge space saver, but more work than doing bulk. I like it for the consistency of conditions and the size, though.


[PFRed.CL1.MS2]

Also, a bit of philosophy I can't really substantiate with science: I feel that, whether we are conscious of it or not, every cultivation choice we make individually has the potential to be a selective pressure. So, things like the size of chamber/medium you use, the condition of the substrate, the degree (or lack) of competition, etc... all put certain pressures on a multispore population that will define what constitutes "fitness," and influence what you are likely to see in a MS grow. A sort of artificial evolutionary bottleneck.

I think that small substrates are one of those pressures that selects for specific metabolic profiles that either acquire real estate faster, or more efficiently utilize available resources. I'll use larger containers when I'm running isolates, to see how they utilize larger resources. But for running through phenotypes, I'd rather do 10-20 of these pint cups than 1-2 32 qt tubs.

Muad.Dweeb said:

I want to talk today about di-mon mating, aka the Buller Phenomenon.

Prerequisites:

You need a monokaryon, derived from any of the usual methods.

You need a clone.


So, the usual methods are serial dilution and plate streaking. These procedures are pretty straight forward. For the serial dilution, you need something small you can sterilize, like a 15 mL centrifuge tube; you need water, distilled and sterilized; you need some clean syringes or measuring pipettes. And agar. Of course, agar.

Everything sterilized, you add your spore sample (say, a 1 cm square) to the first tube, and 10 mL of water and put the cap on. Now add 9 mL each to tubes for dilutions you want to make.

Shake the like the DT's- a back massager can help break up the spores. The goal is to make sure no spores are clumped. You cannot over-do agitating the tube, and it's really, REALLY frustrating when you think you have a monokaryon, but it was two spores stuck together that germinated at different times.

I use 3 mL syringes in this set up. Draw up 1 mL of the well-agitated 10 mL solution, and add it to a tube with 9 mL of water. Cap it. Label it "1/10". Agitate thoroughly again.

With a fresh syringe, draw up 1 mL of the well-agitated 1/10 dilution, and add it to another 9 mL tube. Label it "1/100". Etc..."1/1000", "1/10,000"...

Without a microscope and a means of spore counting, you'll have to ball-park and play around to get the ideal dilution of your sample, so do a single drop streak on at least 1 plate per dilution to calibrate. You're looking for 1-5 colonies germinating per plate. Knowing what dilution to use, you can then start making germination plates to harvest your monokaryons from.

You can try to "skip" the dilution route with careful control of how much spore you pick up and a streak method alone. Things that I have found help are using very light prints (1-2 hours) and a thin loop, cooling my sterilized loop in the agar and literally just dabbing the print. you can get an incredibly small sample with some practice, which makes finding monokaryons right off the bat possible.

To acquire the clone... clone it, then clean it up. Moving along...


*    *    *    *    *

I like stark contrast. Call me extra, IDEK. But the strong contrast has a practical application here. It, again, is best to view this for yourself with a Clone and Monokaryon from strikingly different varieties.

Stick them together on the same growing medium, like agar. When I'm doing di-mon breeding, I make a plate with one sample of each about 2 cm apart and just let them grow. Eventually, it will pin. Then I clone a pin from the (labled) monokaryon side and grow it out to see what it looks like. Of course, once you have the clean cultures of both, you can put them together to fruit on any media, like grains to bulk. But agar is most convenient IMO.

This is something anyone can try out, no need for a microscope even. It certainly helps to reassure by looking at the hyphae under a scope to be sure you have a monokaryon (monokaryons don't have "clamps" between the cells), but a monokaryon will not fruit on its own. So, no matter how long you leave your harvested "monos" out on the shelf, you shouldn't see knots or pins. You can run a control plate of just the monokaryon side by side with the di-mon plate and use it as confirmation IF pins appear on the mon-side of the di-mon plate, but not the mon plate.

You will also notice there is a distinct morphological change to the monokaryotic mycelium once it is impregnated with a doner nucleus. It becomes more robust and dense. Monokaryons are often whispy, cotton-mold-looking sadbois.


[Koh Samui Squat clone x Leucistic India Orissa Mk1 and Mk2]

Why does this work?

When genetically distinct dikaryons are close to each other, they exchange pheromones through excretion. This will tend to form an "inhibitory zone" between two dikaryons (what we call sectoring), but monokaryons can detect a potential mate and both will continue growing into the other. Plasma is exchanged when they physically touch and attempt a fusion. If the mating factors are compatible, the dikaryon donates one of its nuclei to the monokaryon and it becomes a "hybrid" of that dikaryon and the monokaryon. In some species, there is a nuclear preference, and only one nuclei will be donated, even through repeat trials. I don't know if such data is out there for cubes, but in theory, this arrangement could produce two distinct "hybrid" genotypes.


[Allowing the plate to pin]

Why use this approach?

For one, it's a classic. It is part of the "parasexual" behavior that makes fungi so fascinating, and watching it in real time is a special feeling that doesn't lend itself to description well.

For another, you know 2/3rds of the genetic material at play, at least casually, from the start. It makes viewing the way traits blend, dominate and obfuscate very dramatic. There are also methods for splitting dikaryons into monokaryons, but this is sort of a short cut around that.


[Squat pins in the center strip; Beefy, not-squats, not-orissa in the monokaryon sides]

And because it's fucking cool. Do it.

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Muad.Dweeb said:So, ask me anything.

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Muad.Dweeb said:
I can't be the only one to have noticed that some of these varieties have been inbred consistently for decades, right? Lots of unusual "mutations" lately, right?

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Muad.Dweeb said:

And then we have, of course, Enigma. The ultimate sign to some people that the genes have degraded so much that it can't produce spore bearing structures (I dub these blobs "pseudocarps"). To others, Enigma is a mysterious, miracle mutation. I claim it is neither

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Muad.Dweeb said:

I have the same thinking. Last year I started using what I think is a killer combo, though I'm still working on getting the FAE just right. I use the Betty Crocker Easy Seal pint cups and Surefresh 1L cups that I find at a local dollar store. The 1L fits right over the threaded lip of the pint cup. I'm using a modified PF Tek substrate, which is basically just BRF and CV sub mixed 1:3. I cut crockpot liners down and use them as liners, otherwise it's terribly difficult to avoid side pinning. I modified the lids with filter patches and omit the dry verm layer, since I'm working in front of an FFU. It is a huge space saver, but more work than doing bulk. I like it for the consistency of conditions and the size, though.


[PFRed.CL1.MS2]

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Muad.Dweeb said:

Also, a bit of philosophy I can't really substantiate with science: I feel that, whether we are conscious of it or not, every cultivation choice we make individually has the potential to be a selective pressure. So, things like the size of chamber/medium you use, the condition of the substrate, the degree (or lack) of competition, etc... all put certain pressures on a multispore population that will define what constitutes "fitness," and influence what you are likely to see in a MS grow. A sort of artificial evolutionary bottleneck.

I think that small substrates are one of those pressures that selects for specific metabolic profiles that either acquire real estate faster, or more efficiently utilize available resources. I'll use larger containers when I'm running isolates, to see how they utilize larger resources. But for running through phenotypes, I'd rather do 10-20 of these pint cups than 1-2 32 qt tubs.

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Fried_Sushi said:

Oh, I notice! Imagine that. Me, a noob that just started reading on shroom cultivation a month ago.

Wouldn't this be due to taking clones from clones from clones through multi-generations? One of the first things I noticed in shroom cultivation culture is the fashionable attitude that working with spores is so distasteful, due to the "crapshoot" of phenotypes and so on. Many say that spores don't germinate well on agar then go on to group shame people for germinating in liquid culture. My point is that there is a big groupthink push toward cloning. And my first thought in reaction is, "Isn't this a form of inbreeding to cause undesirable mutations?" I notice there seems to be a lot of blobs among albinos.

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Are not albinos undesirable mutations? Aparantly not when the potency is high. I think they are ugly and I gravitate toward the pigmented. Although I am going to get a couple of TAT genotypes, but I like the pigmented MVP best of the bunch.

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And the sterility factor! Is the high potency of PE varieties a result of energy not focused on reproduction?

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Yes Enigma, an offshoot of Tidal Wave. Tidal Wave was the first species I decided to order. My understanding is that it (Tidal Wave) is unstable because it was put to market at F2-F3 or something and is prone to blobs and varied pheno characteristics.

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So if I want to stabalize my Tidal Wave and carry it forward for my personal grows, I should grow from the favored spores 3 or 4 more generations, correct? My instincts tell me this should be done from a mix between 2 different caps since spores from a single cap is a form of inbreeding; is this correct?

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Muad.Dweeb said:

I want to talk today about di-mon mating, aka the Buller Phenomenon.

Prerequisites:

You need a monokaryon, derived from any of the usual methods.

You need a clone.

...you know 2/3rds of the genetic material at play, at least casually, from the start. It makes viewing the way traits blend, dominate and obfuscate very dramatic. There are also methods for splitting dikaryons into monokaryons, but this is sort of a short cut around that.

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lothar48 said:

A few Qs about this.

With the process you describe, you end with two dikaryons, yes?

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Presumably they're "close" to each other, awash in the afterglow. How do you separate them so you know you've isolated them from each other? First guess is take samples from the far side of the growth, but I wonder if there is there a way to be certain.

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In the case where you are starting with spores, is it better to just isolate a few monokaryons instead?

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On that topic, when isolating monokaryons, how do you *know* that's what you have? How do you know when you have a dikaryon? I ask because I just plated some GT spores, guessed at a few monokaryons I thought I saw, but really not sure without seeing if I can fruit them.

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Vast218 said:

is it possible to breed two different phenotype using the cloning method? Say you put the bits of stems from two strains next to each other on agar hoping for the primary myceliums to merge?

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