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OfflineElrik
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Re: Monokaryons [Re: trubblesome]
    #26458430 - 01/29/20 11:58 AM (4 years, 19 days ago)

Yeast extract is often used in agar to give the nutrients needed by fungi. Mushrooms eating dead yeast is like people eating monkeys.
It is conceivable that mushroom spores could convey some growth factor that yeast might not.

It has been demonstrated in genus Agaricus that growing mycelium can emit volatile hormones that promote spore germination on the other side of the agar plate.
Its possible these hormones might be produced better by monokaryons. When clearing plates I've noticed that cubensis monokaryons often have a more fruity or almondy odor than standard dikaryotic mycelium. But I haven't tracked it to see if that really is a monokaryotic thing.


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Edited by Elrik (01/29/20 12:01 PM)


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26458481 - 01/29/20 12:39 PM (4 years, 19 days ago)

oh that's interesting! I've got all these split plates hanging around, maybe when I pick the project back up I'll use some of those with that purpose in mind - cube myc on one side, single spore on the other


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Offlinesporecap
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Re: Monokaryons [Re: trubblesome]
    #26458660 - 01/29/20 02:58 PM (4 years, 19 days ago)

My experiment did not turn out very conclusive unfortunately.
What I did was take two cultures which I expected to be monokaryotic since
they grew from ~1.5 years old sporeprints and they stayed very tomentose even after ~6 transfers
and never showed any kind of rhizo growth. Even after changing for example from MEA to grain water agar
and increasing nutrient content.

Here is what I started out with, two different cultures but from the same spore print


Right before they grow into each other


They meet! I was excited whether this change in structure would actually now indicate the "mating" of mycelium and it would start to show some rhizo growth


A couple of days later it's not very conclusive


There's definitely some rhizo structures forming at the boundary, which these cultures never showed before.
But I cannot exclude that this might be caused because they became stressed when "mating" or reaching the edge of the agar plate?
Do you think it would be interesting to take a small wedge from the center where it's still tomentose and one from the rhizomorphic edge and try to grow them out? Or has the full culture now become dikaryotic in case this really happened?

Just for comparison this is how other cultures from this spore print can look like, from a pinning agar dish which showed almost the same rhizo growth


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OfflineElrik
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Re: Monokaryons [Re: sporecap]
    #26459299 - 01/29/20 10:27 PM (4 years, 18 days ago)

Without a microscope you'll likely never really be sure the tomentose isolates really are monokaryotic.
If I were to attempt to tell if they were I might colonize two grain jars with each and hybridize one of each using spores, then try to fruit everything. True monos would never fruit, but crosses with spores should have no trouble.

Dikaryotization should proceed through the colonies if two sexually compatible monokaryons meet. This isn't instant, it takes time.
This plate comes from crossability tests I did with confirmed monokaryons I derived from a PE6 bloodline that isn't very rhizomorphic:

After they grew together and had their fun for some time I confirmed that clamp connections were present at the union between the two colonies. I then re-wraped the plate just to see what they would do. As you can see, pins formed along the outer border of both colonies indicating that the dikaryotization spread all the way around.
Neither ever did go rhizomorphic, damnit :laugh:
Some day I might refine this PE6 bloodline.


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InvisibleBabuFrik
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Re: Monokaryons [Re: Elrik]
    #26463570 - 02/01/20 12:17 PM (4 years, 16 days ago)

Elrik: Thank you for posting this and responding with so much detail. I am really enjoying it and learning a lot!


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Edited by BabuFrik (07/14/20 04:10 PM)


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OfflineSolipsis
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Re: Monokaryons [Re: BabuFrik]
    #26466855 - 02/03/20 01:54 PM (4 years, 14 days ago)

Yeah your contributions are much appreciated!

I recently streaked Pleurotus citrinopileatus to try and get monokaryons but not sure if i did it quite right. If you do 3 streakings and turn the plate, arent you supposed to get rid of the spores on your loop and only cross your previous path a little to achieve the dilution? Otherwise if you accidentally had too many spores on your loop idk if you lose enough of them when you drag..

I did my best with that :wink: and should just review the protocols and methods well.

Will read these posts better too - just really busy.


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26467701 - 02/03/20 10:51 PM (4 years, 13 days ago)

What you describe is the method used for bacteria.
I've heard of it being used for mushroom spores.
Numerous times I tried it, swiping a line on one side, turning the plate and swiping a more or less over-lapping path, and again, etc. until I had either a square or pentagonal layout. Always I had lots of germinations on the first path, a couple on the second, and nothing on the rest.
For me spores just don't act like bacteria and all I have to do is spread out a line I rubbed down the center.
Try out different methods and see what works for you.

My last hunt was another success, I now have six tentatively verified Penis Envy T2 monokaryon colonies and a tub of PE6 ready to go into fruiting to give me clean spores. Back-cross is officially in the works! :smile:


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26468155 - 02/04/20 09:40 AM (4 years, 13 days ago)

yeah, I could see that method working for spore syringe solution maybe but mostly is for bacteria in suspension.

The method that worked for me:

"I lightly drew my loop across some of the areas of my print with a finer spore load, then carefully turned the loop so that those spores were facing "up" on the loop. Then I streaked the plate with the bottom side of the loop touching the agar, so that the movement would more lightly draw spores off of the loop and increase my chances of finding single spore locations to cut from later. These were very sucessful, I found several locations with individual spores.

this last time i streaked back and forth across the whole plate, then again perpendicular to that streak. it left grids of streak lines that helped me extract the spores that much easier without magnification once they were identified and marked with sharpie"

I verified the individual spores with a scope, but if you don't have a scope, basically if you cant see any groups of spores with the naked eye it's probably worked well.


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OfflineSolipsis
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Re: Monokaryons [Re: trubblesome]
    #26576728 - 04/04/20 05:57 AM (3 years, 10 months ago)

So in the meanwhile thanks to info from this thread, I have been streaking spores and with P. caerulescens have gotten many monokaryons from 2 dishes.
9 in fact although I only checked 3 under the microscope and couldn't find clamp connections for the life of me, whereas with some other dikaryon culture it was easy to find them.

I transfered 9 monokaryons and put them in sets of 3 on plates. On a plate i got them from though it does not appear like there have been any successful matings among them yet.

Does anyone know if ALL Psilocybes are bifactorial (so 25% chance of matching mating types)?

And can someone show me a plate of successful mating and what that looks like with the resulting sector and the lack of a dead demarcation zone?


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26577013 - 04/04/20 10:33 AM (3 years, 10 months ago)

These were confirmed cubensis monokaryons made to mate

Somehow I still have that plate over 2 months later and that line of separation between the two is still visible.

Oh, Alan has said nearly all Psilocybes are bifactorial. I never found a list of exceptions, though.


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The Nook


Edited by Elrik (04/04/20 10:34 AM)


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OfflineSolipsis
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Re: Monokaryons [Re: Elrik]
    #26578689 - 04/05/20 04:08 AM (3 years, 10 months ago)

Thnx good to know about the bifactorial thing.

About the plate: appreciate you showing it. I didn't know there would still be such a demarcation line. I wonder if it is also present with di-mon matings.

How come no extra sectors are visible on your plate?


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26579642 - 04/05/20 02:27 PM (3 years, 10 months ago)

You noticed that. I did too, several times.
I think the reason mon X mon crosses do not sector is because there is no self-other incompatibility. Assuming no mutation, every strain that appears is either half identical or identical to every cell it meets.
Say you try to cross MK4 and MK 5. Here are the options:
• They don't cross, each stays as it is. No new strain to sector, though in some almost-crosses the cells can 'fight' where they meet.
• MK4 sees MK5 and mates with it like a drunken college student, and vice versa.
• MK4 sees other MK4 cells and sees itself, same for MK5.
• MK4 X MK5 sees MK4 and sees a 100% genetically compatible sub-component of itself, same if it sees MK5.
Outside of mutations, those are the only options and none would lead to recognition of a foreign and/or incompatible strain. So no sectoring.

Sectoring will happen with di X di crosses and, I think, mon X di crosses. In both of those scenarios there would be strains that dont recognize each other and cant mate by the standard reproductive mechanism.

I've never seen demarcation lines completely vanish. Every time they appeared to it was just one colony growing over the other, rather than a fusion [inter-species cross attempts tend to do that]. I have seen demarcation lines much more severe than the above example, those tend to be incompatible cross attempts.


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The Nook


Edited by Elrik (04/05/20 02:30 PM)


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OfflineSolipsis
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Re: Monokaryons [Re: Elrik]
    #26580521 - 04/05/20 09:09 PM (3 years, 10 months ago)

Increasingly interesting, increasingly confusing.. :laugh: (on par for mycology I guess)...

I follow most of what you describe about the different scenarios or phenomena taking place.

A question about that first: afaik from di-mon interactions you get a nucleus donated by the dikaryon to the monokaryon which is not exactly random. If MK4 x MK5 forms and it sees either mono MK4 or MK5, that would mean the dikaryon MK4 x MK5 will donate the complementary nucleus to whichever of the two it encounters, and it seems like necessarily both are (separately so to speak) encountered pretty much right away.

That would amount to a sort of wave backwards of nucleus donations so that the rest of both monokaryons get dikaryotized. On a sidenote, I assume these donated nuclei are just being copied where it is convenient. Earlier I was imagining them migrating.. from... where exactly? Storage unit?

Anyway I digress, seems to make sense if that is true / if you agree.

Also interesting, i didn't know di-di fusion was possible. Never seen a mechanism explained really. What kind of exchange happens? (Yes yes off-topic! lol)

Don't you find it a little weird if through mating it all basically can become the same dikaryon, but it will still refuse to fill in that demarcation line?
I guess mycelium dies and perhaps even dies back in the demarcation zone and apparently this leaves some sort of signal for the culture to permanently quarantine that shit. Growing right *over* it ah ok i guess that is a workaround for that.

I would like to asap show 2 dishes, one is a dish from a friend with what seems like 2 cubes that i personally thought were monokaryons that mated, and a third sector formed.



And another is my own dish right now, caerulescens with a whole bunch of monokaryons present pretty discretely it looks like. Most encounters do not look like they resulted in mating but indeed, some have much more subtle demarcation lines and are even being readily overgrown.



I have a slightly more recent pic which is less than a day later, but I think it can all be seen a bit clearer here.

BTW I appreciate that i just gotta take a microscope to verify a dikaryon, but ofc thats not what this is about.

I have looked around in articles and theses and you name it, and indeed I'm not seeing much about third or even fourth sectors, not unless it's indeed as you say di-di then you can get a new sector. Not sure about di-mon, maybe that is happening on the plate from AFOAF after all.


Edited by Solipsis (04/05/20 09:55 PM)


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26580747 - 04/05/20 11:08 PM (3 years, 10 months ago)

Quote:

Solipsis said:If MK4 x MK5 forms and it sees either mono MK4 or MK5, that would mean the dikaryon MK4 x MK5 will donate the complementary nucleus to whichever of the two it encounters


That is my understanding as well. A mon X mon cross is a mon X mon cross where they first touch and then it moves backwards as a mon X di cross.
Quote:

Solipsis said:On a side note, I assume these donated nuclei are just being copied where it is convenient. Earlier I was imagining them migrating.. from... where exactly? Storage unit?


I really have no idea how nuclei are signaled to migrate and then do the actual migration :wink: They also must be signaled to duplicate the needed nucleus so one is available to donate.
Quote:

Solipsis said:i didn't know di-di fusion was possible. Never seen a mechanism explained really. What kind of exchange happens?


My understanding is this [and take it with a grain of salt, it may only be partly correct] When a dikaryotic cell divides the new cell receives just one nucleus, apparently often with a ~90% preference for one nucleus type over the other, the new daughter cell then gets the second nucleus donated to it by the parent cell via a clamp connection. Thus explaining why all dikaryotic cells have clamp connections. This results in the single cell leading edge of a dikaryotic culture being transiently monokaryotic. If this transiently monokaryotic cell runs into another sexually compatible cell, be it actually monokaryotic or transiently monokaryotic, then there is a chance it will pick up its second nucleus from the 'foreign' strain rather than its parent strain.
In this way, in a manner of speaking, all mon X di and di X di crosses are technically mon X mon crosses.
That's what I've pieced together from various bits of information from books, mycology papers, and Alan Rockefellers posts.


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OfflineSolipsis
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Re: Monokaryons [Re: Elrik]
    #26580981 - 04/06/20 03:33 AM (3 years, 10 months ago)

Wow :thumbup::thumbup:

I guess there must be other genetic effects going on between them as I read about merely some genes being exchanged between di-di, but not sure if this has anything to do with horizontal gene transfer or anything like that.

It would be fascinating to learn about what kind of things can be done to increase the chance of di-di fusion.  I found this:

https://www.differencebetween.com/wp-content/uploads/2020/01/Difference-Between-Dikaryotic-and-Diploid_1-e1578369540335.png

I thought from your explanation it would be the hyphal tip to be the temporarily monokaryotic one but maybe that would create problems far too often.
Spitballing here. Can't find much on di-di pairing so far.

Anyway... the nice thing about my mess caerulescens plate above full of mono's is being able to see something (probably) about the mating and that sooner or later a dikaryon virtually must be formed which then should cause extensive migrations of nuclei dikaryotizing a lot, most or all of the plate I guess.
Unfortunately it may get difficult for me to see which monokaryons that I transferred tiny pieces of and put together on plates, should actually be compatible with one another as a sort of preview.

I will be sure to save wedges of the monokaryons plated in triangular patterns and def hope that at least there i should be able to see which mated.
I consider this practice. The idea of making mating testers intrigues me: figuring out by putting different combinations together which have complementary mating types. Call the monokaryons something like a, A, b, and B (if that is not confusing).
But I don't think that if a x A would form a complementary pair and b x B do too.. that there is a meaningful or testable relationship between say a and b. I guess that's arbitrary but i could be wrong there.

Mating testers as reference in case of caerulescens has very limited use (its practice) but it seems pretty cool for cubensis or certain gourmet species.

Its all good fun but probably all has limited potential and after that i should probably look into mechanical dedikaryotization and much later protoplasts which if nothing else seems like an expensive occasion.


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Re: Monokaryons [Re: Solipsis]
    #26581118 - 04/06/20 07:13 AM (3 years, 10 months ago)

Thread is 👍. Carry on.


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Offlinepoisoned
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Re: Monokaryons [Re: Smartattack]
    #26581485 - 04/06/20 11:24 AM (3 years, 10 months ago)

:threadmonitor:


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OfflineSolipsis
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Re: Monokaryons [Re: Elrik]
    #26663955 - 05/11/20 07:24 AM (3 years, 9 months ago)

Hey all, and Elrik :smile:

I was talking with a friend and we were wondering about di-mon matings and what happens when there is only half compatibility, idk about the differences in that given between unifactorial and bifactorial but lets just focus on bifactorial cause at least for me that is what the fungi i work most on are...
A prof was not able to give a straight answer (yet)...

Aside from that there is another thing which is my mating experiments. So far I have made monokaryons and I have confirmed that caerulescens MS dikaryon does have clamp connections but my monokaryons do not.
I put bunches of monokaryons together on agar and it gave both a bunch of sectoring at least apparently, but also it just seemed like the myc was possibly very brittle and i just lost pieces of it during transfer (which did not happen during initial transfers), so i have a bunch of satellite colonies.

My method which i don't know if is been done before coincidentally but i (also) came up with this: to stack the agar of one or more dikaryons upside down on agar with MS of another variety or species. The top one should only contain monokaryon, no matter how many. Plates fully grown as possible.
Hopefully gravity will give me plenty of contact, and di-mon mating should take place. The verification would be that dikaryon surfaces and it should not be the bottom dikaryon(s) as it shouldn't pass through.
I imagine there are tons of patterns imaginable for both the layers, MS will be a bit messy usually... and the monokaryon layer either just one multiple ones clearly visible as marked off island cultures.

I started out with the idea of just using one monokaryon and MS plate. The upside to this method would be to be able to give multiple strains the opportunity to mate di-mon, giving a multi-strain top layer - all crossed. I do have plates with 3 monokaryons on a single plate too so i figure this should give even more diversity as it is not limited to 1 blindly chosen monokaryon as parent.
What i am curious about is: is there 'someone out there for everybody' and can you breed something ok with a parent that wasnt properly selected?

Anyway i am almost ready to sandwich. :laugh:

It is possible to mechanically turn a dikaryon (say an isolate for simplicity and usefulness sake)... but i wonder if you even get nice genes from only one nucleus of a cool isolate. My guess is that you should get "healthy" genes because some wellbred isolate should generally have healthy genes, but other than that it seems to me it guarantees very little if you make a cross with it. Yes it can carry recessive traits but just going through spores would do that too?
I guess my point is i don't really see the advantage of having a nucleus from an wellbred isolate rather than just the meiosis.


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OfflineSolipsis
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Re: Monokaryons [Re: Solipsis]
    #26917574 - 09/04/20 02:37 PM (3 years, 5 months ago)

So, I have since learned what some drawbacks are of that sandwich method ^
Very simply put: MS cannot really be tracked, so depending on what the breeding plan is, the disadvantage is you don't have the parent isolated.



In any case... I wanted to report that all signs point towards having made my first successful cross (di-mon), and i wanted to thank elrik for the helpful contributions.

I put a monokaryon and dikaryon together kind of on the edge of a plate, not that far apart.. the idea being that there would be a lot further to go for them to interact.

Having reached the other side it appears like on the monokaryon side there are sort of radial sectors, a bit like on the edge of a roulette spinner. I allowed some time to pass and those sectors now clearly show clamp connections.

I figure there will only be two strains created with the cross since the dikaryon could donate two different nuclei. And i figure as they grew together, they isolated from each other so I would expect them to be alternating strains (like ABABAB etc).

The varieties I crossed (RustyWhyte x Orissa) while intentionally having noticeably different phenotypes, were a bit of a random choice of what became available.

It will be interesting to see at least, if the offspring F1 looks normal since the leucism and rusty spore color are probably recessive at least. I would need to go to F2.

Of course I will need to grow it out to really verify what happened but I think so far my checks such as microscopically checking for clamps have given results 100% as expected.

Encouraging to proceed to more interesting crosses and attempt hybridization in gourmets.

<3


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Re: Monokaryons [Re: Solipsis]
    #26968891 - 10/04/20 10:45 AM (3 years, 4 months ago)

thanks, so much good informations.

Is anyone counting spores? 

the route i'm taking, instead of streaking plates and searching for monospores i'll consider dilution. Dilute down to 10 spores per plate.. you can count the spore load under a slide then squirt onto agar when happy.  you can very quickly do 10 plates like this, you'll have a lot more monos to choose from and they will be easy to grab.


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