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OfflineElrik
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Re: Monokaryons [Re: Darkslide] * 1
    #26397218 - 12/23/19 11:09 AM (4 years, 1 month ago)

My first one was accidental too, when I was fighting to isolate cube mycelium away from mould. I didn't even recognize what it was at first, just that it was fluffy, aggressive, 100% non-rhizo, not sectoring, but clearly cubensis. In the end 60% of the isolates I got from that print were monokaryotic simply because there was so much mould! To actually grow the mushrooms I just put all cleaned dis and monos on a plate together so I could mix them for injecting into jars.
My Psi. tamp. 'Pollock' was similar, the print was very bacterial. I wasnt trying to isolate monos, just to get clean tamp transfers. I put all my isolates under the scope hours before I took 9 grams of cubes, seeing that I had managed to rescue quite a few monos made that one seriously enjoyable trip!

It wouldn't surprise me if unrecognized accidental monokaryotic isolates are not uncommon. Some of those non-fruiting strains people find on agar may simply be monokaryotes. It could even add to the explanation of why rhizomorphic growth is a sought after trait, because sometimes those fluffy non-rhizo isolates just wont fruit. Because some are monokaryotes :laugh:


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Offlinesporecap
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Re: Monokaryons [Re: Elrik]
    #26397555 - 12/23/19 01:54 PM (4 years, 1 month ago)

Wow, this is really interesting. Right now I also have a culture of cubes on Petri dishes from a 1.5years old spore print, which I streaked very diluted (many nonviable spores+dilution). It stayed tomentose even after 5 transfers but looks healthy and very homogeneous. Still, a dish which by now is about 2 months old didn't pin yet. I have two jars of it which are completely colonized, now I'm very interested to see whether they are going to produce some fruits or not... Too bad I don't own a microscope:frown:


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OfflineElrik
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Re: Monokaryons [Re: sporecap]
    #26398385 - 12/23/19 10:39 PM (4 years, 1 month ago)

Quote:

sporecap said:...I have two jars of it...


I know what I would do. Spawn one jar out and try to fruit it. If it doesn't fruit in a month shake the second jar and inject some spores from something else, give it 10-12 days, and then spawn it 1:2 to coir.
Colonized jars can sit there consolidating for quite a while and still perform when spawned out [Link]


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OfflineMuad.Dweeb
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Re: Monokaryons [Re: Elrik]
    #26398409 - 12/23/19 11:24 PM (4 years, 1 month ago)

My streak method is to load a swab with minimal spores from a print. Around the center of the plate, I make a circle of looping motions in the center. Then I rotate the swab to the clean side, and zig zag the full width of the plate, starting at the center and going all the way to to top of the plate. Then from center to the bottom. Then I rotate the dish 90° and zigzag from top to bottom. If I were more on the ball about transfers, I could probably catch monos on demand. I have several plates I suspect are, but they sit in my fridge because I have more pressing projects (dedikaryotization to back-engineer clones).



I do want to see if the mitochondria have any significant influence on fruiting though. I'll be generating monokaryotic cultures from spore to make sure the mitochondria is intact, then test compatibility and cross. After nuclear migration, cultures will be separated and confirmed for clamps and run side by side.


Edited by Muad.Dweeb (12/23/19 11:25 PM)


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OfflineeLeSDenes
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Re: Monokaryons [Re: Muad.Dweeb]
    #26398563 - 12/24/19 03:26 AM (4 years, 1 month ago)

I just use the scalpel handle, flame it, let it cool down then just barely touch the spores and swipe. Managed to get lots of monokaryons that way.


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OfflineSolipsis
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Re: Monokaryons [Re: eLeSDenes]
    #26414017 - 01/03/20 10:05 AM (4 years, 1 month ago)

Hey guys,

thanks that is very encouraging! I was on a little hiatus :}

idk if its really that things were forgotten in a decade or however long it was. :smile: I am just quite on my own and not really that in touch and i wasn't finding the Workman stuff i was looking for.
Also just interested in engaging in discussion about current thoughts etc about the matter.

What about the surfactants such as tween? does it have a bioactive effect on germination? Thanks for the tip about agar. clever!

Just today i was hypothesizing to a friend about possibly germination may very well or likely involve various chemical-binding receptors since isnt this how it always goes basically? with ratios of usually mostly polysaccharides but importantly proteins working togethers.
So my idea was: isnt the reason for activated carbon to help in germination generally (aside from mycorrhizal benefits) that spores adsorb to the AC and this messes with these proteins on the exospore structure which may improperly trigger germination?

Anyway that's another thread.
Still i'm curious if tween is bad because it more negatively interferes with those proteins or shields them from detecting anything at all? Just a little bit too much clingy clingy enthousiasm.

The dedikaryotization seems very interesting by the way and something i wanna work on with a friend, but it seems mostly useful for sporeless cultivars and rather long term advanced breeding projects.

But blood, idk your method vs the textbook streaking method of swiping, turning 60 degrees, streaking out the end bit of the previous pattern and then repeat one more time. Or another 2 more times perhaps, not textbook but i wouldn't arrest you for trying that. Your way definitely spreads stuff around but not in careful steps of order of magnitude.

I'll consider all of these mentioned methods and implement what i will after i am done with maintenance.

Also i have to do a quick grow of a species to get its spores.


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26414388 - 01/03/20 01:29 PM (4 years, 1 month ago)

Concern was raised over the size of mycelial colonies needed for visualization without a scope and practical transfer of colonies to fresh plates.
I have some plates germinating right now so I took this picture of a colony I would transfer if doing it today [I'll wait until tomorrow so more spores can sprout first, hopefully in less spore-dense areas].
This is the size of colonies I can see with my naked eye, the image is of a region 1,13mm wide and 0,85mm tall and encompasses the whole colony.

Sorry about it being blurry and not dyed, its still sealed in glass.
My transfers are kept as close to 1mm X 1mm as possible, but in practice they tend to be more like 1,5mm X 1,5mm.

Tiny, tiny wedges are key for high success in monokaryon isolation in most cases.
Though not all.
If you have a scope you should check any isolates that stay tomentose, even if you weren't seeking monokaryons. You might be surprised.


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26418865 - 01/06/20 07:27 AM (4 years, 1 month ago)

so I'm just barely getting started in this game, but my sense is that there is something about yeah, groups of spores aiding in each other's germination. an entourage effect of sorts, whether it's the actual physical aspect of them being next to each other causing capillary action between them to draw water out of the agar/whatever surface they're on, already germinated spores waking up other spores, or what. I'm tracking several "isolated" spores (within millimeters of each other) daily on a dish, though I haven't seen a peep from any of them yet (it's only been 5 days). have any of you working with scopes and searching for monokaryons seen a single spore under the scope and come back later to actually see mycelium sprouting forth? It seems possible something could be added to stimulate them in the same way being around a clump does, like Solipsis says, some proteins or polysaccharides to tell them to wake up and get to mating, there's water, AND genetics available nearby!

these are the 2 spots I'm tracking right now, the single spore has been isolated to it's own dish, the other 5 are on their own as well, though having read RR's statement on spore viability in the months post print I'm growing doubtful any of these random 6 out of thousands will germinate:





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InvisiblebodhisattaMDiscordReddit
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Re: Monokaryons [Re: trubblesome]
    #26418876 - 01/06/20 07:33 AM (4 years, 1 month ago)

RRs statement about spore vitality seems to be completely wrong in practice. I've witnessed nothing remotely close to what he said for spores.


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Offlinetrubblesome
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Re: Monokaryons [Re: bodhisatta]
    #26418901 - 01/06/20 07:51 AM (4 years, 1 month ago)

gotcha, good to know. guess I'll keep checking back on them and a few others then.

finally realized I can get the 10x objective down comfortably by...flipping the plate upside down :facepalm3:

better pics to come


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OfflineSolipsis
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Re: Monokaryons [Re: trubblesome]
    #26428160 - 01/11/20 01:49 PM (4 years, 1 month ago)

Actually Elrik.. while i have been generally enthousiastic and believe in a broad sense that one can get monokaryons since there are also just too many people working on this stuff who do have training and meeans of verifications... that post earlier about the PE6 strains did prove nothing i believe, on the contrary.

It would be good to actually see two mating monokaryons or at least a mono and dikaryon mating, and not the sort of absence of evidence is proof kind of thing.

TBH on a hunch i did try putting two strains of Cordyceps militaris together on plate.. and while i am tired and lazy now i dont mind showing the plate. I have been back and forth about it and now realize it has not mated despite one spore culture looking a lot like a mono - and with the weirdness of Asco's i admit i dont understand i thought fuck it i wanna see what happens.

Well a bit less inhibition between the culture happened but still no real action, they just kinda alpha blend into each other but i see now its nothing.

That has nothing to do with dilutions and streaking however i was just playing out a hunch/curiosity.

If nothing else, mating a mon-mon or di-mon decently is something i suggest is a challenge for us all, with reasonable means so not micromanipulation or anything like that.

I realize not all mon-mon or di-mon may even be compatible per se but i think that would be whats to focus on and not just indirect evidence of having a monokaryon, regardless.

I am still waiting on some fruiting to gather the spores i need to start on real crossing experiments. Not cubes btw.

Just trying to keep this in the middle here, all sides get skepticism. This is about the science and not about conviction. So let's science each other better.


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OfflineElrik
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Re: Monokaryons [Re: Solipsis]
    #26428906 - 01/11/20 10:37 PM (4 years, 1 month ago)

I truly don't get why people think this is prohibitively advanced.
I will freely agree that, for hobby mushroom growing, isolating and breeding with monokaryons is advanced work and I will not fault anyone who doesn't choose to go that far. But in true mycology, as a scientific discipline; isolating, verifying, and breeding with monokaryons is beginner level fundamental work. Its whats taught in the first year of college. Literally. I sadly never took the wild-eyed lunatics classes but I knew my colleges professor of mycology and this is exactly what he taught to first and second year college students in the lab course of his beginner mycology classes.
Now, I do at least partially agree with the spirit of your point. I dont know why you have the impression I have only indirect evidence I was even working with a monokaryon. As I said before. Be cautious, be suspicious, be attentive and aware, re-confirm everything. But breeding different varieties within a species is as easy as you can get. When I made two crosses with PE6 I wasn't trying to scientifically prove that monokaryons exist or that Psi. cubensis can cross with Psi. cubensis. I was just making a cross. It worked too, every shred of evidence indicates they were valid crosses.
There are times for careful mon-mon crosses on petris. I'll give some of my own examples:
•I recently isolated and thoroughly verified three Psi. mexicana 'Chicon Nindo' monokaryotic isolates numbered 1.2, 2.1, and 3.4. I bred these to each other on petris. The first two formed clamp connections with 3.4 but 1.2 and 2.1 would not cross with eachother indicating sexual incompatibility, so now I know that. And now I have 1.2 X 3.4 F1 and 2.1 X 3.4 F1 that I can grow out. If 1.2 X 3.4 yields better or is more potent than the other than I'll know that the monokaryotic strain 1.2 is better for breeding than 2.1. Stuff like that is a good use for mon-mon crosses.
•I recently [before verifying the Chicon Nindo 3.4 isolate] also isolated three Atl #7 monokaryons. Unlike a cube X cube cross, a Psi. mexicana 'Chicon Nindo' X Psi. tampanensis 'Atl#7' cross would be news! I was also very dubious about it even being possible. I crossed my two Chicon Nindo monos with my three Atl#7 monos by every combination. Sadly, even after a good amount of time, no clamp connections formed at the interfaces of any of the six colony pairs. No cross. If I had got clamp connections on a pair I would have redone that test after intensive re-verification of the monokaryotic lines and I would have grown out both resulting crosses scrutinizing them for intermediate or mixed morphology with as much suspicion as I could muster.

To be fair, another recent incident shows why I am emphatic about being cautious and reconfirming monokaryon status and why I put more work into verifying them than many would. It also shows why I am sympathetic to skepticism:
I was reviving an old Transkei print with the hope of hunting monos as well as starting up a few jars. I streaked 4 plates thinly. Spore germination was horribly low and I wasn't home in the right window to catch any of the germinated spores just as they become visible, as I like to when hunting monos. Fresh spores really work best for finding monos. So I just let them grow. I soon had 1cm colonies that looked like potential monokaryons on plates ready to be turned into inocula for my grain jars. I mounted some mycelium from the colonies and saw that they were monokaryotic, I couldnt find clamps anywhere. Bonus! I took two transfers each from the healthiest looking three seemingly monokaryotic colonies. But as I said, be suspicious, re-verify. I waited. Both the transfers from one colony soon turned dikaryotic, then one transfer from the second colony did, both transfers from the third colony stayed monokaryotic. I'll never know if a few spores germinated late on the spore-laden transfers or if the colonies were just beginning to undergo dikaryotization and I just wasn't lucky enough to see one of the sparse clamp connections as that occurred. I'm still going to re-re-confirm them before putting them to slants and grain.

When I say hunting and taming monokaryons isn't hard I don't mean it can be done with frivolous or poorly developed technique. Don't let your guard down, monokaryons are as sneaky about sex as teenagers.
But it's not hard.
It just requires skill, precision, and lots of attention.


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26435847 - 01/15/20 11:00 PM (4 years, 1 month ago)

so it is possible:



I have 4, maybe 5? single spores on their own plates now. The one above (Plate #4) is the only one to have germinated, but they were printed and streaked Sunday 1/12.




lets see what happens


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Offlinetrubblesome
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Re: Monokaryons [Re: trubblesome]
    #26438043 - 01/17/20 08:37 AM (4 years, 1 month ago)

...or is it?

not satisfied with being unable to see the spore/growth, I dicked around a bit looking for it this morning and found it - still ungerminated, which is clear at 100x:




so I guess we wait.


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OfflineElrik
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Re: Monokaryons [Re: trubblesome]
    #26438288 - 01/17/20 10:30 AM (4 years, 1 month ago)

You're braver than I.
When over 10% of spores are viable I'm shocked.
When 0.1% of spores are viable I'm not surprised.
When 0.01% of spores are viable it must be monday.
I wouldn't put one spore per plate.
I might put 10 spores on a plate in a grid if I had a laminar flow hood, fresh spores, and nothing to do.

Mycelium doesn't need to have a witness to be monokaryotic, it just needs to consistently have a total absence of clamp connections through multiple transfers. The smaller the wedges, the better.

To find things on petris it helps to write on the bottom of the petri with a super-fine point permanent marker. Just use 90% isopropanol to clean the markings off if you reuse.
On a crowded plate that's a great way to point out which colonies you intend to transfer. When starting stone formers I did MS plates, waited until baby stones were forming, and put elongated arrows pointing at my targets like:
---->-----


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26441327 - 01/19/20 08:44 AM (4 years, 29 days ago)

i still feel compelled to witness it though, for my own probably insane reasons. im just going to leave these plates alone for a while and if i come back in a week and there is mycelium, great, if not, no big deal. maybe I’ll try again next time I take prints of something. either way it took like ten minutes total between streaking and making transfers. seems easier than properly looking for clamp connections or obsessively checking plates. without sampling and staining and 400x magnification ive never been able to see them well enough anyway.


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InvisibleSmartattack
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Re: Monokaryons [Re: trubblesome]
    #26441368 - 01/19/20 09:09 AM (4 years, 29 days ago)

I was having a thought about this the other day, concerning isolating spores with reasonable distance between them in single form.

I'm wondering if there's a better way to do this right from the start rather than using prints. Here's a thought I had. Rather than taking a cap that is sporulating to make a print, why not place it Gill down on an elevated small grill of some type maybe 3" above your work surface. Given the amount of spores dropped by a cap in short time, maybe taking an agar plate and sliding it under the elevated grill for a short period would catch a few in their natural Cascade out of the cap, I assume they fall out much less clumpy than when in a print and possibly solo much of the time?

Some experimenting may need to be done as far as timing is concerned, like with a steadily dropping cap, maybe you would only want something like 1-2 seconds of exposure under the cap with your plate. I feel like this would be a fairly easy way to keep things clean as well since there is no direct contact other than the spores themselves.


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OfflineElrik
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Re: Monokaryons [Re: Smartattack]
    #26441560 - 01/19/20 11:41 AM (4 years, 29 days ago)

That's a very good idea to try. If someones technique is good enough that taking several transfers from a plate generally wont contam it, I don't imagine just holding a cap over it for 10 seconds would be a problem and, theoretically, it should lay spores down quite well if it was sufficiently active in sporulation.
My main problem is I usually start from a newly acquired print that I haven't grown out yet, so I can develop the monos while doing my first grow of the variety. Sometimes I'm lucky and the print will be new and essentially undried, the spores act like moist flour.
Often the print will be desiccated dry. Great for long term storage, but its like trying to scrape spray paint from foil.

Today I'm looking at a few very promising PE colonies.
PE genetics will be improved! :smile:

...I just had a wacky thought.
Isolate several monokaryons from a given variety, say PE, get them colonized to grain, and then inject with spores from that same variety. I haven't actually seen anyone doing that.
It wouldn't be a cross, but an isolation of one half of the genetics. You could then investigate the properties of each group and if one was better than the others you could 1) clone and isolate a great strain, 2) print caps and go from there, and 3) know which mono was better for outcrossing to different varieties. All at once.
Dude. Shit, yes. I'm trying this! :cool:


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Edited by Elrik (01/19/20 11:50 AM)


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Offlinetrubblesome
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Re: Monokaryons [Re: Elrik]
    #26441821 - 01/19/20 02:41 PM (4 years, 29 days ago)

im confusing myself with how much ive included in this thread vs my private journal where ive been keeping track of this process but i did something similar in theory to what you posted, smartattack -

“ The less successful plates I lightly drew the loop on the spore print in the same manner, but then I held the loop from above the plate, and used the tip of a flame sterilized scalpel to pull back the loop and then allow it to snap, the idea being a scatter of spores would drift on to the plates. I don't think I let the air hang long enough though for any spores to land on the surface of the agar, and scanning the surface of the plates for spores that could be literally anywhere ended up not being very fun so I'll be focusing on the light streaking method for now”

the method i use instead is:

“ 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, 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

live print dropping is something I’ll probably try at some point but i’d be nervous about all the other shit that would fall on to the plate with the spores that cant be seen at 40x on the plate under the scope. with the light streak method I’m gonna have several spores i can transfer p much right away, plus I can just scan along the streak line when looking for spores. the spores arent always visible in the same plane so without the streak lines to give you a sense of your depth of field its difficult to find anything but the largest clumps under the scope.

my hypothesis about all of this is that because spores rely on hydrophilic sugars to attract water to the basidium and collect enough weight that they can fall off the gills, they will more often fall in clumps and the mechanical action of drawing the loop over them will break them up. moving them back and forth rapidly allows the dryer spores to flitter off on their own on to the plate. I have absolutely zero scientific documentation of this but my intuition says that there is chemical signaling going on that tells spores that are in contact with one another that there are pairs to be made in short distance from them, as well as capillary action and the hydrophilic sugars to provide moisture and an energy source to the growing spores. im kind of thinking that having to plant that one upside down may help in the long run of tricking the spore in to thinking it will get the necessary genes to make fruit with right away


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Re: Monokaryons [Re: trubblesome]
    #26458177 - 01/29/20 08:40 AM (4 years, 19 days ago)

soooo I think I'm going to put my project on this on pause - I just don't see myself having the time to really dedicate myself to it at the moment. It also seems like just selecting fruits for the traits I'm looking for will be way easier to get the ones I want.

Something I was thinking about though, if anybody else feels ambitious, is to make a boat load of prints, and then put the spores in to an agar recipe. They'll be deactivated during the PC cycle but the sugars and chemicals should be preserved and hopefully help initiate germination for spores put to the plate, IF that really is a germination mechanism. Anyway.


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