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Offlinecheor
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Dedikaryotization of Cubensis fruit body clones? * 2
    #27619669 - 01/15/22 10:17 AM (2 years, 4 months ago)

I've been trying to find a way to create monokaryotic mycelium from dikaryotic mycelium obtained from  a clone of the strangest looking cube I've ever seen.

The goal being to then cross the resulting monokaryote with some particularly potent blue meanie cultures I have going....

So far I've come up with agar supplemented with  2-Deoxy-D-glucose at 5mM2

Or possibly performing mechanical dedikaryotization then growing out the fragments in a dextrose/glycine solution?


Does anyone have any input or other techniques they use or know of?

So far as I know these techniques haven't been tried on any species within this family....

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Offlinefossilshark
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: cheor] * 1
    #27637772 - 01/29/22 05:24 PM (2 years, 3 months ago)

You cannot create a monokaryotic culture from a dikaryotic culture, your options are to A) fruit the clone culture so you have spores from it and then serial dilute the spore solution so you have a monokaryotic culture to breed with OR B) clamp the monokaryotic culture of the "blue meanie" to the dikaryotic culture of the clone via bullers phenomenon.

no matter how you breed its important that you have grown out the varietys you are working with enough to be able to visually identify a cross, as the only way you can confirm a cross is a cross is if the fruit carries traits from both parents.


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: cheor] * 1
    #27650663 - 02/08/22 09:54 AM (2 years, 3 months ago)

I've been interested in learning more about Di-Mon pairing.

Commenting to witness follow ups.


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OfflineAlan RockefellerM
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: cheor] * 2
    #27652578 - 02/09/22 04:43 PM (2 years, 3 months ago)

I have heard of a physical method to create monokaryons from a dikaryotic culture - basically put a colonized agar plate and some water into a sterile blender cup (metal ones can be autoclaved), blend it really well, and plate out the results.    Some of the colonies that grow will be monokaryons if you blend it enough to break them apart but not so much that they are completely dead.  I am not sure what the success rate is.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller] * 1
    #27654356 - 02/10/22 08:10 PM (2 years, 3 months ago)

:popcorn: Alan , you should try it and post up for followers:thumbup:
:bigyesnod:


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: cheor] * 1
    #27733880 - 04/14/22 05:36 AM (2 years, 1 month ago)
Log in to view attachment

I'd try a variant of Leal-Lara and Egar-Hummel 1982.

They had some challenges regarding the ratios.
The method employs magnesium and phosphorus deficient media and carefully controlled volumes to prevent overcrowding so that the torn hyphae and their nuclei can recover and become Neohaplonts. If the density is too high they reconnect.

The Peptone P method takes advantage of the high glycine content and the use of simple media made of anhydrous glucose and peptone P or glycine prevents caramelization and or sediment from forming in the media.

This method for production of monokaryonic neohaplonts using dedikaryotization has been employed for decades.

I was able to obtain the 1982 paper for further consideration and have attached it to this post.

Edited by Nillion (04/14/22 05:41 AM)

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OfflinePsilotoad
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Nillion] * 1
    #27748189 - 04/23/22 02:08 PM (2 years, 26 days ago)

Leal-Lara and Egar-Hummel 1982 method worked for me with a Long Ghost isolation and 15 second blend times. I was only able to recover one haplont due to mistakes on my end but the other was there because what wasn't recovered and plated from the solution became dikaryotic again. I am in the process of running it again on a few more strains and will hopefully get symmetrical recovery. Will provide a pictorial when done.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad] * 1
    #27750284 - 04/25/22 06:33 AM (2 years, 24 days ago)

It would be about just as difficult as obtaining. A single spore isolation as well.

You would need to blend it to expose the 2 nuclei that are present in dikaryotic myceliums. And really I doubt a regular blender could do this, I imagine you would need a laboratory grade, razor sharp blender to slice through cell walls.


I was talking with a buddy about this recently. You might even be able to use a cell wall degrading enzyme like in Protoplast Fusion.

Either way. Once you have your nuclei exposed from the cell walls, you may as well have a spore solution as you have a bunch of individual nuclei, monokayotes floating around your solution.

The solution would need to be diluted down to the extent you would dilute a solution for single spore isolation. You need a microscope and hemocytometer for this. And then you would need to go back and identify the growth that's worth transferring ebfor it interacts with another colony.


And really if your trying to cross 2 specimens from the same species you can literally just let the myceliums mate. If you want a higher likelyhood of traits from one than the other use more of that mycelium in the culture.

In theory if you have 2 same species you want to cross, and you want the traights from 1 more than the other, if you say did a 1 to 3 ratio, 3 bring the spawn of the traights you want primarily, say you g2g 2 different spawns of 2 same species specimens together, than in theory you would be more likely to obtain the traights of the one with the higher spawn rate as their ar more nuclei containing the genetic information, increasing the breeding probabilities for those traights.


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OfflinePsilotoad
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: QM33] * 2
    #27781385 - 05/17/22 09:43 AM (2 years, 2 days ago)

Single spore isolation is not as rapid and controllable as dedikayotization. You can buy any eberbach jar and just find a compatible base. Single spore isolation will only yield 1 monokaryon type from the parent and with unknown genetic changes from the parent whereas dedikaryotization can lead to symmetrical recovery of both types allowing the potential of 4 offspring of the same cross from 2 proven parent strains. I performed both and the dedikaryotization is faster and with better results. You can look up SYMMETRICAL RECOVERY OF MONOKARYOTIC COMPONENTS FROM LENTINULA EDODES USING DEDIKARYOTIZATION  R. Ramírez-Carrillo and H. Leal-Lara for further information regarding the advantages of symmetrical dedikaryotization over single spore dilution and other dedikaryotization methods.

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InvisibleQM33
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad]
    #27781520 - 05/17/22 12:12 PM (2 years, 2 days ago)

What kind of blender do you need? You say eberbach jar, that's just a standard lab blender right?


Have you attempted this? Do you know if it damaged the nuclei?

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InvisibleQM33
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: QM33]
    #27781529 - 05/17/22 12:21 PM (2 years, 2 days ago)

All I was able to read was thi, but it did answer my question

""
The dedikaryotization method proposed for Pleurotus spp and other basidiomycetes by Leal-Lara and Eger-Hummel in 1982 was modified in order to recover the monokaryotic components of seven Lentinula edodes strains. By decreasing the time that agar cultures were blended, mycelial death was reduced and more neohaplonts were recovered. If agar culture homogenates were plated on malt extract agar, L. edodes strains barely survived 30 seconds blending. Agar cultures of L. edodes blended for more than 5 seconds did not yield viable mycelium when agar culture homogenates were inoculated in dedikaryotization liquid media. Optimum blending time of agar cultures was determined for each strain. An attempt was made to recover neohaplont types at different stages of the dedikaryotization procedure either directly from agar culture homogenates or from dedikaryotization media inoculated with such homogenates and incubated for 14 days at 24oC or from homogenates incubated for fourteen days in dedikaryotization media. All seven L. edodes strains were successfully dedikaryotized. In all cases, both monokaryotic components were recovered and in six cases, they were isolated in a 1:1 ratio.""




So it doesn't say what type of blender they use.?

What does isolated in a 1 to 1 ratio mean?

And of course you have the perks of the parent fruits.

Your still going to need to make a serial dilutions with a hemocytometer and plate them out and make sure they don't have clamp connection.


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OfflinePsilotoad
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Re: Dedikaryotization of Cubensis fruit body clones? *DELETED* [Re: QM33]
    #28071641 - 11/27/22 02:34 PM (1 year, 5 months ago)

Post deleted by Psilotoad

Reason for deletion: Went too far wasn't constructive

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OfflinePsilotoad
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad]
    #28071646 - 11/27/22 02:35 PM (1 year, 5 months ago)

WhAt iS a 1:1 ratio :rolleyes:

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OfflineAlan RockefellerM
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad] * 13
    #28073380 - 11/28/22 04:52 PM (1 year, 5 months ago)

Julian shared his writeup on Facebook ten days ago:


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OfflineDERRAYLD
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller] * 3
    #28073839 - 11/28/22 11:09 PM (1 year, 5 months ago)

Thanks Alan, and thanks for posting the actual pages here for those of us that aren't on Facebook.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller]
    #28073858 - 11/28/22 11:40 PM (1 year, 5 months ago)

That method sounds so simple. I know it glosses over the mechanics of generating stable and desirable dikaryotic strains, but that is what we all strive to do every day. Once that is done, it really seems as simple as blending the mycelium and confirming that luck (what the paper calls trial and error) played its part in a colony under the microscope?

I would have loved an intuitive explanation with recognizing the difference of nuclei in mon/dikaryons under the scope to fully complete the introduction, but google is full of those. Is this truly as simple as all monkaryons do not present with clamps with differing nuclei and by extension that all dikaryons will have a clamp between each link of two different nuclei to another?

If it is truly this simple. I may be buying a microscope in the future.

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OfflineAlan RockefellerM
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: cooleko] * 1
    #28075423 - 11/30/22 02:36 AM (1 year, 5 months ago)

Quote:

cooleko said:
I would have loved an intuitive explanation with recognizing the difference of nuclei in mon/dikaryons under the scope to fully complete the introduction, but google is full of those. Is this truly as simple as all monkaryons do not present with clamps with differing nuclei and by extension that all dikaryons will have a clamp between each link of two different nuclei to another?

If it is truly this simple. I may be buying a microscope in the future.




Yes that's it.  It's good to let the mycelium grow for a few weeks to make sure it didn't get a nuclei in there somewhere, and the leading edge is sometimes monokaryotic so sample from further in.

Another way to do it is to get a nuclear stain so you can see how many nuclei are present in hypha.  Safranin is one popular one, or you could use DAPI + blue light + orange filter.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller]
    #28150293 - 01/21/23 09:45 AM (1 year, 3 months ago)

Bumping this back to front page but also..


Having a hard time understanding how this consistently results in 2 monokayrotic parent types when fruiting strains can consist of more than 2 parents.


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OfflineAlan RockefellerM
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: AyePlus] * 1
    #28165294 - 01/31/23 12:47 AM (1 year, 3 months ago)

Quote:

AyePlus said:
Having a hard time understanding how this consistently results in 2 monokayrotic parent types when fruiting strains can consist of more than 2 parents.





What makes you think that fruiting strains can consist of more than 2 parents?  Where would the extra nuclei live?

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller]
    #28165510 - 01/31/23 07:20 AM (1 year, 3 months ago)

Quote:

Alan Rockefeller said:
Quote:

AyePlus said:
Having a hard time understanding how this consistently results in 2 monokayrotic parent types when fruiting strains can consist of more than 2 parents.





What makes you think that fruiting strains can consist of more than 2 parents?  Where would the extra nuclei live?




Doesnt  the possibility of a a di-mon pairing or di-di pairing happening mean there can be more than 2 parent genetics?



I thought alot of basidiomycetes are capable of tetrapolar mating systems which i though meant multiple parents

I dont understand fungal breeding all that well butni was under the impression that a fruiting culture can contain multiple strains each with their own set of genetics that can be teased apart, IE sectoring and isolation.
Doesnt that imply multiple sets of genetics IE more than 2?

Or
Is something else happening involving genes being transferred to the parent strain through a different process.


Any reading on the topic would be helpful

I found this but its mostly about cryptococcus and jumps species to species, that gets a bit muddy for me.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025156/


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Edited by AyePlus (02/01/23 07:43 AM)

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InvisibleAyePlus
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: AyePlus]
    #28182194 - 02/11/23 11:16 AM (1 year, 3 months ago)

I’m getting even more confused.


@Alan

Are you saying
Quote:

LtLurker said:
Shrooms Dont have just 2 parents like a punnit square. They have many parent spores, some from the same fruit, so we're talking thousands of parents for each fruit.





Isnt true?


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller]
    #28197415 - 02/21/23 12:27 PM (1 year, 2 months ago)

Quote:

Alan Rockefeller said:
Julian shared his writeup on Facebook ten days ago:






What makes the peptone and glucose solution more dedikaryotizing than just water?

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OfflineAlan RockefellerM
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: wy35]
    #28201677 - 02/23/23 10:57 PM (1 year, 2 months ago)

Quote:

wy35 said:
What makes the peptone and glucose solution more dedikaryotizing than just water?





Maybe they are nutrients to help the mycelium heal after the physical destruction of the blender.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Alan Rockefeller]
    #28202122 - 02/24/23 08:41 AM (1 year, 2 months ago)

Yea, I think it's the recuperation and regrowth, not helping separate nuclei.

Peptone are amino acids and nitrates(and lots of other good stuff,) which are some of the major building blocks of life, and they're mainly/mostly water soluble, so when your blending it's stripping away and diluting it into the water. Adding extra allows them to recuperate/regenerate. Glucose plus nitrates also creates amino acids, glucose is also converted into lipids, and lipids are used for all kinds of stuff, including cell walls. Stuff they normally have to obtain by breaking down stuff which takes energy(and they've already been weakened), this concoction allows it to be readily available.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: wy35]
    #28203660 - 02/25/23 08:25 AM (1 year, 2 months ago)

You could use just water it has no magnesium or phosphorus but the survivability would be lower and the recovery would be much longer. Like Alan said the peptone glucose helps the mycelium grow and recover. Just basically giving it nutes with low magnesium and phosphorus to prevent clamps from forming when submerged in the solution.

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: AyePlus] * 2
    #28203674 - 02/25/23 08:38 AM (1 year, 2 months ago)

When you use multispore each spore that germinates produces monokaryotic hyphae and each successful pairing of monokaryotic strains produces a new dikaryotic strain so when you put down multispore you have many strains of your type. That is why this mentions you want to use an isolated dikaryon. An isolated dikaryon is comprised of 2 genetically distinct types of nuclei one from each parent monokaryon. In di-mon mating only one nuclei type is donated to the monokaryon and this is not a reciprocal exchange. Each dikaryon only has two parent nuclei from their monokaryon parents or mon and di parent but with multispore you are producing many dikaryons. Once you isolate a colony or clone a fruit you have a single dikaryon this dikaryon will be comprised of 2 nuclei one from each parent monokaryon.

Here this includes some more info about nuclei exchange in mon-mon and di-mon pairings

https://www.mdpi.com/2076-2607/9/6/1248/htm

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad]
    #28205446 - 02/26/23 01:00 PM (1 year, 2 months ago)

Not sure if you guys have seen This thread but I seemingly have crossed compatible clones by blending pulverized myc water of two clones and inoculating qts directly with the mixed GLC

It was pretty easy

I actually got the idea to try it based on Alan’s post from FB

If tldr:

Yeti clone


Mak 118 clone


Mixed myc water of both


It’s almost done with the second flush where I’ll be getting spores and doing a heavy ms grow to show the variation in phenos between parents in the f2

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Re: Dedikaryotization of Cubensis fruit body clones? [Re: fahtster]
    #28208092 - 02/28/23 12:11 PM (1 year, 2 months ago)

I found this patent on dedikaryotizing strains: https://patents.google.com/patent/US4242832A/en

Describes the same procedure, including the peptone and glucose solution. Might be helpful for someone looking for the specifics (e.g. what peptone to use).


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad]
    #28213997 - 03/04/23 11:25 AM (1 year, 2 months ago)

Quote:

Psilotoad said:
When you use multispore each spore that germinates produces monokaryotic hyphae and each successful pairing of monokaryotic strains produces a new dikaryotic strain so when you put down multispore you have many strains of your type. That is why this mentions you want to use an isolated dikaryon. An isolated dikaryon is comprised of 2 genetically distinct types of nuclei one from each parent monokaryon. In di-mon mating only one nuclei type is donated to the monokaryon and this is not a reciprocal exchange. Each dikaryon only has two parent nuclei from their monokaryon parents or mon and di parent but with multispore you are producing many dikaryons. Once you isolate a colony or clone a fruit you have a single dikaryon this dikaryon will be comprised of 2 nuclei one from each parent monokaryon.

Here this includes some more info about nuclei exchange in mon-mon and di-mon pairings

https://www.mdpi.com/2076-2607/9/6/1248/htm




Thank you.


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Psilotoad]
    #28437476 - 08/17/23 06:41 PM (8 months, 27 days ago)

Quote:

Psilotoad said:
When you use multispore each spore that germinates produces monokaryotic hyphae and each successful pairing of monokaryotic strains produces a new dikaryotic strain so when you put down multispore you have many strains of your type. That is why this mentions you want to use an isolated dikaryon. An isolated dikaryon is comprised of 2 genetically distinct types of nuclei one from each parent monokaryon. In di-mon mating only one nuclei type is donated to the monokaryon and this is not a reciprocal exchange. Each dikaryon only has two parent nuclei from their monokaryon parents or mon and di parent but with multispore you are producing many dikaryons. Once you isolate a colony or clone a fruit you have a single dikaryon this dikaryon will be comprised of 2 nuclei one from each parent monokaryon.

Here this includes some more info about nuclei exchange in mon-mon and di-mon pairings

https://www.mdpi.com/2076-2607/9/6/1248/htm




Bump.
Everyone should learn this stuff. :cheers:
:takingnotes:


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Re: Dedikaryotization of Cubensis fruit body clones? [Re: tryptkaloids]
    #28438194 - 08/18/23 01:01 PM (8 months, 26 days ago)

I have some single ascospore isolate of Cordyceps militaris:
If i use one isolate to colonize a substrat and after full colonisation i inocule the substrat with a ms liquid culture: only the FIRST  monokaryotic compatible culture from ms go to give his genes to my isolate? Is there the same diversity with a clone or a ms cross with a monokaryotic isolate culture ?
I hope my English is understandable, sorry for any errors (corrections welcome)


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Offlinetrippleblack
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Mushroom love]
    #28440266 - 08/20/23 07:59 AM (8 months, 24 days ago)

Its possible that clones of mushrooms are made up of multiple mycellium colonies; not just two parents. To my knowledge this has not been directly studied in cubensis. i think it's just an assumption that a clone is only comprised of two parents.

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InvisibleMushroom love
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: trippleblack]
    #28440559 - 08/20/23 12:06 PM (8 months, 24 days ago)

Quote:

Psilotoad said:
When you use multispore each spore that germinates produces monokaryotic hyphae and each successful pairing of monokaryotic strains produces a new dikaryotic strain so when you put down multispore you have many strains of your type. That is why this mentions you want to use an isolated dikaryon. An isolated dikaryon is comprised of 2 genetically distinct types of nuclei one from each parent monokaryon. In di-mon mating only one nuclei type is donated to the monokaryon and this is not a reciprocal exchange. Each dikaryon only has two parent nuclei from their monokaryon parents or mon and di parent but with multispore you are producing many dikaryons. Once you isolate a colony or clone a fruit you have a single dikaryon this dikaryon will be comprised of 2 nuclei one from each parent monokaryon.






Quote:

trippleblack said:
Its possible that clones of mushrooms are made up of multiple mycellium colonies; not just two parents. To my knowledge this has not been directly studied in cubensis. i think it's just an assumption that a clone is only comprised of two parents.




Who is right?

So if i do a di-mon cross with a monokaryotic strain and a multispores culture, just the first fastest compatible dikaryons from the ms go to give a nuclei to my monokaryotic culture ?

will it be a means of natural selection of high-performance culture or is it only chance? (the first drop of inoculum to hit the monokaryotic strain)
I think this is the case if the substrate remains whole after colonization and there is a lot of combinations if the grain is broken before (for a multispores inoculum)


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OfflineZwinst
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: Mushroom love] * 1
    #28482325 - 09/25/23 12:08 PM (7 months, 19 days ago)

Quote:

Mushroom love said:

Who is right?




First, my least convincing observation: In a tray there can be multiple different genotypes present. For example when you see both leucistic and coloured fruiting bodys in a tray that came from multispore.
But i guess there could be multiple, distinct dikaryons present. I have observed this multiple times, in different cubensis.

More convincing to me: A single fruiting body can display different traits that don´t necessarily get expressed, when cloned. Those i got described as "chimeras" to me. For example a fruit that has "patched" gills with zones of coloured and coulerless spores. Or If you have a fruit with leucistic traits, clone it and you get different fruits both with and without leucism.

Of course i am talking about mutations that aren´t necessarily stable or even well understood and there could be different mechanisms or factors involved. But judging from observations made by people here and having seen this myself multiple times, i would tend to agree, that multiple parents must be involved.


If you think about it, beeing able to accept more than just 2 nuclei into your organism can have big advantages.

Lets just look at the life of a monokaryon, freshly germinated, stretching it´s hyphae for companionship...
Now it finds a compatible mate east and another one west at the same time. Why should it have to choose? I bet this one mono will just mate with both.
If someone wants to try this on a plate - the way i described it, would be a good starting point, i guess.
My hepa isn´t finished yet and my own 2 leucistic strains aren´t stabilized to the point, where i want to release them or start creating monos for crossing. But it is going to happen! And if noone has made this experiment till then, i´ll have to do it.

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InvisibleNillion
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: wy35]
    #28599977 - 12/27/23 03:56 PM (4 months, 19 days ago)

Quote:

wy35 said:
What makes the peptone and glucose solution more dedikaryotizing than just water?



It is actually slightly deficient in nutrients, which stunts the growth of the mycelia and makes it weaker, leading to increased production of neohaplonts, which can appear and recombine rapidly and thus be difficult to recover in some situations. A key part of the method is also culture density, by slowing growth and avoiding excess density it becomes easier to isolate the neohaplonts.

Also, the blender is nothing more than an agitation method, hand agitation or another method of stirring can work just as well to create the sheer forces needed for this, the blender is just faster. Any blender you can keep sterile works just fine for this.

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InvisibleNillion
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Re: Dedikaryotization of Cubensis fruit body clones? [Re: fahtster] * 1
    #28599983 - 12/27/23 04:01 PM (4 months, 19 days ago)

Quote:

fahtster said:
Not sure if you guys have seen This thread but I seemingly have crossed compatible clones by blending pulverized myc water of two clones and inoculating qts directly with the mixed




Yeah, one doesn't need to isolate neohaplonts to use dedikaryotization for breeding, one can just agitate mixed cultures prior to inoculation and screen the results for the desired phenotypes. It's quite handy actually.

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