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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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InvisibleTheConfluence
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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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InvisiblerhizoRider
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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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InvisibleNillion
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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)
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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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InvisibleQM33
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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.


--------------------
OmManiPadmeHum,OmManiPadmeHum, OmManiPadMeHum...
There are known knowns, there are known unknowns,
          there are also unknown unknowns.
With great privilege comes great responsibility.

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Turn your Swab to a Syringe and Syringe to Multiple Syringes!
No Pours (QuantomStyal)
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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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Invisiblecooleko
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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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InvisibleAyePlus
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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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InvisibleAyePlus
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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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