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Invisiblemorty422
Wuba-luba-dub-dub!
I'm a teapot


Registered: 07/06/16
Posts: 988
On the subject of cloning...
    #23597085 - 08/31/16 10:52 AM (7 years, 4 months ago)

Quote:

FrankHorrigan said:
This is part of my series on how I get things done. I hope it helps!

The easiest way to get a good set of genetics is to clone an ideal looking fruit or cluster from a multispore grow. This process is very simple, and the way I do it is essentially the same way that RR does it.

Some people will try to clone into LC or using a needle, etc. This will almost always fail. Make sure to use agar. Since fruiting is done in open air, you can always assume the fruit is covered in contaminants. This is doubly true for cloning wild mushrooms.

Even though you'll be taking a cut of virgin mycelium that has never been exposed to open air, it is very common to need to transfer your mycelium away from contaminants on the first dish.

Since a flowhood can potentially blow contaminants around from the fruit you are cloning, I recommend working in a still air box.


Start by wearing tyvek sleeves and disposable gloves. Make sure to work quickly and carefully inside the still air box. Use good sterile technique.

Flame sterilize your scalpel and set it down in the still air box where it won't be disturbed. I place it on my receiving petri dish. Do not set it on the floor of your SAB.

Take the mushroom (this is a golden oyster) and pinch it at the base. Rip it down the middle. Do not cut into the stem with your scalpel or you will force contaminants down into it.



Holding part of the fruit with one hand, use your scalpel to gently scrape at the center of the stem, near the base. Try to get a small piece on to the blade. As RR says, a scraping motion will be easier than trying to slice a specific piece.


Once you have a small piece of tissue on the end of your scalpel, transfer it to the agar. It may take a bit of effort to get it off the blade :shrug:


Take as many pieces of tissue as you want to clone. You can flame sterilize between each cut, or just flame sterilize between each petri dish or fruit. I use 3 section dishes so I flame sterilize every three cuts.

That's all there is to it. Wrap the petri dish and let it colonize. Transfer healthy mycelium away from any contaminants as soon as they appear!

Some fruits will give you an isolate right off the bat, but a lot of mushrooms are actually composites of multiple genetics. However, I have seen TCs say that the original clone sectors perform better together than as an isolate of each. So it's up to you if you decide to isolate the clone further :thumbup:


:cheers:





In the bold text of Frank's post above he states that some TC's say the original clone sectors perform better TOGETHER than as an isolate of each...

This might be hard for me to ask - being new to AGAR I may possibly be using the wrong terms. Bare with me, correct me as I go and let me know if I am on the right track with this ideology!

1. The cloned tissue of the best fruit is transferred to agar.

2. The cloned tissue is cleaned up further by transferring a section of clean growth that show no signs of bacteria.

3. The next dish of agar is very clean (if not step 2 can be repeated once more)and can be colonized fully to be dropped into multiple masters or possibly just one jar (such as the tiger drop).

^^^^Let me know if any of this was wrong, even in the slightest- as the next portion of my post and my actual question sort of revolves around the top being true.

That final plate (before the drop into grain, or LC, or whatever) could possibly look like this:



or maybe this:



Etc...

My questions regarding this are:

A: If you are trying to clean up a fruits tissue to clone it, are you also separating and isolating 'away' some of the genetics as you go?

B: If you are "tearing up that fruits genetics" are you now diminishing the chance that your cloned grow will grow that type of fruits characteristics?

C: (I think this is where Frank's quote comes into play...)
Is the monoculture what we are after? If question A is true, is it best to isolate sectors as little as possible to keep that fruits genetic code present in your grow?

D: Is a good goal to continue with multiple grows of the same mycelium and continue to clone fruits off the same genetics you cleaned up before? Is there anything that could even come of that-that is worth the time?

E: Is all this shit nebulous and I just need to grab some good-clean rhyzo myc and roll that bitch out?

Thanks!


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Invisible36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
Trusted Cultivator
Re: On the subject of cloning... [Re: morty422]
    #23597321 - 08/31/16 12:37 PM (7 years, 4 months ago)

Quote:

morty422 said:
A: If you are trying to clean up a fruits tissue to clone it, are you also separating and isolating 'away' some of the genetics as you go?




Potentially, but probably not.

Quote:

B: If you are "tearing up that fruits genetics" are you now diminishing the chance that your cloned grow will grow that type of fruits characteristics?




I've never seen that happen.

Quote:

C: (I think this is where Frank's quote comes into play...)
Is the monoculture what we are after? If question A is true, is it best to isolate sectors as little as possible to keep that fruits genetic code present in your grow?




A monoculture is usually what I'm after. It's your preference.

Quote:

D: Is a good goal to continue with multiple grows of the same mycelium and continue to clone fruits off the same genetics you cleaned up before? Is there anything that could even come of that-that is worth the time?




Clone once. Your myc won't be as vigorous if you keep putting it all the way through the reproductive cycle.

Quote:

E: Is all this shit nebulous and I just need to grab some good-clean rhyzo myc and roll that bitch out?




Yup.


--------------------
Redd Foxx said:
If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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Invisiblemorty422
Wuba-luba-dub-dub!
I'm a teapot


Registered: 07/06/16
Posts: 988
Re: On the subject of cloning... [Re: 36fuckin5]
    #23597404 - 08/31/16 12:59 PM (7 years, 4 months ago)

Thanks for the response. I posted this in cult first and it was viewed but never posted on. I figured advanced myc would be more of a help.

:asianofapproval:


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Invisible36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
Trusted Cultivator
Re: On the subject of cloning... [Re: morty422]
    #23600525 - 09/01/16 09:46 AM (7 years, 4 months ago)

Don't double-post, just be patient. If it takes too long, bump your thread and it'll get answered.


--------------------
Redd Foxx said:
If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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Invisiblemorty422
Wuba-luba-dub-dub!
I'm a teapot


Registered: 07/06/16
Posts: 988
Re: On the subject of cloning... [Re: 36fuckin5]
    #23601017 - 09/01/16 12:37 PM (7 years, 4 months ago)

Quote:

36fuckin5 said:
Don't double-post, just be patient. If it takes too long, bump your thread and it'll get answered.




No problem! Will do!

I didn't intend for a "double post". I just figured Advanced Mycology was the more appropriate forum.

I'll heed your advice next time or ask a mod to move it for me!

Thanks!


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Invisible36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
Trusted Cultivator
Re: On the subject of cloning... [Re: morty422]
    #23601089 - 09/01/16 12:59 PM (7 years, 4 months ago)

No problem. Just trying to catch you before you get an infraction.


--------------------
Redd Foxx said:
If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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Invisiblemorty422
Wuba-luba-dub-dub!
I'm a teapot


Registered: 07/06/16
Posts: 988
Re: On the subject of cloning... [Re: 36fuckin5]
    #23601218 - 09/01/16 01:40 PM (7 years, 4 months ago)

Quote:

36fuckin5 said:
No problem. Just trying to catch you before you get an infraction.



Thanks 36!

:asianofapproval:

-----------------------------

While you're here, what're your thoughts on my questions above?
Am I thinking a little bit too deeply or are these items that veteran growers think about when working with sectors and agar?


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Invisiblec10h12n2o
serial dilutor
 User Gallery


Registered: 01/21/15
Posts: 3,200
Loc: the abyss
Re: On the subject of cloning... [Re: morty422]
    #23601792 - 09/01/16 04:43 PM (7 years, 4 months ago)

Hell yes, GREAT questions mort my man!! I have been wondering about several of those things myself lately

i would love to hear some feedback on how one could preserve the whole spectrum of strains present in a clone without limiting them by transferring or using the initial plate

Wish I could help answer, but I'm eagerly monitoring the thread now :laugh:


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OfflineHomesteader
Student of shroomery
Male User Gallery


Registered: 01/17/16
Posts: 171
Last seen: 5 years, 3 months
Re: On the subject of cloning... [Re: morty422] * 3
    #23658087 - 09/19/16 08:16 AM (7 years, 4 months ago)

Hey morty great post and you are definitely on the right track brother! This topic is one I struggled with most when starting on Agar. What I've noticed is that a clone is usually comprised of multiple isolates fused together through anastamosis. They will hold together as one uniform 'type' of mycelium for several transfers. However, 3-5 transfers (Increase in P value) later I notice sectoring of once stable strains. Transferring these sectors will leave you with an isolate that no longer contains all the genetic material that once made up your cloned fruit and may no longer even have the capacity to fruit. To combat this I keep as low P value as possible and avoid unnecessary transfers. I also change up the nutrient concentrations of my Agar blend and sometimes alternate the actual source of carbohydrates (one time PDA, next time MEA).

The advantage to mycelium of a cloned fruit is the fact that it is an exact replica of the parent. The disadvantage here (so I'm told) is that some or many of the isolates that make up the cloned fruit may in fact only have the ability to grow vegetatively and have no ability to fruit. Thus, robbing nutrients from the isolates that do have the ability to fruit. This should make no difference whatsoever as the strains that make up a clone are anastomosed and can share their nutrients across the entire network. In theory they work together and not against one another.

To answer your question though: yes you are isolating by trying to clean up your plates and yes you could most certainly end up with an isolate that has no ability to fruit. Is this likely to happen? Not really and it depends on how many times you are transferring. Keep in mind, if you are grabbing rhizomorphic sectors of fast and healthy growing mycelium than you will almost always have a fruiting strain.

Sorry if some of this info is redundant but I felt this is an important topic to cover for people getting started on Agar.


--------------------


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Invisiblemorty422
Wuba-luba-dub-dub!
I'm a teapot


Registered: 07/06/16
Posts: 988
Re: On the subject of cloning... [Re: Homesteader]
    #23658591 - 09/19/16 12:09 PM (7 years, 4 months ago)

Quote:

Homesteader said:
Hey morty great post and you are definitely on the right track brother! This topic is one I struggled with most when starting on Agar. What I've noticed is that a clone is usually comprised of multiple isolates fused together through anastamosis. They will hold together as one uniform 'type' of mycelium for several transfers. However, 3-5 transfers (Increase in P value) later I notice sectoring of once stable strains. Transferring these sectors will leave you with an isolate that no longer contains all the genetic material that once made up your cloned fruit and may no longer even have the capacity to fruit. To combat this I keep as low P value as possible and avoid unnecessary transfers. I also change up the nutrient concentrations of my Agar blend and sometimes alternate the actual source of carbohydrates (one time PDA, next time MEA).

The advantage to mycelium of a cloned fruit is the fact that it is an exact replica of the parent. The disadvantage here (so I'm told) is that some or many of the isolates that make up the cloned fruit may in fact only have the ability to grow vegetatively and have no ability to fruit. Thus, robbing nutrients from the isolates that do have the ability to fruit. This should make no difference whatsoever as the strains that make up a clone are anastomosed and can share their nutrients across the entire network. In theory they work together and not against one another.

To answer your question though: yes you are isolating by trying to clean up your plates and yes you could most certainly end up with an isolate that has no ability to fruit. Is this likely to happen? Not really and it depends on how many times you are transferring. Keep in mind, if you are grabbing rhizomorphic sectors of fast and healthy growing mycelium than you will almost always have a fruiting strain.

Sorry if some of this info is redundant but I felt this is an important topic to cover for people getting started on Agar.




Nice post. Thank you! This cleared up a lot of vague things I've seen around town here. :cheers:


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Invisiblec10h12n2o
serial dilutor
 User Gallery


Registered: 01/21/15
Posts: 3,200
Loc: the abyss
Re: On the subject of cloning... [Re: Homesteader]
    #23660093 - 09/19/16 09:24 PM (7 years, 4 months ago)

Quote:

Homesteader said:
Hey morty great post and you are definitely on the right track brother! This topic is one I struggled with most when starting on Agar. What I've noticed is that a clone is usually comprised of multiple isolates fused together through anastamosis. They will hold together as one uniform 'type' of mycelium for several transfers. However, 3-5 transfers (Increase in P value) later I notice sectoring of once stable strains. Transferring these sectors will leave you with an isolate that no longer contains all the genetic material that once made up your cloned fruit and may no longer even have the capacity to fruit. To combat this I keep as low P value as possible and avoid unnecessary transfers. I also change up the nutrient concentrations of my Agar blend and sometimes alternate the actual source of carbohydrates (one time PDA, next time MEA).

The advantage to mycelium of a cloned fruit is the fact that it is an exact replica of the parent. The disadvantage here (so I'm told) is that some or many of the isolates that make up the cloned fruit may in fact only have the ability to grow vegetatively and have no ability to fruit. Thus, robbing nutrients from the isolates that do have the ability to fruit. This should make no difference whatsoever as the strains that make up a clone are anastomosed and can share their nutrients across the entire network. In theory they work together and not against one another.

To answer your question though: yes you are isolating by trying to clean up your plates and yes you could most certainly end up with an isolate that has no ability to fruit. Is this likely to happen? Not really and it depends on how many times you are transferring. Keep in mind, if you are grabbing rhizomorphic sectors of fast and healthy growing mycelium than you will almost always have a fruiting strain.

Sorry if some of this info is redundant but I felt this is an important topic to cover for people getting started on Agar.




now this is exactly what i was looking for, killer post homesteader +5 :mushroom2: :highfive1:

info is absolutely not redundant, its clear and succinctly put, and i'm probably not alone in appreciating it :wink:

i have been ruminating on these questions for a while, and your post confirms a lot of what i had been wondering about.

In making master slants from clones, is there any reliable way to transfer a culture which represents the entire genetic makeup of the fruit? it seems like in cleaning the culture (away from the initial tissue transfer, which often contams eventually), i necessarily limit it by selecting one sector vs another

i have been trying to make slants as early in the clone process as possible, as few transfers in as i can, for this reason. afterward, i have been further isolating the individual sectors that show up in these clone cultures, to compare them against the full-spectrum clone (if i can figure out a way to get the full spectrum of strains present in the fruiting body)


--------------------

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"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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