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Cloning/Isolation/Testing

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#1 peacefrog

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Posted 15 April 2017 - 09:29 PM

I have put up a thread on isolation from spores but have not done one about cloning, isolating and testing, so I decided to write one up.

 

There are 2 ways one can clone from a fruit body; clone a whole pin or a section of the inside tissue of a more mature fruit. I personally prefer taking a clone from a mature fruit body, but pins do recover and revert back to mycelial growth very quickly. I just like seeing the fruit matured before I decide to clone it, but it really is personal preference. In this post, I have shown both ways and will leave it up to the reader to decide which one he or she prefers.

 

Also, there seems to be 2 schools of thought on cloning. The first is to leave a clone as a clone, which means no further isolation past clean growth. The other is isolating the sectors down to a monoculture.

 

A multi-spore cloned mushroom usually is made up of several strains. This is why most MS cloned tissue sectors. Sometimes if you isolate those sectors away from the complete set of the multiple strains, you can lose the ability for great fruiting potential or different phenotypes show up in the subsequent fruit. Some strains may not be able to produce a fruit body at all. But that isn’t always the case, sometimes you find a gem with the characteristics you are seeking. I personally like to isolate to a monoculture after cloning because I like having true monocultures. But it’s imperative that one tests each isolate independently. Sometimes the best results come from the original cloned tissue, one or more transfers in between, a monoculture, OR none of the isolates produces as great as one would want and it’s a bust. However, there will be a much higher chance of obtaining one or multiple fruiting strains since the culture is coming from a set of genetics that has been proven to fruit. This is one of the reasons a lot of growers, including myself, prefer cloning to spore isolation. Plus it is MUCH faster to isolate down to a monoculture verses MS plates. Do this with multiple fruit bodies and test each, and eventually you will find a great culture to store and grow for many years to come.

 

Cloning from a pin:

1.JPG 2.JPG Pins (3).jpg Pins (4).jpg Pins (1).JPG 3.JPG 4.JPG 5.jpg

 

I use tweezers for this that have been flame sterilized to gently pluck a pin from the substrate. One could also use the tweezers to gently hold the pin while a flame sterilized scalpel cuts away from the substrate (recommended when transferring from bulk or cased material). I used pinning agar jars in this instance since I was cleaning up old spores and had several agar jars to let pin. But one could take a pin from any type of grow really. I personally do not sanitize my pins before transferring. They are removed and go straight onto agar. However, one could dip them in a bleach water solution before transferring. I have done it in the past with good results. I just don’t like the extra step with pins, as they aggressively revert back to the vegetative state. And I find the benefit is in the agar’s ability to only colonize in a 2 dimensional plane, which makes it easy to spot clean verses dirty growth.

 

Cloning from a mature fruit body:

Tissue (2).JPG Tissue (6).JPG Tissue (3).JPG Tissue (7).JPG Tissue (4).JPG Tissue (8).JPG Tissue (5).JPG Tissue (1).JPG Tissue (9).JPG

 

After the fruit is harvested, I always decapitate, clean, remove the casing/substrate material and sanitize it with a bleach water sprayed paper towel before placing into the SAB. I also place a separate bleach water sprayed paper towel to set the fruit on inside the SAB. I then gently split the fruit in half with my hands and scrape some tissue from the interior section of the stipe to transfer to awaiting agar jars. 

 

Growth from cloned pins:

6.jpg 8.JPG

 

Growth from inner tissue of a mature fruit:

Cloned Tissue (2).JPG Cloned Tissue (1).JPG

 

Hopefully you can see the sectoring going on in these plates as well as different types of mycelial growth. And although they look pretty clean, it is advisable to make at least one transfer after the tissue grows out, just to make sure there is no hidden contamination tagging along before taking to grain, LC or LI. A couple of transfers will not isolate the strains down that much to notice really IME and I always start “the original cloned genetics” from the clean plate, not ones from the tissue. And if you are lucky or you used a very minute amount of spores to grow this fruit from, you may already have yourself a monoculture, thus expansion on agar will not alter the results what so ever. I have never been so lucky personally, but I streak from prints and most likely have hundreds or thousands of strains growing after germination. Either way, it’s good practice to transfer again just to make sure. Nothing will ruin your day more in this hobby as making a lot of spawn, spawning into a lot of bulk material just to have it all contaminate due to some latent contaminated agar!

 

Cloned tissue after transferring and is now clean and ready to use as inoculant or one could continue isolating down:

9.JPG 10.JPG 11.JPG 12.JPG

 

This is a far as I have taken this grow, as I am unable to spend as much time on this post as I did for “Isolation/Testing/Storing Cultures”, found here: https://mycotopia.ne...oring-cultures/. I just don’t have the time right now for weeks/months of isolation and testing with these guys and only needed to replenish my spore collection. So I have not gone through the entire procedure of isolation to a monoculture and testing all, but at this stage the concept is exactly the same as the above mentioned post. But I have included an example of the end product of utilizing this procedure.

 

This is the only cube culture that I have kept and is Golden Teacher variety. It is a monoculture from an isolated clone that has sat in my refrigerator, badly neglected for 6 years. I thought it was dead, but thankfully after a couple of nail biting transfers, it still lives and produces as well as I remember:

22.JPG 23.jpg

 

Notice how matted and unhealthy the old slant appears compared to the new revived growth. This goes to show how resilient these mushrooms actually are. No wonder they have been on our planet for so long. 

 

And here is the finished product, a beautiful flush of Golden Teachers via a mini-grow:

Flush (1).jpg Flush (2).jpg Flush (3).jpg

 

This procedure normally takes a while, but one could get lucky and harness some great genetics very quickly. The chose is up to the grower if he or she wants to isolate a clone down, but ultimately the only way to get these and better results is to do many clones/isolates and test, test, test. But of course, the testing is the fun part lol.

 

And as always, good vibes and happy growing to all!   

 

 

 

 

 


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#2 tailsmcsnails

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Posted 16 April 2017 - 12:01 AM

Thank you so much for taking the time to do posts like this.

Sent from my A1601 using Tapatalk
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#3 wharfrat

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Posted 16 April 2017 - 12:03 AM

nice write up brother  :thumbs_up:  thanks for sharing 


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#4 Arathu

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Posted 16 April 2017 - 08:38 AM

Excellent instructions and presentation..................

 

Y'all pay attention to the frog...........this is the bomb right here!

 

My lab is coming together hopefully this year and completely dedicated space shall result..........

 

I've been wanting to put some pins on agar..........

 

Great stuff Frog!

 

p.s. Thanks Bat for pointing me here............

 

A


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#5 CatsAndBats

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Posted 16 April 2017 - 10:33 AM

Excellent instructions and presentation..................

 

Y'all pay attention to the frog...........this is the bomb right here!

 

My lab is coming together hopefully this year and completely dedicated space shall result..........

 

I've been wanting to put some pins on agar..........

 

Great stuff Frog!

 

p.s. Thanks Bat for pointing me here............

 

A

 

 

No problem, I make it a habit of "stealing" peacefrog's threads and putting them on my agar threads for reference, he is and was instrumental in my learning agar skills.

 

Thanks @peacefrog, for your continued support of this site through your majestic write ups. Your work has inspired me and countless others I'm sure. Thank you.


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#6 peacefrog

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Posted 16 April 2017 - 12:44 PM

Thank you all very much for the kind words!

And, Cat thank you for the props, your skills know no bounds, brother. You and others like microbe are doing some really great stuff to teach us all some great lessons! Keep up the good work.
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#7 CatsAndBats

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Posted 16 April 2017 - 01:04 PM

kitten_hugs_a_frog_as_dragonfly_buzzes.w


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#8 peacefrog

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Posted 16 April 2017 - 01:06 PM

Lol. Who's the dragon fly?

#9 CatsAndBats

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Posted 16 April 2017 - 01:09 PM

Lol. Who's the dragon fly?

 

Microbe's conscious?  ¯\_(ツ)_/¯


Edited by CatsAndBats, 16 April 2017 - 01:09 PM.

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#10 peacefrog

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Posted 16 April 2017 - 01:22 PM

Haha. I like it, but that cat kind of looks like it might want to eat that pour little helpless frog.

#11 CatsAndBats

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Posted 16 April 2017 - 01:25 PM

Haha. I like it, but that cat kind of looks like it might want to eat that pour little helpless frog.

 

You're biased. :tongue: Some poor moderator is going to have to clean this up (and our other threads) before they archive it. Hahaha.


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#12 peacefrog

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Posted 19 April 2017 - 05:33 PM

Haha, yes there are plenty of threads out there that need cleaning up after we are done with it lol.

Edited by peacefrog, 19 April 2017 - 05:33 PM.

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#13 CatsAndBats

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Posted 19 April 2017 - 06:07 PM

Haha, yes there are plenty of threads out there that need cleaning up after we are done with it lol.

 

For real, between you me and jammer perilously derailing threads. :rolleyes:


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#14 peacefrog

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Posted 19 April 2017 - 06:11 PM

Yea, Jammer has been known to do that pretty well too lol.
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#15 MLBjammer

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Posted Today, 04:04 AM

That is some nice work, PF.  Thanks for sharing.







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