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Cubensis Breeding: A Group Effort


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#21 CatsAndBats

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Posted 20 November 2016 - 03:27 PM

 post-147940-0-72169500-1479673647.jpg

 

Yes, inner stipe is where best to clone from in my opinion. Some teks call for a blunt needle to push through the stem, but that drags any unwanted microorganisms into the sample from the surface. I'm going to soak this guy in ethanol and tear into the stem from the bottom. I'll then use my pointy tweezers (pictured here: https://mycotopia.ne...ain/?p=1294432) to grab a piece from the middle. One can also do this with a scalpel, but I'm too spazzy.

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Edited by catattack, 20 November 2016 - 03:28 PM.


#22 HrVanker

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Posted 20 November 2016 - 03:47 PM



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Yes, inner stipe is where best to clone from in my opinion. Some teks call for a blunt needle to push through the stem, but that drags any unwanted microorganisms into the sample from the surface. I'm going to soak this guy in ethanol and tear into the stem from the bottom. I'll then use my pointy tweezers (pictured here: https://mycotopia.ne...ain/?p=1294432) to grab a piece from the middle. One can also do this with a scalpel, but I'm too spazzy.


I remember when I first started reading about cultivating mushrooms, I found RR's videos and watched them all.

He said exactly what you did: tear from the end to expose the center, take a sample from the center. I've heard of people just soaking/rinsing in peroxide prior to tearing.

I may have to rewatch some of his advanced techniques videos... I imagine there are some useful nuggets of info that I don't remember.

#23 Microbe

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Posted 20 November 2016 - 05:27 PM

I have a question/s. Are the amount of genetics in say a cloned fruit body quantifiable? Is the smallest tissue sample one can grab going to show say 4 sets or am I way off on that number? Agar guys, I'm looking at you.

Haploid is a single set of unpaired chromosomes while diploid contains 2 sets of chromosomes, one from each parent. I think.

Look at my randoms thread, the 3 plates that you couldnt see any mycelium on, was enough to grab both sets lol.

A single cell is all thats needed to grab the gens

I don't think you understood my question. I'm about to grab clone material from a fruit, how many sets of genes will be on that "plate" I make? Is it at least quantifiable? 4? 20? Over 20? This would probably be better suited for my agar thread but it applies here as well.
I dont think you understood my answer......

#24 Needles

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Posted 21 November 2016 - 12:47 AM

This subject has been very interesting and one that I have been working on for a while. I liked the idea that colchicine could be a agent in genetic alterations in mushrooms. Back in the 70s I used my grandpas colchicine for his gout to mutate marijuana. I was to young and inexperienced and equipment for that matter for any positive results but the idea was still there. Where I live I have crazy friends that hunt rattle snakes. With the proper license you can harvest two snakes per year. I asked one person if I could get some venom. Apparently milking a rattlesnake is a crime, even though you are going to harvest the snak anyway. So I'm still working on that idea. I've also read about gamma rays to mutate spores, but really...
I'm following along and plan on doing other research as well. I hope I can contribute to this thread........
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#25 Needles

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Posted 24 November 2016 - 09:32 PM

Just added a new member to the family. Meet the Nikon alphaphot YS. image.jpeg
Hopefully it will help in isolation of single spores for this genetic mutation project.
I think I my have other valuable information and materials coming, will just have to wait and see.....
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#26 CatsAndBats

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Posted 29 November 2016 - 07:01 PM

More reading:

 

https://mycotopia.ne...ssibly-falbino/



#27 Needles

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Posted 07 December 2016 - 12:01 PM

I know the subject is cube breeding but I have fresh Hypsizygus marmoreus and H. tessulatus printed on microscope slides.
H. marmoreus print close upimage.jpeg
This will be a total long shot getting aseptic results but I plan on trying to isolate one single spore from each species and put them on a sterilized slide coated with malt agar and see if they will mate. First thing to do is mix up a small 100ml batch of agarimage.jpeg
I'm only trying five slides. In the past I have successfully germinated spores this way but never isolated just one spore. Tools for this experiment image.jpeg
Being sterilized.
Microscope slides
Cover slips
Pipettes
2cc syringe
Small amount of water.
Agar
Like I said it's a long shot but still worth a try.......
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#28 Needles

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Posted 07 December 2016 - 11:05 PM

Well wrangling a spore off a glass slide wasn't as easy as I thought. I was able to grab a spore but who knows what happened in between the microscope and my safety cabinet. This could be a ongoing process. Even if there is growth it would be hard to tell if I didn't just get two spores of one species until it's lifecycle was completed.

I was also wondering if mycelium from two strains were blended and chopped into pieces then placed into sterilized water would they just continue to grow separately or would they join together and continue to grow. Kinda like grafting fruit trees. Guess that will be the next experiment......
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#29 Microbe

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Posted 10 January 2017 - 08:03 PM

Where you at @HrVanker?

#30 HrVanker

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Posted 10 January 2017 - 10:36 PM

Where you at @HrVanker?

Hey guys!

Sorry for dropping off there... I've been finishing up my first grow. Got infected with vert again, but I wanted some fruits, so I let it go. Not the best decision, but it is what it is. I stopped misting to help prevent spores from flying (just did mini dunks on pinning cakes), and will have to do a good disinfection of the area. I know where the contam came from this round (the air tubes never got disinfected), so that should help on the next round.

Speaking of which, my Lipa Yai WBS jars are chugging along.

Right now, I need to clean, organize and build a better glovebox. Then I should he back. I've just been busy and organization has never been a strong suit of mine. But we all have to grow up sometime! Lol

I was also wondering if mycelium from two strains were blended and chopped into pieces then placed into sterilized water would they just continue to grow separately or would they join together and continue to grow. Kinda like grafting fruit trees. Guess that will be the next experiment......


This has been an ongoing debate between myself and @CatAttack... I believe that two genetically different samples (whether both Maz, Thai, etc.) will fight for dominance. Two different species will definitely compete. I don't know about identical cultures... how can they recognize each other? Or is it more like a compatability thing? E.g. Do hyphae from different genes have different diameters and can only connect with identical hyphae?

I once did some research on dying mycelium, but it doesn't seem too effective. If we could do that, you could potentially see red myc fight with blue myc. However, I believe that agar could help us. One could pour a nute rich agar and a low nute agar on the same plate (would involve some jerry rigging). Transfer one sample to each side and see what happens when they meet. Will the nute rich culture feed the nute poor culture? To fully test, we would need a several itterations with varying parameters, and I'm nowhere near ready to get that deep yet.
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#31 Needles

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Posted 27 January 2017 - 05:15 PM

Another learning curve for me. The glass slides that I used dried out very quickly. My new approach to this will be done in a 150mm glass petri plate. I'm working on getting the required chemicals and venoms to try to break down hyphae and merge two strains for a medicinal "super strain" so to speak. But for now I'm just messing with agar on microscope slides. The same results could be achieved by using plates but I am hoping to be able to use my bright field microscope to get a closer look at mycelium as it encounters one another. A inverted microscope would be the ticket for this analysis. Hopefully by summer I can ad one to the family. For now this is the plan,
Sterilize slides and cover slips then cut a piece of agar from a dish. After placing wedge on slide introduce mycelium from two different strains one on each side. Here's my plates
image.jpeg image.jpeg
I added 15ml of water to a cardboard disk that was placed on the bottom of the plate. That should be enough to keep the humidity up. I found a stainless steel container that works well to hold the plates while sterilization.image.jpeg image.jpeg
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#32 Needles

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Posted 29 January 2017 - 01:44 PM

A very interesting result to this experiment. I used mycelium from Hypsyzigus maramoures on the left and Hypsizygus tessulatus on the right. They are not rejecting each other but intertwining. Very cool to look at under the microscope.image.jpeg
This is a close up of my wedge transfer
image.jpeg

This micrograph was taken through the glass dish and cover slip so not as sharp as it could be.
image.jpeg
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#33 Needles

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Posted 29 January 2017 - 02:04 PM

I was getting into looking at this culture and took another micrograph. I found it interesting how the top right culture has built a sector (9 o'clock position) , aggressive to the opposing strain maybe?
image.jpeg
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#34 CatsAndBats

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Posted 29 January 2017 - 02:11 PM

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666th post!

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Edited by catattack, 29 January 2017 - 02:12 PM.

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#35 Needles

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Posted 29 January 2017 - 02:20 PM

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666th post!

It's the devil post and on a Sunday of all days lol...
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#36 Alder Logs

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Posted 29 January 2017 - 02:25 PM

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666th post!

Posted at 11:11 AM!


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#37 Microbe

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Posted 30 January 2017 - 01:21 AM

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.

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Edited by Microbe, 30 January 2017 - 01:22 AM.


#38 Microbe

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Posted 30 January 2017 - 02:02 AM

https://www.ncbi.nlm...les/PMC3385130/

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#39 Needles

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Posted 30 January 2017 - 02:06 AM

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
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No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....

#40 Microbe

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Posted 30 January 2017 - 02:06 AM

In your pictures they do not look to be evading each other or even retreating. The hyphae have certainly passed the natural zone and i speculate that each will halt or overrun the other. Break out one of your other bad ass scopes and capture the clamp connection.

Yeah you will.....

Edited by Microbe, 30 January 2017 - 02:07 AM.

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