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 CaptainAhab
Registered: 12/25/09
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 Got some rattlesnake venom? Trying to make a new strain. Project in the works 
#14372553 - 04/29/11 03:07 PM (2 years, 21 days ago) |
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I'm interested in experimenting and creating a new strain, like RR did with PE6. The problem is that what he used to degrade the spore cell wall and expose the DNA, rattlesnake venom, is tightly controlled by the powers that be. So, how can you create hybrid strains without it? I read that the creation of Falbino was due to mixing liquid cultures from PF Albino and F+, so it seems possible without it. That's leaving a lot to chance, though. Anyone have a tried and true method?
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 Fungal growth
Lootinint
 
Registered: 03/21/10
Posts: 3,536
Loc: under a rock in your yard
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14372560 - 04/29/11 03:08 PM (2 years, 21 days ago) |
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y not go catch a rattlesnake?
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CaptainAhab
Registered: 12/25/09
Posts: 1,815
Last seen: 15 hours, 22 minutes
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Fungal growth]
#14372574 - 04/29/11 03:11 PM (2 years, 21 days ago) |
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Quote:
Fungal growth said:
y not go catch a rattlesnake?
I saw a how to snake charm youtube video, but the guys turned out to be arabs just selling stuff. Think it's fake
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thenpc
Glass Artist
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Loc: NE USA
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14372581 - 04/29/11 03:13 PM (2 years, 21 days ago) |
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You don't charm rattle snakes, you grab them and milk them. Someone does it, why not you?
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Flowing
Monkey
Registered: 08/16/09
Posts: 319
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14372594 - 04/29/11 03:15 PM (2 years, 21 days ago) |
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You can cross cubensis pretty easily without snake venom.
the real reason to use venom is for cross-species couplings like his ethereal Pan/cubensis cross.
You can't use regular venom, you'd need to vacuum distill it.
Dikaryotic strains of cubensis will mate if they are compatible, use a petri dish and let them meet, either they'll grow over each other or a third strain will come out of the coupling.
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Sillicybin
Registered: 02/14/05
Posts: 2,134
Loc: 
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14372602 - 04/29/11 03:16 PM (2 years, 21 days ago) |
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It seems to me that there should be a wide variety of compounds that could be used instead. Chitinase isolated from Trichoderma might be a workable candidate.
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CaptainAhab
Registered: 12/25/09
Posts: 1,815
Last seen: 15 hours, 22 minutes
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Flowing]
#14372624 - 04/29/11 03:21 PM (2 years, 21 days ago) |
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Quote:
Flowing said:
You can cross cubensis pretty easily without snake venom.
the real reason to use venom is for cross-species couplings like his ethereal Pan/cubensis cross.
You can't use regular venom, you'd need to vacuum distill it.
Dikaryotic strains of cubensis will mate if they are compatible, use a petri dish and let them meet, either they'll grow over each other or a third strain will come out of the coupling.
Interesting. I've got the ebook of DMT the spirit molecule if you want it. I notice that you have a quote from it in your sig.
Anyhow, would it be possible to identify the new strain after the 2 strains meet macroscopically? I don't exactly have a lab with compound microscopes.
You mentioned a panaeolus/cubensis hybrid? Reminds me of the KKL01 strain that tangich is working on
http://www.shroomery.org/forums/showflat.php/Number/14317732
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CaptainAhab
Registered: 12/25/09
Posts: 1,815
Last seen: 15 hours, 22 minutes
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Sillicybin]
#14372640 - 04/29/11 03:26 PM (2 years, 21 days ago) |
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Quote:
Sillicybin said:
It seems to me that there should be a wide variety of compounds that could be used instead. Chitinase isolated from Trichoderma might be a workable candidate.
I'll look into that. I'm reading about basidiospores, now (the classification given to mushroom spores).
http://en.wikipedia.org/wiki/Basidiospore
Cell wall made of chitin, yes? Chitinase ought to work, then. Some interesting info for anyone else reading this: "Basidiospores typically each contain one haploid nucleus that is the product of meiosis, and they are produced by specialized fungal cells called basidia."
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Sillicybin
Registered: 02/14/05
Posts: 2,134
Loc:
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14372686 - 04/29/11 03:46 PM (2 years, 21 days ago) |
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Quote:
CaptainAhab said:
Cell wall made of chitin, yes? Chitinase ought to work, then. Some interesting info for anyone else reading this: "Basidiospores typically each contain one haploid nucleus that is the product of meiosis, and they are produced by specialized fungal cells called basidia."
http://en.wikipedia.org/wiki/Cell_wall#Fungal_cell_walls
I've always understood it to be chitin, but looking at this link on wiki, it appears that it is actually multiple layers, with chitin being the inner-most. I do not know if chitinase would work on the other layers but my guess is probably not. Someone with more experience might be able to chime in on what it takes to get through the other two.
EDIT: Unless I'm mistaken, the reason that the venom would work is because it acts as a hemolytic to break down the walls of blood cells in mammals. There should be more than a few other compounds that would perform the same function, hopefully without causing complete cell death.
Edited by Sillicybin (04/29/11 03:53 PM)
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CaptainAhab
Registered: 12/25/09
Posts: 1,815
Last seen: 15 hours, 22 minutes
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Sillicybin]
#14372714 - 04/29/11 03:54 PM (2 years, 21 days ago) |
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Something else about the cell wall thickness:
"A mushroom spore cell consists of a cell wall that encloses a mass of cytoplasm and a nucleus. Some spores germinate quickly; others remain dormant for long periods. Active spores are produced in large numbers and may be dispersed over great distances by wind, water, or animals. They have thin cell walls that allow them to germinate quickly. Dormant spores have strong cell walls that protect them from environmental extremes and prevent them from germinating immediately."
http://www.*******.com/articles/mushroom-spores non sponsor, oops
Paging RR to line 1, Paging RR to line 1
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Edited by CaptainAhab (04/29/11 03:55 PM)
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CaptainAhab
Registered: 12/25/09
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Last seen: 15 hours, 22 minutes
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14375749 - 04/30/11 03:25 AM (2 years, 20 days ago) |
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Strain Hybridization
Great Thread on Strain Hybridization
http://www.shroomery.org/forums/showflat.php/Number/650041#650041
"In essence hybrids can be formed between substrains of a single strain or between different strains of the same species...
Anastomosis has been studied extensively in the edible mushroom world. Agaricus bisporus is homothallic and two spored, not four. Each and every spore it produces already contains both haploid nuclei to make a dikaryon. But fusion(Anastamosis) between these dikaryons produces more productive Dikaryons!!! The majority of High yielding bisporous are a result of HYBRIDIZATION within a STRAIN or between Strains of this species. So if it occurs between DIKARYONS of this species,it has been viewed occuring between monokaryons of other species, WHY WOULD anyone Doubt that it can occur within the Species Psilocybe cubensis.
The major factor to overcome with mating monokaryons is the proximity with which they germinate. Spores tend to clump. So simply placing spores of two different strains in a single syringe, will not overcome the clumping of spores of like strains, and hence their close proximity to each other upon germination.
Probably still occurs, when injected into a substrate, and some of the resulting fruitbodies might actually be hybrids, between Strains, and the resulting offspring (spores) from that mushroom will be different looking then both the mushroom it came from, and all of the mushrooms from both the Donor strains.
Much easier to DILUTE spores from each strain seperately, plate them, isolate slower growing monokaryons, and try mating as many of these from each strain as possible, with as many as possible from the other strain. All matings that fruit, are hybrids!!! If they produce spores, you now have a new STRAIN. Simply cloning the original matings that fruited, will be a Hybrid as well, but not a true hybrid, becasue their has been NO Recombination between the two strains, NO MIXING OF GENES. There has simply been a successful coexistence of two haploid nuclei, one from each Strain, acting independently, but together to create fruits!!! The real genetic swap occurs during Karyogomay and the subsequent meiosis."
... from the same guy:
"Monokaryon mating is the way to go. Not random spore mixtures of two different strains.
The true hybrid strain is going to come from the SPORES of the original Dikaryon formed via anastamosis or monokaryon mating. Until spores occur, the life cycle of the fungus is one of cooperation between two SEPERATE nuclei housed under a single roof(hyphae) acting in a coordinated effort. The spores produced from this coordinated effort, will be the hybrid SPORES.
Hybridization frequency is greatest between monokaryons. Succesful hybridization is more then likely going to occur in isolation, as opposed to concentration. A single germinated spore from one strain placed next to a single germinated spore from the other strain. Doing this with as many monokaryons as you can isolate from each strain.
Hybridization occuring between different species of fungi is highly unlikely to occur this way."
How To Create a Hybrid
http://www.shroomery.org/forums/showflat.php/Number/561706#561706
"There are two ways to make a hybrid without the addition of BIOTECHNOLOGY.
1. Make a very dilute spore solution. Drop onto agar plates and isolate monokaryons. Do this for the two strains you want to hybridize. Place individual monokaryons onto plate with individual monokaryons. One from each strain, and see if they are compatible. Grow out all suspected crosses, and test for fruiting.
2. Take dikaryons that are pure, and of know fruitability. One from each strain you want to hybridize, and place them on a single plate. The two will grow towards eachother, and if you are VERY LUCKY, a third strain might appear sectoring out of the area of interaction between the two known strains. This is called anastomosis.
When viewing a single Spore race, from a multispore germination, on a single petri plate. You will actually see incompatibility between the Dikaryons originating from this single print. Anastomosis will occur at a higher rate between Dikaryons from the single strain, then between dikaryons from different strains.
The second one is easier to try, but will have less chance to succeed. The first type involving monokaryon breeding, is far more tedious, and time consuming, but will result in Higher frequency of hybridization. You have to isolate individual monokaryons!!! This means you have to really dilute the spores, if two compatible monokaryons are near eachother on a plate, they will mate. You have to transfer quickly, and label your plates VERY WELL. LOTS OF PLATES.
According to Stametes, most hybrids are less vigorous then their donor strains."
Species Hybridization
RR's Hybrid Cubensis/Panaeolus That Actually Fruited
RR's Opinion on Hybrid Techniques w/ and w/out Rattlesnake Venom
http://www.shroomery.org/forums/showflat.php/Number/8967425#8967425
Basically what RR says is that it takes hundreds of plates and a lot of venom to get a successful hybrid between two different species. According to him, other methods don't work or aren't published, yet. He, himself, was (or is?) working on a method that he wanted to publish. I haven't seen it posted, so I'm assuming that it's old news, unuseful, or never happened.
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Well kids, that's all I got for now. Looks like I won't be trying to catch any snakes. As far as making a dilute spore solution and isolating monokaryons, then getting them to hybridize on agar, well, let's just say that looks like fun  Now, who wants to donate an albino penis envy to the cause?
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Luger0815
noob
Registered: 12/22/10
Posts: 1,677
Loc: @ Home
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14375895 - 04/30/11 04:30 AM (2 years, 20 days ago) |
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RoyalPalm
a passerby
Registered: 03/02/11
Posts: 157
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Luger0815]
#14377015 - 04/30/11 01:02 PM (2 years, 20 days ago) |
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dosent the venom have to be sterile ?
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RogerRabbit
Bans for Pleasure
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: RoyalPalm]
#14377087 - 04/30/11 01:18 PM (2 years, 20 days ago) |
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The quote above is horribly inaccurate. It presumes that the name some fool wrote on a print constitutes a strain, which it doesn't. A strain is a pairing of compatible hyphae. One can easily cross spores from prints labeled ecuador and cambodian for example by simply placing a cap to print on top of the print made by the first, and then using agar or a spore syringe. This would not be a hybrid any more than someone named Smith would have a hybrid baby from somebody named Jones.
RR
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2jew4u
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: RogerRabbit]
#14377145 - 04/30/11 01:34 PM (2 years, 20 days ago) |
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So RR how did the Hybrid work? Or did it, The two mycelium crossed, so was just curious- Redboy worked- so wonder what the new (2 years old experiment) strain is? Was it sterile or could it reproduce?
Edited by 2jew4u (04/30/11 01:35 PM)
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Ajaxx
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: 2jew4u]
#14377161 - 04/30/11 01:39 PM (2 years, 20 days ago) |
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wasnt PF albino created using black lights as lights over a certain number of generations?
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2jew4u
Stranger
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Ajaxx] 1
#14377170 - 04/30/11 01:41 PM (2 years, 20 days ago) |
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Quote:
Ajaxx said:
wasnt PF albino created using black lights as lights over a certain number of generations?
Thought that it was made by UVB- but maybe not
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flameclown
totality is amust
Registered: 04/04/04
Posts: 920
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: Flowing]
#14377234 - 04/30/11 01:54 PM (2 years, 20 days ago) |
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Quote:
Flowing said: vacuum distill it.
I assume you mean freeze dry it? If so you could just drop it in some LN2 and pull a vacuum..
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CaptainAhab
Registered: 12/25/09
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: RogerRabbit]
#14377611 - 04/30/11 03:08 PM (2 years, 20 days ago) |
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Quote:
RogerRabbit said:
The quote above is horribly inaccurate. It presumes that the name some fool wrote on a print constitutes a strain, which it doesn't. A strain is a pairing of compatible hyphae. One can easily cross spores from prints labeled ecuador and cambodian for example by simply placing a cap to print on top of the print made by the first, and then using agar or a spore syringe. This would not be a hybrid any more than someone named Smith would have a hybrid baby from somebody named Jones.
RR
That's funny, Roadkill, your buddy, called the quote on strain hybridization " [a] Very good post Teonan!!
one of the better posts that I have read lately."
http://www.shroomery.org/forums/showflat.php/Number/650288#650288
In your "strain" argument, what is "horribly inaccurate" about stating that "2. Take dikaryons that are pure, and of know fruitability. One from each strain you want to hybridize, and place them on a single plate." ??
Didn't you, yourself, create the PE6 hybrid?
Why discourage people from trying to do the same thing that you did?
I would think that peer review is important to making progress in mycology, not guarding secrets or discouraging discussion. That's the scientific equivalent of eating a pie and not sharing it. And then throwing the crusts at people.
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Edited by CaptainAhab (04/30/11 03:15 PM)
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overstand
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Re: Got some rattlesnake venom? Trying to make a new strain. Project in the works [Re: CaptainAhab]
#14377725 - 04/30/11 03:30 PM (2 years, 20 days ago) |
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Awesome discussion! I would love to see more research on the subject.
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