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Mycostotle
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Snake venom agar tek
#26939480 - 09/16/20 05:19 PM (3 years, 6 months ago) |
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I have lately found a website selling rattlesnake venom for 110$ per gram and Im considering a few hybridization attempts.
How much rattlesnake venom should be put to a petri dish?
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mycorry
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Re: Snake venom agar tek [Re: Mycostotle]
#26940506 - 09/17/20 10:52 AM (3 years, 6 months ago) |
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I cant quite remember the exact amount but I remember finding the answer to that question somewhere in the search function. I think maybe it was one drop per plate but that is hardly a reliable metric. I believe RogerRabbit is the one who used to use that tek a lot.
I also vaguely remember finding out that it is possible for someone to milk rattlesnakes themselves then filter sterilize the venom for use in agar. It would then be mixed into the warm agar right before pouring into plates. My memory is shakey on this one.
Hope that helps!
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Edited by mycorry (09/17/20 10:53 AM)
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Jakeoncid419
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Re: Snake venom agar tek [Re: mycorry] 1
#26941358 - 09/17/20 09:02 PM (3 years, 6 months ago) |
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10mg:300ml -30mg:300ml (Venom to sterile water) made a few sterile syringes up. I cut in .01mg of venom into 3 ml of sterile water. I then added to the culture where the growths intersected, I also added about 50 ml of reagent to the agar as I poured my dishes
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MycoTricho
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That's interesting. What would be the benefit of this? Is it nutritious for fungus?
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Jakeoncid419
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Re: Snake venom agar tek [Re: MycoTricho]
#26941525 - 09/17/20 11:29 PM (3 years, 6 months ago) |
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No the Hemotoxic venom Weakens the cell walls allowing genetic material to pass through them without clamp connections needing to be made.
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MycoTricho
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Wow, that's fascinating. I have so much to learn.
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bodhisatta
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Re: Snake venom agar tek [Re: MycoTricho]
#26943999 - 09/19/20 12:00 PM (3 years, 6 months ago) |
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RR used venom, supposedly, on old redboy spores that wouldn't germinate. Not for any hybridization attempts. John Holiday has some story about using it, but he's about as trustworthy as Stamets which isn't saying a lot. They both made a career out of scamming investors.
https://www.shroomery.org/forums/showflat.php/Number/25973517#25973517 And the replies
https://www.shroomery.org/forums/showflat.php/Number/25613736#25613736 And the replies
There are chemicals mycologists actually use for protoplast fusion though. I would start there if you want to have any success.
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Solipsis
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Re: Snake venom agar tek [Re: bodhisatta]
#26944206 - 09/19/20 01:39 PM (3 years, 6 months ago) |
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Trichoderma species actually make enzymes that do this, i think those also break down cell membranes like cytolytic venom does. The difference is fungal cell walls may have bilipid layers but they also are composed of chitin, polysaccharides and mannoproteins, an actual cell wall animals don't have. So there are definitely differences and i have never seen a snake bite a mushroom...
Am not an expert on this at all tho, am curious what it takes for protoplast fusion.
There's also spheroplasts apparently which don't have the entire cell wall removed.
Seems tho that the cell membrane must NOT be ruptured otherwise there is nothing containing the organelles of the cell and just spills its guts then nobody wins, strange if this is what snake venom breaks down...
IIRC Trichoderma enzymes are or include chitinases, not sure if this forms a matrix with the polysaccharides or if they have enzymes breaking that down to but makes a lot more sense all of that strips fungal cells down to their membrane.
IF snake venom can work i suppose it achieves somatic fusion not through protoplasts perhaps? The enzymes modulating cell membranes first leading to other components that form cell walls to reorder and if you're lucky it could happen with different homokaryons, fusing no more and no less than two of them.
just a thought
snake venom is interesting but besides the cost i think you also need to have it shipped cooled which brings additional cost.
Trichoderma now wouldn't be cool if we could exploit the bastards? something i am all for.
But probably the easiest is getting synthetic enzymes used for protoplast induction - at least if you take into consideration that there may be differences between different kingdoms of organisms and their cell anatomy (unless i am a noob moron - but seems like bodh's links answers all that). I've come across these products before that seem regularly used in research but forgot the/a name. Sorry also dont know if the stuff still requires cooling during shipping.
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Jakeoncid419
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Re: Snake venom agar tek [Re: Solipsis]
#26944849 - 09/19/20 08:09 PM (3 years, 6 months ago) |
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Yeah I mean while my reagent did work and I was able to force hybridization between two Dikaryotic cultures of different pan species (cyan and trop) I was also able to breed them together traditionally (although I had to use monokaryotic cultures of course) it did take a few attempts to get the reagent right but I will eventually do another forced hybridization project, It just kind of lost some momentum when I discovered I was able to easily mate different pan sub species traditionally. I would look to take advice from people who have bread more than just cubes if that is what you are going for as there are many nuances in practice and little tricks that can help make it more likely to succeed however from what I have experienced so far the process in theory is very similar.
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Edited by Jakeoncid419 (09/22/20 09:29 PM)
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Jakeoncid419
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Keeping it cold is important that’s another reason why I haven’t ran another project I ran out of liquid nitrogen and lost the last of my supply I can get more anytime I just don’t want to order it until I’m ready to run the project and my mycology work always slows down in the summer I am replacing all my filters and preparing for mushroom season now, I should have my greenhouse is winterized in the next two weeks and I’ll be back in the swing of things
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san1
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If you can't test what you're buying don't do it. Otherwise they'll send you snake oil water.
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Solipsis
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Re: Snake venom agar tek [Re: san1]
#26948012 - 09/21/20 03:33 PM (3 years, 6 months ago) |
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very interesting JOC
can i ask how did you confirm the forced di-di hybridization? (was the trop RDU by any chance?)
and would you shine a light on what happened with 'getting the reagent right'?
in the OP was asked how to dose the venom, but i figure you guys would first need to confirm you even got it from the same species of snake cause there may be difference in effectiveness so that data might not be so useful, however it could be a reference point to start.
vetting the source is ofc not a bad point, there must be ways tho.
there is still the question of whether there aren't better options for this.
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Jakeoncid419
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Re: Snake venom agar tek [Re: Solipsis]
#26948518 - 09/21/20 09:09 PM (3 years, 6 months ago) |
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Yes so that cross was actually pretty easy to confirm since I did use the pan Trop red spore X with pan cyan hausteca (black spore) The resulting purple spore was a pretty easy identifier however Alan and I Ran it thru microscopic key and I’ve ran PCR on it, He still has not sent off his large collection of pcr’d samples for Sequencing yet (he’s waiting till he gets a certain amount to get a discount) I also know he’s had a bunch of other stuff going on I should ask him what’s up with all that but microscopically it is unique.. I’ve never had either hausteca or pan Australia to drop purple spores only the cross does that, The fruit bodies also look very unique from each other to the naked eye
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Solipsis
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Fantastic
We so rarely hear of actual hybridization.
And yes it is confusing that mating compatibility is not linked to species definition. Apparently you found compatible different species, i hope we find a bunch more. Maybe it makes most sense for those with reproductive isolation, found in parts of the world separate from each other but more when the isolation has not happened that long ago? idk with your pans if that checks out
I tried to mate different varieties of caerulescens (even that is disputed, the vars), however all attempts have been unsuccessful it seems. It's generally possible different varieties are not compatible as I believe the different tampanensis varieties aren't. But in my case i might have been a little out of my league and will step by step verify my methods.
Anyway on-topic: also, this suggests or claims various storage conditions are actually not necessarily so detrimental to the stability of rattlesnake venom, largely unaffected actually:
https://www.unco.edu/nhs/biology/about-us/mackessy-stephen/documents/1998-Venom-stability-Munekiyo-and-Mackessy.pdf
lyophilization may actually be harmful.
i personally find the Trichoderma option to be the most fascinating.
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jason9086
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Re: Snake venom agar tek [Re: bodhisatta] 1
#26949974 - 09/22/20 07:31 PM (3 years, 6 months ago) |
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yes, snake venom breeding seems like a load of bullshit to me. What kind of scientist literally does no genetic analysis of their supposed hybrid to confirm it? I think RR drank the kool aid on that one, and a large number of people on the shroomery followed suit.
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Jakeoncid419
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Re: Snake venom agar tek [Re: jason9086]
#26950025 - 09/22/20 08:08 PM (3 years, 6 months ago) |
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I mean it definitely worked, phv is microscopically Unique and it has been PCR’d Alan is Just waiting until he gets enough PCR samples to send them in for sequencing that he gets a bulk discount but we already know it’s unique since we microscopically analyzed it and both parents and it is unique. Also the purple spores are a pretty big giveaway as they are stable and are not found in either parent strain. I have grown multiple flushes of both and never have a gotten a random purple print the purple spores are only on the resulting hybrid
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Edited by Jakeoncid419 (09/22/20 08:12 PM)
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bodhisatta
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I wouldn't be the least bit surprised if the cross was varietal because the two parent species were actually the same just phenotypically different. But time will tell. In the meanwhile I wonder why the coolest experimentation has the absolute least documentation....
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Jakeoncid419
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Re: Snake venom agar tek [Re: bodhisatta]
#26950124 - 09/22/20 09:12 PM (3 years, 6 months ago) |
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What you mean pan Australia tropicalis and pan cyan hausteca? Alan has keyed the pan aus in as tropicalis and he collected the hausteca cyan....
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Jakeoncid419
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Even if they were both the same species (doubtful imo) both parents were dikaryotic Cultures so the venom reagent Would still have had to have done it’s job in order for breeding to take place PHV looks nothing like either parent culture and like I said I’ve ran both parent cultures at least 50 times each and have never gotten anything close to the PHV phenotype from them only the PHV hybrid produce is purple spores
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Edited by Jakeoncid419 (09/22/20 09:43 PM)
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Alan Rockefeller
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Re: Snake venom agar tek [Re: jason9086]
#26950148 - 09/22/20 09:24 PM (3 years, 6 months ago) |
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Quote:
jason9086 said: yes, snake venom breeding seems like a load of bullshit to me. What kind of scientist literally does no genetic analysis of their supposed hybrid to confirm it? I think RR drank the kool aid on that one, and a large number of people on the shroomery followed suit.
What sort of genetic analysis would be used to confirm it?
And what should be looked for in the sequence data to confirm that they have crossed?
The PHV cross has a spore size that is in between the size of the spores of the two parent species, spore color is in between as well. The fruits look macroscopically much different than either parent.
PHV spores measure (10.4) 11.5 – 12.9 (13.3) × (6.5) 9.2 – 11 (11.4) µm, which is midway in between the spore size of Panaeolus tropicalis and Panaeolus cyanescens.
PHV parent 1: https://images.mushroomobserver.org/1280/949765.jpg PHV parent 2: https://mushroomobserver.org/images/1280/1150852.jpg PHV fruits: https://mushroomobserver.org/images/1280/1188752.jpg
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bodhisatta
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Quote:
Jakeoncid419 said: Even if they were both the same species (doubtful imo) both parents were dikaryotic Cultures so the venom reagent Would still have had to have done it’s job in order for breeding to take place PHV looks nothing like either parent culture and like I said I’ve ran both parent cultures at least 50 times each and have never gotten anything close to the PHV phenotype from them only the PHV hybrid produce is purple spores
People have been making crosses by putting two dikayotic cultures on the same plate with no venom. You don't need Venom to make varietal hybrids or even monokaryotic mycelium.
There's cubes with purple spores, brown spores, albino spores, and even cubes that look like dicks. All same species but big phenotype difference.
Alan knows more about speciation than I do. what I do know is venom and monokaryotic cultures are absolutely not necessary to cross varieties of the same species
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Jakeoncid419
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Re: Snake venom agar tek [Re: bodhisatta]
#26950965 - 09/23/20 12:07 PM (3 years, 6 months ago) |
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Well first of all they are not the same species, Second of all I have never had success trying to mate cultures by just throwing two Dikaryotic cultures on a dish next to each other, I did that along side my venom dishes as a control and you could clearly see the division between the growth of the two cultures I cut it out on the divide anyway transferred and grew out and the result was one of either parent strain, no cross or alteration of traits happened. A Dikaryotic culture Has already made clamp connections so it would not be able to make these connections with a different culture. I can’t say it’s 100% impossible because as of a year ago breeding different pan species was “impossible” But I have never seen it done and I don’t understand how it could happen either. However Most of my hybridization work is done with pans. cubes I’ve always just made monocultures and they have bred super easily for me so I haven’t really tried mating Dikaryotic cubes. However I have accidentally mixed two different cube strains of spawn in a tub together and I did not get hybrid fruits I got a mix of both strains, I’ve also seen other people do the same thing and get the same results I don’t see why it would be any different from trying to mate them on a dish. Again not saying it’s impossible however if clamp connections have already been made I don’t understand how the genetic material would exchange
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Mycostotle
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1 gram of western diamond back rattlesnake venom has been ordered now
Im doing this out of curiosity with very little expectations, the venom will used to attempt hybridize cubensis with semilanceata and the results will be posted here
I think I have a source for semilanceata spores
I have these cubensis trains: ape, pe6, red boy, mckennai, b+, GT, melmac and mexican
Choose the one I should use in my attempts 😆
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bodhisatta
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Quote:
Jakeoncid419 said: Well first of all they are not the same species, Second of all I have never had success trying to mate cultures by just throwing two Dikaryotic cultures on a dish next to each other, I did that along side my venom dishes as a control and you could clearly see the division between the growth of the two cultures I cut it out on the divide anyway transferred and grew out and the result was one of either parent strain, no cross or alteration of traits happened. A Dikaryotic culture Has already made clamp connections so it would not be able to make these connections with a different culture. I can’t say it’s 100% impossible because as of a year ago breeding different pan species was “impossible” But I have never seen it done and I don’t understand how it could happen either. However Most of my hybridization work is done with pans. cubes I’ve always just made monocultures and they have bred super easily for me so I haven’t really tried mating Dikaryotic cubes. However I have accidentally mixed two different cube strains of spawn in a tub together and I did not get hybrid fruits I got a mix of both strains, I’ve also seen other people do the same thing and get the same results I don’t see why it would be any different from trying to mate them on a dish. Again not saying it’s impossible however if clamp connections have already been made I don’t understand how the genetic material would exchange
You do know there's more than a couple shroomery made crosses that are circulating and some are even sold by vendors now that were made by people putting spores of two varieties on a petri dish than carefully breeding the resultant cross back with itself over several generations to stabilize.
Ask pasty where rustywhyte came from. Look up mudafuka's ESS. Etc..
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Jakeoncid419
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Re: Snake venom agar tek [Re: bodhisatta]
#26951448 - 09/23/20 04:47 PM (3 years, 6 months ago) |
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Well sure when you put spores on to a petri dish there is a chance that you will get two different spores to land side-by-side thus the monocultures will clamp connect with each other. it’s success rate is low because most of the time the spores cluster and they breed with themselves before they are able to breed with the other. But that’s not putting too Dikaryotic cultures That have already clamped side by side on a dish and breeding them. . you can ensure this will happen by making serial dilutions spreading the spores out then transfer the mono growth before it clamps to anything, The only difference here is you are lucking out and landing two different spores close enough that they clamp with the other strain before anything else
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bodhisatta
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people put spores on opposite sides of dishes. There's nothing monokaryotic left after a milliliter of growth with a drop of spore solution let alone a few centimeters
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Alan Rockefeller
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Re: Snake venom agar tek [Re: Mycostotle]
#26951492 - 09/23/20 05:19 PM (3 years, 6 months ago) |
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Quote:
Mycostotle said: Im doing this out of curiosity with very little expectations, the venom will used to attempt hybridize cubensis with semilanceata and the results will be posted here
I think I have a source for semilanceata spores
Sounds like a good experiment!
PM me if your semilanceata spore source doesn't come through.
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Jakeoncid419
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How are they clamping if they are already clamped? And they why couldn’t you just mix different types of spawn together to make crosses if this works? I’m not saying I don’t believe you I’m just curious as so how that is possible without clamps. It’s also interesting than pans will not do this at least not that I have seen. Making serial dilutions is pretty easy and you can then verify with a scope that it worked, that’s y I’ve done more traditional mating with pans because it’s easier to verify
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Alan Rockefeller
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Perhaps if they are already clamped they don't need to clamp more, and can exchange genetic information.
Maybe if they are already dikaryotic they are less likely to cross, but still do occasionally.
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Jakeoncid419
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How would that work tho? When I put two different Dikaryotic cultures On a dish I can see the clear division where the two cultures meet it’s not like they meld into each other... Perhaps certain cultures naturally suffer from weak cell membranes and “venom bleed effect” happens on its own
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Alan Rockefeller
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Quote:
Jakeoncid419 said: How would that work tho? When I put two different Dikaryotic cultures On a dish I can see the clear division where the two cultures meet it’s not like they meld into each other... Perhaps certain cultures naturally suffer from weak cell membranes and “venom bleed effect” happens on its own
Maybe sometimes a third mycelium emerges where the two meet.
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Jakeoncid419
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It would be interesting to watch it breed like this under a scope
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bodhisatta
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How do all the strains you get when you shoot tens of thousands of spores into a single cake or grain jar get along? They don't know they're different if they're compatible mating types.
MS grows become homogeneous substrates not just little islands of individual strains.
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Jakeoncid419
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Re: Snake venom agar tek [Re: bodhisatta]
#26951605 - 09/23/20 06:15 PM (3 years, 6 months ago) |
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Well like I said they mate with each other when you inject the cake, this still does not explain how two Dikaryotic cultures would mate
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Jakeoncid419
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Also the cultures are somewhat divide with MS grows that’s why isolated Dikaryotic cultures do a better job of putting up full canopies
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jason9086
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Quote:
Alan Rockefeller said:
Quote:
jason9086 said: yes, snake venom breeding seems like a load of bullshit to me. What kind of scientist literally does no genetic analysis of their supposed hybrid to confirm it? I think RR drank the kool aid on that one, and a large number of people on the shroomery followed suit.
What sort of genetic analysis would be used to confirm it?
And what should be looked for in the sequence data to confirm that they have crossed?
The PHV cross has a spore size that is in between the size of the spores of the two parent species, spore color is in between as well. The fruits look macroscopically much different than either parent.
PHV spores measure (10.4) 11.5 – 12.9 (13.3) × (6.5) 9.2 – 11 (11.4) µm, which is midway in between the spore size of Panaeolus tropicalis and Panaeolus cyanescens.
PHV parent 1: https://images.mushroomobserver.org/1280/949765.jpg PHV parent 2: https://mushroomobserver.org/images/1280/1150852.jpg PHV fruits: https://mushroomobserver.org/images/1280/1188752.jpg
Well we would first have to establish a marker. For instance, say the its region is distinguishable between all the nuclei involved which it likely is at least in the spacer regions, then you could sequence this of both patents and resultant sector to see if there is a new pair of nuclei (one from each parent dikaryon) or if a marker cant be established perhaps more intensive next gen sequencing would be in order. Maybe mat genes would be good as well. but i think just seeing a new sector pop up and assuming breeding occured (when i am unsure is even possible with two dikaryons, anyone with knowledge of published research on this please inform me) is not very solid.
I dont know though.. im not an expert on basidiomycetes so info on this would be quite helpful to me. Im trying to figure out the process of these so called dikaryotic crosses.
And just to be clear, im not saying it didnt happen, im saying the evidence isnt clear. I want someone to dunk on me with info. The PHV cross was done with dikaryons? Whats the history on that one?
Edited by jason9086 (09/23/20 10:53 PM)
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Alan Rockefeller
Mycologist
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Re: Snake venom agar tek [Re: jason9086]
#26952086 - 09/24/20 12:51 AM (3 years, 6 months ago) |
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Quote:
jason9086 said: The PHV cross was done with dikaryons? Whats the history on that one?
https://mushroomobserver.org/404923
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Solipsis
m̶a̶d̶ disappointed scientist
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Fascinating! I'm looking into this but it seems to be possible when there is 1) vegetative compatibility in the first place [,similar enough cytoplasm and lots more] ;
then 2) also compatible interactions between mating factor expressions;
yet 3) having different mating types, and then bifactorial fungi should have 25% compatibility.
Apparently it is called hyphal anastomosis fusion, I would love to get a clearer visual image of the dance the nuclei perform in such an encounter.
But just scratching the surface here i may easily be wrong... idk what is required more exactly that makes it possible between those Pans but generally imposes limits on crossing of (e.g. cube variety) dikaryons being fine requiring no monokaryon at all although having but 25% odds. I cannot find the reason why you couldn't just cross isolates of different varieties of e.g. cubes with those odds - at first glance that seems to check all the boxes but I guess there is a lot going on and one of the compatibility tests prohibits it.
It has apparently been tested in oyster species and resulted in hybrids - somehow or another considerable difference could make it quite favorable. Perhaps also because in other words species relatively related to each other but growing geographically isolated from each other do not have the same incompatibility safeguards that are in place to carefully regulate incompatibility of relatively much more related strains of the same species?
Hoping to get insights from a more knowledgeable friend in the coming days.
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