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Offlinekronosofvegas
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Creating a new strain
    #18788454 - 09/02/13 06:24 AM (10 years, 4 months ago)

I heard roger rabbit created a strain(pe6 or something) how is this done?

i'd love to try crossing a few good strains that i have


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OfflineRatatoskr
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Re: Creating a new strain [Re: kronosofvegas]
    #18788555 - 09/02/13 07:15 AM (10 years, 4 months ago)

Rattle snake venom?


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Offlinekronosofvegas
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Re: Creating a new strain [Re: Ratatoskr]
    #18789503 - 09/02/13 12:19 PM (10 years, 4 months ago)

is there really nothing except that??? that's what i read, but is there some type of acid that would work aswell - there must be. 


I have no idea where to get rattlesnake venom except from finding a snake.


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Offlineomegafaust
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Re: Creating a new strain [Re: kronosofvegas]
    #18791038 - 09/02/13 05:44 PM (10 years, 4 months ago)

You two are confusing strain and species.


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Offlinekronosofvegas
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Re: Creating a new strain [Re: omegafaust]
    #18792994 - 09/03/13 02:24 AM (10 years, 4 months ago)

no i believe we're right crossing species would be p.cubensis vs p.cyanescens

I want to x the certain strain of a species(or maybe it's called sub species in mycology)


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InvisiblePestile

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Re: Creating a new strain [Re: kronosofvegas]
    #18793234 - 09/03/13 06:26 AM (10 years, 4 months ago)

Quote:

RogerRabbit said:
A few PE 6 prints were stuffed into an envelope and donated along with a bunch of others to mycotopia when I was still active over there.  It was one of the contests or something, I don't remember.  I had no idea those prints were in the bunch.  I must have gotten distracted and grabbed a big pile without going through them or something.  The first I knew was someone posting a PEG grow.  He thought the 6 was a G, and asked what strain PEG was. 

A single sector fruiting isolate of PE, and another of TEX were placed on a petri dish with rattlesnake venom added to the agar.  The snake venom helped the two dikaryotic strains share genetic information to form a third sector, which was which was labeled PE6.  Other Isolates of the PE were labeled PE 1-7, and apparently, some of the 6, which was the cross, were in the same batch of prints. The idea was to get a good spore producing PE, but I never had time to stabilize the strain due to accidentally releasing it early.  That's why sometimes they look like texans and sometimes very much like PE.
RR




Quote:

RogerRabbit said:
Quote:

Is the rattle snake venom a joke?



It's a well-established procedure for hybridization in mycology.  Google it.
RR






http://www.shroomery.org/forums/showflat.php/Number/7614007#7614007


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Offlineomegafaust
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Re: Creating a new strain [Re: Pestile]
    #18793808 - 09/03/13 10:37 AM (10 years, 4 months ago)

Yes I believe you are,  strains are of the same species, erego to cross them you only need isolates from each and they will grow together.  the tricky part is isolating the traits you want.

rr was using rattlesnake venom to cross a cube and a cyan.  he has even stated combining myclium on agar will result in a 1 in 4 chance of crossing strains.

The rattlesnake venom is used specifically to deteriorate the cell walls allowing a "fusion" of mycelium.

this is completely unnecessary for crossing strains.


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OfflineAlan RockefellerM
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Re: Creating a new strain [Re: omegafaust]
    #18794368 - 09/03/13 01:32 PM (10 years, 4 months ago)

Quote:

omegafaust said:
rr was using rattlesnake venom to cross a cube and a cyan.  he has even stated combining myclium on agar will result in a 1 in 4 chance of crossing strains.





I doubt that was successful, he probably ended up with pissed off cubes or Panaeolus.  As far as I know the resulting culture was never checked microscopically or sequenced.


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InvisiblePestile

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Re: Creating a new strain [Re: Alan Rockefeller]
    #18794458 - 09/03/13 01:53 PM (10 years, 4 months ago)

Quote:

Alan Rockefeller said:
Quote:

omegafaust said:
rr was using rattlesnake venom to cross a cube and a cyan.  he has even stated combining myclium on agar will result in a 1 in 4 chance of crossing strains.





I doubt that was successful, he probably ended up with pissed off cubes or Panaeolus.  As far as I know the resulting culture was never checked microscopically or sequenced.




cyan doesn't necessarily have to be Panaeolus.
Just sayin...


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Offlineomegafaust
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Re: Creating a new strain [Re: Pestile]
    #18794523 - 09/03/13 02:06 PM (10 years, 4 months ago)

The fact is that's what he was using it gor.  cross species.

alan.  is rattlesnake venom necessary to cross two cubes strains?


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OfflineMonkeyJesusFresco
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Re: Creating a new strain [Re: omegafaust]
    #18795085 - 09/03/13 04:15 PM (10 years, 4 months ago)

hey!

I was under the impression you had to get one spore from both strains to germinate individually, combine them, and if by some miracle they were sexually compatable they then make the sweet love and you've got yourself a dikaryotic cross between the two strains, and if that
cross fruited and sporulated, at that point you would be able to multi-spore fruit and then begin your multi generational selective breeding program, or MGSBP for short (cause, why not right?)


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Offlineomegafaust
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Re: Creating a new strain [Re: MonkeyJesusFresco]
    #18795104 - 09/03/13 04:18 PM (10 years, 4 months ago)

Exactly ^


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InvisibleinskiM
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Re: Creating a new strain [Re: MonkeyJesusFresco]
    #18797734 - 09/04/13 01:54 AM (10 years, 4 months ago)

Quote:

omegafaust said:
The fact is that's what he was using it gor.  cross species.

alan.  is rattlesnake venom necessary to cross two cubes strains?



No, in Roger's case the venom was only used in an attempt to cross two species belonging to different genera, an intergeneric hybrid, I agree with alan and don't believe the cross was successful.

I believe it is possible to cross two species belonging to the same genus if they are very closely related without using venom.

Crossing distinct strains of the same species can result in some interesting new varieties which can be considered hybrids, venom is not necessary..

There is two ways of making these types of crosses, the easiest is by placing two or more distinct strains of dikaryotic mycelium on an agar filled petri dish, as the strains grow together anastomosis may or may not occur, if it does a new "hybrid" is created, it must be tested for fruiting capabilities.
The other method is to isolate single spore germinations usually by diluting a spore solution making it easier to isolate monokaryotic cultures, to check if you have a monokaryon it is necessary to look at the mycelium under a microscope and be certain that there is no clamp connections, when you have monokaryotic cultures two must be grown together on agar, if they are compatible a dikaryotic culture will be formed and that culture can then be tested for fruiting capabilities.

Quote:

MonkeyJesusFresco said:
hey!

I was under the impression you had to get one spore from both strains to germinate individually, combine them, and if by some miracle they were sexually compatable they then make the sweet love and you've got yourself a dikaryotic cross between the two strains, and if that
cross fruited and sporulated, at that point you would be able to multi-spore fruit and then begin your multi generational selective breeding program, or MGSBP for short (cause, why not right?)




...So as explained above, it is possible to create hybrids of the same species using monokaryotic or dikaryotic mycelium.

I doubt very much that it would be possible to cross Psilocybe cubensis with P. cyanescens because they are not so closely related although using venom may make it possible!
On the other hand, a great experiment would be to try hybridising P. weraroa with P. subaeruginosa and P. "subsecotioides":cool:


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OfflineNobitte
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Re: Creating a new strain [Re: inski]
    #18797992 - 09/04/13 04:39 AM (10 years, 4 months ago)

Quote:

inski said:
I doubt very much that it would be possible to cross Psilocybe cubensis with P. cyanescens because they are not so closely related although using venom may make it possible!
On the other hand, a great experiment would be to try hybridising P. weraroa with P. subaeruginosa and P. "subsecotioides":cool:




Agree!

Have any studies been done to date that you're aware of, amateur or otherwise?


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Offlineomegafaust
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Re: Creating a new strain [Re: Nobitte]
    #18798572 - 09/04/13 10:02 AM (10 years, 4 months ago)

So, op you don't need venom.  :p. you should make it into a journal if you attempt it


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InvisibleinskiM
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Re: Creating a new strain [Re: Nobitte]
    #18799036 - 09/04/13 12:30 PM (10 years, 4 months ago)

Quote:

Nobitte said:
Quote:

inski said:
I doubt very much that it would be possible to cross Psilocybe cubensis with P. cyanescens because they are not so closely related although using venom may make it possible!
On the other hand, a great experiment would be to try hybridising P. weraroa with P. subaeruginosa and P. "subsecotioides":cool:




Agree!

Have any studies been done to date that you're aware of, amateur or otherwise?



I should have made it clearer that I was referring to Psilocybe cyanescens in my above statement.

Yes, many mating compatibility studies have been done, those experiments were routinely done to check if two separate collections were of the same taxon, now DNA sequencing is the preferred and more accurate (in some cases) method, all methods of observation are useful tools for taxonomy, macroscopic, microscopic, mating compatibility testing and DNA sequencing, all should be used in conjunction in order to gain a better understanding of the organisms studied.


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OfflineNobitte
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Re: Creating a new strain [Re: inski]
    #18799735 - 09/04/13 03:30 PM (10 years, 4 months ago)

Sorry, i was similarly unclear there, i was just expressing excitement at the idea of studying the three Australasian saprotrophs you mentioned. I figured the hybridization of two disparate species(like cyanescens and cubensis) would be an unlikely prospect.


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Edited by Nobitte (09/04/13 03:35 PM)


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Offlinekronosofvegas
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Re: Creating a new strain [Re: Nobitte]
    #18800292 - 09/04/13 05:51 PM (10 years, 4 months ago)

yeah i only want to cross strains(only cubes at this point)(not species) like PES Hawaiian, PE, Creepers, not species

so from what i've read i would need the venom to break down the genes so they can hybridize. 

but does anyone know of anything else that would work any kind of acid?


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OfflineMonkeyJesusFresco
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Re: Creating a new strain [Re: kronosofvegas]
    #18800358 - 09/04/13 06:09 PM (10 years, 4 months ago)

no, typically, you won't need the venom for strains of the same species

edit:

if you're trying to cross cubensis, just culture two strains on two separate petris,
isolate single sectors of each, put 'em both on a new petri, and where those two come together and sector off, ta-da! cross?

edit edit:

good luck on getting your hands on rattlesnake venom!


for more info on that use the search function for "Venom" and Posted By "RogerRabbit"


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Edited by MonkeyJesusFresco (09/06/13 01:58 AM)


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OfflineRogerRabbitM
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Re: Creating a new strain [Re: MonkeyJesusFresco]
    #18805414 - 09/05/13 09:59 PM (10 years, 4 months ago)

The venom is just one tool available, and was used for crossing two isolated single sector fruiting strains of cubensis.

Any two compatible spores will form a strain.  A strain is not the name someone wrote on a print, so taking a spore from two different prints and creating a strain from them is not a cross.
RR


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OfflineMonkeyJesusFresco
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Re: Creating a new strain [Re: RogerRabbit]
    #18806072 - 09/06/13 01:57 AM (10 years, 4 months ago)

Quote:

The venom is just one tool available, and was used for crossing two isolated single sector fruiting strains of cubensis.




oooooooooooooooooooooooooooohhhhhhh fuckme:facepalm: yeah, thank you for that clarification, I see now where I wasn't exactly pay'n attention :ashamed:


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Invisiblewildernessjunkie
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Re: Creating a new strain [Re: RogerRabbit]
    #18806205 - 09/06/13 04:45 AM (10 years, 4 months ago)

Quote:

RogerRabbit said:
Any two compatible spores will form a strain.  A strain is not the name someone wrote on a print, so taking a spore from two different prints and creating a strain from them is not a cross.
RR




I think Ive seen you write that in a hundred different posts. And each time I see it, I have this vision of you banging your head against a brick wall.


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OfflineAce1928
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Re: Creating a new strain [Re: wildernessjunkie]
    #18806253 - 09/06/13 05:26 AM (10 years, 4 months ago)

Is rattlesnake venom the only thing that will work?
What about a chitinase enzyme? That should achieve the same outcome yes?

I ask because im in Australia and from what I can find rattlesnake venom is stupidly hard to acquire.


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Re: Creating a new strain [Re: Ace1928] * 1
    #18810136 - 09/07/13 12:54 AM (10 years, 4 months ago)

There's a good half-dozen commercial chitinases available.

What you're talking about is protoplast fusion.  There's plenty of literature on the subject out there.  Mushrooms breeders have done plenty of successful cross species crosses.  It's not particularly difficult given enough effort and money.

Squirting rattlesnake venom on a dish probably doesn't have much chance of success.

The normal procedure is to suspend the cells in an osmotically supportive solution (mineral salts added) and add in a chitinase and a fusing agent (high MW PEG).  After the right amount of time you plate out the solution.

Cells on a plate will just leak all over and die when the cell wall is dissolved.  That's why they must be suspended in a proper density, osmotically balanced solution.  This way the cell wall dissolves and the contents come out in nice round spheres, AKA protoplasts.

When you have PEG in the solution this causes the protoplasts to fuse together when they touch instead of just floating around separately.  Then you just have to plate them out in the proper strength agar and some of them will live long enough to regenerate their cell wall and they'll start growing normally again.

There's lots of factors involved.  An in depth review can be found in "Fungal Protoplast: A Biotechnological Tool".

Overall there's nothing particularly difficult or expensive involved, but you have to tune each parameter to optimize the procedure, then repeat it enough to be successful, and then there's no guarantee you'll get anything worthwhile out of it.  You might get non-fruiting fusants, or they might shed one or another chromosome during division until you end up with one of the parental types again.

The biggest problem is separating parental types from actual fusants.  You'll end up with lots of unfused protoplasts, and fusants of a single parental type.  There are a few methods to overcome this, mainly using complementary auxotrophs.  Most of the chemical methods I'm aware of require at least one of the parents to be an auxotroph.


-FF


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OfflineAce1928
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Re: Creating a new strain [Re: fastfred]
    #18810533 - 09/07/13 04:57 AM (10 years, 4 months ago)

Awesome answer thank you.
I understand plant somatic fusion for creating protoplasts but was missing a few things for fungi.
Cheers :-D


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Invisiblefastfred
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Re: Creating a new strain [Re: Ace1928]
    #18814046 - 09/08/13 05:01 AM (10 years, 4 months ago)

Our main enemy, trich, produces a chitinase that is one of the best enzymes for creating protoplasts.  You could order some up or possibly extract your own.

PEG = Poly Ethylene Glycol.  It's cheap and easy to come by, and used for quite a number of things.

All the rest is just standard lab stuff you probably already have on hand.

The toughest part is doing microscopic examinations to optimize everything, and optimizing all the steps.  Other than that it's just brute force dish work.

Assuming you don't find complementary auxotrophs of each parental species, or at least one combined with chemical methods, then you can probably replace that with sheer numbers in the dish work.

One thing is for sure, you'll be getting lots of crazy regenerated fusants.  And who knows, maybe just melding a bunch of cells together and letting recombination and cell wall regeneration take place will give you some fun genetics to work with.

Grasses, like corn, have many levels of polyplodity.  Who's to say a tetroploid cube wouldn't be something interesting, especially if it had a multi-species origin?

-FF


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Re: Creating a new strain [Re: fastfred]
    #18814919 - 09/08/13 11:56 AM (10 years, 4 months ago)

Exactly same logic as what I've been thinking. Genetics are wonderful and playing with them can be a lot of fun.

I never knew that trich made a particularly good chitinase. I'm going to research that further.

Thanks for all the info. Greatly appreciate it. Looking forward to having some fun with protoplasts


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Offlinekronosofvegas
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Re: Creating a new strain [Re: Ace1928]
    #18814978 - 09/08/13 12:09 PM (10 years, 4 months ago)

thanks for the great answer exactly what i was looking for aswell!


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OfflineStromriderM
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Re: Creating a new strain [Re: kronosofvegas]
    #18818026 - 09/09/13 07:29 AM (10 years, 4 months ago)

I very much enjoyed reading this thread. Some good info here. Fungus absolutely fascinates me!


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Offlinecaricapapaya
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Re: Creating a new strain [Re: Stromrider]
    #18862957 - 09/19/13 12:56 PM (10 years, 4 months ago)

FF: have you ever read or heard about using an anti-mitotic (such as colchicine) on fungi?


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OfflineAlan RockefellerM
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Re: Creating a new strain [Re: kronosofvegas]
    #18862997 - 09/19/13 01:06 PM (10 years, 4 months ago)

Quote:

kronosofvegas said:
yeah i only want to cross strains(only cubes at this point)(not species) like PES Hawaiian, PE, Creepers, not species

so from what i've read i would need the venom to break down the genes so they can hybridize. 

but does anyone know of anything else that would work any kind of acid?





That is easy, you do not need venom or anything else.    You just dilute the spore solution enough that it creates colonies of monokaryotic mycelium, then let the two monokaryotic colonies of your choice join together on an agar dish.   

They will make a dikaryotic mycelium that is a cross between the strains.  Whether or not they have the traits you want is another question...


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Re: Creating a new strain [Re: caricapapaya]
    #18863725 - 09/19/13 04:27 PM (10 years, 4 months ago)

Quote:

caricapapaya said:
FF: have you ever read or heard about using an anti-mitotic (such as colchicine) on fungi?




I don't think it would be very effective.  Fungi is a bit different than plants in that the nuclei migrate through the network.  You can usually find multinucleate cells in culture already.


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InvisibleSporeo

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Re: Creating a new strain [Re: fastfred]
    #18934932 - 10/05/13 06:41 AM (10 years, 3 months ago)

Isn't possible to get monokaryotic myc by putting spores on agar, then make a transfer as soon as the myc begins to grow, Xfer to a new plate with another variety(using the same tek as above) of your choice? I've read this before but I'm unsure where. Still rusty and getting back to the hobby.


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OfflineRogerRabbitM
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Re: Creating a new strain [Re: Sporeo]
    #18934986 - 10/05/13 07:16 AM (10 years, 3 months ago)

Any single spore which germinates forms monokaryotic mycelium.  Simply dilute the spore solution enough to swipe individual spores with a space around them so they're easy to grab.

An easier method is to simply make a print with one cap, and then set the cap from the other 'strain' on top of it, to print both caps on the same medium. Then use multispore inoculation and look later for the cross you were hoping for after fruits develop.
RR


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OfflineHerbal Ways

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Re: Creating a new strain [Re: RogerRabbit]
    #18963500 - 10/11/13 11:22 AM (10 years, 3 months ago)

RR. You make so much make sense, had a question, read all your posts, answered question.. now if more people can do that we`d have a clean forum without all the repeat questions lol.


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