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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, 5 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, 5 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, 5 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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Invisiblefastfred
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Re: Creating a new strain [Re: Ace1928] * 1
    #18810136 - 09/07/13 12:54 AM (10 years, 5 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, 5 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, 5 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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OfflineAce1928
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Re: Creating a new strain [Re: fastfred]
    #18814919 - 09/08/13 11:56 AM (10 years, 5 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, 5 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, 5 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, 5 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, 5 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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Invisiblefastfred
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Re: Creating a new strain [Re: caricapapaya]
    #18863725 - 09/19/13 04:27 PM (10 years, 5 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

Registered: 10/04/13
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Re: Creating a new strain [Re: fastfred]
    #18934932 - 10/05/13 06:41 AM (10 years, 4 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, 4 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, 4 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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