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Offlinetonpole
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Re: recommended # trial isolates for trait selection? [Re: 36fuckin5]
    #22012873 - 07/29/15 12:41 AM (8 years, 6 months ago)

Ive done many grows from multispores isolations, from my experience the difference mostly is neglible but i have seen as much as a 50% increase in weight between the highest and lowest performing, however pinning, shroom size, and timings always remained the same across them. Isolations do cause them to flush at the same time which is nice, clones dont really offer anything over an isolation as they are an isolation themselves and produce the same result.

If you are hoping to get a superior genetic result like larger or denser mushrooms you are wasting your time isolating and cloning them, you will need a strain that does this naturally via multispore and then work towards achieving optimal environmental conditions.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: tonpole]
    #22012923 - 07/29/15 12:57 AM (8 years, 6 months ago)

Vigor always wins. :Awemush:


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InvisibleToadstool5
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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22012939 - 07/29/15 01:01 AM (8 years, 6 months ago)

Quote:

clones dont really offer anything over an isolation as they are an isolation themselves and produce the same result.




Clones are not true isolates they are only partial isolates of several different mycelial cords felted together inside the stipe.

Quote:

If you are hoping to get a superior genetic result like larger or denser mushrooms you are wasting your time isolating and cloning them




Yeah, no. Isolation of superior cultures is essential to biological efficiency and adaptations such as leucistic mutations or high-temperature fruiting varieties.

Auxotrophic mutants can lead to the ability of digesting different substrates that where previously indigestible. Morphological mutants (what we typically isolate) can lead to faster colonization through stronger anastomosis and thicker rhizomorphs. Then there are fruiting and fruit body mutations to help with pin set timing and phenotypical traits such as color.

Quote:

you will need a strain that does this naturally via multispore




Mutations can be induced through many different methods such as beta-radiation, chemical mutagenics, or UV radiation. Spontaneous mutation rates are also quite high in species so diverse as psilocybe so you can literally grow a culture out and over time it will eventually mutate.

Selective breeding methods are viable options to creating better cultures but not the only way, i wouldnt even consider them comparable to mutagenesis and proper isolation and maintenance of monocultures.


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OfflineKizzle
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22013119 - 07/29/15 02:32 AM (8 years, 6 months ago)

Quote:

36fuckin5 said:
CLones definitely sector and are composed of different strains. Isolate some out (keeping ALL of them, you'll need a case of petris) and fruit them individually and you'll see that.



Well strain stability in itself is a consistent trait of a strain. The sectoring isn't the result of multiple strains being present in the mushroom. It's the result of a single strain reacting to being placed on a high nutrient medium.

Quote:

The existence and phenotypic expression of strain instability in fungal isolates that are repeatedly subcultured in the laboratory are a recurring theme in the industrial microbiological literature. Numerous studies have shown that filamentous fungi grown in nutritionally rich laboratory media exhibit  extremely high levels of morphological and physiological variation and a high frequency of instability is often encountered in the absence of any external mutagenic agent.



http://www.ncbi.nlm.nih.gov/pmc/articles/PMC201660/?page=1


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Offlinetonpole
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Re: recommended # trial isolates for trait selection? [Re: Toadstool5]
    #22014642 - 07/29/15 11:56 AM (8 years, 6 months ago)

Quote:


Spontaneous mutation rates are also quite high in species so diverse as psilocybe so you can literally grow a culture out and over time it will eventually mutate.





Youre referring to a plate mutating on its own over time? How often does this occur, plates dont have that long a lifespan.

Quote:


Mutations can be induced through many different methods such as beta-radiation, chemical mutagenics, or UV radiation.





Thats interesting, whats your experience with this? How would one go about using something like UV-C light to mutate a strain without killing it?


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InvisibleToadstool5
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Re: recommended # trial isolates for trait selection? [Re: tonpole]
    #22015138 - 07/29/15 01:47 PM (8 years, 6 months ago)

Quote:

Bulk fungal tissues, cords, and membranes, such as those of mushrooms and lichens, are mainly composed of felted and often anastomosed hyphae.




21st century guide to fungi, david moore.

Clones of a fruiting body are not monocultures. Unless you have induced monokaryotic fruiting (normally impossible) it should have rhizomorphed or anastomosed more than one sector together in a felt-like mesh. This is why morphological mutants are so hard to isolate without special forceps and microscopes or several transfers.

Quote:

Mutagenic Treatment: Ultraviolet
For most investigators seeking to obtain mutants of fungi, it is simpler to use ultraviolet (UV) light
than X rays, because a simple, inexpensive, germicidal UV lamp is available. The germicidal effect is
related to the absorption maximum of nucleic acid at 260 nm (1 nanometer = 10 angstroms, symbolized
Å), which is included in the wavelengths delivered by the germicidal lamp. Actually, most germicidal
lamps deliver a broader range of wavelengths, but experimentation has demonstrated that 260 nm is
the effective wavelength. This wavelength has also been shown to increase mutation frequency.
Both the germicidal (lethal) effect and the mutagenic effect are believed to result from the
formation of covalent bonds between adjacent pyrimidine nucleotides, generally thymine but
sometimes cytosine. These linked pyrimidine nucleotides are referred to as dimers, either thymine dimers or cytosine dimers, depending on the specific nucleotides that have bonded together. The
consequence of dimer formation is an inhibition of normal DNA synthesis.
It has also been demonstrated that there is a mechanism that can repair the UV-induced damage.
The mechanism requires exposure to wavelengths in the approximate range of 360 to 480 nm. Light
in this range has the effect of activating an enzyme that splits the dimer, thus permitting the return
to normal DNA synthesis and lack of mutagenicity. This light repair is called photoreactivation.
In experimental work in which UV is being used to obtain mutants, it is important that cells not be
exposed to the wavelengths inducing photoreactivation. This can be accomplished by illuminating
the work area with yellow light, which does not transmit wavelengths causing photoreactivation.
Mutations are caused by UV through dimer formation, which is followed by replication of
DNA. The dimers are responsible for gaps in the strands of DNA that are synthesized. This method
of mutagenesis is different from the mode of action of ionizing radiation, e.g., X rays, which cause
alteration in the bases of the DNA and breaks in the DNA strands.




I wouldn't use a lamp, its too messy for my likes and melanoma is real :lol: i would use nitrous acid to change a DNA-base. If using a UV light, restict the photoreactive wavelengths and dont expose it to UV until the nucleus bursts and it will mutate without dying.

Quote:

Nitrous acid is one of the compounds that act directly on DNA to produce a change in one of
the bases. It does this by reacting with primary amino groups to give hydroxyl groups. For example,
adenine becomes hypoxanthine, guanine becomes xanthine, and cytosine becomes uracil. Subse-
quently, base pair changes of the transition type (purine for purine, and pyrimidine for pyrimidine)
are produced, which are responsible for the mutations.




Then once a mutation is present selectively breed it or isolate and test until you have a stabilized variety.

Quote:

Youre referring to a plate mutating on its own over time? How often does this occur, plates dont have that long a lifespan.




Transfers, and even the short 2" distance is enough to cause sectoring and mutations, this is definitely true after a few transfers. Depends on the species, some have a fast spontaneous mutation rate and some take months. The nutritional aspect of the media being grown on also contributes to how fast it mutates.

Quote:

The existence and phenotypic expression of strain instability in fungal isolates that are repeatedly subcultured in the laboratory are a recurring theme in the industrial microbiological literature. Numerous studies have shown that filamentous fungi grown in nutritionally rich laboratory media exhibit  extremely high levels of morphological and physiological variation and a high frequency of instability is often encountered in the absence of any external mutagenic agent.




This is very true, thats why even if somehow you create a true isolate from the stipe you still have to maintain it and restrict the number of transfers done. Isolating is a never-ending job. :shrug:

You can even do this purposly to induce and isoate auxotrophic mutants

Quote:

The principle of the rescue method is to add nutrients in some manner to a minimal medium upon
which prototrophic cells have already formed colonies after a period of incubation. Colonies that
develop after the addition of the supplementary nutrients are likely to be auxotrophs. This technique
can be utilized for the selection of specific auxotrophs by use of specific supplementary nutrients.




Edited by Toadstool5 (07/29/15 02:14 PM)


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OfflineKizzle
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Re: recommended # trial isolates for trait selection? [Re: Toadstool5]
    #22016094 - 07/29/15 05:11 PM (8 years, 5 months ago)

Quote:

Clones of a fruiting body are not monocultures



How exactly would you define a monoculture?

I just have to say how difficult it is to have a meaningful discussion when everyone has there own little definition of things and I'm not blaming anyone because in many cases there are multiple definitions or they're only vaguely defined and it's impossible to conform to everyones definition at once. So what is a monoculture? Is it a culture where every single fungal cell has the exact same DNA? Since that definition pretty much will never be true on the scale of an agar plate I'm guess not.

Since most people don't have magic strain-o-vision goggles when they isolate their MS cultures yet they always know it's a monoculture it can't be that, a single strain culture.

Then surely it must be uniform appearing culture and that's what I meant by getting a monoculture from a clone.

What about a strain? When two monokaryons combine into a dikaryon does that make a strain? If that dikaryon undergoes further anastomosis does that make it a new strain or is it the same strain? If that strain produces two sectors on agar does that make two strains then?

Lets look in the dictionary is it...

The collective descendants of a common ancestor; a race, stock, line, or breed.

or

A group of cultivated plants or domestic animals of the same species that have distinctive characteristics but are not considered a separate breed or variety.

or

A group of organisms within a species or variety, distinguished by one or more minor characteristics

or

A variety of bacterium or fungus, esp one used for a culture

or

A group of organisms of the same species, sharing certain characteristics not typical of the entire species but minor enough not to warrant classification as a separate breed or variety

:blowmybrainsout:


Edited by Kizzle (07/29/15 06:01 PM)


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InvisibleToadstool5
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22016332 - 07/29/15 06:09 PM (8 years, 5 months ago)

A monoculture in the sense that most of us are used to is a morphological mutant that has been isolated from the other hyphae inside the rhizomorphic growth. If there is any sectoring then its not a monoculture. Mating type is still random in our "monocultures" same with phenotype.

When you clone from a stipe there are several differentiated morphologies and mating types and when you grow them out they sector. Thats why it is considered an isolate of several morphologies but not a true isolate of a single type (monoculture).

See Isolates vs Clones:

https://www.shroomery.org/forums/showflat.php/Number/17947323#17947323

Quote:


Quote:

Hyphaeoid said:
So, a clone isn't really neccessarily a clone the way most of us would think it would be?  It would still be similar to multi-spore performance, except with far LESS variance?


Yes, exactly....basically your just taking a shortcut to get down to isolates, but ones you know are most likely fruiters.

Quote:

Hyphaeoid said:
that is to say, instead of 1,000 different ways to perform, it might have 10, where as the isolate would have exactly 1? am i on the right track?


It could be more than 20 different sets of genetics in a single MS fruit, but yea, same concept




There are several trusted cultivators that will back me up on this, a cloned fruit is an isolate but not a monoculture.

Basically the definition:

Quote:

A group of organisms of the same species, sharing certain characteristics not typical of the entire species but minor enough not to warrant classification as a separate breed or variety





In the context of mycelial morphology is the best way to describe it.

Mushrooms - Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact second edition by shu-ting chang has a great chapter on different aspects of isolation, breeding, genetics, and mutations that explains it good.

If you are isolating for mating type, phenotypes, fruiting mutations, etc. then you must use more advanced methods more comparable to selective breeding.


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Edited by Toadstool5 (07/29/15 06:19 PM)


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Offlinekmetric
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Re: recommended # trial isolates for trait selection? [Re: Toadstool5]
    #22016789 - 07/29/15 07:37 PM (8 years, 5 months ago)

Solid info toad :thumbup:


Quote:

a cloned fruit is an isolate but not a monoculture





Seems like very often isolate and monoculture are used interchangeably, and shouldn't be


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InvisibleToadstool5
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Re: recommended # trial isolates for trait selection? [Re: kmetric]
    #22016836 - 07/29/15 07:47 PM (8 years, 5 months ago)

Yeah it can become confusing because saying isolate doesnt denote how isolated it is.

Kizzle makes a good point too, when you are discussing another type of mutation other than mycelial morphology then what we call a monoculture or isolate is not really applicable because of the different DNA of mating types still causing further evolution of certain characteristics like fruit body.

Genetics are extra complicated in filamentous fungi :lol: i have had so much fun reading about genetics and isolation methods.


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Offlinetonpole
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Re: recommended # trial isolates for trait selection? [Re: Toadstool5]
    #22021436 - 07/30/15 06:42 PM (8 years, 5 months ago)

Quote:


I wouldn't use a lamp, its too messy for my likes and melanoma is real :lol: i would use nitrous acid to change a DNA-base. If using a UV light, restict the photoreactive wavelengths and dont expose it to UV until the nucleus bursts and it will mutate without dying.





Where can one find nitrous acid locally? Is it available online to us common serfs or do we have to be an institution? The companies i see selling it all require the latter.


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InvisibleToadstool5
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Re: recommended # trial isolates for trait selection? [Re: tonpole]
    #22021649 - 07/30/15 07:22 PM (8 years, 5 months ago)

You wont find it locally, it is produced commercially using amberlite resin columns and nitrite solutions. Its also very unstable in storage. Most labs make it as they need it for mutagenesis.

A crude, not-pure solution can be made at home.

Add about 100ml of DI water in a borosilicate beaker, dissolve about 0.7g of sodium nitrite. In a separate beaker dilute some sulfuric acid down to about 5% and chill in the fridge also chill the sodium nitrite solution.

Finally put the NaNO2solu on an icebath and stir,, slowly add the sulfuric acid until a light blue hue is created. This is very crude Nitrous acid, it can be used but the amberlite column stuff is way cleaner and better for biological work.

Be careful when you add the sulfuric! It will emit brown fumes.

Heres a video if i described it badly:



I remember there being a cleaner reaction but it eludes my memory. Some type of catalytic reaction with sodium nitrite too


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If you do not know where the mushroom products you are consuming are grown, think twice before eating them. :badshroom:
- Paul Stamets

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OfflineOysterFace
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Re: recommended # trial isolates for trait selection? [Re: Toadstool5]
    #22022017 - 07/30/15 08:56 PM (8 years, 5 months ago)

usually just z swap some spores to agar,  itll fuzz up then show a few signs of sectoring as rhizomorphic growth, after 2-4 transfers it is single isolate.  Then test as many strains as there are spots for tubs in the ol FC.  compare each one based on colonization speed, pin density, fruit characteristics(size,shape, BEAUTY), and potency. 

this usually comes out to 10-12 strain comparisons.  pick the top 2.  simple really


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: OysterFace]
    #22022237 - 07/30/15 09:46 PM (8 years, 5 months ago)

And that's what I was looking for. :raisemyglass:


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