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OfflinePrimalSoup
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recommended # trial isolates for trait selection?
    #21948857 - 07/15/15 06:42 PM (8 years, 6 months ago)

Just wondering if people have a preference when testing isolates for traits.  My plans are to select out and isolate about 10 substrains from a PE swab I've saved in the fridge for a couple years (those are the original fruits in my sig).  The swab germinated contam free in around 5 days the last time I put it to agar.  The genetic variance is what I'm looking to capture of course, in this case likely potency but more along the lines of just the kind of trip I like. :thumbup:

An ancillary question is this - IME it takes a little while for dikaryotic myc to show up on the innoc plate, but without resorting to microscopy on the plates is there a "best time" to start isolating from germination?  As soon as growth starts or as it begins to proliferate?


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Edited by PrimalSoup (07/18/15 01:14 AM)


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OfflineKizzle
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Re: recommended # trial isolates for trait selection [Re: PrimalSoup]
    #21957009 - 07/17/15 05:05 PM (8 years, 6 months ago)

It doesn't really matter unless you're wanting to isolate the monokaryotic mycelia for breeding which should be done early on.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection [Re: Kizzle]
    #21957192 - 07/17/15 05:44 PM (8 years, 6 months ago)

Thanks, that's what I thought.


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Re: recommended # trial isolates for trait selection [Re: PrimalSoup]
    #21958316 - 07/17/15 09:52 PM (8 years, 6 months ago)

A preference for what when testing isolates? How many to test at a time, best way to test, substrate, etc?

I don't try to keep up with more than 10 at a time. I make containers that require no maintenance per my sig, easier than cakes, and let the iso run through whatever sub I will eventually use to grow it regularly if it turns out to be a winner.

Testing potency takes a while since you really should have a single individual testing and have to account for serotonin levels and tolerance.

Or......are you simply asking what traits are desirable for an isolate?


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: maddchef]
    #21958870 - 07/18/15 12:24 AM (8 years, 6 months ago)

"Recommended # trial isolates for trait selection?" just your preferred number to run not how to do it. :shrug:

I'm looking for a number that gets a good cross section of variability without going to far to extremes - somewhere between 10 to 100 I expect. :lol:


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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22004116 - 07/27/15 11:46 AM (8 years, 6 months ago)

If you want the best one, run them all.

Personally, I just do a MS run, take a clone then isolate from there. You'll have a lot less strains to work with, and they'll all usually be decent if not great.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: 36fuckin5]
    #22004169 - 07/27/15 12:02 PM (8 years, 6 months ago)

Well, since I always isolate based on the myc appearance on the plate, I'll take anything interesting and see how it grows there.  But what I don't and haven't done is to give less spectacular myc a chance, but my experience over the years selecting vigorous growing sectors suggests that that's the best way to do it in any case.  Tomentose cultures (that remain that way) rarely have done well for me - I have one now from a free PFC syringe that is just this side of a complete failure.

My clones usually contam so I'm not really into that. :shrug:


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Invisible36fuckin5
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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22004255 - 07/27/15 12:21 PM (8 years, 6 months ago)

Well yeah, if you're gonna isolate from spores you'd obviously take the best-looking, most aggressive growth. And then I'd grow ALL of those out.

If your clones are all contaminating you're doing something wrong. They're about as likely to contam as starting from spores IME. I won't do less than 5 plates from tissue, then isolate from there.


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Redd Foxx said:
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Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: 36fuckin5]
    #22005985 - 07/27/15 06:19 PM (8 years, 6 months ago)

No I just don't work with clones much, my experience hasn't been good so I never bother. :lol:

My spore plates germinate contam free most of the time, taking something out of the open air and extracting a contam free portion doesn't work for me.


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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22007829 - 07/28/15 02:14 AM (8 years, 6 months ago)

Quote:

PrimalSoup said:
No I just don't work with clones much, my experience hasn't been good so I never bother. :lol:

My spore plates germinate contam free most of the time, taking something out of the open air and extracting a contam free portion doesn't work for me.



You can take the pins from a MS agar plate when it starts pinning so there's a higher chance of getting a sterile culture. Probably a more vigorous culture too in terms of senescence compared to cloning pins from a fruiting bulk substrate.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22009387 - 07/28/15 12:31 PM (8 years, 6 months ago)

Yeah, I've been considering that.  But is there an advantage to cloning pins (at the end of the growth cycle regardless) over just selecting rhyzo myc for vigor?  Perhaps the speed to pinning?


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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22009728 - 07/28/15 01:36 PM (8 years, 6 months ago)

So you can get a genetically homogeneous strain. It's only way get one from a MS culture with complete certainty. Even though MS cultures produce sectors of a sort, sectoring between genetic individuals only occurs between incompatible single spore monokaryotic colonies.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22009773 - 07/28/15 01:44 PM (8 years, 6 months ago)

But any given fruit from MS is just as likely to be a mosaic, SFAIK, regardless of whether it occurs on a plate or in a fruiting sub.  If you know that after this step the resulting culture exhibits all the traits of an isolate then I'm interested.

My experience has always been that repeated selection of a sectoring plate always results eventually in a homogeneous culture that does everything an isolate is supposed to do... :shrug:


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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22009975 - 07/28/15 02:26 PM (8 years, 6 months ago)

You usually don't need any extra transfers with a sterile clone. Here's an agar pin clone. It may not look like a monoculture because of the asymmetry, that's in part because it's adapting to a new growth medium, but given enough space it grows into a perfect circle. Not that a perfectly circular colony is necessary to ensure consistency between substrains of a culture but it's a sign that there are no hidden contaminants.



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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22010112 - 07/28/15 02:49 PM (8 years, 6 months ago)

Not about contaminents IME - the even circular growth is a culture that doesn't sector because it can't.  I generally transfer until I have regular growth - my trial experiment here (which I've never done, which is why I'm asking) is for PE isolation and testing. - but not necessarily until it's completely regular.


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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22010500 - 07/28/15 04:01 PM (8 years, 6 months ago)

Actually I'm not even entirely sure that two dikaryotic strains can't grow together without causing any sectoring.

What I am sure about is despite what a lot of people on this forum believe, and I used to be one them, a mushroom is composed of a single strain. So there's no need to keep doing transfers with it until the sectoring stops to know you have a single strain like you would if you were trying to isolate a strain from the mycelium or to know that it's not contaminated like you would with a nonsterile clone.

Although it's possible to wind up with multiple strains from a clone if it is sporulating or if you clone tissue from the very base of the stem.


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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22010872 - 07/28/15 05:19 PM (8 years, 6 months ago)

Quote:

Actually I'm not even entirely sure that two dikaryotic strains can't grow together without causing any sectoring.


:orly: 

I may have to give that triple negative some thought.  But, I'll grant that if they were very similar it would be hard to pick them apart on a plate.  Which brings me to the next step, I suppose, mating individual monokaryotes with the aid of magnification.

For the other though, I can sort of accept that, given the way pins form.  But I'll have to succeed at cloning sometime to see it work. :lol:

FWIW I had a similar sort of situation come up some years ago while attempting to ressurect a stored mutant Ps. cyan print (15 years in the fridge).  After some soaking I got sparse germination, with what appeared to be no more than a few germinations and only a couple of rhyzomorphic growths.  I transferred these to the same plate to see what would happen.  Got a beautiful example of incompatible growth, all from the same parent print:



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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup]
    #22011523 - 07/28/15 07:11 PM (8 years, 6 months ago)

It also seems kind of self-defeating to try and attain a vigorous strain by using a method that requires a crapload of transfers. Each transfer brings you one step closer to strain degradation. A couple inches of growth may not seem like much but it's about the distance the mycelium has colonize after a g2g.


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OfflinePrimalSoup
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Re: recommended # trial isolates for trait selection? [Re: Kizzle]
    #22012444 - 07/28/15 10:11 PM (8 years, 6 months ago)

The transfers take only short periods of growth IME to show sectoring, less than you'd let a plate grow out.  I finally have a use for my glass petris again 'cause I'll be making a lot of plates serially to process this stuff.  :lol:

I haven't done it but forcing a pin to go back to vegetative growth would seem to exact a toll on vigor as well.  But senescence (if your talking about that) doesn't ever seem much of a factor in plating.  Again that's just my experience.


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Invisible36fuckin5
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Re: recommended # trial isolates for trait selection? [Re: PrimalSoup] * 1
    #22012512 - 07/28/15 10:32 PM (8 years, 6 months ago)

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.


--------------------
Redd Foxx said:
If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


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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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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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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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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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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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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
Ã…), 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, 6 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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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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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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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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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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