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Offlinemycot
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Breeding and Selection for secondary metabolite production
    #18391476 - 06/09/13 08:03 AM (10 years, 11 months ago)

Mods move this on if this is the wrong forum.
A subject which I'd like some discussion is on whether one might breed and select for high potentcy in a species.
As an example :- say one had a species of relatively low potentcy such as P.subbalteatus. Could one through selection breed something as strong as or even stronger than cubes.
If this is feasible then how might one go about such a thing and what would be the necessary technology in order to do this.
Quantittative analytic chemistry of small amounts of material may be involved, as well as unpredictable elements in alkaloid production.

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Invisiblemicro
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Re: Breeding and Selection for secondary metabolite production [Re: mycot]
    #18391666 - 06/09/13 09:50 AM (10 years, 11 months ago)

If certain strains are more potent than others, it would follow genetics can influence potency. You will reach a limit though; I doubt you are going to breed any sort of "super-variety." You wouldn't need much to try and breed them, but extraction and HPLC equipment, or whatever you are using to do your quantitative analysis might be a challenge.

I also remember reading that adding metabolic precursors (i.e. tryptamine) to the substrate can influence potency as well (some precursors can also trigger down-regulation of production). There is a paper that did a quantitative analysis on this floating around somewhere.

Another thing to keep in mind is that smaller mushrooms are usually more potent by weight.

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OfflineRogerRabbitM
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Re: Breeding and Selection for secondary metabolite production [Re: micro]
    #18391876 - 06/09/13 10:55 AM (10 years, 11 months ago)

Quote:

secondary metabolite production




I think the word you were looking for is alkaloid, not metabolite.
RR


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Offlinemycot
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Re: Breeding and Selection for secondary metabolite production [Re: RogerRabbit]
    #18396076 - 06/10/13 06:37 AM (10 years, 11 months ago)

I thought that alkaloids were often considered as secondary metabolites.
Whatever the case, for this thread I'm primarily interested in the selection and breeding of very highly desirable strains of any particular species, the desire-ability accruing from the maximization of the selection for high alkaloid production.
So to make much of this practical, one must have an understanding of some of the hurdles to be overcome.
Things such as:-
Alkaloid content varying from flush to flush.
Smaller shrooms more potent by weight making meaningful measurements more difficult.
HPLC equiptment is expensive and outside the reach of most folks.
Alternative analytic approaches may have to be considered especially if many and multiple samples require testing.
So we're looking at gameplans towards acheiving the results required. :loveeyes:
And selecting for size. Would the selections keep in succeeding generations or would they quickly revert ?
Sorry, I'm a bit ignorant on some of this. :frown:

Edited by mycot (06/10/13 07:17 AM)

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InvisibleSatanschild
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Re: Breeding and Selection for secondary metabolite production [Re: mycot]
    #18396990 - 06/10/13 11:59 AM (10 years, 11 months ago)

Simplest way would be to pick mushrooms about the same size and weight. Spore print, and then cut the caps to equal weight.
Extract the psilocybin and compare the weight. I don't know about the efficiency of extractions, but I assume this method should be effective enough in the long run. You'll need a pretty good scale though and a lot of patience.

"Would the selections keep in succeeding generations or would they quickly revert?"
As you will select from spores the genetics will vary very much, but this is what you want when selecting.
When you start getting good results you should start making clones. As spores are random and clones are clones.

The game-plan is simple, but a project like this might take years.

Edit:
To speed up things a little this should also be possible when using mycelium only, right?
Just colonize a lot of boxes, clone and extract. Then fruit the best clone and use the spores for the next round.

Edited by Satanschild (06/10/13 12:16 PM)

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Offlinethiotimoline
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Re: Breeding and Selection for secondary metabolite production [Re: Satanschild]
    #18397941 - 06/10/13 03:36 PM (10 years, 11 months ago)

Quote:

Satanschild said:
Extract the psilocybin and compare the weight. I don't know about the efficiency of extractions, but I assume this method should be effective enough in the long run. You'll need a pretty good scale though and a lot of patience.




I get the impression that obtaining sufficient purity for weight to be a good measure is not practical, in that it requires chromatography of bulk quantities and non-thin-layer chromatography is a huge pain in the ass. Reacting a standardized extract (e.g., with Ehrlich's reagent (in excess, otherwise increasing alkaloid content will not increase the quantity of colored product)) to produce a colored product, and then performing spectrophotometry, may be more convenient. Given the fraction of light transmitted at the maximum-absorption wavelength for each sample, Beer's Law can be used to compare concentrations. A spectrophotometer and cuvettes should be under $150 on eBay.

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InvisibleSatanschild
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Re: Breeding and Selection for secondary metabolite production [Re: thiotimoline]
    #18401229 - 06/11/13 02:13 AM (10 years, 11 months ago)

Don't the genetics vary too much when using bulk quantities?

I expected that a spectrophotometer would be too expensive. At that price that would definitely be a better method. :thumbup:

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Invisibletrade_om
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Re: Breeding and Selection for secondary metabolite production [Re: Satanschild]
    #18402703 - 06/11/13 11:58 AM (10 years, 11 months ago)

I highly doubt you can find a digital spectrophotometer for 150$ let alone the software it usually requires which is sometimes more expensive than a used spectrometer.
See the attached "article" regarding digital quantification of 5-meo-dmt with TLC and a digital camera. There is excellent information there suitable for your needs.

Also, to minimize oxidation and degradation of the active materials, you will need to conduct the extraction and evaporation of solvent in the dark and under an inert atmosphere (argon, nitrogen, helium, etc).
I think it would be most suitable to extract the dry biomass in dry methanol or ethanol, evaporate to a small volume and than standardize to a specific volume.

If you dont have a standard it will be hard and expensive to get exact concentration data. However, for your task relative concentration is good enough and you could easily tell the most potent samples from the other samples.

EDIT : hmm, anyone can help me to upload a PDF file here? I could give a link to it online, but its on drug sites I dont like to use.


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Edited by trade_om (06/11/13 11:58 AM)

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Invisiblewildernessjunkie
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Re: Breeding and Selection for secondary metabolite production [Re: trade_om]
    #18404020 - 06/11/13 04:20 PM (10 years, 11 months ago)

:popcorn:

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Offlinethiotimoline
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Re: Breeding and Selection for secondary metabolite production [Re: trade_om]
    #18404660 - 06/11/13 06:25 PM (10 years, 11 months ago)

Quote:

trade_om said:
Also, to minimize oxidation and degradation of the active materials, you will need to conduct the extraction and evaporation of solvent in the dark and under an inert atmosphere (argon, nitrogen, helium, etc).




It's not necessary to do this if all you care about is relative quantities. Just make sure that, whatever procedure you end up using, you do it exactly the same way every time (under the same atmosphere (actually I would worry that if you tried to use an inert atmosphere then occasional leaks would lead to inconsistent oxygen exposure, adding extra noise to the data), same extraction time, same temperature, same light level, etc.).

It's true that I have no idea whether the cheap spectrophotometer listings are any good.

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Invisibletrade_om
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Re: Breeding and Selection for secondary metabolite production [Re: thiotimoline]
    #18406119 - 06/11/13 11:29 PM (10 years, 11 months ago)

Quote:

thiotimoline said:

It's not necessary to do this if all you care about is relative quantities. Just make sure that, whatever procedure you end up using, you do it exactly the same way every time (under the same atmosphere (actually I would worry that if you tried to use an inert atmosphere then occasional leaks would lead to inconsistent oxygen exposure, adding extra noise to the data), same extraction time, same temperature, same light level, etc.).

It's true that I have no idea whether the cheap spectrophotometer listings are any good.




You are correct in a way, but scientific experiments should be conducted under the most rigorous conditions to avoid any false data. "Leak" from the vessel doesnt happen, at least if you know what you are doing. Also keeping the same light level in a non controlled environment is going to be hard.


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Offlinethiotimoline
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Re: Breeding and Selection for secondary metabolite production [Re: trade_om]
    #18406467 - 06/12/13 01:01 AM (10 years, 11 months ago)

:shrug: I'm trying to give suggestions that minimize pain-in-the-ass (which is why I am not advising chromatography + weighing in the first place). Working under inert atmosphere is a pain in the ass and I don't want to assume too much about the lab skills or equipment quality of some random person on the internet.

Working at night with the same lighting every time is not so bad. Though darkness + dim red light is obviously best.

Or if our oxidized product *is* the colored product we are measuring then things get even simpler and all we have to do is make sure everything gets completely oxidized! (Assuming the blue stuff is stable and water-soluble. Does anyone know its solubility, or whether it degrades into something less colorful?)

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OfflinePrimalSoup
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Re: Breeding and Selection for secondary metabolite production [Re: mycot]
    #18425766 - 06/16/13 12:09 AM (10 years, 10 months ago)

Quote:

A subject which I'd like some discussion is on whether one might breed and select for high potentcy in a species.




Certainly you can, or at least you can try. Just like anything else. :popcorn:

But you'll want a way to effect mutations in the alkaloid production pathways, and SFAIK those aren't understood beyond the chemical precursor chain - at a genetic level it's a bit of a black box.

Heat at near heat death tolerance can bring on interesting mutations (I've experienced this firsthand with a Ps. cyanescens strain in domestic cultivation).  The other usual mutagenic methods might be employed with some success.  But since it's all hit or miss, your original caveats about being able to quantify results definitely apply, as this might be a process very close to a random walk otherwise. :thumbup:

:peace:PS


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