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OfflineOrangeJ
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Strain degeneration in cloning vs. isolates, and how to make isolates last
    #22429832 - 10/24/15 10:26 PM (8 years, 3 months ago)

Hi there, I have some questions about strain degeneration.

I've read that repeatedly cloning shrooms causes the strain to degenerate due to lack of genetic variance. To my understanding, this is not as much of a problem with isolates because you keep going back to the same "master culture" instead of using the DNA from newer generations of cloned (basically inbred) shrooms.

What I'm wondering is: (1)Is strain degeneration inevitable after a long period of time, given a closed population of genes such as one would find in the average homegrower's spore/isolate/shroom collection? and (2) What can I do to make a good isolate last as long as possible, WITHOUT causing further degeneration?

Thanks in advance for any tips/answers!


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OfflineOrangeJ
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22429851 - 10/24/15 10:29 PM (8 years, 3 months ago)

Oh, also: is there a way to actually add genetic variance to your shrooms' gene pool without buying extra spores or isolates from an outside source?


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OfflineRoker
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22429916 - 10/24/15 10:43 PM (8 years, 3 months ago)

growing from a tissue isolate will eventually lead to senescence. Senescence will be avoided anytime you grow from spores.

There is research that indicates senescence can be reset with an electrical discharge in the same way that a female egg with an inserted nucleus can be kickstarted into mitosis as in mammalian cloning.

Perhaps you could try pumping a few thousand volts at milliamp range through a tissue sample and see if you can revive a tired strain, a la Frankenstein?


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OfflineOrangeJ
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22429981 - 10/24/15 10:57 PM (8 years, 3 months ago)

Quote:

Roker said:
growing from a tissue isolate will eventually lead to senescence. Senescence will be avoided anytime you grow from spores.




What if I use the spores from a senescent mushroom? Will the senescence carry over to the shrooms grown from those spores, or will things "reset"?

Quote:

Roker said: Perhaps you could try pumping a few thousand volts at milliamp range through a tissue sample and see if you can revive a tired strain, a la Frankenstein?




lol :smile: That is an interesting idea, but I lack the equipment.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22430091 - 10/24/15 11:30 PM (8 years, 3 months ago)

The function of spores is to avoid senescence. A mushroom is the fruit body of two or more spores that have fused. Spores are like sperm and eggs, no two are identical and each fusion creates a fresh life with renewed vigor. I'm not an expert on this, you will get more indepth info at advanced mycology (whoops, just realized this is the advanced mycology forum). but culturing from tissue is like marrying your sister, sooner or later things start to slow down or go wrong. Spores keep things mixed up and fresh.



Edited by Roker (10/24/15 11:34 PM)


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OfflineRoker
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22430113 - 10/24/15 11:37 PM (8 years, 3 months ago)

you can also store desirable strains in distilled water. I've kept tissue samples this way and revived them after a number of years.


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OfflineOrangeJ
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22430233 - 10/25/15 12:38 AM (8 years, 3 months ago)

Ah, ok - after thinking about your explanation and reading some other threads, I've formed this rough impression of what's going on:

When a mycelium is created as a result of "spore sex", it's kind of like a newborn baby - all energetic and fresh. Then the mycelium does a whole bunch of cell divisions in order to spread itself and grow. The "age" of the mycelium, which is one of the factors that determine its vigor/speed/fruitfulness, corresponds to how many cell divisions it goes through. After going through many cell divisions and presumably growing/spreading a lot, the mycelium starts getting tired and old. This is senescence. The reason that repeated tissue cloning/transfer becomes less effective over successive generations is that you are essentially using mycelium with an ever-increasing number of cell divisions; we can think of each transfer as being a part of the same mycelial "network", only we are taking sections that are more and more on the "outside, used-up" parts of the network rather than the "central, fresh/vigorous" parts of it. So the key fact here is that the repeatedly tissue-transferred or tissue-cloned mycelium you are working with is not a "descendant" of your original culture, but is instead like an older, tired version of it!

So really, it might not be a genetic problem (or at least, not as much of a genetic problem as I thought). Instead it is all about the "tiredness" or "age" of the mycelium, which increases as the number of cell divisions increases, and as the mycelium grows. The way to renew the energy and vigor of the tired old mycelium is to create a new mycelium altogether - and this is done by sexual reproduction in the form of spores!


Of course, the above explanation by me might just be bunk. But still, it seems consistent with what many of the posts I've read on this site have been saying. I guess this is what I will go along with, unless/until experience proves me wrong.


Edited by OrangeJ (10/25/15 12:44 AM)


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OfflineOrangeJ
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22430248 - 10/25/15 12:51 AM (8 years, 3 months ago)

Quote:

Roker said:
you can also store desirable strains in distilled water. I've kept tissue samples this way and revived them after a number of years.




This sounds like a neat way of storing stuff. Does this method involve freezing? What kinds of containers did you use, and how did you get the mycelium in there? Also, how does the revival work? Where did you get distilled water?

(BTW, sorry if it seems like I am peppering you with questions. But I am trying to soak up knowledge like a sponge! :grin:)


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Offlinepsyder
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22430895 - 10/25/15 08:26 AM (8 years, 3 months ago)

You might want to take your clones from a very small spore-inoculated substrate.

After that do not clone new fruits, rather when inoculating use the master culture from your first grow and always go back to that dish. make new dishes for inoculating from the master.

Keep it wrapped in fridge and transfer to new petri every 6 or so months (had success with 1 year and 4 months old petris.)

Search for slants, they should keep it at least for a year if done properly.

And last, not the least, you can take peace of mycelium from agar(but not the agar, scrape it off) and put it in distilled water. keep in fridge (5 years max i think) Airtight container necessary.

Also you might add 15% glycerin to distilled water and freeze the culture, given low temp fluctuations and undisturbed storage it may last for a decade or so at -20 celsius

Just download lots of pdf-s titled "culture storage techniques" or something similar.

Peas!


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: psyder]
    #22431943 - 10/25/15 01:25 PM (8 years, 3 months ago)

I have read a few things regarding the distilled water storage and I am very intrigued by this storage method for LONG term storage!


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OfflineOrangeJ
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: psyder]
    #22435644 - 10/26/15 11:44 AM (8 years, 3 months ago)

Quote:

psyder said:
You might want to take your clones from a very small spore-inoculated substrate.

After that do not clone new fruits, rather when inoculating use the master culture from your first grow and always go back to that dish. make new dishes for inoculating from the master.

Keep it wrapped in fridge and transfer to new petri every 6 or so months (had success with 1 year and 4 months old petris.)

Search for slants, they should keep it at least for a year if done properly.

And last, not the least, you can take peace of mycelium from agar(but not the agar, scrape it off) and put it in distilled water. keep in fridge (5 years max i think) Airtight container necessary.

Also you might add 15% glycerin to distilled water and freeze the culture, given low temp fluctuations and undisturbed storage it may last for a decade or so at -20 celsius

Just download lots of pdf-s titled "culture storage techniques" or something similar.

Peas!





Thanks! This sounds like some good advice.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22437319 - 10/26/15 07:19 PM (8 years, 3 months ago)

For stuff that grows on logs, you can inoculate a couple of big logs and then clone the fruits every two or three years then put it on another log.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22438712 - 10/27/15 02:20 AM (8 years, 3 months ago)

Quote:

OrangeJ said:
Quote:

Roker said:
you can also store desirable strains in distilled water. I've kept tissue samples this way and revived them after a number of years.




This sounds like a neat way of storing stuff. Does this method involve freezing? What kinds of containers did you use, and how did you get the mycelium in there? Also, how does the revival work? Where did you get distilled water?




I use baby food jars and fill them with distilled water (supermarket or auto parts store should have this) pressure cook for 10-15 minutes. Add a small sample of mycelium or a piece of mushroom tissue to each jar when things have cooled down (keep things as clean as you can). Label and date each jar and leave in a cool dark place until you want to revive them. It will look like a gelatinous blob when it has been sitting in water for a few years but can still be grown out again with a bit of luck.

the tissue goes into a dormant state when it runs out of food and that happens pretty quickly in distilled water. Once you place the tissue onto agar it wakes up after a few days and you can grow it out as per usual.

I've never tried freezing wet mycelium so I can't discuss that, but I have kept dried mushroom tissue in the freezer and revived it after a couple of years.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22438816 - 10/27/15 03:24 AM (8 years, 3 months ago)

I wouldn't recommend freezing.

The sharp crystalline structure of frozen water will lyse hyphal cells.


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Offlinepsyder
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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: micro]
    #22439732 - 10/27/15 11:04 AM (8 years, 3 months ago)

^^ I agree, some manuals do list freezing as viable option. Glycerin is there to dehydrate the mycelium just for the reason you mentioned above. I need to try it yet tho.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: psyder]
    #22439837 - 10/27/15 11:30 AM (8 years, 3 months ago)

Hmm, that could work.

Are you sure it said to dehydrate the living cells?

I don't think glycerine would make a very good desiccant; it's hydroscopic but it's thick like syrup and I'd think it would cover up the water.

I wouldn't be surprised if freezing them in it worked though and for some reason I recall glycerine and freezing cells.

edit: yeah, it's on fischer's website which is why i remembered it

apparently dmso can be used, as well.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: Roker]
    #22441961 - 10/27/15 08:27 PM (8 years, 3 months ago)

Quote:

Roker said:
I use baby food jars and fill them with distilled water (supermarket or auto parts store should have this) pressure cook for 10-15 minutes. Add a small sample of mycelium or a piece of mushroom tissue to each jar when things have cooled down (keep things as clean as you can). Label and date each jar and leave in a cool dark place until you want to revive them. It will look like a gelatinous blob when it has been sitting in water for a few years but can still be grown out again with a bit of luck.

the tissue goes into a dormant state when it runs out of food and that happens pretty quickly in distilled water. Once you place the tissue onto agar it wakes up after a few days and you can grow it out as per usual.

I've never tried freezing wet mycelium so I can't discuss that, but I have kept dried mushroom tissue in the freezer and revived it after a couple of years.




Thanks!


Quote:

micro said:
The sharp crystalline structure of frozen water will lyse hyphal cells.



Quote:

psyder said:
Glycerin is there to dehydrate the mycelium just for the reason you mentioned above. I need to try it yet tho.



Quote:

micro said:
[...]
edit: yeah, it's on fischer's website which is why i remembered it

apparently dmso can be used, as well.




Would there be a possible contam/cleanliness problem with the glycerin that one could buy at a local drugstore (say, Walgreens)?


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22443530 - 10/28/15 07:02 AM (8 years, 3 months ago)

Hmm... I would have thought the suppositories they sell would be sterile but I guess not.

Since I wasn't sure if sterilizing it is even safe I had to look this up:

http://www.researchgate.net/post/can_glycerol_be_sterilized

Quote:

Luisa F. Jimenez
Hi!
For freezen my Stocks, I make a solution of 70% Glycerol, autoclave it and then mix it 1:1 with my fluid cultures that I wan to freeze (E.coli only!)
As Allan Pang told you, you can autoclave in a glassware. Just make sure that the volume of glycerol does not go over the half of the maximum volume of the flask...
good luck




So, I guess it is safe at least to those specs to sterilize it yourself.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: micro]
    #22447523 - 10/29/15 01:01 AM (8 years, 3 months ago)

Quote:

micro said:
Hmm... I would have thought the suppositories they sell would be sterile but I guess not.

Since I wasn't sure if sterilizing it is even safe I had to look this up:

http://www.researchgate.net/post/can_glycerol_be_sterilized

Quote:

Luisa F. Jimenez
Hi!
For freezen my Stocks, I make a solution of 70% Glycerol, autoclave it and then mix it 1:1 with my fluid cultures that I wan to freeze (E.coli only!)
As Allan Pang told you, you can autoclave in a glassware. Just make sure that the volume of glycerol does not go over the half of the maximum volume of the flask...
good luck




So, I guess it is safe at least to those specs to sterilize it yourself.




Thanks for the info! I think I will use my pressure cooker as sort of an improvised autoclave then, after I mix up the 70% glycerin solution.

For detailed instructions on sterilizing using a pressure cooker, I found this helpful link: http://www.umsl.edu/~microbes/sterilizing.pdf . Sterilization directions are under the subtitle "Sterilizing with Moist Heat and Pressure". It seems that in both the link you sent and the one I found, it is important to make sure that the volume of the container is twice the volume of the glycerin/water you're using.


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Re: Strain degeneration in cloning vs. isolates, and how to make isolates last [Re: OrangeJ]
    #22449724 - 10/29/15 03:44 PM (8 years, 3 months ago)

Correct; it'll boil pretty vigorously and you don't want it to boil over.

Especially with glycerin; that could make a mess x.x


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