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OfflineSkysTheLimit
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Cloning from dried mushroom tissue techniques?
    #18929216 - 10/04/13 01:02 AM (10 years, 4 months ago)

Okay, has anyone SUCCESSFULLY cloned from dried tissue and what method did they use?

I tried looking for RR's method but the original post was deleted.

I read alot of posts of people claiming its impossible but I know that commercial laboratory's deliberately dehydrate mushroom fruit bodies and reactivate it years later to reclaim the parent strains.

Thanks in advance!


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OfflineSkysTheLimit
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Re: Cloning from dried mushroom tissue techniques? [Re: SkysTheLimit]
    #18929323 - 10/04/13 01:54 AM (10 years, 4 months ago)

Okay well, currently I am trying to clone dried shiitake tissue (dried in dirty air) by dipping in 1% peroxide for different intervals (5 secs, 30 secs, 1 min, 2 min) and placing it in either a PDY LC or just in a empty test tube. The chunks were a bit too large (tip of finger sized) and taken from every part of the mushroom (it was too hard to cut dessicated mushroom).


2nd day and no visible mycelial growth.


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OfflineRogerRabbitM
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Re: Cloning from dried mushroom tissue techniques? [Re: SkysTheLimit]
    #18929570 - 10/04/13 03:35 AM (10 years, 4 months ago)

Peroxide is just going to damage the mycelium right when you want it to grow.

Use distilled water with no nutrients.  Wash a dozen or so small chunks about 2mm in diameter in a jar of distilled water.  Strain off and repeat the wash with fresh, sterilized distilled water. Repeat a few times and then leave the pieces floating after the last rinse in a jar with proper filter.

Wait for a piece or two to fuzz up, and then transfer to antibiotic agar.

Transfer as necessary until you have a clean culture.
RR


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Offlinehidyn
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18929677 - 10/04/13 04:47 AM (10 years, 4 months ago)

Interesting! Does this mean that I could recover a viable culture from the dried mushrooms available in grocery stores? My local Asian market stocks a wide variety.


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OfflineSkysTheLimit
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18929931 - 10/04/13 07:01 AM (10 years, 4 months ago)

Rogerrabbit: Ahh ok so that's the post you made, thanks alot!
I'll try this tonight

Hidyn: Yeah im trying that with a grocery strain as well, provided it hasn't been sterilised by heat or gamma rays then the fruit body cells should still be 'alive'.


Edited by SkysTheLimit (10/04/13 07:11 AM)


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Offlinej_db69
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Re: Cloning from dried mushroom tissue techniques? [Re: SkysTheLimit]
    #18937520 - 10/05/13 07:11 PM (10 years, 4 months ago)

one more to add to hidyn's question: if, in theory, the moisture content of a fruit body was 0%, would it be possible to bring it back to it's full "vigor"?  How long at 0% until it would not grow (if at all)?


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OfflineRogerRabbitM
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Re: Cloning from dried mushroom tissue techniques? [Re: j_db69]
    #18938947 - 10/06/13 05:09 AM (10 years, 4 months ago)

I have a 150 year old mushroom growing book and in it they describe saving dried spawn for years.  They hadn't yet learned sterile technique in those days so they would dig up some mycelium from the mushroom beds, dry it, and then use it to spawn new beds later, often much later.
RR


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Offlinej_db69
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18939333 - 10/06/13 09:09 AM (10 years, 4 months ago)

Would you not say that "dried spawn" would consists of >1% moisture though?  Would the theoretically 0% spawn or body re-animate?

and thanks for the idea skysthelimit :thumbup:  I too will be looking over the supermarket selections with a new eye


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OfflineRogerRabbitM
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Re: Cloning from dried mushroom tissue techniques? [Re: j_db69] * 1
    #18939669 - 10/06/13 10:50 AM (10 years, 4 months ago)

It would be awfully hard for the spawn to be much less than the ambient humidity level. It will absorb moisture from the air so it's never at zero moisture.

Don't waste too much time on grocery store specimens.  If the grower was worth his salt he already expanded his spawn as far as practicable before inoculating the bulk substrate to grow those mushrooms.  If you create spawn from them, you start out with geriatric cell lines before you do your first grain to grain tranfer.
RR


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semper in excretia sumus solim profundum variat

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Offlinej_db69
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18941281 - 10/06/13 05:03 PM (10 years, 4 months ago)

damn, master slants are more valuable than I had thought.

I need to read up and see what the "magic" number is for most fungus' generations. thanks for the read.  I guess the more rare, the further you let the gens go, compromising "vigor" for safety...


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OfflineRogerRabbitM
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Re: Cloning from dried mushroom tissue techniques? [Re: j_db69]
    #18941551 - 10/06/13 06:04 PM (10 years, 4 months ago)

A grain to grain transfer is not a generation.
RR


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Offlinehidyn
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18948617 - 10/08/13 08:59 AM (10 years, 4 months ago)

Quote:

RogerRabbit said:
If the grower was worth his salt he already expanded his spawn as far as practicable before inoculating the bulk substrate to grow those mushrooms.  If you create spawn from them, you start out with geriatric cell lines before you do your first grain to grain tranfer.
RR




Could you please expand on this?

Are you saying that the grower would just keep dividing their spawn until they had a pile of spawn bags full of mycelium that just didn't want to colonize anymore, then fruited them?

If a mycelial network can in fact 'die of old age', do you have any sorts of information as to how long this can take? How do you reset the clock? Can you just germinate spores from the fruiting bodies? If so, is there enough genetic material in the spores to start anew? Is letting multiple spores germinate and mesh together enough, or would you eventually need to cross your strains with someone else's?


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Invisiblehamloaf
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Re: Cloning from dried mushroom tissue techniques? [Re: hidyn]
    #18949202 - 10/08/13 11:43 AM (10 years, 4 months ago)

Mycelium can only divide it's cells so many times before it gets "geriatric", yes. It's called senescence.

If the farmer is doing it right they're expanding their culture to point of senescence so that their strains are not worth the effort of cloning or they have to expand their cultures out so far out of necessity in order to support the size of their grows. Hit up a sponsor or something and get yourself some fresh live cultures delivered to your door.


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Re: Cloning from dried mushroom tissue techniques? [Re: hamloaf]
    #18949835 - 10/08/13 02:22 PM (10 years, 4 months ago)

Hrm. Thank you!

I've begun work with agar and I'm quite enthusiastic about developing my own selective breeding program.

I'm going to look a little closer into how long I can maintain a tissue bank in my current situation.

Thanks for the info!


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InvisibleGimpCollector
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Re: Cloning from dried mushroom tissue techniques? [Re: RogerRabbit]
    #18967661 - 10/12/13 09:44 AM (10 years, 4 months ago)

I should be good as RR mentioned a G2G is not a generation. At what point does a action become a "generstion"
Clone,  grow out clone, clone from grown out fruit,  equal 1 generation cycle?

Im a bit confused with the geriatric comment. I don't do agar work. I've selected a specimen to clone and popped a chunk into a jar of grains. From this ive been doing g2g transfers. Am I going to hit a wall with the genetics and waste my time this way.  I only work with one type of strain so agar and slants never worked into my lab. 1 jar becomes 10. Rinse and repeat. Is that bad


Edited by GimpCollector (10/12/13 09:59 AM)


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OfflineSkysTheLimit
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Re: Cloning from dried mushroom tissue techniques? [Re: GimpCollector]
    #18969611 - 10/12/13 07:31 PM (10 years, 4 months ago)

hey, here's to clarify, a generation is when sexual reproduction has occurred. In relatioin to mushrooms, thats when they form a fruiting body and sporulate. Generation 1 is the mycelium you originally had, and when the mycelium produce spores and those spores produce mycelium, that myclium is called generation 2.

However, continuously expanding the same generation can cause the mycelium to lose vigor as well.


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InvisibleGimpCollector
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Re: Cloning from dried mushroom tissue techniques? [Re: SkysTheLimit]
    #18969630 - 10/12/13 07:41 PM (10 years, 4 months ago)

Hmmm, so it's a fine line one has to walk. Guess when your chosen mycelium runs out of steam you look for a new donor. Thanks for your reply.


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Offlinej_db69
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Re: Cloning from dried mushroom tissue techniques? [Re: GimpCollector]
    #18972673 - 10/13/13 03:47 PM (10 years, 4 months ago)

Is there a curve one follows, especially with commercial growing, that represents the life cycle of fungus?  As in, does the "vigor" remain constant until a certain point?
OR does it steadily improve to a point, then decline from there?  A graph would be really cool.

What other outside forces effect "vigor"? stress? consuming certain materials?


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OfflineSkysTheLimit
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Re: Cloning from dried mushroom tissue techniques? [Re: j_db69]
    #18973943 - 10/13/13 09:00 PM (10 years, 4 months ago)

A graph of the timeline of the vigor of strain would be a very slow but steady decline starting from germination.
This is because over 50% of the spores produced are less vigorous than the last generation, and because growers can't pick out the best spores (in nature the less vigorous spores get out competed, leaving the more vigorous ones to grow) the strain slowly becomes less vigorous because the nature of sexual reproduction.

There should be outside forces affecting vigor, stress would be one. In humans, high caloric intake results in faster 'expenditure' of our cells.


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Invisiblefastfred
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Re: Cloning from dried mushroom tissue techniques? [Re: SkysTheLimit]
    #18977278 - 10/14/13 03:56 PM (10 years, 4 months ago)

Senescence is not a problem for growers unless they ignore all common sense and are completely ignorant of the phenomenon.

When you isolate a good specimen you grow it out a little and preserve it (refrigerate, etc., etc.).  For each grow you go back to the original sample.

Only a dumbass would try starting over each time with older tissue.  I'm not even sure how someone would do that.  Trying to start over from spent substrate or some other foolery I guess.  Otherwise it's not something you need to worry about.


-FF


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