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Invisibleteknix
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Wild Reishi Clone Grow. * 1
    #27002833 - 10/25/20 02:07 PM (3 years, 5 months ago)

So I found these Reishi in the Wild and decided to clone it and cultivate it.



I had dried them all at low temps around 90F, and stored them for a few months in sandwich bags until I felt like taking on the project.

For cloning I just poured a 10% bleach/water solution in one jar and plain water in another jar for rinsing. I took the softest tissue from the specimen I had dried and dunked it in the bleach water. I poked the tissue into the water, stabbing it down with the forceps to bbbreak the water tension on the surface of the tissue. I let it soak up bleach from 2 minutes to probably about 7 minutes, which was the length of the process.

After all the tissue I was going to put to agar was sitting in the bleech solution for 2 minutes I started the process by using forceps to take out a tissue sample and dry it on a paper towel, I then dunked it in the glass of water and then again dried it on a paper towel, (which I had set out in advance). I then put the tissue onto agar. I did this for the rest of the tissue until I had made a total of 8 plates.



(The plates are fully colonized now and in a fridge minus a few that got contaminated, which was acceptable considering I was open air inoculating and using a time spread for the bleach soak.)


I then moved some wedges from a couple of plates to sterilized WBS and allowed it to colonize. (fresh out of popcorn t.t)



I then proceeded to spawn the WBS to sterilized fuel pellet/sawdust blocks with about 10% verm added and a sprinkling of calcium carbonate. I used roughly 4 teaspoons of spawn per bag, so 1 quart spawned all 7 bags. There is roughly 3 quarts of substrate per bag.



Now I wait :smile:

Edited by teknix (10/25/20 03:47 PM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003151 - 10/25/20 05:08 PM (3 years, 5 months ago)

Awesome, I love wild clone grows!

Do you know what species you're working with?


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27003208 - 10/25/20 05:33 PM (3 years, 5 months ago)

Best guess is Ganoderma Sessile, considering the G. Lucidum that I have has growth characteristics that is much different. I might do some taxonomy if I get some decent results to find out for certain. It is definitely KOH positive for Ganoderic Acid tho.

I had spread a G. Lucidum Slurry around the area the previous years, but this mycelium grows quite a bit different from the G. Lucidum mycelium. I didn't particularly inoculate the tree so I'm guessing it's not the G. Lucidum.

I also have a run of G. Lucidum incubating to compare it to later.

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003217 - 10/25/20 05:38 PM (3 years, 5 months ago)

Nice, looking forward to see how it fruits.  Ganoderma is one of my favorites to watch :smile:


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Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003223 - 10/25/20 05:41 PM (3 years, 5 months ago)

The G. Lucidum are almost done incubating so, unfortunately, I won't be able to do a true side by side.

But the substrate and spawn ratios are very similar.


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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27003244 - 10/25/20 05:53 PM (3 years, 5 months ago)

Here are a few specimen I dried and kept from my first and last grows, can you tell which is which? :P



Hopefully I can get some conks to sporalate again. Since the spores contain the highest concentrations of Ganoderic Acid(?), that is most desirable to me.

Edited by teknix (10/25/20 07:11 PM)

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27006592 - 10/27/20 03:34 PM (3 years, 4 months ago)

Great overview of your methods - thank you for sharing!  Will come back and update here if I try it.  :smile:

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27016804 - 11/02/20 06:03 AM (3 years, 4 months ago)

Quote:

teknix said:

Hopefully I can get some conks to sporalate again. Since the spores contain the highest concentrations of Ganoderic Acid(?), that is most desirable to me.




Just remember you'd have to crack the spores to get that stuff out of them. It's not easy, but there's some threads around on methods


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27018312 - 11/02/20 09:01 PM (3 years, 4 months ago)

Haha yeah, that's why you do the alcohol extraction first right? :smile:

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27019928 - 11/03/20 05:44 PM (3 years, 4 months ago)

I'm pretty sure even alcohol doesn't get much of the stuff inside spores when they're not cracked.  Cracking them requires either germination (which reishi has a terribly low germination rate), or some pretty intense mechanical methods.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27019995 - 11/03/20 06:38 PM (3 years, 4 months ago)

It looks like you are right about the insolubility of chitin in alcohol. I found a decent write-up that tested the solubility of chitin in various solutions.

https://www.intechopen.com/books/solubility-of-polysaccharides/solubility-of-chitin-solvents-solution-behaviors-and-their-related-mechanisms

I'm not sure if the Ganoderic Acid in a spore would become bio-available through digestion? I'll have to look for more research about that.

Evidently we can break down the chitin cell walls through digestion, but increasing the bioavailability would definitely be better.

https://www.researchgate.net/publication/6249199_Human_Gastric_Juice_Contains_Chitinase_That_Can_Degrade_Chitin

Looks like trichoderma has the chitinase enzyme as well:

https://www.mybiosource.com/enzyme/chitinase-trichoderma-viride/653950

So techinically we could use trichoderma to increase the bioavailability of ganoderic acid in Reishi spores? However I am currently at a loss on the procedure we would use to do so. Chitinase with the spores could cause a slow-release of ganoderic acid, maybe making more of it useful in the long run. You get your instant GAE from the extraction and save some in the spores to be released later with the enzymes.

Thanks for bringing this up Forrester, I appreciate it.

Edited by teknix (11/03/20 07:10 PM)

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27020428 - 11/03/20 10:19 PM (3 years, 4 months ago)

Also chitinase in soybean seed coats.

https://academic.oup.com/jxb/article/52/365/2283/543833

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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27021991 - 11/04/20 05:45 PM (3 years, 4 months ago)

No, I wasn't referring to chitin being insoluble.  We can still get the good stuff out of mushrooms via alcohol despite that.

I was talking about what's inside spores themselves, it's an entirely different thing.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27022090 - 11/04/20 06:43 PM (3 years, 4 months ago)

Interesting, so the higher concentration of G. Acid are largely bio-unavailable in spores:

Zhou et al. (2012) observed that dietary SGL (2 g/kg) had no effect on oxidative stress and mitochondrial dysfunction in the hippocampus of rats. The activities of spores are closely related to the status of the sporoderm, and sporoderm-broken spores of G. lucidum (SSGL) are more effective in modulating the immune responses in rats than sporoderm-unbroken spores (Yue et al., 2008).

The spores were dried at 55 Β°C for 24 h and then were broken by a supercritical fluid extraction device according to Fu et al. (2009) and Li et al. (2011). Briefly, approximately 150 g of the spores was loaded into a steel cylinder equipped with mesh filters on both ends. Liquefied CO2 was pumped into the vessel, and the pressure was raised to 35 MPa. The temperature was controlled at 25 Β°C during the processing. The pressure was released within 1 min at the end of the 4-h process. During the process of rapid depressurization, the resistance of the sporoderm created a pressure difference inside and outside the sporoderm, and the spores were broken when CO2 burst out of the sporoderm. Sporoderm-broken spores of G. lucidum were collected and stored at βˆ’20 Β°C before adding into the diets.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082644/

I saw this guy (NileRed) on youtube build a supercritical CO2 chamber to make aerogel. But it cost about $2k to build.



Here is another one, just using dry ice.


Edited by teknix (11/05/20 01:49 AM)

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Offlinedeadmandave
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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27022212 - 11/04/20 07:40 PM (3 years, 4 months ago)

So much easier to just grow more reishi and make more tincure than do anything to the spores. But it is interesting nonetheless.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: deadmandave]
    #27022274 - 11/04/20 08:13 PM (3 years, 4 months ago)

Deinitely, there are also ways to increase ganoderic acid production, that are much simpler like in this paper where they use Aspirin in an LC (submerged culture):

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053616

But they degrade the cell walls prior to extraction with enzymes:

Fungal cells of G. lucidum were treated with aspirin for 16 hr, fixed with 4% paraformadehyde for 1 hr, washed with PBS, and then digested with cell wall degrading enzymes (0.5 U mLβˆ’1 of driselase, 1050 U mLβˆ’1 of Ξ²-glucaronidase, 81.25 U mLβˆ’1 of lyticase, 5 mg mLβˆ’1 of lysing enzyme, and 0.015 U mLβˆ’1 of chitinase) for 30 min.

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Re: Wild Reishi Clone Grow. [Re: deadmandave]
    #27026078 - 11/07/20 04:07 AM (3 years, 4 months ago)

Quote:

deadmandave said:
So much easier to just grow more reishi and make more tincure than do anything to the spores. But it is interesting nonetheless.




^^ this.


--------------------
Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here.
-------------------

Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: Forrester]
    #27026839 - 11/07/20 01:14 PM (3 years, 4 months ago)

Update 13 days since spawn:

Growth seems pretty slow and a weaker mycelial mat in comparison to the G. Lucidum. I didn't mix the spawn around at all, I just left the blocks as tightly compact as they were when they came out of the pc and put the spawn on top of it.



Here is the G. Lucidum under lights now:


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Re: Wild Reishi Clone Grow. [Re: teknix]
    #27027393 - 11/07/20 06:35 PM (3 years, 4 months ago)

cool!  ya prob loose all the antioxidant effects getting micro dosages of paraformaldehyde and whatever extras are in the solution form manufacture... 

if get any amount of spores. maybe just ferment and all teh microbes would break down cell walls eventually. or put in disitlled water and do a couple freeze thaw cycles. maybe even with dried ice?? i dunno space and spores as they say but water and ice have a good mechanical action and knock over mountains..

one question what time frame for getting reishi to fruit?  in my mind it was 6 months + to get decent fruits but likely not the case. thanks!

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Invisibleteknix
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Re: Wild Reishi Clone Grow. [Re: the man]
    #27027829 - 11/08/20 12:53 AM (3 years, 4 months ago)

It depends largely on the strain you are working with and if you are shaking/mixing the spawn/sub halfway through, as well as the amount of spawn that you use and even the supplementation. I think the G. Lucidum is a total of about 3 months for me from inoculation of grains to fruit. It's a rough estimate, but about 2-4 weeks to colonize grains, 2-4 weeks to colonize the substrate and 3+ weeks to fruit.

I'm using little amounts of spawn and not shaking/mixing, and the wild clone is slow to colonize so it will take longer in my case.

The ones in the humidity tent are pinning, I've been spraying them with a J.Crows Lugol's Solution dilute and it isn't looking too great.


Edited by teknix (11/08/20 01:38 AM)

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