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InvisibleLa_Magdalene
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tissue and grain
    #1233925 - 01/20/03 10:26 AM (21 years, 4 months ago)

I was wondering if it is possible to put live tissue from a fruitbody back to grain (millet) successfully??


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Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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OfflineOn_the_Down-Low
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Re: tissue and grain [Re: La_Magdalene]
    #1233939 - 01/20/03 10:31 AM (21 years, 4 months ago)

Do you want to clone it or grow it from spores??
The easiest way is to put some of the fresh gill material into the jar to be inoculated.

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OfflineAzmodeus
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Re: tissue and grain [Re: On_the_Down-Low]
    #1233971 - 01/20/03 10:43 AM (21 years, 4 months ago)

WTF!!!? That works?! I always put the tissue in honey water, let it grow out and then innoculate rye jars. But if you can put the tissue directly on the rye and it colonises, thats even better!!


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Lest we forget. "

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OfflineJssMthrFcknChrst
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Re: tissue and grain [Re: Azmodeus]
    #1234205 - 01/20/03 12:18 PM (21 years, 4 months ago)

Gill material would contain sprores. If you get innoculated with spores, then the jars wouldnt contain the same genetics, unlike a honeywater innoculation which creates clones.


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InvisibleLa_Magdalene
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Re: tissue and grain [Re: JssMthrFcknChrst]
    #1234223 - 01/20/03 12:26 PM (21 years, 4 months ago)

What if one were to omit the honey and just use sterile water with the live tissue? Suck up the blended H2O/tissue mixture and use to inject jars of millet? Does this sound viable?


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Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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OfflineOn_the_Down-Low
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Re: tissue and grain [Re: La_Magdalene]
    #1234604 - 01/20/03 03:45 PM (21 years, 4 months ago)

That's basically how you make a spore syringe.  :blush: 

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Invisibledeanofmean
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Re: tissue and grain [Re: La_Magdalene]
    #1234719 - 01/20/03 04:29 PM (21 years, 4 months ago)

as stated above, fresh gills can be used instead of a spore print .
for cloning of live tissue, you would need to use a mycelium culture .i dont think you can just skip this step . that would be like putting a stick in the ground and expecting it to grow a tree . i'm not trying to be cruel here, getting the point across . :smile:
http://www.shroomery.org/index/par/9 

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Offlinethe spiral
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Re: tissue and grain [Re: deanofmean]
    #1235068 - 01/20/03 07:27 PM (21 years, 4 months ago)

i'm surprised at some of the answers to this post.

a tissue transfer is a perfectly viable way to clone your existing fungus. If you work in suitably sterile conditions, a piece of fruitbody or colonized grain/birdseed/whatever can be dropped into a freshly cooked bag of substrate, and it will grow - much faster than if you'd used spores, because you don't have to wait for germination.

there's no need to go through a liquid mycelium stage. Direct transfer of mycelium is a very effective, very common method.


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"A celibate clergy is an especially good idea, because it tends to suppress any hereditary propensity toward fanaticism." - Carl Sagan

Edited by the spiral (01/20/03 07:39 PM)

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Invisibledeanofmean
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Re: tissue and grain [Re: the spiral]
    #1235130 - 01/20/03 08:14 PM (21 years, 4 months ago)

Quote:


there's no need to go through a liquid mycelium stage. Direct transfer of mycelium is a very effective, very common method.




yes, but that was not the question .
please correct me if i'm wrong, but the tissue of a fruit is not the same as mycelium .
it will produce mycelium, but in its first stages of growth would not be strong enough to colonize grain at a rate that would be productive .
but you are welcome to try .i would love to be shown how it could be done .

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OfflineAnnoA
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Re: tissue and grain [Re: deanofmean]
    #1235153 - 01/20/03 08:55 PM (21 years, 4 months ago)

>but in its first stages of growth would not be strong enough to colonize grain at a rate that would be productive

It would and it is actually.

Read http://www.shroomery.org/index/par/7923/pag/6 .

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InvisibleG a n j a
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Re: tissue and grain [Re: deanofmean]
    #1235193 - 01/20/03 09:55 PM (21 years, 4 months ago)

Quote:

. that would be like putting a stick in the ground and expecting it to grow a tree . 




This does actualy work with many tree species :tongue: infact the largest "cutting" ive seen was a 20f willow branch that snapped of in a storm from over 10 foot above the ground,landed snaped end first and grew on as a tree in its own right :smile:

Bit of toppic but interessting :smile:



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Invisibledeanofmean
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Re: tissue and grain [Re: Anno]
    #1235207 - 01/20/03 10:16 PM (21 years, 4 months ago)

oh, i've seen that tec .i guess it didn't occur to me it would be an efficient way to inoculate millet .  :cool: thanx
  which method would win a race i wonder ? 

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Invisibledeanofmean
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Re: tissue and grain [Re: G a n j a]
    #1235216 - 01/20/03 10:46 PM (21 years, 4 months ago)

>This does actualy work with many tree species
yes in know, with the right conditions, but its not always that easy .
i was reaching for a graphic exsample .lol maybe it was a bad one  :tongue:
have you ever tried to grow boganvilla from cuttings ? :laugh: i had some actually starting to root once, but i couldn't keep the ducks out of the vermiculite . :grin:
lol happy shrooms 

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InvisibleLa_Magdalene
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Re: tissue and grain [Re: the spiral]
    #1235482 - 01/21/03 03:13 AM (21 years, 4 months ago)

Thank you Harry Manback....this was the answer that I was looking for. I am fully compitent in making clones (from back in the PF jar days) ...but was wondering how clone tissue would colonize millet grain? If the tissue would be strong enough to innoculate an entire quart?


--------------------
Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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InvisibleLa_Magdalene
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Re: tissue and grain [Re: La_Magdalene]
    #1235488 - 01/21/03 03:17 AM (21 years, 4 months ago)

The reason I ask this Q is because I have discovored what a flop I am at making spore syringes.  Must say is the most difficult thing encountered thus far.  I am essentially at a dead end with my G2G's....on G2 now and am worried about deteriorating the strain.  I understand that cloning is not a viable option for long...but would buy me enough time to figure something else out...aside from spore syringes.  :confused: 


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Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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OfflineJssMthrFcknChrst
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Re: tissue and grain [Re: La_Magdalene]
    #1235570 - 01/21/03 04:01 AM (21 years, 4 months ago)

Can you really deteriorate a strain when using grain to grain transfers? I could understand if you used live tissue to make a clone, because you would be going from fruit body back to vegetative. Kinda like pot. If you keep using the same mother without flowering it, the strain stays strong.

Am I off track here, can you stretch your g2g clones too far?

-jssmthrfcknchrst


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Offlinedebianlinux
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Re: tissue and grain [Re: JssMthrFcknChrst]
    #1235601 - 01/21/03 04:20 AM (21 years, 4 months ago)

yes you can go too far. you will know too. the mycelium gets pretty wimpy.

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OfflineJssMthrFcknChrst
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Re: tissue and grain [Re: debianlinux]
    #1235611 - 01/21/03 04:27 AM (21 years, 4 months ago)

Why is this? Is it because the mycelium just can't take the stress of being broken up and spread out? I would think that it would just keep growing like in nature. huh


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OfflineOn_the_Down-Low
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Re: tissue and grain [Re: La_Magdalene]
    #1235638 - 01/21/03 04:36 AM (21 years, 4 months ago)

So what's the problem with the syringes?? Not viable or contams?????

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Re: tissue and grain [Re: JssMthrFcknChrst]
    #1235646 - 01/21/03 04:39 AM (21 years, 4 months ago)

can't say I know specific reasons why but let's see how well you play basketball when your 65 years old. now if you reproduce and have a kid when your 40 how well will your 25 year old play basketball compared to you at 65? it's called a lifecycle and a lifespan, all living things have them.

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OfflineJssMthrFcknChrst
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Re: tissue and grain [Re: debianlinux]
    #1235757 - 01/21/03 05:12 AM (21 years, 4 months ago)

alright, i can see the logic in that.

now, if you slowed down the life cycle of the fungus by storing grain jars in the fridge, would you be able to stretch the strain further. This is done with cultures, keeping them viable for years.

Example:
Innoculate 12qt jars w/ spores
Choose healthiest looking one and g2g into 12 more jars, use others as substrate
g2g 1 of the new jars to 12 more jars, store 11 in frige
Use 12 jars as bulk spawn
Go back to other 11 to spawn more jars.
When you're down to 1 jar in fridge, g2g 12 more, save 11.

Hope you could follow my logic here. Also, would this work by freezing?

jssmthrfcknchrst


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Re: tissue and grain [Re: JssMthrFcknChrst]
    #1235778 - 01/21/03 05:16 AM (21 years, 4 months ago)

yes

edit: had to rethink that. in theory it works. on agar it works. as far as colonized grain jars it sounds right but only an experiment would tell as I have not tried that (my jars are used much more efficiently if I'm colonizing and reusing versus just storing)
edit2: also looking at your example I would suggest storing one or more of the 1st set of 12 quart jars (as opposed to the 2nd generation) so your stopping the aging process at an earlier point
edit 3 (:)) revised example

inoculate 12 qts, colonize, weed out any weak looking ones, refrigerate all but 1
use the 1 to inoculate 12 more. use the 12 to inoc 144 or just fuit from these 12. once these are down go back to the fridge and get the next jar to span out. the combinations are endless but it goes back to my suggestion of refrigerating the 1st generation.

Edited by debianlinux (01/21/03 05:24 AM)

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Invisiblemycofile
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Re: tissue and grain [Re: JssMthrFcknChrst]
    #1235871 - 01/21/03 05:44 AM (21 years, 4 months ago)

Quote:

which method would win a race i wonder ?



in general culture transfers are faster than spores innoculations. Of course within reason, 500 ccs of spores might outrace a 1 cm piece of shroom stem. The fastest to colonize will always be a liquid innoculum. In this case, baring liquid cultures like honey tek, the fastest way would be a blended fruitbody used as liquid innoculum (see 9er's tek, or shroomdadi's mycomachine).

Quote:

I am essentially at a dead end with my G2G's....on G2 now and am worried about deteriorating the strain



and
Quote:

Why is this? Is it because the mycelium just can't take the stress of being broken up and spread out



I think people worry far too much about strain degeneration. Cultures are suspected to degrade in this sense due to rare random mutations. These mutations eventually build up over time. A certain number of cell divisions is usually the determining factor (well into the millions of cell divisions). Refrigeration won't stop this without stopping growth. Keeping a stock of cultures as close to the original clone in cell divisions is the way to keep this in check. For example, when making the original clone, make several copies and store these in the fridge for later use. Then you can go back to a young cell line when you need to.

Now to address the G2G concerns. I wouldn't worry about making several generations as long as the first one is started from a relatively young culture (meaning it hasn't been cloned and fruited and cloned again etc.) Think about this to give you an idea of how many cell divisions it takes to encounter senescence. In GG&MM, stamets recomends that 1 petri be used for liquid culture which then through liquid innoculation of grain, grain to grain transfers (up to 3 generations if sterility is maintained) and finally spawning of bulk sustrates, that one petri can effectively fill an entire warehouse with mycelium. Stamets recomends this method and never once mentions senescence in regards to this. The number of cell divisions in this scenario is far more than you will encounter in simply 3 or 4 generations of g2g even if you take the 4th generation to a bulk substrate because most home cultivators use much higher spawn rates than the pros use, therefore meaning you aren't stretching the spawn with as many cell divisions.

I've seen a 2nd gen qts innoculate 2 gallon bags and the bags then innoculate 3 cubic feet of bulk substrate with absolutely no decline in vigor. I've even cloned from the resulting fruits and repeated the entire process with no decline. In other words, I wouldn't worry about strain degradation until you see it, then back off accordingly.


--------------------
"From a certain point of view"
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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: tissue and grain [Re: mycofile]
    #1235904 - 01/21/03 06:11 AM (21 years, 4 months ago)

Quote:

ln GG&MM, stamets recomends that 1 petri be used for liquid culture which then through liquid innoculation of grain, grain to grain transfers (up to 3 generations if sterility is maintained) and finally spawning of bulk sustrates,




um, does that not support my argument? (it is mentioned in TMC as well as he DOES mention senescence becoming apparent after G3), In my experience senscence will be pronounced in G4 and the mycelium is practically useless in G5.

I'm also confused about your comment concering refridgeration.
Quote:

A certain number of cell divisions is usually the determining factor (well into the millions of cell divisions). Refrigeration won't stop this without stopping growth



that IS the point of refridgerating. the jars will cease (mostly) to grow and divide and therefore age. they will grow just fine when normal tempratures are restored. I have proven this with experiments as well.

edit: maybe I'm just confused and thought you were arguing against my posts...

Edited by debianlinux (01/21/03 06:13 AM)

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InvisibleLa_Magdalene
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Re: tissue and grain [Re: mycofile]
    #1236252 - 01/21/03 08:53 AM (21 years, 4 months ago)

Essentially, according to your theory Mycofile, I should be able to make MANY jars of G3 from one of my G2's without experiencing any adverse effects to the strain?  If that is the case, then I am fine for a while on my fridge full of G3's....keeping my last G2 for usage at a latter time?
As for the spore syringes....I found that sucking the spores INTO the syringe to be a rough task.  I would scrape many spores with the steralized needle tip into the shot glass, but then they would all clump together, stick to the outside of the needle and such.  I'm afraid not many spores made it into the syringe.  Also...they leak when I shake them.  The needle is in position correctly, this I know.  However, theoretically...if water can leak out, contams surely can leak in, so I am even afraid to wast a jar of grain on the shotty syringes.  Perhaps one day when I am bored, maybe try them out to my amusement??  See just how fast green can grow! LOL :grin: 


--------------------
Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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Anonymous

Re: tissue and grain [Re: La_Magdalene]
    #1236950 - 01/21/03 12:52 PM (21 years, 4 months ago)

When storing in the future try and store the youngest pure cultures or jars you can. Since you already have G2 and want to store G3, go for it. Just in the future it would be better to store the G1 jars.

Degeneration does happen. With plants as well, when cloning. Just might take some time. Home cultivators don't have the similar investment laid out into a crop, that a commercial Mushroom Grow op has. When your trays go bad, you didn't have a warehouse full of them.


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Re: tissue and grain [Re: La_Magdalene]
    #1237916 - 01/21/03 05:46 PM (21 years, 4 months ago)

Try the liquid culture via syringe. Either the honey tek or the 'blender tek'.

I think that 'reverting' back to a spore print from a cloned shroom would effectively restart the clock , so to speak. As long as you are trying to just preserve a strain, and not improve/select it.

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Invisiblemycofile
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Re: tissue and grain [Re: debianlinux]
    #1238491 - 01/22/03 03:18 AM (21 years, 4 months ago)

I wasn't trying to argue against your posts, just sharing my experience.

Senescence does happen, and stamets does mention it. I really don't think that he warns against it in G3's in TMC. If I remember he warns against G3's because of the ample opportunities contams have had to infiltrate the culture. Basically that most cultivators see dramatic increases in contam rates on G3. Could be wrong though. I do know that I never saw senescence on G3 or 4. Although I rarely did a G4, and my third generation usually used a whole quart to spawn a 1-2 gallon bag (a higher spawn rate than the 10% usually used in G2G) perhaps that has something to do with it?

If you've seen pronounced senescence in G4's, then perhaps that is more common than my results which were limited with G4's. Perhaps this variation is due to substrate, strain, inc temps or something we could figure out? Either way, I think the best way for a home cultivator to deal with senescence is to wait until they see it, then scale back. Worrying about it before you run into it may limit you. Especially in this case where he's having trouble growing anything else.

On the fridge thing, your right. I just thought I read a post that seemed like it was saying basically "grow the jars in the fridge and they won't go bad". I was just reinforcing that low temps keep the mycelium from growing, they don't allow the mycelium to grow, while reducing the random mutations which cause strain degredation.

Since you sound like you're experienced with fridge storage, what is the longest you've been able to store grain in the fridge? Did you find it more effective to refrigerate at a particular stage? Fully colonized, just spawned, or somewhere in the middle?


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

Edited by mycofile (01/22/03 04:04 AM)

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Invisiblemycofile
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Re: tissue and grain [Re: La_Magdalene]
    #1238549 - 01/22/03 03:54 AM (21 years, 4 months ago)

Not in my theory, IME. I've had success using the standard 10% spawn rate to make G3's. Your best bet is to always stay as young as possible in regards to cell divisions. So, you've got a fridge full of G2's now, right? How about just using one of those to make 10 G3's and see how the G3's grow?

If you do have 12 G2's (which is what I understand), then save those as long as you can. If the G3's work well for you (and I think they will), then over time as you need them you can make up to 110 more G3's. Alternatively, if you do have some trouble with the G3's, use a lower spawn rate and see if that helps (20, even 50%). That should last you more than enough time for you to get the spore syringe process worked out.

Tip, try a very tiny amount of sex lube in your syringe water, it keeps spores from clumping. ForPlay sex lubricant has been recomended. ( http://www.nansnook.com/forum/index.php?act=ST&f=21&t=415&hl=sex+lube&s=43ae57ea9bc2614585df17fac4af9647 ) Anno uses a tiny amount of dish soap, but that's not as fun! Also, the best little known spore trick is to blend your spore solution (see blender usage in 9er's tek) I'm not sure if I'd use soap in the blender though. Any of these tricks will help with clumpy spores, and blending will allow your spores to stretch into even more solution and still remain "hot". (thanks to Nan for the sex lube and blender tips)


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

Edited by mycofile (01/22/03 04:06 AM)

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Re: tissue and grain [Re: mycofile]
    #1239083 - 01/22/03 07:48 AM (21 years, 4 months ago)

I have some experience with refridgeration but more out of necessity than in experimentation. The necessity being my limited number of jars and incubating/grow space. I do keep logs to track everything but I am still ironing out the way I want to track (it can be infinitely complex) and I have not really considered experimenting with optimal times to refridgerate. My general procedure is to refridgerate fully colonized G1 quarts. The G1's are used to spawn G2 as needed. The G2's are used 50/50 to spawn G3 and trays for fruiting. I am relatively new to doing a lot of this so I am open for correction and advice.

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Invisiblemycofile
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Re: tissue and grain [Re: debianlinux]
    #1245236 - 01/24/03 06:20 AM (21 years, 4 months ago)

Quote:

but I am still ironing out the way I want to track (it can be infinitely complex)



I know how you feel. I filled a 150 page notebook and a 100 page graph pad in the first 6 months I grew!


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: tissue and grain [Re: La_Magdalene]
    #1246613 - 01/24/03 03:46 PM (21 years, 4 months ago)

Quote:

Also...they leak when I shake them. The needle is in position correctly, this I know. However, theoretically...if water can leak out, contams surely can leak in, so I am even afraid to wast a jar of grain on the shotty syringes.




That's why you shouldn't shake around your syringes without the cap on.


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"A celibate clergy is an especially good idea, because it tends to suppress any hereditary propensity toward fanaticism." - Carl Sagan

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InvisibleLa_Magdalene
Redeemed

Registered: 01/20/03
Posts: 33
Loc: En La' Magdala
Re: tissue and grain [Re: the spiral]
    #1268425 - 02/01/03 01:42 PM (21 years, 4 months ago)

The caps were on.  :smile: 


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Near the cross of Jesus stood his mother and Mary Magdalene. When Jesus saw his mother and the disciple HE LOVED standing nearby, he said to his mother, "Dear woman, here is your son."

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