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OfflineMych
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senescence .. need some answers.
    #4456656 - 07/26/05 07:23 PM (12 years, 3 days ago)

1:
alright, it seems that the opinion of reaching senescence is via the number of transfers you do. However, wouldn't it be the volume you have colonized? I mean if you only were transfering at a constant 1:5 ratio for 10 "generations" compared to transfering at a constant 1:20 ratio for the same number of generations.. wouldn't the 1:20 ratio transfers be "older" by the time you got down to that 10th generation?

2:
now about cloning.. when taking a sample from a fruit and dropping it into say.. Liquid Culture, does the senescence follow with it? As in: if you clone a fruit from a 1st "generation" cake vs a fruit from a 20th "genration" cake.. whill the 20th gen Liquid Culture be weaker? Is the fruit somehow immune to senescence transfer? if not.. then up comes question #3

3: if fruits transfer senescene just as much as mycellium.. well: only a tiny part of a fruit is needed for cloning it to enough liquid culture to make 100 jars. You could use mycellium the same way (however the genetic isolation isnt happening) Following that same math, couldn't you take a single 1st generation jar and turn it into THOUSANDS of 2nd generation jars via Liquid Culture of equivalent method? that would translate to a near infinate supply of mycellium before hitting senescene (assuming "generations" define senescence and not volume)


4:
now about fruiting.. does fruiting the mycellium cause it to age at all? Say i fruit a cake for 2 flushes, then spawn the bag to some WBS... would there be any aging to worry about? granted the mycellium will be a bit weaker.. but is that permanent or will it "revive" once it hits fresh nutes? it almost doesn't make any sense to NOT fruit your cakes prior to doing a G2G transfer (contam issues asside) unless it just isnt worth your time.


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Re: senescence .. need some answers. [Re: Mych]
    #4456712 - 07/26/05 07:42 PM (12 years, 3 days ago)

While this doesn't really answer - your questions.

It was ANNO - who said it best. Something on the order of:

Young, vigorous mycelium does far better than old mycelium.

The same as a young healthy athlete will do far better than an elderly geriatric athlete in the same contest.


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OfflineMych
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Re: senescence .. need some answers. [Re: agar]
    #4456802 - 07/26/05 08:03 PM (12 years, 3 days ago)

Quote:

agar said:
While this doesn't really answer - your questions.

It was ANNO - who said it best. Something on the order of:

Young, vigorous mycelium does far better than old mycelium.

The same as a young healthy athlete will do far better than an elderly geriatric athlete in the same contest.




ok, but is senescence "formed" by the number of generations or is it really by the volume of substrate it has covered? or is it actually by time itself?


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Re: senescence .. need some answers. [Re: Mych] * 1
    #4456948 - 07/26/05 08:54 PM (12 years, 3 days ago)

IMHO, it is "time", not volume.

For instance, if a human fetus remained in the womb - 10 times longer than normal. That would certainly have adverse effects on it.

Myc is genetically set to go from spores to fruits in a single season & the spore release propagates the next generation, for the upcoming season.

Throw that cycle to far out of whack, and problems occur.


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OfflineMych
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Re: senescence .. need some answers. [Re: agar]
    #4456993 - 07/26/05 09:09 PM (12 years, 3 days ago)

Quote:

agar said:
IMHO, it is "time", not volume.

For instance, if a human fetus remained in the womb - 10 times longer than normal. That would certainly have adverse effects on it.

Myc is genetically set to go from spores to fruits in a single season & the spore release propagates the next generation, for the upcoming season.

Throw that cycle to far out of whack, and problems occur.




that i can work with. now obviously putting mycellium in the fridge will "slow time down" for it but.. at incubation/fruiting temp range.. what is the time (from spore) that it takes beofre mycellium starts to exhibit noticable senescence?


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Re: senescence .. need some answers. [Re: Mych]
    #4457134 - 07/26/05 09:40 PM (12 years, 3 days ago)

My experiance is usualy mild mutation, excess aborts, lousy pinsets, weird fruits & sterile caps sets in after G4 on G2G transfers, over a period averaging 6 weeks from G1 mother jars.


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Re: senescence .. need some answers. [Re: Mych]
    #4459596 - 07/27/05 11:54 AM (12 years, 2 days ago)

ME differs from Agars. I often incubate grain for a month or three with few noticable differences (I notice an increase in potency, but that's in other threads.

1. It's not specifically transfers. Usually it's cell divisions. It can be others, see below.

2. Yes, the 20th generation cake clone will show senescence sooner than the 1st gen.

3. yes, start young and multiply it thousands of times

Here's what is going on, at least as I understand it (mostly from stamets). It's about randomn mutations that show up after lots of cell divisions. Think of old people. They start as a fetus, a few million cell divisions later they are young people and everything is ok. A few million cell divisions later, randomn mutations start to show up. They start to get weird blotches on their skin, shit like that. Then a few million more cell divisions later, all kinds of randomn mutations show up, and they start to fall apart all over. Same with mycelium. 2 spores germinate, then mate and form a dikaryon. A few thousand cell diivisions later the petri dish is covered with young vibrant mycelium. Hundreds of cell divisions later you've colonized grain, done a G2G and fruited. The mycelium is still vigorous. You clone it, but you aren't starting at zero, the clone is still millions of cell divisions in "age". IMO, it's not quite middle aged yet and will likely still perform as the young strain did. Especially on the scale that most home cultivators grow on. Pros go through 1000 times the number of cell divisions, turning a single petri dish into TONS or colonized fruiting substrate. Most of us put our strains through about 1/10 the number of divisions as the pros, thus a even a standard bulk grown strain is 1/10 the "age" as it would have been if grown to it's maximum. Cakes are even "younger". I've personally cloned, shot cakes, fruited, cloned, shot cakes repeat for nearly a year before the strain showed any decline.

Senescence, or something very similar can occur due to environmental conditions. Caramelized sugars (exceeding 250 deg f when sterilizing) increase the number of randomn mutations, making a culture senescent sooner. In the same way, UV light, chemicals and lots of other environmental conditions can "age" a culture faster than it would otherwise. Refer to PF's sterile albino mutation which took generations to manifest.


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OfflineMych
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Re: senescence .. need some answers. [Re: mycofile]
    #4459815 - 07/27/05 01:16 PM (12 years, 2 days ago)

good posts!! while the theories do not match, they each make sense.  It is good to hear other ideas while trying to figure out how it works. Maybe i will start seeing some senescence from my 4th gen poo that was spawned from spent cakes!! :smile:


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Re: senescence .. need some answers. [Re: Mych]
    #4460294 - 07/27/05 03:52 PM (12 years, 2 days ago)

There may be something to what agar says, but I don't think it's what's referred to as senescence, although the symptoms are right. Most of the over-incubated grain I've used was used as spawn. My experience simply fruiting the substrate is minimal although it has been done (first time I used old spawn I didn't know it would be fine as spawn so I cased and fruited it directly. Now if Agar is seeing these troubles when fruiting the substrate, that could jive with what I've seen. Perhaps the mycelium goes into wacky mode when sitting around, but gets reinvigorated when given new substrate. Or maybe some of it senesces, but the parts that don't colonize the bulk substrate and hide the senescent properties of the wacky parts.

There may be some straight up age factor, but the bit I gave about randomn mutations is fact (whether it is the sole cause of senescense is up in the air though, as the creationists say, it's just a theory). Let me expand on the randomn mutations if you didn't study biology much in school.

When an organism grows it does so by dividing existing cells through a process known as mitosis. The general process of mitosis is that the chromosomes of DNA copy themselves, then split, then the copies spread further apart to opposite sides of the cell, then the cell wall pinches shut between them yielding 2 cells which (should be) exact copies of each other (genetically speaking, they can do different things depending on which genes get turned on or off, but they have the same genetic code. This is what I meant by cell divisions.

This doesn't always work exactly right. There is a small margin of error. It works pretty well. Let's arbitrarily say it works 99.99% of time. This means that statistically by the 1000th division, a mistake has been made in one cell. You would never notice this unless you looked at that one cell. Even then, it can be an error that doesn't matter. It can be in a part of the genetic code that isn't used, or it could be an error that actually codes for the same thing. Say the code is ABA, but it writes it backwords giving ABA, doesn't matter. But lets say it coded BAB on accident, in one cell. This won't matter yet. But a thousand cell divisions later, that one cell has reproduced several times, and another mistake has occurred somewhere else. Now there can be, say 101 cells with mistakes. Another thousand divisions gives 202 cells with mistakes at this rate. By a million divisions there are lots of cells with mistakes. Some of these mistakes may be significant. Also, some of the cells with mistakes may have reproduced with further errors magnifying the mistake.

You can see how these randomn mutations accumulate and become worse with age, measured in cellular divisions.

Anyway, that's the way that randomn mutations build up, and that is the mechanism most widely thought to be responsible for genetic senescence. It's why Stamets talks so much about keeping a strain library with the minimum number of cell divisions. He rates his strains using the P-value which is the number of times the strain has multiplied across the surface of a petri dish since the isolation of the strain and uses the P-value as an estimate of the number of cell divisions that have occurred. Clearly the P-value doesn't apply to growth in media, which would be many many times the number of cell divisions across a 2 dimensional petri dish.


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Re: senescence .. need some answers. [Re: mycofile]
    #4462572 - 07/28/05 02:13 AM (12 years, 2 days ago)

Mycofile,

I don't disagree - either. In fact - time - is what allows for more cell divisions.

BTW agar = 6Tango (from another life)


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Offlineblackout
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Re: senescence .. need some answers. [Re: agar]
    #4463348 - 07/28/05 05:20 AM (12 years, 1 day ago)

Quote:

agar said:
The same as a young healthy athlete will do far better than an elderly geriatric athlete in the same contest.



I use primarily LC's, do you think there is an optimum time to use it. A very young athlete will not be as strong as one at peak age, so can an LC be used too soon. Will cooling stop more cell divisions (I guess it will). Would it be best to chill the LC before it has fully used up all the nutrients. Will a fully colonised LC that has no nutrients left suffer much as it will no longer have cell division (or will it?)


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Re: senescence .. need some answers. [Re: blackout]
    #4463402 - 07/28/05 06:24 AM (12 years, 1 day ago)

My experience with LC's is that once you have what you consider to be thick healthy growth inside a container. If you refrigerate it to stall growth. It will give good results for months.

I have one large (started as 1 liter) LC that is 7 months old. It was refrigerated after about 10 days growth. I take syringes from it, allow them to come up to room temps & have inoculated both spawn jars & bags. Last crop - was as good as the first crop & showed no ill effects - what-so-ever.


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Re: senescence .. need some answers. [Re: agar]
    #4465470 - 07/28/05 07:08 PM (12 years, 1 day ago)

Agar, I know who you are silly. I recognized your "accent" shortly after your started posting again. I even sent you a PM to make sure if you remember. Sorry if I sounded testy, I was 48 hours + sleep deprived, wasn't trying to be, sometimes it just comes out like that. Anyway,

I've had good luck storing LC too, just so long as it has something more than just plain sugar in it. If I'm not mistaken agar uses Malt Extract in his LC which would be much better than just dextrose or karo or such.

My experience with purified sugar based LC's is that much more than a month in the fridge will show declines in vigor.


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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: senescence .. need some answers. [Re: agar]
    #15845489 - 02/22/12 01:23 AM (5 years, 5 months ago)

agar whats the max number of g2g transfers u would suggest and still avoid se┬Ěnes┬Ěcence


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Re: senescence .. need some answers. [Re: soicyboy121] * 1
    #15845563 - 02/22/12 01:42 AM (5 years, 5 months ago)

Wow....a six year old thread!

I look forward to meeting the zombies.

There were some cool cultivators in this thread.

JD


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Re: senescence .. need some answers. [Re: Mych]
    #15846317 - 02/22/12 08:50 AM (5 years, 5 months ago)

This (and his other posts) from John Holliday of Aloha Medicinals may help a bit:

Regarding stored cultures: The answer to how long cultures can be stored is very much strain related. I know of Morels that were frozen whole and kept for extended periods, and when thawed out were viable. Others cultures, like Volvariella, tend to die no matter what you do to them. The way we store cultures is in test tubes or petri dishes, on agar, well para-filmed, in a refrigerated container that maintains at about 36-38 degrees F. The same reefer stores large qty's of spawn, with the resulting side effect that the O2 content is about 50% of atmospheric (remainder N, CO and CO2). Some of the cultures are indeed 30 yrs old. Another trick I like is to get the wooden stir sticks that are used for coffee, cut them to the right length, and put one into the test tube before you pour slants. They should stick up well above the agar surface. That way, the fungus will colonize the wood, and even after the agar culture is long dead, the wood is still viable. Just take a scraping, or stick the whole thing into another dish of agar, or even directly into a bag of grain as in the old Amycell spawn technique. As far as sterile water storage I tried that with some strains a few years ago and as I recall it worked out OK. I don't normally try new stuff because what we are doing seems to work OK. As for spore storage, most spores will store for long periods of time, like longer than you and I will be around. A note on collecting spores: Make it easy on yourself and collect sterilly. As in, sterilize the collection surface, filter paper or glass, etc, or use a pre-sterile petri dish for collecting. You can put a cleaned cap in a dish and close it up, leave in the hood for 24 hrs, then remove the cap, parafilm and store away. Always put plates inside of plastic bags, like zip-locks. The parafilm is not always dependable. And always wrap at least two layers of parafilm over the joint. If collecting on paper, sterilize the paper first in a clean gallon jar with a filter disc on the lid, then open in the hood and place the cap inside the jar, re-close, and leave in hood for 24 hrs. Then you have a sterile spore print in the jar. Keep in mind always, sterile techniques. You cannot be Too Sterile. It is like being too rich or too beautiful, just doesn't happen. Another good collection surface for spore prints is glass plates, then place two plates together and tape the edges all around. Stored this way, spore prints are good for a LONG time. See the photos at: http://fruit.naro.affrc.go.jp/kajunoheya/epfdb/method/spore1.htm

http://www.shroomery.org/forums/dosearch.php?namebox=150667

And...a few comments from RR:

Normally, the traits in a fruit will carry over if you clone it. If you clone a mutant, you'll get harvests of mutants, provided the mutation was genetic in nature. If you clone from a cluster of large fruits, your future crops will develop clusters of large fruits. Senescence is always a problem if you try to keep a fungal cell line going for too long. It isn't necessary though. Instead of constantly cloning the clones, clone the first one and then make a few master culture slants in test tubes to store the mycelium. You can then take it out of the refrigerator and use it for years to come without degradation. Damage as the pins are forming, or for another example, damage from chemicals containing petroleum can be mistaken for mutations. I doubt there's an environmental condition you can easily create that would cause mutations. CLONING

A grain to grain transfer is done with young, rapidly growing mycelium. In no way is that the same as cloning. I would not clone a clone. It isn't necessary. If you have young cell lines, simply store them in the refrigerator for years and keep using them. You keep the original master culture slant in the refrigerator. When you need mycelium, you take a tiny piece from the culture slant and transfer it to a petri dish, then return the slant to the refrigerator, where the mycelium slowly grows to replace what you cut out. Use the petri dish(es) to start the next batch. By doing this, your master slant slows way down on cell division, thus preserving the culture without senescence. When the culture slant goes into the refrigerator, it essentially stops cell division, putting it into stasis. This way, you can take it out of the fridge, get a small piece, and then return the slant to the refrigerator. Using this system, a 20 year old culture might only have six weeks or less of growth, thus senenscence isn't a problem. CLONE A CLONE

You would still be at first generation after ten, or even fifty grain to grain transfers, but senescence would definitely be a factor. A clone keeps mycelium from a particular cell line growing. After about three months of rapid cell division, most mycelium will give up the ghost and performance will be poor. This is why we recommend no more than three grain to grain transfers. The reason is to make sure that once the mycelium from the 3rd G2G colonizes the bulk substrate, it still has the energy to produce a nice flush. A generation is just like with humans or other animals. If you do a grow from spores, it's first generation, no matter how long you grow it or how many transfers you make, just like you are your mom and dad's son, no matter how long you live. Once you fruit it, take the spores and germinate them, this new cell line is what would be 2nd generation, and so on. Mycelium can go for many, many generations. The PE strain of cubensis is living proof of this. It's degrading, but still viable after all these years. My PE6 and Workman's work with PE albino, etc., are efforts to infuse fresh genetics into the old strain. GENERATIONS

Guys,
This is old science. It's been done for many years in the commercial mushroom industry. It's the standard way they keep and trade isolated strains of edible mushrooms. They desiccant dry the fruitbodies, then powder them. They store the powder until ready for use, then sprinkle it on agar to regenerate the strain. It stops senescence in its tracks. Try it without soaking in peroxide first. That only weakens the mushroom myc so it doesn't take off as fast. Watch for the first signs of myc growth, and immediately transfer that to a new petri dish. Do this a time or two and you'll have a clean specimen, even from years old dried fruits. It works, trust me. DRIED MUSHROOMS

From:  http://www.shroomery.org/forums/showflat.php/Number/8524930#8524930


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Edited by OICU812 (02/22/12 09:08 AM)


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Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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