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Jesse James (Spacecowboy)
Posted on Wednesday, August 27, 2003 - 05:00 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

This is how I would go about it, but I am new to mycology so I could be way off. Clone the rhizomorphic mycelium, collect spores, and then repeat the process. Eventually a super strain will evolve for your controlled conditions, from which you can then apply the Stamets’ “P-value” scale.

The following is the outline of my proposed experiment, with some background info. I have chosen to use the PF Tek as the main model as it is a proven cultivation method, and very versatile. The experiment could also be done with agar plates for strain isolation, and then grain spawn for bulk substrate inoculation. Please leave feedback and suggestions:

First start with a spore print, or Parent strain termed G-0, and do a multi-spore injection with a PF cake. After germination, isolate each of the rhizomorphic growing myceliums from the cake. Each of these mycelium isolations are the first generation offspring of the parent strain, or G-1 since they are the first sexually produced offspring of the parent strain which has been given the term G-0. Be sure to label each of these isolations for selection later on (i.e. G-1a. G-1b, G-1c…).

Next grow each of the G-1 isolates up individually on a slurry, since it can be easily re-suspended in a syringe, and then use it as spawn to inoculate a second PF cake, one cake for each isolate. Do not inoculate a cake with more than one isolate, as they will fight each other for control of the substrate. Observe the colonization speed and fruiting attributes of each of the G-1 isolates, while at the same time collecting spores from the largest, most healthy and attractive fruits. Then select which G-1 isolate to use spores from based on the speed of colonization, fruiting yield, and quality of fruits.

After selecting which G-1 spores to use, perform another multi-spore injection with a PF cake. Again, isolate each of the rhizomorphic growing myceliums from the cake. These isolates are designated G-2, since they are the second generation of offspring produced by sexual reproduction since the parent strain. Grow up each isolate, and use them as spawn like you did with the G-1 generation. Observe each of the G-2 isolates growing characteristics, while at the same time collecting spores like before. Lastly, select which G-2 isolate to use spores from.

The spores selected for are once again used for a multi-spore inoculation, and the isolated offspring myceliums are designated G-3. Keep repeating the above process and eventually you will have a super strain for PF cakes. This process may take awhile, or it may happen right away. There is also the chance that it may not happen at all, as the genes may not be present for creating a super strain. In this case you will need to breed the characteristics into the genome either symmetrically or asymmetrically, thus creating a hybrid strain (http://archives.mycotopia.net/discus/messages/5/29498.html?1059106285).

When doing a multi-spore inoculation hundreds, if not thousands, of new genomes are created for that parent strain. Most call these sub strains of the parent strain. By selecting a certain substrate/environment and breeding a mushroom on it, one can eventually select for a sub strain that performs exceptionally well in that given situation. The offspring of that sub strain should also thrive in that type of environment. Nature accomplishes this through the process of natural selection, or survival of the fittest. Starting with a multi spore inoculation, and isolating the most aggressive mycelium with the desired fruiting properties mimics nature’s method. It can take a very long time to isolate a PF cake super strain, as it takes about 1.5 months from inoculation to the first spore collection.

For basic cloning purposes, Paul Stamets has developed a scale called the P-value (http://fungi.com/cultures/cultures.html):

“Each strain is designated with the Stamets "P-value" scale, signifying the expansion of mycelium covering a 100 x 15mm petri dish (approximately 1,000-2,000 cell divisions). The first time a wild species is tissue cultured; the age is denoted as P-O. Thereafter each successive growth over the petri dish's surface is described in increments of P-l, etc. This library tries to maintain strains closest to their wild origins or closest to their peak fruiting potentials.”

Lets apply this to the PF Tek. Imagine that you start with a spore syringe (PF-0), and you use it to inoculate a PF cake. The cake colonizes, and produces fruit, from which you take a tissue sample from the biggest fruit body formed for cloning (PF-1). From this you grow up mycelium, and use it to inoculate another PF cake. This second cake produces all large fruit similar to the fruit body from which the tissue was taken, since all the mycelium tissue is of the same cloned genetics. Once again you take a tissue sample from a fruit body for cloning (PF-2). Mycelium is grown up and used to inoculate another PF cake, which produces fruit. Again, you take a tissue sample from a fruit body for cloning (PF-3). However, this time the tissue produces mycelium that undergoes senescence. The cloned tissue has strayed too far from its wild origins, or peak fruiting potential for optimal performance on PF cakes, or the mushroom has been cloned to death.

This PF cake model is similar to Stamets’ Petri dish model. P-0 is like PF-1, and supposedly the tissue clone will under go senescence after a set number of cell divisions. Allowing the mycelium to grow to fruit body stage before cloning a tissue sample speeds up the onset of senescence greatly, due to the exponential process of cell division. However, I have read that changing substrates helps to prevent the onset of senescence (http://www.stainblue.com/kitchen.html):

“For long-term health and maintenance of sacred strains, alternate any two of the above media. This will help prevent senescence, which can occur when cultures are grown on one medium only. Store strains at 35° - 40°F; transfer every six months. In this manner, sacred strains can be maintained for years.”

Therefore, once a super strain is isolated, apply the Stamets’ "P-value" scale. Store the super strain at 35° - 40°F and rotate your plate media every six months. It only takes a small piece of tissue to grow up spawn, but as the cells divide you move further and further away from the wild type origin and peak fruiting potential. Otherwise you could use a tissue culture to grow up fruit, and repeat the process indefinitely by rotating substrates.
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Hippie3 (Admin)
Posted on Wednesday, August 27, 2003 - 03:19 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

you could rotate different additives in your pf cakes, such as flax one time, rye another, etc. just a tablespoon or 2 per cake added to the mix should help.
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rodger rabbit (Skyypilot)
Posted on Wednesday, August 27, 2003 - 03:36 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

If you're taking a sporeprint every second flush or so as you should, then senescense won't be a problem. Just constantly be starting your strains over again on petri dishes to isolate the best substrains, then transfer to grain. Once you transfer to grain, simply label the petri dishes, and stick what's left of them into the refrigerator. That way, when flushing starts and you determine the best strain, go back to the fridge and get the appropriate petri dish out, only to start over....And on and on it goes. . .
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xylem flow (Xylem)
Posted on Thursday, August 28, 2003 - 04:22 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

good idea, only one problem i see, but i'm not a pro mycologist or geneticist myself either, just a newbie. the problem i see is that the phenotypes you are trying to exaggerate have nothing to do with potency, and what effect do the exaggerated phenotypes have on potency? or is that even a consideration?
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Jesse James (Spacecowboy)
Posted on Thursday, August 28, 2003 - 10:34 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Hip, I was thinking something similar, like rotating BRF and Rye flour.

Rodger, I had planned on second flush spore prints, since this flush seems to produce the highest quality mushrooms, but why the senescence issue? I am hoping to work up to a very aggressive and high yielding strain created by sexual reproduction of spores. I am trying to do exactly what you described with the refrigerated petri dishes. Once I have isolated the super strain I would like to store it as a petri dish culture, and use it to generate spawn using the Stamets’ P-value theory. My biggest issue is avoiding senescence. I haven’t taken any of Stamets’ seminars yet, and I have read that you have. Have you taken his advanced seminar, and are their any pointers you can give me in maintaining a strain for spawn generation and viable spores?

Xylem, potency is not a consideration at this time. I am only interested in aggressive mycelium with high fruiting yields, and spores that actively maintain these characteristics. It would be nice to select for potency, but I do not know of any inexpensive ways to test for levels of potency, other than blueing and bioassays which are not that reliable. I would also like to apply this experiment to other mushrooms such as oysters, shiitakes, and reishi with the addition of sawdust to the substrate.

Thanks for the quick feedback. I hope to isolate some very strong strains with this idea, and I can use all the positive and negative feedback I can get. I foresee this experiment as being a long and drawn out task, with the rewards coming in the long run. I don’t want to proceed into it, only to find out it was flawed from the start.

Does anyone see any more pros, cons, ideas, or pitfalls that may arise for this experiment (like things I should play particularly close attention too)?
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munks (Munks)
Posted on Friday, August 29, 2003 - 12:59 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

i'm new to mycology so i'm talking out my arse, but dont mushrooms reproduce asexually? the purpose of that sort of breeding is to bring out the best combination of recessive genes that were present in the host parents, but i have no clue what the genetic structure and reproduction methods of mushrooms is like and how genetic material is passed on, i just thought i would ask...
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Jesse James (Spacecowboy)
Posted on Friday, August 29, 2003 - 01:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I guess your right in the asexual part. When I talk about sexual mushroom reproduction, I am referring to spores mating and producing a new genetic offspring. There is no real male or female part involved that I know of.

I made a reference to an article I picked up at MushWorld.com on breeding in my first post. I am also concerned with the recessive genes becoming dominant too. But take the PF Classic for example, as far as I can tell, it was the same mushroom grown over and over again by multi-spore inoculation, and over the years it became the dominant mushroom adapted for growth on PF cakes.
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rodger rabbit (Skyypilot)
Posted on Friday, August 29, 2003 - 02:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Mushroom sex is somewhat more boring than we're used to, but it's sex just the same. Not in the male/female respect, but in a pairing up of like mycelia. (I won't get technical...it's not important for this discussion) Senescence won't be a problem if you are taking sporeprints the way you should. Agar culture is absolutely necessary if you want to develop a good strain. The petri dishes in the refrigerator idea I expressed above is only used to save the 'mother' culture until fruiting, when you determine the best strain. Then, transfer some of that mycelium from the best fruiting strain into a test tube slant for long term storage. This way, you can always go back to it later. A petri dish will dry out too soon. Test tubes can be stored in the refrigerator for years.
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munks (Munks)
Posted on Monday, September 01, 2003 - 05:50 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

then how does mushroom sex help genetic variety, as most cultures are formed from a mass spore colonization that came from a single cap? there is no introduction of fresh genetic material into the cycle, it just doesnt make sense to me
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Jesse James (Spacecowboy)
Posted on Monday, September 01, 2003 - 09:13 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I have been thinking that too Munks, and I am curious if it would be better to mix spores from two different selected isolated strains during each rotation to try and keep variety in the genome. However, I also am thinking that this might disrupt the forced selection process for aggressive mycelium with high fruit yield.
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munks (Munks)
Posted on Tuesday, September 02, 2003 - 04:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

i guess i need to know more about mushroom sex to understand the whole thing, but personally i'd be more selective towards potency than fruit yield

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