Breeding project

Discussion in 'ADVANCED MYCOLOGY' started by cyanara, May 2, 2012.

  1. cyanara

    cyanara Moderator Mushroom Doctor

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    Ok, so the day has finally arrived that I'm ready to take the next step. I dont have a microscope or hundreds of plates to blow thru so monokariotic breeding is not in the near future..
    I do however have seen tons of threads suggesting that dikariotic x'ing is possible as long as the culture is a true monoculture.
    My pride and joy, crown jewel the Blue Magnolia will be candidate number 1. reason being,
    A) this is a first generation from the wild, so lots of genetic goodies
    B) true iso, true monoculture, stable, and at a P-2
    C) Potent, slow growing, and desease resistant
    The other strain of choice will be P.Cubensis Brazilian, reasons will be as follow.
    A) P-3
    B) fast growing, potent, very stable being that you will see very small veriability between 20 plates.
    C) monoculture, and is a very easy strain to maintain.
    My reason beind this project is to challenge myself on a flow bench..
    If a third sector has develolped then i know that the dikariotic mycelia has fused and a cross acheived..
    I remember seeing a post from Blimmey Grimmey and Lipa saying something about rotating Prints so that the spores from two different varieties would be of the same age and have abetter chance of germinating at the same time and fusing. I cannot however find the post and will have to go on this venture instead.
    I'm hoping now that experienced peeps like Alan R. RR and such are here, I can get some more indepth insight.
    Cy
     
  2. PE-n-hed

    PE-n-hed Well-Known Member Moderator Mushroom Doctor Cannabis Doctor Supporter

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    what about the snake venom?
     
  3. harponet

    harponet Well-Known Member

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    Great! I can't wait! Your projects are always interesting. I have just 2 days ago been reading about crossing dikariotic in TMC i believe (i can't even say which of Stamets book i am reading since i bought all 3 and am reading them interchangeably)
     
  4. the_chosen_one

    the_chosen_one there are no answers.. only choices Moderator Mushroom Doctor

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    if i can do it so can you my friend. you don't need all the fancy equipment to isolate a monokaryon. i've done it several times with only a few plates. having a monokaryon isolate will eliminate most of the problems that arise using other methods.

    good stuff! thanks for starting this!
     
  5. Major Myc

    Major Myc pasture pirate Mushroom Doctor

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    i can't wait to see how this goes, i'd like to see pe crossed with something in the future. good luck man!
     
  6. the_chosen_one

    the_chosen_one there are no answers.. only choices Moderator Mushroom Doctor

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    jeeze, i figured it would be at least a year before i quote myself here lol, but here's some ramblings from a pm topic.

    "yep. a monokaryon will not fruit. they tend to be faster growing than dykaryons and they do look a little different. it was the look that got my attention on my first one. looked more silky and less branches. extremely thin compared to dykaryon myc. i had to use a scope to verify the first one before i bred Falbino with it, but since then i have been able to simply identify by eye.

    although, my first one was an accident, i have used a few different methods to acquire them with success. they key is dilutiuon and seperation of spores... bottom line. workmans isolation tek on agar works very well. if you have agar skills i would mostly recommend this. dilution with water can also work. i was doing my own dilution work before i found stamets version lol.

    just a side note.. workman and i both noticed that albino (white) spores tend not to clump as bad as pigmented spores.

    hey, i'm gonna copy these ramblings to the thread! it'd be great to get everyone involved and it saves my busy but some typing lol.

    hope this helps, just let me know or post if you have any questions.

    :hippie:"
     
  7. Professor PinHead

    Professor PinHead Lost in the Tek.... Administrator Mushroom Doctor Cannabis Doctor Supporter

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    You def don't need snake venom,.

    Shit, if you were taking about breeding from spore you wouldn't need anything more than a scalpel, a pile of plates, and a pair of prints. :hehehe:

    Breeding is a lot of work though. It also takes a lot of time.
    :time:

    I will be jumping in on this one in the next couple weeks when I am little less busy.
     
  8. treewood

    treewood mediocre mycologist Moderator Mushroom Doctor Supporter

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    How powerful of microscope do you need to view mycelium?
     
  9. harponet

    harponet Well-Known Member

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    Treewood I need one too - or as Prof already said: The second one:)
     
  10. cyanara

    cyanara Moderator Mushroom Doctor

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    TCO, can you link workmans tek?
    do you have pics of your monokariotic myc?
    I'm intersted in trying this, but while I'm getting advice from you and compiling data, I'm going to attempt dikariotic x-ing.
    I've also had some recent interesting development with possible genetic recombination on my PPE2 plate. If this is the case and I have a hunch what often time we think is a monoculture is infact poly culture that have combined which would infact explain sectoring when tranfered a few more time and recombining. It seems that the culture has produce new mycelium and if so could bypass spore by subculturing these genetically compatible strains from the same culture.
    I will be including these experiments in this thread as I feel it pertains to the topic at hand.
    Cy
     
  11. treewood

    treewood mediocre mycologist Moderator Mushroom Doctor Supporter

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    idk if this is the tek he is talking about.
    -workmans-psilocybe-cubensis-breeding-method-

    don't hate me for linkin to the "other" site. haha
     
    Last edited by a moderator: May 11, 2012
  12. nomendubium

    nomendubium scraping by, since '97 Expert Identifier

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    hmmm, that was certainly an interesting read, thanks treewood! :thumbup: obviously, I have a lot to learn in the genetics department;
    1. I'm assuming when a dikaryotic mycelia meets a monocaryon, the dikaryon donates the dna and the moncaryons dna is lost?
    2. Ive seen people use 2 different types of spawn in the same tub, and it looks like by the second or third flush, the mushrooms have homogynized in appearance....Is there a reason why 2 dikaryons can't anastomize and there-for essentially have mated? I don't understand why people talk about using snake venom to break dna ect, doesn't it naturally combine and become homogynous through anastomosis?
    3. why couldn't I transfer 2 dicayrons to the same plate, observe the zone of uncolonization, and transfer a small peice from where they meet and call that a hybrid?
     
  13. treewood

    treewood mediocre mycologist Moderator Mushroom Doctor Supporter

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    1. I'm assuming when a dikaryotic mycelia meets a monocaryon, the dikaryon donates the dna and the moncaryons dna is lost?
    Not exactly, you start out with a monocaryon in a jar (A). then scrape spores into the jar (B). Those spores will germinate (there still monocaryon). Then (B) will connect with the monocaryon in jar(A). If the spores "connect" with themself(B to B) they will supposedly die because the monocaryon A will have all the nutrients taken up and there for you should have a hybrid. It won't be stable of course.

    (A) is your known monocaryon
    (B) is your spores
    question 2. IDK


    3. I think you have to single sector both A and B and put them in a jar and look for a 3rd sector. It could take 1000 times but I guess it is possible.


    I hope everything in question 1 makes since and if I said anything wrong I hope a higher up will come and correct me.
     
    Last edited: May 4, 2012
  14. nomendubium

    nomendubium scraping by, since '97 Expert Identifier

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    everything in 1 makes sense to workmans post, but I just meant in general, not in workmans tek. he is counting on the grain to be unavailable to the new moncaryons formed, but I'm asking if I had a dicaryon and a mono, what happens?
     
  15. treewood

    treewood mediocre mycologist Moderator Mushroom Doctor Supporter

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  16. nomendubium

    nomendubium scraping by, since '97 Expert Identifier

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    don't feel bad, old boy, I've been trying to pin down the answer to that question for about a year. I've heard that the dikaryotic mycelia donate dna to the monocaryon, but then what happens, it eats the mono dna? :shrug: I don't know. And if 2 dikaryons meet up, what determines what dna gets used/doesn't get used? Tough questions, I reckon. Maybe we are not supposed to know
     
  17. the_chosen_one

    the_chosen_one there are no answers.. only choices Moderator Mushroom Doctor

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    quoting Workman -

    "Workman's Psilocybe Cubensis Breeding Method



    By multiple requests, the hybrization method. This is the highly simplified version with as few technical terms as I can muster. It still requires some agar work and only works within a single species. In this example we are crossing strains of Psilocybe cubensis

    Classic mushroom breeding requires the isolation and germination of single spores of both parents and then letting the mycelium from both parents cross in a petri dish. This is a precise and controlled breeding method but it is tedious and time consuming. It requires the isolation of many single spores and many petri dishes in the hopes of a few viable crosses.


    This simplified method still requires the isolation of at least one monokaryotic mycelium from a single spore. To get the single spore mycelium I just streak an agar petri dish with a small amount of spores (serial dilution as described in The Mushroom Cultivator (Stamets) also works). At the end of the streak there are usually just a few spores on the agar. As soon as the tiniest bit of germination is noticed, transfer the smallest bit of mycelium to a fresh plate. Select single isolated germination points far from any clusters. The mycelium from a single spore grows slow, isn't rhizomorphic and can't fruit. You can confirm that the mycelium is monokaryotic by looking at a bit of the mycelium under the microscope (monokaryotic mycelium lacks clamp connections), but its usually pretty obvious by the way the mycelium grows. If you don't have a microscope you can skip the confirmation step.


    Now that you have your petri dish of monokaryotic mycelium, the hard part is over. The next step is to innoculate some spawn with this mycelium and let it grow to near completion. Once the spawn is fully run with mycelium but not completely white you can proceed to the next step. Don't expect the jar to colonize as fast as multispore or fruiting clones.


    Once your monokaryotic jar is ready you can add parent #2 in the cross. The nearly colonized jar should be fairly contaminate resistant at this stage so you don't have to be exceptionally careful now. Open the spawn jar lid and scrape in some spores of parent #2 or make a fresh syringe and inject some spores. The syringe should be freshly made as spontaneous germination can deflower the spores within. Shake the jar and let incubate for at least a week or two.


    What is Happening?

    What we started with was a jar of monokaryotic mycelium but when we added the parent #2 spores, some of them germinated and fused with the monokaryotic mycelium which becomes a dikaryotic mycelium by nuclear migration. Essentially the entire monokaryotic culture becomes dikaryotic by replicating and moving nuclei in a sort of a chain reaction through the already existing mycelial network. Since there isn't any available uncolonized substrate left in the spawn, any spores of parent #2 that fuse with each other won't have enough resources to produce any mushrooms.


    Fruit the spawn directly (don't add to bulk substrate) and all the mushrooms produced should be varietal hybrids. The beauty of this method is you (or a friend) only have to produce a single petri dish of monokaryotic mycelium to make as many crosses as you like. You will want to start with your best performer for parent #1 and then you can easily make crosses with any prints you have around for parent #2. Maintain the parent monokaryotic mycelium with periodic transfers to new plates. Its also a good idea to use a very distinct strain for parent #1 since many cubensis look similar and it may not be visually obvious if the cross worked. In my example I used the distinct Falbino strain as the monokaryotic parent #1. Its pretty obvious when the jar produces pigmented mushrooms that the cross was successful.


    It is important to realize that the F1 mushrooms are 50% of each parent at this stage but the spores they produce are genetically recombined. This means the prints are not going to breed true and any prints you distribute at this stage won't often produce mushrooms that look like the F1 generation. Only clones of the F1 mushrooms will be the same. To stabilize a new strain you need to grow out the F1 prints to produce F2 prints with selection of the mushrooms that have the traits you want. You need to do this for 5 or 6 sequential generations with selection before the strain can be considered stable."
     
    Last edited: May 11, 2012
  18. the_chosen_one

    the_chosen_one there are no answers.. only choices Moderator Mushroom Doctor

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    100x is best. i can't remember if i used my 40x as well, but i think i did. it works for clamp connections, but to see the nucleus the 100x is a must.
     
  19. mycborg

    mycborg Well-Known Member Supporter

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    Nice reading Obi-One! :super:
     
  20. the_chosen_one

    the_chosen_one there are no answers.. only choices Moderator Mushroom Doctor

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    my old "tek".. brings back memories :sigh:

    A microscope is a must for true precision but not completely needed. Basically you start with two syringes from two strains. The lighter the better. 3cc is usually best and you will need several empties. First shake it well and drop 1 drop on a slide. Then using another empty (or part full of sterile water) syringe with the needle tip cut off suck up a small portion of the drop. Then take that syringe and add (or more) water. The step can be repeated several times until eventually the dilution isolates only a few spores. This can then be proven under the mscope otherwise it's a gut feel. This should be done to both parent strains. Then a syringe can be made that is a mix of the two.
    This is what I meant by right place at the right time. There will be only a few spores from both strains thus narrowing the chances of clamp connections being formed too soon. It's kinda like letting the monokaryotic myc run further thus improving the chances of the other strain crossing it and decreasing the chances of the same strain connecting. Isolates can be taken from the resulting fruits. It's totally theory but I believe to have had some success at least with the PF family shrooms. Then one encounters the problem of identifying what actually happened if anything .

    quoted from a long time ago..