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OfflineSillypcybin
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Req: Petri Dish Pics With Following Content...
    #4723693 - 09/28/05 03:37 AM (11 years, 2 months ago)

Does anyone have a picture or a series of pictures of a petri dish with 2 strains of cubensis colonizing it? I'm mainly interested in seeing what happens at the meeting point between the two colonies.

Let me know. Thanks!


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: Sillypcybin]
    #4724405 - 09/28/05 10:43 AM (11 years, 2 months ago)

Here's two strains meeting on the same petri dish both from the front and from the back while held up to a light. A "line of inhibition" develops between the two strains. Sometimes along this line, a third sector will begin to grow. This third sector would be a hybridized version of the two dikaryotic strains.
RR


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Offlineblackout
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4724437 - 09/28/05 10:52 AM (11 years, 2 months ago)

Nice pics!
Have you any pics of these hybrid third sectors?
I thought a hybrid was a cross of 2 species rather than say 2 cubensis strains.


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Edited by blackout (09/28/05 10:53 AM)


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: blackout]
    #4724569 - 09/28/05 11:31 AM (11 years, 2 months ago)

Stamets calls it 'hybridization between dikaryons' but it is correctly a 'cross' and not a hybrid. A true hybrid would be a cross species mating. No, I don't have a picture of a third sector opening up. I'll try to get one next time. It's very rare for them to cross on a dish like that. Perhaps one in a thousand.
RR


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OfflineSillypcybin
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4724603 - 09/28/05 11:43 AM (11 years, 2 months ago)

Yeah! Thats EXACTLY what I was looking for! And a cross is exactly what I was wondering about. I want that to be a side project of mine, but what advantages would a cross bring?


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: Sillypcybin]
    #4724628 - 09/28/05 11:56 AM (11 years, 2 months ago)

Fresh genetics to both strains.
RR


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Offlineblackout
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4724773 - 09/28/05 12:32 PM (11 years, 2 months ago)

Could possibly get the good genetic trait from each strain, like in cannabis breeding, people striving for the sativa high and the indicas bushiness and high resin count. I would like a strain that produced sclerotia with the alleged non-waviness high of PE, and alkaloid content of azures :smile:


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OfflineHypha
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Re: Req: Petri Dish Pics With Following Content... [Re: blackout]
    #4725317 - 09/28/05 02:37 PM (11 years, 2 months ago)

(My first post, woot.)

I've been interested in creating crosses between different strains/variants/isolates myself. I'm by no means an expert. In fact, my only useful ability is to read large amounts of text I can't understand without my eyes glazing over. (I think this is due, in part, to my work with computers.)

At first, when I read this post I tried looking up some information in a mycology book I was reading that mentioned something about hyphae from different variants of a species merging together and gave some clues as to how the process might work, but, alas, I could not find it. I did, however, search the internet and I found a fairly good site which may provide you with information you already know, hopefully not:
http://bugs.bio.usyd.edu.au/Mycology/Reprodn_Dispersal/compatibility.shtml

If you're going to start breeding different variants of a relatively unstudied species (due to the legality in some countries) it would help to know as much as you possibly can. While the link doesn't give step-by-step instructions on a viable method of crossing variants, it does give a summary of the process. The site has other information you might find useful so click around. Pay special attention to anything talking about Basidiomycota, which is basically the "mushroom" division.

If you're already well educated in the process and theory behind inter variant reproduction, I'd love to hear about it. I've poked around on the forums and tried to find what I could on the net (and I'll try going to the nearby university library later today) but I still don't know as much as I'd like to.


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Invisiblemycofile
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Re: Req: Petri Dish Pics With Following Content... [Re: Sillypcybin]
    #4725982 - 09/28/05 05:11 PM (11 years, 2 months ago)

Quote:

Sillypcybin said:
but what advantages would a cross bring?



bragging rights more likely than anything else. And a lot of flames from people who didn't believe you. Right roger?


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: mycofile]
    #4726005 - 09/28/05 05:16 PM (11 years, 2 months ago)

No shit...lol Even when you show them a picture.
RR


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OfflineJeremy_Davis
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4733977 - 09/29/05 11:56 PM (11 years, 2 months ago)

Here's some info on how to use rattle snake venom to cause non-self recognition of same species or interspecies crosses leading to the hybrid strain. The techniques are a bit out of reach for most of us, but it's good to know someone's out there doing it. This is from Aloha Medicinals, and I actually bought some of the hybrid cordyceps after reading the entire article a fewe year back, good stuff. I still use it.



http://www.nwbotanicals.org/nwb/lexicon/hybridcordyceps.htm

SNAKE VENOM AS A HYBRIDIZATION AGENT:

We used purified snake venom from the Western Diamondback Rattlesnake (Crotalus atrox see illustration 2) [Sigma Scientific, St Louis Missouri, USA] for our hybridization techniques. The snake venom is added to the agar medium in quantities that alters the growth but does not prove toxic to the strain in question. This range of snake venom is from 10 mg to 30 mg per 300 ml of agar medium. The venom is not heat stable and must be added aseptically after sterilization of the medium. The agar used for this hybridization is an Aloha Medicinals Inc. proprietary agar named R7 Agar, consisting of malt extract, activated carbon, minerals and humus ? the carbon-rich ash residue from a coal burning industrial process. For the exact recipe see table 4. Other agars could probably be used as well. This just happens to be our production agar that we use everyday, and once we found that it also worked with the snake venom for hybridization, we found no reason to experiment with any other agar.

TABLE 4 ? R7 AGAR RECIPE
2.1 L
Distilled Water

50 g
Light Malt Extract

34 g
Agar

10 g
Humus

5 g
Activated carbon

1 g
MgSO4

10 ml
1% KOH solution


ILLUSTRATION 2

Crotalus atrox - Western Diamondback Rattlesnake

HYBRIDIZATION TECHNIQUE:

Petri dishes of this R7 agar medium are inoculated with mycelium from two different strains of the Cordyceps genus. These are usually two varieties of C. sinensis, although we have also crossbred C. sinensis with other Cordyceps species such as C. militaris, C. sobolifera and C. ophioglosoides. These different strains when inoculated together onto one petri dish will normally grow towards each other until they almost meet, at which point they form a zone of inhibition, where neither strain can grow. Eventually, one strain may prove stronger than the other and overgrow the plate, but they will remain genetically distinct; two different cultures residing in the same petri dish.

With the addition of a sufficient quantity of snake venom to the agar, we found that what happens is the two cultures grow towards each other until they meet and form their mutual zone of inhibition. This period of inhibition is short lived however, for in only about 2 or 3 hours the colonies each start sending out mycelial strands into this no-mans land, the zone of inhibition. These strands grow together and exchange nuclear material through their venom-weakened cell walls. They form a hybrid strain at this point of mutual contact. A new strain, one that is distinctly different from either of the parent strains. Within about 4 hours after first forming the zone of inhibition, the hybridization is complete and the colonies resume rapid growth towards each other. They become three colonies rather than the original two. There then exist in the same plate the original two colonies and a genetically distinct third?The Hybrid.

A section of the newly formed hybrid is carefully removed from the original zone of inhibition at the precise time that the colonies begin to fuse. That is during hour 3-4 after the initial meeting of the colonies. The hybrid is transferred to a new petri dish containing normal (non-snake venom) agar. Our quick method of determining hybridization is to inoculate a new dish containing normal agar with all three strains, the original two and the suspected hybrid. If the hybridization has in fact taken place, these are now three distinct colonies, and will form a mutual three-way zone of inhibition. If hybridization has failed to occur, then the suspected hybrid will readily fuse with either one or the other of the original colonies. This proves that our suspected hybrid is not genetically distinct from the original and we start anew. See Illustration 3

Once a hybrid is confirmed, it is tested for growth parameters. If it appears to be a vigorous and hardy grower on our substrate of choice, we grow out a quantity of mycelium, harvest it and analyze it for active ingredients. Through repeated testing in this way we were able to create the hybrid strain shown in Plot 6; a hybrid strain that is easily grown in solid substrate culture, with a potency greater than any other cultivated strain and at least equal in potency to the highest quality wild Cordyceps. We are referring to this new strain as Cordyceps sinensis Alohaensis. We are presently continuing this hybridization work with other species of Cordyceps, for the production of very specific target compounds


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Jeremy Davis
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Check out the ECHO mushroom blog page to see our lab, growing facility, and more-www.echotech.org/greta


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OfflineHypha
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Re: Req: Petri Dish Pics With Following Content... [Re: Jeremy_Davis]
    #4737129 - 09/30/05 03:55 PM (11 years, 2 months ago)

This looks very useful and informative, thanks for the link. I'm going to go stroll around outside now and try and find some rattle snakes.


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... *DELETED* [Re: Hypha]
    #4737224 - 09/30/05 04:13 PM (11 years, 2 months ago)

Post deleted by RogerRabbit

Reason for deletion: Mis-posted



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Invisiblemycofile
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4739519 - 10/01/05 02:16 AM (11 years, 2 months ago)

Hey roger, at what temp does that stuff break down? The paper says it can't be sterilized with the media, and the storage recomendations from a supplier say something like -5 million degrees. Do you just mix it in the agar just before it cools, like around 115 - 120 degrees? Thanks man.


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"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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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: mycofile]
    #4740125 - 10/01/05 07:01 AM (11 years, 2 months ago)

Yes. Add it at the very last moment when the agar is cool enough to handle without gloves or other protection for your hands, but still liquid enough to pour. If you add it even a little too early, the heat will ruin it.
RR


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OfflineJeremy_Davis
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4740790 - 10/01/05 02:10 PM (11 years, 2 months ago)

So Roger, can you detail your experiences with this? I'm so excited to see you with that snake venom (Why? God help me!)! I'm interested to know how you use it, because in the paper, they are making a transfer of the hybrid in a window of an hour or so from the fusion of the protoplasts from the two integrating species. So do you try to be this specific, doing this microscopically, or have you developed a more "community-friendly" version?
This could be a great new avenue for people to explore!
Please keep us posted!
Light and Love,
Jeremy Davis


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Jeremy Davis
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Check out the ECHO mushroom blog page to see our lab, growing facility, and more-www.echotech.org/greta


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OfflineRogerRabbitM
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Re: Req: Petri Dish Pics With Following Content... [Re: Jeremy_Davis]
    #4742565 - 10/01/05 11:07 PM (11 years, 2 months ago)

That one hour window can be extended by making a couple of transfers instead of just one. The reason for the very limited window is because the hybridized strain, being brand new and not established will be quickly 'swallowed up' by the two parent strains which are rapidly growing.

The procedure I've developed calls for making a transfer when the two parent strains are about 1/4" apart, before the very first mycelial strands drift into the zone of inhibition. Take a small square of agar, but be sure to get the very smallest amount of each parent strain as possible. By doing this, there is not a large mass from the parents waiting to overtake any possible dikaryotization(when two binucleate mycelial cells exchange genetic material as opposed to two mononucleate cells) between the two strains that may have occured. Remember, there are millions of cells in each parent strain by the time they meet up, and only one or two cells are likely to pass genetic material between them, forming the cross. Watch very carefully over the next few days for a third segment of growth to emerge. As soon as it's noticed, remove it to a dish of it's own.(a jewlers microscope is handy for this) Once the new growth gets established, test for hybridization by placing it on a dish with two equal sized mycelial wedges from the two parents. If a three way line of inhibition forms, you now have a third, independent strain that is a cross between the two parents. Bear in mind, even using toxicated agar, this will only happen once in 50 to 100 petri dish you perform the experiment on. Get ready for a lot of frustration before you see your first success. Then, you must do it again and again until you actually get a fruiting strain. It also sucks when you're away at work during the two to three hour window you have to make the transfers. It's amazing how much closer two growths can get to each other during ten to twelve hours while you're gone to work. It's like approaching a car head on, the speed of closing is twice that of either growth. Also, it's important to know the toxin breaks down very quickly, so don't put the two growths too far apart on the agar. You only have 48 hours or so for them to grow together before the toxin degrades, so you have to have all your ducks in a row before you start.
RR


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OfflinePsiloman
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Re: Req: Petri Dish Pics With Following Content... [Re: RogerRabbit]
    #4744462 - 10/02/05 08:56 AM (11 years, 2 months ago)

That is actually very interesting Roger! I have been following this thread and im actually amazed,since i thought that the only viable option for this kind of hybridisation would be protoplast fusion which recquires some enzymes to break down the "cell wall" (i think chitinase,they have chitin on the outside surface dont they?) and they doing some merging either with electric current or PEG (polyethylene glycol ,easily found). Correct me if im wrong ,but is mycelium a syncytium,meaning that all the cells are kind of joind together with free "moving" nuclei?

Some rather basic questions here as well :

1)With this method apparently one can cross only STRAINS of the same species ,right? One cant cross lets say Psilocybe cubensis with Psilocybe azuresence,right?

2) Seeing that you apparently bought a jar of this venom ,and you have quite some experience in using it (you gave even advice on the trasfers) im itching to ask : On the macroscopical ,fruiting level, did you get anything interesting with your procedures? If so ,what?


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OfflineKotton
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Re: Req: Petri Dish Pics With Following Content... [Re: Sillypcybin]
    #4765311 - 10/06/05 08:06 PM (11 years, 2 months ago)

I was never able to get growth like that on my petri dish's. I bought some premade stuff and when i injected spores on them i would just get some mushy cum looking stuff never any rhiz growth. The cum looking stuff would just expand everywhere....it sucked. lol


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OfflineJohnHolliday
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Re: Req: Petri Dish Pics With Following Content... [Re: Psiloman]
    #4765757 - 10/06/05 09:40 PM (11 years, 2 months ago)

John Holliday here - I did the original work and wrote the papers on the snake venom hybridization techniques. I would be glad to share my data and techniques with anyone intereted. This hybridization work grew out of Doc Stoller's work of the 70's and 80's on Agaricus hybridization. I have Doc's original papers still, and there is a ton of good info on hybridization. As well as some practical techniques. (such as laying a sterile microscope slide down the middle of the petri dish and inoculating the two strains on each side of the slide. The hyphae grow across the glass, slowly, and allow more time to capture the hybrids - longer than the 2-3 hrs mentioned in the paper. I didn't know about this trick until after I wrote the original paper. Just a jem I found in Doc's notes.)I would be pleased to provide you with copies - just ask. The question is often asked, can different species be crossed? Yes. Not always, but sometimes. Are there other hybridization triggers besides snake venom? Sure, lots. It is nice to know somebody is actually reading the papers we write. Thanks!


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