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OfflineCloneufc
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Crossing P. Mexicana (sclerotia) and psilocybe cubensis!
    #11269658 - 10/18/09 06:11 AM (8 years, 1 month ago)

I want to try crossing these species! I understand the process of doing it but lack snake venom. Im comming into some money soon, so I will try and order snake venom at a later date. This is a cheap hobby to me compared to my auto racing hobby.
Will anything else work in damaging the spore cell wall allowing for DNA exchange? Acids, or are they still experimental?

Speculate the outcome if these two species were to cross? Surface sclerotia jk.


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Invisiblebadman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11269771 - 10/18/09 07:22 AM (8 years, 1 month ago)

First off good luck with this hybrid project and obtaining snake venom, I expect you'll find both quite a challenge. If not impossible.


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Offlinerope

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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: badman]
    #11269896 - 10/18/09 08:47 AM (8 years, 1 month ago)

I wish you the best of luck man, and no harm in trying... but like badman said... its going to probably be hard... very hard. I don't even really understand the whole proccess, but have read some of the work for crossing penis envy and pf albino to make APE. But that is crossing two same species... I think to cross mexicana and cubensis is a whole new ball game.

Give it a go, and see what happens. Maybe give a shout out to workman and get some info on it. He seems to know his stuff.


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OfflinemuleV
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: rope]
    #11270175 - 10/18/09 11:10 AM (8 years, 1 month ago)

Why?  better chance to get a mating pair from Mexicana and or tampanensis or ATL#7. I think while there is a vast difference in these, there is a underlining genetic base. Which if I am right would make a cross possible. Was thinking of working on this awhile ago, just to see if it's possible. As far as mutagens (aka Snake venom), would first sit down and start by obtain mono cultures and work out from there, with out their use. Any way be prepared for a lot of work and keep detail notes, the chances are slim but fun to try.


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Offlineblackout
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: rope]
    #11270944 - 10/18/09 01:55 PM (8 years, 1 month ago)

Quote:

rope said:I think to cross mexicana and cubensis is a whole new ball game.



It is. If you want experiments there are loads of other easier things to try out, suited to beginners. I am surprised at the lack of experimentation going on.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: blackout]
    #11272419 - 10/18/09 06:57 PM (8 years, 1 month ago)

Im also trying to cross a Cube with a Pan Cyan. I read that RR pulled this off, so I know that combination is possible. I shouldnt have a problem getting snake venom. I have a squeaky clean record and if it were $5,000 a bottle I could care less. I just wish I knew before hand what hoops I have to jump through to get it. Im experimenting with Salicylic Acid and it hasent been working. I have read that nicotinic acid agar works and Im going to try that out before I even attempt to get snake venom. I just have to make sure that Im using the right chemical and not some derivative.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11272858 - 10/18/09 08:21 PM (8 years, 1 month ago)

Interesting and worth reading.


HYBRIDIZATION:

Once we had developed the substrate and growth parameters to optimize the target compounds, we started looking into the chemical profile differences from different strains of Cordyceps sinensis. Since there were so many strains of Cordyceps, and each strain has its own unique chemical profile, we tested all of the strains we were able to obtain. None of the known strains was shown to produce nearly the quantities of active ingredients found in the wild Cordyceps. So we started experimenting with ways to quantitatively increase the target compound production through the hybridization of Cordyceps strains; to cross breed them in order to gain greater production of target compounds. This was quite a challenge. Since spore collection and separation is very time consuming and results in entirely too much unknown variations, we felt this method would take too much time before we had reliable results. Rather we took a novel approach. We experimented with various ways to get different strains of the fungi to perform their own nuclear fusion. There are several chemicals known to trigger this exchange of genetic material between unlike cells. Nicotinic acid for instance, can be used to create hybridized mycelium. This compound is difficult to use and yields unreliable results. After trying several different compounds to trigger this fusion, what we settled on was snake venom.

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.

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


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. Top quality Cordyceps is no longer a health supplement only for the very rich. By utilizing these new methods of cultivation, the best quality Cordyceps is now within economic reach of even the common man throughout the world.




(FREEZE DRIED) SNAKE VENOM

US$ PRICE PER GRAM

Aspidelaps scutatus Shield Nose Snake  1800.00

Bitis arietans Puff Adder 180.00

Bitis caudalis Horned Adder 1500.00

Bitis gabonica Gaboon Adder 210.00

Bitis rhinoceros West African Gaboon Adder 210.00

Causus rhombeatus Night Adder 230.00

Dendroaspis angusticeps Green Mamba 510.00

Dendroaspis polylepis Black Mamba 495.00

Dendroaspis jamesoni Jameson's Mamba 850.00

Dendroaspis viridis West African Green Mamba 1010.00

Dispholidus typus Boomslang 2 400.00

Echis Pyramidium North East Carpet Viper 3250.00

Echis Ocellatus W.A. Carpet Viper 3350.00

Echis Coloratus Painted Carpet Viper 3400.00

Hemachatus Haemachatus Rinkhals 370.00

Naja annulifera Snouted Cobra 190.00

Naja melanoleuca Forest Cobra 190.00

Naja pallida Red spitting Cobra 210.00

Naja mossambica Mozambique Spitting Cobra 200.00

Naja Nubiae Nubian Spitting Cobra 210.00

Naja Nivea Cape Cobra 425.00

Naja Naja Kaouthia Monocle Cobra 100.00

Naja Haje Haje Egyptian Cobra 245.00

Naja Nigricollis Black-necked Spitting Cobra 200.00

Proatheris Superciliaris Swamp Viper 2000.00

Rhamphiophis Rostratus Rufous Beaked Snake 2000.00

Trimerusurus Okinawnesis Okinawa Habu 250.00



Edited by Cloneufc (10/18/09 08:32 PM)


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InvisibleAuxin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11275598 - 10/19/09 05:31 AM (8 years, 1 month ago)

Has anyone any info on use of nicotinic acid in promoting interspecific hybridizations? Specifically I'm looking for the concentration used in the nicotinic acid hybridization agar that is sometimes referenced.
I've used the search function and found nil, I am aware of InvisibleBlimeyGrimey's post from 15 months ago that refered to an agar recipe but that was just for avoiding niacin deficiency in auxotrophic mutants (it supplied 2 ppm niacin). Google scholar has not yielded any useful numbers.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Auxin]
    #11275668 - 10/19/09 06:47 AM (8 years, 1 month ago)

Potato Dextrose Yeast Agar:
10 grams agar
10 gram dextrose
500 ml potato water
1 gram nutritional yeast
Concentration 2.0 mg/mL add 0.5mL to 1.0 mL Nicotinic acid


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InvisibleAuxin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11276856 - 10/19/09 02:00 PM (8 years, 1 month ago)

Thats it? just 2-4 parts per million of a vitamin thats produced by mushrooms themselves? Kind of surprising that that might be enough to short out the sectorizing mechanism and allow them to cross.
Have you personally successfully used that recipe?


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Invisiblefastfred
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Auxin]
    #11276944 - 10/19/09 02:13 PM (8 years, 1 month ago)

Why not just get raw snake venom from a snake enthusiast and filter sterilize it?

As long as you did it quickly and properly stored it I wouldn't think you'd need to worry about purifying and freeze drying it.


-FF


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: fastfred]
    #11277506 - 10/19/09 03:40 PM (8 years, 1 month ago)

That concentration of nicotinic acid has been used in mycology and proven to work. Nicotinic acid itself is hit or miss. The rest of the agar recipe I got here and its proven to work.Snake Venom on the other hand is a reliable means of crossing fungal species.  I just hope that I can get it to work. It is vitamin B3 and you can get it at places like GNC. Just make sure its real niacin and not non flushing. Niacin is water soluble but the pills have a filler in them. It can be extracted from pill form, into a liquid with alcohol. I wanted to buy it but all I come up with is suppliers from China and you have to buy several kilos.

I might try the filter sterilize method. That is if Nicotinic acid doesnt work for me. Theres no shortage of Diamondbacks around here, you'd be surprised at how many people keep rattlesnakes as pets.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11279055 - 10/19/09 08:01 PM (8 years, 1 month ago)

I just found another site that sells it and its much cheaper than sigma aldrich. Its only $45 for 1 gram of Western Diamondback venom and it sterilized. Wow they want $1000 dollars for 1 gram of Mojave green rattle snake venom. They are all over the place where I live and one of the most deadly snakes. They have both a hemo toxin and neuro toxin.

From the site. Venom is collected under stringent laboratory conditions using disposable labwear for each extraction. Venom is collected into sterile plastic cups with parafilm covering. Snakes are allowed to bite into the parafilm diaphram and venom glands are not massaged. Immediately following collection, each venom sample is clarified by centrifugation at 500 x g for 5 minutes to remove cellular debis and frozen at -90 C until lyophilized.


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InvisibleBlimeyGrimey
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11291552 - 10/21/09 02:01 PM (8 years, 1 month ago)

Quote:

Cloneufc said:
I just found another site that sells it and its much cheaper than sigma aldrich. Its only $45 for 1 gram of Western Diamondback venom and it sterilized.




Now convince them to sell it to you. If you have success, pass the info along. I've wanted to get my hands on some venom for quite awhile. Though within the next year I'll have access to equipment capable of protoplast fusion.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: BlimeyGrimey]
    #11358419 - 10/31/09 05:55 PM (8 years, 1 month ago)



Well here is my nicotinic acid agar. I made about 20 of these. I used agar wedges of psilocybe cubensis (PESA) and Psilocybe galindoi (Alt#7). The agar recipe I used is not one posted. I also extracted nicotinic acid from pill form to liquid form. The mycelium seems to grow slower than normal on the nicotinic acid agar. Im just waiting for the mycelium to grow some more.


Edited by Cloneufc (10/31/09 06:01 PM)


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OfflineLadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11358689 - 10/31/09 06:50 PM (8 years, 1 month ago)

Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate? 
:jimmorrison:


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OfflineLucienis
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
    #11359776 - 10/31/09 10:08 PM (8 years, 1 month ago)

Quote:

LadyLittleZeppelin said:
Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate? 
:jimmorrison:




Wow for someone with 7 posts you sure seem to know the value of science!
Why waste time with experiments that might contribute something to the field when we can do the same things everyone else has done a million times that we KNOW will work. Where's the pay off in that?

Jerk.


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OfflineCloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Lucienis]
    #11360484 - 11/01/09 01:26 AM (8 years, 1 month ago)

RR crossed a cube with a pan cyan.  I dont see why this combination isnt possible. Im going to try and cross a cube and pan cyan also. I have pans but I want to cross pan goliath with a cube.  Im not sure as to why RR didnt release prints of it but my guess is that because it supposely looked to much like a cube, it would cause a rukus.


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InvisibleinskiM
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
    #11360848 - 11/01/09 03:57 AM (8 years, 1 month ago)

I don't think Roger Rabbit successfully fruited a cross between two different genera, what he got was P. cubensis.
inski.


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Invisiblebadman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: inski]
    #11361218 - 11/01/09 08:55 AM (8 years, 1 month ago)

Quote:

inski said:
I don't think Roger Rabbit successfully fruited a cross between two different genera, what he got was P. cubensis.
inski.




:highfive:


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