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 Cloneufc
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 Crossing P. Mexicana (sclerotia) and psilocybe cubensis! 
#11269658 - 10/18/09 04:11 AM (1 month, 3 days ago) |
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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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 badman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11269771 - 10/18/09 05:22 AM (1 month, 3 days ago) |
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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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rope
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: badman]
#11269896 - 10/18/09 06:47 AM (1 month, 3 days ago) |
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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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mule
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: rope]
#11270175 - 10/18/09 09:10 AM (1 month, 3 days ago) |
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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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Agar
"Successful cultivation is based on a degree of knowledge, ability, circumstance, means & art. Normally, no two cultivation circumstance are identical. Meaning, everyone?s circumstance differs to the degree - what works for one person, doesn?t for another. Which creates inherent difficulties in answering individuals questions."
"The ART of the matter is FINDING WHAT WORKS FOR YOU - IN YOUR INDIVIUAL CIRCUMSTANCE. Even though the method may differ from others, or even the majority."
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blackout
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: rope]
#11270944 - 10/18/09 11:55 AM (1 month, 3 days ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: blackout]
#11272419 - 10/18/09 04:57 PM (1 month, 3 days ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11272858 - 10/18/09 06:21 PM (1 month, 3 days ago) |
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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
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Edited by Cloneufc (10/18/09 06:32 PM)
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Auxin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11275598 - 10/19/09 03:31 AM (1 month, 2 days ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Auxin]
#11275668 - 10/19/09 04:47 AM (1 month, 2 days ago) |
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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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Auxin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11276856 - 10/19/09 12:00 PM (1 month, 2 days ago) |
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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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fastfred
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Auxin]
#11276944 - 10/19/09 12:13 PM (1 month, 2 days ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: fastfred]
#11277506 - 10/19/09 01:40 PM (1 month, 2 days ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11279055 - 10/19/09 06:01 PM (1 month, 2 days ago) |
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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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BlimeyGrimey
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11291552 - 10/21/09 12:01 PM (1 month, 13 hours ago) |
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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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: BlimeyGrimey]
#11358419 - 10/31/09 03:55 PM (21 days, 9 hours ago) |
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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.
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Edited by Cloneufc (10/31/09 04:01 PM)
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LadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11358689 - 10/31/09 04:50 PM (21 days, 9 hours ago) |
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Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate?
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Lucienis
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11359776 - 10/31/09 08:08 PM (21 days, 5 hours ago) |
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Quote:
LadyLittleZeppelin said:
Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate?
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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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Lucienis]
#11360484 - 10/31/09 11:26 PM (21 days, 2 hours ago) |
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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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inski
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11360848 - 11/01/09 01:57 AM (20 days, 22 hours ago) |
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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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badman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: inski]
#11361218 - 11/01/09 06:55 AM (20 days, 17 hours ago) |
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Quote:
inski said:
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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German Kahuna
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: badman]
#11361345 - 11/01/09 07:52 AM (20 days, 17 hours ago) |
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Quote:
it supposely looked to much like a cube
That's because it was a cube.
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LadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Lucienis]
#11361443 - 11/01/09 08:27 AM (20 days, 16 hours ago) |
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Quote:
Lucienis said:
Quote:
LadyLittleZeppelin said:
Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate?
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.
For someone with a whole 71 posts you really seem to know a bunch too 
It was hard enough to cross Penis Envy with PF Albino. What he is trying to do seems almost completely scientifically impossible 
Thus, it is a waste of his time.
Edited by LadyLittleZeppelin (11/01/09 08:31 AM)
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11362913 - 11/01/09 02:05 PM (20 days, 10 hours ago) |
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Read here were RR crossed a Pancyan and cube. It was a cross, why would it magically be missing the veil? He also said the prints were viable.
http://www.shroomery.org/forums/showflat.php/Number/9421501#9421501
THOSE WHO SAY ITS IMPOSSIBLE NEED TO STAY OUT OF THE WAY OF THOSE WHO ARE DOING IT.
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Edited by Cloneufc (11/01/09 02:13 PM)
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badman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11363540 - 11/01/09 03:46 PM (20 days, 9 hours ago) |
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Im not saying its impossible but Im finding it VERY hard to believe, even more so when it says to offspring were fertile, someone should analyse the spores.
Where is this hybrid? There are no pics and its very easy to remove a veil from a cube when its breaking, and was the veil-less trait the only genetic trait passed down from the Pan genes, only one characteristic passed down... .
Way too bait IMO
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inski
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11363710 - 11/01/09 04:18 PM (20 days, 8 hours ago) |
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I will wait for the scientific paper that describes this successful cross and the results from the DNA or rDNA tests before congratulating Roger Rabbit, I hope he was successful, it would be an amazing achievement, good luck to everyone attempting these experiments!
I believe it's possible to cross different Psilocybe species and would like to see it done with proof to show the methods used!
Of particular interest to me would be the successful crossing of Weraroa novae-zelandiae with other Psilocybe species that are closely related like P. subaeruginosa.
inski.
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: inski]
#11368835 - 11/02/09 12:25 PM (19 days, 12 hours ago) |
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Real mycologists like Doc holiday cross species all the time. For legal reasons they only cross edibles. You can clearly see the cross that RR had going. He's a standup guy and to confirm it really was a cross he is going to send it for genetic testing. Give the guy some credit, for one he's the first person I have heard of trying to cross an active species.
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troncotron
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11370212 - 11/02/09 03:28 PM (19 days, 9 hours ago) |
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Why donīt you try isolating monokarions? i consider this technique much more convincing. I think the experiments using snake venom or nicotinic acid will throw different results than using monokarions (and less reproducible). With these products, you are assuming that two dikaryotic strains are exchanging exactly half part of their genome via recombination.. and thatīs pretty much to assume, in my opinion. Moreover, each cell may exchange different quantities of DNA resulting something like a.. chimeraŋ?.Itīs almost impossible to know what you have without using DNA testing. I think the only way to assure a real hybridization is by crossing monokarions (and is the method that researchers use for improving strains).
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badman
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11370260 - 11/02/09 03:35 PM (19 days, 9 hours ago) |
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Theres nothing like cold hard data to prove this.
If someone is willing to post the data I'll believe it.
No data, no proof the harder you will find it to convince people that you are correct.
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LadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: badman]
#11372276 - 11/02/09 08:09 PM (19 days, 4 hours ago) |
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Quote:
badman said:
Theres nothing like cold hard data to prove this.
If someone is willing to post the data I'll believe it.
No data, no proof the harder you will find it to convince people that you are correct.
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LitCloset
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11372447 - 11/02/09 08:26 PM (19 days, 4 hours ago) |
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i say if your having any succes aquiring the required materials, there is no harm at all in trying it out.
regardless of how hard it is, it also has a lot of luck involved. so do as many as you can, vary concentration etc and let us know about the results.
what are you using for a petri dish there? it looks like a jar, if it is i would advise getting some cheapo dishes.
it would not hurt at all to try this with some easier to do species (as well) for practice. try two cube "strains" like rr did.
hope you have results!
everyone else whos being negative, lets try to give some constructive advice instead.
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11372614 - 11/02/09 08:48 PM (19 days, 4 hours ago) |
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Quote:
LadyLittleZeppelin said:
Quote:
Lucienis said:
Quote:
LadyLittleZeppelin said:
Excuse me Cloneufc but isn't there better things to do than cross mushrooms that has a almost 0% success rate?
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.
For someone with a whole 71 posts you really seem to know a bunch too
It was hard enough to cross Penis Envy with PF Albino. What he is trying to do seems almost completely scientifically impossible
Thus, it is a waste of his time.
How do you figure it was hard to cross Penis Envy and PF Albino?
All Psilocybe Cubensis strains will cross without the use of any chemicals. Its also not a true hybrid. A true hybrid is a species cross not a sub strain cross. Your a noob and have nothing to contribute here.
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LadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11373217 - 11/02/09 10:05 PM (19 days, 2 hours ago) |
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I've read up enough to know that what you're attempting to do is almost scientifical impossible. When you prove me wrong and show me the evidence than I will stand corrected 
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troncotron
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11381999 - 11/04/09 07:18 AM (17 days, 17 hours ago) |
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Who says itīs almost impossible? itīs possible to cross even two distinct genera through protoplast fussion, there are many examples free in the web. In this case, you are talking about crossing two distinct species of the same genera, i think IS possible but difficult to do at home. And also think the methodology is wrong. You want a 50/50 hybrid, not a weird chimeric specimen. My suggestion is to isolate lots of monokarions of each strain and confront all of them covering all possibilities. I say itīs difficult because it requires lot of time and money, you may have to isolate and test 100, 200 monokarions before reaching something interesting.. or maybe only 10, who knows..
Buy you have to work with monokarions!
Edited by troncotron (11/04/09 07:19 AM)
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: troncotron]
#11382901 - 11/04/09 11:00 AM (17 days, 13 hours ago) |
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I know diluting spores on agar with a zigzag pattern can give you monokaryotic mycelium. You have to look for the first signs of germination with a microscope. I dont have a microscope. How can I do this without a microscope?
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Auxin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: troncotron]
#11383436 - 11/04/09 12:16 PM (17 days, 12 hours ago) |
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Quote:
troncotron said:.... you are assuming that two dikaryotic strains are exchanging exactly half part of their genome via recombination.. and that�s pretty much to assume, in my opinion.... I think the only way to assure a real hybridization is by crossing monokarions (and is the method that researchers use for improving strains).
I find it odd that such an emphasis is on exact 50/50 crosses. I've done hybridizations before, in my case I was working with plants but I never had a percentage of genetic hybridization as my ultimate goal, I had the sharing of specific traits between two or more species or cultivars as my goal. Therefore, in the case of mushrooms, given that dikaryotic crosses seem to be much easier for the home hobbyist to achieve- why not do it that way to achieve a cross with at least some of the traits your after? You could always do another cross to refine it more or then move to a backcross of monokaryons to get the desired trait into a hybrid thats much closer to one species than another.
For example: say you have a small tasty mushroom you want bigger and you have a bigger less tasty mushroom you think might be able to hybridize. Do a series of dikaryotic crosses and grow out all resultant hybrids, select hybrids that are large and show significant expression of other traits from the small tasty mushroom, then use spores from those to do monokaryon backcrosses with the original small mushroom. Not all spore grown monokaryons will have your desired trait but some will. The results would be hybrids with at least 75% the DNA of the small tasty mushroom and a good chance of having the large-mushroom trait and less work because half the process was done the easier simpler way. Anyway, its more likely that after one or two generations of dikaryon crosses you'll be satisfied with the result enough to not bother with any more difficult breeding steps.
Doesnt that seem logical? I just dont get the 50/50 fetish lol
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inski
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11385869 - 11/04/09 05:14 PM (17 days, 7 hours ago) |
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You can dilute spore solutions down until there are very few in a solution, then it is a lot easier to isolate monokaryons, vacutainers are extremely useful for this type of work.
inski.
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: inski]
#11386716 - 11/04/09 07:04 PM (17 days, 5 hours ago) |
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Without a Microscope I cant check for clamp connections. What kind of microscope will I need for checking monokaryotic mycelium?
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LadyLittleZeppelin
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11386837 - 11/04/09 07:20 PM (17 days, 5 hours ago) |
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*Edit
40x won't get it but 400 will, as the poster below me mentioned, and I found a site that sells those kind of microscopes.
http://fieldmicroscopes.com/0053000a.html
Fail or not it's worth a try I guess!
Edited by LadyLittleZeppelin (11/04/09 08:00 PM)
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inski
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11387059 - 11/04/09 07:51 PM (17 days, 5 hours ago) |
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I quite often view clamp connections using 400x magnification, I was able to clearly see the clamp connections and asexual conidia forming on the hyphae of the tissue culture I grew this Psilocybe species from!
inski.
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: inski]
#11388581 - 11/05/09 12:17 AM (17 days, 37 minutes ago) |
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Im gonna have to buy one now. I'll probably get one with a usb camera on it so I can post pics. I will be looking for mycelium with the absence of clamp connections. So far the 20 petri dishes have failed using dikaryotic mycelium. They either grew over each other or created a barrier against one another.
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troncotron
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: Cloneufc]
#11389089 - 11/05/09 04:23 AM (16 days, 20 hours ago) |
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You donīt need USB camera, look at this photos, i took them with a normal digital camera. In ebay you have 400X microscopes for 70 euros, it will work for your purpose.
 
 
Auxin, i prefer working with monokarions, for various reasons:
- Once meiosis and spore production has take place , you are going to have a huge diversity in the F1 descendant as a result of recombination, so you can choose and select your desired traits. If you are looking for a specific trait, working with dykarions is like a lotery. example: You will never isolate a red mushroom if the region of the crhomosome involved has not recombined,even in 10th generations... you will be loosing your time. If you cross monokarions you have much more gene pool, the "red trait" already IS in someplace, and you could isolate it by doing many generations
If you work with dykarions, you canīt know how much cells have recombined and how much recombination has occured. You may have products with a 0,000001% of recombination that will never show traits of one parental.
Another problem i see is that you may isolate a product with cells that have recombined and others that not.
Please, correct me if iīm wrong, thatīs only my opinion
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BlimeyGrimey
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: LadyLittleZeppelin]
#11390633 - 11/05/09 12:31 PM (16 days, 12 hours ago) |
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Quote:
LadyLittleZeppelin said:
It was hard enough to cross Penis Envy with PF Albino. What he is trying to do seems almost completely scientifically impossible
Thus, it is a waste of his time.
How was injecting a jar of monokaryotic PFA mycelium with PE spores hard enough? Quite simple actually. The hardest part was getting a monokaryotic culture.
Quote:
troncotron said:
Auxin, i prefer working with monokarions, for various reasons:
- Once meiosis and spore production has take place , you are going to have a huge diversity in the F1 descendant as a result of recombination, so you can choose and select your desired traits. If you are looking for a specific trait, working with dykarions is like a lotery. example: You will never isolate a red mushroom if the region of the crhomosome involved has not recombined,even in 10th generations... you will be loosing your time. If you cross monokarions you have much more gene pool, the "red trait" already IS in someplace, and you could isolate it by doing many generations
If you work with dykarions, you can�t know how much cells have recombined and how much recombination has occured. You may have products with a 0,000001% of recombination that will never show traits of one parental.
Another problem i see is that you may isolate a product with cells that have recombined and others that not.
Please, correct me if i�m wrong, that�s only my opinion
I haven't started my recombinant DNA classes yet, so I can't make a truly educated response, but I mostly agree with what you are saying.
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Cloneufc
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Re: Crossing P. Mexicana (sclerotia) and psilocybe cubensis! [Re: troncotron]
#11392202 - 11/05/09 04:40 PM (16 days, 8 hours ago) |
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Quote:
troncotron said:
You don?t need USB camera, look at this photos, i took them with a normal digital camera. In ebay you have 400X microscopes for 70 euros, it will work for your purpose.
Auxin, i prefer working with monokarions, for various reasons:
- Once meiosis and spore production has take place , you are going to have a huge diversity in the F1 descendant as a result of recombination, so you can choose and select your desired traits. If you are looking for a specific trait, working with dykarions is like a lotery. example: You will never isolate a red mushroom if the region of the crhomosome involved has not recombined,even in 10th generations... you will be loosing your time. If you cross monokarions you have much more gene pool, the "red trait" already IS in someplace, and you could isolate it by doing many generations
If you work with dykarions, you can?t know how much cells have recombined and how much recombination has occured. You may have products with a 0,000001% of recombination that will never show traits of one parental.
Another problem i see is that you may isolate a product with cells that have recombined and others that not.
Please, correct me if i?m wrong, that?s only my opinion
I agree. Monokarions are the way to go. I started out with Dykarions because thats all I have to work with at the moment.
Mushroom genetics are still not fully understood. There has been more work done with plants. Thats were most confusion takes place is because people compare mushroom genes to plant genes and they are not the same at all.
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