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 shroomsynergy
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 Cubensis Cross Breeding? 
#6612513 - 02/26/07 01:19 PM (17 years, 1 month ago) |
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I just got my new Microscope set up (up to 2000x), and was able to isolate a few spores onto a slide, but was wondering if there is a good source of info on how i would go about crossing one strain with the other.
would it be possible to make a karo solution, place a drop on a slide, and isolate a couple spores from 2 strains into a near vicinity with them able to germinate on the slide, and then grow on agar???
sooo many questions about this now. any info is awsome!!
P.S. I hope that my shitty spelling doesn't offend anyone. i type fast, don't proof, and suck at spelling. i love ya though!!!!
edited just for you!!!!!
Edited by shroomsynergy (03/02/07 12:16 AM)
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Workman
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Picking out individual spores for germination isn't practical, since not all spores germinate and its difficult to do cleanly.
It is better to do serial dilutions with sterile water and place drops of different spore concentrations on several agar plates. At the very earliest signs of germination on a properly diluted plate, transplant the smallest bit of mycelium to a new plate. This will likely be mycelium generated from one spore. To confirm, take a bit of the mycelium after it is well grown out on its new plate and place it on a slide. Look for clamp connections and if you don't see any, you are golden. Good luck!
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RogerRabbit
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Re: Cubensis Cross Breading? [Re: Workman]
#6612900 - 02/26/07 02:46 PM (17 years, 1 month ago) |
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Quote:
Workman said:
Picking out individual spores for germination isn't practical, since not all spores germinate and its difficult to do cleanly.
It is better to do serial dilutions with sterile water and place drops of different spore concentrations on several agar plates. At the very earliest signs of germination on a properly diluted plate, transplant the smallest bit of mycelium to a new plate. This will likely be mycelium generated from one spore. To confirm, take a bit of the mycelium after it is well grown out on its new plate and place it on a slide. Look for clamp connections and if you don't see any, you are golden. Good luck!
The voice of experience. 
RR
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Thomas Edison
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: RogerRabbit]
#6613517 - 02/26/07 05:39 PM (17 years, 1 month ago) |
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awsome advice, i'll get started that way also.
What does a clamp connection look like? is that when the hypa connects to another? Also, how long will that grow for before needing to connect to another? ...and does the mycelium avoid the other strains, or will they easily connect? I have these little 'miniplates' that i was going to use (they are PC'able pill containers) because they are smaller, and would waste less agar; do you think that is feasable???
thanx for the help guys!! Hopefully if this works out i'll have a cross between the OI, and PE strains!! that would be odd, lol
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Edited by shroomsynergy (02/26/07 05:41 PM)
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 fastfred
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Registered: 05/17/04
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I'm sure others will have better answers for your questions about times and alternate methods.
But here's a picture of a clamp connection I took.
It's slightly to the right of center. They're not all that easy to spot, but with an experienced eye you'll start picking them out.
-FF
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RogerRabbit
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Re: Cubensis Cross Breading? [Re: fastfred]
#6613614 - 02/26/07 06:13 PM (17 years, 1 month ago) |
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The best place to start is to get a copy of stamets "The Mushroom Cultivator" and read the first few chapters ten times. You can get it from fungi.com or amazon.com. There's no way you can start out without basic mycology knowledge to do something like what you're wanting. We'll be here to help with the advanced stuff that you don't get from his book.
RR
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semper in excretia sumus solim profundum variat
"I've never had a failed experiment. I've only discovered 10,000 methods which do not work."
Thomas Edison
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: RogerRabbit]
#6615330 - 02/27/07 01:06 AM (17 years, 1 month ago) |
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i am planning to get that book, but my new scope set my bank to ZeRo for a while, and well.... you may know how that goes.
thanx for the picture Fast!!!
again, many thanx for info~~!!!!
...Also, if you have a sec...please list any crossbreeds that you've heard of!! ( i know of PE uncut, and the PE6 (which i am skeptical)) I'm talking the REAL DEAL, not that "every strain that we know of today" crap, but true man made crosses, like Purple Haze/Bubblegum style!!! this ought to keep it interesting!
thanx!!!!
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CureCat
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Registered: 04/19/06
Posts: 14,058
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Quote:
shroomsynergy said:
...Also, if you have a sec...please list any crossbreeds that you've heard of!! ( i know of PE uncut, and the PE6 (which i am skeptical)) I'm talking the REAL DEAL, not that "every strain that we know of today" crap, but true man made crosses, like Purple Haze/Bubblegum style!!!
I'm not sure what you mean... The only selectively "bred" strains of cubensis that are available are the various Penis Envy strains, and the Albinos (the real albinos- not the "albino" strains sold by some vendors, which are simply light coloured, purple spored mushrooms- i'm talking about the all white fruits, with colourless spores), as far as I am aware.
All of the other strains available are not hand selected with the expectation of any particular consistent traits being passed on by the spores. Generally, each print should still represent the region that the original fruit was harvested from.
I'd be interested to know what strains of cubensis the Albino and PE were bred from... Anyone know??
Here is a thread by Workman, of his project with crossing the two "man-made" strains:
http://www.shroomery.org/forums/showflat.php/Number/5194890#5194890
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RogerRabbit
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Re: Cubensis Cross Breading? [Re: CureCat]
#6616102 - 02/27/07 09:56 AM (17 years, 1 month ago) |
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Spores from different strains are totally compatible, just as pollen from bubblegum can easily cross with purple haze. There is no challenge or or anything special about 'man made' strains. It happens every time you squirt spores into a jar. A strain is made when compatible monokaryons cross paths and exchange genetic information. It mattes little what the 'name' on the print was, or even if the monokaryons came from separate prints. Dikaryons can easily cross too via anastomosis. You need to pick up that book and do some reading. It gives a great starting point to build from and will dispel your early misunderstandings.
RR
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"I've never had a failed experiment. I've only discovered 10,000 methods which do not work."
Thomas Edison
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CureCat
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 Re: Cubensis Cross Breading? [Re: RogerRabbit]
#6617004 - 02/27/07 02:52 PM (17 years, 1 month ago) |
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*EDIT - Nope. not talking to me. Nevermind.*
I never said that strains were incompatible. All strains of Psilocybe cubensis are the same species, and thus, they can cross.
Maybe you were talking to shroomsynergy.
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Edited by CureCat (02/27/07 10:59 PM)
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: CureCat]
#6625796 - 03/01/07 07:48 PM (17 years, 1 month ago) |
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Hey agar, i just ordered that book!!!!! i'll get it and memorize it soon as i can!
i took WORKMAN's advice, and placed two different myceliums from different strains myc. without clamp connections into the same plate. they are just now meeting.
this ought to be cool!!!
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RogerRabbit
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An alternative is to isolate a couple of strains down to single sector dikaryons, and then place those on a petri and allow them to run together. If a third sector opens up and starts growing, isolate it. You can then test for the cross by placing all three on a new dish. A line of isolation should develop between all three.
RR
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Download Let's Grow Mushrooms
semper in excretia sumus solim profundum variat
"I've never had a failed experiment. I've only discovered 10,000 methods which do not work."
Thomas Edison
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: RogerRabbit]
#6626500 - 03/01/07 11:06 PM (17 years, 1 month ago) |
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cool, i think that if this works, i'll be able to tell pretty easy by the fruits, considering that one would be PE, and the other OI. i guess that only time will tell. I just hope that my efforts go to fruit, since i sat there, and checked them like every hour until i saw germination (which happened alot quicker than i'm used to) but my spores were new.
...anyways, if i dont see a totally unusual fruit produce from one of the dishes after i re-apply the combined mycelium to master dishes, and fruit to grain spawn, i'll try, try again. this microscope is freaking sick. the only hard part is steadying my hand to get a good sample (which still looks like i have a convulsion while taking samples. lololol)
sorry if my lack of terminology is pissing anyone off, especially RR, but it's the skill, luck and technique that really matters, right???? lolololol, just kidding, i know that some peeps work very hard at getting their degrees at microbiology, and mycology.
thanx for all of your help, and if this works, i'll be sure to send the people who help me some nice crossed prints. ...if this works. lol
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RogerRabbit
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It sounds strange, but if you'll sip a glass of wine or a single shot of vodka, it will steady your hands for precision work. In fact, at shooting competitions, they test them for alcohol for this very reason.
You'll want to duplicate your efforts several times over to increase the chances for success. Cubes have tetrapolar basidia, so hyphae from individual spores won't be compatible with all hyphae from other spores. It's the A, B, A1, B1 thing. I'm not sure paul covers that in TMC, but you might find some information on it at Tom Volks website. I know when he travels around, he gives an excellent presentation on exactly what you're trying to do.
RR
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Download Let's Grow Mushrooms
semper in excretia sumus solim profundum variat
"I've never had a failed experiment. I've only discovered 10,000 methods which do not work."
Thomas Edison
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Cubensis Cross Breading? [Re: RogerRabbit]
#6626713 - 03/02/07 12:27 AM (17 years, 1 month ago) |
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Hopefully these diagrams will help you to understand what goes on when monokaryons meet.
-FF
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: fastfred]
#6626813 - 03/02/07 01:00 AM (17 years, 1 month ago) |
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(FIRST PHOTO)
so, on the first pair, does the nuclear migration change the genetic code for that pair, or simply attach two strands into one genetically indifferent being? If not, what are the byproducts of such a union? would that stimulate a more variable outcome if that were to happen before isolation???
the second pic was too jumbled to read, although it looks like some good info!!
thanx man!!
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Workman
1999 Spore War Veteran
Registered: 03/01/01
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Quote:
shroomsynergy said:
cool, i think that if this works, i'll be able to tell pretty easy by the fruits, considering that one would be PE, and the other OI.
Actually, you may not be able to tell from the F1 generation. The Penis shaped cap is a recessive trait and the F1 generation will only have one PE nuclei. Expect all of the F1 mushrooms to look like normal cubes. If you see PE caps in the F1 generation, the cross has failed. Only after recombination in the basidia will the genes be shuffled into the spores. Multispore F2 cultures generated from the F1 spores is where the action is and all sorts of recessive traits can be revealed.
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CureCat
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Re: Cubensis Cross Breading? [Re: Workman]
#6675408 - 03/16/07 12:28 AM (17 years, 1 month ago) |
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Quote:
Workman said:
Actually, you may not be able to tell from the F1 generation. The Penis shaped cap is a recessive trait and the F1 generation will only have one PE nuclei. Expect all of the F1 mushrooms to look like normal cubes. If you see PE caps in the F1 generation, the cross has failed. Only after recombination in the basidia will the genes be shuffled into the spores. Multispore F2 cultures generated from the F1 spores is where the action is and all sorts of recessive traits can be revealed.
Mendelian inheritance is fun. 
Can't wait till I get moved and can set up lab in my room.
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shroomsynergy
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Re: Cubensis Cross Breading? [Re: CureCat]
#6676774 - 03/16/07 01:02 PM (17 years, 1 month ago) |
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thats very good to know. I am still colonizing the master grain jar, but i followed peoples advice, and took mycelium without clamps (at least to my knowledge) and combined them. then grew that out until i could get enough to use for inoculation. I made sure that the combined myceliums had clamps before i used it as an inoculum, and did stamets advice of cutting a wedge in half, and putting each half on opposite ends of a small sterile stick. that kicked ass!!!!
..anywho, hopefully this works, but i would still feel that it would be beginners luck. I hope to get a larger version of the PE. I used OI with the PE. if it works i'll call it PE - Ron Jeremy. lololol j/k
...Question though, after growing out the mycelium (OI after i took my samples) i noticed that it is of the cottony variety, and not rhizomorphic. is that bad for the F1??? or just for the pure OI culture???
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fastfred
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> so, on the first pair, does the nuclear migration change the genetic code for that pair,
No. Fungi have a different life cycle from mammals. When spores germinate they produce haploid organisms. That means that they have one set of chromosomes rather than one set of chromosome pairs.
When a haploid mycelium meets another haploid mycelium of the same species, and they are sexually compatible, the two mycelia join together and each cell receives a nucleus from the other mycelium. They are then diploid organisms, meaning that they have pairs of chromosomes in each cell. However, the nuclei don't fuse together as they do in plants and animals, they stay separate.
Only just before meiosis (for spore production) do the nuclei fuse. Then recombination occurs and the nucleus divides twice to produce four haploid spores, and the cycle starts again.
> i noticed that it is of the cottony variety, and not rhizomorphic. is that bad for the F1?
The same isolate will produce different morphology during different conditions or stages in their life cycle. The same genetics will exhibit different morphology. I would not worry about this. You could continue to grow out your isolate and it will likely become rizomorphic before long.
-FF
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