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 troncotron
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 Cross breeding experiments (pics) 
#11842133 - 01/16/10 06:06 PM (3 years, 4 months ago) |
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Here is the work i´ve been doing in cubensis breeding:
I bought a basic microscope enough for watching spores, hyfaes and clamp connections and started the agar work. I first attempted crossing with B+ and GT spores, but quickly realized that I needed some phenotypic marker to look for in the segregation, and B+ or GT wouldn´t work in that sense. So I took Penis envy and Shooting stars for my experiments:
Penis Envy
 
Shooting stars
 
First of all, a set of petri dishes were inoculated in a flow hod with a spore dilution of Penis envy strain. 48h later I could see some monokaryotic colonies starting to grow. I isolated 3 of these colonies and let them grow for a week, checking every day the absence of clamps. The growth was absolutely fluffy and cottony. Then I inoculated a new dish with a drop of a Shooting Stars spore dilution and let 24h to start germination. After that, I re-inoculated the dish with a piece of P.Envy monokaryon and let it for 5 days. Assuming there are 2 genes controlling mating type, there should be a 25% mating probability once anastomosis takes place, so that I tough this method would be more efficient than confronting monokaryotic colonies. The idea is waiting until that piece of P.E mats with the first compatible monokaryon it meets and then remove the initial piece when becomes dikaryotic.
Penis envy monokaryotic
After that, I took the same piece of mycelium and checked it with the microscope. It was full of clamps, so I remove the piece and put in other dish. It started to grow aggressive and rhizomorphic.
In order to avoid scapes, I inoculated one jar with the monokaryotic mycelium and three with the dikaryotic one. After seven days, the suspected hybrid had a really nice rhizomorphic growing.
This photo shows the differences in growth parameters in grain.
 
The P.Envy cottony never colonized the full jar. It just stopped growing. I tried several times and it couldn´t advance neither in grain or straw bulk.
Finally, the F1 results:
   
I will update when F2 is ready...
Edited by troncotron (01/17/10 06:00 AM)
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RogerRabbit
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11843816 - 01/16/10 11:06 PM (3 years, 4 months ago) |
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Clamp connections can be very hard to spot unless you got a pretty good scope. I'd suggest counting nuclei in each cell to ensure there's only one. How much did you dilute the spores? If you're just dumping a single drop from a commercial spore syringe, I'd suspect you're placing a few thousand spores on the dish, which would explain the fuzzy mycelium.
The bumps in your second picture look a lot more like metabolite oozing rather than clamps.
RR
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troncotron
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Re: Cross breeding experiments (pics) [Re: RogerRabbit]
#11845420 - 01/17/10 05:55 AM (3 years, 4 months ago) |
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Hello, RogerRabbit. I diluted the PE spore solution until 50-100 spores for drop. Then, took a drop and deposited in zigzag with a inoculation loop. The picture with fuzzy mycelium is the dish with the three isolated monokaryons of PE. I took them when they have just germinated an i could only see them with the scope and with a 10X magnifying glass. The PE dish photo in previous post was taken much later so they had already met.
About clamp connections, it is difficult to take decent photos with the camera attached to the ocular with tape , but i´m sure they are, i can clearly see them in each cell connection and i´m familiarized with them.
i´ll try countig nuclei, maybe with some tinction..
thanks
And excuse my English, i´m still learning
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 Pinback
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11845717 - 01/17/10 09:01 AM (3 years, 4 months ago) |
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Very interesting, thanks for posting! I'm looking forward to the update.
Did you use any surfactant to keep the spores from clinging to each other?
How would you grade the possibility that the strains didn't combine, and you have fruited Shooting Stars only?
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caricapapaya
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Re: Cross breeding experiments (pics) [Re: Pinback]
#11848596 - 01/17/10 07:51 PM (3 years, 4 months ago) |
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> How would you grade the possibility that the strains didn't combine, and you have fruited Shooting Stars only?
After the putative mating, he removed mycelia from the PE monokaryon (now a dikaryon) to grow out. After mating two monokaryons, the nuclei will migrate throughout the hyphal network of each individual, so removing material from the edge of the colony furthestr from the point of contact will give you a good chance that you are getting what you think.
But you are right, it would be a good idea to grow out each of the parent strains along side the putative hybrid to compare them under the same conditions.
Good job, by the way, this is the kind of experimentation I like to see!
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mule
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Re: Cross breeding experiments (pics) [Re: caricapapaya]
#11849394 - 01/17/10 10:17 PM (3 years, 4 months ago) |
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A little congo red will make clamp connections easier to see, but will kill the mycelia, if possible take a small sample from a mass, and stain it while prepping your slide, usually if you find one, there is more in the mass the sample came from.
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troncotron
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Re: Cross breeding experiments (pics) [Re: mule]
#11851977 - 01/18/10 11:18 AM (3 years, 4 months ago) |
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Thank you for the advices
the F1 has a huge spore production, i will inoculate soon some jars with a multispore solution.
 
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RogerRabbit
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11855000 - 01/18/10 08:15 PM (3 years, 4 months ago) |
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Another way to ensure you're getting the genetics you want is to cross two dikaryotic known fruiting strains. It fails about 90% of the time or more, but that's still probably better odds than breeding monokaryons, and then waiting to see if it's a good fruiter with the qualities you desire.
Put the two strains on the same petri dish and let them run together. If a third sector opens up along the line of isolation, it's a third strain. You can test it by placing on a petri dish with the two originals. If they develop a line of isolation between all three, you know you've succeeded.
RR
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troncotron
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Re: Cross breeding experiments (pics) [Re: RogerRabbit]
#11863891 - 01/20/10 04:38 AM (3 years, 3 months ago) |
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I found this method very interesting and confusing at the same time, I read J.Holliday paper, and I can´t understand what´s going on in this kind of somatic hybridization. If plasmogamy occurs between 2 dikaryotic strains and they can exchange his DNA trough recombination, the result would be a 4n organism, unless a meiotic stage takes place. But I suppose it would be a great discovery, cause as far as I know meiosis only takes place in the basidia.
Nevertheless, I find more useful working with monokaryons in a breeding program. A Bulk Segregant Analysis assisted with molecular markers will give you a lot of information, and once you have mapped the traits and mating type and have markers associated with them, you just have to isolate some monokaryons and look for the desired traits with a simple assay before crossing.
Edited by troncotron (01/20/10 04:40 AM)
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caricapapaya
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11864873 - 01/20/10 12:15 PM (3 years, 3 months ago) |
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keep us posted. dont let this thread die out.
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RogerRabbit
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Re: Cross breeding experiments (pics) [Re: caricapapaya]
#11865113 - 01/20/10 01:01 PM (3 years, 3 months ago) |
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Quote:
you just have to isolate some monokaryons and look for the desired traits with a simple assay before crossing.
Unfortunately it isn't that simple. The more we learn about fungi, the more we learn how little we actually do know. Nothing is simple. Please keep us posted on your progress.
RR
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Smokey the Bear
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11869337 - 01/21/10 12:10 AM (3 years, 3 months ago) |
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Interesting work and you wrote it up very well
Edited by Smokey the Bear (01/21/10 12:10 AM)
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fastfred
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Nice work and writeup! The monokaryotic pic looks a little weird though. What are the dots or speckles you see?
-FF
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troncotron
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Re: Cross breeding experiments (pics) [Re: fastfred]
#11877527 - 01/22/10 11:07 AM (3 years, 3 months ago) |
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Hello fastfred,
I think the pots appearing in the picture of monokaryotic micelium are media components. I took the picture over the petri dish, I mean without isolating a piece of it in a glass slide. This might also contribute to distort the image.
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Lennybernadino
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11880339 - 01/22/10 06:56 PM (3 years, 3 months ago) |
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If they can use somew rattlesnake venom to make a hybird of two seperate species from two seperate genera , then It would not be unimainable to think that a bit of rattlesnake venom might help teh process of getting two strains to combine . Just a thought, like hybridising P.cubensis and Stropharia roguso-anulata, that would be something!
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troncotron
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I suppose snakevenome increases the possibility that horizontal transfer takes place. Is the only way I can imagine. Two dikaryotic cells in contact may exchange some genes through recombination if the sequences involved are homologous enough. The resultant strain should be a non balanced hybrid, not a 50/50 hybrid.
Here, a better picture of clamp connections of the suspected hybrid
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fastfred
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11896147 - 01/25/10 04:08 AM (3 years, 3 months ago) |
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> Two dikaryotic cells in contact may exchange some genes through recombination if the sequences involved are homologous enough.
How would that happen? Recombination usually only happens during meiosis.
-FF
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troncotron
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Re: Cross breeding experiments (pics) [Re: fastfred]
#11896441 - 01/25/10 06:24 AM (3 years, 3 months ago) |
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I think it would be extremely rare, I was imaging something similar to illegitimate recombination in agrobacterium mediated transformations, but there are some models explaining homologous recombination in bacteria.
http://www.biorom.uma.es/contenido/av_bma/apuntes/T8/t8_recomb.htm
I don´t know if it can be spread to eukaryotic organisms, but if a trait passes from one strain to another, I suppose that recombination has to occur somehow. What do you think about it, Fastfred?
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fastfred
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Re: Cross breeding experiments (pics) [Re: troncotron]
#11897898 - 01/25/10 02:17 PM (3 years, 3 months ago) |
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If a nuclei did happen to migrate into the other strain it might end up getting destroyed. If a DNA fragment happened to contain a NLS it could make it to the nucleus and be integrated into a host chromosome.
However all of that is highly unlikely. And unless it conferred a highly advantageous trait it would go nowhere. Even if it did it would have to happen at a hyphal tip or it would end up being just a single cell in the myc.
-FF
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anonjon
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Re: Cross breeding experiments (pics) [Re: fastfred]
#11903824 - 01/26/10 08:52 AM (3 years, 3 months ago) |
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This is a good picture. I have a dumb question. These strands are hyphae and the little half circle bumps are where the clamps are? Or these strands are the clamp connections?
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The above post is fictional, hypothetical, or downright nonsensical.
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