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covertjoy

Registered: 07/09/23
Posts: 272
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Can monokaryotic mycelium take a nucleus from an ungerminated spore?
#28471792 - 09/17/23 12:39 AM (4 months, 8 days ago) |
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A thought I had. I have heard about mycelium cannibalising other spores. Can they take a nucleus in the process?
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MagicDane
Experimenter

Registered: 09/19/23
Posts: 9
Loc: Denmark
Last seen: 2 months, 2 days
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: covertjoy] 1
#28475433 - 09/19/23 06:17 PM (4 months, 6 days ago) |
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Do you mean share DNA similar to what bacteria do with plasmids, or just consume the entire foreign nucleus into their cell?
I couldn’t find any literature about this subject, but I don’t think “normal” mushrooms (meaning basidiomycete fungi) can do this. I can’t say for all species of fungi, but most basidiomycetes have a very specific process they have to undergo when mating, where two genetically compatible homokaryotic hyphae meet and undergo plasmogamy (mate), and create a new cell that has both nuclei separate inside; a new heterokaryotic mycelium.
When they divide they make a copy of both nuclei, now have 4 nuclei (2 and 2 identical) and send 2 and sometimes 3 of the 4 nuclei into the new cell. In the case of one of the old nuclei ending up in the new cell, they then use something called a clamp connection to transport the duplicate nucleus backwards to the old cell, so it also contains 2 genetically different nuclei. Clamp connections are like a vein that connect parent and child cells, and can only be used once for this specific purpose.
They have to do this, because the cells are separated by septa (walls) that allow for the transport of cytoplasm between cells, but not nuclei.
A fun thing about clamp connections is that you can use a microscope and look at a mycelium to see if it has mated. It is useful when you are growing from spores on a Petri dish, or when you are crossbreeding strains or selectively breeding one strain for certain traits. 
Edit: I forgot to write that most “normal” mushroom forming fungi digest their food outside of their cells, by excreting digestive chemicals which destroys the matter they are targeting. Cannibalization in these fungi probably results in the cells being completely destroyed and the only the nutrients being absorbed.
Edited by MagicDane (09/19/23 06:31 PM)
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Alan Rockefeller
Mycologist

Registered: 03/10/07
Posts: 48,266
Last seen: 11 hours, 27 minutes
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: MagicDane] 1
#28479944 - 09/23/23 12:42 PM (4 months, 2 days ago) |
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Quote:
MagicDane said: I couldn’t find any literature about this subject, but I don’t think “normal” mushrooms (meaning basidiomycete fungi) can do this.
I agree. While spores do have nuclei, I don't think they are likely to find their way into a monokaryon. Stranger things have happened though.
Quote:
MagicDane said: A fun thing about clamp connections is that you can use a microscope and look at a mycelium to see if it has mated. It is useful when you are growing from spores on a Petri dish, or when you are crossbreeding strains or selectively breeding one strain for certain traits. 
This is really cool, though it has some limitations. Not all basidiomycetes form clamp connections, not all septa of dikaryons have clamps and it can take some time for dikaryotic mycelium to develop clamps. I wonder if it would be better to use a stain that stains the nucleus so they can be observed directly via fluorescence microscopy. It is relatively easy to convert a normal scope to a fluorescence scope with a UV or blue LED + filter above the objective. DAPI is the classic stain used for this but it is kind of expensive and I think there are some better options.
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: Alan Rockefeller]
#28482046 - 09/25/23 06:22 AM (4 months, 16 hours ago) |
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Quote:
Alan Rockefeller said:
Quote:
MagicDane said: I couldn’t find any literature about this subject, but I don’t think “normal” mushrooms (meaning basidiomycete fungi) can do this.
I agree. While spores do have nuclei, I don't think they are likely to find their way into a monokaryon. Stranger things have happened though.
Thanks for the replies regarding this. Have been thinking about how to test this experimentally.
And perhaps there could be some chemical agent which would facilitate a monokaryotic mycelium in taking a nucleus from an ungerminated spore. If so I wonder if the mating type would still apply.
Quote:
Alan Rockefeller said:
Quote:
MagicDane said: A fun thing about clamp connections is that you can use a microscope and look at a mycelium to see if it has mated. It is useful when you are growing from spores on a Petri dish, or when you are crossbreeding strains or selectively breeding one strain for certain traits. 
This is really cool, though it has some limitations. Not all basidiomycetes form clamp connections, not all septa of dikaryons have clamps and it can take some time for dikaryotic mycelium to develop clamps. I wonder if it would be better to use a stain that stains the nucleus so they can be observed directly via fluorescence microscopy. It is relatively easy to convert a normal scope to a fluorescence scope with a UV or blue LED + filter above the objective. DAPI is the classic stain used for this but it is kind of expensive and I think there are some better options.
Agree that searching for clamp connections under the microscope has limitations. Have done it in the past however have found that they can be elusive and ambiguous and as such, searching for the lack of them frustrating and inconclusive.
I've been looking into converting my microscope to fluorescence as per your recommendation. From what I've seen so far, people with more experience than me are getting quite unimpressive results in doing so. Also it seems like converting for blue fluorescence is the most difficult which would rule out using DAPI. Hoechst 33342 sounds good as it is more membrane permeable however also emits blue fluorescence.
Any resources or recommendations in regards to converting a microscope for fluorescence? I have a Motic B3 which has brightfield, darkfield, phase contrast and polarisation.
I wonder if observing the nuclei is possible without fluorescence microscopy?
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Alan Rockefeller
Mycologist

Registered: 03/10/07
Posts: 48,266
Last seen: 11 hours, 27 minutes
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: covertjoy]
#28482259 - 09/25/23 11:15 AM (4 months, 11 hours ago) |
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DAPI excites best at 359 nanometers, so a bright 365 nanometer LED should work well. I would add a ZWB2 filter because these LED's make a bit of aqua light.
You can get a 10 watt 365 nm LED for $17, I have one that I have been meaning to try but haven't yet. You could also get a 30 watt for $60 or 100 watt for $150. https://www.ebay.com/itm/175666297009
It would be a good idea to add a filter that blocks UV in the nosepiece to protect your eyes, polycarbonate looks like it would be a good choice.
I use a 100 watt white high CRI LED to light my microscope, thermal glued to a brick of aluminum. Smaller wattage LED's would not need as large of a heatsink.
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: Alan Rockefeller]
#28483279 - 09/26/23 02:44 AM (3 months, 30 days ago) |
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Thanks.
I've ordered a 10W 360nm LED to replace the illuminator bulb. Looks like it should be a fairly simple task to remove the mirror and attach the LED in it's place:

The voltage of the original bulb is 12V where as the voltage for the LED is 9-11V. So I also ordered some 1N4007 diodes. Putting two of these diodes in series ahead of the LED should drop the voltage across the LED to around 10.8V, if I understand the internet correctly. I'll test it with a voltmeter.
I'm thinking to place a single narrow angle white LED above the 360nm LED wired in parallel and drill some switches into the side of the microscope so I can turn fluorescence and normal brightfield on and off easily. But that's something I'll experiment with later.
I also ordered a 42mm ZWB2 filter to place over the illuminator iris:

If I remove the head from the microscope there is a place to put a filter. It's where the polarization analyzer goes. I measure the filter to be 22mm diameter. I may be wrong but 360nm is in the UVA spectrum so a clear acrylic filter should do the job of filtering this while allowing the 460nm blue light emitted by the Hoechst 33342? So I ordered a 22mm diameter 2mm thick acrylic disc. Perhaps 2mm is too thin. I also ordered some vintage 22mm glass UV filter. I'll experiment with both using UV detection cards.

Should I be using any special objective for this?
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Alan Rockefeller
Mycologist

Registered: 03/10/07
Posts: 48,266
Last seen: 11 hours, 27 minutes
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: covertjoy] 1
#28484083 - 09/26/23 06:49 PM (3 months, 30 days ago) |
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To power a LED, it is the current that needs to be regulated. If you just supply the right voltage without regulating the current it will pull too much power and fry the LED. I suggest using a 10 watt LED driver. If you don't find one easily you could message the seller of the LED and ask which driver they recommend.
This looks good, and you might want the lenses / heat sink too: https://www.ebay.com/itm/231941822121
The lenses would likely help feed more of the LED light into where it needs to go.
I am not sure if it is best to replace that halogen light, or just leave it there and put the LED under the stage. I would power the LED up and move it around and see how the image looks best.
I would just leave the bulb / mirror in place,leave the door on the back of the scope open and mount the LED(s) there, that way you can just take the LED's away and close the door if you want to put the scope back to halogen lighting.
Regular objectives should be fine.
You can shine the LED through the acrylic on to some white paper to see how much UV the acrylic blocks.
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rocky_raccoon
Not a number


Registered: 12/12/22
Posts: 94
Last seen: 4 days, 11 hours
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Re: Can monokaryotic mycelium take a nucleus from an ungerminated spore? [Re: covertjoy]
#28487586 - 09/30/23 10:04 AM (3 months, 26 days ago) |
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Acetocarmine is a common brightfield stain for DNA and RNA. Nuclei stain red, no fluorescence is needed and it is non-toxic. It works well on plant nuclei so I guess it should be the same for mushrooms.
https://www.k-state.edu/wgrc/electronic_lab/aceto_stain.html
-------------------- Nothing in life is to be feared, it is only to be understood. -Marie Curie
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