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Offlinedenger
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Hybrid selection methods
    #8962364 - 09/21/08 09:17 AM (15 years, 6 months ago)

We have a few discussions about hybridizing and methods pop up here, but very little on ways to distinguish hybrids from parents.

Irregardless of a hybridization method, it is crucial to be able to select the hybrids out of a mix of organisms resulting from a hybridization procedure.

Normally you would get a whole lot of unaltered parents and very few viable hybrids, which will be lost in this pool of parents.

In one of the ideal systems, you would have one parent that is not growing in presence of one chemical (such as an antibiotic for bacteria) or in absence of a chemical (such as a specific amino acid), and another parent doing the same but with a different chemical. Some percentage of hybrids would survive on a media selecting for both factors, but none of the parents will.

In a less then ideal system you would have one chemical selector and one phenotypical selector, i.e. one parent will not survive on a given media, and also exhibits an easily observed characteristics (for example chartreuse mycelia :laugh: ). Once hybridized, the results are plated on a selective media which eliminates one parent and by observing colonies retaining the special phenotypical characteristics, hybrids can be found.

In even less then ideal system, you would have two parents with distinctly different phenotypical characteristics. For example, a chartreuse mycelia of one and lumpy growth pattern of another. Then, one will be looking for lumpy chartreuse colonies among all the parental colonies after hybridization attempt. One will have to have a lot of plates and a lot of patience for this method.

Now, my question to all of you here, is do you know of any strain of any easily cultivated mushroom which either has a deficiency (needs special chemical to grow), or has a resistance to a chemical, or has a special easily distinguishable characteristics of the mycelial growth?

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OfflineRogerRabbitM
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Re: Hybrid selection methods [Re: denger]
    #8962666 - 09/21/08 10:25 AM (15 years, 6 months ago)

That's not how you identify hybrids.  They're a distinct sector on a petri dish that is separate from either parent. 

For example, in the petri dish below, small samples of two species were placed about 1 inch apart on the same petri dish of venomated agar.  They were Pan cyan on the right, and P cubensis on the left.  As you can see, when the two cultures met, a third sector grew from the meeting point.  This sector was a hybrid of the two species.  Actually, they were of different genera according to mycologists who classify such things.

The fruits grown from this culture displayed properties of both species.
RR


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Offlinedenger
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Re: Hybrid selection methods [Re: RogerRabbit]
    #8962748 - 09/21/08 10:43 AM (15 years, 6 months ago)

RR, you'r talking about hybridizing two strains on a dish. I was talking of any method that produces a large number of individuals, such as PEG-induced hybridization of protease treated cells.

Your example is interesting, but not applicable to a large scale experiments.

Having a system which simplifies hybrid selection can lead to all kinds of wonderful things.

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OfflineRogerRabbitM
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Re: Hybrid selection methods [Re: denger]
    #8962801 - 09/21/08 10:52 AM (15 years, 6 months ago)

I guess you're the expert. For your information however, those are two distinct species, not strains.
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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Offlinecaricapapaya
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Re: Hybrid selection methods [Re: RogerRabbit]
    #8962824 - 09/21/08 10:58 AM (15 years, 6 months ago)

You can induce mutations (randomly) using a chemical mutagen or uv (or other methods) and select for mutants that are dependant on the inclusion of a certain chemical (amino acid, etc) Auxotrophic mutants are commonly used as markers in hybridizing things.

then when you have the mutant monokaryons you can use them in breeding programs and use minimal media as well as media supplemented with the "special" ingredient to select for hybrids.

good luck

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InvisibleAdoreChampignons
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Re: Hybrid selection methods [Re: denger]
    #8962926 - 09/21/08 11:16 AM (15 years, 6 months ago)

Quote:

denger said:
...Now, my question to all of you here, is do you know of any strain of any easily cultivated mushroom which either has a deficiency (needs special chemical to grow), or has a resistance to a chemical, or has a special easily distinguishable characteristics of the mycelial growth?




The kind of strains that you're asking for are most likely not those that are normally available to the public.  If a strain requires special chemicals or has certain deficiencies that need to be addressed in order for it to grow, it would be a strain that could not normally survive as a wild type.  I do not know of any strains that fit the description of what you're asking for.  However, I'd check with research institutions.

However, if you're just looking for a more definitive way of selecting out hybrids, there are a number methods.  It really depends on the level of sophistication that you want to invest in.  At the lower end, one could just buy finer measuring and weighing tools to make finer distinctions between mushrooms.  At the molecular level, you could do chromatographic separation tests on mushroom proteins to see what strains produce specific proteins.  At the genetic level, it is possible to isolate mushroom DNA and run it through a gel electrophoresis separation procedure.  A lot of these things can be done in a home laboratory.  However, one needs to really check the MSDS safety sheets on some of the compounds that you may be exposing yourself to.  A few of them are fairly carcinogenic.


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Offlinedenger
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Re: Hybrid selection methods [Re: caricapapaya]
    #8963102 - 09/21/08 11:50 AM (15 years, 6 months ago)

Quote:

caricapapaya said:
You can induce mutations (randomly) using ...




Yes, but I was hoping to skip this tedious and lengthly step - thus the question if anyone knows or (ideally) has strains of this sort.

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Offlinecaricapapaya
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Re: Hybrid selection methods [Re: denger]
    #8964393 - 09/21/08 04:31 PM (15 years, 6 months ago)

You know, thinking more about it, I think RR's response is probably the most amenable to everday homebrew breeding, and will yield results. The more complicated methods require much greater investment of time and $$.

good luck

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Offlinedenger
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Re: Hybrid selection methods [Re: caricapapaya]
    #8964616 - 09/21/08 05:27 PM (15 years, 6 months ago)

I am not disputing that this is a good method for what it does, but it is not what I was looking for. This method will not allow me to screen thousands of hybrids, its for a one-off.
I have no problem with obtaining special media, amino acids, antibiotics or PEG, or whatever Sigma will not sell to mortals without SWTG license.
I have no problem paying for the eq if I feel I need it.

I do have a problem with no access to strains, and it seems like I'm not alone.

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InvisibleAdoreChampignons
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Re: Hybrid selection methods [Re: denger]
    #8965027 - 09/21/08 07:09 PM (15 years, 6 months ago)

Quote:

denger said:
I am not disputing that this is a good method for what it does, but it is not what I was looking for. This method will not allow me to screen thousands of hybrids, its for a one-off.
I have no problem with obtaining special media, amino acids, antibiotics or PEG, or whatever Sigma will not sell to mortals without SWTG license.
I have no problem paying for the eq if I feel I need it.

I do have a problem with no access to strains, and it seems like I'm not alone.




Check with the American Type Culture Collection.  Their website is www.atcc.org .  I've ordered from them years ago when I was in high school.  I do not what what kind of regulations that they follow now.  Never the less, they are well known among molecular biologist.  If they cannot sell to you a culture, you might be able to find someone who works in a research laboratory that can order your cultures for you on their own department purchase order.  Check with your local agriculture department too.  Even though some strains of fungi are completely innocuous, certain states have agricultural regulators that don't have a background in microbiology or even biology.  As a consequence, when they see that you're shipping in a culture of bacteria, they literally flip out.


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InvisibleinskiM
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Re: Hybrid selection methods [Re: RogerRabbit]
    #8967065 - 09/22/08 06:36 AM (15 years, 6 months ago)

Hi RogerRabit,
In my recent post about hybrids you mention that it would be unlikely to have any success with hybridizing different genera, your culture image and comments above seem to contradict this, do you have images of the fruitbodies obtained from this culture?
I would be very interested in seeing them!
inski..


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OfflineRogerRabbitM
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Re: Hybrid selection methods [Re: inski]
    #8967425 - 09/22/08 09:55 AM (15 years, 6 months ago)

Yes, it's very difficult and out of hundreds of experiments, this is the only one that crossed. A nearly lethal dose of rattlesnake venom was used in the agar. I have pictures, but they're going into a paper I'll be writing for publication in the IJMM when I get time, possibly this winter. 

I'm also working on another hybridization technique that is showing early promise.  If it works, I'll post the results.  If not, this is the last you'll hear of it.  I don't post ideas or failures.
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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OfflineJuke Adro
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Re: Hybrid selection methods [Re: RogerRabbit]
    #8967897 - 09/22/08 12:06 PM (15 years, 6 months ago)

Quote:

RogerRabbit said:
That's not how you identify hybrids.  They're a distinct sector on a petri dish that is separate from either parent. 

For example, in the petri dish below, small samples of two species were placed about 1 inch apart on the same petri dish of venomated agar.  They were Pan cyan on the right, and P cubensis on the left.  As you can see, when the two cultures met, a third sector grew from the meeting point.  This sector was a hybrid of the two species.  Actually, they were of different genera according to mycologists who classify such things.

The fruits grown from this culture displayed properties of both species.
RR





When was that done? I never heard of it till now, I'm actually really excited to see this, its amazing, can anyone expect to see this in the near future in regards to microscopy?.


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OfflineRogerRabbitM
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Re: Hybrid selection methods [Re: Juke Adro]
    #8968019 - 09/22/08 12:48 PM (15 years, 6 months ago)

It's from a few years ago, immediately after working on the Redboy strain.  I made literally hundreds of cross-species attempts before getting this one.  I'm fairly certain I sent pictures to John Allen for verification on the ID at the time, but he's the only one.  I still have the culture though and will eventually get it back out.  Once I get the paper written up, I'll release it.  Until then, I'd prefer to keep it low key. 

You can clearly see the third sector that grew from the 12 o'clock position where the two species met. Those of you who are familiar with the looks of pan cyan and cubensis mycelium will clearly see the two in that picture.

I have another technique that was showing much better results than snake venom in crossing otherwise non-compatible strains, so if it works to create interspecies hybrids, I'll get a paper written on it. 

However, having moved to a new location high in the mountains near the Canada border this past spring, all mycology projects are on hold while we get our cabin ready for winter.  It's snowing like mad right now, and we've already had our wood stove running for the last couple of weeks, since late August when we got our first hard freeze.  I just cut two more cords of wood over the weekend to make sure we stay warm until mid-June, when we won't have to heat anymore.
RR


--------------------
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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OfflineJuke Adro
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Re: Hybrid selection methods [Re: RogerRabbit]
    #8968095 - 09/22/08 01:03 PM (15 years, 6 months ago)

Yeah I sure can see the different myceliums in their, the third "the cross" even has its own unique look, very cool indeed :wink:.

Well good luck with your cabin and the cold brrrrrrr, and I look forward to seeing some more of your work in the future :wink:


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Re: Hybrid selection methods [Re: RogerRabbit]
    #8968561 - 09/22/08 02:50 PM (15 years, 6 months ago)

It's a shame that the IJMM cost so damn much!

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Invisiblewisp

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Re: Hybrid selection methods [Re: RogerRabbit]
    #8971763 - 09/23/08 06:47 AM (15 years, 6 months ago)

RogerRabbit, what is the purpose of you crossing species, or rather, more specifcally, why do you do it?

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OfflineRogerRabbitM
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Re: Hybrid selection methods [Re: wisp]
    #8973951 - 09/23/08 04:14 PM (15 years, 6 months ago)

Quote:

why do you do it?




To see if I can.  It's the same reason I climb at least one mountain per week, even in winter.
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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Offlinelibertaire
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Re: Hybrid selection methods [Re: RogerRabbit]
    #11441533 - 11/12/09 03:02 PM (14 years, 4 months ago)

Quote:

RogerRabbit said:
It's from a few years ago, immediately after working on the Redboy strain.  I made literally hundreds of cross-species attempts before getting this one.  I'm fairly certain I sent pictures to John Allen for verification on the ID at the time, but he's the only one.  I still have the culture though and will eventually get it back out.  Once I get the paper written up, I'll release it.  Until then, I'd prefer to keep it low key. 

You can clearly see the third sector that grew from the 12 o'clock position where the two species met. Those of you who are familiar with the looks of pan cyan and cubensis mycelium will clearly see the two in that picture.

I have another technique that was showing much better results than snake venom in crossing otherwise non-compatible strains, so if it works to create interspecies hybrids, I'll get a paper written on it. 

However, having moved to a new location high in the mountains near the Canada border this past spring, all mycology projects are on hold while we get our cabin ready for winter.  It's snowing like mad right now, and we've already had our wood stove running for the last couple of weeks, since late August when we got our first hard freeze.  I just cut two more cords of wood over the weekend to make sure we stay warm until mid-June, when we won't have to heat anymore.
RR




Sorry to bump an old thread, but any updates on the progress of this ambitious project?  I don't doubt the fact that you did it, but I'd definitely like to see the results of a project like this, the prospects are crazy to think about.  I mean, if anything, send the culture to someone who has the time to work with it like workman, it's something that I think needs to get out there.  That would obviously be up to your discretion though.  I understand why you would want to take your time with it.

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OfflineMycopathic
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Re: Hybrid selection methods [Re: libertaire]
    #11449727 - 11/13/09 06:36 PM (14 years, 4 months ago)

cool

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