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InvisibleHeruuka
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Registered: 10/16/99
Posts: 313
Interspecific Hybridization by Protoplast Fusion
    #2411784 - 03/09/04 07:49 PM (13 years, 5 months ago)

Now this post must be brief, but i recently stumbled upon a textbook on mushroom breeding in my library. It has a chapter on the aformentioned topic, and looks like a very interesting emerging science in mycology. It allows for the breeding of incompatible (i.e. different species) basidiomycetes, through the isolation, adulteration through the use of a "fusagen", and hybridization of protoplasts. The resulting fusion products of these hybridizations often have a unique phenotype, and display characteristics of both parent species. The procedure doesn't seem to be all that complicated, and i'll go into further detail on thursday when i finally get a day off!

just think ==>  King Stropharia X P. cubensis  !!!!:eyemouth:

Cheers,
H


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Anonymous

Re: Interspecific Hybridization by Protoplast Fusion [Re: Heruuka]
    #2411812 - 03/09/04 07:56 PM (13 years, 5 months ago)

Obtaining the neccessary enzyme/chemicals for protoplast isolation is difficult without a commercial/research lab, and all the paperwork that implies.

If you find a source let me know.


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InvisibleHeruuka
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Re: Interspecific Hybridization by Protoplast Fusion [Re: ]
    #2411891 - 03/09/04 08:15 PM (13 years, 5 months ago)

err... this is Advanced cultivation, is it not?


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Anonymous

Re: Interspecific Hybridization by Protoplast Fusion [Re: ]
    #2413434 - 03/10/04 02:59 AM (13 years, 5 months ago)

Yes it is. Yes I am interested in the content of your thread. Most importantly I am interested in a source to obtain the cellulase and chitinase, and all the necessary media ingredients to isolate and maintain and then fuse the protoplasts, without owning a commercial lab or a research lab, and living in the USA.  I find it difficult just to get media for germinating fern spores and orchid seeds. Sigma doesn't sell certain things too anyone who asks to buy.

If these things are easy to obtain for anyone here please PM me.

I dig the thread. Just have no first hand knowledge to add to it. : :blush:

Most companies are going to question why you want to dissolve cell walls and maintain protoplasts.  And having a shipping adress that doesn't look safe for doing these things.


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Offlineragadinks
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Re: Interspecific Hybridization by Protoplast Fusion [Re: ]
    #2413646 - 03/10/04 05:58 AM (13 years, 5 months ago)

What are the exact names of the product you need ? I could ask around a bit - maybe someone I know ( a microbiologist ) does know how to obtain these things.


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Offlinezeronio
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Re: Interspecific Hybridization by Protoplast Fusion [Re: Heruuka]
    #2413706 - 03/10/04 06:49 AM (13 years, 5 months ago)

Can you give us also the title of the book?


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Offlinezeronio
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Re: Interspecific Hybridization by Protoplast Fusion [Re: zeronio]
    #2413713 - 03/10/04 06:51 AM (13 years, 5 months ago)

It's probably this one:

Genetics and Breeding of Edible Mushrooms
Chang, Buswell and Miles (Editors)


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InvisibleHeruuka
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Re: Interspecific Hybridization by Protoplast Fusion [Re: Heruuka]
    #2415389 - 03/10/04 03:58 PM (13 years, 5 months ago)

Damn, once again rushed for time.

I'll try and outline the procedure as thoroughly as i can, please feel free to contribute and point out any holes.

From:

Chang, Shu-Ting. et. al. ?Genetics and Breeding of Edible Mushrooms?. Gordon and Breach, 1993.


Simplified steps:

1. Isolation of individual protoplasts using lytic enzyme cocktail.
2. Fusion of protoplasts from different species using "fusagen"
3. Isolation of fusion product colonies (selection of hybrids)


More detail...

1. ISOLATION OF INDIVIDUAL PROTOPLASTS

MYG medium inoclulated with mycelial suspension and incubated for 6 days without shaking. Mycelial mass harvested by filtration, washed with sterile water to remove nutritive media, and incubated with 3ml of lytic enzyme for 1 to 2 hours. Mixture is then filtered again to remove unreacted mycelial strands, and centrifuged to give a sediment of protoplasts.

The lytic enzyme cocktail (Cellulase, Chitinase, etc.) commonly used in this process seems to be the major practical barrier, for reasons mentioned above, though a lytic enzyme preparation derived from Trichoderma(!!) called Usukizyme proved to be effective on its own. I'm looking for information on the preparation of Usukizyme from trich cultures since the stuff is in such startling/dissapointing abundance around these parts. Imagine that, a use for Trich!


2. FROM PROTOPLAST TO HYBRIDIZATION

Protoplasts are suspended in mannitol solution (0.6M) (see below) and incubated until hyphae are observed (as little as 48hr). The rate of protoplast regeneration is low; observed ~1.5% of protoplasts would produce hyphae. Protoplasts from the two parent species are mixed in a solution 35% polyethylene glycol 4000 (fusagen) and buffering agents to maintain osmotic balance, none of which are hard to obtain chemicals. This mixture is allowed to stand at 25C for 15mins, and is then centrifuged and plated onto osmotically balanced MYG agar. Fusion hybrids selected based on ?CLAMP CONNECTION FORMATION AND THE BARRAGE REACTION? (a little help guys?).

Mannitol is a carbohydrate that maintains osmotic balance, we gotta source this shit still, and polyethylene glycol 4000 seems to be pretty easy to get, here is one source

http://www.hamptonresearch.com/hrproducts/2529.html

The rest of the compounds are just common mineral salts like MgSO4, CaCl2 and such.


3.ISOLATION OF FUSION PRODUCT COLONIES

The research trials using Usukizyme with P. ostreatus and P. cornucopiae resulted in 10 identified fusion product colonies out of 300 total colonies. The fusion products produced fruits with the yellowish colour of P. cornucopiae and the morphology of P. ostreatus.

Pretty neat, eh? Anyways, i lifted and paraphrased this entire thread from the above book, so i have to give entire credit to them. I can post the exact media used in each step if people are interested. PEG is used as a laxative if it cant be purchased in pure form.

damn, gotta go.

i'll add more later

cheers

h


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Offlinepluteus
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Re: Interspecific Hybridization by Protoplast Fusion [Re: Heruuka]
    #2416585 - 03/10/04 09:26 PM (13 years, 5 months ago)

PROTOPLAST FUSION FOR AMATEURS

This is a modified version of an email I recently sent to some contacts (amateur cultivators) who were toying with grand ideas of shroom hybridization

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Reading up on protoplast fusion techniques does lead naturally to frankenstienian daydreaming about cooking up supershroom hybrids in your basement. But as a non-scientist, getting hold of the right chems is the least of your worries.

Yes, the required Novozyme, cellulases, mannitol, polyethylene glycol*, buffer salts etc, are all quite inexpensive and widely available to labs. And there will be lab people who are friendly to your cause.

Failing lab contacts, Heruuka?s above suggestion of producing your own purified lytic enzymes from Trichoderma is a good one ? providing, of course, that you are in possession of transformed strains, fermentation vats, and purification facilities (perhaps he/she didn?t realize that the cellulases / chitinases he/she mentioned are also harvested from molds like Trichoderma and Aspergillus, along with a high proportion of all other commercially available lytic enzymes?).

Anyway, imagine you?ve obtained the necessary reagents. Maybe you actually have. But there is still, unfortunately, a long list of essential things you won?t easily be able to achieve outside of a commercial or research laboratory, things like:
- finely controlled centrifugation
- single spore culture isolation to ensure your protoplasts are monokaryotic
- calibration of your lysis medium to achieve optimum conditions for the notoriously unique cell wall properties of each target shroom species
- sterile filtration

Alarmingly even these issues pale into insignificance when compared with the MAJOR problem of hybrid selection.

You?ve created 100000s of protoplasts of each species. A tiny fraction of these are viable, and an even tinier fraction (as low as 0.032% in some studies!) will actually fuse in the presence of a chemical fusogen. So how do you tell the fusion products apart from the unfused protoplasts?

In the protocol posted above, Chang et al recommend identifying products under a microscope according to the presence of clamp connections. This isn?t very sound advice, because many species and hybrids don?t produce clamps! It may be good criteria for Pleurotus, but there?s no reason your fantastic new Psilocybe hybrid will show recognizable mating structures, even if its parent species do. Trying to fruit every single potential fusion product is not an option, unless you wish to devote your entire life to the task. Plus, how will you be confident that failed fruiting trials failed because the strain was not a viable hybrid and not because a viable hybrid had unusual fruiting requirements? You won?t.

Then, how does one identify which protoplasts represent fusions between species? We are now uncomfortably wandering into the realm of auxotrophic strain creation, incomplete media, molecular characterization of nuclei, and fusion product isolation on the scale of thousands. Easy in a lab, not so in your kitchen.

Even if you?re brave enough to take all this on, there?s still little guarantee of success. Reports of successful hybridization mostly involve groups like Pleurotus that are already known for unusual properties of interspecies breeding compatibility. The few instances of intergeneric hybridization have not yielded strains that persist for many generations.

The technical explanation is that protoplast fusion only overcomes barriers to initial cell fusion, while doing nothing to prevent potential postfusion incompatibility (otherwise we?d all already be eating shiitake flavoured giant puffballs, etc.).

However, don?t let me discourage you entirely. Mushrooms are genetically bizarre ? eternally surprising in their sexual trickery ? and the fact that intergeneric hybridization is even possible is highly encouraging for growers and theorists alike. So, best of luck to any amateur attempting this kind of work. You might overcome all technical hurdles, find that Psilocybes love to fuse with King Stropharias ? forming clear clamp connections upon doing so ? and go on to reap a rich crop of amazingly huge/concentrated hybrid fruitbodies!! I?d love my cynicism to be proved excessive.

*An alternative to PEG-induced protoplast fusion is electrofusion, which although even more demanding offers the possibility of greater control over the fusion process.


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Offlinezeronio
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Re: Interspecific Hybridization by Protoplast Fusion [Re: pluteus]
    #2417607 - 03/11/04 02:52 AM (13 years, 5 months ago)

Very good post!
But look - now even artists manipulate genes and make "things" like this cactus growing human hair: :ooo:


( http://www.geocities.com/transgenicart/ )


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InvisibleSpeeker

Registered: 02/11/04
Posts: 649
Re: Interspecific Hybridization by Protoplast Fusion [Re: zeronio]
    #2419647 - 03/11/04 04:44 PM (13 years, 5 months ago)

Fungus obtained by the fusion of Grifola umbelleta and Ganoderma lucidum
LINK

(Yes, its the same link I posted in that "Reishi question" thread.)

As this patent shows all you need to make a fusion fungus is a little
bit of dirt, some PEG and the BIGGEST damn mortar you can find.  :lol:


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Offlinepluteus
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Re: Interspecific Hybridization by Protoplast Fusion [Re: Speeker]
    #2419929 - 03/11/04 06:08 PM (13 years, 5 months ago)

rampant dubiousness


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Anonymous

Re: Interspecific Hybridization by Protoplast Fusion [Re: pluteus]
    #2431125 - 03/14/04 02:02 PM (13 years, 5 months ago)

Good post.


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InvisibleMirrorImage
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Registered: 03/19/04
Posts: 50
Re: Interspecific Hybridization by Protoplast Fusion [Re: zeronio]
    #2455411 - 03/20/04 09:15 PM (13 years, 5 months ago)

like the link zeronio


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