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Invisiblemicro
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Colchicine/ Other mutagens
    #1551217 - 05/15/03 03:24 PM (14 years, 1 month ago)

***WARNING: COLCHICINE AND ETBR ARE MUTAGENS. THEY CAN CAUSE CANCER, BIRTH DEFECTS, MULTIPLE APPENDAGES.... BAD STUFF***

Does anyone have any information on the use of colchicine to induce random mutations in cubensis? I would like to make "colchicine plates" but I only have a little bit of colchicine and don't know a working concentration.

Also, anyone ever heard of other mutagens, i.e. ethidium bromide being used?

Thanks,

--
Micro


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Edited by micro (05/15/03 06:04 PM)


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Invisiblemycofile
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Re: Colchicine/ Other mutagens [Re: micro]
    #1551643 - 05/15/03 05:31 PM (14 years, 1 month ago)

Please do not mention colchicine whatsover without strong warnings of the fact that it is a poison, and is potentially dangerous/deadly to work with. Also, please always include the fact that no cultures treated with colchicine (or any other chemical mutagen) should be consumed. One must go back to spores and start a 2nd generation of the mutated fungus before it is safe to consume.

That being said, yes there have been experiments, yes some were successful/interesting. I've posted some rather detailed info over the years, but it may have been in older posts which aren't available any more. Did you try a search, I know it's been discussed serveral times.

What is ethidium bromide, what types of mutations would one expect, and most importantly, what are the safety precautions one must follow if planning to implement it?


--------------------
"From a certain point of view"
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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemicro
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Re: Colchicine/ Other mutagens [Re: mycofile]
    #1551703 - 05/15/03 05:54 PM (14 years, 1 month ago)

Yeah -- you're right, it is dangerous but you need a license to get it (at least in the U.S.) And you're right -- nothing can be consumed, not even from the fruits from innoculating substrate with a mycelial culture that had it in it.

EtBr is a reagent used to stain DNA in PCR analasys. It is dangerous enough that many people refuse to use it even in a lab. I would imagine it could cause random mutations -- it is a mutagen and readily available to me.

I did a search but only came up with something about grapefruit juice.

--
Micro


--------------------
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(Avatar is Maxxy, a character by Mizzyam, RIP)


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Offlinehomebrew
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Re: Colchicine/ Other mutagens [Re: mycofile]
    #1554306 - 05/16/03 04:23 PM (14 years, 1 month ago)

Ethidium Bromide is a DNA intercalator and causes base pair deletions/insertions to DNA when replication occurs.


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Invisiblemycofile
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Re: Colchicine/ Other mutagens [Re: micro]
    #1554337 - 05/16/03 04:38 PM (14 years, 1 month ago)

Now that the safety issues are out of the way, colchicine is easily extracted from the bulbs of the autumn crocus. Experiments are readily available from weed specific sites for making the extract, just a tea really. The tea can be used on plates, and the resulting mycelium will either die (most often) or become mutated (double number of chromosomes). Out of those that survive, most will be worthless freaks of nature, some will fruit with varrying degrees of success. Spores can be taken from this generation and will carry on the diploid characteristics of the treated culture. They will also be "safe" so far as anyone can guess, and carry over no traces of the colchicine. They may entirely possibly be mutated to produce other toxins however, so caution should still be used. You just never know what will happen when start rearranging mother natures code. BTW, nearly every strawberry you eat comes from diploid stock that was originally treated with colchicine years ago, they strive from their diploid status. Little to no research has been done by true mycologists with colchicine and fungi, at least none that I've found. Very few professors of the related subjects have more than the educated guesses an intelligent hobbyist will come up with.

Never heard of EtBr, but seems like I swiped a test tube from a hospital ER one time that had a gel type culture media that contained it. Maybe not, I discarded it years ago, but seems like that's what it was. I never used it.

So far as colchicine goes, experiment with caution, you may end up blind or with polyploid kids. Please keep safety warnings at the bottom of all posts on the subject as some idiot may stumble across one post and not read the entire thread.

BTW, I can go into detail on how colchicine doubles chromosomes if you would like. Basically it just interupts mitosis by dissolving spindle fibers (pahse III?) once they've formed to seperate the newly double chromosomes. Then once out of contact with the colchicine, the cell undergoes normal mitosis, but has a double set of chroms to begin with so it yields two cells with double chromosomes.

It is speculative at best, but it seems like to me that a single spore from a mutated culture would be capable of growing and fruiting without having to mate like normal spores do. The single spore would already contain the normal number of chromes for the species, so it would yield normal fruits if it didn't mate. If it mates, which I would imagine it would, then the culture becomes diploid and would likely express mutations.

Be careful.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemicro
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Re: Colchicine/ Other mutagens [Re: mycofile]
    #1554582 - 05/16/03 06:27 PM (14 years, 1 month ago)

Any ideas on concentration in 1L (cooled) media?

--
Micro


--------------------
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(Avatar is Maxxy, a character by Mizzyam, RIP)


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Offlinehenryp
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Ethidium bromide... [Re: micro]
    #1558891 - 05/18/03 11:40 PM (14 years, 1 month ago)

is a frame-shift mutagen. Leave it alone. If you aren't trained in using it, and don't have a safe way (safe for everyone, not just you) way to dispose of it, leave it be.

Why tempt fate?


--------------------
"You do know how to whistle, don't you, Steve?  You just put your lips together and.....blow"


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Invisiblemicro
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Re: Ethidium bromide... [Re: henryp]
    #1558910 - 05/18/03 11:54 PM (14 years, 1 month ago)

I have a chemical hood that would suck the wind out of you and we get rid of barrels of EtBr waste every week. EtBr can also be absorbed using activated charcoal and bleach will break it down.

It is bad, but I work with stuff like OsO4 -- I'm not too concerned. But for someone outside a lab or who is not skilled, yes -- it's bad stuff.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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Invisiblemycofile
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Re: Ethidium bromide... [Re: micro]
    #1559015 - 05/19/03 12:54 AM (14 years, 1 month ago)

I'd read some of the botanical references and use them as guidlines if you are looking for specific concentrations. That's where I started, too bad I didn't keep that lab book......
BTW, shoudn't be too hard to find. In 9th grade I induced polyploidism into some plants with the help of a text book and the guidance of my freshman biology teacher. Seemed almost common......


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemicro
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Re: Ethidium bromide... [Re: mycofile]
    #1559031 - 05/19/03 01:01 AM (14 years, 1 month ago)

Guess I'll go with 10^(-5) M? This sound about right?

I've read some stuff, but if you could point me in the right direction it would be greatly appreciated....

--
Micro


--------------------
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(Avatar is Maxxy, a character by Mizzyam, RIP)


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Invisiblemycofile
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Re: Ethidium bromide... [Re: micro]
    #1559036 - 05/19/03 01:05 AM (14 years, 1 month ago)

Just cuz you replied so quickly, I'll see what I can do. BTW, thanks for the warning edit in the first post, makes me feel safer.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemycofile
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Re: Ethidium bromide... [Re: mycofile]
    #1559054 - 05/19/03 01:15 AM (14 years, 1 month ago)

This is just something I found, no recomendation here from me as to what to do......
Quote:

Colchicine is a white powder that readily dissolves in water and is obtained from any one of a number of species of plants. When colchicine comes into contact with dividing cells, it alters the mitotic spindle fibers. This is achieved by the application of a 0.2% aqueous solution of colchicine for a period of not less than 48 hours. The chemical acts specifically on the spindle fibers without seemingly to alter the individual chromosomes. When the cell recovers from being treated with an application of colchicine, a new spindle appears and the nucleus undergoes a regular mitosis with the increased number of chromosomes. If sufficient numbers of cells are have been changed from diploid to tetraploid, the new plant tissue is tetraploid.



&
Quote:

Colchicine is a chemical used to create polyploid plants, by interfering with chromosome migration. This should not be attempted by anyone not familiar with laboratory procedures and safety techniques. The chemical is classified highly toxic. To use the Colchicine, laboratory technicians prepare a pre soaking solution of distilled water with about 0.05 % Colchicine. This will cause many of the seeds to die and not germinate, but the ones that do come up will be polyploid plants. This is the accepted difference between such strains as "Redneck Techno" and normal grass. The first generation plants contain residual chemicals, and can be dangerous if smoked. Subsequent generations will be polyploid plants and not contain any residual mutative chemical. Do not try this at home unless you have received proper supervised training!



also found
Quote:

It should be noted that COLCHICINE is a powerful deadly poison that has killed in doses as low as 6mg, though deaths are usually from 20mg or more. And it flies around with the least ammount of static. Also it causes birth defects, even if the exposure is well before impregnation.
My boss never supervised me when I was working with potassium cyanide, but when I was working with colchicine he watched me like a hawk- the stuff is THAT BAD.



http://biotech.icmb.utexas.edu/botany/colch.html
easy enough
Be careful kiddies.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Invisiblemicro
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Re: Ethidium bromide... [Re: mycofile]
    #1559089 - 05/19/03 01:32 AM (14 years, 1 month ago)

Thanks a bunch, (5) shrooms 4u!

I can work it out from there.

--
Micro


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)


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Invisiblemycofile
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Re: Ethidium bromide... [Re: micro]
    #1559907 - 05/19/03 01:49 PM (14 years, 1 month ago)

PM me when you get some results, good or bad.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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