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Offlinemajik_monkey
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Registered: 01/24/03
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Getting a strain isolate without agar ??
    #1304592 - 02/13/03 06:24 AM (13 years, 9 months ago)

one of the alternatives to cloning wiith agar is doing a couple g - g transfers, which I've read allows the stronger, more rhizomorphic mycelleum to dominate.....so with this in mind....say you colonize some WBS....there are noticable differences in mycelleum quality throught the cake surface (from multiple spore germination)....can you cut out pieces that appear to have strong rhizo growth and use them to innoculate spawn?
Anyone tried anything like this before?


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OfflineRaadt
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Re: Getting a strain isolate without agar ?? [Re: majik_monkey]
    #1304612 - 02/13/03 06:30 AM (13 years, 9 months ago)

yes, it will selectively weed out some of the weaker breeding strains. It wont be as efficient as agar, and I wouldn't go past 3 generations of transfers. But it should work as a course prefilter, at least.


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Raadt

-- The information I provide is only information from readings, growing of gourmet mushrooms, and second hand stories--


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Invisibletripndicular
My Minds Eye IsRhizomorphic

Registered: 08/25/02
Posts: 2,791
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Re: Getting a strain isolate without agar ?? [Re: majik_monkey]
    #1304646 - 02/13/03 06:41 AM (13 years, 9 months ago)

As RAADT said so masterfully .....YES is possible
Risky...contam risk is greater , you must man handle grain alot to harvest selected colonies .
Tough...may be small clumps you harvest to do transfer with , thus making agar much more controlled IMHO . If colony you choose is small , may not be quite enough to do "good" transfer . What safe ratio is aprox 25% in G2G transfers ?
But very possible with TLC !

oopppsss sorry brain farted this was using cakes to take isolation from ....anywho good stuff !


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Any information I give is not intended to aide you in the production of potentialy illegal substances !None of my exp comes from growing illegal varities , so take it as you will .
So with that said here is our mission statement .

Then the priest fell into a trance or swoon,& said unto the Queen of heaven ; Write unto us the ordeals; write unto us the rituals; write unto us the law !


Edited by tripndicular (02/13/03 06:45 AM)


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InvisibleIDontGrow
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Registered: 09/23/02
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Re: Getting a strain isolate without agar ?? [Re: tripndicular]
    #1304968 - 02/13/03 09:00 AM (13 years, 9 months ago)

i feel the vendors are shiping very good "isolates" as is....they prity much do all the work for you by sending you the spores of the strains they isolate.


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OfflineRaadt
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Re: Getting a strain isolate without agar ?? [Re: IDontGrow]
    #1305015 - 02/13/03 09:35 AM (13 years, 9 months ago)

spores will germinate and create their own strains, the vendor isolation has nothing to do with anything. Vendors or the original person, can domesticate the strain some, by environmental stressing, and over time the genetics may change. But when spores germinate they each create a dikaryotic strain of mycellium, doesn't matter if it's spores from a cow patty, or from a syringe.


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Raadt

-- The information I provide is only information from readings, growing of gourmet mushrooms, and second hand stories--


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Invisibletripndicular
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Re: Getting a strain isolate without agar ?? [Re: Raadt]
    #1305070 - 02/13/03 10:00 AM (13 years, 9 months ago)

Thanx RAADT , I was about to go into each spore has its own genetic make up , so issolations are still required , if that is the sort of thing you are into .
I love doing agar isolates , almost as much as eating the little fellas !


--------------------
Any information I give is not intended to aide you in the production of potentialy illegal substances !None of my exp comes from growing illegal varities , so take it as you will .
So with that said here is our mission statement .

Then the priest fell into a trance or swoon,& said unto the Queen of heaven ; Write unto us the ordeals; write unto us the rituals; write unto us the law !


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Invisiblepsyphon
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Re: Getting a strain isolate without agar ?? [Re: majik_monkey]
    #1306390 - 02/13/03 06:52 PM (13 years, 9 months ago)

Select a mushroom that shows good characteristics. (large, healthy, fast growing, etc)

Make your grain jars as usual (maybe a little less water).

After pressure cooking and cooling, use a sterile syringe to suck up some hydrogen peroxide (3%) and inject about 5mL (mL = cc) into each jar. Shake the jars well.

Use a sterile scalpel to cut pieces from the inside of the mushroom's stem.

Place these pieces into the jars and shake well.

Colonize as usual.

You have now created a fruiting isolate a.k.a. clone! Case or spawn as usual.

This worked well for some people I know. Thanks to Rush Wayne, Mycofile, Anno and others for helping people with these ideas.


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"The real voyage of discovery consists not in seeking new landscapes but in having new eyes."
- Marcel Proust

I wish you all ceaselessly flowing moments of happiness.


Edited by psyphon (02/13/03 07:02 PM)


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InvisibleJoshua
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Registered: 10/27/98
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Re: Getting a strain isolate without agar ?? [Re: Raadt]
    #1306430 - 02/13/03 07:09 PM (13 years, 9 months ago)

I don't completely agree.

With each clone/isolation you are creating a bottleneck event, thereby reducing the available genetic diversity.

Joshua


--------------------
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OfflineRaadt
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Re: Getting a strain isolate without agar ?? [Re: Joshua]
    #1307634 - 02/14/03 09:44 AM (13 years, 9 months ago)

In the spores?


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Raadt

-- The information I provide is only information from readings, growing of gourmet mushrooms, and second hand stories--


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InvisibleG a n j a
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Re: Getting a strain isolate without agar ?? [Re: Raadt]
    #1307853 - 02/14/03 11:24 AM (13 years, 9 months ago)

I would have though if you fruited an isulate in a casing that all the mycelium would hold the same genetics there for semi stabalising its spores in its fruits due to lack of other germinated spores being present though the isulate will contain differing genes from the origanal multispore you isulted from and if you continued the process would it not be possible to stabalise the strain making even multi spore inoculations stable?


newbie question  :crazy:


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OfflineAzmodeus
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Re: Getting a strain isolate without agar ?? [Re: Joshua]
    #1307912 - 02/14/03 11:48 AM (13 years, 9 months ago)

Josh, what do you mean by bottleneck event? :confused:

And if it reduces the genetic diversity, isn't that good?  Cus the reason you are cloning is to reduce the diversity and have it more or less the same so that the fruits should resemble the parent.

Just curious... :tongue:


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"Know your Body - Know your Mind - Know your Substance - Know your Source.

Lest we forget. "


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Offlinethe spiral
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Re: Getting a strain isolate without agar ?? [Re: Azmodeus]
    #1307927 - 02/14/03 11:54 AM (13 years, 9 months ago)

bottleneck event - you're taking the spores only from the best shrooms, selectively excluding all but the best genetics...


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OfflineAzmodeus
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Re: Getting a strain isolate without agar ?? [Re: the spiral]
    #1307956 - 02/14/03 12:10 PM (13 years, 9 months ago)

bottleneck event - you're taking the spores only from the best shrooms, selectively excluding all but the best genetics...

But weren't we talking about taking a tissue sample from the most charismatic, not spores?
I assume once the mycelium from the tissue fruited you wouldn't harvest those spores but start again from a multispore then pick the best.  The flush from the tissue sample will have all fruits similar, but spores aren't good is this correct? :confused:


--------------------
"Know your Body - Know your Mind - Know your Substance - Know your Source.

Lest we forget. "


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InvisibleZen Peddler
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Re: Getting a strain isolate without agar ?? [Re: Joshua]
    #1308436 - 02/14/03 04:26 PM (13 years, 9 months ago)

Well put Joshua!
Isolating without agar will be so much more difficult because it will be hard to see.


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InvisibleZen Peddler
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Re: Getting a strain isolate without agar ?? [Re: G a n j a]
    #1308444 - 02/14/03 04:28 PM (13 years, 9 months ago)

I dont understand your post.


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InvisibleJoshua
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Re: Getting a strain isolate without agar ?? [Re: G a n j a]
    #1309001 - 02/15/03 12:33 AM (13 years, 9 months ago)

Spores are haploid. This means they carry 1 set of genes.

When spores germinate they produce haploid hyphae. Hyphae are the filaments of cells that make up mycelium. These are labeled + and -. + and - are called mating types. When a haploid hyphae of one mating type crosses path with the opposite mating type. The hyphae fuse and undergo a process called plasmogamy. Plasmogamy is the fusion of the cytoplasm between cells. Once plasmogamy has occured the nuclei fuse and combine chromosomes to form a diploid nuclei. Diploid means the nucleus has two copies of each gene. One copy came from each mating type.

The new diploid cell then divides to form additional diploid cells. It is usually the diploid mycelium that grows faster. When the correct environmental queues are available the diploid mycelium forms a small mass. The is mass is a hyphal knot (from hyphae) and eventually grows into a complete mushroom.

Inside the gills of the mushroom are the terminal ends of the dikaryotic mycelial cells, it is at these cells that karyogamy occurs to form the diploid basidium. The diploid basidium then undergo meiosis to form four haploid nuclei. Each basidium also produces an appendage through which the haploid nucleus travels to form the basidiospore. These basidiospores then break free and are held between the gills by an electric static force. When the conditions are right the haploid basidiospore is released and carried away by air currents to germinate into new mycelium on another substrate.

______________________________________________

Each fruit is made up of a congregation of hyphae. These hyphae have two copies of each gene just like you and I (most of us at least). When you clone a fruit you are selecting a specific set of genes. These genes are present twice with in the culture. When you fruit this clone the spores from it will only be rearrangements of these two sets of genes.

A bottleneck event is an event in which the genetics of a population is reduced greatly. Imagine 100 people are left on the Earth. Only the genes from these 100 people will be present in future generations. If all of these people were Eskimos, the future progeny would appear as Eskimos. You would lose all other races.

When you isolate or clone you are causing the above circumstance. You are allowing only a small number of genes to be passed on to the next flush.

At least that's how I understand the basics. Keep in mind that new genes can arrive through mutation, but the rate of mutation is usually very low. Chromosomes can also undergo crossovers and square dancing....but that's a whole nother story.

Joshua


--------------------
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"Life After Death is Saprophytic!"


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OfflineAnnoA
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Re: Getting a strain isolate without agar ?? [Re: Joshua]
    #1309009 - 02/15/03 12:41 AM (13 years, 9 months ago)

>When you isolate or clone you are causing the above circumstance. You are
>allowing only a small number of genes to be passed on to the next flush.

But, Joshua, you are making this very thing happen also if you give me a print of your mushrooms.
You give me 1 print of 1 mushroom, same as if you had given me 1 clone of 1 mushroom and I grew it out and made prints of this.

Wher?s the difference?


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OfflineTeRzMaStA
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Re: Getting a strain isolate without agar ?? [Re: Anno]
    #1309013 - 02/15/03 12:46 AM (13 years, 9 months ago)

Correct me if I'm wrong here, but mushrooms are made of condensed mycelium. If you clone a mushroom you are only getting the genes from that shroom. If you take a print each spore has different genes. Each spore emits a hypha which branches off and forms a primary haploid mycellium. It eventually meets up with another compatible haploid mycelium and their cell walls break down and they exchange genetic information. Thus a secondary diploid mycelium if formed which is capable of producing fruits.
It would be like the difference of taking a skin sample from me which would contain my exact genetic make up or a semen sample which does only contain the limited amount of genes I could pass on but still has many more genetic variations.
I didn't read all the posts in this thread just the one I replied too, so I could have missed the point entirely now that I think about it. If you're talking about printing the clone or printing a shroom grown from a print. I suppose it's getting to the point where you can just ignore my worthless babbling. It's 4:20 in the morning and I'm sick. Sorry for the inconvenience. You're the man Anno.


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Think for yourself; Question Authority


Edited by TeRzMaStA (02/15/03 01:15 AM)


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InvisibleJoshua
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Re: Getting a strain isolate without agar ?? [Re: Anno]
    #1309026 - 02/15/03 01:08 AM (13 years, 9 months ago)

In the isolation process I have isolated a small fraction of the original genes in the multispore inoculation. The fruits from this isolation will have fewer genes than were present in the original group of spores. With each consecutive isolation/clone you narrow the available gene pool from the print you started with.

In the wild spores from different fruit bodies will mix to propogate genetic variability.

If I gave you a print and you fruited from it and took a single print from that flush and continually repeated this proceedure you would find your genenic variability to be reduced with each consecutive print.

Lets say you started w/ 100,000 available variations of a particular gene. Each variation is present once in each of 100,000 spores you germinate. You germinate these spores at a single point. From this point you sector 25% of the growth, which should contain 25% of the original variation, 25,000 variations. If you then sector 25% of this growth you are down to 6.25% of your original variation w/ 6250 variations.

I'm sure my #'s aren't accurate, but the principle should be sound.

What are your thoughts?

Joshua


--------------------
The Shroomery Bookstore

Great books for inquiring minds!

"Life After Death is Saprophytic!"


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OfflineAnnoA
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Re: Getting a strain isolate without agar ?? [Re: Joshua]
    #1309040 - 02/15/03 01:24 AM (13 years, 9 months ago)

>The fruits from this isolation will have fewer genes than were present in the original group of spores.

The fruits from an isolation are all are genetically identical (if you assume there are no mutations occurring underway), they have the same genes as the mushroom they originated from.

So again:

I harvest one particular mushroom, take a print from it, and clone it.

I then grow this clone out, and bring it to fruit and take a print again from this cloned mushroom.

What I?m saying is: Both prints will be identical.

So why should cloning be a bottleneck event?
It?s not more bottleneck than if I buy a print from a single mushroom.
And if you buy a print, then it?s mostly of one single specimen.


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