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Cubensis Breeding: A Group Effort


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#1 HrVanker

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Posted 14 November 2016 - 06:00 PM

Hello to all!

Several 'topiates have expressed interest in attempting to breed Cubensis. So, as a group we are starting a group thread to discuss and document our efforts.

We like pictures, details, summaries, data, links to relevant articles, etc.

As such, posts should contain info on the agar being used (recipe/list of additives), strain/species being used, and your goal.


Original Thread:
https://mycotopia.ne...-granted/page-5

Reference Material:
Reproduction in Basidiomycota

The 'short and sweet' version

Genetic Recombination

Agar Work
Some basic agar/isolation info

How to tell if your myc is a virgin, and what those clamps are for!

Dissolving spore/cell walls for hybridization

Dying Myc, to see how it interacts with neighbors

So are we going to start a breeding thread?

Of course we are! Whomever is ready first, start it.
Sent from my SM-G935V using Tapatalk

Edited by HrVanker, 15 November 2016 - 12:47 AM.

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#2 catattack

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Posted 14 November 2016 - 07:22 PM

We should cut and paste those and put them here.

 

I'm down to try breeding. I can start soon or tonight. I was going to try culturing a monokaryotic jar of myc and have it fuse with another distinct strain per conversations with @microbe77 and your thread Hrvanker.

 

I'm thinking either

 

PE and redboys (hyph's suggestion)

or

PE and aa+

 

PEs because they're historically sparse, plus easily identifiable.

 

I can also just try seeing if I can grab monokaryotic mycelium by using a new scalpel tip.

 

 

Thoughts?


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#3 HrVanker

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Posted 15 November 2016 - 12:18 AM

Psilocybe Cubensis:The Magic Mushroom

By Luke Bielmeie - University of Wisconsin

The figure below illustrates the Basidiomycota life cycle. Once again, the Basidiomycota phylum includes an enormous amount of other species along with P. cubensis. The text below the illustration further explains the life cycle. For clarity, the red, blue, and green text describes their corresponding areas on the life cycle illustration

webpage1.jpg

Like all other mushrooms, the P. cubensis fruiting body (also basidiocarp, or mushroom) is the only stage of the life cycle that is visible to us in nature. The ploidy of the fruiting body is a peculiar one. Termed as 'dikaryotic, or (n+n)' this ploidy is characterized by the presence of two nuclei in each cell. Each of these nuclei are haploid nuclei, which will be discussed later in this section. In all other known life forms, this stage never occurs so it remains exclusive to the fungi. As a comparison, this would be equivalent to a human sperm uniting with an egg and remaining side by side in the same cell for a length of time. However, like all other animals, the human sperm nucleus fuses with the egg nucleus almost instantaneously upon entering (fertilization). In fungi, this stage is present in almost the entire fruiting body. The underside of the fruiting body is composed of a gilled tissue. The gilled shaped is advantageous to the mushroom because it increases the surface area, which is crucial when considering its function; producing spores. If you've ever picked up a mushroom and examined its underside, you've probably noticed that these gills are paper thin. These paper thin structures give rise to basidia (singular, basidium). Within each basidia, the two nuclei types fuse in a process known as karyogamy, which translates literally to the joining of two nuclei.



After karyogamy occurs, the ploidy of the basidium is now diploid (2n), meaning that the nucleus contains two sets of chromosomes. Humans (animals) are diploid their entire life from the moment of fertilization. Once diploid, the nucleus almost immediately undergoes meiosis to produce 4 haploid (n) nuclei.



Keep in mind that each basidium is extremely small. Its been found that an entire basidiocarp (mushroom) is able to produce up to 4.5 trillion basidia in a single growing season! (Sadava et al.). Recall that the function of the basidiocarp gills is to increase the surface area of the underside. This is exactly why! With so much surface area, the basidiocarp is able to produce an incredible amount of basidiospores, thus increasing its chances of reproduction. The basidiospores are then released from the gilled surface and carried by the wind to a new destination. Keep in mind that each basidiospore contains one haploid nucleus. If a basidiospore happens to be one of the lucky few (quite literally 'one in a million') they germinate and undergo mitosis and develop into fine, branching, tube-like structures known as hyphae (Biology-Online). Together, large amounts of hyphae (singular, hypha) form an underground, net-like structure called a mycelium (plural, mycelia). When this haploid mycelium comes into contact with another mycelium of the opposite mating type, they undergo plasmogamy. This means that their cells fuse, however, their nuclei do not. The two haploid nuclei are now in the same cell, which means the ploidy is now...dikaryotic!!!

This brings us back to the beginning of the cycle. The dikaryotic mycelium continues to grow and will eventually develop into a fruiting body above the surface. The cycle repeats itself!

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#4 HrVanker

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Posted 15 November 2016 - 12:46 AM

Fungi - Basidiomycota, Club Fungi
an uncredited article made available by Net Industries

Species in this phylum reproduce sexually by forming spores on top of club-shaped structures called basidia. The club fungi are believed to be closely related to the sac fungi. Both groups have cells which are separated by septa (walls), and both have a dikaryotic phase in their life cycle; a phase with two haploid nuclei per cell. The septum of the club fungi is somewhat different from those of sac fungi and is referred to as a dolipore septum. The dolipore septum has a bagel-shaped pore in its center.

The club fungi reproduce asexually by producing asexual spores or by fragmentation of mycelium.

The sexual reproduction phase of the club fungi involves three developmental stages of the mycelium. In the primary stage, a haploid spore germinates and grows a germ tube, which develops into mycelium. The mycelium initially contains a single haploid nucleus. Then, its haploid nucleus divides and septa form between the nuclei.

A secondary mycelium forms upon conjugation of two sexually compatible hyphae. The secondary mycelium is dikaryotic, in that it has two haploid nuclei, one from each parent. As the dikaryotic mycelium grows, the cells divide and more septa are formed between the new cells.

Each of the new cells in the secondary mycelium has one haploid nucleus from each parent. This is assured by clamp connections, specialized structures unique to the club fungi. These are loop-like hyphae which connect the cytoplasm of adjacent cells and through which nuclei move during cell division. In particular, during cell division, one nucleus divides directly into the newly formed cell; the other nucleus divides inside the clamp connection and the two daughter nuclei migrate through the clamp connection in opposite directions to the two daughter cells.

The tertiary mycelium is simply an organized mass of secondary mycelium. It is a morphologically complex tissue and forms structures such as the typically mushroom-shaped basidiocarps commonly seen in nature.

Sexual reproduction of the club fungi begins upon fusion of two primary hyphae to form a club-shaped structure, known as a basidium. Second, the two haploid nuclei inside the basidium fuse together to form a diploid zygote. Third, the zygote undergoes meiosis to form two haploid nuclei. Fourth, these two haploid nuclei undergo mitosis to form a total of four haploid nuclei. These four nuclei then migrate into projections, which form on the tip of the basidium. These projections then develop into four separate haploid spores, each with a single nucleus.

In the species of club fungi which are large and fleshy, such as the mushrooms, a mass of basidia form a structure called a basidiocarp. The spores on the basidia are released from the underside of the fleshy gills of the mushroom. The color and shape of the basidiocarp, as well as the color of the spores are often diagnostic for species identification.

This large phylum includes species which are known as mushrooms, toadstools, earthstars, stinkhorns, puffballs, jelly fungi, coral fungi, and many other interesting common names. Some species, such as the rusts and smuts, are pathogens which attack agricultural grains. Other species, such as the fly agaric (Agaricus muscaria) and some species in the genus Psilocybe, produce chemical hallucinogens and have been used by numerous cultures in their religious ceremonies. Another species, Agaricus bisporus, is the common edible mushroom found in supermarkets.

An important aspect of the club fungi is the great diversity of alkaloids and other toxic and psychogenic chemicals produced by some species. For example, Amanita virosa, a mushroom colloquially known as "death angel," is so deadly poisonous that a small bite can kill a person. A related mushroom is Amanita muscaria, known as "fly agaric," which is hallucinogenic. Over the millennia, numerous cultures have eaten the fly agaric as part of their religious ceremonies. For example, R. Gordon Wasson has shown that Amanita muscaria is the hallucinogenic plant referred to as "Soma" throughout Rg Veda, the ancient religious text. According to Rg Veda, the ancient Aryans who invaded India about four millennia ago ingested "Soma" as a euphoriant.

While mushrooms are the best-known club fungi, many other club fungi grow underground as mycorrhizae. Mycorrhizae result from a symbiosis between a plant root and a fungus. In mycorrhizae, the fungus typically supplies nitrogen-containing compounds to the plant, and the plant supplies carbohydrates and other organic compounds to the fungus. Mycorrhizae are very important for the growth of orchids. One reason many orchids are difficult to grow is because they require particular fungal species to form mycorrhizae on their roots.

A recent report investigated a subterranean club fungus, Armillaria bulbosa, which is a pathogen on tree roots. The investigators used molecular biology techniques to demonstrate that a single subterranean "indi vidual" of this species in Northern Michigan was spread out over 37 acres (15 ha) and weighed an estimated 22,000 lb (10,000 kg). Based on the estimated growth rate of this species, of about 0.7 ft (0.2 m) per year, this individual was about 1,500 years old.
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#5 JustAnEyedea

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Posted 15 November 2016 - 12:57 AM

So you can take two pieces of monkaryotic mycelium from different strains, and breed them that way? That's what I'm getting... Sounds really damn interesting. Pulling up a chair.

#6 HrVanker

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Posted 15 November 2016 - 01:00 AM

Basidiomycota: The Club Fungi

KEY POINTS:

The majority of edible fungi belong to the Phylum Basidiomycota.

The basidiomycota includes shelf fungus, toadstools, and smuts and rusts.

Unlike most fungi, basidiomycota reproduce sexually as opposed to asexually.

Two different mating strains are required for the fusion of genetic material in the basidium which is followed by meiosis producing haploid basidiospores.

Mycelia of different mating types combine to produce a secondary mycelium that contains haploid nuclei in what is called the dikaryotic stage, where the fungi remains until a basidiocarp (mushroom) is generated with the developing basidia and finally basidiospores on the gills under its cap.


TERMS:

Basidiomycete
a fungus of the phylum Basidiomycota, which produces sexual spores on a basidium

Basidiocarp
a fruiting body that protrudes from the ground, known as a mushroom, which has a developing basidia on the gills under its cap

Basidiomycota
a taxonomic division within the kingdom Fungi: 30,000 species of fungi that produce spores from a basidium

Basidium
a small structure, shaped like a club, found in the Basidiomycota phylum of fungi, that bears four spores at the tips of small projections

Basidiospore
a sexually-reproductive spore produced by fungi of the phylum Basidiomycota

Text:
The fungi in the Phylum Basidiomycota are easily recognizable under a light microscope by their club-shaped fruiting bodies called basidia (singular, basidium), which are the swollen terminal cell of a hypha. The basidia, which are the reproductive organs of these fungi, are often contained within the familiar mushroom, commonly seen in fields after rain, on the supermarket shelves, and growing on your lawn . These mushroom-producing basidiomyces are sometimes referred to as "gill fungi" because of the presence of gill-like structures on the underside of the cap. The "gills" are actually compacted hyphae on which the basidia are borne. This group also includes shelf fungus, which cling to the bark of trees like small shelves. In addition, the basidiomycota includes smuts and rusts, which are important plant pathogens, and toadstools. Most edible fungi belong to the Phylum Basidiomycota; however, some basidiomycetes produce deadly toxins. For example, Cryptococcus neoformans causes severe respiratory illness.

The lifecycle of basidiomycetes includes alternation of generations . Spores are generally produced through sexual reproduction, rather than asexual reproduction. The club-shaped basidium carries spores called basidiospores. In the basidium, nuclei of two different mating strains fuse (karyogamy), giving rise to a diploid zygote that then undergoes meiosis. The haploid nuclei migrate into basidiospores, which germinate and generate monokaryotic hyphae. The mycelium that results is called a primary mycelium. Mycelia of different mating strains can combine and produce a secondary mycelium that contains haploid nuclei of two different mating strains. This is the dikaryotic stage of the basidiomyces lifecyle and it is the dominant stage. Eventually, the secondary mycelium generates a basidiocarp, which is a fruiting body that protrudes from the ground; this is what we think of as a mushroom. The basidiocarp bears the developing basidia on the gills under its cap.

The Life Cycle of a Basidiomycete
figure-24-02-07.@2x.png

Source: Boundless. “Basidiomycota: The Club Fungi.” Boundless Biology. Boundless, 26 May. 2016.
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#7 Sidestreet

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Posted 15 November 2016 - 05:39 AM

I remember reading that one method for obtaining monokaryotic mycelium involves serial dilution of spore water until there are so few spores that you will only get a handful of germination points on an agar plate.  That way you can keep the mycelium from a single spore separate from the others until you are ready to combine them.


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#8 Microbe

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Posted 15 November 2016 - 05:36 PM

I remember reading that one method for obtaining monokaryotic mycelium involves serial dilution of spore water until there are so few spores that you will only get a handful of germination points on an agar plate. That way you can keep the mycelium from a single spore separate from the others until you are ready to combine them.

This would be my second method of choice in trying to get a monokaryon culture. I think if one would take the tip of nichrome wire and slightly touch the edge of a spore print and then swirl it around in 30 cc's of water then disperse a single drop through a 18 gauge or smaller sharp, i like the probability. As soon as growth is observed then transfer it.

Im hoping to dig my spore prints out and see get something going. I will probably go AT with EC because im limited on my inventory. I was offered a RB print, maybe RB and AT? There was a lot of noise around AT also at another myco site.

This thread is going to be great!
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#9 HrVanker

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Posted 15 November 2016 - 06:00 PM

I remember reading that one method for obtaining monokaryotic mycelium involves serial dilution of spore water until there are so few spores that you will only get a handful of germination points on an agar plate. That way you can keep the mycelium from a single spore separate from the others until you are ready to combine them.

This would be my second method of choice in trying to get a monokaryon culture. I think if one would take the tip of nichrome wire and slightly touch the edge of a spore print and then swirl it around in 30 cc's of water then disperse a single drop through a 18 gauge or smaller sharp, i like the probability. As soon as growth is observed then transfer it.

Im hoping to dig my spore prints out and see get something going. I will probably go AT with EC because im limited on my inventory. I was offered a RB print, maybe RB and AT? There was a lot of noise around AT also at another myco site.

This thread is going to be great!
I've been thinking about what I would like to breed. I'm not sure what the cross will be, but I like the idea of a short mushroom with a large cap... both for looks and potency.

Edit: I was stoned and talking with my sister... didn't finish my thought.

@Microbe77 and SideStreet

I was thinking about a combo of serial dillution, and plate streaking... or basically streaking with well mixed spore water. It may work just as well as using a print, but at least if it's suspended in water --Isn't Astroglide supposed to help prevent clumping?-- You still have less spores by volume. You could dip the nichrome in a drop of spore water right from the syringe!

Once i get going, i may try both methods.

Edited by HrVanker, 15 November 2016 - 09:06 PM.

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#10 Heirloom Spores

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Posted 15 November 2016 - 09:12 PM

This is a nice subject. I have wanted to learn about breeding cubes. I hope to watch it done here. Thanks a lot of nice reading material.


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#11 catattack

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Posted 15 November 2016 - 09:15 PM

These are great too:

 

https://mycotopia.ne...hybrid-strains/

 

https://mycotopia.ne...reeding-method/


Edited by catattack, 15 November 2016 - 09:15 PM.

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#12 coorsmikey

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Posted 15 November 2016 - 09:20 PM

Whoa! Deja Vu :)
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#13 Microbe

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Posted 15 November 2016 - 11:29 PM

Alright Hr you probably dont know me but i want to tell you now that im silly. I am all over the place at times and i am not ready to actual ready to attempt hybridization. I want to get 2 different monocultures each with very obvious phenotypes.

I am aware that hybridization can result and although both parents genotypes are present it is possible that the phenotype may resemble that of a single parent but it is also possible that they can resemble both, which i would hope for. I believe this is a result of the weaker genetics conceding to the more dominant just as humans do. 1 of my kids looks exactly like me while 3 resemble both of us and 1 exactly their mother.

I know making babies isnt hybridization but i use that as a reference for genotypes and phenotypes.

I believe the inly true way to tell if you have successfully mated to monokaryons is to view under a scope and look at the clamp connections. I have the appropriate stains and scope to observe this i think.

Now we could wait and see how it fruits, and if it clearly has the phenotypes of both parents, then you can rule that a success but if they show the phenotype of 1 parent, then you cant exactly rule that a failure without looking at clamp connections.

Im probably going to work in this order, and i will try to shut up after this so we can limit this thread to actual demonstration and practical applications, that is unless everyone is cool with reading through all this.

Anyways here is the order i will be working;

Between all the said-below i will be practicing and performing techniques for monospore germination and isolating a monokaryon and growing it out. As i stated in the other thread monokaryons, at least with cubesis, are very easy to differentiate with the naked eye. I could even take it to another step and do a extremely small microgrow to make sure it doesnt fruit. I have no intentions nof breeding these and they will be released into wild.


I. I will establish 2 mono cultures of the same substrain and each with their own distinguished phenotypes.

II. I will print each one and then probably do a streak and serial dilution method or which ever i was repeatedly successful at during my practice techniques.

III. I will then attempt to breed them. This isnt hybridization and is more along the lines of selective breeding which is the same process but with a much higher success rate then hybridization. On the contrary to what i said-above, a scope should not be needed at least with cubes because it is obvious when you have dikaryons growing across the plate but it will be a best practice for me to observe under a scope the clamp connections to as it will help with my non existent microscopy skills!

IV. Fruit and observe the phenotype(s)

V. Repeat steps I-IV but this time using 2 separate strains to be determined.

VI. Take a monokaryon and induce double haploid production using Colchicine. If this works then it is instant monoculture, there is difference of opinion on rather or not it will sporulate. I think it will but even if it doesnt, and for production purposes, i work with only living cultures anyway. Couple that with the cryoprotectant LC im going to attempt, and im certain it will work, who the hell needs spores? ;)

VII. Im skeptical on being able to cross species and im not sure its possible, but i would like to attempt crossing a species within the same genus such as Psilocybe cubensis with Psilocybe cyanescens. I know wood lover and dung lover, but there is also enzyme adding and all kinds of other stuff that is going to be advanced on this already advanced subject.

Happy hybridization to all of you and best of luck! Lets create some monsters!

Edited by Microbe77, 16 November 2016 - 12:28 AM.

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#14 Microbe

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Posted 17 November 2016 - 11:13 PM

https://www.ncbi.nlm...les/PMC3833681/
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#15 Microbe

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Posted 17 November 2016 - 11:36 PM

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#16 Microbe

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Posted 18 November 2016 - 12:59 AM

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#17 catattack

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Posted 20 November 2016 - 10:49 AM

I have a question/s. Are the amount of genetics in say a cloned fruit body quantifiable? Is the smallest tissue sample one can grab going to show say 4 sets or am I way off on that number? Agar guys, I'm looking at you.


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#18 Microbe

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Posted 20 November 2016 - 12:39 PM

I have a question/s. Are the amount of genetics in say a cloned fruit body quantifiable? Is the smallest tissue sample one can grab going to show say 4 sets or am I way off on that number? Agar guys, I'm looking at you.

Haploid is a single set of unpaired chromosomes while diploid contains 2 sets of chromosomes, one from each parent. I think.

Look at my randoms thread, the 3 plates that you couldnt see any mycelium on, was enough to grab both sets lol.

A single cell is all thats needed to grab the gens

#19 catattack

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Posted 20 November 2016 - 01:03 PM

 

I have a question/s. Are the amount of genetics in say a cloned fruit body quantifiable? Is the smallest tissue sample one can grab going to show say 4 sets or am I way off on that number? Agar guys, I'm looking at you.

Haploid is a single set of unpaired chromosomes while diploid contains 2 sets of chromosomes, one from each parent. I think.

Look at my randoms thread, the 3 plates that you couldnt see any mycelium on, was enough to grab both sets lol.

A single cell is all thats needed to grab the gens

 

 

I don't think you understood my question. I'm about to grab clone material from a fruit, how many sets of genes will be on that "plate" I make? Is it at least quantifiable? 4? 20? Over 20? This would probably be better suited for my agar thread but it applies here as well.



#20 HrVanker

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Posted 20 November 2016 - 02:37 PM

I have a question/s. Are the amount of genetics in say a cloned fruit body quantifiable? Is the smallest tissue sample one can grab going to show say 4 sets or am I way off on that number? Agar guys, I'm looking at you.

Haploid is a single set of unpaired chromosomes while diploid contains 2 sets of chromosomes, one from each parent. I think.

Look at my randoms thread, the 3 plates that you couldnt see any mycelium on, was enough to grab both sets lol.

A single cell is all thats needed to grab the gens
I don't think you understood my question. I'm about to grab clone material from a fruit, how many sets of genes will be on that "plate" I make? Is it at least quantifiable? 4? 20? Over 20? This would probably be better suited for my agar thread but it applies here as well.
If you are cloning from an MS grow, there COULD be a couple scenario. You mentioned in another thread, that when you clone fruit you still have to isolate sectors.

If that is the case, then each unique sector will have two nuclei in each cell, and one set of chromosomes each.

If you have a monoculture, you still have two nuclei per cell and one set of chromosomes per nuclei.

**The number of genes per chromosome varies by species. But there are several hundred to 1000 genes per chromosome. Each gene is an instruction for producing proteins.**

I have not heard of needing to isolate from a clone before you mentioned it though. --I don't have experience to back up my knowledge.-- But when you clone, where do you take the sample from? I hear the stipe interior is the best, and I believe the higher up the stem you go the more likely you are to get a monoculture. If you take from the lower end (closer to the ground) the better chance some myc from another culture in the sub got pulled into the fruit as it expanded.

Anybody else have thoughts?

Edited by HrVanker, 20 November 2016 - 03:40 PM.

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