Would this Work for Creating Hybrid Strains??

[warriorsoul]

Can i make a hybrid by mixing the spores of two different strains in water?

[Aqua Velva]

Originally Posted by warriorsoul

Can i make a hybrid by mixing the spores of two different strains in water?

No.

[warriorsoul]

why not?

[Aqua Velva]

Creating a hybrid takes a little more effort than that. Since I see you posted the same question over at the Shroomery, I'll give you a hint. Use their search feature and search for "hybrids" or "crossing strains" and in the "username" type "workman".

If it were that easy, everyone would be making hybrids.

[warriorsoul]

I have read some of the threads on the subject.
When spores germinate they are monokaryons until they mate with the closet compatible spore and become Dikaryon.
I found this interesting..
System prevents selfing, promotes outcrossing.
As long as the Afactors and B factors are different they mate.
Differences are based on alleles present at each of the A factor loci, and each of the B factor loci.
A(allele set 1)1
A(allele set2)2
B(alllele set1)1
B(allele set2)2

A1B1 mates with A2B2 succesfully. Dikaryon.
A1B1 mating with A2B1 might form clamps but not true clamps, no success.
A2B1 mates with A2B2 no clamps, no success.
Mating between a single strain 1/4 compatability.
Mating between two different strains, that share NO common parents, 100% compatable.
Mating between two strains that share a like A or B factor will result in 3/4 compatability.
If there is no common parent there should be over a 98% of mating. This doesn't apply to mixing mycelium of different strains, though, of course. Only to different spores mating.

[Aqua Velva]

For the paired dikaryon to be a true hybrid, the resulting fruits would have to produce spores. That is the real challenge with creating a true hybrid.

[warriorsoul]

So what % of the mushrooms are hybrids in a cross like this?
Wouldnt you up the odds by mixing the spores a well as possible before they germinate?

[chrissbl]

exactly aqua...... I don't see any spores or veil or anything on these semi-hybrids..... They are much smaller then usual with the Mazatapec(TC) I did , but the Golden Teacher and Treasure Coast. Is a larger mushroom but I see gills but not sure about spores...I shall verify this once I get my sisters microscope

[warriorsoul]

You dont need a microscope to take a spore print

[the_chosen_one]

Aqua Velva - i beg to differ. with the correct dilution ratios it can be accomplished much like in stamets TMC strain dilution and isolation tek. it's a lot more random, but it can be done. i have a short write up in some of the vaulted albino threads.

[chrissbl]

deleted by admin.

[warriorsoul]

Doesn't really apply to what i'm talking about, but thanks!

What would happen if you made two spore prints on top of each other from different strains?

The laws of genetics should favor the crossing of the two strains.
F1 hybrid vigor should be noticed.
F2 recessive phenotypes should also be noticed.

I guess you could call it passive hybridization for lack of a better term.
But i really think it would work..
Time to make some hybrid prints!

[the_chosen_one]

not to say it won't work but, the problem with simply mixing spores is germination timing. chances are that one strain will germinate before the other and within a matter of hours hyphae have already mated.

dilution could improve your chances by stretching things out a bit.. geometrically speaking. as the monokaryons grow longer and there is more space between them, possibly allowing more time for germination of the other strain and extention of hyphae.

dilution will also reduce the number of substrains and it will be easier to isolate the one you are looking for.

many good vibes to you with your experiment!

[warriorsoul]

Thanks!
I have considered the hydration issue.
(As spores age they lose water and take longer to germinate)

I plan to take both prints right after one another.
and on top of each other to maximize contact.
I'm considering using an anti-clumping agent.

If only 1 in 4 hyphae with the same parents can mate,
and 4 out of 4 hyphae from different strains mate.
This favors the hybrid.
Hopefully any small differences in germination times will be nullified by the 100% compatibility of unrelated hyphae.

The key may be to use spores that are the same age with the same hydration.
That should help maximize contact between monokaryotic hyphae.
All the spores wont germinate at the exact same time,
there should be enough overlap in germination rates to give the hybrids a chance.

[the_chosen_one]

now this topic gets really interesting!

Workman and i have discussed anti-clumping agents to a meager extent..

if i may ask, what did you have in mind? if you don't want to disclose i understand

[warriorsoul]

I was thinking of jet-dry.
Looks like my first cross is going to be Falbino x PE6.

[the_chosen_one]

two crosses, both quite solid excellent choice!!! this is great!

many good vibes

[warriorsoul]

I've chosen to overlap the prints so i can take spores from both the Falbino and PE6 strain and the hybrid to compare.

[SporeCrazy]

I'm nnot entirely up on this whole process... but what would be the results if you mixed those spores in an lc? Would the resulting myc be combined or seperate?

[warriorsoul]

The main reason i overlapped the prints is because i wanted at least some of the spores from the two strains to stick together and hopefully increase the chance of mating.
I got four jars of the hybrid and two jars of the original strains to compare growth.

Have to come up with a name if this works..
So far i've got FATE (f+,albino,texan,envy) or F6..
Let me know what you think y'all!

[mockeylock]

Originally Posted by warriorsoul

The main reason i overlapped the prints is because i wanted at least some of the spores from the two strains to stick together and hopefully increase the chance of mating.
I got four jars of the hybrid and two jars of the original strains to compare growth.

Have to come up with a name if this works..
So far i've got FATE (f+,albino,texan,envy) or F6..
Let me know what you think y'all!

Well you're going to have a hell of a time convincing anyone you succeeded even if you did....

What distictive phenotypes are you looking for from each strain? Unfortunately, PE is known to throw out albinos...

[warriorsoul]

The main thing i'll be looking for in the F1 generation is hybrid vigor and dominant gene expression. Albinism and other unfavorable genes are recessive, so i wont choose from those expressing these recessive genes in the F1, as they likely wont be hybrids.
The F1 hybrids will likely have a normal looking phenotype because of Dominant Gene Expression.
The F2 generation should show a much wider range of phenotype expression than either of the original parent strains.
The change in phenotype should be obvious when compared to the Controls.


The hybrid mycelium should also consistently outperform the mycelium of the more homozygous parent strains.

[Mycotope]

dude you have alot of reading to do. get some agar and spend then next two years trying to get two rihzos to merge.

[warriorsoul]

Your not the first person tell me that.
I like the feedback, thanks.
Your misconception on the life process of mushrooms is common, so don't feel too bad.
Welcome.

[warriorsoul]

This is a quote from chat i had with Workman from Sporeworks..

"Mixing ungerminated spores together should allow mating between strains of the same species (avoid mixing premade syringes since the spores can germinate within the syringe before use). Since most cubensis look relatively the same and an individual spore print from a single strain can generate a range of phenotypes, you need to use very different looking strains of cubensis to be certain of hybridization (and yes, I would call these hybrids in the broadest sense, just like hybrid corn which is also not an interspecies or intergeneric cross).

The obvious problem is distinguishing hybrids from selfed strains. Randomly mixing spores is a crude method and you are going to end up with a mixture of the original strains and hopefully some hybrids (assuming they are compatible). The hybrids won't necessarily look intermediate between the two original strains. If the parents were relatively true breeding without much variability, all of the hybrids are going to look about the same as each other (uniform) as they will each get 50% of their genes from each parent. Only later generations from spores will give you that mix of traits you are looking for (see below)

My initial hybrid work was between the PF albino and the PE strains, both true breeding from spores and easily distinguished from other strains. The cross looked like a normal unremarkable cubensis lacking the penis shaped caps and albinism of the parents. In this case the normal appearance helped confirm the cross but you can imagine that a normal looking cubensis isn't going to stand out in most other crosses. Controlled crosses are better in this regard since you can be sure the result is a hybrid no matter what it looks like.

Another issue is that you can't perpetuate the hybrid if you use the hybrid's spores to start the next generation. There will be all sorts of new strains revealed in the F2 generation as the mix of genes in the hybrid are recombined. Only after selecting for the traits you want for about 6 generations will the features you are after stabilize into a distinct strain (I use strain in the broadest sense even if its not technically correct).

The fact that a hybrid won't breed true is a method that vegetable seed suppliers count on for repeated sales and to discourage seed saving (the bastards). A package of hybrid corn seed will generate a superior and uniform corn crop. If you save that corn crop to replant the next season (to save money), you will end up with all sorts of mixed traits and not all will be as good as the original hybrid seed. This is bad for farmers but can be fun for the home gardener.

So the short answer is, yes it will work but you may not be able to tell. Hybrids tend to show hybrid vigor so even if you aren't sure or can't prove you were successful with the hybridization you may still end up with a superior strain. A good clue that a hybrid is successful is if the F2 generation from the hybrid's spores produces a wide range of phenotypes.

Go for it and have fun.

That is a very good point about using spores of the same freshness to help syncronize germination. That is something I didn't even consider and is potentially very important."

[warriorsoul]

Here is part of my journal on breeding distinctive mushrooms, i've adapted it from Marijuana Botany.Robert Clarke
Plants and fungi are different, but the genetic laws are the same.


Many of the random parents from single strain multi-spore inoculations may be undesirable for breeding since they may pass on tendencies such as slow growth, weak activity or retarded maturation.
However, creating hybrids of strains will produce, on the average, larger and more desirable offspring than their homozygous parents. This condition is called hybrid vigor and results from the hybrid crossing of two diverse gene pools. The tendency is for many of the dominant characteristics from both parents to be transmitted to the F1 offspring, resulting in particularly large and vigorous mushrooms. This increased vigor due to recombination of dominant genes may also raise the Psilocybin level of the F1 offspring, hybridization also opens up the possibility that undesirable (usually recessive) genes may form pairs and express their characteristics in the F2 offspring. Hybrid vigor may also mask inferior qualities due to abnormally rapid growth.The possible F2 combinations are tremendous.

With some care the breeder can avoid the hidden dangers of unconscious selection. Definite goals are vital to progress in breeding Psilocybe mushrooms. What qualities are desired in a strain that it does not already exhibit? What characteristics does a strain exhibit that are unfavorable and should be bred out?
Answers to these questions suggest goals for breeding. In addition to a basic knowledge of mushroom botany, propagation, and genetics, the successful breeder also becomes aware of the most minute differences and similarities in phenotype. A sensitive rapport is established between breeder and mushrooms and at the same time strict guidelines are followed. Selection is the first and most important step in the breeding of any mushroom.
One must get clearly in mind the kind of mushroom he wants, then breed and select to that end, always choosing through a series of generations the mushrooms which are approaching nearest the ideal, and rejecting all others.
Proper selection of prospective parents is only possible if the breeder is familiar with the variable characteristics of Psilocybe mushrooms that may be genetically controlled, has a way to accurately measure these variations, and has established goals for improving these characteristics by selective breeding. By selecting against unfavorable traits while selecting for favorable ones, the unconscious breeding of poor strains is avoided.


Essential Points of Mushroom Breeding

1.The genotypes of mushrooms are controlled by genes which are passed on unchanged from generation to generation.
2.Genes occur in pairs, one from each parent spore.
3.When the members of a gene pair differ in their effect upon phenotype, the mushroom is termed hybrid or heterozygous.
4.When the members of a pair of genes are equal in their effect upon phenotype, then they are termed truebreeding or homozygous.
5.Pairs of genes controlling different phenotypic traits are (usually) inherited independently.
6.Dominance relations and gene interaction can alter the phenotypic ratios of the F1, F2, and subsequent generations.

Genotype and Phenotype Ratios

Phenotype and genotype ratios are probabilistic. If recessive genes are desired for three traits it is not
effective to raise only 64 offspring and count on getting one homozygous recessive individual. To increase the probability of success it is better to raise hundreds of offspring, choosing only the best homozygous recessive individuals as future parents. All laws of inheritance are based on chance and offspring may not approach predicted ratios until many more have been phenotypically characterized and grouped than the theoretical minimums.

The genotype of each individual is expressed by a mosaic of thousands of subtle overlapping traits. It is the sum total of these traits that determines the general pheno- type of an individual. It is often difficult to determine if the characteristic being selected is one trait or the blending of several traits and whether these traits are controlled by one or several pairs of genes. It often makes little difference that a breeder does not have mushrooms that are proven to breed true. Breeding goals can still be established. The selfing of F1 hybrids will often give rise to the variation needed in the F2 generation for selecting parents for subsequent gen-
erations, even if the characteristics of the original parents of the F1 hybrid are not known. It is in the following generations that fixed characteristics appear and the breeding of pure strains can begin. By selecting and crossing individuals that most nearly approach the ideal described by the breeding goals, the variety can be continuously improved even if the exact patterns of inheritance are never determined. Complementary traits are eventually combined into one line whose seeds reproduce the favorable parental traits. Inbreeding strains also allows weak recessive traits to express
themselves and these abnormalities must be diligently removed from the breeding population. After five or six generations, strains become amazingly uniform. Vigor is occasionally restored by crossing with other lines or by backcrossing.
Parental mushrooms are selected which most nearly approach the ideal. If a desirable trait is not expressed
by the parent, it is much less likely to appear in the offspring. It is imperative that desirable characteristics be hereditary and not primarily the result of environment and cultivation. Acquired traits
are not hereditary and cannot be made hereditary. Breeding for as few traits as possible at one time
greatly increases the chance of success. In addition to the specific traits chosen as the aims of breeding,
parents are selected which possess other generally desirable traits such as vigor and size. Determinations
of dominance and recessiveness can only be made by observing the outcome of many crosses, although wild traits often tend to be dominant. This is one of the keys to adaptive survival. However, all the possible combinations will appear in the F2 generation if it is large enough, regardless of dominance.

Now, after further simplifying this wonderful system of inheritance, there are additional exceptions to the rules which must be explored. In some cases, a pair of genes may control a trait but a second or third pair of genes is needed to express this trait. This is known as gene interaction. No particular genetic attribute in which we may be interested is totally isolated from other genes and the effects of environment. Genes are occasionally transferred in groups instead of assorting independently. This is known as gene linkage, These genes are spaced along the same chromosome and may or may not control
the same trait. The result of linkage might be that one trait cannot be inherited without another. At
times, traits may be associated with the sex chromosomes and they may be limited to expression in only one sex (sex linkage). Crossing over also interferes with the analysis of crosses. Crossing over is the exchanging of entire pieces of genetic material between two chromosomes. This can result in two genes that are nor- mally linked appearing on separate chromosomes where they will be independently inherited. All of these processes can cause crosses to deviate from the expected Mendelian outcome.
Chance is a major factor in breeding mushrooms, and the more crosses a breeder attempts the higher are the chances of success.
Variate, isolate, intermate, evaluate, multiplicate, and disseminate are the key words in mushroom improvement. A mushroom breeder begins by producing or collecting various prospective parents from which the most desirable ones are selected and isolated. Intermating of the select parents results in offspring which must be evaluated for favorable characteristics. If evaluation indicates that the offspring are not improved, then the process is repeated. Improved offspring are multiplied and disseminated. Further evaluation in the field is necessary to check for uniformity and to choose parents for further intermating. This cyclic approach provides a balanced system of mushroom improvement.
The basic nature of mushrooms make them challenging to breed. Developing a knowledge and feel for the mushroom is more important than memorizing Mendelian ratios. The words of the great Luther Burbank say it well, "Heredity is indelibly fixed by repetition."

List of Favorable Traits of Mushrooms in Which Variation Occurs
1. General Traits
a) Size and Yield
b) Vigor
c) Adaptability
d) Hardiness
e) Disease and Pest Resistance
f) Maturation
g) Mycelium Production
h) Pin set

2. Specific Traits
a) Shape
b) Form
c) Color
d) Psilocybin/Psilocin Level
e) Taste and Aroma
f) Drying Rate
g) Ease of Harvest
h) Spore Characteristics
i) Maturation


feel free to add to or correct this journal..

[~Hippie3]

good luck.
like was said already
the hard part will be making sure it's really a cross
and not just variation within a strain.

[warriorsoul]

Thanks.
I think with most strains this would be hard to do, but falbino and pe6 are unique enough looking to pull it off.
There is no mistaking these two strains for each other when started from multi-spore.
My use of controls will help show any change in phenotype.

[grod31]

Originally Posted by warriorsoul

Thanks.
I think with most strains this would be hard to do, but falbino and pe6 are unique enough looking to pull it off.
There is no mistaking these two strains for each other when started from multi-spore.
My use of controls will help show any change in phenotype.

Do you really think this? There are 100s of substrains in every single multispore inoculation. Even one singe mushroom can have multiple substrians within in.
and you want to use two already crossed strains there are 1000s of sub strains within all those spores and even if you can find a fruit that looks something that you like it will most likely be a variation of one of the four parent strains and a spore print from it would bring you back to square one. Sorry im not trying to be a dick im just thinking to my self there is NO way you will ever know

[warriorsoul]

If i select phenotypes that show dominate traits (large normal looking fruits with hybrid vigor) in the F1 generation, and the F2 generation show greater phenotype variation than either of the parent strains, that will be a very good indication of a hybrid.

I wont be isolating, but comparing multispore from each strain to the f2 generation of the hybrid.
Its important to get to know the phenotype of each strain and be able to document any change.

Anyone thats grown the two parent strains (falbino and pe6), know they have certain traits that are unique unto themselves.
Its all about comparing the ratio of multispore phenotypes from the F2 of the hybrid to the parent strains.
A good eye for detail also helps in the equation.

This method could also be used to fix certain phenotypes by backcrossing and mating homozygous individuals.

[Cuban]

I don't think you really understand what is going on here for you to be so negative. Basically, take two very phenotypically different strains, then using 'mulatto' mushrooms from the F1, and then recrossing them to stabilize certain phenotypes over each subsequent generation, gradually refining and securing different traits. It's really not conceptually difficult.

Originally Posted by grod31

Do you really think this? There are 100s of substrains in every single multispore inoculation. Even one singe mushroom can have multiple substrians within in.
and you want to use two already crossed strains there are 1000s of sub strains within all those spores and even if you can find a fruit that looks something that you like it will most likely be a variation of one of the four parent strains and a spore print from it would bring you back to square one. Sorry im not trying to be a dick im just thinking to my self there is NO way you will ever know

[the_chosen_one]

i would recommend early isolation on agar. make numerous cultures. this will help to seperate the strains being produced, thus making any crosses much easier to identify.

[warriorsoul]

Thanks for your input.

I want to try and avoid isolating early on, and let a larger portion of genotypes have a chance to compete in a multi spore enviroment and show final phenotype.
I could isolate first, but don't see a point in the extra step.
The typical hybrid should outgrow the controls and have a normal cube look.
Not all of the hybrids from the two strains will show this vigor.
But a large enough portion should to notice, and allow isolation of the hybrid.
The much greater variation in the F2 from the controls will confirm the hybrid.

[the_chosen_one]

yeah, ime the hybrid strains do seem to be faster. you should be able to isolate at anytime.. me? i like to bury myself in work "but there would be so many options to work with! hahaha!" holy shit

[bear]

Originally Posted by the_chosen_one

i would recommend early isolation on agar. make numerous cultures. this will help to seperate the strains being produced, thus making any crosses much easier to identify.

If you don't do this it invalidates your results because you can't really be sure. If you keep doing more experiments you might eventually get the evidence you want especially if you're looking for it

Im anxiously awaiting the results

[warriorsoul]

How does it invalidate the results?
Your gonna get more phenotypes to choose from if you let them all express themselves.
its harder to get lucky if your just selecting from a few unknown isolations on agar.

I predict the hybrids will be obvious.
The controls are the proof.

[warriorsoul]

48 hours after inoculation..
Three of the four hybrid jars are showing early growth.

[shroom_seeker]

[warriorsoul]

60 hours after inoculation.
All the jars show nice growth, but the hybrid jars are ahead.

Both Falbino and PE6 are fast strains to begin with, so i was wondering how much hybrid vigor would be noticed...

[righteous]

Figured I would chime in here about hybridization of a different sort. It is known that certain snake venoms in dilute agar can be used to cause two samples of mycelium to exchange genetic information thus producing a merged variant.

An important part of this is that you can use clones that have been selected from good fruiting MS innocs!

It's been discussed and decided in the past that this works on cubensis, but to my knowledge noone is aware of any well documented examples used on this species.
(Oh yeah.... this can also work to hybridize different species, or different strains that would normally be incompatible to fuse via the normal 2 monokaryote route.)


The nice thing about this method is that allows the selection of the particular fruiting substrains to be fused and they need not be monokaryotic. This should also make identification of a true hybrid easier to be sure of, as there should be no signifigant shift from the original clones if there is no hybrid obtained.
The eastern diamondback rattlesnake works as well as the western and this is quite a common snake the the USA.
Anyone have any further info about this, or interest in trying this out for a cross-species psilocybe?

Here is a paper detailling this process:
Pharmaceutical Mushrooms- Cordyceps sinensis

Cmon yall, how about a weilii-cubie cross?!

[warriorsoul]

The venom is super expensive to buy..
PE6 was created using snake venom.
Thats a great link though, thanks

[righteous]

The venom is indeed expensive to buy, and I believe that it degrades with time.....
Additionally you need a permit to possess it in a lot of places(deadly poison ya know)

However, if you've got balls or you feel like channeling Steve Irwin, these snakes are quite cheap to purchase and can be found at just about any reptile/exotic animal tradeshow. A safer alternative would be that you most likely could also pay the snakeowner to milk it for you.

(a lot of poisonous snakehandlers milk and sell venom;
there's a good bit of money to be made this way)

A little venom goes a really long way. (a single drop does multiple agar plates if I calculated right based on that link's methodology)

[warriorsoul]

No go, on snakes in the house.

[warriorsoul]

The jars are ready for birthing. I lost the falbino and pe6 jar to trich. I had couple other cakes going already, so its not a big deal.temps 75-80.17 days.