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Exhaustus
Not-Paul-Stamets



Registered: 07/14/23
Posts: 84
Loc: Tallon IV
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Selective Breeding - A few questions 1
#28443721 - 08/23/23 11:07 AM (5 months, 2 days ago) |
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I figure I have the capabilities to gather spores for selective germination for crossing species. Or to isolate monocultures
If I follow the methods bellow and inoculate two tubes do the Dip/Hap spores grow/expand hypha into the media?


Do I have to transfer them into close proximity or is there growth beyond a few millimeters?


The only other thing I was curious about was maybe terms to find the best gauge needle for isolating the spores. I'm reading that Sabouraud dextrose Agar with a pH of 5.6 is a great media to use because it keeps bacteria from growing as easily. Anyone else able to confirm or have used this?
My first Agar to Agar transfer of cloned material went well so I'm bored enough to venture into. I have a few mutagens on the way to see if I can induce anything goofy.
Thanks for any help with my questions.
-------------------- Rhizomorphically hunting Tomentose Sectors - x3zR Mycofluidics Turgor is Awesome - https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/turgor Shoe Boxes R Us! - Or else!
To attain the paradise all sentient creatures are open to, I train day and night, among mountains and fields!
Edited by Exhaustus (08/23/23 02:18 PM)
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Hap/Dip Mating - A few questions [Re: Exhaustus] 1
#28443746 - 08/23/23 11:32 AM (5 months, 2 days ago) |
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I don't believe there is such thing as 'haploid spores' or 'diploid spores'. As far as I am aware, all spores are haploid. When the spore germinates it creates haploid hyphae/mycelium which then combines with another compatible haploid hyphae/mycelium to create diploid mycelium. That is mon-mon mating (monokaryotic = haploid, dikaryotic = diploid, I believe mono/di-karyotic is the up to date terminology).
Di-mon mating is also possible. Here is a paper about that: https://sci-hub.se/http://www.jstor.org/stable/2439413.
How do you intend to collect the haploid (monokaryotic) culture/s? Are you planning to cross a mono culture with a cloned (dikaryotic) culture? There is also something called dedikaryotization where you turn a dikaryotic culture into a monokaryotic for mating: https://www.shroomery.org/forums/showflat.php/Number/27619669/fpart/all
And what mutagens do you have on the way?
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Eclipse3130
Servant of the Fungi



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Re: Hap/Dip Mating - A few questions [Re: covertjoy] 2
#28443822 - 08/23/23 12:47 PM (5 months, 2 days ago) |
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Selectively crossing 2 monokaryons is an inferior way to breed, because you can't select for vigor or health, and also the gene pool of what you get is extremely tiny - which could result most of the time in very weak genes.
The easiest way to breed is to combine spores of different variety and let them germinate naturally, this way natural selection takes over - the strongest will show itself amongst millions of spores and thus you can select from there.
-------------------- "In The Material World One seeks retirement and grows Old In The Magical World One seeks Enlightenment and grows Wiser In The Miraculous World One seeks nothing and grows Lighter As we all tread the Homeward Path we will explore many Realms And one day... we will all Realize that all experiences are Simply Different ways in which The All-That Is Perceives Itself"
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Hap/Dip Mating - A few questions [Re: Eclipse3130] 1
#28443926 - 08/23/23 02:11 PM (5 months, 2 days ago) |
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I agree with that. I mixed 8 different cubensis spore syringes together and am currently germinating them out to select for best genetics. By my calculations that is a possibility of 8 strains and 24 strain hybrids. I figure by crossing different strains there is a narrow chance of very good genetics, and it becomes a numbers game.
That being said, perhaps a selectively crossed strain hybrid could have health and vigor selected for from the F2 generation onwards? And you can still play the numbers game a bit by creating many monokaryon cultures and permeating them.
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Exhaustus
Not-Paul-Stamets



Registered: 07/14/23
Posts: 84
Loc: Tallon IV
Last seen: 1 month, 4 days
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Re: Hap/Dip Mating - A few questions [Re: covertjoy]
#28443942 - 08/23/23 02:20 PM (5 months, 2 days ago) |
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I don't mind trying to isolate Pre/Post Reproduction I don't mind combining singular spores just to see I hope to just screw around.
DNA alkylating NTG (N′-methyl-N′-nitro-N′-nitrosoguanidine)
Appreciate the links, You're correct on the spore thing. I did also have in mind trying to get a 3-way cross if anything else worked.
-------------------- Rhizomorphically hunting Tomentose Sectors - x3zR Mycofluidics Turgor is Awesome - https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/turgor Shoe Boxes R Us! - Or else!
To attain the paradise all sentient creatures are open to, I train day and night, among mountains and fields!
Edited by Exhaustus (08/23/23 02:30 PM)
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Hap/Dip Mating - A few questions [Re: Exhaustus] 2
#28444001 - 08/23/23 02:59 PM (5 months, 2 days ago) |
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I am with you on wanting to do it for the fun of it.
The method which appeals the most to me is to use a hemocytometer to count the spore density in a spore solution under a microscope and then use serial dilution to dilute the spore solution down let's say 2 spores per 0.05ml (a drop of a pipette). Then put 5 drops onto a petridish and use a spreader and you should have 10 spores spread out across each plate.
Any germinating cultures can be inspected under the microscope to determine if they are coming from a single spore and lack clamp connections. But bear in mind that per spore the germination rate is very low (Stamets estimates 1-5%) and any two given monokaryotic cultures only have a 1 in 4 chance of being compatible mating types.
So you may need to inoculate 3-4 plates with 10 spores each to get a monokaryotic culture and then let's say you get 2 of strain A and 2 of strain B, from 16 germination plates, that gives you 4 possible permutations, on average resulting in 1 compatible pair based on mating type. Whether their genotypes are compatible is another question and then for them to have strong genetics is another.
So you're looking at something like 16 germination plates, 4 isolation plates and 4 crossing plates for 1 strain hybrid with no guarantee of quality. 4 monokaryotic cultures per parent would be 32 germination plates, 8 isolation plates and 16 crossing plates with on average 4 compatible crosses by mating type, which seems like I think would give a reasonable chance of getting a hybrid.
If crossing species then the genotypic compatibility is going to be much lower and most will fail.
These numbers are of course all based on estimates and averages.
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Edited by covertjoy (08/23/23 03:08 PM)
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Exhaustus
Not-Paul-Stamets



Registered: 07/14/23
Posts: 84
Loc: Tallon IV
Last seen: 1 month, 4 days
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Re: Hap/Dip Mating - A few questions [Re: covertjoy]
#28444140 - 08/23/23 04:57 PM (5 months, 2 days ago) |
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Quote:
covertjoy said: I am with you on wanting to do it for the fun of it.
The method which appeals the most to me is to use a hemocytometer to count the spore density in a spore solution under a microscope and then use serial dilution to dilute the spore solution down let's say 2 spores per 0.05ml (a drop of a pipette). Then put 5 drops onto a petridish and use a spreader and you should have 10 spores spread out across each plate.
Any germinating cultures can be inspected under the microscope to determine if they are coming from a single spore and lack clamp connections. But bear in mind that per spore the germination rate is very low (Stamets estimates 1-5%) and any two given monokaryotic cultures only have a 1 in 4 chance of being compatible mating types.
So you may need to inoculate 3-4 plates with 10 spores each to get a monokaryotic culture and then let's say you get 2 of strain A and 2 of strain B, from 16 germination plates, that gives you 4 possible permutations, on average resulting in 1 compatible pair based on mating type. Whether their genotypes are compatible is another question and then for them to have strong genetics is another.
So you're looking at something like 16 germination plates, 4 isolation plates and 4 crossing plates for 1 strain hybrid with no guarantee of quality. 4 monokaryotic cultures per parent would be 32 germination plates, 8 isolation plates and 16 crossing plates with on average 4 compatible crosses by mating type, which seems like I think would give a reasonable chance of getting a hybrid.
If crossing species then the genotypic compatibility is going to be much lower and most will fail.
These numbers are of course all based on estimates and averages.
Beautiful, I have a couple dozen pages on similar dilutions. Breeding pokemon out here :glasses:
Perhaps Ultrasonic blending of liquid culture, dilution, and Loop Dots on plates. Or spore loop dots (in place of streaking the plate, I feel it allows expression of culture at source). I'm not sure if I like Darkfield Imaging better or not yet. My shaky hands ain't helping my success rate either
I have done "node" transfers of tropical plants and have always wanted to see how small of a trimming I could make to produce a new plant. I read into some of your links and got my ticker, ticking 
Late Edit - Should I do the parafin and agar mounting technique with coverslips or just tissue and oil immersion? These old notes of mine are tempting. I wish I had a few more things for gene insertion, I found a sequence code for the "color" of the fruit. I wonder if I could make a "Starry Night" Tidal Wave (They tend to have healthy spores even when bad)
I wonder if there is any micro/macro nutrient data free for full access. I would love to see staged metabolic pathway data similar to the EnteroPluri Testing for bacteria.
-------------------- Rhizomorphically hunting Tomentose Sectors - x3zR Mycofluidics Turgor is Awesome - https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/turgor Shoe Boxes R Us! - Or else!
To attain the paradise all sentient creatures are open to, I train day and night, among mountains and fields!
Edited by Exhaustus (08/23/23 05:20 PM)
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Workman
1999 Spore War Veteran



Registered: 03/01/01
Posts: 3,598
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Re: Hap/Dip Mating - A few questions [Re: Exhaustus] 1
#28444531 - 08/23/23 10:10 PM (5 months, 2 days ago) |
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Here is my breeding method. It only requires a single monokaryotic culture for one parent and some dry spores for the other parent(s).
https://www.shroomery.org/forums/showflat.php/Number/5194890/fpart/1/vc/1
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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Eclipse3130
Servant of the Fungi



Registered: 10/06/13
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Re: Hap/Dip Mating - A few questions [Re: Workman]
#28445082 - 08/24/23 12:54 PM (5 months, 1 day ago) |
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And there you have it the legendary creator of the coveted and well known APE.
-------------------- "In The Material World One seeks retirement and grows Old In The Magical World One seeks Enlightenment and grows Wiser In The Miraculous World One seeks nothing and grows Lighter As we all tread the Homeward Path we will explore many Realms And one day... we will all Realize that all experiences are Simply Different ways in which The All-That Is Perceives Itself"
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Exhaustus
Not-Paul-Stamets



Registered: 07/14/23
Posts: 84
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Re: Hap/Dip Mating - A few questions [Re: Eclipse3130]
#28445485 - 08/24/23 06:37 PM (5 months, 1 day ago) |
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Quote:
Workman said: Here is my breeding method. It only requires a single monokaryotic culture for one parent and some dry spores for the other parent(s).
https://www.shroomery.org/forums/showflat.php/Number/5194890/fpart/1/vc/1
Greatly Appreciated! Wow, reading through and I swear I didn't copy paste you HAHA.
You mostly answered my next question in your link! <3 I guess I just need some guidance once I get to checking for clamp connections.
Quote:
Eclipse3130 said: And there you have it the legendary creator of the coveted and well known APE.
Humbling
-------------------- Rhizomorphically hunting Tomentose Sectors - x3zR Mycofluidics Turgor is Awesome - https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/turgor Shoe Boxes R Us! - Or else!
To attain the paradise all sentient creatures are open to, I train day and night, among mountains and fields!
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Hap/Dip Mating - A few questions [Re: Workman]
#28446163 - 08/25/23 08:05 AM (5 months, 14 hours ago) |
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Quote:
Workman said: Here is my breeding method. It only requires a single monokaryotic culture for one parent and some dry spores for the other parent(s).
https://www.shroomery.org/forums/showflat.php/Number/5194890/fpart/1/vc/1
How long did it take for the monokaryotic mycelium to colonise the grain? And how did you know when the nuclear migration was complete?
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Workman
1999 Spore War Veteran



Registered: 03/01/01
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Re: Hap/Dip Mating - A few questions [Re: covertjoy] 2
#28446309 - 08/25/23 11:16 AM (5 months, 11 hours ago) |
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Quote:
covertjoy said:
Quote:
Workman said: Here is my breeding method. It only requires a single monokaryotic culture for one parent and some dry spores for the other parent(s).
https://www.shroomery.org/forums/showflat.php/Number/5194890/fpart/1/vc/1
How long did it take for the monokaryotic mycelium to colonise the grain? And how did you know when the nuclear migration was complete?
A couple weeks, if I recall correctly, but the important part is that it is only mostly colonized, which you will have to visually judge. I didn't know when the nuclear migration was complete so I just gave it 7-10 days. It can continue to migrate after putting the spawn in a tray. I mentioned it somewhere but you have to fruit the spawn directly and not use it as spawn or you will likely get non-hybrid fruits mixed in.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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Eclipse3130
Servant of the Fungi



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Re: Hap/Dip Mating - A few questions [Re: Workman] 1
#28446370 - 08/25/23 12:31 PM (5 months, 10 hours ago) |
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Is there any benefit to breeding "the long way" utilizing those various methods?
What are there if any downsides to simply combining spores together of two different varieties?
It seems to yield the same results without all the hassle no?
-------------------- "In The Material World One seeks retirement and grows Old In The Magical World One seeks Enlightenment and grows Wiser In The Miraculous World One seeks nothing and grows Lighter As we all tread the Homeward Path we will explore many Realms And one day... we will all Realize that all experiences are Simply Different ways in which The All-That Is Perceives Itself"
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covertjoy

Registered: 07/09/23
Posts: 272
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Re: Hap/Dip Mating - A few questions [Re: Workman]
#28446382 - 08/25/23 12:44 PM (5 months, 9 hours ago) |
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Quote:
Workman said:the important part is that it is only mostly colonized
Why is it important that it is only mostly colonized? Will the nuclear migration not take place otherwise?
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Workman
1999 Spore War Veteran



Registered: 03/01/01
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Re: Hap/Dip Mating - A few questions [Re: covertjoy] 1
#28446487 - 08/25/23 02:07 PM (5 months, 8 hours ago) |
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The added spores probably need a little room to germinate and grow. It might not be totally necessary.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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Workman
1999 Spore War Veteran



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Re: Hap/Dip Mating - A few questions [Re: Eclipse3130]
#28446494 - 08/25/23 02:11 PM (5 months, 8 hours ago) |
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Quote:
Eclipse3130 said: Is there any benefit to breeding "the long way" utilizing those various methods?
What are there if any downsides to simply combining spores together of two different varieties?
It seems to yield the same results without all the hassle no?
It may be difficult to know if you get a successful hybrid if you just mix spores. It should work but unless the parents are visually distinct, the F1 hybrids may not be obvious.
Doing it the long way is more controlled, but you only end up with a single hybrid per cross, which may or may not be the best.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification 
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shroomies4me
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Re: Selective Breeding - A few questions [Re: Exhaustus] 1
#28449716 - 08/28/23 10:29 AM (4 months, 28 days ago) |
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There's a youtube series where the guy goes through the entire process over the course of months with videos each step of the way for breeding multiple gourmet species. Here's the first video where he goes through serial dilutions, but there's a playlist, I just couldn't figure out how to link it. Hope it helps:
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Exhaustus
Not-Paul-Stamets



Registered: 07/14/23
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Re: Selective Breeding - A few questions [Re: Exhaustus]
#28506010 - 10/15/23 08:14 PM (3 months, 11 days ago) |
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Quote:
Exhaustus said: I figure I have the capabilities to gather spores for selective germination for crossing species. Or to isolate monocultures
If I follow the methods bellow and inoculate two tubes do the Dip/Hap spores grow/expand hypha into the media?


Do I have to transfer them into close proximity or is there growth beyond a few millimeters?


The only other thing I was curious about was maybe terms to find the best gauge needle for isolating the spores. I'm reading that Sabouraud dextrose Agar with a pH of 5.6 is a great media to use because it keeps bacteria from growing as easily. Anyone else able to confirm or have used this?
My first Agar to Agar transfer of cloned material went well so I'm bored enough to venture into. I have a few mutagens on the way to see if I can induce anything goofy.
Thanks for any help with my questions.
Some of my clonework is panning out, yet I keep running into only a few within the flush having the original phenotypes. The rest are standard phenotype within the cap, with marginal traits appearing on the stem. I'm not sure if this is common within tissue to agar clones. Any advice?
-------------------- Rhizomorphically hunting Tomentose Sectors - x3zR Mycofluidics Turgor is Awesome - https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/turgor Shoe Boxes R Us! - Or else!
To attain the paradise all sentient creatures are open to, I train day and night, among mountains and fields!
Edited by Exhaustus (10/20/23 11:08 PM)
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