Breeding Mushrooms Part 2: Mating Haploid Isolates to Form Diploid Mycelium
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- เผยแพร่เมื่อ 28 ก.ย. 2024
- Part 2 of Breeding Mushrooms: Mating Haploid Isolates to Form Diploid Mycelium - In this video, Gary with Fresh from the Farm Fungi explains how to cross mycelium from spores to form new, unique strains of gourmet mushrooms! For more information visit freshfromthefar... to purchase our cultures check out our ETSY shop FRESH FUNGI www.etsy.com/s... Thanks for watching along! MUSHLOVE
Gary's Top Picks Amazon: www.amazon.com...
I really appreciate this thorough and methodical display of the practice, thank you!
This is ART, Mr. White!
I get so excited seeing Uni-level stuff that I've done being actively supported and disseminated on youtube! I get so excitd i want to comment in the first 5 minutes of videos but always wait lol
comment away sir! Thanks for watching
17:56 the one time you didn't get it perfect is how my transfers look everytime hahaha
it takes lots of practice and I still am improving - can’t wait to look back in 30 years and see the differences
Thank you for sharing your knowledge!!
I think the term is eukaryotic and dikaryotic mean single celled nucleus and double celled nucleus.
Do you get a new blade every time instead of applying a flame to it?
for breeding yes - if I use a mono culture I will flame but since it’s so sensitive I want to make sure there is no carryover
Please could you explain how that is haploid?
Who taught you all of this? very cool
S Smith I have read a lot of literature and been performing tissue culture for a long time - Also, have a background in clinical mycology/microbiology. I recommend any Paul Stamets books, Tradd Cotter and Peter McCoy have great books as well on mycology - MUSHLOVE
This is how you cross breed lots of different isolates of sepperate strains, love the lab coat and the apple earphone but you're not fooling me. Using 6 different scalpels when you could have used one and flame sterilized it each time which is what you should be doing if you hope to achieve selecting isolates from the agar...
I have yet to purchase a bacticinerator which I would use, but am against open flames since it’s my own lab and operation. For safety reasons I prefer to use a fresh blade each time and get it into liquid culture as soon as possible 👍 MUSHLOVE
Bro, you really counted all the permutations? There's a whole field of math called combinatorics and you can calculate that with a simple equation: N x (N-1) / 2
Is there an ideal amount of space to leave between transfers for them to grow into? Or could they be placed directly next to each other in hopes that they find each other and mate sooner, leaving more room for peripheral growth?
it is important to see if there is a zone of inhibition for non-mating pairs so I wouldn’t place them right next to eachother
I hate to nag, but could you do a lab walk through, with afterthoughts? I'm starting my basement lab and would like tips from someone that's proven successful 👍
I've gotten a reply in regards to the flow hoods too, prices were on point 😊 what width is yours?
Motor Miljø Thanks for following along - I will see what we can do - the lab here is less than ideal and I recommend reading up on Paul Stamets’ literature (Mushroom cultivar and How to grow gourmet and medicinal mushrooms are the go-to guides in mycology) Also, Tradd Cotter’s book and Peter McCoy’s Radical Mycology are great for the technical and more experimental aspects in growing - these books have helped shaped our processes and provide a deeper insight into designing a lab/system that will fit your needs - MUSHLOVE
P.S. Our hood is 8ft dual filter
what are the materials used
it is agar (MEA) and petri dishes
What type of agar is that? It's so pretty
MEA (malt extract agar)
Good job! Please can you tell me souce where I can find information about making powerful mycelium with good crop. I know how to make pure culture on agar media from spor or tissue but how I can know that it's profitable strain?
A lot of it is getting a feel for what is good growth and what is not. Just keep experimenting and observing. Remembering and learning. These videos are really nice because he shows you the growth and talks about it, so you can get a few looks at good stuff here.
it is hard to know before you fruit it out - start with some strong cultures we sell ours here etsy.com/shop/freshfungi
I think I'm finding out that the cotton looking mycelium is from too much moisture . And the (slow growth/growing more in a mound /not stretching) is because u have too much nutrients for the mycelium to eat
it may be that but I have seen rizomorphic mycelium in the same conditions so some mycelium may be more susceptible than others to these morphologies 🍄❤️
@@FreshfromtheFarmFungi I can agree with that ... some times u will plant a seed and the plant pops up deformed.. I'm sure the same thing happens with fungi
I'm still unclear on how you made sure the haploids were truly single haploids and not 2 or more or already fused diploids. Did you just lower the concentration of spores so much that they were too dilute in your streaks to meet and fuse before you separated them?
yes please watch part 1 it will make more sense
@@FreshfromtheFarmFungi I did watch part 1. I left this comment after watching both parts
What results of isolation in part 1?
th-cam.com/play/PLFfnF_UJ5WgKRxZZbixO27fxOi9mEtmTv.html watch this playlist it explains all
Meow.
Damn....what happened to this guy's flow hood? Looks like someone kicked the livin' shit out of it. Disgruntled employee? Bad day at the office? Too much whiskey at the staff Christmas party? 6:01
Ha good observation the cracking is from cooling off substrate blocks stacked up against the plexiglass - caused it to expand and contract and well, crack - we are planning to upgrade the housing to stainless steel when the season slows down 👍😎
@@FreshfromtheFarmFungi Hahaha.....I was just razzin' you a bit. Whatever gets the job done, buddy....that's all that counts. Cheers!
Thank you everyone now go out there and breed some cool strains! MUSHLOVE
Building my little myco-corner right now. Can't wait to finally get started. Been doing a lot of research and am ready to get my hands dirty so to speak
Does this mean you get to name it🤔
Thank you, you sparked my interest and I will be experimenting with your techniques to create new genetic strains. It's crazy that you are the only person on TH-cam who talks about this amazing process. Who would have thought haploid only has one chromosome set and we have the ability to create 2 complete chromosome sets. SO MUCH FUN!
Iv been waiting for this day
This is incredible knowledge you are sharing. I've got what I need now to get started on something I've wondered about for months. Thank you!
OK coming from a small business owner making spawn, some one in mycology for 6 years and some some who have gone through almost everyone on TH-cam with mycology
I take my hat of for you, your methods for explaining is the simplest and easiest
Thanks I appreciate it! Thanks for watching! Coming from someone with so much experience - is there any topic that is being ignored that I should be covering? MUSHLOVE
@@FreshfromtheFarmFungi
Actually yes the exact topics your handling
I haven't watched all of your videos yes but there all really good
Again I take my hat off for you
Fresh from the Farm Fungi LLC .. thanks love these breeding videos, if you ever get time, would love to see any microscopy techniques, such as verifying isolates, and dikaryon crosses; or doing it with pcr. long term storage like using nitrogen or glycerin ect... would be good too. I can only find this info google scholar.
@@mikerizzyraw I actually think this guy is so great he might even be heading/ showing that stuff soon regardless of asking
Again I really love this guys videos
Absolutely awesome mate. Thank you so much for putting this stuff out. Absolute quality!
Damn your good bro! Your agar process may be the tidiest and quickest I’ve seen yet.
Thanks for this Gary! Super interesting and exciting stuff.
Hey Gary, I’ve really been enjoying these videos. You seem to be extremely well educated and a good presenter. May I ask if you know of any books or other robust resources that I could study to learn about this genetic/breeding/microbiology stuff pertaining to our mycology contexts? TH-cam is a great resource, but I really like an authoritative sources like books
Can you just "mate" them when you transfer the agar to spawn bags? i.e. cut up the agar dishes and add at least two types into the same spawn bag?
Hey! I was wondering. Do you also do this for your Cordyceps?
I think this is the way to cross cordyceps genetics to create new strain lineages. Shouldn't matter what Mushroom it is. Should work for all of them ^^
biggest waste of plastic and blades... use pp5 dishes and reuse them !!!
For someone with plenty of time and does small production I agree - this may be overkill. For someone seriously relying on sterility and efficiency, this is a better method. 🍄❤️
@@FreshfromtheFarmFungi efficiency yes....sterile no way... never ever i had problem with pp5 agar dishes .. and they are reusable many many times over.. we waste to much plastic... you also could go for glass petrie dishes...
Keep the videos coming. Thank you.
Gary, you are the man.
Thanks for all the kick ass videos.
I'm learning a phenomenal amount.
Good karma for FFTFF!
Do Cordyceps militaris spores last long? Can you use them to make a spore print on foil and store it for years?
Or should the spores be planted immediately since their shelf life is short?
the cordyceps spores are ascospores and are fragile so they should be used fresh as quickly as possible
This makes me wonder if a microtubulin inhibitor like oryzalin could produce polyploid fungi.
not sure but interested! 👍
I've got oryzalin and was wondering this myself. What would be the best practice for this? Any recommendations?
Do you look for clamp connections under a microscope to make sure u have monokaryotic isolates before introducing them together? Or do u not find it necessary
I hate how life doesn't allow me to be first on these videos 😅
Gary , wonderful Video, i love your organization. you make it seem very easy. THANK YOU
Great content, but honestly I wouldn't waste so many blades. Just flae-sterilize the quickly or with ethanol.
When you weigh sterility x $5 pack of 10 blades w/handle, It's just not worth the risk to waste time with contaminants
How do you know if you have a haploid though what if two spores just stuck together after diluting and you didn't know? Can you tell?
genetic testing would confirm but otherwise just growing it out is the best way - imagine it as strawberry plants that had to meet - there would be patches with berries and patches without so you could decipher
Great video! Quick question will Haploid and Diploid Mycelium act and look the same if grow out the only difference being one can produce fruit? Is there like a certain distance a Haploid can grow out looking for a mate before it stalls or looses viggor? to the naked eye do both behave and look the same and there is no way to tell one from the other?
It can behave and look the same - you can do genetic analysis to differentiate them before fruiting but it’s cheaper and easier in my opinion to just fruit them out unless you have access to PCR, extraction equipment and a way to read and compare genomic sequences
instead of using all those scael blades couldn't you have used flame to sterilize 1 blade? ty
Yes I wanted to guarantee sterility and prefer the sharper blades, but I do flame sterilize sometimes for inoculations
How can I modify the genetics of my mushroom?
there are many techniques- spore breeding, di-mon mating, crispr cas9, physical and chemical mutations. All these require extensive laboratory procedures
Yeah, I'm so lost.
is diploid mycelium and dikaryotic mycelium interchangeable?
Dikariotic refers to the nucleus (two nuclei), diploid refers to the genetic information required to produce a viable ascospore or basidiospore - they are relatively the same but slightly different. At least from my understanding
@@FreshfromtheFarmFungi with every question answered i get at least one more! but anyway, this is helpful, thanks a lot for taking the time, Gary! MUSH Love!
Does each dish with isolates contain dikaryotic mycelium? Looks like those mycelia in dishes come from mushroom body fragments, not spores.
Good information, grettings from Chile!🇨🇱
Hello! Do you think this is possible to do in a still air box?
I've been doing it in open air n into 2oz clear ketchup cups with snap on lids. Small bathroom, no air movement hvac is off, gloves, iso, mask... most importantly you first spray superfine mist of water into corners of room and all around. The mist dropping to ground removes many contaminates from air. I next spray similarly with ISO (careful, you do still need to breath, 😆). Really get all air in small room, from top to bottom, misted to drop all contaminates. This is after I've got everything set up and I'm place.
Using this method, I've been having great success with spore to agar cup as well as spore syringe to agar.
No contaminants yet from almost 40 agar cups over last 2 months, and I've even been able to innoculate grain and LC jars from agar growths, etc. I did have contamination when I tried to innoculate LC with clone tissue. Both LC went cloudy and had contams. 😢 But I've got 7 other LC from spore syringe and, agar inoculations that are doing great.
So... it can be done without laminar hood, it can be done without SAB. Just be sure to wipe and spray with ISO a LOT
Is this the morel mushroom production process?
for cordyceps
Please do a video hybridizing yellow and pink oyster mushrooms!!
I think you're doing that in the video actually. Your use of certain words just throws me off completely. I'm not a scientist.
check out this video th-cam.com/video/S81tML5_R-E/w-d-xo.html
Will you take a transfer of the mated diploids and let that grow out or just put the diploid plate straight to grain? And what do you do w/ the plates that don't mate and remain haploid?
I heard genetics can't cross that way.
The science has been there for decades lol - Look up “Monokaryon mating in filamentous fungi” you will find your answers
Great!
Nice
Do you do microscopy to confirm monokaryons?
microscopy would not confirm - clamp connections are subjective only genetic testing and growing them out confirms 100%
Not me dreaming of breeding mushrooms in the mountains for the rest of my life lol.
Is every colony from spore plate from streaked solution a haploid colony definitely?
not definitely until it is crossed and grown out. There is a chance two spores can stick together or germinate very close and the only way to know for sure is to grow them out or do genetic testing
@@FreshfromtheFarmFungi thanks so much for the swift response. But both haploid and diploid mycelium can fruit? I understood from multi spore I was trying to separate the mycelium to somehow isolate it. Then see if that would fruit. Do I now understand I should make pairings of all the transfers from the multispore plate ? Then fruit ones which combine and create diploid mycelium .or is this essentially pointless without your early extraction of colonies because I can't really know if my plates are infact haploid? I hope that make sense ? 🙏
I often come to a place with people in my line of questioning and hear you even say sometimes that it's purely chance and choice really. And essentially it's much simpler to fruit and clone a phenotype ,so why go through such a process for another phenotype? Maybe I missed in this video but what makes it better that it's worth this or its for the science and exploration. ?
We're all very excited about your chestnuts too.
Ol big brain over here flexing. Many thanks homie 🤙
this is just a random question im sure you get all the time
definately in the mindset of coulda not shoulda
but could you cross breed say pink oyster and a cubensis variety?
would they even be able to breed?
for purely scientific purposes of course
I think anything is possible - I have heard of people using snake venom to hybridize different mushrooms strains but am not very familiar with that procedure - but it’s out there somewhere 🙂❤️🍄
I want to know these referrals and their amounts?
Hey Gary - thanks so much for sharing this! I am bout to nerd out like a boss! 😜😆
Shhhhhh... you're upsetting the agar...
God I'm really scratching my head right now . So I'm watching your videos in for some reason I'm having a hard time grasping and taking it all in maybe I'm stressed. And I've been looking up a lot of research on you know mixing mushrooms and some people say snake that some are even saying utilizing penicillin I believe. I was watching one of your videos and I'm not sure if I grasped it right but it seems like you were taking two different species putting them inside the same petri dish and if the mycelium (connected in the middle You're taking a sample from the connection) And if the two mycelium mats were not compatible clearly divide down the middle. Now with that being said let's say I wanted to crossbreed Panaeolus cyanescens with something like albino penis envy. Could I just take the two mycelium grow them out in the dish and And if it connects in the middle take a sample and put it into a different dish and then grow it out? My question is from that connection sample from ape an pan can is there a chance I could crossbreed or make a hybrid penis envy/pan . Or I might just completely wrong about why you are putting two different mycelial mats together hoping for a connection? I'm sorry I'm just trying to take this all in
These spores are from the same species and each spore contains half of the genetics needed to fruit out a mushroom successfully- that being said there are many theoretical and some proven methods of hybridization between different species but it’s much more complex and less likely to result in successful pairing. Hope that makes sense - I don’t want to discourage people from trying anything but this method described in this series is within the same species.
so that means mushroom throwing spores and they creating haploid colonies then finding each other and create new diploin breed ready for fruiting, right?can we say that diploid colony needed for mycilium to fruit?
thank you so much! i'm really appreciate you sharing such information
yes that is correct for the most part! Some exceptions exist but that’s the rule of thumb for most species
Hey, Thanks for this! quick question - How can I tell if my culture is haploid or diploid without a microscope?
Jordan Jacobs If you fruit it out then it is diploid (some exceptions) Also, if it came from a single spore then it is haploid. In order to obtain a single spore isolate, one should perform serial dilutions and quantitate the spore load/viability and then isolate single spore colonies. Also, genetic testing can be performed (PCR or Gene Sequencing)
@@FreshfromtheFarmFungi Not an expert but i think under a microscope you should only see clamp connections in diploid(mated) mycelium but i'm sure there are exceptions.
How do you know its a haploid, and two didn't mate? Is it very obvious? Does the look vary wildly across species? Thanks
it’s not very obvious without fruiting them out - there is some variation yes but it needs to be crossed and fruited to verify
Have anything against high-temp sterilizing your blade and just using one?
I don’t like open flames and prefer the sharp clean cuts of a new blade. I do have a torch for loops now but prefer new blades especially when breeding to ensure sterility and prevent any cross contam. It’s a higher standard
Providing a link to the conclusion that would be good idea
th-cam.com/video/tkWsv5E5m-8/w-d-xo.html
are the isolates you are working with in this video the same spore dilution plates from video one feels like I skipped a step or video
yes they are the same ones follow the playlist “breeding mushrooms from spores” th-cam.com/play/PLFfnF_UJ5WgKRxZZbixO27fxOi9mEtmTv.html
Awesome video!
OK the numbers are the order in which they formed the colony, what is the letter code?
the letters are indicating the plate of origin from the dilutions
Great content. Thanks for the time🌈😃🤙
Very helpful!
It would help if you showed some results of your experiments
they make it all the way - follow these videos on the playlist “breeding mushrooms from spores” th-cam.com/play/PLFfnF_UJ5WgKRxZZbixO27fxOi9mEtmTv.html
This is awesome... thank you!
Thank you for putting this content out here.
Also, is there a rule of thumb in regards to what species will mix?
Motor Miljø We are always tinkering with the “rules of thumbs” hah experimentation is key! However, in nature, like species mate with like species until small mutations occur and develop sub-species which later become their own species living in their niche
Thank you for sharing your knowledge! I cant wait to try this.
Thanks for watching and following along! 🍄❤️
Question - that was a pretty big gap between video 1 and 2 - how did you isolate the haploid mycelium and know it was haploid? I saw you mention in another question that you either have to fruit it out or genetic sequence. Which did you do? Seems like a lot of work/waste to "fruit" stuff out to see if it doesn't fruit to make sure it's hapoid, especially when you know nothing else about the strain.
In the first video he diluted the spore solutions which I believe is key to the process by ensuring you get roughly 10 (numbers may be way off) spores/plate. This ensures you are getting haploids whereas when you scrape thousands of spores onto a plate it is more of a free for all.
This was my interpretation anyway!
th-cam.com/play/PLFfnF_UJ5WgKRxZZbixO27fxOi9mEtmTv.html watch this playlist - yes it is very wasteful but it’s still the best way because once you find a good one you can keep the haploid and cross again. The hard part is finding them
yes exactly
Beautiful! Thank you!
Отлично брат, ты супер!!!
.
what did you seal your dishes with?
parafilm
Can you ship to Canada?
Sweet Life 529 We only ship within the US right now
Fresh from the Farm Fungi LLC , yeah.....to bad! Would love to order some Chestnuts!
dude, you really need to try and practice more sustainable less wasteful methods. I appreciate your videos, but the amount of plastic waste is just ridiculous
I appreciate this concern and will try to think of something better for this winter
@@FreshfromtheFarmFungi thanks for taking the time to read this and respond 😁 sorry, I don't mean to come off negatively in any way. You make great videos!
@@zuul902 how exactly would you go about reducing the number of plates based on the number of isolates and transfer requirements? Split plates maybe 🤔 but that doesn’t seem viable.
Glass agar dishes that can be reused / sterilized
@@mycomachine9488 glass or autoclavable petris
18:55 yeah close call. I even held my breath when I saw that slip.
and you should close your petri dishes between transfers
Love it. Thank you for your generosity and time
you don't ever worry about contam with handling those with bare hands?
It’s not the best practice but for learning purposes it doesn’t matter - for production it is much more stringent
@@FreshfromtheFarmFungi appreciate the response. I've recently been getting mold on few plates after getting back into agar..showing friends and family etc..
i've been told is likely due to over-handling.
This video got me price checking pyrex petri dishes