|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Clone --> Isolate, senescence?
#26908137 - 08/30/20 03:14 PM (3 years, 4 months ago) |
|
|
Hey, so I grew some PE6 out on BRF, put a clone to agar, and started transferring, U use 100mm dishes and I always transfer once the culture has colonized about 40%-60% of the dish. I'm currently on my 8th transfer and my last bulk run with my T7 dishes all contaminated. That got me thinking, is t possible for a cloned culture to reach senescence before you even reach an isolated set of genetics?
|
Forrester
aspiring sociopath


Registered: 02/05/13
Posts: 9,351
Loc: Northeast USA
Last seen: 24 days, 16 hours
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908348 - 08/30/20 04:43 PM (3 years, 4 months ago) |
|
|
I don't think so. But if it's looking tired, switch up your agar recipe to get some different nutrients into it. Sometimes running across a couple different recipes will even get an old, tired culture going again.
-------------------- Repugnant is a creature who would squander the ability to lift an eye to heaven, conscious of his fleeting time here. ------------------- Have some medicinal mushrooms and want to get the most out of them? Try this double extraction method.
|
jcm4620
Stranger


Registered: 05/26/19
Posts: 6,700
Last seen: 1 year, 11 months
|
Re: Clone --> Isolate, senescence? [Re: Forrester]
#26908367 - 08/30/20 04:54 PM (3 years, 4 months ago) |
|
|
u should be making masters of each transfer and fruiting them all out so you can go back and slant or transfer out further if wanted to narrow it down even more. while keeping good performers and junking the rest. cuz the way u make it sound is that u just keep pulling transfers off the tissue sample plate each time and what ever your fruiting out your just basicly losing them as you go.
thats what it sounds like ur saying to me at least so if im wrong im sorry.
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908443 - 08/30/20 05:33 PM (3 years, 4 months ago) |
|
|
Quote:
jcm4620 said: the way u make it sound is that u just keep pulling transfers off the tissue sample plate each time
That's exactly what I'm doing, I'm only interested in single sets of genetics. I cloned that sample just to narrow down the genetics before I started isolating, because when I start from spore I find reaching an isolate just takes too long, I'm hoping that I can just take a shortcut to an isolate. as I understand it, starting from spore will give me the best shot at an amazing isolate, but that's going to take too long, so I'm doing it this way to look for something that's pretty damn good, maybe not absolutely amazing, but I don't need that. I mainly want one that's going to pin and grow to maturity at basically the same rate. I'll make masters of the isolates, but that's it. I'm growing them out as I go just to get some along the way.
Edited by 3SIXTY5 (08/30/20 05:34 PM)
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: Forrester]
#26908465 - 08/30/20 05:42 PM (3 years, 4 months ago) |
|
|
Quote:
Forrester said: I don't think so. But if it's looking tired, switch up your agar recipe to get some different nutrients into it.
Yeah, it's still growing out fine on dishes I think, they're not in an incubator so it's a little harder to tell with the weather fluctuation so much lately. So could it still grow alright on dishes, and grains, but once it hits substrate it's too tired?
I probably fucked up my bulk subs. I was just thinking maybe it was the culture getting weaker and that's why my subs contaminated, I just didn't want to do another grain spawn and substrate run before I find out if it's the culture and not my substrate process that fucked me Up.
|
Roger Clemency
Smile


Registered: 03/23/20
Posts: 2,005
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908472 - 08/30/20 05:45 PM (3 years, 4 months ago) |
|
|
What was your bulk sub recipe and prep?
-------------------- Sour grapes, sweet revenge Heaven starts right where hell ends
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908477 - 08/30/20 05:48 PM (3 years, 4 months ago) |
|
|
Quote:
3SIXTY5 said:
Quote:
jcm4620 said: the way u make it sound is that u just keep pulling transfers off the tissue sample plate each time
That's exactly what I'm doing, I'm only interested in single sets of genetics. I cloned that sample just to narrow down the genetics before I started isolating, because when I start from spore I find reaching an isolate just takes too long, I'm hoping that I can just take a shortcut to an isolate. as I understand it, starting from spore will give me the best shot at an amazing isolate, but that's going to take too long, so I'm doing it this way to look for something that's pretty damn good, maybe not absolutely amazing, but I don't need that. I mainly want one that's going to pin and grow to maturity at basically the same rate. I'll make masters of the isolates, but that's it. I'm growing them out as I go just to get some along the way.
you already have what you're looking for. transfers from clone plates are to clean up from contams. The genetics are just that - clones, they're narrowed down to just one set.
--------------------
|
jcm4620
Stranger


Registered: 05/26/19
Posts: 6,700
Last seen: 1 year, 11 months
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908479 - 08/30/20 05:49 PM (3 years, 4 months ago) |
|
|
there is no such thing as a shortcut to a true isolate tho. thats not technically an isolate there is a fuck ton of sets of genetics in every transfer you make. all your doing is just taking transfers from a clone. the term isolate is actually used rather loosely by people. just cuz you think your "isolating" one piece of myc dosent make it a true isolate. its far to much information to really explain typing with my thumbs on my phone but if your interested in learning about true isolates and mono and dikaryotic cultures there is plenty of info available. all i was getting at with what you are doing is potentially throwing away your best genetics from your clone since your not taking transfers and making a master to go back to if it ends up being a great fruiter is all i was saying. but no matter how many transfers you make you really still just have a clone
|
jcm4620
Stranger


Registered: 05/26/19
Posts: 6,700
Last seen: 1 year, 11 months
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908482 - 08/30/20 05:51 PM (3 years, 4 months ago) |
|
|
i think jake actually has shit on hear explaining actual isolates very well that dudes the fucking man when it comes to that shit so if u search it toss his name in there also
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908517 - 08/30/20 06:10 PM (3 years, 4 months ago) |
|
|
Quote:
jcm4620 said: i think jake actually has shit on hear explaining actual isolates very well that dudes the fucking man when it comes to that shit so if u search it toss his name in there also
I'll definitely check that out, thanks.
I understand it's not a true isolate, maybe I'm using the wrong terminology, maybe I don't understand the process, but how can a clone be one set of genetics if its still sectoring on a plate? Also I remember reading a post by RR explaining that clones aren't usually single sets of genetics.
I understand monokaryotic mycelium is formed when one spore is germinated and that it's a non fruiting strain without another monokaryon. Is this related to an isolate?
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26908522 - 08/30/20 06:13 PM (3 years, 4 months ago) |
|
|
Quote:
trubblesome said: you already have what you're looking for. transfers from clone plates are to clean up from contams. The genetics are just that - clones, they're narrowed down to just one set.
Really, I've read otherwise, I remember an RR post one clone being made up of multiple sets of genetics.
|
jcm4620
Stranger


Registered: 05/26/19
Posts: 6,700
Last seen: 1 year, 11 months
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908538 - 08/30/20 06:20 PM (3 years, 4 months ago) |
|
|
clones have more genetics than i would ever attempt or want to try to count lol
|
jcm4620
Stranger


Registered: 05/26/19
Posts: 6,700
Last seen: 1 year, 11 months
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908558 - 08/30/20 06:28 PM (3 years, 4 months ago) |
|
|
when working with clones your really looking for traits more than the actual genetics themselves. but yet when u have a good clone its common and often refered to as good genetics lol i know right 😂words are fun arent they😃
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908596 - 08/30/20 06:51 PM (3 years, 4 months ago) |
|
|
Quote:
jcm4620 said: clones have more genetics than i would ever attempt or want to try to count lol
Yes I hear you on that, but my understanding was that when one drop of spore solution or one streak of a loop would have more sets of genetics than a cloned tissue sample from a fruit body, am I wrong about this?
I'm not looking for a true isolate or I would have started from spore, I'm looking for a single set of genetics so the whole substrate will pin and reach maturity within 24 hrs or so, so I don't have a substrate taking up space in my fruiting chamber for too long. Once I have a bunch of single genetic dishes, then I will test them for yield and all that.
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: jcm4620]
#26908601 - 08/30/20 06:53 PM (3 years, 4 months ago) |
|
|
Do you have a source on that? I've never read that anywhere and it doesn't make a lot of sense intuitively based on everything I have read about the life cycle of mushrooms. As I understand, in the flesh of the fruiting body the cells should all be identical and dikaryotic, containing the unfused haploid nuclei from the hyphae that paired and formed the primordia. in the basidia the nuclei fuse to create a diploid set and then undergo meiosis to create haploid spores. I looked for something myself and all I could find was an article about some Armillaria species undergoing a haploidization -> diploidization -> haploidization before producing primordia but that seemed to be unique to them and perhaps a reason for their ability to grow large and old.
my intuition says that differences in fruiting body phenotype would be due to conditions and available resources, not genetics.
--------------------
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
|
Quote:
Roger Clemency said: What was your bulk sub recipe and prep?
SUBSTRATE PREP
I sterilize my bulk substrate in a sort of modified insulated bucket Tek style bin, I boil water in the bin, wait until it's over 200f, dump in my bulk ingredients (780 grams coco + 225 grams verm + a handful of gypsum + a little more than 3 litres of water, per 5lb bag spawn) let it sit for about 2 hours, mix, let it sit for another 2 hours or so, then mix again. The bin stays above 180f for like 12-18hours, I usually have to keep mixing and waiting for 2-3 days before the substrate is back under 100F.
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26908616 - 08/30/20 07:03 PM (3 years, 4 months ago) |
|
|
Quote:
trubblesome said: Do you have a source on that?
RogerRabbit said: Yes. More than one strain can be present in an individual fruit. Thus, a clone is not an isolated strain, unless it was grown from an isolated line of mycelium. RR
I can link you the post if you want, pretty interesting, but a long read.
Edited by 3SIXTY5 (08/30/20 07:11 PM)
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908645 - 08/30/20 07:27 PM (3 years, 4 months ago) |
|
|
yeah, that would be great, thanks
--------------------
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26908646 - 08/30/20 07:28 PM (3 years, 4 months ago) |
|
|
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26908689 - 08/30/20 07:57 PM (3 years, 4 months ago) |
|
|
if he's just pointing to sectoring on the plate as evidence that the mycelium is not actually genetically identical, I don't know, i'm not super convinced.
--------------------
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26908691 - 08/30/20 07:58 PM (3 years, 4 months ago) |
|
|
unless we're using the same words but different understandings. seems like that happens a lot around here
--------------------
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26908706 - 08/30/20 08:07 PM (3 years, 4 months ago) |
|
|
It seems to be a fairly general consensus amongst people on this forum at least. I've heard this same thing many times from other TC's. I'm sure I can find a few more people talking about it if you want.
In my experience, anything I've ever grown from clone --> agar --> grains without transferring until it's no longer sectoring, there's still definitely many different physical expressions of fruit bodies, which I assume are seperate sets of genetics, just like with a multi spore substrate there's different physical expressions of fruit bodies.
The way I understood it was like this, if you grow out a substrate and it produces mushrooms of varying sizes, shades of color, and time from pins to mature mushrooms then it's definitely not a single set of genetics.
Edited by 3SIXTY5 (08/30/20 08:08 PM)
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26909110 - 08/31/20 05:43 AM (3 years, 4 months ago) |
|
|
phenotypic expression isn't a stand in for actual genotype difference and genetic analysis though, especially when we're talking about things like pinning and size and shade of cap, which can all be influenced by various environmental factors, even within the same tub and especially between grows. the only thing referencing scientific articles I can find says that fruiting bodies that are genetic mosaics as RR is describing are rare. see: Are mushrooms genetic individuals or genetic mosaics?
"Within the typical mushroom life cycle, spores and the primary mycelium they give rise to, are haploid in their genetic makeup, meaning that the nucleus present in each cell contains only a single copy of each chromosome (think sperm and eggs here, folks). Depending on their genetic compatibility, when hyphae of the same species encounter one another in the environment, they will fuse and give rise to a new secondary mycelium. The hyphal cells of this secondary mycelium each contain two un-fused, haploid nuclei—one from each strain (again, think eggs and sperm). This condition of two haploid nuclei per cell is termed “dikaryotic.” Mushrooms and other higher fungi are unique in that this dikaryotic phase is believed to persist for an extended portion of the life cycle. In most other organisms, compatible haploid nuclei (usually in the form of gametes) fuse soon after they encounter each other.
When environmental conditions are suitable, the secondary mycelium will form primordia that soon develop into mushrooms.
...
The question remains, however, how widespread is genetic mosaicism within the fungi? So far the presence of an extra-basidial diploidization-haploidization event has only been documented in two species of Armillaria."
if there is a new understanding of how fruit bodies form I'd love to see the study.
--------------------
|
A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 25 minutes, 35 seconds
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26909161 - 08/31/20 06:33 AM (3 years, 4 months ago) |
|
|
This is the same clone but one is from the tissue plate and one was t3 or t4. Huge difference from a few transfers narrowing genetics.
--------------------
LAGM2020     
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26909196 - 08/31/20 07:07 AM (3 years, 4 months ago) |
|
|
I'm not going to argue the scientific reasoning behind why this happens, because I'm not qualified to argue for or against the research done by actual mycologists, like you said earlier maybe we're using the same terminology in different ways.
It could be that they're identical when we're talking about the actual genetic composition of each fruit body that comes from a clone, although I highly doubt that because I've had albinos and regular colored mushrooms grown from the same substrate made from cloned tissue T4 dish.
All I know is that when you take a clone, clean the culture and grow it out, it acts more like a MS substrate than a "true isolate" produced by MS --> AGAR with multiple transfers, without hitting grains until only one single set of genetics are showing in the dish.
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: A.k.a]
#26909199 - 08/31/20 07:09 AM (3 years, 4 months ago) |
|
|
Quote:
A.k.a said: This is the same clone but one is from the tissue plate and one was t3 or t4. Huge difference from a few transfers narrowing genetics.
Is the pic on the left after transfers and the pic on the right before?
|
A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 25 minutes, 35 seconds
|
Re: Clone --> Isolate, senescence? [Re: 3SIXTY5]
#26909219 - 08/31/20 07:26 AM (3 years, 4 months ago) |
|
|
The one on the right was the early plate. The thick stem fruit at the bottom left is almost identical to the one I took the tissue from. A few transfers later and it’s changed to packed with fairly uniform mushrooms instead of the ms look it had before.
Also they were grown at the same time side by side.
--------------------
LAGM2020     
Edited by A.k.a (08/31/20 07:28 AM)
|
trubblesome
Stranger



Registered: 11/09/19
Posts: 406
Last seen: 10 months, 17 days
|
Re: Clone --> Isolate, senescence? [Re: A.k.a]
#26909241 - 08/31/20 07:43 AM (3 years, 4 months ago) |
|
|
right, without actual genetic analysis there's no way to tell by phenotypic expression alone - pic on the right has fewer mushrooms using the substrate for one
maybe I'm mistaken though and hyphal knots are less one on one missionary fucking than they are family orgies.
--------------------
|
3SIXTY5
Stranger

Registered: 03/27/20
Posts: 62
Last seen: 2 years, 3 months
|
Re: Clone --> Isolate, senescence? [Re: trubblesome]
#26909317 - 08/31/20 08:49 AM (3 years, 4 months ago) |
|
|
UPDATE
It definitely wasn't senescence after all lol, 3 of the 10 substrates I made are already 100% colonized, it's only been 6 days since spawning. 7 out of 10 were completely wrecked by a bacterial contamination, and I'm not sure why, at first I thought it was lack of GE, but I don't think it could be because 3 are completely finished.
Maybe I spawned them with the substrate being too warm still and that plus a lack of GE created an ideal environment for bacteria, idk, all I know is that 7 out of 10 didn't grow at all and 3 out of 10 are completely colonized in 6 days lol.
|
|