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 Rotnpins 🤮 Rotten-Pins 🍄     Registered: 01/11/22 Posts: 4,738 Loc: in (front of) the hood Last seen: 3 years, 9 months |
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-------------------- Wire-Lock Challenge Grow Log Spore to Grain H2O Agar Plate Pins: Clone & Test at the same time Updated PF Tek H2O tub Signs of contamination Agar Transfers Surface Conditions
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labbar Stranger  Registered: 10/30/19 Posts: 331 Last seen: 5 months, 6 days |
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 the_chosen_one so called sage  Registered: 09/11/06 Posts: 5,823 Loc: 1984 
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I left off at "Gold Member" lol.. just marking a place. Great work!
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Muad.Dweeb Seer  Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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I have noticed that there isn't a lot of discussion about how to actually handle isolated monokaryons (and cultures generally). This is part of Basic Culture Hygiene.
Context I'll use some monokaryons I just isolated as the illustrative. Currently, I am on F4 of my gold PE type project, my selected clone named Gold Member. I have the clone running to generate clean spores that will be ideal for a MS cake grow; meanwhile, the initial prints have been scoped, streaked, pre-germinated in sterile water, and serial diluted for experiments and demos.  From the initial 4 streak plates (using the cross streak method), I have isolated and confirmed 3 monokaryons. These will be used for di-mon and mon-mon mating experiments/tests, and recreating some classic cross methods, but first they need to be preserved as pure culture (axenic) so I can reset to the original, earliest possible state of the culture if anything bad happens.  on T1 (2 weeks old):  on T2 (just a week old):   Microscopy To be able to view my germ plates in situ, I use plates that are fairly thin. Below is about as thick as I'll use for plates I'll need to scope later:  The thinner your plate is (and the more translucent the agar is), the better resolution you can get, and the higher the magnification you can use. Typically, agar as thick as this will only be able to use the 4x objective. That's only 40x with standard 10x ocular lenses. If you have or purchase 25x ocular or a 3 megapixel ocular cam, that can bump up to 100x total. If the agar is thin enough, you will be able to resolve the 10x objective, which will give you 100x/250x, respectively. To achieve this, we will be viewing the plate from the bottom:  Also, we can do what I call the LayZ Mount directly on the plate, after we have transferred from it, by pressing a cover slip directly onto the leading edge of the culture. This will allow us to use the 40x objective lens and even the 100x oil immersion objective, giving up to 1000x/2500x, respectively:  Inspecting As soon as you see small, translucent growth on the plate (<1mm), try to scope it from the bottom and find the leading edge. Along the leading edge, scroll to the very tips of hyphae and start following them inward, one by one. What we're looking for is a periodic little, dark bump on the hyphae. Even at 100x, this can be difficult to see, so we will double check after we've made our transfers at higher magnification.  These bumps are one of two things, typically- drops of liquid (metabolites or condensation) or clamp connections. The presence of clamp connections indicates a dikaryotic culture, and should be avoided if isolating monokaryons. If not using a scope, you will have to do testing to confirm it is a monokaryon later (discussed in The Buller Phenomenon). Transfer anything you see that looks promising to new agar and seal all the plates back up. Take the smallest possible transfers, trying to leave a small bit on the plate for later inspection. If this isn't feasible, we can use the T1 for confirmation when we take transfers from that. As the T1 grows, check again under the scope for clamps. Any definite clamp identified means you can toss the plate, it's not a monokaryon. Wait until T1 is about 2-2.5cm to take transfers. When you do, you can open the germ plate (if it continued to grow from any of the transfer sites), and scope both at a minimum 400x using the LayZ mount or any traditional slide prep method. Up close, we will easily be able to identify any clamps now and confirm or disqualify our monokaryons:   Clear clamp bumps formed over the septa walls of the hypha, ~1000x.  This could be either a clamp in formation or a "peg," aka, the start of a new hyphal branch. It is pointing toward the apical tip of the hypha, though, so I'm pretty sure it's a peg. Clamps form from the new cell back to the old cell, tightly hugging the septa wall. Without a scope, one of the first indications it is a monokaryon is unusual mycelium. Monokaryons tend to have distinct, individually unique growth patterns. Often wispy, flat, thin, or asymmetical growths, some grow in a distinct shape, or with a specific fuzz matting, while some look like dense tomentose dikaryotic cultures. I've never seen ropey rhizo in a monokaryon, and they tend to grow slower than dikaryotic mycelium, but some can be on par. Most often, they look like a contaminant mold, which was both terrifying and hilarious to discover. I have a Golden Halo monokaryon that always grows in a starburst pattern. However, I also have Koh Samui squat monos that are thick tomentose fluff and look more like a fruiting culture than its parent, so this tell can be a mislead. Culture Hygiene From the point that a monokaryon is isolated, confirmed and clean, make two slants of each- the first slant is a master slant, the second is a "working" slant. This serves as a redundancy and a "restart" kit, to maximize the genetic preservation of the culture. Allow them to colonize just a few millimeters at room temp and refrigerate them around 40F (~4.5C), ideally in separate refrigerators (in case one fails or freezes). Make any "working" plates for short term use at the same time, if you only have one plate. The working plates can be expanded and used until any aberrant behavior or contamination occurs. When a reset is needed for any reason, the slants can be retrieved, allowed to 'wake up' at room temp and used to make more working plates from the Working slant, and making two new slants from the Master slant. Which is Master and Working doesn't matter so much, it is simply a practice of separating the two to minimize contam vectors. This method of Master/Working storage should be practiced with all cultures that you wish to preserve. I use slants, but whatever your long term storage method is (sterile water ampules, for example), this practice accomplishes higher vector control, and maintains your culture at its youngest genetic state, optimizing the potential time it can be preserved. Bonus pic of spores germinating:  -------------------- 
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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Yoooo! Falbino was one of my first studies on breeding! --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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the_chosen_one so called sage Registered: 09/11/06 Posts: 5,823 Loc: 1984
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Absolutely stunning!!!
 Yeah, that was quite the experience lol. Weren't many of us even attempting crossing strains and what not back then.. RR, Shedthemonkey, Workman and myself are all that come to mind right off the bat. Sure is fun watching the Big Guy and several others like yourself carrying on the work.. and play lol. I think we're in good hands with the next generation. 
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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This is an excellent study in inheritance, published in the Brazilian Journal of Microbiology called, “MORPHOLOGY AND MYCELIAL GROWTH RATE OF Pleurotus spp. STRAINS FROM THE MEXICAN MIXTEC REGION.”
Recovery of neohaplonts is something I'm working on. I plan to do a write up after much longer, but this paper goes over it in adequate enough detail that a clever cultivator can easily extrapolate and adapt the process. However, this applies equally to selection when working with monokaryons from spore.
They cloned, cleaned up the two clones until axenic, pure culture was obtained, then used a dedikaryotizatation (dedi) procedure to recover the monokaryotic components.
Here, we have some general characterization of mycelial traits for the recovered monokaryotic strains, which correlate to the parental strains. When pairing monokaryons, these sorts of traits are good to keep track of, because they will grow your understanding of what “traits” are actually multiple genes that inherit separately, what traits are dominant vs. recessive vs. blended, and grant you the ability to make informed, controlled pairings. Utilizing a standardized agar ensures that they will have the same conditions, and that consistency is important to mitigate environmental influence as a variable.
A simple and effective means to keep track of growth rate is to draw an incremental X, Y graph on the bottom of the plate with sharpie, using the epicenter as 0, 0. Use a straight edge like a ruler, and make periodic marks along the X and Y at regular intervals (5mm for example).
This is interesting. We are looking at the individual monokaryotic components of two wild pink oyster strains, one white, the other a light pink. From the nuclear types they present, we can infer that off-white, sparse, low density mycelium is somehow linked to the floccose mycelium. We can also infer that pale pink mycelium is dominant to white, by the fact that the LB-051 specimen was pale pink and yielded white and pink neohaplonts.
This is a controversial debate- does colonization speed affect fruit production? It has been found that in some species it does correlate, albeit variable between species and even strains within a species. What can be positively said for cubes regarding this is that fast colonization, independent of fruiting ability, is still a desirable trait, since clean, colonized spawn/media is our best defense against competitors.
There is some indication here that floccose (and therefore, off-white/low density/sparse) mycelium could be damaged or variant neohaplonts (see Table 2/3). Regardless, it does appear to be heritable.
This is an odd conclusion to me. If we are assuming the off-white neohaplonts always produce off-white dikaryons, regardless of the other type paired, that should indicate it as dominant over pink and white. This is another reason I have inferred that the F-low, off-white, and sparse characteristics are damaged nuclear types. This is the sort of discernment that needs to be cultivated when taking on a breeding or isolation project. --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques Edited by Muad.Dweeb (08/12/22 11:43 AM)
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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Some will probably argue this post belongs in Advanced Mushroom Cultivation. I don't wholly disagree, but for mutational options, this is honestly the safest and easiest route. I'm including it here because excitable noobs that will try things like this anyway need to know how to do it safely. Trust me kids, lung scarring is not fun to live with.
 *THIS TEK IS POTENTIALLY HAZARDOUS!* Do not attempt without proper protection. Ultraviolet exposure can burn skin, cornea, retina, and cause cancer. Additionally, proper UVC lights produce ozone, which can irritate and scar lung tissue and eyes. Do not remain in the same room while this device is running, unless proper fume extraction is in place and operational. Allow for 45-60 minutes after the UV cycle for the ozone to destabilize into oxygen. Failure to follow these precautions can result in injury, blindness, difficulty breathing, loss of smell, or eye irritation. Anyone using the described device and procedure agrees to follow all precautions, and does so at their own risk. The author cannot be held liable for irresponsible use. * An opaque, hinged box with a latching lid (in this design, I used a 12”x5”x4.5” security box) * An in-line power outlet timer with range between 5-60 minutes * A 185nm – 280nm ultraviolet (UVC) light/ballast that can mount to the underside of the box lid (in this design, a 9.25” length tube lamp) * A “stepping” drill bit and drill * A metal file or mini-hacksaw * Silicone sealant * (Optional) Wire mesh, like “hardware cloth”, to keep media off the bottom of the box  1. On the underside of the lid, align the lamp ballast to the center of the lid and mark the area of placement. Take note of where the power source will exit on the hinged side and mark that spot on the lid as well as the box proper. Set the ballast aside. 2. To determine the size of the hole needed, use the end of the cord that plugs into the ballast as a reference against the stepping bit and mark that step with a permanent marker. (The marker can be erased later with some isopropyl alcohol.) 3. Drill out a hole where the lid and box are marked. Make sure that the ballast plug can slip through easily. 4. For the box proper, mark a line from either side of the hole straight up to the edge of the box. Use the metal file or mini-hacksaw to cut along those lines. The finished inner hole should look like a big “U”. 5. Cut a section of the wire mesh the length of the box with shears or tin snips. The width of the mesh should be a couple inches wider than the box. Bend the sides evenly so they can function as legs to hold the mesh up off the bottom of the box. Ensure there will still be enough space to place spores and culture samples.  6. Connect the power cord to the ballast. From the outside with the lid open, fill the through-hole with silicone sealant, keeping the power cord relatively centered. Allow to fully cure for 24 hours before moving. 7. Add a bead of silicone sealant to the underside of the lid, to help form a seal when the lid is closed. Allow to cure 24 hours before using. 8. I recommend gluing the power outlet timer to the ballast power cord after inserting the prongs. This will ensure that the safety mechanism is always connected to the light box.  9. Put Safety Hazard Labeling on the box.  You can skip fabrication and just buy a "UV sterilizer box" like this:  I don't like these, personally. For one, the drawer is translucent. While the plastic will block around 95% of the UVC, it won't block all of it. There is also a black grid between the light and the drawer, blocking a good deal of light transmission. It also just doesn't feel very sturdy. To find out how long you should use the UVO box, you will need to calibrate it with several exposures of varying times, for both prints and cultures. What we are trying to find here is at least the approximate LD50, or the dose of UVC/ozone exposure that is lethal to 50% of the sample. An hour of exposure will probably be beyond any use, so we can test in the shorter 5 minute increments. Each iteration should be performed separately, so that only continuous exposure is applied. Calibration should be used each time a new culture source is used, as tolerance to UV won't be universal, nor will the efficacy of various UVC wavelengths. In practice, the actual LD50 may not be what is used. It will be up to your discretion what level of viability is desirable. 1. Sanitize the inside of the box with a spray bottle of isopropyl alcohol. 2. Place spore/culture sample(s) into the box with nothing to block the UV. 3. Close and latch the box. 4. Plug the timer into a power outlet and set to 5 minutes. 5. Exit the experimental area for the time of the experiment, plus 45-60 minutes. 6. Remove the sample and label it with exposure time. Incubate at room temperature. 7. Repeat for additional exposures with the same culture. 8. Transfer samples to fresh agar, label with exposure times, and observe growth. Once you have the acceptable limits for the culture, continue with cultivation as normal and record the optimum exposure time in a notebook for future reference. Many of the resulting cultures may exhibit undesirable mutations mixed with desirable mutations. The desirable mutations should be isolated by breeding the spores from the mutant culture with a lineage that does not have the deleterious traits the mutant has, and selected for in a standard breeding program. Glass petri dishes with the lid on should be avoided, unless they are fused silica quartz, as standard glass will inhibit most UVB and UVC light, but fused quartz will allow it to pass relatively freely. These are generally very expensive compared to most disposable, or even glass, petri dishes. It is far less expensive to expose the culture/spores without a petri lid obstructing the UV transmission, and to wear gloves (and perhaps tyvek sleeves) sanitized with isopropyl while placing and removing the plates from the box. The hazards cannot be stressed enough for working with UVC and ozone. After starting the exposure, remove yourself from the area. Aside from the hazards, the smell of ozone is rather unpleasant. I strongly recommend setting up in your clean work area (hood, or SAB), then plugging it in to run with the lid closed in an area away from any other cultures (the garage, etc). The chance that ozone will seep out and harm or mutate an unintended culture is only slight, but it is present. It may be safer, albeit less reliable and time consuming, to try and recreate the Psylocybe Fanaticus UV Accident. His hypothesis that using UV black lights cause strange mutations to his PF variety (resulting in PF Redspore and PF Albino) was met with much skepticism. Granted, the scientific disposition should be skepticism until evidence is furnished, I feel there is more than enough research and evidence already available to substantiate this. Also, I know a little secret. The "True Albino Teacher" variety was actually discovered while Jik was using UV A/B reptile lights when TAT popped up. Interesting, huh? This one is super simple, so I'm not writing it up like the UVO Box. All you need are some 10 watt UV reptile CFL lights. You'll find them next to the coir at the pet store, conveniently. Use these lights on a 12/12 or longer timer when you fruit and do your best to shield the light so it's only hitting your fruit. Looking at UV lights is like looking at the sun, don't do it. In plastic tubs, the UVA and UVB should have enough penetration to reach the fruit and mycelium. Position the light within maybe 6 inches of the lid. I would recommend that this be done only with the goal of mutating spores. There are genes involved in "DNA Repair" that regularly monitor the nuclei of cells and either correct damage and mutations, or induce apoptosis (cell suicide). In this set up, all the UV exposure is coming from above, so the caps will eventually be most or all of what is exposed to UV. There is a much higher chance that cells in the cap, particularly the basidia, will pass on uncorrected, mutated genes to the spores. Good Luck! Have Fun! Be Safe! references: UV-induced mutagenesis in Volvariella volvacea to improve mushroom yield The mechanisms of UV mutagenesis - PubMed How UV Light Damages DNA and the Havoc it Can Cause to Your Experiments --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques Edited by Muad.Dweeb (08/16/22 09:46 AM)
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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For those wondering, yes, I have done this (clearly, I have constructed the box). [Note: the entire box (not including the step bit if you have to buy one) was under 60 USD. Not very pricey at all.]
While I haven't done a whole lot of experimentation with this, my best results so far have been with spores exposed for 30 minutes. Here is one example: "Stetson" is an early cross experiment I tried before I really had a grasp of selection practices. I had a party hat pheno of the Koh Samui (Tall Type), which clusters massively even from MS. I wanted to try and improve the yields of APE by infusing these KS genes, hopefully carrying over the party hat. I was only successful at making a short KS (Tall Type) with a fuzzy hat tendency.  Cute, but missing the mark by a few whole targets. It is very predictable in the size of fruit, the shape, the color, the degree of party hats from spore, and decent clustering. So I used Stetson spores as my first test. Below is one of the mutants:   I think that counts as a significant deviation. Currently, I have some UV exposed PF Red Spore, which is a much more established type with less variability overall. I'll be doing more replications with this as time goes on, but I'm trying not to fall into that trap of taking on too much and going through burnout. I'm also trying to replicate the paddy straw mushroom paper above with mycelium (as they did), but the exact time seems to be more delicate. The germination rates I get with 30 min and spore are high enough that I could probably push it another 15 minutes and still have plenty of viable spore. --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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Also, when you hit an effective UV exposure time, things can get... weird.
You may see blobs, mass aborts, non-pinning, sterility, all sorts of undesired mutations. Be Prepared. --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Solipsis m̶a̶d̶ disappointed scientist  Registered: 12/28/09 Posts: 3,398 Loc: the Neitherlands 
Last seen: 3 years, 1 month |
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Hey there, i think i will probably feel right at home here! xD am not on Shroomery all that much but i will try if i can participate, i do work on many related things and I also got a friend who actually finished a master's related to breeding (knows about both plants and also mushrooms which he is really into). Maybe i could consult him on occasion but it seems like a pretty closed field, like where are all the books and textbooks at for breeding? lmao
I havent read the whole thread yet but no it doesnt seem that di-di mating is a thing except for the parasexual cycle but that doesn't really apply to the basidiomycetes we are growing here. Also i saw mention of snake venom and i just wanted to say I think that is nonsense, like there arent really proper examples of where somebody really demonstrated with our basidios afaik esp not in a decent peer reviewed way, and it also doesn't make a whole lot of sense when it comes to the technical details. That said, other methods do involve the weakening (or beyond) of cell walls of course, to mess with genetic information transfer. Methods of (easy) dedikaryotization are i think very intriguing and I know ppl are working on this, ways to do it with like chemicals tho arent really yet available information. Just mechanical that i know of. I have experience with Psilocybe (caerulescens and cubensis) monokaryons popping off oidia as well, that would also be pretty dope if that could be exploited somehow. But ofc that still requires the initial monokaryon which we would preferably like to get around. Some decently well known shroom breeder who i have seriously dislike and disdain for (dont really wanna get into our ridiculous encounters) also apparently sells like a kit for "transformation" without really knowing the first thing about these types of things. But it seems to claim that you can indeed just fuse 2 dikaryons with it. All beyond vague. So yea stay skeptical especially when it is so hard getting verifiable details or experiments on something. Idk if it is possible to basically use certain parts of the protoplasting protocol and just do a quick and dirty form of it where you don't bother to fully induce and fuse them but you try to do it in more of a one-pot synthesis. Also in this respect/context it doesnt seem the same thing at all to attempt if you want to hybridize 2 species that normally really don't accept this to happen vs if you want to fuse 2 dikaryons of the same species. I hope that the minute details become known about like chemically dedikaryotizing, that it might also be tried on a mix / co-culture of 2 dikaryons to see if you can do this instantly, maybe in an LC of 2 unrelated strains, idk. Taking what is already pioneering stuff and trying to hack it lol. -------------------- My trade lists: 
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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Not many I've run into. "Genetics and Breeding of Edible Mushrooms" edited by "A.C. Chang" (around 50 USD for the digital DL, there are unlocked PDF's floating around) I've been trying to find a copy or PDF of John Raper's "Genetics and Sexuality of Higher Fungi" which has proven to be fairly elusive.
Rattle snakes and some other snakes have venom that is a hemolytic protease. It functions by attacking proteins in the cell wall. Chitinase is the most referenced lysing agent for fungal cells, but chitin and chitinase both are diverse related groups and don't have universal efficacy. Trichodermas produce a chitinase particularly effective at attacking the chitin of fungal cell walls, but it's actually not the primary lysing enzyme they produce, glucanases, which is actually ideal because the primary constituent of most fungal cell walls is alpha and beta glucans. The primary issue with proteases, chitinases and/or glucanases is that getting quality material is either very costly, very invasive vetting, or both, making it prohibitive. There are some simple alternatives for someone who doesn't mind a little work though.
I'm going to be working on an idea I had using DMSO to carry molecules into the cell wall. I'm also playing with a lysing cocktail extracted from trichoderma sp. 
Chemical means have a good deal of published information. Dick and Raper have papers on using cholates, in particular, sodium taurocholate (from ox bile). Similar uses of Oxoid (Peptone P) have been published (the exact papers escape me atm). Many "mechanical" methods also use a "chemical" component (as dedikaryotization broth), like Oxoid. One exception is a patent I found where a vortexer was used at something like 240rpm over a two week period. Most of the techniques result in recovery of only one nuclear type (asymmetrical). Most of the procedures capable of symmetrical recovery involve mincing the culture in a blender. I haven't encountered any theory regarding why it works, and I'm still working on it myself (it's a very sensitive operation with variables per species and even strain). However, I think the main mode of action is in the mincing- with one networked culture, you can do one iteration or replicate, but mincing the culture makes the iterations per experiment astronomical. Then, in the presence of a medium that encourages monokaryotic growth, there is a much higher chance of generating the monokaryotic components. There is actually a good deal of debate and, ultimately, still no conclusive answer as to why most dedikaryotization procedures have a nuclear preference, but I believe it has something to do with particular tolerance of one nuclei over another to withstand the procedure.
Lol, I know Doma/MMF and his snake venom "fusion" kit. I introduced the larger community on FB to inductors, and within a few weeks he was making MycoCoil (TM)  I surely wasn't the first person to do that, but I told everyone how to make them. I'm also pretty versed in all the Enigma/Brainiac lore and drama. C'est la vie. The short answer is "no"- there isn't such a thing as "di-di" mating. There are events that can create conditions that look like it though. Some strains produce random monokaryotic filaments that eventually populate with the complementary nucleus. This is noticeable as a "halo" that is decidedly not bacterial (though, bacterial halo is definitely a thing). Certain environmental and chemical exposures can increase the occurrence. The result is what looks like two dikaryotic cultures mating.
Actual protoplast preparation has a lot of applications. Forced mating, passing hard to absorb molecules into cells (i.e. mutagens), using Cas9 to deliver plasmids. All procedures require a lysing agent to break open the cell wall, such as I mentioned earlier. The rest is in making a properly isotonic medium so you don't crush or burst the cell membrane and filtering the hyphal material from liberated protoplasts. Using the lysing agents themselves is the portion you'd want to focus on for a "partial" protoplast prep, i.e. making venom or trich extract agar. All of them are proteins though, and will denature with too much heat (usually over 50C), so using a 0.22 micron syringe filter is a must to make clean serum.
Well, that is the idea. And there isn't any reason you can't use lysing enzymes to force two cultures to mix, it's just how you go about it that makes all the difference. Once I have some solid data to share, I have a method that makes a lysing enzyme unnecessary if you just want the cross and don't want the monokaryotic components. I'm close to sharing my initial findings (waiting on F2 gen), though. --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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I'm currently trying to adapt this procedure for protoplast prep. It seems within the reach of a typical home hobby lab.
  [https://www.ncbi.nlm.nih.gov/pm Here are some osmotic stabilizers that have been tested. However, 0.6M Mannitol and 1M Anyhydrous MgSO4 (baked epsom salt) seem to be the most commonly used for an isotonic solution.  [https://www.researchgate.net/fi Also, for filtration, somewhere between 20 and 40 micron membrane filter (syringe filter) should be able to catch hyphae while allowing liberated protoplasts to pass. For any non-autoclaveable media, a 0.22 micron filter is needed. --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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Update on Pseudocarps (blobs)
In my early obsession with Enigma and PE blobs, I came across this paper, "Polymorphism in Psilocybe Merdaria" [since reclassed as Deconica merdaria]. I was already using the term "pseudocarp" to refer to fruit bodies deformed due to genetic aberration (as opposed to environmental causes). However, the term "carpophoroids" has been in use since at least 1962, so that technical term is available if anyone prefers that. The original use was specifically in reference to "Gasteromycetoid" (or "Gasteroid") fruit bodies (puffball-like). Since this paper, many other examples have been studied, and have had some light shed on them. In all cases, I believe the basic common thread is misuse of fruit body orchestration genes. The most probable causes range from viral infection, mycoparasitism, transcription factor mutations, to tenuously compatible mating types. We can rule out infection from Entoloma abortivum pretty confidently as the cause in P. cubensis. Other known mycoparasites, I feel, would also present complications that would a) present in much less organized form, and b) pass between cultures readily. Not to mention, parasites hijack or use their own reproductive structures, so such modifications would be relatively easy to find under a microscope. Fruits like Enigma and PE blobs don't have such modifications. Viruses can leave lasting effects after an infecting a host, such that effects can even be passed on to offspring and become part of the genome. Without first locating where the genetic loci are that cause pseudocarps, determining if they have viral markers isn't really possible. While future probing may indicate that such traits originate from viral-induced mutations, it's sort of a moot point at the moment. The fact that Enigma came from a cross of PE and B+ - two very unrelated cubes - suggests to me that it isn't really an issue of marginal compatibility, verging on being incompatible. With some of the pseudocarp cultures that have resulted from a single, mostly homozygous variety (Pneuma from B+; Omni from Normak, a Melmak Revert), I could entertain the notion that mating type alleles are close to self-infertility. But it is occurring spontaneously in crosses between unrelated cubes, so that possibility is a much harder sell in my mind. That makes the most likely possibility I can imagine mutations to transcription factors. In simple terms, there are sections of DNA (blueprints) that are instructions for how to use the DNA (a legend). If the legend for the blueprints is inaccurate (mutated), the final building dimensions are going to be much different. In mushrooms, there are a lot of genes used in the formation of fruit bodies. If the cells forming have irregular building instructions, the result may be Fibonacci spirals, or crests of fans, or puffy round blobs, or whatever the instructions tell the mycelium to build. If this is the case, finding specimens with aberrant fruiting traits like my Golog, or like B+ style "hats", should produce a higher frequency of pseudocarp or semi-pseudocarp cultures from self or cross breeding. So, that's what I've been doing. My first investigation was into early generation Tidalwave spores. While some pseudocarps were found, they were only little capless fingers that rarely grew beyond 1 cm tall. Nothing cloned ever did anything more impressive, just fields of tiny fingers. Next, I decided to investigate B+ hats and PE blobs. If tidalwave produced Enigma, but searching early gen spores only produced the fingers, maybe backing up to the parents could illuminate something. What I found in B+, just investigating the heritability of hats, is that it was a heritable trait. Not only that, it is a highly variable heritable trait that can be selected for numerous sub-traits, like density, length, indifference to humidity, etc... While sharing my findings with some friends, one of them found a full pseudocarp in some B+ another friend sent him. Named "Pneuma" after the Tool song, it immediately occurred to me that this was a pivotal piece of data. Not only was it another pseudocarp, it was completely different from Enigma. Enigma will occasionally have a "broccoli" floret style, but is most known for the layered finning and fanning growths, reminiscent of desert rose selenite. Pneuma has an almost exclusive broccoli type growth, in multiple conditions and substrates. Prior, the prevailing assumption was that PE was the culprit for the weirdo genes in Enigma. PE already had blobs. Golog:   Shortly after that, I had found my "Golog" clone in some Melmak (Homestead PE) swabs and decided to try some inheritance tests by crossing them with another clone, by mixing spores. "Stetson," a clone from previous B+ hat experiments, who is a very short guy with very consistent fuzzy pom style hat. The cross I dubbed TBP, or Trailer Park Boys, and a lot of the clones have names that are references to the show (a great mnemonic device). At the same time, roughly, I started a cross between Golog and Golden Halo spores (GOGH), with the goal of a Penis type with gold spores that discharge. Many of the names are references to Van Gogh, or other impressionist art (it's a REALLY good mnemonic device, try it). In the F1 For TPB, I did not find any true pseudocarps. However, I did find one in GOGH. I called him Starry Night:    I just about shit myself when I found this. A year and a half of hoping and searching, and I found a novel pseudocarp. Then I lost him in a major move about six months later :shadenfreude: But I was already on the trail of more. I had several lines from GOGH.Calico that showed a propensity to have transient pseudocarps, suggesting the genes were there but in a heterozygous state that didn't consistently express. Meanwhile, in the TPB line, I was finding semi-pseudocarp clones that consistently produce sporeworks's PE style blobs, but they were fanning florets like Enigma. Meet Sexy Julian:  Since the new year, I have isolated nine possible pseudocarps total from both lines for testing without competition. Two have resulted in Albino (or Leucistic Sterile), these were not pursued further. So far, two have been confirmed as pseudocarps, one from each line. Earlier this year, I isolated GOGH.Calico.F2.CL2, "Cypress":  I currently have several pseudocarps from TPB.SexyJulian on the verge of fruiting. One of them is already well underway. This novel pseudocarp is a gasteromycetoid carpophoroid, that grows balls under balls under balls. The culture's name is Atodaso.    Because, I didn't want to be the one to say atodaso, but fuckin' atodaso!  There will be more pseudocarps to come, and I'll take updated pics of Atodaso as it develops and on larger substrates later (along with the rest when I have a good handful). --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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Jacubey Wanna-be-cultivar  Registered: 08/19/20 Posts: 338 Last seen: 7 months, 19 days |
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I'm assuming this is in reference to epigenetics? Iirc the legend (the epigenome) can change over time within an organism and that's what causes mutations like tumors or strange hair growth in humans. Is there a similar thing in mushrooms? I know you're more talking about the legend in general, and not the legend changing through the lifecycle but I'm pretty sure this still falls under epigenetics Awesome work by the way. Are you going to be publishing your research in any official capacity, or just here? Also are you going to try to make a sporulating pseudocarp? --------------------   
Edited by Jacubey (09/03/22 12:42 PM)
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milkboy Village idiot  Registered: 05/01/21 Posts: 2,733
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Thanks for the link, good read. Learned a new word, "invaginate"
-------------------- Gummies by milkboy Milkboys Public Display of Affection I'm a Semp      
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smalltalk_canceled Babnik  Registered: 07/13/20 Posts: 3,297 Last seen: 6 months, 5 days |
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This thread is the best posted in 2022 prove me wrong
-------------------- Willpower is the one true virtue     
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fahtster Now With 33%More Faht  Registered: 06/17/06 Posts: 10,884
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This thread > Chuck Norris
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HILLBILLY OUTLAW Above And Beyond!  Registered: 04/21/22 Posts: 4,344 Loc: Luckenbach Texas
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--------------------  
🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 TEAM SPREAD THE LOVE! Smellyhobbit said: Embarrassment and bashfulness are leeches on your ability to learn.
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Muad.Dweeb Seer Registered: 04/13/17 Posts: 148 Last seen: 3 years, 1 month |
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I've received a few questions in private and elsewhere regarding the Atodaso cakes.
The yellowish chunks are agar inoculum. I have tried basically every method of inoculating these cakes, and chunked or even whole disk agar (think "autocasing") works surprisingly well. Nothing beats the responsiveness and speed of quality LC. Under that in descending order: using LI, using clean grain spawn, using chunked or whole plate agar, using MSS with obvious signs of germination in situ, applying spores direct from a swab or print. They all have their foibles. Agar is extremely convenient for removing a couple refinement/prep steps and still keeping a reasonable speed of colonization. Sometimes during sterilization, excess moisture between the liner and the cup causes the liner to stick and pull away from the cake. I'm working on resolving this issue, it happens often enough that liners are only 70% effective, generously. ETA: Holy fuck guys, you humble me! I have some amazing shit coming, major breakthroughs in some more advanced techniques that really aren't that hard. Like, literally a noob fucking around could accidentally this shit. But it's been very consistent so far, a couple more tests and a choice F2 specimen and I'll be ready to present it.      --------------------
How to Breed like the Bene Gesserit The Weirding Way - Advanced Bene Gesserit Techniques
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