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Cubensis Breeding: A Group Effort


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#41 Needles

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Posted 30 January 2017 - 02:55 AM

In your pictures they do not look to be evading each other or even retreating. The hyphae have certainly passed the natural zone and i speculate that each will halt or overrun the other. Break out one of your other bad ass scopes and capture the clamp connection.
Yeah you will.....


I have one slide that should be binding tomorrow. I have it sitting on my phase contrast microscope. It is very cool the way the mycelium looks...
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#42 CatsAndBats

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Posted 30 January 2017 - 08:41 AM

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You can play the lotto too.

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Edited by catattack, 30 January 2017 - 08:41 AM.

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#43 Microbe

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Posted 30 January 2017 - 03:42 PM

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
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No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....
868a313f47de87ceb8e48d33e082269d.jpg

Not to chamge the course of this thread but why do dogs eat poop? I really do want take some leather to my dog when she does this but i would never abuse her unless she bit one of us.

#44 CatsAndBats

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Posted 30 January 2017 - 03:50 PM

found here: http://www.dogpoopdi...-dogs-eat-poop/looks like they're selling something so take it with a grain of salt, but I like their first answer

 

 

 

5 Reasons Why Dogs Eat Poop

 

why-do-dogs-eat-poop.png

Like humans, dogs each have their own motives for their individual behavior. Dogs eat poop for various reasons, based on their age, their training, their living conditions and their diet. There are basically 5 different classifications, or reasons that your dog may be eating poop.

 

Your Dog Was Born to Eat Poop!

Before dogs were domesticated they were scavengers, living off of whatever they could find. Dogs commonly fed on the waste of other animals (and other dogs) thousands of years ago. Poop eating may just be a remnant of dog history.

In certain situations, as with a newborn litter of puppies, eating poop is instinctual and completely normal. A mother with pups is wired to keep her den clean so as not to attract predators with scent cues. Thus, she quite often will clean up after her young by consuming their poop.

For households with multiple dogs there is often a pecking order of dominant and submissive roles. Submissive dogs will sometimes eat the stool of their dominant counterparts.

 

Dogs Are “A” Students

Dogs pick up things quickly and will often learn things that you don’t want them to. For instance, consider a dog that is punished for a housebreaking accident. If he is punished by having his nose rubbed in poop (which is absolutely not a good way to deal with the problem) he may try to “dispose of the evidence” the next time around.

If you clean up after your dog while he looks on, he may misunderstand your intent and try to copy your actions in some fashion by “picking up after himself”. Your dog might also see other dogs eating poop and learn the behavior from them.

For puppies, eating feces may simply be a learning experience. Puppies learn things by putting nearly everything that comes in front of them in their mouth. Most puppies will develop a distaste for poop in fairly short order. So, if your dog is a puppy, you can relax… chances are that they will change their behavior in due time. Just make sure you keep an eye on things and try to remove waste whenever possible so that your dog doesn’t develop bad habits.

 

Why You Should Take Your Poop Eating Dog to the Vet

If your dog eats poop, you should make sure it’s not because of a health issue. Some dogs will start eating poop when they aren’t absorbing enough nutrients, they have parasites, or they have issues with their pancreas. All coprophagic dogs should be examined by a veterinarian. Please read my other post on coprophagia and dog health.

Another, rather interesting phenomenon is when multiple dogs are in the same household and one gets sick, the healthy dog will sometimes eat the feces of the unhealthy dog. This may be an instinctual reaction to hide the weaker dog from “predators” much as a mother does with pups (see the section on instinct below).

 

Why Your Dog Sometimes Prefers Poop to Dog Food

A dog’s digestive system is dependent on a specific mix of enzymes to break down carbohydrates, proteins and fats. There is some evidence that suggests that dog digestive systems haven’t quite caught up to modern diets that include less animal protein and far more carbohydrates and plant proteins. Some veterinary nutritionists have suggested that dogs eat stool to replenish enzymes so that they are better prepared to digest their food.

There is also evidence that dogs that aren’t getting enough of certain nutrients will resort to eating poop. A lack of vitamin B is often said to be a cause of coprophagia.

Another common theory is that overfeeding a dog can lead to coprophagia. A dog that is overfed can’t absorb all of the nutrients in his food, and thus may try to “recycle” his nutrient rich waste.

 

Neglectful Parents

In many cases, a dog’s behavior can be linked directly to the owner’s behavior. Many dogs will eat stool simply for the attention that they get from their owner. Negative attention is still attention, and owners who scold their dogs for the behavior will quite often only reinforce it.

Dogs that are bored and lonely may play with and eat stool as a pastime. And, some dogs may resort to eating stool because they are not getting enough real food. If a dog’s living area is not kept clean, some dogs will resort to their own “housekeeping” efforts by eating stool.


Edited by catattack, 30 January 2017 - 03:53 PM.

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#45 Needles

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Posted 31 January 2017 - 01:34 PM

Ok back on track, I believe I located strands of hyphae from both strains of mushrooms connecting.

Pardon the shakie lines I used a stylus and pad. Anyway the green line is H. marmoreus and the blue is H. tessulatus and the red arrow points to the connection. It almost looks like a drop of glue, I believe that is metabolic fluid. There is another spot towards the top that looks like another connection on the same strand. When working with photography like this there is very little depth of field or sharp focus so I did the best I could on this image.
image.jpeg

I am adding a micrograph of the leading edge of hyphae growing out looking for nutrients.
image.jpeg

I have learned a lot from this experiment and am getting ready to do it again. I will come back with a stained sample that may be easier to identify what I believe is metabolic fluid being secreted from the mycelium.
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#46 OysterFarmer

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Posted 20 October 2017 - 03:06 PM

I remember reading that one method for obtaining monokaryotic mycelium involves serial dilution of spore water until there are so few spores that you will only get a handful of germination points on an agar plate.  That way you can keep the mycelium from a single spore separate from the others until you are ready to combine them.

The American Type Culture laboratory I believe sells cultures started from single spores.  Its only a guess but I'm thinking they probably use high powered microscopes and other high tech gadgets to accomplish this.

 

I haven't looked at their site in a while as I found they were out of my price range.


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#47 Microbe

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Posted 21 October 2017 - 12:17 AM

Well wtf? This thread is alive again.....

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#48 OysterFarmer

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Posted 24 October 2017 - 02:01 PM

 

 

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
Sent from my LGLS992 using Tapatalk

No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....
868a313f47de87ceb8e48d33e082269d.jpg

Not to chamge the course of this thread but why do dogs eat poop? I really do want take some leather to my dog when she does this but i would never abuse her unless she bit one of us.

 

Its usually B vitamin deficiency.  Does the dog eat rocks too?  Get some real dog food and not some knock off walmart shit food.  The first three ingredients in dog food should NOT be grain.  Meat, berries, peas.  Things like that.

 

Honestly though I think even walmart carries high end dog food now.  The cost is only a few bucks dif.  For all dogs do for us I say the few bucks is worth it.



#49 OysterFarmer

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Posted 24 October 2017 - 02:04 PM

In theory at least mushrooms aren't all that different from plants.  Plants spread their DNA by seeds.  Mushrooms spread their DNA via spores.  Only dif is you dont' see a lot of plants which produce a billion seeds.

 

Obviously agar is one way to predict your fruiting outcome but in theory at least if you keep taking prints from the best mushrooms in your grows with desirable traits then the progeny should eventually resemble what you are looking for.

 

I've bred plants which people said I could not possibly due for my cold environment.  It took me about five years.  How long would it take for developing my own mushroom strain?  I don't know if I've got that kind of time but it is doable.


Edited by OysterFarmer, 24 October 2017 - 02:05 PM.


#50 HrVanker

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Posted 24 October 2017 - 02:26 PM

Well wtf? This thread is alive again.....

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I was thinking the same thing... just saw the notification.

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#51 CatsAndBats

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Posted 24 October 2017 - 02:56 PM

Well wtf? This thread is alive again.....

Sent from my LGLS992 using Tapatalk

I was thinking the same thing... just saw the notification.

Sent from my SM-G935V using Tapatalk
Welcome home. Now that I have a microscope maybe I can figure out how to move individual spores now that I can see them. Suggestions? :biggrin:

Edited by CatsAndBats, 24 October 2017 - 02:57 PM.


#52 HrVanker

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Posted 24 October 2017 - 03:05 PM

Well wtf? This thread is alive again.....

Sent from my LGLS992 using Tapatalk

I was thinking the same thing... just saw the notification.

Sent from my SM-G935V using Tapatalk
Welcome home. Now that I have a microscope maybe I can figure out how to move individual spores now that I can see them. Suggestions?
Well, last we discussed, I believe we were looking into plate swiping, and volumetric dilution of spores. I haven't really thought about it for a while.

I do recall one video mentioning that you should add some astroglide to your syringes, as it keeps spores from sticking together.

Perhaps mixing a small bit of lube and spores well, then swiping a plate with the tiniest bit of the mixture on a wire?

That's just off the top of my head. I'll keep thinking, and do a little research later.

I'm glad to be back, I'll post a thread with my latest grow soon.

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#53 CatsAndBats

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Posted 24 October 2017 - 03:08 PM

Well wtf? This thread is alive again.....

Sent from my LGLS992 using Tapatalk

I was thinking the same thing... just saw the notification.

Sent from my SM-G935V using Tapatalk
Welcome home. Now that I have a microscope maybe I can figure out how to move individual spores now that I can see them. Suggestions?
Well, last we discussed, I believe we were looking into plate swiping, and volumetric dilution of spores. I haven't really thought about it for a while.

I do recall one video mentioning that you should add some astroglide to your syringes, as it keeps spores from sticking together.

Perhaps mixing a small bit of lube and spores well, then swiping a plate with the tiniest bit of the mixture on a wire?

That's just off the top of my head. I'll keep thinking, and do a little research later.

I'm glad to be back, I'll post a thread with my latest grow soon.

Sent from my SM-G935V using Tapatalk
Good to have you back Jack. Now get to work.

All this talk of lube has me all giddy!

Edited by CatsAndBats, 24 October 2017 - 03:09 PM.

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#54 HrVanker

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Posted 24 October 2017 - 10:48 PM

It would appear that we are on the right track when it comes to isolating spores.

I did some Googling, and while nobody mentioned any lube (they must live boring lives); the two most commonly discussed methods are: serial dilution, and streaking agar plates.

Also, mentioned on Research Gate was filtering the spore solution through MiraCloth or lens cloth to remove spore clumps, then doing a serial dilution. This could work!

Attached Files


Edited by HrVanker, 24 October 2017 - 10:55 PM.

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#55 Microbe

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Posted 25 October 2017 - 09:42 AM

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
Sent from my LGLS992 using Tapatalk

No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....
868a313f47de87ceb8e48d33e082269d.jpg

Not to chamge the course of this thread but why do dogs eat poop? I really do want take some leather to my dog when she does this but i would never abuse her unless she bit one of us.
Its usually B vitamin deficiency. Does the dog eat rocks too? Get some real dog food and not some knock off walmart shit food. The first three ingredients in dog food should NOT be grain. Meat, berries, peas. Things like that.

Honestly though I think even walmart carries high end dog food now. The cost is only a few bucks dif. For all dogs do for us I say the few bucks is worth it.
She gets good brand band, she doesn't eat rocks and have not seen her eat her poop in a long time..,now she eats Tootsie rolls out of the litter box!

Sent from my LGLS992 using Tapatalk

#56 CatsAndBats

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Posted 25 October 2017 - 10:05 AM

 

 

 

 

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
Sent from my LGLS992 using Tapatalk

No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....
868a313f47de87ceb8e48d33e082269d.jpg

Not to chamge the course of this thread but why do dogs eat poop? I really do want take some leather to my dog when she does this but i would never abuse her unless she bit one of us.
Its usually B vitamin deficiency. Does the dog eat rocks too? Get some real dog food and not some knock off walmart shit food. The first three ingredients in dog food should NOT be grain. Meat, berries, peas. Things like that.

Honestly though I think even walmart carries high end dog food now. The cost is only a few bucks dif. For all dogs do for us I say the few bucks is worth it.
She gets good brand band, she doesn't eat rocks and have not seen her eat her poop in a long time..,now she eats Tootsie rolls out of the litter box!

Sent from my LGLS992 using Tapatalk

 

 

 

https%3A%2F%2Fi.cdn.tbs.com%2Fassets%2Fi

 

Do you want to borrow my Adderall?

 

 

206px-Racemic_amphetamine_2.svg.png


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#57 Microbe

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Posted 25 October 2017 - 11:57 AM

Wow! From all that you received the the infamous "666" recognition! You have been around much longer then I so you know thats celebrated around here. Im surprised hyphenation hasnt showed up with his devil tongue gif! Damn i pissed rught now! I need to go punch my fucking dog in the snout right now and tell her to stop eating poop. I will be back.
Sent from my LGLS992 using Tapatalk

No need to kick the dog lol it's all good...... Funny how our pets like to eat poop lol I would watch our jack eat horse poo then I would tell her to go give mommy a kiss lol.....
868a313f47de87ceb8e48d33e082269d.jpg

Not to chamge the course of this thread but why do dogs eat poop? I really do want take some leather to my dog when she does this but i would never abuse her unless she bit one of us.
Its usually B vitamin deficiency. Does the dog eat rocks too? Get some real dog food and not some knock off walmart shit food. The first three ingredients in dog food should NOT be grain. Meat, berries, peas. Things like that.

Honestly though I think even walmart carries high end dog food now. The cost is only a few bucks dif. For all dogs do for us I say the few bucks is worth it.
She gets good brand band, she doesn't eat rocks and have not seen her eat her poop in a long time..,now she eats Tootsie rolls out of the litter box!

Sent from my LGLS992 using Tapatalk


https%3A%2F%2Fi.cdn.tbs.com%2Fassets%2Fi

Do you want to borrow my Adderall?


206px-Racemic_amphetamine_2.svg.png
Please!

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#58 Justintime

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Posted 27 October 2017 - 09:32 AM

Dog hates cat. Goes to cat box to eat its turds. Heh.

Electroporation. It's when a cell is influenced by a very short burst of electricity in an aqueous solution between two electrodes. This causes the cells hydrophobic outer lipid wall structure to momentarily turn in on itself creating an opening or pore hence electroporation, the opening becomes hydrophilic. Readily allowing water molecules carrying alien DNA into the cell. Scientists use this method to introduce new genetic material into cells. This happens in nano seconds. Prolonged electroporation becomes damaging to the cell. That is called irreversible electroporation and the cell is swamped with water and dies.
This effect should be the same with mushroom spores. I imagined rubbing mycelium between fine sheets of sandpaper then moving a small amount to water with spores of another mushroom. Into a very small vessel with two electrodes and introducing pulses of a strong direct current. In theory this should introduce DNA/genetics into the spore of another mushroom. Just a thought.

#59 Justintime

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Posted 27 October 2017 - 09:50 PM

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Journal ListMycobiologyv.43(1); 2015 MarPMC4397374
Logo of mycobiology
Mycobiology. 2015 Mar; 43(1): 1–8.
Published online 2015 Mar 31. doi: 10.5941/MYCO.2015.43.1.1
PMCID: PMC4397374
Current Technologies and Related Issues for Mushroom Transformation
Sinil Kim, Byeong-Suk Ha, and Hyeon-Su Rocorresponding author
Author information ► Article notes ► Copyright and License information ►
This article has been cited by other articles in PMC.
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Abstract
Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Keywords: Agrobacterium, Mushroom, Protoplast, REMI, Transformation
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MUSHROOM LIFE CYCLE AND PERSPECTIVES
Mushrooms are fruiting body-forming fungi normally belonging to Ascomycota or Basidiomycota. These fungi normally grow on plant materials by propagating vegetative mycelia through elongation of hyphae. Mycelia can be either dikaryotic (N + N) or monokaryotic (N). Monokaryotic mycelia are composed of mononuclear cells, which generally appear in most of life-cycle stages of Ascomycota as well as in mycelia generated from basidiospores of Basidiomycota. Dikaryotic myceliua are formed by hyphal fusion (anastomosis) of compatible monokaryotic mycelia. Basidiomycetes spend most of their vegetative lives as dikaryotic mycelia. Mushroom fruiting bodies are masses of dikaryotic mycelia with specialized structures, called basidia (2N), which make sexual spores such as basidiospores and ascospores as a means of sexual reproduction.

From a practical point of view, fruiting bodies are often consumed as food sources due to their unique flavors. Edible mushrooms, including Agaricus bisporus, Flammulina velutipes, Lentinula edodes, Pleurotus eryngii, and P. ostreatus, are readily available at any food market owing to their commercial cultivation. In addition to their nutritional consumption, mushrooms have been recently explored for new applications, including bioremediation, cell factories for foreign protein production [1, 2], value-added material sources [3, 4], and medicines [5, 6].

Mushrooms are ubiquitous organisms found in almost every ecosystem and play central roles in the recycling of organic matter. A considerable amount of literature has been published on the ecology, physiology, genetics, and biotechnology of mushrooms. Moreover, more than 100 genomes of Agaricomycotina alone have been sequenced and are openly available in a public database (http://genome.jgi.do...programs/fungi/). However, knowledge of the molecular cell biology of mushrooms has been restricted to only a few model mushrooms, such as A. bisporus and Coprinopsis cinereus, due to lack of molecular biological tools related to transformation. This review lists current obstacles that hinder the introduction of foreign genes into mushroom cells along with current solutions.

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MAJOR OBSTACLES TO MUSHROOM TRANSFORMATION
Introduction of foreign DNA into mushroom cells should be readily accessible at any stage of the life-cycle in order to better understand mushroom biology as well as generate mushrooms with new industrial applicability. However, transformation of mushrooms is very difficult due to the following reasons.

Connected cells with apical growth

Fungal cells are connected to form haphae, which are highly interconnected and form mycelia. For this reason, isolation of single mushroom cells is impossible. Growth (division) of filamentous fungi only occurs at the tip of mycelia, which means most cellular machineries for growth are concentrated at the apical tip of cells [7]. Moreover, cytoplasms of cells in mycelia are connected by septal pores (dolipores for Basidiomycota), through which molecules and even subcellular organelles can pass so that cells without selective markers survive [8, 9]. Use of germinating spores, finely broken mycelia with regenerated apical tips, or protoplasts can circumvent the issue of connected cells.

Thick cell wall

Fungal cells in vegetative mycelia have thick cell walls, mainly composed of chitin, β-1,3-glucan and β-1,6-glucan, and glycoproteins. Penicillium brevicompactum has a thickness of 149 nm [10, 11], whereas Gram-negative and Gram-positive cell walls have thicknesses of 5~10 nm and 20~80 nm, respectively [12]. Spores of fungi have even thicker walls of approximately 1 µm [13]. At least some cell walls have to be removed to introduce foreign DNA.

Heterokaryosis

In Basidiomycota, vegetative mycelial cells normally contain two different nuclei with compatible mating types [14, 15]. Therefore, if foreign DNA is not integrated into both nuclei at the same time, which is obviously very hard to achieve, then transformant carrying the selective marker will be diluted through the sporulation process during either sexual or asexual life-cycle when there is no selective pressure. Therefore, it is desirable to use monokaryotic cells for maintenance of the transformant.

Few molecular biological tools

There are very few available molecular biological tools for mushroom transformation. Unlike yeast, few natural nuclear plasmids have been discovered in filamentous fungi while plasmids found in filamentous fungi are mostly mitochondrial plasmids [16]. Synthetic plasmids with an autonomous replicative sequence (ARS), promoter, and selective marker from various origins have been developed for many fungi in Ascomycota and some in Basidiomycota [17, 18]. Moreover, vast numbers of hosts for Saccharomyces cerevisiae are available with sets of auxotrophic markers (e.g., leu2-3, 12, trp1-1, ura3-1, ade2-1, and his 3-11, 15 in W303-1A strain [19]) while few auxotrophs have been generated in mushrooms. C. cinereus is one of the most studied model basidiomycetes, and efforts have been made in order to generate mutant strains with selective markers, but few are available, including trp-2 [20, 21], met, his, and cystathionine [22].

Host defense mechanism

DNA double-strand breaks (DSBs) are detrimental and therefore must be repaired. DSBs in the eukaryotic genome are mainly repaired by homologous recombination (HR), single-strand annealing, microhomology-mediated end-joining, and non-homologous end-joining (NHEJ) [23, 24, 25]. These repair systems also play roles in the integration of foreign DNA into eukaryotic genomes mainly via HR and NHEJ. HR is the major player in yeast, and thus yeast transformation can be easily facilitated using homologous regions of the gene of interest [26]. On the contrary, filamentous fungi and higher eukaryotic organisms use NHEJ as their main repair system, such as KU70, KU80, and DNL4 in S. cerevisiae [27]. As a result, integration of exogenous DNA for the transformation of filamentous fungi is considered to be highly difficult since it requires DSBs, which are random and relatively rare [27]. Deletion of MUS-51 and MUS-52, which are homologous to KU70 and KU80, respectively, or MUS-53, which is homologous to DNL4 or Lig4 in humans, highly increases the integration of exogenous DNA through HR in Neurospora crassa [27, 28]. This is also true for basidiomycete fungus C. cinerea. Disruption of Cc.ku70 or Cc.lig4 has been shown to cause high-frequency gene targeting in oidia and mycelia of C. cinerea [29]. Therefore, disruption of NHEJ-related genes in a target host is apparently one of the prerequisite steps in mushroom transformation.

Maintenance of foreign DNA inside mushroom cells

No nuclear or cytoplasmic plasmid DNA has been discovered in mushrooms so far. Maintenance of a synthetic plasmid in mushroom cells is strictly dependent on selective marker. However, maintenance of constant selection pressure is hardly achievable since mushroom cultivation can last for several months in solid medium while producing potent degradative enzymes. Moreover, cell division only occurs at the apical tip of cells. The plasmid has to be multiplied at the tip of the cells and segregate into the dividing cell. Synthetic vector containing mushroom mitochondrial ARS or mycovirus with a target gene can be developed as mushroom vectors but need further development. Direct integration of linear DNA into the host genome appears to be the only way to maintain foreign gene integration for now.

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METHODS FOR MUSHROOM TRANSFORMATION
Mushroom transformation has been performed using various samples at different life cycles with various methods, including Agrobacterium tumefaciens-mediated transformation (ATMT), polyethylene glycol (PEG)-mediated protoplast transformation, restriction enzyme-mediated integration (REMI), electroporation, and ballistic bombardment, along with following questions and solutions:

Removal of thick mushroom cell wall

As described above, mushroom cells are surrounded by a thick cell wall with layers of chitin, β-glucan, and glycoproteins. Removal of the cell wall is a prerequisite for most of the transformation protocols. Several hydrolytic enzymes from a fungal pathogen, Trichoderma harzianum, are available as mixtures of chitinase, β-glucanase, and protease (Table 1) [30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45]. The enzyme reaction with mushroom tissue or mycelia should be conducted in the presence of a high concentration of osmotic stabilizers, including sorbitol [30, 32, 36, 45], mannitol [33, 34, 35, 38], sucrose [42], and MgSO4 [31, 37, 39], since protoplasts and spheroplasts are sensitive to osmotic pressure. The optimal concentration of osmotic stabilizers is around 0.6M.

Table 1
Table 1
Cell wall lysing enzymes and osmotic stabilizer
Permeabilization of mushroom cell membrane

The cell membrane is the primary barrier preventing introduction of foreign DNA into the cytoplasm. Permeabilization of the cell membrane has been performed by various physicochemical methods. In S. cerevisiae, treatment with PEG together with lithium acetate (LiAc) and single strand DNA (ssDNA) enables high efficiency transformation of yeast cells [47]. PEG is known to increase membrane permeability through interaction with membrane lipids [48]. LiAc and ssDNA also increase the permeability of nucleic acids, although the detailed mechanism is not fully understood [49, 50]. PEG treatment has been applied to the transformation of Pleurotus mushrooms using PEG3350 or PEG4000, as shown in Table 2 [38, 39, 41, 43, 44, 51, 52, 53, 54, 55, 56, 57, 58]. Electroporation is a method for local disturbance of the cell membrane by applying electric pulses with a certain electric potential (V) and capacitance (F). Variables in this method are the intensity (ranging 5~10 kV/cm) and duration (few msec) of the electric pulse. The intensity can vary depending on the applied electric potential (V) and the width of the cuvette (0.1 or 0.2 cm). Typical settings for mushroom transformation are summarized in Table 2. Ballistic bombardment is a transformation method using helium pressure to introduce DNA-coated gold particles into cells. This method has been generally applied for the transient expression of genes in plants, and it has been seldom applied for mushroom transformation [57, 58]. Agrobacterium tumefaciens can make holes in the membrane of any living organism. A. tumefaciens produces proteins related to host interaction and gene transfer from the virB operon in Ti-plasmid [59]. The virB2 gene encodes T-pilin protein for assembly of the T-pilus, with which the bacteria attach to hosts cell and function as a channel for T-DNA transfer. Some examples are described in below.

Table 2
Table 2
Techniques for the introduction of foreign DNA to mushroom protoplasts
Agrobacterium tumefaciens-mediated transformation

A. tumefaciens can infect any higher organism and transfers a DNA fragment (transfer DNA, T-DNA) from a tumor-inducing plasmid (Ti-plasmid) into the host genome via activity of vir gene product, which is contained in Ti-plasmid. Using this property, ATMT has been applied to the transformation of a variety of filamentous fungi, including Aspergillus, Fusarium, and Trichoderma [60-63]. Spores, mycelia, protoplasts, and tissues of fruiting bodies from basidiomycete fungi also have been subjected to ATMT (Table 3) [61, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81]. Successful ATMT relies on several factors. A. tumefaciens strains were shown to differentially affect the transformation efficiency of tomato and the copy numbers of genes integrated in the tomato genome [82]. Although the AGL-1 strain shows the best performance for tomato, the effect of the strain can vary depending on the organism and transformation conditions (Table 3). The cultivation conditions for co-culture of the bacterium together with the host fungi are also very important factors. The concentration of acetosyringone, a wound response molecule required for activation of the vir gene [83], is normally maintained at 0.2 mM for most of the mushroom transformation (Table 3). Duration and temperature for co-culture are normally 2~5 days and near 25℃, respectively, but can last up to a month at low temperature for slow-growing mushrooms [74, 75, 78, 80]. Various binary vectors containing T-DNA regions, which are composed of insert DNAs planked by a left board and right board, have been applied to mushroom transformation. The T-DNA regions mostly convey bicistronic genes, one for the selective marker and another for the reporter gene or gene of interest, whose expression is regulated by independent promoters (Fig. 1). The most frequent selective marker is a hygromycin resistant gene (hph), due to lack of auxotrophic mutant strains. C. cinereus is one of the few mushrooms which can use an auxotrophic marker [66, 67]. Expression of the reporter or selective marker gene is regulated by constitutive promoters, such as glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) and cauliflower mosaic virus 35S promoter (PCaMV35S or P35S), and terminators, such as TtrpC and T35S. More rigorous efforts should be made to generate mutant strains with robust selective markers and to identify controllable promoters.

Fig. 1
Fig. 1
T-DNA regions of selected binary vectors. The binary vectors and related citations are described in Table 3. LB and RB are the left board and right board of T-DNA region in Ti plasmid, respectively.
Table 3
Table 3
Selected mushroom transformation by ATMT
Integration of linear DNA by REMI

REMI is a transformation method for introducing linear DNA, which is cut by certain restriction enzyme(s), into the same restriction site in the host genome by including linear DNA together with the restriction enzyme in the transformation mixture [84]. REMI is a powerful tool to transform a variety of filamentous fungi [81, 85] and mushrooms, including C. congregatus [30], F. velutipes [31], P. ostreatus [40, 42], P. eryngii [37], Trametes versicolor [45], Ganoderma lucidum [32], and L. edodes [33, 34, 35, 36]. For example, we previously transformed a dikaryotic strain of P. eryngii with HindIII-digested pAN7-1-ECFP fragment using PEG4000-treated protoplasts in the presence of HindIII [37] (Fig. 2). Although the transformation was successful as shown by the expression of EGFP in the mycelia, we failed to detect EGFP protein in the mature fruiting bodies. The integrant was eventually diluted out during subculture. This may indicate that direct integration of foreign DNA into one of the nuclei in dikaryotic cells generates a fairly unstable transformant, suggesting that it is more desirable to use a monokaryotic strain in any mushroom transformation. Until now, REMI has been conducted on protoplasts of mushroom cells treated with PEG3350 or PEG4000 [30, 31, 32, 33, 34, 35, 36, 37, 40, 42]. Restriction enzymes employed for REMI are mostly six-cutter enzymes, such as EcoRI [30], HindIII [37], and BamHI [42].

Fig. 2
Fig. 2
Schematic description for restriction enzyme-mediated integration (REMI). The figure shows application of REMI to introduce hph-EGFP gene into the genomic DNA of Pleurotus eryngii [37]. EGFP expression was observed under fluorescence microscope.
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CONCLUSIONS
Introduction of a foreign gene into living cells and deletion of a target gene from genomic DNA are essential steps for better understanding cell biology at the molecular level. Nevertheless, mushroom transformation has not been freely accessible partly due to the dikaryotic nature of mushroom mycelia. More importantly, mushroom cells have their own limitations as good hosts for foreign DNA since HR is suppressed in filamentous fungi, whereas NHEJ is the major repair system against DSBs. With NHEJ, integration of foreign DNA has to be random, which makes it difficult to target a specific gene. Therefore, suppression of NHEJ by downregulation or knockout of NHEJ components is necessary and conceivably a prerequisite. Filamentous growth with connected cells is another obstacle since it prevents isolation of single cells from certain experimental treatments and thus causes difficulties in the selection of independent cells with a desirable phenotype. Use of germinating spores, protoplasts, and finely broken mycelia with regenerated apical tips may circumvent this problem. Generation of a variety of auxotrophic mutant strains is also needed to make mushrooms as an efficient host as yeast. In closing this review, more serious efforts should be concentrated on the host engineering for further advances in mushroom science.

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ACKNOWLEDGEMENTS
This research was supported by Golden Seed Project (Center for Horticultural Seed Development, No. 213003-04-1-WTH13), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS). HBS and SIK were supported by a scholarship from the BK 21 Plus Program, the Ministry of Education, Korea.

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Ultrastructural analysis of hyphal tip cell growth in fungi: Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution.
[J Cell Sci. 1981]
Cell-to-cell transport via motile tubules in growing hyphae of a fungus.
[J Cell Sci. 1993]
Ultrastructure, glutathione and low molecular weight proteins of Penicillium brevicompactum in response to cobalt.
[Pol J Microbiol. 2009]
Review Antifungal proteins.
[Appl Environ Microbiol. 2001]
Ultrastructure of a thermotolerant basidiomycete possibly suitable for production of food protein.
[Appl Microbiol. 1974]
Sexual selection in mushroom-forming basidiomycetes.
[Proc Biol Sci. 2011]
Review Mating-type genes for basidiomycete strain improvement in mushroom farming.
[Appl Microbiol Biotechnol. 2001]
Review Natural plasmids of filamentous fungi.
[Microbiol Rev. 1995]
An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency.
[Gene. 1991]
Plasmids for increased efficiency of vector construction and genetic engineering in filamentous fungi.
[Fungal Genet Biol. 2013]
One-step gene disruption in yeast.
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Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration.
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Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance.
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Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA.
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https://www.ncbi.nlm...les/PMC4397374/

#60 happy4nic8r

happy4nic8r

    cyans rule!!

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Posted 05 December 2017 - 07:27 AM

I find this subject fascinating, especially since I accidentally produced a cross breed of two different cubensis strains, redboy, and penis envy. I did this by using the pf Tek, 1/2 pint jars brf verm h20, and shooting them with the redboy. They sat in the cupboard for almost two months and not one produced any signs of white. Since none contaminated either I shot them again with the people, thinking that there might be some chance of them still being useful. This time they all jumped and within three weeks they were ready to birth. I was an absentee father by then, but I paid one last visit to the ex and put the cakes in an aquarium for her. They grew rapidly into the weirdest fruits I have ever seen. Black caps with dark violet streaking on the stems. I got a mutual friend to grab a few pics, and one cap which I printed. That cap grew 20 more, and those prints are right now being grown in 4 dozen jars to see if they retain their characteristic color and size. I am just getting a lab up, and am going to be using this method of experimenting with crossing these black beauties with my cyanescens, as was mentioned earlier. When I figure out a more scientific explanation for this, and I will entertain any thoughts as to what may have happened at the microscopic level. Updates to come.




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