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BlimeyGrimey
Collector of Spores
Registered: 08/24/05
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Loc: Puget Sound
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Fusion of Grifola umbelleta and Ganoderma lucidum!
#8303558 - 04/19/08 05:31 AM (15 years, 10 months ago) |
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Source : http://www.patentstorm.us/patents/5455171-description.html
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a new microorganism useful for preparing medicines.
2. Description of Related Art
Recently, fusion of cells, including those of basidiomycetes, has been accomplished.
However, the frequency of reproducibility is low, and is no more than 10-5 to 10-7 (i.e., is almost impossible).
Furthermore, nuclear combination in cell fusions between different species and genera is also low.
SUMMARY OF THE PRESENT INVENTION
Nevertheless, via cell fusion of basidiomycetes, the present applicants have obtained a cell-body exhibiting special biochemical properties in a hetero-fungus by fusion of two species producing medicinal components.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the shape of a colony on an agar culture medium separated and germinated from the nucleus of a Grifola fungus under the microscope.
FIG. 2 shows the shape of a Ganoderma hypha under the microscope.
FIG. 3 shows the shape of a fusion fungus of the present invention under the microscope, grown on an onion, soy sauce and sesame oil agar culture medium.
FIG. 4 shows the shape observed for the early stage of growth of the fusion fungus under the microscope on a YM-12 flat agar culture medium.
FIG. 5 shows the shape observed during the adolescent growth stage of the fusion fungus under the microscope.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a new microorganism obtained by fusing cells of fungi belonging to the genera Ganoderma and Grifola.
The new microorganism of the present invention, and the mycological properties of the Ganoderma and Grifola species utilized to form said microorganism of the present invention, are described in detail as follows:
MYCOLOGICAL PROPERTIES OF THE GANODERMA
The instant Ganoderma is Ganoderma lucidum.
1) Morphology
This is a mushroom that has a pileus and a stem that is lustrous, as though varnished with lacquer (when steamed). The pileus is a kidney type. Its surface is covered with a shell. Its color is a reddish brown and/or a black violet. Its pulp is corky, and consists of two layers. The upper layer is white. The portion near the spores is light cinnamon color, and the layer length of the tube is 5-10 mm. The tube hole is round, and there are 3 to 4 1 mm holes. The spore is an egg type; its membrane is dual in structure, the outer membrane being nearly colorless, the inner membrane having a weak brown small projection. Said projection is inserted from the inside to the outside. Its dimension is 9 to 11×5.5×7µ. Said shell wraps the pileus, and the stem has a thickness of 30-40µ. A brown cell of a thick club type membrane is arranged on said shell. A varnish-like material is secreted thereon.
2) Separation and Cultivation
Yellow tissue is picked from the ripe tip of the Ganoderma fruiting body, sterilely cut to a size of 3 mm3, inoculated onto an onion, soy sauce, and sesame oil agar culture medium, and cultured at a temperature of 25° C. to 30° C. for germination, resulting in propagation of the first white cotton wool hypha within 7 days. Such germinated hyphae are utilized in fusions with other fungi.
Said fungus can also be propagated on culture media of any composition.
3) Oxygen requirement
Hyphae on agar culture media generate only a small quantity of CO2, require a very small amount of O2, and are aerobic. Since CO2 is generated in large quantities during propagation and cultivation on sawdust, when fruiting bodies are generated, a large amount of O2 is required. Thus, sufficient ventilation is necessary.
4) Growth temperature
The growth temperature is in the range of 5° C. to 30° C., but it differs depending upon the mycetoma, and the optimum temperature is 30° C.
Hyphae can also be grown continuously at 5° C.
5) Growth pH
The growth pH is neutral, as weak alkali and CaCO3 are necessary. At a pH of 7.0 to 8.5, growth on agar culture media is retarded.
6) Antibiotic activity
When a fruiting body is wetted and air is blown thereon, adhesion of molds in the air is excellent in basidiomycetes. When an extract of said fruiting body is allowed to stand, it becomes a good nutrient for molds in the air. Propagation of the molds is good; however, they do not have antibiotic activity.
7) The taste
A water solution of the Ganoderma has an intensive bitter taste.
8) Utilization in Chinese (herb) medicine
In China, Ganoderma is a spirit grass, and is a folk remedy medicine i.e., a hermit medicine, disclosed in "A Shinnong Boncho Kyeong," meaning a hermit agricultural botanical bible.
MYCOLOGICAL PROPERTIES OF THE GRIFOLA
Grifola, as used herein, means the sclerotium in soil of Grifola umbellata. Those utilized for fusion in the present invention are cell bodies of germinated Grifola.
1) Morphology
A sclerotium body produced in Japan is referred to as "Chinjeoryeong", meaning a true Grifola. Its flesh is thin and irregular. A sclerotium body produced in China has thick flesh. In herb medicine, Chinese products are recognized as important medicines.
A fruiting body produced above ground is an annual plant. It is branched from one plant similar to a bush clover mushroom. It is 10-25 cm tall, and its soil sclerotium is a glass bead type or is flat. It is connected in the form of a ginger root, its surface is black or gray-black, and the inner flesh is white and rich.
2) Separation and Cultivation
The inner tissue of Grifola umbellata is cut to a size of 3 mm3. This is inoculated onto a sterile nutrient culture medium containing a boiling water extract of Grifola. It is cultured at 25°-30° C. to produce mycelia.
3) Requirement for O2
It is aerobic.
4) Growth temperature
20°-28° C. Optimum temperature is 22° C. Below 5° C., growth is stopped.
5) Growth pH
pH 6.0-7.0
6) Antibiotic activity
When a dried sclerotium was stored wet, adhesion and germination of molds were not observed for a year.
Further, when an extracted concentrate thereof is heated at 30° C. in an unsterilized state and then stored, antibiotic activity thereof was not observed.
7) Utilization in herb medicine
Referring to the action of Grifola in herb medicine, when 5 g of decocted Grifola is administered to a healthy human, after 6 hours the quantity of urine is increased 62%, and chloride in the urine is increased 45%.
However, 3 g of a decoction of Grifola causes no diuresis.
In an experiment involving a dog having urenia, when 0.25-0.5 g/kg of a decoction of a Grifola is administered by intravenous or intramuscular injection, the diuretic activity thereof is observed; however, by oral or intravenous injection of no more than 0.0048 g/kg of a decoction of Grifola, said activity was not observed.
In toxicity testing in a rabbit, until a dosage for humans by oral or intraperitoneal injection is administered thereto, no effect was observed.
When a water solution of an extract of Grifola extracted with alcohol is administered orally to a mouse, the quantity of urine is increased.
However, in the case of a mouse in which the adrenal bodies are removed, although a decoction of a Grifola and deoxycorticosterone are administered together, the quantity of secretion of urine and chloride from it is unchanged.
An alcohol extract of the Grifola is inhibitory against a yellow Staphylococcus and Escherichia coli, and a water soluble glucan obtained from the Grifola exhibits intensive antitumor activity in a mouse suffering from bladder cancer.
Method for Producing the Present Fusion Fungus--Fusion of Mycelia of Ganoderma lucidum and Grifola umbellata
Mountain soil from a mushroom-growing district is dried, sifted using a 100 mesh sieve to produce microparticles of soil, and the resulting sieved 100 mesh soil is dried and sterilized at 150° C. for two hours to obtain microparticles of soil having a pH of 4.5-5.0. To said obtained soil a 40% aqueous solution of PEG is added with agitation, and the mixture is sterilized in an autoclave at 120° C. for 1.5 hours to give a sterilized, agitated soil-PEG mixture.
A very small amount of the resulting mixture is added to a mixture of mycelia of Ganoderma lucidum and Grifola umbellata in a mortar. This is crushed together, and is inoculated on the surface of agar medium for a mushroom and is placed in a transparent airtight pouch equipped with a stopper.
The mouth of said pouch is bottled up by electrical melt adhesion. The inside of said pouch is evacuated, and CO2 gas is injected into said pouch. The mixture in said pouch is cultured for 30-40 days to produce dark brown mycelia wherein mycelia of Ganoderma lucidum and Grifola umbellata are fused.
Propagation of Said Fused Mycelia on Sawdust Medium
A mixture of 70% sawdust of a broadleaved tree and 30% bran are mixed with water while stirring, and is placed in a heat-resistant glass bottle or tube. This is sterilized under 1.2 kg/cm2 of pressure at 120° C. for two hours, and is cooled. Said fused mycelia are inoculated on the surface of said treated mixture of sawdust and bran placed in the bottle. Said inoculated bottle is placed in a pouch which consists of a polypropylene film layer and a polypropylene nonwoven layer having micropores of 0.5-02.µ to provide sterile air. Then the mouth of said pouch is bottled up by electrical melt adhesion and culturing is carried out at 25° C. for 120 days to propagate black-brown mycelia.
Mycological Properties of the Present Fused Fungus
1) Growth pH of the Fused Fungus
A black-brown fungus is generated in the range of pH 4.5-5.0.
2) O2 Requirement of the Fused Fungus
Since it is propagated in sawdust medium, sufficient O2 should be provided.
3) Antibiotic Activity
Ganoderma or Grifola alone do not exhibit antibiotic activity. However the fused fungus exhibits intensive antibiotic activity.
4) Bactericidal Action
When an extract of the fused fungus is employed to treat cutaneous disease, athlete's foot, purulent matter of the gums, eczema, and dermatophytosis, it is highly effective.
Difference in Properties Between Ganoderma, Grifola, and the Present Fused Fungus
1) Sclerotium of Grifola
Colonies on an agar culture medium separated and germinated from a sclerotium of a Grifola are black-brown or black, and are globular in shape. They are no more than 1/4-1/8 the size of a black sesame oil seed, and are connected by invisible microhyphae.
The connected globular fungus grows in a piled state (tumor-like), while the hyphae are extended on the agar surface. When it grows against the glass wall of a test tube, since its extension is controlled, its tip is inflated to form a globular mass.
Its shape is shown in FIG. 1 as observed microscopically or visibly.
The cell body is an aggregate of globular masses.
When the pH of the culture medium is 4-5.5, its growth is stopped.
2) Ganoderma Fungus
Referring to the properties and shape of hyphae of Ganoderma on an agar surface, it grows in the form of white cotton wool. When the Ganoderma fungus has aged, adhesion of other saprophytic bacteria is great, and thereby the Ganoderma fungus withers and dies.
A water extract from the cell body of Ganoderma has nutrient properties for saprophytic bacteria, and it does not exhibit antibiotic activity.
It grows well on an onion, soy sauce, and sesame oil agar culture medium.
The shape of the Ganoderma hypha is as shown microscopically in FIG. 2, wherein each hypha has alternate projections on it.
3) Properties and Identification of the Present Fused Fungus
(a) It does not entirely adhere or germinate on other saprophytes and the like (opened and cultivated in air), and it has intensive bactericidal action.
(b) The black-brown fungus adheres to and grows on sawdust propagation medium.
(c) It grows well on an onion, soy sauce, and sesame oil agar culture medium (pH 6.0-6.5). It grows as an aggregation of mixed white hyphae and black-brown hyphae, and does not have projections.
(d) The hyphae on YM-12 agar culture medium are disposed with equally spaced black lines (FIG. 4).
It is observed that said fungus becomes black-green due to the pH of YM-12 medium.
(e) The morphology and properties of the present fused fungus are not found in the literature.
Test for Antibiotic Activity of Water Extracts of Each Fungus, (Ganoderma, Grifola, and the present Fused Fungus)
In a test of the antibiotic activity of Ganoderma, many species of molds are generated on an extract of the Ganoderma.
In a test of an extract of a sclerotium of Grifola, there is little antibiotic activity.
A water extract of the sawdust propagation medium used to cultivate the fused fungus is colorless and transparent, and exhibits intensive antibiotic activity.
Summary of the Properties of the Present Fused Fungus
The color of the present fused fungus propagated on sawdust is black-brown.
Its water extract exhibits intensive antibiotic activity. In contrast, the water extract of basidiomycetes is a good nutrient source for molds and the like (cultivation at 32° C. for seven days).
The cell body of Grifola on agar culture media is a globular aggregate, and it has spheroid masses on the outer surface.
Accordingly, it will be appreciated that this is entirely different from the present fused fungus.
Toxicity of the Fused Mycelia (G2 .multidot.sY)
A toxicity test of the fused mycelia was carried out as follows:
BALB/C(.male.) Mouse Experimental Process
A mixture of 100 g of the fusion mycelia propagated on sawdust medium and pure water is poured in a container, boiled, concentrated to a volume of 300 ml, filtered using conventional filter paper to give a flitrate, and then is filtered again using a 0.2 µm filter. Minute particles in the resulting filtrate are removed by centrifugation at 5000 rpm/20 min. The supernatant is sterilized in an autoclave under a pressure of 1.2 kg/cm2 at 120° C. to produce a solution for intravenous injection.
0.2 ml of solution was administered to a mouse, but the mouse no showed physical reaction.
In a test employing rabbits, when 20 ml of solution is injected intravenously into the ear of a Japanese white female rabbit, the rabbits showed no physical reaction.
These results demonstrate that the fusion mycelia causes no physical toxicity at these levels.
Oral Administration Test in Humans
When the fusion mycelia is administered to 200 gastric ulcer patients, all patients were completely cured before or after 10 days.
The method for oral administration of the fusion mycelia to humans is as follows:
1 kg of raw fusion mycelia are extracted with water, concentrated to a volume of 6 liters, and then the resulting extracted solution is administered to the subject 20 ml at a time, ter in die (three times a day), one hour before meals.
Effect on Cancer Patients
The aches of a cancer patient are gone after 3-5 days by administration of 1-2 liters of the fusion mycelia extract.
Utility of the Present Invention
The fused microorganism of the present invention has antibiotic properties, and can be utilized for the preparation of medicines and the like.
Deposit
The present fusion fungus G2 .multidot.sY was deposited under the terms of the Budapest Treaty as accession number FERM BP-3131 on Oct. 12, 1990, at the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukubashi, Ibaraki-ken, 305, Japan.
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fastfred
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: BlimeyGrimey]
#8303577 - 04/19/08 05:45 AM (15 years, 10 months ago) |
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Quote:
It grows well on an onion, soy sauce, and sesame oil agar culture medium.
They sure make it sound tasty.
Quote:
Method for Producing the Present Fusion Fungus--Fusion of Mycelia of Ganoderma lucidum and Grifola umbellata
Mountain soil from a mushroom-growing district is dried, sifted using a 100 mesh sieve to produce microparticles of soil, and the resulting sieved 100 mesh soil is dried and sterilized at 150° C. for two hours to obtain microparticles of soil having a pH of 4.5-5.0. To said obtained soil a 40% aqueous solution of PEG is added with agitation, and the mixture is sterilized in an autoclave at 120° C. for 1.5 hours to give a sterilized, agitated soil-PEG mixture.
A very small amount of the resulting mixture is added to a mixture of mycelia of Ganoderma lucidum and Grifola umbellata in a mortar. This is crushed together, and is inoculated on the surface of agar medium for a mushroom and is placed in a transparent airtight pouch equipped with a stopper.
The mouth of said pouch is bottled up by electrical melt adhesion. The inside of said pouch is evacuated, and CO2 gas is injected into said pouch. The mixture in said pouch is cultured for 30-40 days to produce dark brown mycelia wherein mycelia of Ganoderma lucidum and Grifola umbellata are fused.
Pretty interesting that they didn't have to prepare protoplasts first. They just basically mashed it up in some sterilized dirt with PEG in it. Pretty simple stuff!
-FF
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BlimeyGrimey
Collector of Spores
Registered: 08/24/05
Posts: 3,792
Loc: Puget Sound
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8303909 - 04/19/08 09:46 AM (15 years, 10 months ago) |
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Know where to get 100% PEG? I can only find the 97% stuff with 3% glycerin. Think that stuff would work?
I already have any idea for the CO2 chamber.
I've also thought that maybe some PEG infused agar might also do the trick.
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Workman
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: BlimeyGrimey]
#8303993 - 04/19/08 10:31 AM (15 years, 10 months ago) |
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the shape of a colony on an agar culture medium separated and germinated from the nucleus of a Grifola fungus under the microscope.
FIG. 2 shows the shape of a Ganoderma hypha under the microscope.
FIG. 3 shows the shape of a fusion fungus of the present invention under the microscope, grown on an onion, soy sauce and sesame oil agar culture medium.
FIG. 4 shows the shape observed for the early stage of growth of the fusion fungus under the microscope on a YM-12 flat agar culture medium.
FIG. 5 shows the shape observed during the adolescent growth stage of the fusion fungus under the microscope.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification
Edited by Workman (04/19/08 10:33 AM)
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fastfred
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: Workman]
#8304095 - 04/19/08 11:18 AM (15 years, 10 months ago) |
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> Know where to get 100% PEG? I can only find the 97% stuff with 3% glycerin. Think that stuff would work?
They used 40% aqueous PEG, so that should be plenty fine. No mention of buffers. 30% polyethylene glycol 4000 in 10 mM CaCl2-glycine solution (pH 8.0) is often used for good osmotic stability.
> I already have any idea for the CO2 chamber.
I'm not sure what role the CO2 is supposed to play in this scheme. It seems counter to regenerating fusants IMHO.
> I've also thought that maybe some PEG infused agar might also do the trick.
PEG is a fusant. It causes suspended protoplasts and cell debris to fuse together. Then you regenerate the fusants on agar. Once the cells are fused you no longer need the PEG and having it in agar would do nothing since cells are not mobile enough to fuse on solid media. It's only useful in solutions.
They leave out a lot of possibly important details in this patent and don't seem to cite the prior art well enough. I would like to know where they got this technique, since it doesn't seem like they are patenting it as a process for producing fusants.
Anyone have any more literature on this?
-FF
Edited by fastfred (04/19/08 11:40 AM)
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canid
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8304855 - 04/19/08 03:26 PM (15 years, 10 months ago) |
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they also make poorly worded and ambiguous statements a lot, as though they're more worried about impressing a patent clerk than standing to review.
-------------------- Attn PWN hunters: If you should come across a bluing Psilocybe matching P. pellicolusa please smell it. If you detect a scent reminiscent of Anethole (anise) please preserve a specimen or two for study and please PM me.
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Workman
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8306099 - 04/19/08 09:17 PM (15 years, 10 months ago) |
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I also have serious doubts on this method. Details are lacking and it seems like everything put into the mixture fused as desired. I would have at least expected an isolation step where you weed out the unfused products. I wouldn't be surprised if the dark "fused" mycelium was actually some sort of contaminate.
I didn't carefully read the patent so maybe I missed some things, but it didn't seem worth my time.
I would think that a very small but better fusion success would be had using an osmotically stablized solution of PEG and two colonized petris of agar in a blender.
-------------------- Research funded by the patrons of The Spore Works Exotic Spore Supply My Instagram Reinvesting 25% of Sales Towards Basic Research and Species Identification
Edited by Workman (04/19/08 09:19 PM)
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BlimeyGrimey
Collector of Spores
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: Workman]
#8306438 - 04/19/08 10:40 PM (15 years, 10 months ago) |
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I was thinking the same thing. Simply put some mycelium from both species into a blender with the PEG solution. Then streak the slurry onto petri's. I read another article somewhere that used a similar method (mortar/pestle) but they said that out of 300 or so colonies only about 7 "fused" colonies were identified. That seems more believable to me.
There HAS to be an isolating step somewhere simply because you can't expect everything to fuse.
I also don't understand why they used dirt. What purpose did the dirt particles serve? Any ideas?
I also found this article. http://www.springerlink.com/content/h244p6043k20gtq2/
They claim that they used PEG protoplast fusion to create Volvariella volvacea/Pleurotus florida hybrid colonies and some of the hybrids produced fruits.
Without some type of DNA analysis or successful fruiting I don't see how they (they being the group from the first article) know for sure that a cross happened.
I think, for now atleast, I'll stick with my nicotinic acid experiments.
P.S. Doesn't figure 4 in that picture look like mold? With the segmented hyphae?
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canid
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: BlimeyGrimey]
#8306507 - 04/19/08 10:56 PM (15 years, 10 months ago) |
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the more i read the article the more i come to one primary conclusion. i'm applying for those patents i've been hesitating to as soon as i can. if you can't dazzle them with brilliance, baffle them with bullsh!t...
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fastfred
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: canid]
#8308236 - 04/20/08 04:17 PM (15 years, 10 months ago) |
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Their method may be a little lacking in some respects. But really all you have to do is prepare protoplasts, fuse with PEG (and usually an osmotic stabilizer), then regenerate them.
Protoplasts are usually prepared by enzymatic digest (chitinase, etc.), but can be made using a french press or other mechanical methods. This method seems to add simple maceration. I'd also bet that blenderizing can release protoplasts ok too.
But the problem with that is usually the selection step. Protoplasts don't regenerate as fast as intact cells that are left over, and recombinants don't usually grow as fast as parental cell lines.
So I don't really see how they could have gotten this method to work. They make no mention of it, but perhaps if they used an agar overlay method they might have given their fusants a chance to grow a little before their plates were overgrown. If the morphology (color) was significantly different and they picked the slower growing colonies (presumably regenerants) it might be able to work.
Perhaps the soil acts as something to macerate the myc against. Maybe the soil particles act to pierce the cells and then the nuclear material and cytoplasm congregates with the soil particles, eventually regenerating into cells. I just don't see how they could separate live and vigorously growing cells from the slowly regenerating protoplasts.
Maybe if you took the macerated/blended material and filtered it you could filter out cells and the protoplasts would pass through the filter. Otherwise maybe you could centrifuge out the cell material and then resuspend just the protoplasts. If you could work out the density of the protoplasts and create the proper solution maybe you could centrifuge out the cells while leaving the protoplasts in the top layer.
Anyhow, it would be interesting to see if the procedure can be replicated. In order to be a valid patent it has to disclose a procedure that allows one "skilled in the art" to successfully replicate the procedure. It seems like they are really patenting the fusant species though, rather than the process. I try to stay up on the research and have read a couple books on protoplast fusion in fungi and this method is new to me. I can't seem to find anything on it.
-FF
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canid
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8308455 - 04/20/08 05:59 PM (15 years, 10 months ago) |
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or perhaps their method didn't work exactly as they claim and they just wanted the patent in place and where willing to bullshit their case to get it.
-------------------- Attn PWN hunters: If you should come across a bluing Psilocybe matching P. pellicolusa please smell it. If you detect a scent reminiscent of Anethole (anise) please preserve a specimen or two for study and please PM me.
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Morelman
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! *DELETED* [Re: canid]
#8309919 - 04/21/08 09:00 AM (15 years, 10 months ago) |
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Post deleted by MorelmanReason for deletion: Never again...
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Feelers
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: Morelman]
#8312157 - 04/21/08 08:45 PM (15 years, 10 months ago) |
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Log in to view attachment
You guys will probably be interested the the file I uploaded. It's a protoplast fusion with photos about crossing pink and white oysters, and they clearly succeed.
On other fusion articles... Can anyone get this? http://www.springerlink.com/content/h244p6043k20gtq2/
or this? http://www.springerlink.com/content/h57212400m8868w0/
They are both successful protoplast fusions (I think).
Edited by Feelers (04/21/08 08:52 PM)
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BlimeyGrimey
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: Feelers]
#8312485 - 04/21/08 10:25 PM (15 years, 10 months ago) |
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I posted that first link already in my last post. That pdf you posted was quite helpful. Thanks.
It re-enforced a previous theory I had about using monokaryons vs. dikaryons during fusion. By using monokaryons it would be quite simple to verify a hybrid by simply looking for clamp connections in the regenerants.
If I ever obtain monokaryotic cultures of 2 similar species I might attempt the maceration/blenderize technique using them.
One day, protoplast fusion will be a common method people can do at home. Atleast I hope it will. Look at plant tissue culture. Years ago noone did it at home. Now they sell home plant tissue culture kits online.
I believe within the next year someone on the forum will bring forth a real cross-species attempt.
Hopefully the Biotech course I'm taking this year will help my understand of all this stuff and maybe even help me get some of the materials I need.
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fastfred
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: BlimeyGrimey]
#8312878 - 04/22/08 12:34 AM (15 years, 10 months ago) |
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> Can anyone get this?
Yep.
Quote:
One day, protoplast fusion will be a common method people can do at home.
Seems like this is already happening. If the dirt + myc + PEG + grinding method works well it can't get any more simple than that.
-FF
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8312882 - 04/22/08 12:36 AM (15 years, 10 months ago) |
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Here's the other one.
-FF
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BlimeyGrimey
Collector of Spores
Registered: 08/24/05
Posts: 3,792
Loc: Puget Sound
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: fastfred]
#8313030 - 04/22/08 01:25 AM (15 years, 10 months ago) |
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Thanks for those pdf's!
Back to the dirt thing, I think very fine sieved sand would work just as good if not better than dirt to macerate the cells.
-------------------- Message me for free microscopy services on Psilocybe, Panaeolus, and Gymnopilus species. Looking for wild Panaeolus cinctulus and Panaeolus olivaceus prints.
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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Re: Fusion of Grifola umbelleta and Ganoderma lucidum! [Re: BlimeyGrimey]
#8313224 - 04/22/08 02:57 AM (15 years, 10 months ago) |
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Sand might work, but who knows? Maybe there are particular nutrients or other goodies in the soil.
Trichoderma harzianum is the most easily cultured and abundant fungal organism in the soil. TH produces a number of chitinases and it's enzymes are often used in releasing protoplasts. So maybe that has something to do with it. I'm not sure how those enzymes would survive being denatured during sterilization, but it's possible.
Soil probably also provides some salts and minerals that provide osmotic stability to the solution. Osmotic stabilizers are always used in protoplast fusion experiments as far as I've seen. CaCl2, MgCl2, sucrose, NaCl, and mannitol are commonly used.
If your solution is not the proper osmolarity then protoplasts simply dissolve into the solution, releasing their contents, and you'll have an extremely low regeneration efficiency. Perhaps the soil provides this or else they figured that anyone "skilled in the art" would know to add an osmotic stabilizer.
There may be other ways that are better and just as easy compared to the soil method. Plenty of people here have lots of experience with the mean green (Trichoderma harzianum). All you need to do to replace expensive chitinase enzyme preparations is culture some TH, blend it up, and filter sterilize it and you've got a homebrew chitinolytic mixture for releasing protoplasts. It's been done in at least one paper. They concentrated the enzymes 50X using ammonium sulfate precipitation, which is easy to do. Otherwise researchers have found that chitin (not surprisingly) induces the chitinolytic enzyme system. Culturing TH in the presence of chitin would provide higher enzyme levels.
Anyhow, protoplast fusion is within the reach of at least a handful of mycologists here and maybe this information will encourage people to pursue it.
-FF
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