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 Data
That Guy
Registered: 08/12/08
Posts: 4,035
Loc: Southwestern US 
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 Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants.  2
#823385 - 02/03/17 10:16 AM (7 years, 9 months ago) |
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Hello again,
Section 1: Background
I've posted before about the potential value of Protoplast Fusion (aka somatic fusion) for several applications in the cannabis industry. It can potentially be used to generate novel hybrids between cannabis and related species (such as Hops (Humulus lupulus). I can also be used to generate a wide variety of polyploidy.
As a quick review, protoplast fusion is the process by which plant cells from two different samples are stripped of their cell walls, and are fused to one another via the joining of their cell membranes through various procedures. While this seems simple enough, there is a major hiccup in the process as applied to plants.
The hiccup lies in the way to efficiently select successfully fused cells. If you think about mixing thousands of individual cells from each tissue sample into the same vial and allowing the cells to fuse at random for a fixed period of time, there are several different pathways that each cell can take:
- 1) The cell floats around in the vial, not fusing with anything, and remaining a viable cell that will grow after the fusing session is over.
- 2) The cell fuses with another cell of its own tissue sample, thus generating a polyploid cell of that single sample.
- 3) The cell fuses with a cell of the other tissue sample, thus creating a successful fusant.
- 4) The cell performs any of the prior 3 scenarios, but does so multiple times, thus becoming an atypical fusant that may or may not live.
- 5) The cell fuses successfully just like scenario (3), but upon the first round of cell division, poops out the extra genetic material, resulting in an unsuccessful fusant.
Scenario 2 is just fine for induction of polyploidy, but not good for hybridization of 2 species. Scenario 3, 4, and possibly 5 are good for hybridization, with scenario 5 being less desirable because it does not represent a good datapoint for how the expected hybridization would pan out.
As you can see, Scenarios 1, 2, 3, 5, and possibly 4 can all grow out after the fusion session, thus leading to difficulty in selecting the true fusant cells until many months later, when the plant cell is fully grown out into a plant, and the phenotype is checked visually. This would use up tons of space, petri dishes, nutrient solution, and require a large incubation lab and garden area in order to systematically eliminate non-fusants.
There are some alternative methods that are tedious, error-prone, and inefficent. These involve the use of expensive inverted, phase contrast microscopes and the manual isolation of stained cells using a micropipette and a very steady hand. Another method involves flow cytometry or individual genetic comparison, which is out of the reach of most clandestine cannabis enthusiasts from a monetary perspective. 
Section 2: Auxotrophic Selection
In microbiology (bacteria, protists, fungi), the mutation rate is high enough that a method called "Auxotrophic selection" can be utilized to efficiently select only cells from Scenario 3 and some from Scenario 4, and thus guaranteeing that only fusants carrying the genetics from both samples are carried on to the grow-out phase.
Auxotrophy is a genetic mutation in which a cell lacks some gene that is required for producing an essential compound for life. These cells cannot survive in a culture unless the culture media is supplemented with whatever compound the mutant cell can't produce.
In the case of protoplast fusion for the hybridization of two species, the two parent cell cultures are selected for two separate auxotrophic mutations, one for each cell culture. They can be related to something like the production of biotin (vitamin B7), tryptophan, or histidine...but for the simplicity of the example, we'll call them A and B. So we have the first cell culture that can't produce A, and the 2nd cell culture that can't produce B. We centrifuge each cell sample and resuspend each in a mannitol solution (that isn't supplemented with either A or B) and then combine the cultures into one container. We then initiate a fusion session, and allow the cells to fuse with one another. At the end of the fusion session, we have a mixture of Scenarios 1 through 5. However, the scenarios are now as such:
- 1) Cell A or B float around and do not fuse, and thus still cannot survive without A or B, respectively.
- 2) The cell fuse with another cell of its own kind, creating either an AA or BB polyploid, that still cannot survive without A or B, respectively.
- 3) The cell from the first sample fuses with a cell from the second sample, producing an AB hybrid. The genetics from the A sample can produce the compound that B needs to survive, and the B sample can produce the compound that A needs to survive. This new hybrid can now survive without supplementation of either A or B.
- 4) The cell performs any of the previous scenarios, but does so multiple times. If any of the fusings involves Scenario 3, then the new hybrid cell will probably survive, as long as it is not fused too many times.
- 5) The cell performs any of the previous scenarios, but then poops out the extra genetic material during the first round of cell division. If all extra chromosomes are rejected from the cell, then the resulting daughter cells will be either A or B deficient, and will not be able to survive without A or B supplementation, respectively.
As you can see, we are still left with all 5 scenarios directly after fusion, but if we transfer this combined cell culture to unsupplemented growth media (aka minimal media) the auxotrophic mutations of the parent cells help to eliminate all but the cells that successfully incorporate the genetic material of both parent cell cultures, as they are the only ones that can survive without A or B in the growth media. 
The problem with Auxotrophic selection in plants is that it is an exceedingly rare mutation, and involves both a high volume of seed germination and tight timing (in order to catch a dying seedling, sample the tissue, transfer it to a fully supplemented media, and then going through the tedious process of removing one supplement and checking for a cellular reaction). This makes the isolation of plant-based auxotrophic mutants exceedingly tedious and expensive. This is where technology has opened the door, yet again. 
Section 3: CRISPR/Cas9
The CRISPR/Cas9 system was originally discovered as a way for bacteria to identify, locate, chop up, and inactivate viral genetic material. This system involves a Cas9 protein that actually does the chopping, a guide or target crRNA (in the bacteria, this would be the exact copy of the genetic material to be removed, and a sequence that forms into a hairpin loop, creating an active complex), and a tracrRNA (that binds to the crRNA and forms another active complex, which interacts with the Cas9 protein and initiates the chopping sequence).
This system can be easily used to knock out target genes, or multiple genes at a time. Common plant genes such as the essential biotin genes BIO1 and BIO2 can be easily found (along with their target loci). Additionally, there are many companies out there that can assemble custom sgRNA/Cas9 plasmids, package them into either bacterial or viral vector packages, and send them to you with all the materials needed to successfully transfect and activate the system in vitro for less than 1000 USD. 
Section 4: Summary and Conclusion (TLDR)
To recap, we have previously discussed the method of producing novel cannabis-based interspecies hybrids and polyploid cannabis workhorses, utilizing protoplast fusion as a method that a reasonably intelligent and resourceful cannabis enthusiast can perform in a clandestine biolab, on a budget that is very acheivable relative to the type of work being done.
However, we have not discussed the difficulties in properly identifying and isolating an exceedingly rare set of auxotrophic mutations, which is really the only affordable and efficient way to sort out successful fusants from the thousands or millions of unsuccessful fusants, rendering all previous discussion purely hypothetical and out of reach of the common cannabis enthusiast.
Per the information provide in the above post, it can now be concluded that:
- Isolation (or rather directed production of auxotrophic mutants of known types) is both possible and economically feasible.
- Auxotrophic mutations can be produced with genetic code common to the majority of higher plants, and thus can be performed without leading anyone involved to conclude that cannabis is the intended target for genetic manipulation, allowing this technique to be utilized anywhere.
- Successful protoplast fusion for the purpose of interspecies hybridization and/or induction of various magnitudes of polyploidy can be accomplished on a hobby budget, without the need for a degree in genetics or biology, and with very little experience in a lab.
The time is now, the future of cannabis is upon us. 
Section 6: Useless Babble and Advertisement
At this point, I just need to save up my $$$ for the equipment and the CRISPR/Cas9 kits. I already have some good cannabis genetics available, as well as access to some Hops of the "Nugget" variety. There might be some extra research in order to properly design the sgRNA for a plant application, but I'm pretty sure that the aforementioned companies would be willing to work with me on this.
I will take this time to selfishly endorse myself for my own interest of getting out West, closer to the mountains that I love so much, and into an industry that I feel passionate about. If anyone in any of the legal states works for the cannabis industry, and knows how to contact an owner, I would be happy to whore myself out to said company in exchange for a job, and try to produce some of the first cannabis/hops hybrids, cannabis polyploids (most research points towards the increased expression of genes as the primary manipulation mechanism for current breeding, so the potential for multiple copies of chromosomes may break through the 30% THC ceiling that seems to be forming). Either way, I do intend on moving forward with these projects, it just may happen faster if I can get some start-up capital from an interested investor.
For now, I post my research here, and hope that it motivates someone.
 ,  , & 
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“The Universe is under no obligation to make sense to you” -NDT
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Deadkndys420
Registered: 08/28/12
Posts: 8,703
Loc: █████
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 Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: Data]
#823386 - 02/03/17 10:41 AM (7 years, 9 months ago) |
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Good read.
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 TrueHerbCrystal
Uncertified Oregrowian
Registered: 02/07/09
Posts: 1,352
Loc: Emerald City
Last seen: 6 years, 1 month
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Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: Data]
#823405 - 02/04/17 10:07 AM (7 years, 9 months ago) |
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very cool!
Some heavy stuff man 
I mean, if scientists can make a mouse with biolumiencent genes from jellyfish, surely we can gene splice cannabis with hops!
Hell, someday, we might have cannabis spliced with biolumiencent genes that glow in the dark
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Data
That Guy
Registered: 08/12/08
Posts: 4,035
Loc: Southwestern US
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Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: TrueHerbCrystal] 1
#823410 - 02/04/17 11:39 AM (7 years, 9 months ago) |
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That actually sounds like a neat novelty strain. the CRISPR system can be combined with a recombinant gene, and the cut area has the possibility of incorporating the introduced gene segment during repair.
That might be a neat future project.
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“The Universe is under no obligation to make sense to you” -NDT
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Deadkndys420
Registered: 08/28/12
Posts: 8,703
Loc: █████
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Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: Data]
#823411 - 02/04/17 12:47 PM (7 years, 9 months ago) |
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There's mushrooms that glow in the dark.
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TrueHerbCrystal
Uncertified Oregrowian
Registered: 02/07/09
Posts: 1,352
Loc: Emerald City
Last seen: 6 years, 1 month
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Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: Deadkndys420]
#823413 - 02/04/17 03:06 PM (7 years, 9 months ago) |
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true, but ever seen a marijuana plant glow in the dark?
Imagine Purple Kush that glows purple 
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Data
That Guy
Registered: 08/12/08
Posts: 4,035
Loc: Southwestern US
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Re: Protoplast Fusion: Utilizing the CRISPR system to efficiently generate auxotrophic mutants. [Re: TrueHerbCrystal] 1
#823420 - 02/04/17 04:45 PM (7 years, 9 months ago) |
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It's been done in other plants.
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“The Universe is under no obligation to make sense to you” -NDT
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