How do you feel about GMO Cannabis?

Botany and Cultivation
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Successful tissue culture is often seen as the first step towards genetic modification of a new target organism. Our favourite plant has been amenable to culture for years now, so presumably there are already folks out there using DNA based manipulations to achieve their goals. If not, there are certainly folks seriously considering it.

How do you feel about that? How do you think the technology could/should be used?

Producing THC or CBD in hops should be a single gene transfer. I’ve heard rumours of it having been done already. Certainly the barrier is pretty low at this point. The genes involved have been cloned, sequenced, and expressed in a heterologous system to prove that it is both necessary and sufficient for converting Olivetol to THCA or CBDA.

When I first discussed moving THC production into tomatoes 25 years ago, the tools where available, but the sequence data was not. The tools have improved markedly, and sequence data is accumulating at an incredible rate. The need to bamboozle the feds by producing schedule one narcotics in produce has substantially subsided.

Are there any benefits beyond CBD laced beer to moving cannabinoid production into other organisms? What about the rest of the entourage?

Folks who are making or using isolate might benefit from bioreactor based cannabinoid production, especially if it could be wired as a secretory process. Purification would likely be considerably simpler without all the co-extracting terpenes etc.

How about tweaking the major cannabinoid synthase to make it less prone to side reactions? THC synthase and CBD synthase are allelic (like blue vs brown eyes) and are essentially slightly different versions of the same enzyme. Both seem to be capable of producing THCA, CBDA, or CBCA from CBG. What if we could tighten that up so your “hemp” plant really did make zero THC? Would that be worth the GMO label?

How about just signing your damn name in there? You want folks to know that you made this? Encode your name, address, and today’s date, and slam it in there…

For the phenotype hunters, I would suggest introducing an exogenous transposable element system, although digging through the available sequence data will probably reveal several endogenous candidates for an insertional mutagen.

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“Purification would likely be considerably simpler without all the co-extracting terpenes etc.”

Not sure about that in general, maybe so with respect to selectivity of one terpene vs another. Bioreactor would likely use aqueous media, and organism would be highly inhibited by the metabolites, leading to low yield and titer. Separation is not “easy” from fermentation broth. Cannabis flower has an incredibly high concentration of sought after compounds. I read a thesis posted on this forum suggesting this phenomenon is highly unusual in plants, and a gift from Gaia in terms of separation! Generally, the direct cost of a manufactured chemical is inversely proportional to concentration in raw material.

I’m skeptical that recombinant strains of microbes will be excreting cannabis isolates in the near future. But would love to work on it! Sign me up.

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I want to join this discussion later but for now I recommend checking out Hyasynth Bio.

Generally speaking, having to grow a whole plant because you are interested in producing a single compound is quite inefficient, and will not be the method to scale.

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in this case I have to agree with both of you :slight_smile:

The trichomes really are an incredible structure. if you collect them before extraction you’ve got starting material that can run better than 50% target. That’s gonna be pretty hard to beat with any of the secretion based bio-reactor schemes I’ve seen.

on the other hand, if you’ve got secretion working, and a broth/oil emulsion in your reactor, your purification could be as simple as centrifugation to separate the broth/oil followed by winterization in EtOH to drop the oil and leave your target molecule.

Anyone heard of Cannabisativine?

Rumour has it that it is a cytotoxic alkaloid that comes along for the ride with both light hydrocarbon and ethanol extraction (but not CO2). That alone might be a good reason to use a heterologous expression system.

…and yeah, hyasynth bio looks to be running with it already

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Ran across Cannabisativin(e) here

0103]
It is moreover noted that the primary extracts obtained with the aid of lioophilic solvents contain the alkaloids that are readily soluble in these solvents, such as, cannabisativin which is highly cytotoxic. This alkaloid contamination may very well also still occur in an extract prepared in accordance with WO00/25127 from the primary extract described there, following additional purification and enrichment steps in accordance with WO00/25127 which extract is said to have a 98% content of Δ9-THC.
[0104]
In contrast, already the primary extracts of the invention without any further purification steps—as shown in Table 1—practically do not contain any more cannabisativin.
[0105]
Thus the ethanol extract contains about 200 times more toxic alkaloids, in particular the highly oytotoxic cannabissativin, and the hexane extract in accordance with WO00/25127 even about 350 times more than the CO2 primary extract of the invention.

   TABLE 1
        Primary extracts from industrial hemp with different solvents
        	EtOH 	Hexane primary 	Inventive
        Measured 	primary 	extract* in accordance 	primary CO2
        substance 	extract 	with WO00/25127 	extract
        Chlorophyll 	3.00% 	2.85% 	0.010%
        CBD 	14.50% 	12.40% 	58.000%
        Δ9-THC 	2.30% 	2.30% 	9.500%
        Δ8-THC 	0.00% 	0.00% 	0.000%
        CBN 	0.50% 	0.50% 	0.100%
        Flavonoid 	12.50% 	8.50% 	0.150%
        glycosides
        Alkaloids: 	0.20% 	0.35% 	0.001%
        cannabisativin
        Monoterpenes:
        α-Pinene 	0.02% 	0.03% 	0.001%
        β-Pinene 	0.01% 	0.02% 	0.001%
        Myrcene 	0.02% 	0.02% 	0.001%
        Sesquiterpenes:
        Caryophyllene 	0.53% 	0.45% 	0.020%
        β-Humulene 	0.18% 	0.22% 	0.008%
        Δ-Selinene 	0.10% 	0.15% 	0.004%
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What about canna-canola?

Rape is almost trivial to transform these days, and given that its an oil producer, getting it to store your cannabinoid of choice in a retrievable location should not be particularly difficult.

You’d still need to do some degumming of the cold pressed oil, but could conceptually provide “preformulated” CBD in oil in the 2-3% range with very little other purification.

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I actually got into working in this industry by pitching an idea for transgenic hemp that had the THCA synthase gene knocked out. I am certainly not the only person who has thought of this. A certifiable THC-free hemp. I couldn’t get the idea funded, but did get hired at a medical grow and began to setup a tissue culture and genetics lab. Unfortunately, the R&D went by the wayside as production and management was more important to the CEO and I ended up managing the farm. I left that place a while ago. All the biotech gear I got them is sitting in storage probably. Such a shame.

You would need to add more than just the THCA synthase gene to hops in order to get it to produce THCA most likely, but you could probably shoot it in there and see what happens anyway. Lots of people have now figured out how to do cannabis tissue culture. I was able to induce callus formation (think embryonic stem cells for plants) from leaf cells. That’s the first step, you then mutate those cells with Agrobacterium or, more easily, a gene gun, and then add hormones to cause them to start to make plant structures called “shoots”. I never got my callus cultures to make shoots, and it basically ended there. It became a side project, but was pretty fucking rad.

I have absolutely no qualms about GMO plants as long as they are carefully studied. The strains we smoke are inbred mutants that are nothing like their wild ancestors. Same for the food we eat. Take a look at what corn used to look like (look up Teosinte).

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Just to be clear: knocking out THC synthase wouldn’t get you a THC free CBD line. They are allelic & leaky/promiscuous. If you knock one out, you knock them both out

You’re right. Olivetol => CBG is probably also required.

Preaching to the choir…

Worked in maize for my PhD. Have grown Teosinte. Worked in a tissue culture lab for an number of years. Have a patent on what I consider a more CRISPR friendly gene gun

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Interesting link on hyacinth, thanks. My first hand experience with industrial biotechnology startups like hyacinth, and larger… In theory the bioreactor approach is the most scalable way for so many products/problems, is sooooo promising, but when you actually go to scale innovative advanced industrial biotechnology, it becomes difficult and expensive, real fast, investors give up. Often, the stumbling block is separation, and simple centrifuging will likely not be the entire story… But maybe… Fascinating though. They are a young group of people working there.

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Ice bullet gene gun! Brilliant!

The Biorad hand held gene gun is one of the most overpriced pieces of lab gear I have ever seen. Absolutely ridiculous. It just blows helium through a fucking tube. Something similar could be built for $500. That was my original plan.

Re Hyasynth: Yeah that seemed like a terrible idea to me at the time. That was started years ago and obviously the THC-yeast thing must have turned out to be not a worthy pursuit. It looks like they are just some kind of aimless transgenic yeast company for hire now. They were riding a small wave of biotech startup craze that I got interested in. None of the projects I knew about back then really panned out. The Glowing Plant company was a major fail with I think half a million in kickstarter funds evaporating into thin air.

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Woof! so much for “running with it…”

but glowing plants are easy… especially if Tobbaco or Morning Glory float your boat.

Yeah, using ice got around Du Pont’s patent at the time. It works. but we couldn’t afford more than a Daisy bb-gun for motive power, so we never really got to explore it.

Tried to convince the buddy that came up with it with me to update it to a CRISPR delivery tool (Ice CRISPR) before the patent ran out, but he’s found other things to amuse him.

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so…it would appear that the Scots are the first to acknowledge GMO cannabis.

MacKinnon, L. (2003) Genetic transformation of Cannabis sativa Linn: a multi purpose fibre crop, doctoral thesis, University of Dundee, Scotland.

I’m not sure I see the need for engineered Botrytis resistance in fibre strains…but that trait would sure be helpful here in the Pacific Northwest.

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Oh damn good find! I’ll have to read it later when I hit my THC fraction today…

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She pulled the resistance gene from Raspberry.

Which I find amusing because the first thing our primary investor asked upon learning of my bioengineering background was “can you move cannabinoids into Raspberry?”.

here’s the link. British Library EThOS: Genetic transformation of Cannabis sativa Linn : a multi purpose fibre crop
registration is free.

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The Biotech Institute LLC crew is doing marker assisted selection, if I recall correctly? And judging by their patents they first want to end the high saturation of myrcene dominant cultivars.

https://patents.google.com/patent/US9095554B2/en

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The future is now and there will be small amounts by the end of the summer from recombinant yeast.

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I support GMO cannabis and food to fulfill our ever increasing demands for food and energy!!

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Cool! I, and others, have proposed marker assisted selection breeding strategies. Same old story, the people with money want instant results not long term serious research, so it’s hard to get it funded. Good for them! So much more cool work to do regarding breeding. Looks like I will be sticking to chemical engineering for now.

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@farmerj & @FarmerJoeParker how about controllable male sterility like they play in maize?

although in the case of cannabis it would be a controllable “maleness”.

when you want pollen, you induce it.

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I feel that certain goals can be achieved with genetic modification, and it’s place is needed.
However, anything that can change cannabis genetics forever should not be taken lightly.

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