Overexpression of TPS genes for extream Terpene systhesis

Hi, since we are in the advance growing science thread, I wonder if some of you have applied genomic or genetic engineering approaches to increase plant secondary metabolism (or have an opinion on the subject, and please I don't want to start a debate about GMO, please!).

Are you aware of any work related to the overexpression of genes involved in terpene synthesis, specially monoterpene synthases, a (-)-limonene synthase (CsTPS1) (a sesquiterpene synthase-encoding gene involved in citrus aroma formation) and a (+)-α-pinene synthase (CsTPS2), with 35S CaMV promoter?

At my understanding, all the genomic information regarding the CsTPS1 and CsTPS2 are already accessible through the National Center for Biotechnology Information (Cannabis sativa (-)-limonene synthase mRNA, complete cds, GenBank: DQ839404.1, https://www.ncbi.nlm.nih.gov/nuccore/DQ839404.1). This could lead to the creation of a really tasty strain…

There is a good publication on the subject entitled «Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-a-pinene synthase from Cannabis sativa, Article (PDF Available) in Natural product communications 2(3):223-232 · March 2007.»

I’m also wondering if genetic engineering techniques have already been used to increase substantively the Glandular Trichome procuction of a plant through the overexpression of the homeodomain protein gene analogue GLABRA2 (GL2), leading to the promotion of trichome formation, or through the knockout of R3 MYB genes analogue in cannabis?

Darpa said:

Hi, since we are in the advance growing science thread, I wonder if some of you have applied genomic or genetic engineering approaches to increase plant secondary metabolism (or have an opinion on the subject, and please I don't want to start a debate about GMO, please!). Working on it!!!
Personally, not professionally..
Its not exactly an easy process, but with tech like CRISPR down the road it will become quite commonplace I am sure.


Are you aware of any work related to the overexpression of genes involved in terpene synthesis, specially monoterpene synthases, a (-)-limonene synthase (CsTPS1) (a sesquiterpene synthase-encoding gene involved in citrus aroma formation) and a (+)-α-pinene synthase (CsTPS2), with 35S CaMV promoter?
I doubt many here are! You're making me work more thats for sure. I hadn't given overexpression of terpene synthase genes a thought in awhile though, shit I'm really glad you put it back in my immediate head. I'm gonna have to do some more searching on my own.

At my understanding, all the genomic information regarding the CsTPS1 and CsTPS2 are already accessible through the National Center for Biotechnology Information (Cannabis sativa (-)-limonene synthase mRNA, complete cds, GenBank: DQ839404.1, https://www.ncbi.nlm.nih.gov/nuccore/DQ839404.1). This could lead to the creation of a really tasty strain… YEAH!! But to that point... There are already really tasty strains out there.. What genomics can do is improve those lines, even and especially if clone only, and potentially improve them yes - offering better yeilds, more essential oil production, and of course disease resistance, something still SO overlooked with cannabis breeding.

There is a good publication on the subject entitled «Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-a-pinene synthase from Cannabis sativa, Article (PDF Available) in Natural product communications 2(3):223-232 · March 2007.»
For all the stuff I am often researching, it's amazing what doesn't come up in seach engines when I look. WTF this paper is from 06? How do I miss this! Hm maybe I didn't and I just forgot. LoL I do forget things

I’m also wondering if genetic engineering techniques have already been used to increase substantively the Glandular Trichome procuction of a plant through the overexpression of the homeodomain protein gene analogue GLABRA2 (GL2), leading to the promotion of trichome formation, or through the knockout of R3 MYB genes analogue in cannabis? Pffft! If they are they are too busy to waste time posting online about it! You'd be the one who knows Gotta DAN PENA that shit!
We should team up!

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No one's doing anything like those things.. Most of the people you will find here are ideologically opposed to such science.

I on the other hand would LOVE to take my awesomeness and engineer something even better. Selective breeding is great but it can only take one so far, and I am one who is never opposed to using ALL the tools available to me... Why would I not use shot guns AND rocket launchers?? Harrier jets rule, but helicopters are still more practical. And sometimes you need to launch the stealth bombers.

Darpa said:

I’m also wondering if genetic engineering techniques have already been used to increase substantively the Glandular Trichome procuction of a plant through the overexpression of the homeodomain protein gene analogue GLABRA2 (GL2), leading to the promotion of trichome formation, or through the knockout of R3 MYB genes analogue in cannabis?

Click to expand...

Found this
A regulatory gene induces trichome formation and embryo lethality in tomato.

http://www.pnas.org/content/108/29/11836.full?sid=9cccc4d5-80df-4c7c-af3b-0a060e055480

shaggyballs said:

Found this
A regulatory gene induces trichome formation and embryo lethality in tomato.

http://www.pnas.org/content/108/29/11836.full?sid=9cccc4d5-80df-4c7c-af3b-0a060e055480

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Thanks ShaggyBalls for sharing this info.

Both Arabidopsis and Cannabis belong to the clades of Rosids. However, Tomato plant belong to a different clade (Asterids). Studies suggest that the pathway controlling trichome formation in the clades of Asterids may be different than the one in Rosids.

For that reason, relating to scientific publications associated with the trichome biochemical pathway in a plant model belonging to the clade of Rosid is probably a better start if our goal would be to create a plant that would increase glandular trichomes density, especially capitate-stalked glands, and increase substantially the total cannabinoid content of the plant in the same process.

Cannabologist said:

No one's doing anything like those things.. Most of the people you will find here are ideologically opposed to such science.

I on the other hand would LOVE to take my awesomeness and engineer something even better. Selective breeding is great but it can only take one so far, and I am one who is never opposed to using ALL the tools available to me... Why would I not use shot guns AND rocket launchers?? Harrier jets rule, but helicopters are still more practical. And sometimes you need to launch the stealth bombers.

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Hi Cannabologist, I'm glad that there is people like you that are interested in Biotech. I would have to get a closer look at the CRISPR technique you were referring to, since this technique was fairly new when I graduated and I didn't had the change to explore its possibility since then. I used to do most of my plant transformation via protoplasm electroporation with a BioRad electroporator… but I still have some experience with agrobacterium transformation...

Theres a lot more info out online on CRISPR than I could give you, even I don't know all that much as it has been developed well beyond my academic career at this point.

[FONT=Arial, Helvetica, sans-serif]Agrobacterium transformation of hemp has been performed and I believe is the way to go. Again a little ahead of my expertise but nothing that can't be learned in time. CRISPR apparently is not perfect- there are issues with cutting the DNA at the wrong base for example, and thus getting unwanted effects. There is much more that will come in the coming years, but it is exciting. Personally though on the human end I am more interested in stem cell and regenerative therapies lately, healing traumatic injury, and replenishing cells to vitality and optimum potentiality. Then from there, maintaining, or enhancing.
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I think CRISPRs main advantage is that it is so simple to work, for example with publicly available components (research non-profits at least), and basic lab gear, we could shotgun approach the gene overexpression and rapidly generate literally dozens or hundreds of transformed lines.
Problem as always is transformation and recovery of whole transformed plants. I looked intensively for publications, and though some have succeeded in cell suspension, I could only find a reference to hemp transformation and recovery presentation at a symposium in the 80s, with no details on methods. I see some tissue culture methods out there that do go through indeterminate growth, but efficiency is low. My understanding is that this more basic work would be the rate limiting step moreso than specific transformation technology applied, at least at this point in time...

This topic is very interesting, any more on it?

Well we went from a plant where the only people who didn't smoke it were ones who couldn't handle the pungency, to a plant that needs modification to resemble it's natural self. Yet we have hero's and celebrities in this scene?

Increased Linolenic acid synthesis is my goal,since it's acids that contribute anything worthwhile, not terpenes. Terpenes are the bottom of the chain, byproducts. I dont care if it smells like a Navel vs a Bergamot, if it tastes like perfume instead of an orange. Corporate propaganda is bad science. At the current rate of BS being pumped by future Cannabis producers of America (Philly Morris), sweetness/acidity of tomatoes will be pinned on terpenes rather than sugars and acids in future publications.

There's a lot of work to make this area functionally applied in production. 35s as a viral promoter is dangerous and not usable for production for instance. The promotion is the trick, it's not easy to perform although crispr helps tremendously here. I've worked a bit with research in this field in other crops recently, I wish I knew more about who was doing what with cannabis transformation

Darpa said:

Hi, since we are in the advance growing science thread, I wonder if some of you have applied genomic or genetic engineering approaches to increase plant secondary metabolism (or have an opinion on the subject, and please I don't want to start a debate about GMO, please!).

Are you aware of any work related to the overexpression of genes involved in terpene synthesis, specially monoterpene synthases, a (-)-limonene synthase (CsTPS1) (a sesquiterpene synthase-encoding gene involved in citrus aroma formation) and a (+)-α-pinene synthase (CsTPS2), with 35S CaMV promoter?

At my understanding, all the genomic information regarding the CsTPS1 and CsTPS2 are already accessible through the National Center for Biotechnology Information (Cannabis sativa (-)-limonene synthase mRNA, complete cds, GenBank: DQ839404.1, https://www.ncbi.nlm.nih.gov/nuccore/DQ839404.1). This could lead to the creation of a really tasty strain…

There is a good publication on the subject entitled «Functional expression and characterization of trichome-specific (-)-limonene synthase and (+)-a-pinene synthase from Cannabis sativa, Article (PDF Available) in Natural product communications 2(3):223-232 · March 2007.»

I’m also wondering if genetic engineering techniques have already been used to increase substantively the Glandular Trichome procuction of a plant through the overexpression of the homeodomain protein gene analogue GLABRA2 (GL2), leading to the promotion of trichome formation, or through the knockout of R3 MYB genes analogue in cannabis?

Click to expand...

Darpa in my post pages first thread, forum aims. I cover in great detail about the study of microbes having an effect on cannabinoids. It was determined that the greater the microbial colonies in the soil, the higher the cannabinoids level and phenotypic expressions. Please read all my posts there. The information shared is priceless to cannabis growers.
Peace farmerlion

Hello, I just posted a few days ago about the work verified by a couple other doctors. The cannabinoids percentages rise with the higher percentages of microbial species/colonies in the soil food wed. In the thread Forum Aims I cover in great detail cutting edge techniques that out perform all other types of cannabis cultivation.
peace farmerlion

P. Dondrub said:

I think CRISPRs main advantage is that it is so simple to work, for example with publicly available components (research non-profits at least), and basic lab gear, we could shotgun approach the gene overexpression and rapidly generate literally dozens or hundreds of transformed lines.
Problem as always is transformation and recovery of whole transformed plants. I looked intensively for publications, and though some have succeeded in cell suspension, I could only find a reference to hemp transformation and recovery presentation at a symposium in the 80s, with no details on methods. I see some tissue culture methods out there that do go through indeterminate growth, but efficiency is low. My understanding is that this more basic work would be the rate limiting step moreso than specific transformation technology applied, at least at this point in time...

Click to expand...

when I looked around in the literature for the possibilitires of modification in cannabis a while ago I indeed found the tissue culture step was the problem.
it could be some more private/commercial labs do have working protocols, or there are protocols that work only for some varieties.

but in general what you'll find in the literature is that almost everything worked, but I think shoot regeneration failed (for modification you go back to one/a few undifferentiated cells, callus. after transformation you let those grow, you now have a clump of callus. but now this callus needs to turn back into a plant, so you supply the right hormones for it to first grow a shoot, then change the hormones so it grows a root too). as far as I remember root regeneration did work, it was only the shoot regeneration that has not been figured out yet.

however, there would be ways to get around that, to achieve gmo without going through a tissue culture step. for example with agrobacterium in arabidopsis there's the 'floral dip' method, I think that could work in cannabis too.
I also know of people working on modifying the pollen (then use that for pollination, and you also don't need tissue culture).


btw, regarding the strategy in the OP, I'm not sure overexpression of TPS genes would really be the way to go.
you'd first want to know where in the pathway leading to those terpenes the limiting step is. if for example the limiting step is the substrate the TPS works with, increasing expression of the TPS will have no effect at all.
so you need to dive a bit deeper into the pathway, not consider only the TPS genes.
TPS genes might be interesting to alter with crispr though, since very minor changes could change how the enzyme works (i.e. what terpenes it makes), so you could add specific point mutations with crispr and change their function.

I've recently been researching inducing defense terpenes through both mechanical wounding and hormonal such as methyl jasmonate. Using delta ³ carene as an example here.
Fiber hemp strains from andong korea were noted by hillig in 2004 as being extremely high in carene. Since its a major terp in jack the ripper I started looking into it. I found that korean pine trees are susceptible to specific pine tree nematode. Now defense terpenes can either be constant(deterrant) or reactionary (induced defense). Frankincense is a great example it is procured by physically wounding a boswellia tree and the resulting oleoresin is allowed to dry on bark then scraped off.voila Frankincense
. Now back to the carene. I found a study which exposed a Korean pine tree to said nematodes and the delta carene content exploded going from barely registering to huge content. Now this opens up a whole world of possibilities and also could help a lot with taxonomy. By studying terpene synthase families, samples of known geographic origin, and terpene entourage effects targeted growing strategies could allow for tailored cultivars without genetic manipulation. Landrace microbial soil content, local herbivores, predator attraction of herbivotrd enemies, pheromones for pollination or plant to plant communication the possibilities are quite exciting. Similar to the oolong tea method discoveries. Dna methylation and epigenetic dosage/regulation may be much more important then just mapping chromosomes. Here are the hillig paper and carene pape

https://www.google.com/url?sa=t&source=we b&am p;amp;rc t=j&url=https://static1.squarespace.com/static/5dab51c52920995e635d4295/t/5e148f35553f9b060f30f53a/1578405686727/Hillig2004BiochemSystematEcol. pdf&ved=2ahUKEwiKt 6mpx M_1A hWIl4kEHd2QChMQFnoECAoQAQ& amp; amp; usg=AOvVaw2zzq1XNHEZ7_173MDGX0 9g

https://www.mdpi.com/1999-4907/12/5/514/htm

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Piff, the fertilizer and pesticide empire have steered this specific sect of cannabis breeding for decades. We see the exact same thing everywhere we look. Most Americans will never taste a Haze.


Most Americans will never even taste corn, no matter how often they eat it. Example: American grasshopper spit contains sulfated fatty acids that increase caryophyllene in non American corn. American corn has no caryophyllene gene. American corn also has no flavor. American corn has also no nutritional value. American corn has also no pest defense mechanism. American corn is bred to benefit the fertilizer and pesticide empire. Breeding at its finest in the eyes of many on this site.

Americans have historically stripped the nature out of everything natural, possibly to 'renature' at a later, controlled date? Or maybe to create a customer pool with no connection to or expectations from natural products? Or to patent caryophyllene corn, the natural form removed from Americans minds long ago?