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Cannabis in tissue culture


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#21 drmcnasty

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Posted 22 January 2017 - 06:50 PM

This writeup is very well put together, expecially since English is not your primary language. I will be following this closely and playing along when time permits.

#22 Hash_Man

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Posted 22 January 2017 - 08:15 PM

Does anyone think nano particles of gold could be made by using 2 gold electrodes in distilled water, running like 9 -12 volts DC ...."


Are you wanting to make colloidal Gold?

You will need the following materials.
1) .999 fine gold wire anode, of about 6" to 1 foot in length.
3) Sodium Chloride (Table salt)
3) Sodium Citrate (Also called tri-sodium citrate)
4) Ordinary Corn Syrup.
5) DC Power supply capable of supplying 500ma @ 30volts1
6) 500 ml pyrex beaker
7) Hot plate with magnetic stirrer
8) Distilled water.
9) Lab supplies, scale, graduated cylinders,supports for the electrodes

Well here's the link I've only done colloidal silver many years ago

https://www.cgcsforu...rticles/CG.html
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#23 Heirloom Spores

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Posted 22 January 2017 - 10:35 PM

I've done colloidal silver with only silver electrodes and distilled water . I will find my electrode and show the silver floating on top of the distilled water when let go overnight, a little more time than needed. I should try it with 24 carot gold and see what happens.



#24 Hash_Man

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Posted 23 January 2017 - 06:20 AM

I've done colloidal silver with only silver electrodes and distilled water . I will find my electrode and show the silver floating on top of the distilled water when let go overnight, a little more time than needed. I should try it with 24 carot gold and see what happens.

I DK bro, their saying that you'll need more voltage than 12v
2017_1_23_6_15_56_914_AM.png

Edit: can liken making colloidal Gold to growing mushrooms, in that easy to learn hard to master . . There's some small but important details i.e. spacing of the electrodes the surface area and color etc.
Here's a video and link to a relating thread

https://www.cgcsforu...x.php?topic=7.0

[Direct Link]


Edited by Hash_Man, 23 January 2017 - 10:10 AM.


#25 JanSteen

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Posted 24 January 2017 - 01:00 PM

I have been digging into the gene gun and it's possibilities.. Not long after that, my dreams were shattered by reality. I'm going to do the same to you now. Be prepared, because it might hurt a little. I did not choose my words carefully, because I want this idea out of your systems. Keep that in mind. Also, please, think about what I say. My linguistic skills might not be the best, but it's about the message, the thought process.

Let's do this step by step to see if it works, starting enthusiastically:

Being a little educated in genomics and genetics. I mean, I have done some transformations in the past and it was hell of a job to do it right. I'd say: the idea is nice, but you're wanting to put a specific gene and all of its related pathways(!) into another plant cell. That would work if you know what kind of genes you'd need, and where to get those. That's the mayor hurdle. That information is unavailable at the time. There might have been advances I'm unaware of, forgive me for that. But let's just keep going.

Hurdle 2: First of all, THC for instance, is produced locally in the THC glands (modified trichomes so to speak) by mitochondrial DNA (to my knowledge). How are you going to get something out of a polymerized organic resin globule without damaging the internals all too much? Then, how do you want to find the genes responsible for producing anything in a plant? Do you have a clue? If not, go ahead and try to understand qPCR and related tests, go for some statistics as well, you'll need it.

Hurdle 3: Then, what part of the gene and what other genes are necessary for a plant to produce THC? You'd have to get those genes snipped out of a normal chromosomal strand. You'd need to find markers and the proteins that cut at that location. It might just be the case that you'll have to use entire mitochondria to get things working. How? Beats me!

Hurdle 4: Then you'd need to know what your target is, and where this gene in the host organism can be placed to become and stay functional. For that, you'd need to understand bioinformatics and you'd need the genome or parts of the genome of your original plant. This means you will need to know how to search and use databases, how to target and test for genes, protein composition and functions, pathway principles, gene mapping and loads more.

When you're at that point, you would have spent a few years researching and a couple of thousand dollars. You have everything worked out, from donor to host, from starting point to endpoint in chemical additions and changes caused by all of the related proteins.

Hurdle 5: THEN and only then, it's time to start thinking about a gene gun. Nanoparticles are expensive but can de made from wolfram as well, they would need to bind to the DNA you're using. Add a couple of hundred bucks on a cleanroom and chemicals while you're at it. But, are gene guns better than other methods? I don't think the gene gun is any good compared to other methods.

Hurdle 6: Then, there's something you'd need to do as well: figure out how to recognise/distinguish between the cells that have successfully transformed and those that didn't. There are some antibiotic resistant pieces of DNA readily available. And you would need to build those parts in as well. Otherwise the dominant non-infected cells would overtake and reject the rest, basically resetting all your work.
Random gene transfers have a success rate of something below 1*10^-15 %. Good luck on that!

Hurdle 7: From there on, you'd have to regrow your plant from a single batch of cells. Knowing that some people around here even have trouble with unopened autoclaved mason jars that go haywire, I'm not sure if we would even get to this stage.

Hurdle 8: You will never be able to get credit for it, unless you want to accept a lifetime in jail for producing, manufacturing, selling and owning unregistered GM organisms that possibly pose a threat to native flora and fauna. One of the biggest offences in the biological world. Most biologists and biochemists don't even care all that much about native flora and fauna, but they do have some serious opinions about using GM for something that doesn't necessarily improve the world. Personally you might think it's an improvement, there are about a billion people that think otherwise. People who are actually in politics and decide what kind of decision judges make.

Total costs without extensive prior knowledge: around 40K USD. And 2 years of studying.

It seems easy, but it's a job of nearly 2 years to transform a plant if you want to make it economically worth your while. Economically? Yes, let's talk about that. The current case is: we have plants that yields within 3-4 months, they can grow in various kinds of media, various kinds of pH ranges, various kinds of light levels and it grows on nearly every continent.
Again, I have been thinking about this, and worked it out from A-Z only without knowing the genes, with focus on procedures and costs. The only thing left for me to say is: I have never found a more reliable plant, nor one that grows as fast and is versatile as cannabis. There is literally 0 use of putting interesting cannabis genes into other plants, since it would be a downgrade compared to the original plant. What would you like? Bamboo with THC? Bamboo that takes 3 months to germinate? Or rather keep a quick AF strain that yields within 4 months? Of course, it sounds awesome. It sounds like reinventing the wheel - which is actually a good analogue for making something already existent even better than its predecessors. But do we really have a need for it? Why?

I would love to hear more than 5 reasons, arguments, whatever, to pull this off. I haven't been able to hold and defend 1 good argument to do it. This is possibly because I am an asshat, but I have worked out a few points for you:
- Legality? Meh, it would be illegal by the time the organism is done. Also, my country's law states that any product containing THC is considered illegal, it doesn't matter what organism. Result: 0 value for me to change other organisms.
- Speed? We're talking about a C4 plant that outgrows corn.. Cannabis even outgrows most grass species.
- Yield? Use that 40K USD to expand your fields or grow room and what was that about yield again?
- Quality? Are you going to suggest that top shelf weed can be improved with the taste of something like lettuce? You want more sugars and volatile components in your burning weed, producing chemicals that might harm you?
- Availability? Cannabis is hard to grow in some places, but so are many more natural products. Transport costs are around 15 bucks for nationwide shipping up to a kilogram - enough to last you a year.
- Cost reduction? Come on guys, I plant 10 seeds in my back yard, seeds which I bred myself. Total annual costs: 30 bucks on materials to yield a laundry crate full of ganja. A good commercial grower has a production cost of around 30 cents per gram and profits nearly 300% of that.
Not a single argument can hold. There is no use to it. Think about this: we have the opportunity to improve the cannabis plant, why would we want to put pieces of cannabis in other plants, when we can improve the cannabis itself? Why aren't we reversing the idea?

I mean, I've been doing this all wrong all of my life.. Back in 2008 I was hoping to put psylocin genes in yeast, and I had it all worked out. Then I figured; why not select a more resistant and better producing strain of cubes? If I need psylocin, why not do it the right way? Why make expensive bioreactors and awful extraction protocols which are way more expensive, to get just a little increase in total yield? Is there something to gain compared to regular cubes? Not long after, I found a cheaper rye seller, I found that reusing materials cut costs, and right now I grow my own cubes for less than 10 cents per trip. Do I really need to spend 2 years slimming down that 10 cents to something like 7,5 cents? I'm not even selling drugs! I'm not even making money off of it.. And the one time I did, I was able to sell 3 grams for 55$ already.. That's nearly 54,90 on profit.

If your argument is: I have the money, the time and interest, and I just want to see it done, simply for the sake of it.. Then hire me. For the same amount, I could also do a lot of other things, like producing potable water from sea water with solar power.. If you have that kind of money to spend just for the sake of it, I would also be most happy to revert my current research and improve existing cannabis strains. Or just create a new one from scratch..


Now, back on topic. This is a tissue culture topic, started with the idea of telling and discussing about tissue culturing. The starting post was pretty clear, and I have the feeling the subject is going to be lost if I don't point this out. GM is an entirely different subject, though it's related, it has nothing to do with "in vitro" on it's own. I want to keep those separated, because it might scare some people. It might also give the wrong impression of what I'm trying to describe.
Working in genomics, I found that the majority of the population has no clue of what we did. They thought we were making frankenstein cattle, that we were producing killer algae or plastic munching fungi, when in fact, we were just mapping biodiversity. I want to shed that idea, and let separate things be separated. I simply don't want talk about GM in a tissue culture topic. I am being a bitch about it, I know, but it's for our own interest. If the seed of an idea is planted now, the idea that tissue culture equals GM, then whatever grows out of that seed might work against us in the future. We're far, far away from curing cancer because of some of those seeds. We're on the verge of wiping out Ebola and Zika because of some other seeds.

If you want to talk GM, no offence even though I might/do imply I'm offended, please do that in another topic. Don't forget to mention agrobacterium tumefasciens and CRISPR-CAS9, since those options are economically way more accessible and have a higher success rate than gene guns. Agrobacterium can be found in nearly every forest, for free. Electroporation is even cheaper and easier. Safer as well. As is protoplast fusion, which can be done for a little less than 200 bucks. But not here. Please, not here.

With all due respect, let's keep it about tissue culture, and tissue culture alone.

#26 JanSteen

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Posted 25 January 2017 - 07:31 AM

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See above for starters. All in the first week / day 1

 

 

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gallery_149763_5_71666.jpggallery_149763_5_280545.jpg

Reculturing and whatnot. Media with a green coloration is coconut water, plain, used straight from the carton after sterilizing. Of course I added 1,5% agar.

 

Let's see if these calli will grow up to be plants in plain coconut water.

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And we'll have to see what happens if you don't move away from callus forming media.

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#27 JanSteen

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Posted 25 January 2017 - 07:35 AM

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Establishment of callus culture from seed. You see here that the auxins in the seed itself, are stronger than the hormones in the medium. It will balance out eventually.

 

gallery_149763_5_676018.jpg

Mulitplication media does seem to work; branches are forming. However, there's too much BAP in it. The formation isn't as nice as with lower levels.


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#28 Heirloom Spores

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Posted 25 January 2017 - 01:21 PM

 I offer a sincere apology for posting a tangent in your thread, I meant no offence to you. I am responsible.

Maybe a Mod can remove offending GM talk.


Edited by Heirloom Spores, 25 January 2017 - 01:43 PM.


#29 Hash_Man

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Posted 25 January 2017 - 02:05 PM

I offer a sincere apology for posting a tangent in your threa..."


Yea me too, I was part of all that wow I thought I was contributing, oh well one man's contrabution is an other man's interruption won't let it happen again so sorry no more tangents here.

BTW Heirloom good word 'tangent' , I had to look it up, soon I'll be fully educted . . LOL
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#30 JanSteen

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Posted 26 January 2017 - 10:14 AM

As I said, I'm not offended, though my text might imply that. :) Don't apologize, there's no need for it. I just wanted to make some things very clear, the way I did it, is not the best. But I hope the message has been received. I think it's better to leave this topic as is, because some people might find it useful to read.

Some extra commentary about me flipping out:

It's not that I don't want to discuss GM, in fact, I'd love to! But I want to make it clear that the subjects are separate ones.
GM is 'actively messing with genetic codes' of organisms, tissue culture is 'making good use of the pre-existing genetic code' of organisms. In other words: rebuilding a freak computer with parts from cars and what not, instead of using a store bought computer.

Where me being an ass about it comes from:
I have noticed that a lot of people see the two practices (GM & TC) as one, which in my opinion causes harm to the field of work. I feels like crap to get spray painted, covered in buckets of cow blood by activists when you work at a place that only wants to make crops and livestock more reliable, healthy and safe.
One time I was researching how to improve cattle farms by introducing automated petting machines and something like chill-out-corners (with some objects that could act as a blanket, and a relaxing soft cushion) for veal. It was actually a side project we did as a genetic research center. Some activists cut my tires, cut my brake wires and spray painted my car, simply because they held the idea(!) that I was doing something else (because of the word Genetic).
I was trying to improve the little animals' way of life for as long as it lasted, and some German creeps nearly ruined mine.. Somebody planted an idea there, and somebody else figured it was 'the right thing to do' based on wrong assumptions.. Dealing with that on a daily basis makes me kind of pissed off sometimes.
When we decided it was time to improve disease resistance in crops, based on heirloom genetics (using the wild type which is resistant to mildew for instance to improve domesticated types by crossing them) the same thing happened. We received daily death threats, and nobody could do a thing about it.. Why? Because people seem to think that everything related to tissue culture and genetics automatically means practising GM. They hold scientists responsible for ruining parts of Africa, while the majority is actually trying to undo the damage caused by big a few (two, to be precise) corporations.

The distinction should be made utterly clear in my opinion, because intertwining the two practises has nearly cost me my life on multiple occasions.
I know, you couldn't have known that. I should have mentioned it! I'm to blame here. But let's forgive and forget, and just keep on culturing. That's what it's about here ;-)
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#31 JanSteen

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Posted 30 January 2017 - 10:43 AM

Coconut water proved to be too heavy on the plantlets and calli. I'm keeping some in the medium to see how they manage, next time I'll use 1/2 coconut water and 1/2 demineralised water.

#32 TVCasualty

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Posted 02 February 2017 - 01:03 PM

I have been digging into the gene gun and it's possibilities.. Not long after that, my dreams were shattered by reality. I'm going to do the same to you now. Be prepared, because it might hurt a little. I did not choose my words carefully, because I want this idea out of your systems. Keep that in mind. Also, please, think about what I say. My linguistic skills might not be the best, but it's about the message, the thought process.

 
My comment about the gene gun was more speculative musing than anything else, so I had no real expectations to dash. Still, an imagination that's unencumbered by practical experience can sometimes shatter reality with its dreams rather than the other way around (e.g. Einstein, though I'd grant that that sort of thing happens at about the same rate as beneficial mutations occur in nature, which is almost negligible).
 
Also, I can assure you that I don't conflate genetic modification with tissue culture and didn't mean to give that impression. I tend to dispense with caveats and nuance in replies (to a fault, obviously) since I also tend to write really long posts and being as unambiguous as a Journal article would make them even longer, though less potentially misleading. It's a tricky balance, after all.
 
As to producing THC with callus, I brought it up precisely because I didn't know how realistic/viable of an approach it would be. I have read that callus can produce the secondary metabolites that the actual plant would produce, and that that's how most of the active or presumed-active compounds found in some species (e.g. polysaccarides, various phenolic compounds, and echinacoside) are currently produced (i.e. in vats, not from echinacea flowers). Of course, that doesn't mean that all plants would be able to do so.
 
So if cannabis can't produce THC in culture, can Mimosa hostilis or any of the other DMT-rich species of plant or tree produce DMT in culture? How about Peyote and it's desirable molecules? If so, either one would be revolutionary (literally!).
 
 

 

It seems easy, but it's a job of nearly 2 years to transform a plant if you want to make it economically worth your while. Economically? Yes, let's talk about that. The current case is: we have plants that yields within 3-4 months, they can grow in various kinds of media, various kinds of pH ranges, various kinds of light levels and it grows on nearly every continent.

- Cost reduction? Come on guys, I plant 10 seeds in my back yard, seeds which I bred myself. Total annual costs: 30 bucks on materials to yield a laundry crate full of ganja. A good commercial grower has a production cost of around 30 cents per gram and profits nearly 300% of that.
Not a single argument can hold. There is no use to it. Think about this: we have the opportunity to improve the cannabis plant, why would we want to put pieces of cannabis in other plants, when we can improve the cannabis itself? Why aren't we reversing the idea?

 

That sounds a lot like arguments against installing solar panels back when their cost was so high that one would likely never break even on the investment. But slow, expensive, and difficult gradually develops into faster, cheaper, and easier. It's like the old saying that "pioneers get slaughtered, settlers prosper."

 

So sure, developing something novel and unprecedented is going to be costly, inefficient, and tedious, but that's what scaling is for once a successful approach is found, assuming such is possible (and that often requires a lot of expensive failure along the way).

 


 

With all due respect, let's keep it about tissue culture, and tissue culture alone.

 

That's cool, I didn't mean to send it on a tangent. For what's it's worth, I don't actually intend to attempt to cross cannabis with kudzu.

 

I'm a lot more interested in grafting cannabis onto hops rootstock. And with that, my segue back to the topic is accomplished. Please carry on, I'm learning a lot in this thread and the effort is appreciated.

 


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#33 Heirloom Spores

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Posted 02 February 2017 - 02:34 PM

The gene gun thing started out as more of a joke.

This is good for those who have dreamed of tissue culture.
now they can do it.

Jan you are helping out people who might likely  use tissue culture.

 



#34 Heirloom Spores

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Posted 03 February 2017 - 01:18 PM

I believe this will make it into the Archives.






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