Cannabis tissue culture

Can't express the love I feel for you oregon skunk pharmers. Thanks for the info you guys continually freely provide.

Like gi Joe says... Knowing is half the battle

wow! great info everyone. i love these science threads.

we are fortunate to live in oregon and to have a tight group of scientists working together, the skunk pharm.

our attorney advises that as long as there are no roots, it is not a plant. this gives grow geek some parameters under which to function. i wonder if there were no foliage, only root, if it would be considered a plant? hmmmm........

eloquentsoution said:

our attorney advises that as long as there are no roots, it is not a plant. this gives grow geek some parameters under which to function. i wonder if there were no foliage, only root, if it would be considered a plant? hmmmm........

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Just because it's in vitro doesn't absolve one of section 201.1 of the US Sentencing Guidelines:

"For purposes of the guidelines, a "plant" is an organism having leaves and a readily observable root formation (e.g., a marihuana cutting having roots, a rootball, or root hairs is a marihuana plant.)"

See also U.S. v. Miles, 319 Fed. Appx. 266 (4th Cir. 2009).

If anything TC, just gets you to that 99 plant limit just a little faster. Even callus tissue can have roots. Marijuana is still "considered all parts of the plant Cannabis sativa L.," including seeds.

Now, as for the all roots bit- look up hairy root culture. Of course, given that roots don't produce the compounds of interest, it's a moot point.

AZClones said:

Just because it's in vitro doesn't absolve one of section 201.1 of the US Sentencing Guidelines:

"For purposes of the guidelines, a "plant" is an organism having leaves and a readily observable root formation (e.g., a marihuana cutting having roots, a rootball, or root hairs is a marihuana plant.)"

See also U.S. v. Miles, 319 Fed. Appx. 266 (4th Cir. 2009).

If anything TC, just gets you to that 99 plant limit just a little faster. Even callus tissue can have roots. Marijuana is still "considered all parts of the plant Cannabis sativa L.," including seeds.

Now, as for the all roots bit- look up hairy root culture. Of course, given that roots don't produce the compounds of interest, it's a moot point.

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without doubt, we are outside bounds of federal laws. we are, however, making all efforts to stay within the bounds of oregon law, which is much more flexable.

azclones, are you saying the tissue culture shows root hairs and thus is considered a plant?

AZClones said:

Just because it's in vitro doesn't absolve one of section 201.1 of the US Sentencing Guidelines:

"For purposes of the guidelines, a "plant" is an organism having leaves and a readily observable root formation (e.g., a marihuana cutting having roots, a rootball, or root hairs is a marihuana plant.)"

See also U.S. v. Miles, 319 Fed. Appx. 266 (4th Cir. 2009).

If anything TC, just gets you to that 99 plant limit just a little faster. Even callus tissue can have roots. Marijuana is still "considered all parts of the plant Cannabis sativa L.," including seeds.

Now, as for the all roots bit- look up hairy root culture. Of course, given that roots don't produce the compounds of interest, it's a moot point.

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As you suggest, it is a crapshoot, but our attorney advises us that until it has a definable root structure, it is not legally a plant by Oregon law.

By our process, it will never have a root structure, until it is moved from the growing culture media to the rooting media, so it will never fall under section 201.1 until we have made that move.

Since there is no reason to move plantlets into the rooting media at a rate in violation of Oregon OMMP regulations, it appears easy to keep a library of any number of different strains without being in violation.

As ES notes, the feds dance to their own drummer, regardless of state laws and us having the legal right under state laws is inadmissible evidence in federal court.

By definition, all of us medical marijuana patients are at the very least civilly disobedient at a federal level, so we can either recognize that and back off, or exert our state rights by doing our legal research.

We have simply elected to pursue state legal research within 201.1 guidelines, without engaging in other flagrant violations of both state and federal laws that are more likely to draw LEO fire.

Ohmmmmmmmmmmmmmmmmmm!!!!!!!!!!!!!!!!!!!1

eloquentsoution said:

azclones, are you saying the tissue culture shows root hairs and thus is considered a plant?

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Every plant I've ever maintained in axenic culture has produced at least some root hairs after establishment. You might have to look for them, but they're there. I can only think of a couple of species that produced smooth callus after induction, but most had trichoids that were at least morphologically similar to roots. Admittedly, I didn't put them under the 'scope to see if they were leaf hairs or root hairs. I mean, you get some weird-ass growth out of callus tissue, but plants have to take up nutrients somehow. Cuticular wax doesn't allow for too much of that, so even callus can throw small roots and root hairs.

As a practical matter, I would not consider it unlikely that some clever prosecutor might try to impose a greater sentence on plants in tissue culture, if for no better reason that they would appear to present a greater "threat." From that, they'd say that X plants could be regenerated from a single callus, and therefore the imposed sentence should be greater than if it were a single plant. It'll make for an interesting test case.

since there is no clear answer to whether or not there are root hairs, and the likelyhood that some street officer will pull out a microscope to check for root hairs (or that he would know what to look for) is minimal, we will follow the advised of the most seasoned oregon state mmj defense attorney in this situation.

but i do thank you for the added knowledge and will advise grow geek to get out his best scope to check. we will fill you in when we have additional findings.

AZClones said:

Every plant I've ever maintained in axenic culture has produced at least some root hairs after establishment. You might have to look for them, but they're there.
As a practical matter, I would not consider it unlikely that some clever prosecutor might try to impose a greater sentence on plants in tissue culture, if for no better reason that they would appear to present a greater "threat." It'll make for an interesting test case.

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Thanks for the heads up bro! So far ours do not appear to have anything in the form of root structures, but looking at them under a microscope is a good idea and we will do so.

Valid point on no protection from clever prosecutors, not to mention unscruplous practices by Leo, and as you note, it would make an interesting test case. I hoping not ours.

We did see a cannabis attorney on the subject before experimenting, who opined that it was clearly defined as a non plant and that LEO is more focused on operations whose business practices are in violation of both state and federal law.

Our plan to ride in the center of the posse and keep our heads down, by staying within Oregon state laws and not becoming a thorn in the DEA's side with any other of our practices.

No doubt it is also likely that if cannabis tissue culture becomes widespread, they will simply rewrite the description of a plant.

Very interesting.

I have oft hypothesised whether it would be possible to trick our lovely lady into doing some interesting things in tissue culture.

Not really even if it is possible, because it is, more how and what are the hormones, growth regulators and other conditions required to get a MJ culture to produce large amounts of either THC and cannabinoids or Trichs in culture.

Here is what the meristem slices have turned into in just two weeks.

How should one preserve a tissue sample if they wanted to do this in the future?

Duplicate said:

How should one preserve a tissue sample if they wanted to do this in the future?

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The sample node has to be fresh cut, so I really don't know how you could preserve for treatment at a later date. Quick freezing might work.

I'll ask GG if he has any thoughts and perhaps we can freeze a sample first and then try to culture it after thawing. We might be able to quick freeze one with dry ice or liquid nitrogen, so as to produce smaller ice crystals that might not rupture as many cells.

After culturing, ostensibly it will live forever and continue growing without roots in the culture medium, so that might be a way to preserve it. You just take cuttings off it to subsequently root.

^ Thanks! I did a quick google search earlier and saw some articles about liquid nitrogen but I didn't even bother reading them as that would be above my head.

For long-term stuff, cultures are normally placed on minimal media; those that form tubers are the easiest- put them in a box, no light, and subculture every 6 months. Herbaceous stuff is a little more high-maintenance; minimal media with subculture at some pre-determined period (weeks, months). The main problem is if you have a fungal contamination problem in the lab (and all labs have these eventually), and you lose a lot of valuable stuff.

Perpetual subculture is normally employed versus cryogenic storage; I can think of perhaps fewer than half a dozen lines over >10 years where it was merited. Between the cryoprotectants and having to top off with LN2 every few months, it's a pain in the butt.

There's also suspension culture, but I don't know if anyone's pulled that off with cannabis. I don't know why they would as the benefits would be limited.

Chimera said:

Shoot Culture is neat and all but pretty basic TC wise.

The real fun comes with callous culture and shoot regenation. Callous is the real 'stem cells' or totipotent cells in plants... using callous and regenerating shoots opens up all sorts of possibilities.

Coming soon to a med state/country near you!

-Chimera

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What kind of possibilities, Chim?

The people want their frog eggs!

I've seen articles on cryo protocols for long term storage and I didn't give them much more than a glace because I don't have a -80 freezer or liquid nitrogen at my house.

I have kept cuttings viable for over a week in nothing more than ziplock bags stored in the fridge. One branch per ziplock bag, moist paper towel wrapped around cut end. Blow a bit of air in and close it up all puffed up.

I too have wondered about if there are actually root hairs on a callus or on the stem. I've been looking under a stereoscope and I can't see any obvious root formations. Mind you it was only 20x. I'm going to pull my Zeiss scope out of the closet sometime soon to get a closer look. I'll let you know what I find and also try to post some closeups.

ScrubNinja said:

Very cool, cheers for posting. Do you have a full blown lab, or how much trouble/expense did you go to to create a suitable environment to do this?

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Sorry I missed your post SN! Thanks!

No we don't have a full blown anything, including budget. Except for one student, we are all retired folks or playing at a hobby at our homes.

GG manages to culture just mocking up a sterile enclosure out of a plastic tub in his kitchen, but has gotten one bread mold contamination thus far anyway.

His interests actually lie in plant culture in general, not just with cannabis, so he has started dumping his toy allowance into expanding that hobby.

We looked at building our own laminar flow hood or glove box, but GG has found a used LF hood that he is negotiating for in a reasonable range, so we will wait and see.

Fortunately he is a bachelor and as I understand it, the color even matches his existing kitchen decor and will neatly fit in the dining area in lieu of a table.

AZClones said:

There's also suspension culture, but I don't know if anyone's pulled that off with cannabis. I don't know why they would as the benefits would be limited.

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Laminar flow hoods are readily available for a few thousand dollars- in fact a complete TC lab with flow hood, autoclave, glassware, reagents, supplies etc can be setup for less than 10K provided you have the space. In order to get serious about sterility (which is paramount if we're talking about storing highly valued genotypes in culture)... a flow hood and proper sterile technique across the board to avoid contamination is crucial. Grow Geek has done some great things with his home setup to work out the protocol, but the setup does certainly increase the likelihood of contamination.

The benefits of storage via suspension culture of callous are obvious, given you have a protocol to re-shoot the callous.

People have been culturing cannabis in suspension since the early 70's. Callous from suspension cultures maintained in the dark @ 25 degree Celcius are easily sub-cultured in fresh suspension media without any agar.... Erlenmeyer flasks with liquid suspension media placed an a rotary shaker can be left unattended for a week or more while the callous multiplies, and subculture is very simple involving a splitting of the callous mass under sterile conditions and re-suspension in fresh media.

Callous is akin to stem cells; these cells can become any tissue with the correct PGR regimen... shoots, roots, etc.

Callous cultures also can be maintained and multiplied on agar in petri dishes without the need for light... stack em and pack 'em... we're talking about the ability to keep 100's of unique genotypes in 'stasis' in the size large desk drawer, with only minimal labor costs in terms of re-plating.

Cryo storage doesn't work well with plant cells, due to the formation of ice crystals combined with the rigidity of the plant cell wall doesn't allow for expansion to accommodate the crystals which form and expand when the water inside the cells freezes.


The frogs eggs are a done deal Imona; the only barrier to making these available across the board is a legal one... no current seed sellers are willing to list them as they don't fit the definition of a seed... so the retailer would essentially be violating laws by 'trafficking in cannabis'. Makes international shipping and distribution... problematic.

Grow Geek.. bachelor huh? Sell the bed on craigslist and get rid of the bed and sleep on the couch! You'll have lots of space for the sterile room then... a few pints of blood and your well on your way to a serious TC production facility.

Keep up the great work,

-Chimera

Chimera, I have thought of selling my tv, but not my bed. I have (however) cut back on my scotch intake... Bumped it back to 12yr from 20yr! ah ha ha ha!

I'm slowly getting my culture lab setup. Saving pennies where I can.