I’m a plant scientist in the Los Angeles area doing research on cannabis tissue culture and genetics (mostly hemp for now, but hopefully marijuana in the future). I’ve been browsing around the conversations here and it seems like y’all have yourselves a pretty great community.
The cannabis scene is still pretty new for us scientists, most academic labs are still afraid of getting involved but that’s starting to change now. I just wanted to let everyone know that I’m here and if anyone has any questions regarding cannabis plant science don’t be afraid to ask.
Thank you Lorenzo,
Have you seen any chimeric properties from tissue cultured Cannabis? I am using tissue culture propagations now and have not but I am mad curious.
I’m not 100% sure what you mean by ‘chimeric properties’. In a plant science context a chimera refers to a plant which has 2 or more distinct genomes within the same plant.
Usually, every cell in an organism has the exact same genes, and differences between organs and tissues (eg roots vs leaves) are due to how those genes are expressed. In a chimera, the plant has different DNA in different parts of the plant. There are a few ways that this can occur, the two most common are from grafting and from badly-executed genetic engineering.
If you’re just doing tissue culture propagation, you shouldn’t have any issue with chimeric plants. This is because there is no source of ‘foreign’ DNA which could lead to differences in genetic code between cells. Every propagated plant has just one ‘mother’, so they should be pretty much genetically identical. This has been my experience with tissue culture propagation of cannabis.
I found this when researching TC (see link below). I was curious if you had ever seen this before. I believe your answer say you have not; and that is the good answer I was looking for.
Interesting article, I haven’t seen it before. The article is not so much talking about creating chimeras from tissue culture, but more the impact of tissue culture on plants that are already chimeric. They reference the phenomenon of clonal plants having different characteristics from their ‘mothers,’ due to the mothers being chimeric but the clones have segregated into the two (or more) genotypes found within the mother.
Ultimately the article is exploring how one could use tissue culture to segregate divergent genotypes within a chimera into different plants.
Keep in mind that when they talk about tissue culture in the article they’re not so much referring to propagation, but full regeneration. Regeneration is when you grow an entire plant from a single cell. This makes it possible to separate the different genotypes within a chimera, as you can regenerate two plants from the same chimera that have different DNA makeups as they’d come from single cells that have the different genomes that make it a chimera in the first place.
If you did normal tissue culture propagation on a chimera you’d probably just get more chimeras in most cases, but it would still be possible (with a little luck) to separate the genotypes in this manner.
Can you talk a bit about what kind of tissue culture (micro propagation?) techniques you’re finding to be most successful with cannabis? Is there a medium and set of procedures that you’ve found the greatest success with? How about meristem culture and scrubbing viruses?
There’s so much I still want to learn more about all aspects of cannabis. Thanks for sharing!
Right now, I’ve only done nodal micropropagation for cannabis. It’s pretty similar to normal propagation, but you need less plant material (just a single node + 1-2 cm of stem below).
Take the nodal cutting from a plant (if it’s not already in tissue culture, you’ll have to sterilize it first) and put it into media containing shoot-promoting hormones. After you get good shoot development (a few more nodes develop), you can repeat the process to get more clones or transfer it to media containing root-promoting hormones and wait for roots to develop. Once roots develop, start hardening the plant by cracking open the sterile vessel you were growing the plant in and over the next week keep opening it further and further until the cover is completely off. Then, carefully wash the media away from the roots and transplant it into soil (or equivalent).
I haven’t done meristem culture for cannabis yet, but it requires much more babysitting, as you’re dealing with less overall plant tissue so it’s a lot more fragile. However, that would probably be the way to go for scrubbing viruses, as that less amount of tissue means you have a higher chance of starting with a piece of plant that is virus-free. In theory, you could scrub a virus using the nodal-propagation technique, but you’d have to put it through a lot more propagation cycles (because the starting material is so much larger).
To get into the specifics of materials and such: The media I use is a combination of MS media (with vitamins) and agar (~7g per liter of media). You should use enough agar so that the nodal segments can stand on their own, but you don’t need to press hard to get them into the media. Autoclave it, then mix in whatever hormones you’re using when it cools to 60 C. Finally, pour the media into your vessel of choice (I use magenta boxes) in a laminar flow hood to ensure sterility, and let it cool.
For hormones, I’ve experimented with a bunch of different ones. I’ve found that cannabis is so vigorous that (when using nodal segments) you don’t need a shoot-promoting hormone, you can get good stem/node development just in the plan MS media. For rooting, it’s more difficult. You can use IBA (the active ingredient in Clonex), but I’ve also had success with meta-toplin.
Thanks for the detailed response, @twigresearch. I’ve been slowly building up my supplies and equipment to experiment with tissue culture. Only left to do is get my autoclave plumbed and wired.
Does your nodal propagation technique require any leaf area on the explant? Are you using the standard MS formula, or a modified version?
And thanks for sharing your experience with rooting. I use a powdered IBA for my clones currently and wasn’t sure if it would be needed for growing the plants in vitro.
Are you using tissue culture to expedite any genotyping projects?
No leaf tissue, just the node. The reason is that you want as little tissue as possible so it’s easier to sterilize. But make sure the node has ‘released’, meaning it’s started to grow already. Released nodes are powerhouses of hormone production, which is probably why I haven’t found the need to add any additional shooting hormones. Dormant nodes would probably require extra hormones in the media for shoot proliferation.
Just the standard MS formula (4.43 g per liter), but make sure it includes the vitamins.
At the moment, my lab is working on developing a genetic engineering protocol for cannabis as well as developing a method for cryopreservation of cannabis. These two projects are what we’re using tissue culture for.
What is the end goal your lab is working toward? A large genetic library with many varietals in cryopreservation? A nursery with hundreds of genotypes for customers to choose from to fill their commercial facilities?
This is a company local to me that is doing things very similar to you, I believe.
We’re a research lab, so our main goal is technology development. Once we’ve developed the technology we would either license it to other people or create a new entity to pursue the application of the technology.
For cryopreservation (if we were to keep the technology rather than license it), the goal would be to provide a service for growers to ‘back up’ their prized genotypes. I’ve heard some horror stories of people losing unique genetics to powdery mildew or broad mites. With this service people would send us a plants and we would keep them ‘on ice’ until needed. If they had a catastrophic crop loss we would pull their plant(s) out of cryo-storage and send them one or more clones to restart their grow.
We’re not really looking to be a nursery, as that’s not where our expertise is. We’re sticking pretty much to R&D.
Thanks for the clarification. I think the company I linked above would be of interest to you, as they are currently offering their “biobanking” and “failsafe” services, genetic storage and a full back-up crop kept in vitro respectively.
Yeah, that service seems very similar to the idea behind cryopreservation. Although, keeping plants in tissue culture is much more labor intensive as they need a change of media every month or so. And, in tissue culture there’s always the risk of contamination. But, on the other hand, their turn-around time is much shorter than it would be for cryopreserved plants.