Is there anything like working on agar but with plant cultures?

Milky Weed

Well-Known Member
Is it possible to grow plant cells out on a petri dish like we do with bacteria and fungi to select the most vigorous genetics?

I used to do agar work with mycelium. is this impossible with plants due to how they grow? I know tissue cultures are a thing but I’m not sure how far they actually break the plant down.

Thanks for any and all answers.
 

hotrodharley

Well-Known Member
Is it possible to grow plant cells out on a petri dish like we do with bacteria and fungi to select the most vigorous genetics?

I used to do agar work with mycelium. is this impossible with plants due to how they grow? I know tissue cultures are a thing but I’m not sure how far they actually break the plant down.

Thanks for any and all answers.
Tissue culture. Read about it. Or ask @jimihendrix1
 

jimihendrix1

Well-Known Member

SCJedi

Well-Known Member
Tissue culture takes it back to the very beginning-embryonic stage.
I'd be careful about making this statement without clarifying the differences between nodal tissue and meristematic tissue.

Nodal culture is merely aseptic stem propogation.

Meristematic tissue is not technically connected to the plants vascular system and culturing it can reset the epigenetic clock.
 

Milky Weed

Well-Known Member
I'd be careful about making this statement without clarifying the differences between nodal tissue and meristematic tissue.

Nodal culture is merely aseptic stem propogation.

Meristematic tissue is not technically connected to the plants vascular system and culturing it can reset the epigenetic clock.
Very cool. Thanks. Meristem cultures seem to be the most advanced, taking the plant down to its very essence.

Meristem could not really be used for propagation could it?

what I’m more curious about is if the meristem cultures get broken down to the point of individual cells growing on agar.

I saw a few videos of this, and it looked like meristem cultures were still technically a plant? I want to know how far they can be broken down. Does anyone grow cannabis plant cells on agar in thin sheets for anything useful?
 
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testtime

Well-Known Member
Very cool. Thanks. Meristem cultures seem to be the most advanced, taking the plant down to its very essence.

Meristem could not really be used for propagation could it?

what I’m more curious about is if the meristem cultures get broken down to the point of individual cells growing on agar.

I saw a few videos of this, and it looked like meristem cultures were still technically a plant? I want to know how far they can be broken down. Does anyone grow cannabis plant cells on agar in thin sheets for anything useful?

Vigorous growth that we select for in mycelium is an indicator of how well the whole organism will do. That's meaningless for cannabis. The final trichome production is what we are after. So those little itty bitty cells are used to store large quantities of plant genetics. You can do a breeding program of plants and take samples from them and be able to reproduce that plant if you determine that particular plant is the one you are after. And occasionally pull them out and apply the chemicals that say you grow a leaf and you grow a root and then it becomes a plant.

So you can have thousands of clones going in the refrigerator.

Other than that I don't see any production use for this other than basic biological research.
 

Milky Weed

Well-Known Member
Vigorous growth that we select for in mycelium is an indicator of how well the whole organism will do. That's meaningless for cannabis. The final trichome production is what we are after. So those little itty bitty cells are used to store large quantities of plant genetics. You can do a breeding program of plants and take samples from them and be able to reproduce that plant if you determine that particular plant is the one you are after. And occasionally pull them out and apply the chemicals that say you grow a leaf and you grow a root and then it becomes a plant.

So you can have thousands of clones going in the refrigerator.

Other than that I don't see any production use for this other than basic biological research.
Thanks
 

SCJedi

Well-Known Member
Very cool. Thanks. Meristem cultures seem to be the most advanced, taking the plant down to its very essence.

Meristem could not really be used for propagation could it?

what I’m more curious about is if the meristem cultures get broken down to the point of individual cells growing on agar.

I saw a few videos of this, and it looked like meristem cultures were still technically a plant? I want to know how far they can be broken down. Does anyone grow cannabis plant cells on agar in thin sheets for anything useful?
When you excise the meristematic dome you're working with a cluster of undifferentiated cells about 0.25mm in size. It's maybe a few hundred cells at most.

Cannabis is totipotent which means, theoretically, a single cell can grow into an identical replica of the plant it was sourced from.

Let's stop for a second to talk about somaclonal variation and mutagenesis. If you have a cutting, you have a plantet with most of its organs intact. Leaves and leaves, stems are stems, etc. 100s of thousands of differentiated cells make up that plantlet. The chance that something significant occurs and caused a genetic mutation that affects the genotype of that plant is small. Very small.

Now let's re-visit that small cluster of meristem cells. They aren't organs yet. They are a blob of stem cells waiting to be told what to do through hormone signaling pathways. If something goes wrong and mutated in that teeny cluster of cells, when they differentiate the possibility of an altered genotype expression through chromosomal rearrangement is a possibility. This is called somaclonal variation and that new plant is no longer true to type. In lay terms it means it IS NOT and identical clone anymore. This could be bad, it could be good but if the goal is the reset the epigenetic clock and remain true to type then it is bad. Fingerprint in, fingerprint out. That is how you know you've avoided it.

Finally, there is another way to reset the clock. You can take differentiated cells, like a leaf segment or a cotyedon, and undifferentiate it back into a cluster of cells called callus. Calli can be used for long storage and if you're R&D is on point you can re-differentiate callus back into an organ.

I have attached a picture of Wedding Crasher leaf segments that I turned into callus and then signaled it to start growing into shoots again. This was very difficult to do. It's easier to get callus to grow roots than it is to grow shoots.

20211224_091927.jpg
 

Milky Weed

Well-Known Member
When you excise the meristematic dome you're working with a cluster of undifferentiated cells about 0.25mm in size. It's maybe a few hundred cells at most.

Cannabis is totipotent which means, theoretically, a single cell can grow into an identical replica of the plant it was sourced from.

Let's stop for a second to talk about somaclonal variation and mutagenesis. If you have a cutting, you have a plantet with most of its organs intact. Leaves and leaves, stems are stems, etc. 100s of thousands of differentiated cells make up that plantlet. The chance that something significant occurs and caused a genetic mutation that affects the genotype of that plant is small. Very small.

Now let's re-visit that small cluster of meristem cells. They aren't organs yet. They are a blob of stem cells waiting to be told what to do through hormone signaling pathways. If something goes wrong and mutated in that teeny cluster of cells, when they differentiate the possibility of an altered genotype expression through chromosomal rearrangement is a possibility. This is called somaclonal variation and that new plant is no longer true to type. In lay terms it means it IS NOT and identical clone anymore. This could be bad, it could be good but if the goal is the reset the epigenetic clock and remain true to type then it is bad. Fingerprint in, fingerprint out. That is how you know you've avoided it.

Finally, there is another way to reset the clock. You can take differentiated cells, like a leaf segment or a cotyedon, and undifferentiate it back into a cluster of cells called callus. Calli can be used for long storage and if you're R&D is on point you can re-differentiate callus back into an organ.

I have attached a picture of Wedding Crasher leaf segments that I turned into callus and then signaled it to start growing into shoots again. This was very difficult to do. It's easier to get callus to grow roots than it is to grow shoots.

View attachment 5053988
Now that is what I was looking for. thanks for taking the time to explain what tissue Cultures are capable of.
 

SCJedi

Well-Known Member
What up buddy? You like my setup? I'm going to get my first plants in-vitro this Sunday. I have old school blue dream, apple fritter, Sunday driver & GMO ready to go!
I've cultured 3 of those 4 strains. I have an apple fritter but it hasn't hit my personal threshold of health to take tips from yet. The Santa Cruz cut of Blue Dream was a pain in the ass to figure out.

Tips for your phytotech score pictured above:

Ditch the flip tops unless you modify them for a vent. You'll get some success but you'll be way more likely to see hyperhydricity without vented vessels. These are plants and not fungus so you typically benefit from some gas exchange.

Use the hemp multi kit but know there is not biocide in them so flex your aseptic skills if initiating to them, as opposed to transferring to them from initiating media. Reuse the tubes. I always liked phytotech's PP tubes.

Don't pay that much for sucrose. Just be a caveman and use table sugar, it's way cheaper.

Those MS pouches are convenient but will only allow 1L at a time. You don't really want to use a partial pouch. They should also be refrigerated. 2-8* C

I'm happy to provide some advice or tips if you prefer to do it here or in pm.

You've got a great start as I can see you have a used flow hood which means you are flexing some aseptic skills.

Good luck!
 
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Cool thread, when we going crsipr in some coca genes and make a Jeffery strain. Jk. I always thought microprop was situational. I have the gear but need to fix coolers. May be a solution to my sea of green problem. Hard to get enough clones of one plant at one even stage. Unrelated I bought a biological the other day for fixing N in non legumes, 500 bucks per gallon. Definitely gonna be culturing that shit. May try it on cannabis. Probably make it kinda harsh. Since we all overeducsted in this one, if a bacteria takes N from atmosphere in a sealed room, if it were truly vacuum sealed would we see a drop in pressure as the gas is converted to a solid?
 
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