Umamiseedco
Well-known member
I have been doing tc in house since 2015. I have learned a lot since then and will work to showcase how anyone (including you) can do it too.
Tissue Culture - Umami Style
- Thread starter Umamiseedco
- Start date
Rico D
Active member
Very generous of you to share your knowledge on this. I took a 4 day in person course over the summer and am looking fwd to setting up my own mini lab.
would love to know your experience with meristem culture and if youve personally seen it work miracles on old, tired cuts.
thanks!
would love to know your experience with meristem culture and if youve personally seen it work miracles on old, tired cuts.
thanks!
you.cannabis.do.it
Member
Explant is one of my 2024 goals! Meristem down the road.
Umamiseedco
Well-known member
you totally can. Embryo rescueVery kool!
Now if we could only use TC to pop old seed stock?!
Umamiseedco
Well-known member
Meristem is the only method of culture worth doing. Its not for the faint of heart, it takes me at least a year from start to finish. But cuts come back BANGING
grandpaBREATH82
New member
VERY interesting!
Please more info? Like...how do we do this?
Umamiseedco
Well-known member
the basic steps are to:
1) surface-sterilize the seeds (with bleach) to eliminate any microorganisms present on the seed coat. Make sure to wash the seeds in sterile water at least 3x and then soak the seeds overnight to soften up the outer seed coat.
2) You then dissect the seeds in a sterile environment, like under a laminar flow hood. The embryo has no immune system and is a prime target for bacteria/mold. This step is very VERY delicate as you will need to remove the seed coat to access the embryo inside without damaging or killing it. (no easy task)
3) Next, the embryo must be isolated from the surrounding tissues. Again, this is a delicate process and you must avoid any damage to the embryo.
4) Once you have a clean and (hopefully live and undamaged) embryo, you can transplant into tissue culture media and hope for the best.
This video is a pretty good demo on the excision.
What you are starting with
vs what you are looking to put into culture
Danimal Chin
Member
Looking forward to learning. Thank you.
Umamiseedco
Well-known member
My hood setup. Nothing fancy, just a blower, carbon + HEPA filters, and a uv lamp mounted on a husky 4x4 rack with a stainless steel sheet as my work surface. Mostly built from 2" insulation foam, this whole setup cost me a couple hundred bucks and works just as well as any of the commercial systems.
Sugarcreek
New member
Can we get more info on how you’re doing media prep? Are you creating different media by variety and if so how are you making those determinations? Just trial and error or is there an easier approach?
Umamiseedco
Well-known member
I wish it was easier, but it is a lot of trial and error.
I have found that most cultivars will tolerate most media, but to get them to thrive is another story. And some cultivars do really well in tc while others aren't happy even under ideal conditions. This thesis has several SOPs and formulations for media that I have found work well for the most part.
https://summit.sfu.ca/_flysystem/fedora/2022-11/etd21287.pdf
Umamiseedco
Well-known member
Payzos meristem culture finally showing some green. These start out as a small cluster of transparent cells and are really only visible with a microscope. Excising meristem cells is more art than science, but once you get them into gel, the cells grow super fast and will result in mother plants with crazy vigor.
Anthem27.5
Member
I have studied up on this process some what. I see you doing this in a manner that is designed to create a clean genetic expression of the plant. I have viewed several different attempts at TC that I did not feel were correct. I believe the idea is to start with a very small maristem piece if you are trying to clean up the selection. Most of the documentation I have previously viewed the maristem piece was approximately 2 to 3 inches. I have questioned using such a large sample because if the intent is to clean up the selection the younger and smaller the sample, the better the chance of cleaning up the selection.
I assume using smaller samples creates a much more difficult process of development. Is this your understanding?
Secondly, how much more difficult have you found it to be using small samples over larger samples?
Umamiseedco
Well-known member
If the meristem shows any green coloration (at all) then it is not really a meristem culture. The whole point is to collect only meristematic cells that are free from contamination and regrow the entire plant from that tissue. Those cells multiply incredibly fast and create very vigorous plants that are hopefully free from any past "scars" that the plant may have had.
The meristem is TINY and it is important to remove all other leaf around the cells as they can already be contaminated.
You can see how an infection will travel up the leaf first. You need to remove and culture only the meristem cells if you want a chance at getting a clean culture.
Umamiseedco
Well-known member
In this example, you can actually see the virus as it moves up the stem. The virus grows just as fast as the plant cells do, so you are trying to "chase out" the infected cells.
