Tissue culture cloning

Broseidon

Member
Hey there Bros and Brodettes,

so the new buzz in the bizz seems to be tissue culture cloning.

But it seems there is some gatekeeping going on with that technique at present...
Information is hard to come by and people seem to actively avoid speaking about it in too much detail.

For those of you who aren't all that familiar with this technique (like me) here is a summary of what I could gather so far:

- In tissue cloning you don't need to cut little clones (nodes) off of mother plants but instead a single leaf off of a plant can create several clones, just from the tissue culture of the leaf.
- The way it works (roughly paraphrased) is that tissue samples are taken from plants to the heart's desire (a mother that could yield say 20 or 30 healthy clones previously, can now yield hundreds of clones through tissue culture). These tissue samples are then labeled and stored in liquid nitrogen (this is the first whole I found, can't find information on how exactly they are frozen in liquid nitrogen, I doubt they are just thrown in there. Probably in plastic vials but what is in the vials, nothing? Probably some solution..).
- Once cloning is supposed to take place, the tissue samples are taken out of the liquid nitrogen and are further tampered with through reagents in a very sterile environment and then left to incubate (which reagents, how much, etc. I am unclear at the moment).
- A fully fledged clone will develop in a short time and the main advantage aside from space constraints is that all clones will be exactly the same in size, development stage etc. and there is more uniformity in the grow because of that. This has big implications on timetables etc. etc.


Now I am aware that this technique is not something for us home growers (I mean, who can realistically afford a liquid nitrogen storage solution at home?) but it is highly interesting to me nonetheless. I am always gathering information and looking to prepare for the day comes that things are legal and I might be able to start a business in the field.

And if that day ever comes, I would be highly interested in working with tissue culture cloning as I see immense advantages over classic cloning from the little information I could gather online.

So any and all information on the subject is welcome. Particularly:
- What equipment (liquid nitrogen storage, reagents, etc.) would be required and what would be a rough cost estimate to implement tissue culture cloning in a grow op
- How exactly is it carried out and what potential pitfalls are there (I saw for example that you should do the final step of applying reagents to start the clone incubation, after thawing from the liquid nitrogen storage, under a sterile hood like you find in laboratories in order to avoid contamination. Others just carry an isopropyl alcohol bottle around with them and constantly spray everything down until they are done ... Information welcome


And as always: Of course feel free to discuss anything surrounding this topic.

Stay frosty my Bros and Brodettes

Broseidon
 

Podg

Member
Hey Bro

I've heard some buzz around tissue culture and it does seem to be quite viable in the future. Theres some info out there but if you search tissue culture into google scholar (search google scholar into google) and have a geez at the results. After reading your post i thought you might find this useful.

Take it easy
 

59lespaul

Member
I bought a Tissue Culture kit 20+ years go from Worms Way.
Its really not that hard to do.

The hard part is sterilizing everything, and keeping dust, or anything else out of the tube. If a piece of dust, or anything that may carry a virus, bacteria gets in there, it will replicate first, as it is a more simple organism vs weed, so the tissue will die.

They also cant take any kind of UV. If you walk past a window, with your new cuts, they will be dead. Keep ALL UV away from them at this point, and I mean even a flash of it.

Cultures are then put under fluorescent tubes. I used a combination of cool/warm.

Cool should be used in the 1st stage, and in stages 2 and 3 full spectrum.

This also takes the plant back to the embryonic stage, and will have 0 genetic drift over time, and repeated culturing.

Feds got all my stuff. Along with 1000 clones.
 
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Marcus_in_the_Darkus

Well-known member
Interesting stuff. I want to look into tissue culture cloning for germinating ancient seeds, because I'd like to tease some old landrace Mexican and Colombian genetics out of a batch of very old hippy seed I recently acquired.

Interestingly, for my purposes, the best method may be at the other end of the sterility scale: bio-priming by germinating the old seeds in worm dirt to bathe the seeds in good microorganisms that kill and compete with the bad stuff that otherwise tends to rot old seed embryos.
 

blowingupjake

Mothership Mod
I'm supposed to be helping a Friend build his glove box for his TC setup.

I'll get some info and maybe some pics from him.


Stay hazed
Jake
 

Broseidon

Member
I saw a design for a hood on youtube where they just used a clear plastic storage container, drilled 2 round holes on the sides, fitted them with solo cups on each side which had rubber gloves glued to the end and built a small extraction ventilation with a cpu cooler fan. Then sealed everything up with silicone safe for the lid which I am not sure if they lined the inside with some foam or something or just left it as is.

Seemed alright for a makeshift hood and likely keeps at least most of contaminants out.

I assume none of you or your friends employed liquid nitrogen in the whole process for storing purposes though? Everyone just worked with fresh cuts?
What reagents did you use? Or everyone who did this worked with a "kit" ?
 

59lespaul

Member
The room you want to do it in is preferably void of windows, curtains, carpet. Its best to scrub the room down, and then spray with Lysol all around.
I used a glass aquarium on its side, and a plastic panel, that I cut holes in for my hands/gloves to go inside of. I mounted the plastic to the top of the aquarium and made it air tight.


But you gotta be totally clean when you do it. Have your hair enclosed in something. Not just a hat. Shave face. Don't have a beard ect, and if you do, you better make sure no hair can escape. Make sure no hair is exposed in the box.

Its all about sterile, and no fungus, or virus floating around, or contaminated by your tools, or yourself.
 

Seed Collector

Active member
I found an Ozone generator made a huge difference in eliminating contaminates from the air. If you have one lying around, I would definitely try that ;)

Keeping the air clean really is the hardest part. If you put an ozone generator in a closed bedroom for an hour or two, then aim it right into your hood when ready to work and it creates an airflow that has to go past the uv bulb. Additionally there will be very little contaminates if any left in the air after the hour or two of bathing the room in ozone.

I don't suggest breathing in any more high concentration ozone than necessary however., so work quick (good idea anyway) or wear a mask.
 

Broseidon

Member
Yeah an ozone genny would definitely work for that. But would have to get back in at the right time after running it. Not too early to avoid health risks to yourself and the cuts/tissue cultures but not too late so new contaminants haven't found their way in.

Good info from all.

My question still remains though: What are the required reagents to do this (maybe I find that out when I read the link on the micropropagation technique posted above?)? Everyone with experience in this worked with test kits from online?
And nobody got experience with liquid nitrogen storage, right?
 

Seed Collector

Active member
No experience with liquid nitrogen.
What I did tissue cultures on wasn't cannabis.
Last I looked into this (many years ago) the other problem people couldn't overcome was getting the clones back out of the test tube/petri dish/whatever and into the real world again. It seems the world is a harsh place after becoming accustomed to that environment..
 

PlantManBee

Well-known member
One great tool for clean work: Ozone generator. I used to use one when I was doing mycology work. Be careful though, that stuff is toxic to you as well as microbes.

EDIT: I see that it is already mentioned.
 

59lespaul

Member
At home you also have to think about your clothes ect.

I used a big garbage bag with a hole for my head, and arms. 100% Nylon workout pants, sprayed down with Lysol before I go in room, and after I go in room. Rubber surgical gloves with saran wrap around my arms, sprayed down with Lysol. I used saran wrap on my feet. Sprayed of course.

After sanitizing the room, I also constantly spray the air to weigh down any dust ect, until I start actual work.

All cutting tools, tweezers ect have to be 10000% Sterile. My kit used Agar, and test tubes.

You cut a sq centimeter of plant material. I used leaf, which also has to be processed, and sterilized, and then place it so it is Not Flat on the Agar, but the thin edge buried into the Agar enough to hold it up. Put on the stopper. Wait for plantlets/Bumps.

Then each plantlet has to be separated, and put into a medium, for roots to start to grow. After they get established, they have to be slowly introduced to UV.
 

H.E. Pennypacker

New member
You can't? Can't you just crack the seed and use the embryo/root/cotyledons? Or is it too little material to work with?

Never invested much time reading into it further, the costs vs benefits aren't there compared to just simple cloning, plus, all the extra steps you have to go through to make sure everything is sterile essentially means you pretty much need a dedicated room for it, with all the necessary equipment.

But it's cool though, especially considering you can rejuvenate plants if they're suffering from any bacterial or viral infections.
 

Broseidon

Member
I guess theoretically it is possible.

But to clone a seed... I would imagine just due to size alone all the difficulties and potential pitfalls that could make you fail your attempt would be cranked up to 11 and make it waaaay too costly.

Probably it would always remain cheaper to just grow the seed to a stage where the plant can be cloned.
 

shantibaba

Breeder and moderator
Hi All

there is several companies already doing tissue culture for cultivators.

Segra International | An emerging specialist Research and Development Company focused on plant-based medicines, including medicinal cannabis.

this group is out of Canada but there are groups all over trying to do it with Cannabis in large quantities as it is a great alternative for cloning in quantity...well that is one of the main applications.

Glad to see your innovative ways of diy tissue culture...but sterile environment is the most important factor or else it is a mess.

The disadvantage is it takes quite some time to grow out...would love to see a photo of a home system if anyone wishes to show theirs....

all the best Sb
 

Broseidon

Member
Would you trust such companies with your genetics Shanti?

I mean the whole Equifax thing makes you think, doesn't it?
 
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