Plant in a test tube

The process for making media is relatively straightforward once you know what hormones you need. Different plants prefer different levels of auxins (root) and cytokinins (multiply). I'm using a basic multiplication media, so it'll be high in cytokinins and low in auxins. if I were rooting I would swap those levels and have high auxins and low cytokinins. if I were to keep the levels equal I would be inducing callus cultures which is a rabbit hole for another day.

Today's media consists of 1000ml distilled water, 4.43 g/L Ms media (nutrients plants need to live), 30 g/L sucrose, 1.5mg/L Benzylaminopurine (cytokinin), 0.15mg/L Naphthaleneacetic acid (auxin), 3 g/L gellan gum. half the batch also contains 1g/L charcoal which helps absorb phenols leached out by some plants, also helps with generalized longevity of the culture by absorbing toxins.
all that is mixed up, pH set to 5.8 and put into jars and test tubes. 20ml per tube, 40 ml per jar. it's then all loaded into a pressure cooker where it 'cooks' for 20 min at 15psi to sterilize the media. when it cools the gellan gum will solidify, the jars with charcoal are agitated until it cools to keep the charcoal suspended in the media. now it sits to cool off, and then it'll hang out until it's used.

apocalypse scorpion peppers and aji mango peppers were the plants taken for culture. I've never cultured peppers so we'll see how it does.
few shoots were taken off of the aji, the scorpion peppers were seedlings I had started that had grown several nodes and were cut at the base. all leaves except the top newest growth were removed.
plants were put into a 10%ish bleach solution and agitated for 15 min. for the final 5 min I added a squirt of 3% peroxide. adding an oxidizer to an oxidizer makes a nice bubbly mess to really get into the nooks and crannies.
plants were removed from bleachy bubble bath and rinsed with sterile water 3x.
I cleaned all the petioles off the stems, cut off the bottom on the stem to the next nearest node. (you can see where the bleach kills the tissue, it turns all white, and you just cut back to fresh stem.)
cleaned up explants were then placed into the media. the media is the same in all the jars, the only difference is one has charcoal. I don't know how much phenols the peppers will exude, that's the reason for the charcoal additive in half of them. I don't use charcoal unless it's needed. you can tell when you need it because the media at the base of the plant will start turning grey. charcoal will absorb those toxins, but it doesn't absorb selectively. it'll take your good stuff too, auxins, cytokinins, antibiotics/antifungals (if you use them, I didn't this time). the first time I culture a plant I'll do both and if it's not needed I just don't add it next time. the need for charcoal seems to be cultivar dependent. I have a few different types of coleus (next post will show the exponential multiplication of culture on a coleus) some need charcoal, some don't. sorry, got on a rant there.
after placing explants in the media tops are screwed on and sealed w grafting wrap, labeled and put under a t5 on a heat mat set to 75f.
if there was any contamination you'll know in a few days so for now they sit... thanks for playing, see you next time!!!!

this is a coleus I've had in multiplying media (1.5 mg/L BAP, 0.15 mg/L NAA) for 45ish(?) days. it started as 2 and was turned into 16. those 16 shoots were then placed into fresh multiplication media. if I get the same average of 8 shoots per cutting it'll turn into 128 plants. if I were to put those into multiplication culture I'd end up with 1024. from 2 cuttings to 1024 in 180 days. 🤯(that's 524,288 in a year).

the implications for this in the cannabis industry are massive. how big would your mother plant have to be to pull 1000+ clones in 180days? nevermind half a million/year.
How much space is that taking up?
How much light, food, soil, man hours tending to it?
the culture sat on a shelf forgotten, under a t5 light, in a jar.

What if you could have your favorite strain put in a jar, root it and grow it out whenever you felt like it? you could have a collection that fit in a briefcase.
yes there is a downside. contamination can wipe out the whole jar, but you won't get spider mites...😂. yes you'll have to root it and transition it from a 100% humidity, sterile environment to soil, but that's not hard. yes it'll be small, but as vigorously as it grows, it won't add much time.

I don't know. the possibility is there.

contamination happens...

el Choco red philodendron contaminated with pink slime
philodendron green congo nuclear
spiritus sancti philodendron
monstera adosonii
Rusty coleus (that's been in culture too long.)

few jars have contamination. not surprising since I was taking pictures while I was doing it. see what I sacrifice for you 😆😆. other than that they are just doing their thing sitting under a light. I'll get another round of stuff going eventually. will probably up the bleach to 15% on the next round. culturing is a lot of hurry up and wait and trial and error.

begonia stems are sending out roots, leafs will be next. adosonii cutting is leaf and roots, can be removed and planted or wait and get more shoots. coleus needs to be split and re cultured as it is running out of media. pepper had some good root structure but no shoot growth. big callus on stem is indicative of hormones being too high or wrong type. you're guess is as good as mine because I've never cultured pepper before. may have to reattempt.

1:12 bleach soak for 20 min. added h2o2 for final 5 min. rinsed 3x in sterile water. cultured in 1.5 mg/L benzylaminopurine and 0.15 mg/L Naphthaleneacetic acid. some have 1mg/L charcoal, some don't. all cultures solidified w 3g/L gellan gum.

had to trim out the pepper plant so I decided to culture more shoots.
Dahlia, goldfish plant, aji mango pepper, apocalypse scorpion pepper, and tomato.

no idea what the dahlia or tomato are going to do in culture. the tomato didnt look to good after the bleach bath.

begonia cross section stems were put into a callus culture. the stems developed some roots and now little baby begonias are forming

finally got around to culturing clones (and other stuff). sterilized in 1:10 bleach solution for 20 min. 20 min may have been too long. next time I'll shoot for 15min. there was some damage to the leaf structure from the bleach, but we didn't need that part, hopefully meristems were not damaged from bleach. time will tell. if it all dies, we'll just take more cuttings and try again.
pics: (1) clones. (2)clones chopped up in bleach solution, no need to sterilize all that material, so I remove most fan leaves and chop into sections. (3)clones in culture media. (4) flow hood aftermath (5) green congo nuclear previously in culture, given fresh media. (6) begonia? callus culture. here's the crazy part. most plants are totipotent. each cell has the ability to turn into any other cell and become a new plant. you can do this by adjusting hormone levels in the media. cytokinins and auxins. high cytokinin, low auxin gives you more shoots to form. high auxin, low cytokinin gives roots, equal parts cytokinin and auxins helps promote callus formation. believe it or not, you probably take advantage of totipotency and didn't realize it. every time you root a cutting. Clonex, yup just an auxin. you are adding hormones to the plant to make those cells that used to be stem or branch, turn into root.
this was a leaf cutting that turned into a big pile of differentiated cells (green,/yellow blob and eventually turned into a new plants

find some jars that desperately need to be recultured. this is where it gets crazy.
pics:
1 swedish black ivy. 1 cutting has turned into many nodes. will need to chop up and either multiply again or root via culture or aeroponic. they root easy so I'm leaning toward aeroponic.
pic 2. philodendron minarium. one cutting that has turned into many plantlets. I feel I can get 25 out of this. I'll probably put a bunch into rooting culture and at least a few into multiply again.
pic 3 - 5
rex begonia stems. rooting and forming a callus. (big pile of differentiated cells) from those pile of cells they'll start forming new plants as you can see in pic 4. also I'm pic 5 you can see the callus that is forming. big green bubbly ball o goodness.
anyway that's what I found in the back of the shelf. I have a few jars of media left over from the other night. I'll have to make more this week and get them into it. aaahhhh way to much to do which brings me to my post title, I need an assistant!!!! lol or maybe less of a scatterbrain, that'll work too. happy growing 🤪🤪

few of the cannabis cuttings are growing new shoots. (ignore the bleached out leaves, went a little heavy on the sterilization). eucalyptus is taking really well showing lots of auxiliary bud growth. coleus is already producing roots, I want to look into altering the media to promote flowing. would be interesting to see if I can flower in a jar lol

pics
1 & 2. durban x freak tubes 1 & 3
one node was turned into 4 plantlets for each tube so 8 new clones total. they're small but give em time they'll grow. I put into new media, see what they do. once these settle in and grow a touch I want to isolate one of the plantlets and see what happens if I put it under 12/12 lighting. no reason it shouldn't flower, it's a fully mature clone. nugget in a tube. lol other tubes were not viable.
3. begonia callus culture
looks alien like. roots everywhere, blobs of cells and a few leaf structures starting to form.
4. eucalyptus doing great in culture. will have to cut this up multiply again and see how many I can get to hit the market eventually.
5. purple/black coleus plant took well to culture, new growth looks healthy. will have to chop and multiply at a later date
6 rusty coleus
this one didn't initiate into culture to well. top of plant has died, but bottom nodes are showing shoot growth so I'll keep it around and see if it can pull through.
still need to make more media. hoping to culture a few more things laying around the house.

eucalyptus is due to be chopped and recultured. black coleus is starting to show fungal contaminate. will treat and recultured

decided to keep pheno b. was cutting down pheno a clones and remembered I had a bunch of culture media sitting around. few hours later, cuttings were set into media. used only 10min of bleach wash compared to 20, tissue didn't look damaged like last time. just hope it was enough to kill all bacteria and fungus. in a week we'll know. I'll update if I remember.
pic 1: cuttings
pic2: cuttings cleaned up
pic 3: cuttings in 10% bleach bath
pic 4&5: cuttings introduced into media

so far so good. most of the cultures look promising. one didn't take and a jar got contaminated. usually most contamination shows between days 3-14.
one culture is exuding a fair amount of phenols, I may try and switch it to a charcoal based media but only if I culture some other plants in the near future. it's not really worth the sterilization of the hood and tools just for 1 thing.

pheno b clones were getting unruly so I topped then and trimmed up some growth, took all the good parts and introduced to culture. see what happens!!!

this may be how I keep phenos around. I joke that I'm trying to cryogenically freeze them 🤣. so far things are looking good. I need to dive into some heavy research on what nutrients/hormones work best for cannabis and if I can get those hormones in the usa and what their storage protocol is.

I forgot about all these. Some dead leaves but new green growth, chopping and reculturing is on the to do list. They were introduce into culture some time late October kept then kicking for 2 months. Further testing still needed to see if this is a practical way to keep phenos alive