Tissue Cultured Marijuana ? You Bet !

[zachw]

GM - quick question. Let's say I'd like to eventually try tissue culture but am not ready to do so yet. And let's say I don't want to maintain a desirable plant/clones until that time. Is there any (mostly) idiot-proof way to store the plants genetics for the future?

 

TIA!

[Restorium2]

seeds

[trichcycler]

GM - quick question. Let's say I'd like to eventually try tissue culture but am not ready to do so yet. And let's say I don't want to maintain a desirable plant/clones until that time. Is there any (mostly) idiot-proof way to store the plants genetics for the future?

 

TIA!

idiot proof is the deal breaker. even a test tube with a cfl bulb over it will need some amount of care. If disturbed it will need to be "transplanted" again. the good thing about those is that if an issue arises and caught in time sample offers up an opportunity to take traditional clone cuttings from it. The longest I've stored a cutting without reopening the vessel is 2.5 yrs. The sample invites dust mites like cheese to mice, so eventually something happens generally. Without that the substrate will eventually dry up and wont provide for the cut any longer. I'd say best to accomplish in steps. A prepared space and learning curve will go along way to keeping stored genetics alive forever. The space build is fun, cheap, and quick. The "kits" can be fairly expensive but if 200$ is a deal breaker culturing may not be for the investor. A pressure cooker is the first purchase I rec.

[suneday11]

seeds

I think the topic name should give a good insight that tissue cultures are being discussed, not seeds.

 

Troll elsewhere, amigo.

[zachw]

idiot proof is the deal breaker. even a test tube with a cfl bulb over it will need some amount of care. If disturbed it will need to be "transplanted" again. the good thing about those is that if an issue arises and caught in time sample offers up an opportunity to take traditional clone cuttings from it. The longest I've stored a cutting without reopening the vessel is 2.5 yrs. The sample invites dust mites like cheese to mice, so eventually something happens generally. Without that the substrate will eventually dry up and wont provide for the cut any longer. I'd say best to accomplish in steps. A prepared space and learning curve will go along way to keeping stored genetics alive forever. The space build is fun, cheap, and quick. The "kits" can be fairly expensive but if 200$ is a deal breaker culturing may not be for the investor. A pressure cooker is the first purchase I rec.

 

Cool. If you already answered this, sorry, but I'm lazy: do you have a primary resource for learning about the process?  Don't know if you've seen it, but I have some materials from kitchenculturekit.com still left over from a kid's high school project. Never did get the pressure cooker, however, but it's on the list for a mycology project.

[trichcycler]

The perfect starter for me would have been the book Plants From Test Tubes. Its easy on the eyes and a quick read with the info you will need to culture cannabis from beginning to end, or any other plant.  the instructions included in that kit are a sure thing. I hope you can find them....if not prolly available online somewhere.

 

the fastest way to success is beginning with a few dozen carrot cultures. fast and furious and very active. premixed agar and ferts for the carrot are cheap and readily available. cannabis is the same process but different additives for different tasks.

[trichcycler]

choosing a tissue culture vessel
 
The common assembled kits often contain an à la carte number of  BFJ in the kit. Baby food jars are nice to use for many reasons. Ease of cleaning is at the top and glass is preferred by me over plastic. I've used both glass and plastic and find glass less troublesome, most likely because its easier to be sterilized/cleaned by me.

On a budget though,(most of my tissue culture life) any container will do as long as it can be covered and pressure cooked. Small deli containers used to be my favorites on a shoestring. Mayo, food, pickle jars etc all work well too, depending on the size cultures are allowed to grow.  Shallow is better than deep sometimes.

 

I settled on glass test tubes. They clean easy store perfectly in a small area, and easy to cap. I cap mine with a special breathable cap, but tape all around the seal like all other containers. If you want to see what a dust mite could bring into your culture, don't tape your seams. There are pests that naturally live everywhere that are able to slip right through the threads of a jar and through the seam of the lid, around a rubber stopper somehow.

 

 I keep two faced tape along the whole perimeter of the inside opening of my tissue tent. I spray(Lysol) the entire zipper area all the way around, and the floor in front of the tent before I open it, then immediately spray again inside of the tent, all over the floor, when done, I repeat in reverse. I store the test tube racks on a  stainless steel wire cart. The legs are painted with Tanglefoot and a bunch of "baited" vessels are on the bottom level of the cart. Some on the floor for test. I store everything I use in the same tent, tools, pressure cooker, spare parts, living tissue, etc.

[trichcycler]

I like the no powder latex gloves. I often Lysol spray my own gloved hands when opening vessels or transferring cultures. I own one tyvek suit I've used for a bunch of years, and keep it hung right inside the closet door against the tent. I spray it down before I put it on, and spray the sleeves before I work too. I keep a sprayed chefs hat on while in there to keep my hair out of the mix. Lotion on a clean face will help keep skin flakes and stuff from falling in also, and a face mask is used sometimes.

 

I put all of my tools, paper towels, empty vessels and lids, a jar of filtered water in the pressure cooker before I use them each time. A small alcohol burner next to my work space is a danger but necessary for me to dip/burn tools while in use. Even with all of these precautions I still get mold but getting better every year. In 2015 6 vessels spoiled out of 130 that were "worked" or "new", while 100% of the stored/dividing/rooting ones remain perfect specimens. I do not use a flow hood. I used to long ago but found the environment around me needed to be lab like for success. I culture in a grow room, in a closet, in a tent, its not a lab. I have a small clean space to work on them with no air current around, no filters or hepa.  I do better this way than before surprisingly.

[mrdizee]

Just joined this forum. I live in Ashland, Oregon where med use has been legal for several years and rec use just approved last year. Lots of opportunity for the benefits of TC here and have been working with it for the past 5 months. Very frustrating even with a background in sterile work. This is a whole nuther animal! Or, plant, I should say. Steep learning curve! But, I've brushed up on my college biology and chemistry as I hadn't retained much from the late 70's...was stoned all the time. So, it's ironic that I'd be working with this, lol.

 

For the benefit of all, when grassmatch talks about clean, sterile conditions, it's serious stuff. I had russet mites find their way into my bfj culture vessels. I must have had a contaminated plant come through the house. I'm going to try and rescue the cultures by dipping them in Avid and subbing into new clean jars of media. I'll wrap the jars now, which I haven't been doing. Two steps forward, one step back, as they say.

 

I want to say this is one of the best forums I've read or belonged to...more of a "class act" than most. That speaks highly of its members and admin folks.

 

Peace!

[imiubu]

Welcome

[trichcycler]

Just joined this forum. I live in Ashland, Oregon where med use has been legal for several years and rec use just approved last year. Lots of opportunity for the benefits of TC here and have been working with it for the past 5 months. Very frustrating even with a background in sterile work. This is a whole nuther animal! Or, plant, I should say. Steep learning curve! But, I've brushed up on my college biology and chemistry as I hadn't retained much from the late 70's...was stoned all the time. So, it's ironic that I'd be working with this, lol.

 

For the benefit of all, when grassmatch talks about clean, sterile conditions, it's serious stuff. I had russet mites find their way into my bfj culture vessels. I must have had a contaminated plant come through the house. I'm going to try and rescue the cultures by dipping them in Avid and subbing into new clean jars of media. I'll wrap the jars now, which I haven't been doing. Two steps forward, one step back, as they say.

 

I want to say this is one of the best forums I've read or belonged to...more of a "class act" than most. That speaks highly of its members and admin folks.

 

Peace!

Welcome to the forum and to this thread! Thanks for joining to share your experiences.

First things first right.....do you have any famous Oregon strains in stasis currently ? ( )

 

Avid will kill your cultures, but if you're going to transfer them anyways I suggest another dip in the bleach/alcohol and a fresh trim, then transplant if you believe the sample is viable. If you see mold brought into the vessel by the mite.....sorry, its failed. I've never been able to bring a visibly moldy sample to a usable state unfortunately. Maybe you would have better fortune though.

Those mites, not sure of the different types, are everywhere in the house. dust mites I'll call them. other mites come in on produce, houseplants, houseplant store sections pants etc.. None can resist the sweet nectar of a culture vessel.

 

Glad you see the value in wrapping your lid, I use tape with good results. I'd like to see the critters I catch in the tanglefoot goop on the legs of my culture stand, I bet there's a lot. I can see the fluid running through their bodies with my digi microscope but the tangle foot messes my observations up so I haven't looked yet. I have caught them at the entrance in the double faced tape. nasty borgs, sorry you saw one.

 

Please tell me about the culture scene in your area.  Do you have examples of how an Oregon grower would benefit from culture access? 

 

Is cannabis supply  mostly indoor or outdoors there? quality differences in your opinion?  costs?

 

ok, back to cultures.....will you tell me about your culture space?  I've seen some real smarties putting stellar rooms together.

 

Its not often I get to chat with a fellow culturist. Thank you for your sharing I'm looking forward to more.

[mrdizee]

Thanks, GM, for your willingness in creating this thread and for the time you've put into posting! It's good to find a kindred spirit, culturally speaking ;-)

 

I'll let the readers know up front that I know little about the cannabis business, or even about cannabis in general. I used to smoke it a lot; seldom do anymore, mainly because if what it does to my lungs...even the oils and extracts cause issues. So, I'm content to pursue the line I'm on with the TC. I'm learning a lot and have met some really great people.

 

A friend of mine, knowing I did mushroom culture approached me about doing plant TC. He wanted to save the genetics of some pretty cool medicinal plants that had CBD/THC ratios up to 16/1. I said, Hell yeah, I'll give it a try! So here I am, thousands of dollars and many months of frustration later, and I'm afraid I'm going to lose his cultures. Obviously not the end of the world, but I want to succeed.

 

By "stasis" I assume you mean in a state of dormancy, not growing, but alive and viable. I have lots of strains I'm working with, Sour Diesel, Sour Strawberry, Death Star, Z7, QTZ, Purple Gorrilla, Gorrilla Glue, Game Changer. What most people are looking for in the culture arena is clean starts. I know this is where TC can shine. They're also looking for quantities of clean starts...some of the folks I know cater to the dispensaries that sell starts. If I had my technique down, I could sell 20K starts tomorrow. Theoretically speaking, of course, as the laws changed the first of the year and no one really knows what's going on...even the lawmakers. They're trying to integrate med and rec.

 

I've not had mold from the mites, and I suspect it's due to using PPM in all my mixtures. My contam rate is really low since I started using PPM. I'll take your advice on using Avid on the plantlets and use the alcohol, bleach method. I have rescued some moldy plantlets before by soaking in the rescue mixture recommended in the PPM literature. I also soak explants, as a final step before culturing, in a mixture of 3% PPM with full strength MS and no PS20 or pHing. Kills everything except the explant. Yes, the stuff is a dollar a ml, but to me it's worth it for not having to deal with contams.

 

Russet mites are the bane of the Southern Oregon cannabis grower. They came up here from CA and have adapted to the cooler climate. Do you have them in MI? They're already developing resistance to various pesticides and the natural products are of no use at all. You cannot see them with the naked eye, but they're deadly and pernicious. In addition to Avid, there's a product call Pylon that is being used.

 

Most of the grows here are outdoor. The climate here is superb with the long summer days and high temps. However, indoor is catching on and I've seen some awesome grows indoors. The indoor grows keep the market more steady. Not sure about quality differences...perhaps better yields outdoors, but only one crop per year. Higher costs indoors, of course, but less potential for loss due to weather or theft. Some of the new regs are forcing better security at grow sites...cameras, safe houses.

 

My culture space consists of a 4x8 grow tent with a 48" homemade flow hood. Got the parts on eBay and the melamine board at Home Depot. It is maintained with a positive pressure by an 8 inch centrifugal fan blowing air through a HEPA filter I designed. I have a Bacti-Zapper for my instruments, good fluorescent lighting. I made a door for easy entrance out of PVC tubing. I have an All American 941 pressure cooker that I converted to a sterilizer for autoclaving. At times it's larger than I need but I originally got it for sterilizing sawdust for mushroom growing...I can sterilize 50# of wet sawdust in it! Or a few liters of media for plant TC. It's my workhorse, for sure. I suit up with a Tyvek suit with a hood and booties, face mask, nitrile gloves, lots of alcohol on surfaces. It's all in the garage...not the best place but my wife won't let me use the spare bedroom...go figure!!

 

My multi recipe is 1 liter distilled H2O, 0.5 µM TDZ, 1 ml PPM, 30 g sugar, 4 g Gelzan, pH to 5.7 after Gelzan is completely dissolved in the microwave. If the gel isn't completely dissolved the pH will drop after autoclaving by a couple tenths. Rooting is 2.5 µM IBA with no TDZ or NAA. I also have been adding 500 mg activated carbon lately. However, I've yet to see any roots on anything, with or without AC. These are simply recipes I've seen used in various studies that I'm sure you've seen online. I can grow callus easily with TDZ/NAA mixture, but have not been able to get callus to initiate organogenesis. I did try adding GA3 to multi culture and the cell elongation was insane, so won't do that again.

 

I've also had a lot of issues with vitrification. The only thing I've found that helps is keeping temps to 21 C.

 

Well, that's it for now.

 

Be well!

[mrdizee]

Something I left out of the last post: I use 4.4 g MS salts w/vitamins and 1/2 strength in rooting media. Reading some studies lately I've found they seem to only use the basic MS with out vitamins, or with the Gambourg B5 mixture. Is glycine an issue for cannabis? The formulas w/vitamins and the B5 are very different in the vitamin mixture. This was something I had overlooked. You recommend the carrot mixtures and when I looked that up it seems similar to the Gambourg B5. Any thoughts...?

 

BTW, I do have the Plants...Tubes book...fascinating, but I didn't find a carrot recipe in it, FWIW.

 

Also, there's no "culture community" in this area, so I'm kind of a loner, in that respect.

 

Peace.

[mrdizee]

Trying to post some photos here...hope they come through. Here are some images of what russet mites do to plants...the beginning stages. Also an image of a plantlets in culture, and one of my clean room/tent.

[trichcycler]

good pics, clean hands...awesome!!!! lol

 

have you positively identified a mite on your affected plants?

this dispensary you mention .....do they broker culture starts to their patrons?  what is the end user cost for a start there?  Are they producing their own cultures in house?  rooted? substrate?  in sealed vessels?  tell me about QTZ please?

 

I never saw mites in my garden here but have seen them in others. I hear about them occasionally but talk is slim because it always leads to pesticide allowances and medical growers don't often appreciate chems on meds. I helped eradicate them in a couple gardens using c02 saturation. I couldn't tell you which mite or any details of its features, but it does require some 02  and dies without it.  I did have a thrip issue once and found they also need 02 in the grow room, and die quicker than a mite when limited.

 

While I hold my recipes dear I'll do my best to get you rooted, you're close.  I no longer use ppm or any antibiotic, they spoiled me for years though.  MS packs are the easiest to work with, measuring basal salts in the small volumes is difficult(for me).

I'll private message a suitable rooting recipe today. Keep in mind you'll have to transfer one more time (at least) before exiting the vessel and hardening off.

The biggest disappointment in my culture experience is that NO pre mix I ever used was capable of bringing my start to the veg room. Some tweaking is necessary, unless you love those strange plant anomalies( I do sometimes)

 

I want a zapper

 

I'll find a suitable carrot recipe and share it here this afternoon. I like your room, a lot. My tent is 2x4 so I dont go inside, but work at the door instead.   my wife paid no attention to my culturing.....until I cultured a human specimen, that got her attention quickly, jokes abound.

[trichcycler]

useful links

 

http://deepwater.org/bioteacher/05-Genetics/biotech/biotech%20for%20plants,%20animals,%20and%20environment/PDFFILES/PDF02/LESSON7.PDF

https://www.scribd.com/book/206825681/Plants-from-Test-Tubes-An-Introduction-to-Micropropogation-4th-Edition

 

http://themodern.farm/studies/Tissue%20Culturing/micropropogation-cloning-hemp-shoot-tip-cultures.pdf-(carrotculture recipe too)

[mrdizee]

Good morning!  I'm such a freak about my hands being clean! Imagine the anxiety I suffered when I was a diesel mechanic for many years, lol.

 

No, I have not positively identified a mite in my cultures. I'm going by the plant symptoms, as the leaves are rolling and curling just like an in vivo plant that has mites. But the more I think about it, they probably are not in the cultures because they would contaminate from the spores they brought in; even PPM can be overloaded. That's not happening, so I'm having to re-think the problems that are showing up in the cultures. I've attached a photo of one that's in rooting. It could be vitrification. Any input would be greatly appreciated.

 

Some of the dispensaries sell starts, some do not. I think it may have to do with licensing, but not sure. They sell for 15-20 bucks a piece. Most growers do their own cloning and sell their surplus to the dispensaries, I believe. There are a couple of dispensaries that grow their own starts. It's a crap shoot, though, buying them, from what I've heard. Lots of junk, lots of mold. They are just like a tomato start you'd buy from a greenhouse...ready to put in the ground.

 

I just spoke with the grower who has the QTZ a few minutes ago. I was wrong on the ratio...it's 20:1, not 16:1 CBD:THC. I've got 16 plantlets in rooting culture...some look good, some do not. I would really like to get at least a few to survive so I have some material to work with. I've also got a bunch of callus. That's all I can really tell you about it right now...all I know. I know it does fetch some good $$$ from the dispensaries. I really want to save these genetics! Sorry for the sideways photo...that seems to happen.

 

Thanks for the links...already had two of them, but the one explaining the four stages was new. I've scoured the internet a lot so surprised I hadn't run into it...I'm sure there's a lot out there I haven't seen. I'm wondering if the Stage 1 is where one would do long term storage of an explant for future use? Does callus grow in Stage 1?

 

Yep...I love my zapper! I keep my instruments in 70 iso and then about 5-10 seconds in the zapper and they're clean.

 

I had a 5x5 tent, which was a bit small to work in with a 48" hood, so got the larger one. I actually may put a 3x3 pressurized pre-chamber on it to help keep the tent clean. Room is tight though in my side of the garage and I don't think my wife will park her car outside so I can take over the whole garage.

 

Funny thing about the human tissue...I told a neighbor/friend about doing tissue culture and he said he didn't want to see kids running around with plants coming out of their ears. Have you read a book called Plant Intelligence and the Imaginal Realm, by Bruhner? Awesome book, but he talks about the concept of symbiogenesis...where two symbiotic organisms suddenly unite to form a single organism when they swap DNA. Humans really do have a lot of similarities to plants, so be careful with the human tissue culture Serotonin is a big similarity and necessary for plant neural networking. I wonder what would happen if one was to add it to the culture?

[trichcycler]

miralax, just miralax my friend. the same stuff people are huffing in their nicotine pens is able to break down the dna proteins I've been taught, providing an opportunity for such a marriage. have fun!

 

I think you're arriving at the same conclusions I was getting at with the curled leaves, in and out of culture. Those are most likely not mite caused. I see this in culture experiments often with cannabis. There is no cannabis mix available yet so we use the ones for another plant, like tobacco, carrots, etc.....more or less of all knds of constituents.

I also feed the GFP for some fun. I had to get live jelly fish to obtain this from a diver collector. I've aimed to eliminate hormones form my mix after learning just how closely related ours are and fearing exposure. There are disease markers that show an elevated exposure to the two usual suspects already, most likely coming from our own GMO foods.

I had much fun with the agrobacterium too, necessary to vector in strangers. you can collect it yourself in the woods, snatch an explant of a Crown Gall diseased tree and culture away. Keep it away from anything outside of your tent though. its not even legal to buy without the educational permits and promises. I kill thousands of cultures with odd ball additives. never did try serotonin though.

 

If you've got a successful mix for your callous try to sterilize a small cutting and plant it in the mix in the vessel. this should be what you're looking for. If it survives it may begin to form new callous material at its base, or it may even begin to grow slowly. Either way adjust your mix accordingly for desired effects. The time it takes to "see" results is discouraging but your cuts should do or die in a couple weeks.

 

so, are these "starts" actual cultures or traditional clones?

[mrdizee]

Just very quickly, I'll respond more later, but there is a company that claims to have a product available called TriQ Systems (you can find them online...Maia Culture). The grower I'm working with ordered one of their kits when I was just getting started doing this. It was 165 bucks for a 1 liter kit containing 1L each of Stage I, II, III. Unfortunately the mixer they employed didn't add enough gelling agent and I had to add some agar to get it to gel. They sent me extra formula but being in a learning curve I didn't have much success getting to stage II, even. Their prices are outrageous, but...if it works, and I have no assurances it does, it would be worth it. They claim now to have their mixers straightened out, but I'm not willing to risk the $$$. I've asked to be put in touch with someone who has had success with it, but to this day, no response.

 

The starts are traditional clones. No one's doing culture around here that I'm aware of...except me ;-)

[mrdizee]

I've got about 5 or 6 studies on cannabis culture that I've found online. Problem is, they contradict each other at times. One says "xyz works" and another says, "xyz doesn't work" but "abc does work." I've got an Italian study that bypasses stage 1 and 2 and goes straight to S3 with the explant. However, it's all in Italian except the abstract. It states they've developed an alternative to MS salts that makes a significant difference. I don't know anyone who can translate Italian. At any rate, I don't think the "secrets" are divulged in the article.cannibisItalian2009Casano and Grassi.pdf I'm able to decipher some of the obvious words and there are no numbers that indicate a potential alternative formula.

 

The curled leaves out of culture are definitely caused by russet mites. I saw them under my microscope...adults, larva, as well as eggs. It's the early stages of an infestation and they're on a mom, so I don't have an issue with using something to cure the problem. Would not recommend on a plant intended for harvest. Chlorfenapyr, however, does have a very short half-life...1.5-3 days in the sun; slightly longer indoors.

 

My callus mix doubles the size of the callus in 7-10 days, and it works best in the dark. It's mostly friable callus though, which presents a problem when trying to transfer...it falls apart. Great for cell suspensions though, which I haven't tried yet. Plant to go that route for syn seeds at some point in the future. Cannot get organogenesis with any callus, as of yet.

 

Just looked at my QTZ plantlets in rooting stage and a couple of them have suddenly gotten more vital and lush. I wonder if they've developed some roots and are absorbing more nutrients. I cannot see the base of the plant because of the carbon in the mix, but I'm happy!

 

Will look more closely at the the carrot and tobacco recipes. Can really appreciate the desire to get rid of the syn hormones and go natural all the way. Me...not there yet...need to get more basics down and branch out from there. I've read about the crown getting infected...an area just above the soil line. Agrobacterium can be friend or foe to the trees, depending on where it comes in contact.

 

Thanks for the dialogue!

 

 

[trichcycler]

triq mixes wont bring an explant to the veg room, at least not with the couple hundred bucks I gave them. I took one for the culture team and ordered all of the google results at the time. Best chance to get to the hardening off stage is trial and error. I thing those companies have a sure thing. With so few home cannabis culturists, and wnnabe's, they can tell us it works knowing that only a small percentage of people will even try it after buying it, most of them will be fungi farms and trash, and the few that get a root, and chute probably will kill it before it hardens.  Company remains confidant their mix works, and says we just don't know what we're doing right.

 So far I see nobody willing to share the correct formulas for cannabis. I don't blame them at this time. I don't understand what is happening with your callus, friable? congealed then falls apart?





I would discard the pest infected cultures and start a new sample


I put samples under a microscope before I spend dime on them.....

[zachw]

(snip)

 

I would discard the pest infected cultures and start a new sample

 

 

I put samples under a microscope before I spend dime on them.....

 

Scope recommendations?

[trichcycler]

http://www.celestron.com/browse-shop/microscopes/digital-microscopes/lcd-digital-microscope-ii

 

I got this one some years ago. Probably overkill for a mite check, but invaluable in culture. It hooks up to the puter for a full show, makes movies, etc. I got mine for the built in light filters so I could detect miniscule plant/animal/fungi fluorescence. When I discovered I could watch the hydraulics of living insects with it I was sold! a cheap little radio shack scope will show mites/eggs though, so the cost could be prohibitive for the occasional user. It's paid for itself many time over for me, I love it, if it broke I'd have another next day air. For the money there may be better ones, this one fit the bill for me, and I'm past the learning curve now with this model.

 

if you get one I hope you enjoy yours as much as I have mine.

[mrdizee]

20 bucks at the local grow shop and works surprisingly well.

[mrdizee]

 

 So far I see nobody willing to share the correct formulas for cannabis. I don't blame them at this time. I don't understand what is happening with your callus, friable? congealed then falls apart?

 

 

 

 

 

 

I don't blame them for not sharing, either. Makes me wonder if there is something intentionally left out of what is published in the studies.

 

The callus is often very delicate and crumbly, like trying to pick up a glob of cooked rice. To quote from Plants from Test Tubes: "Callus can be friable or firm textured and it can be green, cream, yellowish, or brown colored." It seems to vary with media composition and plant variety.