Tissue culture and cannabis?

[Uncle Ben]

thank

Thanks ya the down side is you need money for the equipment and steril everything air ppm purity is expensive and so is the flo hood and all the parts and pieces pluss the room its realy easy its all mostly flora nova.....

IOW, it's not practical and the success rate is very low with cannabis as opposed to orchids where the success rate is very high. You adapt to whatever methods works best for you regarding cost and being reasonable.

 

[thump easy]

im gona try it at our new place im gona invest in all the steril equipment and just for shits and gigles im gona try to market them lolz who knows it might work as far as genetics go sounds like another project new place almost done

 

[eyes13]

who knows it might work as far as genetics go

it's basically for preserving genetics. If you want 1000 genetics in a small warehouse, tissue culture is where it's at. It works great. I haven't done this myself, but I know people who have. I wouldn't take a tissue culture for something you want to flower next month though, but it does have it's use.

Ideally I want an incubator in addition to the same veg space I use now, so just eliminating a dedicated mother space for ~20 strains I'd wish to hold on to. Does that sound like a reasonable application here? Glove box, filter etc are within reach.

 

[AlphaPhase]

Ideally I want an incubator in addition to the same veg space I use now, so just eliminating a dedicated mother space for ~20 strains I'd wish to hold on to. Does that sound like a reasonable application here? Glove box, filter etc are within reach.

That sounds reasonable. I wouldn't expect much success at the first attempts but practice makes perfect. I wish I had experience with the method because I had to cull many plants that I really wish I had kept for future grows but just didn't have the space to keep them around. Like mentioned above, if the room, tools and equipment are surgery kind of sterile, you'll have much better success. Good luck and keep me updated with how it turns out, it's an interesting topic.

 

[AlphaPhase]

One tip I'd like to mention, even if the equipment is sterile.. there still can be contaminants in the air. Lysol the hell out of the area you plan to work in (spray the air, kill airborne pathogens) and work quickly, this will better the success rate, you wouldn't believe the stuff floating in the air that can contaminate things.

 

[thump easy]

i know it sounds crazzy but just spray a branch with silver spray and seed up a branch cover and tie it off you can save those for years wish i could go in depth but im retired!!!!

 

[GrowerGoneWild]

i got the vitamins its so easy your gona be like what the fuck its that easy yes i did the work and im not a high school grad im a GED kid lolz its realy easy i gota find the notes ill give you guys the recipe its so fucken easy no more than 50 buck the jars and the lids probley the most expensive part.. or wait the presure cooker was the expensive part.

Would you mind posting your notes on your mix for the medium?.

I got a few mixes i'm gonna try, it looks like you've had success, I'm interested in the final agar mix for rooting.

Any thoughts on the hardening off process?

 

[GrowerGoneWild]

One tip I'd like to mention, even if the equipment is sterile.. there still can be contaminants in the air. Lysol the hell out of the area you plan to work in (spray the air, kill airborne pathogens) and work quickly, this will better the success rate, you wouldn't believe the stuff floating in the air that can contaminate things.

I do something similar when working with mushrooms when inoculating jars, Not like I have a laminar flow hood.. yet.

 

[GrowerGoneWild]

Well I was poking around and heres a kit..

http://phytotechlab.com/media/downloads/16477/C1850- Hemp (Cannabis) Multiplication Kit.pdf

Somebody posted the PDF of the 2009 Wang formulation for the TC. Its a commercial kit, now I admit its expensive, but it might be worthwhile to experiment with a kit till you get familar with the process.

I just finished a book on Tissue Culture, and I'll have to post that formula here.. I'm just trying to decipher some of the abbrevieations for the the hormones.

 

[thump easy]

Would you mind posting your notes on your mix for the medium?.

I got a few mixes i'm gonna try, it looks like you've had success, I'm interested in the final agar mix for rooting.

Any thoughts on the hardening off process?

well the ppm you gota get it from a chemical lab its real expesive.. that thier alone is worth it if you dont have a steril 99.0 invirement i can if you give me a week or so but the kit i got originaly was the same incraments i only did a few drops of the other stuff lett me look back thier on you tube you have to sit for a few hours to listen to the proffesors in univerities give their lecture on TC listen for the key ingredients and go to the hydro store and look on the lables on the back you'll find the ingredents i gave up because i have dogs and thier in and out all the time most my cultures would eventualy get lost infected with mold spores.... the original kit changed its chemicals and the guy waited for you to call him so you had one last crack at it the beta commerial was pritty fucked up excuse the aunguage.... thow even the instructions didnt mach.. 250 pluss shipping the world is made of gimiks, its realy easy you do need the presure cooker and you do need lysol you need clean equipment and the augar ill see if i can im pritty bizzy person i wont promise you i will but i tell you just youtube a few lectures and its that easy... Dont rely on anyone dude realy fucken easy im a joe smoe if i did it you can do it. but realy in my experience its not worth it... just s1 you genetix and you have a freezer that what i do... but fo the sake of fun and spending 1000$ go for it if you have the money.. It is very fun i do things for fun even if they cost money.. just fo the sake of doing it good man im shure you,ll get it.

 

[GrowerGoneWild]

well the ppm you gota get it from a chemical lab its real expesive.. that thier alone is worth it if you dont have a steril 99.0 invirement i can if you give me a week or so but the kit i got originaly was the same incraments i only did a few drops of the other stuff lett me look back thier on you tube you have to sit for a few hours to listen to the proffesors in univerities give their lecture on TC listen for the key ingredients and go to the hydro store and look on the lables on the back you'll find the ingredents i gave up because i have dogs and thier in and out all the time most my cultures would eventualy get lost infected with mold spores.... the original kit changed its chemicals and the guy waited for you to call him so you had one last crack at it the beta commerial was pritty fucked up excuse the aunguage.... thow even the instructions didnt mach.. 250 pluss shipping the world is made of gimiks, its realy easy you do need the presure cooker and you do need lysol you need clean equipment and the augar ill see if i can im pritty bizzy person i wont promise you i will but i tell you just youtube a few lectures and its that easy... Dont rely on anyone dude realy fucken easy im a joe smoe if i did it you can do it. but realy in my experience its not worth it... just s1 you genetix and you have a freezer that what i do... but fo the sake of fun and spending 1000$ go for it if you have the money.. It is very fun i do things for fun even if they cost money.. just fo the sake of doing it good man im shure you,ll get it.

Thats cool, whenever you can. I just wanna take a peek at your formula, I just want a ballpark idea of what I need for the TC. I'm looking at the chinese formula, and thats available in the kit link mentioned above. And theres a formula by Melinda in her book on Micropropagation.

Making the agar is easy, what goes into the agar is the question.

I guess I could use S1's to bank the seeds, but I'm looking at it from a different view. For example: Dark Heart Nursery is looking hard at TC. I bet there is a heavy demand on clones thats why they want to use TC.

 

[thump easy]

Thats cool, whenever you can. I just wanna take a peek at your formula, I just want a ballpark idea of what I need for the TC. I'm looking at the chinese formula, and thats available in the kit link mentioned above. And theres a formula by Melinda in her book on Micropropagation.

Making the agar is easy, what goes into the agar is the question.

I guess I could use S1's to bank the seeds, but I'm looking at it from a different view. For example: Dark Heart Nursery is looking hard at TC. I bet there is a heavy demand on clones thats why they want to use TC.

that would be asome but its faster on the money side to clone out... tc is a hole lab and workers and months before you get a well astablished plant that you could sell a patient if i know money and i know the back side of bizzness its not good for them unless they up the price it takes longer to propigate and longer process to root and get shoots and then to aclimate and then turn around and sell the product!!! if someone really got down testest verifired the quility of a strain it be on like donky kong but clone people sercum to greed or sercumstances letting the quility of the clone go to shit or god know what i have never ran into a consistant great clone place its hard!!!!! good luck and may the force be with you on you quest i hope it works out for you ill post what i can find im cleaning house ill try to get you the info..

 

[EverythingsHazy]

Generally, TC works best if:
-You need a massive amount of identical clones.
-You need to clone a plant that doesn't root well with traditional methods.
-You need to get rid of a plant virus that is harming one of your strains.
-The plant you want to multiply takes a long time to become mature enough to flower and set seed.

Most cannabis sellers don't fit into those categories, but that doesn't mean it won't benefit some. Do keep in mind, that it is tedious and somewhat difficult (difficulty level varies depending on skill, technique, and the tissue you are trying to culture). You will probably have lots of failures in the beginning, and you just have to learn from them instead of getting discouraged, or ignoring them and making the same errors over and over.

 

[GrowerGoneWild]

Generally, TC works best if:
-You need a massive amount of identical clones.

Yes, this is my motivation for TC. And i'm trying to exploit a loophole with unrooted cultures not falling under any legal definition of "cuttings". TC could help me out. Colorado has an 8" rule for cuttings.. etc..

Another thing I would like to fix is the rather slow time I use to isolate clones to prevent infection. I currently use 2 weeks to make sure the plant is free from infection, pests and stuff. And then it goes to mom.. And that takes time too. I figgure in the same ammount of time I should be able to have at least 20 clones in the same time I'm ready to take clones from a mother plant.

 

[GrowerGoneWild]

Well here is one possible formulation for Tissue Culture for Cannabis.

I would suggest reading "Plants from Test Tubes" by Holly Scoggins, its a good primer for TC.
Then read "Marijuana from Test Tubes" by Melinda Davis.

I would suggest reading these books in that order, it will help you to mix the agar plates as well as help with the basic concepts of TC. What light to use, hardening process.. etc.

MS = Murashgie & Skoog
Dont ask me what formulation of MS, there seems to be many variations of it in the Sigma Aldrich catalog for TC. I'll update this when I get things figgured out. And it seems to be a MS version on Amazon oddly enough.

There are a few versions, of TC in vitro but here's Melinda's Davis formula.

Verbatim.. (My kindle is on the fritz, Its from notes I scribbled down.)
Stage 1 from cutting / Stage 2 Divisions
1 MS with vitamins
2 Tablespoons sugar (sucrose)
1 ML PPM
.5ml IBA at 1mg/ml
9g/l Agar.
30 g/l Sucrose

Stage 3 Rooting
1 MS with vitamins
2 tablespoons of sugar
1 ML PPM
1 ML IBA 1mg/ml
1 ML TDZ @ .1mg/ml
9g/l Agar
30g/l sucrose
.5G/L Activated Charcoal.

I'll post something about the Chinese work (Wang 2009 Microprop of Hemp.) Be patient, but the formula is commercialy available.. just got to find that link again.

Now I understand there's quite a few kits out there, I'm trying to see if I can formulate it from Melindas formula, and this also helped me understand that the TC involves "stages". With 3rd being the rooting stage.

And I'm cheap, I would think that it would be more economical to make my own...

 

[Voidling]

The reason I want to tissue culture is to have a library backup of mothers. I have lost some wonderful plants. I was even sharing with two friends so we each had cuts and we had a couple where we all lost our cut of. It wouldn't do away with mothers for me because my grow isn't that large.

That being said I believe a way to reduce mold and bacteria take over, plus improved hardening off would be in a form of tissue culture called
photoautotrophic propagation

I have studied on tc a lot but life took me away from it for a while. Unfortunately for now I have no money for such things, but when I'm done setting up my business I can write up all the equipment as a business expense for cloning other kinds of plants.

 

[GrowerGoneWild]

I dont know how long explants last or if they could be used as a "library". My motivation for TC is to produce large volumes of clones and clone autflower plants. Would it not be easier to store an F1 or S1 seeds of your mothers plants?.

I looked up photoautotropic propagation, it seems to be different from the TC i'm working on, with the MS medium with sucrose. I tried to read the abstract on Photo TC, but I didn't want to pay the 30 bucks to look at it. Care to comment on why you pick that version of TC?

I want to pick Dark Heart Nursery's brains on what version of TC they are using. As of now the only versions I know that are workable are Melinda's and Wang's TC. I'm more or less finishing up the abstract on Wang's TC, And more or less trying to understand MS formulations, I'm kinda stuck on if the optimum formula was MS, with Gamborg vitamins, and I'll still have to find the formula in Sigma Aldritch catalog.

I'm pretty close to getting my first TC's going.. Mebby in a week I'll buy some medium.

 

[Voidling]

Just waking up so groggy. I'll try to remember to look up the pdf I have Ann's try uploading somewhere.

It uses a sugarless medium so less chance of nasty things taking hold. Second part is I believe there is a better rate of survival in the hardening off stage. I believe it is similar to a method to certain mushroom cultivation where you have air flowing through an aquarium.

It may be too complicated or expensive for a home setup. I haven't been able to set up yet to try it yet unfortunately.

People learned how to germinate orchids seeds in vitro back in 1900 and have experimented since to perfect it and move forward with micropropagation.

I have read that every Cavendish banana is tissue cultured. These are the bananas on shelves world wide. Not surprising, they were bred to have no functional seeds. I'm not sure about other commercial crops.

Venus fly traps and the like ate popular for tc, at least amongst hobbyists and they seem to have that down part as well.

The lack of study on marijuana is because it and hemp have been illegal for so long. There is the one Chinese paper I know of that tried it with hemp. I forget their success rate.

There's only a couple of reasons I see it could be worthwhile other than just liking to tinker and experiment.

1) Keep a back up library of many strains. This in case the mother dies. Or in case you are short on room and want to try new strains still, but don't want to lose other strains you'd need to cull to make room.

2) You're running a large commercial grow. This way you don't need tons of identical mothers to keep product consistency. Even this way you can get variations in size I'd think. My understanding on tissue culture is it's rather uniform in growth when everything is consistent. You wouldn't have to root in multiple batches as the mothers degree enough.

3) Better spread of clone only strains, or maybe hard to clone strains. It theoretically would allow shopping if the person on the other end knew what they were doing. Or to this end possibly the practice of artificial seeds will be perfected for marijuana.

I would say I'm surprised the operations in Amsterdam never picked this up for crop uniformity but sadly I'm not. Those seed companies don't even bother stabilizing their lines. Just grab any two "strains" also them together, also a new name to it and ship it right out the door

 

[Voidling]

F1 isn't stable genetics and would leave you pheno hunting for something similar but would never be exact. This is why there's big variation when buying seeds from seedbanks

F1? Is that femenizing the seeds? Some believe this is not a good practice for various reasons. Some will say not to breed from a feminized seed. I don't know if these people are any smarter than me but it's not an area I Ann interested in researching things like tc. I'm also not sure how much variation you get this way.

As for explants they should go on forever provided a few things. They will have to be removed, divided, and placed in new agar mix periodically. During the time they are open it must be a sterile environment. My understanding of good Petri dishes is they seal completely to keep out contamination in lab cultures.

If you search here for tissue culture you will find a couple of threads. There it's probably a small amount of good information on each. Unfortunately they invariably get somebody talking about how they've done it for decades and it's so easy but never have proof or give out actual information. One went as far as to claim he had an article all about it in the upcoming skunk magazine but as far as I could tell they had no such article the month he said, nor the next. They disappeared and the threads die

 

[GrowerGoneWild]

F1 isn't stable genetics and would leave you pheno hunting for something similar but would never be exact. This is why there's big variation when buying seeds from seedbanks

F1? Is that femenizing the seeds? Some believe this is not a good practice for various reasons. Some will say not to breed from a feminized seed. I don't know if these people are any smarter than me but it's not an area I Ann interested in researching things like tc. I'm also not sure how much variation you get this way.

As for explants they should go on forever provided a few things. They will have to be removed, divided, and placed in new agar mix periodically. During the time they are open it must be a sterile environment. My understanding of good Petri dishes is they seal completely to keep out contamination in lab cultures.

If you search here for tissue culture you will find a couple of threads. There it's probably a small amount of good information on each. Unfortunately they invariably get somebody talking about how they've done it for decades and it's so easy but never have proof or give out actual information. One went as far as to claim he had an article all about it in the upcoming skunk magazine but as far as I could tell they had no such article the month he said, nor the next. They disappeared and the threads die

No I'm talking about S1 a selfed female to limit varation. F1's could be stable if the result was a homozygous allele.
Perhaps it doesn't even matter, it would be ideal to grow out each one to pick desired traits. As much as I would like to keep certian phenotype expressions, I have to realize that cannabis hybrids will continue to evolve, what was awesome yesterday can be plain vanilla today..

Yeah I'm thinking it could be possible to "continue" the culture, vs a mom in a bucket scenario. If one was to have proper equipment like a laminar flow hood.. I could see this as possible, It just comes down to economics, Its relatively low cost and simple to maintain a mom.

Yeah I dont really care about RIU contributions, the overall quality has gone down, I'm more of an ICmag person myself, I'd rather spend time looking an actual abstract than going through the threads here. I think I post stuff here mainly as a way to think out loud.