Cannabis tissue culture

[Hidyn]

Oh crap. I wish I hadn't found this thread. I can tell already that I'll need to find space for culturing gear amongst my all my other hobby areas

Damn you curiosity, you'll be the end of me!

 

[Mia]

Not sure what company you are using, but I know the guy that makes these out of Mountain View California. This guy really knows his shit and makes a great product.

Lol, you must be referring to the infamous Bill. Bills cool, always trying to sell you something. Old school Pure food in Mountain View FTW!lol
Awesome thread gray wolf can't believe I missed it.

 

[jump /injack]

Cannabis propagation in China

Cannabis propagation in China

If anyone knows of any published studies or threads talking about shoot differentiation from callus please point me in the right direction. My google-fu is weak it seems.

This is a site I ran into a few years ago about cannabis in vitro propagation in China, everything you need. There has been some other studies since then, this will get you started. Go to the web site below and download the 6 page pdf.

http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf

This study describes the standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from shoot tips of Hemp (Cannabis sativa L.). Hemp seedlings were germinated on half-strength 1/2 MS medium supplemented with 10 g·L-1sucrose, 5.5 g·L-1agar at a pH of 6.8 under light for 16 h per day. MS medium containing 0.2 mg·L-1TDZ, 0.1 mg·L-1NAA supported the maximal auxiliary bud multiplication rate of 3.22 per shoot tip. The proliferated buds were successfully rooted on MS medium supplemented with 0.1 mg·L-1IBA and 0.05 mg·L-1NAA resulting in 85% of the plantlets rooting. The procedure requires a 54 days cycle for the In vitro clonal propagation (14 days for shoot multiplication and 40 days for root induction) which includes 35-42 days for acclimatized plantlet production.

 

[OrganicMilitia]

This would be a wonderful way to preserve genetics if they could be kept in a dormant state that could be multiplied at a later time. Just imagine how many different tissue cultures I could sent to someone on the other side of the world! It would be a wonderful way to spread some of those medical strains to regions where they are needed. This could be the basis of a new international Care giving crew. If done correctly it could revolutionize genetics if everyone shared and was Irie about their genetics. If it was technologically advanced enough we could sequence the DNA of everyone genetics so any future Hybrids could be distinguishable as coming from a Breeder thus still giving credit where it is due.

 

[Green Supreme]

"The procedure requires a 54 days cycle for the In vitro clonal propagation (14 days for shoot multiplication and 40 days for root induction) which includes 35-42 days for acclimatized plantlet production. "

Always wondered at this part. Thanks. Peace GS

 

[Dripperz]

and r.e. the pathogens. the theory is that viruses either cannot or do not penetrate into the meristematic tissues of the plants (because if they did they would kill the plant way faster) so you can TC the meristematic tissues and hopefully will clone off virus free growth.

agrobacterium... taking multiple gene from different strain and cross genetic... take some of your favorites strains, make callus from it, use a bacteria to infest the callus and absorbs its trait by infestation for a day or two, then culture tho gene imprinted bacteria, propagate them into a new strain callus, as it infect the new strain it will also incorporate it'S gene, then kill the bacteria with it's antibiotic, and you'll have your self a new strain, if on where to have a libraries of most plant families and species on hand, one could someday make strawberries high in thc, and that would be the end of all illigal issues related to cannabis, since thc could be found in all living plant... (of topic) but hey, that was my dream when a was young, have on hand over 2500 strain of cannabis and make high in thc fruit, tho i do not consume cannabis' i still love fruit and consider myself a cannabis passionate

 

[indocult]

Hey all I thought I'd chime in....just finished our micro propagation section at school, did ky14 tobacco and begonia leaf cuttings. All work was done in flow hoods, utensils flame sterilized, hands sprayed with 70% ethanol solution. Proper techniques for working in a flow hood include keeping all utensils/petri dishes towards the back where air is cleanest, always open lids away from you towards the clean air, work in a vertical line of stacked petri dishes so your hands are never positioned over either petri dish/test tube, whatever you want to use. Parafilm works best to seal your growing chambers but saran wrap is a cheap alternative.

The media we work with is the basis of all micro-propagation agars, known as the Murashige and Skoog media, MS for short. Basically they analyzed tobacco leaves, drew a line of best fit through the nutrients found, and transferred it to an agar recipe. A hot plate, preferably with magnetic stirrer, and a pressure cooker will be needed for fix this.

1. Weigh 30 g sucrose
2. Measure out 800 mL double distilled water into 1000mL beaker, combine, this is where a magnetic stirrer/hot plate comes in handy
3. Add 1 mL vitamin B(unsure of exact concentration)
4. Add 10 mL NaFeEDTA
5. Add 20 mL nitration solution
6. Add 10 mL Halide
7. Add 10 mL Myoisotol
8. Add 10 mL Sulfat
9. Add 10 mL PbMO
10. Add BA, cytokinin
11. Add NAA, auxin
12. pH 7.7-7.75, adjusted with varying concentrations of NaOH
13. 8g agar is then added
14. Top to 1000 mL double distilled water
15. Autoclave/pressure cooker for 15 minutes at 20 psi
16. Pour into petri dishes/test tubes, cover and wait to cool.

I realize this isn't a complete formula, I am missing exact concentrations and I forgot how much BA and NAA was added.

As I am sure everyone realizes, the benefits of micro-propagation are immense. I created 6 african violet plants with a leaf about the size of my little thumb and it took less than 5 minutes. Plants have what is known at totipotency, which is basically the ability to form an entire plant with just a single cell of material. It's not an overnight process, the cells must first undifferentiate and form a callus tissue, similar to a tumor. It is from this callus tissue the cells re-differentiate and roots/stems begin to form. Its pretty cool holding entire plants that are sealed in test tubes. I'll post pics of final results tomorrow. I could take a large cannabis leaf and cut in into 75-100 pieces, placed into a few petri dishes, and within a matter of 4-6 weeks have 75-100 newly forming plants. All from 1 leaf.
It really starts getting interesting when they start inserting genes during this process, that's a whole other topic and ethical discussion. I think the US has a hard enough time getting parents to give their children regular vaccines but somehow its O.K. for companies to test bananas on children in Africa that have had the immunizations spliced into the fruit. Or the new corn with Bt inserted into it. I saw a new tomato species that had some desert plants genes inserted into so that it stopped responding to heat and drought stress. Even at like 10 days of 100F+ days, 90F+ nights, healthy and green as could be while the controls were a crisp.

Anyways sorry for the incomplete formula, it should be enough for most of the people actually interested in this to accomplish their goal. Its really not hard, just stay sterile, and use

Thanks man, I just found a .pdf with this recipe

It mentions BAP for Shoot formation and NAA for root formation. I'm guessing these are the same hormones you are mentioning, or similar/with similar functions. It says to add them each to the rest of the recipe in separate containers, at a rate of
BAP 2mg/liter
NAA .1 mg/liter (to do this you make a concentrate with the hormones suspended in liquid (water?) at that concentration, and add the liquid to your media.

I've never gone to college or taken any courses in this, just learned online stuff, done a lot of myco work so I am very familiar with laminar flow hoods, still air boxes (better than glove box), sterile technique, Agar media, etc.

I'm thinking the tissue culture can't be that much different than myco culture work, just different recipes, and as I am seeing with the hormone thing, a different regime to get it going. (of course it's plants, not fungus).

Growgeek's words are inspiring saying his 6 year old goddaughter takes culture of african violets and such, It's a shame all kids aren't taught this stuff instead of b/s other things that aren't useful or practical. I am bias here though, maybe things are different in some places

I have a really awesome smelling/looking plant deep into flower now that I realize I forgot to clone. I am hoping I can come up with a solution with this Tissue culture thing rather than having to keep a bud and try to re veg..

 

[Team Microbe]

Has anyone had success with the TC kit that Monster Garden sells?

https://www.monstergardens.com/Plant-Tissue-Culture-Microclone-Kit?filter_name=tissue culture

 

[Team Microbe]

bump

 

[ghostmade]

How did i miss this.subbed

 

[ganjourno]

So I tried all this shit many many times, never got it to work. Maybe with lab grade equipment it's possible, but even being extremely careful and hosing my kitchen down in alcohol, using a dust hood, steam-cleaned and flamed scalpels - there was always contamination after a couple weeks. I managed to get some plantlets established but even then, could not get them to root again using IBA.

It's a nice idea but not achievable for the layperson I think. Definitely not as simple, effective, or practical as old fashioned moms and clones.

 

[Only Ornamental]

Yea, sterile work requires some practice and routine. If you want to learn it by yourself, you're predisposition to fail cause there are so many things you won't think of and which are in pure contradiction to the way of handling things in everyday life.

Just as an example:
- Alcohol kills no spores
- Dust hood? Like a cover out of plastic sheets or serious air filtration and a laminar flow?
- Stem cleaners, unless industrial ones which nearly melt your stuff, are a prime source of contamination and seldom do what they're supposed to do but make things worse instead.
- It needs some trial and error to figure out the ratios of plant hormones added (there's more than just IBA). Can't do that if you only have a very few ones which don't get mould...

 

[Team Microbe]

Yea, sterile work requires some practice and routine. If you want to learn it by yourself, you're predisposition to fail cause there are so many things you won't think of and which are in pure contradiction to the way of handling things in everyday life.

Just as an example:
- Alcohol kills no spores
- Dust hood? Like a cover out of plastic sheets or serious air filtration and a laminar flow?
- Stem cleaners, unless industrial ones which nearly melt your stuff, are a prime source of contamination and seldom do what they're supposed to do but make things worse instead.
- It needs some trial and error to figure out the ratios of plant hormones added (there's more than just IBA). Can't do that if you only have a very few ones which don't get mould...

Valid points made here O.O. - I'm very curious about this for obvious reasons - mainly the possibility to take more cuttings in a smaller space.

What would you recommend to the newbies looking to get into tissue culture to read/watch/do in order to start off on a good foot?

Are there any common mistakes people often make in the beginning that can save us time in the trial and error phase?

I'm wondering if this is something I can utilize for size-able outdoor grows, this would save a lot of space in my room and still let me put out the kind of numbers I'd like to plant outside... Thanks for any insight man

 

[Only Ornamental]

...
What would you recommend to the newbies looking to get into tissue culture to read/watch/do in order to start off on a good foot?

Are there any common mistakes people often make in the beginning that can save us time in the trial and error phase?
...

Training, training, training!! Best and in most cases only thing that you really need apart from a sense of order and a steady hand is someone who can actually show you how to do it and even more important who watches over your shoulder for a few times.
The thing is less the general rules, you find them posted everywhere (check out laminar flow manufacturers home pages) but the small things. How you organise your stuff, how quickly you move, how long you need to open and close a sterile jar, your sleeves touching things without you noticing it, you watching your right hand whilst your left hovers over sterilised equipment... there's unfortunately a ton of things not really in a book that count as much.
What I've seen/read people tend to combine 'sterilisation' techniques hoping that more is more but have no idea what exactly they do. There's for example no point in using H2O2 and bleach one after the other...
A minimum requirement SOP is quickly a few Word pages long but will only cover the basic routines. These are important, no doubt, but people are no robots and tend to introduce their 'personal style'. This is as good as it's bad: Personalise your routine makes it more handy and practical for you but there's no telling if it's a good or bad routine if you don't watch the person perform it.

Try to hire someone or take a seminar if you have the needed pennies. Else, it might be possible to do it per Skype with a small but good resolution remote camera (I don't like computers/laptops in such an environment, they have fans and the keyboard is... disgusting, to say the least). Sure it won't capture every angle (which is important) but it'll be better than nothing. You just have to find someone willing to do so (PMing me might help but I'm not at liberty to discuss such things here, the TOU, you know...).

 

[Only Ornamental]

Oh, forgot, one little trick for those who have no permanent 'sterile' bench:
Use cling film (saran wrap)! It's produced at high temperature and great speed and hence is nearly free of dust and microbes. With common household equipment, you're unlikely to get something as sterile unless you put it in the oven or drown it in bleach or hydrogen peroxide (which require ventilation you shouldn't use right then). Quickly wrapping a rack and your table à la Dexter Morgan is the fastest and cheapest way of creating something similar to a disposable sterile hood. It's not completely sterile and won't remain for long but might still come in handy when you have no serious lab equipment.

 

[ghostmade]

Google diy glove box(should take you to shoomery,mycotopia).make use of your old storeage bin.also lysol is your best freind in this situation. Ive also made quick use of a cardboard box and cling wrap.not ideal but i have done alot of successful inoculation,grain to grwin transfers,and shroom cloning this way

 

[Only Ornamental]

I hate to work with glove boxes (too small and confined) but true, they're easier to keep clean and tolerate more mistakes. Thanks for bringing this up.

 

[ganjourno]

So after your comments, my failure was likely due to the sterilant I used (90% alcohol).

I used a clear storage tote with the opening closed off with cling film except for the bottom few inches where I got my (gloved) hands through. All the equipment and water was sterilized in a pressure cooker prior to use. All windows were closed to ensure zero air movement and the whole room was sprayed down with alcohol prior to beginning. But of course since the alcohol was not the right thing, I was doomed from the start.

For the ex-plant preparation I cut new growth tips and sterilized them in a 1:10 household bleach/distilled water solution in a magnetic mixing beaker. I did another wash with a tissue culture specific sterilant as well, which I forget the name to. It was some three letter acronym that started with M, like MEP or somesuch. It was expensive to procure.

The medium was an agar mix with pre-weighed nutrient salts froma tissue culture supply place, mixed and poured into tubes then pressure cooked for a while.

After the explants were sterilized I moved them into the shrouded working area and trimmed them down to core meristematic tissue and then implanted into the media. The tubes were sealed with some thin strips of cling wrap.

So it seems like the alcohol is where I went wrong. After 1-2 weeks most of my cultures developed moulds.

 

[jimbop]

I did another wash with a tissue culture specific sterilant as well, which I forget the name to. It was some three letter acronym that started with M, like MEP or somesuch. It was expensive to procure.

"PPM," or "plant preservative mixture." PPM is comprised of isothiazolone biocides, used in (among other things) cosmetics and shampoos to keep them from rotting in the bottle. While they can be used for acute disinfection in the same manner as bleach, the concentration needs to be high and the duration needs to be several hours to a day or more. They are normally added to the medium to inhibit growth of microorganisms, and they're not very good at that. I've never worked in a commercial lab that used PPM; it's too expensive for mass propagation.

Having spent more than a decade working in tissue culture, I would be happy to answer specific questions about tissue culture. For the most part, TC isn't useful for marijuana as it's much easier to propagate by cuttings.

 

[ganjourno]

PPM! That's the stuff!

THanks for making your knowledge accessible! I agree that it's way easier to take a few hundred cuttings - the TC advantage isn't until you need to make tens of thousands of identical starts which at this point noone is doing.

But what about for having a library of strains? For example if I wanted to have 25 strains on standby, without keeping mothers. Could I keep these in culture in a relatively stable fashion, and then divide and root the callous tissue into new starts as needed? How long do you think it would take to go from callous tissue to a hardened start suitable for planting?

And for the purposes of a library, how long will a sterile callous culture last? Months? Years? What is the maintenance like?

I'm also interested in those cultured seeds as well