Stinky's Adventures in Micropropagation...

[stinkyattic]

Hey all,
I just went out and finally got a digicam with flash so I can document my latest folly.

I've decided to let my plants be guinea pigs for a go at tissue culture, which I'm just going to call TC from now in because I type really not so fast! :D

Here's what I am trying to find out in these experiments:

1) What media/additive combination works best for shoot formation in Cannabis.

2) What media/additive combination works best for rooting.

3) What part(s) of a Cannabis plant are appropriate for TC:
-Nodes
-Internodal stem segments
-Growing tip tissue
-Leaves
-Roots (I might not use roots, depends how much space I have in the incubator)

4) What sort of growth pattern do you get- Can you get a single-stemmed plant out of a mass of TC'd organisms without damaging them too much?

5) How long does this take?

I'm working on a trial media and have come up with a list of components that several different sources that all agree on:
-A gelling agent
-A sugar
-A nutrient source
-One or more growth regulating substances:
-----inositol
-----B1 Thiamin
-----NAA
-----coconut milk (seriously)
-----Kinetin
-----IBA

So I'm putting this all together.

I found Agar locally at a Chinese market as an ingredient in a dessert mix... but it was almond-flavored and already had the sugar added, so no control over that.
GNC stores carry powdered inositol and Thiamin.
Coconut milk comes from coconuts.
NAA and IBA are found in rooting agents but I want them individuially and might have to go to a biological supply store for that.
I don't know where in the heck I'm going to easily find kinetin!

Keep ya posted.

[turtle420]

This will definitely be a Favorite thread.

So far, 1st place in the Science Fair! :)

[downundadude]

i agree you know your stuff.... good luck ill wait for futher posts definatly

[stinkyattic]

Still trying to assemble ingredients for my media. Thank you for your patience!

Although I do have my tools lined up:

Containers will be half-pint jelly jars, because they're the right size and can take autoclaving.

My sterile cabinet will be a clear plastic rubbermaid-type tote with hand-holes cut in it.

I've got access to a nice analytical balance accurate to 0.0001 gram :D yay.

Sterilizing agents will be bleach spray and a spirit lamp for flaming off tools.

The autoclave is a beefy old pressure cooker that has a HUGE capacity, but it's at work and I'm still hunting around salvation army locations for a cheap canning processor.

I've narrowed my plant-parts choices to leaf segments and nodes.
We all know that nodes are the source of good totipotent cells when it comes to cloning.
But I want to try leaves as well because obviously the plant has plenty of leaves and not so many 'extra' nodes. Leaf sections will be 1cm square and placed cuticle-side down on the medium so the stomata can still breathe.

[UnitedParcelSecrets]

Are you making any progress, Stink?

[stinkyattic]

Yeah, I FINALLY found a product that contains cytokinin.
It's Bonide Tomato Blossom Set Spray.
So add that to my supplies-on-hand list.

[locomark]

Sticky,

This shit is a little over my head but I'm watching you.

Loco

[stinkyattic]

It'll be a while. I had to put this project on the back burner. The new autoclave we're ordering at work hasn't arrived yet.

[harris7]

Stinky does that make you a tatto artist?

[stinkyattic]

Nope, a chemist.

[harris7]

oh, interesting. I myself am quite apt in the field of chemistry. what kind of chemist are you?

[opabud]

hey i was wondering of another way to preserve -for like a year-
i have been ziplocking, and freezing
but i heard about mason jars, but need more info on that, but if anyone has any other ideas ???
thanks
opabud

[opabud]

also, i have aquired mold/fungi in some of my tomatoes, and was told to now throw away the ones that house this lovely pain in the ass. its gonna kill me to throw away this at one time beautiful substance, don't know how it got there, its friends don't show this ???

[stinkyattic]

hey i was wondering of another way to preserve -for like a year-
i have been ziplocking, and freezing
but i heard about mason jars, but need more info on that, but if anyone has any other ideas ???
thanks
opabud

opabud, you need to start your own thread, bud.

[stinkyattic]

oh, interesting. I myself am quite apt in the field of chemistry. what kind of chemist are you?

inorganic/environmental.
water and sediments, nutrient load, road salts, heavy metals in fish tissues, YUCK!!!!
You haven't lived until you've processed mouse fat samples to extract mercury from them.

[ericwt]

Have you done anything with this lately? I have recently become interested in Micropropagation. I ordered a Micropropagation kit recently.

Keep us updated.

[foghead]

[attachment=o253496]

This thread looks like it died a few years ago but I thought I would put in my two cents worth if any one is still interested. I was given the protocol attached above from someone who is doing this successfully. I have not attempted this yet but will be in the next month. I would recommend purchasing a kit. This where I got mine >>Home Tissue Culture Group Catalog - Kits (http://www.hometissueculture.org/catalogkits.htm)

Comes with basic instructions. Sterilization and sanitation seems to be the key.
Then it is just a matter of finding the right recipe for the plant species you are culturing. The recipe in the above PDF file is working for my friend. One thing that is missing is the addition of 30 grams of sugar per liter.

[caligrown82807]

dang wat happnd stink :wtf:

[lenoxluda]

Hey fog head I am making an attempt at micropropagation. The pdf file doesnt work. Im having a hard time finding information on this, any help would be greatly appreciated. Thanks
[attachment=o253496]

This thread looks like it died a few years ago but I thought I would put in my two cents worth if any one is still interested. I was given the protocol attached above from someone who is doing this successfully. I have not attempted this yet but will be in the next month. I would recommend purchasing a kit. This where I got mine >>Home Tissue Culture Group Catalog - Kits (http://www.hometissueculture.org/catalogkits.htm)

Comes with basic instructions. Sterilization and sanitation seems to be the key.
Then it is just a matter of finding the right recipe for the plant species you are culturing. The recipe in the above PDF file is working for my friend. One thing that is missing is the addition of 30 grams of sugar per liter.

[thrive]

any conclusions?
Thanks!!

[oldmac]

Hello thrive,

I mastered African Violets, but was never able to work out the needed medium/hormones for mj.

And StinkyAttic has just been gone for awhile now.

OM

[AZClones]

Hello thrive,
I mastered African Violets, but was never able to work out the needed medium/hormones for mj.


I do tissue culture for a living. Maybe I could lend a hand.

[Prodaytrader]

well shit...now I am interested too.

Am I to take it that tissue culturing in terms of cannabis would allow us to clone from a flowering plant? Or more likely store the sample for a length of time to later use? How's about some of you brainiacs dumb it down for us business majors. Is there a process/procedure we can do at home?

[AZClones]

well shit...now I am interested too.

Am I to take it that tissue culturing in terms of cannabis would allow us to clone from a flowering plant? Or more likely store the sample for a length of time to later use? How's about some of you brainiacs dumb it down for us business majors. Is there a process/procedure we can do at home?

The ability to clone flowering plants in order to produce "new" plants- that's tricky; I've never tried it, so the best analogy might be with bamboo, which dies once it's done flowering. Unfortunately, I've never worked with bamboo in tissue culture, so I'm not sure if it's a determinate plant in that context.

TC of cannabis offers clear utility with the propagation of large numbers of clonal propagules; most growers wouldn't need more than can be produced by cuttings, although at substantial cost to the parent clone.

As for the DIY thing- it's there, but it requires a sterile prep area (which can be as simple as an aquarium tipped on its side with a sheet of plastic over the front), equipment for sterilizing media (a pressure cooker), culture vessels (plastic or glass), a handful of tools, and chemicals. It's about 60% technique, 35% know-how, and 5% materials. However, few growers have reason to try tissue culture as existing propagation techniques are adequate for most purposes. It's more of a parlor trick than anything else; if it weren't, it would be much more widespread by now- like LEDs for indoor growth. Really- nobody is buying $1200 LED illuminators for growing carrots. It's all market-driven.

There might be some application in cloning high-percentage ruderalis plants, but that's theoretical.