Tissue Culture- Let's discuss!

I want to get a tissue culture discussion going here!
It's my newest little geek obsession...

Here's what I've got so far:

Good points:
You can get thousands of exact genetic replicas from one donor plant with minimum stress to the donor.
You don't need to use a slip with multiple nodes!
You could overnight someone an envelope with a few leaves and it's like getting a clone, you just have to do the rest of the work. So spreading your favorite genetics to your buds (hee hee) would be VERY easy.

Bad points:
Takes 8 weeks
Requires you to be a wicked nerd with a degree in geekiness and thick glasses and no social life (Um, I made that one up)
Requires sterile conditions
Requires media and chemicals that aren't all that easy to find

My questions:
Have any of you happy gardeners done tissue culture on any plant at all?
If so, have you done it on cannabis?
If so, is there a specific supplement that you add to your agar?

Please peeps don't tell me this is a bogus subject since taking cuttings is so easy. Yeah, yeah, yeah, I know, whatever. I'm not arguing that. I mean, f'real, if you want low tech, plant a seed outdoors. Wait for it to rain! This whole hobby is high tech and I think tissue culture is, frankly, COOL.

Oh I agree! Unfortunately, I'm nowhere near geek enough to actually be able to do it, but I think it's awesome. Have YOU done it? And if you have, how'd it turn out? Was it worth the work?

You could be the resident DNA guru (kind of already are).

I haven't done it and I'm trying to assemble enough rudimentary knowledge to try.

Here's something interesting!
DYI media recipe included!

http://www.une.edu.au/agronomy/AgSSrHortTCinfo.html

It seems to me, that by doing this ... you could theoretically make a plant that would have many many main colas, or something similar. Couldn't you?

You absolutely have to try this, Stinky!!! :D I'm so curious.

I 'm assembling all the media, etc as we speak. Some is kind of hard to get your hands on, and I have to wait for my boss to get back from vacation and use his lab account to order some of the stranger chemicals!
One thing I found by doing a literature review is that there is a lot of research being done on micropropagation of hops because it is such a disease-prone genus. Hops, well we all know that's one of the family Cannabacea so I assume the methods are cross-applicable. I'm definitely going to have to kind of wing it on media based on info for hops and info that I found about pot growth regulators.

This is quite an interesting subject.From my experience with shrooms, contamination was the biggest problem.

Why even use things from "work". This can only bring questions. If not from your boss, maybe his bosses boss.....all it takes is one accountant seeing a strange order/expenditure, and unless you've started a business with "cloned" houseplants, only bad things can be assumed.

Actually, there are plenty of companies out there selling home culture kits, and with the "magic" stuff like growth promoters and such that you'll need.

I've done a bunch of tissue culture, and even protoplast fusion, but that was in University.

But I've never tried just sitting there with a paper puch, holing up some nice, squat indicas to get a bunch of sweet "clones".

And tissue culture IS the same as conventional cloning....same thingy. What will you do when you have lots of callous, though...?

The original plan was just to keep dividing the callous and putting bits of it in a root-promoting media as needed, and keeping the rest of it essentially in suspended animation on shoot-promoting media so it is sort of a genetic archive. I think it's likely I will break down and order a kit... in the meantime I got an ez-clone and am having fun with that.

What types of cells are you going to try starting from. Root, stem leaf? are you going to try all of em for fun?

is cannabis different than other plants in the sence that you can clone from any tissue?

You can get many of the chemicals and materials you need from Carolina Biological or Fisher Scientific (education section). I would also check techniques used in orchids....the most info on cloning in tissue culture is on these plants. Current methods can allow you to do gene transfers and get plants to produce many medical compounds. I have access to all necessary equipment including ultra low temp storage, sterile hoods, autoclaves....I'm looking into it. Imagine being able to place the gene that produces THC into another plant...what would the Feds do then? Probably prosecute for some new law....or better yet, add a promotor gene to the THC genes, producing massive quantities....:smokin: :thumbsup: I always knew this degree would come in handy for something ....

Quote:

---

Originally Posted by machinehead83

Imagine being able to place the gene that produces THC into another plant...

---

Is that even possible? o.O Hot damn, science is amazing.

I wouldn’t smoke it, genes react in unpredictable ways.

Harris... planning on trying all of them to see which works fastest... but hoping for results with leaves especially since there are so many of them...

Machinehead... I've got access to that stuff too, cool. Just trying to figure out if orchid media would be appropriate... I'd love to get the THC gene into hops... it's a vine and a perennial and close already!

Afghooey... yes, I think you have to use bacterial or fungal carriers, but that's a whole field I don't know a damn about!

Harris... I wouldn't smoke it either. Oh well, nice to dream anyway!

i accutaly know quite a bit about GM and yes bacterial fungal viral or the gene gun. which shoots little pieces of gold soaked in DNA into te cells

Quote:

---

Originally Posted by stinkyattic

Harris... planning on trying all of them to see which works fastest... but hoping for results with leaves especially since there are so many of them...

Machinehead... I've got access to that stuff too, cool. Just trying to figure out if orchid media would be appropriate... I'd love to get the THC gene into hops... it's a vine and a perennial and close already!

Afghooey... yes, I think you have to use bacterial or fungal carriers, but that's a whole field I don't know a damn about!

Harris... I wouldn't smoke it either. Oh well, nice to dream anyway!

---

Why are there all these people afraid of genes???:dance:

The hops idea is a good one Stinkyattic....I like the idea of inserting a high copy, powerful promotor in to control the gene...I'll bet some pubmed lookups will find the sequence for the gene, even bet there are companies that sell it ready to go....it's REALLY big now to design drugs based on MJ....that is why they will fight legalization with every lawyer and breath in their bodies....I have 1 friend working for a Big Pharma company that has been getting strains from Amsterdam for 7 years and isolating the compounds of different medical strains....finding the best ones for pain and making a drug out of it...etc....

My doc budies are using marinol for MS with GREAT success, but says the patients tell him the real deal works much better....actually stops the autoimmune distruction of mylen sheaths....probably through a very slight inhibition of immune function (remember them trying to blame HIV on MJ? it does lower some T cell function).....OK, I've gotten WAY too geeky....LOL

love to compare notes with you Stinkyattic....:thumbsup:

by the way, I would use an electroporator for the gene delivery.....no chemicals and doesn't kill the cells like ...also MUCH cheaper than a BioRad gene gun!!!! Think you can't even buy them, have to lease it with a service contract....and since I have several of their "high tech" molecular bio equipment, like a real time PCR....have to say, it's not worth the hassle...electroporators work great and can be found cheaply on LabX....although I already have one...and a good one at that....:)

is that were they pass the current through the cells which make them more permeable?


i'm not afraid of genes i'm afraid of uncontrolled genetic Engineering mainly in our food supply. I support it in controlled lab settings to create drugs. As they are controlled. But with all this shit Monsanto is doing they released practically untested, as the US gov doesn’t require it, into the environment.

Yes, an electric pulse is sent through the media the cells are in and carries DNA or RNA into the cell. Also used to get proteins into cells.

As far as plant genetic engineering...you are listening to too much propaganda....there have been NO wierd products produced in this area....they use identified genes for different properties such as frost resistance, sweetness, nutritional value factors....I could see someone with ill intentions using it for some horrible effect, but that is true with anything. You can't prevent every nut out there from doing nasty things. I can also bet that any plant that has been altered required many months if not years in the lab environment just to get it to grow properly. Now if you were talking about insects, animals, humans, etc...I have a REAL problem with messing with that genetic make up. Although it's already being done with farm animals (in labs) in an attempt to make their organs acceptable for transplantation into humans...they put human genes in that express the same HLA antigens as humans so not rejected. These are known genes. You should see all the transgenic animals already available!!! Insects, bacteria, viruses are what you have to worry about, TRUST ME. When you mess with them, that's where the problems begin. messing with plants and farm animals will not put anyone in danger, unless they make a fire breathing mad cow with wings (not possible).

Also, you should know that in the past, regular breeding techniques in insects have created killer bees among other screw ups....we didn't need genetic engineering to do that!

Don't worry, be happy......:rasta:

i dont know what you mean by weird products. I am talking about normal food crops ie canola, corn, soy. Which are all the largest crops in north america and are now GM (not all but lots. all in the case of canola and soy. the only non gm soy is grown in asia)

I can bring up many examples of how they fucked up. Dont tell me i'm listening to propaganda. How about GE corn. which has caused infertility in pigs. And a big warning went out to European farmers telling them not to feed GE corn to pigs they want to breed. Hmm a mammal and what are we... mammals. Yet no warning for us.

It is an incorrect assumption to think that these are being tested properly. They are done by American corporations. And what was Monsanto’s comment to this

Something like: as a corporation we are concerned with selling as much of our product as possible, it is not our responsibility to find out the safety of the products. This is the FDA’s job.

And what did the FDA say. Hmm I believe 15 out of 17 FDA scientists voted againsted releasing these products YET the American government overruled them and released them.

I am willing to speak to this. But I don’t think it is appropriate to discuss in this thread.

oh yea and your wrong to think that they modify food crops for sweetness, frost etc... To date only one product has any benefit to consumers. Which was the flavor saver tomato (you know the one which caused stomach lesions in rats). It was recalled because they didn’t store well. All other products are generally roundup ready resistant.

They are made so monsanto can sell more round up.

who is listening to the propaganda

The genetic modifications to staple crops- soy, corn, etc. benefits one entity, frickin' monsanto, ADP, DuPont, the whole seed production industry.
Don't get me started.
And monocultures.... brrrr... gives me chills.

TC, isnt that where you take a small section of the plant and make it grow?? It sounds hard, my friend, took a bud from his plant, planted it, and it grew roots, and is just a tiny bud plant, i was so angry when i found out, i sit around working out shit for my plants all day long,(i do get better crop alot of the time :)) and he just waters his plants and doesnt really care about them, and he managed to make a bud grow roots in driveway soil, i dont know how he does it, he must have lady luck on his side i guess.... oh well, sorry if im completely off subject haha, just ignore me...

Quote:

---

Originally Posted by Abattoir Dream

TC, isnt that where you take a small section of the plant and make it grow?? ...

---

Yup, like 1 square cm of leaf.

Quote:

---

Originally Posted by Abattoir Dream

he just waters his plants and doesnt really care about them, and he managed to make a bud grow roots in driveway soil, i dont know how he does it, ...

---

I'm sorry, but that is hilarious. You should go steal some soil from his driveway if it's that magical! :D

yeh, i would but knowing me im not as neglectfull as him so i wont get the same results lol

I looked into this a couple of years back- Getting root growth is apparantly pretty easy, but this particular plant has an atypical response to shoot-induction regulators.

Of course that could have been the particular cultivar they were working with in the research I saw- It was intended to prop Fun-free stock for the fiber industry.

Lemme look around and see if I still have the referances.

well i just found out that i have access to all the equipment to do tissue samples (except an autoclave) but i might have access to one of them too.

hmmm, I see a science project coming on.

I wish i had a kid, i could teach them how to do a tissue culture for grade 7 he he

Quote:

---

Originally Posted by harris7

well i just found out that i have access to all the equipment to do tissue samples (except an autoclave) but i might have access to one of them too.

hmmm, I see a science project coming on.

I wish i had a kid, i could teach them how to do a tissue culture for grade 7 he he

---

LOL Awesome. Borrow a neighbor's kid!
You can use a regular canning pressure cooker. Ask your grandmama or your old auntie Bernice! Tell her you are making jam.

Hello every one I am new here and I am Stash 1, I was Stash but I got a new email and forgot my password a long time ago. This thread caught my eye. I gotta get in on this but it's been a few months since the last post. I hope all here have not lost all interest. I am an old school mushroom grower so I know almost all that is needed to know and have all the equipment stored away. I am also the guy that discovered using Colloidal Silver to change sex in pot plants. I don't want to do this all alone. Is this thread dead?

Naw it isn't, and since I just had to ditch my grow because of my snitch ex, I have all sorts of time on my hands to experiment... and we just got a spankin new laminar flow hood with UV sterilization at work... I will be practicing on legal plants of course so my boss doesn't hit the ceiling :D

Stinkyattic, It's very good meet you. One thing that I used in the past that is very important is using H2O2 (Hydrogen Peroxide) in the agar. Agar turns solid at (if I remember ) 110 deg - 130 deg. Over 120 deg H2O2 turns into water and hydrogen. The H2O2 must be added to the agar at around 120 deg. H2O2 stops anything from germinating (a great help). In mushrooms you can use this when cloning but not when germinating spores. The way you do this is to leave your boilling flask of agar in the pressure cooker. Remove the lid every now and then to take the temp of the water inside with a thermometer (keeping pressure cooker inside the air hood or if it's to big keep it in the air flow). When temp hits 120 deg pour the H2O2 into the flask, add enough H2O2 to make 10% of total volume. Move flask into hood and put cooker away. Pour agar into petri dishes, put covers on and let cool. Then take a new box of small zip lock baggies. Spray it down with rubbing alcohol and put it into the hood. Put cooled petri diskes into baggies. Spray down a new box of large zip lock baggies and put smaller bagges inside. Store away in frig until needed. Ware rubber gloves and respray hands with rubbing alcohol before putting them into the hood every time. (Don't Smoke) I have alot of agar on hand but it's the wrong mix for plants. I'll have to do some reading. Later, Stash