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Offlinejust me
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Cloning Plant Tissue on Agar
    #14605872 - 06/13/11 12:06 PM (12 years, 7 months ago)

could yous guys n gals help me understand this :lol:

curious mainly for the sake of saving genetics long term

and also mainly focused on marijuana

although veggies would be nice too.

can anyone link me, or shed their personal knowledge on this subject.

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ill just add links/quotes here in case anyone wants to read on similar findings

Troncotron's thread on Marijuana tissue culturing(looks GOOD!)
http://www.shroomery.org/forums/showflat.php/Number/10803981#10803981

Quote:

DjCorbetto said:
I had a recipie for shoots for cannabis, and it just said: M&S media with 15g/litre of sucrose. when i spoke to carol at home tissue culture she reccomended that i got the M&S media with vitamins as its same price but better, ive read that if you add a multivitamin tablet to the solution that helps too

The media is M&S, with 15 g/L of sucrose, pH 5.8 before sterilizing (balanced with KOH) and 2mg/L kinetin, 0,2 mg/L IAA and 10 g/L agar.

it doesnt specify whether its for roots or shoots but then im still looking at his follow up as he got shoots on the plant material but so fare dont see a bit on roots.

Link: The media is M&S, with 15 g/L of sucrose, pH 5.8 before sterilizing (balanced with KOH) and 2mg/L kinetin, 0,2 mg/L IAA and 10 g/L agar.
http://www.lycaeum.org/mv/anagrams/cannabis_tissue_culture.html





Quote:

Burbles said:
Check out http://www.bananas.org/f260/

Shows a lot of people making smaller banana, pineapple and other interesting plants via TC

I think we should all work together and try to get a pretty serious tissue culture community going somewhere that is welcoming of ethnobotanicals, and other "questionably legal" plants...




Doc-T's thread on micro-propagation
http://www.shroomery.org/forums/showflat.php/Number/14013111#14013111 ;

Quote:

Burbles said:
Yes - this actually makes sense, that is the main sugar used within plants, and that also explains why sucrose is so available, we get it from plants.
Other than that, I don't know why, but it is what I have read.

I was thinking today in class that propel - that gaterade stuff, might actually be a good thing to add to these experiments, as it has EDTA - which is a common chelating agent and would help the metals stay in solution better, in addition it also has small amounts of various B vitamins (some are needed by plants, IIRC - like thiamine?) and has other things like citric acid (its used in MicroP for various reasons, as a nutrient, pH regulation, etc) and ascorbic acid (another chelating agent, if I remember correctly - though probably would decompose on PCing)

Might be worth a shot, I don't know.. I think EDTA is important though, and is hard to find OTC unless it is in one of those gunky EDTA supplements...

Also:
Link & description about Banana micropropagation and other exotic fruit/plants
http://www.thelaboratory.org/talk/index.php?topic=134.0
Amamature Micropropagation thread in general.. has some useful info...
http://www.thelaboratory.org/talk/index.php?topic=18.0
First attempt at micropropagation... using potato cuttings, guano tea
coconut and sea water
http://www.thelaboratory.org/talk/index.php?topic=202.0
And also there is a bit more about growing out cacti callus cultures, etc.. but i don't want to spam my site - but there is a pretty good amount of micro/plant tissue culture info there
Also check out these micropropagation videos, great for beginners using the Home plant tissue culture materials..
http://www.youtube.com/user/fbt2007

glad you are trying some stuff out!





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-pEaCeLoVeGoDbLeSs-

"The Downfall of Mankind; is Believing He Has Limitations."


Edited by just me (06/13/11 02:05 PM)


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Invisiblepuff4200
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Re: Cloning Plant Tissue on Agar [Re: just me]
    #14605879 - 06/13/11 12:07 PM (12 years, 7 months ago)

:popcorn:


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Invisiblewanderingmarlin
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Re: Cloning Plant Tissue on Agar [Re: puff4200]
    #14605901 - 06/13/11 12:10 PM (12 years, 7 months ago)

As far as I understand, a piece of tissue is placed on an agar medium that contains all the required nutrients and hormones for growth.  Search this forum for tissue culture, there's some great threads on it floating around.


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InvisibleDoc_T
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Re: Cloning Plant Tissue on Agar [Re: wanderingmarlin]
    #14605913 - 06/13/11 12:13 PM (12 years, 7 months ago)

I tried in a halfassed way this winter, and got some interesting results.
Enough that I'd try it again sometimes with better temps and preparation.


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: Doc_T]
    #14606001 - 06/13/11 12:33 PM (12 years, 7 months ago)

doc, what was your "test subject"

ill search around in the forums, and link anything i find that seems useful

but id definitely like to hear some first hand responses

and wanderingmarlin do you know the required nutrients and hormones?

ill have a look!


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"The Downfall of Mankind; is Believing He Has Limitations."


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Re: Cloning Plant Tissue on Agar [Re: just me]
    #14606030 - 06/13/11 12:40 PM (12 years, 7 months ago)

I believe standard cacti nutes (2-10-10 or so) are used.  As for hormones, I think the bare minimum would be cytokinin (6-BAP) and a rooting hormone like IAA or IBA.  Gibberellic acid could also work, but I'm basing that off botanical theory, not actual evidence.


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: wanderingmarlin]
    #14606133 - 06/13/11 01:02 PM (12 years, 7 months ago)

i dont have any cacti atm
this would be more specific for MMJ
and vegetables down the road sometime

Quote:

As for hormones, I think the bare minimum would be cytokinin (6-BAP) and a rooting hormone like IAA or IBA.  Gibberellic acid could also work, but I'm basing that off botanical theory, not actual evidence.




Thanks! ill look into these


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"The Downfall of Mankind; is Believing He Has Limitations."


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Invisiblefngbronco
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Re: Cloning Plant Tissue on Agar [Re: wanderingmarlin]
    #14606134 - 06/13/11 01:02 PM (12 years, 7 months ago)

:popcorn:


--------------------
I challenge you to challenge yourself more! When you feel complacent and ready to hang it up, challenge yourself to get over it! If you fail, don't look at it as you didn't succeed, look at it as you would a rock face you're trying to climb. Stand back, wayyyy back, and look at it and plot another path. If you can't find one, shuffle down the way a little, a little change of scenery or a view from a different angle may give you the insight you need.

Anything I state is relayed information from a friend of a friend and should be viewed as completely fictitious. I do not partake in any illegal or grey-area-of-the-law activities, but do have lots of friends who may or may not.    -fngbronco

Pill Divider Agar Tek


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InvisibleDoc_T
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Re: Cloning Plant Tissue on Agar [Re: fngbronco]
    #14606188 - 06/13/11 01:14 PM (12 years, 7 months ago)

I tried cannabis seedlings on dilute Miracle Gro agar.
Chopped up seedlings onto agar, some sort of grew.


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: Doc_T]
    #14606208 - 06/13/11 01:18 PM (12 years, 7 months ago)

hahaha:lol:

roots?

dilute miracle grow agar? miracle grow fertilizer, or miracle grow soil/agar dilution :what:

it would be amazing if theres some greenhouse garden ways of doing this, as apposed to in the lab funny business :crazy2:

im getting an image of what ill use as containers, which will allow room for deep medium(increasing the exp. date)??? and filtered GE if necessary


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Re: Cloning Plant Tissue on Agar [Re: just me]
    #14606253 - 06/13/11 01:26 PM (12 years, 7 months ago)

Agar made with dilute Miracle Gro instead of water.
It was winter time like I said, so it's not really a fair trial.

Here's a link to a post w/pic within the thread: http://www.shroomery.org/forums/showflat.php/Number/14061459#14061459


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: Doc_T]
    #14606328 - 06/13/11 01:41 PM (12 years, 7 months ago)

:thumbup: thanks

no worries man, im starting from ground zero, im sure itll take me a while to get started, let alone successfully shit talking


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"The Downfall of Mankind; is Believing He Has Limitations."


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Offlinebrshroomer
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Re: Cloning Plant Tissue on Agar [Re: Doc_T]
    #14606401 - 06/13/11 01:53 PM (12 years, 7 months ago)

Doc_T, long time since we talked(haven't been up lately)

i was inspired by your experiment and tried something similar. it's currently running for the last 24h and i should see some results soon.

media used was a mix of commercially available chemical nutrients with micronutrients, sugar, agar and algae extract('rooting extract')

we'll see if it works in a few days, i have a wedge of pereskiopsis there.

one thing i noticed is that there's a lot of precipitate or undissolved nutrients, adding EDTA would probably help keep these dissolved.


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OfflineBurblesV
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Re: Cloning Plant Tissue on Agar [Re: brshroomer]
    #14607961 - 06/13/11 06:35 PM (12 years, 7 months ago)

It seems most people jump in to do it before they really understand the purpose, what is necessary, and costs of doing the project.

It won't cost thousands to do like many claim; but it will cost at least 250 if you are savvy and hours and hours of research to find the right hormone ratio and nutrients needed for any specific plant, including the trial and errors. I have posted tons about cacti mixtures involving coconut milk but I haven't had the opportunity to go for this full on the people who CAN do this do not share their info (though, I am pretty sure I cracked the code, its not that hard to UTFSE and get all the info you need from people with journal access)

However! since its literally just a a type of agar different than mycology; most of us are there. You just need to buy the MS media, hormones and be damn good at sterilizing the plant tissue, using the teks (alcohol, soap, distilled and sterile water). I don't think cutting corners is the best idea to begin a relativly new hobby; buy from the current and only plant tissue organization on the web for amateurs, get in contact with them, and see what advice they can give you.

I will be taking on this project at some point with Salvia divinorum when I get the funds and time to do so... and you can trust me I will document every inch of it in pictures, and excessive detail.

I even recently bought a series 1 laminar flow hood to spearhead the TC and keep up the quality of mushroom cultures and already have the culture media, hormones, culture tubes, sterilizers, and all that jazz...


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Offlinebrshroomer
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Re: Cloning Plant Tissue on Agar [Re: Burbles]
    #14611140 - 06/14/11 10:03 AM (12 years, 7 months ago)

well, you're probably right, cutting corners is a bad idea, except i'm not cutting corners, i'm rather experimenting to see how simple a medium can be made to get some growth out of plant tissue.

coconut milk in media is a VERY classic approach, about when plants started to be cultured. but it wasn't their first implement and plants have been grown sucessfully on nutrient agar before that or any complex mixtures where used. if i remember correctly the first reported culture was a carrot callous culture.

you don't have to bash me or Doc_T for doing this saying that we could use the search engine, i'm not a noob and neither is he. i'm actually currently studying biochemistry in college, which has a specific section on plant micropropagation and such.

i know i would be better off starting with murashige and skoog media and working from that to get a better media, and i plan on doing so.

i'm currently stocking the reagents to make it, but cobalt isn't something particularly easy to find OTC. the other stuff seems easily available to me and rather cheap. the most weight of MS0 media is due to ammonium nitrate and potassium nitrate, both easily available.

i hope to see your work posted soon, it would help MANY of us. i'll be looking for it.


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: Burbles]
    #14612784 - 06/14/11 03:28 PM (12 years, 7 months ago)

Quote:

Burbles said:
It seems most people jump in to do it before they really understand the purpose, what is necessary, and costs of doing the project.

* but it will cost at least 250 if you are savvy and hours and hours of research to find the right hormone ratio and nutrients needed for any specific plant

* You just need to buy the MS media, hormones and be damn good at sterilizing the plant tissue, using the teks

* buy from the current and only plant tissue organization on the web for amateurs, get in contact with them, and see what advice they can give you.





well this is kinda where im at right now

btwn a rock and a hard place :lol:

Burbles suggests it takes at least $250 investment, with hours of research
and can really only get the GOODS from one company. (and even still i have to amend it to get the hormones and nutrient levels correct)
the problem here being, no $money$ (time, willingness to fail or succeed and technique i have DOWN :P )

Doc_T and brshroomer are trying a more OTC/DIY approach
more like the way i would like to start this endeavor
ive never been a lab coat and goggles kinda guy.
more like the, "ooooh whats this nice piece of trash? i could use that somehow!" kinda guy
just because micropropagation is semi new, doesnt mean it has to be done by the specs of the only ppl doing it

i would like to do it RIGHT the first time
but i would be MORE APT to try the whole project if it didnt involve spending a small fortune to a company that can charge that because theyre pioneers in plant tissue culturing


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"The Downfall of Mankind; is Believing He Has Limitations."


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OfflineBurblesV
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Re: Cloning Plant Tissue on Agar [Re: just me]
    #14613464 - 06/14/11 05:36 PM (12 years, 7 months ago)

Sorry wasn't trying to bash, more so just saying a warning for others.. I've talked to a lot of people that are doing *facepalm* approaches.. so to speak?

If you want some M&S media - I have some, and will send you a few grams if you want (enough to make a liter)
also have some IAA and 6-BAP...
PM me if interested.

I got it from here, its the cheapest place:
http://www.caissonlabs.com/

No need for DIY M&S media when you can get it for http://www.caissonlabs.com/catalog.php?srch=MSP06 its dirt cheap... 1 liter worth of powder for less than a dollar, and 50 litters for about 25 dollars.


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Re: Cloning Plant Tissue on Agar [Re: Burbles]
    #14613492 - 06/14/11 05:40 PM (12 years, 7 months ago)

the 250 is assuming you don't have the culture tubes, agar, etc -- which you do. So its much less - probably  bout' 50 bucks if you happen to already do mycology...

http://www.hometissueculture.org/
and of course check out the YT videos about it...


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Offlinejust me
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Re: Cloning Plant Tissue on Agar [Re: Burbles]
    #14613506 - 06/14/11 05:42 PM (12 years, 7 months ago)

Burbles, ill talk with you bout the trade :smile:

Quote:

If you want some M&S media - I have some, and will send you a few grams if you want (enough to make a liter)
also have some IAA and 6-BAP...
PM me if interested.



Quote:

No need for DIY M&S media when you can get it for http://www.caissonlabs.com/catalog.php?srch=MSP06 its dirt cheap... 1 liter worth of powder for less than a dollar, and 50 litters for about 25 dollars.




...and this is all i need to get my foot in the door?

C, im doing this ATM solely for marijuana
i imagine its one of the easiest plants to work with
and i have plenty of room for error. im not going to lose the plants if i cant get them on agar.
i just travel a lot, and i would love to take strains with me wherever, and not ALWAYS have to depend on seed that i may or may not have successfully pollinated the parent :lol:


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"The Downfall of Mankind; is Believing He Has Limitations."


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OfflineBurblesV
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Re: Cloning Plant Tissue on Agar [Re: Burbles]
    #14613584 - 06/14/11 06:02 PM (12 years, 7 months ago)

Well you'll need:
1. Freshly grown tips of plants, ideally from indoors where there is less spores, bacteria, etc.

2. a good method of sterilising the tips without killing them; so say 60% alcohol, sterile water, and a good way to cut them up for multiple plantlets... H2O2 is not a good option. IME


3. The right nutrient mixture for the plant at various phases; http://www.lycaeum.org/mv/anagrams/cannabis_tissue_culture.html
Hormones are the most expensive; you will also probably want the expensive, unnecessary, but very very helpful PPM "plant persarvative mixture" as it does a great job at fighting/preventing bacterial and fungal contaminations...

4. Fantastic sterile technique... Bacteria is way more prone to grow on M&S media than it is on PDA, or other... due to pH and what not.

5. Good containers; you can get reuseable PP ones at that home tissue culture thing... just contact carol and she'll sell you some  or use baby food jars.
Gotta pressure cook the stuff and what not.

Also less agar per liter of water aids in the bacteria spreading faster; for whatever reason? IIRC plant tissue culture has less agar per liter by about half than does when working with mycelium - probably because its better for the plant, and more agar when working with agar wedges is desirable for strength.

Also when you transplant them I'd suggest sterilizing the soil first, and have it very neutrient poor to prevent any rot as they will need to be kept very humid at the start.

I have been messing around with psudo-micro-propagation a bit; just seeing how small of cuttings I can take and still have them root without using anything fancy - but keeping them in a culture tube with water, or water + agar (no neutrients) and of course in high humidity - I have not had any results I want to see from that... but small chunks of salvia will root... seems like its pretty hard to get them to grow into plants after as the new shoots don't survive for some reason (probably bacteria or some sort of water damage)


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