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Experiments with plant tissue culture
    #10803981 -

Hello, i´ve been doing some experiments with cannabis tissue culture and other species, this is the first attempt but seems to work , no contamination!
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.
The sterile work was done in front of a homemade flowhood.
This are the results:


Edited by troncotron (08/11/09 07:28 AM)

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10804087 -

Lookin' good. What other species have you tried?

I used to do a bit of that when at university but not with cannabis.


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My Ethnobotanical Growing TeksWhere to Start Flow Chart, QnA, and helpful links

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10804445 -

nice work, they look good.
try sally next.


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Rodney Brooks on Robots
Nalim said: "Quoting yourself is retarded."

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Re: Experiments with plant tissue culture [Re: Nalim]
    #10804974 -

Very nifty.


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The Plant Mage Guild

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Re: Experiments with plant tissue culture [Re: Horticulture]
    #10806127 -

I'm  very impressed....He says staring dumbly at the acrynoms with no idea what they could even be possibly referring to.


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Friendships have come and gone but I always regret the girls I didn't bang




I just dont really care what you think

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10808011 -

interesting stuff :thumbup:


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View My Gallery

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Re: Experiments with plant tissue culture [Re: Dr. uarewotueat]
    #10809731 -

Hello, thank you. Karode13, i´ve done also with other ornamental species as Scirpus aureus, as i said is the first attempt at home, before at the university i did direct and indirect organogenesis (tomato, melon, kalanchoe, potatoe), somatic embryogenesis (carrot), and transformation techniques using Agrobacterium (tomato). Now, i´m going to experiment with Salvia divinorum explants (if i can finally found it, it´s difficult here in Spain) and with some cactus seeds that i´ve recently ordered (Lophophora and trichocereus).



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Plant tissue culture
Crossbreeding experiments

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10809783 -

I'd happily swap a rooted Salvia cutting for a clear writeup on how to do make this stuff up aand do this procedure. Just have to get some cuttings rooted :-). Or just plant material as its only 2 days or so shipping from Ireland to spain.


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Friendships have come and gone but I always regret the girls I didn't bang




I just dont really care what you think

Edited by Fleadh (08/06/09 06:34 AM)

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Re: Experiments with plant tissue culture [Re: Fleadh]
    #10809843 -

You sound way more advanced than anything I have done, things look clean and healthy. :thumbup:

I have only done a crash course in plant organogenesis/micro-propagation techniques, species like Zandtedeschia, Metrosideros, Calla and Solanum.

Good luck with your experiments,


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My Ethnobotanical Growing TeksWhere to Start Flow Chart, QnA, and helpful links

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10813353 -

Given that you got nicely down the "hard part" for many (agarwork, hormone,sterility and above all devotion : getting to try it rather than discussing it), why dont you try further experiments such as polyploidy? Well, and that doesnt mean using colchicine :wink: 

A thread i started on it ,to give some ideas can be found at the shroomery , here.

I wonder if a plant calus could be subjected to treatments i propose in the thread.

Good work, keep it up Troncotron!

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Re: Experiments with plant tissue culture [Re: Psiloman]
    #10816295 -

That´s interesting, Psiloman, but i still have to improve the method. I want to achieve a good method for regenerating plants from callus, it´s complicated and the results i´ve seen in some papers are very low regenerating ratios. I also want to do some research in somatic embryogenesis.
Flehad, i´m afraid i can´t offer this clear explanation cause each plant has different medium requirements, and i´ve never experimented with salvia divinorum before. Otherwise, it seems to be easy to multiplicate in vitro via "single node culture" and i would like to try it.


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Plant tissue culture
Crossbreeding experiments

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10826357 -

New adventitious shoots appearing via direct organogenesis in Scirpus aureus stem. Only two of eight explants have shown this response, something expectable cause hormone concentration was the same as for meristem culture. Doing some research and reaching the optimun concentration you can obtain much more quantity of plants faster than the other method. You can obtain shoots from stem, roots, cotyledons, leafs, etc..



The other shoot keeps growing fast



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Plant tissue culture
Crossbreeding experiments

Edited by troncotron (08/09/09 07:14 AM)

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10835098 -

Those cultures look great troncotron! Also, thanks for posting your recipe! I figured I might as well contribute some of my own photos - I've been working on loph. tissue culture, all of these jars were produced from one cultured seed about 5 months ago [I started with one jar, then divided it up]. Ignore the brown phenolic extrusions/bruising in some of the photos, it was a problem with my early media formulation + dull razors, not contamination.


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Re: Experiments with plant tissue culture [Re: sushi]
    #10835152 -

I was wondering about TC-ing cacti. Great to see the pictures!


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The Plant Mage Guild

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Re: Experiments with plant tissue culture [Re: sushi]
    #10835378 -

That is awesome Sushi!

Have you hardened any down to living on their own roots yet?


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Re: Experiments with plant tissue culture [Re: Cactusdan]
    #10844038 -

Hey sushi, nice work:thumbup:

I´ve recently received some lophophora and trichocereus seeds that i´m going to inoculate today. I´ll post results soon. The problem with growing cactus in vitro seems to be the tissue vitrification. Here´s an interesting paper that explains how to overcome this problem in some species controlling air exchange.

http://www.springerlink.com/content/r204287p16w66x10/fulltext.pdf


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Plant tissue culture
Crossbreeding experiments

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10851765 -

Here´s some pictures of the process. I´ve inoculated only ten seeds in the first round in order to test if the surface sterilization was right.




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Plant tissue culture
Crossbreeding experiments

Edited by troncotron (08/13/09 12:25 PM)

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Re: Experiments with plant tissue culture [Re: troncotron]
    #10851850 -

What recipe are you using for the cacti guys(if you are willing to give it up that is)? This looks funky! Is it true the cacti grow faster in vitro?

are you guys using a MS medium similar to what these people did
http://www.acgssr.org/BioTechnology/V8N1January2005/abstract/English/e15.pdf ?

Edited by smaerd (08/13/09 01:42 PM)

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Re: Experiments with plant tissue culture [Re: smaerd]
    #10855525 -

troncotron: I was having problems with vitrification in my old media [the pictures I posted show some evidence of this] but I've since refined my recipe and the problem seems gone now, I also have been getting good hair [or whatever you'd call it, the tufts of stuff older peyote plants get] growth on the peyote in culture the new recipe while before as you can see in the pictures I posted the plants were hairless. I love your idea of using filter paper to sterilize the seeds, I've been using this convoluted mesh strainer I built out of a plastic test tube and it's really not ideal. You need to keep me updated as to your success, I find with cactus seeds other then peyote I can get them to germinate fine but then the seedlings just stall and I have no idea why!? In fact I haven't been able to get reasonable growth rates from any columnar cacti - I can induce pupping just fine but having the pups elongate is another story!

I'm going to post my media recipes for cacti culture on the condition that if anyone uses any of these they'll report back with any improvements/observations/suggestions/whatever. All my media is solidified with agar, I haven't tried any other gelling agents.

Loph Media/Pedro pupping [my original formula, causes a little vitrification]:
1/2 MS w/ vit./L
0.7mg/L PPM [trying 1.0 mg/L]
5.0mg/L BAP
0.5mg/L NAA
25g/L sucrose [trying 20g sucrose + 5g dextrose]
~5.80 pH adjusted w/ H2SO4

Loph Media [my newest recipe, no vitrification and stronger growth]:
1/2 MS w/ vit./L
1.0mg/L PPM
5.0mg/L BAP
0.5mg/L NAA
20g/L sucrose
5g/L dextrose
~5.80 pH adjusted w/ H2SO4

So far, this is the best recipe I've been able to find for actual shoot elongation in columnar cacti [still far from ideal, growth is very slow]:

1/4 MS w/ vit./L
0.8mg/L PPM
2.0mg/L BAP
0.1mg/L IBA [this seems to be key, swapping IBA for NAA pretty much stops all growth, if anyone is experimenting with this you might want to pay special attention here]
25g/L sucrose
~5.80 pH adjusted w/ H2SO4

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Re: Experiments with plant tissue culture [Re: sushi]
    #10855591 -

Thanks so much for that sushi, I know some people don't like giving out their recipe's. If I do end up finding something to improve this I certainly will let you know!(P.S. I gave you a nice rating :laugh:)

Edited by smaerd (08/13/09 10:30 PM)

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Mushrooms, Mycology and Psychedelics > The Ethnobotanical Garden
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