tissue culture

[Reeferman]

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Home / All Titles / In Vitro Cellular and Developmental Biology - Plant / November 2003 / pg(s) 578-585
In Vitro Cellular and Developmental Biology - Plant
Published by: Society for In Vitro Biology

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In Vitro Cellular and Developmental Biology - Plant 39(6):578-585. 2003
doi: 10.1079/IVP2003454

TISSUE CULTURE AND AGROBACTERIUM-MEDIATED TRANSFORMATION OF HEMP (CANNABIS SATIVA L.)
M. FEENEYa and Z. K. PUNJAab
aCentre for Environmental Biology, Department of Biological Sciences, 8888 University Drive, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6
bAuthor to whom correspondence should be addressed: punja@ sfu.ca
P. Ozias-Akins
Summary
Hemp (Cannabis sativa L.) is cultivated in many parts of the world for its fiber, oil, and seed. The development of new hemp cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 µM kinetin, 3% sucrose, and 8 g l−1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium containing 2.5 µM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed. Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines ranged from one to four.

Received: August 20, 2002; Accepted: April 30, 2003
Keywords: callus, suspension culture, Agrobacterium tumefaciens, mannose selection, phosphomannose isomerase, regeneration

 

[Reeferman]

I am trying to learn as much as I can about TC before my initial attempt I am going to call skunkman and see if he can offer me any assistance on medium hormone levels etc
Reef

 

[FastForward]

Check this out, posted by AquaLab, if you've not already seen it:

 

[hererisssh]

I don't know how you get on with Obsoul33t reef, but he's alluded to continuing success with tissue cultures without a crazy lab setup. Hit him up, he posted some basics in this thread:

 

[bageledj]

ya deff go talk to OB.he does well with the tissue cultures.he dosnt have a lab but he built himself a nice little "glovebox" to not have any contamination problems

 

[Reeferman]

Thanks guys he was big help thanks absol !

 

[Jalisco Kid]

Thats the direction to be going in. This way the elite will be for the masses.

 

[xchaoticmetalx]

I am very interested in learning more about this myself. My boy has schematics for a sweet small cleanbox he uses for shrooms, I think im going to have him build me one so I can get my hands on learning started. Looks to be a very interesting technique.

 

[edelephant]

there isnt a way to upload PDF files, but i sent you something of interest.

enjoy

 

[Reeferman]

The tc iis not as easy as it looks and mailing it is not going to happen its not there yet .
Reef

 

[xchaoticmetalx]

The tc iis not as easy as it looks and mailing it is not going to happen its not there yet .
Reef

Yeah, lol, that would be some intense, and prob expensive shipping. Even when worked out.

 

[Reeferman]

Female seeds will have to do it friends !

 

[nurlord]

Reef.

Your killing me slow maaaaaaaaaan. Can we get a list of the fem beans in the works?

 

[Motarebel]

Wasn't Breeder Steve and his crew collecting clones to do tc right before CannabisWorld went down? Never heard anything about it after that so it must of went tits up.

 

[nurlord]

I remember that even though I was a little confused as to whether it was tissue culture. I believe he said that there wouldn't be any legalities involved as there wasn't any plant material. Don't quote me on that though.

 

[matamus]

any news on this

The tc iis not as easy as it looks and mailing it is not going to happen its not there yet .
Reef

very intriguing, but this thread died real fast, reeferman did you ever have any luck doing this. My mind is now working in all sorts of directions.

 

[zoeronerer]

i have been doing TC since the 90's ...im an avid aquarium keeper and its really common in the biz for keeping different species and color variety's ...

the main thing that most if not all of you will have trouble with is sterile culture most of you are dirty bastards .. constantly touching dirty dirty things.

take your time in prep and cut your fingernails short. wash hands to your elbows you dirty fukerz.

change your shoes , spray lysol be extra careful.

tc the only way to keep large numbers of genetics in stasis with no bugs.
if you do it correctly you can even cure the plant of long term disease like pm or plant virus .... most people will fail the first few tries . stick with it and good luck u dirty boys and girls...

 

[bud good]

nice nice ive always wanted to but dont have the time right now to dedicate to sterility and perfecting the method but heres a web site with a kit www.planttc.com i think its 200-250 dollars less shipping its real fucking neat i did a little mush once and read up on cloning id take sterility tips from the shroom guys everything zoeronerer said is dead on man i dont wanna pry in to your personal but could you post some pics of you baby jars im so interested in the subject id love to see pic's

thanks man

 

[gorillaseeds]

it took me a few times to get the hormones right for cannibis now im in the process keeping my mother in tc. Zoeronerer you allways impress me. i wasted my money on planttc the hormones are not right and you can get better equipment way cheaper.

google is your guide

good luck

 

[thefabman]

A buddy has been doing well and seems to have it down now, so my first attempts will be comming shortly. Can't wait!!