Anyone cloning via Tissue Culture? opinions welcome

[AOD2012]

hey everyone. just read some stuff in my biology class that got me thinking, so i started to do some research. at my school, the biology department has all these heirloom plant species in tissue culture thingys. basically you take a soft cutting, wash it off with soap and water and alcohol to make sure everything is sterile, then you put it in a little jar with the gelatin that has nutes and hormones or whatever. and from this you can make multiple clones, in a much smaller of an area, and you also dont have to water or anything. only drawback is that they take a lot longer to root. i just like the fact that you can keep literally hundreds of different strains in a very small area.


well anyway, just wanted to see what peoples opinions on this were, and if anyone has used is successfully. thanks a lot.


aod

 

[HighDesertJoe]

It's a great idea I saw it being done in the 70's at Cal Poly, always thought it'd work great for Herb I wouldn't be surprised if big MJ seed company's aren't doing it all ready.

 

[nsaneone]

In my experience, its really useful when you are in a situation where you are considering a re-veg. Tissue culture works at any stage of growth. Also, if you needed to create a couple million clones. It'd be great. It's really easy to do. If you want to get started, you don't need a laminar flow hood or anything, you can have reasonable success with stuff you have at home already. I don't know if it's cool to post links to other sites, but this place has all the stuff you need to get up and running.

http://www.kitchenculturekit.com

I've flowered tiny roses in vitro. It might be fun to grow a tiny nug sometime. Good luck on this one man.

 

[nsaneone]

I have a nice tissue culture tutorial. I will try to find it. It's from the University I attended, so I'll have to edit it a bit to remove evidence of my location.

 

[HighDesertJoe]

In my experience, its really useful when you are in a situation where you are considering a re-veg. Tissue culture works at any stage of growth. Also, if you needed to create a couple million clones. It'd be great. It's really easy to do. If you want to get started, you don't need a laminar flow hood or anything, you can have reasonable success with stuff you have at home already. I don't know if it's cool to post links to other sites, but this place has all the stuff you need to get up and running.

http://www.kitchenculturekit.com

I've flowered tiny roses in vitro. It might be fun to grow a tiny nug sometime. Good luck on this one man.

I Bookmarked it all ready

 

[The_Grim_Reefer]

I have a nice tissue culture tutorial. I will try to find it. It's from the University I attended, so I'll have to edit it a bit to remove evidence of my location.

That would be appreciated

 

[nsaneone]

[FONT=Times New Roman, serif]There might be a couple of images that don't show up, but here it is. You can just substitute cannabis for tobacco, or whatever the hell we used. I would consider building some sort of clean box and dousing it with 70% isopropyl alcohol and doing your work in there. Much higher rate of success. You'll need to sterilize your own water which can be done with a microwave or a pressure cooker (you totally have one of these if you've done any at home mycology ) Without further ado, here's the tutorial.[/FONT]


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[FONT=Times New Roman, serif][/FONT]
[FONT=Times New Roman, serif]Please follow the directions! Do not start or do any of the tissue culture procedure without first being instructed to do so![/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Do Not Start [/FONT][FONT=Times New Roman, serif]the lab procedures[/FONT][FONT=Times New Roman, serif] until instructed to do so. Work will be done in groups of 3-4.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Each group of students needs to collect and bring back to their table:[/FONT][/FONT]
   
   1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Three baby food jars of water, labeled ‘W’. [/FONT][FONT=Times New Roman, serif]Do not open![/FONT][/FONT]
   2. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]One baby food jar of 10% bleach Clorox® – for surface disinfesting your leaf. And one small squirt bottle of [/FONT][FONT=Times New Roman, serif]70% ETOH (ethanol)[/FONT][FONT=Times New Roman, serif] for cleaning your work area and hands. Note: Clorox solution can damage clothing.[/FONT][/FONT]
   3. [FONT=Times New Roman, serif]One baby food jar of 70% alcohol (ethanol).[/FONT]
   4. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Six petri dishes of prepared medium* and one empty petri dish. [/FONT][FONT=Times New Roman, serif]Do Not Open! [/FONT][FONT=Times New Roman, serif] The petri dishes are labeled A, B, and C, you will have two of each, one for the light treatment and one for the dark treatment (*see table on 5-c for medium information).[/FONT][/FONT]
   5. [FONT=Times New Roman, serif]Several paper towels.[/FONT]
2. [FONT=Times New Roman, serif]On your worktable, you will find: forceps, tweezers, scalpels, dissecting needles, magic markers and an alcohol burner.[/FONT]

1. [FONT=Times New Roman, serif]With your marker, label:[/FONT]
   
   1. 1. [FONT=Times New Roman, serif]Your water jars: W-1, W-2, W-3.[/FONT]
      2. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Spray bottle with 70% ETOH (ethanol)[/FONT][/FONT]
      3. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Your bleach jar[/FONT][FONT=Times New Roman, serif]: B-2 (10% bleach solution (Clorox®)).[/FONT][/FONT]
      4. [FONT=Times New Roman, serif]Your ethyl alcohol: 70%.[/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Wait until all the students are back at their tables before proceeding. [/FONT][FONT=Times New Roman, serif](This reduces air movement, which helps to reduce contamination.)[/FONT][FONT=Times New Roman, serif] Moisten a paper towel with 70% ETOH in spray bottle and wipe your hands. Then wash your working area, the outside of all containers and tools.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Collect [/FONT][FONT=Times New Roman, serif]2[/FONT][FONT=Times New Roman, serif] [/FONT][FONT=Times New Roman, serif]tobacco [[/FONT][FONT=Times New Roman, serif]Nicotiana benthamiana[/FONT][FONT=Times New Roman, serif] Domin.] (or petunia)[/FONT][FONT=Times New Roman, serif] leaves, selecting fully expanded healthy-looking [/FONT][FONT=Times New Roman, serif]leaves with petioles[/FONT][FONT=Times New Roman, serif], or other plant materials as directed. [/FONT][FONT=Times New Roman, serif]Use smaller to mid sized leaves.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Place the plant material in your ‘B-2’ container. 2: Gently shake the container to keep the solution moving in the container. 3: Gently poke down the plant material with your forceps or dissecting needle to keep the leaves submerged in the solution. Do this for [/FONT][FONT=Times New Roman, serif]15 minutes[/FONT][FONT=Times New Roman, serif]. You have now initiated the disinfestation procedure.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]During your 15 minutes’ wait, practice flaming your tools:[/FONT][/FONT]

1. [FONT=Times New Roman, serif]Light the alcohol burner[/FONT]
2. [FONT=Times New Roman, serif]Dip your forceps in the ethanol flask.[/FONT]
3. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Holding the forceps [/FONT][FONT=Times New Roman, serif]horizontally[/FONT][FONT=Times New Roman, serif], place the tips of the forceps in the flame. Hold over the flame only long enough to ignite the ethanol. [/FONT][FONT=Times New Roman, serif]Do Not [/FONT][FONT=Times New Roman, serif]tip (cutting end)[/FONT][FONT=Times New Roman, serif] the points of the forceps upward as the flaming alcohol is almost invisible and you can be burned easily. [/FONT][FONT=Times New Roman, serif]PLEASE BE CAREFUL!!![/FONT][/FONT]

1. [FONT=Times New Roman, serif]Practice your flaming techniques again, using the scalpel.[/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]With your freshly flamed and cooled forceps, remove your plant material from the bleach solution and place in ‘W-1’ for five minutes and gently agitate, then transfer to ‘W-2’ for five minutes (gently agitate) and finally to ‘W-3’ for five minutes (gently agitate). [/FONT][FONT=Times New Roman, serif]Three (3)[/FONT][FONT=Times New Roman, serif] rinses are necessary to remove the bleach solution from the plant material since long exposure to bleach is toxic to plants.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Working as a pair, one student should open the empty petri dish slightly, and the other student should remove a [/FONT][FONT=Times New Roman, serif]tobacco[/FONT][FONT=Times New Roman, serif] leaf and place into the opened petri dish. The inside of this dish is your sterile cutting surface.[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Flame and cool your scalpel and a dissecting needle. While one student is holding the petri dish slightly open, the other student should cut the leaf into strips about 4-5 mm wide [/FONT][FONT=Times New Roman, serif](see sketch).[/FONT][FONT=Times New Roman, serif] Use the dissecting needle to hold the leaf while cutting.[/FONT][/FONT]

[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] [/FONT][FONT=Times New Roman, serif]Sketch information:[/FONT][/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] [/FONT][FONT=Times New Roman, serif]Plant Propagation – tips for keeping plant materials sterile[/FONT][/FONT]
[FONT=Times New Roman, serif] -After Clorox® (bleach) treatment plant materials is to be kept sterile as possible –[/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] -Tools dipped in 70% ETOH should be placed in (or on) sterile PLASTIC PLATE. Be sure to flame the tools often.[/FONT][/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] -Instructions for the cutting surface for making leaf pieces (explants): use the sterile plates (Petri dishes).[/FONT][/FONT]
[FONT=Times New Roman, serif] [/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] -Using the sterile blade and forceps cut the leaves into narrow strips [[/FONT][FONT=Times New Roman, serif]4-5 mm[/FONT][FONT=Times New Roman, serif]] across the veins (perpendicular to the mid vein). The veins are where most of the new growth activity will occur.[/FONT][/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] [/FONT][FONT=Times New Roman, serif]See below picture. [/FONT][/FONT]
[FONT=Times New Roman, serif] [/FONT]
[FONT=Times New Roman, serif] [/FONT]
[FONT=Times New Roman, serif]Making the perpendicular cuts across the mid vein of the leaf.[/FONT]
Mid vein of the leaf



[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]If a cut portion of the leaf was exposed to the bleach treatment - make a fresh new cut. As the bleach has burn the exposed cells and tissues and damaging them.[/FONT][/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]-As quickly as possible put the cut leaf strips in the PLASTIC sterile cutting plate and [/FONT][FONT=Times New Roman, serif]KEEP[/FONT][FONT=Times New Roman, serif] the plate [/FONT][FONT=Times New Roman, serif]COVERED[/FONT][FONT=Times New Roman, serif].[/FONT][/FONT]
[FONT=Times New Roman, serif]- When placing the cut leaves on the media plates keep the media plate covered between placements.[/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Place two leaf pieces into each petri dish. When placing the leaf pieces into the dishes, open them only enough to slip in your [/FONT][FONT=Times New Roman, serif]leaf pieces[/FONT][FONT=Times New Roman, serif] (explants). Do not expose the nutrient medium surface to the air any longer than necessary. You may want to compare the growth of older leaves* with younger leaves. You can do this by placing a leaf piece from both types in each petri dish and then label the cover of the petri dish to indicate the location of each age leaf piece.[/FONT][/FONT]

[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif] [/FONT][FONT=Times New Roman, serif]* Good idea[/FONT][/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Seal the edges of the petri dishes with parafilm or tape. [/FONT][FONT=Times New Roman, serif]Be sure to have no gaps in the tape to let air into the plate.[/FONT][/FONT]
2. [FONT=Times New Roman, serif]Remember to label your dishes with your name, date, kind of plant and any other pertinent information.[/FONT]

1. [FONT=Times New Roman, serif]Clean up your area.[/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Place your cultures in a warm (70-80ºF) area with appropriate lighting. Placing these cultures 12-15 inches below ‘growth lights.’ In home setting, household fluorescent lights work best, but any well-lighted areas will do. The light bank provided is set to a 16 hour photoperiod. [/FONT][/FONT]

1. [FONT=Times New Roman, serif]Wait! After 4-6 weeks, new plantlets should form in some of the petri dishes. [/FONT]

[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Also observe any contamination that may occur, often observable within a few days of establishing your culture. Generally a fuzzy contamination is a fungus and a slimy contamination is bacterial. Destroy and discard any contaminated cultures after taking notes. Summarize the major differences that you observed among treatments. When longest shoots reach 2-3 cm in length, take data for all treatments on shoot number per explant, mean shoot length, root number and length, and callus occurrence, if any. [/FONT][FONT=Times New Roman, serif](See Table 1 for media information.) [/FONT][/FONT]






[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Table 1: Medium Information[/FONT][/FONT]
[FONT=Times New Roman, serif]Murashige & Skoog Basal Medium w/ Vitamins[/FONT]
[FONT=Times New Roman, serif]Plate “A”[/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif].2 mg. BA per L [/FONT][/FONT]
[FONT=Times New Roman, serif]Plate “B”[/FONT]
[FONT=Times New Roman, serif].1 mg. NAA per L[/FONT]
[FONT=Times New Roman, serif]Plate “C”[/FONT]
[FONT=Times New Roman, serif].2 mg. BA per L + .1 mg. NAA per L [/FONT]



[FONT=Times New Roman, serif]Plate “A”[/FONT]
[FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif].2 mg. BA per L [/FONT][/FONT]
[FONT=Times New Roman, serif]Plate “B”[/FONT]
[FONT=Times New Roman, serif].1 mg. NAA per L[/FONT]
[FONT=Times New Roman, serif]Plate “C”[/FONT]
[FONT=Times New Roman, serif].2 mg. BA per L + .1 mg. NAA per L [/FONT]











[FONT=Times New Roman, serif]To transfer these new plantlets to the soil, you will need:[/FONT]

1. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Containers (pots) with drainage holes [/FONT][/FONT]
2. [FONT=Arial Unicode MS, sans-serif][FONT=Times New Roman, serif]Sterile potting soil such as Redi-earth or Jiffy mix[/FONT][/FONT]
3. [FONT=Times New Roman, serif]Plastic bags[/FONT]
4. [FONT=Times New Roman, serif]Labels[/FONT]
5. [FONT=Times New Roman, serif]Spray bottle with water[/FONT]

 

[AOD2012]

thanks a lot for all the info. i am really interested in this, and as soon as i get anything up, i will post it. thanks alot.


aod

 

[Voidling]

What would the law be on having undifferentiated cells with mmj dna ? I'm curious as to if one was moving cross country and wanted to keep their genetics or mailing to someone. They aren't seeds and they aren't plants at that point...

 

[Pompey_Monkey]

What would the law be on having undifferentiated cells with mmj dna ? I'm curious as to if one was moving cross country and wanted to keep their genetics or mailing to someone. They aren't seeds and they aren't plants at that point...

Here in the UK, if the cultures could not be legally classified at pot plants, I expect you could be done for "conspiracy to produce" or similar. The bastards will usually find a way to get you.

 

[danimarie193]

There was a write up in High Times last year about how to do this. I am sure you can look it up on there website.

 

[AOD2012]

yea dani thats actually where i found it. i just wanted to get some opinions from other people, because HT was steering me towards one brand, and i figured they probably got paid to do that. anyways, thanks a lot for the response.

aod

 

[Dopeboy668]

I've always been curious about this as well. Has anyone here actually done it succesfully with cannabis? Any pics?

 

[bobblehead]

this is cool. Thanks for sharing the info.

 

[Stress_test]

I bought a couple different kits a couple years ago and was playing with the idea.
Then there wasn't really much information about the concept and applying it to MJ. I ended up with an aquarium full of stuff and not alot of information available to work out a viable technique. (BTW ebay usually has kits for around $50-$60 US).

However in the last couple of months I have been getting newsletters from sites that I had joined then about advances in the area of micropropagation.

I will say that one of the very first things I learned was to use a large aquarium for a work station (WS). Sterilize everything before placing it in the WS and don't even think for a second that you can cheat, or shortcut sterilizing, because you will grow all kinds of scary shit.

One aspect of micropropagation that really is interesting is the idea of breeding 2 or more strains during in vitro. Using what they call "somatic hybridization", in theory it is possible to create genetic crosses without going through the conventional breeding process. The idea of "somatic hybridization" is that when cells are stripped of their cell walls and brought into close contact, they tend to fuse with each other. So if you use the cell tissue of let's say a WR and a LR2 then "in theory" you could run several test sets of different material combination's and choose the one that best suits your goals. Which could then be divided into hundreds or thousands of plants.

 

[Dopeboy668]

One aspect of micropropagation that really is interesting is the idea of breeding 2 or more strains during in vitro. Using what they call "somatic hybridization", in theory it is possible to create genetic crosses without going through the conventional breeding process. The idea of "somatic hybridization" is that when cells are stripped of their cell walls and brought into close contact, they tend to fuse with each other. So if you use the cell tissue of let's say a WR and a LR2 then "in theory" you could run several test sets of different material combination's and choose the one that best suits your goals. Which could then be divided into hundreds or thousands of plants.

Thats amazing! I never heard of that but I could imagine how usefull it could be.

 

[jump /injack]

I'm glad to see this site come up, have book marked it. There is some discussion groups on Yahoo Group on micropropagation that will be of interest to you, there is nothing about our particular herb of interest [in fact they shy away from MJ on purpose] but the idea is there and how to proceed with other plants. I found a site in China that is doing research on cannabis and will post the information that I found. Every plant has a particular nutrient that it does best in and I could never find the formulation until just this last month out of this group of biologists doing research with hemp. http://www.pakbs.org/pjbot/PDFs/41(2)/PJB41(2)603.pdf This is the site that I've referred to and its the only one that I could find with a cannabis specific formulation. Lets get something going on this. The site mentioned above has all of the necessary tools for little money, http://www.kitchenculturekit.com/ and I've also found that EBay has medical equipment for sale cheap. The book to get for more information is: "Plants from Test Tubes" by Kyte and Kleyn, it is widely hailed as the best for starters, it had everything but the formulation of plant nutrients and I think that one above is what everyone needs and its available on Amazon, thats where I bought it. You have to get the sterilization aspects down pat or you'll be growing a lot of things you won't like.

 

[AOD2012]

nice everybody, im happy we are getting some conversation about the subject. im going to look online, and try and order a kit off of ebay, and give it a go next cloning round.

 

[HighDesertJoe]

I'm really surprised that the MJ Seed company's haven't been doing this for years or maybe they have been.
Hell Monsanto's been doing it I bet.

 

[jump /injack]

Propagation utilizing tissue culture is a huge global business but since MJ is considered a class 1 drug only those with the proper and legal authorization could be doing it I believe. The Chinese have been using hemp for a thousand years and see no problem with it. Here is another site that you can take a look at: http://tech.groups.yahoo.com/group/hometissueculture/ Lots of people doing tissue culture for flowers, tree's, veggie's etc. and most likely its still being done in the United States at certain universities like where Government Mule was developed.