# Growing plants, Tissue Culture?



## Zefrik (Oct 23, 2011)

I have been growing aquarium plants now for a few years and I have now come to a point where I want to get a bit more advanced. I have recently been researching plant tissue culture. It is a little over my head though at times. Has anybody on here tried to grow any plants like this? I would like to try but I can't find a good or slightly dumbed down tutorial on how to do it. I know that you take a cutting or a piece from a plant and disinfect it. Then you place it in a bacteria free medium. You give it what it needs and it should start to develop into a clone. I know it is far more advanced than that but I believe that is similar to how it works. 

I have read that you need some kind of gel, like agar gel. Isn't that the stuff they put plants in those tubes you find at petco? Could something as simple as gelatin be used to make something similar? If so would it be a solid piece or in chunks? I also know that you have to have an almost 100% disinfected, bacteria free environment. I have looked and all the things you need seem hard to find. Are there substitutes with stuff you could just find at a store or on the internet?

I don't know if I am ready for this yet. I might need to take multiple biology courses just to understand everything!:icon_redf

Any tutorials or how to's?


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## Bushkill (Feb 15, 2012)

I'm coming along for the ride on this one. I think I'm right about where you are with this, but I may draw the line at the Bio course, lol!


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## Da Plant Man (Apr 7, 2010)

As I'm sure other people will recommend, check out this website, its really helpful. 

Another is simply youtube: 'How to tissue culture" I'd reccomend trying african violets before doing anything expensive, as its one of the easiest to propogate. I have done multiple days of research, even bought most the stuff needed, but I never actually took the plunge. Once I move, I really want to get into it. 

For mediums, a cheap alternative to Agar Agar, is a corn starch based medium. Although, for our purposes, cheap agar, is fine. 

You'll most definitely need a precise scale. 

Basically, research, research, and research some more. There are some great  books out there too. I have read the one I linked to and its quite an interesting read. 

For everything needed to get going, its going to be around ~$250 excluding the actual plants (could be more or less, depending on the deals you get and if you want some more "luxury" items). HOWEVER! You'll get thousands of plants that normally grow slow. So you'll make that back in a month and then its cash in the pocket (or aquariums) from there on


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## BIG_Z (Dec 7, 2011)

http://www.youtube.com/user/fbt2007?feature=watch

guy has an entire intro course into tissue culture, he also suggests tons of sites for you to get supplies and info. Also join the yahoo tissue culture group he talks about..tons of good info their.


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## Jeffww (Aug 6, 2010)

It's like a 50 buck start up cost. It doesn't need to be fancy. I've currently got one C. wendtii tropica in culture right now.


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## Zefrik (Oct 23, 2011)

So...

1. Get a sterile container.
2. Get agar gel into container.
3. Get a portion of a plant sterilized and set in the gel.
4. Cytokinins are added to the container.

Questions...

1. What kind of cutting do you get? A leaf or stem or root?
2. Cytokinins, I read that you can use coconut milk. Is that true? If so how much?


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## BIG_Z (Dec 7, 2011)

for crypts the best cuttings are the thick root aka future plants. as far as coconut milk I have no clue.


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## Jeffww (Aug 6, 2010)

More like: 

1. Prepare media with all nutrients and cytokinins. I'm using benzylamino purine, agar, potassium salt of napthalene acetic acid and full strength Murashige and skoog. 
2. Pour into containers 
3. Sterilize via microwave or pressure cooker or autoclave if you're loaded or a lab tech. 
4. Isolate explant tissue
5. Sterilize ( hardest part in the whole deal) 
6. Plate
7. Let grow
8. Transfer to multiplication media for rapid in vitro multiplication.... 
9. Transfer into in situ conditions. 

Good luck I have a journal but since nothing much as happened so far I haven't got much else to say. I do suggest that you do not grow your crypts in soil but instead opt for a cleaner medium such as rockwool or sphagnum. Also while it IS possible to use submersed tissue for culture it's a tough job getting it sterilized. I got 100% contamination off of submersed anubias. 

Explants should be new growth. Leaves may be used as well but I haven't read any papers on it. Everything suggests rapidly dividing aerial shoots. 

I missed my chance on using a cryptocoryne spathe to try multiplication the other month and now I'm waiting for another one.


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## Zefrik (Oct 23, 2011)

What does the sixth step "plate" mean.:icon_roll


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## Zefrik (Oct 23, 2011)

Jeffww said:


> More like:
> 
> 1. Prepare media with all nutrients and cytokinins. I'm using benzylamino purine, agar, potassium salt of napthalene acetic acid and full strength Murashige and skoog.


Where do you get this stuff? I have found some online but not all of it. I have a feeling I won't get any positive reactions if I go strolling into local shops asking for this stuff. People will think I am crazy. I already get enough flack getting my co2 filled for my tank! Haha:icon_redf


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## Jeffww (Aug 6, 2010)

http://phytotechlab.com/ 

I use it for all my materials.


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## kuro (May 21, 2010)

I think it going to be a waste of time unless you have access to sterilize hood to work in. However if you gonna try it remember to seal the cover and plate with breathable tape if you gonna try it, it will help prevent fungus from getting in.


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## Zefrik (Oct 23, 2011)

I am now going to make a list of what I think I need, or a list to all of the links of things I need. Could you tell me if I have the correct stuff or do I need anything else?


- Agar - http://www.phytotechlab.com/detail.aspx?ID=70
- BENZYLAMINOPURINE - http://www.phytotechlab.com/detail.aspx?ID=141
- MURASHIGE & SKOOG - http://www.phytotechlab.com/detail.aspx?ID=452
- Container - http://www.phytotechlab.com/detail.aspx?ID=804

I could not find the potassium salt of napthalene acetic acid. Could you give me a link?

Thanks


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## Zefrik (Oct 23, 2011)

Also, I am kind of a visual learner, so I drew out a picture of a plant to ask which part of the plant is used as an explant.


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## keithy (Jun 8, 2010)

Jeffww said:


> http://phytotechlab.com/
> 
> I use it for all my materials.



as soon as I saw this post, I instinctively thought about your journal. Maybe you guys should start a club and share thoughts, ideas and supplies within a "tissue culture club". The more people who knows about it, the better as more people share their success/failure, more ideas will be flowing for better protocol.


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