# tissue culturing various plants



## ravenc83 (May 26, 2006)

well this week I contacted a friend that works in the lab at a local college. I had been reading about tissue culturing, so I asked if she had a recipe for the media. maybe I'll be posting it later on for those who get intrested. so far all that has been done is the mounting of the samples : CP: pitcher plant, venus fly trap, butterwart. and a black jewel orchid, fire ball bromelad and another brom that i had around. 
the best way to get started is to find friends that have plants you like, and ask to "barrow" a sample. The next part is a bit harder, find a recipe or someone that knows how. prepair a medium, and sanitize the sample with dish soap (i used joy) wash off the soap, then put it in a 10% bleach bath ( the more delecate the tissue the less time) for about 3 min. then wash that, keep everything as sterile as possible.

unfortunatly, i don't know how to post pictures, so someone please pm me instructions. thanks. 
I'll post pics as soon as possible.


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## defaced (May 23, 2005)

I'm very interested. I've been wanting to read up on this but haven't had time to. 

To post pictures, first start a gallery here. Upload your pictures. When you are viewing your pictures, right click on the larger version on it, select properties, and copy the URL. It'll be something like

```
http://www.dendroboard.com/coppermine/albums/userpics/number/filename.jpg
```
Now go to make your post and use th Img button right above this text window to make your images. It'll look like this:

```
[img]http://www.dendroboard.com/coppermine/albums/userpics/number/filename.jpg[/img]
```
Hit preview to make sure it's all working and there ya go. If something gets messed up and you can't fix it, make the post anyway and someone will see that it's messed and post the images for you.


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## ravenc83 (May 26, 2006)

*first pics*









a picture of the pertidish with no cp.








the picher plant culture








self explanitory








a butter wart








you have to keep the plant cultures lit with uv light and at around 70ish degrees.
the bottle at the bottom is a stock culture, hopefuly in a week or so I'll be posting images of the cell growth.


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## KeroKero (Jun 13, 2004)

Media for all the plants can be the same? I'd be interested in trying out the media if you're willing to give out the recipe. I've been thinking about trying "kitchen" tissue culturing for a while.


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## Ryan (Feb 18, 2004)

I am also interested, I am curious abou tusing Hydrogen Peroxide as a sterilizing media, i know they have started using it for mushroom cultivation. I am also interested in tissue culture for the sake of growing orchids from seed.


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## stchupa (Apr 25, 2006)

*Re: first pics*

Very interesting methods. I'm confused though.
Never heard of anyone allowing light to a new culture, if you have the right agar it will provide all the carbs/sugars/ribulose needed, bypassing the photo process. Also the tissue you're supposed to harvest doesn't make use of light in such an early stage.
I also noticed with the fly trap, it looks like you took a sample of already differentiated tissue. I've never done fly traps but what you seem to be doing with those is just cloning? Tissue culture must always be done with parenchyma.

Soap? Never heard of this. I think you might have some contaminantion. Why not autoclave the media/dishes instead?

I am very intrigued, and want to hear about any success. You should know by next week. 

Let us know one way or the other. Good luck to ya.


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## stchupa (Apr 25, 2006)

*Re: first pics*

One more thing what are you doing to keep your transfer sterile. Bunsin veil method?


How did you sterilize the samples you used. You can't core snippings.


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## Guest (Jun 4, 2006)

*Re: first pics*



stchupa said:


> Very interesting methods.
> 
> Soap? Never heard of this. I think you might have some contaminantion. Why not autoclave the media/dishes instead?
> 
> ...


I agree with stchupa on everything I quoted. I bet your friend has access to an autoclave and maybe as a favor can sterilize everything for you. But I suppose hydrogen peroxide would be good enough for those who don't have access to an autoclave. Wouldnt you have to wash the soap off? And if so, how would you keep everything sterile once you have to wash the soap off? And the soap would have to be pretty strong to keep everything sterile.


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## ravenc83 (May 26, 2006)

*reply to most answers*

as far as the sterilization, the first step for sterilizing the tissue was to use a dish soap that kills bacteria, then you wash it off(using a five bath system or rinceing it off with distilled water. after that you soak the sampels in alcohal, or something like it, i think we used denatured alc. you soak it for 2-3 min.



> Soap? Never heard of this. I think you might have some contaminantion. Why not autoclave the media/dishes instead?


now as to the autoclaving, yes we did use an autoclave to sterlized the media. and a sterile hood to trasnfer the tissue from the tubes to the petri dish.

as far as useing Hy Prox. to seterile, in therory it should work. but otherwise i realy done know.

another method to sterlized every thing that isn't metal is to get one of those microwave sterolizing steam bags that they use to clean up baby stuff.


> Media for all the plants can be the same?


the media is all the same for these plants, it was taken from a prosses to culture a plant that is very difficult to do. we figured that it should work for plants that are easy to do.


> Never heard of anyone allowing light to a new culture, if you have the right agar it will provide all the carbs/sugars/ribulose needed, bypassing the photo process.


the light is manily to help incubate the samples by providing heat.
thats all for now, i went to magic mountain yesterday, and now i feel like I got into a fight with a sumo guy.[/quote]


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## Grassypeak (Jun 14, 2005)

This is very interesting. I’ve always wanted to try “Kitchen Tissue Culturing”, but I’m confused by your choice of cuttings. I thought that meristematic tissue was needed where new plants were desired. Perhaps this is what Stchupa was asking about. If so, I would think that for bromeliads you would have to harvest the tip of a newly growing pup stalk before it opens into a rosette. Bromeliads cannot grow pups from leaf tissue (as far as I know, at least). For the Jewel Orchid I would think that a stem section would work, as new plants can grow from such tissue. 

Good luck, regardless of my ramblings, and please keep us posted. I’d be very interested in knowing your media recipe.


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## ravenc83 (May 26, 2006)

its really just an experiment to see if it will work.


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## ravenc83 (May 26, 2006)

well i got some containation on two plates. crap!!!


















the rest look great, only time will tell


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## Guest (Jun 8, 2006)

Hope for the best!


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## Grassypeak (Jun 14, 2005)

From what I understand it sometimes takes considerable effort to get a plant into tissue culture. Once you get it there life is a lot easier. Some plants have bacterial symbionts that are hard to get rid of. 

I’ve done a lot of reading on this subject, and from this, I would think you would be better off trying one type of plant per culture. This way if one plant proves to be difficult to clean you don’t lose everything that you try. I would also try to use tissue that is capable of producing plantlets. A jewel orchid stem with at least one leaf node, a begonia stem with a least one leaf node, an African violet sucker, etc.

It also seems as though it is necessary to use two different mediums. The first has hormones for plantlet growth, the second has hormones for root growth. Once you produce sufficient plantlets on the first medium you replate the plantlets onto the rooting medium. You also have to be careful about how long you leave the plantlets on any medium, as waste products build up and eventually poison the culture.

Thanks for the images. Please keep us updated on your progress.


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## ravenc83 (May 26, 2006)

that is very intresting info grassypeak. now after this try is done, i;ll try other things untill something works. any medium recipes that you think would work please post! I only found one.


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## ravenc83 (May 26, 2006)

*disaster!!!!!!!!!!!!!!!!!!!!*

damn! i got something other than what i wanted to grow, white green and pink fuzzy crap!!! well i guess i'll have to give it another try, unfortunatly, i think i ran out of plants.
:evil:


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## Grassypeak (Jun 14, 2005)

Ravence,

Carolina has plant specific mediums available and some plants already in tissue culture (no sterilizing necessary). There are some online resources for mediums and biocides which can be added to the medium when a symbiotic bacterium is giving you a hard time. Try googling “Kitchen Tissue Culture”. Personally I enjoy reading about the various glove box designs that people come up with. I’ve always wanted to make one and do some tissue culturing but there are so many other projects and so little room in my new fish room.


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## stchupa (Apr 25, 2006)

> Grassypeak said:
> 
> 
> > From what I understand it sometimes takes considerable effort to get a plant into tissue culture. Once you get it there life is a lot easier. Some plants have bacterial symbionts that are hard to get rid of.
> ...


There is no applicable way to sanitize the exterior of a plant (cuticle of the leaf, stem, root) without killing the external cells needed to be in contact with the medium. Then again I can't understand why you would want to.
The interior of plant is self sterile. Getting to it and keeping it that way is a learning process and the technique differs from plant to plant.

Anything exterior is considered cloning and agar is not required.
Shouldn't have come to this point after explaining all other. 
Now I can expect confusion?
Nodes produce undifferentiated tissue, but can't be used due to size and contact to outside polutants.

Again what is being demonstrated hear is not tissue culture (sorry Grassy/who else, but this must be set straight for any success to be expected and prevent anyone reading from becoming disuaded through unfavorable results in their efforts), just a more elaborate way of cloning.

If you're talking jewels, begonias, or violets, the time and materials needed in tissue culture would not be feesable/practical for these plants which can easily be propagated without much of an attempt (in the way stated above, but without the need of sterilization or a prepared agar, only the right parts [in some cases any part] introduced directly into to the right and conditioned material needed for that particular plant [basically the consistant and preferable moisture levels of the substrate])



> It also seems as though it is necessary to use two different mediums. The first has hormones for plantlet growth, the second has hormones for root growth. Once you produce sufficient plantlets on the first medium you replate the plantlets onto the rooting medium. You also have to be careful about how long you leave the plantlets on any medium, as waste products build up and eventually poison the culture.


I don't know what would produce poisons in a successful culture? certainly not the plant being cultured, and what else would be in it?
The only reason for transfers is the agar retains the required amount of water, as the the plant uses the nutrients/moisture it's in direct contact with, the moisture transpires through the plant leaving the solids in the agar (dry). Intiation medium is first then growth/development/root, then comes the shoot media transfer for last petri application. After that point no attentive care needs to be given towards sterility. The only reason to keep a sterile culture is because any undesirable inoculants compete with the plant for the sugar in the agar (fencing), they dont parasitize the plant itself.
Transfers would need to be done daily costing a lump sum=an incredable amount $
Contaminants normally congregate on the sample taken because of either the methods of extraction/transfer or contact with the air/person. Keeping the agar in the dish sterile is no great art, just common sense.



> Thanks for the images. Please keep us updated on your progress.


[/quote:r3gavnvl]


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## ravenc83 (May 26, 2006)

uuummmm......ok.


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## stchupa (Apr 25, 2006)

Yeah, that's what I thought.

Better stick with root crops, like carrots.


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## ravenc83 (May 26, 2006)

well life is a learning process, and you can't learn unless you try right?


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## stchupa (Apr 25, 2006)

Couldn't agree more. 
Who's to say you can only know/learn so much, availability is endless, continually created, knowledge evolves and converts with time.
But people have done this and why not learn through their mistakes and save you the trouble?
Typing is a pain for me but might be worth it to someone else.
Then again, unless they don't get it, right?


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