# Some questions on Tissue Culture



## Xema

Hello guys,

I am very interesting in tissue culture of crypts. It´s difficult find info in my mother language so i was looking for in english infomation about tissue. i found some good info from Kitchenculturekit.com, but i am not familiar with the terms used, so i want ask you about the meaning of PPM.

Anyone have experience with crypts tissue?

I am succed with my primitive tissue culture way, based in generate seedling from fragment of rhizomes in moss media.










I am thinking try a true tissue culture to spread some uncommon crypts here.

Greetings from Spain


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## gnatster

PPM - Parts Per Million

In the APC Library you can find a transcript of a chat presented by Art on Tissue Culture


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## Xema

Thanks for you reply, gnatster... in this case PPM is not part per million.... i found the meanig into the kitchencultirekit site, it is Plant Preservative Mixture, a product to sterilized the growing media.

That transcription is quite tedious for me, my english level is so poor...

Any practice case?.... maybe HeyPK? 

Any "homemade" alternative to PPM?


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## Happy Camper

Xema said:


> Thanks for you reply, gnatster... in this case PPM is not part per million.... i found the meanig into the kitchencultirekit site, it is Plant Preservative Mixture, a product to sterilized the growing media.
> 
> That transcription is quite tedious for me, my english level is so poor...
> 
> Any practice case?.... maybe HeyPK?
> 
> Any "homemade" alternative to PPM?


Hi there
There is a TC mailing list available.
Send a mail to: [email protected] with subscribe in the subject or body of the mail (try both as I can't remember which). There are many folks from all over the world on this list. They will better inform you of where to buy equipment and such in your area. Good luck


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## pineapple

Xema,

If you wish to try some Plant Preservative Mixture, I have 30ml sitting in cool storage (about 3 months old). The one and only supplier charges USD1.00 per ml. I can mail you the sample providing you pay the mailing charge (or do a trade of some type). Please PM me if interested.

Andrew Cribb


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## Xema

Before an overseas shipment i will try get some sustitutive for PPM, maybe in that mailing list thare are people from more near countries.

Thanks!!!


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## pineapple

Thanks for the reply.

Andrew Cribb


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## ruben

*Hola Xema*

Saludos, yo estoy en Mexico y tambien he intentado el cultivo celular ( soy laboratorista y tengo experiencia con cultiuvo celular pero de animales, la tecnica es totalmente diferente), los medios son efectivamente dificiles de conseguir, he intentado sustituirlos por fertilizantes de uso agriciola con regulares resultados, mi problema recurrente es la contaminacion con hongos, esto a partir del tejido, la esterilizacion con cloro y alcohol no me funciona muy bien (mis placas testigo siguen esteriles), por alli encontre que el cloro de alberca funciona mejor, espero tener algo de tiempo para probar con esto.
Ojala y pudieramos mantener el contacto

Saludos
Ruben Echeveste


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## ruben

ups I'm sorry this was a PM but I got confused with the buttons


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## Xema

Hola, Ruben

Intenta la esterilizacion con una disolucion de Hipoclorito Sodico al 1% y una gotas de jabon para platos

Mi problema es la esterilizacion del medio de cultivo, y la introduccion del material vegetal en los medios de cultivo en un entorno esteril.

English:

Try sterilization with a 1% sodium hypochlorite disolution and some drops detergent.

my main problem is get sterilized medium, and putting vegetal stuff in medium under strilized atmosphere.


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## MiamiAG

Sorry for being late to this.

You don't need PPM to do tissue culture. It is a relatively new anti-fungal chemical designed to reduce your losses. With proper sterilization, you don't need it.

The foremost expert on this topic is Dr. Michael Kane of the University of Florida. He has written several articles on this that you may be able to get. I was successful in tissue culturing common Cryptocoryne following his techniques.

If you are interested in this area, I highly recommend that you purchase Plants from Test Tubes: An Introduction to Micropropagation. It will walk you through the equipment you need, how to setup a work area, and the stages of tissue culture. The book is truly the bible for tissue culture.

Let me know if you have any other questions on this topic.

Saludos,


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## Raul-7

Art, have you tried this on stem plants? Is this a better and faster substitute to reproducing plants?


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## MiamiAG

Raul,

I assume you mean reproduction by cutting or runner. There is no doubt that micropropagation will lead to many more plants than any other method. That is why nuseries are moving to this form of growing. So, if you ask, "What is the fastest way for me to get 1,000 clones of X plant without having to purchase 1,000, the answer is micropropagation (tissue culture). In addition, microporpagation will result in disease-free, strong plants. This is not always the case using other methods.

However, micropropagation is very difficult to do without a) a knowledge of the protocol for each plant and b) the right equipment. As a result, it is harder to do than other methods. This is why not all nusery plants are grown this way.

So, based on the above, I'm not sure it's better. It depends on your objectives, time and cash.


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## Xema

Sorry.... i forget this thread...

My first one attempt was a failure... medium gots contaminate.

Now i am testing a new technic, with rhizomes the "moss bed" way, goes good. My next objetive is regenerate a new plant starting for a leaf.

Trebol gots good results with the way used in terrestrial gardening.









So, i am testing this way with crypts.... i think in a few weeks i would speak aobut the results of this experience.


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## MiamiAG

Xema,

I'd be interested to follow your project. There is a name (that I can't remember right now) for plants that can be cloned from leaf parts. Unfortunately, most aquatic plants don't fall within that group of plants. Therefore, you need to go get good clean apical meristems from shoots. These are the cells growing at the tip of the shoots. They are your best bet in getting good clones.


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## AaronT

About how much money did you spend on your setup Art? This sounds like an intriguing endeavor down the road.


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## MiamiAG

I think I was about $75-$100 into it when I got started. The costly part is when you start to grow out plants. You need to keep them somewhere!


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## AaronT

Art_Giacosa said:


> I think I was about $75-$100 into it when I got started. The costly part is when you start to grow out plants. You need to keep them somewhere!


That's a lot less than I anticipated. I'll definitely have to give this the 'ole college try sometime.

Obviously I'll try it on cheap plants at first, but cloning the right plant could easily pay for itself and more.


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## Phil Edwards

I've done quite a bit of propegation of Coleus at the greenhouse by taking cuttings. The only really important part is getting some sort of antifungal agent on the open cut prior to putting in the medium. For what it's worth, we use Perlite for all of our cutting propegetion and it works very well. Drainage is one of the most important things to have when doing cutting propegation, if the medium stays saturated it will get fungi and all sort of other nasties growing in it.


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## MiamiAG

Just to be clear. There is a difference in propogation from cuttings and micropropogation. Which are you attempting Xema?


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## Trebol-a

Hi,
in my case i practice "micro-cuttings" not micropropagation and tall plants (¿?) like rotala, limnophila, not crypts, and the successfully is good.

In the upper picture you can see *limnophila aromatica* and *Glossostigma*, i probe too *Bacopa* and *lysimachia*.



> There is a name (that I can't remember right now) for plants that can be cloned from leaf parts.


i use leafs full, not parts, but i have very interesting in this. Why you can not do micropropagation with aquatics plants?


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## MiamiAG

You can do micropropagation of aquatic plants. For most, however, you cannot us leaf parts as the initial explant. You need to use basal or meristem tissue that has the needed cells that will ultimately multiply into many plants.


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## Phil Edwards

Trebol-a,

If you wanted to do true micropropegation you would need tissue from either the very tip of the plant, some tissue from the leaf axil, or from the growing tip of roots. Those are the only places in the plant where the meristematic tissue Art mentioned can be found. 

What you're doing with the leaves right now is getting enough of an undeveloped axillary bud to germinate into a plant. Rather than using a leaf, you could just as easily cut the stem above and below of the leaf axils, trim the leaves, and put that little tissue disc in medium and grow it out that way. The leaves just give the plant an immediate source of photosynthesis which is probably helping you out a lot. 

Best,
Phil


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## SugarLEVI

As I don't feel like making a new thread for these questions I'll just put them here, these are questions I've been wondering about for some time, and I am just hoping maybe someone tried already.

The thing I was wondering about is if watering your emerse culture with some coconut milk will that make them multiply faster? 
And would the rooting media be a good media to grow out small clippings? As these haven't rooted either yet?

And oh yeah, is Tonina a good plant to try tissue culture on?
As I had some growing emerse, but most of the stalks have decided to die on me all of the sudden.


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## MiamiAG

SugarLEVI said:


> The thing I was wondering about is if watering your emerse culture with some coconut milk will that make them multiply faster?
> And would the rooting media be a good media to grow out small clippings? As these haven't rooted either yet?
> 
> And oh yeah, is Tonina a good plant to try tissue culture on?
> As I had some growing emerse, but most of the stalks have decided to die on me all of the sudden.


I've not used coconut milk but I've heard of its use. I'm not sure it will grow faster. I think it has some disinfectant properties.

Also, unfortunately, I have not information on the micropropagation of Tonina. Sorry.


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## SugarLEVI

Thanks for you answers Art, I'll just try and see what happens.


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## Xema

Reporting me...

Micropropagation on crypts based in adult leaf not succed. Glossostigma based in leaf was very successfull.

In a few days i will show to you some pictures of the result to micropropagation based in rhizomes on crypts... I got some good results with wendtii and becketii var. and cordata.

Greetings from Spain


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## SugarLEVI

I am very interested in your experiences, how did you do it exactly, what did you use? As I am still very anxious to start experimenting on my own.


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## Xema

Results and methodology...

First step... get the vegetal stuff, in this case Cryptocoryne cordata rhizomes. Divide the rhizome with sterile knife (or someone which can cut it).










step two, showing the pots, soil, and the way to put the rhizomes on the substrate. I used in this case 50% sand and 50% compost mix, covering the rhizomes (or plantlets) with a layer of sand.









In futures pictures, i will show to you the different results on growth using differents soil like 50/50 sand/compost mix, coconut litter, and 25/25/25/25 sepiolite/akadama/compost/sand mix.

Greetings from Spain


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## Xema

The pics of the results...

100 days after plantation on the original soil (compost and sand)










The same, but with pure coconut litter soil










The same, but with sand, compost and clay mix soil









Greetings from Spain


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## Trebol-a

Excelent report & photos Xema.
What temperature you set the pots?


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## Xema

thanks Trebol!!

Temperatura is over 18ªC.. around 20-25ºC.


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