noksu

Hello everybody....!

First i have to say, I´m Sorry for my bad english.It´s only a little bit school english that i´ve learned and this is 15 years ago .
So here is my problem:
My first try of cultivating Bananas in Vitro was terrible.
I´ve started with 15 Culture Vessels and all of them shows contamination
after 7-10 days.
I put the medium in the culture Vessels and placed them for 25 minutes in the Autoclave.Then i dipped the washed plant material for 10 seconds in
Ethanol 70 % and after that for 20 minutes in NaClO 1 %.
After that i rinsed it 3 Times with destilled water.
I worked under a laminar flow hood.The only reason for the contamination
in my point of view is the Surface Sterilization.I think there is anywhere a mistake, but where ?

What do you mean,was the NaClO 1 % solution to low ?
Or should i let the material longer in the solution ?

What´s the best way for the Surface Sterilization of plant material ?

I don´t want to loose all my little suckers of my banana plants.

So how i make it right in the future ?

Maybe anyone of you can help me.

Thanks a lot.
greetings from Germany.......

jmoore

Hi

I'm no expert, but your bleach is too low, it should be 10% and dipped for 10 minutes and the rinse sould be in sterile distilled water. You then cut the outside of your explant off. The alcohol should be iso propyl alcohol >70% (propan-2-ol) and not ethanol and this should be after the bleach.

This should help, and only try 1 first to save all your pups.

Gabe15

James (jmoore) has the right idea.

It seems that it possibly came from the distilled water. Just because water is distilled, it does not mean it is sterile. To ensure it is sterile, it must be autoclaved and in a wrapped vessel, and then poured from that same wrapped vessel in the hood. However, I do not think this step is important.

What you may want to try is this: cut down your tissue piece to about 3-4X larger than you want in the vessel. Then, sterilize that piece with the bleach and/or alcohol (I find it doesn't matter too much, just as long as it works!). You may also want to try the bleach solution with a drop or two or dish soap, this will help to coat the tissue surface better. After it is soaked in the bleach or alcohol solution, carefully (with sterilized tools on a new sterile surface, either a sterile petri dish or autoclaved paper), cut off all of the outside tissue little by little, and with each layer removed move the desired piece to a new sterile surface. You can kill the outside layers of the tissue during the initial sterilization process, because you will be removing them and exposing only a very small, clean piece of tissue to the medium in the culture vessel.

You can start the sterilization with a meristem-containing tissue piece about 4x4x4cm and cut it down to something only about 1x1x1cm.

__________________
Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

noksu

Hi guys....!!!!

Thanks a lot four your quick reply and your help.

@ Gabe15 I took the distilled Water,boiled it and the i put it in the autoclave for 30 minutes.

The next week i will start my second try.
I will try it with a higher concentration of NaCIO.
The only problem is that you can here in Germany buy NaCio concentrate only
with a concentration lower than 15 % for a private person.
So i think i will try it with 15 % NaCIO for 30 minutes.
Maybe it will be enough.
I will post it here if it works.
Thanks for your help.

If you want i will post some pics of my little new homelab.

jmoore

15% is the free chlorine concentration and doesn't refer to the NaClO concentration. Don't use the bleach undiluted it will kill your explant.

You want 10% by volume of whatever bleach you have. so 1000 cm3 requires 900 cm3 of water and 100 cm3 of bleach and so on. It can be the cheapest you can buy.

I hope this makes sense

ImperialExotics

To expand a little on what Gabe said........

Sterilize your initial explant. After it is fully sterilized remove another complete layer of that explant, and go through the process of sterilization AGAIN.

Then remove a second complete layer of the explant and it is ready for innoculation.

I also read a paper suggesting treating the tissue for an 18 hour soak in Bavistin to kill fungus and fungal spores, prior to the sterilization and trimming procedure.

Gabe15

Something else you should also consider trying is to use a new sterilized set of tools to remove each outer layer of the initial tissue, or flame sterilize the set between each cut, and also move the explant to a new sterile surface each time. The initiation step is normally the hardest to keep sterile, and extra (sometimes seemingly unnecessary) steps often help to ensure the meristem remains sterile. Once you have sterile cultures established, you can be less careful to an extent.

__________________
Growing bananas in Colorado, Washington, Hawaii since 2004. Commercial banana farmer, 200+ varieties.

noksu

Hi,
here i am again

@ jmoore


ok, for an example :
I take Commercial household bleach which contains 5% sodium hypochlorite.
I have to mix 1 part bleach with 9 parts water and i get a 10 % solution.
so far so good.....but it means the solution contains only
0,5 % sodium hypochlorite. ?
Is this right ?
And that will it be enough ???