Tissue Culture revisited
 

This weekend, I went out to an old lady in Trondheim and bought an almost new pressure cooker. Apart from cooking lamb the old fashioned way, I am extremely happy to now have a reliable sterilizer for banana tissue culture! :2223:I think this is what made me fail before, - unsanitary lab-conditions.

Beeing a very simple minded guy, I am going to try 2 approaches:

1. Germinating seeds and chopping the shoots off. Sterilizing these, make incisions, and putting them in multiplication media.

2. The "regular" shoot tip culture from a sucker.

Any ideas/ comments to this approach?

Erlend

Re: Tissue Culture revisited
 

Richard Wallace gave a presentation at the last Southeastern Palm Society meeting in Savannah a little over a week ago, and discussed tissue culturing. They are actually multiplying embryos first, before germinating them, which I thought was really cool.

If you're going to do it the first way, let your plant get a few leaves first.

Unsanitary lab conditions = failure for tissue culture. Before I knew much about it or tried it myself at school, I thought that the instructors and/or grad students were just being a little anal about the whole thing. That is, until I found a nice colony of fast-growing black mold growing in my first try! LOL!!

Re: Tissue Culture revisited
 

Now I know I have lots to learn. I would have thought putting shoots in a pressure cooker would make great dish of steamed shoots. I need to do more research.
NanaNut2

Re: Tissue Culture revisited
 

I see how I made myself a little diffuse here, The pressure cooker is for sterilizing utensils and media, not for the banana shoots themselves. Although that would be something to try too!

Bigdog; out of curiousity, why do the plants need to have leaves before they can be tissue cultured? I would think newly "hatched" banana seedlings had a high density of meristematic tissue? Am I wasting my time chopping these shoots off of the seeds and incubating them in MS multiplication media?

Thanks again;
Erlend

Re: Tissue Culture revisited
 

Thank you for clearing that up Erland. I must have been hungry when I read your post ... lol. It makes much more sense to steralize the equipment. But, I may eat Chinese take out tomorrow night.
NanaNut2

Re: Tissue Culture revisited
 

You don't need to sterilize your manipulation tools in the pressure cooker, I would recommend heating them over a flame just prior to use.

As for the tissue to use, I've been able to germinate embryos on the basic multiplication medium and it's not very hard to take out the embryos with a bit of practice. You may be able to put the seedlings directly into culture but I'm wondering how well they will be able to stand being submerged in bleach with such little tissue between the environment and the meristem, it's worth a try I suppose.

Re: Tissue Culture revisited
 

Gabe, did you use this mix for your embryos: PhytoTech Lab E-Commerce - Detail

Also, did you add any more chemicals, or just water & fun?

Erlend

Re: Tissue Culture revisited
 

We just mix our own with basically the same stuff in it (MS, sugar, thiamin HCl, gelling agent and BA depending on use), but we make our own so we can modify it has needed. In the premade media from PhytoTech Labs, it has BA in it which is the chemical responsible for shoot multiplication, however it also inhibits shoot elongation and root formation (which is ideal for subculturing when multiplying your stock). So what we do is cycle the explants through the medium with the BA in it, and when the time comes to root the explants, they are switched to a similar medium which has no BA in it, this allows the shoots to elongate, roots to form and no longer induces extra shoot proliferation. It is possible to root explants in soil that come directly from a high BA medium, but this is usually only done to try and save plants which have been contaminated in culture, its much easier to have them root in vitro. I also use a low BA medium to store explants in for culture at a later date.

For the embryos specifically, I just used our high BA content medias (standard "multiplication medium"). I haven't tried much with a low BA or zero BA media yet. There are also other specific formulations for embryo culture, but I have not been able to try those yet.

Re: Tissue Culture revisited
 

Erlend, I believe the higher mass helps with the survival of the meristem when it is undergoing the sterilization process (like Gabe said, submerged in a bleach solution). The newly sprouted seedlings are very tender, and the bleach could actually kill them.