Fern cultures

Several people have written to me for information relating to fern spore cultures. I am not an expert at this phase of micropropagation and have only a vague familiarity with this type of cultured plants but here is what I do know. Hopefully it is of use to you.

Although there are currently at least 20 different species of ferns which are found here in America, I would venture to say that the same process could easily work with any of them. So without any further wait here is the information.

The fern spores are usually collected in the late autumn from mature fern plants. We will use the domestic “Platycerium superbum” as an example. The spores would naturally have to be sterilized and this is usually accomplished by way of a centrifuging technique. The spores are quickly shaken in a test tube using sterile water with a couple of drops of Tweeen 20 added. If you have access to a centrifuge you could place the tube in it and rotate it at the rate of 2800 RPM for 5 minutes. This will settle the spores to the bottom of the test tube. Next, discard the liquid in the tube and replace it with clean sterile water. Once again shake and let the tube sit at room temperature for a 24 hour period.

The following day re-centrifuge the tube and discard the water again. Now add 10ml of common household bleach. Let the bleach remain in the test tube for approximately 5 minutes. Finally, centrifuge again and discard the solution and place the aseptically clean plant spores into a media as described below.

These spores you will note were planted into the media without a final wash with sterile water. The cultures were then incubated for 16 hours. The temperature should be kept at about 25 degrees C.

This is a slow growth method and your spores will start to germinate and produce prothallus in about three month’s time. You will likely need to sub-divide these early cultures as they start to form. In a future article I shall cover construction of a home made centrifuge as well as an incubator.

This media should be successful for just about any fern cultures that you wish to try it on.

300 mg MS salts
500 mg NaH2PO4
5 mg Inositol
20000 mg Sucrose
8000 mg agar

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Sterilization of TC Containers by Microwave

I generally purchase my tissue culture containers by the case and it goes without saying that they can get rather expensive. The particular ones that I like to use are made out of plastic which tends to make them slightly more affordable then those created from pure glass.

I am especially fond of recycling whenever it is feasibly possible and it seems like such a waste to toss these perfectly good containers into the trash when I am finished with them. In the past the major disadvantage of the plastic containers has been the inability to properly decontaminate them and to be able to reuse it for additional cultures. By being able to do so greatly cuts the cost of the tissue culture containers down to a minimum.

With this thought in mind I decided it was time to reuse these vessels once again. Naturally, they would need to be in a sterile condition in order to prevent contamination of the next series of explants that would be developing within them.

It obviously seemed like a very good idea to use the microwave oven for this exact purpose. The microwave can effectively kill any type of bacteria present and successfully decontaminate the plastic containers for further tissue culture work. Tests have shown that our common home microwave is well capable of decontaminating these containers with only a 3 minute cycle.

Initially cleaning the plastic tissue culture containers is fairly easy. One merely uses their usual dish detergent and washes them well followed by a rinse in fresh water. Generally, this will take most of the residue out of the containers and provide you with a clean container for reuse. It is the sterilization of the containers that used to pose you the greatest difficulty however with the use of the small microwave ovens as described above you are well on your way to an effective means of sterilizing these containers quickly and inexpensively.

So as a home lab user it is now possible to save a lot of money with not having to repurchase your cloning vessels after each and every batch.

Copyright @ 2009 Joseph Parish
http://cloningforthecommonman.blogspot.com/

Tissue Culture Part II

Dr. Sandy Wilson, Associate Professor of Horticulture at the University of Florida lectures on the topics of Tissue Culture and Micropropagation.

Tissue Culture Part I

Dr. Sandy Wilson, Associate Professor of Horticulture at the University of Florida lectures on the topics of Tissue Culture and Micropropagation.