tissue culture!

lindsey.podd podd at botany.unp.ac.za
Fri Aug 23 04:30:56 EST 1996

>Its difficult to answer your question if you dont mention your explant 
source or kind of infection you found >on your medium (probably its only a 
fungal infection as you use fungicides).>There are some important rules to 
follow if you want to retrieve sterile explants from any plant in vitro.>-   
Most important, always start with healthy, good growing plant material. A 
sickly plant from a window sill >   wont do>-   Try different plants and 
keep them separated during the sterilisation process>-   Try sterilising 
different explant sources (like flowers, seeds, stems, leafs, roots).Not all 
explant sources I >   mentioned will give rise to direct regeneration, but 
sometimes adventitious regeneration could be >   possible. Keep these 
different explant sources separated during the sterilisation process. If you 
get less >   or no infection by using a certain explant source you should 
work with that source.>-   Trying the above, a NaOCl concentration of 1% (v/
v) should do (approx. 15 min). A concentration of 3.5% >   is too much, no 
wonder your plant material is being killed. I am not familiar with Nepenthes 
alata, but a >   15 second dip in ethanol (96% v/v) preceding the 
sterilisation in NaOCl and adding 1 mg/l Tween-20 to >   the NaOCl solution 
will add to the sterilisation effect.The alcohol dip removes fatty 
substances from the >   explant surface thus enhancing the following NaOCl 
sterilisation (It also has a little sterilisation >   effect).Tween will 
enhance the contact of the NaOCl to the surface of the explant. (avoid bubble >   formation!)>-   If the infection is internally you could try meristem culture, using a dissection microscope.>-   Alternatively you could try to manipulate your plant materi

>Probably most of the above-mentioned information is known to you, but I still hope it will help. It is most 
>important to retrieve the source of the infection (internal in the plant/on the plant surface) and, if possible, 
>the kind of infection (fungus or yeast species, bacterial strain)
>Please let us know what did work...
>Wiel Mattheij
>The Netherlands
>e-mail: wielmm at worldaccess.nl

I have always used Tween 20, sorry if I didn't mention it originally.  I 
have tried all the plant parts, and get the stems remaining green on 
occasion and the leaves very occasionally, all other plants parts (
tendrils, pitchers, flower parts are killed in even the weekest 
sterilants).  The 3.5% NaOCl may indeed be to strong for the plant, but 
lowering the concentration won't help because even with an weekly spraying 
with Benlate, a benlate dip, an ethanol dip and sterilization (with Tween 
20) the plants are still not coming up sterile.  The plant is insectivorous 
and grows in damp conditions...  and nothing seems to kill the fungus (and 
by the way it's not my bench technique before any one suggests it) - so what 
I am really looking for is unusal and effective ways of sterilizing them, 
for example has anyone had sucess with ultrsound?

Ta Ta

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