Mature ex-plants have lot of hair resulting in fungal contamination. Even after using 1% sodium hypochlorite and 70% ethanol, it doesn't work.
a novel two-step sterilization method, using acidified bleach(ph-4.5), benlate, and an overnight period, produced 90% ‘clean’ live leaf explants and 30-43% ‘clean’ live nodal explants. This method is being used in our laboratory to routinely obtain ‘clean’ and growing nodal and leaf explants of these two species and is being extended to other medicinal and tree species
We had contamination problems with micropropagation of elm trees and used PPM (Plant Preservative mixture) and got rid of it.. You can also use it for Explant sterilisation.. Maybe it helps. It is not very expensive
Just a side note on this:
In case all the sterilisation results in contaminated plant parts - check if it is the same kind of fungus over and over again that makes the contamination. If that is the case you might deal with endophytes that grow from within the plant tissue.
Hii Jimmy ,,,Which plant you are dealing with? apart from establishing sterlized conditions (reagents and other stuff), try to use HgCl2 followed by washing with sterile water containing few drops of Twene 20.
a rapid, efficient desinfection protocol was published by Marchionni Basté et al. (2007):
Genetic analysis of the in vitro culture response in tomato
E.. Marchionni Basté, G. R. Pratta, R. Zorzoli
Plant Cell Tiss Organ Cult (2007) 88:233–239
DOI 10.1007/s11240-006-9195-y
regards,
Tatiana
Wash ur explants with 0.1% mercuric chloride for 5 mins. And then wash with water for 5 to 6 times.
Or if u still want to use hypochlorite, add few drops of tween 20 to the stock bottle and then do the sterilisation.
I recomend you to use vacuum during surface sterilization to avoid bubles of air that could be connected with hairs and caused problems . The most simple way with small explants is to use a syringe (put explants inside).
Tween is also a good idea. If I were you, I would also try higher concentration and longer time. Before sterilisation process I recommend washing under running water after use a bit of detergent. Good luck!
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