Are you sure you have the contamination rather than inner infection. May try to add antibiotics into the culture medium ?

She can try ultrasounds. It helps with the surface disinfection.

In this paper is described the protocol that I used.

http://link.springer.com/article/10.1007%2Fs11240-012-0276-9

She can also try the protocol described in a paper by Watt et al., 2003 (see link below)where they collected material from trees and was able to succesfully propagate it in vitro. We have also used this protocol with varied success. It also helps if the motherstock is treated with a broad spectrum fungicide weekly for a month before collection. http://link.springer.com/article/10.1023%2FA%3A1025819826742#

When i worked on Stevia i used the protocol described in the paper we published. Please find the link as follow

https://www.researchgate.net/publication/233859881_An_efficient_callus_initiation_and_direct_regeneration_of_Stevia_rebaudiana

Article An efficient callus initiation and direct regeneration of St...

Thanks everybody

Fungal contamination in the plant tissue cultures is a common problem when we collect explants directly from field. There are many factors which are responsible for this. Proper sterilization of media, proper surface sterilization procedure, donor plant with minimu inoculum load, etc are key. You can test/use a pre-treatment solution of broad spectrum fungicide (e.g. Bavistin) prior to final surface sterilization. In addition the state of donor plant is also important. Try to collect juvenile or growing tip. If the problem persists you can spray fungicide onto the donor plant before collection of explant so as to reduce to inoculum load. Please also check is there any endophytic fungi from explant, etc.

The best way to avoid fungal contamination is to harvest explants from in vitro grown seedlings.

Try RAN mixture (Rifamycine, ampicillin and Neomycine)

Para evitar ou reduzir a contaminação tenho usado como fonte de explant a parte aérea de miniestacas da planta de interesse. Essas miniestacas são cultivadas em vermiculita em uma sala no próprio laboratório, por ex: sala de lavagem. Após a retirada dos explants, realizar a assepsia normal: lavagem em água corrente, uso de NaOCl 0.5 a 1.0% por 15 minutos e 4 lavagens em água destilada.

To avoid or reduce contamination have as a source of explant, the shoot cuttings of the plant of interest. These cuttings are grown in vermiculite in a laboratory room itself, for instance the wash room. After the removal of the explant perform a normal aseptic: Washing in tap water, use of 0.5 to 1.0% NaOCl for 15 minutes and washed four times in distilled water.

During washing use any antifungal agent i.e. bavistin it is a broad spectrun antifungal agent.First wash ur explant with 1% bavistin with soft shaking for 15-20 mins and wash it throughly.Then wash with 5% teepol for 20-30 mins.Washing of explant with 0.1% HgCl2 should be performed in laminar air flow chamber with distilled autoclaved water.We generally apply this protocol for explant surface sterilisation.Hope this will work out.

I also want to stress you that you should consider several factors such as external environment in the culture area, mother plants, sterilization method, handling of cultures etc. I agree with Zakia's protocol to minimize fungal contamination. If you still have contaminations, it's better to prepare initiation medium supplemented with 1% of fungicide.

Thank you very much to all

Other than adding RAN you can add the defined fungicide in appropriate concentration to the medium. Make sure that the fungicide added to the medium does not interfere with the growth of the tissue

After using bleach for explant sterilization do final 1-2 hours wash with 5%PPM (biocide). Also incorporate PPM into media you are using at 2-3 ml/l. Do transfer each 1-2 weeks

It is essential to ensure that the plants from where you are selecting the explants should be free from any disease especially fungal diseases. If it is infected already, then no matter how many surface sterilizing agents you use for cleaning the explants.

We have some good results with nanosilver in our lab (either you use it in addition to bleach/NaOCl or you add a lower concentration to the media). In any case, as previously mentioned here, the condition of the source material is most important.

Remove the explant ,keep in mercuric chloride solution 10% or 1N for 3 minuets shaking the tube continuously but very slowly with hand,do not use shaker. Wash the explant with double distilled water twice by shaking. Keep the explant on a sterilized filter paper for 30 sec. and culture,you will get some good results.

It is best to use plant material that has been developed under controlled conditions, for example in a culture chamber, in a laboratory or in a nebulization chamber and then continue with the sterilization protocols (alcohol, bleach, etc)

Keeping the ex plants in a controlled condition is must,like lamminer flow or culture chamber.

increase the concentration of Mercuric chloride for surface sterilization...