what is best method of surface sterilization of explant in tissue culture ? and how to minimize fungal contamination in tissue culture ?
Can I use Fungicide for surface sterilization of explant ?
Which fungicide and Quantity ?
Dear Sagar Jadav you must to consider several factors. The kind of explants: seeds are easier to disinfect than nodal segments. The origin: nodal explants from outgrowth plants are worse to disinfect than those from greenhouse. Usually explants from trees are worse to disinfect than those from vegetables. There many other factors; but always depends from your particular conditions. The most used sterilant is NaOCl. However, combining various sterilants (alcohol, H2O2,...) with NaOCl are the most frequent disinfection protocols. You must also to considered the concentrations and the duration of treatment. It would be recommendable to use autoclaved water for all steps; however, it's only mandatory for washing the treated materials. Antibiotics and mercurial substances are also used; however, I would try to avoid their use. Hope it help you. Good luck. Oh, patience is also a good element to obtain success.
I must first say that the most important issue is to identify the source of the contamination. Contamination can always have several causes, but it is usually either due to the mistake of the culture operator or due to environmental contamination (laminar hood, autoclave, blade, plate, glass or petri dish, and growth chamber) or explants (failure of sterilization or endogenous fungal contaminants).
Overall, there are two types of contaminants (Exogenous and endogenous fungal contaminants). Exogenous fungal pathogens are easy to eliminate from the stock plants using many fungicides such as systemic fungicides (e.g. Benomyl) or other disinfectants (sodium hypochlorite). Endogenous or endophytic fungi become pathogenic to the host plants when the plants are stressed, for example, when the cell walls are weakened or under other unfavorable in vitro conditions. Mature stock plants are highly loaded with fungal pathogens and spores, but used due to their maturity, especially in fruit trees.
For further guidance, some of these chemicals include: 1. Nystatin (toxic to fungi ); 2. Copper oxychloride (fungicide ); 3. Sodium & calcium hypochlorite; 4. Formaldehyde (aldehyde); 5. Benomyl and Captain; 6. Teepol (detergent). 7. Silver nanoparticles; 8. Titanium dioxide (TiO2); 9. Ampicillin 10. Alcohol (70%) and so on.
Also, you can use meristem tip culture technique with several sub-culturing.
I hope the content is effective and useful.
With luck
Reza Ghahremani
Also, the concentration and duration of treatment in each plant is different and, since there is no information about the type of plant you are studying, it is necessary to search articles of fungicides [available for you] for reaching a sterilization protocol. In my experience, Silver nanoparticles is successful at 200 ppm concentration for 10 minutes. Also, I had a successful experience in this treatment [Sodium hypochlorite (20% with TWEEN® 20; 20 min)+ Alcohol (70%; 60 second)+ twice washing with sterille-distilled water (3 and 5 min, respectively)].
First of all you should make sure that everything (all the apparatus) you use during tissue culture are properly sterilized and there are different protocols for treating explants based on the explant that you are using
To prevent fungal contamination you can use fungicides such as bavistin in very low concentrations like 0.1% to treat explant for 5-10 min you can also use sodium hypochlorite
You can also try dipping in 0.1% mercuric chloride too
Hope this helped you
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