Fungal spores on surface of explants can be eradicated by surface sterilization with a fungicide. But, how to control endophytic fungal contamination form explants during plant tissue culture. Please suggest any chemical which can be added in tissue culture media or any method to control endophytic fungi from explants.
Hi Sathish,
you could make PCR with ITS-Primers.
You should look for primers that amplify diverse lenght for your plant and potential funghi. If you get only the product for your plant there should be no contamination. If you get diverse products you would have a contamination. By sequencing of the products you could determine the funghi.
regards
Stephan
Article Fungal-specific PCR primers developed for analysis of the IT...
Sathish Dorairaj endophytic fungi certainly can be released to tissue culture; however, in my experience I have never "seen" them in tissue culture. More frequently are bacteria and probably some yeasts. It's difficult to recommend a biocide that act against fungi. Although it's important to determine what kind of microorganisms are, sometimes tissue culturists have to responde quickly, therefore most of time we've to act in a generalistic way because we only have few days, even hours, to save our cultures.
Sathish Dorairaj are you sure that contamination is endophytic? It's a critical step. Can you share some picture.
Sathish Dorairaj, i have also faced the same problem. Antimicrobial supplement was commercialized at that time. optimize the concentration of that supplement according to your plant. Simultaneously, isolate and identify the endophytic fungus and report it to NCBI.
Article Biochemical Characterization of Fungus Isolated during In vi...
@Ricardo Julian Licea-Moreno , Thanks for your valuable time. Fungal contaminants started growing after some 2 weeks of explant inoculation and inhibited the growth of explants. One of my colleagues who work in mycology said that it as endophytic fungi. Unfortunately, I have not taken a photo of it. Thank you.
Then have you lost the materials? If not, try with a gently disinfection, I mean, low NaOCl concentration during few minutes. In this case it's preferable apply the disinfection 2 or 3 times instead an accute treatment.
Hope these articles will help you.
Article Biotic Contamination and Possible Ways of Sterilization: A R...
Chapter Micropropagation
https://phytotechlab.com/media/documents/TechnicalLiterature/ControllingContamination.pdf
Chapter Bacterial Endophytes in Plant Tissue Culture: Mode of Action...
https://www.jstor.org/stable/4293434
mostly, contaminations are coming from 3 ways.
1- from a worker who is making the tissue culture. supposably, you know all this but it can be good to remember. you have to wash your hands with 70% EtOH, and wipe out the laminar hood before you work. also, you should put all the materials under UV light for 15-20 minutes (except plant materials). you have to be sure you burn your forceps/scissors in a bunsen burner. last but not least, you should be sure your plate/magenta is closed very well.
2- from the media you plant your explants. you should be sure that the autoclaving time, temperature and the pressure of the autoclave machine. If you are not sure, you can wait your media (its lid should be closened) under UV light for 15-20 minutes
3- from the explant. although it depends on which explant you used, you can use triton x-100, 5% bleach, 30% hydrogen peroxide or fungicide. But, you have to be careful because these chemicals are most probably will damage your explants. you can increase the time when the explants/seeds get harder.
I hope these will be helpful in your studies. good luck with your experiments
Hello Satish,
Try adding NaDCC (Sodium dichloroisocyanurate) in the media once you are sure that the surface sterilization is done properly as suggested by others.
Article Sterilisation of explants and cultures with sodium dichloroi...
Hi Vivek Rakshit Sonawane, Thank you for your suggestions. The article which you attached says that NaDCC is effective for bacterial contaminants. Will it work for endophytic fungi?
Of course, 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)].
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