Dear fellows
I am into micropropagation of calladium. we didn't observe any bacterial contamination during the initiation and first subculture stages. the second subculture was done last week and found that all my cultures are bacterial contamination after one week.
the media contained 2% PPM also. is there any advice so I can save my explants?
or all should be autoclaved?
I am really devastated by this bacterial contamination we frequently face.
It is almost impossible to rid cultures of contaminants. The best approaches are to start by having very vigorous mother plants and a clean growing environment. Only take explants from rapidly growing plants. You might try to treat the explants with ppm before placing them in culture. See Kushnarenko et al 2022. Then monitor the new explants on nutrient agar (see Reed et al publications). By selecting clean explants early in initiation, you can sometimes get a few clean propagules from dirty mother plants and multiply from them.
For your contaminated explants, try removing them from culture and shaking them in ppm solution, then replant on medium. It might not work, but will at least slow down the contamination.
PPM dosnt really kill microbes, just prevents them from growing/multiplying as fast. If the cultures are critical, I would try to identify the bacteria and select an antibiotic that will kill that species to treat the plants with. Some antibiotics are toxic to plant tissue, so this is not a fool proof approach, but probably better than PPM.
Ultimately, I agree with Barbara that it is better to aim for clean and vigorous explants in the first place, but that is not always possible.
I agree with Barbara's recommendations.
Just one more point is to apply vacuum pump during the sterilization. A liquid type vacuum pump (not gas type) facilitates penetration of ethanol and NaOCl into the explants.
Anyone may agree that once contaminants occurs, none of the methods are very effective.
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