What is the most effective protocol to sterilise maize seeds in the lab?
We have tried few common procedures but with no luck (still get fungus contamination on agar plates):
1)10% sodium hypochlorite solution for 10 min
2)15%bleach + 0.01% triton x-100 for 15 min
3) 50% bleach + 0.1% Triton x-100 for 30 minutes, then in 70% ethanol for 30 seconds
4) 70% ethanol for 1 min, followed by 10 min in 10% bleach
5) 95% ethanol then water for 10 min or more to germinate the fungal spores; then 20-50% bleach + soap for 15 min.
Any other idea? We tried multiple batches of seeds, from more than one origin, but we don't seem to be able to have clean seeds: they germinate on MS agar plates (with 0.5% sucrose), but after few days the roots develop fungus contamination.
I think It depends on what would you like to use maize seeds for after the sterilization process.
Because the endophytic populations of fungi and bacteria are prevalent surface infestation does not eliminate their development en toto.
You biggest issure is probably Fusarium moniliforme which is endophytic parasite. To remove that population the use of systemic fungicide would be able to eliminate a large proportion of endophytic population.
I would suggest using a systemic fungicide seed treatment then selecting the aspetic ones without colonies for you continuiing work.
Thank you!
Could perhaps recommend a specific systemic fungicide seed treatment for this case?
Cheers
To sterilize your seeds, you make them in water with 20% bleach you let them for 20min then rinse your seeds for four times with water only and use them.
You can sterilize the seeds by soaking them in Sodium hypoclorite solution with concentration of %6 for 2-3 minutes and then rinse them with distilled water three times to remove the sterilization residues and then dry them with filter papers.
I think It depends on what would you like to use maize seeds for after the sterilization process.
All of those preparation techniques seem plausible (although the bleach concentrations seem fairly high).
- Are you sure it is the seeds that is the source of the fungus?
- Have you tried the same methods with different seed species? If you get the same results (fungal infection) perhaps it isn't the sterilization process but your plating/agar mix/sterile technique?
- Are you preparing the agar plates in a laminar flow hood or just on the laboratory bench?
- Could it be the case that after germination, the roots are starting to release exudates (creating a rhizosphere) and that is what is allowing the fungus to colonise the plates?
Why are you adding 0.5% sucrose to the agar? Plants photosynthesize sugars and don't usually absorb it through the roots. I suspect you have created optimum fungal habitat.
If all else fails, perhaps you can contact someone from Kew or Rothamstead who might have some more experience with germination techniques.
fungus is transmitted vertically and horizontally to the next generation of plants via clonal infection of seeds and plant debris. Horizontal infection is the manner by which this fungus is spread contagiously and through which infection occurs from the outside that can be reduced by application of certain fungicides. The endophytic phase is vertically transmitted. This type infection is important because it is not controlled by seed applications of fungicides, and it remains the reservoir from which infection and toxin biosynthesis takes place in each generation of plants. Thus, vertical transmission of this fungus is just as important as horizontal transmission. A biological control system using an endophytic bacterium, Bacillus subtilis, has been developed that shows great promise for reducing mycotoxin accumulation during the endophytic (vertical transmission) growth phase. Because this bacterium occupies the identical ecological niche within the plant, it is considered an ecological homologue to F. moniliforme, and the inhibitory mechanism, regardless of the mode of action, operates on the competitive exclusion principle. In addition to this bacterium, an isolate of a species of the fungus Trichoderma shows promise in the postharvest control of the growth and toxin accumulation from F. moniliforme on corn in storage. See this once it will be really helpful https://doi.org/10.2307/3435026
It seems you've used freshly harvested (this year) seeds for sterilization. I had similar situation in my experiments with canola and buckwheat. It was useful to rinse the seeds with running water for 1 - 1,5 hours and then sterilize them as usually (etanol+Na hypochlorite or only Na hypochlorite + distilled water).
Best wishes
Treating with 1% solution of sodium hypochlorite and washing with sterilized distilled water, I think, is enough to sterilise the seeds.
1.Dip seeds overnight in cotton cloth in running water overnight
2. Wash in bleaching powder for 15 minutes with suction
3. Treat with 0.01 percent HgCl2 for 15 sec to two minutes
4. Wash in sterilized distilled water three times in separate petri plates
5. Plant of the medium and you will get no contamination
من خلال نقع البذور في محلول هايبوكلورايت الصوديوم 6% لمده ٣ دقائق وثم تغسل بالماء المطر ثلاث مرات
Article Effect of seed size on in vitro seed germination, seedling g...
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