Has anyone any experience with cleaning up material prior to using for tissue culture purposes?
The pathogen culture plates are indicating fusarium sp. I can see that there are many instances of control in the growing areas but cannot find information for cleaning prior to micropropagation.
Any assistance would be most appreciated
Hi Karen Brock,
1) Put used explant/cutting on a runner water at least 1-2 hours for reducing contamination.
2) sterilize the medium, plates .. etc well.
3) If it needs, use an appropriate antibiotic to avoid growing a pathogen with medium.
Good luck!
Thanks Ehab
We ran under water for 1/2 hour so maybe we could change this up
No issue with the medium or plates
Sterilised with ethanol and NaDcc
Did not use an antibiotic medium- Could you recommmend which one is suitable? for ginger or similar sp
may be that the infection comes from plant material
for the plant material, you can proceed as follows:
- Dip the explants in 70% ethanol for 20 seconds (alcohol will act here as a wetting agent).
-Transfer The explants in a sodium hypochlorite solution (bleach) 0.5% sterilized by microfiltration to prevent browning regions sectioned explants. The explants should remain in this solution (in agitation or rotation) for 5 minutes.
-The Explants are transferred into three pots of sterile water for a period of 5 minutes each.
-After The third and final rinsing in sterile water, the explants were transferred to Petri dishes containing a solution of ascorbic acid (1g / l) sterilized by microfiltration (0,20μm) to prevent the browning level sectioned areas in plant tissue.
best regards
I never worked with ginger, therefore my response will be very general. If you have knowledge, which kind of pathogen (here Fusarium) you have in vitro, use the same biocides as for field protection. In case of fusarium, better if you know also the species, use any fungicide, which is provided for this pathogen in your country. The most important is to have pathogen-free donor plants, so use fungicide on the plants in the greenhouse, where your donors grow. Then, together with sterilization using NaDCC, apply additional sterilisation with fungicide. To test, if your explant is fungus free, use for little samplke of your explant fungi specific medium. Usually the best way to get fungus-free explants is to isolate possibly small tip, if not meristem.
Good luck, Teresa Orlikowska
List the pathogens to ginger then we can give suggestions, because the agents vary with the location. . The infection can comes through plant material, so apply pre treatment before planting. If fusarium there are about 14 forms which show different symptoms.
Hi Karen Brock,
First, you must follow as Mohamed Nacer Mekahlia mentioned above. If it is not success, use antibiotics like tobramycin, moxifloxacin or mystatin (.25 g/L-1) for Fusarium infection (if you assure "it is Fusarium").
Good luck,
Mahdy, Ehab
Thank you for you supportive answers.
It is definitely fusarium as I have had a pathology laboratory do analysis to a certain point. Looks like further testing using PCR to identify the exact form may be required.
I will most certainly follow the advice of Mohamed and then Ehab's recommendation if this still is not successful
Blessings to you all
Ginger throughout the world suffers from
1 . bacterial wilt caused by ralstonia solaneserum
2. Fungus phythium apharnidermatum
3. Fungus fusarium oxyspofium fs zingiberi
4.Nematodes like pratylenchus, melodogyne, & helycotylenchus
Getting disease free explant has to be ensured first which has been expaine by few above.
Regards and best wishes
Hi karen. The tips that mentioned by Teresa is important for establishment of a clean tissue culture. 1- pathogen-free donor plants 2- Sterilization procedure. We follow this methods in our lab and to remove the contamination in the culture medium, we use antibiotic. To control fungi we apply Amphotericin b in the medium in case there is no choice to remove before culture. Regards
Use the freshly grown shoot tips or rhizome explant. In the active season tissue growth is faster than the spread of infection
Fusarium is a plant pathogen causing systemic diseases but it also has some saprofitic species. You should treat donor plants with a systemic fungicide first (benomyl, carbendazim are good options) for at least a month before taking explants from them. In our lab we have reduced fungi contamination by treating plant material with a fungicide containing sulphur for one hour before final disinfection. Antibiotics are not necessary since Fusarium is a fungus and not a bacterium.
HI Maria
Successfully treated mother plants with cardendazim and ridomil in alternation ( we are not allowed to use benomyl here). We had these tested at plant pathology laboratory before progressing any further. So huge thanks for the heads up on this.
Your sulphur treatment - can you advise on this, as I have not used this technique before?
I"m glad to know that you succesfully eliminated Fusarium. Sulfphur is a broad spectrum fungicide and it eliminates mites also, another problem in biotech labs. We treat some explants by dipping them in a sulphur fungicide, 1g/l, for one hour, before the last disinfection process. It has been helpful eliminating some mite and fungal contamination that we used to encounter. It was great with strawberry tissu culture experiments.
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