Hi,
I am working with wild relatives of rice.
I have been facing an issue with the germination as the seeds are highly contaminated.
I tried to use ethanol 70% 2 minutes, bleach 4% for 30 minutes, mercury chloride 0.1% for 3 minutes.
Still, the blueish fungi appear on the seeds (in the petri dish).
Any recommendations? maybe try to use vaccum infiltration in order to help penetrate the seed's wax? Have anybody done it before?
Many thanks in advance
Please have a look at this useful link.
http://pubs.acs.org/doi/abs/10.1021/acsami.6b04555?journalCode=aamick
Hi, I have expeience with wild rice, u can try with 2M nitric acid treatment. 10H treatment is enough. All the best
Hi: I dont know what it the purpose of your experiment, but you can use a systemic fungicide to treat the seed before grow it.
We've had good success controlling in vitro fungal contaminants from citrus node cultures (field collected) by incorporating benzyl ammonium chloride into the culture medium. Fungal contamination is virtually eliminated and there is no phytotoxicity on the citrus plants. We use a product called Floriculture PronTech at 400-800 ppm (the product is 40% BAC where the BAC is chelated to urea). Control of bacteria, at least the types we run into, is not very good.
@ Randall P Niedz,
1. When you included PronTechTM Floriculture (benzyl ammonium chloride) into the medium, how did you sterilize it? By filter sterilization?
2. If the control of this product is not so good on bacteria, did you include other antibiotics in the medium with BAC to eliminate bacteria?
"1. When you included PronTech Floriculture (benzyl ammonium chloride) into the medium, how did you sterilize it? By filter sterilization?" Autoclaving. One of the first tests we ran was the effect of autoclaving vs filter sterilization on controlling contamination - no difference. Now we just add into the medium prior to autoclaving. One thing I have not tested is if the material is inherently sterile and could be added directly to sterilized media.
One thing about PronTech the benzyl ammonium chloride (BAC) is chelated to urea. I think this form of BAC allows for much higher levels of BAC to be used (PronTech is 40% BAC - this is much higher than the BAC levels in cleaning products I have looked at).
"2. If the control of this product is not so good on bacteria, did you include other antibiotics in the medium with BAC to eliminate bacteria?" Streptomycin. We tested a number of antibiotics and found that streptomycin worked well (and was the cheapest of the antibiotics that worked).
@ Randall,
1. Thanks for the good and detailed answers.
2. As you said "One thing I have not tested is if the material is inherently sterile and could be added directly to sterilized media.", I think it would be good if the product can be directly added into autoclaved media. The vapor from autoclaving this product can be harmful for people around. The website says that it is 'low in toxicity', not toxicity-free ( http://www.upitrading.com/english/17/prontech_tm_-floriculture/ ).
Try seed treatment with some broad spectrum systemic fungicide like bavistin
I had worked with heavily fungal contaminated rice seeds for tissue culture. The following steps work sufficiently, and there is no need for mercuric chloride.
- Follow rigorous agitation during all the washes (70% ethanol wash, 4% NaOCl + 0.1% Tween 20 wash, and autoclaved water). You can use Falcon tubes or beads for agitation, as these are more effective than sterilizing in conical flasks.
- You will observe whitish peel-like layers coming off the rice seeds. Wash thoroughly with autoclaved water until this debris is removed.
- Make sure there is no contamination from other sources like poorly autoclaved materials (edges of bottles, forceps, corners), hand sterilization, the potency of bleach (as older bleach stocks are less effective), amount of detergent, etc.
Best of luck!
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