I would like wo quantify the H2O2 concentration in pea leaves after infestation by a pathogenic fungi. As I want the method to be quick and accurate I found some methods after some literature search and conversations with colleagues.
Staining with several agents and sucsequent microscopy with colorimetric quantification of pictures would be one option. However, I think this method is not quick enough as I have about 80 samples to measure it would take the whole day and H2O2 content will change during this time period. Still, Do you think this is appropriate and what stainind agent fits best?
Second, I consider a photometric assay which is probabaly the qickest:
Grinding of leaf tissues in TCA -> centrifugation -> pH adjustment to 7.0 (with phosphate buffer) -> measureing absorbance at 390 nm. (like in publication attached)
However, I heard that qunatificatin with TCA assay is not too accurate. Still, when I found values in literature this measurement seems to be quite stable. Is it stable and accurate enough for my purpose? Do you have any experience on measuring H2O2 in leaves and what would you prefer in my case?
Article Role of catalase, H2O2 and phenolics in resistance of pigeon...
hi, you can use Peroxide Test Strips 0.5-25 ppm or use a Peroxide Assay ELISA Kit its more sensitive
Hi,
for quntification Test stripes may not do the job. A Peroxide Assay ELISA Kit sounds expensive!? How about quantification with that assay?
hi. TCA based spectrophotometric analysis is also reliable for quantification of H2O2. Many of the researchers used this method. You can see one recent paper "Microbial Consortium-Induced Changes in Oxidative Stress Markers in Pea Plants Challenged with Sclerotinia sclerotiorum" see the attachment. This paper is related to your work, may this help you...
Hi Renhard,
What about floating leaf disks on solutions of xylenol orange? (Bindschedler et al, 2002, 2006)
Luminol assay based are sensitive and specific. There might be alternative with leaf disks, and Glo-MAx systems from promega, that are luminol based, and are very specific and sensitive.
Hi Laurence,
thank you for your reply. I looked up the publication from 2006: The method with xylenol seems straight forward. Since I posted my question some time ago, I already made my decision back then on the TCA assay, which works fine in my experience, however, for H2O2 measurement it is maybe still a bit too time consuming if you want to sample in a timeseries of minutes including several replicates.
In such case (timeseries with many replicates) the xylenol orange assay might be more handy. I saw that in your study you used elicitor solution of F. oxysporum. Unfortunately, we did not have elicitor of Didymella pinodes ready, what would probably be troublesome with this assay. But treatment with conidia solution might work as well, just with delayed response (oxidative burst).
Thank you again for your answer. I will keep this in mind for follow up experiments.
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