Stomatal aperture assays in abaxial epidermal peelings of tobacco leaves were done as described in Allen et al. (1999) and He et al. (2013). Peelings were induced to close all stomata in dark condition, and then a part of samples were kept in darkness, a part of samples were put in light condition to induce stomatal opening, and some samples were put in light with 50 µM of abscisic acid to induce stomatal closing (Schemes of the assay are in the following attached picture). Then I obtained microscopy images (10x, 20x and 40x) at three condition times (2 hours, 4h and 6h). I want to determinate the degree of stomatal aperture at those times in each treatment, but I don't know what kind of parameters I need to measure, and if there is any kind of equation that integrate those parameters. I imagine that important parameters can be: stomatal length and width, and stomatal pore length and with, but how can I end this story?
Thank you very much indeed.
JD
Some toxin like FC can increase to a max stomatal opening http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02120.x/pdf. Opening is measured by pore area or ratio of width to lenght and the value can be obtained by a variety of image analysis, Image J in https://www.colby.edu/academics_cs/courses/BI214/upload/lab5-stomata.pdf
Dear Guido Bongi, thank you very much for your quick answer. I'm doing some measurements right now following your recommendations
You can also measure whether the stomata are sunken or raised by Image J software.
As Guido Bongi suggested you can also measure the stomatal aperture area or stomatal length to weight ratio.
You can see the method in the following publication
If I you have questions in this regards I would be happy to help you
The best
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