I am trying to observe stomata by microscopy and want to measure stomatal aperture as well. My goal is to observe stomata opening and closing upon drought and cold stress on Pooideae grasses (e.g. Brachypodium).
1. Please suggest me some good literature for this method and tips on peeling (as the grasses are really difficult to peel). 2. How to measure stomatal aperture?
I have tried the same steps as Sam. Very easy after the third sample. Good luck
1) The following links contain information regarding epidermal peels, one is in a textbook so may be hard to find for free. In general it is basically a case of practice. Set aside a morning and spend it making epidermal peels and by the end you should be substantially better at it.
http://www.cerealsdb.uk.net/cerealgenomics/CerealsDB/PDFs/fpls-06-00533.pdf
https://academic.oup.com/jxb/article-lookup/doi/10.1093/jxb/32.4.837
2) Stomatal aperture is measured on ImageJ. I would strongly recommend getting the objectJ plugin, it is simple to use and will save you a ton of time.
There is some debate on what to measure exactly for stomatal aperture, for kidney shaped stomata (e.g. in Arabidopsis then usually length and width are measured and then an ellipse is fitted). In Brachypodium I would suggest using the polygon tool and tracing the pores. It is probably worth also tracing the stomatal complex, as pore size as a function of stomatal area can be useful information.
I have tried the same steps as Sam. Very easy after the third sample. Good luck
Epidermal peels are useful for studying in vitro interactions with salts and growth regulators. Commelina has been the subject of several studies as in Enid Macrobbie literature, but for measuring stomatal reactionin vivo I suggest to apply gas exchange by a field porometer. The ratio of transpiration to vapor pressure deficit is conductance and it is related to stomatal density and aperture. You may try several conditions on the same spot in vivo.
I could peel some hard leaves using Jeffrey´s solution: immersion of the leaf, time to be tested for each species. Or, accordingly, try using other solutions used to obtain wood macerates; try to use more diluted solutions and test the time of immersion Obviously it will be a dead epidermis.
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