I have more than 300 genotypes and I want to measure CC in the field on these genotypes which are many. I can I do this in two subsequent days.
SPAD is a fast instrument (you can take one reading in about 20 sec) and easy to manage in a day for 300 reading. Anyway, if the climatic conditions are not changing substantially, you can, For safe side, mark some leaves of the last day SPAD reading and check variation in it. That will help you exact situation. All the best
Chlorophyll content mg/g has to be calculated in spectrophotometer. 652 nm for total chlorophyll,645nm for chlorophyll A and 663 for chlorophyll B.And the 300 samples are easy to do in one day.Pluck the leaves take the wight and put it to 80% ACETONE solvent,keep in dark with room temperature. Extract and finish the whole work by 2-3 days.
An hole puncher can take samples for aceton-water control, nut take care about leaf age effect, by using leaf expensin as clock, like the same leaf position and the same delay from unfolding. Chloroplyll is a measure of chloroplast number and any age or light change would have an effect.
This is a constant discussion between ecologists and plant biochemists or biophysicists.
Naturally, an accurate measurement of the chlorophyll content would need careful pigment extraction followed by spectrophotometric measurements. Using equations from the literature, you can calculate concentrations and knowing the volumes and the mass data (fresh or dry) of the leaf pieces, you can calculate the pigment contents. (However, when using 80 % acetone, the maxima where you measure should be 663 nm for chlorophyll a and 645 for chlorophyll b - not the opposite). On the other hand, this needs time and you may have difficulties if you run field works.
Ecologists therefore often use other methods, like SPAD what is very fast. There are lots of theoretical questions what should be clarified. The leaf anatomy (wax, cuticule, hairs, venation) may cause strong light scattering giving false values. The thickness of the leaf is also a factor (optical path) what should be considered. A serious problem is that chlorophylls are in vivo in chlorophyll-protein complexes, the LHC-II is a large aggregate. This means that their electron systems are in excitonic interactions thus one does not know extinction coefficients for them. Consequently, even when using calibration curves you should prepare a curve for each plant species (probably for the same age of leaves) and you can get only a rough estimation. Depending on the research problem, however, this estimation can be enough in supraindividual works.
Would only add that chl content is a proxy for herbicides, age, macro or micro nutrients and light. Then to get control you should be aware of the main factor in action in your case. Imaging of chl fluorescence is also possibly done on several plants at once in a tray up to field at satellite levels.
Dear Gregor,
It is very difficult to measure chlorophyll for 300 genotypes with SPAD in same day. You can,t measure them in same time in same SPAD. And You can,t control on the climate conditions (it is changeable). I suggest using spectrophotometer method as suggested by Utpala Parthasarathy ·(above). All the best.
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