I am looking at chemicals released during the germination process and I want to correlate the release of some of the chemicals to the greening process of the seedling. Can anyone suggest protocols for extracting chlorophyll from seedlings?
Here are some "classic references":
Holden, M. 1976. Chlorophylls. In: Chemistry and Biochemistry of Plant Pigments, Vol. 2 (Ed, Goodwin, T. W.) Academic Press, London, pp. 1-37.
Lichtenthaler, H.K. 1987. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. In: Methods in Enzymology, Pacher, L. & Douce, R. (eds.), Academic Press, New York, 148:35-382.
Porra, R.J., Thompson, W.A., and Kriedemann, P.E. 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975: 384-394
Wellburn, A.R. 1994. The spectral determination of chlorophylls a and b, as well as ttal carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144: 307-313.
for chlorophyll extraction according to Arnon (1949). One gram leaf sample was macerated with 80% (v/v) acetone and pinch of calcium carbonate. The homogenate obtained was centrifuged at 5000 rpm for 10 mins. Then the supernatant obtained was made to known volume with 80% acetone. Finally the absorbance of the supernatant obtained was taken at 645nm and 663 nm. Note all the steps should be performed under dim light.
Chlorophyll contents were eluted from the leaf FW samples (0.05 g) with 5 ml of 80% acetone at and determined using the methods by Porra et al (1989). Samples were then centrifuged at 13,000 g for 5 min. Supernatants were tested to determine the absorbances of Chl a and Chl b in acetone as measured with a spectrophotometer at wavelengths of 663.6, 646.6 respectively. Concentrations (μg g-1 FW) of Chl a and Chl b were determined using the following equations:
Chl a = (12.25 × OD663.6 – 2.55 × A646.6) × volume of supernatant (ml) / sample weight (g)
Chl b = (20.31 × A646.6 – 4.91 × A663.6) × volume of supernatant (ml) / sample weight (g)
Porra R. J., Thompson W. A. and Kriedemann P. E.(1989) Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll-a and chlorophyll-b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy, Biochimica Et Biophysica Acta, 975(3), 384-394 .
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