Chlorophyll Determination in Intact Tissues Using N,N-Dimethylformamide

Abstract

Photosynthetic pigments from etiolated cucumber (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera) cotyledons were extracted by direct immersion of the intact cotyledons into the solvent N,N-dimethylformamide (DMF). The solvent is especially efficient when pigment concentration is low; time and tools are saved and the loss of pigment that usually occurs in more complicated extraction procedures is prevented. The specific absorption coefficient of chlorophyll a in DMF was also deternined.Problems in extracting sufficient pigment are often encountered when working with etiolated plants where Chl concentration is low, especially if material available for sampling is limited.In studies on Chl determination (1, 3, 4) extraction of pigments usually involves a number of procedural steps which inevitably results in loss of material. Furthermore, grinding and centrifugation of tissues require a relatively high minimum volume of solvent which in effect lowers the concentration of pigment in the final solution. DMF', an organic solvent in which Chl is soluble (6), was used to extract Chl from an alga (8) and from leaves of higher plants without the benefit of grinding (2). In the present work, DMF was used to extract photosynthetic pigments from intact etiolated cucumber cotyledons. Extraction with DMF is simpler than with acetone and the results are comparable. MATERIALS AND METHODSCotyledons were excised from dark-grown 3-to 4-day-old cucumber seedlings (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera). Both DMF and 80% acetone extracts were prepared for Pchl determination by direct immersion of the cotyledons in the solvents. The ratio for the extraction was 5-10% (w/ v). Working volumes ranged between 2 and 10 ml. Extracts for Chl a determination were prepared in the same manner from seedlings which had been exposed to 10 min white light prior to removing their cotyledons. Chl b is not present in detectable amounts in etiolated seedlings within the first hours following light exposure (7). Extracts were also prepared by grinding 10 pairs of cotyledons whose approximate weight was 500 mg in a Sorvall Omni-Mix for 3 min and then centrifuging at 27 x 103g for 10 min without repetition. All operations were carried out under a green safelight. The extracts were stored in the dark for 24-48 h at 4 C prior to spectroscopic examination.To compare absorption spectra of Chl a in the two solvents, approximately 1.0 mg of pure Chl a (Sigma) was first dissolved in 2 ml of 100% acetone. Aliquots of 0.1 ml were transferred to both 'Abbreviation: DMF: N,N-dimethylformamide. 6 ml of 80% acetone and 6 ml of DMF so that the final absorbance of these two solutions ranged between 0.1 and 1.0. The concentration of 100%1o acetone in the solutions was only 1.6% and therefore its effect on the spectrum was considered negligible.The various preparations were examined by means of a Varian Techtron U.V.-V.S. model 635 scanning spectrophotometer using the 0.2 nm bandwidth-measuring beam and a l-ml cuvette having a ...

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