A Rapid and Nondestructive Method to Determine Chlorophyll in Intact Leaves

Abstract

The important role that chlorophyll (CHL) plays in plants necessitates its estimation in various types of studies. Singh and Anantrao (6) realized the need for a reasonably simple, rapid, and accurate method of CHL determination capable of accommodating small quantities of leaf tissue and a large number of quantitative estimates in a short time, and thus, in 1937, they developed a photoconductive photometer for quantifying CHL in 80% methanol. Since then, CHL determination techniques have improved considerably, but complete extraction of CHL is laborious, slow, and inconvenient in some plant species (3). Conventional methods used for isolating and measuring chlorophyll in aqueous acetone or similar organic solvents are sometimes cumbersome and slow, and always destructive to leaf tissue (1, 4, 7). Furthermore, steps involved in sample preparation, pigment extraction, and dilution result in pigment loss and contribute to variability. Moran (5) developed an efficient method for extracting small quantities of CHL from intact cotyledons with N , N -dimethyl-formamide (DMF). Inskeep and Bloom (3) further improved this technique by using extinction coefficients of chlorophyll a and b extracted in DMF. Evans (2) extracted CHL in 80% acetone and adapted Amon's modification of the method of McKinney (5), who provided equations (μmol CHL/1 = 22.22 D645 + 9.057 D663) to evaluate the molar concentrations of total chlorophyll (CHL a and b ) in the tissue extracts. Nevertheless, these procedures are still time consuming since they require tissue extraction and spectrophotometric measurement. The objective of this investigation was to determine area concentrations of total chlorophyll (CHL a + b ) using a portable chlorophyll meter (SPAD-501) and to compare and correlate these data with area concentrations of total CHL obtained by conventional methods.