Simple Linear Regression and Reflectance Sensitivity Analysis Used to Determine the Optimum Wavelengths for the Nondestructive Assessment of Chlorophyll in Fresh Leaves Using Spectral Reflectance

The accuracy of nondestructive optical methods for chlorophyll (Chl) assessment based on leaf spectral characteristics depends on the wavelengths used for Chl assessment. Using spectroscopy, the optimum wavelengths (OW) for Chl assessment were determined by using 1-year-old almond (Prunus dulcis), poplar (Populus trichocarpa · P. deltoides), and apple (Malus ·domestica) trees grown at different rates of nitrogen fertilization to produce leaves with different Chl concentrations. Spectral reflectance of leaf discs was measured using a spectroradiometer (300 to 1100 nm at 1-nm intervals), and total Chl concentration in leaf discs was extracted and determined in 80% acetone. The OW for nondestructive Chl assessment by reflectance spectroscopy was estimated using 1) the coefficient of determination (r) from simple linear regression; 2) reflectance sensitivity analysis (a measure for changes of spectral reflectance on unit change in leaf Chl concentration); and 3) the first spectral derivative method. Our results indicated that the first derivative method can be used only to identify OW in the red edge region of the spectrum, whereas r and reflectance sensitivity analysis can be used to identify the OW in both the red edge and green regions. Our results indicate that using simple linear r in combination with reflectance sensitivity and/or the first derivative analyses is a reliable method for determining OW in plant leaves tested. Two optimum wavebands with larger r, smaller root mean square error, and higher reflectance sensitivity were found in red edge (700 to 730 nm) and green (550 to 580 nm) regions, respectively, which can be used as common OW for Chl reflectance assessment in poplar, apple, and almond leaves tested. Single-wavelength indices if developed with OW were even more accurate than those more wavelength indices that developed without using OW. The accuracy of indices can be further improved if indices developed by using one OW and one Chl-insensitive wavelength from near infrared (NIR) (750 to 1100 nm) in the form of RNIR/ROW or (RNIR – ROW)/(RNIR + ROW). The chlorophylls, chlorophyll a (Chl a) and chlorophyll b (Chl b), are essential pigments for the conversion of light energy to stored chemical energy in plants and their presence and function is important from both physiological and applied perspectives (Buschmann et al., 1994; Carter, 1998; Gitelson et al., 2003; Pinar and Curran, 1996; Richardson et al., 2002). As much as 75% of the total nitrogen (N) in a plant is required for normal chloroplast formation (Kutik et al., 1995) and synthesis of components of the photosynthetic apparatus, including thylakoid membranes and photosynthetic enzymes (Evans, 1989); therefore, Chl concentration gives an indirect estimation of plant N status and photosynthetic potential (Filella et al., 1995; Moran et al., 2000). Leaf Chl concentration is often closely related to plant stress and can be used as an indicator of plant stress (Carter, 1993, 1994; Carter and Knapp, 2001; Peñuelas and Filella, 1998). Traditionally, leaf Chl was extracted with organic solvents and measured using a spectrophotometer (Lichtenthaler, 1987; Lichtenthaler and Wellburn, 1983). Recently, alternative Table 1. Effect of nitrogen concentration in fertigation solution on chlorophyll concentration in apple, poplar, and almond leaves.