Non-Destructive Determination of Chlorophyll Content of Leaves of a Green and an Aurea Mutant of Tobacco by Reflectance Measurements

Reflectance spectra from 400 to 800 nm with a spectral resolution of 1 nm and the content of photosynthetic pigments were acquired for leaves of an aurea mutant and a green tobacco (Nicotiana tabacum L.) covering a range of chlorophyll a content from 7 to 30.8 μ cm-2 and total chlorophyll a + b level from 8.4 to 41.4 μg cm-2 leaf area. At a chlorophyll a content of more than 7 μg cm-2 leaf area the reflectance near 670 to 680 nm was not sensitive to a variation in chlorophyll content due to saturation of the relationship ≪absorption vs. chlorophyll content≫. The wavelength position of the red reflectance minimum (Rmin) in the main red absorption bands of in vivo chlorophyll a proved to be independent of the chlorophyll content of leaves and thus is not suitable for chlorophyll determination. Maximum sensitivity of leaf reflectance to variation in chlorophyll content was, however, found in a wide spectral range from 530 to 630 nm and near 700 nm. The wavelength of the red edge position of the reflectance spectrum (inflection point IP) in the range of 694 to 706 nm correlated very closely with the leaves' chlorophyll content in a curvilinear manner. The Ip is also closely correlated in a linear manner with the reflectance signals at 550 and 700 nm. Reflectances near 700 nm and 550 nm were found to be sensitive indicators of the red edge position and the chlorophyll content of leaves as well. The ratios of reflectances in the near infra-red range of the spectrum (above 750 nm) to that at 700 nm and at 550 nm, RNiR/R700 (or R750/R700) and Rnir/R550 (or R750/R550), were directly proportional (r2>0.93) to the leaves' chlorophyll content. These two novel indices for chlorophyll determination (R750/R700 and R750/R550) allowed the estimation of leaf chlorophyll content with an error of less than 2.1 μg cm-2. The two new vegetation indices, R750/R700 and R750/R550, are linearily correlated to the chlorophyll content. They exhibit a more than 6 times wider dynamic range than the widely used Normalized Difference Vegetation Index NDVT. The new vegetation index ratios are very sensitive to changes in chlorophyll content at low, medium and high chlorophyll content which is not the case for the NDVI. This suggests the application of the new indices R750/R700 and R750/R550 in the near and remote sensing of the chlorophyll content of terrestrial vegetation.