Green Index: a widely accessible method to quantify greenness of photosynthetic organisms

Abstract Plant phenotyping involves the quantitative determination of complex plant traits using image analysis. One important parameter is how green plant tissues appear to the observer, which is indicative of their health and developmental stage. Various formulas have been developed to quantify this by calculating leaf greenness scores. We have developed a revised formula called “Green Index” (GI) devised out of the need to quantitatively assess how the apparent greenness of seedlings changes during de-etiolation. The GI calculation is simple, uses widely available RGB values of pixels in images as input, and does not require commercial software platforms or advanced computational skills. In this study we describe the conception of the GI formula, compare it with other widely used greenness formulas, and test its wider application in plant phenotyping using the open source free software platform RawTherapee. We demonstrate the utility of the GI in addressing common issues encountered in assessing plant biology experiments, underscoring its potential as a reliable and accessible tool. Finally, we explore the correlation between GI and chlorophyll content, assess its reliance on different types of photography, and summarizes the key steps for its effective utilization.