Abstract
This study focuses on optimizing chlorophyll extraction techniques, in which leaf discs are cut from places on the leaf blade to enhance chlorophyll concentration in sesame (Sesamum indicum L.) leaves. Thirty sesame genotypes, categorized into light green (LG), middle green (MG), and deep green (DG) pigment groups based on leaf coloration, were selected from a larger pool of field-grown accessions. The investigation involved determining optimal Soil Plant Analysis Development (SPAD) value index measurements, quantifying pigment concentrations, exploring extraction solvents, and selecting suitable leaf disk positions. Significant variations in chlorophyll content were observed across genotypes, greenness categories, and leaf disk positions. The categorization of genotypes into DG, MG, and LG groups revealed a correlation between leaf appearance and chlorophyll content. The study highlighted a consistent relationship between carotenoids and chlorophyll, indicating their role in adaptation to warm environments. An examination of leaf disk positions revealed a significant chlorophyll gradient along the leaf blade, emphasizing the need for standardized protocols. Chlorophyll extraction experiments identified DMSO and 96% ethanol, particularly in those incubated for 10 min at 85 °C, as effective choices. This recommendation considers factors like cost-effectiveness, time efficiency, safety, and environmental regulations, ensuring consistent and simplified extraction processes. For higher chlorophyll extraction, focusing on leaf tips and the 75% localization along the sesame leaf blade is suggested, as this consistently yields increased chlorophyll content. Furthermore, our examination revealed significant anatomical variations in the internal structure of the mesophyll tissue leaves between deep green and light green sesame plants, primarily linked to chloroplast density and pigment-producing structures. Our findings, therefore, provide insightful knowledge of chlorophyll gradients and encourage the use of standardized protocols that enable researchers to refine their experimental designs for precise and comparable chlorophyll measurements. The recommended solvent choices ensure reliable outcomes in plant physiology, ecology, and environmental studies.