Isolation and characterization of blackish-brown BY2-melanin accumulated in cultured tobacco BY-2 cells

The tobacco BY-2 cell line is one of the most utilized plant cell lines. After long-term culture, the cells turn brown to black, but the causal pigment is unknown. We successfully isolated a blackish-brown pigment from BY-2 cells cultured for 3 weeks. Morphological and spectroscopic analyses indicated that the pigment had similar features to a melanin-like substance reported previously. Furthermore, physicochemical analyses revealed that this pigment possessed most of the properties of melanin-like pigments. In addition, the high nitrogen content suggested that it differed from common plant melanins classified as allomelanins, suggesting a novel eumelanin-like pigment: "BY2-melanin". This is the first example showing that eumelanin-like pigments are produced in the cultures of plant cells for which the accumulation of melanin has not been reported. This tobacco BY-2 cell culture technique may represent a customizable and sustainable alternative to conventional melanin production platforms, with significant potential for industrial and pharmacological applications.