Targeted modification of medium composition to enhance production of anthraquinone-type natural dye pigments in Rubia tinctorum cell cultures

Abstract With the increasing demand for eco-friendly textiles, plant cell culture offers a promising alternative to traditional dye sources. In this study, in vitro cell suspension cultures of Rubia tinctorum were investigated as a sustainable source of anthraquinone pigments. The effects of varying concentrations of KNO₃, NH₄NO₃, KH₂PO₄, and vitamins in Murashige and Skoog (MS) medium, as well as the initial medium pH, were evaluated for their influence on biomass accumulation and pigment production. The highest fresh weight (57.45 g FW) and dry weight (2.78 g DW) were achieved in the media supplemented with 0.5× and 2.0× vitamins, respectively. The highest alizarin content (718.42 µg·g⁻¹ DW; 13.6-fold higher than the control) was obtained in the MS medium supplemented with 1× KNO₃ and 3× NH₄NO₃, which also resulted in a 51% lower biomass yield (22.75 g FW) compared to the control (46.01 g FW). The maximum purpurin content (1122.49 µg·g⁻¹ DW; 16.9-fold higher than the control) was recorded in the MS medium supplemented with 1× KNO₃ and 2× NH₄NO₃, while maintaining a comparable biomass yield to the control. Pigment extracts from selected cultures were used to dye wool fabrics without mordanting. Colorimetric analyses showed that dyed fabrics exhibited acceptable K/S values (> 0.5), along with commercially viable L*, a*, and b* values. In vitro-derived extracts produced brighter shades than field-grown root extracts, with L* values ranging from 70.25 to 82.76, compared to 60.13 in the field-grown sample. Additionally, all dyed samples demonstrated acceptable wash and light fastness without the use of mordants. Although the color strength of in vitro-derived dyes was lower than that of three-year-old field-grown roots, the short culture duration (2 weeks) and the controlled production system offer significant advantages. This study provides the first report on the use of in vitro-grown R. tinctorum cells for dyeing textile materials, highlighting their potential as a scalable and sustainable source of natural dyes.