Shikonin was the first phytoconstituent to be commercially produced from tissue culture. It is a nathaquinone dye with anti-bacterial, anti-inflammatory properties and has been used in the treatment of burns, wounds, and hemorrhoids. A valuable vegetable dye, it gives a spectrum of colours ranging from red, purple, to blue. It is obtained from Lythospermum erythrorhizon, a plant that has been extensively harvested to the verge of extinction in its native habitat in Japan, China, and Korea. The plant takes 3–4 years to yield roots before they can be collected and the shikonin content of the reddish purple roots is only 1–2%. In 1983, Scientists at Mitsui Petrochemical Company developed a commercially successful tissue culture process for the production of shikonin from cell cultures of Lithospermum erythrorhizon. Such a product derived from tissue culture was commercialized for sale as a natural colour in soaps, lipsticks, and other cosmetic products.

Commercialization of shikonin production was possible largely because of selection of high-yielding cell lines and optimizing culture conditions to enhance yield to 15% of the biomass. Thus cost of production became economically feasible considering the fact that the plant population is declining in its native countries, making tissue culture technique the method of choice for its production.

Selecting high yielding cell lines is an important first step to enhance productivity. This is because the explant that initiates a cell culture is heterogenous with respect to metabolic productivity of its component cells. When cultures are derived from cells grown from it, due to various genetic and other conditions, they are exposed to in cultures and clusters of cells show great variation in secondary metabolite accumulation. There will be high- and low-yielding cells and therefore selecting and cloning high yielding cells in media that maximizes product formation is very important. Such selected cell lines must be genetically stable over several cycles of aggregate sub culturing and/or single cell cloning. In some cases as in Datura innoxia suspension cultures, it has been observed that gross chromosomal changes occur in the cultured cells. Cells were noted to be tetraploid, haploid, or of variable ploidy in different proportions. While sometimes as in protoplast-derived Hyoscyamus muticus cultures, the haploid plants were richer in hyoscine than diploid plants, maintenance of uniform genetic characteristics among high yielding cells is a significant prerequisite.

The fact that media composition is the most important determinant for optimum production of secondary metabolites has been illustrated by the pioneering work of Zenk, Fujita, Tabata, Gamborg, Blaydes, and Nitsch among others. In general, it was observed that both in the whole plant and in culture, cell growth and secondary metabolite production do not often happen together. When media composition is conducive to optimum cell growth, secondary metabolite production starts happening in the late stationary phase when the growth has become static. Thus came about the two-culture system, where the first culture media’s composition was optimized to support cell growth. Once the optimum level of biomass got accumulated, the cells were transferred to a different medium that is favorable for product yield. First used by Zenk et al., in 1977 for indole alkaloid production by Catharanthus roseus cells, it was also used for shikonin production. Here the change was from growth supporting Linsmaier and Skoog medium to nitrate rich White’s medium. The type of nitrogen affected shikonin production and the nitrate had to be the sole source of nitrogen. Even little replacement with others such as ammonium salt greatly reduced shikonin production.

The right media composition that supports production of secondary metabolites varies with different cultures and even different metabolites of a particular cell line. For example, for maximum ajmalicine production by C. roseus cell cultures, glucose concentration is critical at 500 mM, whereas for optimum tryptamine production, the media has to have lower glucose concentration of 100 mM.

Many cell cultures can be optimized for growth, and secondary metabolite production in a single medium. For example, LS medium supplemented with 10 times the usual copper concentration increased berberine production by 20–30% in cell cultures of Coptis japonica without adversely affecting biomass production.