Influence of medium and elicitors on the production of cocaine, amino acids and phytohormones by Erythroxylum coca calli

Erythroxylum coca (Erythroxylaceae) is the source of the tropane alkaloid cocaine. Several lines of evidence suggest that tropane alkaloid biosynthesis in E. coca differs from that in solanaceous species, but there are many gaps in our understanding of the pathways in both groups. The development of an E. coca cell culture that produces cocaine could provide a reproducible model system for discovering novel biosynthetic genes and study pathway regulation. Calli cultures were successfully established from young leaf explants on three different media: Anderson's Rhododendron, Gamborg B5, and modified Murashige-Tucker, all supplemented with growth regulators: 2,4-D (0.6 mg L-1), indole butyric acid (0.06 mg L-1), and benzylaminopurine (0.5 mg L-1). All accumulated cocaine and cinnamoylcocaine at levels of 0.05-0.5 nmol per gram dry weight, as determined by LC-MS, several orders of magnitude below the concentration found in the intact plant. Anderson's Rhododendron medium supported the highest level of tropane alkaloid production, as well as the highest level of the amino acids arginine, glutamate, proline and phenylalanine, all thought to be precursors of cocaine, but contained generally lower levels of hydroxycinnamate-quinate esters, such as chlorogenic acid. These differences may be ascribed to its relatively low content of nitrate or salts, or its high content of adenine. Addition of 100 mu M salicylic acid or coronalon, an analog of the bioactive jasmonic acid-isoleucine conjugate, did not result in any increase in tropane alkaloid production. These E. coca calli could provide valuable material for studies on tropane alkaloid biosynthesis and regulation.