IN VIVO AND IN VITRO PRODUCTION OF ALKALOIDS IN THEOBROMA CACAO L.

MARCOS PAIVA, Purdue University

Abstract

Theobromine and caffeine, the major alkaloids in seeds of Theobroma cacao L., were quantified by liquid chromatography (HPLC) and followed during embryo development and seed germination. Alkaloid content was highest in seedlings 3 days after germination. Alkaloids were measured in seed non-sacrificially by sampling a portion of cotyledons prior to germination. Alkaloid content (in mg/g dry weight) of individual defatted seeds within 35 clones of cacao varied from 13.5 to 45.0, mean 29.8; theobromine from 10.5 to 41.0, mean 25.2; and caffeine from a trace to 23.9, mean 4.6. Four clones (A43, B38, 'Catongo Blanco' and a 'Catongo Blanco' cross) had low caffeine content varying from 0.4-0.6 mg/g. Although 'Catongo Blanco' is an albino mutant no relation was found between anthocyanin and caffeine in Criollo clones segregating for seed color. There was an inverse relationship between theobromine and caffeine content (r = -.47) and a positive relationship between xanthine and theobromine (r = .35), but no relationship between xanthine and caffeine. Analysis of alkaloid content in seeds of 2 clones showed that seed-to-seed variation is a consequence of genetic differences between seeds. Alkaloid accumulation in asexual embryos cultured in vitro for 90 days in liquid media (10 days in 3% sucrose, 2 days each in 9%, 15%, 21%, and 74 days in 27% sucrose) increased up to 70 days with a maximum of 5.6 mg/g theobromine and 0.9 mg/g caffeine, indicating that the embryo is the site of synthesis of alkaloids and not merely a site of alkaloid accumulation. Increasing sucrose but not glucose concentrations elicited the production of lipids, anthocyanins, and alkaloids in immature zygotic or asexual embryos cultured in vitro; phosphorus, potassium, or gibberellic acid concentrations did not affect alkaloid accumulation. The concommitant development of lipids, anthocyanins, and alkaloids suggests that the development of these metabolites involves a common regulatory pathway that is elicited by high concentrations of sucrose. Asexual embryos initiated from clones varying in caffeine and anthocyanin production in vivo behaved similarly in vitro, suggesting the feasibility of selecting zygotic embryos that are high producers of desirable metabolites such as flavor components and underproducers of undesirable substances such as caffeine for production of cacao embryos in an in vitro system.

Degree

Ph.D.

Subject Area

Agriculture

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