George Brian Lockwood, Supawan Bunrathep, Thanapat Songsak, Nijsiri Ruangrungsi

Abstract: Callus and cell suspension cultures of Citrus japonica Thunb. were established from seedlings. Chitosan was used as an elicitor for improving d-limonene content in cell suspension cultures. The effect of chitosan concentrations on cell growth and d-limonene content was studied. The optimum concentration of chitosan for d-limonene production by C. japonica Thunb. was 200 mg/L which, yielded 42.5 mg/L d-limonene at 12 days. Moreover, at 15 days, α-terpineol, a metabolite of d-limonene also reached the maximum content at 29.2 mg/L. At 21 days, both d-limonene and α-terpineol contents were 14.3 and 18.9 mg/L, respectively, which were increased 17 and 15 fold, respectively, higher than individual content without elicitation.

Chitosan, is a linear polyaminosaccharide found in a wide range of natural sources, eg. crustaceans, fungi, and insects (1). It has been reported to be a general biotic elicitor for suspension cultures of plants (2-5). The mechanism of action of chitosan on secondary metabolite production is complicated by the fact that chitosan is also a permeabilising agent (6-8). At high concentrations of chitosan, cells are permeabilised leading to decreased cell viability, and cell death. At the optimum concentration and optimum time of elicitation, chitosan has proved to be effective at producing volatile substances in suspension cultures from Mentha piperita (5), Vanilla planifolia (6), and Abies grandis Lindl. (9). Citrus japonica Thunb. (syn. Fortunella japonica Swingle) is an evergreen shrub or small tree of the family Rutaceae. It is native to South China, and cultivated throughout subtropical regions of the world (10). It has been widely used as antiphlogistic, carminative, deodorant, and stimulant (11). Its essential oil, namely kumquat oil, has a typical citrus oil character and has been obtained from its peel by steam distillation or simultaneous purging/extraction (SPE) apparatus, in which the most abundant compound was d-limonene (87-97%) (12,13). d-Limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinoma, and its dose for clinical activity is well tolerated in patients (14). To our knowledge, this is the first report on the cultivation of undifferentiated C. japonica, although there have been reports of essential oil components in suspension cultures derived from three other species of Citrus (15). In an attempt to produce d-limonene by this culture, we have selected chitosan as elicitor for improvement of its production.