Effects of Macronutrients on Anthocyanin Production in Roselle (Hibiscus sabdariffa L.) Callus Cultures.

Abstract

The effects of various macronutrients on growth and anthocyanin formation in callus cultures of roselle (Hibiscus sabdaritfa L.) were investigated. Of the nutritional factors examined the type and concentration of carbon and nitrogen sources and phosphate concentration showed marked effects on the growth and anthocyanin production. Utilization of an optimized medium based on the results obtained in the present investigation resulted in a 2.5 fold increase in the anthocyanin content Potential exists for application of a two-stage culture method for the production of anthocyanin pigment Introduction Calyces of roselle (Hibiscus sabdariffa L.) contain cyanidin and delphinidin glycosides1) and have been used for making jelly, jams, beverages and food colorants2). Cultured roselle cells might potentially be a suitable source for large scale production of anthocyanin pigments. In a previous paper3) we reported that callus tissues derived from seedlings of roselle could accumulate anthocyanin pigments tentatively identified as cyanidin-3-monoglucoside (major pigment) and cyanidin-3-xylosylglucoside. Anthocyanin formation in the callus markedly enhanced by 2, 4-D and inhibited by gibberelic acid. The present paper describes the effects of macronutrients on cell growth and anthocyanin production of roselle callus as well as an optimized growth and production medium based on the results obtained in this study. Materials and Methods Plant material and culture method. Callus tissues derived from seedlings of roselle were subcultur-ed at 1-month intervals on Linsmaier and Skoog (LS) basal agar medium4) supplemented with l p M 2, 4-D and l p M kinetin at 25 under 3, 000 lux illumination (16 hr/day). For investigating the influence of various macronutrients, callus tissues (ca 0.2 g) were transferred onto 20 ml of test medium in 50 ml Erlenmeyer flasks and incubated at 25 under illumination for four weeks before harvest. All the test media contained both 2, 4-D and kinetin at 1 uM level. Extraction and quantitative analysis of anthocyanin. Fresh callus tissues were homogenized with 1% methanolic HCl in a mixer. The homogenate was allowed to stand overnight at 4 and then filtered. Absorbance of the filtrate was measured at 530 nm and the anthocyanin content was calculated as a percentage of fresh weight of callus using the molecular extinction coefficient (log E 4.47) for cyanidin-3-monoglucoside5). Results and Discussion Effects of carbon source Although sucrose as a carbon source can support both growth and secondary metabolite production in callus and suspension cultures of plant cells, other carbon sources can also be effective. The effects of different carbon sources at 3% concentration on the growth and anthocyanin production of roselle callus are shown in Fig, 1. As regards anthocyanin formation glucose was as effective as sucrose, whereas fructose and maltose could support anthocyanin formation to a limited extent. In contrast, sucrose was most effective for cell growth; glucose, fructose and maltose were much inferior to sucrose. Other carbon sources such as xylose, galactose, mannose and lactose could support neither cell growth nor anthocyanin formation. Increase in the sucrose concentration above the 2-3% normally used in the basal medium has been reported to enhance the production of various phenolic compounds, including anthocyanin6-8). The optimal concentration of sucrose for the growth and anthocyanin production of roselle callus cultures was examined by varying the sucrose concentration from 1% to 8% in LS medium. Average callus fresh weight per flask was highest with 3% sucrose concentration. Above or below this concentration cell growth tended to decrease rather markedly. In contrast, the anthocyanin content was highest with 3-5% sucrose concentrations (data not shown). Therefore, the optimal