Anthocyanin biosynthesis in roselle (Hibiscus sabdariffa L.): in vitro culture establishment and isolation and characterization of anthocyanidin synthase gene

Hibiscus sabdariffa var. sabdariffa (Malvaceae), a shrub locally known as roselle has been valued for its vibrant red colored calyces that are used as food colorant and health drink. Its anthocyanin content has been known to have health promoting effects like antioxidant activity, antimicrobial and anti-cancer, among others. With its several uses, roselle is considered as one plant source with high potential in the world market. Therefore, there is a need to understand the mechanisms involved in anthocyanin production to address any limitations in meeting consumers' demand. Anthocyanin profiling, tissue culture establishment and molecular analysis of authocyanidin synthase gene were done to understand the factors that affect anthocyanin production. TLC of the extracts of freeze dried calyses detected the presence of monoglucosides: delphinidin and cyanidin (Rf=0.35). Delphinidin (758.09 ppm) in the freeze dried roselle calyzx and cyanidin (38.30 ppm) in the oven dried were detected using HPLC. Absorption maxima was obtained at 516-518 mm at pH 2. In terms of plant sample preparation, freeze drying kept the stability of calyx anthocyanins better than oven dying. Decreasing the pH of the milieu kept anthocyanin integrity. Callus induction was done best using explants of aseptically grown seedling of the Thailand accession grown on MS medium with 1ppm 2, 4-D and 2ppm kinetin. Callus formation and emphemeral anthocyanin production were observed 2 weeks after inoculation. Anthocyanins elicitation was done using biotic (yeast extract) and abiotic (exposure to UV-C) elicitors UV-C exposure of callus cultures did not affect growth of cells but no anthocyanins were produced. Addition of yeast extract increased the growth rate up to 10-fold (4g/L). Subsequently transferred to a growth medium without yeast, the callus produced anthocyanins 2 weeks after subculturing. Appearance of anthoyanins was only temporary but this proved the callus is competent of anthocyanin elaboration. Yellow calli were also observed after exposure to yeast extract. TLC profile showed the presence of cholorogenic acids which are possible precursors of anthocyanins. Characterization of anthocyanidin synthase (ANS) gene was done from a partial sequence derived from the roselle callus cultures. This was made up of 345 bp, translated to 115 amino acids. The partial sequence showed significant similarities in ANS gene nucleotide and animo acid sequences to Gossypium hirsutum (84%) and Theobroma cacao (84%). Based on the alignment of amino acid sequences, the partial ANS gene showed homology to the members of the family of 2-oxoglutarate-dependent oxygenases. The information gathered in this study can be used in the improvement of anthocyanin production of roselle in vitro and in the possible molecular modification of the ANS gene for the promotion of its expression.