Selection Of High Production Roselle Lines And Enhancement Of The Product Accumulation

Callus of roselle (Hibiscus sabdariffa L.) in its initial stage was yellow and did not produced anthocyanin. At 25℃, 27.3 umol/s ? m² , 16h/d light regime, the callus was cultivated on B₅ medium (pH5.8) containing lmg/L 2,4 — D and 0.2mg/L KT. Afer sveral generations, red"pigment areas" which were suitable for selecting were appear. Observation of Hibiscus sabdariffa cells under an optical microscope revealed that the occurence of pigment vesicles (anthocyanoplasts) which were orange , reddish orange , pink , red and purple both in the cytoplasm and in the vacuole. In some cases, anthocyanoplasts of purple color even observed in cells with nonpigment vacuoles, thus supporting the thesis according to which anthocyanoplasts would be the site of anthocyanin sythesis, then anthocyanin released into the vacuole.High production anthocyanin roselle cell lines were selected by both single cell clone plate culture and small-cell-aggregate culture. The cell suspension cultures for 8days were most favourable for the formation of cell clones. When PH value was 5.84 as well as CaCl₂ ? 2H₂ O concentration was 750mg/L in B5 medium, the platingefficiency (PE) was intensively increased.There was a lower PE when the cell plating density was no more than 6 ×10 cell/ml. Homogenous and dark color small-cell-aggregates were selected in high-content pigment areas of roselle callus and cultivated in B₅ medium (pH 5.84; CaCl₂ ? 2H₂ O: 750mg/L). Both red cell lines and cellaggregates selected from cultures were subcultured successively for 10 generations. The anthocyanin content in high production cell line was 2.33 % (g/g dw), as high as15.5 times of that of control.When pigment cells of roselle suspension cultures were grown in B₅ medium for 13 days, best production of anthocyanin which as high as 0.25g/L was obtained. The anthocyanin yield of pigmented suspension cultured cells was signifficantly increased if adding 10^-6 mol/L exogenous L-Phe into B₅ medium. When 10^-7 mol/L quercetin was supplied into the medium, the pigment yield was increased 1-3 fold of that of control. Either L-Phe or querectin feeding did not initate anthocyanin accumulation of yellow roselle callus.