Factors affecting the anthocyanin production in callus culture of roselle (Hibiscus sabdariffa L.)

Calli derived from hypocotyl segments of roselle seedling (Hibiscus sabdariffa L.) were brought to study on factors affecting the anthocyanin production. The initiated calli were obtained from modified Linsmaier and Skoog (1965) medium with 2, 4-D/kinetin at the ratio of 1:1, 5:1 and 10:1. The calli were cultured on the same medium supplemented with 0, 1, 5 and 10 micro mole/l 2, 4-D in combination with 1 micro mole/l kinetin and at 25+-2 deg C under white light for 16 hrs/day. After 16 weeks, the highest amount of 80 percent anthocyanous calli was obtained at 1 micro mole/l 2, 4-D. The calli originally induced by 2, 4-D/kinetin at the ratio of 5:1 and 10:1 yield non significant, however, the ratio at 5:1 gave better growth rate and more red pigment. Red calli were subcultured to the same medium for further study on effect of cultural period of 0, 1, 2, 3, 4 and 5 weeks under light condition. The result showed that the optimum harvesting period for anthocyanin production was 4 weeks with highest yield of 19.39 mg/bottle. Red calli were cultured on the same medium under various light sources of white, blue, gro-lux, red light, and dark condition for 4 weeks. The result showed that the illumination was remarkebly promoted the anthocyanin synthesis while inhibited by dark condition. Gro-lux light promoted callus growth and anthocyanin synthesis with maximum yield of 41.52 mg/bottle. Selected calli were treated with 5 level of the medium pH at 4.7, 5.2, 5.7, 6.2 and 6.7 under gro-lux light. At pH 6.2 gave the maximum callus growth and anthocyanin content with the highest yield of 41.59 mg/bottle. The effect of sucrose concentration on anthocyanin production was examined at 1, 3, 5, 7, and 9 percent under gro-lux light and at pH 6.2. Sucrose at 3 percent markedly promoted callus growth and at 5 percent promoted the highest anthocyanin synthesis with maximum yield of 35.76 mg/bottle. But sucrose concentration at 7 and 9 percent inhibited both callus growth and anthocyanin synthesis.