Anthocyanins are major water-soluble and dynamic colouring plant pigment present in plant tissues with the high antioxidant properties. The role of ammonium and potassium nitrate in the culture medium on anthocyanin augmentation is probed thoroughly, but the mechanism of its biosynthesis continues to be unclear. Hence, the present study was undertaken to optimise nitrate ratio in the culture medium for anthocyanin augmentation and examination of its biosynthesis pathway in callus culture of Daucus carota. MS basal medium fortified with various ratio of NH4NO3:KNO3 was employed to find their impact on biomass, anthocyanin augmentation and the expression profile of anthocyanin biosynthesis genes in the callus culture. The data indicated that the highest anthocyanin content (9.30 ± 0.25 mg/100 g FW) was seen in callus grown on the medium supplemented with 20.0 mM NH4NO3:37.6 mM KNO3 and the least was seen in the medium which contained 40.0 mM NH4NO3:18.8 mM KNO3 (2.74 ± 0.27 mg/100 g FW). This indicates an optimal concentration of NH4NO3:KNO3 ratio is essential to produce a higher amount of anthocyanin in in vitro culture. Meanwhile, anthocyanin biosynthesis genes were differentially expressed as confirmed by qRT-PCR in the time interval of 5, 10, 15, 20 and 25 days. The transcript levels of nine anthocyanin biosynthesis genes were increased in the response of varying NH4NO3:KNO3 ratio in the medium. The transcript level of early genes PAL, 4CL, CHS and CHI increased by 19.5, 21.0, 16.2 and 9.98-fold, respectively, compared with control. In addition, late biosynthesis genes LDOX and UFGT resulted in the transcript level of 11.3 and 13.6-fold, respectively.
Keywords: 4-Coumaroyl:CoA-ligase; Cyanidin 3-glucoside; Gene expression; Nitrate ratio; qRT-PCR.