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Differential expression of anthocyanin biosynthesis genes in Daucus carota callus culture in response to ammonium and potassium nitrate ratio in the culture medium

Saad, Kirti R., Kumar, Gyanendra, Giridhar, Parvatam, Shetty, Nandini Prasad
3 Biotech 2018 v.8 no.10 pp. 431
Daucus carota, ammonium, anthocyanins, antioxidant activity, biomass, biosynthesis, callus, callus culture, culture media, gene expression regulation, genes, potassium nitrate, quantitative polymerase chain reaction, reverse transcriptase polymerase chain reaction, water solubility
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.