Medium composition potentially regulates the anthocyanin production from suspension culture of Daucus carota

Abstract

In the present study, an effort has been made to optimize various culture conditions for enhanced production of anthocyanin. Nutrient content of MS medium (ammonium to potassium nitrate ratio and phosphate concentration) had a profound influence on the cell biomass and anthocyanin accumulation in cell suspension cultures of Daucus carota. Suspension cultures were carried out in shake flasks for 18 days and examined for cell growth, anthocyanin synthesis, anthocyanin yield and development of pigmented cells in relation to the uptake of total sugar, extracellular phosphate, nitrate and ammonia. The addition of NH₄NO₃ to KNO₃ ratio (20.0 mM: 37.6 mM) in the suspension culture media resulted in a 2.85-fold increase in anthocyanin content at day 3. Similarly, a lower concentration of KH₂PO₄ (0.45 mM) in the MS medium resulted in 1.63-fold increase in anthocyanin content at day 9. The total sugar uptake was closely associated with a significant increase in anthocyanin accumulation. Total sugar and nitrate were consumed until 9-12 days, while ammonia and phosphate were completely consumed within 3 days after inoculation. After 9 days, cell lysis was observed and resulted in the leakage of intracellular substances. These observations suggest that anthocyanin was synthesized only by viable pigmented cells and degraded rapidly after cell death and lysis. This study signifies the utility of D. carota suspension culture for further up-scaling studies of anthocyanin.

Keywords: Ammonium nitrate; Anthocyanin; Nutrient; Phosphate; Potassium nitrate; Suspension culture.

Fig. 1

a Establishment of Daucus carota seedlings on MS medium (bar = 8 cm). b Callus induction in the medium supplemented with BAP and NAA (bar = 5 cm). c Pigment production in the medium with IAA and Kin (bar = 5 cm) and d Suspension culture (bar = 8 cm)

Fig. 2

HPLC chromatogram of anthocyanin profile a Cyanidin 3-Glucoside b Daucus carota suspension culture

Fig. 3

Growth kinetics of Daucus carota cell suspensions cultured in MS medium with IAA (11.41 µM), Kin (0.93 µM) and 3% sucrose. a Fresh cell weight and dry cell weight. b Total anthocyanin content and c Total anthocyanin yield. The data shown are means of three replications ± standard deviation. Means marked with different letters are significant different according to one-way ANOVA at P < 0.05 (Tukey’s test)

Fig. 4

Effect of the ratio of ammonium nitrate to potassium nitrate on growth and anthocyanin accumulation in suspension cultures of Daucus carota. a Fresh cell weight. b Total anthocyanin content and c Total anthocyanin yield. The data shown are means of three replications ± standard deviation. Results were analyzed by two-way ANOVA, followed by Tukey’s test. *Denotes P < 0.001 as compared to the other groups, ^#denotes P < 0.05 as compared to control

Fig. 5

Effect of phosphate concentrations on growth and anthocyanin accumulation in the suspension cultures of Daucus carota. a Fresh cell weight; b Total anthocyanin content and c Total anthocyanin yield. Each treatment represents the mean of three replications ± standard deviation Results were analyzed by two-way ANOVA, followed by Tukey’s test. *Denotes P < 0.001 as compared to the other groups

Fig. 6

Nutrient depletion during a culture period of 18 days of a MS suspension culture of Daucus carota containing different concentration of NH₄NO₃ and KNO₃. a Total sugar. b External ammonium. c External nitrate and d External phosphate

Fig. 7

Nutrient depletion during a culture period of 18 days of MS suspension culture of Daucus carota containing different concentrations of phosphate. a Total sugar. b External ammonium. c External nitrate and d External phosphate