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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 . 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 NHNO to KNO 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 KHPO (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 suspension culture for further up-scaling studies of anthocyanin.

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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 NH 4 NO 3 :KNO 3 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 NH 4 NO 3 :37.6 mM KNO 3 and the least was seen in the medium which contained 40.0 mM NH 4 NO 3 :18.8 mM KNO 3 (2.74 ± 0.27 mg/100 g FW). This indicates an optimal concentration of NH 4 NO 3 :KNO 3 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 NH 4 NO 3 :KNO 3 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.

Serotonin and melatonin are important signaling and stress mitigating molecules. However, their role and molecular mechanism in the accumulation of isoflavones are not clearly defined. To elucidate their functions, serotonin and melatonin were applied to in vitro cultures of soybean at different concentrations and analyzed to assess the accumulation of isoflavone content followed by transcript levels of biosynthesis genes at different time intervals. Increased total phenolics, total flavonoids, and different forms of isoflavone content were observed in the treatments. Expression levels of critical genes in isoflavone, ethylene, jasmonic acid, abscisic acid, and melatonin biosynthesis and related transcription factor were quantified. A correlation was observed between the expression of ethylene biosynthesis genes (S-adenosylmethionine synthase and 1-aminocyclopropane-1-carboxylate oxidase) and isoflavone biosynthesis genes (chalcone synthase, chalcone reductase, and isoflavone synthase). We hypothesize that, under serotonin and melatonin treatments, ethylene biosynthesis may play a role in the increase/decrease in isoflavone content in soybean culture.

Anthocyanins biosynthesis is a well-studied biosynthesis pathway in Daucus carota. However, the scale-up production at the bioreactor level and transporter involved in accumulation is poorly understood. To increase anthocyanin content and elucidate the molecular mechanism involved in accumulation, we examined D. carota cell culture in flask and bioreactor for 18 days under salt stress (20.0 mM NH4NO3/37.6 mM KNO3) at 3 day intervals. The expression of anthocyanin biosynthesis and putative MATE (multidrug and toxic compound extrusion) transporter expression was analyzed by qRT-PCR. It was observed that there was a significant enhancement of anthocyanin in the bioreactor compared to the control culture. A correlation was observed between the expression of MATE and the anthocyanin biosynthesis genes (CHS, C4H, LDOX, and UFGT) on the 9th day in a bioreactor, where maximum anthocyanin accumulation and expression was detected. We hypothesize the involvement of MATE in transporting anthocyanin to tonoplast in D. carota culture under salt stress.

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Multidrug and toxic compound extrusion (MATE) is a family of membrane transporters that transport ions and secondary metabolites like anthocyanins in plants. Although various research on MATE has been carried out on different plant species, this is the first comprehensive report to exploit the Daucus carota genome to identify the MATE gene family. We investigated the chromosomal distribution, gene duplication, motif identification, cis-element analysis, in vitro cultures, stress treatment, protein docking, and simulation of DcMATEs. Our studies identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from its genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. Further, we quantified anthocyanin content and identified the co-expression of DcMATE21 with anthocyanin biosynthesis genes in in vitro cultures under ABA, SA, MJ, SNP, and PHE stress treatments. In addition, membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket showing extensive H-bond interactions with ten amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. This investigation speculates the involvement of DcMATE21 in anthocyanin accumulation in in vitro cultures of D. carota.