Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation

Abstract

In vitro suspension culture techniques are cost effective for large-scale production of secondary metabolites. In the present study, firstly, suspension cultures of untransformed Lactuca sativa were prepared using different hormonal combinations and were subjected to different pH, temperature and salt concentrations. Maximum biomass was obtained for suspensions supplemented with 1.5 mg/L BAP and 0.1 mg/L NAA, at pH 5.8, temperature 28 °C and 0 mM NaCl concentration. Using these parameters, suspensions were produced for rol ABC- and rol C-transformed lines of L. sativa. All the transgenic lines showed prominent increase in fresh weight (FW) and dry weight (DW) with maximum values for rol ABC2 line producing 169.8 mg/mL FW and 25.3 mg/mL DW. The exudates of transformed and untransformed plants were tested for the antioxidant activity and in vivo assays on rats. Maximum phenolic content (261 μg/mL) and flavonoid content (637.6 μg/mL) were obtained for rol C1 transgenic line. Total antioxidant capacity was found maximum (1451.7 μg/mL) for untransformed lettuce, whereas rol C1 showed maximum total reducing power activity (637.6 μg/mL). In DPPH assay, maximum activity (104.7 μg/mL) was shown by rol ABC3 line. In rats analgesic assay, maximum activity (74.9%) was shown by rol C2. Line rol C1 showed maximum anti-inflammatory activity (69.2%) and maximum antidepressant activity (minimum immobility time of 55 s). Maximum anticoagulant activity was observed for rol ABC2 with maximum clotting time of 130 s. The present study could help in using lettuce suspension culture as platform for the enhanced production of important metabolites.

Keywords: Antioxidants; Cell suspension; Lactuca sativa; Rats; Rol genes; Transgenic plants.

Fig. 1

Callus and cell suspension culture of Lactuca sativa cv. Grand Rapids. a Fresh biomass callus of Lactuca sativa, scale bar = 1 cm, b fresh weight vs time graph of callus (values are mean ± SEM), c cell suspension in liquid MS medium supplemented with 5 mg/L BAP and 2 mg/L NAA, scale bar = 1 cm, d Microscopic view of suspension cells under light microscope, scale bar = 100 µm, e Growth kinetics of fresh weight of transformed and untransformed (unT) exudates and f Growth kinetics of dry weight of transformed and untransformed (unT) exudates

Fig. 2

Results of total phenolic contents (TPC) of Lactuca sativa exudates. Where unT is untransformed exudates and Liq-MS is simple liquid MS supplemented with BAP (1.5 mg/L) and NAA (0.1 mg/L). Values are expressed in mean ± SEM and are *p < 0.05, **p < 0.01 statistically significant

Fig. 3

Results of total flavonoids contents (TFC) of Lactuca sativa exudates. Where unT is untransformed exudates and Liq-MS is simple liquid MS supplemented with BAP (1.5 mg/L) and NAA (0.1 mg/L). Values are expressed in mean ± SEM and are *p < 0.05, **p < 0.01 statistically significant

Fig. 4

Results of total reducing power (TRP) of Lactuca sativa exudates. Where unT is untransformed exudates and Liq-MS is simple liquid MS supplemented with BAP (1.5 mg/L) and NAA (0.1 mg/L). Values are expressed in mean ± SEM and are *p < 0.05, **p < 0.01 statistically significant

Fig. 5

Results of total antioxidant capacity (TAC) of Lactuca sativa exudates. Where unT is untransformed exudates and Liq-MS is simple liquid MS supplemented with BAP (1.5 mg/L) and NAA (0.1 mg/L). Values are expressed in mean ± SEM and are *p < 0.05, **p < 0.01 statistically significant

Fig. 6

Results of DPPH scavenging assay of Lactuca sativa exudates. Where unT is untransformed exudates and Liq-MS is simple liquid MS supplemented with BAP (1.5 mg/L) and NAA (0.1 mg/L). Values are expressed in mean ± SEM and are *p < 0.05, **p < 0.01 statistically significant

Fig. 7

Results of analgesic assay of Lactuca sativa exudates in comparison of transformed and untransformed plants (unT) with the oral dosage of 10 ml/Kg of rat body weight. Values are expressed in mean ± SEM. *p < 0.05, **p < 0.01 statistically significant as compared to control group

Fig. 8

Results of anti-inflammatory assay of Lactuca sativa exudates in comparison of transformed and untransformed plants (unT) with the oral dosage of 10 ml/Kg of rat body weight. Values are expressed in mean ± SEM. *p < 0.05, **p < 0.01 statistically significant as compared to control group where PC is standard drug Diclofenac potassium

Fig. 9

Results of anti-depressant assay of Lactuca sativa exudates in comparison of transformed and untransformed plants (unT) with the oral dosage of 10 ml/Kg of rat body weight. Values are expressed in mean ± SEM. *p < 0.05, **p < 0.01 statistically significant as compared to control group where PC is standard drug Fluoxetine HCl

Fig. 10

Results of anti-coagulant assay of Lactuca sativa exudates in comparison of transformed and untransformed plants (unT) with the oral dosage of 10 ml/Kg of rat body weight. Values are expressed in mean ± SEM. *p < 0.05, **p < 0.01 statistically significant as compared to control group