Interactive effects of zinc oxide nano particles and different light regimes on growth and silymarin biosynthesis in callus cultures of Silybum marianum L.

Figures & data

Figure 1. Effects of different explants with different plant growth regulators on callus induction frequency. Data represent the mean values of triplicates with ± standard error for each treatment in three repeated experiments. Annotation of columns data with different alphabet (s) represents the significance at (p = .05).

Figure 2. Effects of different levels of Zinc oxide nanoparticles and light regimes on callus biomass formation. Data represent the mean values of triplicates with ± standard error for each treatment in three repeated experiments. Annotation of columns data with different alphabet (s) represents the significance at (p = .05).

Figure 3. Interactive effects of ZnO NPs alone or in combination with PGRs under different light regimes on callus biomass formation in S. marianum. A1–A7= Normal light. A1: 0.05 mg/L ZnO NPs, A2: 0.1 mg/L ZnO NPs, A3: 0.15 mg/L ZnO NPs, A4: 2 mg/L BA + 2 mg/L 2,4-D, A5: 0.05 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A6: 0.1 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A5: 0.15 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D. A8-A14= Diffuse light. A8: 0.05 mg/L ZnO NPs, A9: 0.1 mg/L ZnO NPs, A10: 0.15 mg/L ZnO NPs, A11: 2 mg/L BA + 2 mg/L 2,4-D, A12: 0.05 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A13: 0.1 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A14: 0.15 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D. A15-A21= Complete dark. A15: 0.05 mg/L ZnO NPs, A16: 0.1 mg/L ZnO NPs, A17: 0.15 mg/L ZnO NPs, A18: 2 mg/L BA + 2 mg/L 2,4-D, A19: 0.05 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A20: 0.1 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A21: 0.15 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D. A22-A28= Complete dark followed by normal light. A22: 0.05 mg/L ZnO NPs, A23: 0.1 mg/L ZnO NPs, A24: 0.15 mg/L ZnO NPs, A25: 2 mg/L BA + 2 mg/L 2,4-D, A26: 0.05 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A27: 0.1 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D, A28: 0.15 mg/L ZnO NPs+ 2 mg/L BA + 2 mg/L 2,4-D.

Table 1. Interactive effect of ZnO NPs and light treatments on callus morphology of S. marianum.

Media composition	Culture condition
	Normal Light (2000–2500 l×)	Diffused Light (500–1000 l×)	Complete Dark (0 l×)	Shifting of cultures from complete dark to normal light after two weeks
MS + ZnO NPs (0.05 mg/L)	Yellowish, friable	Light yellowish, friable	Yellowish, friable	Light brown, friable
MS + ZnO NPs (0.10 mg/L)	Yellowish, friable	Light yellowish, friable	Yellowish, friable	Light brown, friable
MS + ZnO NPs (0.15 mg/L)	Yellowish, friable	Light brown, friable	Light brown, friable	Light brown, friable
MS + 2,4 D + BA (2, 2 mg/L)	Brown, compact	Yellowish, compact	Yellowish, Friable	Light brown, compact
MS + ZnO NPs (0.05 mg/L ) + 2,4 D + BA (2, 2 mg/L)	Brown, compact	Yellowish, compact	Yellowish, compact	Dark brown, compact
MS + ZnO NPs (0.10 mg/L) 2,4 D + BA (2, 2 mg/L)	Brown, compact	Yellowish, compact	Brown, compact	Dark brown, compact
MS + ZnO NPs (0.15 mg/L) 2,4 D + BA (2, 2 mg/L)	Brown, compact	Yellowish, compact	Brown, compact	Dark brown, compact

Figure 4. Effects of different levels of Zinc oxide nanoparticles and light regimes on secondary metabolites production in callus cultures. Data represent the mean values of triplicates with ± standard error for each treatment in three repeated experiments. Annotation of columns data with different alphabet (s) represents the significance at (p = .05).

Figure 5. Effects of different levels of Zinc oxide nanoparticles and light regimes on the production of pal activity and silymarin content in callus cultures. Data represent the mean values of triplicates with ± standard error for each treatment in three repeated experiments. Annotation of columns data with different alphabet (s) represents the significance at (p = .05).

Figure 6. HPLC chromatograms of the selected plant samples. (a): standard silymarin, (b): Callus raised in vitro at 0.15 mg/L ZNO NPs under complete dark for two weeks’ period followed by transference to normal light, (c): Callus raised in vitro at 0.5 mg/L ZNO NPs under complete dark, (d): Callus raised in vitro at 0.5 mg/L ZNO NPs under diffused light, (e): Callus raised in vitro at 0.5 mg/L ZNO NPs under normal light, (f): Control treatment under complete dark for two weeks’ period followed by transference to normal light, (g): Callus raised in vitro at 0.15 mg/L ZNO NPs + 2 mg/L BA + 2 mg/L BA under diffused light.