Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis

Abstract

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6β-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 μg/g) and hyoscyamine (0.0344μg/g) compared to untreated.

Fig 1. Effect of different concentrations of growth regulators on the fresh and dry weight of callus of Hyoscyamus niger.

The samples were collected after 6 weeks of incubation period. The data represents the mean of three biological replicates. For each replicate, a total of 10 explants were used. An asterisk represents the significant differences between explants treated with different growth regulators at **P<0.05; **P<0.01; ***P<0.001.

Fig 2. In vitro regeneration of H. niger from callus explants.

(A) Callus formation, (B) Shoot incitation from callus explants (C) Shoot multiplication, (D) Shoot elongation, (E) Rooting (F) Hardening.

Fig 3. Effect of EMS on in vitro regeneration potential of callus of H. niger on MS +TDZ (14μM)+IBA(3.5 μM).

(A) Control (0%), (B) 0.03% EMS, (C) 0.05% EMS, (D) 0.08% EMS, (E) 0.1% EMS.

Fig 4. Effect of EMS on rooting from in vitro raised callus of H. niger L.

(A) Control (0%),(B) 0.03% EMS, (C) 0.05% EMS, (D) 0.1% EMS.

Fig 5. Effect of EMS on callus biomass (fresh weight) from in vitro raised callus of H. niger. L.

The samples were collected after 6 weeks of incubation period. Data indicates the mean of three biological replicates. For each replicate, a total of 10 explants were used. An asterisk represents the significant differences between explants treated with EMS compared to control **P<0.05; **P<0.01; ***P<0.001.

Fig 6

Representative image of DNA extraction and PCR analysis of H6H and PMT gene isolated from EMS treated and control explants of H. niger of different samples (500 and 400bp respectively) of (A) DNA extraction isolated from explants treated with different concentrations of EMS and untreated. (B) Depicts the PCR analysis of H6H (500bp) Gene (C) PCR amplification PMT Gene (400bp).

Fig 7. Multiple sequence alignment of samples generated from PMT gene amplification showing various base pair changes (shown by colour change).

The fasta sequences of samples were aligned with the reference sequence of PMT gene available from NCBI website using MEGA-X software.

Fig 8. Multiple sequence alignment of samples generated from H6H gene amplification showing various base pair changes (shown by color change).

The fasta sequences of samples were aligned with the reference sequence of H6H gene available from NCBI website using MEGA-X software.

Fig 9. Transcript expression analysis of PMT and H6H in Hyoscyamus niger callus treated with different concentrations of EMS mutagen.

(A) Expression analysis of PMT (B) expression analysis of H6H. The column represents the mean of three biological repeats. Asterisks represent the statistically significant differences between EMS treated explants and control (untreated) at *P<0.05; **P<0.01; **P<0.001.

Fig 10. HPLC analysis of hyoscyamine and scopolamine extracted from callus treated with different concentrations of EMS.

(A-F) representative peaks of hyoscyamine and scopolamine content in callus treated with 0.0%, 0.01%, 0.02%, 0.03%, 0.04 and 0.05% EMS, respectively.

Fig 11. Conversion of hyoscyamine to scopolamine.

Schematic representation of scopolamine production from hyosyamine.