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.
Highlights
Tropane alkaloid biosynthesis in Hyoscyamus niger begins with the methylation of putrescine to N-methylputrescine by putrescine N-methyltransferase (PMT) as putrescine is the common precursor of tropane alkaloids [1]
The best response for callus formation with Green Compact Nodular Callus (GCNC) was obtained from leaf explants followed by hypocotyl; no growth was recorded in case of stem and cotyledons
Our study showed a maximum increase of 10.3 and 4.8 fold in the expression of hyoscyamine 6β-hydroxylase (H6H) and PMT, respectively, at 0.03% Ethyl Methane Sulfonate (EMS) to control, suggesting that EMS at low concentration leads to an increase in expression, whereas the decrease in expression is seen with a higher concentration of EMS
Summary
Tropane alkaloid biosynthesis in Hyoscyamus niger begins with the methylation of putrescine to N-methylputrescine by putrescine N-methyltransferase (PMT) as putrescine is the common precursor of tropane alkaloids [1]. Scopolamine (6,7-β-epoxide of hyoscyamine) being the final product of this pathway, is formed from hyoscyamine using 6β-hydroxy hyoscyamine followed by intermolecular epoxide formation catalyzed by hyoscyamine 6β-hydroxylase (H6H) [2]. The cDNA of which has been cloned from several tropane alkaloid-containing plants such. Mutational analysis of tropane alkaloid biosynthetic pathway in Hyoscyamus niger L. was performed at Scigenom Laboratory Kochin, India
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