In vitro mutagenesis with special reference to alteration of pmt and h6h genes of tropane alkaloid biosynthetic pathway in Hyoscyamus niger L

Abstract

newlineIn vitro mutagenesis is a potent technique to produce genetic variability in medicinal newlineplants and is a significant tool to create variability in a short period of time and has newlinebeen utilized in improving plant yield, its architecture and secondary metabolite newlinecontent. Hyoscyamus niger L. contains scopolamine and hyoscyamine which are newlinepharmaceutically important tropane alkaloids and extensively used as an newlineanticholinergic agent and have a strong action on neuroreceptors. Here, we report the newlineoverexpression of genes encoding the rate-limiting upstream enzyme putrescine Nmethyltransferase (pmt) and the downstream enzyme hyoscyamine 6and#946;-hydroxylase newline(h6h) of alkaloid biosynthetic pathway in Hyoscyamus niger which had led to increase newlinein scopolamine and hyoscyamine content due to in vitro mutagenesis using Ethyl newlineMethane Sulfonate. During the tissue culture studies, the best growth response was newlineobserved on MS (TDZ+IBA) with maximum average number of shoots 14.4±0.26 in newlinecallus and 13.2±0.35 in nodal explants. Rooted plantlets derived from in vitro culture newlinewere acclimatized with 86.6% and 90% survival from callus and nodal explants newlinerespectively. newlineBesides normal growth regulators, the effect of EMS was found to have significant newlineimpact on various growth parameters (percent survival, germination (%), shoot number, newlineroot number, shoot and root length, plant height, number of primary branches and seed newlineyield) under in vitro conditions as well as in M1 and M2 generation and also on genes newlineencoding secondary metabolites (pmt and h6h). The mutational analysis was detected newlinethrough gene sequencing among the variants and their parents. 0.03% EMS was seen to newlinebe effectively enhancing the average shoot number 14.4±0.30 to 22.2 ±0.77 and root newlinenumber from 8.5±0.31 to 12.8±0.53 in callus explants. 0.04 % EMS was effective in newlineterms of enhancing the shoot number upto 20.1±0.35 in comparison to control newline(13.2±0.25) and root number upto 13.2±0.47 in comparison to control (8.8±0.32) from newlinenodal explants under in vitro conditions. LD50 .....