Metabolite Profiles of Arsenic Tolerant Plants Regenerated from Stem Calli of Andrographis paniculata (Burm.f.) Nees using HPLC and ¹D 1H NMR

Abstract

In vitro culture provide a suitable condition for regeneration of arsenic tolerant plants from stem calli of Andrographis paniculata (Burm.f.) Nees. The regenerated plants could be valuable material applicable to soil remediation. In vitro culture of stem explants on MS basal salts, 3.0% sucrose, 0.8% agar medium fortified with various concentrations of As₂O₃ (0.0-9.0 µM) along with NAA (3.5 mg L^-1) and KIN (1.0 mg L^-1) influenced resistant callus formation. Growth of callus was slightly inhibited with increased resistance up to 22% (53±0.8 mg fwt and 27±1.5 mg dwt) on 7.0 µM As₂O₃ selective medium. The resistant callus line inoculated on MS medium supplemented with different concentrations of As₂O₃ along with BA (2.5 mg L^-1) and NAA (3.0 mg L^-1) induced the development of shoots. Shoot organogenesis was slightly inhibited by arsenic metal stress. However the growth tolerance has increased up to 14.5% on medium with 7.0 µM As₂O₃. The rate of adventitious rooting of plantlets was gradually decreased with more tolerance (11.1±1.4 rootlets per plantlets) in 7.0 µM As₂O₃ selected media. After acclimatization, about 40% of plants were survived as arsenic tolerance than control plants in pots containing soil treated with 7.0 µM As₂O₃ solutions. The level of arsenic detectability was 0.96 ppb and 4.67 ppb in control and 7.0 µM As₂O₃ treated plants, respectively by AAS analysis. Moreover, the production of Andrographolide was found quite high (4.41 mg/g) in tolerant plants grown at 7.0 µM As₂O₃ treatment than control by HPLC analysis. 1D ¹H NMR profile revealed the metabolic changes significantly in control and 7.0 µM As₂O₃ treated plant samples. This is the first report confirming the suitability of in vitro selection for obtaining of vigorous and proliferative clones of A. paniculata plants tolerant to elevated arsenic concentration.