Polyamine elicited aristolochic acid production in in vitro clonally fidel Aristolochia indica L.: An ISSR and RAPD markers and HPTLC based study

Abstract

The present investigation was conducted to find out the influence of different polyamines (PAs) viz. spermine (spm), spermidine (spd) and putrescine (put) and plant growth regulators (PGRs) on multiplication, rooting, callusing and regeneration in Aristolochia indica L. in vitro. Firstly, the A. indica plants were collected, surface sterilized and cultured in Schenk and Hildebrandt (SH) media fortified with various PGRs and PAs in different concentrations and combinations. SH media fortified with benzylaminopurine (BAP) (2.0 mg/l)+put (0.5 mM) and in another experiment, BAP (2.0 mg/l)+spd (1 mM) produced best results with 41.00 and 39.2 axillary shoots/ nodal explant respectively. Following 4-6 weeks of incubation, explants propagated in SH media augmented with various combinations and concentrations of BAP and kinetin (kin) (0.5, 1, 1.5 and 2 mg/l), spm, spd and put (0.5 and 1 mM) generated white friable callus. SH media supplemented with BAP (2.0 mg/l)+spd (0.5 mM) displayed best response with an average number of 47.5 base callus derived shoots . Following 6 weeks of incubation, SH+ indole-3-acetic acid (IAA) (1.0 mg/l)+spd (0.5 mM) resulted maximum average number (7) of roots per shoot. The genetic homogeneity of the mother plant (MP), hardened plant (HP) and in vitro regenerated plants was ascertained employing random-amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. In this present study, in randomly selected A. indica plants, by using 6 ISSR and 5 RAPD primers, 63 scorable bands were derived, 59 of which were recorded as monomorphic with a degree of monomorphism of 90%. High-performance thin layer chromatography (HPTLC) revealed aristolochic acid (AA) content in in vitro grown A. indica roots and the roots from the mother plant (MP) as 6074.54 µg/g and 5891.14 µg/g respectively.