Multi-Elemental Profiling of In Vitro Calliclones vis-à-vis In Vitro and In Vivo Leaf Samples of a Medicinal Herb Hybanthus enneaspermus (L.) F. Muell. Using Energy Dispersive X-Ray Fluorescence Technique

The multi-elemental compositions of leaf tissues of In Vitro regenerated plants and leaf derived callus of Hybanthus enneaspermus, a medicinal herb were compared with leaf samples of In Vivo grown plants of the same species using EDXRF. Trace elements namely Cr, Mn, Fe, Co, Ni, Cu and Zn in addition to three macro-elements K, Ca and Na were identified and quantified in all the three sources. Multiple shoot organogenesis was achieved in mature leaf derived callus of H. enneaspermus. About 93.33% of leaf explants produced green, compact, nodular organogenic callus in Murashige and Skoog (1962) medium (MS) containing 3.0 mg/l α-naphthaleneacetic acid (NAA) in 3 wk following culture initiation. Proliferated calli were transferred to regeneration media with varying concentrations and combinations of plant growth regulators, augmented to MS. N⁶-benzyladenine (BA) (0.25 mg/l)+NAA (0.1 mg/l) was the most effective, where 100% shoot regeneration frequency was achieved with a highest shoot number of 235 per callus derived from single explant. Best rooting was obtained in the medium containing 1.0 mg/l IBA. Rooted plants were acclimatized and subsequently established in the field with 100% survival. Analysis of inorganic elements by atomic absorption spectrometry revealed a close correspondence between leaf tissues of donor and In Vitro regenerated plants vis-à-vis leaf calli with respect to Na, K, Ca, Mg, Mn, Zn, Fe, Cr, Ni, Co, Cu, Pb, Cd and As.