Micropropagation ofAtropa acuminataRoyle ex Lindl. (a critically endangered medicinal herb) through root callus and evaluation of genetic fidelity, enzymatic and non-enzymatic antioxidant activity of regenerants

Atropa acuminataRoyle ex Lindl. (family Solanaceae) is a critically endangered therapeutic herb, known for its immense therapeutic potential against innumerable ailments. In view of protecting this species from brink of extinction, present work focuses upon development of an effectual micropropagation protocol via indirect organogenesis using root-derived callus. Best callusing was observed on Murashige and Skoog (MS) medium augmented with 10 mu M Thidiazuron (TDZ), where 99% of root explants induced whitish-green and compact calluses within 45 days. Upon sub-culturing on same medium (10 mu M TDZ), these callus pieces differentiated an average of 5.54 +/- 0.65 shoots having 2.38 +/- 0.37 cm of length in 71.5% of the explants, after 28 days. MS + 1 mu M indole-3-butyric acid (IBA) resulted in optimum rooting response where an average of 8.08 +/- 0.19 roots having an average length of 7.28 +/- 0.11 cm were produced. Micropropagated plants with well-developed and thick roots were acclimatized under greenhouse conditions. Genetic fidelity of ex vitro acclimatized plants using start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) markers, revealed 96% similarity between mother plant and micropropagated plants. These plants showed higher yield of phenols, flavonoids and exhibited better antioxidant (DPPH and phosphomolybdenum assay) activities in comparison with mother plant. Higher levels of photosynthetic pigments and antioxidant enzymes were also observed in these plants as compared to the mother plant. Besides, quantification of atropine and scopolamine using high-performance liquid chromatography (HPLC) in different plant parts of acclimatized plantlets revealed higher amount of both the alkaloids in leaves. The present protocol shall be gainfully employed for large-scale micropropagation, conservation and extraction of biotherapeutic molecules for commercial purposes.