In vitro selection of elite clone of Withania somnifera against leaf blight disease caused by Alternaria alternata

In vitro selection of Withania somnifera has been incurred against Alternaria alternata by selection pressure of fungal toxin in an innovative way. Solid green callus was maintained in Murashige and Skoog's medium sup-plemented with 6-Benzylaminopurine (2 mgL(-1)). Toxin was partially purified from A. alternata broth culture. Elite clone was generated by gradual intoxication of solid green callus in the toxin containing medium (5-400 mgL(-1)). Three highest dose of toxin tolerance was selected viz. 50 mgL(-1), 100 mgL(-1) and 200 mgL(-1). Plants were successfully regenerated from those toxin selected callus. Eventually, various defense enzymes and total phenolics were measured in regenerated plants and toxin selected callus. Callus selected from 100 mgL(-1) toxin dose and plants obtained from them give the best results among all and showed more than 2-5 fold increase of defense enzyme activities and total phenolics compared to control. Furthermore, those plants when challenge inoculated showed significant reduction (more than 70%) of leaf spot incidence. Augmentation of disease resistance persists for at least two successive generations. Furthermore, the increase in defense molecules also correlated with increased nitric oxide generation (73%). Presence of withaferin A and withanolide A has also been confirmed in the toxin regenerated plants by High-performance thin-layer chromatography (HPTLC) analysis. Over all observations suggests that fungal toxin at a concentration 100 mgL(-1) and by habituation technique can be use as a selection method for the production of disease resistant medicinally important crop like W. somnifera without altering its secondary metabolite production.