Survival selections reveal altered pharmacological phenotypes in nicotiana tabacum var. SR1 activation tagged mutants

This study demonstrates the utility of survival-selection strategies for identifying novel secondary metabolic profiles and accompanying pharmacological activity in activation-tagged mutants generated from Nicotiana tabacum var. SR1. Leaf discs were infected by Agrobacterium tumefaciens strain 3850 harboring an activation tagging vector pPCVICEn4HPT in which four copies of enhancer sequences are located at the right border of T-DNA. After Agrobacterium infection and co-cultivation, the transformed leaf discs were grown on shoot regeneration media containing either ethanol (200 mM) or 4-methyltryptophan (4-MT, 50 mM). The biochemical analysis of ethanol resistant mutants showed the presence of high level of antioxidants in plants. Similarly, the mutants were selected on 4-MT containing media revealed 3-5 fold higher nicotine content compared to wild-type control plants. These studies demonstrate the utility of survival selection for identifying individual mutants with novel pharmacological phenotypes from a large activation-tagged mutant population. Similar selection strategies may be applied to plants of pharmaceutical importance to identify novel phytochemical drug leads, or possibly improve yields of commercially important secondary metabolites.