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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (1) 1064-1072 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)Survival Selections Reveal Altered PharmacologicalPhenotypes in Nicotiana tabacum var. SR1 ActivationTagged Mutants1064S. K. Gunjan 1 *, T. Rogers 1,2 , J. Czarnecki 1 , J. Lutz 1 <strong>and</strong> J. Littleton 1,21Kentucky Tobacco Research <strong>and</strong> Development Center, 2 Department <strong>of</strong> Pharmaceutical Sciences, University <strong>of</strong>Kentucky, Lexington, Kentucky 40546-0236, USA*For correspondence - samir.gunjan@uky.eduAbstractThis study demonstrates the utility <strong>of</strong>survival-selection strategies for identifyingnovel secondary metabolic pr<strong>of</strong>iles <strong>and</strong>accompanying pharmacological activity inactivation-tagged mutants generated fromNicotiana tabacum var. SR1. Leaf discs wereinfected by Agrobacterium tumefaciens strain3850 harboring an activation tagging vectorpPCVICEn4HPT in which four copies <strong>of</strong>enhancer sequences are located at the rightborder <strong>of</strong> T-DNA. After Agrobacteriuminfection <strong>and</strong> co-cultivation, the transformedleaf discs were grown on shoot regenerationmedia containing either ethanol (200 mM) or4-methyltryptophan (4-MT, 50 mM). Thebiochemical analysis <strong>of</strong> ethanol resistantmutants showed the presence <strong>of</strong> high level <strong>of</strong>antioxidants in plants. Similarly, the mutantswere selected on 4-MT containing mediarevealed 3-5 fold higher nicotine contentcompared to wild-type control plants. Thesestudies demonstrate the utility <strong>of</strong> survivalselection for identifying individual mutantswith novel pharmacological phenotypes froma large activation-tagged mutant population.Similar selection strategies may be applied toplants <strong>of</strong> pharmaceutical importance to identifynovel phytochemical drug leads, or possiblyimprove yields <strong>of</strong> commercially importantsecondary metabolites.Key words: 4-methyltryptophan, activationtagging mutagenesis, antioxidants, ethanol,Nicotiana tabaccum.IntroductionThe elucidation <strong>of</strong> plant genomicinformation <strong>of</strong>fers tremendous potential forexp<strong>and</strong>ing use <strong>of</strong> plants as drug discovery <strong>and</strong>manufacturing resources. Generation <strong>of</strong> novelplant compounds or increasing concentrations<strong>of</strong> existing economically valuable plantsecondary metabolites is one immediateapplication <strong>of</strong> our increased underst<strong>and</strong>ing <strong>of</strong>plant genomics. One strategy for applying theadvances in our underst<strong>and</strong>ing <strong>of</strong> plantgenomic is to use mutagenesis to producelarge mutant populations followed bymetabolic pr<strong>of</strong>iling to identify novelmetabolites. Walden <strong>and</strong> colleagues developeda directed way to induce gain-<strong>of</strong>-functionmutations in plants (1) that uses four copies<strong>of</strong> enhancer elements from cauliflower mosaicvirus 35S gene cloned into T-DNA. Thisapproach, known as activation tagging, cancause transcriptional activation <strong>of</strong> genes nearits point <strong>of</strong> insertion into a plant genome.When combined with gene rescue techniques,activation tagging can be a powerful tool foridentifying plant mutants <strong>and</strong> correspondinggenes. The functions <strong>of</strong> several genes haveSurvival selection <strong>of</strong> N. tabaccum activation tagged mutants