Tobacco (Nicotiana tabacum) Leaf Disc Transformation with a Maize-Derived Reporter Gene

Jernigan, Hannah
Bergey, D. R.
Tobacco is a fast-growing viscid plant that is a model species for genetic transformation. Gene manipulation is an expanding field for both research and commercial purposes. Agrobacterium tumefaciens is a soil-borne bacterium that is capable of transferring part of its DNA, via plant wound sites, into plant cells. This transferred DNA is incorporated into the plant genome and expressed by the plant. Modified strains of A. tumefaciens are used to transfer traits of interest into plants. The goal of this study was to insert a maize-derived transcription factor and evaluate its efficiency as a reporter gene in tobacco. All procedures were completed using sterile techniques in laminar air flow hoods. Tobacco leaves were excised from seedlings growing in tissue culture and wounded using tweezers. The leaves were then suspended in a solution of A. tumefaciens carrying a maize regulatory gene (LC) that overexpresses the production of anthocyanin pigments. After co-cultivating bacteria and wounded tobacco leaves for 10 minutes, the leaves were put on callus induction media containing no antibiotics, and cultivated in darkness for 3 days. After one week leaves were transferred to new media containing antibiotics for inhibiting bacterial growth and selection of transformed cells. Red pigmentation was observed in cells of tobacco leaf discs co-cultivated with the LC gene. Within two weeks transgenic red shoots interspersed with non-transformed green color shoots emerged. Subsequent studies include regenerating transgenic plant lines, examining leaf surfaces with scanning electron microscopy (SEM), and using HPLC to analyze anthocyanin composition of transgenic tobacco plants.
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