Gene Insertion and Expression of a MybA1 Transcription Factor in Nicotiana tabacum (tobacco)

Jernigan, Paige
Kandel, R.
Bergey, D. R.
Tobacco is a model plant species in genetic transformation studies due to its ease in genetic manipulation. This characteristic makes it an ideal candidate for gene insertion and expression studies. In this study, a grape-derived MybA1 transcription factor that regulates anthocyanin pigment production was used as a reporter gene to study gene insertion and expression in tobacco cultures and plants. Tobacco seeds were surface-sterilized in 50% bleach solution and germinated on MS medium. Agrobacterium harboring the grape MybA1 and nptII genes was used in transformation studies. Leaf discs obtained from germinated seedlings were wounded with tweezers, and then co-cultivated with Agrobacterium, and then transferred to callus induction medium for 3 days in the dark. Co-cultivated leaf discs were then transferred to callus induction medium containing antibiotics 200 µM cefotaxin, 200 µM Carbenicillin, 100 µM Kanamycin for inhibiting bacterial growth and selection of cells containing the inserted genes. Co-cultivated leaf discs on induction medium produced transgenic shoots that exhibited intense red pigmentation along with non-transformed shoots that were green in color. We are currently recording percent transgenic shoot production from transformed leaf discs. Transgenic plants obtained after rooting of shoots will be hardened and transferred to a greenhouse. Scanning electron microscopy will be used to analyze differences in morphology and anatomy between transgenic and non-transformed plants.
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