Overexpression of LrAN2 Enhances Anthocyanin Content in Tobacco

The MYB transcription factor LrAN2 was first reported by our laboratory. It expressed only in the black fruit of Lycium ruthenicum Murry and the allele variation of LrAN2 is strictly related to the black fruit trait [23 (link)]. In this study, LrAN2 was transformed into tobacco Samsun to obtain transgenic lines with high anthocyanin content. The vector PJAM1502 with a double 35 s promoter was used for the transformation. The construct PJAM1502:LrAN2 was established using a Gateway Cloning Kit (Invitrogen, Carlsbad, CA, USA). The freeze-melt method was employed to transform the binary vectors into Agrobacterium tumefaciens strain GV3101. The leaf disc transformation method was used for the tobacco transformation [31 (link)]. Regeneration tissues were grown on selective media (0.7% (w/v) agar, 3% (w/v) sucrose, 1.0 mg/L 1-naphthaleneacetic acid (NAA), 1.0 mg/L 6-benzylaminopurine (6-BA), and 150 mg/L kanamycin. The positive shoots were planted in the greenhouse under long-day lighting (16 h light/8 h dark). Though 23 positive lines were obtained, only three T3 family lines carrying the objective gene without segregation of character were selected for further experiments.