The first 2000 bp of the biparental LsAPRR2 promoter were cloned (Supplementary Table S1) and subsequently ligated into an expression vector (pBI121) containing a β-Glucuronidase (GUS) reporter gene (Li et al., 2022 (link)). The Agrobacterium tumefaciens GV3101 strain was transformed with the fusion reporter vectors as well as the empty vector. Transformed Agrobacterium cell suspension was injected into tobacco leaves and samples were immersed in X-Gluc buffer 7 days after injection (12 mM potassium ferricyanide, 0.3% (v/v) Triton X-100, 12 mM potassium ferrocyanide and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-glucuronide). The buffer was allowed to penetrate the samples under vacuum, stained overnight at 37°C, decolorized by several washes in 75% (v/v) ethanol, and photographed after complete decolorization of the chlorophyll (Koo et al., 2007 (link)).
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