Figure 2. Analyses of transgenic tobacco BY2 calli.

(a) Images of typical transgenic BY2 calli. WT, wild-type; VC, vector control (pIG121-Hm); TYR, 35Sp-LeTYR line no. 3; DOD, 35Sp-MjDOD line no. 3; TYR/DOD, 35Sp-LeTYR/35Sp-MjDOD line no. 3. (b) Northern blot analysis. Total RNAs were isolated from wild-type (WT) and transgenic BY2 including vector control (VC), and three independent transgenic lines 35Sp-LeTYR (TYR), 35Sp-MjDOD (DOD), and 35Sp-LeTYR/35Sp-MjDOD (TYR/DOD). Total RNAs (5 μg) were separated on an agarose gel and then transferred to a nylon membrane and hybridized with LeTYR or MjDOD probes. Ethidium bromide staining of rRNA bands shows quality and loading controls. (c) Tyrosine-hydroxylating activity and accumulation of LeTYR proteins in transgenic BY2 calli. The activity was determined by CE-MS analysis as described in Materials and methods. Western blot analysis was performed using anti-LeTYR antibody²¹.