Figure 4

Increased GFP expression in transformed cells when selected by a weakly expressed HPT gene controlled by Nos and tCUP1 promoters. A. Images of green fluorescence emitted from callus transformed with HPT-GFP vectors controlled by 35S, Nos and tCUP1 promoters (see Figure 3A) 21 days after the onset of the first round of selection. Single representative calli from two independent transformations are shown under both bright and blue light field. Bar=1 mm. B. HPT mRNA levels in cells transformed with 35S, Nos and tCUP1 promoter controlled HPT-GFP vectors 12 or 15 days after the onset of the second round of selection. Independent secondary resistant calli were collected from 25 primary calli in each of two independent experiments (white and gray bars) for RNA extraction. Relative HPT gene expression levels were analyzed through qRT-PCR by normalizing to the OsActin 1 level as a control. Data are shown as mean ± SD of three technical replicates in each independent experiment. For comparison, the HPT expression in the 35S vector was defined as 100%. C. GFP mRNA levels in the same cells transformed with 35S, Nos and tCUP1 promoter controlled HPT-GFP vectors. For comparison, the GFP expression in 35S vector was defined as 1 fold.