Figure 3. mRNA expression profiles of Os.hsp18, Os.hsp82 and the hptII transgene, driven by the CaMV 35S promoter, in rice var. Senia.

Different stages during transformation with A. tumefaciens, either carrying pCAMBIA1300 (hptII) or no T-DNA (WT), and expression in mature and imbibed embryos, are shown. The following stages of callus are shown: 6 weeks after induction of embryogenic callus; immediately after 15 min infection with A. tumefaciens; after a 3-day incubation in R2CS medium; after a 2-week incubation in R2S selection medium; 3 weeks after transfer to NBS selection medium; after an 8 to 10-day incubation in PR-AG selection medium; 3–4 weeks after induction of regeneration in RN medium; and two weeks after seedling culture in P medium. Embryos extracted from mature seeds, either before or after imbibition in water for two or five hours, were also analyzed. When the hptII DNA cassette was transformed, transgenic cells were selected using hygromycin (asterisk). Three biological replicates, each of five calluses, with two experimental replicates, were analyzed at each stage. The ef1α reference gene (M values <0.5 in these samples) was used for normalization. Dark to light color scale represents high to low expression level. All values were in the 90 to 0.01-fold range. nd: not determined. Striped: underestimated values due to callus samples containing a mixture of transgenic and non-transgenic cells.