Reporter genes using the firefly luciferase coding region and the promoter of either the Bz1 or A1 structural genes were also tested for transactivation by the B-Peru or B-I genes in C1 r b aleurones or embryos. As an internal control for the efficiency of gene transfer a plasmid containing the Adh1 promoter driving the chloramphenicol acetyltransferase (CAT) coding region was included in each DNA sample used for bombardment. The values of B-Peru or B-I transactivation of Bz1- or A1-luciferase are therefore expressed as a ratio of luciferase enzymatic activity to the CAT enzymatic activity generated from the internal control plasmid. Co-bombardment of the Bz1-luciferase reporter construct with the B-Peru gene, or an expressed B-Peru or B-I cDNA resulted in an 80 to 130-fold increase in the ratio of luciferase to CAT activity relative to tissues receiving the reporter plasmids alone. Aleurones and embryos receiving the A1-luciferase reporter plasmid and a B-I expression plasmid contained a 15 to 50 fold higher ratio of luciferase to CAT activity than did tissues receiving only the reporter plasmid alone. The magnitude of induction of the A1 promoter appears lower than that of the Bz1 promoter due to higher basal activity of the A1 promoter. The induced levels of luciferase/CAT from the A1 and Bz1 promoters are comparable.

In an attempt to extend the above described system to more easily obtained tissues, we tested for transactivation of the anthocyanin pathway in embryogenic callus cells and BMS suspension culture cells. Following delivery of either the B-Peru genomic, or an expressed B-Peru or B-I cDNA, no pigmented cells were observed. Since this embryogenic callus was derived from r c1 embryos, we determined the effect of delivering an expressed C1 cDNA with each of the B plasmids. This combination results in the activation of the anthocyanin biosynthetic pathway and pigmented cells were observed. Unlike either aleurones, embryos, or embryogenic callus cells, BMS suspension culture cells were not observed to contain pigmented cells regardless of the construct or combination of constructs delivered. These results demonstrate that the expression of both the B-Peru (or B-I) and C1 genes is required to activate the anthocyanin biosynthetic pathway in r c1 embryogenic callus.

Transactivation of the Bz1 or A1 promoters was also examined in embryogenic callus and BMS suspension culture cells. The B-Peru genomic or expressed B-Peru or B-I cDNAs alone were insufficient to transactivate either the Bz1 or A1 promoters. Transactivation of the Bz1-luciferase or A1-luciferase constructs required cotransformation of one of the B-Peru or B-I genes with an expressed C1 cDNA. In embryogenic callus a 30 to 190 fold transactivation of the Bz1 promoter and a 15 fold transactivation of the A1 promoter was observed. Thus embryogenic callus appears similar to aleurones and embryos in this transactivation assay. In BMS suspension culture cells however, only a 2 to 10 fold transactivation of the Bz1 promoter was observed. This low level induction of the Bz1 promoter is probably too weak an induction to be observed using the anthocyanin pigmentation assay.

The activation of the Bz1 and A1 promoters or the genes for the entire anthocyanin biosynthetic pathway in embryogenic callus tissue indicates that this system is a valid alternative to the time-consuming process of isolating aleurones and/or embryos.

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