Study on mechanisms of specific expression of gibberellin 3-oxidase genes in carrot somatic embryogenesis.

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Aims of this project were (1)detection of gibberellin 3-oxidase-mRNAs (GA3ox-mRNA) in carrot plant, somatic embryo, and zygotic embryo, (2)quantitative (or semi-quantitative) analysis of DcGA3ox-mRNA in both embryos, (3)isolation of 5'-upper regions from those genes for promoter analysis, (4)production of transgenic carrot which was introduced each promoter :: GUS fusion gene, (5)observation of each promoter activity. Carrot (Daucus carota L. cv. US-Harumakigosun) plants and somatic embryos were used as plant materials for these experiments. We isolated three GA3ox-genes named as DcGA3oxl, 2, 3 from carrot somatic embryos. Expression of these genes was dramatically increased in induced somatic embryos if these transcripts were analyzes by semi-quantitative RT-PCR method. qRT-PCR analysis showed these genes were expressed at root, cotyledon and root, respectively in young seedlings. Especially, DcGA3ox3-mRNAs were most abundant mRNA among them in it. Then, amount of DcGA3ox2-mRNA was dr … More astically increased in somatic embryo, and it became higher than that of DcGA3ox3, at least, at heart stage-somatic embryos. Two of those transcripts were detected in epidermal and some endodermis cells at pre-stem region, at least, in torpedo stage-somatic embryos by using in situ hybridization. While, only one transcript was detected at the same region in torpedo stage-zygotic embryos. To be clear why these differences were happened in different embryos, we had introduced promoter :: GUS analysis. At first, more than 2000b of 5'-upper region in these genes were isolated by using genome-walking, and those were fused with GUS gene to produce plasmid-constracts for agrobacterium infection. Transgenic cells were grown up in liquid medium before induction of somatic embryo. Some of positive signals were observed in callus cells and regenerated somatic embryo. However, position of those signals were sometimes different in each embryo. So individual cell cluster has incubated separately to establish each cell-line. Now, next generation of there line are growing in tube. Less