| Summary: | 碩士 === 國立彰化師範大學 === 生物學系 === 104 === The generation of Doubled haploid (DH) from inheritance of uni-parental chromosomes can remarkably accelerate plant breeding. The DH homozygous parent lines are crucial for vegetable biotechnology to generate a collection of varieties. Cultured pollen gametophyte cells and interspecific crosses are currently two methodologies for generating DH lines. However, it remains a labour-intensive and time-consuming to generate DH plant lines. In Arabidopsis thaliana, DH plants can be easily generated through seeds by manipulating a single centromere gene, the centromere-specific histone CENH3 (Centromere-mediated genome elimination technology). As CENH3 is conserved in plants and thus can be extended to produce DH in most plant species. Generation of DH lines by centromere-mediated genome elimination is genetic-modified free due to elimination of one parent genomes (Centromere-mediated genome elimination inducer). In this study, we employed a rapid-cycling doubled haploid Brassica oleracea TO1000 as a centromere-mediated genome elimination inducer for generating of broccoli DH lines. The BoCENH3 gene was isolated from B. oleracea. Gene expression analysis indicated that BoCENH3 was expressed predominately in proliferating tissues such as flower buds and young leaves. Subcellular localization analysis by fusing BoCENH3 to green fluorescence protein showed nuclear localization. BiFC (Bimolecular fluorescence complementation) assays showed BoCENH3 interacted itself to form aggregation. Future work is in progress to generate centromere-mediated genome elimination inducer (B. oleracea TO1000). Taken together, this study will establish a rapid and cost-effective platform for generating broccoli DH lines, leading to accelerate and improve broccoli breeding.
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