Evaluation of somatic embryogenesis potential of citrus callus lines overexpressing Csi-miR390 and Csi-miR156

• 1、Key Laboratory of Horticultural Plant Biology (Ministry of Education),Huazhong Agricultural University,Wuhan 430070

Abstract：In order to validate the potential role of Csi-MIR390 and Csi-MIR156 on citrus somatic embryogenesis, we cloned the precursors of Csi-MIR390 and Csi-MIR156 from embryogenic callus of Valencia sweet orange, which maintains strong embryogenesis capacity, and constructed overexpression vectors out of the precursors, which were then transformed to embryogenic callus of Satsuma mandarin 'Guoqing No.1' (Citrus unshiu) that had lost embryogenesis capacity. In two of the Csi-MIR156 overexpressing lines, the two SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) target transcription factors are down-regulated at different levels; while the only one target gene of csi-miR390, which is annotated as an 'unknown' gene, has no expression level difference in overexpressing lines, compared to wild type or empty vector transformants. Overexpressing Csi-MIR156 and Csi-MIR390 callus lines are unable to produce somatic embryos after being moved onto induction medium, however, they have slower growth rate relative to the wild type or empty vector transformants. By Transmission Electron Microscope (TEM) observation, we found that overexpressing Csi-MIR156 and Csi-MIR39 callus cells showed the tendency to differentiate, instead of the proliferation growth typical of callus; and the starch content in two of the Csi-MIR156 overexpressing callus lines were elevated significantly. These results suggest that miR390 and miR156 play certain roles in citrus somatic embryogenesis.

Keywords： citrus 'Guoqing NO.1' callus microRNA Csi-miR390 Csi-miR156 somatic embryogenesis