Functional Investigation Of Somatic Embryogenesis Related CSI-miR390 And 156 In Calluses Of Satsuma Mandarin ’Guoqing No.1’

Somatic Embryogenesis (SE) is the capacity of plant somatic cells to form embryos by a process resembling zygotic embryogenesis. As such, it is used as a model system for study on embryo development. Embryogenic callus, as an ideal material for in vitro palnt regeneration, could fullfill the process of SE and then develop into whole plant. It, thus, represents a best example for the concept of cellular totipotency, and could be widely used in genetic transformation, germplasm creation and cultivar improvement. However, the embryogenic callus would gradually lose their embryogenic capacity as preservation period prolonged. In citrus, one of such typical examples is the callus of Satsuma mandarin’Guoqing No.1’. Consequently, it is necessary to study the mechanism of citrus somatic embryogenesis. Recently, a series set of microRNAs (miRNAs) were cloned from rice embryogenic callus, including some tissue specific miRNAs. Additionally, our previous study demonstrated that Csi-miR390 and 156 might exert regulatory function during the somatic embryo induction process. In the present investigation, overexpression plasmids carrying Csi-miR390 and 156 genes were transformed into calluses of Satsuma mandarin’Guoqing No.1’, the main results are as follows:1. The Csi-MIR390 and 156 DNA sequences were cloned from embryogenic callus of Valencia sweet orange, which maintains strong somatic embryogenesis capacity after preservation for 27 years. Blast results showed that these pre-miRNA sequences have no similiarities to those of corresponding DNA isolated from Arabidopsis, while they produced the same mature miRNAs.2. Overexpression vectors containing Csi-MIR390 and 156 genes were constructed respectively and then transformed into calluses of Satsuma mandarin’Guoqing No.1’by Agrobacterium-mediated transformation independently. As a result,23 different tranagenic lines including 6 OX-C390 lines,10 OX-C156 lines and 7 empty vector control lines were obtained by GUS and PCR analysis.3. For expression analysis, quantitative Real-Time PCR (qRT-PCR) analysis combined with stem-loop qRT-PCR were performed to screen the transgenic lines, from which 3 Csi-miR390 and 2 Csi-miR 156 overexpression lines were identified. 4. In the transformed calluses of Satsuma mandarin’Guoqing No.1’, no significant phenotypic changes were observed, even though in four SE induction mediums added with different carbon source. However, the ultrastructural observation showed that the transgenic lines exhibited similar subcelluler characteristics to those previously described in the embryogenic callus. These results indicated that Csi-miR390 and 156 may exert positive function in citrus somatic embryogenesis capacity recovery.