Mining And Identification Of Key Genes Involved In Somatic Embryogenesis In Ponkan

Citrus is an important fruit tree in China,and variety improvement is crucial for the development of the citrus industry.However,conventional hybrid breeding faces significant obstacles due to the long juvenile period,polyembryony,and male/female sterility of mandarin.To overcome these barriers,somatic embryogenesis is an important regeneration pathway in biotechnological breeding.Therefore,elucidating the developmental regulatory mechanism of somatic embryogenesis can provide a theoretical and technical basis for improving the efficiency of mandarin regeneration and accelerating biotechnological improvement.In this study,we compared the structural and physiological differences between embryogenic callus(EC)and non-embryogenic callus(NEC)of’ Ponkan’(Citrus reticulata Blanco cv.Ponkan).We identified key regeneration genes through transcriptome sequencing and conducted bioinformatics analysis of the WUSCHEL-related homeobox(WOX)gene family in ’Ponkan’.Finally,we obtained transgenic callus overexpressing CrWOX11 via Agrobacterium-mediated transformation and detected the soluble protein content,H2O2 content,and the activities of peroxidase(POD),superoxide dismutase(SOD),and catalase(CAT)in the transgenic callus.The main research findings are as follows:1.Observation of the microstructure and ultrastructure of the two types of callus revealed that embryogenic callus had denser cytoplasm,larger nuclei,and more organelles compared to non-embryogenic callus,which only had larger vacuoles and minimal cytoplasm and organelles.Additionally,the soluble protein content and activities of three antioxidant enzymes(POD,SOD,and CAT)were significantly higher in embryogenic callus than in non-embryogenic callus,while the level of reactive oxygen species was the opposite.2.Transcriptome sequencing of embryogenic and non-embryogenic callus tissues of pummelo yielded a total of 269.9 million sequencing reads,of which 259.6 million reads were obtained after filtering and alignment.Using a log absolute value≥1 as the threshold,a total of 9076 differentially expressed genes(DEGs)were identified,among which 6036 were up-regulated and 3040 were down-regulated.Eight GO terms were significantly enriched and 1798 DEGs were enriched in 117 KEGG metabolic pathways.Expression verification of 9 genes related to somatic embryogenesis by qRT-PCR showed results consistent with the transcriptome data..3.Using the BLAST and conserved domain identification methods,a total of 11 WOX genes were identified in the mandarin genome,predicted to be located in the cell nucleus.The family genes contain 2-4 exons,1-3 introns,and 2-7 motifs.The results of the systematic evolutionary tree analysis show that the 11 mandarin WOX gene family members and 15 Arabidopsis WOX gene family members are divided into three subgroups.The promoter region contains 27 cis-acting elements.Among the 11 WOX genes,only the CrWOX11 gene is expressed exclusively in embryogenic callus tissue.4.The full-length sequence of the pummelo CrWOX11 gene was cloned,which was 795 bp in length and encoded 265 amino acids.To verify the function of CrWOX11,four overexpression callus lines were obtained through Agrobacterium-mediated transformation,among which overexpression callus line 1 exhibited somatic embryogenesis from somatic cells.The soluble protein content and antioxidant enzyme activity of the overexpression CrWOX11 callus lines were significantly higher than those of the wild-type callus(non-embryogenic),while their reactive oxygen species(ROS)levels were significantly lower than those of the wild-type callus.These research findings provide a theoretical and technical basis for further elucidating the application of WOX in efficient germplasm innovation in citrus and other fruit trees via somatic embryogenesis systems.