Fig. 1 Phylogenetic，conserved domain and cis-acting elements analysis of the WOX，GRF，and GIF gene families in citrus A：Maximum-likelihood（ML）phylogenetic trees of the WOX，GRF，and GIF gene families. Cs：Citrus sinensis；At：Arabidopsis thaliana；Os：Oryza sativa. B：cis-Regulatory element analysis of the WOX，GRF，and GIF gene families in sweet orange. The numbers indicate the quantity of cis-acting elements associated with each gene，and darker colors represent a higher abundance of cis-acting elements

Fig. 2 Expression analysis of WOX，GRF，and GIF gene families during somatic embryogenesis and differentiation in sweet orange suspension cells A：Somatic embryogenesis and differentiation of sweet orange（Citrus sinensis）suspension cells. i：Suspension cells；ii：Globular embryos（green arrows），heart-shaped embryos（yellow arrows），torpedo-shaped embryos（blue arrows），and mature embryos（red arrows）；iii：Further differentiation of mature embryos；iv：Shoot regeneration of somatic embryos；v：Four developmental stages used for expression analysis，where stage 1 corresponds to globular or heart-shaped embryos，stage 2 to cotyledonary embryos，stage 3 to mid-to-late embryos，and stage 4 to late-stage embryos with differentiated tissues such as the embryonic radicle. B：Expression dynamics of WOX，GRF，and GIF gene families during somatic embryogenesis. Gene expression levels were normalized to CsACT2 as the internal reference gene，and the expression level in undifferentiated suspension cells was set to 1. Student’s t-test，with ** P < 0.01

Fig. 3 Efficient activation of target gene expression in citrus protoplasts using the CRISPR/dCas9-TV system A：Schematic diagram of the CRISPR/dCas9-TV system vector. Key elements are labeled：35S（Cauliflower mosaic virus 35S promoter）；HygR （Hygromycin resistance）；ZmUbi-1pro（Zea mays ubiquitin gene-1 promoter）；tRNA（Arabidopsis thaliana glycine tRNA scaffold）；sgRNA （Single-guide RNA）；HSP（Heat shock protein terminator）；AtUBQ10pro（Arabidopsis thaliana ubiquitin 10 promoter）；2x flag-tag（Two copies of flag-tag protein）；NLS（Nuclear localization signal）；TAL（Xanthomonas TALE transcriptional activator）；VP64（Herpes simplex virus VP64 transcriptional activation domain）；NOS（Agrobacterium nopaline synthase terminator）；LB/RB（Left border/Right border of the Agrobacterium T-DNA）. B：Schematic of sgRNA design. Two sgRNAs were designed for each citrus gene to target conserved regions within the promoter. The PTG （Polycistronic tRNA-gRNA）processing system was employed to enable simultaneous expression of multiple sgRNAs from a single vector. C：Validation of transfection efficiency in citrus leaf protoplasts using a 35S：：GFP plasmid. GFP（Green Fluorescent Protein）expression efficiency was observed under a fluorescence microscope 20 hours post-transfection. D：Western blot analysis of Flag-dCas9-TV fusion protein expression. Protein levels were assessed in citrus protoplasts following transfection with the CRISPR/dCas9-TV system. E：Validation of target gene activation by the CRISPR/dCas9-TV system using a citrus protoplast transient expression assay. Relative gene expression levels are shown，with the expression level in protoplasts transfected with the empty vector set to 1. CsACT2 was used as the internal reference gene.** P < 0.01 by Student’s t-test

Fig. 4 Activation of CsWUS promoted differentiation and regeneration of embryogenic callus suspension cells in sweet orange mpi：Months post-infection. No somatic embryos were observed in the empty vector control after 5 months of induction culture. In contrast，protoplasts transfected with the dCas9-TV-CsWUS construct rapidly proliferated and formed new callus tissue（blue arrow）within one month on selection medium. These calli subsequently developed globular embryos（red arrow）by two months，differentiated green buds by four months，and ultimately produced green leaves after five months of culture

Fig. 5 Activation of CpWUS，CpWOX9，and CpGRF4&CpGIF1 improves regeneration of grapefruit epicotyl explants

Fig. 6 Activation of ClWUS&ClWOX9&ClWRKY42 resulted in enhanced transformation and regeneration rates of hairy roots A：Lemon leaves transfected with DR5pro::RUBY induced the formation of purple hairy roots and subsequently regenerated into plantlets. i：Leaf explants inducing hairy roots；ii：Hairy roots excised into 1 cm segments for further tissue culture；iii：Green buds regenerating from the cut ends of hairy root segments；iv：Continued culture resulting in rooted plantlets. B：Compared with the control，lemon leaves transfected with dCas9-TV-ClWUS/ClWOX9/ClWRKY42 produced a significantly higher number of hairy roots，which exhibited a yellow coloration due to carotenoid accumulation. C，D：Statistical analysis of hairy root number and positive transformation rate for the control and experimental groups shown in picture B. E：Relative expression levels of ClWUS，ClWOX9 and ClWRKY42 in transformation-negative and transformation-positive hairy roots. Student’s t-test，* P < 0.05；** P < 0.01