Efficient creation of decorative double‐flowered petunia through CRISPR/Cas9‐mediated simultaneous editing of PMADS3 and FBP6 genes

Double-flowered petunias are highly desirable for their ornamental appeal, yet their availability remains limited. Mutations in the C-class AGAMOUS (AG) gene can cause homeotic transformation, converting stamens into petals and carpels into new floral organs, resulting in double-flowered phenotypes (Bowman et al., 1989). However, as in most plants, petunia harbours two functionally redundant AG orthologues, PETUNIA MADS-BOX GENE3 (PMADS3) and FLORAL BINDING PROTEIN6 (FBP6), making it difficult to obtain complete AG knockout mutants through natural or induced mutations (Angenent et al., 1993; Tsuchimoto et al., 1993). The CRISPR/Cas9 tool provides an opportunity to simultaneously target the PMADS3 and FBP6 genes and create double-flowered petunias (Zhang et al., 2020). We first selected a dicot gene editing vector with the AtU6 promoter for sgRNA expression and a 35S promoter for Cas9 expression (Figure 1a). Next, the rGRF4-GIF1 chimeras, derived from the dicot grape and known to enhance transformation efficiency (Debernardi et al., 2020), were synthesized and subsequently integrated into the dicot gene-editing vector. In the GRF4 sequence, six synonymous mutations were introduced at the miR396 binding sites to further improve transformation efficiency (Appendix S1) (Qiu et al., 2022). We named this vector eDGE1 (enhanced dicot genome editing 1) (Figure 1a). Target sequences for PMADS3 and FBP6 were both designed within the first exon and simultaneously incorporated into the eDGE1 vector using the tRNA-based multiplex system, which has been reported to function in petunia (Figure 1b) (Sun et al., 2018; Xie et al., 2015). The constructed vector was then transformed into the commonly used laboratory Petunia hybrida W115 (Mitchel diploid). Among the 16 obtained petunia plants, the FBP6 gene was successfully edited in all plants, with a homozygous/biallelic mutation rate of 43.75% and a heterozygous/chimeric mutation rate of 56.25% (Figure 1c). Editing of the PMADS3 gene was achieved in 6 of the 16 plants, resulting in an editing efficiency of 37.50% (Figure 1c). Lines #4 and #12 were selected for further phenotypic analysis. In line #4, PMADS3 was wild-type (WT)/+1 bp heterozygous, and FBP6 showed a biallelic −1 bp/−4 bp mutation type (Figure 1d). In line #12, PMADS3 was also WT/+1 bp heterozygous, and FBP6 exhibited a homozygous −1 bp deletion (Figure 1d). Phenotypically, the anthers of lines #4 and #12 have both transformed into petals (Figure 1e). Both the stigma of the carpel in #4 and #12 showed abnormal development. In #4, the stigma split into a linear shape, while in #12, the stigma was enlarged with a clear splitting (Figure 1e). These results suggest that although PMADS3 has not yet been homozygously mutated, the knockout of the FBP6 gene is sufficient to produce weak double-flowered petunia. We furthermore knocked out PMADS3 and FBP6 in a commercial petunia variety, Mirage Rose. By reverse transcribing the mRNA of FBP6 and PMADS3 in Mirage Rose and sequencing, it was found that the sequences of these two genes were identical to those in W115 (Table S1). Therefore, the previously constructed vector was directly applied to this variety (Figure 1a). A total of 120 regenerated lines were obtained for Mirage Rose. Genotypic analysis revealed an editing efficiency of 55.83% for PMADS3, with 10.00% showing homozygous/biallelic mutations, and 85.00% for FBP6, with 74.17% exhibiting homozygous/biallelic mutations (Figure 1c). The efficiency of simultaneous homozygous/biallelic mutations at both loci was 3.33% (Figure 1c). Lines #40, #46 and #105 were selected for phenotypic characterization. Line #40 had a WT/+1 bp heterozygous mutation in the PMADS3 gene and a −2 bp homozygous mutation in FBP6. Line #46 exhibited a +1 bp homozygous mutation in PMADS3 and a −6 bp homozygous mutation in FBP6, which caused a two-amino-acid deletion in the conserved MADS-box domain (Figure 1d, Figure S1). Line #105 showed biallelic mutations in both genes (+1 bp/−1 bp in PMADS3 and −1 bp/−3 bp in FBP6). Phenotypic analysis revealed that none of the three lines displayed significant morphological differences compared to the wild type, but all exhibited double-flowered phenotypes (Figure 1g). While all three lines demonstrated stamen-to-petal transformation, the size of the stamen-derived petals varied significantly (Figure 1h). In #105, the stamen-derived petals were the largest, whereas those in #40 were the smallest (Figure 1h). All three lines displayed a loss of floral determinacy, but the extent of carpel transformation varied among the lines (Figure 1h). In #40, the carpel transformed into a small undeveloped floral organ, while in #46 and #105, it developed into a new floral organ, with the new floral organ in #105 being larger (Figure 1h). Generally, #105 exhibited the most richly layered double-flowered phenotype (Figure 1h). To observe the stability of double-flower phenotype, we propagated lines #40, #46 and #105 through cuttings (Figure S2A). All progeny maintained the double-flowered phenotype of their respective parental lines, indicating that the edited traits are stable and heritable through vegetative propagation (Figure S2B–D). Off-target analysis of several potential sites revealed no detectable off-target effects (Table S2). To elucidate the relationship between floral homeotic genes and the double-flower phenotype in petunia, we analysed gene expression profiles in petals and stamens/carpel or petaloid organs of line #105 and the wild type using RNA-seq (Figures S4–S6, Dataset S1). The resulting data were further examined through gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and weighted gene co-expression network analysis (WGCNA) (Database S2–S4). WGCNA revealed that FBP6 and PMADS3 exhibited the highest co-expression correlation. Moreover, several other MADS-box family genes were found to be highly co-expressed with FBP6 and PMADS3, including the class B gene TM6 and previously uncharacterized members such as AGL19 and MADS9 (Database S4). In line #105, the expression of FBP6 and PMADS3 in petaloid organs was significantly reduced to 6.43% and 3.83% of WT levels, respectively (Figure S7). A-class genes (FBP29, AP2 and FBP26) were expressed in the WT petals, with AP2 showing the highest expression, while their levels in stamens and carpel were much lower or undetectable (Figure S7). In line #105, these genes showed a similar expression level to the wild type in petals but were significantly upregulated in petaloid organs (Figure S4). For class B genes DEF, GLO1 and GLO2 were expressed in both petals and stamens/carpel in the wild type, with higher levels in petals (Figure S7). In line #105, their expression in petaloid organs increased to levels comparable to petals (Figure S7). TM6 exhibited higher expression in stamens/carpels than petals in the wild type, but its expression in line #105 was significantly reduced in petaloid organs, matching that in petals (Figure S7). Furthermore, we confirmed the expression of PMADS3 and FBP6 in lines #40, #46 and #105 using qRT-PCR (Figure 1i). Compared to the wild type, PMADS3 expression decreased by approximately 79.14% in #40, and by 97.66% and 98.02% in #46 and #105, respectively (Figure 1i). The expression of FBP6 decreased by 97.00%, 96.55% and 89.51% in lines #40, #46 and #105, respectively, compared to the wild type (Figure 1i). In summary, we successfully utilized CRISPR/Cas9 technology to simultaneously knock out PMADS3 and FBP6, resulting in stable and decorative double-flower varieties in petunia. This work was funded by the Shanghai Agricultural Science and Technology Innovation Programs (No. 2024-02-08-00-12-F00007 and No. K2023002) and the Chongming District Agricultural Science and Innovation Project (No. 2021CNKC-02-02). All authors declare no competing interests. H.Z. and C.W. designed and supervised the work; Y.X., S.L., J.N., Y.D., and Z.H. performed the research; Y.X., C.W., and R.G. analyzed and investigated the data; H.Z., C.W., and Y.S. wrote and revised the paper. Dataset S1 Differential gene expression calculated by DESeq2. Database S2 GO enrichment analysis of downregulated-DEGs in the petal of gene-edited double-flowered line #105. Database S3 Enrichment analysis of the KEGG pathway of downregulated-DEGs in the petal of gene-edited double-flowered line #105. Database S4 WGCNA analysis identified the genes related to FBP6. Appendix S1 Supplementary sequences, figures, tables, and materials and methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.