The transcriptional activation of genes that encode the ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate oxidase (PhACO3 and PhACO4) during petunia flower senescence has been reported. However, no studies have elaborately investigated their specific roles in ethylene production and flower longevity using genetic manipulation. Hence, we used the CRISPR/Cas9 system to edit the genes (PhACO3 and/or PhACO4) involved in ethylene production and flower longevity in petunia cv. Mirage Rose. The use of the CRISPR/Cas9 system with a sgRNA, which was designed from exon 2 of PhACO3, allows for the specific editing of the genes PhACO3 and/or PhACO4 with high mutation frequency, consequently producing different types of zygotes. The PhACO3 and PhACO4-edited lines 8 and 9 showed remarkably reduced ethylene production (approximately 2.8- to 3.0-fold in corollas and 1.5-fold in pistils) during flowering and extended flower longevity (approximately 9.5 d), while the PhACO3-edited bi-allelic and PhACO4-edited homozygous T₀ mutant lines (14 and 23) showed enhanced flower longevity (approximately 8.0 d) compared with 6.0 d for the WT line. This was associated with reduction of PhACO4 protein levels in PhACO4-edited lines, which was confirmed using Western blot analysis and Image J software. Moreover, there was no undesirable editing effect on the PhACO1 gene. The transmission of the edited alleles to the T₁ generation was also observed, and ethylene production and flower longevity were identical to those of the T₀ mutant lines. Taken together, this study demonstrated not only the single and combined role of PhACO3 and PhACO4 in ethylene production in petunia flowers but also reports improvements in flower longevity by editing of the aforementioned genes using the CRISPR/Cas9 system. Therefore, our study can pave the way for the editing of homologous genes in other ornamental plants using the CRISPR/Cas9 system with a common sgRNA, thus allowing for a time- and cost-effective approach to advancing plant biology and the floricultural industry.

矮牵牛花衰老过程中，编码乙烯生物合成酶1-氨基环丙烷-1-羧酸氧化酶（PhACO3和PhACO4）的基因的转录激活已有报道。但是，尚无研究使用基因操作详细研究其在乙烯生产和花朵寿命中的特定作用。因此，我们使用CRISPR / Cas9系统编辑矮牵牛cv中参与乙烯生产和花寿命的基因（PhACO3和/或PhACO4）。幻影玫瑰。所述CRISPR / Cas9系统与因组，将其从外显子2设计使用PhACO3，允许特定编辑基因PhACO3和/或PhACO4具有高突变频率，因此产生不同类型的合子。的PhACO3和PhACO4 -edited线8,9显示显着降低乙烯产量（花冠约2.8-到3.0倍和在雌蕊1.5倍）开花和扩展长寿花（约9.5 d）期间，而PhACO3 -edited双向-等位基因和由PhACO4编辑的纯合T ₀突变株（14和23）显示出更长的花寿命（约8.0 d），而WT株为6.0 d。这与降低PhACO4中的PhACO4蛋白水平有关编辑的品系，使用Western blot分析和Image J软件确认。而且，对PhACO1基因没有不希望的编辑作用。还观察到编辑的等位基因向T ₁代的传递，并且乙烯的产生和花的寿命与T ₀突变体系相同。综上所述，本研究不仅证明了PhACO3和PhACO4的单一作用，而且还发挥了作用矮牵牛花中乙烯的产生，但也报道了通过使用CRISPR / Cas9系统编辑上述基因来提高花的寿命。因此，我们的研究可以使用具有常见sgRNA的CRISPR / Cas9系统为其他观赏植物中的同源基因编辑铺平道路，从而为开发植物生物学和花卉产业提供了一种既省时又经济的方法。