A novel and efficient <i>Agrobacterium</i>-mediated transient gene expression in citrus epicotyls and mature stem tissues

<i>Agrobacterium</i>-mediated transient gene expression is a powerful technique for rapidly evaluating gene expression in higher plants. This study aimed to improve transient expression levels of T-DNA genes in citrus by investigating the effects of various factors, including seedling age, tissue treatments, <i>Agrobacterium</i> incubation medium and duration, hormone combinations, methylation inhibitors, antioxidants, and more. These parameters were optimized and tested, and significant increases in transient gene expression in juvenile epicotyls of Carrizo citrange and mature stem tissues of Pineapple sweet orange, Valencia orange, and Washington navel orange were observed. The present results demonstrated up to a six-fold increase in transient GUS gene expression, highlighting the effectiveness of these simple and inexpensive treatments. The juvenile and mature citrus explants used in this study displayed high levels of transient expression, which may provide a valuable tool for studying phloem-associated diseases such as Huanglongbing (HLB), facilitating rapid analysis of gene expression involved in <i>Candidatus</i> Liberibacter asiacticus (CLas) pathogenicity. This optimized method may also offer a promising tool for advancing genetic studies and improving the efficiency of <i>Agrobacterium</i>-mediated transgene-free editing in citrus and other economically significant perennial crops.