A simple and efficient agroinfiltration method for transient gene expression in Citrus.

KEY MESSAGE: Microwounding pre-treatment facilitates agroinfiltration and transient gene expression in hard-to-agroinfiltrate citrus varieties. Agrobacterium infiltration is a widely used method for transient expression studies in plants, but this method is not used extensively in citrus because of its low efficiency. In this study, we developed an easy, cheap, and reliable agroinfiltration method for transient gene expression in citrus. A microneedle roller was used to create microscopic wounds in the leaf epidermis to facilitate agroinfiltration. Several optimization parameters were explored in this study, including the density of wounds per cm2 of abaxial leaf area, the leaf maturity grade, the effect of the Agrobacterium strain, and the length of the incubation period. Increasing the density of wounds on the leaf surface had a positive effect on transient expression. Higher transient expression levels were observed in well-expanded young leaves in comparison with older leaves. The Agrobacterium strain GV2260 was the most suitable to express a large amount of recombinant protein, and an eight- to ten-day incubation period resulted in the highest expression. Endoplasmic reticulum and cytoskeleton-targeted GFP were both successfully localized, confirming that this protocol can be used for protein subcellular localization in citrus. Finally, up to 100 ng of GFP per milligram of agroinfiltrated leaf tissue was estimated to be expressed using this method. This protocol was tested for GFP expression in five different citrus varieties with no significant statistical differences among them. This simple and easy method can speed up functional genomic studies in citrus and may be applied to other recalcitrant species with extensive epidermal cuticular wax.