In-planta transient transformation of avocado (Persea americana) by vacuum agroinfiltration of aerial plant parts

Avocado, Persea americana Mill, is one of the most traded tropical fruits in the international market. To date, stable and transient transformation has only been achieved for of zygotic embryos and not of adult plant tissue, which limits functional genomics research. We provide the first transient Agrobacterium-mediated transformation methodology in avocado leaves that overcomes the recalcitrance to transformation of this species. We investigated the effect of Agrobacterium strain, leaf stage, wounding pre-treatment, the phytohormone jasmonic acid, and vacuum infiltration on transient transformation of avocado leaves. Using the Agrobacterium strain LBA4404 and the RUBY reporter a transformation frequency of up to 27% was obtained for avocado detached leaves. The transformation efficiency depended on the age of the leaf, with an intermediate stage of leaf development showing the highest efficiency of transient reporter gene expression. Microwounding pre-treatment facilitates agroinfiltration and coupled with leaf age are the primary factors influencing competence for transient transformation. Jasmonic acid did not significantly affect transient transformation in the absence of microwounding. However, microwounding and 250 µM of jasmonic acid acted synergistically to significantly enhance transient expression. Using this methodology with localized vacuum agroinfiltration, transient transformation of attached avocado leaves was achieved. This method unlocks the use of Agrobacterium-mediated transient transformation as a tool for explore gene function and metabolic pathways in both, detached and attached avocado leaves.