<p>Despite decades of progress in plant biotechnology, a major technical barrier has remained largely unresolved: the lack of a feasible method to agroinfiltrate large woody perennials <i>in planta</i>. This constraint has confined functional genomics mainly to seedlings or detached organs, limiting the analysis of gene regulation in mature leaves, reproductive structures, and developing fruits—key organs for understanding perennial crop biology. This Perspective challenges that paradigm by highlighting a recently developed and experimentally validated innovation: localized vacuum agroinfiltration, a novel sealing interface that enables transient gene expression directly in attached mature organs without the need for oversized vacuum chambers. Rather than representing a minor protocol optimization, this method constitutes a conceptual reframing of how vacuum infiltration can be achieved in woody species. The robustness of the approach has already been demonstrated in two notoriously recalcitrant tropical crops—avocado (<i>Persea americana</i>) and cacao (<i>Theobroma cacao</i>)—through successful <i>in planta</i> transient expression reported in peer-reviewed studies. By preserving vascular connectivity, localized vacuum agroinfiltration provides biologically relevant conditions for probing transcriptional responses, regulatory dynamics, and gene-function relationships in adult organs. This strategy complements in vitro biotechnology by enabling early <i>in planta</i> validation of construct performance prior to regeneration workflows. Its low-cost, adaptable design aligns with open science principles and has the potential to democratize access to functional genomics in perennial crops, particularly in resource-limited research settings. We argue that this innovation can accelerate functional genomics in fruit and forest species and open new research frontiers previously inaccessible in woody perennials.</p>