Functional genomics analyses in planta can be hampered in non-model plants that are recalcitrant to the genetic transformation such as the medicinal plant Catharanthus roseus (Apocynaceae). No stable transformation and regeneration of plantlets have been achieved with a high efficiency in this plant to date. In addition, while virus-mediated transient gene silencing has been reported a decade ago in C. roseus, tools for transient overexpression remain scarce. Here, we describe an efficient and reliable methodology for transiently overexpressing any gene of interest in C. roseus leaves. This protocol combines a vacuum-based Agroinfiltration approach and the high translational efficiency of a deconstructed virus-based binary vector (pEAQ-HT). The described methodology is robust, easy to perform, and results in high amount of transient expression in C. roseus. This protocol is expected to serve as valuable tool to enhance the in planta characterization of gene functions or even transiently knock-in novel enzymatic activities.
Keywords: Agrobacterium tumefaciens; Agroinfiltration; Apocynaceae; Catharanthus roseus; Cowpea mosaic virus HT (CPMV-HT); Functional genomics; Madagascar periwinkle; Recombinant proteins; Transient expression; Vacuum infiltration.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.