ABS 066: Agrobacterium-Mediated in Planta Transformation of the Medicinal Plant Catharanthus roseus

Molly Johnson ¹ , Emma Meehan ² , Dr. Lauren F. Cole-Osborn ² , Natalie Soens ² , Dr. Carolyn W.T. Lee-Parsons ¹ ² ³

¹ Department of Bioengineering Northeastern University
² Department of Chemical Engineering Northeastern University
³ Department of Chemistry & Chemical Biology Northeastern University

Van Wickle (2025) Volume 1, ABS 066

Introduction: Catharanthus roseus (Madagascar periwinkle) synthesizes the valuable anti-cancer alkaloids vinblastine and vincristine via an intricate monoterpene indole alkaloid (MIA) biosynthetic pathway. However, the extremely low natural abundance of these compounds presents a major bottleneck for large-scale pharmaceutical production. To facilitate metabolic engineering aimed at enhancing their biosynthesis, we are employing a tissue culture-independent Agrobacterium tumefaciens-mediated in planta transformation approach to generate transgenic C. roseus lines. Compared to conventional tissue culture-based transformation and regeneration methods, this in planta technique offers a shorter transformation timeline and improved regeneration efficiency. As a proof-of-concept, we constructed a plasmid encoding green fluorescent protein (GFP), enabling the visual identification of transformed cells via fluorescence under ultraviolet (UV) excitation. GFP expression will serve as a marker for evaluating transformation efficiency and refining the protocol. This method provides a rapid and effective platform for stable genome modification in C. roseus, facilitating targeted interventions to boost vinblastine and vincristine production.

Methods: In this study, a plasmid construct was generated using Golden Gate cloning to express green fluorescent protein (GFP) under the control of the nopaline synthase (NOS) promoter and terminated with a NOS terminator. The construct was first introduced into Escherichia coli for plasmid amplification and sequence verification, and subsequently transformed into Agrobacterium tumefaciens strain GV3101. Transformation of Catharanthus roseus seedlings at the cotyledon stage was performed via mechanical wounding of the apical meristem using a sterile scalpel, followed by application of the Agrobacterium suspension. Infected seedlings were cultivated for 3–4 weeks under controlled growth conditions. Successful transformation was initially assessed via fluorescence microscopy using a GFP filter; GFP expression was indicated by green fluorescence comparable to a transiently expressing positive control and absent in wild-type controls. To further validate transgene integration and exclude the presence of residual Agrobacterium, genomic DNA was extracted from fluorescent tissues and subjected to PCR analysis.

Results: Not Published

Discussion: Not Published