Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Titis A.K. Wardhani, Yuda Purwana Roswanjaya, Simon Dupin*, Huchen Li, Sidney Linders, Marijke Hartog, Rene Geurts, Arjan van Zeijl

*Corresponding author for this work

    Research output: Contribution to JournalArticleAcademicpeer-review


    Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

    Original languageEnglish
    Article number59971
    JournalJournal of visualized experiments : JoVE
    Issue number150
    Publication statusPublished - 18 Aug 2019


    The authors like to acknowledge Mark Youles, Sophien Kamoun and Sylvestre Marillonnet for making Golden Gate cloning parts available through the Addgene database. Additionally, we would like to thank E. James, P. Hadobas, and T. J. Higgens for P. andersonii seeds. This work was supported by The Netherlands Organization for Scientific Research (NWO-VICI grant 865.13.001; NWO-Open Competition grant 819.01.007) and The Ministry of Research, Technology and Higher Education of the Republic of Indonesia (RISET-PRO grant 8245-ID).

    FundersFunder number
    The Netherlands Organization for Scientific Research
    Kementerian Riset, Teknologi dan Pendidikan Tinggi


      Dive into the research topics of 'Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii'. Together they form a unique fingerprint.

      Cite this