Efficient Agrobacterium-Mediated Methods for Transient and Stable Transformation in Common and Tartary Buckwheat

Buckwheat is a promising crop with grains that are rich in nutrients and bioactive compounds. Genome sequence data for common and Tartary buckwheat have recently become available. Currently, there is a critical need for the development of a simple and reliable transient gene expression protocol, as well as a stable genetic transformation method, to facilitate metabolic engineering of bioactive compounds, functional analysis of genes, targeted editing, and, in a long-term perspective, to accelerate the breeding process in buckwheat. In this paper, we report optimized methods for Agrobacterium-mediated transient and stable transformation of Fagopyrum esculentum and F. tartaricum. Leaf and cotyledon tissues were infiltrated with an A. tumefaciens-bearing construct containing eGFP and GUS reporter genes. Histochemical staining and Western blotting were used to confirm the expression of reporter proteins. We also demonstrate the usefulness of the developed method for engineering the gramine biosynthetic pathway in buckwheat. HvAMIS and HvNMT genes were transiently expressed in buckwheat leaves, and the de novo production of gramine was confirmed by LC-MS. Moreover, in planta genetic transformation of common and Tartary buckwheat with a reporter gene (eGFP) and selectable marker gene (NptII) was achieved by Agrobacterium-mediated vacuum infiltration. Genomic integration of the construct was confirmed by polymerase chain reaction (PCR), whereas the production of eGFP was confirmed by fluorescence microscopy.