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.

Double haploid technology is a critical tool for accelerating plant breeding. The manipulation of centromere-specific histone H3 (CENH3) has previously been shown to induce haploid progeny in both the model plant Arabidopsis and certain crop species. However, a universally applicable strategy for CENH3-based haploid induction across diverse crop species is still lacking. Here, we demonstrate that extreme asymmetry in centromeric CENH3 levels between parental chromosomes is a causal factor for eliminating chromosomes with ‘weaker’ centromeres during early embryogenesis. Consequently, engineered E3-ligases were utilized to selectively degrade CENH3 in the egg cell to induce wild-type paternal haploids. Also, we attempted to optimize the CENH3 degradation using E3-ligases of plant-origin. Our findings provide valuable insights to enhance the haploid induction efficiency further. The gametophytic CENH3 degradation approach leverages engineering principles and represents a potentially universal strategy for engineering CENH3-based haploid inducers in crop plants.