A Universal Protocol for Faba Bean (Vicia faba L.) Tissue Culture and Transformation

Background Faba bean ( Vicia faba L.) is an important legume that enhances agricultural sustainability through nitrogen fixation and possesses high nutritional value. Breeding of faba bean is laborious and time consuming, and trait improvement could potentially be accelerated by new breeding technologies including gene editing, however tissue culture and genetic transformation are also challenging in V. faba . Results We established a universal protocol for efficient tissue culture and transformation of faba bean cultivars. Various hormone treatments to enhance shoot and root regeneration and development in tissue culture media were tested and optimized in a total of thirty-three faba bean cultivars. Indole-3-acetic acid (IAA) at a concentration of 1.5 mg/L induced effective regeneration among all tested cultivars. Genetic transformation was achieved using particle bombardment, with transformation efficiencies of 75.5% in cultivar Tiffany and 66.66% in cultivar Hedin/2. Successful transformation was validated by expression of green fluorescent protein (GFP) and PCR analysis. Conclusions A universal protocol generally applicable to a broad range of tested cultivars overcomes prior limitations in faba bean tissue culture and transformation, specifically addressing challenges related to phenolic compound production and inconsistencies in root formation. The practical application of this protocol, demonstrated by successful greenhouse acclimatization of regenerated plants, offers promising prospects for faba bean breeding programs. Our findings establish a solid foundation for future breeding and genetic research by improving tissue culture regeneration and transformation efficiency in faba beans. This is the first report of a reliable tissue culture and transformation protocol for V. faba cultivar Tiffany, for which a high-quality reference has been available since 2023.