Fipexide (FPX), a chemical callus inducer, promotes in vitro shoot regeneration and <i>Agrobacterium</i>-mediated genetic transformation in three fruit tree species

For fruit trees, inefficient transformation and prolonged juvenile phase lasting for years to decades remain two major bottlenecks for breeding and functional gene analysis. Recently, fipexide (FPX) has been identified as a novel chemical that enhances callus induction, regeneration, and Agrobacterium-mediated transformation. In this study, we aimed to optimize FPX treatment for improving shoot regeneration rate and transformation efficiency in European pear (Pyrus communis), highbush blueberry (Vaccinium corymbosum), and persimmon (Diospyros kaki). Explants were cultured on media supplemented with various concentrations of FPX for different durations. In European pear ｀La France’, treatment with 3 µM FPX for one week significantly enhanced regeneration compared with the conventional phytohormone condition, whereas higher concentrations (≥10 µM) and extended exposure inhibited regeneration. Similar patterns were observed in European pear ｀Bartlett’, highbush blueberry ｀O’Neal’ and persimmon ｀Jiro’. Furthermore, 10 µM FPX enhanced Agrobacterium-mediated transient GFP expression in all 3 species. We also attempted to introduce Arabidopsis FLOWERING LOCUS T (FT) into persimmon to shorten juvenile phase and induce precocious flowering. Three transgenic ｀Jiro’ lines with AtFT overexpression were successfully obtained using 10 µM FPX. Collectively, our findings demonstrate that FPX is a potent enhancer of regeneration and transformation in multiple fruit trees species, offering a promising strategy for accelerating breeding programs and gene function analysis in recalcitrant woody species.