Establishment of an In Vitro Culture and Genetic Transformation System of Callus in Japanese Apricot (Prunus mume Sieb. et Zucc.)

Japanese apricot (Prunus mume Sieb. et Zucc.) is a dicotyledonous plant from the Rosaceae family that originated in China. Functional genomic studies in Japanese apricot are essential to elucidate the molecular mechanisms underlying key agronomic traits and to accelerate crop improvement. However, the lack of an efficient genetic transformation system has hindered gene function analysis and impeded molecular breeding efforts. Agrobacterium rhizogenes-mediated transformation has emerged as a robust tool for functional gene validation and studying root-specific processes across diverse plant species, due to its simple protocol and rapid turnaround time. Notably, Agrobacterium-mediated transformation remains notoriously recalcitrant in Rosaceae species, particularly in Japanese apricot. Through screening of ten Japanese apricot varieties, we identified ‘Muguamei’ (MGM) as the optimal cultivar for tissue culture. Using its genotype, we established an Agrobacterium rhizogenes-mediated transformation system for Japanese apricot via an in vitro approach. The binary vector incorporated the RUBY reporter for visual selection and eYGFPuv for fluorescent validation of transformation events. Furthermore, CRISPR/Cas9-mediated knockout of PmPDS in ‘Muguamei’ calli generated albino phenotypes, confirming successful genome editing. Through optimization of antibiotics, the study achieved an 80% explant survival rate using Woody Plant Medium (WPM) supplemented with 6-BA (0.5 mg/L) and TDZ (0.05 mg/L). For in vitro micropropagation, we found that ‘Muguamei’ exhibited optimal shoot growth in the presence of 6-BA (0.06 mg/L) and TDZ (0.1 mg/L), and up to 8 bud proliferation lines could be reached under 4.0 mg/L 6-BA. During the rooting of micro shoots, ½MS medium performed better and reached the optimum root length (35.70 ± 4.56 mm) and number (6.00 ± 1.00) under IAA (0.5 mg/L) and IBA (0.4 mg/L). Leaf explants were cultured on WPM supplemented with TDZ (4.0 mg/L) and NAA (0.2 mg/L). 50 mg/L kanamycin concentrations were the suitable screening concentration.