Optimization of in vitro adventitious shoot regeneration and genetic transformation systems in blueberry (Vaccinium corymbosum) using a heterogeneous Ruby1 gene

Abstract Blueberry is an economically important fruit crop, and demand for it is rising. Despite being essential for advancing precision breeding of blueberries, genetic transformation platforms remain limited for transgenic applications. In this study, we used the widely cultivated blueberry cultivar ‘Legacy’ ( Vaccinium corymbosum ) as the experimental material to establish an efficient adventitious shoot-regeneration system and an Agrobacterium -mediated transformation protocol. Adventitious bud development was promoted via a two-step protocol: induction on WPM medium with 2 mg/L TDZ and 0.5 mg/L NAA, followed by elongation on WPM medium with 3 mg/L ZT. Younger leaves from the shoot tips were selected as explants for the regeneration system. Wounding treatment, placing the abaxial (back) side in contact with the medium, and a 12-d dark treatment at the initial regeneration stage significantly improved the adventitious shoot regeneration rate. Building upon this regeneration system, an Agrobacterium -mediated genetic transformation protocol was developed using a vector carrying the reporter genes enhanced green fluorescent protein ( Egfp ) and CgRuby1 , an anthocyanin-regulating transcription factor from purple pummelo ( Citrus grandis ). The optimal parameters included culturing Agrobacterium -mediated in liquid medium, preparing the Agrobacterium suspension with an OD 600 of 0.8, a 1-h vacuum infiltration, 6 d of co-cultivation, and selection on adventitious shoot regeneration medium containing 10 mg/L kanamycin, which enhanced the regeneration rates of resistant shoots. Polymerase chain reaction (PCR) analysis confirmed that transgenic plants carrying CgRuby1 were achieved with a final transformation efficiency of 6.5%. Phenotypic analysis revealed that the transgenic plants accumulated significantly higher anthocyanin levels in their leaves than the wild-type plants. In addition, key blueberry anthocyanin biosynthetic genes, including VcCHS , VcFHT , VcDFR , VcANS , and VcUFGT , were markedly upregulated in the transgenic lines. Taken together, we have successfully established an efficient transformation system that may hold great potential for functional characterization of genes involved in various processes.