A. rhizogenes-mediated strawberry hairy root transformation

Each A. rhizogenes strain was streaked onto LB agar plates supplemented with 50 µg/mL streptomycin and incubated at 28 °C for two days [10]. A single colony was selected into a 5 mL YEP medium containing 50 µg/mL streptomycin, 50 µg/mL kanamycin, and 50 µg/mL chloramphenicol and incubated at 28 °C with agitation (250 rpm) for 24 h [24]. Subsequently, 1 mL of the starter culture was transferred into 50 mL YEP medium for the grown culture. Bacterial cells were centrifuged at 5000 rpm for 8 min and resuspended on varying optical densities OD₆₀₀ of 0.3, 0.5, 0.7, and 0.9 added in MS liquid medium containing 100 µM acetosyringone (AS). A. rhizogenes strain at each OD₆₀₀ was inoculated with at least 20 explants, and three biological replicates were performed.

Various explants were submerged in bacterial solution suspended to a final OD₆₀₀ of 0.7 for varying infection time as follows: 5, 10, 20, and 30 min. After removing excess liquid on filter paper, infected explants were transferred onto filter papers wetted with the 100 µM AS and co-cultivated in darkness at 22 °C for 2, 3, 4, and 5 days, individually. After co-cultivation, the infected explants were washed with sterile water and transferred onto MS medium containing 300 mg/Lcarbenicilin and 300 mg/L timentin to induce hairy roots. Afterward, the explants were sub-cultured every two weeks. Each parameter was treated using 20 explants, and three biological replicates were performed. The numbers of callus-regenerated hairy roots and eGFP-positive roots were finally recorded. We calculated the induction rate of callus (number of explants induced callus/ total number of explants × 100%), regeneration rate of root (number of explants regenerated roots/total number of explants × 100%), and frequency of eGFP-positive root (number of eGFP-positive roots/number of regenerate roots × 100%).