Carbenicillin

The following working concentrations of antibiotics were used: carbenicillin (Solarbio, 50 μg/ml), kanamycin (Solarbio, 50 μg/ml), spectinomycin (Macklin, 50 μg/ml), chloramphenicol (Macklin, 25 μg/ml). PHANTA 2x mix (Vazyme) was used for cloning PCR, and Flash 2x mix (Vazyme) was used for verification PCR and Sanger sequencing (Tsingke Bioscience). All cloning fragments were assembled by Golden Gate assembly (New England Biolabs) or ClonExpress assembly (Vazyme) methods. Plasmids were cloned in DH5α competent cells (HT Health). Synthetic genes were ordered from Tsingke Bioscience. Cloned plasmids were extracted by Tiangen DNA extraction kit. E. coli strain S2060^{55 (link)} was used in all aspects of the EvoScan process, including system construction, evolution, and plaque assays. The DH5α strain was used for flow cytometry experiments. Detailed information on the plasmids and selection phage (SP) used in this work is given in Supplementary Table 6.

The red-fleshed apple callus was transformed as described previously^{19 (link)}. The coding sequences (CDSs) of MdMYB6 and MdTMT1 were ligated into the pRI-101 vector containing GFP and the 35S CaMV promoter, respectively, to generate the 35S:MdMYB6-GFP and 35 S:MdTMT1-GFP plasmids (Supplementary Fig. S1A). The CDS of MdMYB6 was ligated into the pCAMBIA1301 vector, which contained a sequence encoding a His tag as well as the 35S CaMV promoter, to construct the 35S:MdMYB6-His recombinant plasmid (Supplementary Fig. S1E). The recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404. We then infected the red-fleshed calli with the transformed Agrobacterium and cocultured them on MS medium in the dark at 24 °C for 48 h. The resulting calli were then moved onto a screening medium containing 250 mg/L carbenicillin and 50 mg/L kanamycin (Solarbio, Beijing, China) for the MdMYB6- or MdTMT1-overexpressing calli. The screening medium for cultivating the calli cotransfected with MdMYB6 and MdTMT1 contained 250 mg/L carbenicillin, 20 mg/L hygromycin and 50 mg/L kanamycin.