Overexpression of PaMYB10 in Tomato Fruit

The overexpression pMDC32 binary vector49 was constructed as previously described [41 (link), 42 (link)]. The PaMYB10 coding sequence was PCR amplified from ‘JNL’ cDNA using the primers 5′-ATGGAGGGTTATTTCGGTGTGAG-3′ (forward) and 5′-TACGTAGGAGATGTTGACTAGATCATTGC-3′ (reverse) and cloned using the pCR™8/GW/TOPO TA Cloning Kit (Invitrogen). After sequencing confirmation, the CDS was recombined into the pMDC32 binary vector49 using the Gateway LR Clonase II Enzyme mix (Invitrogen) to produce overexpression vectors. The vectors were transformed into Agrobacterium tumefaciens GV3101 and then resuspended at a final O.D. of 0.8 in liquid MS medium. One milliliter of the suspension was evenly injected into the ‘LT’ fruit at commercial mature stage. Thirty fruits were injected as the repeat, and fruits were injected with Agrobacterium carrying an empty vector as a control treatment. At 7–12 days after injection, the fruit peel surrounding the injection sites was collected and immediately frozen in liquid nitrogen and stored at 80 °C for determining the anthocyanin according to the method of above mentioned.