2.6 Agrobacterium-based transient transformation
Assays for transient expression in peach fruit were conducted following protocols described by Zhang et al. (2015) and Li et al. (2017), with minor modifications. To generate the overexpression construct, thePpINH1 CDS was excised from the BiFC fusion vector and ligated into a pBI121 vector, generating pBI121-PpINH1 , which was subsequently transformed into Agrobacterium tumefaciensGV3101. The pBI121 vector was used as a negative control.
A. tumefaciens transformants (1 ml of 3 OD600 units) were injected into mature green ‘Yulu’ peaches. Peaches were sampled 24, 36, 48, and 72 h after inoculation. The area of infection was a radius of about 1 cm around the injection site. At each time point fruits were cut into 1 cm2slices approximately 1-2 mm thick. Expression of the GUS reporter plasmid (co-transformed with PpINH1 ) was assayed using a histochemical assay (RealTimes, Beijing, China). For VIN activity andPpINH1 expression assays, peels were removed with a scalpel, and the infected tissue was cut into pieces of about 0.5 g. Pieces were flash frozen in liquid nitrogen and stored at –80 °C.