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.