Transient expression of CaNAC2c in pepper leaves
For transient expression analysis, GV3101 cells harboring35S:CaNAC2c-GFP (using 35S:GFP as control) was grown
overnight and then resuspended in induction medium (10 mM MES, 10 mM
MgCl2, 200 µM acetosyringone, pH 5.6) to
OD600 = 0.8, approximately 1 ml was infiltrated into the
leaves of pepper plants at the eight-leaf stage using a syringe without
a needle, and then the infiltrated leaves were collected at the
indicated time points for further use.
Generation of transgenicCaNAC2c-overexpressingN. benthaminana plants
Leaf discs of N. benthamiana were transformed with GV3101
harboring 35S:CaNAC2c-GFP vector followed the method of
Regneret al (F et al., 1992). and Bardonnet al (N, F, MA, &
L, 1994). 19 independent T0 transgenic N.
benthamiana lines were selected by hygromycin (5 mg
l-1) selection and further confirmed by PCR and
quantitative real-time RT-PCR (qRT-PCR). The T0 plants
were then self-pollinated and their seeds individually harvested.
Similarly, corresponding seeds of T2 and
T3 were acquired. Two T3 transgenic
lines that exhibited moderate levels of CaNAC2c transcripts
without phenotypic abnormality were selected for further analysis.