Transient expression of LaTPS7, LaTPS8, andLaCYP71D582 in N. benthamiana
Leaves of N. benthamiana collected from 4-week-old plants were
infiltrated with Agrobacterium tumefaciens GV3101 harboring
target genes. Meanwhile, GV3101 was mixed with TPS and CYP at respective
ratios of 1:1 when OD600 of each strain corresponded to
1.0. Leaf tissues were collected
after 5 days of infiltration, examined under UV light to ensure
successful agroinfiltration and gene expression, and then ground in
liquid nitrogen. Leaf powder was stored at
-80°C,
and volatiles were extracted by vortexing in 70% alcohol (1:4 m/v).
Extracts were maintained at
-20°C
and oscillated before analysis via
solid-phase microextraction (SPME, Agilent). The samples were heated in
a water bath at 40°C for 30 min, and then a fibre coated with
polydimethylsiloxane and divinylbenzene was used to collect volatiles
under the same conditions for 30 min according to a previously published
method (Ilc et al. 2017). Volatiles were then analysed using
GC-MS.