Pds 100 he particle delivery system

Microcarriers for particle gun bombardment were prepared with 100 ng of plasmid DNA. A Bio-Rad PDS-100/He particle delivery system was used (Cabrera-Ponce et al., 1997 , 2015 (link)). An equimolar mixture of plasmid PvTRX1hRiA and pWRG1515 (Christou et al., 1991 (link)) was precipitated onto tungsten microprojectiles of 1.0 μm diameter and delivered onto early globular-stage pro-embryogenic callus that had been sub-cultured for 2–3 months. Ten Petri dishes, each containing 16-20 calli for a total of 2 grams of fresh weight, were bombarded. Control calli were transformed with the pWRG1515 vector only. pWRG1515 contains the uidA reporter gene (gusA) and the hptII gene that confers resistance to hygromycin. Transformed callus were selected in EIM plates containing 50 mg/L hygromycin. After bombardment, calli were sub-cultured every 2 weeks for 3 months in fresh EIM plates containing hygromycin. The transgenic clones maintained their embryogenic capacity, while the construct used in this experiment was stably expressed in the embryogenic callus.

The verified construct of pK7WGF2.0-SaREFl for subcellular localization study was used to transform onion epidermal cell layers using Helium Biolistic gene transformation system (Du Pont). Inner epidermal layers were peeled and placed inside up on Petri dishes containing MS basal medium containing 2.5 mg L^-1 amphotericin B (Sigma, St. Louis, MO, USA) and 6% (w/v) agar. DNA samples containing pK7WGF2.0-SaREFl were prepared as described by the manufacturer (Du Pont) which were coated with 1 μM gold particles and followed by the introduction into onion epidermal cells using particle bombardment (PDS-100/He particle delivery system, Bio-rad). Then, Petri dishes were sealed with parafilm and incubated for 18–24 h at 28°C in the dark. After 20 h, fluorescent cells were imagined. The green fluorescence was observed through a laser scanning confocal microscope (LSM510; Karl Zeiss, Jena, Germany) excitated at 488 nm for GFP.

To investigate the subcellular localizations of BcSnRK2 genes, two expression vectors were constructed using a transient expression system in onion epidermal cells. The full-length coding sequences of BcSnRK2.6a were amplified using Gateway-specific primers cloned into an entry vector, and then subcloned into pEarleyGate101 using Gateway technology (Invitrogen, Carlsbad, CA). The yellow fluorescent marker protein (YFP) was fused to BcSnRK2.6a. Gold particles with a diameter of 1 μm coated with 35S BcSnRK2.6a-YFP was introduced into onion epidermal cells using particle bombardment (PDS-100/He particle delivery system; Bio-Rad, Hercules, CA). After incubation at 22°C for at least 12 h under darkness, fluorescence and bright-light images were observed using laser scanning confocal microscopy (Leica, TCS SP2, Wetzlar, Germany).