Biolistic pds 1000 he gene gun system

The ORFs of CaNAC2 cDNAs (without termination codon) were ligated into the pBI221-GFP vector, resulting in an in-frame fusion protein of GFP gene and the CaNAC2 ORFs. The construct (pBI221-CaNAC2) and the control vector (pBI221 -GFP) were transformed into onion epidermal cells by particle bombardment using a Biolistic PDS-1000/He gene gun system (Bio-Rad, Hercules, CA, USA). After 24 h at 25°C incubation of transformed onion epidermal cells, GFP signal was detected by a confocal fluorescence microscope (LSM510 Meta; Zeiss, Jena, Germany). The primers flanked with restriction sites for subcloning are listed in Table 1.

The randomly selected TCS genes were amplified using gene-specific primers (Table S5) and cloned into the pFGC-EGFP plasmids by Xba I and BamH I restriction sites under the control of the 35S cauliflower mosaic virus promoter (35S CaMV). The pFGC:GFP empty vector was used as control. The recombinant vectors were transformed into onion epidermal cells by particle bombardment using the Biolistic PDS-1000/He gene gun system (Bio-Rad, Hercules, CA, USA) [56 ]. After 16–18 h of incubation in darkness, the onion epidermal cell was plasmolyzed in 0.3 g·mL⁻¹ sucrose for 5 min and the fluorescence of GFP was photographed by a Leica DMLE camera (Leica, Wetzlar, Germany).

An expression vector containing a complete encoding region of TaNAC1 cDNA was constructed to confirm nuclear localization of TaNAC1. Kpn I and Xba I linkers were added to the encoding region and the stop codon was deleted by PCR amplification. The forward primer 5′-GGTACCCGATCCGACCGAGAAGATG-3′ (Kpn I site in italics) and reverse primer 5′-TCTAGATGACAAGCCGTTCTCCTG-3′ (Xba I site in italics) were used. High-fidelity DNA polymerase HIFI Taq (TransGen Biotech, Beijing) was used, and the PCR product was cloned into Kpn I and Xba I sites of the binary vector pCaMV35S::GFP to produce fusion vector pCaMV35S::TaNAC1-GFP. The expression plasmid and vector control were transformed into onion epidermal cells by particle bombardment using a Biolistic PDS-1000/He gene gun system (BIO-RAD, Hercules, CA, USA). After 24 h of incubation at 25°C, the fluorescence of DAPI and GFP images of the transformed onion epidermal cells were observed under a confocal microscope (Zeiss LSM 510 Meta Confocal Microscope).