Pds 1000 he particle delivery system

For observing transient expression of OsRAE3 in onion epidermal cells, the fusion protein of OsRAE3(WT)-YFP was constructed under the control of the 35S promoter. The strips of onion scale leaves were subjected to particle bombardment using the biolistic PDS1000/He Particle Delivery System (Bio-Rad). Bombardment was performed with a 1,100 psi rupture disc (#1652329, Bio-Rad) under the condition of 28 inch Hg (vacuum level in chamber), 1,100 psi helium pressure, and 590 MPa pressure. After bombardment with gold particles, samples were incubated at 28 °C for 16 h in the dark. The epidermal layer was peeled off, then observed under confocal laser microscope with a 40× objective (Zeiss, Oberkochen, Germany).

C. quinoa and soybean plants were inoculated with ALSV as described [19 (link), 23 (link), 41 (link)]. The plasmids for ALSV-RNA1 and RNA2 were mixed in equal amounts, and the DNA solution was mechanically inoculated onto the true leaves of C. quinoa using carborundum. Reverse osmosis (RO) water was used for mock inoculation. The inoculated C. quinoa plants were grown for two to three weeks. The upper leaves were sampled and ground in three volumes of extraction buffer (0.1 M Tris-HCl, pH 8.0, 0.1 M NaCl, 5 mM MgCl₂ [23 (link)]). Debris was precipitated by centrifugation, and the supernatants were used for secondary inoculation of C. quinoa. After two to three weeks, the upper leaves were sampled and stored at -80°C. Total RNA, extracted from the upper leaves of C. quinoa plants subjected to a second inoculation, was used as an inoculum for soybean. Biolistic inoculation of germinated soybean seeds was performed using a PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA) and 1.0 Micron Gold Microcarrier (Bio-Rad) particles that had been coated with total RNA from C. quinoa as described [23 (link)]. Seven to ten germinated soybean seeds were placed onto a Petri dish and bombarded twice at 1,100 psi. Approximately 7 μg of total RNA was used per shot.

After coating gold particles with RNA from infected leaves, grapevine seedlings (10–15 plants/each inoculation) were inoculated using the Helios Gene Gun system (Bio-Rad Laboratories, München, Germany) following a previously reported method [43 ]. The air pressure used was 1379 kilopascal (kPa) and each cotyledon was shot once with gold particles. Grapevine in vitro cultures were inoculated using the PDS-1000/He Particle Delivery System (Bio-Rad Laboratories, München, Germany) at 1379 kPa, with two shots per petri dish. The GDS-80 gene gun system (Nepa Gene Co., Ltd., Ichikawa, Japan) was also used for inoculation of in vitro cultures at 207 kPa.

The C3sgRNA-Cas9 and pC3gRNA-rhBMP2 plasmids were combined at a molar ratio of 1:3. Subsequently, 1.5 mg of 1.0 μm gold particles were coated with 10 μg of the mixed plasmid DNA. The immature embryo-derived calli were transferred into half-strength Murashige and Skoog medium supplemented with 0.4% phytagel and bombarded once with 500 μg DNA-coated gold particles using a PDS-1000/He particle delivery system (Bio-Rad, Hercules, CA, USA) at 1100 psi with a target distance of 9 cm as previously described (Lu et al. 2002 (link)). Transformed calli were selected on N6 medium containing 50 mg/L hygromycin B as previously described (Lu et al. 2002 (link)). Hygromycin-resistant calli were used to regenerate T0 transgenic rice plants on a regeneration medium (Ozawa et al. 2003 (link)). These T0 plants were then self-pollinated in a greenhouse to produce T1 seeds. The T1 seeds were subsequently used to induce T1 calli and establish suspension cell cultures.

The full DKXTH1/2 coding sequence (DkXTH1/2Full), DKXTH1 signal peptide (DkXTH1sp), and DKXTH1 sequence without signal peptide (DkXTH1Int) were amplified by PCR. The appropriate restriction enzymes were used to excise the amplified fragments, which are underlined in the primers listed in Table 1. They were then ligated into the pBI 221-GFP vector fused with the GFP gene in the 3' region. DNA plasmids (5 μg) were applied to bombard onion epidermal cells by a biolistic PDS-1000/He particle delivery system (Bio-Rad, Hercules, CA, USA). All bombarded onion epidermal cells were then incubated on Murashige–Skoog medium for 24 h at 22°C in the dark, and analyzed using a confocal laser scanning microscope (A1R; Nikon, Japan). When indicated, cells were plasmolyzed in 400 mM sucrose for 15 min.

The full-length and truncated versions [C-terminal deletion (ΔC) of 165-261 amino acid (aa) for OsHOX24ΔC and 174-276 aa for OsHOX22ΔC] of homeobox genes were PCR amplified using gene-specific primers (Supplementary Table S2) and cloned in psGFPcs vector (Kapoor et al., 2002 (link)) using ApaI and XmaI restriction sites. The N-terminal GFP fusion constructs and empty vector (psGFPcs; experimental control) were transiently transformed in onion epidermal cells via particle bombardment method using PDS-1000 He particle delivery system (Bio-Rad Laboratories, Hercules, CA, USA) as described earlier (Sharma et al., 2014 (link)). The transformed cells were incubated in dark at 23°C for 24 h and onion peels were visualized under confocal microscope (AOBS TCS-SP2, Leica Microsystems, Mannheim) for detection of GFP and DAPI signals.

Tobacco pollen grains were transformed using a PDS-1000/He particle delivery system (Bio-Rad). Pollen grains were collected from freshly dehisced anthers and stored at -80°C until use. For each bombardment, 4 mg of pollen was suspended in 200 μL of pollen germination medium (1 mM CaCl₂, 1 mM Ca(NO₃)₂, 1 mM MgSO₄, 0.01% H₃BO₃, and 10% sucrose, pH 6.5) [28 (link)]. Then, the pollen suspension was placed on a piece of pre-wetted nylon membrane for bombardment. Microprojectiles were prepared by coating 1 mg of gold particles (1.0 μm) with 3 μg of plasmid, and were used for two bombardments per sample. Bombardment was performed with a 28-inch-Hg vacuum using a 1100-psi rupture disk and a 6-cm target distance. Pollen grains were washed from the membrane with 1.6 mL of germination medium onto a 35-mm Petri dish immediately after bombardment and cultured on a rotary shaker at 26°C at 100 rpm. After germination for 4 h, transformed pollen grains showing green fluorescence were visualized and photographed under a Zeiss LSM 700 confocal microscope. Three biological replicates were conducted, and more than 50 transformed pollen grains were scored for the germination rate and pollen tube length per replicate. Statistical analysis was performed using one-way analysis of variance with Tukey’s test (P < 0.05).