The bioluminescent reporter gene, with an effector gene, was introduced into plants through particle bombardment. A total of 8 μl of prewashed gold particle suspension (1 μm diameter; Bio-Rad) in 50% glycerol (60 mg·ml−1) was mixed with 2 μl of reporter DNA (1 μg μl−1), 1 μl effector DNA (1 μg·μl−1), 6 μl of spermidine (0.1 M) and 15 μl of CaCl2 (2.5 M) in a tube. Preparation of the DNA-coated gold particles and the transfection by a helium gun device (PDS-1000/He; Bio-Rad) were conducted as described previously2 (link).
Pds 1000 he
Manufactured by Bio-Rad
Sourced in United States
The PDS-1000/He is a gene delivery device that uses helium gas pressure to propel DNA-coated gold or tungsten particles into cells or tissues. It is designed to facilitate the introduction of genetic material into a variety of target cells or tissues for research applications.
Automatically generated - may contain errors
Lab products found in correlation
Gold particles, by Bio-Rad (5 mentions)
Lsm 510, by Zeiss (5 mentions)
Lsm 510 meta, by Zeiss (4 mentions)
Tcs sp8, by Leica (3 mentions)
Fluoview fv1000, by Olympus (2 mentions)
D luciferin, by Fujifilm (2 mentions)
Tcs sp5, by Leica (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
0.6 μm gold particles, by Bio-Rad (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Hepta adapter, by Bio-Rad (1 mentions)
Mrc 1024, by Bio-Rad (1 mentions)
Hepta adaptor, by Bio-Rad (1 mentions)
Te2000 e, by Nikon (1 mentions)
Protamine, by Merck Group (1 mentions)
1 μm gold particles, by Bio-Rad (1 mentions)
Fv1000, by Olympus (1 mentions)
Csu x1, by Nikon (1 mentions)
Ti e platform, by Agilent Technologies (1 mentions)
63 protocols using pds 1000 he
1
Bioluminescent Gene Expression in Plants
2
Subcellular Localization of ZmEREB156
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The complete coding sequence of ZmEREB156, minus the stop codon, was ligated into the pCAMBIA2300-35S-eGFP vector after amplification by PCR using primer G156 (Supplementary Table S8 ) with BamHI and XbaI restriction sites to create the fusion construct (pCAMBIA2300-ZmEREB156-eGFP). The empty pCAMBIA2300-35S-eGFP vector was used as a control. The fusion construct and the control were introduced into onion (Allium cepa L.) epidermal cells by particle bombardment using PDS-1000/He (Bio-Rad)50 . The transformed cells were incubated on 1/2 MS medium for 24–48 h at 28 °C. The subcellular localization of GFP fusion proteins was visualized with a fluorescence microscope BX61 (Olympus, Japan).
3
Subcellular Localization of AlTMP1 Protein
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The AlTMP1 coding sequence was amplified by PCR using AlTMP1-B and AlTMP1-X primers containing BamHI and XbaI restriction sites (Table S1 ). The amplified fragment was cloned into BamHI/XbaI sites of the binary vector pCAMBIA2300-GFP to generate the fusion genes GFP::AlTMP1 driven by the CaMV-35S promoter. Both constructs pCAMBIA2300-GFP::AlTMP1 and pCAMBIA2300-GFP (as a control) were separately bombarded into a single layer of onion epidermal cells using the PDS-1000/He (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol [50 (link)]. After incubation on MS medium for 36 h at 28 °C in darkness, the GFP signal was monitored, and the microscope imaging was performed at Montpellier RIO Imaging Platform (http://www/mri/cnrs.fr ) using a confocal microscope (LSM 510, Meta; Carl Zeiss MicroImaging, http://www.zeiss.de ).
4
Optimizing Particle Bombardment Conditions
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The bombardments were performed with a particle gun (PDS 1000/He, Bio-Rad). The transformation conditions of the particle gun were optimized using one 2-level factor (precipitation agents) and five 3-level factors (target tissue distance, helium pressure, bombardment times, plasmid DNA concentration, and chamber vacuum pressure). These parameters and the levels of the variables studied are shown in Table 3 . A hybrid orthogonal table L18(21 × 35) was designed according to the above six parameters and different levels of each parameter [55 (link)], as shown in Table 6 , where A to F represent precipitation agents, target tissue distance, helium pressure, number of bombardments, plasmid DNA concentration, and chamber vacuum pressure. Each factor was repeated three times, and the whole set of experiments was repeated twice.
5
Marker-free Lettuce Transformation via Biolistic Bombardment
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The marker‐free expression vectors, pLs‐MFpelB and pLs‐MF pelD, were bombarded into young (3 weeks old) and fully expanded lettuce leaves through biolistic bombardment as described by Ruhlman et al. (2010 ). The adaxial side of lettuce leaves was bombarded using 0.6‐μm gold particles (Bio‐Rad, Hercules, CA) coated with above mentioned vectors, using the biolistic device PDS1000/He (Bio‐Rad), 1100 psi rupture discs and a target distance of 6 cm. Following incubation at 25 °C in the dark for 2 days, the leaves were cut into small (<1 cm2) pieces and placed adaxial side down on the regeneration media. Primary shoots (4–8 weeks) were analysed by PCR using the three pairs of primers, 16s‐F/3M‐R, 16s‐F/atpB‐R and 23s‐F/3UTR‐R. Leaves from the PCR‐positive shoots were again cut into small less 5 mm2 pieces and transferred to regeneration medium containing spectinomycin for the second round of selection and then moved to rooting medium, containing spectinomycin (50 μg/mL). Green shoots that on this selection medium were evaluated by PCR for aadA gene excision, and these shoots were transferred to rooting medium without spectinomycin.
6
Visualization of CAL1 Protein Localization
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The fragment of mRFP was amplified by PCR using primers RFPF and RFPR (Supplementary Table 5 ) and subcloned to generate the construct 35S:: mRFP/PA7. The coding sequence of CAL1 was further amplified using PCR primers 5′CAL1-RFPF and 5′CAL1-RFPR or 3′CAL1-RFPF and 3′CAL1-RFPR (Supplementary Table 5 ), and the resulted fragments were fused in-frame to the 5′- or 3′- terminus of mRFP, respectively, to generate the constructs 35S::CAL1- mRFP/PA7 and 35S:: mRFP-CAL1/PA7. All constructs were then transiently expressed in onion epidermal cells using a particle gun–mediated system (PDS-1000/He; Bio-Rad). The bombarded cells were held in the dark at 28 °C for 16 h followed by mRFP imaging using confocal microscopy (Carl Zeiss; LSM510Meta). Alternatively, the 35S::CAL1-mRFP/pCAMBIA1300 construct was transformed into CJ06, and indicated tissues of the transgenic lines were subjected to mRFP imaging. For protoplast isolation, rice plants were grown to 2 weeks old in hydroponics supplemented with 5 µM Cd. Leaf sheaths from about 30 seedlings were cut into approximately 0.5 mm strips with propulsive force using sharp razors to isolate protoplasts52 (link). Metal concentration in protoplasts were determined by ICP-MS and normalized to corresponding total proteins.
7
Tobacco Plastid Transformation via Gene Gun
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The PDS-1000He (Bio-Rad, Hercules, CA, USA) gene gun with 900 psi was used for the transformation of tobacco leaves through the gene bombardment method. It involved using the pVSR326-IFN-β plasmid DNA-coated gold (0.6 microns) micro-projectile (Daniell, 1997 ). Nearly 100 tobacco leaves were bombarded with the prepared plasmid. Another 100 tobacco leaves were transformed with the native Agrobacterium tumefaciens without any constructs as control for bioactivity assay.
After bombardment, the leaves were placed on the RMOP medium and incubated in dark at 25°C for 48 h under a 16 : 8-h photoperiod. The bombarded leaves were cut into 5 × 5 mm pieces and cultured in RMOP medium containing NAA (0.1 mg/l), BA (1 mg/l), and spectinomycin dihydrochloride (500 mg/l). They were left until a suitable time for the selection and regeneration of transplastomic tobacco cells. Six to eight weeks later, putative transplastomic shoots were identified as resistant to spectinomycin. Three rounds of regeneration involved using small sections of the shoots as explants on the RMOP medium containing spectinomycin. The antibiotic-resistant shoots were further cultured on the basic MS medium in the growth chamber with the 16 : 8-h light/dark photoperiod at 25°C. The rooted transplastomic plants were potted and continued to grow in a growth room at 26°C and a 16 : 8-h photoperiod.
After bombardment, the leaves were placed on the RMOP medium and incubated in dark at 25°C for 48 h under a 16 : 8-h photoperiod. The bombarded leaves were cut into 5 × 5 mm pieces and cultured in RMOP medium containing NAA (0.1 mg/l), BA (1 mg/l), and spectinomycin dihydrochloride (500 mg/l). They were left until a suitable time for the selection and regeneration of transplastomic tobacco cells. Six to eight weeks later, putative transplastomic shoots were identified as resistant to spectinomycin. Three rounds of regeneration involved using small sections of the shoots as explants on the RMOP medium containing spectinomycin. The antibiotic-resistant shoots were further cultured on the basic MS medium in the growth chamber with the 16 : 8-h light/dark photoperiod at 25°C. The rooted transplastomic plants were potted and continued to grow in a growth room at 26°C and a 16 : 8-h photoperiod.
8
Transient Tobacco Pollen Transformation
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Mature pollen grains were collected from four to five tobacco flowers of approximately eight- to nine-week-old plants. The pollens were resuspended in liquid pollen tube growth media [44 (link)], followed by filtering of the pollen grains onto a cellulose acetate filter, before being transferred onto Whatmann paper which was moistened with pollen tube growth media. They were immediately transformed by bombardment with plasmid-coated 1-μm gold particles with a helium-driven particle accelerator (PDS-1000/He; Bio-Rad) using 1350 psi rupture disks and a vacuum of 28 inches of mercury, as previously described [45 (link)]. Prior to the bombardment, gold particles were coated with 4–5 µg of the desired plasmid of interest. After bombardment, the pollen grains were transferred into 300 µL of pollen tube growth media which was then equally divided onto three microscopic glass slides and viewed under the microscope 4−6 h after the bombardment.
9
Subcellular Localization of OsGA2ox5 and OsGHD7
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The coding region of OsGA2ox5 was amplified using the primer pair OsGA2ox5-pA7YFPF and OsGA2ox5-pA7YFPR (sangon) (the specific primers are listed in supplemental Table S1 ) and cloned into pA7-YFP [31] (link), generating the OsGA2ox5-YFP fusion under the control of the CaMV 35S promoter. A previously study demonstrated that OsGHD7 [32] (link) is a nuclei protein and localized in nuclei only, so we used OsGHD7 as a positive control. The OsGHD7 coding sequence was fused in frame to the N-terminus of YFP under the control of the CaMV 35S promoter. Then, OsGA2ox5-YFP, OsGHD7-YFP fused construct and pA7-YFP vectors were used to transiently transform onion epidermal cells by particle bombardment[33] using a particle gun system (PDS-1000/He; Bio-Rad). After 24 h, the epidermal cells were examined for YFP fluorescence under a scanning confocal microscope (Zeiss LSM510; Carl Zeiss Micro-Imaging GmbH, Jena, Germany).
10
CYP Gene Transformation and Analysis
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The fragments of these 10 candidate CYP genes were amplified using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA) and inserted into the RNAi vector pK7GWIWG2D according to the Gateway procedure (Invitrogen, USA), and the resulting vectors were verified by complete sequencing.
Suspension cells in the logarithmic growth phase were chose and precultured on Murashige and Skoog (MS) solid medium containing 0.5 mg L−1 2,4-D, 0.1 mg L−1 KT, 0.5 mg L−1 IBA and 30 g L−1 sucrose (pH = 5.8) for 7 days. Then, the recombinant plasmid DNA mixed with Au microparticles were transformed into the suspension cells through bombardment using a biolistic gene gun (PDS 1000/He, Bio-Rad). Each transformation was carried out two times. The bombarded suspension cells were cultured for another 7 days before harvesting for qRT-PCR and UPLC analysis73 (link).
Suspension cells in the logarithmic growth phase were chose and precultured on Murashige and Skoog (MS) solid medium containing 0.5 mg L−1 2,4-D, 0.1 mg L−1 KT, 0.5 mg L−1 IBA and 30 g L−1 sucrose (pH = 5.8) for 7 days. Then, the recombinant plasmid DNA mixed with Au microparticles were transformed into the suspension cells through bombardment using a biolistic gene gun (PDS 1000/He, Bio-Rad). Each transformation was carried out two times. The bombarded suspension cells were cultured for another 7 days before harvesting for qRT-PCR and UPLC analysis73 (link).
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