Transient expression of GmCOL2, GmCOL5 and GmFTL1 to 6 tagged by YFP in soybean young leaves was performed with a Model PDS-1000/He Biolistic Particle Delivery System (Bio-Rad). 10 micrograms of purified plasmids were coated with 500 μg 1 μm-gold particles, as described by the manufacturer. After bombardment, young soybean leaves were incubated overnight at 25°C on solid 1/2 MS medium. Fluorescent cells were imaged by confocal microscopy (Leica TCS SP5, Leica Microsystem, Wetzlar, Germany). YFP was excited by the 514-nm argon laser line, and PI (Propidium iodide) stain was excited using a 561-nm He-Ne laser. Fluorescence was detected using photomultiplier tube settings as follows: YFP (520 to 560 nm), and PI (570 to 620 nm). At last, post-acquisition image analyzing and processing were performed using MBF ImageJ (version 1.46) (https://www.macbiophotonics.ca/ ).
Model pds 1000 he biolistic particle delivery system
Manufactured by Bio-Rad
Sourced in United States
The Model PDS-1000/He Biolistic Particle Delivery System is a laboratory equipment designed to facilitate the delivery of DNA-coated particles into target cells or tissues. The core function of this system is to provide a controlled and efficient method for the introduction of genetic material into living samples using high-velocity helium gas.
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5 protocols using model pds 1000 he biolistic particle delivery system
1
Transient Expression of Soybean Clock Genes
2
BiFC Assay for Protein-Protein Interactions
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The full-length ORFs of IQM4 and CaM5 were generated by RT-PCR using IQM4-F5/R5 primers and CaM5-F/R primers, respectively (Supplementary Table S1 ), for the BiFC assay using onion epidermal cells. IQM4 ORF was fused into the Hind III and Xma I sites downstream of the nEYFP gene in the pSATN-nEYFP-C1 vector, and CaM5ORF was inserted into the EcoR I and Sal I sites downstream of cEYFP in the pSATN-cEYFP-C1 vector (Citovsky et al., 2006 (link)). The plasmid combinations were introduced into onion epidermal cells with the Model PDS-1000/He Biolistic Particle Delivery System (Bio-Rad) using 1-μm diameter gold particles, as described in the manufacturer’s instructions. After bombardment, the onion epidermal tissues were incubated overnight at 25°C on solid Murashige-Skoog (MS) medium in the dark, and observed with an epifluorescence microscope (TE2000-U, Nikon, Tokyo, Japan).The plasmid combinations were introduced into Arabidopsis mesophyll protoplasts as described by Sheen (2001) (link). After incubating for 18 h at 23°C in the dark, the protoplasts were examined for YFP signal using confocal laser scanning microscopy (LSM 7 DUO, Zeiss).
3
Transient Expression in Onion Epidermal Cells
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Agrobacterium-mediated transformation in Arabidopsis by floral dip and transient expression in tobacco by infiltration were performed, as described previously [28 (link)]. To visualize the subcellular localization of GmCWI4 in onion epidermal cells, a transient expression system was established based on an optimized protocol. Control plasmid (SecGFP) and YFP-fusion plasmid were bombarded with 1.6 μm gold particles while using the Model PDS-1000/He Biolistic Particle Delivery System (Bio-Rad, Berkeley, CA, USA) with 4481 kPa helium pressure, a vacuum of 86 kPa and a distance of 6 cm. Bombarded cells were incubated for 48 h and then visualized by confocal laser scanning microscopy (CLSM) (Zeiss, Germany). GFP fluorescence detection of transformed cells should treat with 20 mm PIPES buffer (pH 7.0) for 3 h after 48h incubation.
4
Expressing GFP-tagged Ccdc42 in Tetrahymena
5
Genetic Modification of C. neoformans
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