Gene gun method is also called microprojectile bombardment, parti-cle bombardment and biolistics. The gunpowder-detonated gene gun was first developed by Sanford et al. (1987) of Conell University in the United States. In 1987, Klein et al. first used onion epidermal cells as materials and tungsten powder as bullets to introduce DNA or RNA into epidermal cells, and foreign genes could be expressed, proving that this method can achieve genetic transformation of foreign genes. Since the genetic transformation of monocots is limited by the Agrobacterium host, the transfer of foreign genes by gene gun method has become the main means of genetic transformation of monocots.
The basic principle is to coat exogenous DNA on the surface of tiny gold or tungsten particles, and then under the effect of gunpowder explosion, high-pressure gas or high-pressure discharge and other high-pressure,the particles are injected into the recipient cells or tissues, and the foreign DNA is randomly integrated into the genome of the host cell and expressed, thereby realizing the transfer of foreign genes.
Characteristics of gene gun transformation of plants: no host restrictions, can transform any plant, especially those monocotyledonous plants that are difficult to regenerate plants from protoplasts and are not sensitive to Agrobacterium infection; wide range of target receptor types, capable of transforming any tissue or cell of the plant; easy and fast operation. Although gene gun transformation technology has achieved gratifying results since its inception, there are still many problems such as low transformation rate that need further research.
1. Preparation of DNA microprojectiles
a. Weigh 50-60 mg of tungsten powder or gold powder (the particle diameter is preferably 1/10 of the cell diameter) and place it in a 1.5 mL sterilized centrifuge tube. Add 1 mL of absolute ethanol, shake and suspend several times, centrifuge at 4,000-10,000 r/min for 10 s, and discard the supernatant.
b. Add 1 mL of sterile water to wash the tungsten powder or gold powder precipitate, shake and centrifuge, and discard the supernatant. Repeat twice, suspend tungsten powder or gold powder in 1 mL sterile water, use now or store at -20°C.
c. Take 50 µL of the particle suspension into a new sterilized centrifuge tube, add 3-5 µg plasmid DNA, 50 µL 2.5 mol/L CaCl2 solution and 20 µL 0.1 mol/L spermidine solution. After mixing, let stand at room temperature for 10 min to allow the DNA to fully settle onto the microsomes. Centrifuge at 10,000~15,000 r/min for 5-10 s and discard the supernatant.
d. Rinse twice with absolute ethanol, add 60 µL absolute ethanol to resuspend the particles, and set aside.
2. Receptor material preparation
Take the young embryos 12-16 d after flowering and disinfect them with 0.1% HgCl2 for 8-15 min. Rinse 3-5 times with sterile water, peel off the young embryos on a clean workbench, inoculate them with the scutellum facing up on callus induction medium, and culture at (27±2)°C in the dark. Wheat embryos precultured for 3 d were transferred to hypertonic medium and used as transformation recipients for transformation 4-6 h later.
3. Loading
a. Use 70% ethanol to clean the vacuum chamber and ultra-clean work surface. Soak the rupture membrane, bombardment membrane and barrier net in 70% ethanol for 15 min, and blow dry for later use.
b. Turn on the power switches of the vacuum pump and gene gun and the helium cylinder valve.
c. Unscrew the cover of the rupture membrane, place the rupture membrane in the center of the cover, and screw the cover back on.
d. Take 10 µL DNA-tungsten powder or gold powder complex and apply it evenly on the bombardment membrane, let it dry and then bombard it.
e. Install the bombardment membrane and blocking net containing DNA particle bombs into the particle launcher.
f. Place the pretreated receptor material in the appropriate position of the bombardment chamber (the distance between the blocking plate and the target cell stage can be 6 cm), and close the door of the bombardment chamber tightly.
4. Bombardment
a. Press the VAC button to vacuum. When the reading on the meter is the required value (88-101 kPa), turn the switch to HOLD.
b. Press the FIER key to bring the helium pressure to the appropriate value (corresponding to the rupture membrane model, 1,100 Pa rupture membrane is optional). When the appropriate pressure is reached, the rupture membrane will burst automatically. Release the FIER key and hold down the VENT key to release the vacuum in the bombardment chamber. Open the door of the bombardment chamber and take out the sample. Usually, bombard each dish twice. Rotate the dish 180° horizontally or turn the receptor material over before the second bombardment.
5. Transitional culture
After bombardment, the immature embryos were inoculated and cultured on hypertonic medium for 24 h, then transferred to callus induction medium without selective pressure, (27±2)°C, and cultured in the dark. The culture time depends on the specific conditions of the explant. The general principle is that bombarded explants should have sufficient time to recover and for foreign genes to enter and express in recipient cells.
6. Selective culture
The explants after transitional culture are inoculated and cultured in a medium adding appropriate selection pressure (such as kanamycin) for selective culture until regenerated plants are obtained.
7. Detection of transgenic plants
a. Histochemical method. GUS gene expression was detected in calli and leaves according to the histochemical method. GUS detection of Kan-resistant callus: Cut the obtained Kan-resistant callus or leaves into small pieces, add them to the prepared X-gluc substrate solution, and incubate at 37°C overnight. Fix with FAA solution for more than 1 h, and decolorize with 70%, 90%, and 100% ethanol in sequence, and visually observe whether there is GUS gene expression on the surface and inside the callus.
b. PCR identification. The genomic DNA of tobacco to-be-transgenic plants was extracted as a template. Design primers based on the selection marker gene or target gene sequence, and conduct PCR amplification detection using the total DNA of non-transgenic plants (negative control), empty strain transformed plants (negative control) and vector plasmid DNA (positive control) as controls to preliminarily identify whether the foreign gene has been integrated into tobacco genomic DNA.
c. Southern blot analysis. The genomic DNA of tobacco to-be-transgenic plants was extracted and double-digested with restriction endonucleases. The processed DNA was electrophoresed on a 0.7%-0.9% agarose gel, and probe labeling, DNA transfer, membrane treatment, and hybridization operations were performed. Further identify whether the foreign gene is integrated into the genomic DNA of tobacco and the copy number of the foreign gene (control selection is the same as above).
d. RT-PCR. Use the Trizol kit to extract the total RNA of tobacco to-be-transgenic plants, and use the reverse transcription PCR kit to perform RT reaction and PCR reaction on the total RNA according to the prescribed procedures. Further identify the expression of exogenous genes at the RNA level (control selection is the same as above).
e. Northern blot analysis. Extract and purify the total RNA or mRNA of tobacco to-be-transgenic plants, conduct 1.2% (W/V) formaldehyde-denaturing agarose gel electrophoresis, and perform probe labeling, RNA transfer, membrane treatment, and hybridization operations. Further identify the expression of foreign genes in plant cells (control selection is the same as above).
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